Extracellular vesicles (EVs) are involved in exercise-induced cardiac protection. However, the effects and underlying mechanisms of tissue-specific molecular cargo packaged within these EVs, including PIWI (P-element induced wimpy testis)-interacting RNA (piRNA), remain poorly understood. In particular, the mechanistic contribution of exercised intracardiac EV-associated piRNAs to doxorubicin-induced cardiotoxicity (DCT) has not been defined. Transgenic reporter mice and a cardiomyocyte-specific Rab27a (RAB27A, member RAS oncogene family) knockout strategy were used to investigate the contribution of cardiomyocyte-derived EVs to exercise-induced cardioprotection against DCT. To screen functionally relevant cardioprotective piRNA cargo, heart tissues from patients with dilated cardiomyopathy and experimental DCT models were analyzed, including cardiomyocyte-specific knockout mice, human embryonic stem cell-derived cardiomyocytes, and primary murine cardiomyocytes. We found that cardiomyocyte-derived EVs post-exercise were enriched for a cardiac-specific protective piRNA (piR-mmu-57256903), designated as an exercise-induced protective piRNA (EPPIR). EPPIR levels were significantly reduced in heart tissue from patients with dilated cardiomyopathy and DCT models. Functionally, EPPIR protects the heart against DCT by regulating KDM6B (lysine [K]-specific demethylase 6B)-H3K27me3 (trimethylated histone H3 at lysine 27)-Dtna epigenetic axis. In addition, EPPIR acts as a cardiomyocyte-specific suppressor of Tp53 (tumor protein p53). We identify a previously unrecognized role for cardiomyocyte-derived EV-associated piRNA EPPIR in mediating exercise-induced cardioprotection. EPPIR exerts its protective effects through coordinated regulation of the KDM6B-Dtna axis and cardiomyocyte-specific suppressor of Tp53, providing mechanistic insight and highlighting a potential therapeutic strategy for DCT.

Micro(nano)plastics (MNPs) are pervasive environmental contaminants, yet their presence in human cardiac tissue and their potential contribution to myocardial fibrosis remain unclear. We investigated whether myocardial MNP burden is associated with fibrosis severity in patients and evaluated mechanistic plausibility in mice. Left atrial appendage tissues were collected from patients undergoing cardiac surgery (n=33). MNP burden and polymer composition were quantified by pyrolysis-gas chromatography/mass spectrometry, and fibrosis was quantified histologically. In mice, 100-nm or 1-µm polystyrene nanoplastics were administered by oral gavage in coexposure and sequential exposure protocols with isoprenaline. Cardiac function was assessed by echocardiography, and fibrosis was evaluated by histology and immunohistochemistry. Transcriptomics, metabolomics, and 16S ribosomal RNA sequencing were performed to identify pathways linked to MNP exposure. MNPs were detected in all human cardiac samples. Patients with high fibrosis exhibited higher total MNP levels than those with low fibrosis (171.74 [95% CI, 158.18-202.39] versus 119.33 [95% CI, 102.75-148.44] µg/g tissue; P=2.5×10-4), driven predominantly by elevated nanoplastics (122.83 [95% CI, 100.10-149.06] versus 86.39 [95% CI, 36.85-103.74] µg/g; P=0.010). Polystyrene and polyvinyl chloride were enriched in high-fibrosis tissues (polystyrene: P=3.3×10-4; polyvinyl chloride: P=0.002). Transcriptomics indicated activation of inflammatory and profibrotic pathways (TNF/NF-κB [nuclear factor-κB], TGF-β [transforming growth factor-beta], and MAPK), supported by increased α-SMA (alpha-smooth muscle actin), COL1 (collagen I), and TGF-β1 immunostaining, while metabolomics suggested perturbations in lipid metabolism and mitochondrial function. In mice, polystyrene exposure exacerbated isoprenaline-induced systolic dysfunction and myocardial fibrosis in both experimental paradigms and recapitulated pathway signatures related to cell-matrix interactions. Myocardial MNP burden, particularly nanoplastics, is associated with greater fibrosis in humans, and experimental polystyrene exposure aggravates stress-induced myocardial remodeling in vivo. Multiomics analyses nominate inflammatory, ECM (extracellular matrix), and metabolic programs as candidate mediators of MNP-associated cardiotoxicity.

