Alterations in ion channel expression and function known as "electrical remodeling" contribute to the development of hypertrophy and to the emergence of arrhythmias and sudden cardiac death. However, comparing current density values - an electrophysiological parameter commonly utilized to assess ion channel function - between normal and hypertrophied cells may be flawed when current amplitude does not scale with cell size. Even more, common routines to study equally sized cells or to discard measurements when large currents do not allow proper voltage-clamp control may introduce a selection bias and thereby confound direct comparison. To test a possible dependence of current density on cell size and shape, we employed whole-cell patch-clamp recording of voltage-gated sodium and calcium currents in Langendorff-isolated ventricular cardiomyocytes and Purkinje myocytes, as well as in cardiomyocytes derived from trans-aortic constriction operated mice. Here, we describe a distinct inverse relationship between voltage-gated sodium and calcium current densities and cell capacitance both in normal and hypertrophied cells. This inverse relationship was well fit by an exponential function and may be due to physiological adaptations that do not scale proportionally with cell size or may be explained by a selection bias. Our study emphasizes the need to consider cell size bias when comparing current densities in cardiomyocytes of different sizes, particularly in hypertrophic cells. Conventional comparisons based solely on mean current density may be inadequate for groups with unequal cell size or non-proportional current amplitude and cell size scaling.

Dystrophin deficiency in cardiomyocytes leads to abnormally reduced Na + currents. These can be rescued by long-term empagliflozin treatment.

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Austrian Science Fund (FWF) Objective Ivabradine (IVA) is indicated in symptomatic treatment of chronic stable angina, heart failure, and also in those who are unable to tolerate or have contraindications to the use of beta-blockers. It has been showed IVA may have cardiovascular benefits in Duchenne muscular dystrophy (DMD) patients. This study was aimed to investigate the sustained impact of chronic ivabradine (IVA) administration on cardiac function and potential underlying mechanism using high throutput proteomic analysis. Methods DMDmdx male and Sprague–Dawley wt (Sprague–Dawley) male rats were randomly allocated to vehicle (n = 6) or IVA (n=6; 10 mg/kg/day via drinking water for four months). Transthoracic echocardiography and unbiased proteomic analysis were performed to assess cardiac function and left ventricular (LV) tissue, respectively. Protein-protein interactions were graphically represented, and cluster analysis with subsequent enrichment analyses was conducted. Results Chronic IVA treatment significantly enhanced the LV ejection fraction (p<0.05 compared to vehicle-treated DMDmdx rats). Proteomic data identified possible transcription factors (SPI1, IRF1, PPARA), consistent with previous studies. The reduction in copper metabolic changes associated with DMD can be attributed to the upregulation of Atox1 following IVA treatment. Mitochondrial dysfunction was effectively mitigated by IVA, as indicated by the different abundance of mitochondrial proteins. In addition, intercellular adhesion molecule 1 showed a reduction in the number of animals treated with ivabradine, indicating a further positive effect of the treatment. Conclusion In summary, this study demonstrated the beneficial effects of IVA in DMDmdx rat hearts. Cluster analysis of proteomic data revealed notable changes in cardiac metabolism, inflammation, and mitochondrial function. Ivabradine has emerged as a potential therapeutic approach to address these shifts, while proteomic pathway analysis may uncover new drugable targets to alleviate cardiomyopathy progression in DMD.Interaction of IvabradinProtein-protein interaction network

Abstract Background Duchenne muscular dystrophy (DMD) is an X-linked hereditary disease triggered by the deficiency of the structural protein dystrophin, which leads to muscular degeneration mainly affecting young males. Major contributors to early death in DMD patients are cardiac arrhythmias and dystrophic cardiomyopathy. One cause for arrhythmias is impaired ventricular impulse conduction, which leads to ventricular asynchrony and reentrant mechanisms. We recently showed that the disruption of dystrophin results in a significant reduction of Na current in ventricular cardiomyocytes (vCMs) of the dystrophin-deficient mdx mouse model for human DMD. Na current reduction provides a mechanistic explanation for the impaired ventricular conduction