Caveolin-3 is required for regulation of transient outward potassium current by angiotensin II in mouse atrial myocytes.

Angiotensin II (AngII) is a key mediator of the renin-angiotensin system and plays an important role in the regulation of cardiac electrophysiology by affecting various cardiac ion currents, including transient outward potassium current, Ito. AngII receptors and molecular components of Ito, Kv4.2 and Kv4.3 channels, have been linked to caveolae structures. However, their functional interaction and the importance of such proximity within 50- to 100-nm caveolar nanodomains remain unknown. To address this, we studied the mechanisms of Ito regulation by AngII in atrial myocytes of wild-type (WT) and cardiac-specific caveolin-3 (Cav3) conditional knockout (Cav3KO) mice. We showed that in WT atrial myocytes, a short-term (2 h) treatment with AngII (5 µM) significantly reduced Ito density. This effect was prevented 1) by a 30-min pretreatment with a selective antagonist of AngII receptor 1 (Ang1R) losartan (2 µM) or 2) by a selective inhibition of protein kinase C (PKC) by BIM1 (10 µM). The effect of AngII on Ito was completely abolished in Cav3-KO mice, with no change in a baseline Ito current density. In WT atria, Ang1Rs co-localized with Cav3, and the expression of Ang1Rs was significantly decreased in Cav3KO in comparison with WT mice, whereas no change in Kv4.2 and Kv4.3 protein expression was observed. Overall, our findings demonstrate that Cav3 is involved in the regulation of Ang1R expression and is required for the modulation of Ito by AngII in mouse atrial myocytes.NEW & NOTEWORTHY Angiotensin II receptor 1 is associated with caveolae and caveolar scaffolding protein caveolin-3 in mouse atrial myocytes that is required for the regulation of Ito by angiotensin II. Downregulation of caveolae/caveolin-3 disrupts this regulation and may be implicated in pathophysiological atrial remodeling.

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

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.

Caveolin-3 KO disrupts t-tubule structure and decreases t-tubular ICa density in mouse ventricular myocytes.

Caveolin-3 (Cav-3) is a protein that has been implicated in t-tubule formation and function in cardiac ventricular myocytes. In cardiac hypertrophy and failure, Cav-3 expression decreases, t-tubule structure is disrupted, and excitation-contraction coupling is impaired. However, the extent to which the decrease in Cav-3 expression underlies these changes is unclear. We therefore investigated the structure and function of myocytes isolated from the hearts of Cav-3 knockout (KO) mice. These mice showed cardiac dilatation and decreased ejection fraction in vivo compared with wild-type control mice. Isolated KO myocytes showed cellular hypertrophy, altered t-tubule structure, and decreased L-type Ca2+ channel current ( ICa) density. This decrease in density occurred predominantly in the t-tubules, with no change in total ICa, and was therefore a consequence of the increase in membrane area. Cav-3 KO had no effect on L-type Ca2+ channel expression, and C3SD peptide, which mimics the scaffolding domain of Cav-3, had no effect on ICa in KO myocytes. However, inhibition of PKA using H-89 decreased ICa at the surface and t-tubule membranes in both KO and wild-type myocytes. Cav-3 KO had no significant effect on Na+/Ca2+ exchanger current or Ca2+ release. These data suggest that Cav-3 KO causes cellular hypertrophy, thereby decreasing t-tubular ICa density. NEW & NOTEWORTHY Caveolin-3 (Cav-3) is a protein that inhibits hypertrophic pathways, has been implicated in the formation and function of cardiac t-tubules, and shows decreased expression in heart failure. This study demonstrates that Cav-3 knockout mice show cardiac dysfunction in vivo, while isolated ventricular myocytes show cellular hypertrophy, changes in t-tubule structure, and decreased t-tubular L-type Ca2+ current density, suggesting that decreased Cav-3 expression contributes to these changes in cardiac hypertrophy and failure.

Cardiomyocyte Membrane Structure and cAMP Compartmentation Produce Anatomical Variation in ß2AR-cAMP Responsiveness in Murine Hearts.

