Journal of Molecular and Cellular Cardiology

Dilated cardiomyopathy (DCM) is a leading cause of heart failure and cardiac transplantation, and pathogenic variants in LMNA are a well-established cause of inherited DCM. The LMNA gene encodes nuclear lamins A/C, which maintain nuclear integrity, regulate gene expression and mediate mechanotransduction. Here, we investigated the pathogenic consequences of the NM_170707.4(LMNA):c.274C>T NP_733821.1:p.(Leu92Phe) variant, previously associated with lipodystrophy features, using patient-derived induced pluripotent stem cells, differentiated into cardiomyocytes and show implication of LMNA in sarcomere remodeling and mitochondria efficiency. We generated iPSC lines from two DCM patients carrying LMNA p.Leu92Phe variant in heterozygous form and a healthy parental control. Cardiomyocytes differentiation efficiency was preserved, however, LMNAp.Leu92Phe iPSC-CMs exhibited laminopathies associated phenotypes, such as nuclear shape abnormalities and lamin A/C aggregation. Moreover, in vitro study revealed that LMNA p.Leu92Phe iPSC-CMs alter sarcomere reformation and decrease mitochondrial respiration after cardiomyocyte remodeling, which is associated with a worsening nuclear shape phenotype. Functional analyses highlight defects in calcium handling, thereby explaining arrhythmia and dilated cardiomyopathy features in patients. Our results show that the LMNA p.Leu92Phe variant compromises nuclear lamina integrity and disrupts functional cardiomyocyte properties, particularly during sarcomere remodeling, highlighting the long-term impact of this specific variant in LMNA-associated DCM.

Mitochondrial transplantation has recently emerged as an alternative treatment for cardiovascular disease (CVD), aimed at increasing mitochondrial number and improving mitochondrial function. Though mitochondrial dysfunction is a key factor in the development of right ventricular hypertrophy/failure, the effects of mitochondrial transplantation on disease mitigation have been largely unexplored. Therefore, this in vitro study aimed to determine whether the transplantation of exogenous L6 myotube mitochondria mitigates negative outcomes in H9C2 cardiomyocytes that were stimulated to hypertrophy with phenylephrine. Control (CTL), control with mitochondrial transplantation (MitoTx), Phenylephrine only (Phe-only), and phenylephrine with mitochondrial transplantation (Phe+MitoTx) treatments were evaluated. Pilot experiments indicated mitochondrial transplantation into healthy cardiomyocytes acutely increased Complex I-linked oxidative phosphorylation (OXPHOS) capacity (p=0.005) and maximal respiratory capacity (p=0.022) within 24 hours, and these data alongside microscopic evidence of fluorescently labeled L6 mitochondria in H9C2 cardiomyocytes suggested successful transplantation. Regarding treatment comparisons, Phe-only showed a significant increase in cell area (p<0.05), while Phe+MitoTx blunted the hypertrophic cardiomyocyte response. Consistent with these results, proteomic analysis of 4,806 proteins showed that transplantation enriched the mitochondrial proteome, impacting pathways including OXPHOS, respiration, fatty acid and amino acid metabolism, while suppressing extracellular matrix remodeling and de-differentiation signatures. This response was observed in healthy and phenylephrine-stressed cardiomyocytes. In conclusion, our in vitro data indicates that transplantation of L6 skeletal muscle mitochondria mitigates negative effects induced by phenylephrine in H9C2 cardiomyocytes. However, more rigorous in vivo studies are needed to determine if this is a suitable approach for disease mitigation.

