Background: Fabry disease (FD) is an inherited X-chromosomal lysosomal storage disorder caused by deficient α-galactosidase A (α-Gal A) enzyme activity. Patients with FD display left ventricular hypertrophy, fibrosis, and ventricular arrhythmias that can cause sudden cardiac death. Since mice with deletion of the α-Gal A gene (GLAko) develop only bradycardia but no further major symptomatic abnormalities, we crossbred GLAko mice with mice overexpressing human Gb3 synthase to obtain G3Stg/GLAko mice. This potentiates Gb3 accumulation, which induces cardiac fibres disarray and death between week 20 to 35 of age of unknown cause. Moribund mice present arrythmia. Here, we determined whether disturbed heart function in FD mouse models is related to defects in excitation-contraction coupling or compromised mitochondrial function.

Methods and results: Isolated cardiac myocytes from 20 weeks old mice were exposed to a physiological stress protocol by pacing at 0.5 Hz, followed by β-adrenergic stimulation with isoproterenol (Iso) and increased stimulation rate at 5 Hz for 3 minutes (Iso/5Hz). While at baseline, fractional sarcomere shortening was comparable in all three genotypes, it was reduced in myocytes from G3Stg/gGLAko (n=14/3), but not GLAko (n=19/3) compared to WT myocytes (n=57/7; p<0.05) at 0.5 Hz/Iso and 5Hz/Iso, respectively. Furthermore, sarcomere relengthening kinetics were faster in G3Stg/GLAko myocytes compared to WT, while during Iso/5Hz, kinetics accelerated and equilibrated between groups. This acceleration of relengthening kinetics may be related to downregulated protein expression of the inhibitory phospholamban (PLN) in hearts of GLAko and G3Stg/GLAko vs. WT mice, respectively, while PLN phosphorylation (related to protein expression) and protein levels of sarcoplasmic reticulum (SR) Ca²⁺ ATPase were unchanged. The mitochondrial redox states of NAD(P)H/NAD(P)⁺ and FADH₂/FAD were unchanged between groups, despite a trend towards more oxidized NAD(P)H/NAD(P)⁺ in G3Stg/gGLAko at baseline. We also measured single-cell force in pre-stretched cardiac myocytes together with cytosolic Ca²⁺ using Indo-1. Systolic force development was similar between groups, however, diastolic and systolic Ca²⁺ concentrations and amplitudes of Ca²⁺ transients were increased in GLAko compared to WT myocytes. In isolated cardiac mitochondria, basal and maximal ADP-stimulated respiration (supported by pyruvate/malate or fatty acids) and mitochondrial Ca²⁺ uptake were unchanged between GLAko and WT. In contrast, pyruvate/malate-linked state 3 respiration was substantially reduced by 40% (at 1 mM ADP) in isolated cardiac mitochondria from G3Stg/GLAko vs. WT mice.