Permeabilized Rat Cardiomyocyte Response Demonstrates Intracellular Origin of Diffusion Obstacles

Abstract

Intracellular diffusion restrictions for ADP and other molecules have been predicted earlier based on experiments on permeabilized fibers or cardiomyocytes. However, it is possible that the effective diffusion distance is larger than the cell dimensions due to clumping of cells and incomplete separation of cells in fiber preparations. The aim of this work was to check whether diffusion restrictions exist inside rat cardiomyocytes or are caused by large effective diffusion distance. For that, we determined the response of oxidative phosphorylation (OxPhos) to exogenous ADP and ATP stimulation in permeabilized rat cardiomyocytes using fluorescence microscopy. The state of OxPhos was monitored via NADH and flavoprotein autofluorescence. By varying the ADP or ATP concentration in flow chamber, we determined that OxPhos has a low affinity in cardiomyocytes. The experiments were repeated in a fluorometer on cardiomyocyte suspensions leading to similar autofluorescence changes induced by ADP as recorded under the microscope. ATP stimulated OxPhos more in a fluorometer than under the microscope, which was attributed to accumulation of ADP in fluorometer chamber. By calculating the flow profile around the cell in the microscope chamber and comparing model solutions to measured data, we demonstrate that intracellular structures impose significant diffusion obstacles in rat cardiomyocytes.

Real-time determination of sarcomere length of a single cardiomyocyte during contraction

“…Cardiomyocytes are isolated as described by Sepp et al (20) with modifications from Jepihhina et al (8). …”

Respiration of permeabilized cardiomyocytes from mice: no sex differences, but substrate-dependent changes in the apparent ADP-affinity

“…But the situation in permeabilized cardiomyocytes is not straightforward, because it depends not only on enzyme kinetics but also on the rate of diffusion from the external medium to the adenine nucleotide translocase (ANT) in the inner membrane of all mitochondria. The diffusion is affected by hitherto unidentified barriers inside the cells 39,40 , and only ~2% of VDACs in the outer mitochondrial membrane is accessible to external ADP 22 . Removal of the myofilaments as in so-called „ghost fibres“ does not affect the ADP-affinity 41 .…”

Tight Coupling of Na+/K+-ATPase with Glycolysis Demonstrated in Permeabilized Rat Cardiomyocytes

“…The calcium tolerant cardiomyocytes were isolated using Langendorff perfusion following the procedure described previously in [13] with slightly attuned composition of solutions as listed in [9] and below to give a concise overview.…”

“…In addition to skinned fibers, it is possible that isolated cardiomyocytes – an alternative preparation to study intracellular bioenergetics – could be affected by clumping of cells and formation of micro-aggregates leading to increase of effective diffusion distances [8] . We recently tested this hypothesis by comparing cell population and single cell kinetics on isolated permeabilized rat cardiomyocytes [9] . The results show that diffusion restrictions do have an intracellular origin and are not an artifact of the cell preparation.…”