Caveolin-3 differentially orchestrates cholinergic and serotonergic constriction of murine airways

Keshavarz, Maryam; Skill, Melanie; Hollenhorst, Monika I.; Maxeiner, Stephan; Walecki, Magdalena; Pfeil, Uwe; Kummer, Wolfgang; Krasteva‐Christ, Gabriela

doi:10.1038/s41598-018-25445-1

Cited by 5 publications

(3 citation statements)

References 65 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As with other Cav3 knockout mouse strains that have been reported in the literature, 33,34 the mice used in the current study were generated by targeting exon-2 of the Cav3 gene using the gene targeting approach detailed previously. 35 Mice were held according to the German guidelines for the care and use of laboratory animals and experiments approved by the Saarland's institutional Animal Care and Use Committee. The phenotype of Cav3 À/À mice has been well documented, 9 but adult mice exhibit myopathic changes resembling those described in patients with LCMD1C.…”

Section: Animalsmentioning

confidence: 99%

Caveolin‐3 deficiency associated with the dystrophy P104L mutation impairs skeletal muscle mitochondrial form and function

Shah

Nisr

Stretton

et al. 2020

J cachexia sarcopenia muscle

View full text Add to dashboard Cite

Background Caveolin‐3 (Cav3) is the principal structural component of caveolae in skeletal muscle. Dominant pathogenic mutations in the Cav3 gene, such as the Limb Girdle Muscular Dystrophy‐1C (LGMD1C) P104L mutation, result in substantial loss of Cav3 and myopathic changes characterized by muscle weakness and wasting. We hypothesize such myopathy may also be associated with disturbances in mitochondrial biology. Herein, we report studies assessing the effects of Cav3 deficiency on mitochondrial form and function in skeletal muscle cells. Methods L6 myoblasts were stably transfected with Cav3P104L or expression of native Cav3 repressed by shRNA or CRISPR/Cas9 genome editing prior to performing fixed/live cell imaging of mitochondrial morphology, subcellular fractionation and immunoblotting, or analysis of real time mitochondrial respiration. Skeletal muscle from wild‐type and Cav3−/− mice was processed for analysis of mitochondrial proteins by immunoblotting. Results Caveolin‐3 was detected in mitochondrial‐enriched membranes isolated from mouse gastrocnemius muscle and L6 myoblasts. Expression of Cav3P104L in L6 myoblasts led to its targeting to the Golgi and loss of native Cav3 (>95%), including that associated with mitochondrial membranes. Cav3P104L reduced mitochondrial mass and induced fragmentation of the mitochondrial network that was associated with significant loss of proteins involved in mitochondrial biogenesis, respiration, morphology, and redox function [i.e. PGC1α, succinate dehyrdogenase (SDHA), ANT1, MFN2, OPA1, and MnSOD). Furthermore, Cav3P104L myoblasts exhibited increased mitochondrial cholesterol and loss of cardiolipin. Consistent with these changes, Cav3P104L expression reduced mitochondrial respiratory capacity and increased myocellular superoxide production. These morphological, biochemical, and functional mitochondrial changes were phenocopied in myoblasts in which Cav3 had been silenced/knocked‐out using shRNA or CRISPR. Reduced mitochondrial mass, PGC1α, SDHA, ANT1, and MnSOD were also demonstrable in Cav3−/− mouse gastrocnemius. Strikingly, Cav3 re‐expression in Cav3KO myoblasts restored its mitochondrial association and facilitated reformation of a tubular mitochondrial network. Significantly, re‐expression also mitigated changes in mitochondrial superoxide, cholesterol, and cardiolipin content and recovered cellular respiratory capacity. Conclusions Our results identify Cav3 as an important regulator of mitochondrial homeostasis and reveal that Cav3 deficiency in muscle cells associated with the Cav3P104L mutation invokes major disturbances in mitochondrial respiration and energy status that may contribute to the pathology of LGMD1C.

show abstract

Section: Animalsmentioning

confidence: 99%

Caveolin‐3 deficiency associated with the dystrophy P104L mutation impairs skeletal muscle mitochondrial form and function

Shah

Nisr

Stretton

et al. 2020

J cachexia sarcopenia muscle

View full text Add to dashboard Cite

show abstract

“…Cav3 knockout (Cav3KO) mice were produced by targeting exon-2 of the Cav3 gene using the gene targeting approach detailed previously. 19 Mice were maintained according to the German guidelines for the care and use of laboratory animals and experiments approved by the Saarland's Institutional Animal Care and Use Committee. Mice were anaesthetized using isoflurane and killed by severing the aorta within the thoracic cavity.…”

Section: Animalsmentioning

confidence: 99%

Caveolin‐3 loss linked with the P104L LGMD‐1C mutation modulates skeletal muscle mTORC1 signalling and cholesterol homeostasis

