Aberrant Activation of Notch1 Signaling in Glomerular Endothelium Induces Albuminuria.

Li L, Liu Q, Shang T, Song W, ... Lobe CG, Liu J

Glomerular capillaries are lined with a highly specialized fenestrated endothelium and contribute to the glomerular filtration barrier (GFB). The Notch signaling pathway is involved in regulation of GFB, but its role in glomerular endothelium has not been investigated due to the embryonic lethality of animal models with genetic modification of Notch pathway components in the endothelium.To determine the effects of aberrant activation of the Notch signaling in glomerular endothelium and the underlying molecular mechanisms.We established the ZEG-Notch1 intracellular domain (NICD1)/Tie2-tTA/Tet-O-Cre transgenic mouse model to constitutively activate Notch1 signaling in endothelial cells of adult mice. The triple transgenic mice developed severe albuminuria with significantly decreased VE-cadherin expression in the glomerular endothelium. In vitro studies showed that either NICD1 lentiviral infection or treatment with Notch ligand DLL4 markedly reduced VE-cadherin expression and increased monolayer permeability of human renal glomerular endothelial cells (HRGECs). In addition, Notch1 activation or gene knockdown of VE-cadherin reduced the glomerular endothelial glycocalyx. Further investigation demonstrated that activated Notch1 suppression of VE-cadherin was through the transcription factors SNAI1 and ERG, which bind to the -373 E-box and the -134/-118 ETS element of the VE-cadherin promoter, respectively. Our results reveal novel regulatory mechanisms whereby endothelial Notch1 signaling dictates the level of VE-cadherin through the transcription factors SNAI1 and ERG, leading to dysfunction of GFB and induction of albuminuria.



Circ Res: 12 Jan 2021; epub ahead of print

Adiponectin, Leptin and Cardiovascular Disorders.

Zhao S, Kusminski CM, Scherer PE

The landmark discoveries of leptin and adiponectin firmly established adipose tissue as a sophisticated and highly active endocrine organ, opening a new era of investigating adipose-mediated tissue crosstalk. Both obesity-associated hyperleptinemia and hypoadiponectinemia are important biomarkers to predict cardiovascular outcomes, suggesting a crucial role for adiponectin and leptin in obesity-associated cardiovascular disorders. Normal physiological levels of adiponectin and leptin are indeed essential to maintain proper cardiovascular function. Insufficient adiponectin and leptin signaling results in cardiovascular dysfunction. However, a paradox of high levels of both leptin and adiponectin is emerging in the pathogenesis of cardiovascular disorders. Here, we (1) summarize the recent progress in the field of adiponectin and leptin and its association with cardiovascular disorders, (2) further discuss the underlying mechanisms for this new paradox of leptin and adiponectin action, and (3) explore the possible application of partial leptin reduction, in addition to increasing the adiponectin/leptin ratio as a means to prevent or reverse cardiovascular disorders.



Circ Res: 07 Jan 2021; 128:136-149

MTORC1-Regulated Metabolism Controlled by TSC2 Limits Cardiac Reperfusion Injury.

Oeing CU, Jun S, Mishra S, Dunkerly-Eyring B, ... Ranek MJ, Kass DA

The mechanistic target of rapamycin complex-1 (mTORC1) controls metabolism and protein homeostasis, and is activated following ischemic reperfusion (IR) injury and by ischemic preconditioning (IPC). However, studies vary as to whether this activation is beneficial or detrimental, and its influence on metabolism after IR is little studied. A limitation of prior investigations is their use of broad gain/loss of mTORC1 function, mostly applied prior to ischemic stress. This can be circumvented by regulating one serine (S1365) on tuberous sclerosis complex (TSC2) to achieve bi-directional mTORC1 modulation but only with TCS2-regulated co-stimulation.We tested the hypothesis that reduced TSC2 S1365 phosphorylation protects the myocardium against IR and IPC by amplifying mTORC1 activity to favor glycolytic metabolism.Mice with either S1365A (TSC2; phospho-null) or S1365E (TSC2; phosphomimetic) knock-in mutations were studied ex vivo and in vivo. In response to IR, hearts from TSC2 mice had amplified mTORC1 activation and improved heart function compared to WT and TSC2 hearts. The magnitude of protection matched IPC. IPC requited less S1365 phosphorylation, as TSC2 hearts gained no benefit and failed to activate mTORC1 with IPC. IR metabolism was altered in TSC2, with increased mitochondrial oxygen consumption rate and glycolytic capacity (stressed/maximal extracellular acidification) after myocyte hypoxia-reperfusion. In whole heart, lactate increased and long-chain acyl-carnitine levels declined during ischemia. The relative IR protection in TSC2 was lost by lowering glucose in the perfusate by 36%. Adding fatty acid (palmitate) compensated for reduced glucose in WT and TSC2 but not TSC2 which had the worst post-IR function under these conditions.TSC2-S1365 phosphorylation status regulates myocardial substrate utilization, and its decline activates mTORC1 biasing metabolism away from fatty acid oxidation to glycolysis to confer protection against IR. This pathway is also engaged and reduced TSC2 S1365 phosphorylation required for effective IPC.



Circ Res: 05 Jan 2021; epub ahead of print

Radiation Impacts Early Atherosclerosis by Suppressing Intimal LDL Accumulation.

Ikeda J, Scipione CA, Hyduk S, Althagafi MG, ... MacParland SA, Cybulsky MI

Bone marrow transplantation (BMT) is used frequently to study the role of hematopoietic cells in atherosclerosis, but aortic arch lesions are smaller in mice after BMT.To identify the earliest stage of atherosclerosis inhibited by BMT and elucidate potential mechanisms.mice underwent total body γ-irradiation, bone marrow reconstitution and 6-week recovery. Atherosclerosis was studied in the ascending aortic arch and compared to mice without BMT. In BMT mice neutral lipid and myeloid cell topography were lower in lesions after feeding a cholesterol-rich diet (CRD) for 3, 6 and 12 weeks. Lesion coalescence and height were suppressed dramatically in mice post-BMT, whereas lateral growth was inhibited minimally. Targeted radiation to the upper thorax alone reproduced the BMT phenotype. Classical monocyte recruitment, intimal myeloid cell proliferation and apoptosis did not account for the post-BMT phenotype. Neutral lipid accumulation was reduced in 5-day lesions, thus we developed quantitative assays for LDL accumulation and paracellular leakage using DiI-labeled human LDL and rhodamine B-labeled 70kD dextran. LDL accumulation was dramatically higher in the intima ofrelative tomice, and was inhibited by injection of HDL mimics, suggesting a regulated process. LDL, but not dextran, accumulation was lower in mice post-BMT both at baseline and in 5-day lesions. Since the transcript abundance of molecules implicated in LDL transcytosis was not significantly different in the post-BMT intima, transcriptomics from whole aortic arch intima, and at single cell resolution, was performed to give insights into pathways modulated by BMT.Radiation exposure inhibits LDL entry into the aortic intima at baseline and the earliest stages of atherosclerosis. Single cell transcriptomic analysis suggests that LDL uptake by endothelial cells is diverted to lysosomal degradation and reverse cholesterol transport pathways. This reduces intimal accumulation of lipid and impacts lesion initiation and growth.



Circ Res: 04 Jan 2021; epub ahead of print

Single-Cell Analysis of Blood-Brain Barrier Response to Pericyte Loss.

Andaloussi Mäe M, He L, Nordling S, Vazquez-Liebanas E, ... Keller A, Betsholtz C

Pericytes are capillary mural cells playing a role in stabilizing newly formed blood vessels during development and tissue repair. Loss of pericytes has been described in several brain disorders, and genetically induced pericyte deficiency in the brain leads to increased macromolecular leakage across the blood-brain barrier (BBB). However, the molecular details of the endothelial response to pericyte deficiency remain elusive.To map the transcriptional changes in brain endothelial cells resulting from lack of pericyte contact at single-cell level, and to correlate them with regional heterogeneities in BBB function and vascular phenotype.We reveal transcriptional, morphological and functional consequences of pericyte absence for brain endothelial cells using a combination of methodologies, including single-cell RNA sequencing, tracer analyses and immunofluorescent detection of protein expression in pericyte-deficient adult Pdgfbret/ret mice. We find that endothelial cells without pericyte contact retain a general BBB-specific gene expression profile, however, they acquire a venous-shifted molecular pattern and become transformed regarding the expression of numerous growth factors and regulatory proteins. Adult Pdgfbret/ret brains display ongoing angiogenic sprouting without concomitant cell proliferation providing unique insights into the endothelial tip cell transcriptome. We also reveal heterogeneous modes of pericyte-deficient BBB impairment, where hotspot leakage sites display arteriolar-shifted identity and pinpoint putative BBB regulators. By testing the causal involvement of some of these using reverse genetics, we uncover a reinforcing role for angiopoietin 2 at the BBB.By elucidating the complexity of endothelial response to pericyte deficiency at cellular resolution, our study provides insight into the importance of brain pericytes for endothelial arterio-venous zonation, angiogenic quiescence and a limited set of BBB functions. The BBB-reinforcing role of ANGPT2 is paradoxical given its wider role as TIE2 receptor antagonist and may suggest a unique and context-dependent function of ANGPT2 in the brain.



Circ Res: 29 Dec 2020; epub ahead of print

Inositol Trisphosphate Receptors and Nuclear Calcium in Atrial Fibrillation.

Qi XY, Vahdahi Hassani F, Hoffmann D, Xiao J, ... Dobrev D, Nattel S

The mechanisms underlying atrial fibrillation (AF), the most common clinical arrhythmia, are poorly understood. Nucleoplasmic Ca regulates gene-expression, but the nature and significance of nuclear Ca-changes in AF are largely unknown.To elucidate mechanisms by which AF alters atrial cardiomyocyte (CM) nuclear Ca ([Ca]) and Ca/calmodulin-dependent protein kinase-II (CaMKII)-related signaling. Atrial CMs were isolated from control and AF-dogs (kept in AF by atrial tachypacing [600 bpm x 1 week]). [Ca] and cytosolic [Ca] (Ca]) were recorded via confocal microscopy. Diastolic [Ca] was greater than [Ca] under control conditions, while resting [Ca] was similar to [Ca]; both diastolic and resting [Ca] increased with AF. Inositol-trisphosphate-receptor (IPR) stimulation produced larger [Ca] increases in AF versus control CMs, and IPR-blockade suppressed the AF-related [Ca]-differences. AF upregulated nuclear protein-expression of IPR-type 1 (IPR1) and of phosphorylated CaMKII (immunohistochemistry and immunoblot), while decreasing the nuclear/cytosolic expression-ratio for histone deacetylase type-4 (HDAC4). Isolated atrial CMs tachypaced at 3 Hz for 24 hours mimicked AF-type [Ca] changes and L-type calcium current (ICaL) decreases versus 1-Hz-paced CMs; these changes were prevented by IP3R knockdown with short-interfering RNA directed against IPR1. Nuclear/cytosolic HDAC4 expression-ratio was decreased by 3-Hz pacing, while nuclear CaMKII and HDAC4 phosphorylation were increased. Either CaMKII-inhibition (by autocamtide-2-related peptide) or IPR-knockdown prevented the CaMKII-hyperphosphorylation and nuclear-to-cytosolic HDAC4 shift caused by 3-Hz pacing. In human atrial CMs from AF patients, nuclear IPR1-expression was significantly increased, with decreased nuclear/non-nuclear HDAC4 ratio. MicroRNA-26a was predicted to target ITPR1 (confirmed by Luciferase assay) and was downregulated in AF atrial CMs; microRNA-26a silencing reproduced AF-induced IP3R1 upregulation and nuclear diastolic Ca-loading.AF increases atrial CM nucleoplasmic Ca-handling by IPR1-upregulation involving miR-26a, leading to enhanced IPR1-CaMKII-HDAC4 signaling and I-downregulation.



Circ Res: 29 Dec 2020; epub ahead of print

Ca-CaM Dependent Inactivation of RyR2 Underlies Ca Alternans in Intact Heart.

Wei J, Yao J, Belke D, Guo W, ... Echebarria B, Chen SW

Ca alternans plays an essential role in cardiac alternans that can lead to ventricular fibrillation, but the mechanism underlying Ca alternans remains undefined. Increasing evidence suggests that Ca alternans results from alternations in the inactivation of cardiac ryanodine receptor (RyR2). However, what inactivates RyR2 and how RyR2 inactivation leads to Ca2+ alternans are unknown.To determine the role of calmodulin (CaM) on Ca alternans in intact working mouse hearts. We used an in vivo local gene delivery approach to alter CaM function by directly injecting adenoviruses expressing CaM-wild type (CaM-WT), a loss-of-function CaM mutation, CaM (1-4), and a gain-of-function mutation, CaM-M37Q, into the anterior wall of the left ventricle of RyR2 WT or mutant mouse hearts. We monitored Ca transients in ventricular myocytes near the adenovirus injection sites in Langendorff-perfused intact working hearts using confocal Ca imaging. We found that CaM-WT and CaM-M37Q promoted Ca alternans and prolonged Ca transient recovery in intact RyR2 WT and mutant hearts, whereas, CaM (1-4) exerted opposite effects. Altered CaM function also affected the recovery from inactivation of the L-type Ca current, but had no significant impact on sarcoplasmic reticulum Ca content. Further, we developed a novel numerical myocyte model of Ca alternans that incorporates Ca-CaM-dependent regulation of RyR2 and the L-type Ca channel. Remarkably, the new model recapitulates the impact on Ca alternans of altered CaM and RyR2 functions under 9 different experimental conditions. Our simulations reveal that diastolic cytosolic Ca elevation as a result of rapid pacing triggers Ca-CaM dependent inactivation of RyR2. The resultant RyR2 inactivation diminishes SR Carelease, which in turn reduces diastolic cytosolic Ca, leading to alternations in diastolic cytosolic Ca, RyR2 inactivation, and SR Ca release (i.e. Ca alternans).Our results demonstrate that inactivation of RyR2 by Ca-CaM is a major determinant of Ca alternans, making Ca-CaM dependent regulation of RyR2 an important therapeutic target for cardiac alternans.



