Contraction-induced changes in skeletal muscle Na+,K+ pump mRNA expression – importance of exercise intensity and Ca2+-mediated signalling

Aim: To investigate if exercise intensity and Ca²⁺ signalling regulate Na⁺,K⁺ pump mRNA expression in skeletal muscle. Methods: The importance of exercise intensity was evaluated by having trained and untrained humans perform intense intermittent and prolonged exercise. The importance of Ca²⁺ signalling was investigated by electrical stimulation of rat soleus and extensor digitorum longus (EDL) muscles in combination with studies of cell cultures. Results: Intermittent cycling exercise at ∼85% of VO_2peak increased (P < 0.05) α1 and β1 mRNA expression ∼2-fold in untrained and trained subjects. In trained subjects, intermittent exercise at ∼70% of VO_2peak resulted in a less (P < 0.05) pronounced increase (∼1.4-fold; P < 0.05) for α1 and no change in β1 mRNA. Prolonged low intensity exercise increased (P < 0.05) mRNA expression of α1 ∼3.0-fold and α2 ∼1.8-fold in untrained but not in trained subjects. Electrical stimulation of rat soleus, but not EDL, muscle increased (P < 0.05) α1 mRNA expression, but not when combined with KN62 and cyclosporin A incubation. Ionomycin incubation of cultured primary rat skeletal muscle cells increased (P < 0.05) α1 and reduced (P < 0.001) α2 mRNA expression and these responses were abolished (P < 0.05) by co-incubation with cyclosporin A or KN62. Conclusion: (1) Exercise-induced increases in Na⁺,K⁺ pump α1 and β1 mRNA expression in trained subjects are more pronounced after high- than after moderate- and low-intensity exercise. (2) Both prolonged low and short-duration high-intensity exercise increase α1 mRNA expression in untrained subjects. (3) Ca²⁺_i regulates α1 mRNA expression in oxidative muscles via Ca²⁺/calmodulin-dependent protein kinase (CaMK) and calcineurin signalling pathways.     

Deficiency of α-actinin-3 is associated with increased susceptibility to contraction-induced damage and skeletal muscle remodeling

Sarcomeric α-actinins (α-actinin-2 and -3) are a major component of the Z-disk in skeletal muscle, where they crosslink actin and other structural proteins to maintain an ordered myofibrillar array. Homozygosity for the common null polymorphism (R577X) in ACTN3 results in the absence of fast fiber-specific α-actinin-3 in ～20% of the general population. α-Actinin-3 deficiency is associated with decreased force generation and is detrimental to sprint and power performance in elite athletes, suggesting that α-actinin-3 is necessary for optimal forceful repetitive muscle contractions. Since Z-disks are the structures most vulnerable to eccentric damage, we sought to examine the effects of α-actinin-3 deficiency on sarcomeric integrity. Actn3 knockout mouse muscle showed significantly increased force deficits following eccentric contraction at 30% stretch, suggesting that α-actinin-3 deficiency results in an increased susceptibility to muscle damage at the extremes of muscle performance. Microarray analyses demonstrated an increase in muscle remodeling genes, which we confirmed at the protein level. The loss of α-actinin-3 and up-regulation of α-actinin-2 resulted in no significant changes to the total pool of sarcomeric α-actinins, suggesting that alterations in fast fiber Z-disk properties may be related to differences in functional protein interactions between α-actinin-2 and α-actinin-3. In support of this, we demonstrated that the Z-disk proteins, ZASP, titin and vinculin preferentially bind to α-actinin-2. Thus, the loss of α-actinin-3 changes the overall protein composition of fast fiber Z-disks and alters their elastic properties, providing a mechanistic explanation for the loss of force generation and increased susceptibility to eccentric damage in α-actinin-3-deficient individuals.     