NaV (voltage-gated sodium) channels drive cardiac excitability. Although NaV1.5 is the primary cardiac isoform, the composition and functional contributions of non-NaV1.5 isoforms in the heart remain unclear. Here, we developed a chemical-genetic mouse model (NaV1.5GX/GX) in which NaV1.5 can be selectively and reversibly inhibited by acyl- and aryl-sulfonamide compounds (GX [acyl- and aryl-sulfonamide compounds typically denoted by the name GX-### and associated items] drugs). Cardiac activity was assessed by electrocardiograms in vivo, and optical mapping was used for imaging of ex vivo hearts. Whole-cell voltage-clamp in tandem with validated toxins and isoform-selective inhibitors were used to examine sodium current composition. NaV1.5GX/GX mice exhibited normal cardiac function at baseline, but acute GX drug administration caused profound conduction defects and arrhythmias. Whole-heart optical mapping revealed dose-dependent chamber-specific sensitivity to NaV1.5 inhibition, with the right ventricle being the most sensitive, followed by the left ventricle, left atrium, and right atrium. Patch-clamp recordings of isolated cardiomyocytes with application of NaV isoform-selective inhibitors showed that NaV1.5 contributed 93% of sodium current in the left ventricle, 79% in the right ventricle, and 78% in the atria. Non-NaV1.5 isoforms were differentially enriched across chambers: NaV1.8 in the left ventricle, NaV1.1/1.3 in the right ventricle, and NaV1.2/1.6/1.7 in the atria. These results reveal a surprising chamber-specific isoform landscape of cardiac sodium currents, which may underlie the right ventricular predominant phenotype of Brugada syndrome. These data highlight non-NaV1.5 isoforms as potential mediators of chamber-specific cardiac pathologies and as pharmacological targets.

Cardiomyocyte mitochondria align with sarcomeres during heart development. Mitochondrial motility is controlled by RHOT (ras homolog family member T) 1 and RHOT2. RHOT1 and RHOT2 are atypical Rho-like small GTPases that are anchored to the outer mitochondrial membrane and couple mitochondria to kinesin and dynein motors. We hypothesized that RHOT protein expression and mitochondrial motility are required for mitochondrial positioning during cardiomyocyte development. We generated mice with cardiomyocyte-selective deletion of Rhot1 and Rhot2 during embryogenesis (cRhot1/2-KO [constitutive and embryonic cardiomyocyte-selective Rhot1/2 knockout]) or tamoxifen-inducible deletion in the adult heart (iRhot1/2-KO [inducible cardiomyocyte-selective Rhot1/2 knockout]) to assess the importance of mitochondrial motility during and after cardiomyocyte maturation. Mitochondrial motility was determined by a motor protein-driven single mitochondria motility assay. Respiratory capacity was measured in isolated mitochondria. Intracellular mitochondrial localization and ATP production in isolated cardiomyocytes were assessed by confocal microscopy and after adenoviral expression of the fluorescence resonance energy transfer-based ATP biosensor ATeam. Cardiac ultrastructure was assessed by electron micrographs; mass spectrometry was used for proteome analysis. cRhot1/2-KO mice developed fatal cardiomyopathy associated with sarcomere disarray and perinuclear accumulation of mitochondria and ATP production. Mitochondria isolated from cRhot1/2-KO hearts exhibited impaired motility but preserved respiratory capacity. Mechanistically, proteome analysis identified that RHOT proteins bind mitochondria to contractile muscle fiber proteins. In contrast, inducible deletion of Rhot1 and Rhot2 in adult iRhot1/2-KO mice did not result in heart failure. Despite impaired motility of isolated mitochondria, intracellular mitochondrial localization, local ATP production, and sarcomere structure were preserved in adult iRhot1/2-KO hearts after cardiomyocyte maturation. RHOT proteins bind mitochondria to contractile muscle fiber proteins and are required for mitochondrial positioning in cardiomyocytes during development. Our study links mitochondrial motility and local ATP production to structural and functional maturation of the heart.

