Molecular and Cellular Biochemistry 102: 1-12, 1991. © 1991 Kluwer Academic Publishers. Printed in the Netherlands. Myocardial Ca-sequestration failure and compensatory increase in Ca-ATPase with congestive cardiomyopathy: kinetic characterization by a homogenate microassay using real-time ratiometric indo-1 spectrofluorometry Peter James O'Brien, Hua Shen, Janice Weiler, Mehdi Mirsalimi and Richard Julian Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, NIG 2W1, Canada Received 13 March 1990; accepted 22 August 1990 Key words: sarcoplasmic reticulum, calcium-sequestration, furazolidone cardiomyopathy, indo-1, spectrofluorometry Abstract A novel, simple, rapid and reproducible microassay is used for kinetic analysis of Ca-sequestration by homogenates of myocardium of turkeys with furazolidone-induced congestive cardiomyopathy. The assay monitors Ca in real-time using dual-emission ratiometric spectrofluorometry and the Ca-indicator dye indo-1. Using this assay and isolated SR studies we make several novel findings regarding the mechanism of SR failure in furazolidone cardiomyopathy. Qualitative differences in Ca-sequestration were not detected between groups. However, compared to controls the furazolidone treatment resulted in: 1) 50% depression in maximal activities (1.54 + 0.36 vs 0.73 ___ 0.12/.tM/sec); 2) 2-fold increases in post-sequestration concentrations of ionized Ca (79 + 23 vs 141 + 13 nmol Ca/L homogenate); 3) 2-fold increases in Ca half-life (415 vs 790 msec); and 4) 25% increased passive Ca-binding capacity of homogenates. The Ca-ATPase specific activity of isolated sarcoplasmic reticulum was 60% increased in congestive cardiomyopathy (543 ___ 140 vs 873 + 108nmol ATP hydrolyzed/min/mg membrane protein) although membrane yield was 20% decreased (0.79 + 0.09 vs 0.63 + 0.03 mg/g heart). The increased ATPase and decreased Ca-uptake activities in combination with the occurrence of 36% cardiac hypertrophy and 19% decreased body weights resulted in estimates of the relative energy cost to the animal for myocardial Ca transport being 5.5-fold increased with cardiomyopathy (20.5 vs 111nmol ATP hydrolyzed per I~M decrease of sarcoplasmic free Ca/kg body weight). These data indicate that congestive cardiomyopathy is associated with markedly increased permeability of sarcoplasmic reticulum to Ca and compensatorily increased Ca-ATPase activity. Accelerated energy con- sumption due to the increased energy cost of Ca transport and increased time of myocyte activation are predicted to predispose the myocardium to fatigue and irreversible failure.