and accompanying arrhythmias in the dystrophic heart. The extracellular matrix protein tenascin-C (TN-C) is a significant remodeling factor in the injured and diseased heart and is strongly upregulated in dystrophic cardiomyopathy. To this date, it is unknown how the upregulation of TN-C in DMD patients affects dystrophic cardiomyopathy. Purpose In this study, we examined the effect of TN-C inhibition on diminished Na currents in dystrophin-deficient vCMs. Methods We compared four different mouse genotypes with each other, namely wild-type, dystrophin-deficient mdx, TN-C-deficient and dystrophin- plus TN-C-deficient mice. Furthermore, a cohort of mdx mice was injected with TN-C siRNA twice a week for 9 weeks to investigate the effect of TN-C knockdown. Hearts from adult male mice were enzymatically digested using a Langendorff system to isolate single vCMs. Na currents were then measured with the whole cell patch clamp technique. Results Na current densities were increased in TN-C deficient vCMs compared to wild-type vCMs. Accordingly, 24-hour incubation of wild-type vCMs with human recombinant TN-C resulted in a significant decrease in Na current. Na currents of vCMs from mdx mice were reduced, but restored to the wild-type level in vCMs from TN-C-deficient mdx mice. Moreover, vCMs of TN-C siRNA-treated mdx mice had significantly increased Na currents compared to control mdx vCMs. Conclusion Upregulation of TN-C in dystrophin-deficient vCMs reduces Na currents, whereas inhibition of TN-C prevents this reduction. Therefore, inhibition of TN-C in DMD patients may be considered as a potential new therapeutic strategy to improve ventricular conduction and reduce arrhythmia vulnerability.

The muscular dystrophies caused by dystrophin deficiency, the so-called dystrophinopathies, are associated with impaired cardiac contractility and arrhythmias, which considerably contribute to disease morbidity and mortality. Impaired Ca handling in ventricular cardiomyocytes has been identified as a causative factor for complications in the dystrophic heart, and restoration of normal Ca handling in myocytes has emerged as a promising new therapeutic strategy. In the present study, we explored the hypothesis that ivabradine, a drug clinically approved for the treatment of heart failure and stable angina pectoris, improves Ca handling in dystrophic cardiomyocytes and thereby enhances contractile performance in the dystrophic heart. Therefore, ventricular cardiomyocytes were isolated from the hearts of adult dystrophin-deficient DMDmdx rats, and the effects of acutely applied ivabradine on intracellular Ca transients were tested. In addition, the drug's acute impact on cardiac function in DMDmdx rats was assessed by transthoracic echocardiography. We found that administration of ivabradine to DMDmdx rats significantly improved cardiac function. Moreover, the amplitude of electrically induced intracellular Ca transients in ventricular cardiomyocytes isolated from DMDmdx rats was increased by the drug. We conclude that ivabradine enhances Ca release from the sarcoplasmic reticulum in dystrophic cardiomyocytes and thereby improves contractile performance in the dystrophic heart.

Alterations in cardiac impulse conduction may exert both beneficial and detrimental effects. The assessment of ventricular conduction properties is of paramount importance both in clinical and in experimental settings. Currently the duration of the QRS complex is regarded as hallmark of in-vivo assessment of global ventricular conduction time. In addition, the amplitude of the QRS complex has been suggested to reflect ventricular conduction time in man and in rats. Here, for the first time, we systematically investigated the relationship between QRS duration ("QRS") and QRS amplitude ("RS-height"; RSh) in the murine ECG obtained during anesthesia. In mice harbouring a homozygous knockout of the transmembrane protein podoplanin (PDPN-/-; n = 10) we found both a shorter QRS and a greater RSh than in wild-type animals (n = 13). In both genotypes cumulative i.p. administration of 5 mg/kg and 10 mg/kg of the Na channel blocker flecainide resulted in dose-dependent QRS increase and RSh decrease, whereby the drug-induced changes in RSh were greater than in QRS. In both genotypes the flecainide-induced changes in QRS and in RSh were significantly correlated with each other (R = -0.56, P = 0.004). Whereas dispersion of QRS and RSh was similar between genotypes, dispersion of the ratio QRS/RSh was significantly smaller in PDPN-/- than in wild-types. We conclude that in the murine ECG QRS is inversely related to RSh. We suggest that both parameters should be considered in the analysis of ventricular conduction time in the murine ECG.

