Impaired vascular contractility and blood pressure homeostasis in the smooth muscle alpha-actin null mouse.

The smooth muscle (SM) alpha-actin gene activated during the early stages of embryonic cardiovascular development is switched off in late stage heart tissue and replaced by cardiac and skeletal alpha-actins. SM alpha-actin also appears during vascular development, but becomes the single most abundant protein in adult vascular smooth muscle cells. Tissue-specific expression of SM alpha-actin is thought to be required for the principal force-generating capacity of the vascular smooth muscle cell. We wanted to determine whether SM alpha-actin gene expression actually relates to an actin isoform's function. Analysis of SM alpha-actin null mice indicated that SM alpha-actin is not required for the formation of the cardiovascular system. Also, SM alpha-actin null mice appeared to have no difficulty feeding or reproducing. Survival in the absence of SM alpha-actin may result from other actin isoforms partially substituting for this isoform. In fact, skeletal alpha-actin gene, an actin isoform not usually expressed in vascular smooth muscle, was activated in the aortas of these SM alpha-actin null mice. However, even with a modest increase in skeletal alpha-actin activity, highly compromised vascular contractility, tone, and blood flow were detected in SM alpha-actin-defective mice. This study supports the concept that SM alpha-actin has a central role in regulating vascular contractility and blood pressure homeostasis, but is not required for the formation of the cardiovascular system.     

α-Smooth muscle actin is transiently expressed in embryonic rat cardiac and skeletal muscles

Actin isoform expression may change during development, and in certain physiological, experimental and pathological situations. It is accepted that during sarcomeric (skeletal and cardiac) muscle development, the alpha-skeletal and alpha-cardiac isoforms of actin accumulate rapidly at the onset of muscle fibre formation, while there is a rapid fall in the expression of nonmuscle (beta and gamma) actin isoforms. Here we show that, before birth, both skeletal and myocardial cells express significant amounts of alpha-smooth muscle actin mRNA and protein. This expression is transient and disappears over the 1-7 days following birth. Our findings show that the program regulating actin isoform expression in sarcomeric muscle development is complex and that alpha-smooth muscle actin participates in this process.     

Simultaneous expression of skeletal muscle and heart actin proteins in various striated muscle tissues and cells. A quantitative determination of the two actin isoforms

A procedure was developed to determine the percentage of skeletal muscle actin and cardiac actin present in different striated muscle tissues. The method was applied to 2 mg of actin mixtures isolated from various origins. All samples show simultaneous expression of both striated muscle isoactins, with the cardiac actin being the major form (congruent to 80%) in 11-day-old chick embryonic leg muscle, decreasing to approximately 50% values in the late fetal stage of chicken, mouse, and in fused mouse muscle cell cultures and becoming the minor species (less than 5%) in adult skeletal muscle tissues. We also find a significant amount (up to 20%) of the skeletal muscle isoform in adult heart (ventricle) of porcine, bovine, and human origin and no differences in muscle actin ratios in human atrium and ventriculum cells. Similarly, no significant variation in the actin ratios was observed between a normal heart and a heart from a patient with hereditary obstructive myopathy. For those cells and tissues where comparison with levels of mRNA was possible we mostly find a good correlation between the relative ratios of expression of cardiac and skeletal actin proteins and mRNAs.     

A developmental study of the abnormal expression of alpha-cardiac and alpha-skeletal actins in the striated muscle of a mutant mouse

BALB/c mice possess a 5' duplication of the alpha-cardiac actin gene which is associated with abnormal levels of alpha-cardiac and alpha-skeletal actin mRNAs in adult cardiac tissue. This mutation therefore provides a potential tool for the study of the inter-relationship between the striated muscle actins. We have examined the expression of this actin gene pair throughout the development of skeletal and cardiac muscle in BALB/c mice. During embryonic and fetal development, the expression of these two genes is indistinguishable from that in normal mice, as determined by in situ hybridization. A quantitative postnatal study demonstrates that in the hearts of normal mice the level of alpha-cardiac actin mRNA declines, whereas that of alpha-skeletal actin increases. In mutant mice, these trends are exaggerated so that whereas normal mice have 95.8% alpha-cardiac mRNA and 4.2% alpha-skeletal mRNA in the adult heart, BALB/c mice have 52.4 and 47.6% of these mRNAs, respectively. This difference is also reflected at the protein level. In developing skeletal muscle, the expression of these genes follows kinetics similar to that observed in the heart with a decrease in the relative level of alpha-cardiac mRNA as the muscle matures. Cardiac actin mRNA levels are again lower in the mutant mouse, but here the effect is less striking because skeletal actin is the predominant isoform. These results are discussed in the context of the interaction between this actin gene pair in developing and adult striated muscle.     

Disruption of the sarcoglycan-sarcospan complex in vascular smooth muscle: a novel mechanism for cardiomyopathy and muscular dystrophy.