Circ Res: 29 Dec 2020; epub ahead of print

Different DOACs Control Inflammation in Cardiac Ischemia-Reperfusion Differently.

Gadi I, Fatima S, Elwakiel A, Nazir S, ... Isermann B, Shahzad K

While thrombin is the key protease in thrombus formation, other coagulation proteases, such as fXa or activated protein C (aPC), independently modulate intracellular signaling via partially distinct receptors.To study the differential effects of fXa or fIIa inhibition on gene expression and inflammation in myocardial ischemia-reperfusion injury (IRI).Mice were treated with a direct fIIa inhibitor (fIIai) or direct fXa inhibitor (fXai) at doses that induced comparable anticoagulant effects ex vivo and in vivo (tail bleeding assay and FeCl3-induced thrombosis). Myocardial IRI was induced via LAD ligation. We determined infarct size and in vivo aPC generation, analyzed gene expression by RNAseq, and performed immunoblotting and ELISA. The signaling-only 3K3A-aPC variant and inhibitory antibodies that blocked all or only the anticoagulant function of aPC were used to determine the role of aPC. Doses of fIIai and fXai that induced comparable anticoagulant effects resulted in a comparable reduction in infarct size. However, unbiased gene expression analyses revealed marked differences, including pathways related to sterile inflammation and inflammasome regulation. fXai but not fIIai inhibited sterile inflammation by reducing the expression of proinflammatory cytokines (IL-1beta, IL-6, and TNFalpha) as well as NF-κB and inflammasome activation. This anti-inflammatory effect was associated with reduced myocardial fibrosis 28 days post myocardial IRI. Mechanistically, in vivo aPC generation was higher with fXai than with fIIai. Inhibition of the anticoagulant and signaling properties of aPC abolished the anti-inflammatory effect associated with fXai, while inhibiting only the anticoagulant function of aPC had no effect. Combining 3K3A-aPC with fIIai reduced the inflammatory response, mimicking the fXai-associated effect.We showed that specific inhibition of coagulation via DOACs had differential effects on gene expression and inflammation, despite comparable anticoagulant effects and infarct sizes. Targeting individual coagulation proteases induces specific cellular responses unrelated to their anticoagulant effect.



Circ Res: 22 Dec 2020; epub ahead of print

SIRT6 Protects Smooth Muscle Cells from Senescence and Reduces Atherosclerosis.

Grootaert M, Finigan A, Figg N, Uryga AK, Bennett M

Vascular smooth muscle cell (VSMC) senescence promotes atherosclerosis and features of plaque instability, in part through lipid-mediated oxidative DNA damage and telomere dysfunction. Sirtuin 6 (SIRT6) is a nuclear deacetylase involved in DNA damage response signaling, inflammation and metabolism; however, its role in regulating VSMC senescence and atherosclerosis is unclear.We examined SIRT6 expression in human VSMCs (hVSMCs), the role, regulation and downstream pathways activated by SIRT6, and how VSMC SIRT6 regulates atherogenesis.SIRT6 protein, but not mRNA, expression was markedly reduced in VSMCs in human and mouse atherosclerotic plaques, and in hVSMCs derived from plaques or undergoing replicative or palmitate-induced senescence vs. healthy aortic VSMCs. The ubiquitin ligase CHIP promoted SIRT6 stability, but CHIP expression was reduced in human and mouse plaque VSMCs and by palmitate in a p38- and c-Jun N-terminal kinase-dependent manner. SIRT6 bound to telomeres, while SIRT6 inhibition using shRNA or a deacetylase-inactive mutant (SIRT6) shortened hVSMC lifespan and induced senescence, associated with telomeric H3K9 hyperacetylation and 53BP1 binding, indicative of telomere damage. In contrast, SIRT6 overexpression preserved telomere integrity, delayed cellular senescence, and reduced inflammatory cytokine expression and changes in VSMC metabolism associated with senescence. SIRT6, but not SIRT6, promoted proliferation and lifespan of mouse VSMCs, and prevented senescence-associated metabolic changes. ApoE mice were generated that overexpress SIRT6 or SIRT6H133Y in VSMCs only. SM22alpha-hSIRT6/ApoE mice had reduced atherosclerosis, markers of senescence and inflammation compared to littermate controls, while plaques of SM22alpha-hSIRT6/ApoE mice showed increased features of plaque instability.SIRT6 protein expression is reduced in human and mouse plaque VSMCs and is positively regulated by CHIP. SIRT6 regulates telomere maintenance and VSMC lifespan, and inhibits atherogenesis, all dependent on its deacetylase activity. Our data shows that endogenous SIRT6 deacetylase is an important and unrecognized inhibitor of VSMC senescence and atherosclerosis.



Circ Res: 21 Dec 2020; epub ahead of print

Structural and Functional Characterization of A Nav1.5-Mitochondrial Couplon.

Pérez-Hernández Duran M, Leo-Macias A, Keegan S, Jouni M, ... Rothenberg E, Delmar M

The cardiac sodium channel NaV1.5 has a fundamental role in excitability and conduction. Previous studies have shown that sodium channels cluster together in specific cellular subdomains. Their association with intracellular organelles in defined regions of the myocytes, and the functional consequences of that association, remain to be defined.To characterize a subcellular domain formed by sodium channel clusters in the crest region of the myocytes, and the subjacent subsarcolemmal mitochondria (SSM). Through a combination of imaging approaches including super-resolution microscopy and electron microscopy we identified, in adult cardiac myocytes, a NaV1.5 subpopulation in close proximity to SSM; we further found that SSM preferentially host the mitochondrial Na+/Ca2+ exchanger (NCLX). This anatomical proximity led us to investigate functional changes in mitochondria resulting from sodium channel activity. Upon TTX exposure, mitochondria near NaV1.5 channels accumulated more Ca2+ and showed increased ROS production when compared to interfibrillar mitochondria. Finally, crosstalk between NaV1.5 channels and mitochondria was analyzed at a transcriptional level. We found that SCN5A and SLC8B1 (which encode NaV1.5 and NCLX, respectively) are negatively correlated both in a human transcriptome dataset (GTEx) and in human-induced pluripotent stem cell-derived cardiac myocytes deficient in SCN5A.We describe an anatomical hub (a couplon) formed by sodium channel clusters and SSM. Preferential localization of NCLX to this domain allows for functional coupling where the extrusion of Ca2+ from the mitochondria is powered, at least in part, by the entry of sodium through NaV1.5 channels. These results provide a novel entry-point into a mechanistic understanding of the intersection between electrical and structural functions of the heart.



Circ Res: 20 Dec 2020; epub ahead of print

JNK2, A Newly-Identified SERCA2 Enhancer, Augments an Arrhythmic [Ca] Leak-Load Relationship.

Yan J, Bare DJ, DeSantiago J, Zhao W, ... Chen SW, Ai X

We recently discovered pivotal contributions of stress kinase JNK2 in increased risk of atrial fibrillation (AF) through enhanced diastolic sarcoplasmic reticulum (SR) Ca leak via ryanodine receptors (RyR2). However, the role of JNK2 in the function of the SR Ca -ATPase (SERCA2), essential in maintaining [Ca] cycling during each heartbeat, is completely unknown.To test the hypothesis that JNK2 increases SERCA2 activity [Ca] and exacerbates an arrhythmic [Ca] leak-load relationship.We used confocal Ca imaging in myocytes and HEK cells, biochemistry, dual Ca/voltage optical mapping in intact hearts from alcohol-exposed or aged mice (where JNK2 is activated). We found that JNK2, but not JNK1, increased SERCA2 uptake and consequently elevated [Ca]SR load. JNK2 also associates with and phosphorylates SERCA2 proteins. JNK2 causally enhances SERCA2-ATPase activity via increased Vmax, without altering Ca affinity (Km). Unlike the CaMKII-dependent JNK2 action in SR Ca leak, JNK2-driven SERCA2 function was CaMKII-independent (not prevented by CaMKII inhibition). With CaMKII blocked, the JNK2-driven SR Ca loading alone did not significantly raise leak. However, with JNK2-CaMKII-driven SR Ca leak present, the JNK2-enhanced SR Ca uptake limited leak-induced reduction in SR Ca, normalizing Ca transient amplitude, but at a higher arrhythmogenic SR Ca leak. JNK2-specific inhibition completely normalized SR Ca handling, attenuated arrhythmic Ca activities, and alleviated AF susceptibility in aged and alcohol-exposed myocytes and intact hearts.We have identified a novel JNK2-induced activation of SERCA2. The dual-action of JNK2 in CaMKII-dependent arrhythmic SR Ca leak and a CaMKII-independent uptake exacerbates atrial arrhythmogenicity, while helping to maintain normal levels of Ca transients and heart function. JNK2 modulation may be a novel therapeutic target for AF prevention and treatment.



Circ Res: 17 Dec 2020; epub ahead of print

Klotho Deficiency Causes Heart Aging via Impairing the Nrf2-GR Pathway.

Chen K, Wang S, Sun QW, Zhang B, Ullah MF, Sun Z

Cardiac aging is an important contributing factor for heart failure which affects a large population but remains poorly understood.The purpose of this study is to investigate whether Klotho plays a role in cardiac aging.Heart function declined in old mice (24 months), as evidenced by decreases in fractional shortening, ejection fraction, and cardiac output. Heart size and weight, cardiomyocyte size and cardiac fibrosis were increased in old mice, indicating that aging causes cardiac hypertrophy and remodeling. Circulating Klotho levels were dramatically decreased in old mice, which prompted us to investigate whether the Klotho decline may cause heart aging. We found that Klotho gene mutation (KL-/-) largely decreased serum klotho levels and impaired heart function. Interestingly, supplement of exogenous secreted Klotho prevented heart failure, hypertrophy, and remodeling in both old mice and KL (-/-) mice. Secreted Klotho treatment inhibited excessive cardiac oxidative stress, senescence and apoptosis in old mice and KL (-/-) mice. Serum phosphate levels in KL (-/-) mice were kept in the normal range, suggesting that Klotho deficiency-induced heart aging is independent of phosphate metabolism. Mechanistically, Klotho deficiency suppressed glutathione reductase (GR) expression and activity in the heart via inhibition of transcription factor Nrf2. Furthermore, cardiac-specific overexpression of GR prevented excessive oxidative stress, apoptosis, and heart failure in both old and KL (-/-) mice.Klotho deficiency causes cardiac aging via impairing the Nrf2-GR pathway. Supplement of exogenous secreted Klotho represents a promising therapeutic strategy for aging-associated cardiomyopathy and heart failure.



Circ Res: 17 Dec 2020; epub ahead of print

PPARγ-p53-Mediated Vasculoregenerative Program to Reverse Pulmonary Hypertension.

Hennigs JK, Cao A, Li CG, Shi M, ... Snyder MP, Rabinovitch M

In pulmonary arterial hypertension (PAH), endothelial dysfunction and obliterative vascular disease are associated with DNA damage and impaired signaling of bone morphogenetic protein type 2 receptor (BMPR2) via two downstream transcription factors, PPARγ and p53.We investigated the vasculoprotective and regenerative potential of a newly identified PPARγ- p53 transcription factor complex in the pulmonary endothelium.In this study, we identified a pharmacologically inducible vasculoprotective mechanism in pulmonary arterial (PA) and lung microvascular (MV) endothelial cells (EC) in response to DNA damage and oxidant stress regulated in part by a BMPR2 dependent transcription factor complex between PPARγ and p53. Chromatin immunoprecipitation (ChIP) sequencing (seq) and RNA-seq established an inducible PPARγ-p53 mediated regenerative program regulating 19 genes involved in lung EC survival, angiogenesis and DNA repair including, EPHA2, FHL2, JAG1, SULF2 and TIGAR. Expression of these genes was partially impaired when the PPARγ-p53 complex was pharmacologically disrupted or when BMPR2 was reduced in PAEC subjected to oxidative stress. In EC-Bmpr2-knockout mice unable to stabilize p53 in ECs under oxidative stress, Nutlin-3 rescued endothelial p53 and PPARγ-p53 complex formation and induced target genes, such as APLN and JAG1, to regenerate pulmonary microvessels and reverse pulmonary hypertension. In PAEC from BMPR2 mutant PAH patients, pharmacological induction of p53 and PPARγ-p53 genes repaired damaged DNA utilizing genes from the nucleotide excision repair pathway without provoking PAEC apoptosis.We identified a novel therapeutic strategy that activates a vasculoprotective gene regulation program in PAEC downstream of dysfunctional BMPR2 to rehabilitate PAH PAEC, regenerate pulmonary microvessels and reverse disease. Our studies pave the way for p53-based vasculoregenerative therapies for PAH by extending the therapeutic focus to PAEC dysfunction and to DNA damage associated with PAH progression.