The PPARα agonists fenofibrate and CP-778875 cause increased β-oxidation, leading to oxidative injury in skeletal and cardiac muscle in the rat

Weak peroxisome proliferator-activated receptor (PPAR) α agonists (fibrates) are used to treat dyslipidemia. This study compared the effects of the potent and selective PPARα agonist CP-778875 on peroxisomal β-oxidation and cardiac and/or skeletal muscle injury with those of the weak PPARα agonist fenofibrate. We hypothesized that these muscle effects are mediated through the PPARα receptor, leading to increased β-oxidation and consequent oxidative stress. CP-778875 (5 or 500 mg/kg) and fenofibrate (600 or 2,000→1,200 mg/kg, dose lowered because of intolerance) were administered to rats for six weeks. Standard end points, serum troponin I, heart and skeletal muscle β-oxidation of palmitoyl-CoA, and acyl co-oxidase (AOX) mRNA were assessed. Both compounds dose-dependently increased the incidence and/or severity of cardiomyocyte degeneration and necrosis, heart weight, troponin I, and skeletal muscle degeneration. Mean heart β-oxidation (3.4- to 5.1-fold control) and AOX mRNA (2.4- to 3.2-fold control) were increased with CP-778875 500 mg/kg and both doses of fenofibrate. β-Oxidation of skeletal muscle was not affected by either compound; however, a significant increase in AOX mRNA (1.6- to 2.1-fold control) was observed with CP-778875 500 mg/kg and both doses of fenofibrate. Taken together, these findings were consistent with PPARα agonism and support the link between increased cardiac and skeletal muscle β-oxidation and resultant muscle injury in the rat.     

Caffeine activates preferentially α1-isoform of 5'AMP-activated protein kinase in rat skeletal muscle

Aim: Caffeine activates 5′AMP‐activated protein kinase (AMPK), a signalling intermediary implicated in the regulation of glucose, lipid and energy metabolism in skeletal muscle. Skeletal muscle expresses two catalytic α subunits of AMPK, α1 and α2, but the isoform specificity of caffeine‐induced AMPK activation is unclear. The aim of this study was to determine which α isoform is preferentially activated by caffeine in vitro and in vivo using rat skeletal muscle. Methods: Rat epitrochlearis muscle was isolated and incubated in vitro in the absence or presence of caffeine. In another experiment, the muscle was dissected after intravenous injection of caffeine. Isoform‐specific AMPK activity, the phosphorylation status of AMPKα Thr¹⁷² and acetyl‐CoA carboxylase (ACC) Ser⁷⁹, the concentrations of ATP, phosphocreatine (PCr) and glycogen, and 3‐O‐methyl‐d ‐glucose (3MG) transport activity were estimated. Results: Incubation of isolated epitrochlearis muscle with 1 mm of caffeine for 15 min increased AMPKα1 activity, but not AMPKα2 activity; concentrations of ATP, PCr and glycogen were not affected. Incubation with 3 mm of caffeine activated AMPKα2 and reduced PCr and glycogen concentrations. Incubation with 1 mm of caffeine increased the phosphorylation of AMPK and ACC and enhanced 3MG transport. Intravenous injection of caffeine (5 mg kg^?1) predominantly activated AMPKα1 and increased 3MG transport without affecting energy status. Conclusion: Our results suggest that of the two α isoforms of AMPK, AMPKα1 is predominantly activated by caffeine via an energy‐independent mechanism and that the activation of AMPKα1 increases glucose transport and ACC phosphorylation in skeletal muscle.     

Na+–K+ pump location and translocation during muscle contraction in rat skeletal muscle

