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Small mammals have a higher heart rate and, relative to body mass (Mb), a higher metabolic rate than large mammals. In contrast, heart weight and stroke volume scale linearly with Mb. With mitochondria filling approximately 50% of a shrew cardiomyocyte - space unavailable for myofibrils - it is unclear how small mammals generate enough contractile force to pump blood into circulation. Here, we investigated whether the total number or volume of cardiomyocytes in the left ventricle compensates for allometry-related volume shifts of cardiac mitochondria and myofibrils. Through statistical analysis of data from 25 studies with 19 different mammalian species with Mb spanning seven orders of magnitude (2.2 g to 920 kg), we determined how number, volume density and total volume of cardiomyocytes, mitochondria and myofibrils in the left ventricle depend on Mb. We found that these biological variables follow scaling relationships and are proportional to a power b of Mb. The number [b=1.02 (95% CI: 0.89, 1.14); t-test for b=1: P=0.72] and volume [b=0.95 (95% CI: 0.89, 1.03); t-test for b=1: P=0.18] of cardiomyocytes in the left ventricle increases linearly with increasing Mb. In cardiomyocytes, volume density of mitochondria decreases [b=-0.056 (95% CI: -0.08, -0.04); t-test for b=0: P<0.0001] and that of myofibrils increases [b=0.024 (95%CI: 0.01, 0.04); t-test for b=0: P<0.01] with increasing Mb. Thus, the number or volume of left ventricular cardiomyocytes does not compensate for the higher heart rate and specific metabolic rate of small mammals although a higher mitochondrial and lower myofibrillar volume per cardiomyocyte are present., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2025. Published by The Company of Biologists.)

The human heart regenerates slowly through life, but how new cells are generated is mostly unknown. The atrioventricular junction (AVj) has been indicated as a potential stem cell niche region. Little is known about the protein composition of the human AVj. To map the extracellular matrix (ECM) and expression of stem cell-related biomarkers, this study compares protein and gene expression patterns in AVj and Left Ventricular (LV) tissues. Biopsies were collected from 15 human hearts. Global quantitative proteomics and mRNA sequencing were used to identify differentially expressed proteins and altered genes. Of the total 4904 identified proteins, 1138 were differently expressed between the AVj and LV. While the top proteins in LV were involved in cardiac motor function and energy regulation, the AVj displayed proteins associated with early cardiomyocyte development, differentiation, proliferation, migration, and hypoxia. Furthermore, several developmental signalling pathways, including TGF-β, TNF, WNT, Notch, and FGF, were represented. RNA-seq data verified that the expressed genes were involved with differentiation, cell growth, proliferation, or ECM organization. Immunohistochemistry confirmed the expression of the stem cell-related biomarkers NPPA and POSTN in the AVj, further strengthening the hypothesis of the AVj as a specialized microenvironment conducive to stem cell niche activity.

Several ion currents in the mammalian ventricular myocardium are substantially regulated by the sympathetic nervous system via β-adrenergic receptor activation, including the slow delayed rectifier K + current and the L-type calcium current. This study investigated the downstream mechanisms of β-adrenergic receptor stimulation by isoproterenol (ISO) on the inward rectifier (I K1 ) and the rapid delayed rectifier (I Kr ) K + currents using action potential voltage clamp (APVC) and conventional voltage clamp techniques in isolated canine left ventricular cardiomyocytes. I K1 and I Kr were dissected by 50 µM BaCl 2 and 1 µM E-4031, respectively. Acute application of 10 nM ISO significantly increased I K1 under the plateau phase of the action potential (0-+20 mV) using APVC, and similar results were obtained with conventional voltage clamp. However, β-adrenergic receptor stimulation did not affect the peak current density flowing during terminal repolarization or the overall I K1 integral. The ISO-induced enhancement of I K1 was blocked by the calcium/calmodulin kinase II (CaMKII) inhibitor KN-93 (1 µM) but not by the protein kinase A inhibitor H-89 (3 µM). Neither KN-93 nor H-89 affected the I K1 density under baseline conditions (in the absence of ISO). In contrast, parameters of the I Kr current were not affected by β-adrenergic receptor stimulation with ISO. These findings suggest that sympathetic activation enhances I K1 in canine left ventricular cells through the CaMKII pathway, while I Kr remains unaffected under the experimental conditions used.

Previous studies have observed alterations in excitation-contraction (EC) coupling during end-stage heart failure that include action potential and calcium (Ca 2+ ) transient prolongation and a reduction of the Ca 2+ transient amplitude. Underlying these phenomena are the downregulation of potassium (K + ) currents, downregulation of the sarcoplasmic reticulum Ca 2+ ATPase (SERCA), increase Ca 2+ sensitivity of the ryanodine receptor, and the upregulation of the sodium-calcium (Na = -Ca 2+ ) exchanger. However, in human heart failure (HF), debate continues about the relative contributions of the changes in calcium handling vs. the changes in the membrane currents. To understand the consequences of the above changes, they are incorporated into a computational human ventricular myocyte HF model that can explore the contributions of the spontaneous Ca 2+ release from the sarcoplasmic reticulum (SR). The reduction of transient outward K + current (I to ) is the main membrane current contributor to the decrease in RyR2 open probability and L-type calcium channel (LCC) density which emphasizes its importance to phase 1 of the action potential (AP) shape and duration (APD). During current-clamp conditions, RyR2 hyperphosphorylation exhibits the least amount of Ca 2+ release from the SR into the cytosol and SR Ca 2+ fractional release during a dynamic slow-rapid-slow (0.5-2.5-0.5 Hz) pacing, but it displays the most abundant and more lasting Ca 2+ sparks two-fold longer than a normal cell. On the other hand, under voltage-clamp conditions, HF by decreased SERCA and upregulated I NCX show the least SR Ca 2+ uptake and EC coupling gain, as compared to HF by hyperphosphorylated RyR2s. Overall, this study demonstrates that the (a) combined effect of SERCA and NCX, and the (b) RyR2 dysfunction, along with the downregulation of the cardiomyocyte's potassium currents, could substantially contribute to Ca 2+ mishandling at the spark level that leads to heart failure.

Extensive genetic studies have elucidated cardiomyocyte differentiation and associated gene networks using single-cell RNA-seq, yet the intricate transcriptional mechanisms governing cardiac conduction system (CCS) development and working cardiomyocyte differentiation remain largely unexplored. Here we show that mice deleted for Dhx36 (encoding the Dhx36 helicase) in the embryonic or neonatal heart develop overt dilated cardiomyopathy, surface ECG alterations related to cardiac impulse propagation, and (in the embryonic heart) a lack of a ventricular conduction system (VCS). Heart snRNA-seq and snATAC-seq reveal the role of Dhx36 in CCS development and in the differentiation of working cardiomyocytes. Dhx36 deficiency directly influences cardiomyocyte gene networks by disrupting the resolution of promoter G-quadruplexes in key cardiac genes, impacting cardiomyocyte differentiation and CCS morphogenesis, and ultimately leading to dilated cardiomyopathy and atrioventricular block. These findings further identify crucial genes and pathways that regulate the development and function of the VCS/Purkinje fiber (PF) network., (© 2024. The Author(s).)

Background/aims: Advances in induced pluripotent stem cell (iPSC) technology allow for reprogramming of adult somatic cells into stem cells from which patient- and disease-specific cardiomyocytes (CMs) can be derived. Yet, the potential of iPSC technology to revolutionize cardiovascular research is limited, in part, by the embryonic nature of these cells. Here, we test the hypothesis that decellularized porcine left ventricular extracellular cardiac matrix (ECM) provides environmental cues that promote transcriptional maturation and patterning of iPSC-CMs in culture., Methods: Cardiac progenitor cells were plated on ECM or standard tissue plates (2D monolayer) for 30 days, after which CM orientation and single cell transcriptomics were evaluated using confocal imaging and singe cell RNA-sequencing, respectively., Results: Cardiac progenitors differentiated on left ventricular ECM formed longitudinal fibers that differed quantitatively from progenitors differentiated in standard 2D conditions. Unsupervised clustering of single cell transcriptomics identified a CM cluster expressing a higher level of genes related to CM maturation. CMs differentiated on ECM were overrepresented in this cluster, indicating a bias toward CM maturation, compared to cells differentiated in standard 2D monolayer conditions., Conclusion: Our data suggest that environmental cues related to the left ventricular ECM may promote differentiation to a more mature CM state compared to cells differentiated on a standard 2D monolayer, while facilitating organization into longitudinal micro-fibers. Our study highlights the utility of ECM as a differentiation substrate to promote CM maturation and fiber orientation in vitro ., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

PRDM16 is a transcription factor with histone methyltransferase activity expressed at the earliest stages of cardiac development. Pathogenic mutations in humans lead to cardiomyopathy, conduction abnormalities, and heart failure. PRDM16 is specifically expressed in ventricular but not atrial cardiomyocytes, and its expression declines postnatally. Because in other tissues PRDM16 is best known for its role in binary cell fate decisions, we hypothesized a similar decision-making function in cardiomyocytes. Here, we demonstrated that cardiomyocyte-specific deletion of Prdm16 during cardiac development results in contractile dysfunction and abnormal electrophysiology of the postnatal heart, resulting in premature death. By combined RNA+ATAC single-cell sequencing, we found that PRDM16 favors ventricular working cardiomyocyte identity, by opposing the activity of master regulators of ventricular conduction and atrial fate. Myocardial loss of PRDM16 during development resulted in hyperplasia of the (distal) ventricular conduction system. Hence, PRDM16 plays an indispensable role during cardiac development by driving ventricular working cardiomyocyte identity., (© 2024 Van Wauwe et al.)

