Journal of Molecular and Cellular Cardiology

Pathological and physiological stresses induce diverse forms of cardiac hypertrophy, with distinct manifestations in cardiomyocyte size and shape regulated by still poorly understood signaling networks. Here, we combined high-content morphological profiling, phospho-protein arrays, and systems modeling to characterize the diverse forms of hypertrophy induced by angiotensin II, endothelin-1, insulin growth factor-1, and neuregulin-1. Reverse-phase protein array profiling and partial least squares regression modeling revealed that Akt, GSK3, and MAPK signaling are differentially regulated by hypertrophic agonists and are predictive of distinct phenotypic outcomes. Neuregulin-1 uniquely induced cardiomyocyte elongation in both neonatal rat and human iPSC-derived cardiomyocytes, in addition to increasing cell area. Pharmacological perturbations demonstrated that neuregulin1-induced elongation and area expansion both require PI3K activity, whereas p38 selectively mediates cell area. A logic-based network model incorporating dual-specificity phosphatases were sufficient to capture the amplifying PI3K and transient p38 signaling dynamics driving phenotypic changes. Together, these results identify distinct signaling cascades by which neuregulin-1 coordinates cardiomyocyte size and shape, providing mechanistic insight into how hypertrophic remodeling can be differentially regulated. This systems approach provides new insight into the pathways that drive distinct forms of cardiomyocyte hypertrophy, highlighting opportunities to selectively target maladaptive remodeling in heart failure. Highlights- Reverse-phase protein arrays capture distinct signatures of cellular signaling in response to diverse hypertrophic ligands. - Partial least squares regression model maps proteomic signatures to diverse patterns of cell morphology and gene expression. - Combinatorial ligand-inhibitor screen validates predicted causal regulators of mRNAs and cell morphology - PI3K mediates both neuregulin-1-induced elongation and cell area, validating the PLSR model. In contrast, p38 regulates cell area but not elongation. - Logic-based model demonstrates that the characterized mechanisms are sufficient to predict how distinct PI3K and p38 dynamics drive size and shape.

Hypertrophic cardiomyopathy (HCM) results from pathogenic variants in sarcomeric protein genes, that increase myocyte energy demand and lead to cardiac hypertrophy. But it is unknown whether a common metabolic trait underlies the cardiac phenotype at early disease stage. This study characterized two HCM mouse models (R92W-TnT, R403Q-MyHC) that demonstrate differences in mitochondrial function at early disease stage. Using a combination of cardiac phenotyping, transcriptomics, mass spectrometry-based metabolomics and computational modeling, we discovered allele-specific differences in cardiac structure/function and metabolic changes. TnT-mutant hearts had impaired energy substrate metabolism and increased phospholipid remodeling compared to MyHC-mutants. TnT-mutants showed increased incorporation of saturated fatty acid residues into ceramides, cardiolipin, and increased lipid peroxidation, that could underlie allele-specific differences in mitochondrial function and cardiomyopathy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=172 HEIGHT=200 SRC="FIGDIR/small/564562v1_ufig1.gif" ALT="Figure 1"> O_LINKSMALLFIG WIDTH=185 HEIGHT=200 SRC="FIGDIR/small/564562v1_ufig2.gif" ALT="Figure 1"> O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/564562v1_ufig3.gif" ALT="Figure 1"> View larger version (90K): org.highwire.dtl.DTLVardef@195b1d7org.highwire.dtl.DTLVardef@cead88org.highwire.dtl.DTLVardef@e2bf35org.highwire.dtl.DTLVardef@776765_HPS_FORMAT_FIGEXP M_FIG C_FIG

The study of human cardiac pathologies often relies on research conducted in model organisms to gain molecular insight into disease and to develop novel treatment strategies; however, translating findings from model organisms back to human can present a significant challenge, in part due to a lack of knowledge about the differences across species in cardiac protein abundances and their interactions. Here we set out to bridge this knowledge gap by presenting a global analysis of cardiac protein expression profiles in humans and commonly used model organisms. Using quantitative mass spectrometry-based proteomics, we measured the abundance of ~7,000 proteins in samples from the separate chambers of human, pig, horse, rat, mouse and zebrafish hearts. This knowledgebase of cardiac protein signatures is accessible through an online database at: atlas.cardiacproteomics.com. Quantitative comparison of the protein profiles support the pig as model organism of choice for arrhythmogenic right ventricular cardiomyopathy whereas comparison of profiles from the two-chambered zebrafish heart suggests a better resemblance to the right side of mammalian hearts. This proteomics resource facilitates translational prospect of cardiac studies from model organisms to humans by enabling direct comparison of disease-linked protein networks across species.

