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The giant sarcomere protein titin is a major determinant of cardiomyocyte stiffness and contributor to cardiac strain sensing. Titin-based forces are highly regulated in health and disease, which aids in the regulation of myocardial function, including cardiac filling and output. Due to the enormous size, complexity, and malleability of the titin molecule, titin properties are also vulnerable to dysregulation, as observed in various cardiac disorders. This review provides an overview of how cardiac titin properties can be changed at a molecular level, including the role isoform diversity and post-translational modifications (acetylation, oxidation, and phosphorylation) play in regulating myocardial stiffness and contractility. We then consider how this regulation becomes unbalanced in heart disease, with an emphasis on changes in titin stiffness and protein quality control. In this context, new insights into the key pathomechanisms of human cardiomyopathy due to a truncation in the titin gene (TTN) are discussed. Along the way, we touch on the potential for titin to be therapeutically targeted to treat acquired or inherited cardiac conditions, such as HFpEF or TTN-truncation cardiomyopathy.

Aims Remdesivir is a prodrug of an adenosine triphosphate analogue and is currently the only drug formally approved for the treatment of hospitalised COVID-19 patients. Nucleoside/nucleotide analogues have been shown to induce mitochondrial damage and cardiotoxicity, and this may be exacerbated by hypoxia, which frequently occurs in severe COVID-19 patients. Although there have been few reports of adverse cardiovascular events associated with remdesivir, clinical data are limited. Here, we investigated whether remdesivir induced cardiotoxicity using an in vitro human cardiac model. Methods and Results Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were exposed to remdesivir under normoxic and hypoxic conditions to simulate mild and severe COVID-19 respectively. Remdesivir induced mitochondrial fragmentation, reduced redox potential and suppressed mitochondrial respiration at levels below the estimated plasma concentration under both normoxic and hypoxic conditions. Non-mitochondrial damage such as electrophysiological alterations and sarcomere disarray were also observed. Importantly, some of these changes persisted after the cessation of treatment, culminating in increased cell death. Mechanistically, we found that inhibition of DRP1, a regulator of mitochondrial fission, ameliorated the cardiotoxic effects of remdesivir, showing that remdesivir-induced cardiotoxicity was preventable and excessive mitochondrial fission might contribute to this phenotype. Conclusions Using an in vitro model, we demonstrated that remdesivir can induce cardiotoxicity in hiPSC-CMs at clinically relevant concentrations. These results reveal previously unknown potential side-effects of remdesivir and highlight the importance of further investigations with in vivo animal models and active clinical monitoring to prevent lasting cardiac damage to patients. Translational perspective Adult cardiomyocytes have limited ability to regenerate, thus treatment-induced cardiotoxicity can potentially cause irreparable harm. Remdesivir is currently the only FDA approved treatment for COVID-19 but clinical safety data are limited. Using human pluripotent stem cell-derived cardiomyocytes, we revealed that remdesivir induced persistent mitochondrial and structural abnormalities at clinically relevant concentrations. We advise confirmatory experiments in in vivo animal models, investigations of cardioprotective strategies, and closer patient monitoring such that treatment-induced cardiotoxicity does not contribute to the long term sequelae of COVID-19 patients., Graphical Abstract Graphical Abstract

AIMS: The precise cellular identity and molecular features of non-myocytes (non-CMs) in a mammalian heart at a single-cell level remain elusive. Depiction of epigenetic landscape with transcriptomic signatures using the latest single-cell multi-omics has the potential to unravel the molecular programs underlying the cellular diversity of cardiac non-myocytes. Here, we characterized the molecular and cellular features of cardiac non-CM populations in the adult murine heart at the single-cell level. METHODS AND RESULTS: Through single-cell dual omics analysis, we mapped the epigenetic landscapes, characterized the transcriptomic profiles and delineated the molecular signatures of cardiac non-CMs in the adult murine heart. Distinct cis-regulatory elements and trans-acting factors for the individual major non-CM cell types (endothelial cells, fibroblast, pericytes, and immune cells) were identified. In particular, unbiased sub-clustering and functional annotation of cardiac fibroblasts (FBs) revealed extensive FB heterogeneity and identified FB sub-types with functional states related to the cellular response to stimuli, cytoskeleton organization, and immune regulation, respectively. We further explored the function of marker genes Hsd11b1 and Gfpt2 that label major FB subpopulations and determined the distribution of Hsd11b1+ and Gfp2+ FBs in murine healthy and diseased hearts. CONCLUSIONS: In summary, we characterized the non-CM cellular identity at the transcriptome and epigenome levels using single-cell omics approaches and discovered previously unrecognized cardiac fibroblast subpopulations with unique functional states.

Aims The cardiac natriuretic peptides (atrial natriuretic peptide [ANP] and B-type natriuretic peptide [BNP]) are important regulators of cardiovascular physiology, with reduced natriuretic peptide (NP) activity linked to multiple human cardiovascular diseases. We hypothesized that deficiency of either ANP or BNP would lead to similar changes in left ventricular structure and function given their shared receptor affinities. Methods and results We directly compared murine models deficient of ANP or BNP in the same genetic backgrounds (C57BL6/J) and environments. We evaluated control, ANP deficient (Nppa-/-) or BNP deficient (Nppb-/-) mice under unstressed conditions and multiple forms of pathological myocardial stress. Survival, myocardial structure, function and electrophysiology, tissue histology, and biochemical analyses were evaluated in the groups. In vitro validation of our findings was performed using human derived induced pluripotent stem cell cardiomyocytes (iPS-CM). In the unstressed state, both ANP and BNP deficient mice displayed mild ventricular hypertrophy which did not increase up to 1 year of life. NP-deficient mice exposed to acute myocardial stress secondary to thoracic aortic constriction (TAC) had similar pathological myocardial remodeling but a significant increase in sudden death. We discovered that the NP-deficient mice are more susceptible to stress induced ventricular arrhythmias using both in vivo and ex vivo models. Mechanistically, deficiency of either ANP or BNP led to reduced myocardial cGMP levels and reduced phosphorylation of the cAMP response element-binding protein (CREBS133) transcriptional regulator. Selective CREB inhibition sensitized wild type hearts to stress induced ventricular arrhythmias. ANP and BNP regulate cardiomyocyte CREBS133 phosphorylation through a cGMP-dependent protein kinase 1 (PKG1) and p38 mitogen activated protein kinase (p38 MAPK) signaling cascade. Conclusions Our data show that ANP and BNP act in a non-redundant fashion to maintain myocardial cGMP levels to regulate cardiomyocyte p38 MAPK and CREB activity. Cardiac natriuretic peptide deficiency leads to a reduction in CREB signaling which sensitizes the heart to stress induced ventricular arrhythmias. Translational perspective Our study found that ANP or BNP deficiency leads to increased sudden death and ventricular arrhythmias after acute myocardial stress in murine models. We discovered that ANP and BNP act in a non-redundant fashion to maintain myocardial cGMP levels and uncovered a unique role for these peptides in regulating cardiomyocyte p38 MAPK and CREB signaling through a cGMP-PKG1 pathway. Importantly, this signaling pathway was conserved in human cardiomyocytes. This study provides mechanistic insight into how modulating natriuretic peptide levels in human heart failure patients reduces sudden death and mortality.

The pandemic of coronavirus disease (COVID)-19 is a global threat, causing high mortality, especially in the elderly. The main symptoms and the primary cause of death are related to interstitial pneumonia. Viral entry also into myocardial cells mainly via the angiotensin converting enzyme type 2 (ACE2) receptor and excessive production of pro-inflammatory cytokines, however, also make the heart susceptible to injury. In addition to the immediate damage caused by the acute inflammatory response, the heart may also suffer from long-term consequences of COVID-19, potentially causing a post-pandemic increase in cardiac complications. Although the main cause of cardiac damage in COVID-19 remains coagulopathy with micro- (and to a lesser extent macro-) vascular occlusion, open questions remain about other possible modalities of cardiac dysfunction, such as direct infection of myocardial cells, effects of cytokines storm, and mechanisms related to enhanced coagulopathy. In this opinion paper, we focus on these lesser appreciated possibilities and propose experimental approaches that could provide a more comprehensive understanding of the cellular and molecular bases of cardiac injury in COVID-19 patients. We first discuss approaches to characterize cardiac damage caused by possible direct viral infection of cardiac cells, followed by formulating hypotheses on how to reproduce and investigate the hyperinflammatory and pro-thrombotic conditions observed in the heart of COVID-19 patients using experimental in vitro systems. Finally, we elaborate on strategies to discover novel pathology biomarkers using omics platforms.

Aims The goal of our study was to investigate the heterogeneity of cardiac macrophages (CMφs) in mice with transverse aortic constriction (TAC) via single-cell sequencing and identify a subset of macrophages associated with heart injury. Methods and results We selected all CMφs from CD45+ cells using single-cell mRNA sequencing data. Through dimension reduction, clustering and enrichment analyses, CD72hi CMφs were identified as a subset of proinflammatory macrophages. The pseudotime trajectory and ChIP-Seq analyses identified Rel as the key transcription factor that induces CD72hi CMφ differentiation. Rel KD and Rel-/- bone marrow chimera mice subjected to TAC showed features of mitigated cardiac injury, including decreased levels of cytokines and ROS, which prohibited cardiomyocyte death. The transfer of adoptive Rel-overexpressing monocytes and CD72hi CMφ injection directly aggravated heart injury in the TAC model. The CD72hi macrophages also exerted proinflammatory and cardiac injury effects associated with myocardial infarction (MI). In humans, patients with heart failure exhibit increased CD72hi CMφ levels following dilated cardiomyopathy (DCM) and ischemic cardiomyopathy (ICM). Conclusion Bone marrow-derived, Rel-mediated CD72hi macrophages play a proinflammatory role, induce cardiac injury and, thus, may serve as a therapeutic target for multiple cardiovascular diseases. Translational perspective Heart failure (HF) imposes an enormous clinical and economic burden worldwide and presents limited therapeutic approaches. Given the close association between inflammation and adverse outcomes, proinflammatory immune cells are considered potential therapeutic targets for HF treatment. The present studies identified a specific macrophage subset associated with myocardial injury, which may provide an alternative approach for treating cardiovascular diseases.