Tissues undergo distinct morphogenetic processes to achieve similarly shaped structures. In the heart, cardiomyocytes in both the ventricle and atrium build internal structures for efficient contraction. Ventricular wall formation (trabeculation) is initiated by cardiomyocyte delamination. How cardiomyocytes build the atrial wall is poorly understood. Using longitudinal imaging in zebrafish, we found that at least 25% of the atrial cardiomyocytes elongate along the long axis of the heart. These cell shape changes result in cell intercalation and convergent thickening, leading to the formation of the internal muscle network. We tested factors important for ventricular trabeculation including Nrg/ErbB and Notch signaling and found no evidence for their role in atrial muscle network formation. Instead, our data suggest that atrial cardiomyocyte elongation is regulated by Yap, which has not been implicated in trabeculation. Altogether, these data indicate that distinct cellular and molecular mechanisms build the internal muscle structures in the atrium and ventricle., (© 2024. The Author(s).)

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) have found utility for conducting in vitro drug screening and disease modelling to gain crucial insights into pharmacology or disease phenotype. However, diseases such as atrial fibrillation, affecting >33 M people worldwide, demonstrate the need for cardiac subtype-specific cells. Here, we sought to investigate the base characteristics and pharmacological differences between commercially available chamber-specific atrial or ventricular hiPSC-CMs seeded onto ultra-thin, flexible PDMS membranes to simultaneously measure contractility in a 96 multi-well format. We investigated the effects of GPCR agonists (acetylcholine and carbachol), a Ca 2+ channel agonist (S-Bay K8644), an HCN channel antagonist (ivabradine) and K + channel antagonists (4-AP and vernakalant). We observed differential effects between atrial and ventricular hiPSC-CMs on contractile properties including beat rate, beat duration, contractile force and evidence of arrhythmias at a range of concentrations. As an excerpt of the compound analysis, S-Bay K8644 treatment showed an induced concentration-dependent transient increase in beat duration of atrial hiPSC-CMs, whereas ventricular cells showed a physiological increase in beat rate over time. Carbachol treatment produced marked effects on atrial cells, such as increased beat duration alongside a decrease in beat rate over time, but only minimal effects on ventricular cardiomyocytes. In the context of this chamber-specific pharmacology, we not only add to contractile characterization of hiPSC-CMs but propose a multi-well platform for medium-throughput early compound screening. Overall, these insights illustrate the key pharmacological differences between chamber-specific cardiomyocytes and their application on a multi-well contractility platform to gain insights for in vitro cardiac liability studies and disease modelling., Competing Interests: Declaration of competing interest Authors Bettina Lickiss, Matthias Gossmann, Jan Hunker and Peter Linder are employed by innoVitro GmbH, the manufacturer of the FLEXcyte 96 consumables and co-developer of the FLEXcyte 96 used to compile this manuscript. Authors Ulrich Thomas, Elena Dragicevic and Sonja Stoelzle-Feix are employed by Nanion Technologies GmbH, the manufacturer of the FLEXcyte 96 used to compile this manuscript. Authors Jamie Bhagwan, Hardeep Lotay, Steven Broadbent and Jan Turner are employed by Axol Bioscience Ltd., the manufacturer of axoCells™ Ventricular and Atrial Cardiomyocytes used in this study., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) by traditional methods are a mix of atrial and ventricular CMs and many other non-cardiomyocyte cells. Retinoic acid (RA) plays an important role in regulation of the spatiotemporal development of the embryonic heart., Methods: CMs were derived from hiPSC (hi-PCS-CM) using different concentrations of RA (Control without RA, LRA with 0.05μM and HRA with 0.1 μM) between day 3-6 of the differentiation process. Engineered heart tissues (EHTs) were generated by assembling hiPSC-CM at high cell density in a low collagen hydrogel., Results: In the HRA group, hiPSC-CMs exhibited highest expression of contractile proteins MYH6, MYH7 and cTnT. The expression of TBX5, NKX2.5 and CORIN, which are marker genes for left ventricular CMs, was also the highest in the HRA group. In terms of EHT, the HRA group displayed the highest contraction force, the lowest beating frequency, and the highest sensitivity to hypoxia and isoprenaline, which means it was functionally more similar to the left ventricle. RNAsequencing revealed that the heightened contractility of EHT within the HRA group can be attributed to the promotion of augmented extracellular matrix strength by RA., Conclusion: By interfering with the differentiation process of hiPSC with a specific concentration of RA at a specific time, we were able to successfully induce CMs and EHTs with a phenotype similar to that of the left ventricle or right ventricle., (© 2024. The Author(s).)

The heart beats approximately 100,000 times per day in humans, imposing substantial energetic demands on cardiac muscle. Adenosine triphosphate (ATP) is an essential energy source for normal function of cardiac muscle during each beat, as it powers ion transport, intracellular Ca 2+ handling, and actin-myosin cross-bridge cycling. Despite this, the impact of excitation-contraction coupling on the intracellular ATP concentration ([ATP] i ) in myocytes is poorly understood. Here, we conducted real-time measurements of [ATP] i in ventricular myocytes using a genetically encoded ATP fluorescent reporter. Our data reveal rapid beat-to-beat variations in [ATP] i . Notably, diastolic [ATP] i was <1 mM, which is eightfold to 10-fold lower than previously estimated. Accordingly, ATP-sensitive K + (K ATP ) channels were active at physiological [ATP] i . Cells exhibited two distinct types of ATP fluctuations during an action potential: net increases (Mode 1) or decreases (Mode 2) in [ATP] i . Mode 1 [ATP] i increases necessitated Ca 2+ entry and release from the sarcoplasmic reticulum (SR) and were associated with increases in mitochondrial Ca 2+ . By contrast, decreases in mitochondrial Ca 2+ accompanied Mode 2 [ATP] i decreases. Down-regulation of the protein mitofusin 2 reduced the magnitude of [ATP] i fluctuations, indicating that SR-mitochondrial coupling plays a crucial role in the dynamic control of ATP levels. Activation of β-adrenergic receptors decreased [ATP] i , underscoring the energetic impact of this signaling pathway. Finally, our work suggests that cross-bridge cycling is the largest consumer of ATP in a ventricular myocyte during an action potential. These findings provide insights into the energetic demands of EC coupling and highlight the dynamic nature of ATP concentrations in cardiac muscle., Competing Interests: Competing interests statement:The authors declare no competing interest.

The sodium channel Na V 1.8, encoded by the SCN10A gene, has recently emerged as a potential regulator of cardiac electrophysiology. We have previously shown that Na V 1.8 contributes to arrhythmogenesis by inducing a persistent Na + current (late Na + current, I NaL ) in human atrial and ventricular cardiomyocytes (CM). We now aim to further investigate the contribution of Na V 1.8 to human ventricular arrhythmogenesis at the CM-specific level using pharmacological inhibition as well as a genetic knockout (KO) of SCN10A in induced pluripotent stem cell CM (iPSC-CM). In functional voltage-clamp experiments, we demonstrate that I NaL was significantly reduced in ventricular SCN10A -KO iPSC-CM and in control CM after a specific pharmacological inhibition of Na V 1.8. In contrast, we did not find any effects on ventricular APD 90 . The frequency of spontaneous sarcoplasmic reticulum Ca 2+ sparks and waves were reduced in SCN10A- KO iPSC-CM and control cells following the pharmacological inhibition of Na V 1.8. We further analyzed potential triggers of arrhythmias and found reduced delayed afterdepolarizations (DAD) in SCN10A- KO iPSC-CM and after the specific inhibition of Na V 1.8 in control cells. In conclusion, we show that Na V 1.8-induced I NaL primarily impacts arrhythmogenesis at a subcellular level, with minimal effects on systolic cellular Ca 2+ release. The inhibition or knockout of Na V 1.8 diminishes proarrhythmic triggers in ventricular CM. In conjunction with our previously published results, this work confirms Na V 1.8 as a proarrhythmic target that may be useful in an anti-arrhythmic therapeutic strategy.

