Abstract Body (Do not enter title and authors here): Introduction: Junctophilin-2 (JPH2) plays essential roles in multiple cardiac processes including calcium handling, t-tubule structure, and gene regulation. We recently showed JPH2 also has a mitochondrial role as it binds the mitochondrial protein mitofusin-2 (MFN2) and modulates mitochondrial metabolic function. However, the impacts of JPH2 disease-causing variants on MFN2 binding and subsequent cellular responses to metabolic stressors are unknown.

Methods: Disease-associated variants in the MFN2 binding domain of JPH2 were engineered and recombinantly expressed in E. coli cells. Purified proteins were then subjected to pulldown experiments to assess MFN2 protein binding. Alphafold3 modeled how MFN2 and JPH2 and JPH2 E169K mutants interacted in the presence or absence of fatty acids. Wildtype JPH2 or JPH2 E169K expression was directed by a doxycycline inducible promoter after being cloned into the AAVS1 locus in JPH2 knockout iPSC-cardiomyocytes. In vitro metabolic stress was induced by incubating iPSC-CM with 150 μM oleate, 150 μM palmitate, and 500 μM carnitine overnight. Super resolution confocal microscopy visualized mitochondria integrity, nuclei shape and size, and lipid droplet accumulation and morphology.

Results: In pulldown experiments, the JPH2 E169K mutation disrupted MFN2 binding. Alphafold3-based modeling showed JPH2 E169K disrupted the interaction between the two proteins when modeled with or without lipids (A-D). Control, wild type JPH2, and JPH2 E169K iPSC-CM displayed divergent responses to in vitro metabolic stress. JPH2 E169K cells exhibited pathological mitochondrial network changes characterized by a lower mitochondrial footprint and less network branches (E-G). In addition, the JPH2 E169K iPSC-CM accumulated significantly more lipid droplets in the cytoplasm and the nucleus, and the JPH2 E169K lipid droplets were significantly larger than the other cell types (H-K). Finally, the accumulation of lipid droplets impacted nuclear morphology as the JPH2 E169K cells had nuclear hypertrophy and nuclei were more ellipse-shaped (L-N).

Conclusion: The JPH2 E169K variant disrupts MFN2 binding, which results in heightened metabolic stress characterized by lipid droplet accumulation and mitochondrial and nuclear morphological changes. These results implicate a new pathophysiological mechanism for how the JPH2 E169K mutation causes cardiac dysfunction.