Nature Cardiovascular Research

Vascular disease often begins as a failure to read mechanical force. Cerebral cavernous malformations (CCMs) are leaky, brain-invading capillary clusters that cause stroke and seizures and arise from endothelial loss of the CCM complex. They form exclusively in low-flow vessels but existing models cannot explain this selectivity. We previously established that CCM-mutant endothelium inverts its adhesive balance, weakening cell-cell junctions while building matrix-degrading adhesions. The mechanosensors imposing this switch are unknown. Here we show that the mechanosensitive Ca2+ channels PIEZO1, TRPV2 and TRPV4 are overexpressed in the endothelium of CCM lesions in a 42-patient cohort, and that lesion volume scales with their expression level. Mechanistically, PIEZO1 partners with TRPV4 to destabilise cell-cell junctions and with TRPV2 to reinforce cell-matrix adhesions; silencing TRPV4 restores junctional integrity and abolishes KLF4 induction, whereas silencing TRPV2 or inhibition with tranilast abolishes matrix degradation and invasion. In vivo, trpv4 knockdown or inhibition with GSK2798745 restores atrioventricular canal morphogenesis in ccm2-mutant zebrafish. Gain of mechanosensory channel function is thus a causal step in CCM pathogenesis, upstream of the KLF program. Combined therapeutic targeting of TRPV2 and TRPV4 with inhibitors validated in humans offers a route to therapy.

Cardiovascular disease involves coordinated remodelling across multiple biological processes. Gene-level signatures vary substantially between studies because different combinations of genes can support similar biological processes. Pathway-level analyses can provide more stable representations of these processes but typically consider pathways independently. We hypothesised that grouping related pathways into conserved biological functions and quantifying their relative weighting would reveal a higher-order, transferable property of vascular tissue that we term functional organisation. We quantified the relative weighting of six conserved biological functions across independent transcriptomic datasets spanning human vascular disease, experimental models and therapeutic interventions. Vascular tissues exhibited a reproducible functional organisation defined by the balance of these functions. A dominant remodelling trajectory captured coordinated, nonlinear rebalancing of structural, immune, signalling and metabolic programmes, while a second dimension distinguished contractile/ECM organisation from immune activity. This organisation was preserved across independently reconstructed reference cohorts and remained robust to analytical sensitivity testing. When independent datasets were projected into this fixed framework, biological and clinical phenotypes occupied coherent positions along the remodelling landscape. Plaque-derived vascular, stromal and immune cell populations also occupied ordered functional positions, linking cellular heterogeneity to tissue-level organisation. The same organisational structure generalised across atherosclerosis, peripheral vascular disease and abdominal aortic aneurysm and revealed continuous biological heterogeneity within conventional clinical classifications. Genetic, pharmacological and dietary perturbations reproducibly shifted functional organisation, demonstrating that organisational state is dynamic and biologically responsive. Together, these findings identify functional organisation as a reproducible tissue-level property of vascular remodelling and provide a framework for understanding cardiovascular disease as coordinated rebalancing of biological functions rather than alteration of individual pathways in isolation.

Maintaining cardiac structure and function throughout the lifespan requires a delicate balance in carbon allocation between energetic and biosynthetic processes. At the nexus of this balance are prohibitins-1 and -2 (PHB1, 2) which form a ring-like complex in mitochondrial and plasma membranes responsible for coordinating cellular growth, metabolism and autophagy. Here we describe how ablation of the PHB complex in cardiomyocytes of adult mice (cPHB1KO) causes unrestrained mechanistic target of rapamycin complex 1 (mTORC1) activity and a Warburg-like reprogramming of glucose metabolism in heart toward enhanced de novo amino acid biosynthesis. These changes are accompanied by disruptions in mitochondrial Ca2+ handling and impaired autophagy, leading to severe dilated cardiomyopathy and mortality within 12 weeks. Using pharmacological and nutritional approaches, we further show that mTORC1 inhibition attenuates pathologic cardiac remodeling only in female cPHB1KO mice. Our findings illustrate novel mechanisms linking the PHB complex with altered carbon flux and pathogenesis of cardiomyopathy.