Current density, the membrane current value divided by membrane capacitance (C m ), is widely used in cellular electrophysiology. Comparing current densities obtained in different cell populations assume that C m and ion current magnitudes are linearly related, however data is scarce about this in cardiomyocytes. Therefore, we statistically analyzed the distributions, and the relationship between parameters of canine cardiac ion currents and C m , and tested if dividing original parameters with C m had any effect. Under conventional voltage clamp conditions, correlations were high for I K1 , moderate for I Kr and I Ca,L , while negligible for I Ks . Correlation between I to1 peak amplitude and C m was negligible when analyzing all cells together, however, the analysis showed high correlations when cells of subepicardial, subendocardial or midmyocardial origin were analyzed separately. In action potential voltage clamp experiments I K1, I Kr and I Ca,L parameters showed high correlations with C m . For I NCX , I Na,late and I Ks there were low-to-moderate correlations between C m and these current parameters. Dividing the original current parameters with C m reduced both the coefficient of variation, and the deviation from normal distribution. The level of correlation between ion currents and C m varies depending on the ion current studied. This must be considered when evaluating ion current densities in cardiac cells., (© 2024. The Author(s).)

Doxorubicin (DOX), widely used as a chemotherapeutic agent for various cancers, is limited in its clinical utility by its cardiotoxic effects. Despite its widespread use, the precise mechanisms underlying DOX-induced cardiotoxicity at the cellular and molecular levels remain unclear, hindering the development of preventive and early detection strategies. To characterize the cytotoxic effects of DOX on isolated ventricular cardiomyocytes, focusing on the expression of specific microRNAs (miRNAs) and their molecular targets associated with endogenous cardioprotective mechanisms such as the ATP-sensitive potassium channel (KATP), Sirtuin 1 (SIRT1), FOXO1, and GSK3β. We isolated Guinea pig ventricular cardiomyocytes by retrograde perfusion and enzymatic dissociation. We assessed cell morphology, Reactive Oxygen Species (ROS) levels, intracellular calcium, and mitochondrial membrane potential using light microscopy and specific probes. We determined the miRNA expression profile using small RNAseq and validated it using stem-loop qRT-PCR. We quantified mRNA levels of some predicted and validated molecular targets using qRT-PCR and analyzed protein expression using Western blot. Exposure to 10 µM DOX resulted in cardiomyocyte shortening, increased ROS and intracellular calcium levels, mitochondrial membrane potential depolarization, and changes in specific miRNA expression. Additionally, we observed the differential expression of KATP subunits (ABCC9, KCNJ8, and KCNJ11), FOXO1, SIRT1, and GSK3β molecules associated with endogenous cardioprotective mechanisms. Supported by miRNA gene regulatory networks and functional enrichment analysis, these findings suggest that DOX-induced cardiotoxicity disrupts biological processes associated with cardioprotective mechanisms. Further research must clarify their specific molecular changes in DOX-induced cardiac dysfunction and investigate their diagnostic biomarkers and therapeutic potential.

Endothelial progenitor cells (EPCs) play a critical role in cardiovascular regeneration. Enhancement of their native properties would be highly beneficial to ensuring the proper functioning of the cardiovascular system. As androgens have a positive effect on the cardiovascular system, we hypothesized that dihydrotestosterone (DHT) could also influence EPC-mediated repair processes. To evaluate this hypothesis, we investigated the effects of DHT on cultured human EPCs' proliferation, viability, morphology, migration, angiogenesis, gene and protein expression, and ability to integrate into cardiac tissue. The results showed that DHT at different concentrations had no cytotoxic effect on EPCs, significantly enhanced the cell proliferation and viability and induces fast, androgen-receptor-dependent formation of capillary-like structures. DHT treatment of EPCs regulated gene expression of androgen receptors and the genes and proteins involved in cell migration and angiogenesis. Importantly, DHT stimulation promoted EPC migration and the cells' ability to adhere and integrate into murine cardiac slices, suggesting it has a role in promoting tissue regeneration. Mass spectrometry analysis further highlighted the impact of DHT on EPCs' functioning. In conclusion, DHT increases the proliferation, migration, and androgen-receptor-dependent angiogenesis of EPCs; enhances the cells' secretion of key factors involved in angiogenesis; and significantly potentiates cellular integration into heart tissue. The data offer support for potential therapeutic applications of DHT in cardiovascular regeneration and repair processes.

The heart, which is the first organ to develop, is highly dependent on its form to function 1,2 . However, how diverse cardiac cell types spatially coordinate to create the complex morphological structures that are crucial for heart function remains unclear. Here we integrated single-cell RNA-sequencing with high-resolution multiplexed error-robust fluorescence in situ hybridization to resolve the identity of the cardiac cell types that develop the human heart. This approach also provided a spatial mapping of individual cells that enables illumination of their organization into cellular communities that form distinct cardiac structures. We discovered that many of these cardiac cell types further specified into subpopulations exclusive to specific communities, which support their specialization according to the cellular ecosystem and anatomical region. In particular, ventricular cardiomyocyte subpopulations displayed an unexpected complex laminar organization across the ventricular wall and formed, with other cell subpopulations, several cellular communities. Interrogating cell-cell interactions within these communities using in vivo conditional genetic mouse models and in vitro human pluripotent stem cell systems revealed multicellular signalling pathways that orchestrate the spatial organization of cardiac cell subpopulations during ventricular wall morphogenesis. These detailed findings into the cellular social interactions and specialization of cardiac cell types constructing and remodelling the human heart offer new insights into structural heart diseases and the engineering of complex multicellular tissues for human heart repair., (© 2024. The Author(s).)

The extent of heavy-metal-induced cardiotoxicity is proportional to the levels of metal bioaccumulation, and it was previously assumed that heavy metals accumulate uniformly in the myocardium. Therefore, the aim of this study was to investigate concentrations of metals and metalloids in two distant regions of the left ventricle (LV), the base of the LV, and apex of the LV using inductively coupled plasma mass spectrometry (ICP-MS). We also examined the potential correlation between metal levels and the thickness of the interventricular septum in twenty LV specimens (ten from the base of LV and ten from the apex of LV) from 10 individuals (mean age 75 ± 6 years). We found significantly higher concentrations of arsenic and lead in the LV apex compared to the base of the LV. We also found a positive correlation between the concentrations of arsenic in the myocardium of LV and the thickness of the interventricular septum. Our results indicate that arsenic and lead accumulate to a higher extent in the apex of the LV compared to the base of the LV. Therefore, future studies designed to measure levels of metals in heart muscle should consider non-uniform accumulation of metals in the myocardium.

Background: During the development of heart failure, a fetal cardiac gene program is reactivated and accelerates pathological cardiac remodeling. We previously reported that a transcriptional repressor, NRSF (neuron restrictive silencer factor), suppresses the fetal cardiac gene program, thereby maintaining cardiac integrity. The underlying molecular mechanisms remain to be determined, however., Methods: We aim to elucidate molecular mechanisms by which NRSF maintains normal cardiac function. We generated cardiac-specific NRSF knockout mice and analyzed cardiac gene expression profiles in those mice and mice cardiac-specifically expressing a dominant-negative NRSF mutant., Results: We found that cardiac expression of Gα o , an inhibitory G protein encoded in humans by GNAO1 , is transcriptionally regulated by NRSF and is increased in the ventricles of several mouse models of heart failure. Genetic knockdown of Gnao1 ameliorated the cardiac dysfunction and prolonged survival rates in these mouse heart failure models. Conversely, cardiac-specific overexpression of GNAO1 in mice was sufficient to induce cardiac dysfunction. Mechanistically, we observed that increasing Gα o expression increased surface sarcolemmal L-type Ca 2+ channel activity, activated CaMKII (calcium/calmodulin-dependent kinase-II) signaling, and impaired Ca 2+ handling in ventricular myocytes, which led to cardiac dysfunction., Conclusions: These findings shed light on a novel function of Gα o in the regulation of cardiac Ca 2+ homeostasis and systolic function and suggest Gα o may be an effective therapeutic target for the treatment of heart failure.

