Cysteine post-translational modifications regulate protein interactions of caveolin-3.

Caveolae are small flask-shaped invaginations of the surface membrane which are proposed to recruit and co-localize signaling molecules. The distinctive caveolar shape is achieved by the oligomeric structural protein caveolin, of which three isoforms exist. Aside from the finding that caveolin-3 is specifically expressed in muscle, functional differences between the caveolin isoforms have not been rigorously investigated. Caveolin-3 is relatively cysteine-rich compared to caveolins 1 and 2, so we investigated its cysteine post-translational modifications. We find that caveolin-3 is palmitoylated at 6 cysteines and becomes glutathiolated following redox stress. We map the caveolin-3 palmitoylation sites to a cluster of cysteines in its C terminal membrane domain, and the glutathiolation site to an N terminal cysteine close to the region of caveolin-3 proposed to engage in protein interactions. Glutathiolation abolishes caveolin-3 interaction with heterotrimeric G protein alpha subunits. Our results indicate that a caveolin-3 oligomer contains up to 66 palmitates, compared to up to 33 for caveolin-1. The additional palmitoylation sites in caveolin-3 therefore provide a mechanistic basis by which caveolae in smooth and striated muscle can possess unique phospholipid and protein cargoes. These unique adaptations of the muscle-specific caveolin isoform have important implications for caveolar assembly and signaling.

Nitrative Modification of Caveolin-3: A Novel Mechanism of Cardiac Insulin Resistance and a Potential Therapeutic Target Against Ischemic Heart Failure in Prediabetic Animals.

BACKGROUND: Myocardial insulin resistance is a hallmark of diabetic cardiac injury. However, the underlying molecular mechanisms remain unclear. Recent studies demonstrate that the diabetic heart is resistant to other cardioprotective interventions, including adiponectin and preconditioning. The "universal" resistance to multiple therapeutic interventions suggests impairment of the requisite molecule(s) involved in broad prosurvival signaling cascades. Cav (Caveolin) is a scaffolding protein coordinating transmembrane signaling transduction. However, the role of Cav3 in diabetic impairment of cardiac protective signaling and diabetic ischemic heart failure is unknown. METHODS: Wild-type and gene-manipulated mice were fed a normal diet or high-fat diet for 2 to 12 weeks and subjected to myocardial ischemia and reperfusion. Insulin cardioprotection was determined. RESULTS: Compared with the normal diet group, the cardioprotective effect of insulin was significantly blunted as early as 4 weeks of high-fat diet feeding (prediabetes), a time point where expression levels of insulin-signaling molecules remained unchanged. However, Cav3/insulin receptor-ß complex formation was significantly reduced. Among multiple posttranslational modifications altering protein/protein interaction, Cav3 (not insulin receptor-ß) tyrosine nitration is prominent in the prediabetic heart. Treatment of cardiomyocytes with 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride reduced the signalsome complex and blocked insulin transmembrane signaling. Mass spectrometry identified Tyr73 as the Cav3 nitration site. Phenylalanine substitution of Tyr73 (Cav3Y73F) abolished 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride-induced Cav3 nitration, restored Cav3/insulin receptor-ß complex, and rescued insulin transmembrane signaling. It is most important that adeno-associated virus 9-mediated cardiomyocyte-specific Cav3Y73F reexpression blocked high-fat diet-induced Cav3 nitration, preserved Cav3 signalsome integrity, restored transmembrane signaling, and rescued insulin-protective action against ischemic heart failure. Last, diabetic nitrative modification of Cav3 at Tyr73 also reduced Cav3/AdipoR1 complex formation and blocked adiponectin cardioprotective signaling. CONCLUSIONS: Nitration of Cav3 at Tyr73 and resultant signal complex dissociation results in cardiac insulin/adiponectin resistance in the prediabetic heart, contributing to ischemic heart failure progression. Early interventions preserving Cav3-centered signalsome integrity is an effective novel strategy against diabetic exacerbation of ischemic heart failure.

Differential Expression of Caveolin-3, Suppression of Tumorigenicity 2, and Growth Differentiation Factor-15 Genes and Their Association with Acute Myocardial Infarction: A Cross-Sectional Study in a Multi-Specialty Hospital in Tamil Nadu.