Right ventricular (RV) adaptation to pulmonary hypertension determines outcome in dilated cardiomyopathy (DCM), but the molecular mechanisms of the transition to decompensation remain unclear. We analysed RV tissue from explanted hearts of patients with end-stage DCM using single-nucleus RNA sequencing (n=21), mass spectrometry and Olink Reveal proteomics (both n=44), and integrated these molecular profiles with echocardiographic and right-heart catheterisation measures to identify molecular correlates of RV dysfunction. Mean pulmonary arterial pressure was the dominant correlate of RV transcriptional remodelling, particularly in cardiomyocytes, where higher pressure was associated with contractile remodelling, autophagy, vesicle trafficking and glucose metabolism. In contrast, RV decompensation was characterised by immune activation and reduced oxidative phosphorylation exclusively at the proteomic level. Integrative multi-omics factor analysis (MOFA) further identified fibrosis as the dominant molecular program shared across transcriptomic and proteomic layers. Together, these findings indicate molecular adaptation to pressure load and tissue fibrosis during progression towards RV failure.

Pathogenic variants of COL4A1 and COL4A2, which encode the 1 and 2 chains of type IV collagen in vascular basement membranes, cause cerebral small vessel disease and intracerebral hemorrhage, but the signaling mechanisms that precede vascular damage remain incompletely defined. Here, we investigated cerebral arterial function in mice heterozygous for the Col4a2+/G646D mutation. Cerebral pial arteries from aged (12-month-old), but not young (3-month-old), Col4a2+/G646D mice exhibited impaired pressure-induced myogenic tone, whereas downstream parenchymal arterioles retained normal myogenic responsiveness, revealing a segment-selective vascular defect. The frequency of subcellular Ca2+ signals (Ca2+ sparks) generated by the release of Ca2+ through ryanodine receptors (RyR2s) on the sarcoplasmic reticulum (SR) was increased, and large-conductance Ca2+-activated K+ (BK) channel activity was elevated, in cerebral pial artery smooth muscle cells (SMCs) from aged Col4a2+/G646D mice. SR Ca2+ store load and Ryr2 transcript abundance were unchanged in these cells, but Stimulated Emission Depletion super-resolution microscopy revealed that RyR2 protein clusters were enlarged compared with controls. Protein kinase A (PKA) activation reproduced RyR2 cluster enlargement, whereas PKA inhibition normalized RyR2 cluster organization and BK channel activity in SMCs from aged Col4a2+/G646D mice. These findings identify age-associated, PKA-dependent, nanoscale remodeling of RyR2 clusters as a previously unknown mechanism underlying cerebral arterial dysfunction in COL4A2-related disease.

Inflammatory T-cell infiltration is widely considered a hallmark of cardiac immune-mediated tissue injury, yet whether inflammation alone is sufficient to cause cardiac damage remains unclear. Using a spontaneous genetic model of immune checkpoint inhibitor myocarditis, we show that cardiac immune infiltration and tissue injury are separable processes. CD4+ T-cells promote disease by licensing pathogenic CD8+ T-cell responses through CD40L signaling, whereas perforin-dependent CD8+ cytotoxicity is specifically required to induce cardiomyocyte death and cardiac arrhythmias. Loss of perforin prevented cardiac injury and rescued survival despite persistent myocardial inflammation, demonstrating that inflammatory infiltration is insufficient to produce lethal myocarditis in the absence of a cytotoxic effector program. CD40L blockade similarly attenuated pathogenic CD8+ T-cell activation, myocardial inflammation, and mortality. These findings identify a cooperative CD4-CD8 T-cell circuit that governs autoimmune cardiac injury and establish that acquisition of cytotoxic effector function, rather than inflammation alone, determines the transition from immune infiltration to tissue destruction.

