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Sterile inflammation after myocardial infarction is classically credited to myeloid cells interacting with dead cell debris in the infarct zone1,2. Here we show that cardiomyocytes are the dominant initiators of a previously undescribed type I interferon response in the infarct borderzone. Using spatial transcriptomics analysis in mice and humans, we find that myocardial infarction induces colonies of interferon-induced cells (IFNICs) expressing interferon-stimulated genes decorating the borderzone, where cardiomyocytes experience mechanical stress, nuclear rupture and escape of chromosomal DNA. Cardiomyocyte-selective deletion of Irf3 abrogated IFNIC colonies, whereas mice lacking Irf3 in fibroblasts, macrophages, neutrophils or endothelial cells, Ccr2-deficient mice or plasmacytoid-dendritic-cell-depleted mice did not. Interferons blunted the protective matricellular programs and contractile function of borderzone fibroblasts, and increased vulnerability to pathological remodelling. In mice that died after myocardial infarction, IFNIC colonies were immediately adjacent to sites of ventricular rupture, while mice lacking IFNICs were protected from rupture and exhibited improved survival3. Together, these results reveal a pathological borderzone niche characterized by a cardiomyocyte-initiated innate immune response. We suggest that selective inhibition of IRF3 activation in non-immune cells could limit ischaemic cardiomyopathy while avoiding broad immunosuppression.

Vascular smooth muscle cells (VSMCs) envelop vertebrate brain arteries and play a crucial role in regulating cerebral blood flow and neurovascular coupling. The dedifferentiation of VSMCs is implicated in cerebrovascular disease and neurodegeneration. Despite its importance, the process of VSMC differentiation on brain arteries during development remains inadequately characterized. Understanding this process could aid in reprogramming and regenerating dedifferentiated VSMCs in cerebrovascular diseases. In this study, we investigated VSMC differentiation on zebrafish circle of Willis (CoW), comprising major arteries that supply blood to the vertebrate brain. We observed that arterial specification of CoW endothelial cells (ECs) occurs after their migration from cranial venous plexus to form CoW arteries. Subsequently, acta2+ VSMCs differentiate from pdgfrb+ mural cell progenitors after they were recruited to CoW arteries. The progression of VSMC differentiation exhibits a spatiotemporal pattern, advancing from anterior to posterior CoW arteries. Analysis of blood flow suggests that earlier VSMC differentiation in anterior CoW arteries correlates with higher red blood cell velocity and wall shear stress. Furthermore, pulsatile flow induces differentiation of human brain PDGFRB+ mural cells into VSMCs, and blood flow is required for VSMC differentiation on zebrafish CoW arteries. Consistently, flow-responsive transcription factor klf2a is activated in ECs of CoW arteries prior to VSMC differentiation, and klf2a knockdown delays VSMC differentiation on anterior CoW arteries. In summary, our findings highlight blood flow activation of endothelial klf2a as a mechanism regulating initial VSMC differentiation on vertebrate brain arteries.

During postnatal cardiac hypertrophy, cardiomyocytes undergo mitotic exit, relying on DNA replication-independent mechanisms of histone turnover to maintain chromatin organization and gene transcription. In other tissues, circadian oscillations in nucleosome occupancy influence clock-controlled gene expression, suggesting a role for the circadian clock in temporal control of histone turnover and coordinated cardiomyocyte gene expression. We sought to elucidate roles for the master circadian transcription factor, Bmal1, in histone turnover, chromatin organization, and myocyte-specific gene expression and cell growth in the neonatal period. Bmal1 knockdown in neonatal rat ventricular myocytes decreased myocyte size, total cellular protein synthesis, and transcription of the fetal hypertrophic gene Nppb after treatment with serum or the α-adrenergic agonist phenylephrine. Depletion of Bmal1 decreased the expression of clock-controlled genes Per2 and Tcap, as well as Sik1, a Bmal1 target upregulated in adult versus embryonic hearts. Bmal1 knockdown impaired Per2 and Sik1 promoter accessibility as measured by micrococcal nuclease-quantitative PCR and impaired histone turnover as measured by metabolic labeling of acid-soluble chromatin fractions. Sik1 knockdown in turn decreased myocyte size, while simultaneously inhibiting natriuretic peptide B transcription and activating Per2 transcription. Linking these changes to chromatin remodeling, depletion of the replication-independent histone variant H3.3a inhibited myocyte hypertrophy and prevented phenylephrine-induced changes in clock-controlled gene transcription. Bmal1 is required for neonatal myocyte growth, replication-independent histone turnover, and chromatin organization at the Sik1 promoter. Sik1 represents a novel clock-controlled gene that coordinates myocyte growth with hypertrophic and clock-controlled gene transcription. Replication-independent histone turnover is required for transcriptional remodeling of clock-controlled genes in cardiac myocytes in response to growth stimuli.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are powerful in vitro models to study the mechanisms underlying cardiomyopathies and cardiotoxicity. Quantification of the contractile function in single hiPSC-CMs at high-throughput and over time is essential to disentangle how cellular mechanisms affect heart function. Here, we present CONTRAX, an open-access, versatile, and streamlined pipeline for quantitative tracking of the contractile dynamics of single hiPSC-CMs over time. Three software modules enable: parameter-based identification of single hiPSC-CMs; automated video acquisition of >200 cells/hour; and contractility measurements via traction force microscopy. We analyze >4,500 hiPSC-CMs over time in the same cells under orthogonal conditions of culture media and substrate stiffnesses; +/- drug treatment; +/- cardiac mutations. Using undirected clustering, we reveal converging maturation patterns, quantifiable drug response to Mavacamten and significant deficiencies in hiPSC-CMs with disease mutations. CONTRAX empowers researchers with a potent quantitative approach to develop cardiac therapies.

BackgroundChronic sympathetic stimulation drives desensitization and downregulation of β1 adrenergic receptor (β1AR) in heart failure. We aim to explore the differential downregulation subcellular pools of β1AR signaling in the heart.Methods and resultsWe applied chronic infusion of isoproterenol to induced cardiomyopathy in male C57BL/6J mice. We applied confocal and proximity ligation assay to examine β1AR association with L-type calcium channel, ryanodine receptor 2, and SERCA2a ((Sarco)endoplasmic reticulum calcium ATPase 2a) and Förster resonance energy transfer-based biosensors to probe subcellular β1AR-PKA (protein kinase A) signaling in ventricular myocytes. Chronic infusion of isoproterenol led to reduced β1AR protein levels, receptor association with L-type calcium channel and ryanodine receptor 2 measured by proximity ligation (puncta/cell, 29.65 saline versus 14.17 isoproterenol, P

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 Ca2+ 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

The gastrointestinal system is a hollow organ affected by fibrostenotic diseases that cause volumetric compromise of the lumen via smooth muscle hypertrophy and fibrosis. Many of the driving mechanisms remain unclear. Yes-associated protein-1 (YAP) is a critical mechanosensory transcriptional regulator that mediates cell hypertrophy in response to elevated extracellular rigidity. In the type 2 inflammatory disorder, eosinophilic esophagitis (EoE), phospholamban (PLN) can induce smooth muscle cell hypertrophy. We used EoE as a disease model for understanding a mechanistic pathway in which PLN and YAP interact in response to rigid extracellular substrate to induce smooth muscle cell hypertrophy. PLN-induced YAP nuclear sequestration in a feed-forward loop caused increased cell size in response to a rigid substrate. This mechanism of rigidity sensing may have previously unappreciated clinical implications for PLN-expressing hollow systems such as the esophagus and heart.

BACKGROUND: Dilated cardiomyopathy (DCM) is one of the most common causes of heart failure. Multiple identified mutations in nexilin (NEXN) have been suggested to be linked with severe DCM. However, the exact association between multiple mutations of Nexn and DCM remains unclear. Moreover, it is critical for the development of precise and effective therapeutics in treatments of DCM. RESULTS: In our study, Nexn global knockout mice and mice carrying human equivalent G645del mutation are studied using functional gene rescue assays. AAV-mediated gene delivery is conducted through systemic intravenous injections at the neonatal stage. Heart tissues are analyzed by immunoblots, and functions are assessed by echocardiography. Here, we identify functional components of Nexilin and demonstrate that exogenous introduction could rescue the cardiac function and extend the lifespan of Nexn knockout mouse models. Similar therapeutic effects are also obtained in G645del mice, providing a promising intervention for future clinical therapeutics. CONCLUSIONS: In summary, we demonstrated that a single injection of AAV-Nexn was capable to restore the functions of cardiomyocytes and extended the lifespan of Nexn knockout and G645del mice. Our study represented a long-term gene replacement therapy for DCM that potentially covers all forms of loss-of-function mutations in NEXN.