Background: Acute myocardial infarction (AMI) is one of the world's leading causes of cardiovascular death. Recent studies have reported the influence of the genes caveolin-3 (CAV3), suppression of tumorigenicity 2, and growth differentiating factor-15 in cardiovascular diseases, especially myocardial infarction, but their role and function remain unclear. Hence, this study was designed to evaluate the expression levels of these three genes in AMI and understanding the role of CAV-3 in the pathogenesis of AMI. Methods and Results: Blood samples were collected from 50 AMI patients and 50 non-AMI controls in this cross-sectional study. Relative expression levels of the three genes were performed using real-time PCR. Bioinformatics tools were used for functional gene enrichment and protein-protein interactions. CAV-3 was significantly upregulated among AMI patients compared to controls. In silico analyses identified CAV-3 as playing critical roles in smooth muscle contraction, cardiac conduction, and calcium-mediated transport via binding with essential proteins including dysferlin and annexins Conclusion: This study is a first of its kind, reporting an upregulation of CAV-3 in AMI patients. The expression of all three genes significantly influenced the systolic function of the heart in AMI patients. A more in-depth understanding of CAV-3 in the pathophysiology of AMI is essential and it may prove to be a novel.

Cardiac-targeted PIASy gene silencing mediates deSUMOylation of caveolin-3 and prevents ischemia/reperfusion-induced Nav1.5 downregulation and ventricular arrhythmias.

BACKGROUND: Abnormal myocardial Nav1.5 expression and function cause lethal ventricular arrhythmias during myocardial ischemia-reperfusion (I/R). Protein inhibitor of activated STAT Y (PIASy)-mediated caveolin-3 (Cav-3) SUMO modification affects Cav-3 binding to the voltage-gated sodium channel 1.5 (Nav1.5). PIASy activity is increased after myocardial I/R, but it is unclear whether this is attributable to plasma membrane Nav1.5 downregulation and ventricular arrhythmias. METHODS: Using recombinant adeno-associated virus subtype 9 (AAV9), rat cardiac PIASy was silenced using intraventricular injection of PIASy short hairpin RNA (shRNA). After two weeks, rat hearts were subjected to I/R and electrocardiography was performed to assess malignant arrhythmias. Tissues from peri-infarct areas of the left ventricle were collected for molecular biological measurements. RESULTS: PIASy was upregulated by I/R (P < 0.01), with increased SUMO2/3 modification of Cav-3 and reduced membrane Nav1.5 density (P < 0.01). AAV9-PIASy shRNA intraventricular injection into the rat heart downregulated PIASy after I/R, at both mRNA and protein levels (P < 0.05 vs. Scramble-shRNA + I/R group), decreased SUMO-modified Cav-3 levels, enhanced Cav-3 binding to Nav1.5, and prevented I/R-induced decrease of Nav1.5 and Cav-3 co-localization in the intercalated disc and lateral membrane. PIASy silencing in rat hearts reduced I/R-induced fatal arrhythmias, which was reflected by a modest decrease in the duration of ventricular fibrillation (VF; P < 0.05 vs. Scramble-shRNA + I/R group) and a significantly reduced arrhythmia score (P < 0.01 vs. Scramble-shRNA + I/R group). The anti-arrhythmic effects of PIASy silencing were also evidenced by decreased episodes of ventricular tachycardia (VT), sustained VT and VF, especially at the time 5-10 min after ischemia (P < 0.05 vs. Scramble-shRNA + IR group). Using in vitro human embryonic kidney 293 T (HEK293T) cells and isolated adult rat cardiomyocyte models exposed to hypoxia/reoxygenation (H/R), we confirmed that increased PIASy promoted Cav-3 modification by SUMO2/3 and Nav1.5/Cav-3 dissociation after H/R. Mutation of SUMO consensus lysine sites in Cav-3 (K38R or K144R) altered the membrane expression levels of Nav1.5 and Cav-3 before and after H/R in HEK293T cells. CONCLUSIONS: I/R-induced cardiac PIASy activation increased Cav-3 SUMOylation by SUMO2/3 and dysregulated Nav1.5-related ventricular arrhythmias. Cardiac-targeted PIASy silencing mediated Cav-3 deSUMOylation and partially prevented I/R-induced Nav1.5 downregulation in the plasma membrane of cardiomyocytes, and subsequent ventricular arrhythmias in rats. PIASy was identified as a potential therapeutic target for life-threatening arrhythmias in patients with ischemic heart diseases.

Investigation and modulation of interleukin-6 following subarachnoid hemorrhage: targeting inflammatory activation for cerebral vasospasm.

