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

Abstract 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

A role for cyclooxygenase2-prostaglandin E2 (COX2-PGE2) pathway in fatty acid-induced insulin resistance and mitochondrial dysfunction in L6 rat myotubes

Aims: The mechanisms by which free fatty acids such as palmitate (PA) induce insulin resistance (IR) remain elusive, but we have recently shown that activation of proinflammatory NFκB signalling contributes to IR and mitochondrial dysfunction in rat L6 myotubes. Here, we have explored the role of the COX2-PGE2 pathway, which lies downstream of NFκB in PA-induced IR and mitochondrial dysfunction.Methods: Rat L6 skeletal myotubes were used for Seahorse XF24 mitochondrial function analysis, qPCR and western blotting, 2-deoxyglucose uptake and confocal microscopy.Results: Our data show that PA reduced insulin-stimulated PKB-S473 phosphorylation, glucose uptake, and reduced ATP-linked respiration by ˜50%, whereas mitochondrial superoxide and H2O2 production were increased by two-fold and 30%, respectively. PA also increased expression of COX2. These metabolic perturbations were mitigated using a COX2 inhibitor, celecoxib, which reduced PA-driven IR, inflammation and excessive superoxide generation, but only partially recovered mitochondrial function. Prostaglandin E2, which is synthesised by the COX2 pathway, targets prostanoid (EP) receptors. EP4 is abundantly expressed in L6 myotubes and, significantly, inhibiting EP4 using an EP4 antagonist ONO-AE3-208 partially protected myotubes against PA-induced IR and reduced inflammatory signalling. In contrast, stimulating EP4 using an EP4 agonist TCS 2510 induced IR in myotubes which was attenuated by ONO-AE3-208, without inducing any changes in mitochondrial function.Summary: The COX2-PGE2 pathway contributes to PA-induced IR. PGE2 synthesised by the COX2 pathway may act in an autocrine manner to activate EP4 and contribute to PA-induced IR without affecting mitochondrial function

Cannabinoid 2 receptor (CB2R) activation promotes beneficial effects on metabolic signalling and mitochondrial function in human adipocytes

Aims: Emerging evidence implicates CB2R, a key component of the endocannabinoid signalling (ECS) system, in the control of adiposity and metabolic function by as yet poorly understood mechanisms. Herein, we explore how CB2R modulation in human adipocytes affects processes linked to the activation of AMP-activated protein kinase (AMPK), a key cellular energy sensor and mitochondrial respiratory capacity.Methods: Using human Simpson–Golabi–Behmel syndrome (SGBS) adipocytes as a model system, we examined changes in AMPK-dependent signalling by immunoblotting following provision of a selective CB2R agonist (AM1241) and inverse antagonist (SR144528). In addition, CB2R modulation by these ligands upon radiolabelled 2-deoxyglucose uptake and mitochondrial oxidative capacity, using a Seahorse XF24 Extracellular Flux Analyzer, was determined.Results: Differentiated SGBS adipocytes express CB2R and treatment with AM1241 significantly increased AMPK-Thr172 phosphorylation (1.7-fold), and that of its downstream substrate acetyl-CoA carboxylase (ACC) (2.2-fold), indicative of AMPK activation. Notably, the stimulatory action of AM1241 upon AMPK was found to be dependent on nitric oxide production via nitric oxide synthase (NOS). Allied to this, CB2R stimulation increased glucose uptake (1.3-fold; p < 0.05) and oxygen consumption (1.6-fold; p < 0.05) rates in SGBS adipocytes.Conclusions: Our data implicate CB2R in the modulation of AMPK-dependent signalling in human adipocytes through a NOS-dependent pathway. Associated improvements in adipocyte glucose transport and mitochondrial oxidative capacity suggest that CB2R may be a promising therapeutic target for mitigating obesity-related metabolic dysfunction

A potential role for caveolins in the regulation of mitochondrial function in muscle cells

Aims: Caveolins (Cavs) are critical components of cholesterol-enriched plasma membrane invaginations known as caveolae and have been implicated as signalling scaffolds that regulate diverse aspects of cell function, including insulin signalling. Emerging evidence suggests that Cavs may also localise to membranes of intracellular organelles, such as the mitochondria where they may potentially influence fuel/energy metabolism. In this study, we have investigated the impact of Cav deficiency on mitochondrial integrity/function in skeletal muscle cells.Methods: A lentiviral-based shRNA strategy was deployed to stably silence expression of Cav 1 and 3 in rat L6 skeletal muscle cells. This approach resulted in greater than 90% loss of Cav1 and Cav3 in muscle cells. L6 cells depleted of either Cav1, Cav3 or both isoforms were used for qPCR, microscopy, fluorescence-based assays as well as analysis of mitochondrial function using a Seahorse extracellular flux analyser.Results: Mitochondrial DNA content, which serves as an indicator of mitochondrial mass, was reduced significantly in single and double Cav1 and Cav3 depleted cells. Confocal microscopy using mitotracker green to label mitochondria revealed elongation and fragmentation of mitochondria in Cav1/3 double-silenced cells. This latter observation implies changes in mitochondrial integrity and function. Consistent with this, we observed significant reductions in mitochondrial membrane potential, oxygen consumption, adenosine triphosphate production as well as a decline in production of reactive oxygen species (p < 0.05).Summary: Our data indicate that Cav1 and Cav3 may be novel regulators in mitochondrial integrity and function in skeletal muscle