Background: Human induced pluripotent stem cell-derived cardiomyocytes are valuable for cardiac disease modelling and drug testing but remain limited by immature structural and electrophysiological properties in conventional two-dimensional cultures. Three-dimensional cardiac models, including cardioids, provide a more physiological environment through multicellular organization and enable tissue-level functional analysis. Their electrophysiological properties and maturation, however, remain incompletely characterized. Methods: We performed an integrated electrophysiological characterization of human cardioids over the first three weeks of maturation using sharp microelectrode recordings, calcium transient measurements, whole-cell patch clamp recordings, immunofluorescence, transcriptomic analysis, and pharmacological interventions. Results: During maturation, cardioids exhibited progressive electrophysiological maturation, characterized by more physiological resting membrane potentials (-54.0{+/-}3.1mV to -68.1{+/-}1.2mV), increased action potential amplitude (73.5{+/-}4.8mV to 101.7{+/-}2.1mV) and upstroke velocity (44.3{+/-}5mV/ms to 86.6{+/-}8.4mV/ms), and shorter action potential duration (178.9{+/-}8.7ms to 144.0{+/-}4.8ms at 1Hz stimulation). These changes were associated with progressively increased INa density and enhanced SCN5A mRNA and Nav1.5 protein expression, consistent with progressive maturation of cardiac excitability. IKr current and KCNH2/hERG expression were already present at early maturation stages and remained stable, as evidenced by preserved sensitivity to dofetilide-blockade. Pharmacological inhibition of barium- and HMR-sensitive currents altered resting membrane and/or action potential characteristics in intact cardioids, whereas in isolated cardiomyocytes classical IK1 remained undetectable and IKs was small, highlighting a certain degree of persisting immaturity of the model. Conclusions: These findings demonstrate that human cardioids undergo progressive electrophysiological maturation and provide complementary tissue- and cellular-level insights into cardiac function, establishing their suitability for investigating INa and IKr-related cardiac pathologies.

Background: Arrhythmogenic cardiomyopathy (ACM) is a genetic and phenotypically heterogeneous myocardial disorder characterized by progressive cardiomyocyte loss, immune cell infiltration, and fibro-fatty replacement of the ventricular myocardium, with an increased risk of sudden cardiac death (SCD), predominantly caused by mutations in cardiac desmosomal genes including PKP2, DSP, and DSG2. We previously described the pivotal role of NF{kappa}B and CCR2+ cardiac macrophage-triggered inflammation in ACM pathogenesis. Methods: WT or Dsg2mut/mut mice were treated with either isotype control or anti-CD14 antibody once a week for the described treatment period (4 or 8 weeks). At the end of the treatment period, disease characteristics were assessed by echocardiography, electrocardiography, histology, immunofluorescence, positron emission tomography (PET) imaging and single-cell transcriptomics. Results: Here, we demonstrate the efficacy of neutralizing CD14 signaling, an important mediator of inflammatory macrophage activation, in attenuating ACM disease in vivo. Additionally, we elucidate shifts in the cellular and transcriptomic landscapes that occur following anti-CD14 antibody treatment in a well-established mouse model of ACM and identify substantial reductions in inflammatory macrophage and pro-fibrotic fibroblast populations. Conclusion: This study provides evidence that blockade of CD14 can attenuate the ACM disease phenotype. It may do so by inhibiting macrophage-based activation and inflammation, while also altering the transcriptional state of other important cell types. Collectively, these findings suggest that CD14 inhibition may be an effective option for ACM, which should be explored in early-phase clinical studies.

Aldosterone is a key regulator of systemic sodium, potassium, and fluid homeostasis. Chronic elevation of aldosterone, as observed in primary aldosteronism, chronic heart and kidney diseases, promotes cardiac hypertrophy, inflammation, fibrosis, and heart failure, which are further enhanced in aging. Although these pathological effects are primarily attributed to the mineralocorticoid receptor (MR), emerging evidence suggests roles for nonclassical G protein-coupled receptor (GPCR) signaling. We identified lysophosphatidic acid receptor 2 (LPAR2) as a novel GPCR candidate that mediates aldosterone signaling in cardiomyocytes. Using AC16 human cardiomyocyte cells and reporter gene assays, we demonstrate that aldosterone activates a Gi/o-dependent GPCR pathway. Pharmacological inhibition of LPAR2 internalization confirms its specific reaction to aldosterone. Functional studies revealed that aldosterone-induced cardiomyocyte injury requires both MR and LPAR2, where specific antagonists to both receptors reduced the ability of aldosterone to induce collagen expression and cell death markers. These findings uncover a previously unrecognized non-classical mechanism of aldosterone action in the heart and highlight LPAR2 as a promising therapeutic target for aldosterone-driven cardiovascular disease.