BACKGROUND: Cerebral vasospasm (CV) can contribute to significant morbidity in subarachnoid hemorrhage (SAH) patients. A key unknown is how CV induction is triggered following SAH. METHODS: Human aneurysmal blood and cerebral spinal fluid were collected for evaluation. To confirm mechanism, c57/bl6 wild type and c57/bl6 IL-6 female knockout (KO) mice were utilized with groups: saline injected, SAH, SAH + IL-6 blockade, SAH IL-6 KO, SAH IL-6 KO + IL-6 administration, SAH + p-STAT3 inhibition. Dual-labeled microglia/myeloid mice were used to show myeloid diapedesis. For SAH, 50 µm blood was collected from tail puncture and administered into basal cisterns. IL-6 blockade was given at various time points. Various markers of neuroinflammation were measured with western blot and immunohistochemistry. Cerebral blood flow was also measured. Vasospasm was measured via cardiac injection of India ink/gelatin. Turning test and Garcia's modified SAH score were utilized. P < 0.05 was considered significant. RESULTS: IL-6 expression peaked 3 days following SAH (p < 0.05). Human IL-6 was increased in aneurysmal blood (p < 0.05) and in cerebral spinal fluid (p < 0.01). Receptor upregulation was periventricular and perivascular. Microglia activation following SAH resulted in increased caveolin 3 and myeloid diapedesis. A significant increase in BBB markers endothelin 1 and occludin was noted following SAH, but reduced with IL-6 blockade (p < 0.01). CV occurred 5 days post-SAH, but was absent in IL-6 KO mice and mitigated with IL-6 blockade (p < 0.05). IL-6 blockade, and IL-6 KO mitigated effects of SAH on cerebral blood flow (p < 0.05). SAH mice had impaired performance on turn test and poor modified Garcia scores compared to saline and IL-6 blockade. A distinct microglia phenotype was noted day 5 in the SAH group (overlap coefficients r = 0.96 and r = 0.94) for Arg1 and iNOS, which was altered by IL-6 blockade. Day 7, a significant increase in toll-like receptor 4 and Stat3 was noted. This was mitigated by IL-6 blockade and IL-6 KO, which also reduced Caspase 3 (p < 0.05). To confirm the mechanism, we developed a p-STAT3 inhibitor that targets the IL-6 pathway and this reduced NFΚB, TLR4, and nitrotyrosine (p < 0.001). Ventricular dilation and increased Tunel positivity was noted day 9, but resolved by IL-6 blockade (p < 0.05). CONCLUSION: Correlation between IL-6 and CV has been well documented. We show that a mechanistic connection exists via the p-STAT3 pathway, and IL-6 blockade provides benefit in reducing CV and its consequences mediated by myeloid cell origin diapedesis.

Misregulation of cell adhesion molecules in the Ciona neural tube closure mutant bugeye.

Neural tube closure (NTC) is a complex multi-step morphogenetic process that transforms the flat neural plate found on the surface of the post-gastrulation embryo into the hollow and subsurface central nervous system (CNS). Errors in this process underlie some of the most prevalent human birth defects, and occur in about 1 out of every 1000 births. Previously, we discovered a mutant in the basal chordate Ciona savignyi (named bugeye) that revealed a novel role for a T-Type Calcium Channel (Cav3) in this process. Moreover, the requirement for CAV3s in Xenopus NTC suggests a conserved function among the chordates. Loss of CAV3 leads to defects restricted to anterior NTC, with the brain apparently fully developed, but protruding from the head. Here we report first on a new Cav3 mutant in the related species C. robusta. RNAseq analysis of both C. robusta and C. savignyi bugeye mutants reveals misregulation of a number of transcripts including ones that are involved in cell-cell recognition and adhesion. Two in particular, Selectin and Fibronectin leucine-rich repeat transmembrane, which are aberrantly upregulated in the mutant, are expressed in the closing neural tube, and when disrupted by CRISPR gene editing lead to the open brain phenotype displayed in bugeye mutants. We speculate that these molecules play a transient role in tissue separation and adhesion during NTC and failure to downregulate them leads to an open neural tube.

Caveolin3 Stabilizes McT1-Mediated Lactate/Proton Transport in Cardiomyocytes.

[Figure: see text].

A Role for Caveolin-3 in the Pathogenesis of Muscular Dystrophies.