Approximately 40% of genetic hypertrophic cardiomyopathy cases involve mutations in MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C), a key regulator of sarcomere contractility. The atrial-specific paralog, myosin binding protein-H like (MyBP-HL), has been associated with dilated cardiomyopathy in humans and mice. Both proteins bind to the same binding sites in the thick filament C-zone. In the atria, cMyBP-C and MyBP-HL are found at [~]1:1 ratios, while ventricles only express cMyBP-C, which is found at twice the atrial level, indicating a stoichiometric relationship. In the atria, we hypothesize that missense variants in either gene may cause alterations in thick filament binding affinity and changes in the normal [~]1:1 ratio. Notably, MyBP-HL deletion in atrial myofibrils accelerates relaxation kinetics, suggesting that altered stoichiometry impacts biophysical parameters. We hypothesized that deletion, overexpression, or missense variants in either gene would alter the abundance of the other protein in atrial sarcomeres, affecting sarcomere localization and function. To test this, we engineered two constructs: a mini-C construct comprising thick filament-binding domains of cMyBP-C and a MyBP-HL construct. Selected MYBPC3 and MYBPHL missense variants were introduced and expressed in neonatal rat ventricular cardiomyocytes (NRVMs). Sarcomere localization was assessed by co-localization with endogenous cMyBP-C. MYBPC3 variants were selected across a range of pathogenicity, while MYBPHL variants were based on evolutionary conservation of residues. MYBPHL variants Gly275Ser, Arg285His, and Ala342Thr induced significant sarcomere mislocalization, and MYBPC3 variants Pro1181Ala and Asn1257Lys showed variable effects on sarcomere mislocalization. To assess stoichiometric effects, we developed a T2A/P2A polycistronic construct to co-express mini-C, Td-Tomato, and MyBP-HL. Immunoblotting and mass spectrometry confirmed consistent and reproducible expression. We identified several MYBPC3 and MYBPHL variants that reduced the affinity of their protein for myofilament incorporation. These results suggest that this 2A construct is a useful tool for measuring the effect of myosin binding protein missense variants on sarcomere affinity, with implications for assessing pathogenicity of these variants.

Hypertrophic cardiomyopathy (HCM) is one of the most common heritable cardiovascular diseases and variants of TNNT2 (cardiac troponin T) are linked to increased risk of sudden cardiac arrest despite causing limited hypertrophy. In this study, a TNNT2 variant, R278C+/-, was generated in both human cardiac recombinant/reconstituted thin filaments (hcRTF) and human-induced pluripotent stem cells (hiPSCs) to investigate the mechanisms by which the R278C+/- variant affects cardiomyocytes at the proteomic and functional levels. The results of proteomics analysis showed a significant upregulation of markers of cardiac hypertrophy and remodeling in R278C+/- vs. the isogenic control. Functional measurements showed that R278C+/- variant enhances the myofilament sensitivity to Ca2+, increases the kinetics of contraction, and causes arrhythmia at frequencies >75 bpm. This study uniquely shows the profound impact of the TNNT2 R278C+/- variant on the cardiomyocyte proteomic profile, cardiac electrical and contractile function in the early stages of cardiac development. Translational PerspectiveHypertrophic cardiomyopathy (HCM) is the leading known cause of sudden cardiac arrest in the young. Thin-variant associated HCM variants make up to 15% of familial HCM yet their molecular mechanisms remain less clear relative to thick filament variants. Here, we employ computational modeling, human cardiac recombinant/reconstituted thin filaments (hcRTF), and hiPSC-CMs to study the thin filament TNNT2 R278C+/- variant, revealing its extensive pathogenicity and potential mechanisms by which it can lead to HCM and sudden death. Mavacamten, the recently FDA-approved treatment, was effective in alleviating contractile dysfunction in TNNT2 R278C+/- hiPSC-CMs, positing it as a potential therapy for thin filament HCM. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=117 HEIGHT=200 SRC="FIGDIR/small/542948v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@102a8b0org.highwire.dtl.DTLVardef@190e356org.highwire.dtl.DTLVardef@13d3f9aorg.highwire.dtl.DTLVardef@1a9c33_HPS_FORMAT_FIGEXP M_FIG C_FIG

