Journal: J Mol Cell Cardiol

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Abstract

A junctional cAMP compartment regulates rapid Ca signaling in atrial myocytes.

Brandenburg S, Pawlowitz J, Steckmeister V, Subramanian H, ... Nikolaev VO, Lehnart SE
Axial tubule junctions with the sarcoplasmic reticulum control the rapid intracellular Ca2+-induced Ca2+ release that initiates atrial contraction. In atrial myocytes we previously identified a constitutively increased ryanodine receptor (RyR2) phosphorylation at junctional Ca2+ release sites, whereas non-junctional RyR2 clusters were phosphorylated acutely following β-adrenergic stimulation. Here, we hypothesized that the baseline synthesis of 3\',5\'-cyclic adenosine monophosphate (cAMP) is constitutively augmented in the axial tubule junctional compartments of atrial myocytes. Confocal immunofluorescence imaging of atrial myocytes revealed that junctin, binding to RyR2 in the sarcoplasmic reticulum, was densely clustered at axial tubule junctions. Interestingly, a new transgenic junctin-targeted FRET cAMP biosensor was exclusively co-clustered in the junctional compartment, and hence allowed to monitor cAMP selectively in the vicinity of junctional RyR2 channels. To dissect local cAMP levels at axial tubule junctions versus subsurface Ca2+ release sites, we developed a confocal FRET imaging technique for living atrial myocytes. A constitutively high adenylyl cyclase activity sustained increased local cAMP levels at axial tubule junctions, whereas β-adrenergic stimulation overcame this cAMP compartmentation resulting in additional phosphorylation of non-junctional RyR2 clusters. Adenylyl cyclase inhibition, however, abolished the junctional RyR2 phosphorylation and decreased L-type Ca2+ channel currents, while FRET imaging showed a rapid cAMP decrease. In conclusion, FRET biosensor imaging identified compartmentalized, constitutively augmented cAMP levels in junctional dyads, driving both the locally increased phosphorylation of RyR2 clusters and larger L-type Ca2+ current density in atrial myocytes. This cell-specific cAMP nanodomain is maintained by a constitutively increased adenylyl cyclase activity, contributing to the rapid junctional Ca2+-induced Ca2+ release, whereas β-adrenergic stimulation overcomes the junctional cAMP compartmentation through cell-wide activation of non-junctional RyR2 clusters.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 12 Jan 2022; epub ahead of print
Brandenburg S, Pawlowitz J, Steckmeister V, Subramanian H, ... Nikolaev VO, Lehnart SE
J Mol Cell Cardiol: 12 Jan 2022; epub ahead of print | PMID: 35033544
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Abstract

Shortening the thick filament by partial deletion of titin\'s C-zone alters cardiac function by reducing the operating sarcomere length range.

Methawasin M, Farman GP, Granzier-Nakajima S, Strom J, ... Smith JE, Granzier H
Titin\'s C-zone is an inextensible segment in titin, comprised of 11 super-repeats and located in the cMyBP-C-containing region of the thick filament. Previously we showed that deletion of titin\'s super-repeats C1 and C2 (TtnΔC1-2 model) results in shorter thick filaments and contractile dysfunction of the left ventricular (LV) chamber but that unexpectedly LV diastolic stiffness is normal. Here we studied the contraction-relaxation kinetics from the time-varying elastance of the LV and intact cardiomyocyte, cellular work loops of intact cardiomyocytes, Ca2+ transients, cross-bridge kinetics, and myofilament Ca2+ sensitivity. Intact cardiomyocytes of TtnΔC1-2 mice exhibit systolic dysfunction and impaired relaxation. The time-varying elastance at both LV and single-cell levels showed that activation kinetics are normal in TtnΔC1-2 mice, but that relaxation is slower. The slowed relaxation is, in part, attributable to an increased myofilament Ca2+ sensitivity and slower early Ca2+ reuptake. Cross-bridge dynamics showed that cross-bridge kinetics are normal but that the number of force-generating cross-bridges is reduced. In vivo sarcomere length (SL) measurements revealed that in TtnΔC1-2 mice the operating SL range of the LV is shifted towards shorter lengths. This normalizes the apparent cell and LV diastolic stiffness but further reduces systolic force as systole occurs further down on the ascending limb of the force-SL relation. We propose that the reduced working SLs reflect titin\'s role in regulating diastolic stiffness by altering the number of sarcomeres in series. Overall, our study reveals that thick filament length regulation by titin\'s C-zone is critical for normal cardiac function.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 10 Jan 2022; epub ahead of print
Methawasin M, Farman GP, Granzier-Nakajima S, Strom J, ... Smith JE, Granzier H
J Mol Cell Cardiol: 10 Jan 2022; epub ahead of print | PMID: 35031281
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Abstract

Phosphodiesterase type 4 anchoring regulates cAMP signaling to Popeye domain-containing proteins.

Tibbo AJ, Mika D, Dobi S, Ling J, ... Brand T, Baillie GS
Cyclic AMP is a ubiquitous second messenger used to transduce intracellular signals from a variety of Gs-coupled receptors. Compartmentalisation of protein intermediates within the cAMP signaling pathway underpins receptor-specific responses. The cAMP effector proteins protein-kinase A and EPAC are found in complexes that also contain phosphodiesterases whose presence ensures a coordinated cellular response to receptor activation events. Popeye domain containing (POPDC) proteins are the most recent class of cAMP effectors to be identified and have crucial roles in cardiac pacemaking and conduction. We report the first observation that POPDC proteins exist in complexes with members of the PDE4 family in cardiac myocytes. We show that POPDC1 preferentially binds the PDE4A sub-family via a specificity motif in the PDE4 UCR1 region and that PDE4s bind to the Popeye domain of POPDC1 in a region known to be susceptible to a mutation that causes human disease. Using a cell-permeable disruptor peptide that displaces the POPDC1-PDE4 complex we show that PDE4 activity localized to POPDC1 modulates cycle length of spontaneous Ca2+ transients firing in intact mouse sinoatrial nodes.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 05 Jan 2022; epub ahead of print
Tibbo AJ, Mika D, Dobi S, Ling J, ... Brand T, Baillie GS
J Mol Cell Cardiol: 05 Jan 2022; epub ahead of print | PMID: 34999055
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Abstract

Mitochondrial protein hyperacetylation underpins heart failure with preserved ejection fraction in mice.

Liu X, Zhang Y, Deng Y, Yang L, ... Li Q, Li T
Over 50% of patients with heart failure have preserved ejection fraction (HFpEF), rather than reduced ejection fraction (HFrEF). The prevalence of HFpEF continues to increase, while the pathogenic mechanisms underlying HFpEF remain largely elusive and evidence-based therapies are still lacking. This study was designed to investigate the metabolic signature of HFpEF and test the potential therapeutic intervention in a mouse model. By utilizing a \"3-Hit\" HFpEF mouse model, we observed a global protein hyperacetylation in the HFpEF hearts as compared to the pressure overload-induced HFrEF and adult/aged non-heart failure (NHF) hearts. Acetylome analysis identified that a large proportion of the hyperacetylated proteins (74%) specific to the HFpEF hearts are in mitochondria, and enriched in tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and fatty acid oxidation. Further study showed that the elevated protein acetylation in the HFpEF hearts was correlated with reduced NAD+/NADH ratio, impaired mitochondrial function, and depleted TCA cycle metabolites. Normalization of NAD+/NADH ratio by supplementation of nicotinamide riboside (NR) for 30 days downregulated the acetylation level, improved mitochondrial function and ameliorated HFpEF phenotypes. Therefore, our study identified a distinct protein acetylation pattern in the HFpEF hearts, and proposed NR as a promising agent in lowering acetylation and mitigating HFpEF phenotypes in mice.