Aims Biological sex has fundamental effects on mammalian heart physiology and pathogenesis. While it has been established that female sex is a protective factor against most cardiovascular diseases (CVDs), this beneficial effect may involve pathways associated with cardiac energy metabolism. Our aim was to elucidate the role of transcriptional coactivator PGC-1α in sex dimorphism of heart failure development. Methods and results Here we show that mice deficient in cardiac expression of the peroxisome proliferator-activated receptor gamma (PPAR-γ) coactivator 1α (PGC-1α) develop dilated heart failure associated with changes in aerobic and anaerobic metabolism, calcium handling, cell structure, electrophysiology as well as gene expression. These cardiac changes occur in both sexes, but female mice develop an earlier and more severe structural and functional phenotype associated with dyssynchronous local calcium release resulting from disruption of t-tubular structures of the cardiomyocytes. Conclusions These data reveal that the integrity of the subcellular Ca2+ release and uptake machinery is dependent on energy metabolism and that female hearts are more prone to suffer from contractile dysfunction in conditions with compromised production of cellular energy. Furthermore, these findings suggest that PGC-1α is a central mediator of sex-specific differences in heart function and CVD susceptibility. Translational perspective Biological sex is an important variable in clinical medicine, cardiac physiology, and pathogenesis. However, sex-specific clinical practices or therapies are emerging slowly in the absence of deeper understanding of the specific mechanism behind sex dimorphism in cardiac disease progression. Here, we show that energy metabolism has a central role in sex dimorphism of heart failure progression and that a signalling cascade involving PGC-1α might have a role in it. We provide insights into sex specific mechanisms of heart failure development which are necessary to identify sex specific treatment practices for cardiovascular diseases.

Congenital heart disease (CHD) frequently occurs in newborns due to abnormal formation of the heart or major blood vessels. Mutations in the GATA4 gene, which encodes GATA binding protein 4, are responsible for atrial septal defect (ASD), a common CHD. This study aims to gain insights into the molecular mechanisms of CHD using human-induced pluripotent stem cells (iPSCs) from a family cohort with ASD.Patient-specific iPSCs possess the same genetic information as the donor and can differentiate into various cell types from all three germ layers in vitro, thus presenting a promising approach for disease modelling and molecular mechanism research. Here, we generated a patient-specific iPSC line (iPSC-G4T280M) from a family cohort carrying a hereditary ASD mutation in GATA4 gene (T280M), as well as a human embryonic stem cell line (ESC-G4T280M) carrying the isogenic T280M mutation using the CRISPR/Cas9 genome editing method. The GATA4-mutant iPSCs and ESCs were then differentiated into cardiomyocytes (CMs) to model GATA4 mutation-associated ASD. We observed an obvious defect in cell proliferation in cardiomyocytes derived from both GATA4T280M-mutant iPSCs (iPSC-G4T280M-CMs) and ESCs (ESC-G4T280M-CMs), while the impaired proliferation ability of iPSC-G4T280M-CMs could be restored by gene correction. Integrated analysis of RNA-Seq and ChIP-Seq data indicated that FGF16 is a direct target of wild-type GATA4. However, the T280M mutation obstructed GATA4 occupancy at the FGF16 promoter region, leading to impaired activation of FGF16 transcription. Overexpression of FGF16 in GATA4-mutant cardiomyocytes rescued the cell proliferation defect. The direct relationship between GATA4T280M and ASD was demonstrated in a human iPSC model for the first time.In summary, our study revealed the molecular mechanism of the GATA4T280M mutation in ASD. Understanding the roles of the GATA4-FGF16 axis in iPSC-CMs will shed light on heart development and provide novel insights for the treatment of ASD and other CHD disorders.

Aims Guanylyl cyclase-B (GC-B; natriuretic peptide receptor-B, NPR-B) stimulation by C-type natriuretic peptide (CNP) increases cGMP and causes a lusitropic and negative inotropic response in adult myocardium. These effects are not mimicked by NPR-A (GC-A) stimulation by brain natriuretic peptide (BNP), despite similar cGMP increase. More refined methods are needed to better understand the mechanisms of the differential cGMP signaling and compartmentation. The aim of this work was to measure cGMP near proteins involved in regulating contractility to understand compartmentation of cGMP signaling in adult cardiomyocytes. Methods and results We constructed several fluorescence resonance energy transfer (FRET)-based biosensors for cGMP subcellularly targeted to phospholamban (PLB) and troponin I (TnI). CNP stimulation of adult rat cardiomyocytes increased cGMP near PLB and TnI, whereas BNP stimulation increased cGMP near PLB, but not TnI. The phosphodiesterases PDE2 and PDE3 constrained cGMP in both compartments. Local receptor stimulation aided by scanning ion conductance microscopy (SICM) combined with FRET revealed that CNP stimulation both in the t-tubules and on the cell crests increases cGMP similarly near both TnI and PLB. In ventricular strips, CNP stimulation, but not BNP, induced a lusitropic response, enhanced by inhibition of either PDE2 or PDE3, and a negative inotropic response. In cardiomyocytes from heart failure rats, CNP increased cGMP near PLB and TnI more pronounced than in cells from sham-operated animals. Conclusions These targeted biosensors demonstrate that CNP, but not BNP, increases cGMP near TnI in addition to PLB, explaining how CNP, but not BNP is able to induce lusitropic and negative inotropic responses. Translational perspective Although best known as heart failure biomarkers, natriuretic peptides (ANP, BNP and CNP) are important signaling molecules in the heart and other organs through increasing cyclic GMP (cGMP). Treatment preventing their degradation improves heart failure prognosis. To better understand their cardiac signaling, we employed fluorescent cGMP biosensors targeted to troponin I and phospholamban and found that BNP and CNP increase cGMP differently around these proteins in both normal and failing cardiomyocytes. This may explain the different effects of BNP and CNP on cardiac contractility and relaxation, with possible implications for understanding and treatment of heart failure.

Aims Epidermal growth factor receptor (EGFR) is essential to the development of multiple tissues and organs and is a target of cancer therapeutics. Due to the embryonic lethality of global EGFR deletion and conflicting reports of cardiac-overexpressed EGFR mutants, its specific impact on the adult heart, normally or in response to chronic stress, has not been established. Using complimentary genetic strategies to modulate cardiomyocyte-specific EGFR expression, we aim to define its role in the regulation of cardiac function and remodeling. Methods and results A floxed EGFR mouse model with α-myosin heavy chain-Cre-mediated cardiomyocyte-specific EGFR downregulation (CM-EGFR-KD mice) developed contractile dysfunction by 9 weeks of age, marked by impaired diastolic relaxation, as monitored via echocardiographic, hemodynamic and isolated cardiomyocyte contractility analyses. This contractile defect was maintained over time without overt cardiac remodeling until 10 months of age, after which the mice ultimately developed severe heart failure and reduced lifespan. Acute downregulation of EGFR in adult floxed EGFR mice with adeno-associated virus 9 (AAV9)-encoded Cre with a cardiac troponin T promoter (AAV9-cTnT-Cre) recapitulated the CM-EGFR-KD phenotype, while AAV9-cTnT-EGFR treatment of adult CM-EGFR-KD mice rescued the phenotype. Notably, chronic administration of the β-adrenergic receptor (βAR) agonist isoproterenol effectively and reversibly compensated for the contractile dysfunction in the absence of cardiomyocyte hypertrophy in CM-EGFR-KD mice. Mechanistically, EGFR downregulation reduced the expression of protein phosphatase 2 A (PP2A) regulatory subunit Ppp2r3a/PR72, which was associated with decreased phosphorylation of phospholamban (PLB) and Ca2+ clearance, and whose re-expression via AAV9-cTnT-PR72 rescued the CM-EGFR-KD phenotype. Conclusions Altogether our study highlights a previously unrecognized role for EGFR in maintaining contractile homeostasis under physiologic conditions in the adult heart via regulation of PR72 expression. Translational perspective Our study highlights a previously unrecognized role for EGFR in maintaining contractile homeostasis under physiologic conditions in the adult heart via regulation of PR72, a PP2A regulatory subunit with an unknown impact on cardiac function. Further, we have shown that cardiomyocyte-expressed EGFR is required for the promotion of cardiac hypertrophy under conditions of chronic catecholamine stress. Altogether, our study provides new insight into the dynamic nature of cardiomyocyte-specific EGFR.