BACKGROUNDMYBPC3, encoding cardiac myosin binding protein-C (cMyBP-C), is the most mutated gene known to cause hypertrophic cardiomyopathy (HCM). However, since little is known about the underlying etiology, additional in vitro studies are crucial to defining the underlying molecular mechanisms. Accordingly, this study aimed to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with a polymorphic variant (D389V) in MYBPC3 by using human-induced pluripotent stem cell (hiPSC)-derived cardiac organoids (hCOs). METHODSThe hiPSC-derived cardiomyocytes (hiPSC-CMs) and hCOs were generated from human subjects to define the molecular, cellular, and functional changes caused by the MYBPC3D389V variant. This variant is associated with increased fractional shortening and is highly prevalent in South Asian descendants. Recombinant C0-C2, N-region of cMyBP-C (wildtype and D389V), and myosin S2 proteins were also utilized to perform binding and motility assays in vitro. RESULTSConfocal and electron microscopic analyses of hCOs generated from noncarriers (NC) and carriers of the MYBPC3D389V variant revealed the presence of highly organized sarcomeres. Furthermore, functional experiments showed hypercontractility with increased contraction velocity, faster calcium cycling, and faster contractile kinetics in hCOs expressing MYBPC3D389V than NC hCOs. Interestingly, significantly increased cMyBP-C phosphorylation in MYBPC3D389V hCOs was observed, but without changes in total protein levels, in addition to higher oxidative stress and lower mitochondrial membrane potential ({Delta}{Psi}m). Next, spatial mapping revealed the presence of endothelial cells, fibroblasts, macrophages, immune cells, and cardiomyocytes in the hCOs. The hypercontractile function was significantly improved after treatment with the myosin inhibitor mavacamten (CAMZYOS(R)) in MYBPC3D389V hCOs. Lastly, various in vitro binding assays revealed a significant loss of affinity in the presence of MYBPC3D389V with myosin S2 region as a likely mechanism for hypercontraction. CONCLUSIONSConceptually, we showed the feasibility of assessing the functional and molecular mechanisms of HCM using highly translatable hCOs through pragmatic experiments that led to determining the MYBPC3D389V hypercontractile phenotype, which was rescued by administration of a myosin inhibitor. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIMYBPC3 mutations have been implicated in hypertrophic cardiomyopathy. C_LIO_LID389V is a polymorphic variant of MYBPC3 predicted to be present in 53000 US South Asians owing to the founder effect. D389V carriers have shown evidence of hyperdynamic heart, and human-induced pluripotent stem cells (hiPSC)-derived cardiomyocytes with D389V show cellular hypertrophy and irregular calcium transients. C_LIO_LIThe molecular mechanism by which the D389V variant develops pathological cardiac dysfunction remains to be conclusively determined. C_LI What New Information Does This Article Contribute?O_LIThe authors leveraged a highly translational cardiac organoid model to explore the role of altered cardiac calcium handling and cardiac contractility as a common pathway leading to pathophysiological phenotypes in patients with early HCM. C_LIO_LIThe MYBPC3D389V-mediated pathological pathway is first studied here by comparing functional properties using three-dimensional cardiac organoids differentiated from hiPSC and determining the presence of hypercontraction. C_LIO_LIOur data demonstrate that faster sarcomere kinetics resulting from lower binding affinity between D389V-mutated cMyBP-C protein and myosin S2, as evidenced by in vitro studies, could cause hypercontractility which was rescued by administration of mavacamten (CAMZYOS(R)), a myosin inhibitor. C_LIO_LIIn addition, hypercontractility causes secondary mitochondrial defects such as higher oxidative stress and lower mitochondrial membrane potential ({Delta}{Psi}m), highlighting a possible early adaptive response to primary sarcomeric changes. C_LIO_LIEarly treatment of MYBPC3D389V carriers with mavacamten may prevent or reduce early HCM-related pathology. C_LI GRAPHICAL ABSTRACTA graphical abstract is available for this article. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/596463v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1489301org.highwire.dtl.DTLVardef@1ab60c1org.highwire.dtl.DTLVardef@5d49d1org.highwire.dtl.DTLVardef@9944d6_HPS_FORMAT_FIGEXP M_FIG C_FIG

AimsBats are unique mammals with remarkable adaptations, including powered flight, which demands significant energy expenditure. While previous studies have documented basic structural characteristics of bat hearts, a comprehensive understanding of their response to physiological stress remains unexplored. This study investigates the cardiac adaptations of the cave nectar bat Eonycteris spelaea to elucidate the mechanisms underlying their unique physiological capabilities. Methods and ResultsWe performed RNA sequencing to analyse the cardiac gene expression profile of E. spelaea and 6 other bat species in comparison to mouse and human hearts, revealing enriched transcriptomic signatures related to oxidative phosphorylation and fatty acid metabolism across multiple bat species. Metabolomic profiling compared acylcarnitine metabolites and tricarboxylic acid (TCA) cycle intermediates between bat and mouse hearts, indicating a distinct acylcarnitine profile and increased levels of TCA cycle intermediates in bats, suggesting enhanced metabolic capacity. Structural adaptations were assessed through anatomical and histological analyses on cardiac tissues, showing thicker left ventricular walls and increased vascular density in bats without pathological hypertrophy. Functional characteristics were evaluated using dobutamine stress echocardiography, demonstrating superior cardiac reserve in bats with significant increases in ejection fraction, stroke volume, and cardiac output under stress conditions. Additionally, isolated cardiomyocytes were treated with Angiotensin II (Ang II) to assess stress responses. Bat cardiomyocytes displayed resistance to Ang II-induced hypertrophy and mitochondrial dysfunction compared to mice, further highlighting their resilience to stress-induced damage. ConclusionThe unique adaptations observed in bat hearts, including enhanced metabolic pathways, structural remodelling, and cellular resilience contribute to their ability to meet the high energy demands of powered flight while maintain cardiac function under stress. These insights into bat cardiac physiology provide valuable information on cardioprotective mechanisms that could be applicable to other species. Translational PerspectiveBats exhibit remarkable cardiac adaptations that sustain the high energy demands of powered flight while resisting stress-induced damage. These insights into evolutionarily conserved cardioprotective mechanisms highlight potential therapeutic pathways for preventing heart failure, including resistance to hypertrophy and mitochondrial dysfunction under stress. Studying non-model organisms like bats may offer innovative approaches to enhance metabolic and stress resilience in human hearts, paving the way for translational research in cardiovascular disease management and treatment.

