Runx2 Deletion in Smooth muscle Cells Inhibits Vascular Osteochondrogenesis and Calcification but not Atherosclerotic Lesion Formation.

Lin ME, Chen TM, Wallingford MC, Nguyen NB, ... Speer MY, Giachelli CM
Vascular smooth muscle cells (SMCs) are major precursors contributing to osteochondrogenesis and calcification in atherosclerosis. Runt-related transcription factor-2 (Runx2) has been found essential for SMC differentiation to an osteochondrogenic phenotype and subsequent calcification in vitro A recent study using a conditional targeting allele that produced a truncated Runx2 protein in SMCs of ApoE(-/-) mice showed reduced vascular calcification, likely occurring via reduction of receptor activator of nuclear factor-κB ligand (RANKL), macrophage infiltration, and atherosclerotic lesion formation. Using an improved conditional Runx2 knockout mouse model, we have elucidated new roles for SMC-specific Runx2 in arterial intimal calcification (AIC) without effects on atherosclerotic lesion size.

Cardiovasc Res: 26 Sep 2016; epub ahead of print

The multifactorial nature of microRNAs in vascular remodelling.

Welten SM, Goossens EA, Quax PH, Nossent AY
Vascular remodelling is a multifactorial process which involves both adaptive and maladaptive changes of the vessel wall through amongst others, cell proliferation and migration, but also apoptosis and necrosis of the various cell types in the vessel wall. Vascular remodelling can be beneficial e.g. during neovascularization after ischemia, as well as pathological e.g. during atherosclerosis and aneurysm formation. In recent years, it has become clear that microRNAs are able to target many genes that are involved in vascular remodelling processes and either can promote or inhibit structural changes of the vessel wall. Since many different processes of vascular remodelling are regulated by similar mechanisms and factors, both positive and negative vascular remodelling can be affected by the same microRNAs.A large number of microRNAs has been linked to various aspects of vascular remodelling and indeed, several of these microRNAs regulate multiple vascular remodelling processes, including both the adaptive processes angiogenesis and arteriogenesis as well as maladaptive processes of atherosclerosis, restenosis and aneurysm formation. Here, we discuss the multifactorial role of microRNAs and microRNA clusters that were reported to play a role in multiple forms of vascular remodelling and are clearly linked to cardiovascular disease. The microRNAs reviewed are miR-126, miR-155 and the microRNA gene clusters 17-92, 23/24/27, 143/145 and 14q32. Understanding the contribution of these microRNAs to the entire spectrum of vascular remodelling processes is important, especially as these microRNAs may have great potential as therapeutic targets for treatment of various cardiovascular diseases.

Cardiovasc Res: 24 Feb 2016; epub ahead of print

Cardiomyocyte exosomes regulate glycolytic flux in endothelium by direct transfer of GLUT transporters and glycolytic enzymes.

Garcia NA, Moncayo-Arlandi J, Sepulveda P, Diez-Juan A
Cardiomyocytes (CM) and endothelial cells (ECs) have an intimate anatomical relationship which is essential for maintaining the metabolic requirements of the heart. Little is known about the mechanisms that regulate nutrient flow from ECs to associated cardiomyocytes, especially in situations of acute stress when local active processes are required to regulate endothelial transport. We examined whether cardiomyocyte-derived exosomes can modulate glucose transport in ECs. In conditions of glucose deprivation, cardiomyocytes increase the synthesis and secretion of exosomes. These exosomes are loaded with functional glucose transporters and glycolytic enzymes, which are internalized by ECs, leading to increased glucose uptake, glycolytic activity and pyruvate production in recipient cells. These findings establish cardiomyocyte-derived exosomes as key components of the cardio-endothelial communication system which, through intercellular protein complementation, would allows a rapid response from ECs to increase glucose transport and a putative uptake of metabolic fuels from blood to cardiomyocytes. This cardiomyocyte-EC protein complementation process might have implications for metabolic regulation in health and disease.

Cardiovasc Res: 25 Nov 2015; epub ahead of print

Old dog, new tricks: novel cardiac targets and stress regulation by protein kinase G.

Rainer PP, Kass DA
The second messenger cyclic guanosine 3\'5\' monophosphate (cGMP) and its downstream effector protein kinase G (PKG) have been discovered more than 40 years ago. In vessels, PKG1 induces smooth muscle relaxation in response to nitric oxide signalling and thus lowers systemic and pulmonary blood pressure. In platelets, PKG1 stimulation by cGMP inhibits activation and aggregation, and in experimental models of heart failure (HF), PKG1 activation by inhibiting cGMP degradation is protective. The net effect of the above-mentioned signalling is cardiovascular protection. Yet, while modulation of cGMP-PKG has entered clinical practice for treating pulmonary hypertension or erectile dysfunction, translation of promising studies in experimental HF to clinical success has failed thus far. With the advent of new technologies, novel mechanisms of PKG regulation, including mechanosensing, redox regulation, protein quality control, and cGMP degradation, have been discovered. These novel, non-canonical roles of PKG1 may help understand why clinical translation has disappointed thus far. Addressing them appears to be a requisite for future, successful translation of experimental studies to the clinical arena.

Cardiovasc Res: 13 Jun 2016; epub ahead of print

Systems Biology - Opportunities and Challenges: The Application of Proteomics to Study the Cardiovascular Extracellular Matrix.

Barallobre-Barreiro J, Lynch M, Yin X, Mayr M
Systems biology approaches including proteomics are becoming more widely used in cardiovascular research. In this review article, we focus on the application of proteomics to the cardiac extracellular matrix. Extracellular matrix remodelling is a hallmark of many cardiovascular diseases. Proteomic techniques using mass spectrometry provide a platform for the comprehensive analysis of extracellular matrix proteins without a priori assumptions. Proteomics overcomes various constraints inherent to conventional antibody detection. On the other hand, studies that use whole tissue lysates for proteomic analysis mask the identification of the less abundant extracellular matrix constituents. In this review, we first discuss decellularization-based methods that enrich for extracellular matrix proteins in cardiac tissue, and how targeted mass spectrometry allows for accurate protein quantification. The second part of the review will focus on post-translational modifications including hydroxylation and glycosylation and on the release of matrix fragments with biological activity (matrikines), all of which can be interrogated by proteomic techniques.

Cardiovasc Res: 15 Sep 2016; epub ahead of print

Keeping up the balance: Role of HDACs in cardiac proteostasis and therapeutic implications for atrial fibrillation.

Zhang D, Hu X, Henning RH, Brundel BJ
Cardiomyocytes are long-lived post-mitotic cells with limited regenerative capacity. Proper cardiomyocyte function depends critically on the maintenance of a healthy homeostasis of protein expression, folding, assembly, trafficking, function and degradation, together commonly referred to as proteostasis. Impairment of proteostasis has a prominent role in the pathophysiology of ageing-related neurodegenerative diseases including Huntington\'s, Parkinson\'s, and Alzheimer\'s disease. Emerging evidence reveals also a role for impaired proteostasis in the pathophysiology of common human cardiac diseases such as cardiac hypertrophy, dilated and ischemic cardiomyopathies and atrial fibrillation (AF). Histone deacetylases (HDACs) have recently been recognized as key modulators which control cardiac proteostasis by deacetylating various proteins. By deacetylating chromatin proteins, including histones, HDACs modulate epigenetic regulation of pathological gene expression. Also, HDACs exert a broad range of functions outside the nucleus by deacetylating structural and contractile proteins. The cytosolic actions of HDACs result in changed protein function through post-translational modifications and/or modulation of their degradation. This review describes the mechanisms underlying the derailment of proteostasis in AF and subsequently focuses on the role of HDACs herein. In addition, the therapeutic potential of HDAC inhibition to maintain a healthy proteostasis resulting in a delay in AF onset and progression is discussed.

Cardiovasc Res: 08 Dec 2015; epub ahead of print

Endothelial Cell-Cardiomyocyte Crosstalk in Diabetic Cardiomyopathy.

Wan A, Rodrigues B
The incidence of diabetes is increasing globally, with cardiovascular disease accounting for a substantial number of diabetes-related deaths. Although atherosclerotic vascular disease is a primary reason for this cardiovascular dysfunction, heart failure in patients with diabetes might also be an outcome of an intrinsic heart muscle malfunction, labeled diabetic cardiomyopathy. Changes in cardiomyocyte metabolism, that encompasses a shift to exclusive fatty acid (FA) utilization, are considered a leading stimulus for this cardiomyopathy. In addition to cardiomyocytes, endothelial cells (EC) make up a significant proportion of the heart, with the majority of ATP generation in these cells provided by glucose. In this review, we will discuss the metabolic machinery that drives energy metabolism in the cardiomyocyte and EC, its breakdown following diabetes, and the research direction necessary to assist in devising novel therapeutic strategies to prevent or delay diabetic heart disease.

Cardiovasc Res: 10 Jun 2016; epub ahead of print

The intestine responds to heart failure by enhanced mitochondrial fusion through glucagon-like peptide-1 signalling.

Naruse G, Kanamori H, Yoshida A, Minatoguchi S, ... Nishigaki K, Minatoguchi S
Aims
Glucagon-like peptide-1 (GLP-1) is a neuroendocrine hormone secreted by the intestine. Its receptor (GLP-1R) is expressed in various organs, including the heart. However, the dynamics and function of the GLP-1 signal in heart failure remains unclear. We investigated the impact of the cardio-intestinal association on hypertensive heart failure using miglitol, an α-glucosidase inhibitor known to stimulate intestinal GLP-1 production.
Methods and results
Dahl salt-sensitive (DS) rats fed a high-salt diet were assigned to miglitol, exendin (9-39) (GLP-1R blocker) and untreated control groups and treated for 11 weeks. Control DS rats showed marked hypertension and cardiac dysfunction with left ventricular dilatation accompanied by elevated plasma GLP-1 levels and increased cardiac GLP-1R expression as compared with age-matched Dahl salt-resistant (DR) rats. Miglitol further increased plasma GLP-1 levels, suppressed adverse cardiac remodelling, and mitigated cardiac dysfunction. In cardiomyocytes from miglitol-treated DS hearts, mitochondrial size was significantly larger with denser cristae than in cardiomyocytes from control DS hearts. The change in mitochondrial morphology reflected enhanced mitochondrial fusion mediated by protein kinase A activation leading to phosphorylation of dynamin-related protein 1, expression of mitofusin-1 and OPA-1, and increased myocardial adenosine triphosphate (ATP) content. GLP-1R blockade with exendin (9-39) exacerbated cardiac dysfunction and led to fragmented mitochondria with disarrayed cristae in cardiomyocytes and reduction of myocardial ATP content. In cultured cardiomyocytes, GLP-1 increased expression of mitochondrial fusion-related proteins and ATP content. When GLP-1 and exendin (9-39) were administered together, their effects cancelled out.
Conclusions
Increased intestinal GLP-1 secretion is an adaptive response to heart failure that is enhanced by miglitol. This could be an effective strategy for treating heart failure through regulation of mitochondrial dynamics.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: [email protected]

Cardiovasc Res: 31 Oct 2019; 115:1873-1885

Role of T-type calcium current subunits in post-myocardial infarction remodelling probed with genetically engineered mice.

Le Quang K, Naud P, Qi XY, Duval F, ... Charpentier F, Nattel S
Aims: Previous studies suggested that T-type Ca(2+)-current (I(CaT))-blockers improve cardiac remodelling, but all available I(CaT)-blockers have non-specific actions on other currents and/or functions. To clarify the role of I(CaT) in cardiac remodelling, we studied mice with either of the principal cardiac I(CaT)-subunits (Cav3.1 or Cav3.2) knocked out. Methods and results: Adult male Cav3.1- or Cav3.2-knockout (Cav3.1(-/-), Cav3.2(-/-)) mice and respective wild-type (WT) littermate controls were subjected to left anterior descending coronary artery ligation to create myocardial infarction (MI). Echocardiography and programmed electrical stimulation were performed at baseline and 4 weeks post-MI. At baseline, Cav3.1(-/-) mice had slowed heart rates and longer PR intervals vs. WT, but no other electrophysiological and no haemodynamic differences. Cav3.2(-/-) showed no differences vs. WT. Contractile indices (left ventricular fractional shortening and ejection fraction) decreased more post-MI in Cav3.1(-/-) mice than in Cav3.1(+/+) (e.g. by 34 and 29% for WT; 50 and 45% for Cav3.1(-/-), respectively; P < 0.05 for each). Cav3.1(-/-) mice had increased ventricular tachycardia (VT) inducibility post-MI (9 of 11, 82%) vs. WT (3 of 10, 30%; P < 0.05). Cav3.2(-/-) mice were not different in cardiac function or VT inducibility vs. WT. Quantitative polymerase chain reaction showed that Cav3.1 is the major I(CaT)-subunit and that no compensatory Cav3.2 up-regulation occurs in Cav3.1(-/-) mice. Cav3.1(-/-) and Cav3.2(-/-) mice had no mRNA expression for the knocked-out gene, at baseline or post-MI. Conclusion: Our findings suggest that, contrary to suggestions from previous studies with (imperfectly selective) pharmacological agents having T-type Ca(2+)-channel-blocking actions, elimination of Cav3.1 expression leads to impaired cardiac function and enhanced arrhythmia vulnerability post-MI, whereas Cav3.2 elimination has no effect.

Cardiovasc Res: 30 May 2011; epub ahead of print

Inhibition of heat shock protein 70 blocks the development of cardiac hypertrophy by modulating the phosphorylation of histone deacetylase 2.

Yoon S, Kim M, Min HK, Lee YU, ... Eom GH, Kook H
Aims
Previously, we reported that phosphorylation of histone deacetylase 2 (HDAC2) and the resulting activation causes cardiac hypertrophy. Through further study of the specific binding partners of phosphorylated HDAC2 and their mechanism of regulation, we can better understand how cardiac hypertrophy develops. Thus, in the present study, we aimed to elucidate the function of one such binding partner, heat shock protein 70 (HSP70).
Methods and results
Primary cultures of rat neonatal ventricular cardiomyocytes and H9c2 cardiomyoblasts were used for in vitro cellular experiments. HSP70 knockout (KO) mice and transgenic (Tg) mice that overexpress HSP70 in the heart were used for in vivo analysis. Peptide-precipitation and immunoprecipitation assay revealed that HSP70 preferentially binds to phosphorylated HDAC2 S394. Forced expression of HSP70 increased phosphorylation of HDAC2 S394 and its activation, but not that of S422/424, whereas knocking down of HSP70 reduced it. However, HSP70 failed to phosphorylate HDAC2 in the cell-free condition. Phosphorylation of HDAC2 S394 by casein kinase 2α1 enhanced the binding of HSP70 to HDAC2, whereas dephosphorylation induced by the catalytic subunit of protein phosphatase 2A (PP2CA) had the opposite effect. HSP70 prevented HDAC2 dephosphorylation by reducing the binding of HDAC2 to PP2CA. HSP70 KO mouse hearts failed to phosphorylate S394 HDAC2 in response to isoproterenol infusion, whereas Tg overexpression of HSP70 increased the phosphorylation and activation of HDAC2. 2-Phenylethynesulfonamide (PES), an HSP70 inhibitor, attenuated cardiac hypertrophy induced either by phenylephrine in neonatal ventricular cardiomyocytes or by aortic banding in mice. PES reduced HDAC2 S394 phosphorylation and its activation by interfering with the binding of HSP70 to HDAC2.
Conclusion
These results demonstrate that HSP70 specifically binds to S394-phosphorylated HDAC2 and maintains its phosphorylation status, which results in HDAC2 activation and the development of cardiac hypertrophy. Inhibition of HSP70 has possible application as a therapeutic.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: [email protected]

Cardiovasc Res: 31 Oct 2019; 115:1850-1860

Imaging the injured beating heart intravitally and the vasculoprotection afforded by haematopoietic stem cells.

Kavanagh DPJ, Lokman AB, Neag G, Colley A, Kalia N
Aims
Adequate microcirculatory perfusion, and not just opening of occluded arteries, is critical to salvage heart tissue following myocardial infarction. However, the degree of microvascular perfusion taking place is not known, limited primarily by an inability to directly image coronary microcirculation in a beating heart in vivo. Haematopoietic stem/progenitor cells (HSPCs) offer a potential therapy but little is known about their homing dynamics at a cellular level and whether they protect coronary microvessels. This study used intravital microscopy to image the anaesthetized mouse beating heart microcirculation following stabilization.
Methods and results
A 3D-printed stabilizer was attached to the ischaemia-reperfusion injured (IRI) beating heart. The kinetics of neutrophil, platelet and HSPC recruitment, as well as functional capillary density (FCD), was imaged post-reperfusion. Laser speckle contrast imaging (LSCI) was used for the first time to monitor ventricular blood flow in beating hearts. Sustained hyperaemic responses were measured throughout reperfusion, initially indicating adequate flow resumption. Intravital microscopy confirmed large vessel perfusion but demonstrated poor transmission of flow to downstream coronary microvessels. Significant neutrophil adhesion and microthrombus formation occurred within capillaries with the latter occluding them, resulting in patchy perfusion and reduced FCD. Interestingly, \'patrolling\' neutrophils were also observed in capillaries. Haematopoietic stem/progenitor cells readily trafficked through the heart but local retention was poor. Despite this, remarkable anti-thromboinflammatory effects were observed, consequently improving microvascular perfusion.
Conclusion
We present a novel approach for imaging multiple microcirculatory perturbations in the beating heart with LSCI assessment of blood flow. Despite deceptive hyperaemic responses, increased microcirculatory flow heterogeneity was seen, with non-perfused areas interspersed with perfused areas. Microthrombi, rather than neutrophils, appeared to be the major causative factor. We further applied this technique to demonstrate local stem cell presence is not a pre-requisite to confer vasculoprotection. This is the first detailed in vivo characterization of coronary microcirculatory responses post-reperfusion injury.

© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 31 Oct 2019; 115:1918-1932

Long noncoding RNA NEAT1 modulates immune cell functions and is suppressed in early onset myocardial infarction patients.