Freshly isolated primary cardiomyocytes (CM) are indispensable for cardiac research. Experimental CM research is generally incompatible with life of the donor animal, while human heart samples are usually small and scarce. CM isolation from animal hearts, traditionally performed by coronary artery perfusion of enzymes, liberates millions of cells from the heart. However, due to progressive cell remodeling following isolation, freshly isolated primary CM need to be used within 4-8 h post-isolation for most functional assays, meaning that the majority of cells is essentially wasted. In addition, coronary perfusion-based isolation cannot easily be applied to human tissue biopsies, and it does not straightforwardly allow for assessment of regional differences in CM function within the same heart. Here, we provide a method of multi-day CM isolation from one animal heart, yielding calcium-tolerant ventricular and atrial CM. This is based on cell isolation from cardiac tissue slices following repeated (usually overnight) storage of the tissue under conditions that prolong CM viability beyond the day of organ excision by two additional days. The maintenance of cells in their near-native microenvironment slows the otherwise rapid structural and functional decline seen in isolated CM during attempts for prolonged storage or culture. Multi-day slice-based CM isolation increases the amount of useful information gained per animal heart, improving reproducibility and reducing the number of experimental animals required in basic cardiac research. It also opens the doors to novel experimental designs, including exploring same-heart regional differences.

Intracellular Ca 2+ overload secondary to chronic hemodynamic stimuli promotes the recruitment of Ca 2+ -dependent signaling implicated in cardiomyocyte hypertrophy. The present study tested the hypothesis that sympathetic-mediated hypertrophy of neonatal rat ventricular cardiomyocytes (NRVMs) translated to an increase in calcium influx secondary to the upregulation of Ca V 1.2 channel subunits. Confocal imaging of norepinephrine (NE)-treated NRVMs revealed a hypertrophic response compared to untreated NRVMs. L-type Ca V 1.2 peak current density was increased 4-fold following a 24-h stimulation with NE. NE-treated NRVMs exhibited a significant upregulation of Ca V α2δ1 and Ca V β3 protein levels without significant changes of Ca V α1C and Ca V β2 protein levels. Pre-treatment with the β 1 -blocker metoprolol failed to inhibit hypertrophy or Ca V β3 upregulation whereas Ca V α2δ1 protein levels were significantly reduced. NE promoted the phosphorylation of ERK 1/2, and the response was attenuated by the β 1 -blocker. U0126 pre-treatment suppressed NE-induced ERK1/2 phosphorylation but failed to attenuate hypertrophy. U0126 inhibition of ERK1/2 phosphorylation prevented NE-mediated upregulation of Ca V α2δ1, whereas Ca V β3 protein levels remained elevated. Thus, β 1 -adrenergic receptor-mediated recruitment of the ERK1/2 plays a seminal role in the upregulation of Ca V α2δ1 in NRVMs independent of the concomitant hypertrophic response. However, the upregulation of Ca V β3 protein levels may be directly dependent on the hypertrophic response of NRVMs.

Naturally found chrysosplenol-C (4',5,6-trihydroxy-3,3',7-trimethoxyflavone) increases the contractility of cardiac myocytes independent of β -adrenergic signaling. We investigated the cellular mechanism for chrysosplenol-C-induced positive inotropy. Global and local Ca 2+ signals, L-type Ca 2+ current (I Ca ), and contraction were measured from adult rat ventricular myocytes using two-dimensional confocal Ca 2+ imaging, the whole-cell patch-clamp technique, and video-edge detection, respectively. Application of chrysosplenol-C reversibly increased Ca 2+ transient magnitude with a maximal increase of ∼55% within 2- to 3-minute exposures (EC 50 ≅ 21 μM). This chemical did not alter I Ca and slightly increased diastolic Ca 2+ level. The frequency and size of resting Ca 2+ sparks were increased by chrysosplenol-C. Chrysosplenol-C significantly increased sarcoplasmic reticulum (SR) Ca 2+ content but not fractional release. Pretreatment of protein kinase C (PKC) inhibitor but not Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) inhibitor abolished the stimulatory effects of chrysosplenol-C on Ca 2+ transients and Ca 2+ sparks. Chrysosplenol-C-induced positive inotropy was removed by the inhibition of PKC but not CaMKII or phospholipase C. Western blotting assessment revealed that PKC- δ protein level in the membrane fractions significantly increase within 2 minutes after chrysosplenol-C exposure with a delayed (5-minute) increase in PKC- α levels in insoluble membrane. These results suggest that chrysosplenol-C enhances contractility via PKC (most likely PKC- δ )-dependent enhancement of SR Ca 2+ releases in ventricular myocytes. SIGNIFICANCE STATEMENT: Study shows that chrysosplenol-C, a natural flavone showing a positive inotropic effect, increases SR Ca 2+ releases on depolarizations and Ca 2+ sparks with an increase of SR Ca 2+ loading but not L-type Ca 2+ current in ventricular myocytes. Chrysosplenol-C-induced enhancement in contraction is eliminated by PKC inhibition, and it is associated with redistributions of PKC to the membrane. These indicate that chrysosplenol-C enhances contraction via PKC-dependent augmentations of SR Ca 2+ release and Ca 2+ loading during action potentials., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

A library of well-characterized human induced pluripotent stem cell (hiPSC) lines from clinically healthy human subjects could serve as a useful resource of normal controls for in vitro human development, disease modeling, genotype-phenotype association studies, and drug response evaluation. We report generation and extensive characterization of a gender-balanced, racially/ethnically diverse library of hiPSC lines from 40 clinically healthy human individuals who range in age from 22 to 61 years. The hiPSCs match the karyotype and short tandem repeat identities of their parental fibroblasts, and have a transcription profile characteristic of pluripotent stem cells. We provide whole-genome sequencing data for one hiPSC clone from each individual, genomic ancestry determination, and analysis of mendelian disease genes and risks. We document similar transcriptomic profiles, single-cell RNA-sequencing-derived cell clusters, and physiology of cardiomyocytes differentiated from multiple independent hiPSC lines. This extensive characterization makes this hiPSC library a valuable resource for many studies on human biology., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Mathematical models for the action potential (AP) generation of the electrically excitable cells including the heart are involved different mechanisms including the voltage-dependent currents with nonlinear time- and voltage-gating properties. From the shape of the AP waveforms to the duration of the refractory periods or heart rhythms are greatly affected by the functions describing the features or the quantities of these ion channels. In this work, a mathematical measure to analyze the regional contributions of voltage-gated channels is defined by dividing the AP into phases, epochs, and intervals of interest. The contribution of each time-dependent current for the newly defined cardiomyocyte model is successfully calculated and it is found that the contribution of dominant ion channels changes substantially not only for each phase but also for different regions of the cardiac AP. Besides, the defined method can also be applied in all Hodgkin-Huxley types of electrically excitable cell models to be able to understand the underlying dynamics better.

Isolation and culture of ventricular cardiomyocytes from neonatal rats (NRVMs) is a powerful model to study neonatal cardiac development, cell cycle regulation, and cardiac physiology and pathology in vitro . Here, we present our modified enzymatic digestion protocol followed by two-step discontinuous Percoll gradient centrifugation to isolate a high yield of viable ventricular cardiomyocytes from neonatal rats. Finally, here we describe an immunostaining protocol for cytosolic and nuclear staining of NRVMs. For complete details on the use and execution of this protocol, please refer to Pereira et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Spontaneous AP (action potential) firing of sinoatrial nodal cells (SANC) is critically dependent on protein kinase A (PKA) and Ca 2+ /calmodulin-dependent protein kinase II (CaMKII)-dependent protein phosphorylation, which are required for the generation of spontaneous, diastolic local Ca 2+ releases (LCRs). Although phosphoprotein phosphatases (PP) regulate protein phosphorylation, the expression level of PPs and phosphatase inhibitors in SANC and the impact of phosphatase inhibition on the spontaneous LCRs and other players of the oscillatory coupled-clock system is unknown. Here, we show that rabbit SANC express both PP1, PP2A, and endogenous PP inhibitors I-1 (PPI-1), dopamine and cyclic adenosine 3',5'-monophosphate (cAMP)-regulated phosphoprotein (DARPP-32), kinase C-enhanced PP1 inhibitor (KEPI). Application of Calyculin A, (CyA), a PPs inhibitor, to intact, freshly isolated single SANC: (1) significantly increased phospholamban (PLB) phosphorylation (by 2-3-fold) at both CaMKII-dependent Thr 17 and PKA-dependent Ser 16 sites, in a time and concentration dependent manner; (2) increased ryanodine receptor (RyR) phosphorylation at the Ser 2809 site; (3) substantially increased sarcoplasmic reticulum (SR) Ca 2+ load; (4) augmented L-type Ca 2+ current amplitude; (5) augmented LCR's characteristics and decreased LCR period in intact and permeabilized SANC, and (6) increased the spontaneous basal AP firing rate. In contrast, the selective PP2A inhibitor okadaic acid (100 nmol/L) had no significant effect on spontaneous AP firing, LCR parameters, or PLB phosphorylation. Application of purified PP1 to permeabilized SANC suppressed LCR, whereas purified PP2A had no effect on LCR characteristics. Our numerical model simulations demonstrated that PP inhibition increases AP firing rate via a coupled-clock mechanism, including respective increases in the SR Ca 2+ pumping rate, L-type Ca 2+ current, and Na + /Ca 2+ -exchanger current. Thus, PP1 and its endogenous inhibitors modulate the basal spontaneous firing rate of cardiac pacemaker cells by suppressing SR Ca 2+ cycling protein phosphorylation, the SR Ca 2+ load and LCRs, and L-type Ca 2+ current.