BackgroundPhosphoglucomutase-1 (PGM1) plays a pivotal role in glycolysis, glycogen metabolism, and glycosylation. Pathogenic variants in PGM1 cause PGM1-congenital disorder of glycosylation (PGM1-CDG), a multisystem disorder with cardiac involvement. While glycosylation abnormalities in PGM1-CDG are treatable with galactose, cardiomyopathy does not improve suggesting a glycosylation-independent pathomechanism. Recently, mitochondrial abnormalities have been shown in a heart of a PGM1-deficicient patient and PGM1-mouse model. In addition, PGM1 has been associated with LDB3 (ZASP/Cypher), a sarcomeric Z-disk protein also associated with cardiomyopathy. However, the cardiac-specific role of PGM1 remains poorly understood, and targeted therapies for PGM1-related cardiomyopathy are currently lacking. MethodsInduced pluripotent stem cell-derived cardiomyocytes (iCMs) were generated from PGM1-deficient patient fibroblasts. Multielectrode array (MEA) recordings, untargeted (glyco)proteomics, and pathway analysis were performed to assess functional and molecular changes. Key findings were validated using tracer metabolomics and mitochondrial respiration assays. ResultsPGM1-deficient iCMs exhibited reduced beating frequency, impaired contractility, and prolonged contraction kinetics. Proteomic analyses revealed depletion of Z-disk components, including LDB3. AlphaFold3 structural modeling predicted a direct interaction between PGM1 and LDB3, implicating PGM1 in Z-disk integrity, which was confirmed in vitro. In addition, mitochondrial proteins were severely depleted, prompting us to investigate mitochondrial function. Functional validation confirmed extensive metabolic rewiring, energy depletion, and severely impaired mitochondrial respiration. Finally, the in silico drug repurposing identified possible therapeutic options that could target PGM1-deficient cardiomyopathy. ConclusionPGM1 is a key regulator of cardiomyocyte function, linking sarcomeric Z-disk integrity with mitochondrial metabolism. These mechanistic insights offer a foundation for developing targeted therapies for PGM1-CDG and potentially other cardiomyopathies involving Z-disk dysfunction. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=175 HEIGHT=200 SRC="FIGDIR/small/662580v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@17da449org.highwire.dtl.DTLVardef@1acb669org.highwire.dtl.DTLVardef@1fbbeecorg.highwire.dtl.DTLVardef@b38b6f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Histidyl dipeptides, are synthesized in the heart via enzyme carnosine synthase (Carns), which facilitates glycolysis and glucose oxidation by proton buffering and attenuate ischemia and reperfusion injury. However, a composite understanding of the histidyl dipeptide mediated responses in the heart are lacking. We performed multilayer omics in the cardio specific Carns overexpressing mice, showing higher myocardial levels of histidyl dipeptides lead to extensive changes in microRNAs that could target the expression of contractile proteins and enzymes involved in {beta}-fatty acid oxidation and citric acid cycle (TCA). Similarly, global proteomics showed contractile function, fatty acid degradation and TCA cycle, pathways were enriched in the CarnsTg heart. Parallel with these changes, free fatty acids, and TCA intermediate-succinic acid were lower under aerobic and significantly attenuated under anaerobic conditions in the CarnsTg heart. Integration of multiomics data shows {beta}-fatty acid oxidation and TCA cycle exhibit correlative changes at all three levels in CarnsTg heart, suggesting histidyl dipeptides are critical regulators of myocardial structure, function and energetics.

