NO synthase II in mouse skeletal muscle is associated with caveolin 3.
Ontology highlight
ABSTRACT:
SUBMITTER: Gath I
PROVIDER: S-EPMC1220304 | biostudies-other | 1999 Jun
REPOSITORIES: biostudies-other
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Project description:BackgroundCaveolin-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.MethodsL6 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.ResultsCaveolin-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.ConclusionsOur 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.
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Project description:Here, we examine the role of "non-muscle" caveolins (Cav-1 and Cav-2) in skeletal muscle biology. Our results indicate that skeletal muscle fibers from male Cav-1(-/-) and Cav-2(-/-) mice show striking abnormalities, such as tubular aggregates, mitochondrial proliferation/aggregation, and increased numbers of M-cadherin-positive satellite cells. Notably, these skeletal muscle defects were more pronounced with increasing age. Because Cav-2-deficient mice displayed normal expression levels of Cav-1, whereas Cav-1-null mice exhibited an almost complete deficiency in Cav-2, these skeletal muscle abnormalities seem to be due to loss of Cav-2. Thus, Cav-2(-/-) mice represent a novel animal model-and the first genetically well-defined mouse model-that can be used to study the pathogenesis of tubular aggregate formation, which remains a poorly understood age-related skeletal muscle abnormality. Finally, because Cav-1 and Cav-2 were not expressed within mature skeletal myofibers, our results indicate that development of these abnormalities probably originates in stem/precursor cells, such as satellite cells or myoblasts. Consistent with this hypothesis, skeletal muscle isolated from male Cav-3(-/-) mice did not show any of these abnormalities. As such, this is the first study linking stem cells with the genesis of these intriguing muscle defects.
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Project description:Angiotensin II (ANG II)-induced skeletal muscle wasting is characterized by activation of the ubiquitin-proteasome system. However, the potential involvement of proteolytic system macroautophagy/autophagy in this wasting process remains elusive. Autophagy is precisely regulated to maintain cell survival and homeostasis; thus its dysregulation (i.e., overactivation or persistent suppression) could lead to detrimental outcomes in skeletal muscle. Here we show that infusion of ANG II for 7 days in male FVB mice suppressed autophagy in skeletal muscle. ANG II blunted microtubule-associated protein 1 light chain 3B (LC3B)-I-to-LC3B-II conversion (an autophagosome marker), increased p62/SQSTM1 (an autophagy cargo receptor) protein expression, and decreased the number of autophagic vacuoles. ANG II inhibited UNC-51-like kinase 1 via inhibition of 5'-AMP-activated kinase and activation of mechanistic target of rapamycin complex 1, leading to reduced phosphorylation of beclin-1Ser14 and Autophagy-related protein 14Ser29, suggesting that ANG II impairs autophagosome formation in skeletal muscle. In line with ANG II-mediated suppression of autophagy, ANG II promoted accumulation of abnormal/damaged mitochondria, characterized by swelling and disorganized cristae and matrix dissolution, with associated increase in PTEN-induced kinase 1 protein expression. ANG II also reduced mitochondrial respiration, indicative of mitochondrial dysfunction. Together, these results demonstrate that ANG II reduces autophagic activity and disrupts mitochondrial ultrastructure and function, likely contributing to skeletal muscle wasting. Therefore, strategies that activate autophagy in skeletal muscle have the potential to prevent or blunt ANG II-induced skeletal muscle wasting in chronic diseases. NEW & NOTEWORTHY Our study identified a novel mechanism whereby angiotensin II (ANG II) impairs mitochondrial energy metabolism in skeletal muscle. ANG II suppressed autophagosome formation by inhibiting the UNC-51-like kinase 1(ULK1)-beclin-1 axis, resulting in accumulation of abnormal/damaged and dysfunctional mitochondria and reduced mitochondrial respiratory capacity. Therapeutic strategies that activate the ULK1-beclin-1 axis have the potential to delay or reverse skeletal muscle wasting in chronic diseases characterized by increased systemic ANG II levels.
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