Cardiometabolic diseases (CMDs) refer to a broad spectrum of interconnected disorders, including heart attack, obesity, diabetes, atherosclerosis, and metabolic dysfunction-associated steatohepatitis, which represent the leading cause of mortality worldwide. In recent years, research on the role of gut microbiota in the pathogenesis of CMD has gradually shifted from correlation-based observations to mechanistic explorations. Within this context, microbial enzymes have gained increasing attention as key regulatory factors. These enzymes not only participate in the metabolic regulation of microorganisms themselves but also directly mediate host-microbe interactions, influencing the onset and progression of CMD. Specifically, microbial enzymes play a central role in CMD by modulating the homeostasis of key host metabolites such as cholesterol, generating bioactive molecules with metabolic and immunoregulatory functions, and participating in drug responses and the metabolic transformation of other xenobiotics. These enzymes provide novel and well-defined molecular targets for developing precision intervention strategies targeting the gut microbiota-such as enzyme replacement therapy, the design of enzyme agonists or inhibitors, and in vivo gene editing-thereby holding promise for advancing CMD prevention and treatment strategies toward greater specificity and controllability. This review systematically summarizes key microbial enzymes involved in the metabolism of endobiotics, including amino acids, peptides, and purines, and xenobiotics such as drugs, elucidating their specific mechanisms and functions in the development of CMD, strategies for mining these microbial enzymes, and the challenges and future of microbial enzyme-based interventions.

Pathogenic cardiac hypertrophy, often driven by mechanical stress, is a leading cause of heart failure. However, effective therapeutic targets remain limited. TMC6 (transmembrane channel-like protein 6) is abundant in healthy myocardium but downregulated in hypertrophic hearts; its role in cardiac hypertrophy remains undefined. We combined cardiac-specific Tmc6 knockout mice subjected to transverse aortic constriction surgery, neonatal rat ventricular myocytes, and CRISPR/Cas9-edited human pluripotent stem cell-derived cardiomyocytes to assess hypertrophy and signaling readouts. Subcellular localization, protein-protein interaction, and competitive peptide assays were used to dissect the mechanism. Adeno-associated virus serotype 9 (AAV9)-cTnT (cardiac troponin T)-TMC6 was used for in vivo rescue. TMC6 deficiency increased cardiomyocyte size, fetal gene expression, and adverse remodeling in vivo and in vitro, whereas TMC6 overexpression blunted hypertrophic responses. Full-length TMC6 localized to the endoplasmic reticulum and bound CIB1 (calcium and integrin-binding protein 1) to sequester it in the endoplasmic reticulum, limiting CIB1 access to sarcolemmal Ca2+ microdomains required to scaffold calcineurin and activate NFAT (nuclear factor of activated T cells). A cell-permeable TMC6161-180 peptide competitively displaced CIB1 from TMC6 and augmented hypertrophy in wild-type but not Tmc6 knockout cardiomyocytes, indicating a dominant-negative mechanism. Therapeutically, AAV9-cTnT-TMC6 restored TMC6-CIB1 engagement, suppressed calcineurin/NFAT readouts, and improved function after pressure overload. TMC6 is an endogenous brake on pathological hypertrophy that restrains CIB1-calcineurin/NFAT signaling via endoplasmic reticulum sequestration of CIB1. Restoring full-length TMC6 mitigates pressure-overload remodeling, nominating the TMC6-CIB1 axis as a therapeutic target.

Cardiovascular diseases remain the leading global cause of morbidity and mortality, placing an escalating burden on health care systems and economies. While the gut microbiota is well recognized in atherosclerosis and cardiometabolic disorders, its influence on myocardial injury, repair, and regeneration is only beginning to emerge. Growing evidence reveals that gut microbes and their metabolites regulate myocardial health through intricate cross-organ networks, including the gut-brain-heart, gut-liver-heart, and gut-lung-heart axes. These findings suggest that the heart plays a key role in systemic host-microbe communication. Advances in metagenomics, metabolomics, and single-cell transcriptomics are now defining the molecular and cellular pathways by which microbial metabolites modulate immune tone, endothelial integrity, metabolic resilience, and cardiomyocyte survival. Studies in gnotobiotic models have established causal links between specific microbial taxa and myocardial outcomes while illuminating their roles in fibrosis resolution, angiogenesis, and regeneration. In this review, we synthesize current knowledge on the bidirectional gut-heart dialogue, emphasizing immunometabolic signaling, cross-organ integration, and regenerative mechanisms. We propose that coupling high-resolution multiomics with mechanistic modeling in controlled microbial systems will be pivotal for next-generation, microbiota-informed diagnostics, and therapeutics. We explore the emerging role of the gut-myocardium axis as both a driver of disease and as a promising modifiable therapeutic target and highlight a new frontier in precision cardiovascular medicine, with the potential to transform strategies for prevention, repair, and tissue regeneration.