IntroductionHypertrophic Cardiomyopathy (HCM) is a disease defined by the development of left ventricle hypertrophy. One of the most commonly mutated genes in HCM is cardiac myosin binding protein C (MYBPC3). MYBPC3 protein localizes to the cardiomyocyte sarcomere, but studies have reported detection of both MYBPC3 RNA and protein in non-cardiomyocyte cell populations. Therefore, it was unclear if MYBPC3 expression in non-cardiomyocyte cell populations altered the development of cardiomyopathy caused by MYBPC3 protein deficiency. MethodsWe utilized genetically modified murine models with germline deletion of Mybpc3 exons 3 to 5 (Mybpc3-/-) or cardiomyocyte specific deletion of Mybpc3 exons 3 to 5 (Mybpc3fl/fl; Myh6-Cre). Gene expression was assessed using quantitative RT-PCR. Whole tissue protein levels were assessed using immunoblots. Immunohistochemistry and proximity ligation assays were performed to evaluate in situ protein expression. Echocardiography was utilized to measure left ventricular structure and function. ResultsMybpc3 mRNA was detected in multiple organs including the heart, lung and blood from both humans and mice. Utilizing transgenic murine models with germline or cardiomyocyte specific deletion of Mybpc3 exons 3-5, we discovered that the Mybpc3 mRNA detected in extracardiac locations originated primarily from cardiomyocytes. Likewise, MYBPC3 protein was identified in myocardial tissue but not in other organs and cardiomyocytes were the only cell population in myocardial tissue that had detectable MYBPC3 protein. Importantly, cardiomyocyte deletion of Mybpc3 caused similar pathological myocardial remodeling and alterations in left ventricular function compared to germline deletion of Mybpc3 in all cell populations. ConclusionsOur results show that cardiomyocytes are the primary cell source of Mybpc3 mRNA detected in extracardiac organs and they are the principal cell type responsible for the cardiomyopathy caused by MYBPC3 protein deficiency. These results suggest that selective targeting of cardiomyocytes should be the most efficient approach to treat cardiomyopathies associated with MYBPC3 deficiency.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for cardiac regenerative medicine and disease modeling. However, hiPSC-CMs generated through conventional in vitro differentiation exhibit immature, fetal-like phenotypes. While in vivo bioincubation in neonatal rodent hearts promotes hiPSC-CM maturation toward adult-like phenotypes, studies in large animal models remain limited, particularly with detailed morphological characterization. In this study, we investigated bioincubation of fluorescently labeled hiPSC-CMs in both neonatal rat and pig hearts. Human iPSCs were differentiated into cardiomyocytes expressing GFP or RFP reporters and subsequently injected intramyocardially into neonatal rats (GFP-labeled) and pigs (RFP-labeled). After 4-8 weeks of bioincubation, fluorescent hiPSC-CMs were isolated using large-particle fluorescence-activated cell sorting (COPAS), which preserves cellular morphology of adult-like cardiomyocytes. Immunostaining for cardiac troponin T revealed well-organized sarcomeric structures in multinucleated hiPSC-CMs. Bioincubated hiPSC-CMs displayed rod-shaped morphology with binucleation, characteristic features of mature adult cardiomyocytes. Quantitative analysis demonstrated that bioincubated hiPSC-CMs from rat hearts exhibited sarcomere length and cell circularity comparable to native rat adult cardiomyocytes, though with higher intra-cellular variability in sarcomere organization. Histological examination confirmed successful engraftment of RFP-positive hiPSC-CMs within pig myocardium, with engrafted cells also displaying mature adult-like features. These findings provide critical proof-of-concept data for bioincubation in large animal models and support further investigation for disease modeling, drug screening, and regenerative cell therapies. SIGNIFICANCE STATEMENTHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer tremendous potential for cardiac disease modeling and regenerative therapies, but their clinical application is limited by their immature characteristics. Here we show that in vivo bioincubation in neonatal rat hearts enables hiPSC-CMs to achieve structural maturity, exhibiting features of adult cardiomyocytes, including organized sarcomeres, rod-shaped morphology, and multinucleation. We further provided proof-of-concept evidence for engraftment in neonatal pig hearts for maturation, supporting feasibility in large animal models. The use of large-particle cell sorting enables recovery of intact, adult-sized cardiomyocytes for subsequent analysis. These findings establish a practical and scalable platform for generating structurally mature human cardiomyocytes through in vivo bioincubation.

The adult mammalian heart has limited regenerative capacity following injury, leading to progressive heart failure and mortality. Recent studies have identified the spiny mouse (Acomys) as a unique model for mammalian cardiac regeneration, exhibiting enhanced recovery after myocardial infarction compared to commonly used laboratory mouse strains. However, the cellular and molecular mechanisms underlying this regenerative response remain poorly understood. In this study, we performed a comprehensive characterization of the metabolic adaptations to ischemic injury in cardiomyocytes of Acomys in comparison to the non-regenerative Mus Musculus. To investigate the transcriptomic and metabolomic profiles of cardiomyocytes in response to myocardial infarction, we utilized single-nucleus RNA sequencing (snRNA-seq) in sham-operated animals and 1, 3, and 7 days post-myocardial infarction. Complementary targeted metabolomics, stable isotope-resolved metabolomics, and functional mitochondrial assays were performed on heart tissues from both species to validate the transcriptomic findings and elucidate the metabolic adaptations in cardiomyocytes following ischemic injury. Transcriptomic analysis revealed that Acomys cardiomyocytes upregulate genes associated with glycolysis, the pentose phosphate pathway, and glutathione metabolism while downregulating genes involved in oxidative phosphorylation following injury. These metabolic changes were linked to decreased production of reactive oxygen species and increased antioxidant capacity, evidenced by the upregulation of genes such as Prdx1, Sod1, Sod2, and G6pd. Our targeted metabolomic studies supported these findings, showing a shift from fatty acid oxidation to glycolysis and ancillary biosynthetic pathways in Acomys cardiomyocytes post-injury. Functional mitochondrial studies indicated a higher reliance on glycolysis in Acomys compared to Mus, underscoring the unique metabolic adaptations of Acomys cardiomyocytes. Stable isotope tracing experiments confirmed a shift in glucose utilization from oxidative phosphorylation in Acomys. In conclusion, our study identifies unique metabolic adaptations in Acomys cardiomyocytes that contribute to their enhanced regenerative capacity following myocardial infarction. These findings provide novel insights into the role of metabolism in regulating cardiomyocyte proliferation and cardiac repair in adult mammals. By targeting the specific metabolic pathways and regulators identified in Acomys, such as glycolytic enzymes and PCK2, we may be able to develop innovative therapies to promote cardiac regeneration in patients with ischemic heart disease. Our work highlights the importance of metabolic flexibility in determining cardiomyocyte regenerative responses and establishes Acomys as a valuable model for studying cardiac regeneration in adult mammals. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/595229v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@dba53aorg.highwire.dtl.DTLVardef@3c8a74org.highwire.dtl.DTLVardef@a60251org.highwire.dtl.DTLVardef@e2142c_HPS_FORMAT_FIGEXP M_FIG C_FIG