Copyright © 2022. Published by Elsevier Ltd.

J Mol Cell Cardiol: 04 Jan 2022; 165:76-85
Liu X, Zhang Y, Deng Y, Yang L, ... Li Q, Li T
J Mol Cell Cardiol: 04 Jan 2022; 165:76-85 | PMID: 34998831
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Abstract

Epigallocatechin gallate facilitates extracellular elastin fiber formation in induced pluripotent stem cell derived vascular smooth muscle cells for tissue engineering.

Ellis MW, Riaz M, Huang Y, Anderson CW, ... Gibson KH, Qyang Y
Tissue engineered vascular grafts possess several advantages over synthetic or autologous grafts, including increased availability and reduced rates of infection and thrombosis. Engineered grafts constructed from human induced pluripotent stem cell derivatives further offer enhanced reproducibility in graft production. One notable obstacle to clinical application of these grafts is the lack of elastin in the vessel wall, which would serve to endow compliance in addition to mechanical strength. This study establishes the ability of the polyphenol compound epigallocatechin gallate, a principal component of green tea, to facilitate the extracellular formation of elastin fibers in vascular smooth muscle cells derived from human induced pluripotent stem cells. Further, this study describes the creation of a doxycycline-inducible elastin expression system to uncouple elastin production from vascular smooth muscle cell proliferative capacity to permit fiber formation in conditions conducive to robust tissue engineering.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 30 Dec 2021; epub ahead of print
Ellis MW, Riaz M, Huang Y, Anderson CW, ... Gibson KH, Qyang Y
J Mol Cell Cardiol: 30 Dec 2021; epub ahead of print | PMID: 34979103
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Abstract

Hypoxia signaling and oxygen metabolism in cardio-oncology.

Baik AH
Cardio-oncology is a rapidly growing field in cardiology that focuses on the management of cardiovascular toxicities associated with cancer-directed therapies. Tumor hypoxia is a central driver of pathologic tumor growth, metastasis, and chemo-resistance. In addition, conditions that mimic hypoxia (pseudo-hypoxia) play a causal role in the pathogenesis of numerous types of cancer, including renal cell carcinoma. Therefore, therapies targeted at hypoxia signaling pathways have emerged over the past several years. Though efficacious, these therapies are associated with significant cardiovascular toxicities, ranging from hypertension to cardiomyopathy. This review focuses on oxygen metabolism in tumorigenesis, the role of targeting hypoxia signaling in cancer therapy, and the relevance of oxygen metabolism in cardio-oncology. This review will specifically focus on hypoxia signaling mediated by hypoxia-inducible factors and the prolyl hydroxylase oxygen-sensing enzymes, the cardiovascular effects of specific cancer targeted therapies mediated on VEGF and HIF signaling, hypoxic signaling in cardiovascular disease, and the role of oxygen in anthracycline cardiotoxicity. The implications of these therapies on myocardial biology and cardiac function are discussed, underlining the fine balance of hypoxia signaling in cardiac homeostasis. Understanding these cardiovascular toxicities will be important to optimize treatment for cancer patients while mitigating potentially severe cardiovascular side effects.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 30 Dec 2021; epub ahead of print
Baik AH
J Mol Cell Cardiol: 30 Dec 2021; epub ahead of print | PMID: 34979102
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Abstract

GALNT4 primes monocytes adhesion and transmigration by regulating O-Glycosylation of PSGL-1 in atherosclerosis.

Ye Z, Guo H, Wang L, Li Y, ... Chen Z, Huang R
Atherosclerosis is a major underlying cause of cardiovascular disease. Genome wide association studies have predicted that GalNAc-T4 (GALNT4), which responsible for initiating step of mucin-type O-glycosylation, plays a causal role in the susceptibility to cardiovascular diseases, whereas the precise mechanism remains obscure. Thus, we sought to determine the role and mechanism of GALNT4 in atherosclerosis. Firstly, we found the expression of GALNT4 and protein O-glycosylation were both increased in plaque as atherosclerosis progressed in ApoE-/- mice by immunohistochemistry. And the expression of GALNT4 was also increased in human monocytes treated with ACS (acute coronary syndrome) sera and subjected to LPS and ox-LDL in vitro. Moreover, silencing expression of GALNT4 by shRNA lentivirus alleviated atherosclerotic plaque formation and monocyte/macrophage infiltration in ApoE-/- mice. Functional investigations demonstrate that GALNT4 knockdown inhibited P-selectin-induced activation of β2 integrin on the surface of monocytes, decreased monocytes adhesion under flow condition with P-selectin stimulation, as well as suppressed monocytes transmigration triggered by monocyte chemotactic protein- 1(MCP-1). In contrast, GALNT4 overexpression enhanced monocytes adhesion and transmigration. Furthermore, Vicia Villosa Lectin (VVL) pull down and PSGL-1 immunoprecipitation assays showed that GALNT4 overexpression increased O-Glycosylation of PSGL-1 and P-selectin induce phosphorylation of Akt/mTOR and IκBα/NFκB on monocytes. Conversely, knockdown of GALNT4 decreased VVL binding and attenuated the activation of Akt/mTOR and IκBα/NFκB. Additionally, mTOR inhibitor rapamycin blocked these effects of GALNT4 overexpression on monocytes. Collectively, GALNT4 catalyzed PSGL-1 O-glycosylation that involved in P-selectin induced monocytes adhesion and transmigration via Akt/mTOR and NFκB pathway. Thus, GALNT4 may be a potential therapeutic target for atherosclerosis.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 29 Dec 2021; epub ahead of print
Ye Z, Guo H, Wang L, Li Y, ... Chen Z, Huang R
J Mol Cell Cardiol: 29 Dec 2021; epub ahead of print | PMID: 34974060
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Abstract

MiR-185-5p regulates the development of myocardial fibrosis.

Lin R, Rahtu-Korpela L, Szabo Z, Kemppi A, ... Junttila J, Kerkelä R
Background
Cardiac fibrosis stiffens the ventricular wall, predisposes to cardiac arrhythmias and contributes to the development of heart failure. In the present study, our aim was to identify novel miRNAs that regulate the development of cardiac fibrosis and could serve as potential therapeutic targets for myocardial fibrosis.
Methods and results
Analysis for cardiac samples from sudden cardiac death victims with extensive myocardial fibrosis as the primary cause of death identified dysregulation of miR-185-5p. Analysis of resident cardiac cells from mice subjected to experimental cardiac fibrosis model showed induction of miR-185-5p expression specifically in cardiac fibroblasts. In vitro, augmenting miR-185-5p induced collagen production and profibrotic activation in cardiac fibroblasts, whereas inhibition of miR-185-5p attenuated collagen production. In vivo, targeting miR-185-5p in mice abolished pressure overload induced cardiac interstitial fibrosis. Mechanistically, miR-185-5p targets apelin receptor and inhibits the anti-fibrotic effects of apelin. Finally, analysis of left ventricular tissue from patients with severe cardiomyopathy showed an increase in miR-185-5p expression together with pro-fibrotic TGF-β1 and collagen I.
Conclusions
Our data show that miR-185-5p targets apelin receptor and promotes myocardial fibrosis.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 28 Dec 2021; epub ahead of print
Lin R, Rahtu-Korpela L, Szabo Z, Kemppi A, ... Junttila J, Kerkelä R
J Mol Cell Cardiol: 28 Dec 2021; epub ahead of print | PMID: 34973276
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Abstract

Tissue-engineered vascular grafts and regeneration mechanisms.

