INTRACELLULAR CA2-PERFUSED RAT-HEART - MEASUREMENT USING THE FLUORESCENT INDICATOR FURA-2( TRANSIENTS IN ISOLATED)AM/

We have investigated the nature of Fura-2/AM loading into isolated per fused rat heart and the temporal and kinetic relationship between left ventricular [Ca2+](i) dependent fluorescence and isovolumic pressure. The contribution of hydrolysed mitochondrial matrix Fura-2 fluorescen ce to that measured from the surface of the heart was estimated to be 43.9 +/- 5.5% by the addition of 100 mu M Mn2+ to the perfusate. Maxim um endothelial Fura-2 fluorescence ratio, estimated by the addition of 3 mu M bradykinin to the perfusate, was found to constitute 33.6 +/- 2.7% of the maximum myocardial Fura-2 fluorescence ratio. Approximatel y 11.2% of the 340 nm surface fluorescence was insensitive to 20 mM Mn 2+ in the presence of ionomycin (3 mu M) and therefore indicates the d egree of partial hydrolysis of Fura-2/AM. Thus, depending on the contr ibution of endothelial Fura-2 fluorescence at a physiological endothel ial calcium concentration, cytosolic fluorescence may comprise between 11-45% of the total cellular fluorescence at 340 nm. Net tissue inter ference of the Fura-2 fluorescence ratio by NADH emission and myoglobi n absorption remained unaltered, providing the oxygenation state of th e tissue was unaltered throughout the experiment. The [Ca2+](i) depend ent fluorescence decay from peak systole was best fitted to a biexpone ntial decay with fast and slow rate constants of 18.08 +/- 1.97 s(-1) and 0.23 +/- 0.02 s(-1), respectively. In addition, a phase shift was observed between temporal and kinetic measurements of the left ventric ular isovolumic pressure and calcium dependent fluorescence traces dur ing a contraction-relaxation cycle. We conclude that despite imperfect Fura-2/AM loading, the temporal and kinetic characteristics of intrac ellular [Ca2+] transients in normal isolated perfused rat heart are si milar to those reported in more controlled preparations such as isolat ed myocytes and cardiac trabeculae.