Muscle contraction may up-regulate the number of Na(+)-K(+) pumps in the plasma membrane by translocation of subunits. Since there is still controversy about where this translocation takes place from and if it takes place at all, the present study used different techniques to characterize the translocation. Electrical stimulation and biotin labeling of rat muscle revealed a 40% and 18% increase in the amounts of the Na(+)-K(+) pump alpha(2) subunit and caveolin-3 (Cav-3), respectively, in the sarcolemma. Exercise induced a 36% and 19% increase in the relative amounts of the alpha(2) subunit and Cav-3, respectively, in an outer-membrane-enriched fraction and a 41% and 17% increase, respectively, in sarcolemma giant vesicles. The Na(+)-K(+) pump activity measured with the 3-O-MFPase assay was increased by 37% in giant vesicles from exercised rats. Immunoprecipitation with Cav-3 antibody showed that 17%, 11% and 14% of the alpha(1) subunits were associated with Cav-3 in soleus, extensor digitorum longus, and mixed muscles, respectively. For the alpha(2), the corresponding values were 17%, 5% and 16%. In conclusion; muscle contraction induces translocation of the alpha subunits, which is suggested to be caused partly by structural changes in caveolae and partly by translocation from an intracellular pool.     

The effects of β-adrenergic stimulation and exercise on NR4A3 protein expression in rat skeletal muscle

β-Adrenergic stimulation and exercise up-regulate the mRNA expression of nuclear receptor NR4A3, which is involved in the regulation of glucose and fatty acid utilization genes in skeletal muscle. The objective of our study was to examine the effects of β-adrenergic stimulation and exercise on the expression of NR4A3 protein in rat skeletal muscle. A single subcutaneous injection of clenbuterol, which is a β2-adrenergic receptor (β2-AR) agonist, increased NR4A3 mRNA and protein expression in the fast-twitch glycolytic triceps muscle. On the other hand, an acute 3-h session of either treadmill running or swimming did not increase the NR4A3 protein level in the exercised muscle, although both treadmill running and swimming increased NR4A3 mRNA. Finally, loss of postural contractile activity because of hindlimb immobilization reduced NR4A3 mRNA and protein in the slow-twitch oxidative soleus muscle. These results suggest that: β-adrenergic stimulation up-regulates not only NR4A3 mRNA but also NR4A3 protein in fast-twitch glycolytic muscle; exercise may increase NR4A3 mRNA but not NR4A3 protein in skeletal muscle; and local postural contractile activity plays a crucial role in maintaining NR4A3 protein expression level in postural muscle.     

Exercise with low glycogen increases PGC-1α gene expression in human skeletal muscle

Recent studies suggest that carbohydrate restriction can improve the training-induced adaptation of muscle oxidative capacity. However, the importance of low muscle glycogen on the molecular signaling of mitochondrial biogenesis remains unclear. Here, we compare the effects of exercise with low (LG) and normal (NG) glycogen on different molecular factors involved in the regulation of mitochondrial biogenesis. Ten highly trained cyclists (VO_2max 65 ± 1 ml/kg/min, W _max 387 ± 8 W) exercised for 60 min at approximately 64 % VO_2max with either low [166 ± 21 mmol/kg dry weight (dw)] or normal (478 ± 33 mmol/kg dw) muscle glycogen levels achieved by prior exercise/diet intervention. Muscle biopsies were taken before, and 3 h after, exercise. The mRNA of peroxisome proliferator-activated receptor-γ coactivator-1 was enhanced to a greater extent when exercise was performed with low compared with normal glycogen levels (8.1-fold vs. 2.5-fold increase). Cytochrome c oxidase subunit I and pyruvate dehydrogenase kinase isozyme 4 mRNA were increased after LG (1.3- and 114-fold increase, respectively), but not after NG. Phosphorylation of AMP-activated protein kinase, p38 mitogen-activated protein kinases and acetyl-CoA carboxylase was not changed 3 h post-exercise. Mitochondrial reactive oxygen species production and glutathione oxidative status tended to be reduced 3 h post-exercise. We conclude that exercise with low glycogen levels amplifies the expression of the major genetic marker for mitochondrial biogenesis in highly trained cyclists. The results suggest that low glycogen exercise may be beneficial for improving muscle oxidative capacity.     

