Abstract 19455: A Novel Caveolin-1 Frameshift Mutation Shows Critical Regulation of Mitochondrial Structure, Function and Extracellular Transport

Abstract

Introduction: Caveolins act as molecular scaffolds for signal transduction. A number of caveolin-1 (Cav-1) mutations are associated with pulmonary hypertension with one recent frameshift mutation in exon 3 (c.478_479delTT) resulting in progeroid-like phenotype, lipodystrophy, and pulmonary hypertension. The cellular etiology of these diverse phenotypes is unknown. We have recently shown a critical relationship between caveolins in regulation of mitochondrial function. Hypothesis: As mitochondria are a key to cellular homeostasis, we hypothesize that a Cav-1 mutation (CM) can alter mitochondrial structure and function. Methods and Results: We obtained fibroblasts from a patient containing the CM as well as controls and utilized a variety of microscopy, cell biology, and biochemical techniques to study the role of Cav-1 in mitochondrial regulation. Transmission electron microscopy revealed dysmorphic nuclei, enlarged and increased number of endoplasmic reticulum, and increased number of smaller, swollen, and rounded mitochondria compared to normal cells. Mitochondrial DNA content was increased in CM cells with immunoblots showing decreased mitochondrial fission protein DRP1. Mitotracker staining showed large pools of aggregated mitochondria and DCFDA assay showed 3-fold increase in production of reactive oxygen species in CM cells compared to normal cells (p<0.0001, n=8). Seahorse metabolic flux assay showed lower maximal respiration rates in CM cells compared to normal cells (p=0.0035, n=24). Mitochondrial complex II activity was impaired as addition of succinate failed to evoke any response to ADP in CM cells compared to normal cells. Live 3D microscopy with mitotracker staining showed extracellular mitochondria being transported along structured nanotubes between normal cell; however, impaired extracellular mitochondrial cargo arrangement and movement was observed in cells with the CM. Rescue experiments with Cav-1 overexpression resulted in improved mitochondrial respiration in CM cells. Conclusion: Together the results indicate that Cav-1 may be a critical regulator of mitochondrial structure, function, and transport and mutations in Cav-1 may explain the mitochondrial etiology of a variety of diseases.