Gast M, Rauch BH, Haghikia A, Nakagawa S, ... Zeller T, Poller W
Aims
Inflammation is a key driver of atherosclerosis and myocardial infarction (MI), and beyond proteins and microRNAs (miRs), long noncoding RNAs (lncRNAs) have been implicated in inflammation control. To obtain further information on the possible role of lncRNAs in the context of atherosclerosis, we obtained comprehensive transcriptome maps of circulating immune cells (peripheral blood mononuclear cells, PBMCs) of early onset MI patients. One lncRNA significantly suppressed in post-MI patients was further investigated in a murine knockout model.
Methods and results
Individual RNA-sequencing (RNA-seq) was conducted on PBMCs from 28 post-MI patients with a history of MI at age ≤50 years and stable disease ≥3 months before study participation, and from 31 healthy individuals without manifest cardiovascular disease or family history of MI as controls. RNA-seq revealed deregulated protein-coding transcripts and lncRNAs in post-MI PBMCs, among which nuclear enriched abundant transcript (NEAT1) was the most highly expressed lncRNA, and the only one significantly suppressed in patients. Multivariate statistical analysis of validation cohorts of 106 post-MI patients and 85 controls indicated that the PBMC NEAT1 levels were influenced (P = 0.001) by post-MI status independent of statin intake, left ventricular ejection fraction, low-density lipoprotein or high-density lipoprotein cholesterol, or age. We investigated NEAT1-/- mice as a model of NEAT1 deficiency to evaluate if NEAT1 depletion may directly and causally alter immune regulation. RNA-seq of NEAT1-/- splenocytes identified disturbed expression and regulation of chemokines/receptors, innate immunity genes, tumour necrosis factor (TNF) and caspases, and increased production of reactive oxygen species (ROS) under baseline conditions. NEAT1-/- spleen displayed anomalous Treg and TH cell differentiation. NEAT1-/- bone marrow-derived macrophages (BMDMs) displayed altered transcriptomes with disturbed chemokine/chemokine receptor expression, increased baseline phagocytosis (P < 0.0001), and attenuated proliferation (P = 0.0013). NEAT1-/- BMDMs responded to LPS with increased (P < 0.0001) ROS production and disturbed phagocytic activity (P = 0.0318). Monocyte-macrophage differentiation was deregulated in NEAT1-/- bone marrow and blood. NEAT1-/- mice displayed aortic wall CD68+ cell infiltration, and there was evidence of myocardial inflammation which could lead to severe and potentially life-threatening structural damage in some of these animals.
Conclusion
The study indicates distinctive alterations of lncRNA expression in post-MI patient PBMCs. Regarding the monocyte-enriched NEAT1 suppressed in post-MI patients, the data from NEAT1-/- mice identify NEAT1 as a novel lncRNA-type immunoregulator affecting monocyte-macrophage functions and T cell differentiation. NEAT1 is part of a molecular circuit also involving several chemokines and interleukins persistently deregulated post-MI. Individual profiling of this circuit may contribute to identify high-risk patients likely to benefit from immunomodulatory therapies. It also appears reasonable to look for new therapeutic targets within this circuit.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: [email protected]

Cardiovasc Res: 31 Oct 2019; 115:1886-1906

Enhanced expression of DYRK1A in cardiomyocytes inhibits acute NFAT activation but does not prevent hypertrophy in vivo.

Grebe C, Klingebiel TM, Grau SP, Toischer K, ... Hasenfuss G, Seidler T
Aims The calcineurin and nuclear factor of activated T cells (NFAT) pathway can mediate pro-hypertrophic signalling in the heart. Recently, it has been shown that dual-specificity tyrosine-phosphorylation regulated kinase 1A (DYRK1A) phosphorylates NFAT, which limits calcineurin/NFAT signal transduction in T cells and hypertrophy in cultured cardiomyocytes. The hypothesis tested in this study was that DYRK1A prevents calcineurin/NFAT-mediated cardiac hypertrophy in vivo. Methods and Results In cultured rat cardiomyocytes, adenovirus-mediated overexpression of DYRK1A antagonised calcineurin-mediated nuclear NFAT translocation and the phenylephrine-induced hypertrophic growth response. To test the ability of DYRK1A to reduce hypertrophic cardiac growth in vivo, we created tetracycline-repressible Dyrk1a transgenic mice to avoid the cardiac developmental defects associated with embryonic DYRK1A expression. However, in the mouse model, histological determination of myocyte diameter, heart weight/body weight ratio and echocardiographic measurements revealed that myocardial expression of DYRK1A failed to reduce hypertrophy induced via aortic banding or coexpression of calcineurin. This discrepancy is explained, at least in part, by insufficient long-term inhibition of NFAT and the activation of DYRK1A-resistant maladaptive genes in vivo. Conclusion Isolated augmentation of DYRK1A can be compensated for in vivo, and this may significantly limit anti-hypertrophic interventions aimed at enhancing DYRK1A activity.

Cardiovasc Res: 28 Jan 2011; epub ahead of print

Cardiac Salvage by Tweaking with Beta3-adrenergic Receptors.

Balligand JL
Overstimulation of the orthosympathetic system leads to cardiovascular cell and tissue damage through prolonged activation of beta1-2 adrenergic receptors (BARs). The more recent identification of the third isotype of BAR (B3AR) in cardiac myocytes and endothelial cells with a distinctive coupling and effect on cardiac function and remodeling introduced a new facet to this paradigm. In particular, B3AR is upregulated in cardiac disease and less prone to homologous desensitization, which may reinforce its influence on the diseased myocardium. Mice with transgenic cardiac-specific expression of the human B3AR are protected from cardiac hypertrophy and fibrosis in response to neurohormonal stimulation. B3AR has also been implicated in cardiac protection after ischemia-reperfusion and the benefits of exercise on the heart. Many of these salvage mechanisms are mediated by B3AR coupling to nitric oxide synthase (eNOS and nNOS) and downstream cGMP/protein kinase G signaling. Notably, B3AR exerts antioxidant protective effects on these and other signaling elements, which may subserve its protective properties in the setting of chronic heart failure. Additional vasorelaxing properties and paracrine NO-mediated signaling by B3AR in endothelium, together with systemic metabolic effects on beige/brown fat complete the pleiotropic protective properties of this new therapeutic target.

Cardiovasc Res: 21 Mar 2016; epub ahead of print

A sequential interferon gamma directed chemotactic cellular immune response determines survival and cardiac function post-myocardial infarction.

Finger S, Knorr M, Molitor M, Schüler R, ... Karbach S, Wenzel P
Aims
Myelomonocytic cells are critical in injury and healing post-myocardial infarction (MI). Mechanisms of regulation, however, are incompletely understood. The aim of the study was to elucidate the role of interferon gamma (IFN-γ) in the orchestrated inflammatory response in a murine model of MI.
Methods and results
MI was induced in 8- to 12-week-old male mice (C57BL/6 background) by permanent ligation of the left anterior descending (LAD) coronary artery. Lysozyme M (LysM)+ cell-depleted LysMiDTR transgenic mice displayed a reduced influx of CD45.2+/CD3-/CD11b+/Gr-1high neutrophils into infarcted myocardium 1 day post-MI compared with infarcted controls, paralleled by decreased cardiac mRNA levels of IFN-γ and tumour necrosis factor alpha (TNF-α). Mortality after MI was significantly increased in LysM+ cell-depleted mice within 28 days post-MI. To more specifically address the role of neutrophils, we depleted C57BL/6 mice with a monoclonal anti-Gr-1 antibody and found increased mortality, deteriorated cardiac function as well as decreased cardiac IFN-γ mRNA expression early after MI. Ccl2, Cxcl1, Cx3cl1, and Il12b mRNA were reduced 3 days after MI, as was the amount of CD11b+/Ly-6G-/Ly-6Chigh inflammatory monocytes. LAD-ligated Cramp-/- mice lacking cathelicidin important in neutrophil-dependent monocyte chemotaxis as well as IFNγ-/- and TNFα-/- mice phenocopied Gr-1+ cell-depleted mice, supporting a regulatory role of IFN-γ impacting on both the sequence of inflammatory cell invasion and cardiac outcome early after MI. The use of conditional IFN-γ receptor deficient mice indicated a direct effect of IFN-γ on LysM+ cells in cardiac injury post-MI. Using IFN-γ reporter mice and flow cytometry, we identified cardiac lymphoid cells (CD4+ and CD8+ T cells and natural killer cells) as primary source of this cytokine in the cardiac inflammatory response post-MI.
Conclusion
IFN-γ directs a sequential chemotactic cellular immune response and determines survival and cardiac function post-MI.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: [email protected]

Cardiovasc Res: 31 Oct 2019; 115:1907-1917

Pentraxin 3 deficiency protects from the metabolic inflammation associated to diet-induced obesity.

Bonacina F, Moregola A, Porte R, Baragetti A, ... Garlanda C, Norata GD
Aims
Low-grade chronic inflammation characterizes obesity and metabolic syndrome. Here, we aim at investigating the impact of the acute-phase protein long pentraxin 3 (PTX3) on the immune-inflammatory response occurring during diet-induced obesity.
Methods and results
PTX3 deficiency in mice fed a high-fat diet for 20 weeks protects from weight gain and adipose tissue deposition in visceral and subcutaneous depots. This effect is not related to changes in glucose homeostasis and lipid metabolism but is associated with an improved immune cell phenotype in the adipose tissue of Ptx3 deficient animals, which is characterized by M2-macrophages polarization and increased angiogenesis. These findings are recapitulated in humans where carriers of a PTX3 haplotype (PTX3 h2/h2 haplotype), resulting in lower PTX3 plasma levels, presented with a reduced prevalence of obesity and decreased abdominal adiposity compared with non-carriers.
Conclusion
Our results support a critical role for PTX3 in the onset of obesity by promoting inflammation and limiting adipose tissue vascularization and delineate PTX3 targeting as a valuable strategy for the treatment of adipose tissue-associated inflammatory response.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: [email protected]

Cardiovasc Res: 31 Oct 2019; 115:1861-1872

Succinate metabolism: a new therapeutic target for myocardial reperfusion injury.

Pell VR, Chouchani ET, Frezza C, Murphy MP, Krieg T
Myocardial ischemia/reperfusion (IR) injury is a major cause of death worldwide and remains a disease for which current clinical therapies are strikingly deficient. While the production of mitochondrial reactive oxygen species (ROS) is a critical driver of tissue damage upon reperfusion, the precise mechanisms underlying ROS production have remained elusive. More recently it has been demonstrated that a specific metabolic mechanism occurs during ischemia that underlies elevated ROS at reperfusion, suggesting a unifying model as to why so many different compounds have been found to be cardioprotective against IR injury. This review will discuss the role of the citric acid cycle intermediate succinate in IR pathology focussing on the mechanism by which this metabolite accumulates during ischemia and how it can drive ROS production at complex I via reverse electron transport (RET). We will then examine the potential for manipulating succinate accumulation and metabolism during IR injury in order to protect the heart against IR damage and discuss targets for novel therapeutics designed to reduce reperfusion injury in patients.

Cardiovasc Res: 18 May 2016; epub ahead of print

Effect of S-adenosylmethionine on neointimal formation after balloon injury in obese diabetic rats.

Lim S, Moon MK, Shin H, Kim TH, ... Kim YB, Park KS
Aims: The association between hyperhomocysteinaemia and cardiovascular disease has been attributed to low levels of S-adenosylmethionine (SAM), a metabolic intermediate of homocysteine. However, the role of SAM in the development of restenosis has not been explored. Therefore, we investigated the effects of SAM on neointimal formation after balloon injury in obese diabetic rats and cultured cells. Methods and results: Otsuka Long-Evans Tokushima fatty rats were divided into three groups: control (normal saline), SAM15, and SAM30 (15 and 30 mg/kg per day, respectively; n = 10 per group). SAM was administered orally from one week before carotid injury to two weeks after it. SAM treatment for three weeks caused a significant dose-dependent reduction in the intima-to-media ratio (IMR). SAM treatment significantly reduced the proliferation of vascular smooth muscle cells (VSMCs) and induced more apoptosis than was observed in the controls. This effect was accompanied by reduced circulating levels of high-sensitivity C-reactive protein and monocyte chemoattractant protein-1, reduced urine 8-hydroxy-2\'-deoxyguanosine (8-OHdG), and increased adiponectin. IMR correlated significantly with the levels of inflammatory markers, adiponectin, and 8-OHdG. In vitro experiments demonstrated that VSMC proliferation and migration and the adhesion of monocytes decreased in response to SAM. SAM treatment also reduced tumor-necrosis-factor-α- induced reactive oxygen species and tunicamycin-induced GRP78 expression in VSMCs. Conclusions: These findings suggest that SAM exerts protective effects against restenosis after balloon injury in a rat model of type 2 diabetes by reducing the proliferation and inducing the apoptosis of VSMCs, modifying the inflammatory processes and reducing oxidative and endoplasmic reticulum stresses.

Cardiovasc Res: 19 Jan 2011; epub ahead of print

Biology of PCSK9: beyond LDL cholesterol lowering.

Norata GD, Tavori H, Pirillo A, Fazio S, Catapano AL
Proprotein convertase subtilisin kexin 9 (PCSK9) is a key regulator of low density lipoprotein receptor (LDLR) levels and LDL-C levels. Loss-of-function mutations in PCSK9 gene are associated with hypocholesterolemia and protection against cardiovascular disease, identifying PCSK9 inhibition as a valid therapeutic approach to manage hypercholesterolemia and related diseases. Although PCSK9 is expressed mainly in the liver, it is present also in other tissues and organs with specific functions, raising the question of whether a pharmacological inhibition of PCSK9 to treat hypercholesterolemia and associated cardiovascular diseases might be helpful or deleterious in non-hepatic tissues. For example, PCSK9 is expressed in the vascular wall, in the kidneys, in the brain, where it was proposed to play a role in development, neurocognitive process and neuronal apoptosis. A link between PCSK9 and immunity was also proposed as both sepsis and viral infections are differentially affected in the presence or absence of PCSK9. Despite the increasing number of observations, the debate on the exact roles of PCSK9 in extrahepatic tissues is still ongoing, and as very effective drugs that inhibit PCSK9 have become available to the clinician, a better understanding of the biological roles of PCSK9 is warranted.

Cardiovasc Res: 05 Aug 2016; epub ahead of print

Pathways for salvage and protection of the heart under stress: novel routes for cardiac rejuvenation.

Cannatà A, Camparini L, Sinagra G, Giacca M, Loffredo FS
The world population is aging, and by 2017, there will be more people over the age of 65 than under age 5, and by 2050, two billion of the estimated nine billion people on Earth will be older than 60. Aging itself is a major cardiovascular risk factor, affecting morbidity and mortality of the aging population. At the same time, aging increases the likelihood of the presence of other risk factors. The aged myocardium is characterized by several structural and functional progressive changes that impair its ability to respond appropriately to stressful conditions. Although some progress to understand the complex mechanisms that underlie these phenotypic changes, the molecular pathways that determine the balance between aging and rejuvenation in the aged myocardium still remain elusive. In this article, we review molecular mechanisms responsible for the phenotypic changes observed with aging in the heart, providing insight into molecular pathways and pharmacological interventions that may rejuvenate the aged myocardium. A better understanding of these pathways is essential for determining their therapeutic potential in humans, improving the possibility that the increase in life expectancy that we are observing will be accompanied by a parallel increase in healthspan.

Cardiovasc Res: 01 Jul 2016; epub ahead of print

Maternal-fetal epigenetic interactions in the beginning of cardiovascular damage.

Napoli C, Infante T, Casamassimi A
Several studies indicate that impaired fetal growth, and in utero exposure to risk factors, especially maternal hypercholesterolemia, may be relevant for the early onset of cardiovascular damage. The exact molecular mechanisms of such fetal programming are still unclear. Epigenetics may represent one of the possible scientific explanations of the impact of such intrauterine risk factors for the subsequent development of cardiovascular disease (CVD) during adult age. Translational studies support this hypothesis, however a direct causality in humans cannot be ascertained. This task could be investigated in primates and in human post-mortem fetal arteries. Importantly, some studies also suggest the transgenerational transmission of epigenetic risk. The recently launched International Human Epigenome Consortium and the NIH Roadmap Epigenomics Mapping Consortium will provide the rationale for a useful clinical scenario for primary prevention and therapy CVD. Despite the heritable nature of epigenetic modification, the clinically relevant information is that it could be reversible through therapeutic approaches including histone deacetylase inhibitors, histone acetyltransferase inhibitors and commonly used drugs such as statins.

Cardiovasc Res: 18 Jul 2011; epub ahead of print

Congenital coronary artery anomalies: a bridge from embryology to anatomy and pathophysiology-a position statement of the development, anatomy, and pathology ESC Working Group.

Pérez-Pomares JM, de la Pompa JL, Franco D, Henderson D, ... van den Hoff M, Basso C
Congenital coronary artery anomalies are of major significance in clinical cardiology and cardiac surgery due to their association with myocardial ischaemia and sudden death. Such anomalies are detectable by imaging modalities and, according to various definitions, their prevalence ranges from 0.21 to 5.79%. This consensus document from the Development, Anatomy and Pathology Working Group of the European Society of Cardiology aims to provide: (i) a definition of normality that refers to essential anatomical and embryological features of coronary vessels, based on the integrated analysis of studies of normal and abnormal coronary embryogenesis and pathophysiology; (ii) an animal model-based systematic survey of the molecular and cellular mechanisms that regulate coronary blood vessel development; (iii) an organization of the wide spectrum of coronary artery anomalies, according to a comprehensive anatomical and embryological classification scheme; (iv) current knowledge of the pathophysiological mechanisms underlying symptoms and signs of coronary artery anomalies, with diagnostic and therapeutic implications. This document identifies the mosaic-like embryonic development of the coronary vascular system, as coronary cell types differentiate from multiple cell sources through an intricate network of molecular signals and haemodynamic cues, as the necessary framework for understanding the complex spectrum of coronary artery anomalies observed in human patients.

Cardiovasc Res: 25 Jan 2016; 109:204-16

The value of basic research insights into atrial fibrillation mechanisms as a guide to therapeutic innovation: A critical analysis.