Cardiomyocytes from the apex but not the base of the heart increase their contractility in response to ß2-adrenoceptor (ß2AR) stimulation, which may underlie the development of Takotsubo cardiomyopathy. However, both cell types produce comparable cytosolic amounts of the second messenger cAMP. We investigated this discrepancy using nanoscale imaging techniques and found that, structurally, basal cardiomyocytes have more organized membranes (higher T-tubular and caveolar densities). Local membrane microdomain responses measured in isolated basal cardiomyocytes or in whole hearts revealed significantly smaller and more short-lived ß2AR/cAMP signals. Inhibition of PDE4, caveolar disruption by removing cholesterol or genetic deletion of Cav3 eliminated differences in local cAMP production and equilibrated the contractile response to ß2AR. We conclude that basal cells possess tighter control of cAMP because of a higher degree of signaling microdomain organization. This provides varying levels of nanostructural control for cAMP-mediated functional effects that orchestrate macroscopic, regional physiological differences within the heart.

Caveolin 3 deficiency myopathy associated with dyslipidemia: Treatment challenges and possible pathophysiological association.

We report the case of a patient treated at the lipid clinic because of high cholesterol levels with consistently elevated creatine kinase concentrations that precluded statin treatment. Electromyography showed a rippling muscle disease pattern. A muscle biopsy confirmed caveolin 3 deficiency, and a missense mutation in the CAV3 gene was identified. The patient could be properly managed with ezetimibe and cholestyramine, which reduced the low-density lipoprotein cholesterol by 30%. He remains asymptomatic after 10 years of follow-up. Caveolae and caveolins are essential to membrane integrity, and their deficit has been associated with insulin resistance and hypercholesterolemia in animal models. Therefore, a putative pathophysiological association between myopathy and lipid metabolism mediated by functional alterations of membrane receptors is considered.

Heterozygous caveolin-3 mice show increased susceptibility to palmitate-induced insulin resistance.

Insulin resistance and diabetes are comorbidities of obesity and affect one in 10 adults in the United States. Despite the high prevalence, the mechanisms of cardiac insulin resistance in obesity are still unclear. We test the hypothesis that the insulin receptor localizes to caveolae and is regulated through binding to caveolin-3 (CAV3). We further test whether haploinsufficiency forCAV3 increases the susceptibility to high-fat-induced insulin resistance. We used in vivo and in vitro studies to determine the effect of palmitate exposure on global insulin resistance, contractile performance of the heart in vivo, glucose uptake in the heart, and on cellular signaling downstream of theIR We show that haploinsufficiency forCAV3 increases susceptibility to palmitate-induced global insulin resistance and causes cardiomyopathy. On the basis of fluorescence energy transfer (FRET) experiments, we show thatCAV3 andIRdirectly interact in cardiomyocytes. Palmitate impairs insulin signaling by a decrease in insulin-stimulated phosphorylation of Akt that corresponds to an 87% decrease in insulin-stimulated glucose uptake inHL-1 cardiomyocytes. Despite loss of Akt phosphorylation and lower glucose uptake, palmitate increased insulin-independent serine phosphorylation ofIRS-1 by 35%. In addition, we found lipid induced downregulation ofCD36, the fatty acid transporter associated with caveolae. This may explain the problem the diabetic heart is facing with the simultaneous impairment of glucose uptake and lipid transport. Thus, these findings suggest that loss ofCAV3 interferes with downstream insulin signaling and lipid uptake, implicatingCAV3 as a regulator of theIRand regulator of lipid uptake in the heart.

Caveolinopathies in Greece.

INTRODUCTION: Mutations in the CAV3 gene are usually inherited in an autosomal dominant manner and lead to distinct disorders including limb-girdle muscular dystrophy 1C, rippling muscle disease, and isolated creatine kinase elevation. PATIENTS AND METHODS: The features of the first patients with caveolin-3 deficiency from Greece are presented. Patients' phenotypes ranged from asymptomatic creatine kinase elevation to severe weakness of lower extremities. Clinical evaluation disclosed muscle hypertrophy in 2 patients, whereas percussion-induced muscle mounding was a consistent finding in all of them. Muscle histopathology was variable and unrelated with disease severity. The diagnosis was based on the immunohistochemical study of caveolin-3 expression and molecular analysis of the caveolin-3 gene. CONCLUSIONS: Clinical manifestations and histochemical findings in caveolinopathy patients may be mild or nonspecific or overlapping with features of other muscular dystrophies. Immunohistochemical study of caveolin-3 expression on muscle biopsy should be routinely performed when investigating isolated hyperCKemia or undetermined myopathy especially in the presence of percussion-induced muscle mounding.