Circ Res: 15 Dec 2020; epub ahead of print

RYR2 Channel Inhibition Is the Principal Mechanism 0f Flecainide Action in CPVT.

Kryshtal DO, Blackwell D, Egly C, Smith AN, ... Laver DR, Knollmann BC

The class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive cardiac ryanodine receptor (RyR2) calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro, reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide\'s efficacy in CPVT.To determine whether RyR2 block independently contributes to flecainide\'s efficacy for suppressing spontaneous sarcoplasmic reticulum (SR) Ca release and for preventing ventricular tachycardia in vivo.We synthesized N-methylated flecainide analogues (QX-FL and NM-FL) and showed that N-methylation reduces flecainide\'s inhibitory potency on RyR2 channels incorporated into artificial lipid bilayers. N-Methylation did not alter flecainide\'s inhibitory activity on human cardiac sodium channels expressed in HEK293T cells. Antiarrhythmic efficacy was tested utilizing a calsequestrin knockout (Casq2-/-) CPVT mouse model. In membrane-permeabilized Casq2-/- cardiomyocytes - lacking intact sarcolemma and devoid of sodium channel contribution - flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2-/- cardiomyocytes pretreated with tetrodotoxin (TTX) to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous SR Ca release, while QX-FL and NM-FL did not. In vivo, flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2-/- mice, whereas NM-FL had no significant effect on arrhythmia burden, despite comparable sodium channel block.Flecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone did not prevent ventricular tachycardia. Hence, RyR2 channel inhibition likely constitutes the principal mechanism of antiarrhythmic action of flecainide in CPVT.



Circ Res: 09 Dec 2020; epub ahead of print

Identification and Characterization of Plasmin-Independent Thrombolytic Enzymes.

Hassan MM, Sharmin S, Kim HJ, Hong ST

Current thrombolytic agents activate plasminogen to plasmin which triggers fibrinolysis to dissolve thrombi. Since plasmin is a nonspecific proteolytic enzyme, all of the current plasmin-dependent thrombolytics lead to serious hemorrhagic complications, demanding for a new class of fibrinolytic enzymes independent from plasmin activation and undesirable side effects. We speculated that the mammalian version of bacterial heat shock proteins could selectively degrade intravascular thrombi, a typical example of a highly aggregated protein mixture. The objective of this study is to identify enzymes that can dissolve intravascular thrombi specifically without affecting fibrinogen and fibronectin so that the wound healing processes remains uninterrupted and tissues are not damaged. In this study, high-temperature requirement A (HtrA) proteins were tested for its specific proteolytic activity on intravascular thrombi independently from plasmin activation.HtrA1 and HtrA2/Omi proteins, collectively called as HtrAs, lysed ex vivo blood thrombi by degrading fibrin polymers. The thrombolysis by HtrAs was plasmin-independent and specific to vascular thrombi without causing the systemic activation of plasminogen and preventing non-specific proteolysis of other proteins including fibrinogen and fibronectin. As expected, HtrAs did not disturb clotting and wound healing of excised wounds from mouse skin. It was further confirmed in a tail bleeding and a rebleeding assay that HtrAs allowed normal clotting and maintenance of clot stability in wounds, unlike other thrombolytics. Most importantly, HtrAs completely dissolved blood thrombi in tail thrombosis mice, and the i.v. injection of HtrAs to mice with pulmonary embolism completely dissolved intravascular thrombi and thus rescued thromboembolism.Here, we identified HtrA1 and HtrA2/Omi as plasmin-independent and highly specific thrombolytics which can dissolve intravascular thrombi specifically without bleeding risk. This work is the first report of a plasmin-independent thrombolytic pathway, providing HtrA1 and HtrA2/Omi as ideal therapeutic candidates for various thrombotic diseases without hemorrhagic complications.



Circ Res: 08 Dec 2020; epub ahead of print

Arterial Stiffness Preceding Diabetes: A Longitudinal Study.

Zheng M, Zhang X, Chen S, Song Y, ... Gao X, Wu S
Rationale
Previous studies on the relationship between diabetes and arterial stiffness were mostly cross-sectional. A few longitudinal studies focused on one single direction. Whether the association between arterial stiffness and diabetes is bidirectional remains unclear to date.
Objective
To explore the temporal relationship between arterial stiffness and fasting blood glucose (FBG) status.
Methods and results
Included were 14 159 participants of the Kailuan study with assessment of brachial-ankle pulse wave velocity (baPWV) from 2010 to 2015, and free of diabetes, cardiovascular and cerebrovascular diseases, and chronic kidney disease at baseline. FBG and baPWV were repeatedly measured at baseline and follow-ups. Cox proportional hazard regression model was used to estimate hazard ratios and 95% confidence intervals (CIs) of incident diabetes across baseline baPWV groups: <1400 cm/s (ref), 1400≤ baPWV <1800 cm/s, and ≥1800 cm/s. Path analysis was used to analyze the possible temporal causal relationship between baPWV and FBG, among 8956 participants with repeated assessment of baPWV and FBG twice in 2010 to 2017. The mean baseline age of the observed population was 48.3±12.0 years. During mean 3.72 years of follow-up, 979 incident diabetes cases were identified. After adjusting for potential confounders, the hazard ratio (95% CI) for risk of diabetes was 1.59 (1.34-1.88) for the borderline arterial stiffness group and 2.11 (1.71-2.61) for the elevated arterial stiffness group, compared with the normal ideal arterial stiffness group. In the path analysis, baseline baPWV was associated with follow-up FBG (the standard regression coefficient was 0.09 [95% CI, 0.05-0.10]). In contrast, the standard regression coefficient of baseline FBG for follow-up baPWV (β=0.00 [95% CI, -0.02 to 0.02]) was not significant.
Conclusions
Arterial stiffness, as measured by baPWV, was associated with risk of developing diabetes. Arterial stiffness appeared to precede the increase in FBG.



Circ Res: 03 Dec 2020; 127:1491-1498

Ionic Mechanisms of Impulse Propagation Failure in the FHF2-Deficient Heart.

Park DS, Shekhar A, Santucci J, Redel-Traub G, ... Goldfarb M, Fishman GI
Rationale
FHFs (fibroblast growth factor homologous factors) are key regulators of sodium channel (Na) inactivation. Mutations in these critical proteins have been implicated in human diseases including Brugada syndrome, idiopathic ventricular arrhythmias, and epileptic encephalopathy. The underlying ionic mechanisms by which reduced Na availability inknockout () mice predisposes to abnormal excitability at the tissue level are not well defined.
Objective
Using animal models and theoretical multicellular linear strands, we examined how FHF2 orchestrates the interdependency of sodium, calcium, and gap junctional conductances to safeguard cardiac conduction.
Methods and results
mice were challenged by reducing calcium conductance (gCa) using verapamil or by reducing gap junctional conductance (Gj) using carbenoxolone or by backcrossing into a cardiomyocyte-specific Cx43 (connexin 43) heterozygous background. All conditions produced conduction block inmice, withwild-type () mice showing normal impulse propagation. To explore the ionic mechanisms of block inhearts, multicellular linear strand models incorporating FHF2-deficient Na inactivation properties were constructed and faithfully recapitulated conduction abnormalities seen in mutant hearts. The mechanisms of conduction block in mutant strands with reduced gCa or diminished Gj are very different. Enhanced Na inactivation due to FHF2 deficiency shifts dependence onto calcium current (I) to sustain electrotonic driving force, axial current flow, and action potential (AP) generation from cell-to-cell. In the setting of diminished Gj, slower charging time from upstream cells conspires with accelerated Na inactivation in mutant strands to prevent sufficient downstream cell charging for AP propagation.
Conclusions
FHF2-dependent effects on Na inactivation ensure adequate sodium current (I) reserve to safeguard against numerous threats to reliable cardiac impulse propagation.



Circ Res: 03 Dec 2020; 127:1536-1548

Tamoxifen Accelerates Endothelial Healing by Targeting ERα in Smooth Muscle Cells.

Zahreddine R, Davezac M, Smirnova N, Buscato M, ... Arnal JF, Fontaine C
Rationale
Tamoxifen prevents the recurrence of breast cancer and is also beneficial against bone demineralization and arterial diseases. It acts as an ER (estrogen receptor) α antagonist in ER-positive breast cancers, whereas it mimics the protective action of 17β-estradiol in other tissues such as arteries. However, the mechanisms of these tissue-specific actions remain unclear.
Objective
Here, we tested whether tamoxifen is able to accelerate endothelial healing and analyzed the underlying mechanisms.
Methods and results
Using 3 complementary mouse models of carotid artery injury, we demonstrated that both tamoxifen and estradiol accelerated endothelial healing, but only tamoxifen required the presence of the underlying medial smooth muscle cells. Chronic treatment with 17β-estradiol and tamoxifen elicited differential gene expression profiles in the carotid artery. The use of transgenic mouse models targeting either whole ERα in a cell-specific manner or ERα subfunctions (membrane/extranuclear versus genomic/transcriptional) demonstrated that 17β-estradiol-induced acceleration of endothelial healing is mediated by membrane ERα in endothelial cells, while the effect of tamoxifen is mediated by the nuclear actions of ERα in smooth muscle cells.
Conclusions
Whereas tamoxifen acts as an antiestrogen and ERα antagonist in breast cancer but also on the membrane ERα of endothelial cells, it accelerates endothelial healing through activation of nuclear ERα in smooth muscle cells, inviting to revisit the mechanisms of action of selective modulation of ERα.



Circ Res: 03 Dec 2020; 127:1473-1487

ATAC-Seq Reveals an Enhancer That Regulates Sinoatrial Node Development and Function.

Galang G, Mandla R, Ruan H, Jung C, ... Black BL, Vedantham V
Rationale
Cardiac pacemaker cells (PCs) in the sinoatrial node (SAN) have a distinct gene expression program that allows them to fire automatically and initiate the heartbeat. Although critical SAN transcription factors, including Isl1 (Islet-1), Tbx3 (T-box transcription factor 3), and Shox2 (short-stature homeobox protein 2), have been identified, the -regulatory architecture that governs PC-specific gene expression is not understood, and discrete enhancers required for gene regulation in the SAN have not been identified.
Objective
To define the epigenetic profile of PCs using comparative ATAC-seq (assay for transposase-accessible chromatin with sequencing) and to identify novel enhancers involved in SAN gene regulation, development, and function.
Methods and results
We used ATAC-seq on sorted neonatal mouse SAN to compare regions of accessible chromatin in PCs and right atrial cardiomyocytes. PC-enriched assay for transposase-accessible chromatin peaks, representing candidate SAN regulatory elements, were located near established SAN genes and were enriched for distinct sets of TF (transcription factor) binding sites. Among several novel SAN enhancers that were experimentally validated using transgenic mice, we identified a 2.9-kb regulatory element at thelocus that was active specifically in the cardiac inflow at embryonic day 8.5 and throughout later SAN development and maturation. Deletion of this enhancer from the genome of mice resulted in SAN hypoplasia and sinus arrhythmias. The mouse SAN enhancer also directed reporter activity to the inflow tract in developing zebrafish hearts, demonstrating deep conservation of its upstream regulatory network. Finally, single nucleotide polymorphisms in the human genome that occur near the region syntenic to the mouse enhancer exhibit significant associations with resting heart rate in human populations.
Conclusions
(1) PCs have distinct regions of accessible chromatin that correlate with their gene expression profile and contain novel SAN enhancers, (2) -regulation ofspecifically in the SAN depends upon a conserved SAN enhancer that regulates PC development and SAN function, and (3) a corresponding humanenhancer may regulate human SAN function.



Circ Res: 03 Dec 2020; 127:1502-1518

Genetic Regulation of Atherosclerosis-Relevant Phenotypes in Human Vascular Smooth Muscle Cells.

Aherrahrou R, Guo L, Nagraj VP, Aguhob A, ... Berliner JA, Civelek M
Rationale
Coronary artery disease (CAD) is a major cause of morbidity and mortality worldwide. Recent genome-wide association studies revealed 163 loci associated with CAD. However, the precise molecular mechanisms by which the majority of these loci increase CAD risk are not known. Vascular smooth muscle cells (VSMCs) are critical in the development of CAD. They can play either beneficial or detrimental roles in lesion pathogenesis, depending on the nature of their phenotypic changes.
Objective
To identify genetic variants associated with atherosclerosis-relevant phenotypes in VSMCs.
Methods and results
We quantified 12 atherosclerosis-relevant phenotypes related to calcification, proliferation, and migration in VSMCs isolated from 151 multiethnic heart transplant donors. After genotyping and imputation, we performed association mapping using 6.3 million genetic variants. We demonstrated significant variations in calcification, proliferation, and migration. These phenotypes were not correlated with each other. We performed genome-wide association studies for 12 atherosclerosis-relevant phenotypes and identified 4 genome-wide significant loci associated with at least one VSMC phenotype. We overlapped the previously identified CAD loci with our data set and found nominally significant associations at 79 loci. One of them was the chromosome 1q41 locus, which harbors . The G allele of the lead risk single nucleotide polymorphism (SNP) rs67180937 was associated with lower VSMCexpression and lower proliferation. Lentivirus-mediated silencing of MIA3 (melanoma inhibitory activity protein 3) in VSMCs resulted in lower proliferation, consistent with human genetics findings. Furthermore, we observed a significant reduction of MIA3 protein in VSMCs in thin fibrous caps of late-stage atherosclerotic plaques compared to early fibroatheroma with thick and protective fibrous caps in mice and humans.
Conclusions
Our data demonstrate that genetic variants have significant influences on VSMC function relevant to the development of atherosclerosis. Furthermore, highexpression may promote atheroprotective VSMC phenotypic transitions, including increased proliferation, which is essential in the formation or maintenance of a protective fibrous cap.