Heijman J, Algalarrondo V, Voigt N, Melka J, ... Dobrev D, Nattel S
Atrial fibrillation (AF) is an extremely common clinical problem associated with increased morbidity and mortality. Current antiarrhythmic options include pharmacological, ablation and surgical therapies, and have significantly improved clinical outcomes. However, their efficacy remains suboptimal and their use is limited by a variety of potentially serious adverse effects. There is a clear need for improved therapeutic options. Several decades of research have substantially expanded our understanding of the basic mechanisms of AF. Ectopic firing and reentrant activity have been identified as the predominant mechanisms for arrhythmia initiation and maintenance. However, it has become clear that the clinical factors predisposing to AF and the cellular and molecular mechanisms involved are extremely complex. Moreover, all AF-promoting and maintaining mechanisms are dynamically regulated and subject to remodeling caused by both AF and cardiovascular disease. Accordingly, the initial presentation and clinical progression of AF patients is enormously heterogeneous. Improved understanding of arrhythmia mechanisms is likely needed to facilitate therapeutic innovation, but this assumption has not to our knowledge been carefully examined. Here, we review recent insights into the basic mechanisms of AF, critically analyze the role of basic-research insights in the development of presently-used anti-AF therapeutic options, and assess the potential value of contemporary experimental discoveries for future therapeutic innovation. Finally, we highlight some of the important challenges to the translation of basic science findings to clinical application.

Cardiovasc Res: 24 Dec 2015; epub ahead of print

Branched-chain amino acid metabolism in heart disease: an epiphenomenon or a real culprit?

Huang Y, Zhou M, Sun H, Wang Y
Metabolic remodelling is an integral part of the pathogenesis of heart failure. Although much progress has been made in our current understanding of the metabolic impairment involving carbohydrates and fatty acids in failing hearts, relatively little is known about the changes and potential impact of amino acid metabolism in the onset of heart diseases. Although most amino acid catabolic activities are found in the liver, branched-chain amino acid (BCAA) catabolism requires activity in several non-hepatic tissues, including cardiac muscle, diaphragm, brain and kidney. In this review, the new insights into the regulation of cardiac BCAA catabolism and functional impact on cardiac development and physiology will be discussed along with the potential contribution of impairment in BCAA catabolism to heart diseases. A particular focus will be the new information obtained from recently developed genetic models with BCAA catabolic defects and metabolomic studies in human and animal models. These studies have revealed the potential role of BCAA catabolism in cardiac pathophysiology and have helped to distinguish BCAA metabolic defects as an under-appreciated culprit in cardiac diseases rather than an epiphenomenon associated with metabolic remodelling in the failing heart.

Cardiovasc Res: 19 Apr 2011; 90:220-3

Epicardial adipose stem cell sheets results in greater post-infarction survival than intramyocardial injections.

Hamdi H, Planat-Benard V, Bel A, Puymirat E, ... Agbulut O, Menasché P
Aims: Intramyocardial injections of cells can damage tissue and enhance dissociation-induced cell death. We assessed whether epicardial delivery of cell sheets could overcome these issues in a rat model of chronic myocardial infarction. Methods and results: Eighty-two rats that had undergone coronary ligation and simultaneous harvest of fat tissue to yield the adipose-derived stromal cell (ADSC) fraction were randomized 1 month after infarction to receive injections of either control medium (n= 24) or 10 × 10(6) autologous ADSC (n= 37) or the epicardial deposit, onto the infarcted area, of a trilayered ADSC sheet (10 × 10(6), n= 21) prepared by culturing cells on temperature-sensitive dishes. Some treated rats received green fluorescent protein labelled ADSC. Survival, function, and cell engraftment were blindly assessed after 2 months. Prior to implantation, cell sheets and suspended cells were assessed for the expression of extracellular matrix constituents and molecules involved in angiogenesis and cardiac remodelling. The survival rate of rats receiving the cell sheets was significantly higher than after cell injections (73 vs. 41%, P = 0.01). This correlated with the absence of left ventricular (LV) remodelling in the cell sheet group, as end-diastolic volume only increased by 2.8% compared with baseline [95% confidence interval (CI): -18.7%; +30.0%, P = 0.81] vs. increases of 25.9% (-0.4%; +59.2%, P = 0.05) and 51.2% (+18.6%; +92.8, P = 0.001) in the cell and medium injection groups, respectively. Sheets also resulted in a greater cell engraftment possibly related to the greater expression of extracellular matrix constituents. Conclusion: The better preservation of LV geometry afforded by ADSC sheets is associated with increased survival and engraftment, which supports the concept of an epicardial delivery of cell-seeded biomaterials.

Cardiovasc Res: 13 May 2011; epub ahead of print

Cardiac adipose tissue and atrial fibrillation: the perils of adiposity.

Hatem SN, Redheuil A, Gandjbakhch E
The amount of adipose tissue that accumulates around the atria is associated with the risk, the persistence and the severity of atrial fibrillation. A strong body of clinical and experimental evidences indicates that this relationships is not an epiphenomenon but is the result of complex cross-talks between the adipose tissue and the neighboring atrial myocardium. For instance, epicardial adipose tissue is a major source of adipokines, inflammatory cytokines or reactive oxidative species which can contribute to the fibrotic remodeling of the atrial myocardium. Fibro fatty infiltrations of the subepicardium could contribute also to the functional disorganization of the atrial myocardium. The observation that obesity is associated with distinct structural and functional remodeling of the atria has opened new perspectives of treating AF substrate with aggressive risk factor management. Advances in cardiac imaging should lead to an improved ability to visualize myocardial fat depositions and to localize AF substrates.

Cardiovasc Res: 20 Jan 2016; epub ahead of print

Common Variation in Atrial Fibrillation: Navigating the Path from Genetic Association to Mechanism.

Tucker NR, Clauss S, Ellinor PT
Atrial fibrillation (AF) is the most common cardiac arrhythmia with well-established clinical and genetic risk components. Genome wide association studies (GWAS) have identified 17 independent susceptibility signals for AF at 14 genomic regions, but the mechanisms through which these loci confer risk to AF remain largely undefined. This problem is not unique to AF, as the field of functional genomics, which attempts to bridge this gap from genotype to phenotype, has only uncovered the mechanisms for a handful of GWAS loci. Recent functional genomic studies have made great strides towards translating genetic discoveries to an underlying mechanism, but the large scale application of these techniques to AF has remain limited. These advances, as well as the continued unresolved challenges for both common variation in AF and the functional genomics field in general, will be the subject of the following review.

Cardiovasc Res: 05 Jan 2016; epub ahead of print

A role for coagulation factor Xa in experimental pulmonary arterial hypertension.

Delbeck M, Nickel KF, Perzborn E, Ellinghaus P, ... Schermuly RT, von Degenfeld G
Aims Anticoagulation with warfarin is recommended for the treatment of patients with pulmonary arterial hypertension. However, the therapeutic benefit of anticoagulation has not yet been demonstrated experimentally or clinically. Here, rivaroxaban, an oral, direct factor Xa inhibitor, was compared with warfarin and enoxaparin in the prevention of right ventricular dysfunction and hypertrophy in the monocrotaline model of pulmonary hypertension. Methods and results Sprague-Dawley rats (n = 10 per group) were randomized to receive rivaroxaban, warfarin, enoxaparin or placebo before receiving a subcutaneous injection of monocrotaline 60 mg/kg or saline. Rivaroxaban and enoxaparin were administered for 28 days starting 4 hours before monocrotaline injection; warfarin was given for 35 days initiated 7 days before monocrotaline injection. Right ventricular haemodynamics and hypertrophy were assessed 28 days after monocrotaline administration. Rivaroxaban dose-dependently reduced systolic and end-diastolic right ventricular pressure increase and right ventricular hypertrophy. Warfarin reduced right ventricular pressure increase only. Enoxaparin had no effect on either parameter. Severe bleeding occurred in four and five rats treated with warfarin and enoxaparin, respectively, whereas no overt bleeding was observed in rats treated with rivaroxaban. Conclusion Selective, direct inhibition of Factor Xa by rivaroxaban effectively prevented right ventricular dysfunction and hypertrophy in monocrotaline-injected rats, indicating a role for coagulation factors in experimental pulmonary hypertension. Clinical investigation of the impact of early and continued administration of a specific factor Xa inhibitor such as rivaroxaban on the course of pulmonary arterial hypertension should be considered.

Cardiovasc Res: 16 Jun 2011; epub ahead of print

Two Distinct Phases of Calcium Signaling under Flow.

Liu B, Lu S, Zheng S, Jiang Z, Wang Y
Aims: High shear stress (HSS) can have significant impact on angiogenesis and arthrosclerosis in collateral arteries near the bifurcation and curvature regions. Here we investigate the spatiotemporal pattern of HSS-induced intracellular calcium alteration. Methods and results: Genetically-encoded biosensors based on fluorescence resonance energy transfer (FRET) were targeted in the cytoplasm and the endoplasmic reticulum (ER) to visualize the subcellular calcium dynamics in bovine aortic endothelial cells (BAECs) under HSS (65 dyn/cm(2)). Upon HSS application, the intracellular Ca(2+) concentration ([Ca(2+)](i)) increased immediately and maintained a sustained high level, while the ER-stored calcium had a significant decrease only after 300 sec. The perturbation of calcium influx across the plasma membrane by the removal of extracellular calcium or the blockage of membrane channels inhibited the early phase of [Ca(2+)](i) increase upon HSS application, which was further shown to be sensitive to the magnitudes of shear stress and the integrity of cytoskeletal support. In contrast, Src, phospholipase C(PLC), and inositol 1,4,5-trisphosphate receptor (IP(3)R) can regulate the late phase of HSS-induced [Ca(2+)](i) increase, via the promotion of the ER calcium efflux. Conclusion: HSS-induced [Ca(2+)](i) increase consists of two well-coordinated phases with different sources and mechanisms: (1) an early phase due to the calcium influx across the plasma membrane which is dependent on the mechanical impact and cytoskeletal support; (2) a late phase originated from the ER-calcium efflux which is regulated by the Src, PLC, and IP(3)R signaling pathway. Therefore, our work presented new molecular-level insights into systematic understanding of mechanotransduction in cardiovascular systems.

Cardiovasc Res: 02 Feb 2011; epub ahead of print

Atrial metabolism and tissue perfusion as determinants of electrical and structural remodeling in atrial fibrillation.

Opacic D, van Bragt KA, Nasrallah HM, Schotten U, Verheule S
Atrial fibrillation (AF) is the most common tachyarrhythmia in clinical practice. Over decades of research, a vast amount of knowledge has been gathered about the causes and consequences of AF related to cellular electrophysiology and features of the tissue structure that influence the propagation of fibrillation waves. Far less is known about the role of myocyte metabolism and tissue perfusion in the pathogenesis of AF. However, the rapid rates of electrical activity and contraction during AF must present an enormous challenge to the energy balance of atrial myocytes. This challenge can be met by scaling back energy demand and by increasing energy supply, and there are several indications that both phenomena occur as a result of AF. Still, there is ample evidence that these adaptations fall short of redressing this disbalance, that may represent a driving force for atrial electrical as well as structural remodeling. In addition, several \'metabolic diseases\' such as diabetes, obesity and abnormal thyroid function precipitate some well-known \'culprits\' of the AF substrate such as myocyte hypertrophy and fibrosis, while some other AF risk factors, such as heart failure, affect atrial metabolism. This review provides an overview of metabolic and vascular alterations in AF and their involvement in its pathogenesis.

Cardiovasc Res: 19 Jan 2016; epub ahead of print

Detrimental effect of fractalkine on myocardial ischemia and heart failure.

Xuan W, Liao Y, Chen B, Huang Q, ... Bin J, Kitakaze M
Aims Fractalkine (FKN) is a newly identified membrane-bound chemokine, its role in myocardial ischemia and heart failure is largely unknown. We attempted to investigate the role of FKN in myocardial ischemia and ischemia or pressure overload-induced ventricular remodeling and heart failure. Methods and Results FKN-induced changes of heart failure-related genes in cultured rat cardiac cells and the effect of FKN on cultured cardiomyocyte injury during anoxia-reoxygenation were examined. The direct influence of FKN neutralization on heart failure and the potential mechanism was also investigated. In mice with failing hearts, myocardial FKN expression was correlated with the lung weight/body weight ratio, left ventricular fractional shortening, and BNP expression. In cultured rat cells, exposure to FKN increased ANP expression in cardiomyocytes, matrix metalloproteinase-9 expression in fibroblasts, and intercellular adhesion molecule-1expression in microvascular endothelial cells. FKN also promoted cardiomyocyte damage during anoxia-reoxygenation and neutralizing FKN antibody treatment improved heart failure induced by myocardial infarction or pressure overload. Neutralizing FKN or its receptor inhibited the activation of mitogen-activated protein kinases in hypoxic cardiomyocytes or ischemic myocardium. Conclusion FKN promotes myocardial injury and promotes heart failure, which is associated with the activation of mitogen-activated protein kinases.

Cardiovasc Res: 15 Aug 2011; epub ahead of print

Ablation of Nkx2-5 at mid-embryonic stage results in premature lethality and cardiac malformation.

Terada R, Warren S, Lu JT, Chien KR, Wessels A, Kasahara H
Aims Human congenital heart disease linked to mutations in the homeobox transcription factor, NKX2-5, is characterized by cardiac anomalies including atrial and ventricular septal defects, as well as conduction and occasional defects in contractility. In the mouse, homozygous germline deletion of Nkx2-5 gene results in death around E10.5. It is however not established whether Nkx2-5 is necessary for cardiac development beyond this embryonic stage. Because human NKX2-5 mutations are related to septum secundum type atrial septal defects, we hypothesized that Nkx2-5 deficiency during the processes of septum secundum formation may cause cardiac anomalies; thus we analyzed mice with tamoxifen-inducible Nkx2-5 ablation beginning at E12.5 when the septum secundum starts to develop. Methods and Results Using tamoxifen-inducible Nkx2-5 gene targeted mice, this study demonstrates that Nkx2-5 ablation beginning at E12.5 results in embryonic death by E17.5. Analysis of mutant embryos at E16.5 shows arrhythmias, contraction defects and cardiac malformations including atrial septal defects. Quantitative measurements using serial section histology and 3-dimensional reconstruction demonstrate growth retardation of the septum secundum and enlarged foramen ovale in Nkx2-5 ablated embryos. Functional cardiac defects may be attributed to abnormal expression of transcripts critical for conduction and contraction including cardiac voltage-gated Na(+) channel pore-forming α-subunit (Na(v)1.5-α), gap junction protein connexin40, cardiac myosin light chain kinase, and sarcolipin within 4 days after tamoxifen-injection. Conclusion Nkx2-5 is necessary for survival after the mid-embryonic stage for cardiac function and formation by regulating expression of its downstream target genes.

Cardiovasc Res: 02 Feb 2011; epub ahead of print

Enhanced activation of p21-activated kinase 1 in heart failure contributes to dephosphorylation of connexin 43.

Ai X, Jiang A, Ke Y, Solaro JR, Pogwizd SM
Aims: We previously showed decreased cellular coupling and dephosphorylation of the gap junctional protein connexin 43 (Cx43) in left ventricular (LV) myocytes from an arrhythmogenic rabbit model of non-ischaemic heart failure (HF) that was associated with a 2.5-fold increase in the amount of protein phosphatase type 2A (PP2A) co-localized with Cx43. Here, we further explore the molecular mechanisms of enhanced dephosphorylation of Cx43 in HF. p21-activated kinase 1 (PAK1) is a serine-threonine protein kinase that has been shown to activate PP2A. Methods and results: We found that total PAK1 and activated PAK1 (PAK1-P(Thr423)) were both increased in HF rabbit LV (vs. controls). PAK1 co-immunoprecipitated (co-IP\'d) with Cx43 protein and, with HF, co-IP\'d PAK1 and PAK1-P(Thr423) were increased. With failing human LV, PAK1 total protein and PAK1-P(Thr423) were also increased globally and locally (co-IP\'d with Cx43). To further explore the role of PAK1 in modulating Cx43 dephosphorylation and intercellular coupling, we overexpressed active PAK1 in isolated LV myocytes from control rabbits and in HEK293 cells with genetically modified overexpression of Cx43 (HEK293-Cx43). PAK1 overexpression in both rabbit myocytes and HEK293-Cx43 cells significantly increased PP2A activity (globally and at the level of Cx43), increased dephosphorylated Cx43, and markedly reduced intercellular dye coupling. These effects were attenuated with PP2A inhibition using okadaic acid (10 nM). Conclusions: PAK1 and PP2A are integral components of a macromolecular complex with cardiac Cx43, and increased activation of associated PAK1 can contribute to enhanced Cx43 dephosphorylation and impaired intercellular coupling that may underlie slow conduction in HF.

Cardiovasc Res: 05 Jul 2011; epub ahead of print

Cytokines Present in Smokers\' Serum Interact with Smoke Components to Enhance Endothelial Dysfunction.

Barbieri SS, Zacchi E, Amadio P, Gianellini S, ... Weksler BB, Tremoli E
Aims Cigarette smoking engenders inflammation and endothelial dysfunction, processes implicated in atherothrombotic disease. We hypothesized that an interaction between inflammatory cytokines in smokers\' blood and circulating components of cigarette smoke is necessary to induce reactive oxygen species (ROS) and cyclooxygenase-2 (COX-2) in endothelium. We then explored the molecular mechanisms involved in these effects. Methods and results Serum from 9 healthy active smokers (AS) compared with serum from 9 non-smokers (NS) showed higher levels of interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) and greater ability to induce ROS production, p47phox translocation to the plasma membrane, and COX-2 mRNA and protein expression in endothelial cells (EC). Similar results were obtained in vivo and in vitro after treatment with aqueous extracts of cigarette smoke plus IL-1β and TNF-α (TS/IL-1β/TNF-α). In EC increased ROS production and COX-2 mRNA induced by serum from AS correlated positively with their serum levels of IL-1β and TNF-α. Moreover, a positive correlation was observed between ROS generation and COX-2 mRNA. Simultaneous immuno-neutralization of IL-1β and TNF-α prevented endothelial dysfunction induced by serum from AS. Inhibitors of NADPH-oxidase and/or p47phox siRNA diminished ROS production and COX-2 expression as well as phosphorylation of p38 mitogen activated protein kinase (p38MAPK) and Akt mediated either by AS serum or by TS/IL-1β/TNF-α. Finally, direct inhibition of p38MAPK and Akt activity also abolished COX-2 expression mediated by both types of stimuli. Conclusion Our results suggest a crucial role played by interactions between inflammatory cytokines and tobacco smoke in the induction of endothelial dysfunction.