Aims Direct remuscularization with pluripotent stem cell-derived cardiomyocytes (PSC-CMs) seeks to address the onset of heart failure post-myocardial infarction (MI) by treating the persistent muscle deficiency that underlies it. However, direct remuscularization with PSC-CMs could potentially be arrhythmogenic. We investigated two possible mechanisms of arrhythmogenesis-focal vs reentrant-arising from direct remuscularization with PSC-CM patches in two personalized, human ventricular computer models of post-MI. Moreover, we developed a principled approach for evaluating arrhythmogenicity of direct remuscularization that factors in the VT propensity of the patient-specific post-MI fibrotic substrate and use it to investigate different conditions of patch remuscularization. Methods & results Two personalized, human ventricular models of post-MI (P1 & P2) were constructed from late gadolinium enhanced (LGE)-magnetic resonance images (MRI). In each model, remuscularization with PSC-CM patches were simulated under different treatment conditions that included patch engraftment, patch myofibril orientation, remuscularization site, patch size (thickness and diameter), and patch maturation. To determine arrhythmogenicity of treatment conditions, VT burden of heart models was quantified prior to and after simulated remuscularization and compared. VT burden was quantified based on inducibility (i.e., weighted sum of pacing sites that induced) and severity (i.e., the number of distinct VT morphologies induced). Prior to remuscularization, VT burden was significant in P1 (0.275) and not in P2 (0.0, not VT inducible). We highlight that reentrant VT mechanisms would dominate over focal mechanisms; spontaneous beats emerging from PSC-CM grafts were always a fraction of resting sinus rate. Moreover, incomplete patch engraftment can be particularly arrhythmogenic, giving rise to particularly aberrant electrical activation and conduction slowing across the PSC-CM patches along with elevated VT burden when compared to complete engraftment. Under conditions of complete patch engraftment, remuscularization was almost always arrhythmogenic in P2 but certain treatment conditions could be anti-arrhythmogenic in P1. Moreover, the remuscularization site was the most important factor affecting VT burden in both P1 and P2. Complete maturation of PSC-CM patches, both ionically and electrotonically, at the appropriate site could completely alleviate VT burden. Conclusion We identified that reentrant VT would be the primary VT mechanism in patch remuscularization. To evaluate the arrhythmogenicity of remuscularization, we developed a principled approach that factors in the propensity of the patient-specific fibrotic substrate for VT. We showed that arrhythmogenicity is sensitive to the patient-specific fibrotic substrate and remuscularization site. We demonstrate that targeted remuscularization can be safe in the appropriate individual and holds the potential to nondestructively eliminate VT post-MI in addition to addressing muscle deficiency underlying heart failure progression. Translational perspective If safety from ventricular arrhythmias can be addressed, direct remuscularization with PSC-CMs-achieved either through engineered myocardial patches or intramyocardial injections-holds the potential to halt heart failure progression post-MI. Using personalized 3 D models of the post-MI ventricles derived from LGE-MRI, we provide evidence that arrhythmogenesis following remuscularization with PSC-CM patches is driven by a reentrant as opposed to focal VT mechanism. Moreover, the existing patient-specific fibrotic substrate together with the remuscularization site were primary determinants of arrhythmogenesis. These results suggest that the clinical safety of remuscularization can be achieved through patient-specific optimization guided in-part by computational modeling.

Aims Cardiomyocyte Ca2+ homoeostasis is altered with ageing and predisposes the heart to Ca2+ intolerance and arrhythmia. Transient receptor potential vanilloid 4 (TRPV4) is an osmotically activated cation channel with expression in cardiomyocytes of the aged heart. The objective of this study was to examine the role of TRPV4 in Ca2+ handling and arrhythmogenesis following ischaemia–reperfusion (I/R), a pathological scenario associated with osmotic stress. Methods and results Cardiomyocyte membrane potential was monitored prior to and following I/R in Langendorff-perfused hearts of Aged (19–28 months) male and female C57BL/6 mice ± TRPV4 inhibition (1 μM HC067047, HC). Diastolic resting membrane potential was similar between Aged and Aged HC at baseline, but following I/R Aged exhibited depolarized diastolic membrane potential vs. Aged HC. The effects of TRPV4 on cardiomyocyte Ca2+ signalling following I/R were examined in isolated hearts of Aged cardiac-specific GCaMP6f mice (±HC) using high-speed confocal fluorescence microscopy, with cardiomyocytes of Aged exhibiting an increased incidence of pro-arrhythmic Ca2+ signalling vs. Aged HC. In the isolated cell environment, cardiomyocytes of Aged responded to sustained hypoosmotic stress (250mOsm) with an increase in Ca2+ transient amplitude (fluo-4) and higher incidence of pro-arrhythmic diastolic Ca2+ signals vs. Aged HC. Intracardiac electrocardiogram measurements in isolated hearts following I/R revealed an increased arrhythmia incidence, an accelerated time to ventricular arrhythmia, and increased arrhythmia score in Aged vs. Aged HC. Aged exhibited depolarized resting membrane potential, increased pro-arrhythmic diastolic Ca2+ signalling, and greater incidence of arrhythmia when compared with Young (3–5 months). Conclusion TRPV4 contributes to pro-arrhythmic cardiomyocyte Ca2+ signalling, electrophysiological abnormalities, and ventricular arrhythmia in the aged mouse heart.

Acute myocardial infarction causes lethal cardiomyocyte injury during ischaemia and reperfusion (I/R). Histones have been described as important Danger Associated Molecular Proteins (DAMPs) in sepsis. Aims The objective of this study was to establish whether extracellular histone release contributes to myocardial infarction. Methods and results Isolated, perfused rat hearts were subject to I/R. Nucleosomes and histone H4 release was detected early during reperfusion. Sodium-β-O-Methyl cellobioside sulfate (mCBS), a newly developed histone-neutralising compound, significantly reduced infarct size whilst also reducing the detectable levels of histones. Histones were directly toxic to primary adult rat cardiomyocytes in vitro. This was prevented by mCBS, or HIPe, a recently described, histone-H4 neutralizing peptide, but not by an inhibitor of TLR4, a receptor previously reported to be involved in DAMP-mediated cytotoxicity. Furthermore, TLR4-reporter HEK293 cells revealed that cytotoxicity of histone H4 was independent of TLR4 and NF-κB. In an in vivo rat model of I/R, HIPe significantly reduced infarct size. Conclusion Histones released from the myocardium are cytotoxic to cardiomyocytes, via a TLR4-independent mechanism. The targeting of extracellular histones provides a novel opportunity to limit cardiomyocyte death during I/R injury of the myocardium. Translational perspective Acute myocardial infarction causes lethal cardiomyocyte injury during ischaemia and reperfusion (I/R). New approaches are needed to prevent cardiomyocyte injury and limit final infarct size. We show that histones released from damaged cells, and histone-H4 in particular, causes rapid cardiomyocyte death during I/R. mCBS, a compounds targeting histones non-specifically, was cardioprotective in ex vivo rat hearts, while HIPe, a targeting histone H4 specifically, was cardioprotective in an in vivo rat model. HIPe may have potential as a therapeutic agent in the setting of acute myocardial infarction.

Aims The precise cellular identity and molecular features of non-myocytes (nonCM) in a mammalian heart at a single cell level remain elusive. Depiction of epigenetic landscape with transcriptomic signatures using the latest single-cell multi-omics has the potential to unravel the molecular programs underlying the cellular diversity of cardiac non-myocytes. Here, we characterized the molecular and cellular features of cardiac nonCM populations in the adult murine heart at the single cell level. Methods and results Through single-cell dual omics analysis, we mapped the epigenetic landscapes, characterized the transcriptomic profiles and delineated the molecular signatures of cardiac nonCMs in the adult murine heart. Distinct cis-regulatory elements and trans-acting factors for the individual major nonCM cell types (endothelial cells, fibroblast, pericytes and immune cells) were identified. In particular, unbiased sub-clustering and functional annotation of cardiac fibroblasts (FB) revealed extensive FB heterogeneity and identified FB subtypes with functional states related to cellular response to stimuli, cytoskeleton organization and immune regulation, respectively. We further explored the function of marker genes Hsd11b1 and Gfpt2 that label major FB sub-populations and determined the distribution of Hsd11b1+ and Gfp2+ FBs in murine healthy and diseased hearts. Conclusions In summary, we characterized the nonCM cellular identity at the transcriptome and epigenome levels using single-cell omics approaches and discovered previously unrecognized cardiac fibroblast subpopulations with unique functional states. Translational perspective Our research identified discrete cell types of nonCM in the heart and differentially expressed genes with regulatory factors. Unveiling the heterogeneity of nonCMs and molecular signatures of each cell type or subtypes allows for study, precise capture and manipulation of specific cell type(s) in heart and will provide insights into the development of therapeutics for cardiovascular diseases.

Aims Macrophages (MΦ), known for immunological roles such as phagocytosis and antigen presentation, have been found to electrotonically couple to cardiomyocytes (CM) of the atrio-ventricular node via Cx43, affecting cardiac conduction in isolated mouse hearts. Here, we characterise passive and active electrophysiological properties of murine cardiac resident MΦ, and model their potential electrophysiological relevance for CM. Methods and results We combined classic electrophysiological approaches with 3 D florescence imaging, RNA-sequencing, pharmacological interventions and computer simulations. We used Cx3cr1eYFP/+ mice wherein cardiac MΦ were fluorescently labelled. FACS-purified fluorescent MΦ from mouse hearts were studied by whole-cell patch-clamp. MΦ electrophysiological properties include: membrane resistance 2.2 ± 0.1 GΩ (all data mean±SEM), capacitance 18.3 ± 0.1 pF, resting membrane potential -39.6 ± 0.3 mV, and several voltage-activated, outward or inwardly-rectifying potassium currents. Using ion channel blockers (barium, TEA, 4-AP, margatoxin, XEN-D0103, DIDS), flow cytometry, immuno-staining and RNA-sequencing, we identified Kv1.3, Kv1.5 and Kir2.1 as channels contributing to observed ion currents. MΦ displayed four patterns for outward and two for inward-rectifier potassium currents. Additionally, MΦ showed surface expression of Cx43, a prerequisite for homo- and/or heterotypic electrotonic coupling. Experimental results fed into development of an original computational model to describe cardiac MΦ electrophysiology. Computer simulations to quantitatively assess plausible effects of MΦ on electrotonically coupled CM showed that MΦ can depolarise resting CM, shorten early and prolong late action potential duration, with effects depending on coupling strength and individual MΦ electrophysiological properties, in particular resting membrane potential and presence/absence of Kir2.1. Conclusions Our results provide a first electrophysiological characterisation of cardiac resident MΦ, and a computational model to quantitatively explore their relevance in the heterocellular heart. Future work will be focussed at distinguishing electrophysiological effects of MΦ-CM coupling on both cell types during steady-state and in patho-physiological remodelling, when immune cells change their phenotype, proliferate, and/or invade from external sources. Translational perspective Cardiac tissue contains resident macrophages (MΦ) which, beyond immunological and housekeeping roles, have been found to electrotonically couple via connexins to cardiomyocytes (CM), stabilising atrio-ventricular conduction at high excitation rates. Here, we characterise structure and electrophysiological function of murine cardiac MΦ and provide a computational model to quantitatively probe the potential relevance of MΦ-CM coupling for cardiac electrophysiology. We find that MΦ are unlikely to have major electrophysiological effects in normal tissue, where they would hasten early and slow late CM-repolarisation. Further work will address potential arrhythmogenicity of MΦ in patho-physiologically remodelled tissue containing elevated MΦ-numbers, incl. non-resident recruited cells.

