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176


Single-molecule Localization of Nav1.5 Reveals Different Modes of Reorganization at Cardiomyocyte Membrane Domains

Vermij, Sarah H; Rougier, Jean-Sébastien; Agulló-Pascual, Esperanza; Rothenberg, Eli; Delmar, Mario; Abriel, Hugues
Background - 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. Methods - We used novel single-molecule localization microscopy (SMLM) and computational modeling to define nanoscale features of Nav1.5 localization and distribution at the lateral membrane (LM), the LM groove, and T-tubules (TT) in cardiomyocytes from wild-type (N = 3), dystrophin-deficient (mdx; N = 3) mice, and mice expressing C-terminally truncated Nav1.5 (ΔSIV; N = 3). We moreover assessed TT sodium current by recording whole-cell sodium currents in control (N = 5) and detubulated (N = 5) wild-type cardiomyocytes. Results - We show that Nav1.5 organizes as distinct clusters in the groove and T-tubules which density, distribution, and organization partially depend on SIV and dystrophin. We found that overall reduction in Nav1.5 expression in mdx and ΔSIV cells results in a non-uniform re-distribution with Nav1.5 being specifically reduced at the groove of ΔSIV and increased in T-tubules of mdx cardiomyocytes. A TT sodium current could however not be demonstrated. Conclusions - Nav1.5 mutations may site-specifically affect Nav1.5 localization and distribution at the lateral membrane and T-tubules, depending on site-specific interacting proteins. Future research efforts should elucidate the functional consequences of this redistribution.
PMID: 32536203
ISSN: 1941-3084
CID: 4484432

Desmosome-Dyad Crosstalk: An Arrhythmogenic Axis in Arrhythmogenic Right Ventricular Cardiomyopathy [Editorial]

Delmar, Mario; Alvarado, Francisco J; Valdivia, Héctor H
PMID: 32364772
ISSN: 1524-4539
CID: 4429862

Ankyrin-G mediates targeting of both Na+ and KATP channels to the rat cardiac intercalated disc

Yang, Hua-Qian; Pérez-Hernández, Marta; Sanchez-Alonso, Jose; Shevchuk, Andriy; Gorelik, Julia; Rothenberg, Eli; Delmar, Mario; Coetzee, William A
We investigated targeting mechanisms of Na+ and KATP channels to the intercalated disk (ICD) of cardiomyocytes. Patch clamp and surface biotinylation data show reciprocal downregulation of each other's surface density. Mutagenesis of the Kir6.2 ankyrin binding site disrupts this functional coupling. Duplex patch clamping and Angle SICM recordings show that INa and IKATP functionally co-localize at the rat ICD, but not at the lateral membrane. Quantitative STORM imaging show that Na+ and KATP channels are localized close to each other and to AnkG, but not to AnkB, at the ICD. Peptides corresponding to Nav1.5 and Kir6.2 ankyrin binding sites dysregulate targeting of both Na+ and KATP channels to the ICD, but not to lateral membranes. Finally, a clinically relevant gene variant that disrupts KATP channel trafficking also regulates Na+ channel surface expression. The functional coupling between these two channels need to be considered when assessing clinical variants and therapeutics.
PMID: 31934859
ISSN: 2050-084x
CID: 4263232

Transcriptomic Coupling of PKP2 With Inflammatory and Immune Pathways Endogenous to Adult Cardiac Myocytes