The Na+-Ca2+ exchanger (NCX1) is the dominant Ca2+ extrusion mechanism in cardiac myocytes. NCX1 activity is inhibited by intracellular Na+ via a process known as Na+-dependent inactivation. A central question is whether this inactivation plays a physiological role in heart function. Using CRISPR/Cas9, we inserted the K229Q mutation in the gene (Slc8a1) encoding for NCX1. This mutation removes the Na+-dependent inactivation while preserving transport properties and other allosteric regulations. NCX1 mRNA levels, protein expression, and protein localization are unchanged in K229Q male mice. However, they exhibit reduced left ventricular ejection fraction and fractional shortening, while displaying a prolonged QT interval. K229Q ventricular myocytes show enhanced NCX1 activity, resulting in action potential prolongation, higher incidence of aberrant action potentials, a faster decline of Ca2+ transients, and depressed cell shortening. The results demonstrate that NCX1 Na+-dependent inactivation plays an essential role in heart function by affecting both cardiac excitability and contractility.

BackgroundRecently shown to regulate cardiac development, the secreted axon guidance molecule SLIT3 maintains its expression in the postnatal heart. Despite its known expression in the cardiovascular system after birth, SLIT3's relevance to cardiovascular function in the postnatal state remains unknown. As such, the objectives of this study were to determine the postnatal myocardial sources of SLIT3 and to evaluate its functional role in regulating the cardiac response to pressure overload stress.MethodsWe performed in vitro studies on cardiomyocytes and myocardial tissue samples from patients and performed in vivo investigation with SLIT3 and ROBO1 (roundabout homolog 1) mutant mice undergoing transverse aortic constriction to establish the role of SLIT3-ROBO1 in adverse cardiac remodeling.ResultsWe first found that SLIT3 transcription was increased in myocardial tissue obtained from patients with congenital heart defects that caused ventricular pressure overload. Immunostaining of hearts from WT (wild-type) and reporter mice revealed that SLIT3 is secreted by cardiac stromal cells, namely fibroblasts and vascular mural cells, within the heart. Conditioned media from cardiac fibroblasts and vascular mural cells both stimulated cardiomyocyte hypertrophy in vitro, an effect that was partially inhibited by an anti-SLIT3 antibody. Also, the N-terminal, but not the C-terminal, fragment of SLIT3 and the forced overexpression of SLIT3 stimulated cardiomyocyte hypertrophy and the transcription of hypertrophy-related genes. We next determined that ROBO1 was the most highly expressed roundabout receptor in cardiomyocytes and that ROBO1 mediated SLIT3's hypertrophic effects in vitro. In vivo, Tcf21+ fibroblast and Tbx18+ vascular mural cell-specific knockout of SLIT3 in mice resulted in decreased left ventricular hypertrophy and cardiac fibrosis after transverse aortic constriction. Furthermore, α-MHC+ cardiomyocyte-specific deletion of ROBO1 also preserved left ventricular function and abrogated hypertrophy, but not fibrosis, after transverse aortic constriction.ConclusionsCollectively, these results indicate a novel role for the SLIT3-ROBO1-signaling axis in regulating postnatal cardiomyocyte hypertrophy induced by pressure overload.

Human induced pluripotent stem cell (hiPSC) to cardiomyocyte (CM) differentiation has reshaped approaches to studying cardiac development and disease. In this study, we employed a genome-wide CRISPR screen in a hiPSC to CM differentiation system and reveal here that BRD4, a member of the bromodomain and extraterminal (BET) family, regulates CM differentiation. Chemical inhibition of BET proteins in mouse embryonic stem cell (mESC)-derived or hiPSC-derived cardiac progenitor cells (CPCs) results in decreased CM differentiation and persistence of cells expressing progenitor markers. In vivo, BRD4 deletion in second heart field (SHF) CPCs results in embryonic or early postnatal lethality, with mutants demonstrating myocardial hypoplasia and an increase in CPCs. Single-cell transcriptomics identified a subpopulation of SHF CPCs that is sensitive to BRD4 loss and associated with attenuated CM lineage-specific gene programs. These results highlight a previously unrecognized role for BRD4 in CM fate determination during development and a heterogenous requirement for BRD4 among SHF CPCs.

The heart, which is the first organ to develop, is highly dependent on its form to function1,2. 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.

Cerebrospinal fluid (CSF) in the subarachnoid space around the brain has long been known to drain through the lymphatics to cervical lymph nodes1-17, but the connections and regulation have been challenging to identify. Here, using fluorescent CSF tracers in Prox1-GFP lymphatic reporter mice18, we found that the nasopharyngeal lymphatic plexus is a major hub for CSF outflow to deep cervical lymph nodes. This plexus had unusual valves and short lymphangions but no smooth-muscle coverage, whereas downstream deep cervical lymphatics had typical semilunar valves, long lymphangions and smooth muscle coverage that transported CSF to the deep cervical lymph nodes. α-Adrenergic and nitric oxide signalling in the smooth muscle cells regulated CSF drainage through the transport properties of deep cervical lymphatics. During ageing, the nasopharyngeal lymphatic plexus atrophied, but deep cervical lymphatics were not similarly altered, and CSF outflow could still be increased by adrenergic or nitric oxide signalling. Single-cell analysis of gene expression in lymphatic endothelial cells of the nasopharyngeal plexus of aged mice revealed increased type I interferon signalling and other inflammatory cytokines. The importance of evidence for the nasopharyngeal lymphatic plexus functioning as a CSF outflow hub is highlighted by its regression during ageing. Yet, the ageing-resistant pharmacological activation of deep cervical lymphatic transport towards lymph nodes can still increase CSF outflow, offering an approach for augmenting CSF clearance in age-related neurological conditions in which greater efflux would be beneficial.

Cardiac dysfunction is a hallmark of aging in humans and mice. Here we report that a two-week treatment to restore youthful Bridging Integrator 1 (BIN1) levels in the hearts of 24-month-old mice rejuvenates cardiac function and substantially reverses the aging phenotype. Our data indicate that age-associated overexpression of BIN1 occurs alongside dysregulated endosomal recycling and disrupted trafficking of cardiac CaV1.2 and type 2 ryanodine receptors. These deficiencies affect channel function at rest and their upregulation during acute stress. In vivo echocardiography reveals reduced systolic function in old mice. BIN1 knockdown using an adeno-associated virus serotype 9 packaged shRNA-mBIN1 restores the nanoscale distribution and clustering plasticity of ryanodine receptors and recovers Ca2+ transient amplitudes and cardiac systolic function toward youthful levels. Enhanced systolic function correlates with increased phosphorylation of the myofilament protein cardiac myosin binding protein-C. These results reveal BIN1 knockdown as a novel therapeutic strategy to rejuvenate the aging myocardium.

Cells interpret a variety of signals through G-protein-coupled receptors (GPCRs) and stimulate the generation of second messengers such as cyclic adenosine monophosphate (cAMP). A long-standing puzzle is deciphering how GPCRs elicit different physiological responses despite generating similar levels of cAMP. We previously showed that some GPCRs generate cAMP from both the plasma membrane and the Golgi apparatus. Here we demonstrate that cardiomyocytes distinguish between subcellular cAMP inputs to elicit different physiological outputs. We show that generating cAMP from the Golgi leads to the regulation of a specific protein kinase A (PKA) target that increases the rate of cardiomyocyte relaxation. In contrast, cAMP generation from the plasma membrane activates a different PKA target that increases contractile force. We further validated the physiological consequences of these observations in intact zebrafish and mice. Thus, we demonstrate that the same GPCR acting through the same second messenger regulates cardiac contraction and relaxation dependent on its subcellular location.