Caveolae are the cholesterol-rich small invaginations of the plasma membrane present in many cell types including adipocytes, endothelial cells, epithelial cells, fibroblasts, smooth muscles, skeletal muscles and cardiac muscles. They serve as specialized platforms for many signaling molecules and regulate important cellular processes like energy metabolism, lipid metabolism, mitochondria homeostasis, and mechano-transduction. Caveolae can be internalized together with associated cargo. The caveolae-dependent endocytic pathway plays a role in the withdrawal of many plasma membrane components that can be sent for degradation or recycled back to the cell surface. Caveolae are formed by oligomerization of caveolin proteins. Caveolin-3 is a muscle-specific isoform, whose malfunction is associated with several diseases including diabetes, cancer, atherosclerosis, and cardiovascular diseases. Mutations in Caveolin-3 are known to cause muscular dystrophies that are collectively called caveolinopathies. Altered expression of Caveolin-3 is also observed in Duchenne's muscular dystrophy, which is likely a part of the pathological process leading to muscle weakness. This review summarizes the major functions of Caveolin-3 in skeletal muscles and discusses its involvement in the pathology of muscular dystrophies.

Caveolin-3 protects diabetic hearts from acute myocardial infarction/reperfusion injury through ß2AR, cAMP/PKA, and BDNF/TrkB signaling pathways.

Diabetes mellitus (DM) might increase the incidence and mortality of cardiac failure after acute myocardial infarction (AMI) in patients. We attempted to investigate whether Caveolin-3 showed beneficial effects in DM patient post-MI injury through the cAMP/PKA and BDNF/TrkB signaling pathways. The activity of ADRB2 and cAMP/PKA signaling were impaired in nondiabetic ischemia-reperfusion (I/R) group compared with the sham and DM groups and were more impaired in diabetic I/R group than in the I/R group. In H9C2 cells, high-glucose (HG) stimulation further enhanced H/R injury by promoting cell apoptosis, inhibiting cell viability, and suppressing TrkB and Akt signaling; in contrast, the ADRB2 agonist isoprenaline (ISO) significantly attenuated the above-described effects of HG stimulation. Caveolin-3 overexpression promoted the localization of ADRB2 on the membrane of the HG-stimulated H9C2 cells, subsequently inhibiting apoptosis and promoting cell viability. Under HG stimulation, Caveolin-3 overexpression enhanced the activity of the cAMP/PKA and BDNF/TrkB signaling pathways, whereas ADRB2 silencing reversed the effects of Caveolin-3 overexpression. In conclusion, ADRB2 agonist promoted the activity of the BDNF/TrkB and cAMP/PKA signaling pathways, mitigating the HG-aggravated H/R injuries in H9C2 cells. Caveolin-3 exerts a protective effect on diabetic hearts against I/R damage through the ß2AR, cAMP/PKA, and BDNF/TrkB signaling pathways.

Unique cysteine-enriched, D2L5 and D4L6 extracellular loops in CaV3 T-type channels alter the passage and block of monovalent and divalent ions.

Invertebrate LCaV3 shares the quintessential features of vertebrate CaV3 T-type channels, with a low threshold of channel activation, rapid activation and inactivation kinetics and slow deactivation kinetics compared to other known Ca2+ channels, the CaV1 and CaV2 channels. Unlike the vertebrates though, CaV3 T-type channels in non-cnidarian invertebrates possess an alternative exon 12 spanning the D2L5 extracellular loop, which alters the invertebrate LCaV3 channel into a higher Na+ and lower Ca2+ current passing channel, more resembling a classical NaV1 Na+ channel. Cnidarian CaV3 T-type channels can possess genes with alternative cysteine-rich, D4L6 extracellular loops in a manner reminiscent of the alternative cysteine-rich, D2L5 extracellular loops of non-cnidarian invertebrates. We illustrate here that the preferences for greater Na+ or Ca2+ ion current passing through CaV3 T-type channels are contributed by paired cysteines within D2L5 and D4L6 extracellular loops looming above the pore selectivity filter. Swapping of invertebrate tri- and tetra-cysteine containing extracellular loops, generates higher Na+ current passing channels in human CaV3.2 channels, while corresponding mono- and di-cysteine loop pairs in human CaV3.2 generates greater Ca2+ current passing, invertebrate LCaV3 channels. Alanine substitutions of unique D2L5 loop cysteines of LCaV3 channels increases relative monovalent ion current sizes and increases the potency of Zn2+ and Ni2+ block by ~ 50× and ~ 10× in loop cysteine mutated channels respectively, acquiring characteristics of the high affinity block of CaV3.2 channels, including the loss of the slowing of inactivation kinetics during Zn2+ block. Charge neutralization of a ubiquitous aspartate residue of calcium passing CaV1, CaV2 and CaV3 channels, in the outer pore of the selectivity filter residues in Domain II generates higher Na+ current passing channels in a manner that may resemble how the unique D2L5 extracellular loops of invertebrate CaV3 channels may confer a relatively higher peak current size for Na+ ions over Ca2+ The extracellular loops of CaV3 channels are not engaged with accessory subunit binding, as the other Na+ (NaV1) and Ca2+ (CaV1/CaV2) channels, enabling diversity and expansion of cysteine-bonded extracellular loops, which appears to serve, amongst other possibilities, to alter to the preferences for passage of Ca2+ or Na+ ions through invertebrate CaV3 channels.