Sarcolemmal cholesterol and caveolin-3 dependence of cardiac function, ischemic tolerance, and opioidergic cardioprotection.

Cholesterol-rich caveolar microdomains and associated caveolins influence sarcolemmal ion channel and receptor function and protective stress signaling. However, the importance of membrane cholesterol content to cardiovascular function and myocardial responses to ischemia-reperfusion (I/R) and cardioprotective stimuli are unclear. We assessed the effects of graded cholesterol depletion with methyl-ß-cyclodextrin (MßCD) and lifelong knockout (KO) or overexpression (OE) of caveolin-3 (Cav-3) on cardiac function, I/R tolerance, and opioid receptor (OR)-mediated protection. Langendorff-perfused hearts from young male C57Bl/6 mice were untreated or treated with 0.02-1.0 mM MßCD for 25 min to deplete membrane cholesterol and disrupt caveolae. Hearts were subjected to 25-min ischemia/45-min reperfusion, and the cardioprotective effects of morphine applied either acutely or chronically [sustained ligand-activated preconditioning (SLP)] were assessed. MßCD concentration dependently reduced normoxic contractile function and postischemic outcomes in association with graded (10-30%) reductions in sarcolemmal cholesterol. Cardioprotection with acute morphine was abolished with ≥20 µM MßCD, whereas SLP was more robust and only inhibited with ≥200 µM MßCD. Deletion of Cav-3 also reduced, whereas Cav-3 OE improved, myocardial I/R tolerance. Protection via SLP remained equally effective in Cav-3 KO mice and was additive with innate protection arising with Cav-3 OE. These data reveal the membrane cholesterol dependence of normoxic myocardial and coronary function, I/R tolerance, and OR-mediated cardioprotection in murine hearts (all declining with cholesterol depletion). In contrast, baseline function appears insensitive to Cav-3, whereas cardiac I/R tolerance parallels Cav-3 expression. Novel SLP appears unique, being less sensitive to cholesterol depletion than acute OR protection and arising independently of Cav-3 expression.

Traumatic brain injury enhances neuroinflammation and lesion volume in caveolin deficient mice.

BACKGROUND: Traumatic brain injury (TBI) enhances pro-inflammatory responses, neuronal loss and long-term behavioral deficits. Caveolins (Cavs) are regulators of neuronal and glial survival signaling. Previously we showed that astrocyte and microglial activation is increased in Cav-1 knock-out (KO) mice and that Cav-1 and Cav-3 modulate microglial morphology. We hypothesized that Cavs may regulate cytokine production after TBI. METHODS: Controlled cortical impact (CCI) model of TBI (3 m/second; 1.0 mm depth; parietal cortex) was performed on wild-type (WT; C57Bl/6), Cav-1 KO, and Cav-3 KO mice. Histology and immunofluorescence microscopy (lesion volume, glia activation), behavioral tests (open field, balance beam, wire grip, T-maze), electrophysiology, electron paramagnetic resonance, membrane fractionation, and multiplex assays were performed. Data were analyzed by unpaired t tests or analysis of variance (ANOVA) with post-hoc Bonferroni's multiple comparison. RESULTS: CCI increased cortical and hippocampal injury and decreased expression of MLR-localized synaptic proteins (24 hours), enhanced NADPH oxidase (Nox) activity (24 hours and 1 week), enhanced polysynaptic responses (1 week), and caused hippocampal-dependent learning deficits (3 months). CCI increased brain lesion volume in both Cav-3 and Cav-1 KO mice after 24 hours (P < 0.0001, n = 4; one-way ANOVA). Multiplex array revealed a significant increase in expression of IL-1ß, IL-9, IL-10, KC (keratinocyte chemoattractant), and monocyte chemoattractant protein 1 (MCP-1) in ipsilateral hemisphere and IL-9, IL-10, IL-17, and macrophage inflammatory protein 1 alpha (MIP-1α) in contralateral hemisphere of WT mice after 4 hours. CCI increased IL-2, IL-6, KC and MCP-1 in ipsilateral and IL-6, IL-9, IL-17 and KC in contralateral hemispheres in Cav-1 KO and increased all 10 cytokines/chemokines in both hemispheres except for IL-17 (ipsilateral) and MIP-1α (contralateral) in Cav-3 KO (versus WT CCI). Cav-3 KO CCI showed increased IL-1ß, IL-9, KC, MCP-1, MIP-1α, and granulocyte-macrophage colony-stimulating factor in ipsilateral and IL-1ß, IL-2, IL-9, IL-10, and IL-17 in contralateral hemispheres (P = 0.0005, n = 6; two-way ANOVA) compared to Cav-1 KO CCI. CONCLUSION: CCI caused astrocyte and microglial activation and hippocampal neuronal injury. Cav-1 and Cav-3 KO exhibited enhanced lesion volume and cytokine/chemokine production after CCI. These findings suggest that Cav isoforms may regulate neuroinflammatory responses and neuroprotection following TBI.