Circ Res: 03 Dec 2020; 127:1552-1565

Genome-Wide Analysis Identifies an Essential Human TBX3 Pacemaker Enhancer.

van Eif VWW, Protze SI, Bosada FM, Yuan X, ... Scott IC, Christoffels VM
Rationale
The development and function of the pacemaker cardiomyocytes of the sinoatrial node (SAN), the leading pacemaker of the heart, are tightly controlled by a conserved network of transcription factors, including TBX3 (T-box transcription factor 3), ISL1 (ISL LIM homeobox 1), and SHOX2 (short stature homeobox 2). Yet, the regulatory DNA elements (REs) controlling target gene expression in the SAN pacemaker cells have remained undefined.
Objective
Identification of the regulatory landscape of human SAN-like pacemaker cells and functional assessment of SAN-specific REs potentially involved in pacemaker cell gene regulation.
Methods and results
We performed Assay for Transposase-Accessible Chromatin using sequencing on human pluripotent stem cell-derived SAN-like pacemaker cells and ventricle-like cells and identified thousands of putative REs specific for either human cell type. We validated pacemaker cell-specific elements in theandloci. CRISPR-mediated homozygous deletion of the mouse ortholog of a noncoding region with candidate pacemaker-specific REs in thelocus resulted in selective loss ofexpression from the developing SAN and embryonic lethality. Putative pacemaker-specific REs were identified up to 1 Mbp upstream ofin a region close toharboring variants associated with heart rate recovery after exercise. The orthologous region was deleted in mice, which resulted in selective loss of expression offrom the SAN and (cardiac) ganglia and in neonatal lethality. Expression ofwas maintained in other tissues including the atrioventricular conduction system, lungs, and liver. Heterozygous adult mice showed increased SAN recovery times after pacing. The human REs harboring the associated variants robustly drove expression in the SAN of transgenic mouse embryos.
Conclusions
We provided a genome-wide collection of candidate human pacemaker-specific REs, including the loci of , , and , and identified a link between human genetic variants influencing heart rate recovery after exercise and a variant RE with highly conserved function, driving SAN expression of .



Circ Res: 03 Dec 2020; 127:1522-1535

Lipid Profiles and Heart Failure Risk: Results from Two Prospective Studies.

Wittenbecher C, Eichelmann F, Toledo E, Guasch-Ferre M, ... Martínez-González MA, Hu FB

Altered lipid metabolism has been implicated in heart failure (HF) development, but no prospective studies have examined comprehensive lipidomics data and subsequent risk of HF.We aimed to link single lipid metabolites and lipidomics networks to the risk of developing heart failure.Discovery analyses were based on 216 targeted lipids in a case-control study (331 incident HF cases and 507 controls, matched by age, sex, and study center), nested within the PREDIMED study. Associations of single lipids were examined in conditional logistic regression models. Furthermore, lipidomics networks were linked to HF risk in a multi-step workflow, including machine learning-based identification of the HF-related network-clusters, and regression-based discovery of the HF-related lipid patterns within these clusters. If available, significant findings were externally validated in a subsample of the EPIC-Potsdam cohort (2414 at-risk-participants, including 87 incident HF-cases). After confounder-adjustments, two lipids were significantly associated with HF risk in both cohorts: ceramide 16:0 (HR per SD in PREDIMED 1.28, 95%CI 1.13, 1.47) and phosphatidylcholine 32_0 (HR per SD in PREDIMED 1.23, 95%CI 1.08, 1.41). Additionally, lipid patterns in several network clusters were associated with HF risk in PREDIMED. Adjusted for standard risk factors, an internally cross-validated score based on the significant HF-related lipids that were identified in the network analysis in PREDIMED was associated with a higher HF risk (20 lipids, HR per SD 2.33, 95%CI 1.93, 2.81%). Moreover, a lipid score restricted to the externally available lipids was significantly associated with HF incidence in both cohorts (6 lipids, HRs per SD 1.30, 95%CI 1.14, 1.47 in PREDIMED, and 1.46, 95%CI, 1.17, 1.82 in EPIC-Potsdam).Our study identified and validated two lipid metabolites and several lipidomics patterns as potential novel biomarkers of HF risk. Lipid profiling may capture preclinical molecular alterations that predispose for incident HF.



Circ Res: 03 Dec 2020; epub ahead of print

Endothelial S1P Signaling Counteracts Infarct Expansion in Ischemic Stroke.

Nitzsche A, Poittevin M, Benarab A, Bonnin P, ... Hla T, Camerer E

Cerebrovascular function is critical for brain health, and endogenous vascular-protective pathways may provide therapeutic targets for neurological disorders. Sphingosine 1-phosphate (S1P) signaling coordinates vascular functions in other organs, and S1P receptor-1 (S1P) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P modulation in stroke.To address roles and mechanisms of engagement of endothelial cell (EC) S1P in the naïve and ischemic brain and its potential as a target for cerebrovascular therapy.Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P in the mouse brain. With an S1P signaling reporter, we reveal that abluminal polarization shields S1P from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar ECs. S1P signaling sustains hallmark endothelial functions in the naïve brain, and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by EC-selective deficiency in S1P production, export, or the S1P receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P provides modest protection only in the context of reperfusion. In the ischemic brain, EC S1P supports blood-brain barrier (BBB) function, microvascular patency, and the rerouting of blood to hypo-perfused brain tissue through collateral anastomoses. Selective S1P agonism counteracts cortical infarct expansion after middle cerebral artery occlusion by engaging the endothelial receptor pool after BBB penetration.This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with BBB-penetrating S1P agonists.



Circ Res: 01 Dec 2020; epub ahead of print

Cell - ECM Interactions Play Multiple Essential Roles in Aortic Arch Development.

Warkala M, Chen D, Ramirez A, Jubran A, ... Zhao H, Astrof S

Defects in the morphogenesis of the 4th pharyngeal arch arteries (PAAs) give rise to lethal birth defects. Understanding genes and mechanisms regulating PAA formation will provide important insights into the etiology and treatments for congenital heart disease.Cell-ECM interactions play essential roles in the morphogenesis of PAAs and their derivatives, the aortic arch artery (AAA) and its major branches; however, their specific functions are not well-understood. Previously, we demonstrated that integrin α5β1 and fibronectin (Fn1) expressed in the Isl1 lineages regulate PAA formation. The objective of the current studies was to investigate cellular mechanisms by which integrin α5β1 and Fn1 regulate AAA morphogenesis.Using temporal lineage tracing, whole-mount confocal imaging, and quantitative analysis of the second heart field (SHF) and endothelial cell (EC) dynamics, we show that the majority of PAA EC progenitors arise by E7.5 in the SHF and contribute to pharyngeal arch endothelium between E7.5 and E9.5. Consequently, SHF-derived ECs in the pharyngeal arches form a uniform plexus of small blood vessels, which remodels into the PAAs by 35 somites. The remodeling of the vascular plexus is orchestrated by signals dependent on the pharyngeal ECM microenvironment, extrinsic to the endothelium. Conditional ablation of integrin α5β1 or Fn1 in the Isl1 lineages showed that signaling by the ECM regulates AAA morphogenesis at multiple steps: 1) accumulation of SHF-derived ECs in the pharyngeal arches, 2) remodeling of the uniform EC plexus in the 4th arches into the PAAs; and 3) differentiation of neural crest-derived cells adjacent to the PAA endothelium into vascular smooth muscle cells.PAA formation is a multi-step process entailing dynamic contribution of SHF-derived ECs to pharyngeal arches, the remodeling of endothelial plexus into the PAAs, and the remodeling of the PAAs into the AAA and its major branches. Cell-ECM interactions regulated by integrin α5β1 and Fn1 play essential roles at each of these developmental stages.



Circ Res: 29 Nov 2020; epub ahead of print

Beat-By-Beat Cardiomyocyte T-Tubule Deformation Drives Tubular Content Exchange.

Rog-Zielinska EA, Scardigli M, Peyronnet R, Zgierski-Johnston CM, ... Sacconi L, Kohl P

The sarcolemma of cardiomyocytes contains many proteins that are essential for electro-mechanical function in general, and excitation-contraction coupling in particular. The distribution of these proteins is non-uniform between the bulk sarcolemmal surface and membrane invaginations known as transverse tubules (TT). TT form an intricate network of fluid-filled conduits that support electro-mechanical synchronicity within cardiomyocytes. Although continuous with the extracellular space, the narrow lumen and the tortuous structure of TT can form domains of restricted diffusion. As a result of unequal ion fluxes across cell surface and TT membranes, limited diffusion may generate ion gradients within TT, especially deep within the TT network and at high pacing rates.We postulate that there may be an advective component to TT content exchange, wherein cyclic deformation of TT during diastolic stretch and systolic shortening serves to mix TT luminal content and assists equilibration with bulk extracellular fluid.Using electron tomography, we explore the 3D nanostructure of TT in rabbit ventricular myocytes, preserved at different stages of the dynamic cycle of cell contraction and relaxation. We show that cellular deformation affects TT shape in a sarcomere length-dependent manner and on a beat-by-beat time-scale. Using fluorescence recovery after photobleaching microscopy, we show that apparent speed of diffusion is affected by the mechanical state of cardiomyocytes, and that cyclic contractile activity of cardiomyocytes accelerates TT diffusion dynamics. Our data confirm the existence of an advective component to TT content exchange. This points towards a novel mechanism of cardiac autoregulation, whereby the previously implied increased propensity for TT luminal concentration imbalances at high electrical stimulation rates would be countered by elevated advection-assisted diffusion at high mechanical beating rates. The relevance of this mechanism in health and during pathological remodelling (e.g. cardiac hypertrophy or failure) forms an exciting target for further research.



Circ Res: 23 Nov 2020; epub ahead of print

Heterodimerization with 5-HTR Is Indispensable for βAR-mediated Cardioprotection.

Song Y, Xu C, Liu J, Li Y, ... Woo AY, Xiao RP

The β-adrenoceptor (β-AR), a prototypical G protein-coupled receptor (GPCR), couples to both G and Gproteins. Stimulation of theβ-AR is beneficial to humans and animals with heart failure presumably because it activates the downstream G-PI3K-Akt cell survival pathway. Cardiac β-AR signaling can be regulated by crosstalk or heterodimerization with other GPCRs, but the physiological and pathophysiological significance of this type of regulation has not been sufficiently demonstrated.Here, we aim to investigate the potential cardioprotective effect of β-adrenergic stimulation with a subtype-selective agonist, (R,R\')-4-methoxy-1-naphthylfenoterol (MNF), and to decipher the underlying mechanism with a particular emphasis on the role of heterodimerization of β-ARs with another GPCR, 5-hydroxytryptamine receptors 2B (5-HTRs).Using pharmacological, genetic and biophysical protein-protein interaction approaches, we studied the cardioprotective effect of the β-agonist, MNF, and explored the underlying mechanism in both in vivo in mice and cultured rodent cardiomyocytes insulted with doxorubicin (Dox), hydrogen peroxide (HO) or ischemia/reperfusion. In Dox-treated mice, MNF reduced mortality and body weight loss, while improving cardiac function and cardiomyocyte viability. MNF also alleviated myocardial ischemia/reperfusion injury. In cultured rodent cardiomyocytes, MNF inhibited DNA damage and cell death caused by Dox, HO or hypoxia/reoxygenation. Mechanistically, we found that MNF or another β-agonist zinterol markedly promoted heterodimerization of β-ARs with 5-HTRs. Upregulation of the heterodimerized 5-HTRs and β-ARs enhanced β-AR-stimulated Gi-Akt signaling and cardioprotection while knockdown or pharmacological inhibition of the 5-HTattenuated β-AR-stimulated G signaling and cardioprotection.These data demonstrate that the β-AR-stimulated cardioprotective Gi signaling depends on the heterodimerization of β-ARs and 5-HTRs.



Circ Res: 18 Nov 2020; epub ahead of print

Intracellular β1-Adrenergic Receptors and Organic Cation Transporter 3 Mediate Phospholamban Phosphorylation to Enhance Cardiac Contractility.