Wei Y, Wang F, Guo Z, Qiang Z
Cardiovascular diseases (CVDs) are life-threatening diseases with high morbidity and mortality worldwide. Vascular bypass surgery is still the ultimate strategy for CVD treatment. Autografts are the gold standard for graft transplantation, but insufficient sources limit their widespread application. Therefore, alternative tissue engineered vascular grafts (TEVGs) are urgently needed. In this review, we summarize the major strategies for the preparation of vascular grafts, as well as the factors affecting their patency and tissue regeneration. Finally, the underlying mechanisms of vascular regeneration that are mediated by host cells are discussed.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 27 Dec 2021; epub ahead of print
Wei Y, Wang F, Guo Z, Qiang Z
J Mol Cell Cardiol: 27 Dec 2021; epub ahead of print | PMID: 34971664
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Abstract

Stabilin-1 mediates beneficial monocyte recruitment and tolerogenic macrophage programming during CVB3-induced viral myocarditis.

Carai P, Papageorgiou AP, Van Linthout S, Deckx S, ... Jones EAV, Heymans S
Pathological innate and adaptive immune response upon viral infection may lead to cardiac injury and dysfunction. Stabilin-1 is a scavenger receptor that regulates several aspects of the innate immunity. Whether stabilin-1 affects the inflammatory response during viral myocarditis (VM) is entirely unknown. Here, we assess the role of stabilin-1 in the pathogenesis of VM and its suitability as a therapeutic target. Genetic loss of stabilin-1 increased mortality and cardiac necrosis in a mouse model of human Coxsackievirus B3 (CVB3)-induced myocarditis. Absence of stabilin-1 significantly reduced monocyte recruitment and strongly reduced the number of alternatively activated anti-inflammatory macrophages in the heart, enhancing a pro-inflammatory cardiac niche with a detrimental T lymphocyte response during VM. Yeast two-hybrid screening, confirmed by affinity chromatography, identified fibronectin as a stabilin-1 interacting partner. Absence of stabilin-1 specifically decreased monocyte adhesion on extracellular fibronectin in vitro. Loss of Type III repeats Extra Domain A (EDA) of fibronectin during VM also increased the mortality and cardiac necrosis as in stabilin-1 knockout mice, with reduced monocytic cardiac recruitment and increased T lymphocyte response. Collectively, stabilin-1 has an immune-suppressive role of limiting myocardial damage during VM, regulating anti-inflammatory monocyte-recruitment to the site of inflammation.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 26 Dec 2021; epub ahead of print
Carai P, Papageorgiou AP, Van Linthout S, Deckx S, ... Jones EAV, Heymans S
J Mol Cell Cardiol: 26 Dec 2021; epub ahead of print | PMID: 34968453
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Abstract

Systematic transcriptomic and phenotypic characterization of human and murine cardiac myocyte cell lines and primary cardiomyocytes reveals serious limitations and low resemblances to adult cardiac phenotype.

Onódi Z, Visnovitz T, Kiss B, Hambalkó S, ... Giricz Z, Varga ZV
Background
Cardiac cell lines and primary cells are widely used in cardiovascular research. Despite increasing number of publications using these models, comparative characterization of these cell lines has not been performed, therefore, their limitations are undetermined. We aimed to compare cardiac cell lines to primary cardiomyocytes and to mature cardiac tissues in a systematic manner.
Methods and results
Cardiac cell lines (H9C2, AC16, HL-1) were differentiated with widely used protocols. Left ventricular tissue, neonatal primary cardiomyocytes, and human induced pluripotent stem cell-derived cardiomyocytes served as reference tissue or cells. RNA expression of cardiac markers (e.g. Tnnt2, Ryr2) was markedly lower in cell lines compared to references. Differentiation induced increase in cardiac- and decrease in embryonic markers however, the overall transcriptomic profile and annotation to relevant biological processes showed consistently less pronounced cardiac phenotype in all cell lines in comparison to the corresponding references. Immunocytochemistry confirmed low expressions of structural protein sarcomeric alpha-actinin, troponin I and caveolin-3 in cell lines. Susceptibility of cell lines to sI/R injury in terms of viability as well as mitochondrial polarization differed from the primary cells irrespective of their degree of differentiation.
Conclusion
Expression patterns of cardiomyocyte markers and whole transcriptomic profile, as well as response to sI/R, and to hypertrophic stimuli indicate low-to-moderate similarity of cell lines to primary cells/cardiac tissues regardless their differentiation. Low resemblance of cell lines to mature adult cardiac tissue limits their potential use. Low translational value should be taken into account while choosing a particular cell line to model cardiomyocytes.

Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 23 Dec 2021; 165:19-30
Onódi Z, Visnovitz T, Kiss B, Hambalkó S, ... Giricz Z, Varga ZV
J Mol Cell Cardiol: 23 Dec 2021; 165:19-30 | PMID: 34959166
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Abstract

Computational modeling of mitochondrial K- and H-driven ATP synthesis.

Cortassa S, Aon MA, Juhaszova M, Kobrinsky E, Zorov DB, Sollott SJ
ATP synthase (F1Fo) is a rotary molecular engine that harnesses energy from electrochemical-gradients across the inner mitochondrial membrane for ATP synthesis. Despite the accepted tenet that F1Fo transports exclusively H+, our laboratory has demonstrated that, in addition to H+, F1Fo ATP synthase transports a significant fraction of ΔΨm-driven charge as K+ to synthesize ATP. Herein, we utilize a computational modeling approach as a proof of principle of the feasibility of the core mechanism underlying the enhanced ATP synthesis, and to explore its bioenergetic consequences. A minimal model comprising the \'core\' mechanism constituted by ATP synthase, driven by both proton (PMF) and potassium motive force (KMF), respiratory chain, adenine nucleotide translocator, Pi carrier, and K+/H+ exchanger (KHEmito) was able to simulate enhanced ATP synthesis and respiratory fluxes determined experimentally with isolated heart mitochondria. This capacity of F1Fo ATP synthase confers mitochondria with a significant energetic advantage compared to K+ transport through a channel not linked to oxidative phosphorylation (OxPhos). The K+-cycling mechanism requires a KHEmito that exchanges matrix K+ for intermembrane space H+, leaving PMF as the overall driving energy of OxPhos, in full agreement with the standard chemiosmotic mechanism. Experimental data of state 4➔3 energetic transitions, mimicking low to high energy demand, could be reproduced by an integrated computational model of mitochondrial function that incorporates the \'core\' mechanism. Model simulations display similar behavior compared to the experimentally observed changes in ΔΨm, mitochondrial K+ uptake, matrix volume, respiration, and ATP synthesis during the energetic transitions at physiological pH and K+ concentration. The model also explores the role played by KHEmito in modulating the energetic performance of mitochondria. The results obtained support the available experimental evidence on ATP synthesis driven by K+ and H+ transport through the F1Fo ATP synthase.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 22 Dec 2021; 165:9-18
Cortassa S, Aon MA, Juhaszova M, Kobrinsky E, Zorov DB, Sollott SJ
J Mol Cell Cardiol: 22 Dec 2021; 165:9-18 | PMID: 34954465
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Abstract

A roadmap for the characterization of energy metabolism in human cardiomyocytes derived from induced pluripotent stem cells.