Investigation of changes in skeletal muscle α-actin expression in normal and pathological human and mouse hearts

We have developed a quantitative antibody-based assay to measure the content of skeletal muscle α-actin relative to cardiac α-actin. We found 21 ± 2% skeletal muscle α-actin content in normal heart muscle of adult man and mouse. In end stage failing heart 53 ± 5% of striated actin was skeletal muscle α-actin and in samples of inter-ventricular septum from patients with hypertrophic obstructive cardiomyopathy (HOCM) skeletal muscle α-actin was 72 ± 2% of sarcomeric actin. Thin filaments containing actin isolated from normal and HOCM heart muscle were functionally indistinguishable when studied by quantitative in vitro motility assay. We also found elevated skeletal muscle α-actin (60 ± 7%) in a mouse model of dilated cardiomyopathy.     

Calcineurin A and CaMKIV transactivate PGC-1α promoter, but differentially regulate cytochrome c promoter in rat skeletal muscle

In skeletal muscle, slow-twitch fibers are highly dependent on mitochondrial oxidative metabolism suggesting the existence of common regulatory pathways in the control of slow muscle-specific protein expression and mitochondrial biogenesis. In this study, we determined whether peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α) could transactivate promoters of nuclear-encoded mitochondrial protein (cytochrome c) and muscle-specific proteins (fast troponin I, MyoD). We also investigated if calcineurin A (CnA) and calcium/calmodulin kinase IV (CaMKIV) were involved in the regulation of PGC-1α and cytochrome c promoter. For this purpose, we took advantage of the gene electrotransfer technique, which allows acute expression of a gene of interest. Electrotransfer of a PGC-1α expression vector into rat Tibialis anterior muscle induced a strong transactivation of cytochrome c promoter (P < 0.001) independent of nuclear respiratory factor 1. PGC-1α gene electrotransfer did not transactivate fast troponin I promoter, whereas it did transactivate MyoD promoter (P < 0.05). Finally, whereas electrotransfers of CnA or CaMKIV expression vectors transactivated PGC-1α promoter (P < 0.001), gene electrotransfer of CaMKIV was only able to transactivate cytochrome c promoter. Taken together, these data suggest that CnA triggers PGC-1α promoter transactivation to drive the expression of non-mitochondrial proteins.     

CL 316,243, a selective β3-adrenergic agonist, inhibits protein breakdown in rat skeletal muscle

The in vitro effect of CL 316,243 (CL), a selective ₃-adrenoceptor agonist in the rate of overall proteolysis, the activity of proteolytic systems (lysosomal, Ca²⁺-dependent, ATP-dependent, and ATP-independent) and in the process of protein synthesis was investigated in rat skeletal muscles. The rate of overall proteolysis in soleus muscle from rats incubated with CL (10^–4 and 10^–5 M) or epinephrine (10^–5 M) was significantly decreased. In vitro rates of maximal activity of Ca²⁺-dependent proteolysis in soleus muscles were decreased by about 41% in the presence of 10^–5 M CL. No change was observed in the activities of the lysosomal, ATP-dependent or ATP-independent proteolytic systems. The anti-proteolytic effect of CL or epinephrine was partially prevented by 10^–5 M SR 59230A, a selective ₃-adrenoceptor antagonist. The increase of proteolysis induced by food deprivation in soleus was abolished by in vitro addition of 10^–5 M CL. No change in proteolysis was observed in extensor digitorum longus (EDL) muscles incubated with any concentration of the ₃-adrenoceptor agonist tested. Rates of protein synthesis were not affected by 10^–4 M CL neither in soleus nor EDL. The data suggest that a ₃-adrenoceptor-mediated inhibition of Ca²⁺-dependent proteolysis participates of the antiproteolytic effect of catecholamines in oxidative muscles.     