Aims We prospectively isolate and characterize first and second heart field- and nodal-like cardiomyocytes using a double reporter line from human embryonic stem cells. Our double reporter line utilizes two important transcription factors in cardiac development, TBX5 and NKX2-5. TBX5 expression marks first heart field progenitors and cardiomyocytes while NKX2-5 is expressed in nearly all myocytes of the developing heart (excluding nodal cells). We address the shortcomings of prior work in the generation of heart-field specific cardiomyocytes from induced pluripotent stem cells and provide a comprehensive early developmental transcriptomic as well as electrophysiological analyses of these three populations. Methods and results Transcriptional, immunocytochemical, and functional studies support the cellular identities of isolated populations based on the expression pattern of NKX2-5 and TBX5. Importantly, bulk and single-cell RNA sequencing analyses provide evidence of unique molecular signatures of isolated first and second heart-field cardiomyocytes, as well as nodal-like cells. Extensive electrophysiological analyses reveal dominant atrial action potential phenotypes in first and second heart fields in alignment with our findings in single-cell RNA sequencing. Lastly, we identify two novel surface markers, POPDC2 and CORIN, that enables purification of cardiomyocytes and first heart field cardiomyocytes, respectively. Conclusions We describe a high yield approach for isolation and characterization of human embryonic stem cell-derived heart field specific and nodal-like cardiomyocytes. Obtaining enriched populations of these different cardiomyocyte subtypes increases the resolution of gene expression profiling during early cardiogenesis, arrhythmia modeling, and drug screening. This paves the way for the development of effective stem cell therapy to treat diseases that affect specific regions of the heart or chamber-specific congenital heart defects. Translational perspective Myocardial infarction leads to irreversible loss of cardiomyocytes and eventually heart failure. Human embryonic stem cells (hESCs) can be differentiated to cardiomyocytes and are considered a potential source of cell therapy for cardiac regeneration. However, current differentiation strategies yield a mixture of cardiomyocyte subtypes and safety concerns stemming from the use of a heterogenous population of cardiomyocytes have hindered its application. Here, we report generation of enriched heart field-specific cardiomyocytes using a hESC double reporter. Our study facilitates investigating early human cardiogenesis in vitro and generating chamber-specific cardiomyocytes to treat diseases that affect specific regions of the heart.

Aims After a myocardial infarction, the adult human heart lacks sufficient regenerative capacity to restore lost tissue, leading to heart failure progression. Finding novel ways to reprogram adult cardiomyocytes into a regenerative state is a major therapeutic goal. The epicardium, the outermost layer of the heart, contributes cardiovascular cell types to the forming heart and is a source of trophic signals to promote heart muscle growth during embryonic development. The epicardium is also essential for heart regeneration in zebrafish and neonatal mice and can be reactivated after injury in adult hearts to improve outcome. A recently identified mechanism of cell–cell communication and signalling is that mediated by extracellular vesicles (EVs). Here, we aimed to investigate epicardial signalling via EV release in response to cardiac injury and as a means to optimize cardiac repair and regeneration. Methods and results We isolated epicardial EVs from mouse and human sources and targeted the cardiomyocyte population. Epicardial EVs enhanced proliferation in H9C2 cells and in primary neonatal murine cardiomyocytes in vitro and promoted cell cycle re-entry when injected into the injured area of infarcted neonatal hearts. These EVs also enhanced regeneration in cryoinjured engineered human myocardium (EHM) as a novel model of human myocardial injury. Deep RNA-sequencing of epicardial EV cargo revealed conserved microRNAs (miRs) between human and mouse epicardial-derived exosomes, and the effects on cell cycle re-entry were recapitulated by administration of cargo miR-30a, miR-100, miR-27a, and miR-30e to human stem cell-derived cardiomyocytes and cryoinjured EHM constructs. Conclusion Here, we describe the first characterization of epicardial EV secretion, which can signal to promote proliferation of cardiomyocytes in infarcted mouse hearts and in a human model of myocardial injury, resulting in enhanced contractile function. Analysis of exosome cargo in mouse and human identified conserved pro-regenerative miRs, which in combination recapitulated the therapeutic effects of promoting cardiomyocyte proliferation., Graphical Abstract

Aims The transcription factor Tbx5 controls cardiogenesis and drives Scn5a expression in mice. We have identified two variants in TBX5 encoding p. D111Y and p. F206L Tbx5, respectively, in two unrelated patients with structurally normal hearts diagnosed with long QT (LQTS) and Brugada (BrS) syndrome. Here, we characterized the consequences of each variant to unravel the underlying disease mechanisms. Methods and results We combined clinical analysis with in vivo and in vitro electrophysiological and molecular techniques in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs), HL-1 cells, and cardiomyocytes from mice trans-expressing human wild-type (WT) or mutant proteins. Tbx5 increased transcription of SCN5A encoding cardiac Nav1.5 channels, while repressing CAMK2D and SPTBN4 genes encoding Ca/calmodulin kinase IIδ (CaMKIIδ) and βIV-spectrin, respectively. These effects significantly increased Na current (INa) in hiPSC-CMs and in cardiomyocytes from mice trans-expressing Tbx5. Consequently, action potential (AP) amplitudes increased and QRS interval narrowed in the mouse electrocardiogram. p. F206L Tbx5 bound to the SCN5A promoter failed to transactivate it, thus precluding the pro-transcriptional effect of WT Tbx5. Therefore, p. F206L markedly decreased INa in hiPSC-CM, HL-1 cells and mouse cardiomyocytes. The INa decrease in p. F206L trans-expressing mice translated into QRS widening and increased flecainide sensitivity. p. D111Y Tbx5 increased SCN5A expression but failed to repress CAMK2D and SPTBN4. The increased CaMKIIδ and βIV-spectrin significantly augmented the late component of INa (INaL) which, in turn, significantly prolonged AP duration in both hiPSC-CMs and mouse cardiomyocytes. Ranolazine, a selective INaL inhibitor, eliminated the QT and QTc intervals prolongation seen in p. D111Y trans-expressing mice. Conclusions In addition to peak INa, Tbx5 critically regulates INaL and the duration of repolarization in human cardiomyocytes. Our original results suggest that TBX5 variants associate with and modulate the intensity of the electrical phenotype in LQTS and BrS patients.

Aims Angiotensin (Ang) II signalling has been suggested to promote cardiac fibrosis in inflammatory heart diseases; however, the underlying mechanisms remain obscure. Using Agtr1a-/- mice with genetic deletion of angiotensin receptor type 1 (ATR1) and the experimental autoimmune myocarditis (EAM) model, we aimed to elucidate the role of Ang II-ATR1 pathway in development of heart-specific autoimmunity and post-inflammatory fibrosis. Methods and results EAM was induced in wild-type (WT) and Agtr1a-/- mice by subcutaneous injections with alpha myosin heavy chain peptide emulsified in complete Freund’s adjuvant. Agtr1a-/- mice developed myocarditis to a similar extent as WT controls at day 21 but showed reduced fibrosis and better systolic function at day 40. Crisscross bone marrow chimaera experiments proved that ATR1 signalling in the bone marrow compartment was critical for cardiac fibrosis. Heart infiltrating, bone-marrow-derived cells produced Ang II, but lack of ATR1 in these cells reduced transforming growth factor beta (TGF-β)-mediated fibrotic responses. At the molecular level, Agtr1a-/- heart-inflammatory cells showed impaired TGF-β-mediated phosphorylation of Smad2 and TAK1. In WT cells, TGF-β induced formation of RhoA-GTP and RhoA-A-kinase anchoring protein-Lbc (AKAP-Lbc) complex. In Agtr1a-/- cells, stabilization of RhoA-GTP and interaction of RhoA with AKAP-Lbc were largely impaired. Furthermore, in contrast to WT cells, Agtr1a-/- cells stimulated with TGF-β failed to activate canonical Wnt pathway indicated by suppressed activity of glycogen synthase kinase-3 (GSK-3)β and nuclear β-catenin translocation and showed reduced expression of Wnts. In line with these in vitro findings, β-catenin was detected in inflammatory regions of hearts of WT, but not Agtr1a-/- mice and expression of canonical Wnt1 and Wnt10b were lower in Agtr1a-/- hearts. Conclusion Ang II-ATR1 signalling is critical for development of post-inflammatory fibrotic remodelling and dilated cardiomyopathy. Our data underpin the importance of Ang II-ATR1 in effective TGF-β downstream signalling response including activation of profibrotic Wnt/β-catenin pathway., Graphical Abstract

Aims Diabetic hyperglycaemia is associated with increased arrhythmia risk. We aimed to investigate whether hyperglycaemia alone can be accountable for arrhythmias or whether it requires the presence of additional pathological factors. Methods and results Action potentials (APs) and arrhythmogenic spontaneous diastolic activities were measured in isolated murine ventricular, rabbit atrial, and ventricular myocytes acutely exposed to high glucose. Acute hyperglycaemia increased the short-term variability (STV) of action potential duration (APD), enhanced delayed afterdepolarizations, and the inducibility of APD alternans during tachypacing in both murine and rabbit atrial and ventricular myocytes. Hyperglycaemia also prolonged APD in mice and rabbit atrial cells but not in rabbit ventricular myocytes. However, rabbit ventricular APD was more strongly depressed by block of late Na+ current (INaL) during hyperglycaemia, consistent with elevated INaL in hyperglycaemia. All the above proarrhythmic glucose effects were Ca2+-dependent and abolished by CaMKII inhibition. Importantly, when the repolarization reserve was reduced by pharmacological inhibition of K+ channels (either Ito, IKr, IKs, or IK1) or hypokalaemia, acute hyperglycaemia further prolonged APD and further increased STV and alternans in rabbit ventricular myocytes. Likewise, when rabbit ventricular myocytes were pretreated with isoproterenol or angiotensin II, hyperglycaemia significantly prolonged APD, increased STV and promoted alternans. Moreover, acute hyperglycaemia markedly prolonged APD and further enhanced STV in failing rabbit ventricular myocytes. Conclusion We conclude that even though hyperglycaemia alone can enhance cellular proarrhythmic mechanisms, a second hit which reduces the repolarization reserve or stimulates G protein-coupled receptor signalling greatly exacerbates cardiac arrhythmogenesis in diabetic hyperglycaemia.