Milrinone Attenuates Heart and Lung Remote Injury after Abdominal Aortic Cross-Clamping.

BACKGROUND: Phosphodiesterase enzymes play a pivotal role in the pathogenesis of ischemia/reperfusion (IR). We examined the role of milrinone (MIL), a phosphodiesterase 3 inhibitor, on remote injury of the heart and lung after abdominal aortic cross-clamping. DESIGN: Experimental study. METHODS: Twenty-one Wistar rats were divided into 3 groups: (1) control (C, n = 7), underwent laparotomy and exploration of abdominal aorta only; (2) IR (n = 7), normal saline was applied intraperitoneally (i.p) before IR induced by clamping of the abdominal aorta for 1 hr and then allowing reperfusion for 1 hr; and (3) MIL + IR (n = 7), MIL was given (0.5 mg/kg, i.p) before IR. After sacrification, the lungs and hearts were taken out for analyses and the tissue malondialdehyde (MDA) and glutathione (GSH) were studied. All tissues were examined under light microscopy and transmission electron microscopy (TEM). Expressions of caveolin (Cav)-1 in the lung and Cav-1 and Cav-3 in the heart were examined immunohistochemically. RESULTS: The MIL + IR group had significantly a lower magnitude of oxidative stress than the IR group both in the lung and heart (lung: P = 0.03 for MDA and 0.001 for GSH and heart: P = 0.002 for MDA and 0.000 for GSH). In light microscopy, the MIL + IR group had statistically a lower total injury score than the IR group for both the lung and heart tissue (P = 0.03 and P = 0.04, respectively). In TEM, regression of mitochondrial degeneration and lamellar bodies in type II pneumocytes in the lungs and obvious improvements in disruption at the intercalated discs and mitochondrial degeneration in the hearts in the MIL + IR group were detected compared with the IR group. The expression of both Cav-1 and Cav-3 in the MIL + IR group was improved compared with the IR group (P = 0.03 for both). CONCLUSIONS: MIL attenuates remote injury of heart and lung in lower body IR by inhibiting oxidative stress. Moreover, Cav-1 and Cav-3 might have a potential role in MIL-induced cardioprotection.

Caveolin proteins electrochemical oxidation and interaction with cholesterol.

Caveolae consist in lipid raft domains composed of caveolin proteins, cholesterol, glycosphingolipids, and GPI-anchored proteins. Caveolin proteins present three different types, caveolin 1 (CAV-1), caveolin 2 (CAV-2) and caveolin 3 (CAV-3), with a very similar structure and amino acid composition. The native caveolin proteins oxidation mechanism was investigated for the first time, at a glassy carbon electrode, using cyclic, square wave and differential pulse voltammetry. The three native caveolin proteins oxidation mechanism presented only one tyrosine and tryptophan amino acid residues oxidation peak. Denatured caveolin proteins presented also the tyrosine, tryptophan and cysteine amino acid residues oxidation peaks. The reverse cholesterol transport is related to caveolae and caveolin proteins, and CAV-1 is directly connected to cholesterol transport. The influence of cholesterol on the three caveolin proteins electrochemical behaviour was evaluated. In the absence and in the presence of cholesterol, significant differences in the CAV-1 oxidation peak current were observed.

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.

A novel missense mutation in CAV3 gene in an Italian family with persistent hyperCKemia, myalgia and hypercholesterolemia: Double-trouble.

