Cardiac-specific overexpression of caveolin-3 induces endogenous cardiac protection by mimicking ischemic preconditioning.

BACKGROUND: Caveolae, lipid-rich microdomains of the sarcolemma, localize and enrich cardiac-protective signaling molecules. Caveolin-3 (Cav-3), the dominant isoform in cardiac myocytes, is a determinant of caveolar formation. We hypothesized that cardiac myocyte-specific overexpression of Cav-3 would enhance the formation of caveolae and augment cardiac protection in vivo. METHODS AND RESULTS: Ischemic preconditioning in vivo increased the formation of caveolae. Adenovirus for Cav-3 increased caveolar formation and phosphorylation of survival kinases in cardiac myocytes. A transgenic mouse with cardiac myocyte-specific overexpression of Cav-3 (Cav-3 OE) showed enhanced formation of caveolae on the sarcolemma. Cav-3 OE mice subjected to ischemia/reperfusion injury had a significantly reduced infarct size relative to transgene-negative mice. Endogenous cardiac protection in Cav-3 OE mice was similar to wild-type mice undergoing ischemic preconditioning; no increased protection was observed in preconditioned Cav-3 OE mice. Cav-3 knockout mice did not show endogenous protection and showed no protection in response to ischemic preconditioning. Cav-3 OE mouse hearts had increased basal Akt and glycogen synthase kinase-3beta phosphorylation comparable to wild-type mice exposed to ischemic preconditioning. Wortmannin, a phosphoinositide 3-kinase inhibitor, attenuated basal phosphorylation of Akt and glycogen synthase kinase-3beta and blocked cardiac protection in Cav-3 OE mice. Cav-3 OE mice had improved functional recovery and reduced apoptosis at 24 hours of reperfusion. CONCLUSIONS: Expression of caveolin-3 is both necessary and sufficient for cardiac protection, a conclusion that unites long-standing ultrastructural and molecular observations in the ischemic heart. The present results indicate that increased expression of caveolins, apparently via actions that depend on phosphoinositide 3-kinase, has the potential to protect hearts exposed to ischemia/reperfusion injury.

Caveolin-3 expression and caveolae are required for isoflurane-induced cardiac protection from hypoxia and ischemia/reperfusion injury.

Volatile anesthetics protect the heart from ischemia/reperfusion injury but the mechanisms for this protection are poorly understood. Caveolae, sarcolemmal invaginations, and caveolins, scaffolding proteins in caveolae, localize molecules involved in cardiac protection. We tested the hypothesis that caveolae and caveolins are essential for volatile anesthetic-induced cardiac protection using cardiac myocytes (CMs) from adult rats and in vivo studies in caveolin-3 knockout mice (Cav-3(-/-)). We incubated CM with methyl-beta-cyclodextrin (MbetaCD) or colchicine to disrupt caveolae formation, and then exposed the myocytes to the volatile anesthetic isoflurane (30 min, 1.4%), followed by simulated ischemia/reperfusion (SI/R). Isoflurane protected CM from SI/R [23.2+/-1.6% vs. 71.0+/-5.8% cell death (assessed by trypan blue exclusion), P<0.001] but this protection was abolished by MbetaCD or colchicine (84.9+/-5.5% and 64.5+/-6.1% cell death, P<0.001). Membrane fractionation by sucrose density gradient centrifugation of CM treated with MbetaCD or colchicine revealed that buoyant (caveolae-enriched) fractions had decreased phosphocaveolin-1 and caveolin-3 compared to control CM. Cardiac protection in vivo was assessed by measurement of infarct size relative to the area at risk and cardiac troponin levels. Isoflurane-induced a reduction in infarct size and cardiac troponin relative to control (infarct size: 26.5%+/-2.6% vs. 45.3%+/-5.4%, P<0.01; troponin: 27.7+/-4.4 vs. 77.7+/-11.8 ng/ml, P<0.05). Isoflurane-induced cardiac protection was abolished in Cav-3(-/-) mice (infarct size: 53.4%+/-6.1% vs. 53.2%+/-3.5%, P<0.01; troponin: 102.1+/-22.3 vs. 105.9+/-8.2 ng/ml, P<0.01). Isoflurane-induced cardiac protection is thus dependent on the presence of caveolae and the expression of caveolin-3. We conclude that caveolae and caveolin-3 are critical for volatile anesthetic-induced protection of the heart from ischemia/reperfusion injury.