To investigate mechanisms in the pathogenesis of cardiomyopathy associated with mutations of the dystrophin-glycoprotein complex, we analyzed genetically engineered mice deficient for either alpha-sarcoglycan (Sgca) or delta-sarcoglycan (Sgcd). We found that only Sgcd null mice developed cardiomyopathy with focal areas of necrosis as the histological hallmark in cardiac and skeletal muscle. Absence of the sarcoglycan-sarcospan (SG-SSPN) complex in skeletal and cardiac membranes was observed in both animal models. Loss of vascular smooth muscle SG-SSPN complex was only detected in Sgcd null mice and associated with irregularities of the coronary vasculature. Administration of a vascular smooth muscle relaxant prevented onset of myocardial necrosis. Our data indicate that disruption of the SG-SSPN complex in vascular smooth muscle perturbs vascular function, which initiates cardiomyopathy and exacerbates muscular dystrophy.     

A fetal skeletal muscle actin mRNA in the mouse and its identity with cardiac actin mRNA

We compare a recombinant cDNA plasmid (pAF81) complementary to a fetal skeletal muscle actin mRNA with a plasmid (pAM91) complementary to the actin mRNA expressed in adult skeletal muscle. The two mRNAs are significantly diverged in silent nucleotide positions; they are coexpressed in fetal skeletal muscle, and in differentiating muscle cell cultures their accumulation begins coordinately. The sequence of pAF81 shows that the amino acid sequence of mouse fetal skeletal muscle actin is almost identical to that of adult bovine cardiac actin. Hybridization of pAF81 to RNA from different mouse tissues shows that fetal skeletal muscle actin mRNA is very homologous or identical to fetal and adult cardiac actin mRNA. Only one gene homologous to pAF81 is detected on blots of restricted mouse DNA. We conclude that this gene must be expressed both in fetal skeletal muscle and in fetal heart. Whereas mRNA transcribed from this gene is the major actin mRNA species in adult heart, it is present in low amounts, if at all, in adult skeletal muscle.     

Mitochondrial creatine kinase functional development in post-natal rat skeletal muscle. A combined polarographic/31P NMR study

Mitochondrial creatine kinase (Mi-CK) function in viable mitochondria from developing rat skeletal muscle was assessed both by polarographic measurements of creatine-induced respiration and 31P NMR spectroscopy measurements of phosphocreatine (PCr) synthesis. Creatine-induced respiration was observed in very young rats and increased by 50% to 35 days of age. PCr synthesis was present in 7 day old animals and increased by 300% reaching levels measured in 35 day and adult muscle. Unlike reports showing Mi-CK enzymatic activities but no mitochondrial function in several situations, a concomitant progression of enzymatic activity and mitochondrial function was evidenced during the developmental stages of skeletal muscle Mi-CK in altricious animals. These results correlated with the progressive pattern of muscle differentiation during development of motricity in such animals. The observation that Mi-CK is functional in skeletal muscle mitochondria very early after birth, strongly favors the notion that adaptations in skeletal muscle of Mi-CK knock-out mice occur early.     

Phalloidin suppresses force in nebulin-rich lamprey cardiac muscle

We assessed the effect of phalloidin, known to detach nebulin from actin in skeletal myofibrils, on the isometric force of skinned lamprey cardiac muscle, which has nebulin in amounts comparable to that in skeletal muscle. In contrast to mammalian cardiac muscle, which contains much less nebulin and reacts to phalloidin only by an increase in force, the lamprey cardiac muscle responds to phalloidin by a pronounced (～ 50%) reduction in isometric force, thereby resembling the behavior of skeletal muscle. These results support our hypothesis that nebulin detachment from actin underlies phalloidin-induced force loss and suggest a role of actin-nebulin interaction in contractile function.     

The alpha-subunit of AMPK is essential for submaximal contraction-mediated glucose transport in skeletal muscle in vitro

AMP-activated protein kinase (AMPK) is a key signaling protein in the regulation of skeletal muscle glucose uptake, but its role in mediating contraction-induced glucose transport is still debated. The effect of contraction on glucose transport is impaired in EDL muscle of transgenic mice expressing a kinase-dead, dominant negative form of the AMPKalpha(2) subunit (KD-AMPKalpha(2) mice). However, maximal force production is reduced in this muscle, raising the possibility that the defect in glucose transport was due to a secondary decrease in force production and not impaired AMPKalpha(2) activity. Generation of force-frequency curves revealed that muscle force production is matched between wild-type (WT) and KD-AMPKalpha(2) mice at frequencies < or =50 Hz. Moreover, AMPK activation is already maximal at 50 Hz in muscles of WT mice. When EDL muscles from WT mice were stimulated at a frequency of 50 Hz for 2 min (200-ms train, 1/s, 30 volts), contraction caused an approximately 3.5-fold activation of AMPKalpha(2) activity and an approximately 2-fold stimulation of glucose uptake. Conversely, whereas force production was similar in EDL of KD-AMPKalpha(2) animals, no effect of contraction was observed on AMPKalpha(2) activity, and glucose uptake stimulation was reduced by 50% (P < 0.01) As expected, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranosyl 5'-monophosphate (AICAR) caused a 2.3-fold stimulation of AMPKalpha(2) activity and a 1.7-fold increase in glucose uptake in EDL from WT mice, whereas no effect was detected in muscle from KD-AMPKalpha(2) mice. These data demonstrate that AMPK activation is essential for both AICAR and submaximal contraction-induced glucose transport in skeletal muscle but that AMPK-independent mechanisms are also involved.     