Cardiovasc Res: 02 Feb 2011; epub ahead of print

KATP channel-dependent metaboproteome decoded: Systems approaches to heart failure prediction, diagnosis and therapy.

Arrell DK, Zlatkovic Lindor J, Yamada S, Terzic A
Systems biology provides an integrative platform by which to account for the biological complexity related to cardiac health and disease. In this way, consequences of ATP-sensitive K(+) (K(ATP)) channel deficiency on heart failure prediction, diagnosis and therapy were resolved recently at a proteomic level. Under stress-free conditions, knockout of the Kir6.2 K(ATP) channel pore induced metabolic proteome remodeling, revealing overrepresentation of markers of cardiovascular disease. Imposed stress precipitated structural and functional defects in Kir6.2-knockout hearts, decreasing survival and validating prediction of disease susceptibility. In the setting of hypertension, a leading risk for heart failure development, proteomic analysis diagnosed the metabolism-centric impact of K(ATP) channel deficiency in disease. Bioinformatic interrogation of K(ATP) channel-dependent proteome prioritized heart-specific adverse effects, exposing cardiomyopathic traits of aggravated contractility, fibrosis, and ventricular hypertrophy. In dilated cardiomyopathy induced by Kir6.2-knockout pressure-overload, proteomic remodeling was exacerbated, underlying a multifaceted molecular pathology indicating necessity for a broad-based strategy to achieve repair. Embryonic stem cell intervention in cardiomyopathic K(ATP) channel knockout hearts elicited a distinct proteome signature that forecast amelioration of adverse cardiac outcomes. Functional/structural measurements validated improved contractile performance, reduced ventricular size and decreased cardiac damage in the treated cohort, while systems assessment unmasked cardiovascular development as a prioritized biological function in stem cell-reconstructed hearts. Thus, proteomic deconvolution of K(ATP) channel deficient hearts provides definitive evidence for the channel\'s homeostatic contribution to the cardiac metaboproteome, and establishes the utility of systems-oriented approaches to predict disease susceptibility, diagnose consequences of heart failure progression, and monitor therapy outcome.

Cardiovasc Res: 15 Feb 2011; epub ahead of print

Epigenetic Mechanisms in Diabetic Vascular Complications.

Reddy MA, Natarajan R
There has been a rapid increase in the incidence of diabetes as well the associated vascular complications. Both genetic and environmental factors have been implicated in these pathologies. Increasing evidence suggests that epigenetic factors play a key role in the complex interplay between genes and the environment. Actions of major pathological mediators of diabetes and its complications such as hyperglycemia, oxidant stress and inflammatory factors can lead to dysregulated epigenetic mechanisms that affect chromatin structure and gene expression. Furthermore, persistence of this altered state of the epigenome may be the underlying mechanism contributing to a \'metabolic memory\' that results in chronic inflammation and vascular dysfunction in diabetes even after achieving glycemic control. Further examination of epigenetic mechanisms by also taking advantage of recently developed next generation sequencing technologies, can provide novel insights into the pathology of diabetes and its complications and lead to the discovery of much needed new drug targets for these diseases. In this review, we highlight the role of epigenetics in diabetes and its vascular complications, and recent technological advances that have significantly accelerated the field.

Cardiovasc Res: 26 Jan 2011; epub ahead of print

Influence of p53 in the transition of myotrophin-induced cardiac hypertrophy to heart failure.

Das B, Young D, Vasanji A, Gupta S, Sarkar S, Sen S
Aims: Cardiac-specific overexpression of myotrophin (myo) protein in transgenic (myo-Tg) mice results in hypertrophy at 4 weeks that progresses to heart failure (HF) by 36 weeks. Gene profiling showed that p53 expression increases as hypertrophy worsens to HF, suggesting that p53 may influence myo-induced HF. We aimed to define how the p53 signaling cascade affects the spectrum of cardiac hypertrophy (CH)/HF. Methods and results: Immunoblot analysis showed that in myo-Tg mice (Mus musculus), upregulation of p53 occurs only when hypertrophy transitions to HF (16 weeks onward). To elucidate the role of p53, a double-Tg mouse line (p53(-/-)/myo(+/+)) was developed by crossing myo-Tg mice with p53-null mice. A significant reduction in cardiac mass with improved cardiac function was observed in p53(-/-)/myo(+/+) mice, suggesting that absence of p53 prevents hypertrophy from turning into HF. Analysis via real-time reverse-transcription PCR revealed changes in transcripts of the p53 pathway in p53(-/-)/myo(+/+) mice. Ingenuity Pathway Analysis(TM) indicated that cross-talk among several key nodal molecules (e.g., cyclin-dependent kinase inhibitor 1A, caspase-3, nuclear factor kappa-light-chain-enhancer of activated B cells etc.) may play a regulatory role in the transition of CH to HF. Conclusions: Our data provide evidence, for the first time, that the coherence of p53 with myo plays an active role during the transition of CH to HF in a model of HF induced by myo overexpression. Transition from CH to HF can be prevented in the absence of p53 in myo-induced hypertrophy. Therefore, deletion/inhibition of p53 could be a therapeutic strategy to prevent CH from transitioning to HF.

Cardiovasc Res: 05 Mar 2010; epub ahead of print

A key role of TRPC channels in the regulation of electromechanical activity of the developing heart.

Sabourin J, Robin E, Raddatz E
Aims It is well established that dysfunction of voltage-dependent ion channels results in arrhythmias and conduction disturbances in the fetal and adult heart. However, the involvement of voltage-insensitive cationic TRPC (Transient Receptor Potential Canonical) channels remains unclear. We assessed the hypothesis that TRPC channels play a crucial role in the spontaneous activity of the developing heart. Methods and results TRPC isoforms were investigated in isolated hearts obtained from 4-day-old chick embryos. Using RT-PCR, western blot and co-immunoprecipitation, we report for the first time that TRPC1, 3, 4, 5, 6 and 7 isoforms are expressed at the mRNA and protein levels and that they can form a macromolecular complex with the α1C subunit of the L-type voltage-gated calcium channel (Cav1.2) in atria and ventricle. Using ex vivo electrocardiogram, electrogram of isolated atria and ventricle and ventricular mechanogram, we found that inhibition of TRPC channels by SKF-96365 leads to negative chrono-, dromo- and inotropic effects, prolongs QT interval and provokes first and second degree atrioventricular blocks. Pyr3, a specific antagonist of TRPC3, affected essentially atrioventricular conduction. On the other hand, specific blockade of the L-type calcium channel with nifedipine rapidly stopped ventricular contractile activity without affecting rhythmic electrical activity. Conclusions These results give new insights into the key role that TRPC channels, via interaction with the Cav1.2 channel, play in regulation of cardiac pacemaking, conduction, ventricular activity and contractility during cardiogenesis.

Cardiovasc Res: 15 Jun 2011; epub ahead of print

The role of inflammatory biomarkers in developing targeted cardiovascular therapies: lessons from the cardiovascular inflammation reduction trials.

Passacquale G, Di Giosia P, Ferro A
Anti-inflammatory add-on therapy to conventional cardiovascular prophylaxis has been proposed as a novel therapeutic approach to potentially reduce residual cardiovascular risk. This hypothesis has been challenged by a series of unsuccessful Phase III studies testing the impact on clinical outcomes of novel agents with immunomodulatory actions. Specifically, the apparent ability of phospholipase A2 (PLA2) inhibitors and of anti-oxidants to ameliorate inflammation and to reduce coronary disease in Phase II trials did not translate into improved secondary cardiovascular prevention in larger population-based studies. Other anti-inflammatory agents are still under scrutiny. However, studies to date have lacked information on the inflammatory profile of the participants, both at baseline and at follow-up, thereby limiting the possibility of identifying subgroups of patients in whom \'residual inflammation\' can be detected despite optimal conventional therapy, and who could therefore benefit from a cardiovascular prevention strategy specifically targeting inflammation. This has also rendered it difficult to interpret the results as a conclusive demonstration of inefficacy of the tested anti-inflammatory strategies in the treatment of atherosclerosis. We here discuss the importance of better patient characterisation to minimise heterogeneity of the study population, so that effectiveness of different anti-inflammatory strategies can be evaluated in targeted subgroups of patients. We also illustrate how specific inflammatory biomarkers could assist in this process, with particular emphasis on the roles of high sensitivity C-reactive protein and circulating monocyte phenotype.

Cardiovasc Res: 26 Sep 2015; epub ahead of print

Cardiac Mesenchymal Stem Cells Contribute to Scar Formation After Myocardial Infarction.

Carlson S, Trial J, Soeller C, Entman ML
Aims Therapeutic advances in prevention and treatment of myocardial infarction have decreased patient mortality and increased concern about efficient repair and scar formation, processes that are necessary to attenuate complications such as adverse remodeling and heart failure. Since the rapid accumulation and activity of cardiac fibroblasts is critical for proper scar formation, we hypothesized that infarct fibroblasts are generated by a cardiac-resident progenitor cell population. Methods and Results We found that infarct fibroblasts in C57BL/6 mice are generated by a mesenchymal stem cell population that responds robustly to injury by proliferating and accumulating in the infarct. We report that stem cell-derived fibroblasts contribute to the formation of a scar after an infarction by differentiating into matrix-producing fibroblasts closely associated with fibrillar collagen in the infarct. Further characterization of these cells revealed a heterogenous population with expression of both stem cell and canonical cardiac fibroblast markers, suggesting that some have a commitment to the fibroblast phenotype. Our in vitro study of these cells shows that they have extended self renewal capability and express the primitive marker Nanog. In keeping with these observations, we also report that these cells are multipotent and differentiate readily into fibroblasts as well as other mesenchymal lineages. Conclusions Cells with the properties of mesenchymal stem cells participate in wound healing after myocardial infarction in the adult heart.

Cardiovasc Res: 01 Mar 2011; epub ahead of print

The role of Tenascin C in cardiovascular disease.

Golledge J, Clancy P, Maguire J, Lincz L, Koblar S
The extracellular matrix protein tenascin C (TnC) is expressed in a variety of embryonic tissues but its expression in adult arteries is co-incident with sites of vascular disease. TnC expression has been linked to the development and complications of intimal hyperplasia, pulmonary artery hypertension, atherosclerosis, myocardial infarction and heart failure. This review identifies the growing collection of evidence linking TnC with cardiovascular disease development. The transient upregulation of this extracellular matrix protein at sites of vascular disease could provide a means to target TnC in the development of diagnostics and new therapies. Studies in TnC deficient mice have implicated this protein in the development of intimal hyperplasia. Further animal and human studies are required to thoroughly assess the role of TnC in some of the other pathologies it has been linked with, such as atherosclerosis and pulmonary hypertension. Large population studies are also warranted to clarify the diagnostic value of this extracellular matrix protein in cardiovascular disease, for example by targeting its expression using radiolabelled antibodies or measuring circulating concentrations of TnC.

Cardiovasc Res: 29 Jun 2011; epub ahead of print

Role of Sulfhydryl-Dependent Dimerization of Soluble Guanylyl Cyclase In Relaxation of Porcine Coronary Artery to Nitric Oxide.

Zheng X, Ying L, Liu J, Dou D, ... Vanhoutte PM, Gao Y
Aims Soluble guanylyl cyclase (sGC) is a heterodimer. The dimerization of the enzyme is obligatory for its function in mediating actions caused by agents that elevates cyclic guanosine monophosphate (cGMP). The present study aimed to determine whether sGC dimerization is modulated by thiol reducing agents and its influences relaxations to nitric oxide (NO). Methods and results The dimers and monomers of sGC and cGMP-dependent protein kinase (PKG) were analyzed by Western blotting. The intracellular cGMP content was measured by enzyme-linked immunosorbent assay. Changes in isometric tension were determined in organ chambers. In isolated porcine coronary arteries, the protein levels of sGC dimer were decreased by thiol reductants dithiothreitol (DTT), L-cysteine, reduced L-glutathione (GSH) and tris (2-carboxyethyl) phosphine (TCEP). The effect was associated with reduced cGMP elevation and attenuated relaxations to nitric oxide donors. The dimerization of sGC and activation of the enzyme were also decreased by dihydrolipoic acid, an endogenous thiol antioxidant. DTT at concentrations markedly affecting the dimerization of sGC had no significant effect on the dimerization of PKG or relaxation to 8-Br-cGMP. Relaxation of the coronary artery to NO donor was potentiated by hypoxia when sGC was partially inhibited, coincident with an increased sGC dimer and enhanced cGMP production. These effects were prevented by DTT and TCEP. Conclusion These results demonstrate that the dimerization of sGC is exquisitely sensitive to thiol reductants as compared to that of PKG, which may provide a novel mechanism for thiol-dependent modulation of NO-mediated vasodilatation under conditions such as hypoxia.

Cardiovasc Res: 20 Jan 2011; epub ahead of print

Mechanisms of T-box gene function in the developing heart.

Greulich F, Rudat C, Kispert A
The multi-chambered mammalian heart arises from a simple tube by polar elongation, myocardial differentiation and morphogenesis. Members of the large family of T-box (Tbx) transcription factors have been identified as crucial players that act in distinct subprograms during cardiac regionalization. Tbx1 and Tbx18 ensure elongation of the cardiac tube at the anterior and posterior pole, respectively. Tbx1 acts in the pharyngeal mesoderm to maintain proliferation of mesenchymal precursor cells for formation of a myocardialized and septated outflow tract. Tbx18 is expressed in the sinus venosus region and is required for myocardialization of the caval veins and the sinoatrial node. Tbx5 and Tbx20 function in the early heart tube, and independently activate the chamber myocardial gene program, whereas Tbx2 and Tbx3 locally repress this program to favor valvuloseptal and conduction system development. Here, we summarize that these T-box factors act in different molecular circuits and control target gene expression using diverse molecular strategies including binding to distinct protein interaction partners.

Cardiovasc Res: 18 Apr 2011; epub ahead of print

A Brugada syndrome mutation (p.S216L) and its modulation by p.H558R polymorphism: standard and dynamic characterization.

Marangoni S, Di Resta C, Rocchetti M, Barile L, ... Benedetti S, Zaza A
Aims: The Na(+) channel mutation (p.S216L), previously associated with type 3 long-QT syndrome (LQT3) phenotype, and a common polymorphism (p.H558R) were detected in a patient with an intermittent Brugada syndrome (BS) ECG pattern. The study was aimed to assess the p.S216L electrical phenotype, its modulation by p.H558R, and to identify abnormalities compatible with a mixed BS-LQT3 phenotype. Methods and results: The mutation was expressed alone (S216L channels), or in combination with the polymorphism (S216L-H558R channels), in a mammalian cell line (TSA201). Functional analysis included standard voltage clamp and dynamic clamp with endo- and epicardial action potential waveforms. Expression of S216L channels was associated with a 60% reduction in maximum Na(+) current (I(Na)) density, attributable to protein misfolding (rescued by mexiletine pretreatment) and moderate slowing of inactivation. I(Na) density partially recovered in S216L-H558R channels, but I(Na) inactivation and its recovery were further delayed. The persistent component of I(Na) (I(NaL)) was unchanged. Under dynamic clamp conditions, I(Na) decreased in S216L channels and displayed a \'resurgent\' component during late repolarization. In S216L-H558R channels, I(Na) density partially recovered and did not display a resurgent component. I(Na) changes during dynamic clamp were interpreted by numerical modelling. Conclusion: The BS pattern of p.S216L might result from a decrease in I(Na) density, which masked gating abnormalities that might otherwise result in a LQT phenotype. The p.H558R polymorphism decreased p.S216L expressivity, partly by lessening p.S216L effects and partly through the induction of further gating abnormalities suitable to blunt p.S216L effects during repolarization.

Cardiovasc Res: 27 Jun 2011; epub ahead of print

Alterations in adhesion junction precede gap junction remodeling during the development of heart failure in cardiomyopathic hamster.

Yoshida M, Ohkusa T, Nakashima T, Takanari H, ... Mizukami Y, Matsuzaki M
Aims The intercalated disc (ID) contains two complexes, adhesion junction (AJ) and gap junction (GJ). We studied the ID remodeling and its potential role in the arrhythmogenesis, and investigated the effects of olmesartan on ID remodeling during development of heart failure (HF) in UM-X7.1 cardiomyopathic hamster (UM-X7.1). Methods and Results UM-X7.1 showed left ventricular (LV) hypertrophy by age 10~15w and a moderate impairment in LV contractility at age 20w. At age 10~15w, 10~20% of UM-X7.1 died suddenly without HF, and ventricular tachycardia (VT)/ventricular fibrillation (VF) was induced in ~30% hamsters. Electron microscopy showed density linking cell-to-cell adhesion was irregular and unclearly defined, and filamentous structures attached to electron-dense components arranged in disorder. Western blotting showed the total cellular expression level of β-catenin was decreased, and nuclear β-catenin expression, which functions as T cell factor/lymphocyte enhancer binding factor (TCF/LEF) transcriptional activator, was also remarkably decreased. At age 20w, LV Cx43 expression showed remarkable decrease, and VT/VF induction rate was ~90%. In UM-X7.1, olmesartan improved abnormal ID ultrastructural changes, attenuated the decrease of total cellular and nuclear β-catenin expression, decreased VT/VF induction, and improved survival rate. Conclusion These results suggest changes in AJ protein precede Cx43 GJ alterations, and ID remodeling might contribute to arrhythmogenesis during the development of HF. Angiotensin receptor blockade might be a new therapy for lethal ventricular arrhythmia by modulating both AJ and GJ remodeling.

Cardiovasc Res: 22 Jun 2011; epub ahead of print

Presence and Stability of Rotors in Atrial Fibrillation: Evidence and Therapeutic Implications.

