Intracellular diffusion restrictions in isolated cardiomyocytes from rainbow trout

Abstract

BackgroundRestriction of intracellular diffusion of adenine nucleotides has been studied intensively on adult rat cardiomyocytes. However, their cause and role in vivo is still uncertain. Intracellular membrane structures have been suggested to play a role. We therefore chose to study cardiomyocytes from rainbow trout (Oncorhynchus mykiss), which are thinner and have fewer intracellular membrane structures than adult rat cardiomyocytes. Previous studies suggest that trout permeabilized cardiac fibers also have diffusion restrictions. However, results from fibers may be affected by incomplete separation of the cells. This is avoided when studying permeabilized, isolated cardiomyocytes. The aim of this study was to verify the existence of diffusion restrictions in trout cardiomyocytes by comparing ADP-kinetics of mitochondrial respiration in permeabilized fibers, permeabilized cardiomyocytes and isolated mitochondria from rainbow trout heart. Experiments were performed at 10, 15 and 20°C in the absence and presence of creatine.ResultsTrout cardiomyocytes hypercontracted in the solutions used for mammalian cardiomyocytes. We developed a new solution in which they retained their shape and showed stable steady state respiration rates throughout an experiment. The apparent ADP-affinity of permeabilized cardiomyocytes was different from that of fibers. It was higher, independent of temperature and not increased by creatine. However, it was still about ten times lower than in isolated mitochondria.ConclusionsThe differences between fibers and cardiomyocytes suggest that results from trout heart fibers were affected by incomplete separation of the cells. However, the lower ADP-affinity of cardiomyocytes compared to isolated mitochondria indicate that intracellular diffusion restrictions are still present in trout cardiomyocytes despite their lower density of intracellular membrane structures. The lack of a creatine effect indicates that trout heart lacks mitochondrial creatine kinase tightly coupled to respiration. This argues against diffusion restriction by the outer mitochondrial membrane. These results from rainbow trout cardiomyocytes resemble those from other low-performance hearts such as neonatal rat and rabbit hearts. Thus, it seems that metabolic regulation is related to cardiac performance, and it is likely that rainbow trout can be used as a model animal for further studies of the localization and role of diffusion restrictions in low-performance hearts.

ADP Compartmentation Analysis Reveals Coupling between Pyruvate Kinase and ATPases in Heart Muscle

“…In rat heart, the apparent magnitude of diffusion restrictions increases during ontogeny ( 3 ) and is diminished by ischemia-reperfusion damage of the tissue ( 4 ). Similar diffusion restrictions have been identified in cardiomyocytes of ectothermic vertebrates ( 5 ) and have been found to depend on acclimation conditions in turtles ( 6 ). In comparison, cultured cardiomyocytes, which do not contract against a workload and depend more on glycolytic than aerobic metabolism, have almost no diffusion restrictions ( 7 ).…”

“…However, fiber preparations can be heterogeneous in terms of diffusion distance, as compared to a suspension of permeabilized cardiomyocytes. As a result, the use of cardiomyocytes to study intracellular energy transfer has increased recently ( 5,17,18 ). However, a systematic study of intracellular diffusion patterns is needed for input into mathematical models.…”

The effect of temperature on mitochondrial respiration in permeabilized cardiac fibres from the freshwater turtle, Trachemys scripta

“…However, variable thermal sensitivities between permeabilized preparations and isolated mitochondria from the same species have been described before. Permeabilized rainbow trout cardiac fibres and isolated cardiomyocytes were virtually insensitive to temperature, while isolated mitochondria exhibited significant reductions in respiration rates with decreasing temperature (Sokolova et al, 2009). The authors of the study could not give an explanation for the variable effects of temperature in the different preparations.…”

“…These restrictions have been attributed to low permeability of the outer mitochondrial membrane and cytosolic diffusion restrictions (Andrienko et al, 2003;Saks et al, 1995). Additionally, the different K m ADP of permeabilized preparations may simply be a result of insufficient cell separation or clustering of fibres in the respirometer (Sokolova et al, 2009). Ectothermic cardiomyocytes have a relatively simple morphology with low volumes and high surface-area to volume ratios (Galli et al, 2006), but intracellular diffusion restrictions have also been reported in the rainbow trout.…”

“…Ectothermic cardiomyocytes have a relatively simple morphology with low volumes and high surface-area to volume ratios (Galli et al, 2006), but intracellular diffusion restrictions have also been reported in the rainbow trout. For example, the ranges of K m ADP for rainbow trout permeabilized fibres (224-783 mmol l À 1 ) and cardiomyocytes (86-93 mmol l À 1 ) are orders of magnitude higher than that of the mitochondria (6-50 mmol l À 1 ) (Birkedal and Gesser, 2003;Guderley and St Pierre, 1999;Sokolova et al, 2009). In our study, we calculated turtle cardiac fibre K m ADP to be within the range of 100-110 mmol l À 1 , which are values usually associated with cardiomyocyte preparations.…”