Phalloidin suppresses the force in nebulin-rich lamprey cardiac muscle

The effect of phalloidin, an agent detaching nebulin from actin in skeletal muscle, on the isometric force in lamprey skinned cardiac muscle, which has nebulin in amounts comparable to that in skeletal muscle, has been studied. We found that, unlike mammalian cardiac muscle expressing nebulin less abundantly and responding to phalloidin by a force increase, lamprey cardiac muscle responds to phalloidin by a force decrease (approximately 50% decrease), thereby resembling the response of skeletal muscle. These results support our hypothesis that nebulin detachment from actin underlies phalloidin-induced force loss and suggest a role of actin-nebulin interaction in contractile function.     

Respiratory uncoupling in skeletal muscle delays death and diminishes age-related disease

Age-related disease, not aging per se, causes most morbidity in older humans. Here we report that skeletal muscle respiratory uncoupling due to UCP1 expression diminishes age-related disease in three mouse models. In a longevity study, median survival was increased in UCP mice (animals with skeletal muscle-specific UCP1 expression), and lymphoma was detected less frequently in UCP female mice. In apoE null mice, a vascular disease model, diet-induced atherosclerosis was decreased in UCP animals. In agouti yellow mice, a genetic obesity model, diabetes and hypertension were reversed by induction of UCP1 in skeletal muscle. Uncoupled mice had decreased adiposity, increased temperature and metabolic rate, elevated muscle SIRT and AMP kinase, and serum characterized by increased adiponectin and decreased IGF-1 and fibrinogen. Accelerating metabolism in skeletal muscle does not appear to impact aging but may delay age-related disease.     

Expression of the genes coding for the skeletal muscle and cardiac actions in the heart

Several types of evidence indicate that the gene coding for the skeletal muscle actin is expressed in the rat heart: 1) A recombinant plasmid containing an insert with a nucleotide sequence identical to that of the homologous region of skeletal muscle actin gene was isolated from a cDNA library prepared on rat cardiac mRNA template. 2) Using specific probes it was found that the hearts of newborn rats contain a significant amount of skeletal muscle actin mRNA. The quantity of this mRNA in the heart decreases during development. 3) The skeletal muscle actin gene is DNAase I sensitive in nuclei from rat heart tissue. A plasmid containing a cDNA insert homologous to a part of the cardiac actin mRNA was isolated and sequenced. It was found that in spite of the great similarity between the amino acid sequence of the skeletal muscle and cardiac actins, the nucleotide sequences of the two mRNAs are considerably divergent. There is only limited sequence homology between the 3' untranslated regions of the two mRNAs. However, there is an extensive sequence homology between the 3' untranslated regions of the rat and human cardiac mRNAs, suggesting a functional role for this region of the gene or mRNA.     

Autophagic response to exercise training in skeletal muscle with age

Autophagy, a highly conserved quality control mechanism, is essential for the maintenance of cellular homeostasis and for the orchestration of an efficient cellular response to stress. During aging, the efficiency of autophagic degradation declines, and intracellular waste products accumulate. Therefore, in this study, we tested the hypothesis that skeletal muscle from old mice would have decreased autophagosome formation when compared to the muscle from young mice. We also examined whether autophagic regulatory events differ between muscle fiber types and in response to exercise in aged male mice. The extensor digitorum longus (EDL) and gastrocnemius muscles were studied in young and old ICR mice. Exercise was performed by allowing the mice to run on a treadmill with a 5° incline at 16.4 m/min for 40 min/day, 5 days/week for 8 weeks after a 1-week adaptation period. Our results indicated that the levels of microtubule-associated protein 1b light chain 3, a marker of autophagosome formation, were lower in both the EDL and the gastrocnemius muscle of old mice compared to those young mice. To identify the factors related to the changes observed, the expression of autophagy regulatory proteins was examined in the EDL and gastrocnemius muscles. Beclin-1, autophagy-related gene 7 (ATG7), and lysosome-associated membrane protein were found to be lower in the EDL and gastrocnemius muscles of old mice compared to those in the young mice, then Beclin-1, ATG7, and muscle-specific RING finger protein-1 upregulated after regular exercise. Moreover, the muscle weight/body weight was significantly increased only in the gastrocnemius muscle of the old trained mice. These data suggest that autophagy regulatory events are attenuated in old skeletal muscle. However, this effect is upregulated when animals are subjected to exercise training.     