Sevoflurane preconditioning attenuates myocardial ischemia/reperfusion injury via caveolin-3-dependent cyclooxygenase-2 inhibition.

BACKGROUND: The inhaled anesthetic sevoflurane has been demonstrated to protect against myocardial ischemia/reperfusion (MI/R) injury via mechanisms involving AMP-activated protein kinase (AMPK) and caveolin-3 (Cav-3). However, the relative contributions of AMPK and Cav-3 to sevoflurane preconditioning (SF-PreCon)-mediated cardioprotection and their precise underlying mechanisms of action remain incompletely understood. METHODS AND RESULTS: SF-PreCon (consisting of 3 cycles of 15-minute exposure to 2% sevoflurane before 30 minutes of MI) decreased MI/R injury in wild-type mice (caspase-3 activity, -29.1%; infarct size, -20.2%; and left ventricular end diastolic pressure, -33.8%). In cardiac-specific AMPKα2 dominant-negative overexpressing mice, the cardioprotective effect of SF-PreCon was largely retained (caspase-3 activity, -26.7%; infarct size, -16.7%; and left ventricular end-diastolic pressure, -25.9%; P<0.01). In contrast, SF-PreCon failed to significantly protect Cav-3 knockout mice against MI/R injury (P>0.05). SF-PreCon significantly decreased MI/R-induced superoxide generation in wild-type (-43.6%) and AMPK dominant-negative overexpressing mice (-35.5%; P<0.01) but not in Cav-3 knockout mice. SF-PreCon did not affect nicotinamide adenine dinucleotide phosphate oxidase expression but significantly inhibited cyclooxygenase-2 expression in wild-type (-38.7%) and AMPK dominant-negative overexpressing mice (-35.8%) but not in Cav-3 knockout mice. CONCLUSIONS: We demonstrate for the first time SF-PreCon mediates cardioprotection against MI/R injury via caveolin-3-dependent cyclooxygenase-2 inhibition and antioxidative effects.

Palmitate diet-induced loss of cardiac caveolin-3: a novel mechanism for lipid-induced contractile dysfunction.

Obesity is associated with an increased risk of cardiomyopathy, and mechanisms linking the underlying risk and dietary factors are not well understood. We tested the hypothesis that dietary intake of saturated fat increases the levels of sphingolipids, namely ceramide and sphingomyelin in cardiac cell membranes that disrupt caveolae, specialized membrane micro-domains and important for cellular signaling. C57BL/6 mice were fed two high-fat diets: palmitate diet (21% total fat, 47% is palmitate), and MCT diet (21% medium-chain triglycerides, no palmitate). We established that high-palmitate feeding for 12 weeks leads to 40% and 50% increases in ceramide and sphingomyelin, respectively, in cellular membranes. Concomitant with sphingolipid accumulation, we observed a 40% reduction in systolic contractile performance. To explore the relationship of increased sphingolipids with caveolins, we analyzed caveolin protein levels and intracellular localization in isolated cardiomyocytes. In normal cardiomyocytes, caveolin-1 and caveolin-3 co-localize at the plasma membrane and the T-tubule system. However, mice maintained on palmitate lost 80% of caveolin-3, mainly from the T-tubule system. Mice maintained on MCT diet had a 90% reduction in caveolin-1. These data show that caveolin isoforms are sensitive to the lipid environment. These data are further supported by similar findings in human cardiac tissue samples from non-obese, obese, non-obese cardiomyopathic, and obese cardiomyopathic patients. To further elucidate the contractile dysfunction associated with the loss of caveolin-3, we determined the localization of the ryanodine receptor and found lower expression and loss of the striated appearance of this protein. We suggest that palmitate-induced loss of caveolin-3 results in cardiac contractile dysfunction via a defect in calcium-induced calcium release.

