Influence of SERCA and Actomyosin ATPase on Respiration Kinetics in Permeabilized Rat Cardiomyocytes

Compartmentalization of ATP and ADP by intracellular diffusion restrictions plays a role in heart energetics. While specific causes of diffusion restrictions are not known, intracellular structures are speculated to act as diffusion barriers. We did kinetic experiments on permeabilized rat cardiomyocytes under relaxing conditions and analyzed the data by mathematical modeling. We found significant diffusion restriction by the mitochondrial outer membrane and confirmed a functional coupling between mitochondria and a fraction of ATPases. Such coupling indicates restricted diffusion in the cytosol. In addition, we found evidence for a tight coupling between pyruvate kinase (PK) and some ATPases. However, it is not clear which ATPases are coupled with which cellular energetic sites. The aim of this work was to establish the role of SERCA and myosin ATPase as possible candidates for coupling with PK and mitochondria. First, the effect of SERCA was assessed by inhibition with thapsigargin (TG). We found no effect of TG on respiration and no change in ATPase activity was observed spectrophotometrically. This indicates that SERCA activity is minor in our preparation. Second, the role of myosin ATPase was assessed in cells where myosin had been extracted by incubation in high KCl solution. We recorded respiration rate dependence on exogenous and endogenous ADP, inhibition of ATP-stimulated respiration by endogenous and exogenous PK. In spectrophotometer, we measured activity of ATPases and endogenous PK. We found that while ATP respiration kinetics changed, ADP respiration kinetics were the same as control. To establish the role of actomyosin ATPase, the experimental results will have to be analyzed by mathematical models.