Nanoscale Changes in the Organisation of Junctional Proteins in JPH2 Transgenic Mice

Abstract

In cardiomyocytes, transverse tubule (t-tubule) and sarcoplasmic reticulum (SR) membranes form close associations, known as diadic junctions, which are critical for excitation-contraction (E-C) coupling. Ryanodine receptors (RyRs), which are the SR calcium release channels, are concentrated in junctions where they play a key role for calcium release. In human and animal models of heart failure junctions and transverse (t-) tubules often become disorganised but the underlying mechanisms remain unclear. The SR membrane protein junctophilin 2 (JPH2) has been implicated in junction formation and maintenance. In this study, transgenic mouse models were used to investigate the influence JPH2 expression on RyR organisation. Cardiomyocytes from adult mice in which JPH2 had been acutely knocked down or over-expressed and control cells labeled for RyR and JPH2. Confocal laser scanning and optical super-resolution microscopy were used to investigate junctional protein organisation and t-tubule architecture.As previously observed in response to JPH2 knockdown t-tubule organisation was disrupted. Regular t-tubules in line with the z-discs were predominant in the control cardiomyocytes, but greatly reduced and irregular in JPH2 knockdown cells. In JPH2 overexpressing myocytes the t-tubules and longitudinal tubules were more abundant.RyR organisation was also altered in response to changes in JPH2 expression. Large RyR superclusters (>200nm diameter) were present in control cells and more abundant in JPH2 over-expressing cardiomyocytes. Such superclusters were mostly absent from JPH2 knockdown cells in which smaller clusters were observed. These changes only became apparent with the resolution provided by optical super-resolution but were difficult to detect with conventional confocal imaging. Our results indicate that cardiomyocyte organisation is severely affected by altered expression of JPH2 and raise questions on the functional role of RyR superclusters and their effect on local and global Ca2+ release.