Emanuelli G, Zoccarato A, Reumiller CM, Papadopoulos A, ... Streckfuss-Bömeke K, Shah AM
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are an increasingly employed model in cardiac research and drug discovery. As cellular metabolism plays an integral role in determining phenotype, the characterization of the metabolic profile of hiPSC-CM during maturation is crucial for their translational application. In this study we employ a combination of methods including extracellular flux, 13C-glucose enrichment and targeted metabolomics to characterize the metabolic profile of hiPSC-CM during their maturation in culture from 6 weeks, up to 12 weeks. Results show a progressive remodeling of pathways involved in energy metabolism and substrate utilization along with an increase in sarcomere regularity. The oxidative capacity of hiPSC-CM and particularly their ability to utilize fatty acids increased with time. In parallel, relative glucose oxidation was reduced while glutamine oxidation was maintained at similar levels. There was also evidence of increased coupling of glycolysis to mitochondrial respiration, and away from glycolytic branch pathways at later stages of maturation. The rate of glycolysis as assessed by lactate production was maintained at both stages but with significant alterations in proximal glycolytic enzymes such as hexokinase and phosphofructokinase. We observed a progressive maturation of mitochondrial oxidative capacity at comparable levels of mitochondrial content between these time-points with enhancement of mitochondrial network structure. These results show that the metabolic profile of hiPSC-CM is progressively restructured, recapitulating aspects of early post-natal heart development. This would be particularly important to consider when employing these cell model in studies where metabolism plays an important role.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 15 Dec 2021; 164:136-147
Emanuelli G, Zoccarato A, Reumiller CM, Papadopoulos A, ... Streckfuss-Bömeke K, Shah AM
J Mol Cell Cardiol: 15 Dec 2021; 164:136-147 | PMID: 34923199
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Abstract

The role of autophagy in death of cardiomyocytes.

Ikeda S, Zablocki D, Sadoshima J
Autophagy mediates cellular quality control mechanisms and energy homeostasis through lysosomal degradation. Autophagy is typically viewed as an adaptive process that allows cells to survive against stress, such as nutrient deprivation and hypoxia. However, autophagy also mediates cell death during development and in response to stress. Cell death accompanied by autophagy activation and accumulation of autophagosomes has been classified as type II programmed cell death. Compared to the wealth of knowledge regarding the adaptive role of autophagy, however, the molecular mechanisms through which autophagy induces cell death and its functional significance are poorly understood. Autophagy is activated excessively under some conditions, causing uncontrolled degradation of cellular materials and cell death. An imbalance between autophagosome formation and lysosomal degradation causes a massive accumulation of autophagosomes, which subsequently causes cellular dysfunction and death. Dysregulation of autophagy induces a unique form of cell death, termed autosis, with defined morphological and biochemical features distinct from other forms of programmed cell death, such as apoptosis and necrosis. In the heart, dysregulated autophagy induces death of cardiomyocytes and actively mediates cardiac injury and dysfunction in some conditions, including reperfusion injury, doxorubicin cardiomyopathy, and lysosomal storage disorders. The goal in this review is to introduce the concept of autophagic cell death and discuss its functional significance in various cardiac conditions.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 13 Dec 2021; epub ahead of print
Ikeda S, Zablocki D, Sadoshima J
J Mol Cell Cardiol: 13 Dec 2021; epub ahead of print | PMID: 34919896
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Abstract

Activation of the hypoxia response pathway protects against age-induced cardiac hypertrophy.

Röning T, Magga J, Laitakari A, Halmetoja R, ... Koivunen P, Serpi R
Aims
We have previously demonstrated protection against obesity, metabolic dysfunction, atherosclerosis and cardiac ischemia in a hypoxia-inducible factor (HIF) prolyl 4-hydroxylase-2 (Hif-p4h-2) deficient mouse line, attributing these protective effects to activation of the hypoxia response pathway in a normoxic environment. We intended here to find out whether the Hif-p4h-2 deficiency affects the cardiac health of these mice upon aging.
Methods and results
When the Hif-p4h-2 deficient mice and their wild-type littermates were monitored during normal aging, the Hif-p4h-2 deficient mice had better preserved diastolic function than the wild type at one year of age and less cardiomyocyte hypertrophy at two years. On the mRNA level, downregulation of hypertrophy-associated genes was detected and shown to be associated with upregulation of Notch signaling, and especially of the Notch target gene and transcriptional repressor Hairy and enhancer-of-split-related basic helix-loop-helix (Hey2). Blocking of Notch signaling in cardiomyocytes isolated from Hif-p4h-2 deficient mice with a gamma-secretase inhibitor led to upregulation of the hypertrophy-associated genes. Also, targeting Hey2 in isolated wild-type rat neonatal cardiomyocytes with siRNA led to upregulation of hypertrophic genes and increased leucine incorporation indicative of increased protein synthesis and hypertrophy. Finally, oral treatment of wild-type mice with a small molecule inhibitor of HIF-P4Hs phenocopied the effects of Hif-p4h-2 deficiency with less cardiomyocyte hypertrophy, upregulation of Hey2 and downregulation of the hypertrophy-associated genes.
Conclusions
These results indicate that activation of the hypoxia response pathway upregulates Notch signaling and its target Hey2 resulting in transcriptional repression of hypertrophy-associated genes and less cardiomyocyte hypertrophy. This is eventually associated with better preserved cardiac function upon aging. Activation of the hypoxia response pathway thus has therapeutic potential for combating age-induced cardiac hypertrophy.

Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 13 Dec 2021; 164:148-155
Röning T, Magga J, Laitakari A, Halmetoja R, ... Koivunen P, Serpi R
J Mol Cell Cardiol: 13 Dec 2021; 164:148-155 | PMID: 34919895
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Abstract

Hallmarks of exercised heart.

Qiu Y, Pan X, Chen Y, Xiao J
The benefits of exercise in humans on the heart have been well recognized for many years. Long-term endurance exercise training can induce physiologic cardiac hypertrophy with normal or enhanced heart function, and provide protective benefits in preventing heart failure. The heart-specific responses that occur during exercise are complex and highly variable. This review mainly focuses on the current understanding of the structural and functional cardiac adaptations to exercise as well as molecular pathways and signaling proteins responsible for these changes. Here, we summarize eight tentative hallmarks that represent common denominators of the exercised heart. These hallmarks are: cardiomyocyte growth, cardiomyocyte fate reprogramming, angiogenesis and lymphangiogenesis, mitochondrial remodeling, epigenetic alteration, enhanced endothelial function, quiescent cardiac fibroblast, and improved cardiac metabolism. A major challenge is to explore the underlying molecular mechanisms for cardio-protective effects of exercise, and to identify therapeutic targets for heart diseases.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 12 Dec 2021; epub ahead of print
Qiu Y, Pan X, Chen Y, Xiao J
J Mol Cell Cardiol: 12 Dec 2021; epub ahead of print | PMID: 34914934
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Abstract

Multi-omics of a pre-clinical model of diabetic cardiomyopathy reveals increased fatty acid supply impacts mitochondrial metabolic selectivity.