Guillem MS, Climent AM, Rodrigo M, Fernández-Avilés F, Atienza F, Berenfeld O
Rotor-guided ablation has opened new perspectives into the therapy of atrial fibrillation (AF). However, the dynamics and role of rotors in human AF are still controversial. In this review the current knowledge gained through research models and patient data that support the notion that rotors are key players in AF maintenance is summarized. We report and discuss the discrepancies regarding rotor prevalence and stability in the various studies, which can be attributed in part to methodological differences among mapping systems. Validation and improvement of current clinical electrophysiology mapping technologies will be crucial for developing mechanistic based selection and application of the best therapeutic strategy for individual AF patient, being it, pharmaceutical, ablative or other approach.

Cardiovasc Res: 19 Jan 2016; epub ahead of print

Hemin decreases cardiac oxidative stress and fibrosis in a rat model of systemic hypertension via PI3K/Akt signaling.

Morel-Elvis W, Karim B, Pierre B, Patrick V, ... Georges K, Véronique E
Aims Angiotensin II induces cardiac myocyte apoptosis and hypertrophy which contribute to heart failure possibly through enhanced oxidative stress. The aim of this work was to assess the impact of Hemin (Heme oxygenase-1 inducer) on NADPH oxidase activation, cardiac oxidative stress and fibrosis development in a rat model of renovascular hypertensive cardiomyopathy in comparison to an antihypertensive reference treatment by Losartan. Methods and results A three-week Hemin treatment was tested on an angiotensin II-dependent hypertension rat model and a cellular model of neonatal rat cardiomyocytes stimulated by angiotensin II. Our findings demonstrate that Hemin prevented intercellular fibrosis development, collagen 1 expression and intracellular fiber disorganization. Oxidative stress and apoptosis evaluated in hypertensive myocardial tissue were decreased by Hemin. The reference treatment by angiotensin II receptor (AT1) antagonist (Losartan), was less effective than Hemin in fibrosis and oxidative stress prevention, although it was more effective in reducing hypertension. Rac-1 activation and, consecutively, NADPH oxidase activity were more decreased with Hemin than with Losartan. Hemin enhanced the expression of Phosphoinositide3-Kinase p85 regulatory subunit in contrast to Losartan. PI3Kinase/Akt signaling pathway activation by Hemin was related to HO-1 activation and biliverdin reductase increase and its inhibition by LY294002 reversed Hemin effects on collagen-1 and Caspase-3 expression. Finally, Hemin increased Akt activation, and concomitantly decreased RhoA and p38 Mitogen-activated protein Kinase activation. Conclusion We confirmed a positive effect of Hemin on oxidative cardiac damage, apoptosis and fibrosis induced by hypertension by modulating the NADPH oxidase activation through PI3kinase p85 regulatory subunit enhanced expression.

Cardiovasc Res: 16 Mar 2011; epub ahead of print

The Relationship Between Arrhythmogenesis and Impaired Contractility in Heart Failure: Role of Altered Ryanodine Receptor Function.

Belevych AE, Terentyev D, Terentyeva R, Nishijima Y, ... Carnes CA, Gyorke S
Aims In heart failure (HF), abnormal myocyte Ca(2+) handling has been implicated in cardiac arrhythmias and contractile dysfunction. In the present study, we investigated the relationships between Ca(2+) handling, reduced myocyte contractility and enhanced arrhythmogenesis during HF progression in a canine model of nonischemic HF. Methods and Results Key Ca(2+) handling parameters were determined by measuring cytosolic and intra-sarcoplasmic reticulum (SR) [Ca(2+)] in isolated ventricular myocytes at different stages of HF. The progression of HF was associated with an early and continuous increase in ryanodine receptor (RyR2)-mediated SR Ca(2+) leak. The increase in RyR2 activity was paralleled by an increase in the frequency of diastolic spontaneous Ca(2+) waves (SCWs) in HF myocytes under conditions of β-adrenergic stimulation. In addition to causing arrhythmogenic delayed afterdepolarizations, SCWs decreased the amplitude of subsequent electrically-evoked Ca(2+) transients by depleting SR Ca(2+). At late stages of HF, Ca(2+) release oscillated essentially independent of electrical pacing. The increased propensity for generation of SCWs in HF myocytes was attributable to reduced ability of the RyR2 channels to become refractory following Ca(2+) release. The progressive alterations in RyR2 function and Ca(2+) cycling in HF myocytes were associated with sequential modifications of RyR2 by CaMKII-dependent phosphorylation and thiol oxidation. Conclusions These findings suggest that destabilized RyR2 activity due to excessive CaMKII phopshorylation and oxidation resulting in impaired post-release refractoriness is a common mechanism involved in arrhythmogenesis and contractile dysfunction in the failing heart.

Cardiovasc Res: 28 Jan 2011; epub ahead of print

A phospholipase Cγ1-activated pathway regulates transcription in human vascular smooth muscle cells.

Hunter I, Mascall KS, Ramos JW, Nixon GF
Aims Growth factor-induced repression of smooth muscle (SM) cell marker genes is an integral part of vascular SM cell proliferation. This is partly regulated via translocation of extracellular signal-regulated kinase (ERK)1/2 to the nucleus which activates the transcription factor Elk-1. The mediators involved in ERK1/2 nuclear translocation in vascular SM cells are unknown. The aim of this study is to examine the mechanisms which regulate growth factor-induced nuclear translocation of ERK1/2 and gene expression in vascular SM cells. Methods and Results In cultured human vascular SM cells, phospholipase C (PLC)γ1 expression was required for platelet-derived growth factor (PDGF)-induced ERK1/2 nuclear translocation, Elk-1 phosphorylation and subsequent repression of SM α-actin gene expression. The mechanisms of a role for PLCγ1 in ERK1/2 nuclear localization were further examined by investigating interacting proteins. The ERK1/2-binding phosphoprotein, Protein Enriched in Astrocytes-15 (PEA)-15, was phosphorylated by PDGF and this phosphorylation required activation of PLCγ1. In cells pretreated with PEA-15 siRNA, ERK1/2 distribution significantly increased in the nucleus and resulted in decreased SM α-actin expression and increased vascular SM cell proliferation. Overexpression of PEA-15 increased ERK1/2 localization in the cytoplasm. The regulatory role of PEA-15 phosphorylation was assessed. In vascular SM cells overexpressing a nonphosphorylatable form of PEA-15, PDGF-induced ERK1/2 nuclear localization was inhibited. Conclusions These results suggest that PEA-15 phosphorylation by PLCγ1 is required for PDGF-induced ERK1/2 nuclear translocation. This represents an important level of phenotypic control by directly affecting Elk-1-dependent transcription and ultimately SM cell marker protein expression in vascular SM cells.

Cardiovasc Res: 02 Feb 2011; epub ahead of print

Recovery of cardiac calcium release is controlled by sarcoplasmic reticulum refilling and ryanodine receptor sensitivity.

Ramay HR, Liu OZ, Sobie EA
Aims: In heart cells, the mechanisms underlying refractoriness of the elementary units of sarcoplasmic reticulum (SR) Ca(2+) release, Ca(2+) sparks, remain unclear. We investigated local recovery of SR Ca(2+) release using experimental measurements and mathematical modeling. Methods and results: Repeated Ca(2+) sparks were induced from individual clusters of ryanodine receptors (RyRs) in quiescent rat ventricular myocytes, and we examined how changes in RyR gating influenced the time-dependent recovery of Ca(2+) spark amplitude and triggering probability. Repeated Ca(2+) sparks from individual sites were analyzed in the presence of 50 nM ryanodine with: 1) no additional agents (control); 2) 50 µM caffeine to sensitize RyRs; 3) 50 µM tetracaine to inhibit RyRs; or 4) 100 nM isoproterenol to activate β-adrenergic receptors. Sensitization and inhibition of RyR clusters shortened and lengthened, respectively, the median interval between consecutive Ca(2+) sparks (caffeine 239 ms; control 280 ms; tetracaine 453 ms). Recovery of Ca(2+) spark amplitude, however, was exponential with a time constant of ~100 ms in all cases. Isoproterenol both accelerated the recovery of Ca(2+) spark amplitude (τ= 58 ms) and shortened the median interval between Ca(2+) sparks (192 ms). The results were recapitulated by a mathematical model in which SR [Ca(2+)] depletion terminates Ca(2+) sparks, but not by an alternative model based on limited depletion and Ca(2+)-dependent inactivation of RyRs. Conclusions: Together, the results strongly suggest that: 1) local SR refilling controls Ca(2+) spark amplitude recovery; 2) Ca(2+) spark triggering depends on both refilling and RyR sensitivity; and 3) β-adrenergic stimulation influences both processes.

Cardiovasc Res: 26 May 2011; epub ahead of print

ATRA activates and PDGF-BB represses the SM22α promoter through KLF4 binding to, or dissociating from, its cis-DNA elements.

Yu K, Zheng B, Han M, Wen JK
Aims Krüppel-like factor 4 (KLF4) is implicated in all-trans retinoic acid (ATRA)-induced and platelet-derived growth factor-BB (PDGF-BB)-repressed SM22α expression in vascular smooth muscle cells (VSMCs). However, its exact mechanism of action remains unclear. We determined how KLF4 plays different parts in ATRA- and PDGF-BB-dependent regulation of the SM22α gene. Methods and results ATRA and PDGF-BB induced KLF4 expression but exhibited an opposite effect on SM22α expression and VSMC proliferation. Chromatin immunoprecipitation and oligonucleotide pull-down assays showed that KLF4 was directly bound to the KLF4-binding sites 1 ((-263)CACCC(-259)) and 2 ((-136)GTGGG(-132)) of the SM22α promoter. ATRA increased the binding of KLF4 to site 2, whereas PDGF-BB decreased the binding of KLF4 to site 1. ATRA stimulated KLF4 acetylation by inducing KLF4 phosphorylation and increasing its interaction with p300 via activating c-Jun NH(2)-terminal kinase (JNK) and p38 pathways, and acetylated KLF4 increased its binding activity to site 2. PDGF-BB stimulated KLF4 deacetylation by inducing KLF4 dephosphorylation and increasing its interaction with histone deacetylase 2 (HDAC2) via activating extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathways, and deacetylated KLF4 dissociated from site 1. Conclusions In VSMCs, ATRA activates and PDGF-BB represses SM22α expression through KLF4 binding to, or dissociating from, its different cis-elements in an acetylation-dependent manner.

Cardiovasc Res: 21 Jan 2011; epub ahead of print

Developmental Aspects of Cardiac Arrhythmogenesis.

Postma AV, Christoffels VM, Bezzina CR
The rhythmic and synchronized contraction of atria and ventricles is essential for efficient pumping of blood throughout the body. This process relies on the proper generation and conduction of the cardiac electrical impulse. Electrophysiological properties differ in various regions of the heart and these intrinsic heterogeneities between the cardiac compartments are rooted, at least in part, in the regional differences in expression of ion channel genes and gap junction subunit genes. It is becoming increasingly clear that transcriptional regulation orchestrating the development of the heart, and the specification of the different cardiac regions from cardiac precursor cells during embryonic development, plays an essential role in regulation of expression of ion channel and gap junction genes. A causal relation between transcription factors and regionalized expression of genes involved in cardiac electrical activity has been established. This has led to the realization that in some instances, abnormal cardiac electrical function and arrhythmias in the postnatal heart may stem from a developmental abnormality causing maintained (epigenetic) changes in gene regulation. The role of developmental genes in regulation of cardiac electrical function has recently been brought further into focus as genome-wide association studies have provided strong evidence that common genetic variation, at loci harboring these genes, modulates electrocardiographic indices of conduction and repolarization and susceptibility to arrhythmia. Here we discuss recent findings and provide background insight into these complex mechanisms.

Cardiovasc Res: 13 May 2011; epub ahead of print

Cardiac-specific mindin overexpression attenuates cardiac hypertrophy via blocking AKT/GSK3β and TGF-β1-Smad signaling.

Yan L, Wei X, Tang QZ, Feng J, ... Liu PP, Li H
Aims Mindin is a secreted extracellular matrix protein, an integrin ligand, and an angiogenesis inhibitor, other examples of which are all key players in the progression of cardiac hypertrophy. However, its function during cardiac hypertrophy remains unclear. This study was aimed to identify the effect of mindin on cardiac hypertrophy and the underlying mechanisms. Methods and Results A significant downregulation of mindin expression was observed in human failing hearts. To further investigate the role of mindin in cardiac hypertrophy, we used cultured neonatal rat cardiomyocytes with gain and loss of mindin function, and cardiac-specific Mindin-overexpressing transgenic mice. In cultured cardiomyocytes, mindin negatively regulated Angiotensin II (Ang II)-mediated hypertrophic growth, as detected by [(3)H]-Leucine incorporation, cardiac myocyte area, and hypertrophic marker protein levels. Cardiac hypertrophy in vivo was produced by aortic banding (AB) or Ang II infusion in transgenic mice, and their wild type controls. The extent of cardiac hypertrophy was evaluated by echocardiography as well as by pathological and molecular analyses of heart samples. Mindin overexpression in the heart markedly attenuated cardiac hypertrophy, fibrosis and left ventricular dysfunction in mice in response to AB or Ang II. Further analysis of the signaling events in vitro and in vivo indicated that these beneficial effects of mindin were associated with the interruption of AKT/GSK3β and TGF-β1-Smad signaling. Conclusions The present study demonstrates for the first time that mindin serves as a novel mediator that protects against cardiac hypertrophy and the transition to heart failure by blocking AKT/GSK3β and TGF-β1-Smad signaling.

Cardiovasc Res: 02 Jun 2011; epub ahead of print

Systems biology approaches to heart development and congenital heart disease.

Sperling SR
Even though the foundation of systems biology approaches to cardiac function was led more than 50 years ago, there has been slow progression over the last decades. Systems biology studies were mainly focused on lower organisms, frequently on yeast. With the boost of high-throughput technologies, systems-level analyses, building one backbone of systems biology, started to complement the single-gene focus in the fields of heart development and congenital heart disease. A challenge is to bring together the many uncovered molecular components driving heart development, and eventually to establish computational models describing this complex developmental process. Congenital heart diseases represent overlapping phenotypes, reflecting the modularity of heart development. The etiology of the majority of congenital heart disease is still unknown and it is suggestive that understanding the biological network underlying heart development will enhance our understanding for its alteration. This review provides an overview of the framework for systems biology approaches focusing on the developing heart and its pathology. Recent methodological developments building the basis for future studies are highlighted and the knowledge gained is specified.

Cardiovasc Res: 29 Apr 2011; epub ahead of print

Pulmonary hypertension in adult Alk1 heterozygous mice due to oxidative stress.

Jerkic M, Kabir MG, Davies A, Yu LX, ... Belik J, Letarte M
Aim: Mutations in the ALK1 gene, coding for an endothelial-specific receptor of the transforming growth factor-β superfamily, are the underlying cause of hereditary hemorrhagic telangiectasia (HHT) type 2, but are also associated with familial pulmonary hypertension (PH). We assessed the lung vasculature of mice with a heterozygous deletion of Alk1 (Alk1(+/-)) for disease manifestations and levels of reactive O(2) species (ROS) implicated in both disorders. Methods & Results: Several signs of PH, including elevated right ventricular (RV) systolic pressure leading to RV hypertrophy, reduced vascular density, and increased thickness and outward remodeling of pulmonary arterioles were observed in 8-18 week-old Alk1(+/-) mice relative to wild-type littermate controls. Higher ROS lung levels were also documented by several methods. At 3 weeks, Alk1(+/-) mice were indistinguishable from controls and were prevented from subsequently developing PH when treated with the anti-oxidant Tempol for 6 weeks, confirming a role for ROS in pathogenesis. Levels of NADPH oxidases and superoxide dismutases were higher in adults than newborns, but unchanged in Alk1(+/-) mice versus controls. Prostaglandin metabolites were also normal in adult Alk1(+/-) lungs. By contrast, NO production was reduced, while endothelial NO synthase (eNOS)-dependent ROS production was increased in adult Alk1(+/-) mice. Pulmonary near resistance arteries from adult Alk1(+/-) mice showed less agonist-induced force and greater acetylcholine-induced relaxation; the later was normalized by catalase or Tempol treatment. Conclusion: The increased pulmonary vascular remodeling in Alk1(+/-) mice leads to signs of PH, and is associated with eNOS-dependent ROS production, which is preventable by anti-oxidant treatment.

Cardiovasc Res: 23 Aug 2011; epub ahead of print

Transposition of a pericardial-derived vascular adipose flap for myocardial salvage after infarct.

Gálvez-Montón C, Prat-Vidal C, Roura S, Farré J, ... Arís A, Bayes-Genis A
Aims Coronary artery occlusion is associated with the risk of ventricular remodeling, heart failure and cardiogenic shock. Novel strategies are sought to treat these ominous complications. We examined the effect of a pericardial-derived fat flap secured over an acute infarct caused by coronary occlusion. Methods and Results A novel intervention consisting of the pericardial isolation of a vascularized adipose flap and its transposition fully covering acute infarcted myocardium was developed in the swine model of coronary artery ligation (n=52). Left ventricular (LV) ejection fraction and LV end-diastolic and end-systolic volumes were assessed using magnetic resonance imaging (MRI). Infarct size and gene expression analysis were performed on day 6 and 1 month. Histological changes, collagen volume fraction (CVF), and vascular density were also evaluated on postmortem sections. One month after the intervention, a 18.8% increase in LV ejection fraction (P=0.007), and significant reductions in LV end-systolic (P=0.009) and LV end-diastolic volumes (P=0.03) were found in treated animals compared to control-MI group. At day 6, histopathology confirmed a significant infarct size reduction (P=0.018), the presence of vascular connections at the flap-myocardium interface, and less apoptosis in the infarct border zone compared with control animals (P<0.001). Upregulation of genes involved in cell cycle progression, cellular growth and proliferation, and angiogenesis were identified within the flap. Conclusions Our results indicate that a vascular fat flap exerts beneficial effects on LV function and limits myocardial remodeling. Future studies must confirm whether these findings provide an alternative therapeutic approach for myocardial salvage after infarction.