Pérez-Hernández, Marta; Marrón-Liñares, Grecia M; Schlamp, Florencia; Heguy, Adriana; van Opbergen, Chantal J M; Mezzano, Valeria; Zhang, Mingliang; Liang, Feng-Xia; Cerrone, Marina; Delmar, Mario
Plakophilin-2 (PKP2) is classically defined as a component of the desmosome. Besides its role in cell-cell adhesion, PKP2 can modulate transcription through intracellular signals initiated at the site of cell-cell contact. Mutations in PKP2 associate with arrhythmogenic right ventricular cardiomyopathy (ARVC). Recent data demonstrate that inflammation plays a key role in disease progression; other results show an abundance of anti-heart antibodies in patients with confirmed diagnosis of ARVC. Here, we test the hypothesis that, in adult cardiac myocytes, PKP2 transcript abundance is endogenously linked to the abundance of transcripts participating in the inflammatory/immune response. Cardiac-specific, tamoxifen (TAM)-activated PKP2-knockout mice (PKP2cKO) were crossed with a RiboTag line to allow characterization of the ribosome-resident transcriptome of cardiomyocytes after PKP2 knockdown. Data were combined with informatics analysis of human cardiac transcriptome using GTEx. Separately, the presence of non-myocyte cells at the time of analysis was assessed by imaging methods. We identified a large number of transcripts upregulated consequent to PKP2 deficiency in myocytes, inversely correlated with PKP2 abundance in human transcriptomes, and part of functional pathways associated with inflammatory/immune responses. Our data support the concept that PKP2 is transcriptionally linked, in cardiac myocytes, to genes coding for host-response molecules even in the absence of exogenous triggers. Targeted anti-inflammatory therapy may be effective in ARVC.
PMCID:7849609
PMID: 33536940
ISSN: 1664-042x
CID: 4776512

Reevaluation of Genetic Variants Previously Associated with Arrhythmogenic Right Ventricular Cardiomyopathy Integrating Population-based Cohorts and Proteomics Data

Ye, Johan Ziruo; Delmar, Mario; Lundby, Alicia; Olesen, Morten S
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is one of the most common causes of sudden cardiac death in young people. Patients diagnosed with ARVC may experience increased likelihood of development of anxiety and depression, emphasizing the need for accurate diagnosis. To assist future genetic diagnosis and avoidance of misdiagnosis, we evaluated the reported monogenic disease-causing variants in ARVD/C Genetic Variants Database, Human Gene Mutation Database, and ClinVar. Within the aforementioned databases, 630 monogenic disease-causing variants from 18 genes were identified. In the genome Aggregation Database, 226 of these were identified; 68 of which were found at greater than expected prevalence. Furthermore, 37/226 genetic variants were identified amongst the 409 000 UK biobank participants, 23 were not associated with ARVC. Among the 14 remaining variants, 13 were previously found with greater than expected prevalence for a monogenic variant. Nevertheless, they were associated with serious cardiac phenotypes, suggesting that these 13 variants may be disease-modifiers of ARVC, rather than monogenic disease-causing. In summary, more than 10% of variants previously reported to cause ARVC were found unlikely to be associated with highly penetrant monogenic forms of ARVC. Notably, all variants in OBSCN and MYBPC3 were found, making these unlikely to be monogenic causes of ARVC. This article is protected by copyright. All rights reserved.
PMID: 31402444
ISSN: 1399-0004
CID: 4041772

Non-transcriptional disruption of Ca2+i homeostasis and Cx43 function in the right ventricle precedes overt arrhythmogenic cardiomyopathy in PKP2-deficient mice [Meeting Abstract]

Kim, J C; Perez-Hernandez, M; Alvarado, F J; Maurya, S R; Montnach, J; Yin, Y; Zhang, M; Lin, X; Heguy, A; Rothenberg, E; Lundby, A; Valdivia, H H; Cerrone, M; Delmar, M
Background: Plakophilin-2 (PKP2) is classically defined as a protein of the desmosome, an intercellular adhesion structure that also acts as a signaling hub to maintain structural and electrical homeostasis. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy (ARVC). A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events that occur consequent to its mutation. Here we sought to captureearly molecular/cellular events that can act as nascent substrates for subsequent arrhythmic/cardiomyopathic phenotypes.
Method(s): We used multiple quantitative imaging modalities, as well as biochemical and high-resolution mass spectrometry methods to study the functional/structural properties of cells/tissues derived from cardiomyocytespecific, tamoxifen-activated, PKP2 knockout mice ("PKP2cKO"). Studies were carried out 14 days post-tamoxifen injection, a time point preceding an overt electrical or structural phenotype.Myocytes from right or left ventricular free wall were studied separately, to detect functional/structural asymmetries.
Result(s): Most properties of PKP2cKO left ventricular (LV) myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca2+transients, increased frequency of spontaneous Ca2+release events, increased [Ca2+] in the cytoplasm and sarcoplasmic reticulum compartments, and dynamic Ca2+accumulation in mitochondria. In addition, RyR2 in RV presented enhanced sensitivity to Ca2+and preferential phosphorylation in a domain known to modulate Ca2+gating. RNAseq at 14 days post-TAM showed no relevant difference in transcript abundance between RV and LV, neither in control nor in PKP2cKO cells, suggesting that in the earliest stage, [Ca2+]i dysfunction is not transcriptional. Rather, we found an RV-predominant increase in membrane permeability that can permit Ca2+entry into the cell. Cx43 ablation mitigated the increase in membrane permeability, the accumulation of cytoplasmic Ca2+and the early stages of RV dysfunction.
Conclusion(s): Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca2+ dysregulation) that precedes the cardiomyopathy and that is, at least in part, mediated by a Cx43-dependent membrane conduit. Given that asymmetric Ca2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage
EMBASE:630046385
ISSN: 0195-668x
CID: 4245532

