The goal of this grant application is to develop and optimize techniques by which viable cells can be dissociated from hearts of various mammalian species and to define the biochemical, electrophysiological, and morphological characteristics of these cells. We will determine whether specific modifications of the dissociation procedure affect the yield of viable cells in rat. We will determine whether heart cells become hyperpermeable during the dissociation procedure and we will modify the procedure to minimize the period of hypermeability and bolster the metabolic and electophysiological integrity of the cells while they are hyperpermeable. We will develop procedures to yield cells from hearts of other species. Finally, we will seek to achieve an effective dissociation of heart tissue into a representative population of functionally intact cells by enzymatic incubation without a prior (costly) whole organ perfusion. Functional integrity will be monitored by a series of interdisciplinary techniques, with an emphasis on correlating the data from individual cells with the data obtained in large populations. The maintenance of intercellular pH will be monitored in single cells with intracellular ion-selective microelectrodes and by pH-sensitive dyes. In populations, internal pH will be monitored by NMR and pH-sensitive dyes. These data will be correlated with intracellular lactate determination. High energy phosphate levels will be determined in individual cells and compared to values obtained in large populations. Fuctional integrity will also be assayed using electrophysiological techniques. Whole cell recordings using the patch clamp technique will measure membrane conductances, and gap junctional conductance. We will experimentally manipulate high-energy stores and intracellular pH and correlate these changes with the ability to maintain resting potential and low threshold for action potentials. We will compare, using thin section and freeze fracture techniques, morphological changes induced by varying the isolation procedure. We will establish the presence or absence of a glycocalyx and the structural integrity of desmosomes. Morphological comparisons between myocytes from different species will help establish a basis for dissociation procedures. We will extend the electrophysiological, biochemical, and morphological techniques to isolated myocyctes from spontaneously hypertensive rats or rats made diabetic with streptozotocin.
| White, R L; Doeller, J E; Verselis, V K et al. (1990) Gap junctional conductance between pairs of ventricular myocytes is modulated synergistically by H+ and Ca++. J Gen Physiol 95:1061-75 |
| Sorrentino, D; Stump, D; Potter, B J et al. (1988) Oleate uptake by cardiac myocytes is carrier mediated and involves a 40-kD plasma membrane fatty acid binding protein similar to that in liver, adipose tissue, and gut. J Clin Invest 82:928-35 |
| Wittenberg, B A; Wittenberg, J B (1987) Myoglobin-mediated oxygen delivery to mitochondria of isolated cardiac myocytes. Proc Natl Acad Sci U S A 84:7503-7 |
| Wittenberg, B A; White, R L; Ginzberg, R D et al. (1986) Effect of calcium on the dissociation of the mature rat heart into individual and paired myocytes: electrical properties of cell pairs. Circ Res 59:143-50 |
| White, R L; Spray, D C; Campos de Carvalho, A C et al. (1985) Some electrical and pharmacological properties of gap junctions between adult ventricular myocytes. Am J Physiol 249:C447-55 |