R-loops are naturally occurring transcriptional intermediates containing RNA/DNA hybrids. Excessive R-loops cause genomic instability, DNA damage, and replication stress. Senataxin-associated exonuclease (San1) is a protein that interacts with Senataxin (SETX), a helicase resolving R-loops. It remains unknown if R-loops-induced DNA damage plays a role in the heart, especially in the proliferative neonatal cardiomyocytes (CMs). San1-/- mice were generated using the CRISPR/Cas9 technique. The newborn San1-/- mice show no overt phenotype, but their hearts were smaller with larger, yet fewer CMs. CM proliferation was impaired with reduced cell cycle-related transcripts and proteins. S9.6 staining revealed that excessive R-loops accumulated in the nucleus of neonatal San1-/- CMs. Increased γH2AX staining on newborn and adult heart sections exhibited increased DNA damage. Similarly, San1-/- AC16-cardiomyocytes showed cumulative R-loops and DNA damage, leading to the activation of cell cycle checkpoint kinase ATR and PARP1 hyperactivity, arresting G2/M cell-cycle and CM proliferation. Together, the present study uncovers an essential role of San1 in resolving excessive R-loops that lead to DNA damage and repressing CM proliferation, providing new insights into a novel biological function of San1 in the neonatal heart. San1 may serve as a novel therapeutic target for the treatment of hypoplastic cardiac disorders., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Although insulin-induced cardiac hypertrophy is reported, very little information is available on the hypertrophic effect of insulin on ventricular cardiomyocytes and the regulation of sodium and calcium homeostasis. Taurine is a non-essential amino acid synthesized by cardiomyocytes and the brain and is present in low quantities in many foods, particularly seafood. The purpose of this study was to investigate whether chronic exposure to insulin induces hypertrophy of ventricular cardiomyocytes that are associated with changes in Na + and Ca 2+ homeostasis and whether taurine pre-treatment prevents these effects. Our results showed that chronic treatment with insulin leads to cardiomyocyte hypertrophy that is associated with an increase in basal intracellular Na + and Ca 2+ levels. Furthermore, long-term taurine treatment prevents morphological and ionic remodeling induced by insulin. In addition, blocking the Na + -taurine co-transporter prevented the taurine antihypertrophic effect. Finally, the insulin-induced remodeling of cardiomyocytes was associated with a decrease in the ratio of phospho-CREB (pCREB) to total cAMP response element binding protein (CREB); taurine prevented this effect. In conclusion, our results show that insulin induces ventricular cardiomyocyte hypertrophy via downregulation of the pCREB/tCREB level and that chronic taurine treatment prevents this effect.

Congenital heart disease (CHD) is one of the most common birth defects in humans, present in around 40% of newborns with Down's syndrome (DS). The SH3 domain-binding glutamic acid-rich (SH3BGR) gene, which maps to the DS region, belongs to a gene family encoding a cluster of small thioredoxin-like proteins sharing SH3 domains. Although its expression is confined to the cardiac and skeletal muscle, the physiological role of SH3BGR in the heart is poorly understood. Interestingly, we observed a significant upregulation of SH3BGR in failing hearts of mice and human patients with hypertrophic cardiomyopathy. Along these lines, the overexpression of SH3BGR exhibited a significant increase in the expression of hypertrophic markers (Nppa and Nppb) and increased cell surface area in neonatal rat ventricular cardiomyocytes (NRVCMs), whereas its knockdown attenuated cellular hypertrophy. Mechanistically, using serum response factor (SRF) response element-driven luciferase assays in the presence or the absence of RhoA or its inhibitor, we found that the pro-hypertrophic effects of SH3BGR are mediated via the RhoA-SRF axis. Furthermore, SH3BGR knockdown resulted in the induction of apoptosis and reduced cell viability in NRVCMs via apoptotic Hippo-YAP signaling. Taking these results together, we here show that SH3BGR is vital for maintaining cytoskeletal integrity and cellular viability in NRVCMs through its modulation of the SRF/YAP signaling pathways., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Heart failure (HF) is a clinical syndrome caused by impairment of ventricular filling, ejection of blood, or both and is categorized as HF with reduced ejection fraction (HFrEF) or HF with preserved ejection fraction (HFpEF) based on left ventricular function. Cardiac fibrosis contributes to left ventricular dysfunction and leads to the development of HF. Ivabradine, an If current selective specific inhibitor, has been shown to improve the prognosis of patients with HF. However, the effects of ivabradine on cardiac function and fibrosis in HFpEF and HFrEF and the underlying mechanism remain unclear. In the present study, we utilized mouse models to mimic HFpEF and HFrEF and evaluated the therapeutic effects of ivabradine. By treating mice with different doses (10 mg/kg/d and 20 mg/kg/d) of ivabradine for 4 or 8 weeks, we found that a high dose of ivabradine improved cardiac diastolic function in HFpEF mice and ameliorated cardiac diastolic and systolic function and ventricular tachycardia incidence in HFrEF mice. Moreover, ivabradine significantly reduced the activation of cardiac fibroblasts and myocardial fibrosis in mice. Mechanistically, microRNA-133a, which was upregulated by ivabradine, targeted connective tissue growth factor and collagen 1 in cardiac fibroblasts and might contribute to the protective role of ivabradine. Together, our work utilized mouse models to study HFpEF and HFrEF, demonstrated the protective role of ivabradine in HFpEF and HFrEF, and elucidated the potential underlying mechanism, which provides an effective strategy for related diseases., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2021 Shuai Shao et al.)

Cardiovascular diseases are the leading cause of death in the world, cell therapies have been shown to recover cardiac function in animal models. Biomaterials used as scaffolds can solve some of the problems that cell therapies currently have, plasma polymerized pyrrole (PPPy) is a biomaterial that has been shown to promote cell adhesion and survival. The present research aimed to study PPPy nanoparticles (PPPyN) interaction with adult rat ventricular cardiomyocytes (ARVC), to explore whether PPPyN could be employed as a nanoscaffold and develop cardiac microtissues. PPPyN with a mean diameter of 330 nm were obtained, the infrared spectrum showed that some pyrrole rings are fragmented and that some fragments of the ring can be dehydrogenated during plasma synthesis, it also showed the presence of amino groups in the structure of PPPyN. PPPyN had a significant impact on the ARVC´s shape, delaying dedifferentiation, necrosis, and apoptosis processes, moreover, the cardiomyocytes formed cell aggregates up to 1.12 mm 2 with some aligned cardiomyocytes and generated fibers on its surface similar to cardiac extracellular matrix. PPPyN served as a scaffold for adult ARVC. Our results indicate that PPPyN-scaffold is a biomaterial that could have potential application in cardiac cell therapy (CCT)., (© 2021. The Author(s).)