TGFß signaling: its role in fibrosis formation and myopathies.

PURPOSE OF REVIEW: Modifiers of TGFß signaling have been investigated as treatment options for several types of muscle diseases. The purpose of this review is to focus on the most recent studies that have used this treatment strategy for pathological muscle disorders. We also review the recent insight into the mechanistic processes by which TGFß signaling contributes to these pathologies by promoting fibrosis formation. RECENT FINDINGS: Recent research has shed light on the role of TGFß signaling in the regulation of microRNAs associated with fibrosis formation. Inhibition of TGFß signaling by Losartan treatment greatly improved the phenotype of myopathies associated with laminin-α2-deficient congenital muscular dystrophy. Caveolin 3 deficiency was also ameliorated by the use of several different types of TGFß signaling inhibitors. Use of Losartan had dramatically beneficial effects on sarcopenic muscle by improving the regeneration after injury. Pharmacological manipulation to increase muscle mass is an emerging trend in obesity treatment research. New advances in the use of potent myostatin inhibitors have made this an attractive approach for future studies. SUMMARY: An increasing number of skeletal myopathies are demonstrating favorable responses to alterations of the TGFß signaling pathway. However, future research is needed to fully understand the downstream molecular signature associated with this pathway in order to develop more specific targeted therapies.

An inhibitor of transforming growth factor beta type I receptor ameliorates muscle atrophy in a mouse model of caveolin 3-deficient muscular dystrophy.

Skeletal muscle expressing Pro104Leu mutant caveolin 3 (CAV3(P104L)) in mouse becomes atrophied and serves as a model of autosomal dominant limb-girdle muscular dystrophy 1C. We previously found that caveolin 3-deficient muscles showed activated intramuscular transforming growth factor beta (TGF-ß) signals. However, the cellular mechanism by which loss of caveolin 3 leads to muscle atrophy is unknown. Recently, several small-molecule inhibitors of TGF-ß type I receptor (TßRI) kinase have been developed as molecular-targeting drugs for cancer therapy by suppressing intracellular TGF-ß1, -ß2, and -ß3 signaling. Here, we show that a TßRI kinase inhibitor, Ki26894, restores impaired myoblast differentiation in vitro caused by activin, myostatin, and TGF-ß1, as well as CAV3(P104L). Oral administration of Ki26894 increased muscle mass and strength in vivo in wild-type mice, and improved muscle atrophy and weakness in the CAV3(P104L) mice. The inhibitor restored the number of satellite cells, the resident stem cells of adult skeletal muscle, with suppression of the increased phosphorylation of Smad2, an effector, and the upregulation of p21 (also known as Cdkn1a), a target gene of the TGF-ß family members in muscle. These data indicate that both TGF-ß-dependent reduction in satellite cells and impairment of myoblast differentiation contribute to the cellular mechanism underlying caveolin 3-deficient muscle atrophy. TßRI kinase inhibitors could antagonize the activation of intramuscular anti-myogenic TGF-ß signals, thereby providing a novel therapeutic rationale for the alternative use of this type of anticancer drug in reversing muscle atrophy in various clinical settings.

Essential role of caveolin-3 in adiponectin signalsome formation and adiponectin cardioprotection.