BACKGROUND: Drug-induced QT prolongation (diLQT) is a feared side effect that could expose susceptible individuals to fatal arrhythmias. The occurrence of diLQT is primarily attributed to unintended drug interactions with cardiac ion channels, notably the hERG (human ether-a-go-go-related gene) channels that generate the delayed-rectifier potassium current (IKr) and thereby regulate the late repolarization phase. There is an important interindividual susceptibility to develop diLQT, which is of unknown origin but can be reproduced in patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs). We aimed to investigate the dynamics of hERG channels in response to sotalol and to identify regulators of the susceptibility to developing diLQT. METHODS: We measured electrophysiological activity and cellular distribution of hERG channels after hERG blocker treatment in iPS-CMs derived from patients with highest sensitivity (HS) or lowest sensitivity (LS) to sotalol administration in vivo (ie, on the basis of the measure of the maximal change in QT interval 3 hours after administration). Specific small interfering RNAs and CAVIN1-T2A-GFP adenovirus were used to manipulate CAVIN1 expression. RESULTS: Whereas HS and LS iPS-CMs showed similar electrophysiological characteristics at baseline, the late repolarization phase was prolonged and IKr significantly decreased after exposure of HS iPS-CMs to low sotalol concentrations. IKr reduction was caused by a rapid translocation of hERG channel from the membrane to the cytoskeleton-associated fractions upon sotalol application. CAVIN1, essential for caveolae biogenesis, was 2× more highly expressed in HS iPS-CMs, and its knockdown by small interfering RNA reduced their sensitivity to sotalol. CAVIN1 overexpression in LS iPS-CMs using adenovirus showed reciprocal effects. We found that treatment with sotalol promoted translocation of the hERG channel from the plasma membrane to the cytoskeleton fractions in a process dependent on CAVIN1 (caveolae associated protein 1) expression. CAVIN1 silencing reduced the number of caveolae at the membrane and abrogated the translocation of hERG channel in sotalol-treated HS iPS-CMs. CAVIN1 also controlled cardiomyocyte responses to other hERG blockers, such as E4031, vandetanib, and clarithromycin. CONCLUSIONS: Our study identifies unbridled turnover of the potassium channel hERG as a mechanism supporting the interindividual susceptibility underlying diLQT development and demonstrates how this phenomenon is finely tuned by CAVIN1. [ABSTRACT FROM AUTHOR]

Nicotine, a key constituent of tobacco/electronic cigarettes causes cardiovascular injury and mortality. Nicotine is known to induce oxidative stress and mitochondrial dysfunction in cardiomyocytes leading to cell death. However, the underlying mechanisms remain unclear. Pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) is a member of metal-dependent protein phosphatase (PPM) family and is known to dephosphorylate several AGC family kinases and thereby regulate a diverse set of cellular functions including cell growth, survival, and death. Our lab has previously demonstrated that PHLPP1 removal reduced cardiomyocyte death and cardiac dysfunction following injury. Here, we present a novel finding that nicotine exposure significantly increased PHLPP1 protein expression in the adolescent rodent heart. Building upon our in vivo finding, we determined the mechanism of PHLPP1 expression in cardiomyocytes. Nicotine significantly increased PHLPP1 protein expression without altering PHLPP2 in cardiomyocytes. In cardiomyocytes, nicotine significantly increased NADPH oxidase 4 (NOX4), which coincided with increased reactive oxygen species (ROS) and increased cardiomyocyte apoptosis which were dependent on PHLPP1 expression. PHLPP1 expression was both necessary and sufficient for nicotine induced mitochondrial dysfunction. Mechanistically, nicotine activated extracellular signal-regulated protein kinases (ERK1/2) and subsequent eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) to increase PHLPP1 protein expression. Inhibition of protein synthesis with cycloheximide (CHX) and 4EGI-1 abolished nicotine induced PHLPP1 protein expression. Moreover, inhibition of ERK1/2 activity by U0126 significantly blocked nicotine induced PHLPP1 expression. Overall, this study reveals a novel mechanism by which nicotine regulates PHLPP1 expression through ERK-4E-BP1 signaling axis to drive cardiomyocyte injury. [Display omitted] • Nicotine increases PHLPP1 expression in cardiomyocytes leading to increased ROS, mitochondrial dysfunction, and death. • Overexpression of PHLPP1 alone increased ROS and mitochondrial dysfunction in cardiomyocytes. • Removal of PHLPP1 protected cardiomyocytes from nicotine induced oxidative stress, mitochondrial dysfunction, and damage. • Nicotine increased PHLPP1 through ERK/4E-BP1 signaling axis. [ABSTRACT FROM AUTHOR]

Recent reports of myocardial recovery after mechanical unloading with left ventricular assist devices (LVADs) have challenged the prevailing notion that end-stage heart failure (HF) is irreversible. To improve our understanding of this phenomenon, we comprehensively analysed the structural, functional, and energetic changes in failing human cardiomyocytes after LVAD implantation. Based on a prospectively registered protocol (PROSPERO-CRD42022380214), 30 eligible studies were identified from 940 records with a pooled population of 648 patients predominantly with non-ischaemic cardiomyopathy. LVAD led to a substantial regression in myocyte size similar to that of donor hearts (standardised mean difference, −1.29; p<0.001). The meta-regression analysis revealed that HF duration was a significant modifier on the changes in myocyte size. There were some suggestions of fibrosis reversal (−5.17%; p=0.009); however, this was insignificant after sensitivity analysis. Developed force did not improve in cardiac trabeculae (n=5 studies); however, non-physiological isometric contractions were tested. At the myocyte level (n=4 studies), contractile kinetics improved where the time-to-peak force reduced by 41.7%–50.7% and time to 50% relaxation fell by 47.4%–62.1% (p<0.05). Qualitatively, LVAD enhanced substrate utilisation and mitochondrial function (n=6 studies). Most studies were at a high risk of bias. The regression of maladaptive hypertrophy, partial fibrosis reversal, and normalisation in metabolic pathways after LVAD may be a testament to the heart's remarkable plasticity, even in the advanced stages of HF. However, inconsistencies exist in force-generating capabilities. Using more physiological force-length work-loop assays, addressing the high risks of bias and clinical heterogeneity are crucial to better understand the phenomenon of reverse remodelling. [ABSTRACT FROM AUTHOR]

BackgroundNitric oxide (NO) has been identified as a signaling molecule generated during β-adrenergic receptor stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of CaMKIIδ (Ca2+/calmodulin kinase II delta) is emerging. NO donors are routinely used clinically for their cardioprotective effects on the heart, but it is unknown how NO donors modulate the proarrhythmic CaMKII to alter cardiac arrhythmia incidence. We test the role of S-nitrosylation of CaMKIIδ at the Cysteine-273 inhibitory site and cysteine-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during β-adrenergic receptor stimulation.MethodsWe measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at cysteine-273 or cysteine-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 μM), sodium nitroprusside (200 μM), and β-adrenergic agonist isoproterenol (100 nmol/L).ResultsBoth WT and CaMKIIδ-KO cardiomyocytes responded to isoproterenol with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from isoproterenol-induced Ca2+ sparks and waves was mimicked by GSNO pretreatment in WT cardiomyocytes but lost in CaMKIIδ-C273S cardiomyocytes. When GSNO was applied after isoproterenol, this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pretreatment limited isoproterenol-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after isoproterenol sustained or exacerbated arrhythmic events.ConclusionsWe conclude that prior S-nitrosylation of CaMKIIδ at cysteine-273 can limit subsequent β-adrenergic receptor-induced arrhythmias, but that S-nitrosylation at cysteine-290 might worsen or sustain β-adrenergic receptor-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

Aortic aneurysms, which may dissect or rupture acutely and be lethal, can be a part of multisystem disorders that have a heritable basis. We report four patients with deficiency of selenocysteine-containing proteins due to selenocysteine Insertion Sequence Binding Protein 2 (SECISBP2) mutations who show early-onset, progressive, aneurysmal dilatation of the ascending aorta due to cystic medial necrosis. Zebrafish and male mice with global or vascular smooth muscle cell (VSMC)-targeted disruption of Secisbp2 respectively show similar aortopathy. Aortas from patients and animal models exhibit raised cellular reactive oxygen species, oxidative DNA damage and VSMC apoptosis. Antioxidant exposure or chelation of iron prevents oxidative damage in patients cells and aortopathy in the zebrafish model. Our observations suggest a key role for oxidative stress and cell death, including via ferroptosis, in mediating aortic degeneration.

The composite material-like extracellular matrix (ECM) in the sinoatrial node (SAN) supports the native pacemaking cardiomyocytes (PCMs). To test the roles of SAN ECM in the PCM phenotype and function, we engineered reconstructed-SAN heart tissues (rSANHTs) by recellularizing porcine SAN ECMs with hiPSC-derived PCMs. The hiPSC-PCMs in rSANHTs self-organized into clusters resembling the native SAN and displayed higher expression of pacemaker-specific genes and a faster automaticity compared with PCMs in reconstructed-left ventricular heart tissues (rLVHTs). To test the protective nature of SAN ECMs under strain, rSANHTs and rLVHTs were transplanted onto the murine thoracic diaphragm to undergo constant cyclic strain. All strained-rSANHTs preserved automaticity, whereas 66% of strained-rLVHTs lost their automaticity. In contrast to the strained-rLVHTs, PCMs in strained-rSANHTs maintained high expression of key pacemaker genes (HCN4, TBX3, and TBX18). These findings highlight the promotive and protective roles of the composite SAN ECM and provide valuable insights for pacemaking tissue engineering.