Wang Y, Shi Q, Li M, Zhao M, ... Bers DM, Xiang YK
Rationale
β-adrenoceptors (βARs) exist at intracellular membranes and Organic Cation Transporter 3 (OCT3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular βAR in cardiac contractility remains to be elucidated.
Objective
Test localization and function of intracellular βAR on cardiac contractility.
Methods and results
Membrane fractionation, super-resolution imaging, proximity ligation, co-immunoprecipitation and single-molecule pulldown demonstrated a pool of βARs in mouse hearts that was associated with sarco/endoplasmic reticulum Ca-ATPase at the sarcoplasmic reticulum (SR). Local protein kinase A (PKA) activation was measured using a PKA biosensor targeted at either the plasma membrane (PM) or SR. Compared to wild type (WT), myocytes lacking OCT3 (OCT3KO) responded identically to the membrane-permeant βAR agonist isoproterenol in PKA activation at both PM and SR. The same was true at the PM for membrane-impermeant norepinephrine, but the SR response to norepinephrine was suppressed in OCT3KO myocytes. This differential effect was recapitulated in phosphorylation of the SR-pump regulator phospholamban. Similarly, OCT3KO selectively suppressed calcium transients and contraction responses to norepinephrine, but not isoproterenol. Furthermore, sotalol, a membrane-impermeant βAR-blocker suppressed isoproterenol-induced PKA activation at the PM, but permitted PKA activation at the SR, phospholamban phosphorylation and contractility. Moreover, pretreatment with sotatol in OCT3KO myocytes prevented norepinephrine induced PKA activation at both PM and the SR and contractility.
Conclusions
Functional βARs exists at the SR and is critical for PKA-mediated phosphorylation of phospholamban and cardiac contractility upon catecholamine stimulation. Activation of these intracellular βARs requires catecholamine transport via OCT3.



Circ Res: 12 Nov 2020; epub ahead of print

Targeting Mitochondria-Inflammation Circuit by β-Hydroxybutyrate Mitigates HFpEF.

Deng Y, Xie M, Li Q, Xu X, ... Tian R, Li T
Rationale
Over 50% of heart failure patients have preserved, rather than reduced ejection fraction (HFpEF vs. HFrEF). Complexity of its pathophysiology and the lack of animal models hamper the development of effective therapy for HFpEF.
Objective
This study was designed to investigate the metabolic mechanisms of HFpEF and test therapeutic interventions using a novel animal model.
Methods and results
By combining the age, long-term high-fat diet and desoxycorticosterone pivalate challenge in a mouse model we were able to recapture the myriad features of HFpEF. In these mice, mitochondrial hyperacetylation exacerbated while increasing ketone body availability rescued the phenotypes. The HFpEF mice exhibited overproduction of interleukin (IL)-1β/IL-18, and tissue fibrosis due to increased assembly of NLPR3 inflammasome on hyperacetylated mitochondria. Increasing β-hydroxybutyrate (β-OHB) level attenuated NLPR3 inflammasome formation and antagonized proinflammatory cytokines-triggered mitochondrial dysfunction and fibrosis. Moreover, β-OHB downregulated the acetyl-CoA pool and mitochondrial acetylation, partially via activation of citrate synthase and inhibition of fatty acid uptake.
Conclusions
Therefore, we identify the interplay of mitochondrial hyperacetylation and inflammation as a key driver in HFpEF pathogenesis which can be ameliorated by promoting β-OHB abundance.



Circ Res: 11 Nov 2020; epub ahead of print

Machine Learned Cellular Phenotypes Predict Outcome in Ischemic Cardiomyopathy.

Rogers AJ, Selvalingam A, Alhusseini MI, Krummen DE, ... Zaharia M, Narayan SM
Rationale
Susceptibility to ventricular arrhythmias (VT/VF) is difficult to predict in patients with ischemic cardiomyopathy either by clinical tools or by attempting to translate cellular mechanisms to the bedside.
Objective
To develop computational phenotypes of patients with ischemic cardiomyopathy, by training then interpreting machine learning (ML) of ventricular monophasic action potentials (MAPs) to reveal phenotypes that predict long-term outcomes.
Methods and results
We recorded 5706 ventricular MAPs in 42 patients with coronary disease (CAD) and left ventricular ejection fraction (LVEF) {less than or equal to}40% during steady-state pacing. Patients were randomly allocated to independent training and testing cohorts in a 70:30 ratio, repeated K=10 fold. Support vector machines (SVM) and convolutional neural networks (CNN) were trained to 2 endpoints: (i) sustained VT/VF or (ii) mortality at 3 years. SVM provided superior classification. For patient-level predictions, we computed personalized MAP scores as the proportion of MAP beats predicting each endpoint. Patient-level predictions in independent test cohorts yielded c-statistics of 0.90 for sustained VT/VF (95% CI: 0.76-1.00) and 0.91 for mortality (95% CI: 0.83-1.00) and were the most significant multivariate predictors. Interpreting trained SVM revealed MAP morphologies that, using in silico modeling, revealed higher L-type calcium current or sodium calcium exchanger as predominant phenotypes for VT/VF.
Conclusions
Machine learning of action potential recordings in patients revealed novel phenotypes for long-term outcomes in ischemic cardiomyopathy. Such computational phenotypes provide an approach which may reveal cellular mechanisms for clinical outcomes and could be applied to other conditions.



Circ Res: 09 Nov 2020; epub ahead of print

, a Human Plasma Lipid GWAS Locus, Regulates Lipoprotein Metabolism in Mice.

Bi X, Kuwano T, Lee PC, Millar JS, ... Hand NJ, Rader DJ
Rationale
Single-nucleotide polymorphisms near the(inflammation and lipid regulator with ubiquitin-associated-like and NBR1 [next togene 1 protein]-like domains) gene are genome-wide significantly associated with plasma lipid traits and coronary artery disease (CAD), but the biological basis of this association is unknown.
Objective
To investigate the role of ILRUN in plasma lipid and lipoprotein metabolism.
Methods and results
encodes a protein that contains a ubiquitin-associated-like domain, suggesting that it may interact with ubiquitinylated proteins. We generated mice globally deficient forand found they had significantly lower plasma cholesterol levels resulting from reduced liver lipoprotein production. Liver transcriptome analysis uncovered altered transcription of genes downstream of lipid-related transcription factors, particularly PPARα (peroxisome proliferator-activated receptor alpha), and livers from -deficient mice had increased PPARα protein. Human ILRUN was shown to bind to ubiquitinylated proteins including PPARα, and the ubiquitin-associated-like domain of ILRUN was found to be required for its interaction with PPARα.
Conclusions
These findings establish ILRUN as a novel regulator of lipid metabolism that promotes hepatic lipoprotein production. Our results also provide functional evidence thatmay be the casual gene underlying the observed genetic associations with plasma lipids at 6p21 in human.



Circ Res: 05 Nov 2020; 127:1347-1361

Exercise Regulates MicroRNAs to Preserve Coronary and Cardiac Function in the Diabetic Heart.

Lew JK, Pearson JT, Saw E, Tsuchimochi H, ... Katare R, Schwenke DO
Rationale
Diabetic heart disease (DHD) is a debilitating manifestation of type 2 diabetes mellitus. Exercise has been proposed as a potential therapy for DHD, although the effectiveness of exercise in preventing or reversing the progression of DHD remains controversial. Cardiac function is critically dependent on the preservation of coronary vascular function.
Objective
We aimed to elucidate the effectiveness and mechanisms by which exercise facilitates coronary and cardiac-protection during the onset and progression of DHD.
Methods and results
Diabetic db/db and nondiabetic mice, with or without underlying cardiac dysfunction (16 and 8 weeks old, respectively) were subjected to either moderate-intensity exercise or high-intensity exercise for 8 weeks. Subsequently, synchrotron microangiography, immunohistochemistry, Western blot, and real-time polymerase chain reaction were used to assess time-dependent changes in cardiac and coronary structure and function associated with diabetes mellitus and exercise and determine whether these changes reflect the observed changes in cardiac-enriched and vascular-enriched microRNAs (miRNAs). We show that, if exercise is initiated from 8 weeks of age, both moderate-intensity exercise and high-intensity exercise prevented the onset of coronary and cardiac dysfunction, apoptosis, fibrosis, microvascular rarefaction, and disruption of miRNA signaling, as seen in the nonexercised diabetic mice. Conversely, the cardiovascular benefits of moderate-intensity exercise were absent if the exercise was initiated after the diabetic mice had already established cardiac dysfunction (ie, from 16 weeks of age). The experimental silencing or upregulation of miRNA-126 activity suggests the mechanism underpinning the cardiovascular benefits of exercise were mediated, at least in part, through tissue-specific miRNAs.
Conclusions
Our findings provide the first experimental evidence for the critical importance of early exercise intervention in ameliorating the onset and progression of DHD. Our results also suggest that the beneficial effects of exercise are mediated through the normalization of cardiovascular-enriched miRNAs, which are dysregulated in DHD.



Circ Res: 05 Nov 2020; 127:1384-1400

Sox17 Controls Emergence and Remodeling of Nestin-Expressing Coronary Vessels.

González-Hernández S, Gómez MJ, Sánchez-Cabo F, Méndez-Ferrer S, Muñoz-Cánoves P, Isern J
Rationale
The molecular mechanisms underlying the formation of coronary arteries during development and during cardiac neovascularization after injury are poorly understood. However, a detailed description of the relevant signaling pathways and functional TFs (transcription factors) regulating these processes is still incomplete.
Objective
The goal of this study is to identify novel cardiac transcriptional mechanisms of coronary angiogenesis and vessel remodeling by defining the molecular signatures of coronary vascular endothelial cells during these complex processes.
Methods and results
We demonstrate thatandtransgenic mouse lines are novel tools for studying the emergence of coronary endothelium and targeting sprouting coronary vessels (but not ventricular endocardium) during development. Furthermore, we identify Sox17 as a critical TF upregulated during the sprouting and remodeling of coronary vessels, visualized by a specific neural enhancer from thegene that is strongly induced in developing arterioles. Functionally, genetic-inducible endothelial deletion ofcauses deficient cardiac remodeling of coronary vessels, resulting in improper coronary artery formation.
Conclusions
We demonstrated that Sox17 TF regulates the transcriptional activation of \'s enhancer in developing coronary vessels while its genetic deletion leads to inadequate coronary artery formation. These findings identify Sox17 as a critical regulator for the remodeling of coronary vessels in the developing heart.



Circ Res: 05 Nov 2020; 127:e252-e270

Apo AI Nanoparticles Delivered Post Myocardial Infarction Moderate Inflammation.

Richart AL, Reddy M, Khalaji M, Natoli AL, ... Navdaev AV, Kingwell BA
Rationale
Decades of research have examined immune-modulatory strategies to protect the heart after an acute myocardial infarction and prevent progression to heart failure but have failed to translate to clinical benefit.
Objective
To determine anti-inflammatory actions of n-apo AI (Apo AI nanoparticles) that contribute to cardiac tissue recovery after myocardial infarction.
Methods and results
Using a preclinical mouse model of myocardial infarction, we demonstrate that a single intravenous bolus of n-apo AI (CSL111, 80 mg/kg) delivered immediately after reperfusion reduced the systemic and cardiac inflammatory response. N-apo AI treatment lowered the number of circulating leukocytes by 30±7% and their recruitment into the ischemic heart by 25±10% (all <5.0×10). This was associated with a reduction in plasma levels of the clinical biomarker of cardiac injury, cardiac troponin-I, by 52±17% (=1.01×10). N-apo AI reduced the cardiac expression of chemokines that attract neutrophils and monocytes by 60% to 80% and lowered surface expression of integrin CD11b on monocytes by 20±5% (all <5.0×10). Fluorescently labeled n-apo AI entered the infarct and peri-infarct regions and colocalized with cardiomyocytes undergoing apoptosis and with leukocytes. We further demonstrate that n-apo AI binds to neutrophils and monocytes, with preferential binding to the proinflammatory monocyte subtype and partially via SR-BI (scavenger receptor BI). In patients with type 2 diabetes, we also observed that intravenous infusion of the same n-apo AI (CSL111, 80 mg/kg) similarly reduced the level of circulating leukocytes by 12±5% (all <5.0×10).
Conclusions
A single intravenous bolus of n-apo AI delivered immediately post-myocardial infarction reduced the systemic and cardiac inflammatory response through direct actions on both the ischemic myocardium and leukocytes. These data highlight the anti-inflammatory effects of n-apo AI and provide preclinical support for investigation of its use for management of acute coronary syndromes in the setting of primary percutaneous coronary interventions.



Circ Res: 05 Nov 2020; 127:1422-1436

HIV Antivirals Affect Endothelial Activation and Endothelial-Platelet Crosstalk.

Khawaja AA, Taylor KA, Lovell AO, Nelson M, ... Boffito M, Emerson M
Rationale
People living with HIV on effective antiretroviral therapy are at increased risk of cardiovascular complications, possibly due to off-target drug effects. Some studies have associated antiretroviral therapy with increased risk of myocardial infarction and endothelial dysfunction, but a link between endothelial function and antiretrovirals has not been established.
Objective
To determine the effects of antiretrovirals in common clinical use upon in vitro endothelial function to better understand cardiovascular risk in people living with HIV.
Methods and results
Human umbilical cord vein endothelial cells or human coronary artery endothelial cells were pretreated with the antiretrovirals abacavir sulphate (ABC), tenofovir disoproxil fumarate, or tenofovir alafenamide. Expression of adhesion molecules, ectonucleotidases (CD39 and CD73), tissue factor (TF), endothelial-derived microparticle (EMP) numbers and phenotype, and platelet activation were evaluated by flow cytometry. TF and ectonucleotidase activities were measured using colourimetric plate-based assays. ABC-treated endothelial cells had higher levels of ICAM (intercellular adhesion molecule)-1 and TF expression following TNF (tumor necrosis factor)-α stimulation. In contrast, tenofovir disoproxil fumarate and tenofovir alafenamide treatment gave rise to greater populations of CD39CD73 cells. These cell surface differences were also observed within EMP repertoires. ABC-treated cells and EMP had greater TF activity, while tenofovir disoproxil fumarate- and tenofovir alafenamide-treated cells and EMP displayed higher ectonucleotidase activity. Finally, EMP isolated from ABC-treated cells enhanced collagen-evoked platelet integrin activation and α-granule release.
Conclusions
We report differential effects of antiretrovirals used in the treatment of HIV upon endothelial function. ABC treatment led to an inflammatory, prothrombotic endothelial phenotype that promoted platelet activation. In contrast, tenofovir disoproxil fumarate and tenofovir alafenamide conferred potentially cardioprotective properties associated with ectonucleotidase activity. These observations establish a link between antiretrovirals and specific functional effects that provide insight into cardiovascular disease in people living with HIV.