AimsHypertrophic cardiomyopathy (HCM) is a genetic heart disease that leads to left ventricular hypertrophy, heart failure, and arrhythmias. Pathogenic missense variants in the gene myosin binding protein C (MYBPC3) cluster within its internal subdomains C3 and C6. The protein, myosin binding protein C (MyBP-C), expressing these variants, normally localizes to the myofilaments, leaving uncertainty regarding the mechanism(s) by which they cause HCM. MethodsWe probed the mechanisms of these variants by analyzing (1) their prevalence in an international registry of patients with HCM, (2) total MyBP-C levels and the allelic fraction of mutant MyBP-C in human left ventricular myectomy heart tissue, and (3) performing flag-immunoprecipitation and proximity labeling mass spectrometry of wild-type MyBP-C and four pathogenic missense variants (Arg495Gln, Arg502Trp-C3 subdomain, Trp792Arg, Arg810His-C6 subdomain) to determine the change in MyBP-C interacting and proximity proteins induced by these variants. ResultsWe found that in patients with HCM who had any MYBPC3 pathogenic variant, 17.9% of them had a missense variant localized to the C3 or C6 subdomain. Unlike truncating variants, missense variants did not reduce MyBP-C content relative to myosin. The mutant allelic fraction of MyBP-C varied from 10-67.0% across samples. Flag-immunoprecipitation mass spectrometry identified 252 MyBP-C interacting proteins. Pathogenic missense variants disrupted 23 MyBP-C protein interactions, including lysosomal Ragulator-Rag complex proteins (Rraga, Rragc, LAMTOR4). Proximity labeling mass spectrometry was more sensitive, identifying 3,240 MyBP-C proximity proteins. Pathogenic missense variant (s) altered proximity of 789 proteins (69.4% increased and 30.5% decreased relative to wild-type MyBP-C). Proteins that were increased in proximity to missense MyBP-C were enriched for proteins within thin filament. ConclusionPathogenic MYBPC3 missense variants within the C3 and C6 subdomains are present in a substantial subset of patients with HCM. Our findings implicate unique mechanisms of these variants distinct from haploinsufficiency, potentially driven in part by enhanced protein-protein interactions with the thin filament within the sarcomere. Translational perspectivePathogenic missense variants in the gene myosin binding protein C (MYBPC3) cause hypertrophic cardiomyopathy via an unknown mechanism. These variants exhibit normal cellular localization and stability. We have demonstrated that these variants are present in 17.9% of patients with HCM and a pathogenic MYBPC3 variant and do not reduce the level of total MyBP-C, the protein encoded by MYBPC3, within human left ventricular tissue. Using Flag-immunoprecipitation and proximity labeling mass spectrometry, we identified differential interacting proteins and proximity proteins, respectively, with proximity labeling mass spectrometry displaying much higher sensitivity. This was particularly true for the detection of potential gain-of-function shifts in MyBP-C environment within the sarcomere. Three of the four pathogenic missense proteins evaluated demonstrated increased proximity to thin filament proteins. This may suggest that particular missense variants within C3 and C6 internal subdomains result in a conformational change that favors thin filament binding and activation. These findings have important implications for the development of sarcomeric modulators that address the underlying mechanism HCM caused by MYBPC3 missense variants.

Allele-specific RNA silencing has been shown to be an effective therapeutic treatment in a number of diseases, including neurodegenerative disorders. Studies of allele-specific silencing in hypertrophic cardiomyopathy to date have focused on mouse models of disease. Here, we investigate two methods of allele-specific silencing, short hairpin RNA (shRNA) and antisense oligonucleotide (ASO) silencing, using a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of disease. We used cellular micropatterning devices with traction force microscopy and automated video analysis to examine each strategys effects on contractile defects underlying disease. We find that shRNA silencing ameliorates contractile phenotypes of disease, reducing disease-associated increases in cardiomyocyte velocity, force, and power. We find that ASO silencing, while better able to target and knockdown a specific disease-associated allele, showed more modest improvements in contractile phenotypes. We find a dissociation between allelic-specificity and functional improvements between the two tested therapeutic strategies, suggesting a more complex method of allelic control underlying HCM-associated transcripts.\n\nAuthor summaryAllele-specific silencing, whereby a therapeutic molecule is used to lower the expression of just one of the two copies or alleles of a gene, may be a potential therapeutic strategy in diseases caused by a single mutation. In this paper, we examine two such strategies in hypertrophic cardiomyopathy, a disease characterized by an overgrowth of the left-ventricular heart muscle as well as contractile dysfunction. We used a human cell model of disease, creating induced pluripotent stem cell derived cardiomyocytes from a patient with HCM caused by a single base pair change in just one allele of the gene MYH7. We used two strategies to silence the disease-associated copy of MYH7, both focused on reducing RNA expression from the mutated allele, as well as state-of-the-art biophysical techniques for measuring contractility. We found that one silencing strategy, which reduced expression of both the disease-associated and the healthy alleles of MYH7, showed great improvements in contractility between treated and untreated cells. Our second strategy, which silenced only the disease-associated copy of MYH7, showed more modest improvements in contractility. This suggests that the disease mechanism underlying this type of hypertrophic cardiomyopathy may be more complex than just presence or absence of the mutated RNA.

