Adult cardiac muscle cells in long-term serum-free culture: Myofibrillar organization and expression of myosin heavy chain isoforms

Abstract

A culture system for adult rat cardiac muscle cells has been established without exposure of cells to serum at any step of the procedure. The methodology has been standardized and optimized to obtain better quality and high yield of cells and culture. Subsequent to enzyme perfusion, the release of myocytes from enzyme-perfused tissues was carried out in enzyme-free Joklik's medium instead of exposing cells to proteolytic enzyme(s) as done previously. Approximately 5 million cylindrical muscle cells per ventricle were obtained. The culture medium contained Eagle's minimum essential medium with Earle's salts, basic fibroblast growth factor, epidermal growth factor, insulin, transferrin, selenium, norepinephrine, triiodothyronine (T3), bovine serum albumin, nonessential amino acids, and ascorbic acid. The plating efficiency of the experimental cultures was comparable to that of the control cultures grown in the presence of serum. The cells in the serum-free medium contained myofibrillar and myosin isoforms characteristics of the adult myocytes. The cells underwent cellular reorganization comparable to that of the controls. The initial phase of reorganization involved the breakdown of myofibrils and extrusion of mitochondria, degraded myofibrils, and other cellular organelles. The latter phase of reorganization included myofibrillogenesis and organellogenesis resulting in the development of myofibrillar apparatus with cellular organelles. Myocytes were contractile throughout the culture period. Cardiac myocytes grown in serum-free medium expressed the predominant myosin isoform V1 similar to their counterparts in vivo. T3 is essential for the expression of isomyosin V1.(ABSTRACT TRUNCATED AT 250 WORDS)