MicroRNA-206 is highly expressed in newly formed muscle fibers: implications regarding potential for muscle regeneration and maturation in muscular dystrophy.

miR-1, miR-133a, and miR-206 are muscle-specific microRNAs expressed in skeletal muscles and have been shown to contribute to muscle development. To gain insight into the pathophysiological roles of these three microRNAs in dystrophin-deficient muscular dystrophy, their expression in the tibialis anterior (TA) muscles of mdx mice and CXMD(J) dogs were evaluated by semiquantitative RT-PCR and in situ hybridization. Their temporal and spatial expression patterns were also analyzed in C2C12 cells during muscle differentiation and in cardiotoxin (CTX)-injured TA muscles to examine how muscle degeneration and regeneration affect their expression. In dystrophic TA muscles of mdx mice, miR-206 expression was significantly elevated as compared to that in control TA muscles of age-matched B10 mice, whereas there were no differences in miR-1 or miR-133a expression between B10 and mdx TA muscles. On in situ hybridization analysis, intense signals for miR-206 probes were localized in newly formed myotubes with centralized nuclei, or regenerating muscle fibers, but not in intact pre-degenerated fibers or numerous small mononucleated cells, possibly proliferating myoblasts and inflammatory infiltrates. Similar increased expression of miR-206 was also found in C2C12 differentiation and CTX-induced regeneration, in which differentiated myotubes or regenerating fibers showed abundant expression of miR-206. However, CXMD(J) TA muscles contained smaller amounts of miR-206, miR-1, and miR-133a than controls. They exhibited more severe and more progressive degenerative alterations than mdx TA muscles. Taken together, these observations indicated that newly formed myotubes showed markedly increased expression of miR-206, which might reflect active regeneration and efficient maturation of skeletal muscle fibers.

Class Specificity of Avian and Mammalian Sera in Regards to Myogenic Cell Growth in vitro: POSSIBLE ROLE OF TRANSFERRIN IN THE SPECIFICITY (class specificity of serum/cell growth/culture medium/muscle cell culture).

Chick myogenic cells grew in the presence of a small amount of avian serum in a culture medium composed of Eagle's minimum essential medium (MEM) and horse serum. Mammalian sera, except for fetal bovine serum at high concentrations, could not substitute for the avian serum. Rat myogenic cells grew in the presence of a small amount of mammalian serum in a culture medium composed of MEM and chick serum: avian sera, except for dove serum at high concentrations, could not substitute for the mammalian serum. Serum from animals of the class from which the myoblasts were obtained was needed for cell growth. It is thus concluded that there is a class specificity among sera in regards to myogenic cell growth. The only exceptions to this hypothesis found so far were fetal bovine and dove sera.

A New Method for Fibroblast-less Primary Skeletal Muscle Cell Culture by the Use of Hydroxyurea: (skeletal muscle cell culture/hydroxyurea/muscle cell differentiation/myosin heavy chain/dystrophin).

A method was developed to suppress growth of fibroblasts in chicken and mouse primary skeletal muscle cell cultures. Addition of hydroxyurea to the culture medium at appropriate time and concentrations suppressed the proliferation of fibroblasts whereas leaving myotubes grow and differentiate. The most favorable time for the addition was soon after myotube formation. The optimal concentrations for our purpose ranged from 0.5 to 1.0 mM. In the presence of hydroxyurea at these concentrations, myotubes grew larger and well differentiated, whereas fibroblasts remained in the suppressed state. In chicken myotubes cultured with hydroxyurea, cross-striations, spontaneous twitching and myosin heavy chain appeared as in myotubes without hydroxyurea. In mouse myotubes cultured with hydroxyurea, myosin heavy chain and dystrophin appeared, as in control myotubes.

Vimentin Expression Pattern is Different between the Flank Region and Limb Regions of Somatopleural Mesoderm in the Chicken Embryo: (vimentin/chicken embryo/limb bud/limbness/mesoderm).

It is known that the chicken flank somatopleure also has a limb-forming potential at early stages of development, but loses this potential later. Molecular changes during this process is, however, not well known. We obtained a monoclonal antibody which reacts to the flank somatopleure, but not to the wing bud, the leg bud and the neck somatopleure in the stage 22 chicken embryo. Further study revealed that this antibody is specific to vimentin. Time course of vimentin expression in the somatopleural mesoderm during the development was studied. It was revealed to be biphasic. Somatopleural mesoderm expressed vimentin at stage 10, but not at stage 16. Flank somatopleural mesoderm began to express vimentin again at stage 18, whereas limb bud mesenchymal cells did not until stage 27. The earlier re-expression of vimentin at the flank somatopleure suggests that certain physiological changes take place in cells at this region.

Chick Embryo Extract, Muscle Trophic Factor and Chick and Horse Sera as Environments for Chick Myogenic Cell Growth.

Chick myogenic cells grew in a medium composed of Eagle's minimum essential medium (MEM), horse serum (HS), and one of the essential factors needed for myogenic cell growth (EFMG), that is, chick embryo extract (EE), chick serum (CS), or the muscle trophic factor (MTF). But they did not grow in the absence of the EFMG. In the absence of HS, they scarcely grew in a medium composed of MEM, and EE or MTF. They grew in a medium composed of MEM and CS; they grew much better in a medium composed of MEM, CS, and HS. In the presence of one of the EFMG, the optimal HS concentration for growth varied depending on its lot. At higher HS concentrations, growth was suppressed. Further, it was suggested that an inhibitory substance(s) for myogenic cell growth was present in HS. The inhibitory effects can usually be minimized by diluting the serum with an artificial medium.