OBJECTIVE: Adiponectin (APN) system malfunction is causatively related to increased cardiovascular morbidity/mortality in diabetic patients. The aim of the current study was to investigate molecular mechanisms responsible for APN transmembrane signaling and cardioprotection. METHODS AND RESULTS: Compared with wild-type mice, caveolin-3 knockout (Cav-3KO) mice exhibited modestly increased myocardial ischemia/reperfusion injury (increased infarct size, apoptosis, and poorer cardiac function recovery; P<0.05). Although the expression level of key APN signaling molecules was normal in Cav-3KO, the cardioprotective effects of APN observed in wild-type were either markedly reduced or completely lost in Cav-3KO. Molecular and cellular experiments revealed that APN receptor 1 (AdipoR1) colocalized with Cav-3, forming AdipoR1/Cav-3 complex via specific Cav-3 scaffolding domain binding motifs. AdipoR1/Cav-3 interaction was required for APN-initiated AMP-activated protein kinase (AMPK)-dependent and AMPK-independent intracellular cardioprotective signalings. More importantly, APPL1 and adenylate cyclase, 2 immediately downstream molecules required for AMPK-dependent and AMPK-independent signaling, respectively, formed a protein complex with AdipoR1 in a Cav-3 dependent fashion. Finally, pharmacological activation of both AMPK plus protein kinase A significantly reduced myocardial infarct size and improved cardiac function in Cav-3KO animals. CONCLUSIONS: Taken together, these results demonstrated for the first time that Cav-3 plays an essential role in APN transmembrane signaling and APN anti-ischemic/cardioprotective actions.

Caveolin-3 is a direct molecular partner of the Cav1.1 subunit of the skeletal muscle L-type calcium channel.

Caveolin-3 is the striated muscle specific isoform of the scaffolding protein family of caveolins and has been shown to interact with a variety of proteins, including ion channels. Mutations in the human CAV3 gene have been associated with several muscle disorders called caveolinopathies and among these, the P104L mutation (Cav-3(P104L)) leads to limb girdle muscular dystrophy of type 1C characterized by the loss of sarcolemmal caveolin. There is still no clear-cut explanation as to specifically how caveolin-3 mutations lead to skeletal muscle wasting. Previous results argued in favor of a role for caveolin-3 in dihydropyridine receptor (DHPR) functional regulation and/or T-tubular membrane localization. It appeared worth closely examining such a functional link and investigating if it could result from the direct physical interaction of the two proteins. Transient expression of Cav-3(P104L) or caveolin-3 specific siRNAs in C2C12 myotubes both led to a significant decrease of the L-type Ca(2+) channel maximal conductance. Immunolabeling analysis of adult skeletal muscle fibers revealed the colocalization of a pool of caveolin-3 with the DHPR within the T-tubular membrane. Caveolin-3 was also shown to be present in DHPR-containing triadic membrane preparations from which both proteins co-immunoprecipitated. Using GST-fusion proteins, the I-II loop of Ca(v)1.1 was identified as the domain interacting with caveolin-3, with an apparent affinity of 60nM. The present study thus revealed a direct molecular interaction between caveolin-3 and the DHPR which is likely to underlie their functional link and whose loss might therefore be involved in pathophysiological mechanisms associated to muscle caveolinopathies.

Atelocollagen-mediated systemic administration of myostatin-targeting siRNA improves muscular atrophy in caveolin-3-deficient mice.

Small interfering RNA (siRNA)-mediated silencing of gene expression is rapidly becoming a powerful tool for molecular therapy. However, the rapid degradation of siRNAs and their limited duration of activity require efficient delivery methods. Atelocollagen (ATCOL)-mediated administration of siRNAs is a promising approach to disease treatment, including muscular atrophy. Herein, we report that ATCOL-mediated systemic administration of a myostatin-targeting siRNA into a caveolin-3-deficient mouse model of limb-girdle muscular dystrophy 1C (LGMD1C) induced a marked increase in muscle mass and a significant recovery of contractile force. These results provide evidence that ATCOL-mediated systemic administration of siRNAs may be a powerful therapeutic tool for disease treatment, including muscular atrophy.

Opioid-induced preconditioning is dependent on caveolin-3 expression.

We tested the hypothesis that caveolin-3 (Cav-3) is essential for opioid-induced preconditioning in vivo. Cav-3 overexpressing mice, Cav-3 knockout mice, and controls were exposed to myocardial ischemia/reperfusion (I/R) in the presence of SNC-121 (SNC), a δ-selective opioid agonist, or naloxone, a nonselective opioid antagonist. Controls were protected from I/R injury by SNC. No protection was produced by SNC in Cav-3 knockout mice. Cav-3 overexpressing mice showed innate protection from I/R compared with controls that was abolished by naloxone. Our results show that opioid-induced preconditioning is dependent on Cav-3 expression and that endogenous protection in Cav-3 overexpressing mice is opioid dependent.