Myocardial infarction is a leading cause of death and disability worldwide. Ischemic injury leads to irreversible loss of cardiomyocytes, the contractile cells of the heart, and formation of a fibrotic scar. After infarction, macrophages massively infiltrate the heart and orchestrate the tissue repair process by removing dead cells and modulating fibroblast activation for scar formation. We previously demonstrated that diverse monocyte-derived macrophage populations dynamically accumulate in the heart following myocardial infarction, notably a pro-repair Trem2hi subset. In this study, we leveraged spatial transcriptomics, single-cell RNA-seq, and functional assays to elucidate the role of TREM2 in driving macrophage-mediated cardiac tissue repair post-infarction. We show that Trem2hi macrophages localize in scarring areas of the infarcted myocardium in the vicinity of collagen-producing myofibroblasts. In Trem2-/- mice, cardiac accumulation of monocyte-derived macrophages with a pro-scarring matrisome-associated macrophage signature was reduced. TREM2 deficiency was functionally associated with reduced fibroblast proliferation, accumulation of myofibroblasts, decreased collagen deposition in the infarcted heart, and increased infarct size. In vitro, we show that TREM2 mediates efferocytosis-induced pro-fibrotic gene expression and promotes macrophage ability to induce fibroblast migration. IL-4 priming of bone marrow-derived macrophages further increased the pro-fibrotic response in macrophages, suggesting that IL-4 and efferocytosis act synergistically to drive this phenotype. Altogether, our results show that TREM2 is essential for the accumulation and function of pro-scarring monocyte-derived macrophages in the infarcted myocardium.

Aortic stenosis (AS) is a heterogeneous disease of aging characterized by valvular calcification and distinct structural, electrical, and hemodynamic remodeling that are incompletely captured by any single diagnostic measure. Here we show that three AI-derived digital biomarkers resolve AS-related remodeling into complementary structural (cine-CMR Digital AS Severity Index, DASSi), electrical (AI-ECG), and hemodynamic (phase-contrast CMR peak aortic velocity) axes. Among 68,714 UK Biobank participants, all three biomarkers were independently associated with prevalent AS and prospectively predicted aortic valve replacement. Genetic and transcriptomic analyses of the digital phenotypes revealed partially distinct, heritable architectures: peak aortic velocity aligned closely with clinical AS genetics, whereas DASSi and AI-ECG defined a shared myocardial-remodeling axis largely independent of clinical AS susceptibility. These findings support AS as a multidimensional remodeling syndrome and establish a novel digital phenotyping framework for dissecting complex cardiovascular disease into complementary, biologically informative axes.

Background: Macrophages participate in atherosclerotic plaque development, progression and resolution, integrating lipid and inflammatory cues to balance tissue damage and repair. Despite considerable understanding of macrophage function in atherosclerosis, the underlying regulatory mechanisms governing these responses continue to evolve. Emerging evidence implicates epitranscriptomic gene regulation by the RNA modification N6-methyladenosine (m6A) in control of context specific immune responses, but its role in atherosclerosis is not well-defined. Methods: We integrated m6A quantitative trait loci (m6A QTLs) with Coronary Artery Disease (CAD) GWAS summary statistics. Using Oxford Nanopore direct RNA sequencing and MeRIP-seq, we constructed a high-resolution map of m6A modifications in macrophages. To determine the functional consequences of myeloid m6A loss in-vivo, we generated a myeloid-specific loss-of-function mouse model of m6A (Mettl14f/f LysMCre) and performed atherosclerosis studies using bone marrow transplantation into irradiated Ldlr-/- mice. Atherosclerosis lesions were analyzed using light microscopy and immunofluorescence imaging, with complementary in-vitro mechanistic studies performed in murine bone marrow derived macrophages (BMDMs). Results: m6A quantitative trait loci (m6A-QTLs) were enriched in CAD GWAS loci relevant to immune and lipid function, implicating m6A regulation in human CAD genetics. Using Oxford Nanopore direct RNA sequencing in conjunction with public MeRIP-sequencing data, we generated a single-base resolution, stoichiometric map of m6A modification in macrophages, nominating genes central to macrophage biology in atherosclerosis as high-confidence m6A targets. Myeloid specific deletion of the m6A "writer" complex subunit Mettl14 drove the development of larger, more necrotic and unstable atherosclerotic plaques on an Ldlr-/- background compared with Mettl14f/f controls, establishing an atheroprotective role for myeloid m6A. Loss of myeloid Mettl14 impaired macrophage efferocytosis in atherosclerotic plaques, in a dexamethasone-induced apoptosis in the thymus and in-vitro. Molecularly, m6A methylation of the "don't eat me" antiphagocytic receptor SIRP RNA suppressed its protein expression. Conclusions: These findings identify the myeloid m6A - SIRP axis as a novel homeostatic regulator of efferocytosis and establish epitranscriptomic remodeling as a crucial layer of macrophage regulation in atherosclerosis.

