Confocal Simultaneous Assessment of Calcium and Contractility Dynamics in Single Ventricular Myocytes of a Rat Model of Isoproterenol-Induced Cardiomyopathy

Abstract

Stress-induced cardiomyopathy (SIC), also known as takotsubo syndrome, results from the catecholaminergic discharge upon strong emotional or physical stress. Characteristically, SIC patients present acute left ventricle apical hypokinesia, despite of unobstructed coronary circulation, and may lead to arrhythmias and acute heart failure. Although, most patients recover, this could be slow, and recurrence or death may occur. Despite that SIC common denominator, is the large catecholamine discharge, its pathophysiological mechanisms are incompletely understood. Since apical myocytes have the highest density of β-adrenergic receptors, it is thought that catecholamine-induced overstimulation has direct cytotoxicity on myocytes, causing acute Ca2+ overload and oxidative stress, which cause myocyte stunning or death. Nevertheless, most ventricular Ca2+ assessments have been indirect, and the time course of Ca2+ dynamics in intact ventricular myocytes, and their contribution to contractile dysfunction, has not been documented, which would be important to uncover the cellular and molecular hypokinesia basis, which persists hours or days after the acute phase subsides. Therefore, in single Fluo-4-loaded myocytes from rats receiving an isoproterenol (ISO) overdose (OV; 67 mg/kg body weight), which mimic SIC development, we aimed to simultaneously characterize, with confocal imaging, Ca2+ and shortening dynamics. The studies were performed during 1 to 15 days, and ISO-OV myocytes showed contractile dysfunction; peak shortening decreased, while shortening development and relaxation significantly slowed. All these correlated with systolic Ca2+ dysregulation; peak Ca2+ transient and the rate of SR Ca2+ release decreased, while SR Ca2+ reuptake slowed. Furthermore, RyR2 were hyperactive, displaying reactivation after Ca2+ transient peak and increased diastolic spark frequency. These alterations worsened or maintained for 15 days, explaining slow recovery. Therefore, we conclude that the main determinant of hypokinesia is blunted cellular contractility due to Ca2+ dysfunction.