Caveolins are essential proteins in caveolae architecture, small plasma membrane invaginations that play a key role in a variety of cellular processes, including vesicular trafficking and signal transduction. Mutations in the gene encoding caveolin-3 (CAV3) cause a broad spectrum of clinical phenotypes, ranging from isolated hyperCKemia to most severe limb girdle muscular dystrophy and cardiomyopathy. We report a novel heterozygous p.Val44Met (c.130G > A) CAV3 mutation in two brothers presenting with persistent elevation of serum creatine kinase, myalgia and hypercholesterolemia. Immunofluorescence study with anticaveolin-3 antibodies on muscle biopsy of the proband confirmed a reduced immuno-reactivity of caveolin-3 on the sarcolemma. This findings support the pathogenic effect of this novel mutation and extend the genotypic and clinical spectrum of Caveolinopathies. Finally, we discuss the hypothesis that the association between CAV3 mutations and hypercholesterolemia may not be coincidental.

Cardiac-Specific Caveolin-3 Overexpression Prevents Post-Myocardial Infarction Ventricular Arrhythmias by Inhibiting Ryanodine Receptor-2 Hyperphosphorylation.

INTRODUCTION: Ventricular arrhythmia is the most important risk factor for sudden cardiac death (SCD) after acute myocardial infarction (MI) worldwide. However, the molecular mechanisms underlying these arrhythmias are complex and not completely understood. OBJECTIVE: Here, we evaluated whether caveolin-3 (Cav3), the structural protein of caveolae, plays an important role in the therapeutic strategy for ventricular arrhythmias. METHODS: A model of cardiac-specific overexpression of Cav3 was established to evaluate the incidence of ventricular arrhythmias after MI in mice. Ca2+ imaging was employed to detect the propensity of adult murine cardiomyocytes to generate arrhythmias, and immunoprecipitation and immunofluorescence were used to determine the relationship of proteins. Additionally, qRT-PCR and western blotting were used to detect the mRNA and protein expression. RESULTS: We found that cardiac-specific overexpression of Cav3 delivered by a recombinant adeno-associated viral vector reduced the incidence of ventricular arrhythmias and SCD after MI in mice. Ca2+ imaging and western blotting revealed that overexpression of Cav3 reduced diastolic spontaneous Ca2+ waves by inhibiting the hyperphosphorylation of ryanodine receptor-2 (RyR2) at Ser2814, rather than at Ser2808, compared to in rAAV-red fluorescent protein control mice. Furthermore, we demonstrated that Cav3-regulated RYR2 hyperphosphorylation relied on plakophilin-2 in hypoxia-stimulated cultured cardiomyocytes by western blotting, immunoprecipitation, and immunofluorescence in vitro. CONCLUSIONS: Our results suggested a novel role for Cav3 in the prevention of ventricular arrhythmias, thereby identifying a new target for preventing SCD after MI.

Metabolic syndrome diminishes insulin-induced Akt activation and causes a redistribution of Akt-interacting proteins in cardiomyocytes.

Metabolic syndrome (MetS) is a cluster of cardiometabolic risk factors, with insulin resistance as a critical component for its development. Insulin signaling in the heart leads to Akt (also known as PKB) activation, a serine/threonine protein kinase, which regulates cardiac glucose metabolism and growth. Cardiac metabolic inflexibility, characterized by impaired insulin-induced glucose uptake and oxidation, has been reported as an early and consistent change in the heart of different models of MetS and diabetes; however, the evaluation of Akt activation has yielded variable results. Here we report in cardiomyocytes of MetS rats, diminished insulin-induced glucose uptake and Akt activation, evaluated by its impaired mobilization towards the plasma membrane and phosphorylation, and reflected in a re-distribution of its interacting proteins, assessed by label-free mass spectrometry (data are available via ProteomeXchange with identifier PXD013260). We report 45 proteins with diminished abundance in Akt complex of MetS cardiomyocytes, mainly represented by energy metabolism-related proteins, and also, 31 Akt-interacting proteins with increased abundance, which were mainly related to contraction, endoplasmic reticulum stress, and Akt negative regulation. These results emphasize the relevance of Akt in the regulation of energy metabolism in the heart and highlight Akt-interacting proteins that could be involved in the detrimental effects of MetS in the heart.

Hyperglycemia-Induced Oxidative Stress Abrogates Remifentanil Preconditioning-Mediated Cardioprotection in Diabetic Rats by Impairing Caveolin-3-Modulated PI3K/Akt and JAK2/STAT3 Signaling.

