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Abstract

RATIONALE: CAV3 (caveolin3) variants associated with arrhythmogenic cardiomyopathy and muscular dystrophy can
disrupt post-Golgi surface trafficking. As CAV1 (caveolin1) was recently identified in cardiomyocytes, we hypothesize
that conserved isoform-specific protein/protein interactions orchestrate unique cardiomyocyte microdomain functions.
To analyze the CAV1 versus CAV3 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 CAV3 interactor in gene-edited human induced pluripotent stem cell
cardiomyocytes.
OBJECTIVE: To identify differential CAV1 versus CAV3 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 CAV3 interactors in living cardiomyocytes. Isoform-specific affinity proteomic and co-immunoprecipitation
experiments confirmed the monocarboxylate transporter McT1 (monocarboxylate transporter type 1) versus aquaporin1,
respectively, as CAV3 or CAV1 specific interactors in cardiomyocytes. Superresolution stimulated emission depletion
microscopy showed distinct CAV1 versus CAV3 cluster distributions in cardiomyocyte transverse tubules. CRISPR/
Cas9 (clustered regularly interspaced short palindromic repeats/Cas9 nuclease)-mediated CAV3 knockout uncovered
a stabilizing role for McT1 surface expression, proton-coupled lactate shuttling, increased late Na + currents, and early
afterdepolarizations in human induced pluripotent stem cell-derived cardiomyocytes. Complexome profiling confirmed that
McT1 and the Na,K-ATPase form labile protein assemblies with the multimeric CAV3 complex.
CONCLUSIONS: Combining the strengths of proximity and affinity proteomics, we identified isoform-specific CAV1 versus
CAV3 binding partners in cardiomyocytes. McT1 represents a novel class of metabolically relevant CAV3-specific
interactors close to mitochondria in cardiomyocyte transverse tubules. CAV3 knockout uncovered a previously unknown
role for functional stabilization of McT1 in the surface membrane of human cardiomyocytes. Strikingly, CAV3 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/CAV3 interactions may provide rationales to target this muscle-specific assembly function
therapeutically.