Compartmentation of cAMP signaling is a critical factor for maintaining the integrity of receptor-specific responses in cardiac myocytes. This phenomenon relies on various factors limiting cAMP diffusion. Our previous work in adult rat ventricular myocytes (ARVMs) indicates that PKA regulatory subunits anchored to the outer membrane of mitochondria play a key role in buffering the movement of cytosolic cAMP. PKA can be targeted to discrete subcellular locations through the interaction of both type I and type II regulatory subunits with A-kinase anchoring proteins (AKAPs). The purpose of this study is to identify which AKAPs and PKA regulatory subunit isoforms are associated with mitochondria in ARVMs. Quantitative PCR data demonstrate that mRNA for dual specific AKAP1 and 2 (D-AKAP1 & D-AKAP2), acyl-CoA-binding domain-containing 3 (ACBD3), optic atrophy 1 (OPA1) are most abundant, while Rab32, WAVE-1, and sphingosine kinase type 1 interacting protein (SPHKAP) were barely detectable. Biochemical and immunocytochemical analysis suggests that D-AKAP1, D-AKAP2, and ACBD3 are the predominant mitochondrial AKAPs exposed to the cytosolic compartment in these cells. Furthermore, we show that both type I and type II regulatory subunits of PKA are associated with mitochondria. Taken together, these data suggest that D-AKAP1, D-AKAP2, and ACBD3 may be responsible for tethering both type I and type II PKA regulatory subunits to the outer mitochondrial membrane in ARVMs. In addition to regulating PKA-dependent mitochondrial function, these AKAPs may play an important role by buffering the movement of cAMP necessary for compartmentation., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

In conditions with abnormally increased activity of the cardiac ryanodine receptor (RyR2), Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) can contribute to a further destabilization of RyR2 that results in triggered arrhythmias. Therefore, inhibition of CaMKII in such conditions has been suggested as a strategy to suppress RyR2 activity and arrhythmias. However, suppression of RyR2 activity can lead to the development of arrhythmogenic Ca 2+ alternans. The aim of this study was to test whether the suppression of RyR2 activity caused by inhibition of CaMKII increases propensity for Ca 2+ alternans. We studied spontaneous Ca 2+ release events and Ca 2+ alternans in isolated left ventricular cardiomyocytes from mice carrying the gain-of-function RyR2 mutation RyR2-R2474S and from wild-type mice. CaMKII inhibition by KN-93 effectively decreased the frequency of spontaneous Ca 2+ release events in RyR2-R2474S cardiomyocytes exposed to the β-adrenoceptor agonist isoprenaline. However, KN-93-treated RyR2-R2474S cardiomyocytes also showed increased propensity for Ca 2+ alternans and increased Ca 2+ alternans ratio compared with both an inactive analog of KN-93 and with vehicle-treated controls. This increased propensity for Ca 2+ alternans was explained by prolongation of Ca 2+ release refractoriness. Importantly, the increased propensity for Ca 2+ alternans in KN-93-treated RyR2-R2474S cardiomyocytes did not surpass that of wild type. In conclusion, inhibition of CaMKII efficiently reduces spontaneous Ca 2+ release but promotes Ca 2+ alternans in RyR2-R2474S cardiomyocytes with a gain-of-function RyR2 mutation. The dominant effect in RyR2-R2474S is to reduce spontaneous Ca 2+ release, which supports this intervention as a therapeutic strategy in this specific condition. However, future studies on CaMKII inhibition in conditions with increased propensity for Ca 2+ alternans should include investigation of both phenomena. NEW & NOTEWORTHY Genetically increased RyR2 activity promotes arrhythmogenic Ca 2+ release. Inhibition of CaMKII suppresses RyR2 activity and arrhythmogenic Ca 2+ release. Suppression of RyR2 activity prolongs refractoriness of Ca 2+ release. Prolonged refractoriness of Ca 2+ release leads to arrhythmogenic Ca 2+ alternans. CaMKII inhibition promotes Ca 2+ alternans by prolonging Ca 2+ release refractoriness.

Myocardial ischemia/reperfusion (I/R) injury induces cardiomyocyte apoptosis to deteriorate heart function. Thus, how to inhibit cardiomyocyte apoptosis is the focus of recent researches. Proteasome family member PSMB4 (proteasome subunit beta type-4) promotes cell survival. The relationship between PSMB4 and cardiomyocyte apoptosis during myocardial I/R is unknown. In this study, PSMB4 expression increased in rat myocardial I/R model, positively correlated with cleaved caspase-3 expression, negatively correlated with Bcl-2 expression. In vitro, neonatal ventricle cardiomyocyte hypoxia/reoxygenation (H/R) model was constructed to mimic myocardial I/R. PSMB4 silence promoted cardiomyocyte apoptosis and IκBα expression, inhibited the activation of NF-κB. On the contrary, PSMB4 overexpession inhibited cardiomyocyte apoptosis and IκBα expression, promoted the activation of NF-κB. Additionally, PSMB4-IκBα interaction was identified, suggesting that PSMB4 might participate in the proteasome dependent degradation of IκBα. The data indicates that PSMB4 inhibits cardiomyocyte apoptosis via activating NF-κB signaling pathway during myocardial I/R, which can supply novel molecular target for the treatment of ischemic heart disease., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The pig is commonly used as an experimental model of human heart disease, including for the study of mechanisms of arrhythmia. However, there exist differences between human and porcine cellular electrophysiology: The pig action potential (AP) has a deeper phase-1 notch, a longer duration at 50% repolarization, and higher plateau potentials than human. Ionic differences underlying the AP include larger rapid delayed-rectifier and smaller inward-rectifier K+-currents (IKr and IK1 respectively) in humans. AP steady-state rate-dependence and restitution is steeper in pigs. Porcine Ca2+ transients can have two components, unlike human. Although a reliable computational model for human ventricular myocytes exists, one for pigs is lacking. This hampers translation from results obtained in pigs to human myocardium. Here, we developed a computational model of the pig ventricular cardiomyocyte AP using experimental datasets of the relevant ionic currents, Ca2+-handling, AP shape, AP duration restitution, and inducibility of triggered activity and alternans. To properly capture porcine Ca2+ transients, we introduced a two-step process with a faster release in the t-tubular region, followed by a slower diffusion-induced release from a non t-tubular subcellular region. The pig model behavior was compared with that of a human ventricular cardiomyocyte (O'Hara-Rudy) model. The pig, but not the human model, developed early afterdepolarizations (EADs) under block of IK1, while IKr block led to EADs in the human but not in the pig model. At fast rates (pacing cycle length = 400 ms), the human cell model was more susceptible to spontaneous Ca2+ release-mediated delayed afterdepolarizations (DADs) and triggered activity than pig. Fast pacing led to alternans in human but not pig. Developing species-specific models incorporating electrophysiology and Ca2+-handling provides a tool to aid translating antiarrhythmic and arrhythmogenic assessment from the bench to the clinic., Competing Interests: The authors have declared that no competing interests exist.

Forced expression of core cardiogenic transcription factors can directly reprogram fibroblasts to induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. This cardiac reprogramming approach provides a proof of concept for induced heart regeneration by converting a fibroblast fate to a cardiomyocyte fate. However, it remains elusive whether chamber-specific cardiomyocytes can be generated by cardiac reprogramming. Therefore, we assessed the ability of the cardiac reprogramming approach for chamber specification in vitro and in vivo. We found that in vivo cardiac reprogramming post-myocardial infarction exclusively induces a ventricular-like phenotype, while a major fraction of iCMs generated in vitro failed to determine their chamber identities. Our results suggest that in vivo cardiac reprogramming may have an inherent advantage of generating chamber-matched new cardiomyocytes as a potential heart regenerative approach.

Early afterdepolarizations (EADs) are abnormal depolarizations during the repolarizing phase of the action potential, which are associated with cardiac arrhythmogenesis. EADs are classified into phase-2 and phase-3 EADs. Phase-2 EADs occur during phase 2 of the action potential, with takeoff potentials typically above -40 mV. Phase-3 EADs occur during phase 3 of the action potential, with takeoff potential between -70 and -50 mV. Since the amplitude of phase-3 EADs can be as large as that of a regular action potential, they are also called triggered activities (TAs). This also makes phase-3 EADs and TAs much more arrhythmogenic than phase-2 EADs since they can propagate easily in tissue. Although phase-2 EADs have been widely observed, phase-3 EADs and TAs have been rarely demonstrated in isolated ventricular myocytes. Here we carry out computer simulations of three widely used ventricular action potential models to investigate the mechanisms of phase-3 EADs and TAs. We show that when the T-type Ca 2+ current (I Ca,T ) is absent (e.g., in normal ventricular myocytes), besides the requirement of increasing inward currents and reducing outward currents as for phase-2 EADs, the occurrence of phase-3 EADs and TAs requires a substantially large increase of the L-type Ca 2+ current and the slow component of the delayed rectifier K + current. The presence of I Ca,T (e.g., in neonatal and failing ventricular myocytes) can greatly reduce the thresholds of these two currents for phase-3 EADs and TAs. This implies that I Ca,T may play an important role in arrhythmogenesis in cardiac diseases., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

During cardiac excitation contraction coupling, the arrival of an action potential at the ventricular myocardium triggers voltage-dependent L-type Ca 2+ (Ca V 1.2) channels in individual myocytes to open briefly. The level of this Ca 2+ influx tunes the amplitude of Ca 2+ -induced Ca 2+ release from ryanodine receptors (RyR2) on the junctional sarcoplasmic reticulum and thus the magnitude of the elevation in intracellular Ca 2+ concentration and ultimately the downstream contraction. The number and activity of functional Ca V 1.2 channels at the t-tubule dyads dictates the amplitude of the Ca 2+ influx. Trafficking of these channels and their auxiliary subunits to the cell surface is thus tightly controlled and regulated to ensure adequate sarcolemmal expression to sustain this critical process. To that end, recent discoveries have revealed the existence of internal reservoirs of preformed Ca V 1.2 channels that can be rapidly mobilized to enhance sarcolemmal expression in times of acute stress when hemodynamic and metabolic demand increases. In this review, we provide an overview of the current thinking on Ca V 1.2 channel trafficking dynamics in the heart. We highlight the numerous points of control including the biosynthetic pathway, the endosomal recycling pathway, ubiquitination, and lysosomal and proteasomal degradation pathways, and discuss the effects of β-adrenergic and angiotensin receptor signaling cascades on this process.

