Biology subjects

Delmar, M.

Publications and source records attributed to Delmar, M..

5 recordsLinked to original sources

bioRxiv2025

Electrophysiological abnormalities associated with a CACNA1D variant are rescued by AAV6-Cav1.3-C-terminus gene therapy in patient-iPSC-CMs

Inherited arrhythmia syndromes are caused by genetic variants that alter cardiac ion channel function. We investigated a complex presentation in a pediatric patient with ventricular tachycardia and conduction abnormalities, harboring a de novo CACNA1D (c.3786G>T) variant, and two inherited variants, the SCN5A (c.2618C>G), and a DSP desmosome (c.1582C>G). The CACNA1D variant, which encodes Cav1.3 L-type calcium channel is the focus of this study, because the C-terminus fragment of Cav1.3 has recently been identified as a transcription auto-enhancer of its own gene and able to prevent arrhythmic events in a mouse model of ischemic heart failure. Leveraging this intrinsic property, we hypothesized that the Cav1.3-C-terminus could reverse the arrhythmic events associated with the CACNA1D variant. Patch-clamp and optical mapping experiments demonstrated a loss of Cav1.3 function, characterized by reduced L-type calcium current densities, and decrease of conduction velocity, leading to inducible re-entrant arrhythmias in human induced pluripotent stem cell-cardiomyocytes (hiPSC-CMs). RNA sequencing confirmed this loss-of-function via the downregulation CACNA1D gene expression. Interestingly, Cav1.3-C-terminus treatment of hiPSC-CMs successfully normalized Cav1.3 gene expression, restored calcium currents, and conduction velocity, and prevented the susceptibility to arrhythmias. These findings highlight the electrophysiological consequences resulting from a de novo Cav1.3 variant and demonstrate an important transcriptional role of Cav1.3-C-terminus as a transcriptional regulator and as a promising therapeutic tool to restore normal electrical properties in patients with calcium channels loss of function.

physiology↗

bioRxiv2025

Cardiomyocyte-specific plakophilin-2 loss is sufficient to induce aging and senescence of nonmyocytes. Relevance to arrhythmogenic cardiomyopathy.

IntroductionPathogenic variants in PKP2 are the most common cause of familial arrhythmogenic right ventricular cardiomyopathy (ARVC). ObjectiveTo test whether PKP2 deficiency only in cardiomyocytes is sufficient to provoke premature aging and pro-inflammatory senescence in non-myocytes, cardiac resident cells. MethodsWe studied mice with cardiomyocyte-specific, tamoxifen-activated loss of PKP2 (PKP2cKO) using conventional and multiplex imaging, cytokine arrays, epigenetic clocks, spatial transcriptomics, expansion and structured illumination microscopy, and correlative data analysis. We examined non-myocytes and cardiomyocytes for premature aging and senescence. ResultsWe observed senescence-associated heterochromatin foci (SAHFs) and p21 staining in non-myocytes. Cytokines in media of non-myocyte cells were consistent with senescence-associated secretory phenotype (SASP). Epigenetic clocks identified premature aging. Multiplex immunohistochemistry showed non-myocyte cells in niches, intermingled with cardiomyocytes. Spatial transcriptomics showed over-representation of SASP-related transcripts, predominantly in myocyte-rich areas of the left ventricle. SAHFs, p21 staining and increased epigenetic age were not found in cardiomyocytes from PKP2cKO hearts, though we observed structural features associated to premature aging. Cross-reference analysis showed correlation between the PKP2cKO cardiac proteome and that of mice 5 or 6 times their chronological age, as well as transcriptional signatures of neurodegenerative diseases. ConclusionLoss of PKP2 expression only in adult cardiac myocytes is sufficient to induce pro-inflammatory senescence in non-myocytes, and overall premature cardiac aging. This is the first study to intersect cellular senescence and premature aging in desmosomal arrhythmogenic cardiomyopathies. We speculate that cell-agnostic molecular signatures, biomarkers, and pharmacology of senescence and of neurodegenerative diseases may be relevant to diagnose or treat PKP2-ARVC. UNSTRUCTURED ABSTRACTPathogenic variants in PKP2 are the most common cause of familial arrhythmogenic right ventricular cardiomyopathy. We used the PKP2cKO model (cardiomyocyte-specific, tamoxifen-activated PKP2 knockout) to examine whether PKP2 deficiency only in cardiomyocytes is sufficient to provoke premature aging and pro-inflammatory senescence in non-myocyte, cardiac resident cells. Through a variety of methods, we identified senescence-associated heterochromatin foci (SAHFs), p21 staining, and cytokines consistent with senescence-associated secretory phenotype (SASP) in cells and media of non-myocytes. Epigenetic clocks identified premature aging. Spatial transcriptomics showed over-representation of SASP-related transcripts, predominantly in myocyte-rich areas of the left ventricle. SAHFs, p21 staining and epigenetics suggesting advanced age were not found in cardiomyocytes, though we observed structural features associated to premature aging. Cross-reference analysis showed correlation between the PKP2cKO cardiac proteome and that of mice 5 or 6 times their chronological age, as well as transcriptional signatures of neurodegenerative diseases. HIGHLIGHTSO_LIPKP2-ARVC is a leading cause of sudden unexpected death in the young. C_LIO_LIThe molecular path from the variant of a gene to the clinical disease remains unclear. An inflammatory component has been postulated. C_LIO_LIWe show that loss of PKP2 only in myocytes is sufficient to induce a pro-inflammatory senescence in non-myocytes and premature aging of cardiac cells. C_LIO_LISimilarities to the molecular profile of neurodegenerative diseases and novel paths to therapy are discussed. C_LI

physiology↗

bioRxiv2025

Epicardial contributions to fibro-inflammatory signaling in a Pkp2-deficient arrhythmogenic cardiomyopathy model