Pathological cardiac fibrosis and hypertrophy, key contributors to adverse remodeling that drives heart failure (HF), are not adequately targeted by current therapies. We identified tRNA-Asp-GTC-3'tDR, an ischemia-induced tRNA-derived small RNA (tDR), as an endogenous regulator of both remodeling processes across cardiac fibroblasts and cardiomyocytes. In murine models of myocardial ischemia- and pressure overload-induced HF, cardiac delivery of tRNA-Asp-GTC-3'tDR reduced fibrosis and hypertrophy, preserved cardiac function, and improved survival, with limited three doses conferring durable protection after ischemia, whereas silencing it worsened both. Strikingly, tRNA-Asp-GTC-3'tDR acts through distinct mechanisms in different cell types: in fibroblasts, it assembles stress granules that sequester ribosome-loaded profibrotic transcripts, whereas in cardiomyocytes it engages pseudouridine synthase PUS7 to activate RNA autophagy. Its regulation and functions were confirmed in human cardiac cells and tissues. These findings demonstrate that a single tDR coordinates cell-type-specific protective programs in the heart, suggesting a new therapeutic axis to mitigate HF progression.

BackgroundAdipose tissue surrounding the heart and vasculature plays critical roles in cardiovascular homeostasis and disease, yet the cellular and molecular milieu of these depots at single-cell resolution remains incompletely characterized. Understanding how regional adipocytes differ transcriptionally and communicate with neighboring cardiovascular cells is essential for developing targeted therapeutic strategies. MethodsWe performed single-nucleus RNA sequencing (snRNA-seq) on human adipose tissue from four anatomically distinct depots: ascending aorta, left atrium, right coronary artery, and subcutaneous fat. We characterized cellular composition, adipocyte and progenitor heterogeneity, depot-specific transcriptional programs, and intercellular communication networks. We further examined signaling remodeling in disease contexts, including atrial fibrillation and aortic aneurysm. ResultsWe identified six transcriptionally distinct adipocyte subpopulations and six adipocyte stromal and progenitor cell (ASPC) subpopulations were shared across depots but showed marked differences in abundance and gene expression reflecting developmental imprinting, including HOX family genes and anterior-posterior patterning programs. Intercellular communication analysis revealed depot-specific ligand-receptor interactions, with EPHA signaling identified as selectively enriched in the left atrial adipose depot. Disease-state analyses demonstrated extensive change in cell-cell communication in atrial fibrillation and aortic aneurysm, with differential regulation of FN1, EGF, SLIT, NOTCH, and CD46 signaling pathways. ConclusionsOur study reveals that cardiac and vascular adipose depots harbor transcriptionally specialized adipocytes and progenitors with distinct intercellular communication programs that are remodeled in atrial fibrillation and aortic aneurysm.

Endothelial senescence is increasingly recognized as a driver of vascular pathology, while immunoglobulin G (IgG) has recently been reported to accumulate in aging tissues and induce senescence in macrophages and microglia. In cerebral cavernous malformations (CCMs), IgG accumulation has been obviously observed in CCM lesions, but the contribution of IgG to endothelial injury remains unclear. Using multi-omic profiling, endothelial models, and CCM mice, we identified IgG-secreting plasma cells enriched in lesions associated with endothelial senescence, hemorrhage, and disease severity. CCM loss-associated mTOR activation impaired lysosomal acidification and IgG processing, promoting intracellular IgG accumulation. IgG, in turn, induced NF-kB-dependent endothelial senescence. In vivo, BCMA-mediated plasma cell depletion attenuated lesion progression, whereas IgG supplementation partially restored disease severity. Anti-CD38 treatment likewise reduced IgG accumulation, endothelial senescence, hemorrhage, and lesion progression. These findings identify lysosomal dysfunction-mediated IgG as a pathogenic trigger of endothelial senescence and support targeting the plasma cell-IgG axis in CCM.