Compact cardiomyocytes that make up the ventricular wall of the adult heart represent an important therapeutic target population for modeling and treating cardiovascular diseases. Here, we established a differentiation strategy that promotes the specification, proliferation and maturation of compact ventricular cardiomyocytes from human pluripotent stem cells (hPSCs). The cardiomyocytes generated under these conditions display the ability to use fatty acids as an energy source, a high mitochondrial mass, well-defined sarcomere structures and enhanced contraction force. These ventricular cells undergo metabolic changes indicative of those associated with heart failure when challenged in vitro with pathological stimuli and were found to generate grafts consisting of more mature cells than those derived from immature cardiomyocytes following transplantation into infarcted rat hearts. hPSC-derived atrial cardiomyocytes also responded to the maturation cues identified in this study, indicating that the approach is broadly applicable to different subtypes of the heart. Collectively, these findings highlight the power of recapitulating key aspects of embryonic and postnatal development for generating therapeutically relevant cell types from hPSCs.

A de novo missense variant in Rag GTPase protein C (RagC S75Y ) was recently identified in a syndromic dilated cardiomyopathy (DCM) patient. However, its pathogenicity and the related therapeutic strategy remain unclear. We generated a zebrafish Rragc S56Y (corresponding to human RagC S75Y ) knock-in (KI) line via TALEN technology. The KI fish manifested cardiomyopathy-like phenotypes and poor survival. Overexpression of RagC S75Y via adenovirus infection also led to increased cell size and fetal gene reprogramming in neonatal rat ventricle cardiomyocytes (NRVCMs), indicating a conserved mechanism. Further characterization identified aberrant mammalian target of rapamycin complex 1 (mTORC1) and transcription factor EB (TFEB) signaling, as well as metabolic abnormalities including dysregulated autophagy. However, mTOR inhibition failed to ameliorate cardiac phenotypes in the RagC S75Y cardiomyopathy models, concomitant with a failure to promote TFEB nuclear translocation. This observation was at least partially explained by increased and mTOR-independent physical interaction between RagC S75Y and TFEB in the cytosol. Importantly, TFEB overexpression resulted in more nuclear TFEB and rescued cardiomyopathy phenotypes. These findings suggest that S75Y is a pathogenic gain-of-function mutation in RagC that leads to cardiomyopathy. A primary pathological step of RagC S75Y cardiomyopathy is defective mTOR-TFEB signaling, which can be corrected by TFEB overexpression, but not mTOR inhibition.

Cardiac arrhythmias are the most common cause of sudden cardiac death worldwide. Lengthening the ventricular action potential duration (APD), either congenitally or via pathologic or pharmacologic means, predisposes to a life-threatening ventricular arrhythmia, Torsade de Pointes. I Ks (KCNQ1+KCNE1), a slowly activating K + current, plays a role in action potential repolarization. In this study, we screened a chemical library in silico by docking compounds to the voltage-sensing domain (VSD) of the I Ks channel. Here, we show that C28 specifically shifted I Ks VSD activation in ventricle to more negative voltages and reversed the drug-induced lengthening of APD. At the same dosage, C28 did not cause significant changes of the normal APD in either ventricle or atrium. This study provides evidence in support of a computational prediction of I Ks VSD activation as a potential therapeutic approach for all forms of APD prolongation. This outcome could expand the therapeutic efficacy of a myriad of currently approved drugs that may trigger arrhythmias., Competing Interests: Competing interest statement: J.S. and J.C. are cofounders of a startup company VivoCor LLC, which is targeting IKs for the treatment of cardiac arrhythmia.

Sarcolemmal α2 adrenoceptors (α2-AR), represented by α2A, α2B and α2C isoforms, can safeguard cardiac muscle under sympathoadrenergic surge by governing Ca 2+ handling and contractility of cardiomyocytes. Cardiomyocyte-specific targeting of α2-AR would provide cardiac muscle-delimited stress control and enhance the efficacy of cardiac malfunction treatments. However, little is known about the specific contribution of the α2-AR subtypes in modulating cardiomyocyte functions. Herein, we analyzed the expression profile of α2A, α2B and α2C subtypes in mouse ventricle and conducted electrophysiological antagonist assay evaluating the contribution of these isoforms to the suppression of L-type Ca 2+ current ( I CaL ). Patch-clamp electro-pharmacological studies revealed that the α2-agonist-induced suppression of I CaL involves mainly the α2C, to a lesser extent the α2B, and not the α2A isoforms. RT-qPCR evaluation revealed the presence of adra2b and adra2c (α2B and α2C isoform genes, respectively), but was unable to identify the expression of adra2a (α2A isoform gene) in the mouse left ventricle. Immunoblotting confirmed the presence only of the α2B and the α2C proteins in this tissue. The identified α2-AR isoform-linked regulation of I CaL in the mouse ventricle provides an important molecular substrate for the cardioprotective targeting.

Aims/introduction: Abnormalities in Ca 2+ signaling have a key role in hemodynamic dysfunction in diabetic heart. The purpose of this study was to explore the effects of streptozotocin (STZ)-induced diabetes on Ca 2+ signaling in epicardial (EPI) and endocardial (ENDO) cells of the left ventricle after 5-6 months of STZ injection., Materials and Methods: Whole-cell patch clamp was used to measure the L-type Ca 2+ channel (LTCC) and Na + /Ca 2+ exchanger currents. Fluorescence photometry techniques were used to measure intracellular free Ca 2+ concentration., Results: Although the LTCC current was not significantly altered, the amplitude of Ca 2+ transients increased significantly in EPI-STZ and ENDO-STZ compared with controls. Time to peak LTCC current, time to peak Ca 2+ transient, time to half decay of LTCC current and time to half decay of Ca 2+ transients were not significantly changed in EPI-STZ and ENDO-STZ myocytes compared with controls. The Na + /Ca 2+ exchanger current was significantly smaller in EPI-STZ and in ENDO-STZ compared with controls., Conclusions: STZ-induced diabetes resulted in an increase in amplitude of Ca 2+ transients in EPI and ENDO myocytes that was independent of the LTCC current. Such an effect can be attributed, at least in part, to the dysfunction of the Na + /Ca 2+ exchanger. Additional studies are warranted to improve our understanding of the regional impact of diabetes on Ca 2+ signaling, which will facilitate the discovery of new targeted treatments for diabetic cardiomyopathy., (© 2020 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