Cardiomyocyte hypertrophy is a leading clinical predictor of heart failure, yet newly identified candidate genes often remain disconnected from the signaling mechanisms that govern cardiomyocyte growth. We developed a computational-experimental pipeline that integrates genome-scale mouse knockout phenotypes with a logic-based differential equation model of hypertrophic signaling. Among 9,605 genes evaluated by the International Mouse Phenotyping Consortium, 939 knockout lines induced abnormal heart morphology. Directional curation of hypertrophy-related sub-phenotypes followed by interaction-based network expansion mapped 37 genes to the signaling model. Virtual knockdown screening identified five candidates with concordant in vivo and in silico effects: LRIG1 and CBL as predicted negative regulators and VAV2, RASA1, and LEPR as predicted positive regulators. Mechanistic subnetwork analysis linked these candidates to distinct receptor-proximal, Ras, PI3K-AKT, and MAPK signaling axes. In neonatal rat cardiomyocytes, siRNA-mediated depletion of VAV2, RASA1, or LEPR reduced phenylephrine-induced cell growth, supporting their cell-autonomous contribution to hypertrophy. Quantitative phenotyping further validated the predicted decreased cardiac hypertrophy for VAV2 and LEPR knockouts but identified potential age-dependent mechanisms for RASA1 knockout. Overall, this study establishes the application of network models to translate from in vivo phenotypic screens into pathway mechanisms.

Background Takotsubo cardiomyopathy (TTC) is a stress-induced cardiomyopathy characterized by acute cardiac dysfunction, inflammation, and hypertrophy, yet no therapy directly targets the underlying myocardial injury. Clinical studies have reported elevated circulating Angiotensin II (Ang II) levels in TTC patients, suggesting dysregulation of the renin-angiotensin system (RAS). Given the established cardioprotective effects of Angiotensin-(1-7) [Ang-(1-7)] in other cardiovascular diseases, we hypothesized that oral administration of Ang-(1-7) could attenuate acute cardiac remodeling during TTC. Methods Female C57BL/6J mice were subjected to a single high-dose isoproterenol (ISO) injection (300 mg/Kg, intraperitoneal) to induce TTC and received daily oral treatment with Ang-(1-7) complexed with hydroxypropyl {beta}-cyclodextrin [Ang-(1-7)-CD] or vehicle (CD), for 4 days, beginning one day before ISO administration. Cardiac proteome alterations were characterized by tandem mass tag (TMT)-based quantitative proteomics. Cardiac hypertrophy was assessed by heart weight normalized to tibial length and cardiomyocyte cross-sectional area. Myocardial inflammation was evaluated by histopathology, CD68 immunostaining, and flow cytometric analysis of infiltrating leukocyte populations. Results We observed a distinct cardiac proteome signature in Ang-(1-7)-treated ISO mice, predicting inhibition of pathways associated with cardiac hypertrophy and adverse remodeling. These molecular changes were accompanied by reduced heart weight normalized to tibial length and smaller cardiomyocyte cross-sectional area compared with vehicle-treated ISO mice. Oral Ang-(1-7) also attenuated myocardial inflammation, as evidenced by lower inflammatory scores, reduced CD68+ macrophage infiltration, decreased numbers of CD45+ leukocytes, and marked suppression of Ly6G+ neutrophil and CCR2+ monocyte recruitment to the heart. These protective effects were associated with improved survival following ISO challenge. MS data are available via ProteomeXchange (PXD082872). Conclusions Oral administration of Ang-(1-7) attenuated acute cardiac hypertrophy and inflammation in experimental TTC while inducing cardiac proteome changes consistent with reduced adverse remodeling. These findings identify activation of the protective RAS axis as a promising therapeutic strategy for limiting myocardial injury during TTC.

