Caveolin3 Stabilizes McT1-Mediated Lactate/Proton Transport in Cardiomyocytes

Rationale: Caveolin3 variants associated with arrhythmogenic cardiomyopathy and muscular dystrophy can disrupt post-Golgi surface trafficking. As Caveolin1 was recently identified in cardiomyocytes, we hypothesize that conserved isoform-specific protein/protein interactions orchestrate unique cardiomyocyte microdomain functions. To analyze the Caveolin1 versus Caveolin3 interactome, we employed unbiased live-cell proximity proteomic, isoform-specific affinity, and complexome profiling mass spectrometry techniques. We demonstrate the physiological relevance and loss-of-function mechanism of a novel Caveolin3 interactor in gene-edited human iPSC-cardiomyocytes. Objective: To identify differential Caveolin1 versus Caveolin3 protein interactions and to define the molecular basis of cardiac CAV3 loss-of-function. Methods and Results: Combining stable isotope labeling with proximity proteomics, we applied mass spectrometry to screen for putative Caveolin3 interactors in living cardiomyocytes. Isoform-specific affinity proteomic and co-immunoprecipitation experiments confirmed the monocarboxylate transporter McT1 versus aquaporin1, respectively, as Caveolin3 or Caveolin1 specific interactors in cardiomyocytes. Superresolution STED microscopy showed distinct Caveolin1 versus Caveolin3 cluster distributions in cardiomyocyte transverse tubules. CRISPR/Cas9-mediated Caveolin3 knock-out uncovered a stabilizing role for McT1 surface expression, proton-coupled lactate shuttling, increased late Na+ currents, and early afterdepolarizations in human iPSC-derived cardiomyocytes. Complexome profiling confirmed that McT1 and the Na,K-ATPase form labile protein assemblies with the multimeric Caveolin3 complex. Conclusions: Combining the strengths of proximity and affinity proteomics, we identified isoform-specific Caveolin1 versus Caveolin3 binding partners in cardiomyocytes. McT1 represents a novel class of metabolically relevant Caveolin3-specific interactors close to mitochondria in cardiomyocyte transverse tubules. Caveolin3 knock-out uncovered a previously unknown role for functional stabilization of McT1 in the surface membrane of human cardiomyocytes. Strikingly, Caveolin3 deficient cardiomyocytes exhibit action potential prolongation and instability, reproducing human reentry arrhythmias in silico. Given that lactate is a major substrate for stress adaption both in the healthy and the diseased human heart, future studies of conserved McT1/Caveolin3 interactions may provide rationales to target this muscle-specific assembly function therapeutically.