Expression of caveolin family proteins in serum of patients with systemic lupus erythematosus.

OBJECTIVES: Caveolin family proteins, including caveolin-1 (Cav-1), caveolin-2 (Cav-2), and caveolin-3 (Cav-3), are identified as the principal protein components of caveolae in mammalian cells. Circulating form of caveolin family proteins can be used as a good potential biomarker for predicting disease. METHODS: To investigate the clinical significance of the serological levels of caveolin family proteins in patients with systemic lupus erythematosus (SLE), we evaluated the soluble serum levels of caveolin family proteins in patients with SLE by enzyme-linked immunosorbent assay (ELISA) and assessed their associations with various known clinical variables. RESULTS: The major findings of our study are as follows: Cav-2 was not detected in serum of SLE patients and normal controls (NCs). Serum Cav-1 and Cav-3 levels were higher in SLE patients compared with NCs. There were no significant correlations between serum Cav-1 and Cav-3 levels and SLE disease activity. Further analysis showed that serum Cav-3 may be more valuable as a marker than serum Cav-1 in SLE patients. CONCLUSION: Serum levels of Cav-1 and Cav-3 might have a diagnostic role in patients with SLE. However, their predictive and prognostic value was not determined. Further studies are necessary to determine the potential clinical significance of these assays in SLE.

MENS-associated increase of muscular protein content via modulation of caveolin-3 and TRIM72.

Microcurrent electrical neuromuscular stimulation (MENS) is known as an extracellular stimulus for the regeneration of injured skeletal muscle in sports medicine. However, the effects of MENS-associated increase in muscle protein content are not fully clarified. The purpose of this study was to investigate the effects of MENS on the muscular protein content, intracellular signals, and the expression level of caveolin-3 (Cav-3), tripartite motif-containing 72 (TRIM72) and MM isoenzyme of creatine kinase (CK-MM) in skeletal muscle using cell culture system. C2C12 myotubes on the 7th day of differentiation phase were treated with MENS (intensity: 10-20 microA, frequency: 0.3 Hz, pulse width: 250 ms, stimulation time: 15-120 min). MENS-associated increase in the protein content of myotubes was observed, compared to the untreated control level. MENS upregulated the expression of Cav-3, TRIM72, and CK-MM in myotubes. A transient increase in phosphorylation level of Akt was also observed. However, MENS had no effect on the phosphorylation level of p42/44 extracellular signal-regulated kinase-1/2 and 5'AMP-activated protein kinase. MENS may increase muscle protein content accompanied with a transient activation of Akt and the upregulation of Cav-3 and TRIM72.

Exendin-4, glucagon-like peptide-1 receptor agonist, enhances isoflurane-induced preconditioning against myocardial infarction via caveolin-3 expression.

OBJECTIVE: To investigate the cardioprotective effects of isoflurane and exendin-4 against myocardial ischemia/reperfusion injury and the signaling pathways through which these effects are mediated. MATERIALS AND METHODS: For infarct size measurements, anesthetized mice were subjected to 30 min of coronary artery occlusion followed by 2 h of reperfusion. Wild-type or caveolin-3 knockout mice received isoflurane, exendin-4, or isoflurane with exendin-4 before ischemia index determination. Caveolin-3 expression in the heart was measured by immunoblotting. RESULTS: Myocardial infarct size was smaller in the isoflurane- [1.0 minimum alveolar concentration (MAC)] or exendin-4- (30 ng/kg i.v.) treated groups than the controls. Infarct size was not affected by isoflurane at 0.5 MAC or 3 ng/kg i.v. exendin-4, but the combination of these treatments reduced infarct size. Pharmacological preconditioning (isoflurane at 1.0 MAC, 30 ng/kg i.v. exendin-4, or isoflurane at 0.5 MAC with 3 ng/kg i.v. exendin-4) increased caveolin-3 protein expression in the heart after infarct induction. The cardioprotective effects of isoflurane, exendin-4, and isoflurane with exendin-4 were abolished in caveolin-3 knockout mice. CONCLUSIONS: The combination of isoflurane and exendin-4 reduced infarct size, but it was not more effective than either agent alone, and the cardioprotective effects of these agents are mediated by caveolin-3 expression.

Caveolin contributes to the modulation of basal and ß-adrenoceptor stimulated function of the adult rat ventricular myocyte by simvastatin: a novel pleiotropic effect.