Background: Milrinone, a phosphodiesterase-3 (PDE-3) inhibitor, is widely used to improve cardiac output in pediatric and adult patients. Yet, developmental differences in myocardial responsiveness to milrinone remain incompletely understood. In this study, we examined the impact of postnatal maturation on the acute cardiac effects of milrinone using an intact guinea pig heart model. Methods: Neonatal (0-2 days), juvenile (4-10 days), and adult (> 6 months) guinea pig hearts were excised, Langendorff-perfused, and cardiac metrics were evaluated under basal conditions and in response to acute milrinone treatment (15 minutes, 10 and 100 M sequential concentrations). Pseudo-electrocardiograms were recorded continuously and left ventricular pressure measurements were performed under sinus rhythm and in response to external pacing. Post-rest potentiation was used to assess contractile reserve, and optical action potentials and calcium transients were recorded. Results: Baseline left ventricular developed pressure (LVDP), action potential duration (APD), and calcium transient duration (CaD) increased with postnatal maturation, consistent with developmental cardiomyocyte remodeling and refinement of excitation-contraction coupling. Milrinone increased heart rate in all age groups, with the greatest chronotropic response at 100 M. Milrinone also increased ventricular contractility and relaxation across developmental stages, but the magnitude of the response varied with age and was attenuated during high frequency pacing. Adult hearts had the greatest increase in LVDP during sinus rhythm and robust post-rest potentiation, which were less pronounced in neonatal hearts. APD and CaD were shortened in neonatal and adult hearts, but were minimally affected in juveniles, indicating a non-linear developmental pattern. Conclusions: Milrinone exerts positive chronotropic, inotropic, and lusitropic effects throughout development, but the magnitude and frequency dependence of these responses vary with age. These findings suggest that postnatal maturation influences the cardiac response to PDE3 inhibition. These developmental differences highlight the importance of considering age as a biological factor in pediatric drug selection and dosing.

Background: Barth syndrome (BTHS) is an X-linked mitochondrial disorder caused by loss-of-function mutations in TAFAZZIN (TAZ), resulting in defective cardiolipin (CL) remodeling, cardiomyopathy, and premature death. The mechanisms linking TAZ deficiency to impaired mitochondrial quality control remain incompletely understood, and there are no disease-modifying therapies available for BTHS. Here, we investigated the role of Acyl-CoA Lysocardiolipin Acyltransferase-1 (ALCAT1), a stress-inducible phospholipid-remodeling enzyme, in BTHS cardiomyopathy. Methods: Inducible TAZ knockdown (TAZKD) and cardiomyocyte-specific TAZ knockout (TAZcKO) mice were used to determine the effects of genetic ALCAT1 deletion or pharmacological inhibition with Juvenatin, a highly selective small molecule ALCAT1 inhibitor. Cardiac function, exercise capacity, mitochondrial function, lipid remodeling, lysosomal function, and mitophagic flux were assessed. Results: ALCAT1 was markedly upregulated in TAZ-deficient hearts. Genetic ALCAT1 deletion attenuated cardiac dysfunction in TAZKD mice. Juvenatin similarly improved cardiac function in TAZKD mice and reversed established cardiomyopathy in TAZcKO mice. Genetic or pharmacological ALCAT1 inhibition improved mitochondrial ultrastructure and respiration and reduced mitochondrial oxidative stress. These benefits occurred without correcting the characteristic CL abnormalities caused by TAZ deficiency. Instead, ALCAT1 deletion or inhibition corrected pathological phosphatidylinositol remodeling, normalized phosphoinositide signaling, and restored lysosomal function and mitophagic flux, leading to improved mitochondrial quality control in TAZ-deficient cells. Conclusions: ALCAT1 contributes to BTHS cardiomyopathy through a CL-independent pathway involving dysregulated phosphatidylinositol/phosphoinositide signaling, lysosomal dysfunction, and impaired mitophagy. Genetic and pharmacological targeting of ALCAT1 restores mitochondrial and cardiac function despite persistent CL abnormalities, establishing ALCAT1 as a promising disease-modifying therapeutic target for BTHS.

