The effect of ventricular microvascular dysfunction on cardiomyocyte excitation-contraction coupling and mitochondria

Abstract

Abstract Introduction Microvascular dysfunction is a hallmark feature of cardiac allograft vasculopathy in HTx patients and associated with heart failure and coronary artery disease. On a cellular level, impaired mitochondrial respiration has been associated with the disease, however the exact mechanisms remain elusive. Objective The aim of this study was to determine the effect of microvascular dysfunction on functional and structural properties of cardiomyocytes isolated from ventricular biopsies of HTx patients. Methods We functionally assessed coronary artery microvasculature using guidewire based coronary flow reserve(CFR)/microvasculatory resistance (IMR) measurements (Abbott) in 13 post-HTx patients with angiographically normal coronary arteries or normal FFR. Ventricular (RV septum) myocardial biopsies were obtained for in-vitro measurements and single ventricular cardiomyocytes were isolated using enzymatic digestion. Cells were electrically stimulated (1 Hz) and subcellular Ca2+ signalling during excitation-contraction coupling was measured using confocal imaging (Fluo-4 AM). In a subset of cardiomyocytes mitochondrial density was quantified upon staining with Mitotracker Red FM (otsu thresholding). Results Microvascular dysfunction (MVD) measured by IMR was present in 6 of 13 patients with a mean IMR of 56±11 and 13±3 in MVD and CTRL, respectively. CFR did not differ between MVD and CTRL. Ca2+ transients during excitation-contraction coupling in isolated ventricular cardiomyocytes from a subset of patients showed unaltered amplitudes (1.2±0.01 vs. 1.3±0.05; a.u. F/F0). In addition, Ca2+ release (i.e. time to peak Ca2+) and Ca2+ removal (i.e. tau) were not significantly different between MVD and CTRL. However, mitochondrial density was significantly increased in MVD vs. CTRL, indicating subcellular changes associated with MVD. Conclusion In-vivo ventricular microvascular dysfunction is associated with preserved excitation-contraction coupling in-vitro, potentially owing to compensatory changes on the mitochondrial level.