The number of people taking statins is increasing across the globe, highlighting the importance of fully understanding statins' effects on the cardiovascular system. The beneficial impact of statins extends well beyond regression of atherosclerosis to include direct effects on tissues of the cardiovascular system ('pleiotropic effects'). Pleiotropic effects on the cardiac myocyte are often overlooked. Here we consider the contribution of the caveolin protein, whose expression and cellular distribution is dependent on cholesterol, to statin effects on the cardiac myocyte. Caveolin is a structural and regulatory component of caveolae, and is a key regulator of cardiac contractile function and adrenergic responsiveness. We employed an experimental model in which inhibition of myocyte HMG CoA reductase could be studied in the absence of paracrine influences from non-myocyte cells. Adult rat ventricular myocytes were treated with 10 µM simvastatin for 2 days. Simvastatin treatment reduced myocyte cholesterol, caveolin 3 and caveolar density. Negative inotropic and positive lusitropic effects (with corresponding changes in [Ca2+]i) were seen in statin-treated cells. Simvastatin significantly potentiated the inotropic response to ß2-, but not ß1-, adrenoceptor stimulation. Under conditions of ß2-adrenoceptor stimulation, phosphorylation of phospholamban at Ser16 and troponin I at Ser23/24 was enhanced with statin treatment. Simvastatin increased NO production without significant effects on eNOS expression or phosphorylation (Ser1177), consistent with the reduced expression of caveolin 3, its constitutive inhibitor. In conclusion, statin treatment can reduce caveolin 3 expression, with functional consequences consistent with the known role of caveolae in the cardiac cell. These data are likely to be of significance, particularly during the early phases of statin treatment, and in patients with heart failure who have altered ß-adrenoceptor signalling. In addition, as caveolin is ubiquitously expressed and has myriad tissue-specific functions, the impact of statin-dependent changes in caveolin is likely to have many other functional sequelae.

Caveolin and ß1-integrin coordinate angiotensinogen expression in cardiac myocytes.

BACKGROUND: The cardiac renin-angiotensin system (RAS) has been implicated in mediating myocyte hypertrophy and remodeling, although the biochemical mechanisms responsible for regulating the local RAS are poorly understood. Caveolin-1 (Cav-1)/Cav-3 double-knockout mice display cardiac hypertrophy, and in vitro disruption of lipid rafts/caveolae using methyl-ß-cyclodextrin (MßCD) abolishes cardiac protection. METHODS: In this study, neonatal rat ventricular myocytes (NRVM) were used to determine whether lipid rafts/caveolae may be involved in the regulation of angiotensinogen (Ao) gene expression, a substrate of the RAS system. RESULTS: Treatment with MßCD caused a time-dependent upregulation of Ao gene expression, which was associated with differential regulation of mitogen-activated protein (MAP) kinases ERK1/2, p38 and JNK phosphorylation. JNK was highly phosphorylated shortly after MßCD treatment (2-30 min), whereas marked activation of ERK1/2 and p38 occurred much later (2-4h). ß1D-Integrin was required for MßCD-induced activation of the MAP kinases. Pharmacologic inhibition of ERK1/2 and JNK enhanced MßCD-induced Ao gene expression, whereas p38 blockade inhibited this response. Adenovirus-mediated expression of wild-type p38α enhanced MßCD-induced Ao gene expression; conversely expression of dominant negative p38α blocked the stimulatory effects of MßCD. Expression of Cav-3 siRNA stimulated Ao gene expression, whereas overexpression of Cav-3 was inhibitory. Cav-1 and Cav-3 expression levels were found to be positively regulated by p38, but unaffected by ERK1/2 and JNK. CONCLUSION: Collectively, these studies indicate that lipid rafts/caveolae couple to Ao gene expression through a mechanism that involves ß1-integrin and the differential actions of MAP kinase family members.

ß-Adrenergic receptor antagonists ameliorate myocyte T-tubule remodeling following myocardial infarction.

ß-Adrenergic receptor (AR) blockers provide substantial clinical benefits, including improving overall survival and left ventricular (LV) function following myocardial infarction (MI), though the mechanisms remain incompletely defined. The transverse-tubule (T-tubule) system of ventricular myocytes is an important determinant of cardiac excitation-contraction function. T-tubule remodeling occurs early during LV failure. We hypothesized that ß-AR blockers prevent T-tubule remodeling and thereby provide therapeutic benefits. A murine model of MI was utilized to examine the effect of ß-AR blockers on T-tubule remodeling following LV MI. We applied the in situ imaging of T-tubule structure from Langendorff-perfused intact hearts with laser scanning confocal microscopy. We found that MI caused remarkable T-tubule remodeling near the infarction border zone and moderate LV remodeling remote from the MI. Metoprolol and carvedilol administered 6 d after MI for 4 wk each increased the T-tubule integrity at the remote and border zones. At the molecular level, both ß-AR blockers restored border and remote zone expression of junctophilin-2 (JP-2), which is involved in T-tubule organization and formation of the T-tubule/sarcoplasmic reticulum junctions. In contrast, ß-AR blockers had no significant effects on caveolin-3 expression. In summary, our data show that ß-AR antagonists can protect against T-tubule remodeling after MI, suggesting a novel therapeutic mechanism of action for this drug class. Preservation of JP-2 expression may contribute to the beneficial effects of metoprolol and carvedilol on T-tubule remodeling.

