Cardiac E-C Coupling Protein Junctophilin-2 Sequesters Immunogenic Double-Stranded RNA within Protein-RNA Condensates

Cardiomyocytes are susceptible to innate immune activation due to their high mitochondrial content, which increases the risk of mitochondrial double-stranded RNA (dsRNA) escaping into the cytosol. To prevent detrimental immune activation, cardiomyocytes should require mechanisms to sequester baseline dsRNA immunogenicity. Junctophilin-2 (JPH2) is traditionally known as a structural protein involved in cardiac Excitation-Contraction (E-C) coupling. However, we have identified a novel function of JPH2 as a dsRNA-binding protein regulating innate immune response in cardiomyocytes. We found that JPH2 interacts with dsRNA sensor MDA5, and this interaction was enhanced by upregulating cytosolic dsRNA. Overexpression of JPH2 reduced MDA5-dsRNA binding in mouse cardiomyocytes, while JPH2 deficiency resulted in increased MDA5-dsRNA interaction, MAVS-TBK1 interaction, and spontaneous interferon-beta (IFNβ1) production, indicating that loss of JPH2 drives innate immune activation even in the absence of pathogens. Consistently, JPH2 knockout in mouse hearts led to upregulation of the dsRNA-induced immune signaling cascade. Mechanistically, JPH2 directly binds dsRNA sequences, including poly(I:C) and those derived from bidirectional transcription of mitochondrial DNA, as demonstrated by gel-shift assays. Upon binding dsRNA, JPH2 undergoes condensation and aggregation, characterized by reduced molecular mobility and solvent insolubility. Accordingly, the presence of JPH2 reduced the accessibility of dsRNA, as revealed by RNASE protection assay, suggesting that JPH2 sequesters dsRNA within condensates to shield it from cytosolic dsRNA sensors, thereby reducing dsRNA immunogenicity. Our findings uncover a previously unrecognized role of JPH2 in regulating cardiomyocyte immune homeostasis through its ability to bind dsRNA and form protective condensates. This work highlights JPH2 as a critical mediator in preventing spontaneous activation of innate immune responses in the heart. This work was supported by NIH (5R03AG077141-02) and ND EPSCoR. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.