Circ Res: 05 Nov 2020; 127:1365-1380

Microanatomy of the Human Atherosclerotic Plaque by Single-Cell Transcriptomics.

Depuydt MAC, Prange KHM, Slenders L, Örd T, ... de Winther MPJ, Pasterkamp G
Rationale
Atherosclerotic lesions are known for their cellular heterogeneity, yet the molecular complexity within the cells of human plaques has not been fully assessed.
Objective
Using single-cell transcriptomics and chromatin accessibility, we gained a better understanding of the pathophysiology underlying human atherosclerosis.
Methods and results
We performed single-cell RNA and single-cell ATAC sequencing on human carotid atherosclerotic plaques to define the cells at play and determine their transcriptomic and epigenomic characteristics. We identified 14 distinct cell populations including endothelial cells, smooth muscle cells, mast cells, B cells, myeloid cells, and T cells and identified multiple cellular activation states and suggested cellular interconversions. Within the endothelial cell population, we defined subsets with angiogenic capacity plus clear signs of endothelial to mesenchymal transition. CD4 and CD8 T cells showed activation-based subclasses, each with a gradual decline from a cytotoxic to a more quiescent phenotype. Myeloid cells included 2 populations of proinflammatory macrophages showing IL (interleukin) 1B or TNF (tumor necrosis factor) expression as well as a foam cell-like population expressing TREM2 (triggering receptor expressed on myeloid cells 2) and displaying a fibrosis-promoting phenotype. ATACseq data identified specific transcription factors associated with the myeloid subpopulation and T cell cytokine profiles underlying mutual activation between both cell types. Finally, cardiovascular disease susceptibility genes identified using public genome-wide association studies data were particularly enriched in lesional macrophages, endothelial, and smooth muscle cells.
Conclusions
This study provides a transcriptome-based cellular landscape of human atherosclerotic plaques and highlights cellular plasticity and intercellular communication at the site of disease. This detailed definition of cell communities at play in atherosclerosis will facilitate cell-based mapping of novel interventional targets with direct functional relevance for the treatment of human disease.



Circ Res: 05 Nov 2020; 127:1437-1455

Clonal Hematopoiesis-Driver DNMT3A Mutations Alter Immune Cells in Heart Failure.

Abplanalp WT, Cremer S, John D, Hoffmann J, ... Zeiher AM, Dimmeler S
Rationale
Clonal hematopoiesis (CH) driven by mutations of DNA methyltransferase 3a (DNMT3A) is associated with increased incidence of cardiovascular disease and poor prognosis of patients with chronic heart failure (HF) and aortic stenosis. Although experimental studies suggest that DNMT3A CH-driver mutations may enhance inflammation, specific signatures of inflammatory cells in humans are missing.
Objective
To define subsets of immune cells mediating inflammation in humans using single-cell RNA-sequencing.
Methods and results
Transcriptomic profiles of peripheral blood mononuclear cells were analysed in N=6 HF patients harboring DNMT3A CH-driver mutations and N=4 patients with HF and no DNMT3A mutations by single-cell RNA-sequencing. Monocytes of HF patients carrying DNMT3A mutations demonstrated a significantly increased expression of inflammatory genes compared to monocytes derived from HF patients without DNMT3A mutations. Among the specific up-regulated genes were the prototypic inflammatory interleukin (IL) IL1B, IL6, IL8, the inflammasome NLRP3, and the macrophage inflammatory proteins CCL3 and CCL4 as well as resistin, which augments monocyte-endothelial adhesion. Silencing of DNMT3A in monocytes induced a paracrine pro-inflammatory activation and increased adhesion to endothelial cells. Furthermore, the classical monocyte subset of DNMT3A mutation carriers showed increased expression of T-cell stimulating immunoglobulin superfamily members CD300LB, CD83, SIGLEC12, as well as the CD2 ligand and cell adhesion molecule CD58, all of which may be involved in monocyte-T cell interactions. DNMT3A mutation carriers were further characterized by increased expression of the T-cell alpha receptor constant chain and Th1, Th2, Th17, CD8+ effector, CD4+ memory and Treg specific signatures.
Conclusions
This study demonstrates that circulating monocytes and T-cells of HF patients harboring CH-driver mutations in DNMT3A exhibit a highly inflamed transcriptome, which may contribute to the aggravation of chronic heart failure.



Circ Res: 05 Nov 2020; epub ahead of print

Eosinophils Protect Mice from Angiotensin-II Perfusion-Induced Abdominal Aortic Aneurysm.

Liu CL, Liu X, Zhang Y, Liu J, ... Zhang JY, Shi GP
Rationale
Blood eosinophil (EOS) count and EOS cationic protein (ECP) associate with human cardiovascular diseases (CVD). Yet, whether EOS play a role in CVD remains untested. The current study detected EOS accumulation in human and murine abdominal aortic aneurysm (AAA) lesions, suggesting EOS participation in this aortic disease.
Objective
To test whether and how EOS affect AAA growth.
Methods and results
Population-based randomized clinically controlled screening trials revealed higher blood EOS count in 579 male AAA patients than in 5,063 non-AAA control (0.236{plus minus}0.182 vs 0.211{plus minus}0.154, 109/L, P<0.001). Univariate (OR=1.381, P<0.001) and multivariate (OR=1.237, P=0.031) logistic regression analyses indicated that increased blood EOS count in AAA patients served as an independent risk factor of human AAA. Immunostaining and immunoblot analyses detected EOS accumulation and EOS cationic protein expression in human and murine AAA lesions. Results showed that EOS deficiency exacerbated AAA growth with increased lesion inflammatory cell contents, matrix-degrading protease activity, angiogenesis, cell proliferation and apoptosis, and smooth muscle cell (SMC) loss using angiotensin-II perfusion-induced AAA in Apoe and EOS-deficient Apoe;∆dblGATA mice. EOS deficiency increased lesion chemokine expression, muted lesion expression of IL4 and EOS-associated-ribonuclease-1 (mEar1, human ECP homolog), and slanted M1 macrophage polarization. In cultured macrophages and monocytes, EOS-derived IL4 and mEar1 polarized M2 macrophages, suppressed CD11b+Ly6Chi monocytes, and increased CD11bLy6C monocytes. mEar1 treatment or adoptive transfer of EOS from WT and Il13 mice, but not EOS from Il4 mice, blocked AAA growth in Apoe ∆dblGATA mice. Immunofluorescent staining and immunoblot analyses demonstrated a role for EOS IL4 and mEar1 in blocking NF-κB activation in macrophages, SMCs, and endothelial cells.
Conclusions
EOS play a protective role in AAA by releasing IL4 and cationic proteins such as mEar1 to regulate macrophage and monocyte polarization and to block NF-κB activation in aortic inflammatory and vascular cells.



Circ Res: 05 Nov 2020; epub ahead of print

Loss of Endogenously Cycling Adult Cardiomyocytes Worsens Myocardial Function.

Bradley LA, Young A, Li H, Billcheck HO, Wolf MJ
Rationale
Endogenously cycling adult cardiomyocytes (CMs) increase after myocardial infarction (MI) but remain scare, and are generally thought not to contribute to myocardial function. However, this broadly held assumption has not been tested, mainly because of the lack of transgenic reporters that restrict Cre expression to adult CMs that reenter the cell cycle.
Objective
We created and validated a new transgenic mouse, αMHC-MerDreMer-Ki67p-RoxedCre::Rox-Lox-tdTomato-eGFP (denoted αDKRC) that restricts Cre expression to cycling adult CMs and uniquely integrates spatial and temporal adult CM cycling events based on the DNA specificities of orthologous Dre- and Cre recombinases. We then created mice that expressed an inducible Diphtheria toxin (DTA), αDKRC::DTA mice, in adult cycling CMs and examined the effects of ablating these endogenously cycling CMs on myocardial function after Ischemic-Reperfusion (I/R) MI.
Methods and results
A tandem αDKRC transgene was designed, validated in cultured cells, and used to make transgenic mice. The αDKRC transgene integrated between MYH6 and MYH7 and did not disrupt expression of the surrounding genes. Compared to controls, αDKRC::RLTG mice treated with Tamoxifen expressed tdTomato+ in CMs with rare Bromodeoxyuridine (BrdU)+, eGFP+ CMs, consistent with reentry of the cell cycle. We then pre-treated αDKRC::RLTG mice with Tamoxifen to activate the reporter before sham or reperfusion (I/R) myocardial infarction (MI) surgeries. Compared to Sham surgery, the I/R MI group had increased single and paired eGFP+ CMs predominantly in the border zones (5.8 {plus minus} 0.5 vs. 3.3 {plus minus} 0.3 CMs per ten-micron section, N = 8-9 mice per group, n = 16-24 sections per mouse), indicative of cycled CMs. The single to paired eGFP+ CM ratio was ~9 to 1 (5.2 {plus minus} 0.4 single vs. 0.6 {plus minus} 0.2 paired CMs) in the I/R MI group after MI, suggesting that cycling CMs were more likely to undergo polyploidy than replication. The ablation of endogenously cycling adult CMs in αDKRC::DTA mice caused progressive worsening left ventricular chamber size and function after I/R MI, compared to controls.
Conclusions
Although scarce, endogenously cycling adult CMs contribute to myocardial function after injury, suggesting that these cells may be physiologically relevant.



Circ Res: 03 Nov 2020; epub ahead of print

Live Imaging of Intracranial Lymphatics in the Zebrafish.

Castranova D, Samasa B, Venero Galanternik M, Jung HM, Pham VN, Weinstein BM
Rationale
The recent discovery of meningeal lymphatics in mammals is reshaping our understanding of fluid homeostasis and cellular waste management in the brain, but visualization and experimental analysis of these vessels is challenging in mammals. Although the optical clarity and experimental advantages of zebrafish have made this an essential model organism for studying lymphatic development, the existence of meningeal lymphatics has not yet been reported in this species.
Objective
Examine the intracranial space of larval, juvenile, and adult zebrafish to determine whether and where intracranial lymphatic vessels are present. Using high-resolution optical imaging of the meninges in living animals, we show that Methods and Results:Using high-resolution optical imaging of the meninges in living animals, we show that zebrafish possess a meningeal lymphatic network comparable to that found in mammals. We confirm that this network is separate from the blood vascular network and that it drains interstitial fluid from the brain. We document the developmental origins and growth of these vessels into a distinct network separated from the external lymphatics. Finally we show that these vessels contain immune cells and perform live imaging of immune cell trafficking and transmigration in meningeal lymphatics.
Conclusions
This discovery establishes the zebrafish as a important new model for experimental analysis of meningeal lymphatic development, and opens up new avenues for probing meningeal lymphatic function in health and disease.



Circ Res: 01 Nov 2020; epub ahead of print

Genetic Dissection of a Super Enhancer Controlling the Nppa-Nppb Cluster in the Heart.

Man JC, van Duijvenboden K, Krijger PH, Hooijkaas IB, ... Boukens BJ, Christoffels VM

Atrial and B-type natriuretic peptide, encoded by the clustered genes Nppa and Nppb, are important prognostic, diagnostic and therapeutic proteins in cardiac disease. The spatio-temporal expression pattern and stress-induction of the Nppa and Nppb are tightly regulated, possibly involving their co-regulation by an evolutionary conserved enhancer cluster. To explore the physiological functions of the enhancer cluster and elucidate the genomic mechanism underlying Nppa-Nppb co-regulation in vivo.By analyzing epigenetic data we uncovered an enhancer cluster with super enhancer characteristics upstream of Nppb. Using CRISPR/Cas9 genome editing, the enhancer cluster or parts thereof, Nppb and flanking regions or the entire genomic block spanning Nppa-Nppb, respectively, were deleted from the mouse genome. The impact on gene regulation and phenotype of the respective mouse lines was investigated by transcriptomic, epigenomic and phenotypic analyses. The enhancer cluster was essential for prenatal and postnatal ventricular expression of Nppa and Nppb, but not of any other gene. Enhancer cluster-deficient mice showed enlarged hearts before and after birth, similar to Nppa-Nppb compound knockout mice we generated. Analysis of the other deletion alleles indicated the enhancer cluster engages the promoters of Nppa and Nppb in a competitive rather than a cooperative mode, resulting in increased Nppa expression when Nppb and flanking sequences were deleted. The enhancer cluster maintained its active epigenetic state and selectivity in when its target genes are absent. In enhancer cluster-deficient animals, Nppa was induced but remained low in the post-myocardial infarction border zone and in the hypertrophic ventricle, involving regulatory sequences proximal to Nppa.Coordinated ventricular expression of Nppa and Nppb is controlled in a competitive manner by a shared super enhancer, which is also required to augment stress-induced expression and to prevent premature hypertrophy.



Circ Res: 26 Oct 2020; epub ahead of print

Multiscale Analysis of Extracellular Matrix Remodeling in the Failing Heart.