Left ventricular dysfunction in murine models of heart failure and in failing human heart is associated with a selective decrease in the expression of caveolin-3.

BACKGROUND: Caveolins are scaffolding proteins that are integral components of caveolae, flask-shaped invaginations in the membranes of all mammalian cells. Caveolin-1 and -2 are expressed ubiquitously, whereas caveolin-3 is found only in muscle. The role of caveolin-3 in heart muscle disease is controversial. METHODS AND RESULTS: The present study was undertaken to assess the effects of left ventricular dysfunction on the expression of caveolin proteins using 2 well characterized models of murine heart failure and failing human heart. Transgenic mice with constitutive overexpression of A(1)-adenosine receptor (A(1)-TG) demonstrated cardiac dilatation and decreased left ventricular function at 10 weeks of age. This was accompanied by a marked decrease in caveolin-3 mRNA and protein levels compared with non-TG control mice. The change in caveolin-3 expression was selective, because levels of caveolin-1 and -2 did not change. Confocal imaging of myocytes isolated from A(1)-TG mice demonstrated a loss of the plate-like appearance of T tubules. Caveolin-3 levels were also reduced in hearts from mice overexpressing tumor necrosis factor α. There was a direct relationship between caveolin-3 expression and fractional shortening in all mice that were studied (r = 0.65; P < .001). Although we could not demonstrate a significant decrease in caveolin-3 levels in failing human heart, we did find a direct correlation (r = 0.7; P < .05) between levels of caveolin-3 protein and Ca(2+)-adenosine triphosphatase, a marker of the heart failure phenotype. CONCLUSIONS: These results suggest a relationship between left ventricular dysfunction and caveolin-3 levels and suggest that caveolin-3 may provide a novel target for heart failure therapy.

The rate of loss of T-tubules in cultured adult ventricular myocytes is species dependent.

In this study, we compared the rate of detubulation of adult mouse and rat ventricular myocytes over a 72 h culture period. The T-tubule density was measured in the following two ways: (i) as whole-cell capacitance in voltage-clamped myocytes relative to cell area; and (ii) using di-8-ANEPPS staining and confocal microscopy. In adult rat ventricular myocytes, whole-cell capacitance/area was significantly reduced from 47 +/- 3 fF microm(2) (mean +/- s.e.m.; n = 16) in freshly isolated (control) cells to 36 +/- 2 fF microm(2) (n = 20) after 72 h in culture. The T-tubular density, as assessed optically using di-8-ANEPPS staining, at 48 h was significantly reduced to 70 +/- 7% (n = 14) compared with control cells. The T-tubular density was further reduced after 72 h in culture to 43 +/- 7% (n = 10) compared with control cells. In contrast, in mouse myocytes neither whole-cell capacitance relative to cell area nor optical assessment of T-tubules showed any significant reduction in capacitance/cell area or T-tubule density after 72 h of culture. Expression of caveolin-3 (CAV-3) (a marker of T-tubule development) was also measured, and a significant reduction was observed in CAV-3 expression in rat myocytes at 48 (80 +/- 5.5%; n = 6) and 72 h (66 +/- 9.5%; n = 6) compared with control cells. The expression of CAV-3 in mouse myocytes was not significantly reduced even at 72 h. When rat ventricular myocytes were paced in culture for 72 h they exhibited no significant improvement in T-tubule density or CAV-3 expression compared with non-paced cultured cells. In rat myocytes, sarcomere length shortening was significantly reduced in myocytes cultured for 48 (4.96 +/- 0.72%; n = 26) and 72 h (4.32 +/- 0.80%; n = 26) compared with freshly isolated cells (7.12 +/- 0.56%; n = 18). Mouse myocytes, after 24 h in culture, were unable to follow external pacing. These results suggest that detubulation in quiescent culture is slower in the mouse than the rat and that this loss of T-tubules profoundly affects excitation-contraction coupling in rat myocytes.

Modulation of the dystrophin-associated protein complex in response to resistance training in young and older men.