Inefficiency of regeneration underlies many of the pathologies associated with heart injury and disease. Ventricular diploid cardiomyocytes (CMs) are a candidate population that may have enhanced proliferative and regenerative properties [1-3], but subpopulations of diploid CMs and their regenerative capacities are not yet known. Here, using the expression marker Cntn2-GFP and the lineage marker Etv1CreERT2, we demonstrate that peripheral ventricular conduction CMs (Purkinje CMs) are disproportionately diploid (35%, vs. 4% of bulk ventricular CMs). However, this lineage had no enhanced competence to support regeneration after adult infarction. Furthermore, the CM-specific kinase Tnni3k, which strongly influences bulk ventricular CM ploidy [3] and is also associated with conduction system defects [4], had no influence on the ploidy or organization of the ventricular conduction system. Unlike the bulk diploid CM population, a significant fraction of conduction CMs remain diploid by avoiding neonatal cell cycle activity, likely contributing to these properties.

We have previously demonstrated that pacing the failing sheep heart with respiratory heart rate variability (RespHRV), a natural variability in the heart rate that is linked to respiration on a breath-by-breath basis, improves cardiac output dramatically. In this study, we used proteomics and super-resolution microscopy to explore the role of energetics and T-tubule cellular remodelling in response to ResPHRV pacing in an ischaemic ovine model of heart failure (HF). After 2 weeks of RespHRV pacing, cardiac output improved by 1.1 {+/-} 0.2 L/min (**p=0.003). Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS) was used to probe differences between three groups: HF without any intervention, HF with RespHRV pacing and a healthy control group. Orthogonal Partial Least Squares (OPLS) discriminant analysis demonstrated a clear separation of all three groups by T score (***p<0.001) with the HF+RespHRV pacing group placed intermediate between the HF and control groups. The top 50 proteins negatively correlated with T score (down in HF, restored after RespHRV) were dominated by mitochondrial proteins, as confirmed by Pathway Enrichment Analysis (***p<0.001). Multiple Reaction Monitoring Mass Spectrometry (MRM-MS) analysis confirmed this finding in selected targets (ACAA2, ACADS, CRAT, NDUFA8, and SUCLG1, *p<0.05). STimulated Emission Depletion (STED) microscopy identified a disruption of mitochondria structure in HF (*p<0.05) that was restored in the HF+R group (p=0.051). The area of mitochondria labelling was increased in the HF+RespHRV group compared to HF (**p=0.005). Many cytoskeletal proteins linked to mitochondria regulation and T-tubule remodelling were upregulated in HF and were reduced by RespHRV. MRM-MS was able to confirm these findings for selected targets (ANAXA2, CAVIN2, SPTBN1, TUBA4A). STED microscopy of collagen VI and the ryanodine receptor revealed cellular hypertrophy and remodelling of the T-tubules and cardiac junctions in HF sheep (*p<0.05), RespHRV showed a trend for reversing these structural changes. These data support the hypothesis that within the first two weeks of RespHRV pacing, there is an increase in mitochondrial repair and function coupled with re-organisation of the cellular cytoskeleton, which is consistent with the improvement in cardiac pump function.

The right and left ventricle of the heart have distinctly different developmental origins and are affected differently by similar pathological stimuli. Though it is well established that the heart relies almost entirely on mitochondrial function to sustain energy production, it remains unclear whether bioenergetics differ in the two ventricles. Herein, we define a novel methodology to optimize the isolation of intact cardiomyocytes from the right versus the left ventricle. We demonstrate that this segmental Langendorff-free methodology yields viable cardiomyocytes with intact mitochondrial function. Further, we compare bioenergetics in right versus left ventricle cardiomyocytes and show that cardiomyocytes from the right ventricle have a greater maximal capacity for respiration and enhanced glycolytic rate. This increase in respiration was concomitant with increased fatty acid oxidation and levels of fatty acid oxidation proteins, but no change in mitochondrial electron transport complex expression. These data validate a potentially powerful tool to evaluate differences in right and left ventricular function and advance the understanding of cardiac bioenergetic differences. These data will be discussed in the context of differential responses by the right versus ventricle in pathology.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are widely used to model cardiovascular disease, yet numerous differentiation protocols generate cardiomyocytes with heterogeneous molecular and functional properties, complicating experimental design. Here we systematically compare sixteen commonly used cardiomyocyte differentiation protocols and characterize their resulting cell states using single-nucleus RNA sequencing, functional phenotyping and computational integration with human genetic data. Despite similar cardiomyocyte yields, protocols produced distinct transcriptional programs, subtype compositions and physiological properties. By integrating protocol-specific gene expression signatures with genome-wide association studies of cardiovascular traits, we identify cardiomyocyte states enriched for genetic architectures underlying specific diseases. These analyses accurately predict protocols most suitable for modelling particular disease contexts, including electrophysiological defects associated with Brugada syndrome and metabolic vulnerability relevant to myocardial infarction. Our results demonstrate that differentiation protocols encode biologically distinct cardiomyocyte states with differential disease relevance and establish a framework for aligning stem-cell differentiation strategies with human complex trait genetics to guide model selection. This approach enables rational design of iPSC-based disease models and highlights how population-scale genetic data can inform experimental systems in stem cell biology.