Objective: Mitochondrial oxidative capacity in striated muscle is governed by the PGC-1/ERR transcriptional axis, which controls not only steady-state respiration but the capacity to expand energy production when demand rises. Whether this reserve function requires dedicated downstream effectors, and whether its loss is detectable at rest, remains unclear. We asked whether Perm1, a striated muscle-specific PGC-1/ERR-induced regulator, is required cell-autonomously for cardiac bioenergetic reserve. Methods: We generated cardiomyocyte-specific Perm1 knockout mice using alphaMHC-nCre, complemented by an adult tamoxifen-inducible alphaMHC-MerCreMer model, and assessed cardiac development, structure, and function longitudinally through 12 months of age in both sexes. We evaluated mitochondrial function by high-resolution respirometry, transmission electron microscopy, and molecular profiling. We tested energetic capacity under load by treadmill exercise with tissue ATP quantification and by beta-adrenergic stimulation of cultured neonatal cardiomyocytes. Results: Perm1 deletion did not perturb embryonic cardiac development, and neither constitutive nor adult-onset deletion impaired cardiac structure, systolic function, or diastolic function through 12 months. Despite this preserved resting phenotype, mitochondrial respiration was reduced by 20 - 25% across complex I- and complex I+II-supported conditions, without changes in citrate synthase activity, mtDNA copy number, or OXPHOS subunit abundance. Instead, the MICOS components IMMT (MIC60) and CHCHD3 (MIC19) were selectively reduced, and mitochondrial subpopulations shifted toward fewer subsarcolemmal and more interfibrillar mitochondria, with unchanged total mitochondrial density. Functionally, Perm1-deficient mice of both sexes showed reduced treadmill capacity at 6 and 12 months and failed to maintain cardiac ATP during exercise at a submaximal workload, while skeletal muscle ATP and plasma lactate were unaffected. This deficit was cardiomyocyte-autonomous: beta-adrenergic stimulation increased ATP content in control cardiomyocytes, but Perm1 knockdown reduced this response. Conclusions. Perm1 is a cardiomyocyte-intrinsic determinant of bioenergetic reserve, dispensable for resting cardiac function but required to expand ATP production under increased demand. These findings identify a mode of metabolic regulation that is invisible to resting functional phenotyping.

Background: Loss- and gain-of-function variants in SCN5A, encoding the cardiac sodium channel Nav1.5, are major causes of inherited cardiac channelopathies. Their pathophysiology has almost exclusively been attributed to altered cardiomyocyte excitability and cardiac conduction. However, growing evidence indicates that autonomic dysfunction contributes to these disorders, raising the possibility that Nav1.5 dysfunction may also affect the intrinsic cardiac nervous system. Objectives: We investigated whether Nav1.5 is functionally expressed in murine intrinsic cardiac neurons and evaluated its contribution to neuronal excitability. Methods: Scn5a expression was investigated by digital PCR and immunocytochemistry in isolated murine intracardiac neurons. Whole-cell patch-clamp recordings were used to characterize sodium currents and action potential properties in wild-type and Scn5a+/- mice. Results: Scn5a transcripts and Nav1.5 protein were detected in intrinsic cardiac neurons. Voltage-clamp recordings identified a tetrodotoxin-resistant sodium current accounting for approximately 8% of the total sodium current and displaying pharmacological and biophysical properties consistent with Nav1.5. Activation and inactivation analyses indicated that Nav1.5 is the predominant sodium conductance available at most polarized membrane potentials. Accordingly, Scn5a haploinsufficiency selectively altered action potential threshold, maximal upstroke velocity, overshoot and duration in neurons resting below -60 mV, whereas more depolarized neurons were essentially unaffected. No structural or neurochemical remodeling of intrinsic cardiac ganglia was detected. Conclusion: Nav1.5 is a functional determinant of intrinsic cardiac neuronal excitability. These findings identify the intrinsic cardiac nervous system as a new cellular target of SCN5A dysfunction and suggest that inherited cardiac sodium channelopathies could involve alterations in both myocardial and intrinsic neuronal electrophysiology.

Extracellular vesicles (EVs) are membrane-bound particles secreted by all cells and serve as critical mediators of intercellular communication in both physiological and pathological contexts. However, elucidating the precise functions of endogenous EVs remains challenged by a fundamental technical bottleneck: current isolation methodologies depend on shared physical properties or generic surface markers, preventing the selective enrichment of cell-type-specific EVs within complex tissues. Consequently, EVs recovered from biofluids or tissue extracts represent heterogeneous populations, confounding the direct attribution of biochemical signatures and biological activities to distinct cellular origins. Here, we describe a versatile genetic-labeling and affinity-based purification strategy that enables the selective isolation of cell-specific primary EV. Using cardiomyocyte-derived EV (CM-EVs) as a proof-of-concept, we crossed Myh6-Cre mice with the ROSA26-mTmG reporter line to restrict membrane-targeted GFP expression to cardiomyocytes. Following myocardial infarction, cardiac EV were recovered and GFP-positive CM-EVs were selectively purified by anti-GFP nanobody-based magnetic pulldown. This approach yields highly enriched, functional cardiomyocyte-derived extracellular vesicles (CM-EVs). To accommodate diverse analytical needs, we established two distinct elution workflows: (i) a denaturing protocol optimized for downstream proteomics and immunoblotting, and (ii) a gentle, non-denaturing protocol that preserves vesicle integrity and biological activity for downstream in vitro and in vivo functional assays. Using this method, we characterized CM-EVs size distribution and proteomic signature, and demonstrated their capacity to transfer functional cargo to target cells in vitro. Because the purification approach relies solely on targeting a genetically restricted membrane tag rather than assumed surface markers, it is immediately adaptable to virtually any cell type for which a validated Cre driver exists. This method removes a long-standing bottleneck in EV research and provides a broadly applicable platform for deciphering the biochemical composition and in-vivo functions of cell-type-specific primary EV in physiological and pathological settings.

