Cardiac arrhythmias significantly contribute to mortality in Duchenne muscular dystrophy (DMD), a disease caused by dystrophin deficiency¹. A major source of arrhythmias in DMD patients is impaired ventricular impulse conduction, which predisposes to ventricular asynchrony and 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. Whereas animal studies of AAV µDys therapy yielded substantial structural and functional improvements in the dystrophic heart, 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 Purkinje fibers.

Male mdx-Cx40^eGFP/+ mice² received a single tail vein injection of AAV9 µDys vector. µDys contained the N-terminal and cysteine-rich domains, hinges 1 and 4, and spectrin-like repeats 16 to 19 of human dystrophin. 12 weeks post-injection, mice were anaesthetized (2% isoflurane, 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 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-Cx40^eGFP/+ mice. All procedures had local approval (BMWFW-66.009/0175-WF/V/3b/2015) and conformed to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.

Twelve weeks after AAV µDys vector application to mdx-Cx40^eGFP/+ mice, we observed robust µDys expression in the heart. The Na current density in Purkinje fibers isolated from these hearts (-72 ± 6 pA/pF, mean ± S.E.M, 20 cells from 6 animals) was restored to the wild-type level (-74 ± 4 pA/pF, 58 cells from 8 animals; p=0.86, nested statistical analysis respecting the hierarchical data structure⁴). Impaired Na channel inactivation, represented by a moderately slowed current decay in mdx compared to wild-type fibers, was also rescued by µDys therapy.

Na channel activity in the Purkinje fiber membrane determines ventricular conduction velocity². Thus, by restoring wild-type Na current properties in dystrophic Purkinje fibers, we have corrected the molecular underpinning of impaired ventricular conduction in the dystrophic heart. Further development of this therapeutic strategy may prevent or treat fatal arrhythmias in patients with DMD.

Abbreviations: adeno-associated virus, AAV; Duchenne muscular dystrophy, DMD; micro-dystrophin, µDys