Ventricular Dysfunction and Calcium Handling Derangements in Isolated Hearts From Duchenne Muscular Dystrophy (DMD mdx‐4CV ) Mice Following Sustained Ventricular Preload

Abstract

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy and is caused by mutations in the dystrophin gene. Subclinical signs of cardiac disease present early and usually progress to dilated cardiomyopathy in late stage DMD patients. Dystrophin deficiency associates with stretch-induced myocyte calcium entry, adverse calcium overload, and cardiomyocyte damage. In this investigation we test the hypothesis that dystrophin deficiency leads to ventricular dysfunction and calcium handling abnormalities following sustained ventricular preload challenge. Hearts of 3-10 month male Control and Dmdmdx-4CV dystrophic mice were isolated, cannulated via both the aorta and left atrium, and perfused with physiological saline solution in working heart mode. Following elevation in preload to 20 mmHg, hearts of Control and Dmdmdx-4CV mice exhibited similar systolic maximum pressure development (PMax: 105±13 mmHg Control versus 102±7 mmHg Dmdmdx-4CV). However, while hearts of Control mice maintained systolic performance following the 30 minute pre-load challenge (PMax, 30 minutes - PMax, peak response: -6±3 mmHg, N=5), hearts of Dmdmdx-4CV mice exhibited a marked decline in systolic performance (PMax,30 minutes - PMax, peak response: -29±6 mmHg, N=7, P<0.01 vs. Control). The Dmdmdx-4CV hearts also had visible signs of damage following preload challenge. Using Control and Dmdmdx-4CV mice with transgenic, cardiac-specific expression of the GCaMP6f calcium indicator (MerCreMer-GCaMP6f), we examined incidence of calcium overload behavior following preload challenge. Isolated working hearts were subjected to 20 mmHg preload in working heart mode for 30 minutes, switched to Langendorff mode with physiological saline containing 40 uM blebbistatin, and imaged using laser-scanning confocal fluorescence microscopy. Calcium overload behavior was observed in 21% of cardiomyocytes of Dmdmdx-4CV hearts (n=30/140 cells equally sampled from N=4 hearts) compared to 1% of cardiomyocytes of Control hearts (n=2/140 cells from N=4 hearts). Taken together, these data indicate susceptibility to stretch-induced calcium overload, ventricular damage, and ventricular dysfunction in male Dmdmdx-4CV hearts.