Indispensability of Iron for the Growth of Cultured Chick Cells: (iron/transition metal/transferrin/chick embryonic cell/myogenesis).

In order to clarify the role of iron in the growth promoting effect of transferrin (Tf), the effects of the following substances were examined in cultured chick skeletal myogenic cells: transition metal ions (Fe2+ , Fe3+ , Cr3+ , Cu2+ , Mn2+ , Co2+ , Cd2+ , Zn2+ and Ni2+ ), Tf complexes with these metals and metal-free apoTf. The cells did not grow well when incubated in a culture medium composed of Eagle's minimum essential medium and horse serum. But they grew well in the presence of Fe2+ or Fe3+ (10-100 µM) or iron-bound Tf (10-500 nM) in the medium. None of the transition metal ions other than iron was effective. Neither apoTf nor Tf complexes with these metals showed the growth promoting effect. The generality of the requirement of iron for cell growth was ascertained in the primary culture of other types of chick embryonic cells: fibroblasts, cardiac myocytes, retinal pigment cells and spinal nerve cells. The results show that iron is one of the indispensable substances for cell growth and suggest that Tf protein plays a role in facilitating the transport of iron into the cells.

Molecular and cell biology of the sarcoglycan complex.

The original sarcoglycan (SG) complex has four subunits and comprises a subcomplex of the dystrophin-dystrophin-associated protein complex. Each SG gene has been shown to be responsible for limb-girdle muscular dystrophy, called sarcoglycanopathy (SGP). In this review, we detail the characteristics of the SG subunits, and the mechanism of the formation of the SG complex and various molecules associated with this complex. We discuss the molecular mechanisms of SGP based on studies mostly using SGP animal models. In addition, we describe other SG molecules, epsilon- and zeta-SGs, with special reference to their expression and roles in vascular smooth muscle, which are currently in dispute. We further consider the maternally imprinted nature of the epsilon-SG gene. Finally, we stress that the SG complex cannot work by itself and works in a larger complex system, called the transverse fixation system, which forms an array of molecules responsible for various muscular dystrophies.

Immunolocalization of caveolin-1 and caveolin-3 in monkey skeletal, cardiac and uterine smooth muscles.

Caveolin, a 20-24 kDa integral membrane protein, is a principal component of caveolar domains. Caveolin-1 is expressed predominantly in endothelial cells, fibroblasts, and adipocytes, while the expression of caveolin-3 is confined to muscle cells. However, their localization in various muscles has not been well documented. Using double-immunofluorescence labeling and confocal laser microscopy, we examined the localization of caveolins-1 and 3 in adult monkey skeletal, cardiac and uterine smooth muscles and the co-immunolocalization of these caveolins with dystrophin, which is a product of the Duchenne muscular dystrophy gene. In the skeletal muscle tissue, caveolin-3 was localized along the sarcolemma except for the transverse tubules, and co-immunolocalized with dystrophin, whereas caveolin-1 was absent except in the blood vessels of the muscle tissue. In cardiac muscle cells, caveolins-1 and -3 and dystrophin were co-immunolocalized on the sarcolemma and transverse tubules. In uterine smooth muscle cells, caveolin-1, but not caveolin-3, was co-immunolocalized with dystrophin on the sarcolemma.

Insulin resistance in skeletal muscles of caveolin-3-null mice.

Type 2 diabetes is preceded by the development of insulin resistance, in which the action of insulin is impaired, largely in skeletal muscles. Caveolin-3 (Cav3) is a muscle-specific subtype of caveolin, an example of a scaffolding protein found within membranes. Cav is also known as growth signal inhibitor, although it was recently demonstrated that the genetic disruption of Cav3 did not augment growth in mice. We found, however, that the lack of Cav3 led to the development of insulin resistance, as exemplified by decreased glucose uptake in skeletal muscles, impaired glucose tolerance test performance, and increases in serum lipids. Such impairments were markedly augmented in the presence of streptozotocin, a pancreatic beta cell toxin, suggesting that the mice were susceptible to severe diabetes in the presence of an additional risk factor. Insulin-stimulated activation of insulin receptors and downstream molecules, such as IRS-1 and Akt, was attenuated in the skeletal muscles of Cav3 null mice, but not in the liver, without affecting protein expression or subcellular localization. Genetic transfer of Cav3 by needle injection restored insulin signaling in skeletal muscles. Our findings suggest that Cav3 is an enhancer of insulin signaling in skeletal muscles but does not act as a scaffolding molecule for insulin receptors.