Opening of the mitochondrial permeability transition (PT) pore is an important step in necrotic cell death in various pathologic conditions such as ischemia-reperfusion injury. Methylglyoxal (MGO) is a byproduct of glycolysis that covalently modifies proteins (i.e., glycation) and is widely regarded as toxic (glycative stress), contributing to the pathology of diabetes. Paradoxically, MGO has been shown to inhibit PT pore opening, but the mechanism of this effect is poorly understood. Herein we show that glycation is elevated in various cardioprotective scenarios. Furthermore, building on a serendipitous finding of the ability of phosphate to block MGO's effect on the PT pore, we used proteomics to map potential sites mediating this effect. From this analysis, mitochondrial creatine kinase (CK) emerged as a phosphate-sensitive MGO target with links to the PT pore. Although MGO inhibited CK activity, this did not appear to be important for its effect on the PT pore. Rather, MGO modified sites on CK that are important for its membrane binding, which likely mediates the combined effects of MGO and phosphate on the PT pore. Furthermore, a consensus sequence for MGO modifications in mitochondria is reported.

Background: KCNH2 encodes Kv11.1 channel proteins that conduct the rapidly activating delayed-rectifier K+ current (IKr), which is critical for cardiac repolarization. KCNH2 encodes two functional isoforms, Kv11.1a and Kv11.1b, via alternative transcription start sites. Kv11.1a is the principal determinant of cardiac IKr and ventricular repolarization. The circadian clock, a transcriptional-translational feedback loop that cycles with a period of ~24 hours and drives the circadian expression of many genes, including Kcnh2 in the mouse heart. Because daily body temperature rhythms provide a systemic signal that synchronizes cardiac circadian clocks, we tested whether physiological temperature cycles drive the circadian promoter activity of the cloned human KCNH2 (hKCNH2) promoter. Hypothesis: hKCNH2 is a direct transcriptional target of the circadian clock, with temperature driving its promoter activity through BMAL1:CLOCK acting at a conserved tandem E-box. Methods: We cloned the conserved proximal promoter of KCNH2 (-1631 bp upstream of Kv11.1a exon 1) to generate hKCNH2 promoter luciferase reporter constructs. Constructs were transfected into C2C12 myotubes and synchronized by serum shock (static 37{degrees}C) or temperature cycling (36.5-38.5{degrees}C). Bioluminescence was recorded and assessed for period, phase, and amplitude. BMAL1:CLOCK dependence was tested via dominant-negative CLOCK{Delta}19 co-expression. Results: Temperature cycling did not exhibit the rapid damping characteristic of serum-shock-synchronized oscillations, consistent with continuous entrainment by an external zeitgeber rather than a free-running oscillator. Deletion analysis identified a conserved tandem E-box required for oscillation under both serum shock and temperature cycling, and for BMAL1:CLOCK-dependent transactivation (1.75 {+/-} 0.21 vs. 0.86 {+/-} 0.06 RLU, p = 0.0038). CLOCK{Delta}19 reduced hKCNH2 promoter amplitude under temperature cycling without altering period. Conclusion: The circadian clock regulates KCNH2 promoter activity through daily temperature rhythms.