Aims Myocarditis is associated with formidable symptoms and increased risk of adverse outcomes. Current approaches mostly rely on symptomatic treatments, warranting novel concepts for clinical practice. The aim of this study was to investigate the microRNA (miRNA) expression profile of Balb/c mice with experimental autoimmune myocarditis (EAM), choose a representative miRNA to antagonize after review of available literature and test its effects on myocardial inflammation in vitro and in vivo. Methods and results Methods: Phase 1: EAM was induced in 12 male Balb/c mice, 10 animals served as controls. After sacrifice, next-generation sequencing (NGS) of the microRNA expression profile was performed. Based on these results, H9C2 cells and human ventricular cardiac fibroblasts exposed to lipopolysaccharide (LPS) were treated with the selected candidate antagomiR-21a-5p. Phase 2: EAM was induced in 48 animals. Thereof, 24 animals were either treated with antagomiR-21a-5p or negative control oligonucleotide in a nanoparticle formulation. Transthoracic echocardiography (TTE) was performed on days 0,7,14 and 21. Histopathological examination was performed after sacrifice. Results: Phase 1: EAM resulted in a significant upregulation of 27 miRNAs, including miR-21a-5p (log2FC: 2.23, adj. p = 0.0026). Transfection with antagomiR-21a-5p resulted in a significant reduction of TNFα, IL-6 and collagen I in vitro. Phase 2: Treatment with antagomiR-21a-5p, formulated in polymeric nanoparticles for systemic injection, significantly attenuated myocardial inflammation (p = 0.001) and fibrosis (p = 0.013), as well as myocardial "hypertrophy" on TTE. Conclusions Silencing of miR-21a-5p results in a significant reduction of the expression of pro-inflammatory cytokines in vitro, as well as a significant attenuation of inflammation, fibrosis and echocardiographic effects of EAM in vivo. Translational perspective Based on the findings of our study, silencing of miR-21a-5p by synthetic antagomiR could constitute a novel therapeutic approach in myocarditis. Hence, research on this matter certainly warrants further scientific attention and investigation.

CD36, also known as the scavenger receptor B2, is a multifunctional receptor widely expressed in various organs. CD36 plays a crucial role in the uptake of long-chain fatty acids, the main metabolic substrate in myocardial tissue. The maturation and transportation of CD36 is regulated by post-translational modifications, including phosphorylation, ubiquitination, glycosylation, and palmitoylation. CD36 is decreased in pathological cardiac hypertrophy caused by ischaemia–reperfusion and pressure overload, and increased in diabetic cardiomyopathy and atherosclerosis. Deficiency of CD36 alleviates diabetic cardiomyopathy and atherosclerosis, while overexpression of CD36 eliminates ischaemia–reperfusion damage, together suggesting that CD36 is closely associated with the progression of cardiovascular diseases and may be a new therapeutic target. This review summarizes the regulation and post-translational modifications of CD36 and evaluates its role in cardiovascular diseases and its potential as a therapeutic target.

Myocardial fibrosis, the expansion of the cardiac interstitium through deposition of extracellular matrix proteins, is a common pathophysiologic companion of many different myocardial conditions. Fibrosis may reflect activation of reparative or maladaptive processes. Activated fibroblasts and myofibroblasts are the central cellular effectors in cardiac fibrosis, serving as the main source of matrix proteins. Immune cells, vascular cells and cardiomyocytes may also acquire a fibrogenic phenotype under conditions of stress, activating fibroblast populations. Fibrogenic growth factors (such as transforming growth factor-β and platelet-derived growth factors), cytokines [including tumour necrosis factor-α, interleukin (IL)-1, IL-6, IL-10, and IL-4], and neurohumoral pathways trigger fibrogenic signalling cascades through binding to surface receptors, and activation of downstream signalling cascades. In addition, matricellular macromolecules are deposited in the remodelling myocardium and regulate matrix assembly, while modulating signal transduction cascades and protease or growth factor activity. Cardiac fibroblasts can also sense mechanical stress through mechanosensitive receptors, ion channels and integrins, activating intracellular fibrogenic cascades that contribute to fibrosis in response to pressure overload. Although subpopulations of fibroblast-like cells may exert important protective actions in both reparative and interstitial/perivascular fibrosis, ultimately fibrotic changes perturb systolic and diastolic function, and may play an important role in the pathogenesis of arrhythmias. This review article discusses the molecular mechanisms involved in the pathogenesis of cardiac fibrosis in various myocardial diseases, including myocardial infarction, heart failure with reduced or preserved ejection fraction, genetic cardiomyopathies, and diabetic heart disease. Development of fibrosis-targeting therapies for patients with myocardial diseases will require not only understanding of the functional pluralism of cardiac fibroblasts and dissection of the molecular basis for fibrotic remodelling, but also appreciation of the pathophysiologic heterogeneity of fibrosis-associated myocardial disease.

Aims Dilated cardiomyopathy (DCM) is associated with mutations in many genes encoding sarcomere proteins. Truncating mutations in the titin gene TTN are the most frequent. Proteomic and functional characterizations are required to elucidate the origin of the disease and the pathogenic mechanisms of TTN-truncating variants. Methods and results We isolated myofibrils from DCM hearts carrying truncating TTN mutations and measured the Ca2+ sensitivity of force and its length dependence. Simultaneous measurement of force and adenosine triphosphate (ATP) consumption in skinned cardiomyocytes was also performed. Phosphorylation levels of troponin I (TnI) and myosin binding protein-C (MyBP-C) were manipulated using protein kinase A and λ phosphatase. mRNA sequencing was employed to overview gene expression profiles. We found that Ca2+ sensitivity of myofibrils carrying TTN mutations was significantly higher than in myofibrils from donor hearts. The length dependence of the Ca2+ sensitivity was absent in DCM myofibrils with TTN-truncating variants. No significant difference was found in the expression level of TTN mRNA between the DCM and donor groups. TTN exon usage and splicing were also similar. However, we identified down-regulation of genes encoding Z-disk proteins, while the atrial-specific regulatory myosin light chain gene, MYL7, was up-regulated in DCM patients with TTN-truncating variants. Conclusion Titin-truncating mutations lead to decreased length-dependent activation and increased elasticity of myofibrils. Phosphorylation levels of TnI and MyBP-C seen in the left ventricles are essential for the length-dependent changes in Ca2+ sensitivity in healthy donors, but they are reduced in DCM patients with TTN-truncating variants. A decrease in expression of Z-disk proteins may explain the observed decrease in myofibril passive stiffness and length-dependent activation., Graphical Abstract

Aims Emipagliflozin (EMPA) is a potent inhibitor of the renal sodium-glucose co-transporter 2 (SGLT2) and an effective treatment for type-2 diabetes. In patients with diabetes and heart failure, EMPA has cardioprotective effects independent of improved glycaemic control, despite SGLT2 not being expressed in the heart. A number of non-canonical mechanisms have been proposed to explain these cardiac effects, most notably an inhibitory action on cardiac Na+/H+ exchanger 1 (NHE1), causing a reduction in intracellular [Na+] ([Na+]i). However, at resting intracellular pH (pHi), NHE1 activity is very low and its pharmacological inhibition is not expected to meaningfully alter steady-state [Na+]i. We re-evaluate this putative EMPA target by measuring cardiac NHE1 activity. Methods and results The effect of EMPA on NHE1 activity was tested in isolated rat ventricular cardiomyocytes from measurements of pHi recovery following an ammonium pre-pulse manoeuvre, using cSNARF1 fluorescence imaging. Whereas 10 µM cariporide produced near-complete inhibition, there was no evidence for NHE1 inhibition with EMPA treatment (1, 3, 10, or 30 µM). Intracellular acidification by acetate-superfusion evoked NHE1 activity and raised [Na+]i, reported by sodium binding benzofuran isophthalate (SBFI) fluorescence, but EMPA did not ablate this rise. EMPA (10 µM) also had no significant effect on the rate of cytoplasmic [Na+]i rise upon superfusion of Na+-depleted cells with Na+-containing buffers. In Langendorff-perfused mouse, rat and guinea pig hearts, EMPA did not affect [Na+]i at baseline nor pHi recovery following acute acidosis, as measured by 23Na triple quantum filtered NMR and 31P NMR, respectively. Conclusions Our findings indicate that cardiac NHE1 activity is not inhibited by EMPA (or other SGLT2i’s) and EMPA has no effect on [Na+]i over a wide range of concentrations, including the therapeutic dose. Thus, the beneficial effects of SGLT2i’s in failing hearts should not be interpreted in terms of actions on myocardial NHE1 or intracellular [Na+]., Graphical Abstract

Proper cardiac Ca2+ homeostasis is essential for normal excitation–contraction coupling. Perturbations in cardiac Ca2+ handling through altered kinase activity has been implicated in altered cardiac contractility and arrhythmogenesis. Thus, a better understanding of cardiac Ca2+ handling regulation is vital for a better understanding of various human disease processes. ‘Striated muscle preferentially expressed protein kinase’ (SPEG) is a member of the myosin light chain kinase family that is key for normal cardiac function. Work within the last 5 years has revealed that SPEG has a crucial role in maintaining normal cardiac Ca2+ handling through maintenance of transverse tubule formation and phosphorylation of junctional membrane complex proteins. Additionally, SPEG has been causally impacted in human genetic diseases such as centronuclear myopathy and dilated cardiomyopathy as well as in common acquired cardiovascular disease such as heart failure and atrial fibrillation. Given the rapidly emerging role of SPEG as a key cardiac Ca2+ regulator, we here present this review in order to summarize recent findings regarding the mechanisms of SPEG regulation of cardiac excitation–contraction coupling in both physiology and human disease. A better understanding of the roles of SPEG will be important for a more complete comprehension of cardiac Ca2+ regulation in physiology and disease.

