Intracellular diffusion restrictions in isolated cardiomyocytes from rainbow trout

Abstract

Background: Restriction 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 degrees C in the absence and presence of creatine.

Results: Trout 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.

Conclusions: The 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.

Figure 1

Comparison of trout and rat cardiomyocytes. (A) Transmission image of trout and rat cardiomyocytes next to each other in the same solution. Trout cardiomyocytes can be very long, and only half of the trout cardiomyocyte was within the camera field of view. (B, C) Deconvolved confocal images of rat cardiomyocyte (B) and trout cardiomyocyte (C) labeled with di-8-ANEPPS (green; labeling sarcolemma) and mitotracker Red CMXRos (red; labeling mitochondria). The upper image shows one image from a confocal z-stack, and lower image shows re-constructed cross section. Note different size of the scale bars.

Figure 2

Example of respiration of permeabilized cardiomyocytes. Representative example recorded at 10°C in the absence of creatine showing the respiration rate of permeabilized trout cardiomyocytes (CM) during stepwise increases in ADP-concentration as indicated. Note that the respiration rate was relatively stable at each step of the ADP-titration, that cytochrome c did not increase respiration rate, and that atractyloside brought respiration rate down to the same level as the basal respiration rate before addition of ADP.

Figure 3

Apparent ADP-affinity in fibers, cardiomyocytes and mitochondria. The apparent K_{M ADP} of permeabilized fibers, permeabilized cardiomyocytes and isolated mitochondria as obtained by fitting the data with a single hyperbolic equation. All experiments were done at 10, 15 and 20°C, and experiments on fibers and cardiomyocytes were performed in the absence and presence of creatine as indicated below the columns. Notice the different scales on the y-axes.