Mammals have limited capacity for heart regeneration, whereas zebrafish have extraordinary regeneration abilities. During zebrafish heart regeneration, endothelial cells promote cardiomyocyte cell cycle reentry and myocardial repair, but the mechanisms responsible for promoting an injury microenvironment conducive to regeneration remain incompletely defined. Here, we identify the matrix metalloproteinase Mmp14b as an essential regulator of heart regeneration. We identify a TEAD-dependent mmp14b endothelial enhancer induced by heart injury in zebrafish and mice, and we show that the enhancer is required for regeneration, supporting a role for Hippo signaling upstream of mmp14b. Last, we show that MMP-14 function in mice is important for the accumulation of Agrin, an essential regulator of neonatal mouse heart regeneration. These findings reveal mechanisms for extracellular matrix remodeling that promote heart regeneration.

Despite numerous efforts to generate mature human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), cells often remain immature, electrically isolated, and may not reflect adult biology. Conductive polymers are attractive candidates to facilitate electrical communication between hPSC-CMs, especially at sub-confluent cell densities or diseased cells lacking cell-cell junctions. Here we electrospun conductive polymers to create a conductive fiber mesh and assess if electrical signal propagation is improved in hPSC-CMs seeded on the mesh network. Matrix characterization indicated fiber structure remained stable over weeks in buffer, scaffold stiffness remained near in vivo cardiac stiffness, and electrical conductivity scaled with conductive polymer concentration. Cells remained adherent and viable on the scaffolds for at least 5 days. Transcriptomic profiling of hPSC-CMs cultured on conductive substrates for 3 days showed upregulation of cardiac and muscle-related genes versus non-conductive fibers. Structural proteins were more organized and calcium handling was improved on conductive substrates, even at sub-confluent cell densities; prolonged culture on conductive scaffolds improved membrane depolarization compared to non-conductive substrates. Taken together, these data suggest that blended, conductive scaffolds are stable, supportive of electrical coupling in hPSC-CMs, and promote maturation, which may improve our ability to model cardiac diseases and develop targeted therapies.

In arterial myocytes, the canonical function of voltage-gated CaV1.2 and KV2.1 channels is to induce myocyte contraction and relaxation through their responses to membrane depolarization, respectively. Paradoxically, KV2.1 also plays a sex-specific role by promoting the clustering and activity of CaV1.2 channels. However, the impact of KV2.1 protein organization on CaV1.2 function remains poorly understood. We discovered that KV2.1 forms micro-clusters, which can transform into large macro-clusters when a critical clustering site (S590) in the channel is phosphorylated in arterial myocytes. Notably, female myocytes exhibit greater phosphorylation of S590, and macro-cluster formation compared to males. Contrary to current models, the activity of KV2.1 channels seems unrelated to density or macro-clustering in arterial myocytes. Disrupting the KV2.1 clustering site (KV2.1S590A) eliminated KV2.1 macro-clustering and sex-specific differences in CaV1.2 cluster size and activity. We propose that the degree of KV2.1 clustering tunes CaV1.2 channel function in a sex-specific manner in arterial myocytes.

AimsAtrial fibrillation (AF), the most prevalent clinical arrhythmia, is associated with atrial remodelling manifesting as acute and chronic alterations in expression, function, and regulation of atrial electrophysiological and Ca2+-handling processes. These AF-induced modifications crosstalk and propagate across spatial scales creating a complex pathophysiological network, which renders AF resistant to existing pharmacotherapies that predominantly target transmembrane ion channels. Developing innovative therapeutic strategies requires a systems approach to disentangle quantitatively the pro-arrhythmic contributions of individual AF-induced alterations.Methods and resultsHere, we built a novel computational framework for simulating electrophysiology and Ca2+-handling in human atrial cardiomyocytes and tissues, and their regulation by key upstream signalling pathways [i.e. protein kinase A (PKA), and Ca2+/calmodulin-dependent protein kinase II (CaMKII)] involved in AF-pathogenesis. Populations of atrial cardiomyocyte models were constructed to determine the influence of subcellular ionic processes, signalling components, and regulatory networks on atrial arrhythmogenesis. Our results reveal a novel synergistic crosstalk between PKA and CaMKII that promotes atrial cardiomyocyte electrical instability and arrhythmogenic triggered activity. Simulations of heterogeneous tissue demonstrate that this cellular triggered activity is further amplified by CaMKII- and PKA-dependent alterations of tissue properties, further exacerbating atrial arrhythmogenesis.ConclusionsOur analysis reveals potential mechanisms by which the stress-associated adaptive changes turn into maladaptive pro-arrhythmic triggers at the cellular and tissue levels and identifies potential anti-AF targets. Collectively, our integrative approach is powerful and instrumental to assemble and reconcile existing knowledge into a systems network for identifying novel anti-AF targets and innovative approaches moving beyond the traditional ion channel-based strategy.

After heart injury, dead heart muscle is replaced by scar tissue. Fibroblasts can electrically couple with myocytes, and changes in fibroblast membrane potential can lead to myocyte excitability, which suggests that fibroblast-myocyte coupling in scar tissue may be responsible for arrhythmogenesis. However, the physiologic relevance of electrical coupling of myocytes and fibroblasts and its impact on cardiac excitability in vivo have never been demonstrated. We genetically engineered a mouse that expresses the optogenetic cationic channel ChR2 (H134R) exclusively in cardiac fibroblasts. After myocardial infarction, optical stimulation of scar tissue elicited organ-wide cardiac excitation and induced arrhythmias in these animals. Complementing computational modeling with experimental approaches, we showed that gap junctional and ephaptic coupling, in a synergistic yet functionally redundant manner, excited myocytes coupled to fibroblasts.

BackgroundCalcium (Ca) sparks are elementary units of subcellular Ca release in cardiomyocytes and other cells. Accordingly, Ca spark imaging is an essential tool for understanding the physiology and pathophysiology of Ca handling and is used to identify new drugs targeting Ca-related cellular dysfunction (eg, cardiac arrhythmias). The large volumes of imaging data produced during such experiments require accurate and high-throughput analysis.MethodsWe developed a new software tool SparkMaster 2 (SM2) for the analysis of Ca sparks imaged by confocal line-scan microscopy, combining high accuracy, flexibility, and user-friendliness. SM2 is distributed as a stand-alone application requiring no installation. It can be controlled using a simple-to-use graphical user interface, or using Python scripting.ResultsSM2 is shown to have the following strengths: (1) high accuracy at identifying Ca release events, clearly outperforming previous highly successful software SparkMaster; (2) multiple types of Ca release events can be identified using SM2: Ca sparks, waves, miniwaves, and long sparks; (3) SM2 can accurately split and analyze individual sparks within spark clusters, a capability not handled adequately by prior tools. We demonstrate the practical utility of SM2 in two case studies, investigating how Ca levels affect spontaneous Ca release, and how large-scale release events may promote release refractoriness. SM2 is also useful in atrial and smooth muscle myocytes, across different imaging conditions.ConclusionsSparkMaster 2 is a new, much-improved user-friendly software for accurate high-throughput analysis of line-scan Ca spark imaging data. It is free, easy to use, and provides valuable built-in features to facilitate visualization, analysis, and interpretation of Ca spark data. It should enhance the quality and throughput of Ca spark and wave analysis across cell types, particularly in the study of arrhythmogenic Ca release events in cardiomyocytes.