Li DK, Smith LE, Rookyard AW, Lingam SJ, ... Cordwell SJ, White MY
The incidence of type 2 diabetes (T2D) is increasing globally, with long-term implications for human health and longevity. Heart disease is the leading cause of death in T2D patients, who display an elevated risk of an acute cardiovascular event and worse outcomes following such an insult. The underlying mechanisms that predispose the diabetic heart to this poor prognosis remain to be defined. This study developed a pre-clinical model (Rattus norvegicus) that complemented caloric excess from a high-fat diet (HFD) and pancreatic β-cell dysfunction from streptozotocin (STZ) to produce hyperglycaemia, peripheral insulin resistance, hyperlipidaemia and elevated fat mass to mimic the clinical features of T2D. Ex vivo cardiac function was assessed using Langendorff perfusion with systolic and diastolic contractile depression observed in T2D hearts. Cohorts representing untreated, individual HFD- or STZ-treatments and the combined HFD + STZ approach were used to generate ventricular samples (n = 9 per cohort) for sequential and integrated analysis of the proteome, lipidome and metabolome by liquid chromatography-tandem mass spectrometry. This study found that in T2D hearts, HFD treatment primed the metabolome, while STZ treatment was the major driver for changes in the proteome. Both treatments equally impacted the lipidome. Our data suggest that increases in β-oxidation and early TCA cycle intermediates promoted rerouting via 2-oxaloacetate to glutamate, γ-aminobutyric acid and glutathione. Furthermore, we suggest that the T2D heart activates networks to redistribute excess acetyl-CoA towards ketogenesis and incomplete β-oxidation through the formation of short-chain acylcarnitine species. Multi-omics provided a global and comprehensive molecular view of the diabetic heart, which distributes substrates and products from excess β-oxidation, reduces metabolic flexibility and impairs capacity to restore high energy reservoirs needed to respond to and prevent subsequent acute cardiovascular events.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 23 Nov 2021; 164:92-109
Li DK, Smith LE, Rookyard AW, Lingam SJ, ... Cordwell SJ, White MY
J Mol Cell Cardiol: 23 Nov 2021; 164:92-109 | PMID: 34826416
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Abstract

Improved epicardial cardiac fibroblast generation from iPSCs.

Whitehead AJ, Hocker JD, Ren B, Engler AJ
Since the initial isolation of human embryonic stem cells and subsequent discovery of reprogramming methods for somatic cells, thousands of protocols have been developed to create each of the hundreds of cell types found in-vivo with significant focus on disease-prone systems, e.g., cardiovascular. Robust protocols exist for many of these cell types, except for cardiac fibroblasts (CF). Very recently, several competing methods have been developed to generate these cells through a developmentally conserved epicardial pathway. Such methods generate epicardial cells, but here we report that prolonged exposure to growth factors such as bFGF induces fibroblast spindle-like morphology and similar chromatin architecture to primary CFs. Media conditions for growth and assays are provided, as well as suggestions for seeding densities and timepoints for protein harvest of extracellular matrix. We demonstrate marker expression and matrix competency of resultant cells as shown next to primary human cardiac fibroblasts. These methods provide additional guidance to the original protocol and result in an increasingly stable phenotype.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 23 Nov 2021; 164:58-68
Whitehead AJ, Hocker JD, Ren B, Engler AJ
J Mol Cell Cardiol: 23 Nov 2021; 164:58-68 | PMID: 34826415
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Abstract

Endothelial contribution to COVID-19: an update on mechanisms and therapeutic implications.

Ma Z, Yang KY, Huang Y, Lui KO
The global propagation of SARS-CoV-2 leads to an unprecedented public health emergency. Despite that the lungs are the primary organ targeted by COVID-19, systemic endothelial inflammation and dysfunction is observed particularly in patients with severe COVID-19, manifested by elevated endothelial injury markers, endotheliitis, and coagulopathy. Here, we review the clinical characteristics of COVID-19 associated endothelial dysfunction; and the likely pathological mechanisms underlying the disease including direct cell entry or indirect immune overreactions after SARS-CoV-2 infection. In addition, we discuss potential biomarkers that might indicate the disease severity, particularly related to the abnormal development of thrombosis that is a fatal vascular complication of severe COVID-19. Furthermore, we summarize clinical trials targeting the direct and indirect pathological pathways after SARS-CoV-2 infection to prevent or inhibit the virus induced endothelial disorders.

Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 23 Nov 2021; 164:69-82
Ma Z, Yang KY, Huang Y, Lui KO
J Mol Cell Cardiol: 23 Nov 2021; 164:69-82 | PMID: 34838588
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Abstract

Impact of mitochondria on local calcium release in murine sinoatrial nodal cells.

Takeda Y, Matsuoka S
Roles of mitochondria in sinoatrial nodal cells (SANCs) have not been fully clarified. We have previously demonstrated that mitochondrial Ca2+ efflux through the Na+-Ca2+ exchanger, NCXm, modulates sarcoplasmic reticulum (SR) Ca2+ content and automaticity of HL-1 cardiomyocytes. In this study, we extended this line of investigation to clarify the spatial and functional association between mitochondria and local calcium release (LCR) from the SR in murine SANCs. High-speed two dimensional (2D) and confocal line-scan imaging of SANCs revealed that LCRs in the early phase of the Ca2+ transient cycle length (CL) appeared with a higher probability near mitochondria. Although LCR increased toward the late phase of CL, no significant difference was noted in the occurrence of late LCRs near and distant from mitochondria. LCRs, especially in the late phase of CL, induced temporal and spatial heterogeneity of the Ca2+ transient amplitude. Attenuating mitochondrial Ca2+ efflux using an NCXm inhibitor, CGP-37157 (1 μM), reduced the amplitude, duration and size of LCR. It also attenuated early LCR occurrence, and simultaneously prolonged LCR period and CL. Additionally, CGP-37157 reduced caffeine-induced Ca2+ transient. Therefore, the inhibitory effect on LCR was attributable to the reduction of the SR Ca2+ content through NCXm inhibition. No obvious off-target effects of 1 μM CGP-37157 were found on T- and L-type voltage-gated Ca2+ currents and hyperpolarization-activated inward current. Taken together, these results suggest that mitochondria are involved in LCR generation by modulating the SR Ca2+ content through NCXm-mediated Ca2+ efflux in murine SANCs.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 22 Nov 2021; 164:42-50
Takeda Y, Matsuoka S
J Mol Cell Cardiol: 22 Nov 2021; 164:42-50 | PMID: 34826768
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Abstract

Inducing I and phase 1 repolarization of the cardiac action potential with a Kv4.3/KChIP2.1 bicistronic transgene.