BackgroundArrhythmogenic Cardiomyopathy (ACM) is an inherited disease that is characterized by lethal ventricular arrhythmias stemming from myocyte dysfunction. ACM is associated with considerable subepicardial fibrosis and inflammation with right ventricle predominance. Most cases of gene positive ACM are caused by a desmosome protein mutation, with plakophilin-2 (Pkp2) mutations being most common. We hypothesized Pkp2-deficiency in epicardium-derived cells (EPDCs) contributes to fibro-inflammatory signaling and ACM pathogenesis. MethodsWe developed transgenic mice that lack Pkp2 in cardiomyocytes (Pkp2-cKO) or in both cardiomyocyte and EPDC (Pkp2-ceKO) via the tissue-specific expression of tamoxifen-inducible Cre recombinase. Non-myocyte populations were isolated 21 days post-tamoxifen injection for single cell RNA-sequencing (scRNA-seq). Immunohistochemistry, flow cytometry, qRT-PCR, and echocardiography were used to interrogate cardiac physiology and cellular composition. ResultsWe identified a population of epicardium-derived fibroblasts characterized by the expression of Ccl2, Ccl7, Thbs1, and Ptx3 that accumulated upon Pkp2 deletion. While pro-inflammatory EPDCs are found in Pkp2-cKO mice, they become significantly enriched in Pkp2-ceKO mice. Pro-inflammatory fibroblasts acquired the senescence-associated secretory phenotype (SASP), correlating with elevated Senescence Associated (SA)-{beta}gal staining in the right ventricle. Gene expression, flow cytometry, and histological data also revealed an exaggerated inflammatory response in Pkp2-ceKO mice, that progresses from right to bi-ventricular predominance. Importantly, macrophages and B cells accumulate in both Pkp2-cKO and Pkp2-ceKO mice compared to controls. Antibody-mediated B cell depletion delays the early inflammatory and fibrosis response but did not significantly alter end-stage cardiac physiology. ConclusionPkp2 deletion in EPDC facilitates the emergence of a fibro-inflammatory phenotype that may contribute to ACM pathogenesis.

cell biology↗

bioRxiv2023

AAV-mediated Delivery of Plakophilin-2a Arrests Progression of Arrhythmogenic Right Ventricular Cardiomyopathy in Murine Hearts: Preclinical Evidence Supporting Gene Therapy in Humans

BackgroundPathogenic variants in plakophilin-2 (PKP2) cause arrhythmogenic right ventricular cardiomyopathy (ARVC), a disease characterized by life-threatening arrhythmias and progressive cardiomyopathy leading to heart failure. No effective medical therapy is available to prevent and/or arrest the disease. We tested the hypothesis that AAV-mediated delivery of the human PKP2 gene to an adult mammalian heart deficient in PKP2 can arrest disease progression and significantly prolong survival. MethodsExperiments were carried out using a cardiac-specific, tamoxifen (TAM)-activated PKP2 knockout murine model (PKP2-cKO). The potential therapeutic, AAVrh.74-PKP2a (RP-A601), is a recombinant AAVrh.74 gene therapy viral vector encoding the human PKP2 variant A (PKP2a). AAVrh.74-PKP2a was delivered to adult mice by a single tail vein injection either before or after TAM-activated PKP2-cKO. PKP2 expression was confirmed by molecular and histopathologic analyses. Cardiac function and disease progression were monitored by survival analyses, echocardiography and electrocardiography. ResultsConsistent with prior findings, loss of PKP2 expression caused 100% mortality within 50 days after TAM injection. In contrast, AAVrh.74-PKP2a-mediated PKP2a expression resulted in 100% survival for more than 5 months (at study termination). Echocardiographic analysis revealed that AAVrh.74-PKP2a prevented right ventricle dilation, arrested left ventricle functional decline, and mitigated arrhythmia burden. Molecular and histological analysis showed AAVrh.74-PKP2a- mediated transgene mRNA and protein expression and appropriate PKP2 localization at the cardiomyocyte intercalated disc. Importantly, therapeutic benefit was shown in mice receiving AAVrh.74-PKP2a after disease onset. ConclusionThese preclinical data demonstrate the potential for AAVrh.74-PKP2a (RP-A601) as a therapeutic for PKP2-related ARVC in both early and more advanced stages of disease.

physiology↗

bioRxiv2019

Single-molecule localization of Nav1.5 reveals different modes of reorganization at the lateral membrane and T-tubules of cardiomyocytes

Mutations in the gene encoding the sodium channel Nav1.5 cause various cardiac arrhythmias. This variety may arise from different determinants of Nav1.5 expression between cardiomyocyte domains. At the lateral membrane and T-tubules, Nav1.5 localization and function remain insufficiently characterized. We used novel single-molecule localization microscopy (SMLM) and modeling to define nanoscale features of Nav1.5 localization and distribution at the lateral membrane, groove, and T-tubules in wild-type, dystrophin-deficient (mdx) mice, and mice expressing C-terminally truncated Nav1.5 ({Delta}SIV). We show that Nav1.5 organizes as distinct clusters in the groove and T-tubules which density and distribution partially depend on SIV and dystrophin. We found that overall reduction in Nav1.5 expression in mdx and {Delta}SIV cells results in a non-uniform redistribution with Nav1.5 being specifically reduced at the groove of {Delta}SIV and increased in T-tubules of mdx cardiomyocytes. Nav1.5 mutations may therefore site-specifically affect Nav1.5 localization and distribution depending on site-specific interacting proteins.

physiology↗