Aims Peripartum cardiomyopathy (PPCM) is a life-threatening heart disease occurring in previously heart-healthy women. A common pathomechanism in PPCM involves the angiostatic 16 kDa-prolactin (16 kDa-PRL) fragment, which via NF-κB-mediated up-regulation of microRNA-(miR)-146a induces vascular damage and heart failure. We analyse whether the plasminogen activator inhibitor-1 (PAI-1) is involved in the pathophysiology of PPCM. Methods and results In healthy age-matched postpartum women (PP-Ctrl, n = 53, left ventricular ejection fraction, LVEF > 55%), PAI-1 plasma levels were within the normal range (21 ± 10 ng/mL), but significantly elevated (64 ± 38 ng/mL, P Conclusion In PPCM patients, circulating and cardiac PAI-1 expression are up-regulated. While circulating PAI-1 may add 16 kDa-PRL to induce vascular impairment via the uPAR/NF-κB/miR-146a pathway, experimental data suggest that cardiac PAI-1 expression seems to protect the PPCM heart from fibrosis. Thus, measuring circulating PAI-1 and miR-146a, together with an uPAR/NF-κB-activity assay could be developed into a specific diagnostic marker assay for PPCM, but unrestricted reduction of PAI-1 for therapy may not be advised.

In western countries, cardiovascular (CV) disease and cancer are the leading causes of death in the ageing population. Recent epidemiological data suggest that cancer is more frequent in patients with prevalent or incident CV disease, in particular, heart failure (HF). Indeed, there is a tight link in terms of shared risk factors and mechanisms between HF and cancer. HF induced by anticancer therapies has been extensively studied, primarily focusing on the toxic effects that anti-tumour treatments exert on cardiomyocytes. In this Cardio-Oncology update, members of the ESC Working Groups of Myocardial Function and Cellular Biology of the Heart discuss novel evidence interconnecting cardiac dysfunction and cancer via pathways in which cardiomyocytes may be involved but are not central. In particular, the multiple roles of cardiac stromal cells (endothelial cells and fibroblasts) and inflammatory cells are highlighted. Also, the gut microbiota is depicted as a new player at the crossroads between HF and cancer. Finally, the role of non-coding RNAs in Cardio-Oncology is also addressed. All these insights are expected to fuel additional research efforts in the field of Cardio-Oncology.

Aims Heart failure with preserved LV ejection fraction (HFpEF) is a serious health problem worldwide, as no effective therapy is yet available. We have previously demonstrated that our low-intensity pulsed ultrasound (LIPUS) therapy is effective and safe for angina and dementia. In this study, we aimed to examine whether the LIPUS therapy also ameliorates cardiac diastolic dysfunction in mice. Methods and results Twelve-weeks-old obese diabetic mice (db/db) and their control littermates (db/+) were treated with either the LIPUS therapy (1.875 MHz, 32 cycles, Ispta (spatial peak temporal average intensity) 117-162 mW/cm2, 0.25 W/cm2) or placebo procedure 2 times a week for 4 weeks. At 20-week-old, transthoracic echocardiography and invasive hemodynamic analysis showed that cardiac diastolic function parameters, such as e', E/e', end-diastolic pressure-volume relationship, Tau, and dP/dt min, were all deteriorated in placebo-treated db/db mice compared with db/+ mice, while systolic function was preserved. Importantly, these cardiac diastolic function parameters were significantly ameliorated in the LIPUS-treated db/db mice. We also measured the force (F) and intracellular Ca2+ ([Ca2+]i) in trabeculae dissected from ventricles. We found that relaxation time and [Ca2+]i decay (Tau) were prolonged during electrically stimulated twitch contractions in db/db mice, both of which were significantly ameliorated in the LIPUS-treated db/db mice, indicating that the LIPUS therapy also improves relaxation properties at tissue level. Functionally, exercise capacity was also improved in the LIPUS-treated db/db mice. Histologically, db/db mice displayed progressed cardiomyocyte hypertrophy and myocardial interstitial fibrosis, while those changes were significantly suppressed in the LIPUS-treated db/db mice. Mechanistically, Western blot showed that the eNOS-NO-cGMP-PKG pathway and Ca2+-handling molecules were up-regulated in the LIPUS-treated heart. Conclusions These results indicate that the LIPUS therapy ameliorates cardiac diastolic dysfunction in db/db mice through improvement of eNOS-NO-cGMP-PKG pathway and cardiomyocyte Ca2+-handling system, suggesting its potential usefulness for the treatment of HFpEF patients. Translational perspective Although HFpEF is a serious health problem worldwide, no effective treatment is yet available. We have previously demonstrated that our low-intensity pulsed ultrasound (LIPUS) therapy is effective and safe in animal models of angina and dementia. In this study, we examined whether our LIPUS therapy is also effective to improve cardiac diastolic dysfunction in mice. We found that the LIPUS therapy ameliorated myocardial structures and Ca2+-handling proteins, resulting in the improvement of cardiac diastolic functions and exercise tolerance in a mouse model of HFpEF. These results suggest that the LIPUS therapy is useful for the treatment of HFpEF in humans.

Aims To examine whether transient over-expression of angiopoietin-1 (Ang-1) increases the potency of hiPSC-CMs for treatment of heart failure. Methods and results Atrial hiPSC-CMs (hiPSC-aCMs) were differentiated from hiPSCs and purified by lactic acid and were transfected with Ang-1 (Ang-1-hiPSC-aCMs) plasmid using lipoSTEM. Ang-1 gene transfection efficiency was characterized in vitro. Gene transfected CMs (1x106) were seeded into a fibrin/thrombin patch and implanted on the rat infarcted left ventricular anterior wall after myocardial infarction (MI). Echo function was determined at 1- and 6- weeks post-MI. Immunohistochemistry study was performed at week-6 post-MI. Ang-1 (20 and 40 ng/ml) protected hiPSC-aCMs from hypoxia through up-regulating pERK1/2 and inhibiting Bax protein expressions. Ang-1-hiPSC-aCMs transiently secreted Ang-1 protein up to 14 days, with peak level on day-2 post-transfection (24.39±13.02 ng/ml) in vitro. Animal study showed that transplantation of Ang-1-hiPSC-aCM seeded patch more effectively limited rat heart apoptosis at 1-day post-MI as compared with LipoSTEM-Ang-1 or hiPSC-aCMs transplantation. Ang-1-hiPSC-aCMs transplantation induced host (rat) and donor (human) CM mitosis and arteriole formation, improved cell engraftment rate, more effectively limited left ventricle (LV) dilation (EDV=460.7±96.1 μl and ESV=219.8±72.9 μl) and improved LV global pump function (EF = 53.1±9%) as compared with the MI (EDV=570.9±91.8 μl, p = 0.033; ESV=331.6±71.2 μl, p = 0.011; EF = 42.3±4.1%, p = 0.02) or the LipoSTEM-Ang-1 injected (EDV=491.4±100.4 μl, p = 0.854; ESV=280.9±71.5 μl, p = 0.287; EF = 43.2±4.6, p = 0.039) or hiPSC-CM transplanted (EDV=547.9±55.5 μl, p = 0.095; ESV=300.2±88.4 μl, p = 0.075; EF = 46±10.9%, p = 0.166) animal groups at 6 weeks post-MI and treatment. Conclusions Transient overexpression of Ang-1 enhanced hiPSC-aCM mitosis and engraftment and increased the reparability potency of hiPSC-aCMs for treatment of MI. Translational perspective Cellular cardiomyoplasty using hiPSC-CMs alone may be insufficient to recover the structure and function of infarcted myocardium in which blood vessels and perfusion are damaged. A combinational approach of myogenesis with vasculogenesis to repopulate CMs with functional neovascular network is necessary to recover LV structure and function post-MI. This is the first report showing evidence that transplantation of hiPSC-CM transiently overexpressing Ang-1 had better cell engraftment and improved heart structure and function post-MI. It highlights that transient genetic modification of hiPSC-CMs with Ang-1 using non-viral vectors offers a better CM based angiomyogenesis therapy for treatment of MI.