Right ventricular (RV) trabeculation may reflect adaptation to loading conditions and imprinting of early developmental processes, yet its genetic determinants and clinical relevance in adults remain poorly understood. In contrast to the left ventricle, the RV has distinct developmental origins, geometry and loading conditions, suggesting chamber-specific mechanisms of trabecular remodelling. Using deep learning-based image segmentation and fractal dimension analysis, we quantified RV trabecular complexity in diastole and systole in 48,118 UK Biobank participants with genetic data. RV trabecular morphology was associated with systolic function, as well as cardiometabolic and respiratory conditions. Genetic analyses across the allele frequency spectrum identified 52 common loci and 45 genes with a burden of protein-altering variants, implicating sarcomeric function, cytoskeletal organisation, and early cardiac patterning. Although many loci showed shared effects across both ventricles, we also identified RV-specific genetic associations linked to congenital heart disease and respiratory phenotypes. Notably, associations at CFTR suggest a connection between airway-associated mucus regulation and RV remodelling, whereas MYH6 implicates sarcomeric and developmental mechanisms in adult RV patterning. Together, these findings establish RV trabeculation as a trait that captures both shared and chamber-specific mechanisms relevant to cardiovascular health and disease.

Pathological cardiac hypertrophy represents a common maladaptive response to cardiovascular stress and constitutes a major harbinger of heart failure. Although S-palmitoylation--a reversible post-translational modification--critically governs protein localization, trafficking, and stability, its involvement in cardiac hypertrophy remains poorly characterized. In this study, we aimed to explore the role and regulatory mechanism of a palmitoyltransferase, zinc finger DHHC-type palmitoyltransferase 14 (ZDHHC14), in cardiac hypertrophy. We found that ZDHHC14 was significantly upregulated in cardiac hypertrophy tissues from both human patients and mouse models. Cardiomyocyte-specific ZDHHC14 knockdown ameliorated transverse aortic constriction (TAC)-induced cardiac hypertrophy and dysfunction in male mice, whereas cardiac-specific ZDHHC14 overexpression via AAV9 exacerbated these pathological phenotypes. Mechanistically, TANK-binding kinase 1 (TBK1) was identified as a novel substrate of ZDHHC14 through interactomic screening. ZDHHC14 catalyzed TBK1 palmitoylation at cysteine 267, which in turn facilitated TBK1 phosphorylation and subsequent activation of type I interferon (IFN-I) signaling, ultimately promoting cardiac hypertrophy. Importantly, our findings demonstrate that the TBK1-C267S mutation rectifies ZDHHC14 overexpression-induced exacerbation of cardiac hypertrophy. This study illustrated a ZDHHC14-TBK1-IFN-I axis in regulating cardiac hypertrophy, which may provide a potential therapeutic target to ameliorate pathological cardiac hypertrophy.

Cardiac neural crest-derived glial cells (CNGs) are recently identified glial cells that support cardiac autonomic innervation and maintain sympathetic-parasympathetic balance. Their fate in myocardial infarction (MI) is unknown. Here, we analyzed 16 human cardiac spatial transcriptomes spanning normal myocardium (n=4), infarct zone (n=5), border zone (inner, n=1; outer, n=3), and remote zone (n=3). CNGs (S100B+/GFAP+, CD68- spots) were selectively lost along a spatial gradient from healthy tissue to the infarct core (Spearman rho = -0.92, p = 4.5 x 10-7). Tissue-area-corrected analysis confirmed that CNG loss (26% retained in infarct zone vs. normal; p = 0.016) vastly exceeded overall tissue loss, indicating selective vulnerability rather than bulk tissue destruction. Mechanistically, oxidative phosphorylation (OXPHOS) activity in CNG-positive spots correlated positively with CNG survival (partial rho = 0.65, p = 0.009 after controlling for spot composition), consistent with a high metabolic demand underlying their ischemic sensitivity. Pyroptosis - but not ferroptosis or apoptosis - signatures correlated with CNG loss (partial rho = -0.67, p = 0.006). These findings identify CNGs as selectively vulnerable cellular components of the cardiac autonomic system in MI and nominate metabolic failure and pyroptosis as candidate mechanisms, providing a spatially resolved foundation for future mechanistic and interventional studies.