MAP kinase-activated protein kinase-5 (MK5) plays an important role in cardiac fibroblast function. Although p38 MAPK and atypical MAPKs and ERK3 and ERK4 have been identified as activators of MK5, the kinases that activate MK5 remain controversial. Here we examined the expression, subcellular distribution, and regulation of MK5 in cardiac ventricular myofibroblasts and myocytes. The copy numbers for MK5 and ERK4 mRNA were comparable in myocytes and myofibroblasts, whereas that of ERK3 was much higher in myofibroblasts. Interestingly, MK5 and ERK3 immunoreactivity was detected in myofibroblasts but not myocytes whereas ERK4 immunoreactivity was detected in myocytes: treating in myocytes with a proteasome inhibitor or hypertrophic agonists failed to rescue MK5 immunoreactivity. In myofibroblasts, MK5 and ERK3 immunoreactivity was predominantly nuclear and cytosolic, respectively. In serum-starved cardiac myofibroblasts, phosphothreonine-182 MK5 (pT182-MK5) immunoreactivity was predominantly nuclear but increased in intensity and relocated to the cytoplasm in response to serum, sorbitol, angiotensin II, TGF{beta}, or H2O2 and this was prevented by inhibition of p38/{beta}. Phos-tag SDS-PAGE revealed multiple slower migrating bands of MK5 immunoreactivity, indicating phosphorylation of MK5 at multiple sites. Phos-tag PAGE also revealed MK5 phosphorylation was increased with fibroblast activation and in hearts exposed to a chronic increase in afterload. MK5 and ERK3 co-immunoprecipitated and proximity ligation assays revealed ERK3 and MK5 in close proximity in myofibroblast cytoplasmic compartment. Furthermore, p38/{beta} inhibition decreased the abundance of MK5 immunoreactivity in ERK3 immunoprecipitates. Finally, deleting MK5 did not reduce the abundance of ERK3 immunoreactivity. These observations suggest that p38 and/or p38{beta} are the primary mediators of T182-MK5 phosphorylation and hence MK5 activation in cardiac myofibroblasts.

BackgroundAlternative splicing is a critical determinant of protein diversity in the heart, where it drives the postnatal functional maturation of cardiomyocytes and specifies the ion-channel and calcium-handling isoforms required for mature contractile function; dysregulated splicing programs have in turn been implicated in cardiomyopathies and arrhythmias. However, the splicing regulators that control cardiomyocyte calcium handling remain largely unknown. ObjectiveWe systematically screened 276 splicing factor genes in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to identify regulators of calcium handling, and we selected SF3B2, a core U2 snRNP spliceosome component, for detailed follow-up based on complex-level enrichment and prior identification by Murphy et al. 2021 [1]. MethodsA high-throughput siRNA screen targeting 276 splicing factor genes was performed in hiPSC-CMs using 384-well calcium transient imaging. SF3B2 knockdown was followed by deep bulk RNA-seq analysis (n = 3 per group) for differential gene expression with DESeq2 and alternative splicing quantification with rMATS. RNA immunoprecipitation sequencing (RIP-seq) using an epitope-tagged SF3B2 construct was performed to identify direct mRNA binding targets of SF3B2. ResultsThe screen identified multiple U2 snRNP components, including SF3A2, SF3B1, and SF3B4--as regulators of calcium transient duration. SF3B2 was previously identified as a regulator of contractility in this screen [1], and the enrichment of its complex partners above the significance threshold in the current CTD75 analysis supported its selection for follow-up characterization. SF3B2 knockdown resulted in 2,683 differentially expressed genes (adjusted p < 0.05), with downregulated genes enriched in cell cycle and DNA replication pathways. Alternative splicing analysis revealed significant changes across all five rMATS event types in 36 genes within the cardiac muscle cell action potential involved in contraction gene ontology term (GO:0086002), including calcium channel (CACNA1C, CACNA1D, CACNA2D1), potassium channel (KCNH2, KCNQ1), and sodium channel (SCN5A) genes, although the large number of affected genes is consistent with broad spliceosomal perturbation and a formal enrichment test would be needed to determine whether cardiac action potential genes are preferentially affected. Integration of RIP-seq data (597 SF3B2-enriched transcripts, FDR < 0.05) with splicing analysis identified CACNA2D1, which encodes an auxiliary subunit of L-type calcium channels, as both directly bound by SF3B2 and alternatively spliced upon knockdown. ConclusionThese findings identify SF3B2 as a regulator of cardiomyocyte calcium handling and suggest that SF3B2-dependent missplicing of CACNA2D1 may link core spliceosome function to the splicing programs underlying cardiomyocyte functional maturation. Significance StatementThis study provides the first systematic functional screen of splicing factors in cardiomyocyte calcium handling and identifies SF3B2, a U2 snRNP subunit, as a regulator of cardiac ion channel splicing. Transcriptomic, splicing, and RNA binding data converge on CACNA2D1, identifying a route by which a core splicing factor shapes cardiac contractility, extending cardiac splicing regulation beyond the accessory RBPs studied to date.