Examining the Function of a Novel Protein in the Cardiac Junctional Membrane Complex

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PROJECT SUMMARY/ABSTRACT Incidence of heart failure (HF) remains on the rise in the United States despite advances in treatment and prevention. On a cellular level during HF, excitation-contraction coupling and calcium signaling in the heart becomes severely impaired. These mechanisms occur in junctional membrane complexes (JMCs) or cardiac dyads in cardiomyocytes. Cardiac JMCs are the ultrastructure juxtaposed between the transverse tubules (T- tubules) and the sarcoplasmic reticulum (SR) and are integral for facilitating excitation-contraction coupling and calcium signaling. Junctophilin family proteins (JPs) are critical in establishing and modulating JMCs. There are four isoforms of JPs found in excitable tissues (JP1-4). While JP2 has been established as the primary JP isoform expressed in cardiac muscle, our lab has recently found that JP1, normally in skeletal muscle, is also expressed in cardiac muscle. Through examining mouse cardiac tissue, I found that JP1 protein is expressed primarily in the ventricles of the heart and increases during postnatal development until maturity. I have also found that JP1 localizes with JP2 and RyR2, the primary calcium release channel, in the Z-disc. Using a cardiomyocyte specific JP1 knockout mouse model, I found that JP1 knockout mice have a shortened lifespan, reduced cardiac function, and increased incidence of fibrosis and HF compared to control mice. Despite genetic ablation of JP1 in these mice, JP2 protein expression does not change indicating that JP2 is not sufficient to overcome the loss of JP1. To examine whether JP1 expression changes during cardiac stress I used a model of pressure overload-induced cardiac hypertrophy in mice and found that JP1 protein expression decreases with increased cardiac stress. To investigate whether this downregulation could be due to proteolysis by calpain, a stress induced, calcium-dependent protease, I subjected purified recombinant JP1 protein to an in vitro calpain cleavage assay and found JP1 cleaved between amino acid 505 and 510. My primary hypothesis is that JP1 is functionally distinct from JP2 in cardiomyocytes within the cardiac dyad at baseline and in the nucleus after stress-induced cleavage. To test the hypothesis, I will use two novel mouse models developed in our lab: a cardiomyocyte specific knockout of JP1 (JP1cKO), and a global knock-in of 3xHA-tagged JP1. In Aim 1, I plan to examine the functional role of JP1 in cardiac muscle by (1) determining the consequences of depleting JP1 from JMCs by analyzing cardiomyocytes from JP1cKO mice and (2) examining JP1-interacting molecular targets and the potential signaling pathways using proteomics-based approaches and RNA-sequencing. Aim 2 will explore the mechanism of downregulation of JP1 during cardiac stress by further investigating the molecular consequence of calpain proteolysis of JP1. This work will have broad implications for the mechanism of cardiac muscle contraction and calcium signaling in the heart by characterizing a previously unrecognized protein, JP1, in cardiac muscle.