Caveolae in Rabbit Ventricular Cardiomyocytes: Distribution and Dynamic Diminution after Cell Isolation
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Under temporary indefinite embargo pending publication by the Biophysical Society
Introduction: Caveolae are signal transduction centres, yet their subcellular distribution and preservation in cardiac myocytes after cell-isolation are not well documented. Here, we quantify caveolae, located within 100 nm of the outer cell surface membrane, in rabbit single ventricular cardiomyocytes over 8h post-isolation and relate this to caveolae presence in intact tissue. Methods: Hearts from New Zealand white rabbits were either chemically fixed by coronary perfusion, or enzymatically digested to isolate ventricular myocytes that were subsequently fixed at 0h, 3h and 8h post-isolation. In live cells, the patch-clamp technique was used to measure whole-cell plasma membrane capacitance, and in fixed cells caveolae were quantified by transmission electron microscopy (TEM). Changes in cell surface topology were assessed using scanning electron microscopy (SEM). In fixed ventricular myocardium, dual-axis EM tomography (ET) was used for three-dimensional (3D) reconstruction and analysis of caveolae in situ. Results: Surface-sarcolemmal caveolae presence and distribution in freshly isolated cells matches that of intact myocardium. With time, the number of surface-sarcolemmal caveolae decreases in isolated cardiomyocytes. This is associated with a gradual increase in whole-cell membrane capacitance. Concurrently, there is a significant increase in area, diameter and circularity of sub-sarcolemmal mitochondria, indicative of swelling. In addition, ET data from intact heart illustrate the regular presence of caveolae not only at the surface sarcolemma, but also on T-tubular (T-tub) membranes in ventricular myocardium. Conclusions: Caveolae are dynamic structures, present both at surface sarcolemmal and Ttub membranes. After cell isolation, surface-sarcolemmal caveolae numbers decrease significantly, within a time-frame relevant for single cell research. The concurrent increase in cell capacitance suggests that membrane incorporation of surface-sarcolemmal caveolae underlies this, but internalization and/or micro-vesicle loss to the extracellular space may also contribute. Given that much of the research into cardiac caveolae-dependent signalling utilises isolated cells, and since caveolae-dependent pathways matter for a wide range of other study targets, analysis of isolated cell data should take the time post-isolation into account.
This research was supported by the BBSRC grant #BB/I012117-1. RABB holds a Sir Henry Dale Royal Society and Wellcome Trust Fellowship ((109371/Z/15/Z) and acknowledges support from the Nuffield Benefaction for Medicine and the Wellcome Institutional Strategic Support Fund (ISSF) Oxford and Medical Research Council; EAR-Z is an Immediate PostDoctoral Fellow of the British Heart Foundation (BHF); ADC acknowledges the support of the Worshipful Company of Scientific Instrument Makers. PK is a Senior Fellow of the BHF. PK acknowledges support by the ERC Advanced Grant CardioNECT. Tomography was performed at the Boulder Laboratory for 3D Electron Microscopy of Cells, University of Colorado, Boulder, Colorado (supported by P41GM103431 from the National Institute of Health, and by the Magdi Yacoub Institute).
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