IMMUNOHISTOCHEMICAL ANALYSIS OF THE ADAPTATION OF ADULT GUINEA-PIG CARDIOMYOCYTES IN LONG-TERM CULTURES AND IN COCULTURES WITH CARDIAC NEURONS - A NOVEL MODEL FOR STUDIES OF MYOCARDIAL-FUNCTION

In this study, we used laser confocal scanning microscopy and immunofl uorescent markers to describe the establishment of long-term cultures of adult guinea-pig cardiomyocytes and their cocultures with adult int rinsic cardiac neurons. We have also investigated the effect of platin g density on the adaptation of the myocytes in culture. Providing that the preparation of freshly isolated cardiomyocytes consists mostly (> 80%) of rod-shaped, Ca-tolerant, and quiescent cells and these are pl ated under optimal conditions and density (10(5)/cm(2)), these myocyte s have the following characteristics: (1) they remain elongated with r egular ultrastructural characteristics and quiescent for several days; (2) within 10-14 days, they reestablish their intercellular contacts and resume contractile activity, which becomes synchronous all through the confluent layers; (3) they retain their regular myofibrilar stria tion all through the adaptation to culture conditions without any sign of dedifferentiation or redifferentiation; (4) these characteristics are lost when the cells are plated-at too low (< 10(4)/cm(2)) or too h igh (2 x 10(5)/cm(2)) a density and they exhibit signs of dedifferenti ation; (5) the adult ventricular myocytes appear to retain their abili ty to express atrial natriuretic peptide (ANP), as indicated by immuno reactivity to anti-ANP antibody; (6) this activity seems to be directl y related to the surface area of the myocytes in contact with the subs trate (i.e. to the 'stretch' of the myocytes); (7) the intrinsic cardi ac neurons grow intricate networks of neurites, which form a free-endi ng type of contact with the cocultured myocytes. Long-term cultures of adult guinea-pig ventricular myocytes, alone or in their cocultures w ith cardiac neurons in which both are fully active functionally, provi de a valuable experimental model which opens new possibilities for stu dying the cellular and molecular regulation of myocardial function und er acute or chronic effects of various intrinsic and/or extrinsic fact ors, including neuroregulation.