Isolation and Characterization of Myocytes from the Adult Rat Heart

Abstract

Spontaneously contracting myocytes were isolated from ventricles of the adult rat heart. Hearts were perfused retrogradally via the aorta for 30 minutes at 37°C with Ca²⁺ -free phosphate-buffered saline containing collagenase and hyaluronidase. The ventricles were divided into pieces and incubated 15 minutes with the enzymes. Dislodged cells were decanted, diluted with cold buffer and allowed to settle. The washed cells were then sedimented through 3%Ficoll. This procedure yielded approximately 50 mg of protein from 1 gm of heart. Viability measured by trypan-blue exclusion is 90–95%. Approximately 80% of the cells were beating. Scanning electron microscopic studies suggest that the isolated myocytes are morphologically intact. The cells oxidize glucose, pyruvate, citrate and palmitate to C0₂ and synthesize protein and RNA. Uptake of glucose, 2-deoxyglucose, leucine and taurine was saturable. Glucose uptake was stimulated by insulin. The cells retained LDH and CPK as well as their capacity to oxidize substrates after 24 hours at 4°C or 4 hours at 37°C. After 24 hours at 4°C the cells resumecontracting when returned to room temperature. The procedure reported here for the isolation of spontaneously contracting, adult, rat heart myocytes provides cells with a high index of viability and greater yield than previously reported methods. The cells retain metabolic activity and withstand storage for longer periods than other described preparations.