0514 : Isolation of cardiac myocytes from human heart

Abstract

The investigation of single cardiac myocytes from healthy and diseased hearts of various species is a valuable tool to explore cardiac physio/ pathophysiology. The application of cell isolation to human donor tissue has been proofed to be difficult due to the limited amount of human tissue (mainly human right atrial appendages during cardiac surgery). Another limitation is the low viability of cardiomyocytes after isolation. In this study, we present a method to obtain single cardiac myocytes from different regions of human heart. Human hearts rejected for transplantation were obtained from Bordeaux hospital. This protocol was approved by the Agence de la Biomédecine. Left atrial (LA) and ventricular (LV) myocytes were obtained by enzymatic dissociation. The ventricles and right atrium were removed and used for other studies (e.g. high resolution optical mapping). LA was cannulated by the circumflex artery and mounted into a Langendorff perfusion system after suture of the leaky atrial branches. LA was perfused with a Ca2+-free solution (~10 min), then collagenase and protease solution (0.08 mM Ca2+) and recirculated for ~25 min. Enzymes were washed out with a 0.2 mM Ca2+ solution. LA was separated into 4 regions: Endocardium, Epicardium, roof and pulmonary vein; LV myocytes were also obtained. Cells were re-suspended into a 1.8 mM Ca2+ solution by steps. Ca2+ transients were recorded (Fura-2, field stimulation) using an IonOptix system and cell membrane was stained with di-8 ANEPPS and visualized under confocal microscopy. Ca2+ tolerant myocytes were obtained from the 4 LA regions and LV. Human cardiac myocytes respond to electrical stimulation and Ca2+ transient can be recorded. Analysis of functional and structural data will be presented. Isolation of single cardiac myocytes from human samples is a tedious task, but we present data showing reliable method to obtain functional and structural insights. The author hereby declares no conflict of interest