Cardiovasc Res: 17 May 2011; epub ahead of print

Simulation of multiple ion channel block provides improved early prediction of compounds\' clinical torsadogenic risk.

Mirams GR, Cui Y, Sher A, Fink M, ... Gavaghan DJ, Noble D
Aims: The level of inhibition of the human Ether-à-go-go Related Gene (hERG) channel is one of the earliest preclinical markers used to predict the risk of a compound causing Torsade-de-Pointes (TdP) arrhythmias. Whilst avoiding the use of drugs with maximum therapeutic concentrations within 30-fold of their hERG IC50 values has been suggested, there are drugs which are exceptions to this rule: hERG inhibitors that do not cause TdP, and drugs that can cause TdP but are not strong hERG inhibitors. In this study, we investigate whether a simulated evaluation of multichannel effects could be used to improve this early prediction of TdP risk. Methods and results: We collected multiple ion channel data (hERG, Na, L-type Ca) on 31 drugs associated with varied risks of TdP. To integrate the information on multi-channel block we have performed simulations with a variety of mathematical models of cardiac cells (for rabbit, dog and human ventricular myocyte models). Drug action is modelled using IC50 values and therapeutic drug concentrations to calculate the proportion of blocked channels and the channel conductances are modified accordingly. Various pacing protocols are simulated, and classification analysis is performed to evaluate the predictive power of the models for TdP risk. We find that simulation of action potential duration prolongation, at therapeutic concentrations, provides improved prediction of the TdP risk associated with a compound, above that provided by existing markers. Conclusion: The suggested calculations improve the reliability of early cardiac safety assessments, beyond those based solely on a hERG block effect.

Cardiovasc Res: 08 Feb 2011; epub ahead of print

MODULATION OF CONDUCTIVE ELEMENTS BY PITX2 AND THEIR IMPACT ON ATRIAL ARRHYTHMOGENESIS.

Franco D, Chinchilla A, Daimi H, Dominguez JN, Aránega A
The development of the heart is a complex process during which several types of cells progressively contribute to shape a four-chambered pumping organ. Over the last decades our understanding of the specification and transcriptional regulation of cardiac development has been greatly augmented and progressively also our understanding of the functional bases of the cardiac electrophysiology during embryogenesis. The nascent heart gradually acquires distinct cellular and functional characteristics, such as the formation of contractile structures, the development of conductive capabilities and soon thereafter the coordinated conduction of the electrical impulse, in order to fulfill its functional properties. Over the last decade we have learnt about the consequences of impairing cardiac morphogenesis, which in many cases leads to congenital heart diseases, however we are not aware yet of the consequences of impairing electrical function during cardiogenesis. The most prevalent cardiac arrhythmia is atrial fibrillation, although its genetic etiology remains rather elusive. Recent genome wide association studies have identified several genetic variants highly associated with atrial fibrillation. Among them, genetic variants located in chromosome 4q25 are adjacent to PITX2, a transcription factor known to play a critical role in left-right asymmetry as well as during cardiogenesis. We provide herein insights into the cellular and molecular links between PITX2 and atrial fibrillation.

Cardiovasc Res: 23 Mar 2011; epub ahead of print

Parathyroid hormone is a DPP-IV inhibitor and increases SDF-1-driven homing of CXCR4+ stem cells into the ischemic heart.

Huber BC, Brunner S, Segeth A, Nathan P, ... Gerbitz A, Franz WM
Aims: Parathyroid hormone (PTH) has been shown to promote stem cell mobilization into peripheral blood. Moreover, PTH treatment after myocardial infarction (MI) improved survival and myocardial function associated with enhanced homing of bone marrow-derived stem cells (BMCs). To unravel the molecular mechanisms of PTH mediated stem cell trafficking, we analyzed wildtyp (wt) and green fluorescent protein (GFP)-transgenic mice after MI with respect to the pivotal stromal cell-derived factor-1 (SDF-1)/chemokine receptor type 4 (CXCR4) axis. Methods and results: WT and GFP-transgenic mice (C57BL/6J) were infarcted by coronary artery ligation and PTH (80 μg/kg/d) was injected for 6 days afterwards. Number of BMCs was analyzed by flow cytometry. SDF-1 protein levels and activity of dipeptidyl peptidase IV (DPP-IV) were investigated by ELISA and activity assay. Functional analyses were performed at day 30 after MI. PTH treated animals revealed an enhanced homing of CXCR4(+) BMCs associated with an increased protein level of the corresponding homing factor SDF-1 in the ischemic heart. In vitro and in vivo, PTH inhibited the activity of DPP-IV, which cleaves and inactivates SDF-1. Functionally, PTH significantly improved myocardial function after MI. Both, stem cell homing as well as functional recovery were reversed by the CXCR4 antagonist AMD3100. Conclusion: In summary, PTH is a DPP-IV inhibitor leading to an increased cardiac SDF-1 level, which enhances recruitment of CXCR4(+) BMCs into the ischemic heart associated with attenuated ischemic cardiomyopathy. Since PTH is already clinically used our findings may have direct impact on the initiation of studies in patients with ischemic disorders.

Cardiovasc Res: 19 Jan 2011; epub ahead of print

Long-Distance Intercellular Connectivity between Cardiomyocytes and Cardiofibroblasts Mediated by Membrane Nanotubes.

He K, Shi X, Zhang X, Dang S, ... Fang X, Zhang Y
Aims Intercellular interactions between cardiomyocytes (CMs) and cardiofibroblasts (FBs) are important in the physiologic and pathophysiologic heart. Understanding such interactions is important for developing effective heart disease therapies. However, until recently, little has been known about these interactions. We aimed to investigate structural and functional connections between CMs and FBs that are distinct from gap junctions. Methods and results By membrane dye staining, we observed long, thin membrane nanotubular structures containing actin and microtubules that connected neonatal rat ventricular CMs and FBs. By single-particle tracking, we observed vehicles moving between CMs and FBs within the membrane nanotubes. By dual color staining, confocal imaging and flow cytometry, we observed mitochondria exchange between CMs and FBs in a coculture system. By combined atomic force microscopy (AFM) and confocal microscopy, we observed calcium signal propagation from AFM-stimulated CM (or FB) to unstimulated FB (or CM) via membrane nanotubes. By membrane and cytoskeleton staining, we observed similar nanotubular structures in adult mouse heart tissue, which suggests their physiological relevance. Conclusions As a novel type of CM to FB communication, membrane nanotubes observed in vitro and in vivo provide structural and functional connectivity between CMs and FBs over long distances.

Cardiovasc Res: 01 Jul 2011; epub ahead of print

Up-regulation of type 2 iodothyronine deiodinase in dilated cardiomyopathy.

Wang YY, Morimoto S, Du CK, Lu QW, ... Takahashi-Yanaga F, Sasaguri T
Aims Thyroid hormone (TH) has prominent effects on the heart, and hyperthyroidism is an occasional culprit of dilated cardiomyopathy (DCM). We aim to explore the potential role of TH in the pathogenesis of DCM. Methods and Results The pathophysiological role of TH in the heart was investigated using a knock-in mouse model of inherited DCM with a deletion mutation DeltaK210 in the cardiac troponin T gene. Serum triiodothyronine (T(3)) levels showed no significant difference between wild-type (WT) and DCM mice, whereas cardiac T(3) levels in DCM mice were significantly higher than those in WT mice. Type 2 iodothyronine deiodinase (Dio2), which produces T(3) from thyroxin, was up-regulated in the DCM mice hearts. The cAMP levels were increased in DCM mice hearts, suggesting that transcriptional up-regulation of Dio2 gene is mediated through the evolutionarily-conserved cAMP response element site in its promoter. Propylthiouracil (PTU), an antithyroid drug, prevented the hypertrophic remodeling of the heart in DCM mice and improved their cardiac function and life expectancy. Akt and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation increased in the DCM mice hearts and PTU treatment significantly reduced the phosphorylation levels, strongly suggesting that Dio2 up-regulation is involved in cardiac remodeling in DCM through activating the TH-signaling pathways involving Akt and p38 MAPK. Dio2 gene expression was also markedly up-regulated in the mice hearts developing similar eccentric hypertrophy after myocardial infarction. Conclusions Local hyperthyroidism via transcriptional up-regulation of the Dio2 gene may be an important underlying mechanism for the hypertrophic cardiac remodeling in DCM.

Cardiovasc Res: 10 May 2010; epub ahead of print

Na+-dependent SR Ca2+ overload induces arrhythmogenic events in mouse cardiomyocytes with a human CPVT mutation.

Sedej S, Heinzel FR, Walther S, Dybkova N, ... Kockskämper J, Pieske B
Aims: Mutations in the cardiac ryanodine receptor Ca(2+) release channel, RyR2, underlie catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited life-threatening arrhythmia. CPVT is triggered by spontaneous RyR2-mediated sarcoplasmic reticulum (SR) Ca(2+) release in response to SR Ca(2+) overload during beta-adrenergic stimulation. However, whether elevated SR Ca(2+) content - in the absence of protein kinase A activation - affects RyR2 function and arrhythmogenesis in CPVT remains elusive. Methods and Results Isolated murine ventricular myocytes harbouring a human RyR2 mutation (RyR2(R4496C+/-)) associated with CPVT were investigated in the absence and presence of 1 micromol/L JTV-519 (RyR2 stabilizer) followed by 100 micromol/L ouabain intervention to increase cytosolic [Na(+)] and SR Ca(2+) load. Changes in membrane potential and intracellular [Ca(2+)] were monitored with whole-cell patch-clamping and confocal Ca(2+) imaging, respectively. At baseline, action potentials (APs), Ca(2+) transients, fractional SR Ca(2+) release and SR Ca(2+) load were comparable in wild-type (WT) and RyR2(R4496C+/-) myocytes. Ouabain evoked significant increases in diastolic [Ca(2+)], peak systolic [Ca(2+)], fractional SR Ca(2+) release and SR Ca(2+) content that were quantitatively similar in WT and RyR2(R4496C+/-) myocytes. Ouabain also induced arrhythmogenic events, i.e. spontaneous Ca(2+) waves, delayed afterdepolarizations and spontaneous APs, in both groups. However, the ouabain-induced increase in the frequency of arrhythmogenic events was dramatically larger in RyR2(R4496C+/-) as compared with WT myocytes. JTV-519 greatly reduced the frequency of ouabain-induced arrhythmogenic events. Conclusion(s) The elevation of SR Ca(2+) load - in the absence of beta-adrenergic stimulation - is sufficient to increase the propensity for triggered arrhythmias in RyR2(R4496C+/-) cardiomyocytes. Stabilization of RyR2 by JTV-519 effectively reduces these triggered arrhythmias.

Cardiovasc Res: 18 Jan 2010; epub ahead of print

Angiotensin II increases periostin expression via Ras/p38 MAPK/CREB and ERK1/2/TGF-β1 pathways in cardiac fibroblasts.

Li L, Fan D, Wang C, Wang JY, ... Zhou Y, Wu LL
Aims Angiotensin II (AngII) is involved in extracellular matrix (ECM) accumulation contributing to heart failure. Periostin, a 90-kDa ECM protein, is a key regulator of cardiac fibrosis, and its expression is significantly higher in failing hearts. We determined the modulatory effect of AngII on periostin level and explored the possible signal transduction mechanism. Methods and Results AngII (400 ng/kg/min) or normal saline was infused subcutaneously for 28 days into rats; AngII antagonism was with losartan (10 mg/kg/day orally). AngII infusion induced cardiac fibrosis and increased periostin expression, which was attenuated by losartan. In cultured adult rat cardiac fibroblasts, AngII promoted the mRNA and protein expression of periostin. AngII provoked activation of cAMP response element-binding protein (CREB), and CREB small interfering RNA (siRNA) suppressed AngII-induced periostin expression. Inhibition of p38 mitogen-activated protein kinase (p38 MAPK) with SB202190 attenuated AngII-induced CREB activation and periostin expression. Transfection with RasGRP1 siRNA or RasN17 dominant-negative plasmid prevented AngII-induced p38 MAPK phosphorylation and periostin expression. Transforming growth factor (TGF)-β1 antibody decreased the stimulatory effect of AngII on periostin expression. The extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor PD98059 attenuated AngII-induced TGF-β1 expression, Smad2/3 nuclear accumulation, and periostin expression. Conclusion The activation of the Ras/p38 MAPK/CREB pathway is required for AngII-induced periostin expression. ERK1/2 also participates in AngII-induced periostin expression by regulating TGF-β1/Smad signaling.

Cardiovasc Res: 03 Mar 2011; epub ahead of print

Human C-reactive protein exacerbates metabolic disorders in association with adipose tissue remodeling.

Kaneko H, Anzai T, Nagai T, Anzai A, ... Ogawa S, Fukuda K
Aims C-reactive protein (CRP) expression is increased with metabolic alterations. We sought to clarify the effect of CRP on the development of obesity-induced metabolic disorders using human CRP-overexpressing transgenic mice (CRPTG). Methods and Results CRPTG and their nontransgenic littermates (CON) were fed a standard diet (STD) or a high-fat diet (HFD) from 6 weeks of age. Oral glucose tolerance and intraperitoneal insulin tolerance tests 12 weeks after starting the diets showed deterioration of glucose tolerance and insulin sensitivity in HFD/CRPTG than those in HFD/CON. Hepatocellular ballooning, oil droplets, and perisinusoidal fibrosis were more prominent in HFD/CRPTG than in HFD/CON. In HFD/CRPTG, hepatic triglyceride content was higher and serum adiponectin level was lower than in HFD/CON. Epididymal adipose tissue (EAT) mRNA expression of Emr1, MCP-1, and TNF-α in HFD/CRPTG was upregulated compared to that in HFD/CON. Immunohistochemical staining of EAT showed that number of Mac-3(+) macrophages was higher in HFD/CRPTG than in HFD/CON. Conclusion Human-CRP overexpression facilitated the development of insulin resistance and hepatosteatosis under HFD, in association with adiponectin downregulation and enhancement of macrophage infiltration and expression of proinflammatory cytokines in EAT, suggesting its pathogenic role in the development of obesity-induced metabolic disorders.

Cardiovasc Res: 30 Mar 2011; epub ahead of print

Additive cardioprotection by pharmacological postconditioning with hydrogen sulfide and nitric oxide donors in mouse heart: S-sulfhydration vs S-nitrosylation.

Sun J, Aponte AM, Menazza S, Gucek M, Steenbergen C, Murphy E
Hydrogen sulfide (H2S), as a gaseous signaling molecule, has been found to play important roles in postconditioning (PostC)-induced cardioprotection. Similar to nitric oxide (NO)-mediated protein S-nitrosylation (SNO), recent studies suggest that H2S could regulate protein function through another redox-based post-translational modification on protein cysteine residue(s), i.e., S-sulfhydration (SSH). In this study, we examined whether there are changes in protein SSH associated with cardioprotection induced by treatment with H2S on reperfusion. In addition, we also examined whether there is cross talk between H2S and NO. Compared to control, treatment on reperfusion with NaHS (H2S donor, 100 µmol/L) significantly reduced post-ischemic contractile dysfunction and infarct size. A comparable cardioprotective effect could be also achieved by reperfusion treatment with SNAP (NO donor, 10 µmol/L). Interestingly, simultaneous reperfusion with both donors had an additive protective effect. In addition, C-PTIO (NO scavenger, 20 µmol/L) eliminated the protection induced by NaHS and also the additive protection by SNAP+NaHS together. Using a modified biotin switch method, we observed a small increase in SSH following NaHS treatment on reperfusion. We also found that NaHS treatment on reperfusion increases SNO to a level comparable to that with SNAP treatment. In addition, there was an additive increase in SNO but not SSH when SNAP and NaHS were added together at reperfusion. Thus part of the benefit of NaHS is an increase in SNO, and the magnitude of the protective effect is related to the magnitude of the increase in SNO.

Cardiovasc Res: 23 Feb 2016; epub ahead of print

Preventing Progression of Cardiac Hypertrophy and Development of Heart Failure by Paricalcitol Therapy in Rats.

Bae S, Yalamarti B, Ke Q, Choudhury S, ... Thadhani R, Kang PM
Aims Vitamin D deficiency is associated with cardiac hypertrophy and heart failure, and vitamin D therapy prevents the progression of cardiac hypertrophy in animal models. Here, we examine whether vitamin D therapy prevents progression of pre-existing cardiac hypertrophy, and development of heart failure. Methods and Results When male Dahl salt sensitive (DSS) rats are fed a high salt (HS) diet, all rats develop cardiac hypertrophy after 5 weeks (H). Thereafter, rats were treated with vehicle (V), paricalcitol (PC, an active vitamin D analog at 200ng, IP 3x/wk), enalapril (EP, 90μg/day), and PC+EP. All groups were continued on the HS diet and evaluated after 4 weeks of therapy. The PC and PC+EP, but not the V and EP only groups, showed significant prevention of progression of pre-existing cardiac hypertrophy. The signs of decompensated heart failure were evident in the vehicle treated group; these heart failure parameters significantly improved with PC, EP or PC+EP therapy. The expression of PKCα which is regulated by Ca(2+)and known to stimulate cardiac hypertrophy, was significantly increased in the vehicle group, and PC, EP or PC+EP effectively decreased PKCα activation. We also observed normalization of genetic alterations during progression to heart failure with PC treatment. Conclusions PC treatment resulted in both the prevention of progression of pre-existing cardiac hypertrophy, and the development of heart failure, compared to improvement in progression to heart failure by EP alone. These beneficial findings in heart were associated with inhibition of PKCα activation, and reversal of gene alterations.

Cardiovasc Res: 13 May 2011; epub ahead of print

Micro-RNAs in abdominal aortic aneurysms: insights from animal models and relevance to human disease.

Raffort J, Lareyre F, Clement M, Mallat Z
Abdominal aortic aneurysm is a major health concern and may be associated with high rates of mortality linked to acute complications. Diagnosis and treatment are respectively based on imaging and surgical techniques. Drug-based therapies are still mostly ineffective, which highlights a real unmet need. Major pathophysiological mechanisms leading to aneurysm formation involve inflammatory processes, degradation of the extracellular matrix, and loss of smooth muscle cells. However, the precise cellular and molecular pathways are still poorly understood. Recently, microRNAs have emerged as major intracellular players in a wide range of biological processes, and their stability in extracellular medium within microvesicles has led to propose them as mediators of intercellular crosstalk and as potential biomarkers and therapeutic targets in a variety of disease settings. To date, several studies have been performed to address the involvement of micro-RNAs in aneurysm formation and complications. Here we discuss the roles and implications of micro-RNAs in animal models and their relevance to human abdominal aortic aneurysm.