T-type Ca channels are strongly expressed and important in the developing heart. In the adult heart, these channels play a significant role in pacemaker tissues, but there is uncertainty about their presence and physiological relevance in the working myocardium. Here, we show that the T-type Ca channel isoforms Cav3.1 and Cav3.2 are expressed at a protein level in ventricular cardiomyocytes from healthy adult C57/BL6 mice. Myocytes isolated from adult wild-type and Cav3.2 KO mice showed considerable whole cell T-type Ca currents under beta-adrenergic stimulation with isoprenaline. We further show that the detectability of basal T-type Ca currents in murine wild-type cardiomyocytes depends on the applied experimental conditions. Together, these findings reveal the presence of functional T-type Ca channels in the membrane of ventricular myocytes. In addition, electrically evoked Ca release from the sarcoplasmic reticulum was significantly impaired in Cav3.2 KO compared to wild-type cardiomyocytes. Our work implies a physiological role of T-type Ca channels in the healthy adult murine ventricular working myocardium.

HomeCirculation: Arrhythmia and ElectrophysiologyVol. 15, No. 8Microdystrophin Therapy Rescues Impaired Na Currents in Cardiac Purkinje Fibers From Dystrophin-Deficient Mdx Mice Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBMicrodystrophin Therapy Rescues Impaired Na Currents in Cardiac Purkinje Fibers From Dystrophin-Deficient Mdx Mice Janine Ebner, Xiufang Pan, Yongping Yue, Spyridon Sideromenos, Jessica Marksteiner, Xaver Koenig, Karlheinz Hilber and Dongsheng Duan Janine EbnerJanine Ebner Department of Neurophysiology & Neuropharmacology, Center for Physiology & Pharmacology, Medical University of Vienna, Vienna, Austria (J.E., S.S., J.M., X.K., K.H.). , Xiufang PanXiufang Pan https://orcid.org/0000-0002-2594-5911 Department of Molecular Microbiology & Immunology (X.P., Y.Y., D.D.). , Yongping YueYongping Yue https://orcid.org/0000-0002-1640-2106 Department of Molecular Microbiology & Immunology (X.P., Y.Y., D.D.). , Spyridon SideromenosSpyridon Sideromenos Department of Neurophysiology & Neuropharmacology, Center for Physiology & Pharmacology, Medical University of Vienna, Vienna, Austria (J.E., S.S., J.M., X.K., K.H.). , Jessica MarksteinerJessica Marksteiner Department of Neurophysiology & Neuropharmacology, Center for Physiology & Pharmacology, Medical University of Vienna, Vienna, Austria (J.E., S.S., J.M., X.K., K.H.). , Xaver KoenigXaver Koenig https://orcid.org/0000-0002-2423-4966 Department of Neurophysiology & Neuropharmacology, Center for Physiology & Pharmacology, Medical University of Vienna, Vienna, Austria (J.E., S.S., J.M., X.K., K.H.). , Karlheinz HilberKarlheinz Hilber Correspondence to: Karlheinz Hilber, PhD, Department of Neurophysiology and Neuropharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Schwarzspanierstraße 17, 1090 Vienna, Austria. Email E-mail Address: [email protected] https://orcid.org/0000-0002-3033-0874 Department of Neurophysiology & Neuropharmacology, Center for Physiology & Pharmacology, Medical University of Vienna, Vienna, Austria (J.E., S.S., J.M., X.K., K.H.). and Dongsheng DuanDongsheng Duan https://orcid.org/0000-0003-4109-1132 Department of Molecular Microbiology & Immunology (X.P., Y.Y., D.D.). Department of Neurology, Department of Biomedical Sciences, and Department of Biological & Chemical Engineering, The University of Missouri, Columbia, MO (D.D.). Originally published2 Aug 2022https://doi.org/10.1161/CIRCEP.122.011161Circulation: Arrhythmia and Electrophysiology. 