Detection of calcium release from individual RyR2 clusters in cardiomyocites
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Cardiac ryanodine receptors (RyR2s) are Ca2þ release channels clustering in the sarcoplasmic reticulum membrane. These clusters are believed to be the elementary units of Ca2þ release. The distribution of these Ca2þ release units plays a critical role in determining the spatio-temporal profile and stability of sarcoplasmic reticulum Ca2þ release. RyR2 clusters located in the interior of cardiomyocytes are arranged in highly ordered arrays. However, little is known about the distribution and function of RyR2 clusters in the periphery of cardiomyocytes. Here, we used a knock-in mouse model expressing a green fluorescence protein (GFP)-tagged RyR2 to localize RyR2 clusters in live ventricular myocytes by virtue of their GFP fluorescence. Confocal imaging and total internal reflection fluorescence microscopy was employed to determine and compare the distribution of GFP-RyR2 in the interior and periphery of isolated live ventricular myocytes and in intact hearts. We found tightly ordered arrays of GFP-RyR2 clusters in the interior, as previously described. In contrast, irregular distribution of GFP-RyR2 clusters was observed in the periphery. Time-lapse total internal reflection fluorescence imaging revealed dynamic movements of GFP-RyR2 clusters in the periphery, which were affected by external Ca2þ and RyR2 activator (caffeine) and inhibitor (tetracaine), but little detectable movement of GFP-RyR2 clusters in the interior. Furthermore, simultaneous Ca2þ- and GFP-imaging demonstrated that peripheral RyR2 clusters with an irregular distribution pattern are functional with a Ca2þ release profile similar to that in the interior. These results indicate that the distribution of RyR2 clusters in the periphery of live ventricular myocytes is irregular and dynamic, which is different from that of RyR2 clusters in the interior.
CitationNolla-Colomer, C. [et al.]. Detection of calcium release from individual RyR2 clusters in cardiomyocites. "Biophysical journal", Febrer 2020, vol. 118, núm. 3, p. 343-354.