Shah,

Nisr,

Krasteva‐Christ

et al. 2023

J cachexia sarcopenia muscle

View full text Add to dashboard Cite

BackgroundCaveolins are the principal structural components of plasma membrane caveolae. Dominant pathogenic mutations in the muscle‐specific caveolin‐3 (Cav3) gene isoform, such as the limb girdle muscular dystrophy type 1C (LGMD‐1C) P104L mutation, result in dramatic loss of the Cav3 protein and pathophysiological muscle weakness/wasting. We hypothesize that such muscle degeneration may be linked to disturbances in signalling events that impact protein turnover. Herein, we report studies assessing the effects of Cav3 deficiency on mammalian or mechanistic target of rapamycin complex 1 (mTORC1) signalling in skeletal muscle cells.MethodsL6 myoblasts were stably transfected with Cav3P104L or expression of native Cav3 was abolished by CRISPR/Cas9 genome editing (Cav3 knockout [Cav3KO]) prior to performing subcellular fractionation and immunoblotting, analysis of real‐time mitochondrial respiration or fixed cell immunocytochemistry. Skeletal muscle from wild‐type and Cav3−/− mice was processed for immunoblot analysis of downstream mTORC1 substrate phosphorylation.ResultsCav3 was detected in lysosomal‐enriched membranes isolated from L6 myoblasts and observed by confocal microscopy to co‐localize with lysosomal‐specific markers. Cav3P104L expression, which results in significant (~95%) loss of native Cav3, or CRISPR/Cas9‐mediated Cav3KO, reduced amino acid‐dependent mTORC1 activation. The decline in mTORC1‐directed signalling was detected by immunoblot analysis of L6 muscle cells and gastrocnemius Cav3−/− mouse muscle as judged by reduced phosphorylation of mTORC1 substrates that play key roles in the initiation of protein synthesis (4EBP1S65 and S6K1T389). S6K1T389 and 4EBP1S65 phosphorylation reduced by over 75% and 80% in Cav3KO muscle cells and by over 90% and 30% in Cav3−/− mouse skeletal muscle, respectively. The reduction in protein synthetic capacity in L6 muscle cells was confirmed by analysis of puromycylated peptides using the SUnSET assay. Cav3 loss was also associated with a 26% increase in lysosomal cholesterol, and pharmacological manipulation of lysosomal cholesterol was effective in replicating the reduction in mTORC1 activity observed in Cav3KO cells. Notably, re‐expression of Cav3 in Cav3KO myoblasts normalized lysosomal cholesterol content, which coincided with a recovery in protein translation and an associated increase in mTORC1‐directed phosphorylation of downstream targets.ConclusionsOur findings indicate that Cav3 can localize on lysosomal membranes and is a novel regulator of mTORC1 signalling in muscle. Cav3 deficiency associated with the Cav3P104L mutation impairs mTORC1 activation and protein synthetic capacity in skeletal muscle cells, which may be linked to disturbances in lysosomal cholesterol trafficking and contribute to the pathology of LGMD‐1C.

show abstract

“…For that purpose, three different lysis buffers were tested in parallel according to previously reported CAV3 immunoprecipitation protocols in heterologous cell systems. 135,136,123 Specifically, three whole ventricular tissues were lysed with either octyl β-D-glucopyranoside (OGP), sodium deoxycholate (DCA) or 3-((3cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS), and CAV3 affinity purified interactors were quantified by Sequential Window Acquisition of All Theoretical Mass Spectra (AP/SWATH-MS) (Figure 5.1 A). Mass spectrometry was carried out by the proteomic service unit (Dr. Christof Lenz, Institute of Clinical Chemistry, University Medical Center Göttingen, Göttingen).…”

Section: Validation Of Cav Immunoprecipitation Conditionsmentioning

confidence: 99%

Proximity and Affinity based Analysis of Cardiac Caveolin Protein Interactions

Peper¹

View full text Add to dashboard Cite

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 coimmunoprecipitation 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 Abstract XIV spectrometry and STED microscopy confirmed abundant CAV1 expression in mouse ventricular cardiomyocytes. Interestingly, CAV1 clusters were juxtapositioned in proximity to CAV3 clusters in transverse tubules. Immunoblotting of atrial cardiomyocytes revealed distinct α and β CAV1 forms, while vent...

show abstract

Caveolin-3 differentially orchestrates cholinergic and serotonergic constriction of murine airways

Cited by 5 publications

References 65 publications

Caveolin‐3 deficiency associated with the dystrophy P104L mutation impairs skeletal muscle mitochondrial form and function

Caveolin‐3 deficiency associated with the dystrophy P104L mutation impairs skeletal muscle mitochondrial form and function

Caveolin‐3 loss linked with the P104L LGMD‐1C mutation modulates skeletal muscle mTORC1 signalling and cholesterol homeostasis

Proximity and Affinity based Analysis of Cardiac Caveolin Protein Interactions

Contact Info

Product

Resources

About