Aims Cardiac hypertrophy is a compensatory response to pressure overload, leading to heart failure. Recent studies have demonstrated that Rho is immediately activated in left ventricles after pressure overload and that Rho signalling plays crucial regulatory roles in actin cytoskeleton rearrangement during cardiac hypertrophic responses. However, the mechanisms by which Rho and its downstream proteins control actin dynamics during hypertrophic responses remain not fully understood. In this study, we identified the pivotal roles of mammalian homologue of Drosophila diaphanous (mDia) 1, a Rho-effector molecule, in pressure overload-induced ventricular hypertrophy. Methods and results Male wild-type (WT) and mDia1-knockout (mDia1KO) mice (10–12 weeks old) were subjected to a transverse aortic constriction (TAC) or sham operation. The heart weight/tibia length ratio, cardiomyocyte cross-sectional area, left ventricular wall thickness, and expression of hypertrophy-specific genes were significantly decreased in mDia1KO mice 3 weeks after TAC, and the mortality rate was higher at 12 weeks. Echocardiography indicated that mDia1 deletion increased the severity of heart failure 8 weeks after TAC. Importantly, we could not observe apparent defects in cardiac hypertrophic responses in mDia3-knockout mice. Microarray analysis revealed that mDia1 was involved in the induction of hypertrophy-related genes, including immediate early genes, in pressure overloaded hearts. Loss of mDia1 attenuated activation of the mechanotransduction pathway in TAC-operated mice hearts. We also found that mDia1 was involved in stretch-induced activation of the mechanotransduction pathway and gene expression of c-fos in neonatal rat ventricular cardiomyocytes (NRVMs). mDia1 regulated the filamentous/globular (F/G)-actin ratio in response to pressure overload in mice. Additionally, increases in nuclear myocardin-related transcription factors and serum response factor were perturbed in response to pressure overload in mDia1KO mice and to mechanical stretch in mDia1 depleted NRVMs. Conclusion mDia1, through actin dynamics, is involved in compensatory cardiac hypertrophy in response to pressure overload.

Background During heart failure, the levels of circulatory HSPD1 (HSP60) increase. However, its underlying mechanism is still unknown. The apical domain of HSPD1 is conserved throughout evolution. We found a point mutation in HSPD1 in a familial dilated cardiomyopathy (DCM) patient. A similar point mutation in HSPD1 in the zebrafish mutant, nbl, led to loss of its regenerative capacity and development of pericardial edema under heat stress condition. In this study, we aimed to determine the direct involvement of HSPD1 in the development of DCM. Methods and results By Sanger method, we found a point mutation (Thr320Ala) in the apical domain of HSPD1, in one familial DCM patient, which was four amino acids away from the point mutation (Val324Glu) in the nbl mutant zebrafish. The nbl mutants showed atrioventricular block and sudden death at eight months post-fertilization. Histological and microscopic analysis of the nbl mutant hearts showed decreased ventricular wall thickness, elevated level of reactive oxygen species (ROS), increased fibrosis, mitochondrial damage, and increased autophagosomes. mRNA and protein expression of autophagy-related genes significantly increased in nbl mutants. We established HEK293 stable cell lines of WT, nbl-type, and DCM-type HSPD1, with tetracycline-dependent expression. Compared to WT, both nbl- and DCM-type cells showed decreased cell growth, increased expression of ROS and autophagy-related genes, inhibition of the activity of mitochondrial electron transport chain complexes III and IV, and decreased mitochondrial fission and fusion. Conclusions Mutations in HSPD1 caused mitochondrial dysfunction and induced mitophagy. Mitochondrial dysfunction caused increased ROS and cardiac atrophy. Translational perspective The aged heart is more susceptible to stress despite the increased compensatory chaperones/co-chaperones activity. Here, we identified a point mutation in HSPD1 in a human DCM family. Using zebrafish, we demonstrated that functional inactivation of HSPD1 resulted in increased ROS level and mitophagy, thereby resulting in heart failure at a relatively early age. Inhibition of ROS activity by antioxidants decreased cell death and mitophagy. This work identifies the key role of HSPD1 in cardiac muscle protection and suggests the supplementation of antioxidants may improve the cardiac function through the mitochondrial ROS pathway in patients with chronic heart failure.

Advanced age is a major predisposing risk factor for the incidence of coronary syndromes and comorbid conditions which impact the heart response to cardioprotective interventions. Advanced age also significantly increases the risk of developing post-ischaemic adverse remodelling and heart failure after ischaemia/reperfusion (IR) injury. Some of the signalling pathways become defective or attenuated during ageing, whereas others with well-known detrimental consequences, such as glycoxidation or proinflammatory pathways, are exacerbated. The causative mechanisms responsible for all these changes are yet to be elucidated and are a matter of active research. Here, we review the current knowledge about the pathophysiology of cardiac ageing that eventually impacts on the increased susceptibility of cells to IR injury and can affect the efficiency of cardioprotective strategies.

Normal cardiac contractile and relaxation functions are critically dependent on a continuous energy supply. Accordingly, metabolic perturbations and impaired mitochondrial bioenergetics with subsequent disruption of ATP production underpin a wide variety of cardiac diseases, including diabetic cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, anthracycline cardiomyopathy, peripartum cardiomyopathy, and mitochondrial cardiomyopathies. Crucially, there are no specific treatments for preventing the onset or progression of these cardiomyopathies to heart failure, one of the leading causes of death and disability worldwide. Therefore, new treatments are needed to target the metabolic disturbances and impaired mitochondrial bioenergetics underlying these cardiomyopathies in order to improve health outcomes in these patients. However, investigation of the underlying mechanisms and the identification of novel therapeutic targets have been hampered by the lack of appropriate animal disease models. Furthermore, interspecies variation precludes the use of animal models for studying certain disorders, whereas patient-derived primary cell lines have limited lifespan and availability. Fortunately, the discovery of human-induced pluripotent stem cells has provided a promising tool for modelling cardiomyopathies via human heart tissue in a dish. In this review article, we highlight the use of patient-derived iPSCs for studying the pathogenesis underlying cardiomyopathies associated with metabolic perturbations and impaired mitochondrial bioenergetics, as the ability of iPSCs for self-renewal and differentiation makes them an ideal platform for investigating disease pathogenesis in a controlled in vitro environment. Continuing progress will help elucidate novel mechanistic pathways, and discover novel therapies for preventing the onset and progression of heart failure, thereby advancing a new era of personalized therapeutics for improving health outcomes in patients with cardiomyopathy.

Sodium-glucose co-transporter 2 (SGLT2) inhibitors reduce the risk of serious heart failure events, even though SGLT2 is not expressed in the myocardium. This cardioprotective benefit is not related to an effect of these drugs to lower blood glucose, promote ketone body utilization or enhance natriuresis, but it is linked statistically with their action to increase haematocrit. SGLT2 inhibitors increase both erythropoietin and erythropoiesis, but the increase in red blood cell mass does not directly prevent heart failure events. Instead, erythrocytosis is a biomarker of a state of hypoxia mimicry, which is induced by SGLT2 inhibitors in manner akin to cobalt chloride. The primary mediators of the cellular response to states of energy depletion are sirtuin-1 and hypoxia-inducible factors (HIF-1α/HIF-2α). These master regulators promote the cellular adaptation to states of nutrient and oxygen deprivation, promoting mitochondrial capacity and minimizing the generation of oxidative stress. Activation of sirtuin-1 and HIF-1α/HIF-2α also stimulates autophagy, a lysosome-mediated degradative pathway that maintains cellular homoeostasis by removing dangerous constituents (particularly unhealthy mitochondria and peroxisomes), which are a major source of oxidative stress and cardiomyocyte dysfunction and demise. SGLT2 inhibitors can activate SIRT-1 and stimulate autophagy in the heart, and thereby, favourably influence the course of cardiomyopathy. Therefore, the linkage between erythrocytosis and the reduction in heart failure events with SGLT2 inhibitors may be related to a shared underlying molecular mechanism that is triggered by the action of these drugs to induce a perceived state of oxygen and nutrient deprivation.

The cardiac sodium channel NaV1.5, encoded by the SCN5A gene, is responsible for the fast upstroke of the action potential. Mutations in SCN5A may cause sodium channel dysfunction by decreasing peak sodium current, which slows conduction and facilitates reentry-based arrhythmias, and by enhancing late sodium current, which prolongs the action potential and sets the stage for early afterdepolarization and arrhythmias. Yet, some NaV1.5-related disorders, in particular structural abnormalities, cannot be directly or solely explained on the basis of defective NaV1.5 expression or biophysics. An emerging concept that may explain the large disease spectrum associated with SCN5A mutations centres around the multifunctionality of the NaV1.5 complex. In this alternative view, alterations in NaV1.5 affect processes that are independent of its canonical ion-conducting role. We here propose a novel classification of NaV1.5 (dys)function, categorized into (i) direct ionic effects of sodium influx through NaV1.5 on membrane potential and consequent action potential generation, (ii) indirect ionic effects of sodium influx on intracellular homeostasis and signalling, and (iii) non-ionic effects of NaV1.5, independent of sodium influx, through interactions with macromolecular complexes within the different microdomains of the cardiomyocyte. These indirect ionic and non-ionic processes may, acting alone or in concert, contribute significantly to arrhythmogenesis. Hence, further exploration of these multifunctional effects of NaV1.5 is essential for the development of novel preventive and therapeutic strategies.

Aims In long QT syndrome (LQTS) patients, modifier genes modulate the arrhythmic risk associated with a disease-causing mutation. Their recognition can improve risk stratification and clinical management, but their discovery represents a challenge. We tested whether a cellular-driven approach could help to identify new modifier genes and especially their mechanism of action. Methods and results We generated human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) from two patients carrying the same KCNQ1-Y111C mutation, but presenting opposite clinical phenotypes. We showed that the phenotype of the iPSC-CMs derived from the symptomatic patient is due to impaired trafficking and increased degradation of the mutant KCNQ1 and wild-type human ether-a-go-go-related gene. In the iPSC-CMs of the asymptomatic (AS) patient, the activity of an E3 ubiquitin-protein ligase (Nedd4L) involved in channel protein degradation was reduced and resulted in a decreased arrhythmogenic substrate. Two single-nucleotide variants (SNVs) on the Myotubularin-related protein 4 (MTMR4) gene, an interactor of Nedd4L, were identified by whole-exome sequencing as potential contributors to decreased Nedd4L activity. Correction of these SNVs by CRISPR/Cas9 unmasked the LQTS phenotype in AS cells. Importantly, the same MTMR4 variants were present in 77% of AS Y111C mutation carriers of a separate cohort. Thus, genetically mediated interference with Nedd4L activation seems associated with protective effects. Conclusion Our finding represents the first demonstration of the cellular mechanism of action of a protective modifier gene in LQTS. It provides new clues for advanced risk stratification and paves the way for the design of new therapies targeting this specific molecular pathway.