The dystrophin-associated protein complex (DAPC) is a scaffold of proteins linking the intracellular cytoskeleton with the extracellular matrix that is integral to structural stability and integrity, signaling and mechanotransduction, and force transmission. We hypothesized that the expression of DAPC component proteins would be altered by resistance loading during progressive resistance training (PRT)-mediated myofiber hypertrophy, and we investigated whether aging influenced these changes. Seventeen young (27 yr) and 13 older (65 yr) men completed 16 wk of PRT with muscle biopsies at baseline (T1), 24 h after bout 1 (T2), and 24 h after the final bout at week 16 (T3). Myofiber hypertrophy in the young (type I 31%, P < 0.005; type II 40%, P < 0.001) far exceeded hypertrophy in the old (type II only, 19.5%, P < 0.05). PRT altered protein expression for caveolin-3 (decreased 24% by T3, P < 0.01), alpha(1)-syntrophin (increased 16% by T3, P < 0.05), alpha-dystrobrevin (fell 23% from T2 to T3, P < 0.01), and dystrophin [rose acutely (30% by T2, P < 0.05) and returned to baseline by T3]. The phosphorylation state of membrane neuronal nitric oxide synthase (Ser(1417)) decreased 70% (P < 0.005) by T3, particularly in the old (81%), whereas p38 MAPK phosphorylation increased twofold by T3 in the old (P < 0.01). We conclude that component proteins of the DAPC are modulated by PRT, which may serve to improve both structural and signaling functions during load-mediated myofiber hypertrophy. The blunted hypertrophic adaptation seen in old vs. young men may have resulted from overstress, as suggested by marked p38 MAPK activation in old men only.

Caveolin-1 is required for lateral line neuromast and notochord development.

Caveolae have been linked to diverse cellular functions and to many disease states. In this study we have used zebrafish to examine the role of caveolin-1 and caveolae during early embryonic development. During development, expression is apparent in a number of tissues including Kupffer's vesicle, tailbud, intersomite boundaries, heart, branchial arches, pronephric ducts and periderm. Particularly strong expression is observed in the sensory organs of the lateral line, the neuromasts and in the notochord where it overlaps with expression of caveolin-3. Morpholino-mediated downregulation of Cav1alpha caused a dramatic inhibition of neuromast formation. Detailed ultrastructural analysis, including electron tomography of the notochord, revealed that the central regions of the notochord has the highest density of caveolae of any embryonic tissue comparable to the highest density observed in any vertebrate tissue. In addition, Cav1alpha downregulation caused disruption of the notochord, an effect that was enhanced further by Cav3 knockdown. These results indicate an essential role for caveolin and caveolae in this vital structural and signalling component of the embryo.

Nitric oxide down-regulates caveolin-3 levels through the interaction with myogenin, its transcription factor.

Certain patients suffering from chronic diseases such as AIDS or cancer experience a constant cellular secretion of tumor necrosis factor alpha and other pro-inflammatory cytokines that results in a continuous release of nitric oxide (*NO) to the bloodstream. One immediate consequence of the deleterious action of *NO is weight loss and the progressive destruction of muscular mass in a process known as cachexia. We have previously reported that caveolin-3, a specific marker of muscle cells, becomes down-regulated by the action of *NO on muscular myotubes. We describe herein that the changes observed in caveolin-3 levels are due to the alteration of the DNA binding activity of the muscular transcription factor myogenin. In the presence of *NO, the binding of transcription factors from cell nuclear extracts of muscular tissues to the E boxes present in the caveolin-3 promoter become substantially reduced. When we purified recombinant myogenin and treated it with *NO donors, we could detect its S-nitrosylation by three independent methods, suggesting that very likely one of the cysteine residues of the molecule is being modified. Given the role of myogenin as a regulatory protein that determines the level of multiple muscle genes expressed during late myogenesis, our results might represent a novel mode of regulation of muscle development under conditions of nitric oxide-mediated toxicity.

Increased expression of caveolin-1 and -2 in the hearts of Lewis rats with experimental autoimmune myocarditis.

The expression of caveolin-1, -2 and -3 was studied in the hearts of rats with experimental autoimmune myocarditis (EAM), to elucidate the involvement of caveolins in the pathogenesis of EAM. Western blot analysis showed that levels of caveolin-1 and -2 were significantly increased in the hearts of rats with EAM on day 14 post-immunization (pi), as compared to the hearts of normal controls (p < 0.05, normal controls vs. EAM). Caveolin-3 is already at a high level in control animals, so it does not increase further. Immunohistochemistry showed that caveolin-1 was expressed mainly in ED1-positive macrophages and in some cardiomyocytes and vessels in the EAM lesions. Caveolin-2 was expressed constitutively in the vascular endothelial cells of normal hearts, and its expression was enhanced in EAM rats, as compared with the normal control group. Caveolin-3 was expressed constitutively in the plasma membranes of cardiomyocytes, but not in the vascular endothelial cells and inflammatory cells in the EAM lesions. Our results suggest that the expression of caveolin-1 and -2 is increased in EAM lesions and that the increased expression of caveolin-1 stimulates second signals in affected cells, such as macrophages and some cardiomyocytes, in EAM rats.