2022;15Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: August 2, 2022: Ahead of Print Cardiac arrhythmias significantly contribute to mortality in Duchenne muscular dystrophy (DMD), a disease caused by dystrophin deficiency.1 A major source of arrhythmias in patients with DMD is impaired ventricular impulse conduction, which predisposes for ventricular asynchrony, decreased cardiac output, and the development of reentrant mechanisms. Using the mdx mouse model for DMD, we recently showed that lack of dystrophin causes considerable Na current loss in Purkinje fibers, cardiomyocytes specialized for electrical impulse conduction.2 Our finding provided a mechanistic explanation for ventricular conduction defects and concomitant arrhythmias in the dystrophic heart.Systemic adeno-associated virus (AAV) delivery of microdystrophin (µDys) holds great promise to treat DMD and is currently in human trials.3 Extensive animal studies and early clinical trial data showed encouraging efficacy in skeletal muscle, but beneficial impacts on the heart cannot yet be adequately estimated. Evidence for successful correction of arrhythmia-inducing mechanisms by µDys is completely missing. The aim of the present study was to test whether AAV µDys therapy can rescue Na current loss in dystrophic cardiac Purkinje fibers.All animal studies performed at the Medical University of Vienna coincided with the institutional Animal Welfare Committee rules and had local approval (BMWFW-66.009/0175-WF/V/3b/2015). All procedures also conformed to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Animal studies performed at the University of Missouri were approved by the Institutional Animal Care and Use Committee and were in accordance with the guidelines of the National Institutes of Health. The data that support the findings of this study are available from the corresponding author upon reasonable request.Eight-week-old male mdx (C57BL/10ScSn-Dmdmdx/J)-Cx40eGFP/+ mice2 received a single tail vein injection of AAV9 µDys vector (3×1012 viral genome particles/mouse). µDys contained the N-terminal and cysteine-rich domains, hinges 1 and 4, and spectrin-like repeats 16 to 19 of human dystrophin (Figure [A]). R16-19 is a region implicated in heart protection.4 12 weeks postinjection, mice were anesthetized using isoflurane (2%, inhalation) and euthanized by cervical dislocation. Thereafter, hearts were removed, and single Purkinje fibers were isolated from ventricular tissue according to our published protocol,2 whereby the Cx40eGFP/+ background, i.e., expression of enhanced green fluorescent protein (eGFP) under the control of the connexin 40 gene (Cx40), allowed for unambiguous identification of Purkinje fibers for electrophysiological studies. Na currents of isolated Purkinje fibers were recorded with the whole-cell patch-clamp technique and compared with Na currents of Purkinje fibers isolated from age- and sex-matched untreated mdx-Cx40eGFP/+ and wild-type (C57BL/10ScSnJ)-Cx40eGFP/+ mice.Download figureDownload PowerPointFigure. Full rescue of impaired Na currents in dystrophic Purkinje fibers by microdystrophin (µDys) therapy. A, Full-length dystrophin and µDys structure; C-terminal (CT), cysteine-rich (CR), hinge (H), N-terminal (NT) and rod (R) domains. B, Representative dystrophin immunofluorescence staining photomicrographs in the heart of mdx-Cx40eGFP/+ mice at 12 wk after adeno-associated virus (AAV) µDys injection. Dystrophin R17 was detected with Manex 44A antibody (1:500; gift from Dr Glenn Morris at the Robert Jones and Agnes Hunt Orthopedic Hospital, Oswestry, United Kingdom). Dystrophin CT domain was recognized by Dys-2 antibody (1:20; Novocastra, Newcastle, United Kingdom). C, Representative dystrophin (R17)/laminin (1:200; Sigma, St. Louis, MO) double