TRPV4 ion channels in vascular smooth muscle cells (SMCs) are crucial regulators of blood pressure, and their functional effects are differentially shaped by their signaling partners. However, the mechanisms by which TRPV4 channels are compartmentalized into distinct signaling nanodomains with opposite impacts on blood pressure remain unclear. Here, we identify the scaffolding proteins that compartmentalize TRPV4 channels into discrete nanometer-scale signaling domains at the SMC plasma membrane and define how these nanodomains produce opposing effects on vasoconstriction and blood pressure. We show that AKAP5 anchors a nanodomain linking 1-adrenergic receptors, protein kinase C and TRPV4 channels, thereby driving sympathetic vasoconstriction and blood pressure elevation. In contrast, caveolin-1 promotes a mechanosensitive nanodomain comprising Piezo1, TRPV4, and BK channels that mediates vasodilation and a decrease in blood pressure. In hypertension, AKAP5-dependent constrictor nanodomains are hyperactive, whereas caveolin-1-based dilator nanodomains are hypoactive, shifting the balance toward pathological vasoconstriction. These findings reveal fundamental mechanisms that organize smooth muscle TRPV4 channels into spatially and functionally distinct nanodomains regulating blood pressure and show how disruption of this organization contributes to blood pressure elevation in hypertension.

Atherosclerosis is initiated by circulating low-density lipoprotein (LDL) cholesterol transfer into the artery wall, which is mediated by scavenger receptor class B, type I (SR-BI) in endothelial cells(1). Employing single-cell RNA sequencing in human coronary artery disease (CAD) samples, here we show that endothelial SR-BI expression is increased in atheroma, and in endothelial cells with a transcript signature indicative of responding to disturbed blood flow. In vivo in mice hypercholesterolemia and disturbed blood flow independently upregulate endothelial SR-BI; the flow-related upregulation initiates endothelial cell LDL uptake and atherogenesis. Guided by transcription factor networks, it is revealed that HIF-1 binding to human Scarb1 Intron 1 governs endothelial SR-BI transcription, and in mice HIF-1 drives hypercholesterolemia-related SR-BI upregulation and artery LDL uptake. Thus, the two major instigators of atherosclerotic lesion formation, hypercholesterolemia and disturbed blood flow, both upregulate endothelial SR-BI to drive the LDL transport that underlies the disorder. Targeting the processes regulating endothelial SR-BI potentially represents a new therapeutic strategy against CAD.

Myocardial fibrosis is a common end point of acute and chronic cardiovascular injury, yet the spatially organized fibroblast (Fb) and immune-cell states that sustain fibrotic remodeling across etiologies remain incompletely defined. We applied cyclic in situ hybridization (cISH)-based spatial transcriptomics to myocardial biopsies from patients with acute myocardial infarction (AMI), ischemic cardiomyopathy (ICM), hypertrophic cardiomyopathy (HCM), postviral myocardial fibrosis (PVMF), and systemic sclerosis associated primary heart involvement (SSc-pHI). CODEX-based protein imaging provided orthogonal support for cell subpopulations and cellular neighborhoods. Spatially informed clustering identified six Fb subpopulations: COL1A1hi/POSTNhi, NOTCH3+, TIMP1hi, GSNhi/DCNhi, AP1hi Fbs, and myofibroblasts. COL1A1hi/POSTNhi and NOTCH3+ Fbs were enriched in AMI, whereas GSNhi/DCNhi Fbs and myofibroblasts predominated in chronic fibrotic remodeling. These subpopulations displayed distinct extracellular matrix, inflammatory, and vasoregulatory programs. Infarction-associated reparative niches were enriched in COL1A1hi/POSTNhi or NOTCH3+ Fbs and SPP1+ or APOE+ macrophages (M{Phi}), whereas chronic fibroinflammatory niches were enriched in GSNhi/DCNhi or AP1hi Fbs or myofibroblasts with C1Q+ M{Phi} and plasma cells. The GSNhi/DCNhi Fb niche marked areas of subendocardial fibrosis, whereas the AP1hi Fb niche localized to perivascular fibrosis. In SSc-pHI, the actively fibrogenic COL1A1hi/POSTNhi Fb state characteristic of infarct scar formation was present at higher frequency than in other chronic entities and was individually associated with cardiac dysfunction, whereas the chronic GSNhi/DCNhi Fb niche was associated with the extent of interstitial fibrosis and anti-topoisomerase I autoantibodies. These data show that human myocardial fibrosis is organized into spatially distinct Fb subpopulations that assemble into context-specific fibroinflammatory niches across chronic fibrotic remodeling and repair after AMI.

