The isolation of populations of cardiac myocytes should prove to be useful for the study of any number of functional and structural interactions in normal and hypertrophied hearts. Isolated myocytes, like other isolated cell systems, offer the advantage of being free of th hormonal, neuronal, and tissue interactions found in vivo; however, a reproducible method for the isolation and characterization of myocytes is not availabale. In this proposal, we focus on 3 principal aspects of methodology essential to the production of isolated cardiac myocytes. Isolation: 1) Essential to the isolation of reproducible preparations of myocytes will be the characterization of the enzymes used in isolation. This will include purifying the collagenase so that it is free of proteases, evaluation of the purified collagenase and purified collagenase containing known amounts of specific proteases. This will result in a uniform, reproducible """"""""cocktail"""""""" for cell isolation. 2) Methods will be developed for the separation of calcium stable rod-shaped myocytes from injured round myocytes and contaminating stromal cells. Characterization of isolated myocytes: 1) The viability of isolated myocytes will be assessed by testing their stability in physiological concentrations of calcium, measurement of lactate dehydrogenase release, assaying for ATP and creatine phosphate, and trypan blue exclusion as measures of intact sarcolemma. 2) Oxygen consumption will be measured and compared to the values for the intact heart. 3) Immunohistochemistry and conventional electron microscopy (including morphometric analysis) will be used to evaluate the internal filament network. 4) The ability of the isolated myocytes to contract upon electrical stimulation will be evaluated. 5) Changes in calcium levels in response to chemical stimulation will be measured. 6) The presence of intact Beta-receptors that respond to chemical stimulation with an appropriate increase in cyclic nucleotides will be demonstrated. 7) The ability of the cells to recognize and bind to extracellular matrix components will be used as an indication of the lack of damage to the cell surface by the enzymatic digestion used in isolation of the cells. Culture of myocytes: 1) Techniques will be developed for the short term (24-72hr) maintenance of large numbers of rod-shaped myocytes. 2) Myocytes will be established in longterm (72hr to 1 month) culture. In addition, using the above described techniques, cells from experimentally induced hypertrophic hearts will be isolated and compared to age matched normal hearts.