immunostaining photomicrographs illustrating saturated µDys expression in the heart of injected mice. D, Quantification of dystrophin positive cardiomyocytes. For each group, hearts from 8 mice (4 randomly chosen sections per heart) were used. E, µDysWestern blot using the Manex 44A antibody (against dystrophin R17, 1:100); Filled triangle, µDys (132 kD); Open triangle, alpha-tubulin (50 kD). F, Typical original Ca current (ICa) traces of a wild-type (wt)–Cx40eGFP/+ cell, elicited by the pulse protocol displayed on top. The respective current density–voltage relationship at the bottom shows the presence of considerable T-type Ca current, typical for Purkinje fibers but not ventricular cardiomyocytes. A series of control experiments revealed that 25 out of 26 tested wt-Cx40eGFP/+ cells, and all 18 tested mdx-Cx40eGFP/+ cells, had a T-type Ca current amplitude of at least 33 % when compared with the respective cells' L-type Ca current amplitudes, confirming the eGFP fluorescence signal as robust indicator of Purkinje fiber identity. G, Typical whole-cell Na currents (INa) recorded from a wt, an untreated mdx, and a µDys-treated mdx Purkinje fiber at room temperature (pulse protocol, inset). The bath solution contained (in mM): 5 NaCl, 135 N-Methyl-D-glucamin, 2.5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES; pH=7.4, adjusted with HCl. Pipette solution: 5 NaCl, 110 caesium (Cs) fluoride, 10 EGTA, 10 HEPES; pH=7.3, adjusted with CsOH. H, Current density–voltage relationships (left), and current density values at −38 mV (right); n=58 cells from 8 wt hearts; n=37 cells from 6 mdx hearts; n=20 cells from 6 mdx µDys hearts. I, Representative peak amplitude-normalized Na current decay (left), and comparison of decay half-times (right) at −38 mV. Decay half-time represents the time period between the current peak and the time points at which the current had decayed to 50% (see arrows). J, Comparison of cell capacitance values for cell size estimation. Data are given as means±SE. Statistical comparisons were performed using a nested analysis respecting the hierarchical data structure (measurements of n cells from m animals) detailed in Sikkel et al 2017.5. *P<0.05, **P<0.01, ***P<0.001; P>0.05, not significant.Twelve weeks after AAV µDys vector application to mdx-Cx40eGFP/+ mice, we observed robust µDys expression in the heart (Figure [B] through [E]). Quantitative analyses suggested that nearly 100% of the cardiomyocytes expressed µDys (Figure [D]). Purkinje fiber identity of eGFP-positive cells was confirmed in separate control experiments by detection of significant T-type Ca current (Figure [F], details given in legend). Whereas Purkinje fibers isolated from hearts of untreated mdx mice showed abnormally reduced Na currents, the Na current density in Purkinje fibers from AAV µDys vector-treated mdx mice was restored to the wild-type level (Figure [G], [H], and [J]). Impaired Na channel inactivation, represented by a moderately slowed current decay in mdx compared to wild-type fibers, was also rescued by µDys therapy (Figure [I]). We believe that the Na current rescue in dystrophic Purkinje fibers was caused by treatment-induced µDys expression in this cell type. A paracrine signal originating from µDys-positive ventricular cardiomyocytes acting on Purkinje fibers is a conceivable alternative cause that can currently not be ruled out. Na channel activity in the Purkinje fiber membrane is a major determinant of ventricular conduction velocity. Thus, by restoring wild-type Na current properties in dystrophic Purkinje fibers, we have corrected the molecular underpinning of impaired ventricular conduction and concomitant