Disruption of Ca2+i Homeostasis and Cx43 Hemichannel Function in the Right Ventricle Precedes Overt Arrhythmogenic Cardiomyopathy in PKP2-Deficient Mice

Kim, Joon-Chul; Pérez-Hernández Duran, Marta; Alvarado, Francisco J; Maurya, Svetlana R; Montnach, Jerome; Yin, Yandong; Zhang, Mingliang; Lin, Xianming; Vasquez, Carolina; Heguy, Adriana; Liang, Feng-Xia; Woo, Sun-Hee; Morley, Gregory E; Rothenberg, Eli; Lundby, Alicia; Valdivia, Hector H; Cerrone, Marina; Delmar, Mario
BACKGROUND:Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy (ARVC). A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. METHODS:We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice ("PKP2cKO") 14 days post-tamoxifen (post-TAM) injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. RESULTS:homeostasis. Similarly, PKC inhibition normalized spark frequency at comparable SR load levels. CONCLUSIONS:handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.
PMID: 31315456
ISSN: 1524-4539
CID: 3977952

Plakophilin-2 Haploinsufficiency Causes Calcium Handling Deficits and Modulates the Cardiac Response Towards Stress

van Opbergen, Chantal J M; Noorman, Maartje; Pfenniger, Anna; Copier, Jaël S; Vermij, Sarah H; Li, Zhen; van der Nagel, Roel; Zhang, Mingliang; de Bakker, Jacques M T; Glass, Aaron M; Mohler, Peter J; Taffet, Steven M; Vos, Marc A; van Rijen, Harold V M; Delmar, Mario; van Veen, Toon A B
Human variants in plakophilin-2 (PKP2) associate with most cases of familial arrhythmogenic cardiomyopathy (ACM). Recent studies show that PKP2 not only maintains intercellular coupling, but also regulates transcription of genes involved in Ca2+ cycling and cardiac rhythm. ACM penetrance is low and it remains uncertain, which genetic and environmental modifiers are crucial for developing the cardiomyopathy. In this study, heterozygous PKP2 knock-out mice (PKP2-Hz) were used to investigate the influence of exercise, pressure overload, and inflammation on a PKP2-related disease progression. In PKP2-Hz mice, protein levels of Ca2+-handling proteins were reduced compared to wildtype (WT). PKP2-Hz hearts exposed to voluntary exercise training showed right ventricular lateral connexin43 expression, right ventricular conduction slowing, and a higher susceptibility towards arrhythmias. Pressure overload increased levels of fibrosis in PKP2-Hz hearts, without affecting the susceptibility towards arrhythmias. Experimental autoimmune myocarditis caused more severe subepicardial fibrosis, cell death, and inflammatory infiltrates in PKP2-Hz hearts than in WT. To conclude, PKP2 haploinsufficiency in the murine heart modulates the cardiac response to environmental modifiers via different mechanisms. Exercise upon PKP2 deficiency induces a pro-arrhythmic cardiac remodeling, likely based on impaired Ca2+ cycling and electrical conduction, versus structural remodeling. Pathophysiological stimuli mainly exaggerate the fibrotic and inflammatory response.
PMID: 31438494
ISSN: 1422-0067
CID: 4047002

Beyond the One Gene-One Disease Paradigm: Complex Genetics and Pleiotropy in Inheritable Cardiac Disorders

