Remodelling of Ca2+ handling organelles in adult rat ventricular myocytes during long term culture

It is well known that for cardiomyocytes, isolation and culturing induce largely unknown remodelling processes. We analysed changes in the structure of cell compartments with optical techniques such as confocal microscopy and fluorescence redistribution after photobleaching employing adenoviral-mediated transduction of targeted fluorescent proteins and small molecule dyes. We identified characteristic remodelling processes: the T-tubular membrane system was gradually lost by a process referred to as “sequential pinching off”, in an outward direction. Mitochondria fell in one of three classes, very small (0.9 μm length), medium long (1.8 μm) or extended shape (3.6 μm) organelles. Over the culturing time mitochondria gradually fused. Bleaching of individual mitochondria revealed association between apparently separated mitochondria by “tunnelling” via sub-resolution organelle-tubes. This tunnelling process was increasing over the culturing time. A gradual loss of the cross-striation arrangement in the endoplasmic/sarcoplasmic reticulum was visualised. Analysis of large populations of Ca2+ sparks by video-rate confocal 2D-scanning revealed significant albeit small changes of these elementary SR-Ca2+ release events in adult cardiomyocytes that could be related to changes in SR-Ca2+ content rather than resting Ca2+ concentration. In conclusion, primary isolated cardiomyocytes from adult hearts undergo a well-defined, but reproducible subcellular remodelling during optimised long term culture.

Research Highlights
► Long term culture of cardiac myocytes provide an opportunity to study chronic changes
► Subcellular remodelling during cell culture can be observed by optical techniques
► T-tubular membrane system undergoes gradual loss by sequential pinching off
► Mitochondria gradually fuse during culturing period of adult cardiomyocytes
► Controlled cultured adult cardiac myocytes provide a basis for high content screening.