Caveolin3 Stabilizes McT1-Mediated Lactate/Proton Transport in Cardiomyocytes

Abstract

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.

The role of junctophilin proteins in cellular function

“…Early work suggested that JPH2 might associate with the atypical, partly membrane-integral and cytosolic muscle-specific caveolin-3 protein ( 111 ). Caveolin-3 functions as a cholesterol-binding multimeric scaffolding complex, which stabilizes the omega-shaped membrane invaginations known as caveolae ( 112 ). A JPH2 interaction with the cardiac Ca V 1.2-α1C channel and caveolin-3 was proposed based on cultured isolated adult rat cardiomyocyte immunostaining, proximity ligation, and confocal imaging.…”

“…Proximity based ligation of the antibody-labeled JPH2 and caveolin-3 established a nanometric protein association in cultured rat cardiomyocytes ( 113 ). Smaller caveolin-3 scaffolds stabilize the function of multiple membrane transporters including the voltage-dependent cardiac Na V 1.5 channel and the monocarboxylate lactate/pyruvate shuttle McT1 in ventricular cardiomyocytes ( 112 ). Immunoprecipitation experiments confirmed multiple caveolin-3-specific transmembrane protein interactions, raising the possibility that JPH2 is additionally anchored at the cytosolic PM leaflet through direct or indirect binding to caveolin-3 scaffolds.…”

The RyR2-R2474S Mutation Sensitizes Cardiomyocytes and Hearts to Catecholaminergic Stress-Induced Oxidation of the Mitochondrial Glutathione Pool

“…Ventricular cardiomyocytes were isolated and quality controlled as described previously ( Wagner et al, 2014 ; Peper et al, 2021 ). Isolated cardiomyocyte suspensions were plated onto laminin-coated glass coverslips (Imaging Dish CG 1.0, Zellkontakt, Nörten-Hardenberg, Germany) at room temperature.…”

Application of Patient-Specific iPSCs for Modelling and Treatment of X-Linked Cardiomyopathies

“…Since iPSCs are human in origin and pluripotent, they have enabled the development of patient-specific cell models to recapitulate the pathophysiology of human diseases [ 8 ]. This facilitates the study of inherited diseases, since the derived iPSCs and their differentiated cells will contain the same genomic profile as their patient somatic cell origin [ 31 ].…”

“…These cellular and electrophysiological differences may limit the usefulness of rodent models in studying X-linked cardiomyopathy [ 4 ]. With technical advancements in cellular reprogramming, adult somatic cells can now be reprogrammed into pluripotent stem cells [ 5 , 6 , 7 ], and such induced pluripotent stem cells (iPSCs) can be further differentiated into cardiomyocytes for disease modelling and drug testing [ 8 ]. To date, various studies have evaluated the possibility of using patient-specific iPSCs to model different forms of X-linked cardiomyopathy.…”