arrhythmias in the dystrophic heart. We speculate that a similar µDys therapy may restore normal ventricular conduction in human DMD patients.Collectively, our study implies that AAV µDys therapy can rescue impaired Na currents in dystrophic cardiac Purkinje fibers. Further development of this therapeutic strategy may prevent or treat fatal arrhythmias in patients with DMD.Article InformationAcknowledgmentsThe authors thank L. Miquerol (Aix-Marseille University, Centre National de la Recherche Scientifique UMR7288) for providing the eGFP-positive connexin 40 (Cx40eGFP/+) mice and J. Uhrinova (Medical University of Vienna) and Keqing Zhang (University of Missouri) for excellent technical assistance.Sources of FundingThis work was supported by the Austrian Science Fund ([FWF]; P30234-B27 to Dr Hilber); National Institutes of Health (NS-90634 to Dr Duan); Parent Project Muscular Dystrophy (to Dr Duan); Jackson Freel Duchenne muscular dystrophy (DMD) Research Fund (to Dr Duan).Nonstandard Abbreviations and AcronymsµDysmicrodystrophinAAVadeno-associated virusDMDDuchenne muscular dystrophyDisclosures Dr Duan is a member of the scientific advisory board for Solid Biosciences and equity holders of Solid Biosciences. Dr Duan and Y. Yue are inventors on various patents related to adeno-associated virus (AAV) vector and Duchenne muscular dystrophy (DMD) gene therapy. The Duan lab received research support unrelated to this project from Solid Biosciences and Edgewise Therapeutics in the last 3 years. The other authors report no conflicts.Footnotes*J. Ebner and X. Pan contributed equally as first authors.For Sources of Funding and Disclosures, see page 543.Correspondence to: Karlheinz Hilber, PhD, Department of Neurophysiology and Neuropharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Schwarzspanierstraße 17, 1090 Vienna, Austria. Email karlheinz.hilber@meduniwien.ac.atReferences1. Duan D, Goemans N, Takeda S, Mercuri E, Aartsma-Rus A. Duchenne muscular dystrophy.Nat Rev Dis Primers. 2021; 7:13. doi: 10.1038/s41572-021-00248-3CrossrefMedlineGoogle Scholar2. Ebner J, Uhrin P, Szabo PL, Kiss A, Podesser BK, Todt H, Hilber K, Koenig X. Reduced Na current in Purkinje fibers explains cardiac conduction defects and arrhythmias in duchenne muscular dystrophy.Am J Physiol Heart Circ Physiol. 2020; 318:H1436–H1440. doi: 10.1152/ajpheart.00224.2020CrossrefMedlineGoogle Scholar3. Duan D. Systemic AAV micro-dystrophin gene therapy for duchenne muscular dystrophy.Mol Ther. 2018; 26:2337–2356. doi: 10.1016/j.ymthe.2018.07.011CrossrefMedlineGoogle Scholar4. Wasala NB, Shin JH, Lai Y, Yue Y, Montanaro F, Duan D. Cardiac-specific expression of ΔH2-R15 mini-dystrophin normalized all electrocardiogram abnormalities and the end-diastolic volume in a 23-month-old mouse model of duchenne dilated cardiomyopathy.Hum Gene Ther. 2018; 29:737–748. doi: 10.1089/hum.2017.144CrossrefMedlineGoogle Scholar5. Sikkel MB, Francis DP, Howard J, Gordon F, Rowlands C, Peters NS, Lyon AR, Harding SE, MacLeod KT. Hierarchical statistical techniques are necessary to draw reliable conclusions from analysis of isolated cardiomyocyte studies.Cardiovasc Res. 2017; 113:1743–1752. doi: 10.1093/cvr/cvx151CrossrefMedlineGoogle Scholar eLetters(0)eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. 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August 2022Vol 15, Issue 8 Advertisement Article InformationMetrics © 2022 American Heart Association, Inc.https://doi.org/10.1161/CIRCEP.122.011161PMID: 35917466 Originally publishedAugust 2, 2022 Keywordsmicedystrophinmyocytes, cardiacmuscular dystrophy, Duchennearrhythmia, cardiacPDF download Advertisement SubjectsArrhythmiasElectrophysiology