Wang N, Dries E, Fowler ED, Harmer SC, Hancox JC, Cannell MB
The fast transient outward potassium current (Ito,f) plays a key role in phase 1 repolarization of the human cardiac action potential (AP) and its reduction in heart failure (HF) contributes to the loss of contractility. Therefore, restoring Ito,f might be beneficial for treating HF. The coding sequence of a P2A peptide was cloned, in frame, between Kv4.3 and KChIP2.1 genes and ribosomal skipping was confirmed by Western blotting. Typical Ito,f properties with slowed inactivation and accelerated recovery from inactivation due to the association of KChIP2.1 with Kv4.3 was seen in transfected HEK293 cells. Both bicistronic components trafficked to the plasmamembrane and in adenovirus transduced rabbit cardiomyocytes both t-tubular and sarcolemmal construct labelling appeared. The resulting current was similar to Ito,f seen in human ventricular cardiomyocytes and was 50% blocked at ~0.8 mmol/l 4-aminopyridine and increased ~30% by 5 μmol/l NS5806 (an Ito,f agonist). Variation in the density of the expressed Ito,f, in rabbit cardiomyocytes recapitulated typical species-dependent variations in AP morphology. Simultaneous voltage recording and intracellular Ca2+ imaging showed that modification of phase 1 to a non-failing human phenotype improved the rate of rise and magnitude of the Ca2+ transient. Ito,f expression also reduced AP triangulation but did not affect ICa,L and INa magnitudes. This raises the possibility for a new gene-based therapeutic approach to HF based on selective phase 1 modification.

Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 21 Nov 2021; 164:29-41
Wang N, Dries E, Fowler ED, Harmer SC, Hancox JC, Cannell MB
J Mol Cell Cardiol: 21 Nov 2021; 164:29-41 | PMID: 34823101
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Abstract

Production of functional cardiomyocytes and cardiac tissue from human induced pluripotent stem cells for regenerative therapy.

Tani H, Tohyama S, Kishino Y, Kanazawa H, Fukuda K
The emergence of human induced pluripotent stem cells (hiPSCs) has revealed the potential for curing end-stage heart failure. Indeed, transplantation of hiPSC-derived cardiomyocytes (hiPSC-CMs) may have applications as a replacement for heart transplantation and conventional regenerative therapies. However, there are several challenges that still must be overcome for clinical applications, including large-scale production of hiPSCs and hiPSC-CMs, elimination of residual hiPSCs, purification of hiPSC-CMs, maturation of hiPSC-CMs, efficient engraftment of transplanted hiPSC-CMs, development of an injection device, and avoidance of post-transplant arrhythmia and immunological rejection. Thus, we developed several technologies based on understanding of the metabolic profiles of hiPSCs and hiPSC derivatives. In this review, we outline how to overcome these hurdles to realize the transplantation of hiPSC-CMs in patients with heart failure and introduce cutting-edge findings and perspectives for future regenerative therapy.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 21 Nov 2021; 164:83-91
Tani H, Tohyama S, Kishino Y, Kanazawa H, Fukuda K
J Mol Cell Cardiol: 21 Nov 2021; 164:83-91 | PMID: 34822838
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Abstract

Photosynthetic symbiotic therapeutics - An innovative, effective treatment for ischemic cardiovascular diseases.

Zhu Y, Woo YJ
Ischemic heart disease is a major cause of global morbidity and mortality, affecting over 15 million patients in the United States. Recent advances in research and innovation have greatly broadened clinicians\' ability to treatment ischemic heart disease and associated heart failure using various preventive, pharmacologic, and surgical strategies. Specifically, innovative photosynthetic symbiotic systems using Synechococcus elongatus has gained significant attention. S. elongatus is a unicellular cyanobacterium that can carry out oxygenic photosynthesis. Photosynthetic therapies have been developed to rescue ischemic tissue by taking up tissue-derived carbon dioxide and in turn releasing oxygen for sustained aerobic metabolism during ischemia. In this article, we review the application of cyanobacteria, specifically S. elongatus, in the field of biotechnology, ischemic heart disease, and other clinical applications in ischemic diseases. We also address the motivation for innovation and current limitations in the field of S. elongatus photosynthetic therapeutics for ischemic cardiovascular disease.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 19 Nov 2021; 164:51-57
Zhu Y, Woo YJ
J Mol Cell Cardiol: 19 Nov 2021; 164:51-57 | PMID: 34813842
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Abstract

Computational model of brain endothelial cell signaling pathways predicts therapeutic targets for cerebral pathologies.

Gorick CM, Saucerman JJ, Price RJ
Brain endothelial cells serve many critical homeostatic functions. In addition to sensing and regulating blood flow, they maintain blood-brain barrier function, including precise control of nutrient exchange and efflux of xenobiotics. Many signaling pathways in brain endothelial cells have been implicated in both health and disease; however, our understanding of how these signaling pathways functionally integrate is limited. A model capable of integrating these signaling pathways could both advance our understanding of brain endothelial cell signaling networks and potentially identify promising molecular targets for endothelial cell-based drug or gene therapies. To this end, we developed a large-scale computational model, wherein brain endothelial cell signaling pathways were reconstructed from the literature and converted into a network of logic-based differential equations. The model integrates 63 nodes (including proteins, mRNA, small molecules, and cell phenotypes) and 82 reactions connecting these nodes. Specifically, our model combines signaling pathways relating to VEGF-A, BDNF, NGF, and Wnt signaling, in addition to incorporating pathways relating to focused ultrasound as a therapeutic delivery tool. To validate the model, independently established relationships between selected inputs and outputs were simulated, with the model yielding correct predictions 73% of the time. We identified influential and sensitive nodes under different physiological or pathological contexts, including altered brain endothelial cell conditions during glioma, Alzheimer\'s disease, and ischemic stroke. Nodes with the greatest influence over combinations of desired model outputs were identified as potential druggable targets for these disease conditions. For example, the model predicts therapeutic benefits from inhibiting AKT, Hif-1α, or cathepsin D in the context of glioma - each of which are currently being studied in clinical or pre-clinical trials. Notably, the model also permits testing multiple combinations of node alterations for their effects on the network and the desired outputs (such as inhibiting AKT and overexpressing the P75 neurotrophin receptor simultaneously in the context of glioma), allowing for the prediction of optimal combination therapies. In all, our approach integrates results from over 100 past studies into a coherent and powerful model, capable of both revealing network interactions unapparent from studying any one pathway in isolation and predicting therapeutic targets for treating devastating brain pathologies.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 15 Nov 2021; 164:17-28
Gorick CM, Saucerman JJ, Price RJ
J Mol Cell Cardiol: 15 Nov 2021; 164:17-28 | PMID: 34798125
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Abstract

Sex- and age-specific regulation of ACE2: Insights into severe COVID-19 susceptibility.

Viveiros A, Gheblawi M, Aujla PK, Sosnowski DK, ... Kassiri Z, Oudit GY
Aged males disproportionately succumb to increased COVID-19 severity, hospitalization, and mortality compared to females. Angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2) facilitate SARS-CoV-2 viral entry and may have sexually dimorphic regulation. As viral load dictates disease severity, we investigated the expression, protein levels, and activity of ACE2 and TMPRSS2. Our data reveal that aged males have elevated ACE2 in both mice and humans across organs. We report the first comparative study comprehensively investigating the impact of sex and age in murine and human levels of ACE2 and TMPRSS2, to begin to elucidate the sex bias in COVID-19 severity.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 10 Nov 2021; 164:13-16
Viveiros A, Gheblawi M, Aujla PK, Sosnowski DK, ... Kassiri Z, Oudit GY
J Mol Cell Cardiol: 10 Nov 2021; 164:13-16 | PMID: 34774871
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Abstract

Bone marrow- or adipose-mesenchymal stromal cell secretome preserves myocardial transcriptome profile and ameliorates cardiac damage following ex vivo cold storage.