Cardiovasc Res: 10 Mar 2016; epub ahead of print

Transforming growth factor-β and oxidative stress mediate tachycardia-induced cellular remodeling in cultured atrial-derived myocytes.

Yeh YH, Kuo CT, Chan TH, Chang GJ, ... Nattel S, Chen WJ
Aims Atrial fibrillation (AF), a common tachyarrhythmia in clinical practice, is associated with increased oxidative stress. Structural remodeling in atrial myocytes, including myofibril degradation, is an important characteristic of AF. However, the mechanism underlying AF-induced cellular structural remodeling remains unclear. The aim of this study was to investigate the role of oxidative stress and related factors in tachycardia-induced atrial structural remodeling Methods and results Cultured atrial-derived myocytes (HL-1 cell line) were subjected to electrical stimulation. Immunofluorescence and immunoblotting were used to evaluate oxidative stress, myofibril degradation, and transforming growth factor-β (TGF-β) expression. Tachypacing in HL-1 cells induced TGF-β expression, pronounced oxidative stress including up-regulation of NADPH oxidases (Nox2/4), and degradation of myofibril. Oxidative stress scavenger, NADPH oxidase inhibitors, and small-interfering RNAs for Nox2/4 blocked tachypacing-induced myofibril degradation, suggesting that Nox-derived oxidative stress may lead to tachycardia-induced myofibril degradation. Blockade of TGF-β signaling by neutralizing TGF-β antibodies attenuated myofibril loss in response to tachypacing, implicating autocrine and/or paracrine roles for TGF-β in such effects. Tachypacing also induced the activation of p-Smad3 (an effective mediator of TGF-β) and small-interfering RNAs for Nox2/4 attenuated its activation, supporting a crosstalk between both signaling pathways. Furthermore, TGF-β expression, oxidative stress, and myofibril loss were greater in the atria of patients with AF than those with sinus rhythm. Conclusions Rapid activation in atrial myocytes promotes myofibril degradation through autocrine/paracrine TGF-β signaling and increased oxidative stress. These findings provide an important mechanistic insight into AF-related structural remodeling.

Cardiovasc Res: 03 Feb 2011; epub ahead of print

Apurinic/apyrimidinic endonuclease 1 inhibits protein kinase C-mediated p66shc phosphorylation and vasoconstriction.

Lee SK, Chung JI, Park MS, Joo HK, ... Irani K, Jeon BH
Aims: Phosphorylation of the adaptor protein p66shc is essential for p66shc-mediated oxidative stress. We investigated the role of the reducing protein/DNA repair enzyme apurinic/apyrimidinic endonuclease1 (APE1) in modulating protein kinase CβII (PKCβII)-mediated p66shc phosphorylation in cultured endothelial cells and PKC-mediated vasoconstriction of arteries. Methods and results: Oxidized LDL (oxLDL) induced p66shc phosphorylation at serine 36 residue and PKCβII phosphorylation in mouse endothelial cells. Adenoviral overexpression of APE1 resulted in reduction of oxLDL-induced p66shc and PKCβII phosphorylation. Phorbol 12-myristate 13-acetate (PMA) which stimulates PKCs induced p66shc phosphorylation and this was inhibited by a selective PKCβII inhibitor. Adenoviral overexpression of PKCβII also increased p66shc phosphorylation. Overexpression of APE1 suppressed PMA-induced p66shc phosphorylation. Moreover, PMA-induced p66shc phosphorylation was augmented in cells in which APE1 was knocked down. PMA increased cytoplasmic APE1 expression, compared with basal condition, suggesting the role of cytoplasmic APE1 against p66shc phosphorylation. Finally, vasoconstriction induced by phorbol-12,13, dibutylrate, another PKC agonist, was partially inhibited by transduction of Tat-APE1 into arteries. Conclusion: APE1 suppresses oxLDL-induced p66shc activation in endothelial cells by inhibiting of PKCβII-mediated serine phosphorylation of p66shc, and mitigates vasoconstriction induced by activation of PKC.

Cardiovasc Res: 06 Apr 2011; epub ahead of print

Mitochondrial Adaptations to Physiological versus Pathological Cardiac Hypertrophy.

Abel ED, Doenst T
Cardiac hypertrophy is a stereotypic response of the heart to increased workload. The nature of the workload increase may vary depending on the stimulus (repetitive, chronic, pressure or volume overload). If the heart fully adapts to the new loading condition, the hypertrophic response is considered physiological. If the hypertrophic response is associated with the ultimate development of contractile dysfunction and heart failure, the response is considered pathological. Although divergent signaling mechanisms may lead to these distinct patterns of hypertrophy, there is some overlap. Given the close relationship between workload and energy demand, any form of cardiac hypertrophy will impact the energy generation by mitochondria, which are the key organelles for cellular ATP production. Significant changes in the expression of nuclear and mitochondrially-encoded transcripts that impact mitochondrial function as well as altered mitochondrial proteome composition and mitochondrial energetics have been described in various forms of cardiac hypertrophy. Here, we review mitochondrial alterations in pathological and physiological hypertrophy. We suggest that mitochondrial adaptations to pathological and physiological hypertrophy are distinct and we will review potential mechanisms that might account for these differences.

Cardiovasc Res: 24 Jan 2011; epub ahead of print

Twenty-five years of preconditioning: are we ready for ischaemia? From coronary occlusion to systems biology and back.

Garcia-Dorado D, Barba I, Inserte J
It was 25 years ago that the phenomenon of ischaemic preconditioning was first described. The protection afforded by preconditioning was found to be exceptionally robust and aroused immediate interest amongst the scientific community. During the last quarter century, a large research effort has been made to elucidate its molecular mechanisms with the final aim of using this knowledge to develop new cardioprotective treatments. The scientific impact of the discovery of ischaemic preconditioning has been huge-it has allowed a change of paradigm in the understanding of ischaemia-reperfusion injury, from being a mere consequence of energy deprivation to being a complex, active process taking place to a large extent during the reperfusion phase. However, the clinical impact has been small, and some have anticipated a loss of interest in preconditioning unless this changes in the near future. We propose that the failure to develop clinical applications from ischaemic preconditioning is due in part to the incomplete understanding of its mechanisms and that a new integrative scientific approach should be used to resolve the complex networks of preconditioning protection signalling.

Cardiovasc Res: 10 Jun 2011; epub ahead of print

Tetrahydrobiopterin depletion and NOS2 uncoupling contribute to heart failure-induced alterations in atrial electrophysiology.

Nishijima Y, Sridhar A, Bonilla I, Velayutham M, ... Cardounel AJ, Carnes CA
Aims Heart failure is a common antecedent to atrial fibrillation; both heart failure and atrial fibrillation are associated with increased myocardial oxidative stress. Chronic canine heart failure reduces atrial action potential duration and atrial refractoriness. We hypothesized that inducible NO synthase 2 (NOS2) contributes to atrial oxidative stress and electrophysiologic alterations. Methods and Results A 16 week canine tachypacing model of heart failure was used (n=21). At ten weeks, dogs were randomized to either placebo (n= 12) or active treatment (n = 9) with NOS cofactor, tetrahydrobiopterin (BH(4), 50 mg) and NOS substrate (L-arginine, 3 grams) twice daily for six weeks. A group of matched controls (n=7) was used for comparison. Heart failure increased atrial NOS2, and reduced atrial BH(4), while L-arginine was unchanged. Treatment reduced inducible atrial fibrillation, and normalized the heart failure-induced shortening of the left atrial myocyte action potential duration. Treatment increased atrial [BH(4)] while [L-arginine] was unchanged. Treatment did not improve left ventricular function or dimensions. Heart failure-induced reductions in atrial [BH(4)] resulted in NOS uncoupling, as measured by NO and O(2)(•(-)) production, while BH(4) and L-arginine treatment normalized NO and O(2)(•(-)). Heart failure resulted in left atrial oxidative stress which was attenuated by BH(4) and L-arginine treatment. Conclusions Chronic nonischemic heart failure results in atrial oxidative stress and electrophysiologic abnormalities by depletion of BH(4) and uncoupling of NOS2. Modulation of NOS2 activity by repletion of BH(4) may be a safe and effective approach to reduce the frequency of atrial arrhythmias during heart failure.

Cardiovasc Res: 04 Apr 2011; epub ahead of print

MicroRNA-21 is a key determinant in IL11/STAT-3 anti-apoptotic signaling pathway in preconditioning of skeletal myoblasts.

Haider HK, Idris NM, Kim HW, Ahmed RP, Shujia J, Ashraf M
Aims: We have previously shown that preconditioning of stem and progenitor cells promotes their survival post-engraftment in the infarcted heart. The present study was designed to: 1-delineate the role of microRNA-21 (miR-21) in interleukin-11 (IL11) signaling during preconditioning of skeletal myoblasts (MY) and 2-to study the long-term fate of preconditioned MY ((PC)MY) post-transplantation in the infarcted heart. Methods and Results We report that pharmacological preconditioning of MY with diazoxide (Dz) showed robust expression of IL11 and activation of extra-cellular signal regulated kinase1/2 (Erk1/2) and signal transducers and activators of transcription-3 (Stat3) with concomitantly increased miR-21. These molecular events improved cytoprotection of (PC)MY under oxidant stress in vitro which was compromised by pretreatment of (PC)MY with IL11 specific siRNA, Erk1/2 blocker or anti-miR-21. In vivo studies for sry-gene detection in a female rat heart model of acute myocardial infarction showed 2-fold higher survival of male donor (PC)MY 4 days and 7 days post-engraftment. Long-term fate of the engrafted cells was determined at 4 months after transplantation. Immunohistological studies revealed that in comparison with non-preconditioned MY, (PC)MY improved angiogenic response in the heart which was evident from higher number of blood vessels per surface area (0.155mm(2)) and myogenic differentiation of (PC)MY in the heart. Indices of myocardial contractility including ejection fraction and fractional shortening showed significant improvement in (PC)MY treated animals. Conclusions: miR-21 is key regulator of Erk1/2-Stat3 signaling downstream of IL11 during preconditioning of MY. The therapeutic benefits of (PC)MY were stable and persisted until 4 months of observation.

Cardiovasc Res: 25 May 2010; epub ahead of print

Alterations of Atrial Ca2+ Handling as Cause and Consequence of Atrial Fibrillation.

Greiser M, Lederer WJ, Schotten U
Atrial Fibrillation (AF) is the most prevalent sustained arrhythmia. As the most important risk factor for embolic stroke, AF is associated with a high morbidity and mortality. Despite decades of research, successful (medical and interventional) \'ablation\' of the arrhythmia remains challenging. AF is characterized by a diverse etiology including heart failure, hypertension and valvular disease. Based on this understanding, new treatment strategies that are specifically tailored to the underlying pathophysiology of a certain \'type\' of AF are being developed. One important aspect of AF pathophysiology is altered intracellular Ca(2+) handling. Due to the increase in atrial activation rate and the subsequent initial [Ca(2+)](i) overload, AF induces \'remodeling\' of intracellular Ca(2+) handling. Current research focuses on unraveling the contribution of altered intracellular Ca(2+) handling to different types of AF. More specifically, changes in intracellular Ca(2+) homeostasis preceding the onset of AF, in conditions which predispose to AF (e.g. heart failure), appear to be different from changes in Ca(2+) handling developing after the onset of AF. Here we review and critique altered intracellular Ca(2+) handling and its contribution to three specific aspects of AF pathophysiology, 1. Excitation-transcription coupling and Ca(2+) dependent signaling pathways, 2. Atrial contractile dysfunction and 3. Arrhythmogenicity.

Cardiovasc Res: 16 Dec 2010; epub ahead of print

Matrix aging and vascular impacts: focus on elastin fragmentation.

Duca L, Blaise S, Romier B, Laffargue M, ... Debelle L, Maurice P
Cardiovascular diseases (CVD) are the leading cause of death worldwide and represent a major problem of public health. Over the years, life expectancy has considerably increased throughout the world and the prevalence of CVD is inevitably rising with the growing aging of the population. The normal process of aging is associated with progressive deterioration in structure and function of the vasculature, commonly called vascular aging. At the vascular level, extracellular matrix (ECM) aging leads to molecular alterations of long half-life proteins, such as elastin and collagen, and have critical impacts on vascular diseases.This review highlights the ECM alterations occurring during vascular aging with a specific focus on elastin fragmentation, and the contribution of elastin-derived peptides (EDP) in age-related vascular complications. Moreover, current and new pharmacological strategies aiming at minimizing elastin degradation, EDP generation and associated biological effects are discussed. These strategies may be of major relevance for preventing and/or delaying vascular aging and its complications.

Cardiovasc Res: 23 Mar 2016; epub ahead of print

Establishment of the mouse ventricular conduction system.

Miquerol L, Beyer S, Kelly RG
The ventricular conduction system represents the electrical wiring responsible for the coordination of cardiac contraction. Defects in the circuit produce a delay or conduction block and induce cardiac arrhythmias. Understanding how this circuit forms and identification of the factors important for its development thus provide insights into the origin of cardiac arrhythmias. Recent advances, using genetically modified mouse models, have contributed to understanding how the ventricular conduction system is established during heart development. Transgenic mice carrying different reporter genes have highlighted the conservation of the anatomy and development of the ventricular conduction system between mouse and man. Lineage tracing and retrospective clonal analysis have established the myogenic origin of the ventricular conduction system and determined properties of conductive progenitor cells. Finally, gene knockout models reproducing human cardiac defects, have led to the identification of transcription factors important for the development of the ventricular conduction system. These transcription factors operate at the levels of both conduction system morphogenesis and differentiation by controlling the expression of genes responsible of the electrical activity of the heart. In summary, defects in the ventricular conduction system are a major cause of arrhythmias and deciphering the molecular pathways responsible for conduction system morphogenesis and the differentiation of conductive myocytes furthers our understanding of the mechanisms underlying heart disease.

Cardiovasc Res: 09 Mar 2011; epub ahead of print

Emerging regulators of vascular smooth muscle cell function in the development and progression of atherosclerosis.

Johnson JL
After a period of relative senescence in the field of vascular smooth muscle cell (VSMC) research with particular regards to atherosclerosis, the last few years has witnessed a resurgence, with extensive research re-assessing potential molecular mechanisms and pathways that modulate VSMC behaviour within the atherosclerotic-prone vessel wall and the atherosclerotic plaque itself. Attention has focussed on the pathological contribution of VSMC in plaque calcification; systemic and local mediators such as inflammatory molecules and lipoproteins; autocrine and paracrine regulators which affect cell-cell and cell to matrix contacts alongside cytoskeletal changes. In this brief focused review I will discuss recent insights that have been gained into how a myriad of recently identified factors can influence the pathological behaviour of VSMC and their subsequent contribution to atherosclerotic plaque development and progression. An overriding theme is the mechanisms involved in the alterations of VSMC function during atherosclerosis.

Cardiovasc Res: 22 Jul 2014; epub ahead of print

Shear stress and the endothelial transport barrier.

Tarbell JM
The shear stress of flowing blood on the surfaces of endothelial cells that provide the barrier to transport of solutes and water between blood and the underlying tissue modulates the permeability to solutes and the hydraulic conductivity. This review begins with a discussion of transport pathways across the endothelium and then considers the experimental evidence from both in vivo and in vitro studies that shows an influence of shear stress on endothelial transport properties after both acute (minutes to hours) and chronic (hours to days) changes in shear stress. Next, the effects of shear stress on individual transport pathways (tight junctions, adherens junctions, vesicles and leaky junctions) are described, and this information is integrated with the transport experiments to suggest mechanisms controlling both acute and chronic responses of transport properties to shear stress. The review ends with a summary of future research challenges.

Cardiovasc Res: 14 Jun 2010; epub ahead of print

At the cross-point of connexins, calcium and ATP: blocking hemichannels inhibits vasoconstriction of rat small mesenteric arteries.

Bol M, Wang N, De Bock M, Wacquier B, ... Van de Voorde J, Leybaert L
Connexins form gap-junctions (GJs) that directly connect cells, thereby coordinating vascular cell function and controlling vessel diameter and blood flow. GJs are composed of two hemichannels contributed by each of the connecting cells. Hemichannels also exist as non-junctional channels that, when open, lead to the entry/loss of ions and the escape of ATP. Here we investigated cross-talk between hemichannels and Ca(2+)/purinergic signaling in controlling blood vessel contraction. We hypothesized that hemichannel Ca(2+) entry and ATP release contributes to smooth muscle cell (SMC) Ca(2+) dynamics, thereby influencing vessel contractility. We applied several peptide modulators of hemichannel function and inhibitors of Ca(2+) and ATP signaling to investigate their influence on SMC Ca(2+) dynamics and vessel contractility.

Cardiovasc Res: 27 Sep 2016; epub ahead of print

Heterogeneity in the lymphatic vascular system and its origin.

Ulvmar MH, Mäkinen T
Lymphatic vessels have historically been viewed as passive conduits for fluid and immune cells, but this perspective is increasingly being revised as new functions of lymphatic vessels are revealed. Emerging evidence shows that lymphatic endothelium takes an active part in immune regulation both by antigen presentation and expression of immunomodulatory genes. In addition, lymphatic vessels play an important role in uptake of dietary fat and clearance of cholesterol from peripheral tissues, and they have been implicated in obesity and arteriosclerosis. Lymphatic vessels within different organs and in different physiological and pathological processes show a remarkably plasticity and heterogeneity, reflecting their functional specialization. In addition, lymphatic endothelial cells of different organs were recently shown to have alternative developmental origins, which may contribute to the development of the diverse lymphatic vessel and endothelial functions seen in the adult. Here we discuss recent developments in the understanding of heterogeneity within the lymphatic system considering the organ-specific functional and molecular specialization of lymphatic endothelial cells and their developmental origin.