Altered GLUT4 translocation in skeletal muscle of 12/15-lipoxygenase knockout mice.

We have recently shown that 12(S)-hydroxyeicosatetraenoic acid plays a role in the organization of actin microfilaments in rat cardiomyocytes, and that inhibition of 12-lipoxygenase abrogates insulin-stimulated GLUT4 translocation in these cells. In the present study, we used mice that were null for the leukocyte 12/15-lipoxygenase to explore the implications of this enzyme for insulin action under IN VIVO conditions. Insulin induced a profound reduction in blood glucose in both control and knockout mice. However, significantly higher serum insulin levels were observed in these animals. GLUT4 expression in heart and skeletal muscle was unaffected in KO mice. Insulin-regulated serine phosphorylation of Akt and GSK3alpha and GSK3beta was unaltered in heart and skeletal muscle of knockout mice, suggesting unaltered insulin signaling. Fractionation of hind limb muscles showed that insulin had induced a prominent translocation of GLUT4 to skeletal muscle plasma membranes in control mice. However, this response was largely reduced in knockout animals. Our data show that the lack of leukocyte 12/15-lipoxygenase does not lead to the development of an insulin-resistant phenotype. However, perturbation of GLUT4 translocation in skeletal muscle of knockout mice may indicate latent insulin resistance, and supports our hypothesis that eicosanoids are involved in insulin-mediated regulation of muscle glucose transport.     

Myostatin and MyoD family expression in skeletal muscle of IGF-1 knockout mice

Insulin-like growth factor-1 (IGF-1) is a positive regulator in proliferation and differentiation of skeletal muscle cells, while myostatin (MSTN) is a member of transforming growth factor beta superfamily that acts as a negative regulator of skeletal muscle mass. The present study was performed to detail whether a correlation exists between MSTN and IGF-1 in skeletal muscle of IGF-1 knockout mice (IGF-1(-/-)) and their wild type (WT; i.e., IGF-1(+/+)) littermates. The body weight of IGF-1(-/-) animals was 32% that of WT littermates. The fiber cross-sectional areas (CSA) and number of fibers in M. rectus femoris of IGF-1(-/-) animals were 49 and 59% those of WT animals, respectively. Thus, muscle hypoplasia of IGF-1(-/-) undoubtedly was confirmed. Myostatin mRNA levels and protein levels were similar between M. gastrocnemius of IGF-1(-/-) and WT animals. Myostatin immunoreactivity was similarly localized in muscle fibers of both IGF-1(-/-) and WT M. rectus femoris. The mRNA levels of MyoD family (Myf5, MyoD, MRF4, myogenin) were differentially expressed in IGF-1(-/-)M. gastrocnemius, in which the mRNA expression of MRF4 and myogenin was significantly lower, whereas there were no changes in the mRNA expression of Myf5 and MyoD. These findings first describe that myostatin expression is not influenced by intrinsic failure of IGF-1, although MRF4 and myogenin are downregulated.     

Effect of exercise on muscle fibre composition and enzyme activities of skeletal muscles in young rats

Young Wistar rats underwent dynamic (D) or static (S) exercise from the 5th to 35th day after birth. Histochemical and biochemical analysis were performed in the extensor digitorum longus (EDL) and the soleus muscle (SOL). Lactate dehydrogenase (LDH) (regulating anaerobic metabolism) and citrate synthase (CS) and hydroxyacyl-CoA dehydrogenase (HAD) (both regulating aerobic metabolism) activities were determined spectrophotometrically. An increase of the fast oxidative-glycolytic (FOG) muscle fibres was found in the slow SOL muscle in both trained groups, i.e. by 10% in group D and by 7% in group S in comparison with the C group. The EDL muscle fibre distribution did not differ from those of control animals in respect to the slow oxidative (SO) fibre type. A higher percentage of FOG fibres by 19% was found in group D contrary to a decreased number of the fast glycolytic (FG) muscle fibres in this trained group. The greatest increase of CS (EDL 185%, SOL 176%) and HAD (EDL 83%, SOL 178%) activities were found in group D as compared with control group (C). Only small differences were observed in LDH activity. The values of characteristic enzyme activity ratios show that dynamic training resulted in an elevation of oxidative capacity of skeletal muscle, while the static load led preferentially along the glycolytic pathway. It may be concluded that an adaptive response to the training load during early postnatal development is different due to the type of exercise (dynamic or static) and/or the type of skeletal muscle (fast or slow).