Cerrone, Marina; Remme, Carol Ann; Tadros, Rafik; Bezzina, Connie R; Delmar, Mario
Inheritable cardiac disorders, which may be associated with cardiomyopathic changes, are often associated with increased risk of sudden death in the young. Early linkage analysis studies in Mendelian forms of these diseases, such as hypertrophic cardiomyopathy and long-QT syndrome, uncovered large-effect genetic variants that contribute to the phenotype. In more recent years, through genotype-phenotype studies and methodological advances in genetics, it has become evident that most inheritable cardiac disorders are not monogenic but, rather, have a complex genetic basis wherein multiple genetic variants contribute (oligogenic or polygenic inheritance). Conversely, studies on genes underlying these disorders uncovered pleiotropic effects, with a single gene affecting multiple and apparently unrelated phenotypes. In this review, we explore these 2 phenomena: on the one hand, the evidence that variants in multiple genes converge to generate one clinical phenotype, and, on the other, the evidence that variants in one gene can lead to apparently unrelated phenotypes. Although multiple conditions are addressed to illustrate these concepts, the experience obtained in the study of long-QT syndrome, Brugada syndrome, and arrhythmogenic cardiomyopathy, and in the study of functions related to SCN5A (the gene coding for the α-subunit of the most abundant sodium channel in the heart) and PKP2 (the gene coding for the desmosomal protein plakophilin-2), as well, is discussed in more detail.
PMCID:6697136
PMID: 31403841
ISSN: 1524-4539
CID: 4041912

TARGETING THE MICROTUBULE PLUS-END TRACKING EB1-CLASP2 PROTEIN COMPLEX MODULATES NAV1.5 SPECIFICALLY AT THE INTERCALATED DISC [Meeting Abstract]

Marchal, G A; Portero, V; Podliesna, S; Perez-Hernandez, M; Yu, N; Veerman, C C; Casini, S; Klerk, M; Lodder, E M; Mengarelli, I; Rothenberg, E; Charpentier, F; Redon, R; Verkerk, A O; Delmar, M; Galjart, N; Bezzina, C R; Remme, C A
Background: Nav1.5 is targeted to distinct subcellular microdomains of cardiomyocytes by the microtubule network, with sodium current being largest in the intercalated disc (ID) region. The microtubule plus-end tracking proteins End Binding 1 (EB1) and CLIP-associating protein 2 (CLASP2) are mainly located at the ID and regulate microtubule recruitment and stability. The small molecule SB216763 (SB2) acts on Glycogen synthase kinase 3 beta (GSK3beta) and is known to enhance the EB1-CLASP2 interaction, thereby increasing microtubule stability.
Objective(s): To investigate the effect of targeting EB1-CLASP2 on Nav1.5 localisation and sodium current density (INa) in subcellular microdomains.
Method(s): Patch clamp and Stochastic Optical Reconstruction Microscopy (STORM) imaging experiments were performed on human iPSC-derived cardiomyocytes (hiPSC-CMs) and freshly isolated murine ventricular cardiomyocytes.
Result(s): EB1 overexpression in hiPSC-CMs increased membrane Nav1.5 cluster density and consequently increased whole-cell INa and action potential (AP) upstroke velocity, without affecting INa kinetics or other AP parameters. Increased whole-cell INa was observed in murine cardiomyocytes after 2-4 hours of SB2 treatment (5micro M), while INa kinetics remained unaffected. Macropatch experiments revealed that SB2 specifically increased INa at the ID, while INa at the lateral membrane was unchanged. In contrast, SB2 did not affect INa or Nav1.5 cluster size or density in cardiomyocytes from mice Clasp2-deficient mice.
Conclusion(s): Targeting the plus-end tracking proteins EB1 and CLASP2 resulted in increased whole-cell peak INa in hiPSC-CMs and isolated murine cardiomyocytes. On the subcellular level, INa was specifically increased at the ID after pharmacologically enhancing the CLASP2-EB1 interaction by SB2. Treatment with SB2 in cardiomyocytes lacking CLASP2 did not affect INa or Nav1.5 distribution, demonstrating the central role for CLASP2 in the SB2-mediated effects. Thus, the microtubule-EB1-CLASP2 complex constitutes a promising target for modulating INa in a microdomain-specific manner.
Copyright
EMBASE:2002295991
ISSN: 1556-3871
CID: 4001882