Cardiovasc Res: 29 Jun 2016; epub ahead of print

543miR-19a replacement rescues cardiac and fin defects in zebrafish model of holt-oram syndrome.

Chiavacci E, Cremisi F, Guzzolino E, Cellerino A, ... D\'aurizio R, Pitto L
MicroRNAs (miRNAs) are short, single-stranded RNAs that anneal with complementary sequences in mRNAs thereby suppressing protein expression and often decreasing mRNA stability. Due to the high number of genes that are potentially targeted by one miRNA and to the relatively low number of different miRNAs coded in the genome, miRNAs are the best candidates to orchestrate gene expression in embryonic development and in response to inducing signals, by integrating distinct transcription factors pathways. Indeed, the manipulation of miRNA expression or function can have a profound impact on cellular phenotype. miRNA are critically involved in almost all the biological processes in health and disease including several cardiovascular disorders. Tbx5 is a transcription factor crucial for heart development and implicated in cardiac function. In human, TBX5 mutations are associated with Holt-Oram Syndrome (HOS), which is characterized by upper limb and congenital heart defects. In mouse model of HOS, expression profiling revealed that Tbx5 regulates hundreds of genes through complex transcriptional networks, acting both directly on gene expression and indirectly as "regulating of regulators". Our data indicate that miRNAs can be important effectors of Tbx5. The goal of our project is to better characterize the miRNAs/Tbx5 regulatory network with the aim: a) to understand the molecular roots of the complex HOS phenotype; b) to verify if it might be possible, by miRNA modulation, to restore the aberrant pathways generated by Tbx5 alteration.

Cardiovasc Res: 14 Jul 2014; 103:S99

545MiR-139 expression is detrimental during pressure overload-induced heart failure.

Schroen B, Peters T, Verhesen W, Derks W, ... De Windt L, Heymans S
Cardiac hypertrophy and consequent contractile dysfunction continue to burden Western society. Cardiomyocyte cyclic AMP (cAMP) and calcium are driving forces behind cardiomyocyte contraction. Distortion of their balance may induce HF, but specific therapies aiming at restoring physiological cAMP/calcium signaling are lacking. We recently identified microRNA-139 (miR-139) to be downregulated in failing human hearts. MiR-139 resides in the phosphodiesterase gene PDE2A and is predicted to target several phosphodiesterase messengers. In view of the central role of phosphodiesterases in controlling cardiac cAMP and calcium signaling, we hypothesized that miR-139 may affect HF progression by fine-tuning cAMP and calcium balances. Adeno-associated virus serotype 9 (AAV9), either empty control or expressing pre-miR-139, was administered to male C57Bl/6J mice. After allowing transgene expression for 3 weeks, mice were subjected to sham treatment or 4 weeks of pressure overload by subcutaneous Angiotensin II infusion (AngII, 2,5 mg/(kg·d). MiR-139 overexpression mildly aggravated HF development upon AngII with echocardiographically measured fractional shortening decreasing by 24±7% in AAV9-control AngII and by 46±5% in AAV9-pre-miR-139 AngII (n>11/group; p=0.14). In AAV9-control mice, AngII infusion led to concentric hypertrophy with an increased relative wall thickness (RWT) of 35±6%, whereas mice overexpressing miR-139 showed a rather eccentric form of hypertrophy with an increased RWT of 14±7% (p<0.05). The fraction of unphosphorylated cardiac troponin I (cTnI), a substrate of the cAMP dependent protein kinase A (PKA), tended to increase upon AngII infusion only in mice overexpressing miR-139 (n=4/group; p=0.11), indicating a reduced relaxation rate due to increased calcium sensitivity, a feature commonly observed in HF. Complimentary to these data, in vivo knockdown of mir-139 by cholesterol-tagged antagomiRs (20mg/kg) on three consecutive days before start of AngII infusion dampened the development of pressure overload-induced cardiac hypertrophy (increase in HW/TL: ctrl: 48±10%, n=4; antagomiR: 25±7%, n=7; p=0.16). In conclusion, cardiac downregulation of miR-139 upon pressure overload is a protective response to preserve cardiac function. AAV9-mediated overexpression of miR-139 promotes cardiac dilation and predisposes to HF. Upcoming experiments will aim at defining the molecular mechanism by which miR-139/phosphodiesterase signaling affects cardiac pathophysiology.

Cardiovasc Res: 14 Jul 2014; 103:S99

DNA-dependent protein kinase (DNA-PK) permits vascular smooth muscle cell proliferation through phosphorylation of the orphan nuclear receptor NOR1.

Medunjanin S, Daniel JM, Weinert S, Dutzmann J, ... Zuschratter W, Braun-Dullaeus RC
Being central part of the DNA repair machinery, DNA-dependent protein kinase (DNA-PK) seems to be involved in other signalling processes, as well. NOR1 is a member of the NR4A subfamily of nuclear receptors, which plays a central role in vascular smooth muscle cell (SMC) proliferation and in vascular proliferative processes. We determined putative phosphorylation sites of NDA-PK in NOR1 and hypothesized that the enzyme is able to modulate NOR1 signalling and, this way, proliferation of SMC RESULTS AND Methods: Cultured human aortic SMC were treated with the specific DNA-PK inhibitor NU7026 (or siRNA), which resulted in a 70% inhibition of FCS-induced proliferation as measured by BrdU incorporation. Furthermore, FCS-stimulated upregulation of NOR1 protein as well as the cell cycle promoting proteins proliferating cell nuclear antigen (PCNA), cyclin D1 and hyperphosphorylation of the retinoblastoma protein (RB) were prevented by DNA-PK inhibition. Co-immuno-precipitation studies from VSM cell lysates demonstrated that DNA-PK forms a complex with NOR1. Mutational analysis and kinase assays demonstrated that NOR1 is a substrate of DNA-PK and is phosphorylated in the N-terminal domain. Phosphorylation resulted in posttranscriptional stabilization of the protein through prevention of its ubiquitination. Active DNA-PK and NOR1 were found predominantly expressed within the neointima of human atherosclerotic tissue specimens. In mice, inhibition of DNA-PK significantly attenuated neointimal lesion size 3 weeks after wire-injury.

Cardiovasc Res: 07 Apr 2015; epub ahead of print

Direct and Differential Effects of Stem Cell Factor on the Neovascularization Activity of Endothelial Progenitor Cells.

Kim KL, Meng Y, Kim JY, Baek EJ, Suh W
Aims Previous studies on the role of Stem Cell Factor (SCF) in endothelial progenitor cell (EPC)-mediated neovascularization have focused on the EPC mobilization and homing process. However, the direct effects of SCF on neovascularization activity of EPCs have not been characterized. We sought to determine whether SCF regulates the neovascularization ability of EPCs by comparing its roles in mature endothelial cells. Methods and Results In vitro and in vivo assays revealed that SCF substantially increased the neovascularization activity of human EPCs through c-Kit receptor. Notably, the SCF-induced increase in neovascularization activity was substantially greater in EPCs than that in human umbilical vein endothelial cells (HUVECs). SCF-induced phosphorylation of c-Kit and downstream signaling molecules was consistently found to be more potent and longer-lasting in EPCs than in HUVECs. This high responsiveness of EPCs to SCF was explained by the finding that the cell-surface expression of c-Kit is far higher in EPCs than in HUVECs. A c-Kit promoter assay revealed that the increased expression of c-Kit in EPCs could be attributed to the greater expression of stem cell leukemia, LIM-only 2, and GATA binding protein 2. Conclusion In addition to its documented role in the mobilization and recruitment of EPCs, our findings show that SCF directly enhances the neovascularization activity of EPCs. Furthermore, the present study provides further evidence that EPCs exhibit differentially greater responsiveness to hypoxia-inducible cytokines including SCF than mature endothelial cells, suggesting that EPCs in ischemic tissues function differently from mature endothelial cells, although they exhibit very similar phenotypes.

Cardiovasc Res: 03 Jun 2011; epub ahead of print

Id proteins in the vasculature: from molecular biology to cardiopulmonary medicine.

Yang J, Li X, Morrell NW
The Id proteins belong to the helix-loop-helix group of transcription factors and regulate cell differentiation and proliferation. Recent studies have reported that Id proteins play important roles in cardiogenesis and formation of the vasculature. We have also demonstrated that heritable pulmonary arterial hypertension (HPAH) patients have dysregulated Id gene expression in pulmonary artery smooth muscle cells. The interaction between bone morphogenetic proteins (BMP) and other growth factors or cytokines regulates Id gene expression, which impacts on pulmonary vascular cell differentiation and proliferation. Exploration of the roles of Id proteins in vascular remodelling that occurs in PAH and atherosclerosis might provide new insights into the molecular basis of these diseases. In addition, current progress in identification of the interactors of Id proteins will further the understanding of the function of Ids in vascular cells and enable the identification of novel targets for therapy in PAH and other cardiovascular diseases.

Cardiovasc Res: 01 Oct 2014; epub ahead of print

Thrombomodulin domains attenuate atherosclerosis by inhibiting thrombin-induced endothelial cell activation.

Wei HJ, Li YH, Shi GY, Liu SL, ... Kuo CH, Wu HL
Aims Thrombin modulates the formation of atherosclerotic lesions by stimulating a variety of cellular effects through protease-activated receptor-1 (PAR-1) activation. Thrombomodulin (TM) inhibits thrombin effects by binding thrombin through its domain 2 and 3 (TMD23). We investigated whether recombinant TMD23 (rTMD23) could inhibit atherosclerosis via its thrombin binding ability. Methods and Results Wild-type mouse rTMD23 and 3 mutants with altered thrombin-binding sites, rTMD23 (I425A), rTMD23 (D424A/D426A), and rTMD23 (D424A/I425A/D426A), were expressed and purified in the Pichia pastoris expression system. Wild-type rTMD23 and rTMD23 (D424A/D426A) could effectively bind thrombin, activate protein C, and prolong thrombin clotting time, whereas rTMD23 (I425A) and rTMD23 (D424A/I425A/D426A) lost these functions. Wild-type rTMD23, but not rTMD23 (I425A), decreased both the thrombin-induced surface PAR-1 internalization and the increase of cytoplasmic Ca(2+) concentrations in endothelial cells. Wild-type rTMD23 and rTMD23 (D424A/D426A) also inhibited thrombin-induced adhesion molecules and monocyte chemoattractant protein-1 expression and increased permeability in endothelial cells, whereas rTMD23 (I425A) and rTMD23 (D424A/I425A/D426A) had no such effects. Furthermore, wild-type rTMD23 and rTMD23 (D424A/D426A) were effective in reducing carotid ligation-induced neointima formation in C57BL/6 mice and atherosclerotic lesion formation in apolipoprotein E-deficient mice, whereas rTMD23 with the I425A mutation showed impairment of this function. Wild-type rTMD23, but not rTMD23 (I425A), also markedly suppressed the PAR-1, the adhesion molecules expression, and the macrophage content in the carotid ligation model and apolipoprotein E-deficient mice. Conclusions rTMD23 protein significantly reduces atherosclerosis and neointima formation through its thrombin binding ability.

Cardiovasc Res: 15 Aug 2011; epub ahead of print

P518Biomechanical properties, microstructure and constitutive modeling of human ventricular myocardium.

Sommer G, Schwarz M, Schriefl A, Wolinski H, ... Regitnig P, Holzapfel G
In the multidisciplinary field of heart research it is of utmost importance, for the description of phenomena like mechanoelectric feedback or heart wall thickening, to identify accurate myocardium material properties. Therefore this study aims to determine biaxial tensile and triaxial shear properties of the passive human ventricular myocardium. This novel combination of biaxial and shear testing, together with the investigation of the myocardial microstructure, will yield new innovative and essential information about material properties to fulfil the short term goals of constructing realistic myocardial models capable of capturing the mechanics of the heart, as well as aiding the long term goals of improving methods of medical treatment and quality of life for people suffering from heart diseases. For the biaxial tests, squared specimens (25x25x2mm) were prepared with their sides aligned with the fiber and cross-fiber axis. During the experiments, the specimens were submerged in a cardioplegic solution at physiological conditions and different stretch ratios were applied consecutively. For the triaxial shear testing, three adjoining cubic specimens (4x4x4mm) were prepared with their sides aligned according to the fiber axis, sheet axis and sheet-normal axis. Three cycles of sinusoidal simple shear (0.1-0.5 in 0.1 steps of specimen thickness) were applied to each cubic specimens in two orthogonal directions. A novel combination of optical clearing and multiphoton microscopy was utilized to explore the 3D microstructure of the tissue emphasizing the 3-D orientation and dispersion of the muscle fibers and adjacent collagen fabrics. The tissue showed pronounced nonlinear and highly orientation dependent behavior. The donor\'s age was greatly influencing the mechanical behavior of the myocardial tissue. Microstructural investigations affirmed an orthotropic composition of the investigated tissue and showed highly aligned myofibers with small dispersion in the healthy human myocardium. An invariant-based constitutive model showed the ability to give a good representation of both the biaxial tensile and the triaxial shear responses. The material data from this study is intended to be used in numerical (Finite Element) simulations for better understanding of fundamental underlying ventricular mechanics, a step needed in the improvement of medical treatment of heart diseases.

Cardiovasc Res: 14 Jul 2014; 103:S95

P511Immunomagnetic purification of cardiomyocytes, cardiac fibroblasts and endothelial cells from neonatal mouse and rat hearts.

Christalla P, Kernbach M, Riesen J, Wiencierz AM, Bosio A, Eckardt D
Pure cardiovascular cell lineages are a requisite for an unbiased and cell type specific investigation of (patho-) physiological mechanisms involved in heart failure. Moreover pure heart cells are needed as building blocks for engineered heart mimetics. The latter offers the possibility to replace infarcted myocardium and to investigate heart development and cardiomyogenesis in a simplified in vitro model. However, it has to be acknowledged that strategies capable to specifically purify heart cells are not well established. To overcome these experimental limitations we developed a rapid and automated neonatal heart dissociation protocol enabling an immunomagnetic enrichment of the three major heart cell types, namely cardiomyocytes (CMs), cardiac fibroblasts (Fibs) and endothelial cells (Endos) from both, mouse and rat neonatal hearts. First, we established an enzyme mix optimal for the dissociation of neonatal mouse and rat hearts (P0-P3) by screening our enzyme library. Next, we optimized the dissociation by utilizing our gentleMACS technology resulting in a fast (1h), robust, and fully automated heart dissociation protocol. Analysis of the dissociated mouse and rat heart cells showed: (i) high cell vitalities (>90%), (ii) high frequencies of α-Actinin-positive CMs (>60%) and (iii) vimentin-positive non-CMs with a frequency of >40% (P2). In order to selectively enrich various cell-types from these heterogenous cell populations, we performed a cell-surface marker screen. We identified several candidates for the composition of antibody cocktails enabling selective enrichment of mouse and rat CMs, Fibs or Endos with purities of up to 97%. CMs grown in 2D cultures showed spontaneous beating activity and the expression of sarcomeric proteins in a cross-striated pattern (α-Actinin and cTnT). Additionally, heart cells were cultured on organotypic decellularized (1% SDS) mouse heart slices. Analysis of the slice cultures after recellularization showed (i) partial restoration of synchronous contractions and (ii) orientated and elongated heart cells along the microfibrillar heart structure. Purified Fibs were highly proliferative and could be expanded 34-fold over an analyzed culture period of 15 days. Finally, functionality of the purified endothelial cells was proven by Dil-LDL endocytosis. In summary, we established an automated protocol for the dissociation of neonatal mouse and rat hearts, enabling the subsequent immunomagnetic enrichment of CMs, Fibs and Endos which can readily be utilized for 2D cell culture assays or to generate in vitro heart muscle models and surrogate tissue for myocardial repair.

Cardiovasc Res: 14 Jul 2014; 103:S93

ESC Working Group Cellular Biology of the Heart: Position Paper: Improving the pre-clinical assessment of novel cardioprotective therapies.

Lecour S, Bøtker HE, Condorelli G, Davidson SM, ... Yellon DM, Hausenloy DJ
Ischemic heart disease (IHD) remains the leading cause of death and disability worldwide. As a result, novel therapies are still needed to protect the heart from the detrimental effects of acute ischemia-reperfusion injury, in order to improve clinical outcomes in IHD patients. In this regard, although a large number of novel cardioprotective therapies discovered in the research laboratory have been investigated in the clinical setting, only a few of these have been demonstrated to improve clinical outcomes. One potential reason for this lack of success may have been the failure to thoroughly assess the cardioprotective efficacy of these novel therapies in suitably designed pre-clinical experimental animal models. Therefore, the aim of this Position Paper by the European Society of Cardiology Working Group Cellular Biology of the Heart is to provide recommendations for improving the pre-clinical assessment of novel cardioprotective therapies discovered in the research laboratory, with the aim of increasing the likelihood of success in translating these new treatments into improved clinical outcomes.

Cardiovasc Res: 24 Oct 2014; epub ahead of print

Atrial Fibrillation: Effects Beyond the Atrium?

Wijesurendra RS, Casadei B
Atrial fibrillation (AF) is the most common sustained clinical arrhythmia and is associated with significant morbidity, mostly secondary to heart failure and stroke, and an estimated two-fold increase in premature death. Efforts to increase our understanding of AF and its complications have focused on unravelling the mechanisms of electrical and structural remodelling of the atrial myocardium. Yet, it is increasingly recognised that AF is more than an atrial disease, being associated with systemic inflammation, endothelial dysfunction and adverse effects on the structure and function of the left ventricular myocardium that may be prognostically important. Here we review the molecular and in vivo evidence that underpins current knowledge regarding the effects of human or experimental AF on the ventricular myocardium. Potential mechanisms are explored including diffuse ventricular fibrosis, focal myocardial scarring and impaired myocardial perfusion and perfusion reserve. The complex relationship between AF, systemic inflammation and endothelial/microvascular dysfunction and the effects of AF on ventricular calcium handling and oxidative stress are also addressed. Finally, consideration is given to the clinical implications of these observations and concepts, with particular reference to rate versus rhythm control.

Cardiovasc Res: 13 Jan 2015; epub ahead of print