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.

BackgroundTakotsubo syndrome (TTS) is an acute stress-induced cardiomyopathy characterized by transient left ventricular dysfunction. Despite its reversible nature, TTS is associated with substantial morbidity and mortality in the acute phase, and no specific treatments are available. The precise molecular mechanisms that connect catecholamine stress to reversible myocardial injury are not fully understood. PP2A, a holoenzyme with serine/threonine phosphatase activity, plays a vital role in normal cardiac development, and its dysregulation has been associated with heart disease. Its role in TTS and its potential as a therapeutic target remain completely unknown. MethodsAnalysis of public multi-omics datasets from stress cardiomyopathy (SCM) and experimental models of TTS, along with treatment of cardiomyocytes with human TTS plasma, was used to investigate a potential role for protein phosphatase 2A (PP2A) in stress-induced myocardial injury. To clarify the functional impact of manipulating PP2A activity in TTS, we used a series of disease relevant cell based and in vivo models, leveraging both genetic and pharmacological approaches to modulate PP2A activity in cardiomyocytes and in mice. To gain mechanistic insights into how PP2A influences TTS pathology and downstream signaling pathways, RNA sequencing, stress-responsive iron handling, mitochondrial function, and cardiac phenotypes were thoroughly evaluated in both in vivo and in vitro studies. ResultsPP2A activity was markedly reduced in cardiac tissues from mice with isoprenaline-induced TTS, as well as in isoprenaline-treated cardiomyocytes. Genetic or pharmacological inhibition of PP2A worsened catecholamine-induced cardiac dysfunction and myocardial injury. Most notably, pharmacological activation of PP2A using an orally bioavailable small-molecule activator strongly mitigated myocardial damage and enhanced cardiac function in TTS models. Mechanistically, PP2A inactivation promoted JNK-MAPK signaling and dysregulated stress-responsive iron-handling pathways, leading to ferritinophagy-mediated ferroptosis and mitochondrial dysfunction. Pharmacological JNK inhibition effectively rescued myocardial injury caused by PP2A deficiency in two TTS animal models and in cardiomyocytes. ConclusionsPP2A inactivation is a key molecular event linking catecholamine stress to myocardial injury in TTS. Restoring PP2A activity or inhibiting downstream JNK attenuates ferritinophagy-dependent stress responses and mitochondrial dysfunction, providing a unifying mechanistic framework and highlighting the PP2A-JNK axis as a potential target for short-term intervention during the acute phase of TTS. CLINICAL PERSPECTIVEO_ST_ABSWhat Is New?C_ST_ABS* Protein phosphatase 2A (PP2A) activity is acutely suppressed during the early phase of Takotsubo syndrome (TTS), with preferential involvement of the apical myocardium, closely mirroring the diseases characteristic clinical phenotype. * Downstream of PP2A inactivation, catecholamine stress triggers JNK-MAPK signaling activation and dysregulated stress-responsive iron handling, including ferritinophagy-mediated ferroptosis and mitochondrial dysfunction in cardiomyocytes. * Pharmacological restoration of PP2A activity or downstream inhibition of JNK mitigates acute myocardial injury and preserves cardiac function, thereby establishing the PP2A-JNK axis as a central stress-responsive pathway in TTS. What Are the Clinical Implications?* Identification of PP2A inactivation as a key molecular event highlights a novel opportunity for targeted intervention during the acute phase. * Studies in preclinical animal TTS models provide the first evidence that illuminates the therapeutic potential of a small-molecule PP2A activator as a strategy for acute TTS and may serve as a broad cardioprotective strategy in settings where cardiac dysfunction is implicated. * Targeting downstream JNK signaling, which is activated by PP2A inactivation, emerges as an additional mechanism-based strategy for attenuating acute myocardial injury in TTS.