Distribution of Intracellular ADP Diffusion Restriction in Trout Cardiomyocytes

Cardiomyocytes are compartmentalized by intracellular diffusion restrictions. In our previous study we confirmed that isolated permeabilized cardiomyocytes from rainbow trout (Oncorhynchus mykiss) also display intracellular diffusion restriction (Sokolova et al., BMC Cell Biology, 10:90, 2009). This was surprising, because trout cardiomyocytes are much thinner than those of mammals, lack t-tubules, have a lower sarcoplasmic reticulum density and only a single layer of myofilaments surrounding a central core of mitochondria. However, the extent of diffusion restriction is much smaller than in mammalian cardiomyocytes. The aim of the present study was to find the possible distribution of diffusion restrictions in permeabilized trout cardiomyocytes. We measured mitochondrial respiration stimulated by ADP and ATP, and evaluated the rate of inhibition of ATP stimulated respiration by an ADP trapping system, consisting of phosphoenolpyruvate (PEP) activating endogenous and exogenous pyruvate kinase (PK), which competes with mitochondria for ADP. We found a high activity of hexokinase, which stimulates mitochondrial respiration when activated by glucose. To study its role in more detail, all experiments were performed in the absence and presence of 2 mM glucose. Additionally, we performed ADP titrations on cells alone, in the presence of glucose, and in the presence of creatine kinase and creatine. We found that activation of hexokinase by glucose, but not activation of creatine kinase by creatine, lowered the apparent ADP-affinity in permeabilized trout cardiomyocytes. This is in contrast to the situation in permeabilized mammalian cardiomyocytes, where activation of mitochondrial creatine kinase has a profound effect on the apparent ADP-affinity. This suggests that trout cardiomyocytes have a different ADP-feedback system than mammalian cardiomyocytes. The experimental results will be fitted with a mathematical model (Sepp et al., Biophysical Journal, 98:12, 2010) showing more specifically the distribution and extent of compartmentalization of trout cardiomyocytes.