Micro-dystrophin Therapy Rescues Impaired Na Currents in Cardiac Purkinje Fibers from Dystrophin-deficient Mdx Mice

Cardiac arrhythmias significantly contribute to mortality in Duchenne muscular dystrophy (DMD), a disease caused by dystrophin deficiency¹. 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 conduction². Our finding provided a mechanistic explanation for ventricular conduction defects and concomitant arrhythmias in the dystrophic heart.

Systemic adeno-associated virus (AAV) delivery of micro-dystrophin (μDys) holds great promise to treat DMD and is currently in human trials³. 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)-Cx40^eGFP/+ 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 (Fig. 1A). R16–19 is a region implicated in heart protection⁴. 12 weeks post-injection, 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 protocol², whereby the Cx40^eGFP/+ background 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-Cx40^eGFP/+ and wild-type (C57BL/10ScSnJ)-Cx40^eGFP/+ mice.

Figure 1.

Full rescue of impaired Na currents in dystrophic Purkinje fibers by μDys therapy.

A, Full-length dystrophin and micro-dystrophin structure. B, Representative dystrophin immunofluorescence staining photomicrographs in the heart of mdx-Cx40^eGFP/+ mice at 12 weeks after AAV micro-dystrophin 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, UK). Dystrophin C-terminal domain (CT) was recognized by Dys-2 antibody (1:20; Novocastra, Newcastle, UK). C, Representative dystrophin (R17)/laminin (1:200; Sigma, St. Louis, MO) double immunostaining photomicrographs illustrating saturated micro-dystrophin 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, Micro-dystrophin western blot using the Manex 44A antibody (against dystrophin R17, 1:100); Filled triangle, micro-dystrophin (132 kD); Open triangle, alpha-tubulin (50 kD). F, Typical original Ca current (I_Ca) traces of a wt-Cx40^eGFP/+ 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-Cx40^eGFP/+ cells, and all 18 tested mdx-Cx40^eGFP/+ 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 recorded from a wild-type (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 NMDG, 2.5 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES; pH=7.4, adjusted with HCl. Pipette solution: 5 NaCl, 110 CsF, 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. 2017⁵ (* p<0.05, ** p<0.01, *** p<0.001; p>0.05, ns, not significant).

Twelve weeks after AAV μDys vector application to mdx-Cx40^eGFP/+ mice, we observed robust μDys expression in the heart (Fig. 1B–E). Quantitative analyses suggested that nearly 100% of the cardiomyocytes expressed μDys (Fig. 1D). Purkinje fiber identity of eGFP-positive cells was confirmed in separate control experiments by detection of significant T-type Ca current (Fig. 1F, 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 (Fig. 1G, 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 (Fig. 1I). 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.

Nonstandard Abbreviations and Acronyms:

AAV

adeno-associated virus

I_Ca

Ca current

CT

C-terminal domain

DMD

Duchenne muscular dystrophy

μDys

micro-dystrophin

wt

wild-type