BACKGROUND:Sex and atherosclerotic plaque histology are intertwined, with fibrous plaques being more prevalent in women. Plaque erosion, a significant contributor to acute coronary syndromes, is linked to fibrous plaques and is more prevalent in women than men. We hypothesize that the molecular drivers of histologically determined fibrous plaques differ between men and women.METHODS:Human end-stage atherosclerotic plaques were isolated from carotid endarterectomy patients included in the Athero-Express Biobank. Fibrous plaques were histologically assessed, linked to clinical characteristics, and processed for protein, bulk RNA, single-cell RNA, and DNA methylation data. We leveraged sex-differential gene expression and deconvolution analyses to uncover sex-biased molecular and cellular mechanisms. Spatial transcriptomics localized gene expression patterns in plaques. Furthermore, we studied the female-biased processes in human plaque endothelial cells and vascular smooth muscle cells stimulated with TGF-β (transforming growth factor-β), with or without SMAD3 (SMAD family member 3) inhibition.RESULTS:Of 1889 atherosclerotic plaques (1309 male and 580 female), fibrous lesions were observed in 50% of female and 31% of male patients. Compared with patients with atheromatous plaques (n=494), women with fibrous plaques exhibited a high prevalence of smoking, while men with fibrous plaques presented more often with diabetes. Female fibrous plaques were characterized by smooth muscle cell–driven ECM (extracellular matrix) remodeling, TGF-β response, and endothelial-to-mesenchymal transition, localized to the fibrous cap. Conversely, male plaques were linked to macrophage-mediated inflammation proximal to the core, dependent on diabetes. Finally, we experimentally confirmed these female-biased mechanisms, showing that TGF-β induced endothelial-to-mesenchymal transition in endothelial cells and ECM remodeling in vascular smooth muscle cells, both partly reversed by SMAD3 inhibition.CONCLUSIONS:Women and men with end-stage fibrous atherosclerotic plaques exhibit distinct clinical and molecular profiles. These mechanisms might be candidate pathways to understand plaque erosion from a molecular point of view and may provide promising targets for atherosclerosis therapies, as they account for both sex and plaque phenotype.

Background:Cardiovascular disease remains the leading cause of death worldwide, with coronary artery disease being the primary contributor. Our recent studies suggest that activation of leptin receptors (LepRs) in the brain can improve cardiac function following myocardial infarction. However, the mechanism by which this cardioprotective effect is transmitted from the brain to the heart remains unclear. We hypothesize that brain LepR activation stimulates brown adipose tissue (BAT) to secrete extracellular vesicles (EVs) enriched with cardioprotective factors. These EVs may safeguard the heart by modulating cardiac mitochondrial function and collagen deposition.Methods:Sprague-Dawley rats with BAT intact, BAT ablation, or BAT sympathetic denervation were implanted with an intracerebroventricular (ICV) cannula for continuous leptin or vehicle delivery over 28 days after cardiac ischemia-reperfusion (IR) injury. Cardiac function was assessed weekly via echocardiography and by ventricular catheterization at the end of the protocol. EVs were isolated from BAT for analysis. Rab27a, a protein required for EV release, was knocked down using adeno-associated virus, and EV tracking was conducted using a double fluorescent reporter mouse model.Results:Our findings indicate that BAT ablation or BAT sympathetic denervation diminishes the cardioprotective effects of brain LepR activation. We also observed an increased concentration of EVs within BAT of rats treated with ICV leptin compared to vehicle-treated controls, an effect abolished by BAT denervation. Furthermore, knockdown of Rab27a in BAT reduced the cardioprotective benefits of brain LepR activation. MicroRNA (miR)-29c-3p was identified as a cargo of leptin-stimulated BAT-derived EVs and appears to play a key role in mitigating cardiac fibrosis after IR injury in leptin-treated animals.Conclusion:Activation of LepR in the brain protects the heart after IR injury via sympathetic mediated BAT-derived EVs enriched with miR-29c-3p.