Caveolae are small (50-100 nm) invaginations of the cell membrane present in most cells. Caveolar proteins (caveolins and cavins) aid in the formation of caveolae and assist with the many functions caveolae perform. Current caveolar research focuses on non-muscle cells which lack the muscle-specific caveolin 3 (cav 3) and cavin 4 proteins. Cardiac myocytes express both muscle specific-caveolar proteins, as well as the ubiquitously expressed caveolin 1 (cav 1) and cavin 1. To date we have little understanding of how caveolar proteins are arranged and interact within the cardiac myocyte. Here, we have quantified protein expression and described protein distribution within the cardiac cell using quantitative western blotting (WB) and Airyscan super-resolution microscopy. Ventricular myocytes isolated from male Wistar rats (240-280 g) were either homogenised in Laemmli sample buffer for WB or attached to laminin-coated coverslips for imaging. Quantitative WB was modelled on the DOSCAT system [1] and performed with a capillary-based Protein Simple system. Peptides were designed to include the epitope for each antibody (cav 1, 3, cavin 1, 4) and used as standards (run in parallel with samples) allowing full quantification of protein concentration. For imaging, isolated cells were electrically stimulated at 1 Hz before being fixed in paraformaldehyde. Antibodies against cav 3 and the sodium-calcium exchanger (NCX) were used to map the cell membrane. Cavin 1, cavin 2 and cavin 4 were visualised by immunofluorescence staining in relation to the membrane (Zeiss LSM880 inverted microscope). Quantitative WB revealed that cav 1, cav 3 and cavin 1 are expressed at similar concentrations within cardiac myocytes, with cavin 4 expressed at lower concentrations (26% of cavin 1). The percentage of staining localised within 150 nm of the membrane was significantly greater for cavin 1 (50 ±2.1%) compared with both cavin 4 (39 ±1.6%) and cavin 2 (42 ±1.9%) (mean ± S.E.M; n=9; ANOVA, P<0.05). At the membrane, cavin 2 and 4 were present in more widely-spaced clusters (puncta) compared with cavin 1 (Fig 1). Nuclear staining was evident for all 3 cavin proteins; in the case of cavin 2 two bright spots were typically present in each nucleus. This is the first quantitative measurement of the caveolar proteins in cardiac myocytes. Super-resolution imaging highlights differences in the subcellular distribution of the cavin proteins, consistent with spatially distinct caveolar sub-populations. In a recent proteomics study, cavin 1 and 2 were shown to be translocated to caveolae after β-adrenergic (β-AR) stimulation [2]. We are currently using super-resolution approaches to describe the impact of β-adrenergic stimulation on molecular-scale clustering of the caveolar proteins. Fig 1. Distribution of cavin proteins in the membrane. Line profiles drawn along the length of the t-tubular membrane (black=cavin; grey=NCX + cav 3)