The number and activity of Ca v 1.2 channels in the cardiomyocyte sarcolemma tunes the magnitude of Ca 2+ -induced Ca 2+ release and myocardial contraction. β-Adrenergic receptor ( βAR ) activation stimulates sarcolemmal insertion of Ca V 1.2. This supplements the preexisting sarcolemmal Ca V 1.2 population, forming large "superclusters" wherein neighboring channels undergo enhanced cooperative-gating behavior, amplifying Ca 2+ influx and myocardial contractility. Here, we determine this stimulated insertion is fueled by an internal reserve of early and recycling endosome-localized, presynthesized Ca V 1.2 channels. βAR -activation decreased Ca V 1.2/endosome colocalization in ventricular myocytes, as it triggered "emptying" of endosomal Ca V 1.2 cargo into the t-tubule sarcolemma. We examined the rapid dynamics of this stimulated insertion process with live-myocyte imaging of channel trafficking, and discovered that Ca V 1.2 are often inserted into the sarcolemma as preformed, multichannel clusters. Similarly, entire clusters were removed from the sarcolemma during endocytosis, while in other cases, a more incremental process suggested removal of individual channels. The amplitude of the stimulated insertion response was doubled by coexpression of constitutively active Rab4a, halved by coexpression of dominant-negative Rab11a, and abolished by coexpression of dominant-negative mutant Rab4a. In ventricular myocytes, βAR -stimulated recycling of Ca V 1.2 was diminished by both nocodazole and latrunculin-A, suggesting an essential role of the cytoskeleton in this process. Functionally, cytoskeletal disruptors prevented βAR -activated Ca 2+ current augmentation. Moreover, βAR -regulation of Ca V 1.2 was abolished when recycling was halted by coapplication of nocodazole and latrunculin-A. These findings reveal that βAR -stimulation triggers an on-demand boost in sarcolemmal Ca V 1.2 abundance via targeted Rab4a- and Rab11a-dependent insertion of channels that is essential for βAR -regulation of cardiac Ca V 1.2., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Generating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Current differentiation protocols, however, have limited use due to their production of heterogenous cell populations, primarily consisting of ventricular-like CMs. Here we describe the creation of two chamber-specific reporter hiPSC lines by site-directed genomic integration using CRISPR-Cas9 technology. In the MYL2-tdTomato reporter, the red fluorescent tdTomato was inserted upstream of the 3' untranslated region of the Myosin Light Chain 2 (MYL2) gene in order faithfully label hiPSC-derived ventricular-like CMs while avoiding disruption of endogenous gene expression. Similarly, in the SLN-CFP reporter, Cyan Fluorescent Protein (CFP) was integrated downstream of the coding region of the atrial-specific gene, Sarcolipin (SLN). Purification of tdTomato+ and CFP+ CMs using flow cytometry coupled with transcriptional and functional characterization validated these genetic tools for their use in the isolation of bona fide ventricular-like and atrial-like CMs, respectively. Finally, we successfully generated a double reporter system allowing for the isolation of both ventricular and atrial CM subtypes within a single hiPSC line. These tools provide a platform for chamber-specific hiPSC-derived CM purification and analysis in the context of atrial- or ventricular-specific disease and therapeutic opportunities.

Ca 2+ signaling is critical for heart development; however, the precise roles and regulatory pathways of Ca 2+ transport proteins in cardiogenesis remain largely unknown. Sodium-calcium exchanger 1 (Ncx1) is responsible for Ca 2+ efflux in cardiomyocytes. It is involved in cardiogenesis, while the mechanism is unclear. Here, using the forward genetic screening in zebrafish, we identified a novel mutation at a highly-conserved leucine residue in ncx1 gene (mutant LDD353 /ncx1h L154P ) that led to smaller hearts with reduced heart rate and weak contraction. Mechanistically, the number of ventricular but not atrial cardiomyocytes was reduced in ncx1h L154P zebrafish. These defects were mimicked by knockdown or knockout of ncx1h. Moreover, ncx1h L154P had cytosolic and mitochondrial Ca 2+ overloading and Ca 2+ transient suppression in cardiomyocytes. Furthermore, ncx1h L154P and ncx1h morphants downregulated cardiac transcription factors hand2 and gata4 in the cardiac regions, while overexpression of hand2 and gata4 partially rescued cardiac defects including the number of ventricular myocytes. These findings demonstrate an essential role of the novel 154th leucine residue in the maintenance of Ncx1 function in zebrafish, and reveal previous unrecognized critical roles of the 154th leucine residue and Ncx1 in the formation of ventricular cardiomyocytes by at least partially regulating the expression levels of gata4 and hand2.

Mitochondria are vital organelles inside the cell and contribute to intracellular calcium (Ca2+) dynamics directly and indirectly via calcium exchange, ATP generation, and production of reactive oxygen species (ROS). Arrhythmogenic Ca2+ alternans in cardiac myocytes has been observed in experiments under abnormal mitochondrial depolarization. However, complex signaling pathways and Ca2+ cycling between mitochondria and cytosol make it difficult in experiments to reveal the underlying mechanisms of Ca2+ alternans under abnormal mitochondrial depolarization. In this study, we use a newly developed spatiotemporal ventricular myocyte computer model that integrates mitochondrial Ca2+ cycling and complex signaling pathways to investigate the mechanisms of Ca2+ alternans during mitochondrial depolarization. We find that elevation of ROS in response to mitochondrial depolarization plays a critical role in promoting Ca2+ alternans. Further examination reveals that the redox effect of ROS on ryanodine receptors and sarco/endoplasmic reticulum Ca2+-ATPase synergistically promote alternans. Upregulation of mitochondrial Ca2+ uniporter promotes Ca2+ alternans via Ca2+-dependent mitochondrial permeability transition pore opening. Due to their relatively slow kinetics, oxidized Ca2+/calmodulin-dependent protein kinase II activation and ATP do not play significant roles acutely in the genesis of Ca2+ alternans after mitochondrial depolarization, but their roles can be significant in the long term, mainly through their effects on sarco/endoplasmic reticulum Ca2+-ATPase activity. In conclusion, mitochondrial depolarization promotes Ca2+ alternans acutely via the redox effect of ROS and chronically by ATP reduction. It suppresses Ca2+ alternans chronically through oxidized Ca2+/calmodulin-dependent protein kinase II activation., Competing Interests: The authors have declared that no competing interests exist.

We developed a method to evaluate the activity of the Na + -Ca 2+ exchanger (NCX) and sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA) with fluorescence microscopy in mouse ventricular cardiomyocytes. In non-beating ventricular cardiomyocytes, α-adrenoceptor stimulation by phenylephrine caused a decrease in the cytoplasmic Ca 2+ concentration, which was inhibited by SEA0400, an NCX inhibitor, but not cyclopiazonic acid, a SERCA inhibitor. β-Adrenoceptor stimulation by isoprenaline caused a decrease in the cytoplasmic Ca 2+ concentration, which was inhibited by cyclopiazonic acid but not SEA0400. Ellagic acid, a phenolic phytochemical, also decreased the basal Ca 2+ concentration, which was inhibited by cyclopiazonic acid, but not SEA0400. Thus, this method using fluorescent microscopy and specific inhibitors would be useful for the evaluation of pharmacological agents acting on NCX and SERCA.

The prevalence of arrhythmia in patients with hypertension has gradually attracted widespread attention. However, the relationship between hypertension and arrhythmia still lacks more attention. Herein, we explore the biomechanical mechanism of arrhythmia in hypertensive rats and the effect of amiodarone on biomechanical properties. We applied micro-mechanics and amiodarone to stimulate single ventricular myocytes to compare changes of mechanical parameters and the mechanism was investigated in biomechanics. Then we verified the expression changes of genes and long non-coding RNAs (lncRNAs) related to myocardial mechanics to explore the effect of amiodarone on biomechanical properties. The results found that the stiffness of ventricular myocytes and calcium ion levels in hypertensive rats were significantly increased and amiodarone could alleviate the intracellular calcium response and biomechanical stimulation. In addition, experiments showed spontaneously hypertensive rats were more likely to induce arrhythmia and preoperative amiodarone intervention significantly reduced the occurrence of arrhythmias. Meanwhile, high-throughput sequencing showed the genes and lncRNAs related to myocardial mechanics changed significantly in the spontaneously hypertensive rats that amiodarone was injected. These results strengthen the evidence that hypertension rats are prone to arrhythmia with abnormal myocardial biomechanical properties. Amiodarone effectively inhibit arrhythmia by improving the myocardial biomechanical properties and weakening the sensitivity of mechanical stretch stimulation.

Cardiovascular disease is the leading cause of death worldwide. Advanced insights into disease mechanisms and therapeutic strategies require a deeper understanding of the molecular processes involved in the healthy heart. Knowledge of the full repertoire of cardiac cells and their gene expression profiles is a fundamental first step in this endeavour. Here, using state-of-the-art analyses of large-scale single-cell and single-nucleus transcriptomes, we characterize six anatomical adult heart regions. Our results highlight the cellular heterogeneity of cardiomyocytes, pericytes and fibroblasts, and reveal distinct atrial and ventricular subsets of cells with diverse developmental origins and specialized properties. We define the complexity of the cardiac vasculature and its changes along the arterio-venous axis. In the immune compartment, we identify cardiac-resident macrophages with inflammatory and protective transcriptional signatures. Furthermore, analyses of cell-to-cell interactions highlight different networks of macrophages, fibroblasts and cardiomyocytes between atria and ventricles that are distinct from those of skeletal muscle. Our human cardiac cell atlas improves our understanding of the human heart and provides a valuable reference for future studies.