Diabetic hearts are more vulnerable to ischemia/reperfusion (I/R) injury and less responsive to remifentanil preconditioning (RPC), but the underlying mechanisms are incompletely understood. Caveolin-3 (Cav-3), the dominant isoform of cardiomyocyte caveolae, is reduced in diabetic hearts in which oxidative stress is increased. This study determined whether the compromised RPC in diabetes was an independent manifestation of hyperglycemia-induced oxidative stress or linked to impaired Cav-3 expression with associated signaling abnormality. RPC significantly attenuated postischemic infarction, cardiac dysfunction, myocardial apoptosis, and 15-F2t-isoprostane production (a specific marker of oxidative stress), accompanied with increased Cav-3 expression and enhanced Akt and STAT3 activation in control but not in diabetic rats. Pretreatment with the antioxidant N-acetylcysteine (NAC) attenuated hyperglycemia-induced reduction of Cav-3 expression and Akt and STAT3 activation and restored RPC-mediated cardioprotection in diabetes, which was abolished by cardiac-specific knockdown of Cav-3 by AAV9-shRNA-Cav-3, PI3K/Akt inhibitor wortmannin, or JAK2/STAT3 inhibitor AG490, respectively. Similarly, NAC could restore RPC protection from high glucose and hypoxia/reoxygenation-induced injury evidenced by decreased levels of LDH release, 15-F2t-isoprostane, O2 -, and JC-1 monomeric cells, which were reversed by caveolae disrupter methyl-ß-cyclodextrin, wortmannin, or AG490 in isolated primary cardiomyocytes or siRNAs of Cav-3, Akt, or STAT3 in H9C2 cells. Either methyl-ß-cyclodextrin or Cav-3 knockdown reduced Akt and STAT3 activation. Further, the inhibition of Akt activation by a selective inhibitor or siRNA reduced STAT3 activation and vice versa, but they had no effects on Cav-3 expression. Thus, hyperglycemia-induced oxidative stress abrogates RPC cardioprotection by impairing Cav-3-modulated PI3K/Akt and JAK2/STAT3 signaling. Antioxidant treatment with NAC could restore RPC-induced cardioprotection in diabetes by improving Cav-3-dependent Akt and STAT3 activation and by facilitating the cross talk between PI3K/Akt and JAK2/STAT3 signaling pathways.

ERαΔ4, an ERα splice variant missing exon4, interacts with caveolin-3 and mGluR2/3.

The two isoforms of the nuclear estrogen receptor, ERα and ERß are widely expressed in the central nervous system. Although they were first described as nuclear receptors, both isoforms have also been found at the cell membrane where they mediate cell signaling. Surface biotinylation studies using neuronal and glial primary cultures label an alternatively spliced form of ERα. The 52 kDa protein, ERαΔ4, is missing exon 4 and is highly expressed in membrane fractions derived from cultured cells. In vivo, both full-length (66 kDa) ERα and ERαΔ4 are present in membrane fractions. In response to estradiol, full-length ERα and ERαΔ4 are initially trafficked to the membrane, and then internalized in parallel. Previous studies determined that only the full-length ERα associates with metabotropic glutamate receptor-1a (mGluR1a), initiating cellular signaling. The role of ERαΔ4, remained to be elucidated. Here, we report ERαΔ4 trafficking, association with mGluR2/3, and downstream signaling in female rat arcuate nucleus (ARH). Caveolin (CAV) proteins are needed for ER transport to the cell membrane, and using co-immunoprecipitation CAV-3 was shown to associate with ERαΔ4. CAV-3 was necessary for ERαΔ4 trafficking to the membrane: in the ARH, microinjection of CAV-3 siRNA reduced CAV-3 and ERαΔ4a in membrane fractions by 50%, and 60%, respectively. Moreover, co-immunoprecipitation revealed that ERαΔ4 associated with inhibitory mGluRs, mGluR2/3. Estrogen benzoate (EB) treatment (5 µg; s.c.; every 4 days; three cycles) reduced levels of cAMP, an effect attenuated by antagonizing mGluR2/3. Following EB treatment, membrane levels of ERαΔ4 and mGluR2/3 were reduced implying ligand-induced internalization. These results implicate ERαΔ4 in an estradiol-induced inhibitory cell signaling in the ARH.