Exacerbation of dystrophic cardiomyopathy by phospholamban deficiency mediated chronically increased cardiac Ca²⁺ cycling in vivo

Cardiomyopathy is a significant contributor to morbidity and mortality in Duchenne muscular dystrophy (DMD). Membrane instability, leading to intracellular Ca²⁺ mishandling and overload, causes myocyte death and subsequent fibrosis in DMD cardiomyopathy. On a cellular level, cardiac myocytes from mdx mice have dysregulated Ca²⁺ handling, including increased resting Ca²⁺ and slow Ca²⁺ decay, especially evident under stress conditions. Sarco(endo)plasmic reticulum Ca²⁺ ATPase and its regulatory protein phospholamban (PLN) are potential therapeutic targets for DMD cardiomyopathy owing to their key role in regulating intracellular Ca²⁺ cycling. We tested the hypothesis that enhanced cardiac Ca²⁺ cycling would remediate cardiomyopathy caused by dystrophin deficiency. We used a genetic complementation model approach by crossing dystrophin-deficient mdx mice with PLN knockout (PLNKO) mice [termed double-knockout (DKO) mice]. As expected, adult cardiac myocytes isolated from DKO mice exhibited increased contractility and faster relaxation associated with increased Ca²⁺ transient peak height and faster Ca²⁺ decay rate compared with control mice. However, compared with wild-type, mdx, and PLNKO mice, DKO mice unexpectedly had reduced in vivo systolic and diastolic function as measured by echocardiography. Furthermore, Evans blue dye uptake was increased in DKO hearts compared with control, mdx, and PLNKO hearts, demonstrating increased membrane damage, which subsequently led to increased fibrosis in the DKO myocardium in vivo. In conclusion, despite enhanced intracellular Ca²⁺ handling at the myocyte level, DMD cardiomyopathy was exacerbated owing to unregulated chronic increases in Ca²⁺ cycling in DKO mice in vivo. These findings have potentially important implications for ongoing therapeutic strategies for the dystrophic heart. NEW & NOTEWORTHY This study examined the effects of phospholamban ablation on the pathophysiology of cardiomyopathy in dystrophin-deficient mice. In this setting, contractility and Ca²⁺ cycling were enhanced in isolated myocytes; however, in vivo heart function was diminished. Additionally, sarcolemmal integrity was compromised and fibrosis was increased. This is the first study, to our knowledge, examining unregulated Ca²⁺ cycling in the dystrophin-deficient heart. Results from this study have implications for potential therapies targeting Ca²⁺ handling in dystrophic cardiomyopathy.