Hydrogels are attractive materials for tissue engineering, but efforts to date have shown limited ability to produce the microstructural features necessary to promote cellular self-organization into hierarchical three-dimensional (3D) organ models. Here we develop a hydrogel ink containing prefabricated gelatin fibres to print 3D organ-level scaffolds that recapitulate the intra- and intercellular organization of the heart. The addition of prefabricated gelatin fibres to hydrogels enables the tailoring of the ink rheology, allowing for a controlled sol-gel transition to achieve precise printing of free-standing 3D structures without additional supporting materials. Shear-induced alignment of fibres during ink extrusion provides microscale geometric cues that promote the self-organization of cultured human cardiomyocytes into anisotropic muscular tissues in vitro. The resulting 3D-printed ventricle in vitro model exhibited biomimetic anisotropic electrophysiological and contractile properties.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a severe aortic disease without effective pharmacological approaches. The nuclear hormone receptor LXRα (liver X receptor α), encoded by the NR1H3 gene, serves as a critical transcriptional mediator linked to several vascular pathologies, but its role in AAA remains elusive. METHODS: Through integrated analyses of human and murine AAA gene expression microarray data sets, we identified NR1H3 as a candidate gene regulating AAA formation. To investigate the role of LXRα in AAA formation, we used global Nr1h3-knockout and vascular smooth muscle cell--specific Nr1h3-knockout mice in 2 AAA mouse models induced with angiotensin II (1000 ng·kg·min; 28 days) or calcium chloride (CaCl2; 0.5 mol/L; 42 days). RESULTS: Upregulated LXRα was observed in the aortas of patients with AAA and in angiotensin II-- or CaCl2-treated mice. Global or vascular smooth muscle cell--specific Nr1h3 knockout inhibited AAA formation in 2 mouse models. Loss of LXRα function prevented extracellular matrix degeneration, inflammation, and vascular smooth muscle cell phenotypic switching. Uhrf1, an epigenetic master regulator, was identified as a direct target gene of LXRα by integrated analysis of transcriptome sequencing and chromatin immunoprecipitation sequencing. Susceptibility to AAA development was consistently enhanced by UHRF1 (ubiquitin-like containing PHD and RING finger domains 1) in both angiotensin II-- and CaCl2-induced mouse models. We then determined the CpG methylation status and promoter accessibility of UHRF1-mediated genes using CUT&Tag (cleavage under targets and tagmentation), RRBS (reduced representation bisulfite sequencing), and ATACseq (assay for transposase-accessible chromatin with sequencing) in vascular smooth muscle cells, which revealed that the recruitment of UHRF1 to the promoter of miR-26b led to DNA hypermethylation accompanied by relatively closed chromatin states, and caused downregulation of miR-26b expression in AAA. Regarding clinical significance, we found that underexpression of miR-26b-3p correlated with high risk in patients with AAA. Maintaining miR-26b-3p expression prevented AAA progression and alleviated the overall pathological process. CONCLUSIONS: Our study reveals a pivotal role of the LXRα/UHRF1/miR-26b-3p axis in AAA and provides potential biomarkers and therapeutic targets for AAA. [ABSTRACT FROM AUTHOR]

In obesity, circulating saturated fatty acids (SFAs) and inflammatory cytokines interfere with skeletal muscle insulin signaling, leading to whole body insulin resistance. Further, obese skeletal muscle is characterized by macrophage infiltration and polarization to the inflammatory M1 phenotype, which is central to the development of local inflammation and insulin resistance. While skeletal muscle-infiltrated macrophage–myocyte crosstalk is exacerbated by SFA, the effects of other fatty acids, such as n-3 and n-6 polyunsaturated fatty acids (PUFAs), are less studied. Thus, the objective of this study was to determine the effects of long-chain n-3 and n-6 PUFAs on macrophage M1 polarization and subsequent effects on myocyte inflammation and metabolic function compared to SFA. Using an in vitro model recapitulating obese skeletal muscle cells, differentiated L6 myocytes were cultured for 24 h with RAW 264.7 macrophage-conditioned media (MCM), followed by insulin stimulation (100 nM, 20 min). MCM was generated by pre-treating macrophages for 24 h with 100 µM palmitic acid (16:0, PA–control), arachidonic acid (20:4n-6, AA), or docosahexaenoic acid (22:6n-3, DHA). Next, macrophage cultures were stimulated with a physiological dose (10 ng/mL) of lipopolysaccharide for an additional 12 h to mimic in vivo obese endotoxin levels. Compared to PA, both AA and DHA reduced mRNA expression and/or secreted protein levels of markers for M1 (TNFα, IL-6, iNOS; p < 0.05) and increased those for M2 (IL-10, TGF-β; p < 0.05) macrophage polarization. In turn, AA- and DHA-derived MCM reduced L6 myocyte-secreted cytokines (TNFα, IL6; p < 0.05) and chemokines (MCP-1, MIP-1β; p < 0.05). Only AA-derived MCM increased L6-myocyte phosphorylation of Akt (p < 0.05), yet this was inconsistent with improved insulin signaling, as only DHA-derived MCM improved L6 myocyte glucose uptake (p < 0.05). In conclusion, dietary n-3 and n-6 PUFAs may be a useful strategy to modulate macrophage–myocyte inflammatory crosstalk and improve myocyte insulin sensitivity in obesity. [ABSTRACT FROM AUTHOR]

Purpose: Migraine, a severely debilitating condition, may be effectively managed with topiramate, known for its migraine prevention and weight loss properties due to changes in body muscle and fat composition and improved insulin sensitivity. However, the mechanism of topiramate in modulating insulin response in adipocytes and myocytes remains elusive. This study aims to elucidate these molecular mechanisms, offering insights into its role in weight management for migraine sufferers and underpinning its clinical application. Methods: Insulin resistance improvements were evaluated through glucose uptake measurements in C2C12 muscle cells and 3T3L-1 adipocytes, with Oil red O staining conducted on adipocytes. RNA-seq transcriptome analysis was used to identify the regulatory target genes of topiramate in these cells. The involvement of key genes and pathways was further validated through western blot analysis. Results: Topiramate effectively reduced insulin resistance in C2C12 and 3T3L-1 cells. In C2C12 cells, it significantly lowered SORBS1 gene and protein levels. In 3T3L-1 cells, topiramate upregulated CTGF and downregulated MAPK8 and KPNA1 genes. Changes were notable in nuclear cytoplasmic transport and circadian signaling pathways. Furthermore, it caused downregulation of MKK7, pJNK1/ JNK1, BMAL1, and CLOCK proteins compared to the insulin-resistant model. Conclusion: This study provides preliminary insights into the mechanisms through which topiramate modulates insulin resistance in C2C12 myocytes and 3T3L-1 adipocytes, enhancing our understanding of its therapeutic potential in managing weight and insulin sensitivity in migraine patients. [ABSTRACT FROM AUTHOR]

The article explores the concept of "Athero-Oncology," which examines the similarities between atherosclerosis and cancer. Both diseases share common risk factors and characteristics, such as DNA damage, clonal expansion, hyperproliferation, and chronic inflammation. Smooth muscle cells (SMCs) have been identified as important regulators of plaque progression and stability in atherosclerosis. Recent research suggests that SMCs in atherosclerotic plaques exhibit similarities to cancer cells, including oxidative DNA damage, genomic instability, and activation of cancer-associated signaling pathways. Understanding the role of SMCs and developing preventative and therapeutic strategies is crucial for managing atherosclerosis. A new study found that DNA damage and tumor cell-like features play a significant role in the development of atherosclerosis. The study discovered that DNA damage and sequence variations in SMCs within plaques can lead to the proliferation and migration of these cells, contributing to plaque formation. The study also found that an anticancer drug called niraparib, which inhibits DNA damage repair, can reduce the development and regression of atherosclerosis. However, more research is needed to develop effective therapies for atherosclerosis, as these drugs can have adverse effects. Understanding the similarities and differences between SMCs in atherosclerosis and true tumor cells may provide valuable insights for both diseases. [Extracted from the article]

BACKGROUND: Adult mammalian cardiomyocytes have limited proliferative capacity, but in specifically induced contexts they traverse through cell-cycle reentry, offering the potential for heart regeneration. Endogenous cardiomyocyte proliferation is preceded by cardiomyocyte dedifferentiation (CMDD), wherein adult cardiomyocytes revert to a less matured state that is distinct from the classical myocardial fetal stress gene response associated with heart failure. However, very little is known about CMDD as a defined cardiomyocyte cell state in transition. METHODS: Here, we leveraged 2 models of in vitro cultured adult mouse cardiomyocytes and in vivo adeno-associated virus serotype 9 cardiomyocyte--targeted delivery of reprogramming factors (Oct4, Sox2, Klf4, and Myc) in adult mice to study CMDD. We profiled their transcriptomes using RNA sequencing, in combination with multiple published data sets, with the aim of identifying a common denominator for tracking CMDD. RESULTS: RNA sequencing and integrated analysis identified Asparagine Synthetase (Asns) as a unique molecular marker gene well correlated with CMDD, required for increased asparagine and also for distinct fluxes in other amino acids. Although Asns overexpression in Oct4, Sox2, Klf4, and Myc cardiomyocytes augmented hallmarks of CMDD, Asns deficiency led to defective regeneration in the neonatal mouse myocardial infarction model, increased cell death of cultured adult cardiomyocytes, and reduced cell cycle in Oct4, Sox2, Klf4, and Myc cardiomyocytes, at least in part through disrupting the mammalian target of rapamycin complex 1 pathway. CONCLUSIONS: We discovered a novel gene Asns as both a molecular marker and an essential mediator, marking a distinct threshold that appears in common for at least 4 models of CMDD, and revealing an Asns/mammalian target of rapamycin complex 1 axis dependency for dedifferentiating cardiomyocytes. Further study will be needed to extrapolate and assess its relevance to other cell state transitions as well as in heart regeneration. [ABSTRACT FROM AUTHOR]