Scott SR, March KL, Wang IW, Singh K, ... Sen CK, Wang M
Background
Heart transplantation, a life-saving approach for patients with end-stage heart disease, is limited by shortage of donor organs. While prolonged storage provides more organs, it increases the extent of ischemia. Therefore, we seek to understand molecular mechanisms underlying pathophysiological changes of donor hearts during prolonged storage. Additionally, considering mesenchymal stromal cell (MSC)-derived paracrine protection, we aim to test if MSC secretome preserves myocardial transcriptome profile and whether MSC secretome from a certain source provides the optimal protection in donor hearts during cold storage.
Methods and results
Isolated mouse hearts were divided into: no cold storage (control), 6 h cold storage (6 h-I), 6 h-I + conditioned media from bone marrow MSCs (BM-MSC CM), and 6 h-I + adipose-MSC CM (Ad-MSC CM). Deep RNA sequencing analysis revealed that compared to control, 6 h-I led to 266 differentially expressed genes, many of which were implicated in modulating mitochondrial performance, oxidative stress response, myocardial function, and apoptosis. BM-MSC CM and Ad-MSC CM restored these gene expression towards control. They also improved 6 h-I-induced myocardial functional depression, reduced inflammatory cytokine production, decreased apoptosis, and reduced myocardial H2O2. However, neither MSC-exosomes nor exosome-depleted CM recapitulated MSC CM-ameliorated apoptosis and CM-improved mitochondrial preservation during cold ischemia. Knockdown of Per2 by specific siRNA abolished MSC CM-mediated these protective effects in cardiomyocytes following 6 h cold storage.
Conclusions
Our results demonstrated that using MSC secretome (BM-MSCs and Ad-MSCs) during prolonged cold storage confers preservation of the normal transcriptional \"fingerprint\", and reduces donor heart damage. MSC-released soluble factors and exosomes may synergistically act for donor heart protection.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 10 Nov 2021; 164:1-12
Scott SR, March KL, Wang IW, Singh K, ... Sen CK, Wang M
J Mol Cell Cardiol: 10 Nov 2021; 164:1-12 | PMID: 34774548
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Abstract

Ventricular SK2 upregulation following angiotensin II challenge: Modulation by p21-activated kinase-1.

Yang B, Jiang Q, He S, Li T, ... Lei M, Tan X
Effects of hypertrophic challenge on small-conductance, Ca2+-activated K+(SK2) channel expression were explored in intact murine hearts, isolated ventricular myocytes and neonatal rat cardiomyocytes (NRCMs). An established experimental platform applied angiotensin II (Ang II) challenge in the presence and absence of reduced p21-activated kinase (PAK1) (PAK1cko vs. PAK1f/f, or shRNA-PAK1 interference) expression. SK2 current contributions were detected through their sensitivity to apamin block. Ang II treatment increased such SK2 contributions to optically mapped action potential durations (APD80) and their heterogeneity, and to patch-clamp currents. Such changes were accentuated in PAK1cko compared to PAK1f/f, intact hearts and isolated cardiomyocytes. They paralleled increased histological and echocardiographic hypertrophic indices, reduced cardiac contractility, and increased SK2 protein expression, changes similarly greater with PAK1cko than PAK1f/f. In NRCMs, Ang II challenge replicated such increases in apamin-sensitive SK patch clamp currents as well as in real-time PCR and western blot measures of SK2 mRNA and protein expression and cell hypertrophy. Furthermore, the latter were enhanced by shRNA-PAK1 interference and mitigated by the PAK1 agonist FTY720. Increased CaMKII and CREB phosphorylation accompanied these effects. These were rescued by both FTY720 as well as the CaMKII inhibitor KN93, but not its inactive analogue KN92. Such CREB then specifically bound to the KCNN2 promoter sequence in luciferase assays. These findings associate Ang II induced hypertrophy with increased SK2 expression brought about by a CaMKII/CREB signaling convergent with the PAK1 pathway thence upregulating the KCNN2 promoter activity. SK2 may then influence cardiac electrophysiology under conditions of cardiac hypertrophy and failure.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 10 Nov 2021; 164:110-125
Yang B, Jiang Q, He S, Li T, ... Lei M, Tan X
J Mol Cell Cardiol: 10 Nov 2021; 164:110-125 | PMID: 34774547
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Abstract

Improved nanopore direct RNA sequencing of cardiac myocyte samples by selective mt-RNA depletion.

Naarmann-de Vries IS, Eschenbach J, Dieterich C
RNA sequencing is a powerful tool to analyze gene expression transcriptome wide. However, RNA sequencing in general and especially the recently developed methods of long read RNA sequencing are still low-throughput and cost-intensive. Here, one important design choice is to concentrate the sequencing capacity on specific parts of the transcriptome. Especially, abundant transcripts as ribosomal RNAs may dominate the available sequencing space, if not removed prior to sequencing. Several methods exist to reduce ribosomal RNA read numbers: either based on enrichment of the relevant fraction (polyA+ RNA) or depletion, respectively degradation of ribosomal RNAs. Furthermore, commercial kits are available to deplete globin transcripts from blood samples. However, so far, no solution exists to deal with other tissue-specific highly abundant transcripts. This is especially of interest in the heart and other muscle derived samples, where reads originating from mitochondrial RNAs make up to 30% of reads in polyA+ selected libraries and around 70% in single cell sequencing experiments. We present a simple method to diminish sequencing of mitochondrial RNAs in Oxford Nanopore direct RNA sequencing libraries by RNase H based clipping of the polyA tail. We show that mt-clipping enables enhanced detection of cytoplasmic mRNAs, among them genes involved in heart development and pathogenesis. Mt-clipping may be applied as well to other sequencing protocols that are based on oligo(dT) priming and can be easily adapted to other tissue-specific high-abundant transcripts.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 02 Nov 2021; epub ahead of print
Naarmann-de Vries IS, Eschenbach J, Dieterich C
J Mol Cell Cardiol: 02 Nov 2021; epub ahead of print | PMID: 34742715
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Abstract

Endothelial repair by stem and progenitor cells.

Wang X, Wang R, Jiang L, Xu Q, Guo X
The integrity of the endothelial barrier is required to maintain vascular homeostasis and fluid balance between the circulatory system and surrounding tissues and to prevent the development of vascular disease. However, the origin of the newly developed endothelial cells is still controversial. Stem and progenitor cells have the potential to differentiate into endothelial cell lines and stimulate vascular regeneration in a paracrine/autocrine fashion. The one source of new endothelial cells was believed to come from the bone marrow, which was challenged by the recent findings. By administration of new techniques, including genetic cell lineage tracing and single cell RNA sequencing, more solid data were obtained that support the concept of stem/progenitor cells for regenerating damaged endothelium. Specifically, it was found that tissue resident endothelial progenitors located in the vessel wall were crucial for endothelial repair. In this review, we summarized the latest advances in stem and progenitor cell research in endothelial regeneration through findings from animal models and discussed clinical data to indicate the future direction of stem cell therapy.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 28 Oct 2021; 163:133-146
Wang X, Wang R, Jiang L, Xu Q, Guo X
J Mol Cell Cardiol: 28 Oct 2021; 163:133-146 | PMID: 34743936
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Abstract

Individual variability in animal-specific hemodynamic compensation following myocardial infarction.