Rubina Perestrelo A, Silva AC, Oliver-De La Cruz J, Martino F, ... Nascimento DS, Forte G

Cardiac extracellular matrix (ECM) comprises a dynamic molecular network providing structural support to heart tissue function. Understanding the impact of ECM remodeling on cardiac cells during heart failure (HF) is essential to prevent adverse ventricular remodeling and restore organ functionality in affected patients. We aimed to (i) identify consistent modifications to cardiac ECM structure and mechanics that contribute to HF and (ii) determine the underlying molecular mechanisms.We first performed decellularization of human and murine ECM (dECM) and then analyzed the pathological changes occurring in dECM during HF by atomic force (AFM), two-photon microscopy, high-resolution 3D image analysis and computational fluid dynamics (CFD) simulation. We then performed molecular and functional assays in patient-derived cardiac fibroblasts (CFs) based on YAP-TEAD mechanosensing activity and collagen contraction assays. The analysis of HF dECM resulting from ischemic (IHD) or dilated cardiomyopathy (DCM), as well as from mouse infarcted tissue, identified a common pattern of modifications in their 3D topography. As compared to healthy heart, HF ECM exhibited aligned, flat and compact fiber bundles, with reduced elasticity and organizational complexity. At the molecular level, RNA sequencing of HF CFs highlighted the overrepresentation of dysregulated genes involved in ECM organization, or being connected to TGFß1, Interleukin-1, TNF-alpha and BDNF signaling pathways. Functional tests performed on HF CFs pointed at mechanosensor YAP as a key player in ECM remodeling in the diseased heart via transcriptional activation of focal adhesion assembly. Finally, in vitro experiments clarified pathological cardiac ECM prevents cell homing, thus providing further hints to identify a possible window of action for cell therapy in cardiac diseases.Our multi-parametric approach has highlighted repercussions of ECM remodeling on cell homing, CF activation and focal adhesion protein expression via hyper-activated YAP signaling during HF.



Circ Res: 26 Oct 2020; epub ahead of print

No Evidence for Erythro-Myeloid Progenitor-Derived Vascular Endothelial Cells in Multiple Organs.

Feng T, Gao Z, Kou S, Huang X, ... Lin CP, Zhang H
Rationale
Endothelial cells are thought to emerge de novo from the mesoderm to form the entire circulatory system. Recently, erythro-myeloid progenitors (EMPs) have been proposed to be another remarkable developmental origin for blood vessels in multiple organs, including the hindbrain, liver, lung, and heart, as demonstrated by lineage tracing studies using different genetic tools. These observations challenge the current consensus that intraembryonic vessels are thought to expand solely by the proliferation of preexisting endothelial cells. Resolution of this controversy over the developmental origin of endothelial cells is crucial for developing future therapeutics for vessel-dependent organ repair and regeneration.
Objective
To examine the contribution of EMPs to intraembryonic endothelial cells.
Methods and results
We first used a transgenic mouse expressing a tamoxifen-inducible Mer-iCre fusion protein driven by the Csf1r (colony stimulating factor 1 receptor) promoter. Genetic lineage tracing based on Csf1r-Mer-iCre-Mer showed no contribution of EMPs to brain endothelial cells identified by several markers. We also generated a knock-in mouse line by inserting an internal ribosome entry site-iCre cassette into the 3\' untranslated region ofgene to further investigate the cellular fates of EMPs. Similarly, we did not find any Csf1r-ires-iCre traced endothelial cells in brain, liver, lung, or heart in development either. Additionally, we found that Kit (KIT proto-oncogene receptor tyrosine kinase) was expressed not only in EMPs but also in embryonic hindbrain endothelial cells. Therefore, Kit promoter-driven recombinase, such as Kit-CreER, is a flawed tool for lineage tracing when examining the contribution of EMPs to hindbrain endothelial cells. We also traced CD45 (protein tyrosine phosphatase receptor type C; Ptprc) circulating EMPs and did not find any CD45 lineage-derived endothelial cells during development.
Conclusions
Our study suggested that EMPs are not the origin of intraembryonic endothelial cells.



Circ Res: 22 Oct 2020; 127:1221-1232

Hyperglycemia Induces Myocardial Dysfunction via Epigenetic Regulation of JunD.

Hussain S, Khan AW, Akhmedov A, Suades R, ... Lüscher TF, Cosentino F
Rationale
Hyperglycemia -induced reactive oxygen species are key mediators of cardiac dysfunction. JunD (Jund proto-oncogene subunit), a member of the AP-1 (activator protein-1) family of transcription factors, is emerging as a major gatekeeper against oxidative stress. However, its contribution to redox state and inflammation in the diabetic heart remains to be elucidated.
Objective
The present study investigates the role of JunD in hyperglycemia-induced and reactive oxygen species-driven myocardial dysfunction.
Methods and results
JunD mRNA and protein expression were reduced in the myocardium of mice with streptozotocin-induced diabetes mellitus as compared to controls. JunD downregulation was associated with oxidative stress and left ventricular dysfunction assessed by electron spin resonance spectroscopy as well as conventional and 2-dimensional speckle-tracking echocardiography. Furthermore, myocardial expression of free radical scavenger superoxide dismutase 1 and aldehyde dehydrogenase 2 was reduced, whereas the NOX2 (NADPH [nicotinamide adenine dinucleotide phosphatase] oxidase subunit 2) and NOX4 (NADPH [nicotinamide adenine dinucleotide phosphatase] oxidase subunit 4) were upregulated. The redox changes were associated with increased NF-κB (nuclear factor kappa B) binding activity and expression of inflammatory mediators. Interestingly, mice with cardiac-specific overexpression of JunD via the α MHC (α- myosin heavy chain) promoter (α MHC ) were protected against hyperglycemia-induced cardiac dysfunction. We also showed that JunD was epigenetically regulated by promoter hypermethylation, post-translational modification of histone marks, and translational repression by miRNA (microRNA)-673/menin. Reduced JunD mRNA and protein expression were confirmed in left ventricular specimens obtained from patients with type 2 diabetes mellitus as compared to nondiabetic subjects.
Conclusions
Here, we show that a complex epigenetic machinery involving DNA methylation, histone modifications, and microRNAs mediates hyperglycemia-induced JunD downregulation and myocardial dysfunction in experimental and human diabetes mellitus. Our results pave the way for tissue-specific therapeutic modulation of JunD to prevent diabetic cardiomyopathy.



Circ Res: 22 Oct 2020; 127:1261-1273

Gut Microbiota-Associated Activation of TLR5 Induces Apolipoprotein A1 Production in the Liver.

Yiu JHC, Chan KS, Cheung J, Li J, ... Xu A, Woo CW
Rationale
Dysbiosis of gut microbiota plays an important role in cardiovascular diseases but the molecular mechanisms are complex. An association between gut microbiome and the variance in HDL-C (high-density lipoprotein-cholesterol) level was suggested in a human study. Besides, dietary fat was shown to increase both HDL-C and LDL-C (low-density lipoprotein-cholesterol) levels. We speculate that certain types of gut bacteria responding to dietary fat may help to regulate HDL-C level and potentially affect atherosclerotic development.
Objective
We aimed to investigate whether and how high-fat diet (HFD)-associated gut microbiota regulated HDL-C level.
Methods and results
We found that HFD increased gut flagellated bacteria population in mice. The increase in HDL-C level was adopted by mice receiving fecal microbiome transplantation from HFD-fed mouse donors. HFD led to increased hepatic but not circulating flagellin, and deletion of TLR5 (Toll-like receptor 5), a receptor sensing flagellin, suppressed HFD-stimulated HDL-C and ApoA1 (apolipoprotein A1) levels. Overexpression of TLR5 in the liver of TLR5-knockout mice was able to partially restore the production of ApoA1 and HDL-C levels. Mechanistically, TLR5 activation by flagellin in primary hepatocytes stimulated ApoA1 production through the transcriptional activation responding to the binding of NF-κB (nuclear factor-κB) onpromoter region. Furthermore, oral supplementation of flagellin was able to stimulate hepatic ApoA1 production and HDL-C level and decrease atherosclerotic lesion size in apolipoprotein E-deficient () mice without triggering hepatic and systemic inflammation. The stimulation of ApoA1 production was also seen in human ApoA1-transgenic mice treated with oral flagellin.
Conclusions
Our finding suggests that commensal flagellated bacteria in gut can facilitate ApoA1 and HDL-C productions in liver through activation of TLR5 in hepatocytes. Hepatic TLR5 may be a potential drug target to increase ApoA1.



Circ Res: 22 Oct 2020; 127:1236-1252

High-Density Lipoprotein Carries Markers That Track With Recovery From Stroke.

Plubell DL, Fenton AM, Rosario S, Bergstrom P, ... Fazio S, Pamir N
Rationale
Prospective cohort studies question the value of HDL-C (high-density lipoprotein cholesterol) for stroke risk prediction.
Objective
Investigate the relationship between long-term functional recovery and HDL proteome and function.
Methods and results
Changes in HDL protein composition and function (cholesterol efflux capacity) in patients after acute ischemic stroke at 2 time points (24 hours, 35 patients; 96 hours, 20 patients) and in 35 control subjects were measured. The recovery from stroke was assessed by 3 months, the National Institutes of Health Stroke Scale and modified Rankin scale scores. When compared with control subject after adjustments for sex and HDL-C levels, 12 proteins some of which participate in acute phase response and platelet activation (APMAP [adipocyte plasma membrane-associated protein], GPLD1 [phosphate inositol-glycan specific phospholipase D], APOE [apolipoprotein E], IHH [Indian hedgehog protein], ITIH4 [inter-alpha-trypsin inhibitor chain H4], SAA2 [serum amyloid A2], APOA4 [apolipoprotein A-IV], CLU [clusterin], ANTRX2 [anthrax toxin receptor 2], PON1 [serum paraoxonase/arylesterase], SERPINA1 [alpha-1-antitrypsin], and APOF [apolipoprotein F]) were significantly (adjusted <0.05) altered in stroke HDL at 96 hours. The first 8 of these proteins were also significantly altered at 24 hours. Consistent with inflammatory remodeling, cholesterol efflux capacity was reduced by 32% (<0.001) at both time points. Baseline stroke severity adjusted regression model showed that changes within 96-hour poststroke in APOF, APOL1, APMAP, APOC4 (apolipoprotein C4), APOM (apolipoprotein M), PCYOX1 (prenylcysteine oxidase 1), PON1, and APOE correlate with stroke recovery scores (=0.38-0.73, adjusted <0.05). APOF (=0.73) and APOL1 (=0.60) continued to significantly correlate with recovery scores after accounting for tPA (tissue-type plasminogen activator) treatment.
Conclusions
Changes in HDL proteins during early acute phase of stroke associate with recovery. Monitoring HDL proteins may provide clinical biomarkers that inform on stroke recuperation.



Circ Res: 22 Oct 2020; 127:1274-1287

Association of Habitual Physical Activity With Cardiovascular Disease Risk.

Lin H, Sardana M, Zhang Y, Liu C, ... Murabito JM, McManus DD
Rationale
A sedentary lifestyle is associated with increased risk for cardiovascular disease (CVD). Smartwatches enable accurate daily activity monitoring for physical activity measurement and intervention. Few studies, however, have examined physical activity measures from smartwatches in relation to traditional risk factors associated with future risk for CVD.
Objective
To investigate the association of habitual physical activity measured by smartwatch with predicted CVD risk in adults.
Methods and results
We enrolled consenting FHS (Framingham Heart Study) participants in an ongoing eFHS (electronic Framingham Heart Study) at the time of their FHS research center examination. We provided participants with a smartwatch (Apple Watch Series 0) and instructed them to wear it daily, which measured their habitual physical activity as the average daily step count. We estimated the 10-year predicted risk of CVD using the American College of Cardiology/American Heart Association 2013 pooled cohort risk equation. We estimated the association between physical activity and predicted risk of CVD using linear mixed effects models adjusting for age, sex, wear time, and familial structure. Our study included 903 eFHS participants (mean age 53±9 years, 61% women, 9% non-White) who wore the smartwatch ≥5 hours per day for ≥30 days. Median daily step count was similar among men (7202 with interquartile range 3619) and women (7260 with interquartile range 3068; =0.52). Average 10-year predicted CVD risk was 4.5% (interquartile range, 6.1%) for men and 1.2% (interquartile range, 2.2%) for women (=1.3×10). Every 1000 steps higher habitual physical activity was associated with 0.18% lower predicted CVD risk (=3.2×10). The association was attenuated but remained significant after further adjustment for body mass index (=0.01).
Conclusions
In this community-based sample of adults, higher daily physical activity measured by a study smartwatch was associated with lower predicted risk of CVD. Future research should examine the longitudinal association of prospectively measured daily activity and incident CVD.



Circ Res: 22 Oct 2020; 127:1253-1260

Infarct Collagen Topography Regulates Fibroblast Fate via p38-Yes-Associated Protein Transcriptional Enhanced Associate Domain Signals.