Aims Cardiovascular diseases caused by loss of functional cardiomyocytes (CMs) are a major cause of mortality and morbidity worldwide due in part to the low regenerative capacity of the adult human heart. Human pluripotent stem cell (hPSC)-derived cardiovascular progenitor cells (CPCs) are a potential cell source for cardiac repair. The aim of this study was to examine the impact of extensive remuscularization and coincident revascularization on cardiac remodelling and function in a mouse model of myocardial infarction (MI) by transplanting doxycycline (DOX)-inducible (Tet-On-MYC) hPSC-derived CPCs in vivo and inducing proliferation and cardiovascular differentiation in a drug-regulated manner. Methods and results CPCs were injected firstly at a non-cardiac site in Matrigel suspension under the skin of immunocompromised mice to assess their commitment to the cardiovascular lineage and ability to self-renew or differentiate in vivo when instructed by systemically delivered factors including DOX and basic fibroblast growth factor (bFGF). CPCs in Matrigel were then injected intra-myocardially in mice subjected to MI to assess whether expandable CPCs could mediate cardiac repair. Transplanted CPCs expanded robustly both subcutis and in the myocardium using the same DOX/growth factor inducing regime. Upon withdrawal of these cell-renewal factors, CPCs differentiated with high efficiency at both sites into the major cardiac lineages including CMs, endothelial cells, and smooth muscle cells. After MI, engraftment of CPCs in the heart significantly reduced fibrosis in the infarcted area and prevented left ventricular remodelling, although cardiac function determined by magnetic resonance imaging was unaltered. Conclusion Replacement of large areas of muscle may be required to regenerate the heart of patients following MI. Our human/mouse model demonstrated that proliferating hPSC-CPCs could reduce infarct size and fibrosis resulting in formation of large grafts. Importantly, the results suggested that expanding transplanted cells in situ at the progenitor stage maybe be an effective alternative causing less tissue damage than injection of very large numbers of CMs., Graphical Abstract Graphical Abstract

AimsThe cardioprotective effects of human induced pluripotent stem cell-derived cardiovascular progenitor cells (CPC) are largely mediated by the paracrine release of extracellular vesicles (EV). We aimed to assess the immunological behaviour of EV-CPC, which is a prerequisite for their clinical translation.Methods and resultsFlow cytometry demonstrated that EV-CPC expressed very low levels of immune relevant molecules including HLA Class I, CD80, CD274 (PD-L1), and CD275 (ICOS-L); and moderate levels of ligands of the natural killer (NK) cell activating receptor, NKG2D. In mixed lymphocyte reactions, EV-CPC neither induced nor modulated adaptive allogeneic T cell immune responses. They also failed to induce NK cell degranulation, even at high concentrations. These in vitro effects were confirmed in vivo as repeated injections of EV-CPC did not stimulate production of immunoglobulins or affect the interferon (IFN)-γ responses from primed splenocytes. In a mouse model of chronic heart failure, intra-myocardial injections of EV-CPC, 3 weeks after myocardial infarction, decreased both the number of cardiac pro-inflammatory Ly6Chigh monocytes and circulating levels of pro-inflammatory cytokines (IL-1α, TNF-α, and IFN-γ). In a model of acute infarction, direct cardiac injection of EV-CPC 2 days after infarction reduced pro-inflammatory macrophages, Ly6Chigh monocytes, and neutrophils in heart tissue as compared to controls. EV-CPC also reduced levels of pro-inflammatory cytokines IL-1α, IL-2, and IL-6, and increased levels of the anti-inflammatory cytokine IL-10. These effects on human macrophages and monocytes were reproduced in vitro; EV-CPC reduced the number of pro-inflammatory monocytes and M1 macrophages, while increasing the number of anti-inflammatory M2 macrophages.ConclusionsEV-CPC do not trigger an immune response either in in vitro human allogeneic models or in immunocompetent animal models. The capacity for orienting the response of monocyte/macrophages towards resolution of inflammation strengthens the clinical attractiveness of EV-CPC as an acellular therapy for cardiac repair.

Aims A 25-base pair deletion in the cardiac myosin binding protein-C (cMyBP-C) gene (MYBPC3), proposed to skip exon 33, modifies the C10 domain (cMyBP-CΔC10mut) and is associated with hypertrophic cardiomyopathy (HCM) and heart failure, affecting approximately 100 million South Asians. However, the molecular mechanisms underlying the pathogenicity of cMyBP-CΔC10mutin vivo are unknown. We hypothesized that expression of cMyBP-CΔC10mut exerts a poison polypeptide effect leading to improper assembly of cardiac sarcomeres and the development of HCM. Methods and results To determine whether expression of cMyBP-CΔC10mut is sufficient to cause HCM and contractile dysfunction in vivo, we generated transgenic (TG) mice having cardiac-specific protein expression of cMyBP-CΔC10mut at approximately half the level of endogenous cMyBP-C. At 12 weeks of age, significant hypertrophy was observed in TG mice expressing cMyBP-CΔC10mut (heart weight/body weight ratio: 4.43 ± 0.11 mg/g non-transgenic (NTG) vs. 5.34 ± 0.25 mg/g cMyBP-CΔC10mut, P Conclusion Expression of cMyBP-C protein with a modified C10 domain is sufficient to cause contractile dysfunction and HCM in vivo.

Aims Chronic pressure or volume overload induce concentric vs. eccentric left ventricular (LV) remodelling, respectively. Previous studies suggest that distinct signalling pathways are involved in these responses. NADPH oxidase-4 (Nox4) is a reactive oxygen species-generating enzyme that can limit detrimental cardiac remodelling in response to pressure overload. This study aimed to assess its role in volume overload-induced remodelling. Methods and results We compared the responses to creation of an aortocaval fistula (Shunt) to induce volume overload in Nox4-null mice (Nox4−/−) vs. wild-type (WT) littermates. Induction of Shunt resulted in a significant increase in cardiac Nox4 mRNA and protein levels in WT mice as compared to Sham controls. Nox4−/− mice developed less eccentric LV remodelling than WT mice (echocardiographic relative wall thickness: 0.30 vs. 0.27, P Conclusion Endogenous Nox4 is required for the full development of eccentric cardiac hypertrophy and remodelling during chronic volume overload. Nox4-dependent activation of Akt and its downstream targets S6 and 4E-BP1 may be involved in this effect.

Aims The apelin receptor, a G protein-coupled receptor, has emerged as a key regulator of cardiovascular development, physiology, and disease. However, there is a lack of suitable human in vitro models to investigate the apelinergic system in cardiovascular cell types. For the first time we have used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and a novel inducible knockdown system to examine the role of the apelin receptor in both cardiomyocyte development and to determine the consequences of loss of apelin receptor function as a model of disease. Methods and results Expression of the apelin receptor and its ligands in hESCs and hESC-CMs was determined. hESCs carrying a tetracycline-inducible short hairpin RNA targeting the apelin receptor were generated using the sOPTiKD system. Phenotypic assays characterized the consequences of either apelin receptor knockdown before hESC-CM differentiation (early knockdown) or in 3D engineered heart tissues as a disease model (late knockdown). hESC-CMs expressed the apelin signalling system at a similar level to the adult heart. Early apelin receptor knockdown decreased cardiomyocyte differentiation efficiency and prolonged voltage sensing, associated with asynchronous contraction. Late apelin receptor knockdown had detrimental consequences on 3D engineered heart tissue contractile properties, decreasing contractility and increasing stiffness. Conclusions We have successfully knocked down the apelin receptor, using an inducible system, to demonstrate a key role in hESC-CM differentiation. Knockdown in 3D engineered heart tissues recapitulated the phenotype of apelin receptor down-regulation in a failing heart, providing a potential platform for modelling heart failure and testing novel therapeutic strategies.

Aims In cardiomyocytes, acute disturbances to intracellular pH (pHi) are promptly corrected by a system of finely tuned sarcolemmal acid–base transporters. However, these fluxes become thermodynamically re-balanced in acidic environments, which inadvertently causes their set-point pHi to fall outside the physiological range. It is unclear whether an adaptive mechanism exists to correct this thermodynamic challenge, and return pHi to normal. Methods and results Following left ventricle cryo-damage, a diffuse pattern of low extracellular pH (pHe) was detected by acid-sensing pHLIP. Despite this, pHi measured in the beating heart (13C NMR) was normal. Myocytes had adapted to their acidic environment by reducing Cl−/HCO3− exchange (CBE)-dependent acid-loading and increasing Na+/H+ exchange (NHE1)-dependent acid-extrusion, as measured by fluorescence (cSNARF1). The outcome of this adaptation on pHi is revealed as a cytoplasmic alkalinization when cells are superfused at physiological pHe. Conversely, mice given oral bicarbonate (to improve systemic buffering) had reduced myocardial NHE1 expression, consistent with a needs-dependent expression of pHi-regulatory transporters. The response to sustained acidity could be replicated in vitro using neonatal ventricular myocytes incubated at low pHe for 48 h. The adaptive increase in NHE1 and decrease in CBE activities was linked to Slc9a1 (NHE1) up-regulation and Slc4a2 (AE2) down-regulation. This response was triggered by intracellular H+ ions because it persisted in the absence of CO2/HCO3− and became ablated when acidic incubation media had lower chloride, a solution manoeuvre that reduces the extent of pHi-decrease. Pharmacological inhibition of FAK-family non-receptor kinases, previously characterized as pH-sensors, ablated this pHi autoregulation. In support of a pHi-sensing role, FAK protein Pyk2 (auto)phosphorylation was reduced within minutes of exposure to acidity, ahead of adaptive changes to pHi control. Conclusions Cardiomyocytes fine-tune the expression of pHi-regulators so that pHi is at least 7.0. This autoregulatory feedback mechanism defines physiological pHi and protects it during pHe vulnerabilities.