Diastolic dysfunction and delayed ventricular repolarization are typically observed in the elderly, but whether these defects are intimately associated with the progressive manifestation of the aging myopathy remains to be determined. In this regard, aging in experimental animals is coupled with increased late Na+ current (INa,L) in cardiomyocytes, raising the possibility that INa,L conditions the modality of electrical recovery and myocardial relaxation of the aged heart. For this purpose, aging male and female wild-type (WT) C57Bl/6 mice were studied together with genetically engineered mice with phosphomimetic (gain of function, GoF) or ablated (loss of function, LoF) mutations of the sodium channel Nav1.5 at Ser571 associated with, respectively, increased and stabilized INa,L. At ∼18 mo of age, WT mice developed prolonged duration of the QT interval of the electrocardiogram and impaired diastolic left ventricular (LV) filling, defects that were reversed by INa,L inhibition. Prolonged repolarization and impaired LV filling occurred prematurely in adult (∼5 mo) GoF mutant mice, whereas these alterations were largely attenuated in aging LoF mutant animals. Ca2+ transient decay and kinetics of myocyte shortening/relengthening were delayed in aged (∼24 mo) WT myocytes, with respect to adult cells. In contrast, delayed Ca2+ transients and contractile dynamics occurred at adult stage in GoF myocytes and further deteriorated in old age. Conversely, myocyte mechanics were minimally affected in aging LoF cells. Collectively, these results document that Nav1.5 phosphorylation at Ser571 and the late Na+ current modulate the modality of myocyte relaxation, constituting the mechanism linking delayed ventricular repolarization and diastolic dysfunction. NEW & NOTEWORTHY: We have investigated the impact of the late Na current (INa,L) on cardiac and myocyte function with aging by using genetically engineered animals with enhanced or stabilized INa,L, due to phosphomimetic or phosphoablated mutations of Nav1.5. Our findings support the notion that phosphorylation of Nav1.5 at Ser571 prolongs myocardial repolarization and impairs diastolic function, contributing to the manifestations of the aging myopathy. [ABSTRACT FROM AUTHOR]

We previously showed that the transaminase inhibitor, aminooxyacetic acid, reduced respiration energized at complex II (succinate dehydrogenase, SDH) in mitochondria isolated from mouse hindlimb muscle. The effect required a reduction in membrane potential with resultant accumulation of oxaloacetate (OAA), a potent inhibitor of SDH. To specifically assess the effect of the mitochondrial transaminase, glutamic oxaloacetic transaminase (GOT2) on complex II respiration, and to determine the effect in intact cells as well as isolated mitochondria, we performed respiratory and metabolic studies in wildtype (WT) and CRISPR-generated GOT2 knockdown (KD) C2C12 myocytes. Intact cell respiration by GOT2KD cells versus WT was reduced by adding carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) to lower potential. In mitochondria of C2C12 KD cells, respiration at low potential generated by 1 µM FCCP and energized at complex II by 10 mM succinate + 0.5 mM glutamate (but not by complex I substrates) was reduced versus WT mitochondria. Although we could not detect OAA, metabolite data suggested that OAA inhibition of SDH may have contributed to the FCCP effect. C2C12 mitochondria differed from skeletal muscle mitochondria in that the effect of FCCP on complex II respiration was not evident with ADP addition. We also observed that C2C12 cells, unlike skeletal muscle, expressed glutamate dehydrogenase, which competes with GOT2 for glutamate metabolism. In summary, GOT2 KD reduced C2C12 respiration in intact cells at low potential. From differential substrate effects, this occurred largely at complex II. Moreover, C2C12 versus muscle mitochondria differ in complex II sensitivity to ADP and differ markedly in expression of glutamate dehydrogenase. NEW & NOTEWORTHY: Impairment of the mitochondrial transaminase, GOT2, reduces complex II (succinate dehydrogenase, SDH)-energized respiration in C2C12 myocytes. This occurs only at low inner membrane potential and is consistent with inhibition of SDH. Incidentally, we observed that C2C12 mitochondria compared with muscle tissue mitochondria differ in sensitivity of complex II respiration to ADP and in the expression of glutamate dehydrogenase. [ABSTRACT FROM AUTHOR]

Defining mechanisms of cardiomyocyte proliferation should guide the understanding of endogenous cardiac regeneration and could lead to novel treatments for diseases such as myocardial infarction. In the neonatal heart, energy metabolic reprogramming (phenotypic alteration of glucose, fatty acid, and amino acid metabolism) parallels cell cycle arrest of cardiomyocytes. The metabolic reprogramming occurring shortly after birth is associated with alterations in blood oxygen levels, metabolic substrate availability, hemodynamic stress, and hormone release. In the adult heart, myocardial infarction causes metabolic reprogramming but these changes cannot stimulate sufficient cardiomyocyte proliferation to replace those lost by the ischemic injury. Some putative pro-proliferative interventions can induce the metabolic reprogramming. Recent data show that altering the metabolic enzymes PKM2 [pyruvate kinase 2], LDHA [lactate dehydrogenase A], PDK4 [pyruvate dehydrogenase kinase 4], SDH [succinate dehydrogenase], CPT1b [carnitine palmitoyl transferase 1b], or HMGCS2 [3-hydroxy-3-methylglutaryl-CoA synthase 2] is sufficient to partially reverse metabolic reprogramming and promotes adult cardiomyocyte proliferation. How metabolic reprogramming regulates cardiomyocyte proliferation is not clearly defined. The possible mechanisms involve biosynthetic pathways from the glycolysis shunts and the epigenetic regulation induced by metabolic intermediates. Metabolic manipulation could represent a new approach to stimulate cardiac regeneration; however, the efficacy of these manipulations requires optimization, and novel molecular targets need to be defined. In this review, we summarize the features, triggers, and molecular regulatory networks responsible for metabolic reprogramming and discuss the current understanding of metabolic reprogramming as a critical determinant of cardiomyocyte proliferation. [ABSTRACT FROM AUTHOR]

Pulmonary hypertension (PH) is a condition in which remodeling of the pulmonary vasculature leads to hypertrophy of the muscular vascular wall and extension of muscle into nonmuscular arteries. These pathological changes are predominantly due to the abnormal proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), enhanced cellular functions that have been linked to increases in the cell membrane protein aquaporin 1 (AQP1). However, the mechanisms underlying the increased AQP1 abundance have not been fully elucidated. Here we present data that establishes a novel interaction between AQP1 and the proteolytic enzyme caspase-3. In silico analysis of the AQP1 protein reveals two caspase-3 cleavage sites on its C-terminal tail, proximal to known ubiquitin sites. Using biotin proximity ligase techniques, we establish that AQP1 and caspase-3 interact in both human embryonic kidney (HEK) 293A cells and rat PASMCs. Furthermore, we demonstrate that AQP1 levels increase and decrease with enhanced caspase-3 activity and inhibition, respectively. Ultimately, further work characterizing this interaction could provide the foundation for novel PH therapeutics. NEW & NOTEWORTHY: Pulmonary arterial smooth muscle cells (PASMCs) are integral to pulmonary vascular remodeling, a characteristic of pulmonary arterial hypertension (PAH). PASMCs isolated from robust animal models of disease demonstrate enhanced proliferation and migration, pathological functions associated with increased abundance of the membrane protein aquaporin 1 (AQP1). We present evidence of a novel interaction between the proteolytic enzyme caspase-3 and AQP1, which may control AQP1 abundance. These data suggest a potential new target for novel PAH therapies. [ABSTRACT FROM AUTHOR]

