Caveolae, small ~80 nm membrane invaginations, regulate numerous membrane receptors and signalling pathways within cardiac myocytes. However much of the current knowledge of caveolae is based on experimental evidence from cell lines which do not contain the muscle-specific caveolar proteins (caveolin-3 and cavin-4). Here we examine the populations and protein composition of cardiac caveolae using quantitative protein analysis, protein coat isolation and super-resolution imaging. Cardiac myocytes, isolated from male Wistar rats (230-250 g), were attached to coverslips for super-resolution imaging. Paraformaldehyde-fixed myocytes were labelled with caveolins-3 and cavin-1/cavin-4 antibodies, 10X Expansion Microscopy (ExM) was then performed to achieve ~25nm resolution 1. In cell homogenates caveolar protein abundance was quantified using stable isotope dilution mass spectrometry with custom-made calibration standards 2. HeLa cells and cardiac myocytes were incubated with the cross-linker dithiobis(succinimidyl propionate) (DSP) for isolation of the caveolar coat complex (CCC) on a sucrose velocity gradient 3. Caveolin-3 showed the highest expression within myocytes followed by cavin-1/4 (~60% of caveolin-3). Caveolin-1/2 and cavin-2/3 were detected at lower levels (<20% of caveolin-3)(n = 3 animals). The CCC isolated from HeLa cells was ~80S in size and contained the main ubiquitous isoforms (caveolin-1 and cavin-1)3. In myocytes, the ~80S complex contained only caveolin-1 and cavin-1/4 (n = 4 animals). Despite being the predominant isoform caveolin-3 was absent from these complexes in the cardiac cell. Indeed, caveolin-3 migration in the sucrose gradient did not change with DSP, suggesting that caveolin-3 does not integrate into the CCC in muscle cells as caveolin-1 does in non-muscle cells. Using ExM, caveolin-3 and cavin-1/cavin-4 can clearly be resolved within a single caveola with high levels of co-localisation. Taken together these data highlight distinct differences in caveolae between myocytes and non-muscle cells. Understanding the protein composition of the different caveolae is essential in understanding the multiple functions that caveolae perform. Cavin-1 is lost from the CCC in response to mechanical stimuli (stretch/swelling) in non-cardiac cells and this may impact on caveolar shape and cell function. Whether the same occurs in the cardiac myocyte is not known, despite the key role that mechanotransduction plays in the heart. We are now looking at the composition of the CCC following hypo-osmotic swelling (cells) and stretch (whole heart) using both super-resolution imaging and sub-cellular fractionation.