Bugg D, Bretherton R, Kim P, Olszewski E, ... Kim DH, Davis J
Rationale
Myocardial infarction causes spatial variation in collagen organization and phenotypic diversity in fibroblasts, which regulate the heart\'s ECM (extracellular matrix). The relationship between collagen structure and fibroblast phenotype is poorly understood but could provide insights regarding the mechanistic basis for myofibroblast heterogeneity in the injured heart.
Objective
To investigate the role of collagen organization in cardiac fibroblast fate determination.
Methods and results
Biomimetic topographies were nanofabricated to recapitulate differential collagen organization in the infarcted mouse heart. Here, adult cardiac fibroblasts were freshly isolated and cultured on ECM topographical mimetics for 72 hours. Aligned mimetics caused cardiac fibroblasts to elongate while randomly organized topographies induced circular morphology similar to the disparate myofibroblast morphologies measured in vivo. Alignment cues also induced myofibroblast differentiation, as >60% of fibroblasts formed αSMA (α-smooth muscle actin) stress fibers and expressed myofibroblast-specific ECM genes like Postn (periostin). By contrast, random organization caused 38% of cardiac fibroblasts to express αSMA albeit with downregulated myofibroblast-specific ECM genes. Coupling topographical cues with the profibrotic agonist, TGFβ (transforming growth factor beta), additively upregulated myofibroblast-specific ECM genes independent of topography, but only fibroblasts on flat and randomly oriented mimetics had increased percentages of fibroblasts with αSMA stress fibers. Increased tension sensation at focal adhesions induced myofibroblast differentiation on aligned mimetics. These signals were transduced by p38-YAP (yes-associated protein)-TEAD (transcriptional enhanced associate domain) interactions, in which both p38 and YAP-TEAD (yes-associated protein transcriptional enhanced associate domain) binding were required for myofibroblast differentiation. By contrast, randomly oriented mimetics did not change focal adhesion tension sensation or enrich for p38-YAP-TEAD interactions, which explains the topography-dependent diversity in fibroblast phenotypes observed here.
Conclusions
Spatial variations in collagen organization regulate cardiac fibroblast phenotype through mechanical activation of p38-YAP-TEAD signaling, which likely contribute to myofibroblast heterogeneity in the infarcted myocardium.



Circ Res: 22 Oct 2020; 127:1306-1322

Shaping Waves of Bone Morphogenetic Protein Inhibition During Vascular Growth.

Guihard PJ, Guo Y, Wu X, Zhang L, ... Garfinkel A, Boström KI
Rationale
The BMPs (bone morphogenetic proteins) are essential morphogens in angiogenesis and vascular development. Disruption of BMP signaling can trigger cardiovascular diseases, such as arteriovenous malformations.
Objective
A computational model predicted that BMP4 and BMP9 and their inhibitors MGP (matrix gamma-carboxyglutamic acid [Gla] protein) and CV2 (crossveinless-2) would form a regulatory system consisting of negative feedback loops with time delays and that BMP9 would trigger oscillatory expression of the 2 inhibitors. The goal was to investigate this regulatory system in endothelial differentiation and vascular growth.
Methods and results
Oscillations in the expression of MGP and CV2 were detected in endothelial cells in vitro, using quantitative real-time polymerase chain reaction and immunoblotting. These organized temporally downstream BMP-related activities, including expression of stalk-cell markers and cell proliferation, consistent with an integral role of BMP9 in vessel maturation. In vivo, the inhibitors were located in distinct zones in relation to the front of the expanding retinal network, as determined by immunofluorescence. Time-dependent changes of the CV2 location in the retina and the existence of an endothelial population with signs of oscillatory MGP expression in developing vasculature supported the in vitro findings. Loss of MGP or its BMP4-binding capacity disrupted the retinal vasculature, resulting in poorly formed networks, especially in the venous drainage areas, and arteriovenous malformations as determined by increased cell coverage and functional testing.
Conclusions
Our results suggest a previously unknown mechanism of temporal orchestration of BMP4 and BMP9 activities that utilize the tandem actions of the extracellular antagonists MGP and CV2. Disruption of this mechanism may contribute to vascular malformations and disease.



Circ Res: 22 Oct 2020; 127:1288-1305

Niacin Attenuates Pulmonary Hypertension Through H-PGDS in Macrophages.

Jia D, Bai P, Wan N, Liu J, ... Zhang J, Shen Y
Rationale
Pulmonary arterial hypertension (PAH) is characterized by progressive pulmonary vascular remodeling, accompanied by varying degrees of perivascular inflammation. Niacin, a commonly used lipid-lowering drug, possesses vasodilating and proresolution effects by promoting the release of prostaglandin D (PGD). However, whether or not niacin confers protection against PAH pathogenesis is still unknown.
Objective
This study aimed to determine whether or not niacin attenuates the development of PAH and, if so, to elucidate the molecular mechanisms underlying its effects.
Methods and results
Vascular endothelial growth factor receptor inhibitor SU5416 and hypoxic exposure were used to induce pulmonary hypertension (PH) in rodents. We found that niacin attenuated the development of this hypoxia/SU5416-induced PH in mice and suppressed progression of monocrotaline-induced and hypoxia/SU5416-induced PH in rats through the reduction of pulmonary artery remodeling. Niacin boosted PGD generation in lung tissue, mainly through H-PGDS (hematopoietic PGD synthases). Deletion of H-PGDS, but not lipocalin-type PGDS, exacerbated the hypoxia/SU5416-induced PH in mice and abolished the protective effects of niacin against PAH. Moreover, H-PGDS was expressed dominantly in infiltrated macrophages in lungs of PH mice and patients with idiopathic PAH. Macrophage-specific deletion of H-PGDS markedly decreased PGD generation in lungs, aggravated hypoxia/SU5416-induced PH in mice, and attenuated the therapeutic effect of niacin on PAH.
Conclusions
Niacin treatment ameliorates the progression of PAH through the suppression of vascular remodeling by stimulating H-PGDS-derived PGD release from macrophages.



Circ Res: 22 Oct 2020; 127:1323-1336

Variants of Focal Adhesion Scaffold Genes Cause Thoracic Aortic Aneurysm.

Li Y, Gao S, Han Y, Song L, ... Du J, Li Y

Thoracic aortic aneurysm (TAA) leads to substantial mortality worldwide. Familial and syndromic TAAs are highly correlated with genetics. However, the incidence of sporadic isolated TAA (iTAA) is much higher, and the genetic contribution is not yet clear.Here, we examined the genetic characteristics of sporadic iTAA.We performed a genetic screen of 551 sporadic iTAA cases and 1071 controls via whole-exome sequencing. The prevalence of pathogenic mutations in known causal genes was 5.08% in the iTAA cohort. We selected 100 novel candidate genes using a strict strategy, and the suspected functional variants of these genes were significantly enriched in cases compared with controls and carried by 60.43% of patients. We found more severe phenotypes and a lower proportion of hypertension in cases with pathogenic mutations or suspected functional variants. Among the candidate genes, TESTIN (TES), which encodes a focal adhesion (FA) scaffold protein, was identified as a potential TAA causal gene, accounting for four patients with two missense variants in the LIM1 domain (c.751T > C encoding p.Y251H; c.838T > C encoding p.Y280H) and highly expressed in the aorta. The two variants led to a decrease in TES expression. The thoracic aorta was spontaneously dilated in the Tes knock-in and Tes mice. Mechanistically, the p.Y249H variant or knockdown of TES led to the repression of vascular smooth muscle cell (VSMC) contraction genes and disturbed the VSMC contractile phenotype. Interestingly, suspected functional variants of other FA scaffold genes, including TLN1 and ZYX, were also significantly enriched in patients with iTAA; moreover, their knockdown resulted in decreased contractility of VSMCs.For the first time, this study revealed the genetic landscape across iTAA and showed that the FA scaffold genes are critical in the pathogenesis of iTAA.



Circ Res: 22 Oct 2020; epub ahead of print

Interaction of the Joining Region in Junctophilin-2 with the L-type Ca Channel Is Pivotal for Cardiac Dyad Assembly and Intracellular Ca Dynamics.

Gross P, Johnson J, Romero CM, Eaton DM, ... Chen X, Houser SR

Ca induced Ca release (CICR) in normal hearts requires close approximation of L-type calcium channels (LTCCs) within the transverse tubules (T-tubules), and Ryanodine receptors (RyR) within the junctional sarcoplasmic reticulum (jSR). CICR is disrupted in cardiac hypertrophy and heart failure, which is associated with loss of T-tubules and disruption of cardiac dyads. In these conditions, LTCCs are redistributed from the T-tubules to disrupt CICR. The molecular mechanism responsible for LTCCs recruitment to and from the T-tubules is not well known. Junctophilin-2 (JPH2) enables close association between T-tubules and the jSR to ensure efficient CICR. JPH2 has a so-called Joining region that is located near domains that interact with T-tubular plasma membrane, where LTCCs are housed. The idea that this Joining region directly interacts with LTCCs and contributes to LTCC recruitment to T-tubules is unknown. To determine if the Joining region in JPH2 recruits LTCCs to T-tubules through direct molecular interaction in cardiomyocytes to enable efficient CICR.Modified abundance of JPH2 and redistribution of LTCC were studied in left ventricular hypertrophy in vivo and in cultured adult Feline and rat ventricular myocytes. Protein-protein interaction studies showed that the Joining region in JPH2 interacts with LTCC-α1C subunit and causes LTCCs distribution to the dyads, where they colocalize with RyRs. A JPH2 with induced mutations in the Joining region (mutPG1JPH2) caused T-tubule remodeling and dyad loss, showing that an interaction between LTCC and JPH2 is crucial for T-tubule stabilization. mut caused asynchronous Ca-release with impaired excitation-contraction (EC) coupling after β-adrenergic stimulation. The disturbed Ca regulation in mut overexpressing myocytes caused Calcium/calmodulin-dependent kinase-II activation and altered myocyte bioenergetics.The interaction between LTCC and the Joining region in JPH2 facilitates dyad assembly and maintains normal CIRC in cardiomyocytes.



Circ Res: 22 Oct 2020; epub ahead of print

Adrenergic Ca1.2 Activation via Rad Phosphorylation Converges at α I-II Loop.

Papa A, Kushner JS, Hennessey JA, Katchman AN, ... Johny MB, Marx SO

Changing activity of cardiac Ca1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac CaV1.2 channels are prominently up-regulated via activation of protein kinase A, essential molecular details remained stubbornly enigmatic.The primary goal of this study was to determine how various factors converging at the Ca1.2 I-II loop interact to regulate channel activity under basal conditions, during β-adrenergic stimulation, and in heart failure.We generated transgenic mice with expression of Ca1.2 α subunits with: 1) mutations ablating interaction between α and β subunits; 2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α); or 3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α up-regulated in the hypertrophied heart. Introducing three glycine residues that disrupt a rigid IS6-AID helix markedly reduced basal open probability despite intact binding of Caβ to α I-II loop, and eliminated β-adrenergic agonist stimulation of Ca1.2 current. In contrast, introduction of the exon 9* splice variant in α I-II loop, which is increased in ventricles of patients with end-stage heart failure, increased basal open probability but did not attenuate stimulatory response to β-adrenergic agonists when reconstituted heterologously with β and Rad or transgenically expressed in cardiomyocytes.Ca channel activity is dynamically modulated under basal conditions, during β-adrenergic stimulation, and in heart failure by mechanisms converging at the α I-II loop. Caβ binding to α stabilizes an increased channel open probability gating mode by a mechanism that requires an intact rigid linker between the β subunit binding site in the I-II loop and the channel pore. Release of Rad-mediated inhibition of Ca channel activity by β-adrenergic agonists/PKA also requires this rigid linker and β binding to α.



Circ Res: 21 Oct 2020; epub ahead of print

Mir-30d Regulates Cardiac Remodeling by Intracellular And Paracrine Signaling.

Li J, Salvador AM, Li G, Valkov N, ... Xiao J, Das S

Previous translational studies implicate plasma extracellular microRNA-30d (miR-30d) as a biomarker in left ventricular (LV) remodeling and clinical outcome in heart failure (HF) patients, though precise mechanisms remain obscure.To investigate the mechanism of miR-30d-mediated cardioprotection in HF.In rat and mouse models of ischemic HF, we show that miR-30d gain of function (genetic, lentivirus or agomiR-mediated) improves cardiac function, decreases myocardial fibrosis, and attenuates cardiomyocyte (CM) apoptosis. Genetic or locked nucleic acid (LNA)-based knock-down of miR-30d expression potentiates pathological LV remodeling, with increased dysfunction, fibrosis, and CM death. RNA-seq of in vitro miR-30d gain and loss of function, together with bioinformatic prediction and experimental validation in cardiac myocytes and fibroblasts, were used to identify and validate direct targets of miR-30d. miR-30d expression is selectively enriched in CMs, induced by hypoxic stress and is acutely protective, targeting mitogen-associate protein kinase (MAP4K4) to ameliorate apoptosis. Moreover, miR-30d is secreted primarily in extracellular vesicles by CMs and inhibits fibroblast proliferation and activation by directly targeting integrin α5 in the acute phase via paracrine signaling to cardiac fibroblasts. In the chronic phase of ischemic remodeling, lower expression of miR-30d in the heart and plasma EVs is associated with adverse remodeling in rodent models and human subjects, and is linked to whole blood expression of genes implicated in fibrosis and inflammation, consistent with observations in model systems.These findings provide the mechanistic underpinning for the cardioprotective association of miR-30d in human HF. More broadly, our findings support an emerging paradigm involving intercellular communication of EV-contained miRNAs to trans regulate distinct signaling pathways across cell types. Functionally validated RNA biomarkers and their signaling networks may warrant further investigation as novel therapeutic targets in HF.



Circ Res: 21 Oct 2020; epub ahead of print