Background: It has been reported that botulinum toxin type A (BoNT-A) produces structural changes in masticatory muscles. However, not all histomorphometric parameters affected by BoNT-A parameters have been assessed. This study investigated the histomorphometric changes in the masseter muscle of rats after a single injection of BoNT-A. Methods: Forty-four adult animals were randomly divided into control group (n = 22) and BoNT-A group (n = 22). Controls received a single dose of 0.14 mL/kg of saline in masseter muscles, and the BoNT-A group received a 7 U/Kg of BoNT-A. The groups received the same volume of injected substances. Animals were sacrificed on 7th (n = 5), 14th (n = 5), 21st (n = 5), 28th (n = 4) and 90th (n = 3) days post-treatment. Histological masseter tissue slides were obtained from hematoxylin–eosin treatment and analyzed in optical microscopy regarding muscle cross-sectional area, amount of connective tissue and quantity and diameter of myocytes. For statistical analysis, generalized linear models were used to compare the data (ANOVA). In all test, the significance level of 5% was set. Results: BoNT-A values of cross-sectional area of the masseter muscle were significantly lower than controls (p < 0.01) throughout the study. Regarding myocytes quantity, BoNT-A subgroups presented higher values than controls (p < 0.0001) since the 14th day until the end of the study; however, the diameter of myocytes was smaller in all BoNT-A subgroups (p < 0.0001) in all assessment points. The amount of connective tissue was higher in BoNT-A subgroups (p < 0.0001) throughout the study. Conclusion: A single injection of BoNT-A altered the structure of masseter muscle of rats, regarding its histomorphometric parameters. No Level Assigned: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266. [ABSTRACT FROM AUTHOR]

Connexin-43 (Cx43) is the most abundant protein forming gap junction channels (GJCs) in cardiac ventricles. In multiple cardiac pathologies, including hypertrophy and heart failure, Cx43 is found remodeled at the lateral side of the intercalated discs of ventricular cardiomyocytes. Remodeling of Cx43 has been long linked to spontaneous ventricular arrhythmia, yet the mechanisms by which arrhythmias develop are still debated. Using a model of dystrophic cardiomyopathy, we previously showed that remodeled Cx43 function as aberrant hemichannels (non-forming GJCs) that alter cardiomyocyte excitability and, consequently, promote arrhythmias. Here, we aim to evaluate if opening of remodeled Cx43 can serve as a general mechanism to alter cardiac excitability independent of cellular dysfunction associated with a particular cardiomyopathy. To address this issue, we used a genetically modified Cx43 knock-in mouse (S3A) that promotes cardiac remodeling of Cx43 protein without apparent cardiac dysfunction. Importantly, when S3A mice were subjected to cardiac stress using the β-adrenergic agonist isoproterenol (Iso), they displayed acute and severe arrhythmias, which were not observed in WT mice. Pretreatment of S3A mice with the Cx43 hemichannel blocker, Gap19, prevented Iso-induced abnormal electrocardiographic behavior. At the cellular level, when compared with WT, Iso-treated S3A cardiomyocytes showed increased membrane permeability, greater plasma membrane depolarization, and Ca2+ overload, which likely caused prolonged action potentials, delayed after depolarizations, and triggered activity. All these cellular dysfunctions were also prevented by Cx43 hemichannel blockers. Our results support the notion that opening of remodeled Cx43 hemichannels, regardless of the type of cardiomyopathy, is sufficient to mediate cardiac-stress-induced arrhythmogenicity.

β-adrenergic (β-AR) signaling is essential for the adaptation of the heart to exercise and stress. Chronic stress leads to the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase D (PKD). Unlike CaMKII, the effects of PKD on excitation-contraction coupling (ECC) remain unclear. To elucidate the mechanisms of PKD-dependent ECC regulation, we used hearts from cardiac-specific PKD1 knockout (PKD1 cKO) mice and wild-type (WT) littermates. We measured calcium transients (CaT), Ca2+ sparks, contraction and L-type Ca2+ current in paced cardiomyocytes under acute β-AR stimulation with isoproterenol (ISO; 100 nM). Sarcoplasmic reticulum (SR) Ca2+ load was assessed by rapid caffeine (10 mM) induced Ca2+ release. Expression and phosphorylation of ECC proteins phospholambam (PLB), troponin I (TnI), ryanodine receptor (RyR), sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) were evaluated by western blotting. At baseline, CaT amplitude and decay tau, Ca2+ spark frequency, SR Ca2+ load, L-type Ca2+ current, contractility, and expression and phosphorylation of ECC protein were all similar in PKD1 cKO vs. WT. However, PKD1 cKO cardiomyocytes presented a diminished ISO response vs. WT with less increase in CaT amplitude, slower [Ca2+]i decline, lower Ca2+ spark rate and lower RyR phosphorylation, but with similar SR Ca2+ load, L-type Ca2+ current, contraction and phosphorylation of PLB and TnI. We infer that the presence of PKD1 allows full cardiomyocyte β-adrenergic responsiveness by allowing optimal enhancement in SR Ca2+ uptake and RyR sensitivity, but not altering L-type Ca2+ current, TnI phosphorylation or contractile response. Further studies are necessary to elucidate the specific mechanisms by which PKD1 is regulating RyR sensitivity. We conclude that the presence of basal PKD1 activity in cardiac ventricular myocytes contributes to normal β-adrenergic responses in Ca2+ handling.

Parallel to major changes in fatty acid and glucose metabolism, defect in branched-chain amino acid (BCAA) catabolism has also been recognized as a metabolic hallmark and potential therapeutic target for heart failure. However, BCAA catabolic enzymes are ubiquitously expressed in all cell types and a systemic BCAA catabolic defect is also manifested in metabolic disorder associated with obesity and diabetes. Therefore, it remains to be determined the cell-autonomous impact of BCAA catabolic defect in cardiomyocytes in intact hearts independent from its potential global effects. In this study, we developed two mouse models. One is cardiomyocyte and temporal-specific inactivation of the E1α subunit (BCKDHA-cKO) of the branched-chain α-ketoacid dehydrogenase (BCKDH) complex, which blocks BCAA catabolism. Another model is cardiomyocyte specific inactivation of the BCKDH kinase (BCKDK-cKO), which promotes BCAA catabolism by constitutively activating BCKDH activity in adult cardiomyocytes. Functional and molecular characterizations showed E1α inactivation in cardiomyocytes was sufficient to induce loss of cardiac function, systolic chamber dilation and pathological transcriptome reprogramming. On the other hand, inactivation of BCKDK in intact heart does not have an impact on baseline cardiac function or cardiac dysfunction under pressure overload. Our results for the first time established the cardiomyocyte cell autonomous role of BCAA catabolism in cardiac physiology. These mouse lines will serve as valuable model systems to investigate the underlying mechanisms of BCAA catabolic defect induced heart failure and to provide potential insights for BCAA targeted therapy.

9p21.3 locus polymorphisms have the strongest correlation with coronary artery disease, but as a noncoding locus, disease connection is enigmatic. The lncRNA ANRIL found in 9p21.3 may regulate vascular smooth muscle cell (VSMC) phenotype to contribute to disease risk. We observed significant heterogeneity in induced pluripotent stem cell-derived VSMCs from patients homozygous for risk versus isogenic knockout or nonrisk haplotypes. Subpopulations of risk haplotype cells exhibited variable morphology, proliferation, contraction, and adhesion. When sorted by adhesion, risk VSMCs parsed into synthetic and contractile subpopulations, i.e., weakly adherent and strongly adherent, respectively. Of note, >90% of differentially expressed genes coregulated by haplotype and adhesion and were associated with Rho GTPases, i.e., contractility. Weakly adherent subpopulations expressed more short isoforms of ANRIL, and when overexpressed in knockout cells, ANRIL suppressed adhesion, contractility, and αSMA expression. These data suggest that variable lncRNA penetrance may drive mixed functional outcomes that confound pathology.

Anthracyclines such as doxorubicin (Dox) are effective chemotherapeutic agents; however, their use is hampered by subsequent cardiotoxicity risk. Our understanding of cardiomyocyte protective pathways activated following anthracycline-induced cardiotoxicity (AIC) remains incomplete. Insulin-like growth factor binding protein (IGFBP) 3 (Igfbp-3), the most abundant IGFBP family member in the circulation, is associated with effects on the metabolism, proliferation, and survival of various cells. Whereas Igfbp-3 is induced by Dox in the heart, its role in AIC is ill-defined. We investigated molecular mechanisms as well as systems-level transcriptomic consequences of manipulating Igfbp-3 in AIC using neonatal rat ventricular myocytes and human-induced pluripotent stem cell-derived cardiomyocytes. Our findings reveal that Dox induces the nuclear enrichment of Igfbp-3 in cardiomyocytes. Furthermore, Igfbp-3 reduces DNA damage, impedes topoisomerase IIβ expression (Top2β) which forms Top2β-Dox-DNA cleavage complex leading to DNA double-strand breaks (DSB), alleviates detyrosinated microtubule accumulation-a hallmark of increased cardiomyocyte stiffness and heart failure-and favorably affects contractility following Dox treatment. These results indicate that Igfbp-3 is induced by cardiomyocytes in an effort to mitigate AIC.

