Ryanodine Receptor Staining Identifies Viable Cardiomyocytes in Human and Rabbit Cardiac Tissue Slices.

Abstract

In terms of preserving multicellularity and myocardial function in vitro, the cultivation of beating myocardial slices is an emerging technique in basic and translational cardiac research. It can be used, for example, for drug screening or to study pathomechanisms. Here, we describe staining for viable cardiomyocytes based on the immunofluorescence of ryanodine receptors (RyRs) in human and rabbit myocardial slices. Biomimetic chambers were used for culture and measurements of contractile force. Fixable fluorophore-conjugated dextran, entering cells with a permeable membrane, was used for death staining. RyRs, nuclei and the extracellular matrix, including the t-system, were additionally stained and analyzed by confocal microscopy and image processing. We found the mutual exclusion of the RyR and dextran signals in cultivated slices. T-System density and nucleus size were reduced in RyR-negative/dextran-positive myocytes. The fraction of RyR-positive myocytes and pixels correlated with the contractile force. In RyR-positive/dextran-positive myocytes, we found irregular RyR clusters and SERCA distribution patterns, confirmed by an altered power spectrum. We conclude that RyR immunofluorescence indicates viable cardiomyocytes in vibratome-cut myocardial slices, facilitating the detection and differential structural analysis of living vs. dead or dying myocytes. We suggest the loss of sarcoplasmic reticulum integrity as an early event during cardiomyocyte death.