Caggiano LR, Holmes JW, Witzenburg CM
Ventricular enlargement and heart failure are common in patients who survive a myocardial infarction (MI). There is striking variability in the degree of post-infarction ventricular remodeling, however, and no one factor or set of factors have been identified that predicts heart failure risk well. Sympathetic activation directly and indirectly modulates hypertrophic stimuli by altering both neurohormonal milieu and ventricular loading. In a recent study, we developed a method to identify the balance of reflex compensatory mechanisms employed by individual animals following MI based on measured hemodynamics. Here, we conducted prospective studies of acute myocardial infarction in rats to test the degree of variability in reflex compensation as well as whether responses to pharmacologic agents targeted at those reflex mechanisms could be anticipated in individual animals. We found that individual animals use very different mixtures of reflex compensation in response to experimental coronary ligation. Some of these mechanisms were related - animals that compensated strongly with venoconstriction tended to exhibit a decrease in the contractility of the surviving myocardium and those that increased contractility tended to exhibit venodilation. Furthermore, some compensatory mechanisms - such as venoconstriction - increased the extent of predicted ventricular enlargement. Unfortunately, initial reflex responses to infarction were a poor predictor of subsequent responses to pharmacologic agents, suggesting that customizing pharmacologic therapy to individuals based on an initial response will be challenging.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 28 Oct 2021; 163:156-166
Caggiano LR, Holmes JW, Witzenburg CM
J Mol Cell Cardiol: 28 Oct 2021; 163:156-166 | PMID: 34756992
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Abstract

NAFLD as a continuous driver in the whole spectrum of vascular disease.

Li W, Liu J, Cai J, Zhang XJ, ... Chen S, Li H
Vascular disease is the prime determinant to cardiovascular morbidities and mortalities, which comprises the early vascular damage and subsequent cardiovascular events. Non-alcohol Fatty Liver Disease (NAFLD) is a systemic metabolic disorder that drives the progression of vascular disease through complex interactions. Although a causal relationship between NAFLD and cardiovascular disease (CVD) has not been established, a growing number of epidemiological studies have demonstrated an independent association between NAFLD and early vascular disease and subsequent cardiovascular events. In addition, mechanistic studies suggest that NAFLD initiates and accelerates vascular injury by increasing systemic inflammation and oxidative stress, impairing insulin sensitivity and lipid metabolism, and modulating epigenetics, the intestinal flora and hepatic autonomic nervous system; thus, NAFLD is a putative driving force for CVD progression. In this review, we summarize the clinical evidence supporting the association of NAFLD with subclinical vascular disease and cardiovascular events and discuss the potential mechanisms by which NAFLD promotes the progression of vascular disease.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 27 Oct 2021; 163:118-132
Li W, Liu J, Cai J, Zhang XJ, ... Chen S, Li H
J Mol Cell Cardiol: 27 Oct 2021; 163:118-132 | PMID: 34737121
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Abstract

Inositol 1,4,5-trisphosphate receptor - reactive oxygen signaling domain regulates excitation-contraction coupling in atrial myocytes.

Varma D, Almeida JFQ, DeSantiago J, Blatter LA, Banach K
The inositol 1,4,5-trisphosphate receptor (InsP3R) is up-regulated in patients with atrial fibrillation (AF) and InsP3-induced Ca2+ release (IICR) is linked to pro-arrhythmic spontaneous Ca2+ release events. Nevertheless, knowledge of the physiological relevance and regulation of InsP3Rs in atrial muscle is still limited. We hypothesize that InsP3R and NADPH oxidase 2 (NOX2) form a functional signaling domain where NOX2 derived reactive oxygen species (ROS) regulate InsP3R agonist affinity and thereby Ca2+ release. To quantitate the contribution of IICR to atrial excitation-contraction coupling (ECC) atrial myocytes (AMs) were isolated from wild type and NOX2 deficient (Nox2-/-) mice and changes in the cytoplasmic Ca2+ concentration ([Ca2+]i; fluo-4/AM, indo-1) or ROS (2\',7\'-dichlorofluorescein, DCF) were monitored by fluorescence microscopy. Superfusion of AMs with Angiotensin II (AngII: 1 μmol/L) significantly increased diastolic [Ca2+]i (F/F0, Ctrl: 1.00 ± 0.01, AngII: 1.20 ± 0.03; n = 7; p < 0.05), the field stimulation induced Ca2+ transient (CaT) amplitude (ΔF/F0, Ctrl: 2.00 ± 0.17, AngII: 2.39 ± 0.22, n = 7; p < 0.05), and let to the occurrence of spontaneous increases in [Ca2+]i. These changes in [Ca2+]i were suppressed by the InsP3R blocker 2-aminoethoxydiphenyl-borate (2-APB; 1 μmol/L). Concomitantly, AngII induced an increase in ROS production that was sensitive to the NOX2 specific inhibitor gp91ds-tat (1 μmol/L). In NOX2-/- AMs, AngII failed to increase diastolic [Ca2+]i, CaT amplitude, and the frequency of spontaneous Ca2+ increases. Furthermore, the enhancement of CaTs by exposure to membrane permeant InsP3 was abolished by NOX inhibition with apocynin (1 μM). AngII induced IICR in Nox2-/- AMs could be restored by addition of exogenous ROS (tert-butyl hydroperoxide, tBHP: 5 μmol/L). In saponin permeabilized AMs InsP3 (5 μmol/L) induced Ca2+ sparks that increased in frequency in the presence of ROS (InsP3: 9.65 ± 1.44 sparks*s-1*(100μm)-1; InsP3 + tBHP: 10.77 ± 1.5 sparks*s-1*(100μm)-1; n = 5; p < 0.05). The combined effect of InsP3 + tBHP was entirely suppressed by 2-APB and Xestospongine C (XeC). Changes in IICR due to InsP3R glutathionylation induced by diamide could be reversed by the reducing agent dithiothreitol (DTT: 1 mmol/L) and prevented by pretreatment with 2-APB, supporting that the ROS-dependent post-translational modification of the InsP3R plays a role in the regulation of ECC. Our data demonstrate that in AMs the InsP3R is under dual control of agonist induced InsP3 and ROS formation and suggest that InsP3 and NOX2-derived ROS co-regulate atrial IICR and ECC in a defined InsP3R/NOX2 signaling domain.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 27 Oct 2021; 163:147-155
Varma D, Almeida JFQ, DeSantiago J, Blatter LA, Banach K
J Mol Cell Cardiol: 27 Oct 2021; 163:147-155 | PMID: 34755642
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Abstract

How to repair a broken heart with pluripotent stem cell-derived cardiomyocytes.

Eschenhagen T, Ridders K, Weinberger F
Heart regeneration addresses a central problem in cardiology, the irreversibility of the loss of myocardium that eventually leads to heart failure. True restoration of heart function can only be achieved by remuscularization, i.e. replacement of lost myocardium by new, force-developing heart muscle. With the availability of principally unlimited human cardiomyocytes from pluripotent stem cells, one option to remuscularize the injured heart is to produce large numbers of cardiomyocytes plus/minus other cardiovascular cell types or progenitors ex vivo and apply them to the heart, either by injection or application as a patch. Exciting progress over the past decade has led to the first clinical applications, but important questions remain. Academic and increasingly corporate activity is ongoing to answer them and optimize the approach to finally develop a true regenerative therapy of heart failure.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 19 Oct 2021; 163:106-117
Eschenhagen T, Ridders K, Weinberger F
J Mol Cell Cardiol: 19 Oct 2021; 163:106-117 | PMID: 34687723
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Impact:

This program is still in alpha version.