Background: Activation of the renin-angiotensin system (RAS) contributes to aortic diseases caused by fibrillin-1 haploinsufficiency (Fbn1C1041G/+). However, whether RAS activation influences mitral valve disease associated with FBN1 variants remains undefined. This study determined whether inhibition or augmentation of the RAS affects mitral valve pathology in Fbn1C1041G/+ mice. Methods: Angiotensinogen (AGT) antisense oligonucleotides (ASOs) were administered for 12 weeks to inhibit the RAS in male and female Fbn1C1041G/+ mice. To augment the RAS, mice were infused with angiotensin II (AngII; 1,000 ng/kg/min) for 28 days. Mitral valve and heart function were monitored by ultrasound. At termination, hearts were dissected free and tissue sectioned to assess mitral valve leaflet thickness and pathology. Results: Mitral valve leaflet thickness was significantly increased in Fbn1C1041G/+ mice compared to Fbn1+/+ littermates of both sexes. ASO-mediated AGT inhibition did not alter this phenotype despite markedly reduced plasma AGT concentrations. AngII infusion increased mitral valve leaflet thickness in Fbn1C1041G/+ mice with increased proteoglycan and collagen content, while exerting minimal effects in Fbn1+/+ mice. Twenty-five percent of AngII-infused Fbn1C1041G/+ mice died of non-vascular causes. Ultrasound showed mitral valve dysfunction in AngII-infused Fbn1C1041G/+ mice. Grossly expanded left atria were common in AngII-infused Fbn1C1041G/+ mice. These markedly enlarged left atria contained increased fibrosis, macrophage accumulation, and platelet-rich masses. Conclusions: Mitral valves of Fbn1C1041G/+ mice did not respond to AGT inhibition but were markedly more susceptible to AngII infusion, with greater increases in thickness and compositional changes than mitral valves of Fbn1+/+ mice. In addition, AngII promoted mitral valve dysfunction that were associated with pronounced left atrial pathology.

The adrenergic response is a fundamental mechanism that regulates heart rate (chronotropy), cardiac contractility (inotropy) and relaxation (lusitropy). Adrenergic stress is also a recognized trigger of arrhythmia in disease. Yet, our understanding of the underlying molecular basis remains incomplete. Protein kinase A (PKA) and the calcium/calmodulin-dependent kinase II (CaMKII) phosphorylate multiple targets proposed to participate in the adrenergic response, including the GTP-binding protein Rad, phospholamban (PLB) and ryanodine receptor 2 (RyR2). Here we demonstrate that phosphorylation of both Rad and PLB is necessary for inotropy and lusitropy. We show that changes in cardiac contractility and relaxation are primarily dependent on intracellular calcium handling. Finally, we report that Rad and PLB control stress-induced arrhythmogenesis, despite the phosphorylation of other pro-arrhythmic targets. We have identified the essential molecular components of the adrenergic response, resolving a long-standing debate in cardiac excitation-contraction coupling and refining current models of sympathetic regulation in health and disease.

Aims: Mature, contractile vascular smooth muscle cells (vSMC) in muscular blood vessels can de-differentiate into synthetic vSMC, while human pluripotent stem cells (hPSC) differentiate into proliferating, fetal-like vSMC that can be induced to form contractile vSMC. Given the complicated embryonic origins and conflicting views on vSMC heterogeneity, our aim was to establish how closely hPSC-vSMC of a known germ layer lineage and defined phenotype are representative of vSMC obtained from aorta (AoSMC). Methods and Results: We generated paraxial mesoderm (PM)-derived synthetic and contractile vSMC from hPSC lines and performed phenotypic and mechanical comparisons with AoSMC. Synthetic hPSC-vSMC and cultured AoSMC are proliferative, sensitive to contact inhibition and exhibit phenotype switching. Contractile hPSC-vSMC are non-proliferative, capable of phenotype switching and have elevated levels of contractile proteins (CNN1, TAGLN, ACTA2). These contractile proteins also displayed density-dependent, cell-to-cell heterogeneity in both AoSMC and hPSC-vSMC. Single-cell traction force measurements on micropost array detectors (mPADs) yielded comparable forces per micropost in the nN range across cell types. Quantitative 3D force measurements on humanized smooth muscle cell microtissues (SMTs) developed similar forces per cell. Larger forces were generated by MEKi-treated hPSC-vSMC and AoSMC versus controls. Conclusions: We have systematically studied the properties of hPSC-vSMC and performed comprehensive phenotypic and mechanical comparisons with AoSMC. These results demonstrate that synthetic hPSC-vSMC generated in vitro and cultured AoSMC derived from tissues are comparable and mechanically similar, but results with hPSC-vSMC are more reproducible.