Role of caveolin-3 and glucose transporter-4 in isoflurane-induced delayed cardiac protection.

BACKGROUND: Caveolae are small, flask-like invaginations of the plasma membrane. Caveolins are structural proteins found in caveolae that have scaffolding properties to allow organization of signaling. The authors tested the hypothesis that delayed cardiac protection induced by volatile anesthetics is caveolae or caveolin dependent. METHODS: An in vivo mouse model of ischemia-reperfusion injury with delayed anesthetic preconditioning (APC) was tested in wild-type, caveolin-1 knockout, and caveolin-3 knockout mice. Mice were exposed to 30 min of oxygen or isoflurane and allowed to recover for 24 h. After 24 h recovery, mice underwent 30-min coronary artery occlusion followed by 2 h of reperfusion at which time infarct size was determined. Biochemical assays were also performed in excised hearts. RESULTS: Infarct size as a percent of the area at risk was reduced by isoflurane in wild-type (24.0 +/- 8.8% vs. 45.1 +/- 10.1%) and caveolin-1 knockout mice (27.2 +/- 12.5%). Caveolin-3 knockout mice did not show delayed APC (41.5 +/- 5.0%). Microscopically distinct caveolae were observed in wild-type and caveolin-1 knockout mice but not in caveolin-3 knockout mice. Delayed APC increased the amount of caveolin-3 protein but not caveolin-1 protein in discontinuous sucrose-gradient buoyant fractions. In addition, glucose transporter-4 was increased in buoyant fractions, and caveolin-3/glucose transporter-4 colocalization was observed in wild-type and caveolin-1 knockout mice after APC. CONCLUSIONS: These results show that delayed APC involves translocation of caveolin-3 and glucose transporter-4 to caveolae, resulting in delayed protection in the myocardium.

Caveolin-3 promotes nicotinic acetylcholine receptor clustering and regulates neuromuscular junction activity.

The molecular mechanisms that regulate the organization and activity of the neuromuscular junction remain to be fully identified. Caveolae are invaginations of the plasma membrane. Caveolin-3 is the structural protein component of caveolae in muscle cells. We show that caveolin-3 is expressed at the neuromuscular junction, that it associates with the nicotinic acetylcholine receptor (nAChR), and that a lack of caveolin-3 inhibits clustering of the nAChR in myotubes. At the molecular level, we demonstrate that caveolin-3 is a novel muscle-specific kinase (MuSK) binding protein and that altered nAChR clustering in caveolin-3-lacking myotubes results from inhibition of agrin-induced phosphorylation/activation of MuSK and activation of Rac-1. Functional studies in caveolin-3 null mice show abnormal neuromuscular junction activity that is consistent with altered nAChR localization at the sarcolemma. Together, these data identify caveolin-3 as a critical component of the signaling machinery that drives nicotinic acetylcholine receptor clustering and controls neuromuscular junction function.

Human PTRF mutations cause secondary deficiency of caveolins resulting in muscular dystrophy with generalized lipodystrophy.

Caveolae are invaginations of the plasma membrane involved in many cellular processes, including clathrin-independent endocytosis, cholesterol transport, and signal transduction. They are characterized by the presence of caveolin proteins. Mutations that cause deficiency in caveolin-3, which is expressed exclusively in skeletal and cardiac muscle, have been linked to muscular dystrophy. Polymerase I and transcript release factor (PTRF; also known as cavin) is a caveolar-associated protein suggested to play an essential role in the formation of caveolae and the stabilization of caveolins. Here, we identified PTRF mutations in 5 nonconsanguineous patients who presented with both generalized lipodystrophy and muscular dystrophy. Muscle hypertrophy, muscle mounding, mild metabolic complications, and elevated serum creatine kinase levels were observed in these patients. Skeletal muscle biopsies revealed chronic dystrophic changes, deficiency and mislocalization of all 3 caveolin family members, and reduction of caveolae structure. We generated expression constructs recapitulating the human mutations; upon overexpression in myoblasts, these mutations resulted in PTRF mislocalization and disrupted physical interaction with caveolins. Our data confirm that PTRF is essential for formation of caveolae and proper localization of caveolins in human cells and suggest that clinical features observed in the patients with PTRF mutations are associated with a secondary deficiency of caveolins.