|dc.description.abstracteng||Background and Objectives: The Caveolin (CAV1-3) family contains a unique class of membrane integral proteins with cytosolic termini. CAV1 or CAV3 are essential components of the caveolar core complex, a disc-shaped multimeric macromolecular scaffold, which interacts with membrane lipids and proteins. CAV3 was conceptualized as a muscle-specific and CAV1 alternative isoform. Finally, most CAV3 protein interactions were identified under non-endogenous conditions in heterologous overexpression systems. However, recent quantitative analysis by mass spectrometry demonstrated both CAV1 and CAV3 in the human heart. Therefore, we hypothesized that isoform-specific CAV1 and CAV3 protein interactions provide unique subcellular functions. Therefore, we have targeted the CAV3 complex for live-cell proteomic analysis. Moreover, we developed isoform-specific affinity proteomic approaches to compare CAV1 versus CAV3 interactors. As human CAV3 mutations were associated with action potential prolongation in HEK293 cells, we analyzed the functional impact and pathogenic proteomic mechanisms of the CAV3-F97C and CAV3-S141R mutations in gene edited human induced pluripotent stem cell (iPSC) derived cardiomyocytes.
Methods and Results: In this thesis, an ascorbate peroxidase (APEX2) proximity assay was combined with stable isotope labeling for quantitative CAV3 proximity proteomics. We developed an N-terminally tagged V5-APEX2-CAV3 expression construct for viral transfection of living neonatal rat cardiomyocytes (NRCMs). This assay labels proteins in the proximity of the CAV3 core complex and identified the monocarboxylate transporter (McT1) and the transferrin receptor (TfR1) as novel CAV3 candidate interactors. STED microscopy confirmed the nanometric proximity of McT1 and TfR1 with CAV3 clusters in adult mouse ventricular cardiomyocytes. Affinity proteomics and co-immunoprecipitation of ventricular cardiomyocyte lysates confirmed McT1 and TfR1 as CAV3 interactors, while aquaporin-1 was identified as a novel CAV1 interactor. Importantly, introducing the human mutations in V5-APEX2-CAV3-F97C and V5-APEX2-CAV3-S141R disrupted the proximity of the CAV3 complex with McT1 and TfR1. In addition, V5-APEX2-CAV3-F97C diminished the physiological interactions between essential proteins that constitute the caveolar core complex. CRISPR/Cas9 gene editing was used to generate CAV3 knock-out and CAV3-F97C knock-in human iPSC-derived cardiomyocytes. CAV3 knock-out led to decreased surface expression of both McT1 and TfR1. Importantly, the human CAV3-F97C reduced McT1 surface expression by 97%, destabilizing proton-coupled lactate export and reducing the extracellular acidification, mitochondrial respiration and ATP production. Quantitative mass spectrometry and STED microscopy confirmed abundant CAV1 expression in mouse ventricular cardiomyocytes. Interestingly, CAV1 clusters were juxta-positioned in proximity to CAV3 clusters in transverse tubules. Immunoblotting of atrial cardiomyocytes revealed distinct α and β CAV1 forms, while ventricular cardiomyocytes expressed only the longer CAV1 α-form.
Conclusion: Using a combination of proximity and affinity proteomics, we identified CAV1 and CAV3 isoform-specific protein interactions in cardiomyocytes. McT1 and TfR1 define a new functional group of CAV3 interacting proteins with immediate relevance for cardiac metabolism. CAV3 surface expression was necessary to stabilize McT1 and TfR1 function in the sarcolemmal membrane. Knock-in of F97C in human iPSC-derived cardiomyocytes destabilized McT1 surface expression and lactate-coupled proton export, resulting in depressed mitochondrial respiratory ATP production. These data support a novel pathomechanism for the CAV3-F97C mutation through impaired lactate and proton transport, which may affect mitochondrial function in human cardiomyocytes. Given that lactate is an important energy substrate, the functional stabilization of McT1 provides a novel role of Caveolin3 for cardiac stress adaptation.||de