Background: Aortic stenosis (AS) is a valvular heart disease which imposes chronic pressure overload on the left ventricle (LV). Although LV remodelling may regress following aortic valve replacement, the molecular determinants of reverse remodelling remain poorly understood. This study develops a machine learning-based model using myocardial microRNA (miRNA) expression profiles to predict LV mass normalization one year after valve replacement. Methods: Consensus selection combining Random Forest and Bayesian Network analyses was applied to miRNA sequencing data from LV biopsies of 12 matched patient pairs with AS. Selected miRNAs were qPCR quantified in an extended cohort of 89 AS patients and incorporated into predictive models. Sex-stratified analyses were performed. Model robustness was assessed using bootstrap resampling and cross-validation. Candidate miRNAs and target genes were evaluated in LV from a murine model of pressure overload induced by transverse aortic constriction (TAC). Results: The combined Random Forest-Bayesian Network approach identified a three-miRNA signature (miR-517a-3p, miR-122b-5p, and miR-4683) associated with LV mass normalization after valve replacement. Validation in an extensive cohort demonstrated good overall discrimination and achieved its highest performance in women (AUC >0.80). Functional analyses identified drebrin (DBN1) as a putative target of miR-517a-3p and supported its potential involvement in pressure overload-induced cardiac remodelling. Conclusion: Random Forest and Bayesian Network integrated analyses enabled the identification of a myocardial miRNA signature with sex-dependent predictive value for reverse cardiac remodelling after aortic valve replacement. Functional validation supports the relevance of selected miRNAs and their downstream targets, highlighting their potential as biomarkers and candidate regulators of myocardial recovery.

Instability of the cardiac electrical system is a major cause of life-threatening arrhythmias. Current safety guidelines have traditionally emphasized QT interval prolongation on the electrocardiogram (ECG) as a primary biomarker of proarrhythmic risk. However, this approach does not capture all forms of electrical instability. In this study, we demonstrate that beat-to-beat variability in action potential duration (APD), a cellular surrogate of the QT interval, better stratifies proarrhythmic risk than mean APD prolongation alone. Using a validated computational model of a human ventricular myocyte, we simulated the effects of the {beta}-adrenergic agonist isoproterenol (ISO). Increasing the ISO concentration shortened mean APD but paradoxically increased beat-to-beat APD variability, measured as the standard deviation of APDs. In our simulations, the emergence of early afterdepolarizations (EADs), abnormal oscillations of the membrane potential during the plateau phase of the action potential, at higher ISO concentrations was consistently preceded by a marked rise in APD variability, even while mean APD was reduced. Mechanistic analysis revealed that ISO-induced enhancement of L-type Ca2+ current (ICaL) and slow delayed rectifier K+ current (IKs) steepens the APD restitution slope, which reflects action potential instability, even when the net effect is a reduction in APD. These results support APD variability as a more sensitive and mechanistically plausible marker of proarrhythmic risk than APD prolongation alone. Our findings suggest that QT interval variability may improve risk stratification and provide a more robust mechanistic foundation for drug safety evaluation.

Arrhythmogenic cardiomyopathy (ACM) is an inherited disease characterized by fibrofatty remodeling and sudden cardiac death linked to desmosomal dysfunction. Yet, 40% of cases remain genetically unexplained, and non-desmosomal mutations offer insight into ACM pathophysiology. Myoferlin (MYOF), a calcium (Ca2+)-binding protein in cardiomyocytes, is linked to cardiomyopathy, but its mechanism remains unknown. Whole-exome sequencing identified a heterozygous MYOF missense variant (p.G1654S) in an ACM family; we subsequently found three unrelated p.G1654S cases. We hypothesized that MYOF deficiency promotes Ca2+ handling deficits and arrhythmogenesis in ACM. In patient iPSC-derived cardiomyocytes, MYOF p.G1654S protein dimerized with wild-type protein and underwent accelerated lysosomal degradation, showed disrupted Ca2+ handling, and displayed increased arrhythmia burden, all rescued by genomic correction. MYOF interacted with CaV1.2, and verapamil pharmacologically rescued arrhythmias. Heterozygous Myof knockout mice showed systolic dysfunction and fibrosis. These findings establish MYOF as a causative ACM gene and therapeutic target that mediates arrhythmogenesis by disrupting MYOF-CaV1.2.