Purpose of reviewBioengineering of functional cardiac tissue composed of primary cardiomyocytes has great potential for myocardial regeneration and in vitro tissue modeling. 3D bioprinting was developed to create cardiac tissue in hydrogels that can mimic the structural, physiological, and functional features of native myocardium. Through a detailed review of the 3D printing technologies and bioink materials used in the creation of a heart tissue, this article discusses the potential of engineered heart tissues in biomedical applications.Recent findingsIn this review, we discussed the recent progress in 3D bioprinting strategies for cardiac tissue engineering, including bioink and 3D bioprinting methods as well as examples of engineered cardiac tissue such as in vitro cardiac models and vascular channels. 3D printing is a powerful tool for creating in vitro cardiac tissues that are structurally and functionally similar to real tissues. The use of human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) enables the generation of patient-specific tissues. These tissues have the potential to be used for regenerative therapies, disease modeling, and drug testing.

The fluorescence ubiquitination cell cycle inhibitor (FUCCI) has been introduced to monitor cell cycle activity in living cells, including human induced pluripotent stem cells (hiPSC) and derived cell types. We have recently developed hiPSC with stable expression of dCas9VPR for endogenous gene activation and a Citrine-tagged ACTN2 cell line to monitor sarcomere development and function in muscle cells. Here, we present dual and triple transgenic hiPSC lines developed by genomic integration of FUCCI with and without dCas9VPR into the ROSA26 and AAVS1 loci, respectively, in the previously introduced ACTN2-Citrine line. Functionality of the transgenes was demonstrated in the novel hiPSC line, which we introduce as Myo-CCER and CraCCER.

BackgroundSmall-conductance Ca2+-activated K+ (SK)-channel inhibitors have antiarrhythmic effects in animal models of atrial fibrillation (AF), presenting a potential novel antiarrhythmic option. However, the regulation of SK-channels in human atrial cardiomyocytes and its modification in patients with AF are poorly understood and were the object of this study.MethodsApamin-sensitive SK-channel current (ISK) and action potentials were recorded in human right-atrial cardiomyocytes from sinus rhythm control (Ctl) patients or patients with (long-standing persistent) chronic AF (cAF).ResultsISK was significantly higher, and apamin caused larger action potential prolongation in cAF- versus Ctl-cardiomyocytes. Sensitivity analyses in an in silico human atrial cardiomyocyte model identified IK1 and ISK as major regulators of repolarization. Increased ISK in cAF was not associated with increases in mRNA/protein levels of SK-channel subunits in either right- or left-atrial tissue homogenates or right-atrial cardiomyocytes, but the abundance of SK2 at the sarcolemma was larger in cAF versus Ctl in both tissue-slices and cardiomyocytes. Latrunculin-A and primaquine (anterograde and retrograde protein-trafficking inhibitors) eliminated the differences in SK2 membrane levels and ISK between Ctl- and cAF-cardiomyocytes. In addition, the phosphatase-inhibitor okadaic acid reduced ISK amplitude and abolished the difference between Ctl- and cAF-cardiomyocytes, indicating that reduced calmodulin-Thr80 phosphorylation due to increased protein phosphatase-2A levels in the SK-channel complex likely contribute to the greater ISK in cAF-cardiomyocytes. Finally, rapid electrical activation (5 Hz, 10 minutes) of Ctl-cardiomyocytes promoted SK2 membrane-localization, increased ISK and reduced action potential duration, effects greatly attenuated by apamin. Latrunculin-A or primaquine prevented the 5-Hz-induced ISK-upregulation.ConclusionsISK is upregulated in patients with cAF due to enhanced channel function, mediated by phosphatase-2A-dependent calmodulin-Thr80 dephosphorylation and tachycardia-dependent enhanced trafficking and targeting of SK-channel subunits to the sarcolemma. The observed AF-associated increases in ISK, which promote reentry-stabilizing action potential duration shortening, suggest an important role for SK-channels in AF auto-promotion and provide a rationale for pursuing the antiarrhythmic effects of SK-channel inhibition in humans.

CaV1.2 channels are critical players in cardiac excitation-contraction coupling, yet we do not understand how they are affected by an important therapeutic target of heart failure drugs and regulator of blood pressure, angiotensin II. Signaling through Gq-coupled AT1 receptors, angiotensin II triggers a decrease in PIP2, a phosphoinositide component of the plasma membrane (PM) and known regulator of many ion channels. PIP2 depletion suppresses CaV1.2 currents in heterologous expression systems but the mechanism of this regulation and whether a similar phenomenon occurs in cardiomyocytes is unknown. Previous studies have shown that CaV1.2 currents are also suppressed by angiotensin II. We hypothesized that these two observations are linked and that PIP2 stabilizes CaV1.2 expression at the PM and angiotensin II depresses cardiac excitability by stimulating PIP2 depletion and destabilization of CaV1.2 expression. We tested this hypothesis and report that CaV1.2 channels in tsA201 cells are destabilized after AT1 receptor-triggered PIP2 depletion, leading to their dynamin-dependent endocytosis. Likewise, in cardiomyocytes, angiotensin II decreased t-tubular CaV1.2 expression and cluster size by inducing their dynamic removal from the sarcolemma. These effects were abrogated by PIP2 supplementation. Functional data revealed acute angiotensin II reduced CaV1.2 currents and Ca2+ transient amplitudes thus diminishing excitation-contraction coupling. Finally, mass spectrometry results indicated whole-heart levels of PIP2 are decreased by acute angiotensin II treatment. Based on these observations, we propose a model wherein PIP2 stabilizes CaV1.2 membrane lifetimes, and angiotensin II-induced PIP2 depletion destabilizes sarcolemmal CaV1.2, triggering their removal, and the acute reduction of CaV1.2 currents and contractility.

Cardiomyopathy is a progressive disease of the myocardium leading to impaired contractility. Genotoxic cancer therapies are known to be potent drivers of cardiomyopathy, whereas causes of spontaneous disease remain unclear. To test the hypothesis that endogenous genotoxic stress contributes to cardiomyopathy, we deleted the DNA repair gene Ercc1 specifically in striated muscle using a floxed allele of Ercc1 and mice expressing Cre under control of the muscle-specific creatinine kinase (Ckmm) promoter or depleted systemically (Ercc1-/D mice). Ckmm-Cre+/- ;Ercc1-/fl mice expired suddenly of heart disease by 7 months of age. As young adults, the hearts of Ckmm-Cre+/- ;Ercc1-/fl mice were structurally and functionally normal, but by 6-months-of-age, there was significant ventricular dilation, wall thinning, interstitial fibrosis, and systolic dysfunction indicative of dilated cardiomyopathy. Cardiac tissue from the tissue-specific or systemic model showed increased apoptosis and cardiac myocytes from Ckmm-Cre+/- ;Ercc1-/fl mice were hypersensitive to genotoxins, resulting in apoptosis. p53 levels and target gene expression, including several antioxidants, were increased in cardiac tissue from Ckmm-Cre+/- ;Ercc1-/fl and Ercc1-/D mice. Despite this, cardiac tissue from older mutant mice showed evidence of increased oxidative stress. Genetic or pharmacologic inhibition of p53 attenuated apoptosis and improved disease markers. Similarly, overexpression of mitochondrial-targeted catalase improved disease markers. Together, these data support the conclusion that DNA damage produced endogenously can drive cardiac disease and does so mechanistically via chronic activation of p53 and increased oxidative stress, driving cardiac myocyte apoptosis, dilated cardiomyopathy, and sudden death.

Cardiovascular disease remains the leading cause of mortality worldwide. Cardiomyocytes are irreversibly lost due to cardiac ischemia secondary to disease. This leads to increased cardiac fibrosis, poor contractility, cardiac hypertrophy, and subsequent life-threatening heart failure. Adult mammalian hearts exhibit notoriously low regenerative potential, further compounding the calamities described above. Neonatal mammalian hearts, on the other hand, display robust regenerative capacities. Lower vertebrates such as zebrafish and salamanders retain the ability to replenish lost cardiomyocytes throughout life. It is critical to understand the varying mechanisms that are responsible for these differences in cardiac regeneration across phylogeny and ontogeny. Adult mammalian cardiomyocyte cell cycle arrest and polyploidization have been proposed as major barriers to heart regeneration. Here we review current models about why adult mammalian cardiac regenerative potential is lost including changes in environmental oxygen levels, acquisition of endothermy, complex immune system development, and possible cancer risk tradeoffs. We also discuss recent progress and highlight conflicting reports pertaining to extrinsic and intrinsic signaling pathways that control cardiomyocyte proliferation and polyploidization in growth and regeneration. Uncovering the physiological brakes of cardiac regeneration could illuminate novel molecular targets and offer promising therapeutic strategies to treat heart failure.