Higher TNFα responses in young males compared to females are associated with attenuation of monocyte adenylyl cyclase expression

Tumor necrosis factor α (TNFα) expression is strongly attenuated by the intracellular signaling mediator cyclic adenosine monophosphate (cAMP), which is synthesized by adenylyl cyclase (AC) enzymes. We have compared AC regulation and TNFα production in male and female monocytes, and characterized the role of monocyte AC isoforms in TNFα regulation.Males and females, age groups 20–30 years and 50–70 years donated blood for this study. In lipopolysaccharide-stimulated blood from young male donors, we observed significantly higher TNFα responses (6 h, p = 0.03) compared to females of the same age, a difference not observed in the older donors. Rapid down-regulation of the monocyte AC isoforms AC4, AC7 and AC9 were observed in young males. AC-directed siRNA experiments in the human monocyte cell line THP-1 demonstrated that AC7 and AC9 knock-down significantly induced TNFα release (p = 0.01 for both isoforms). These data indicate that the stronger TNFα-responses in young males may be partly associated with male-specific down-regulation of adenylyl cyclases.     

Ischemia/reperfusion-induced changes in intracellular free Ca2+ levels in rat skeletal muscle fibers – an in vivo study

Accumulation of intracellular free calcium (Ca2+i) may play an essential role in the ischemia/reperfusion injury of skeletal muscle. Although it has been shown that Ca2+i levels significantly increase during ischemia/reperfusion, it is still a matter of debate whether Ca2+i increases during ischemia alone. It was the aim of this study to monitor the in vivo Ca2+i levels in the rat spinotrapezius muscle during ischemia of varying duration and reperfusion, using a ratiometric fluorescence technique, and to investigate the relationship between the postischemic flow patterns and Ca2+i, if any. The muscle was loaded with Indo-1/AM and imaged by a cooled digital camera. Pre- and postischemic tissue perfusion was assessed by means of an analogue camera. Our results show that short-term ischemia (5, 15 and 30 min) and subsequent reperfusion (60 min) does not alter Ca2+i homeostasis and that tissue perfusion promptly recovers after the insult. One or two hours of ischemia resulted in changes in Ca2+i levels, varying from preparation to preparation; increases in some and no changes in others. In these preparations three distinct flow patterns - normal, compromised and no-reflow - could be distinguished during the 60-min reperfusion. Our main conclusion is that in skeletal muscle Ca2+i levels may increase, the increase probably depending on the muscle fiber type exposed.     

Four weeks one-leg training and high fat diet does not alter PPARα protein or mRNA expression in human skeletal muscle

Fatty acid metabolism is influenced by training and diet with exercise training mediating this through activation of nuclear hormone receptor peroxisome proliferator-activated receptor α (PPARα) in skeletal muscle. This study investigated the effect of training and high fat or normal diet on PPARα expression in human skeletal muscle. Thirteen men trained one leg (T) four weeks (31.5 h in total), while the other leg (UT) served as control. During the 4 weeks six subjects consumed high fat (FAT) diet and seven subjects maintained a normal (CHO) diet. Biopsies were obtained from vastus lateralis muscle in both legs before and after training. After the biopsy, one-leg extension exercise was performed in random order with both legs 30 min at 95% of workload max. A training effect was evident as citrate synthase activity increased (P < 0.05) by 15% in the trained, but not the control leg in both groups. During exercise respiratory exchange ratio was lower in FAT (0.86 ± 0.01, 0.83 ± 0.01, mean ± SEM) than CHO (0.96 ± 0.02, 0.94 ± 0.03) and in UT than T legs, respectively. The PPARα protein (144 ± 44, 104 ± 28, 79 ± 15, 79 ± 14, % of pre level) and PPARα mRNA (69 ± [2, 2], 78 ± [7, 6], 92 ± [22, 18], 106 ± [21, 18], % of pre level, geometric mean ± SEM) expression remained unchanged by diet and training in FAT (UT, T) and CHO (UT, T), respectively. After the training and diet CS, HAD, PPARα, UCP2, UCP3 and mFABP mRNA content remained unchanged, whereas GLUT4 mRNA was lower in both groups and LDHA mRNA was lower (P < 0.05) only in FAT. In conclusion: 4 weeks one leg knee extensor training did not affect PPARα protein or mRNA expression. Furthermore, higher fat oxidation during exercise after fat rich diet was not accompanied by an increased PPARα protein or mRNA expression after 4 weeks.     

Progressive arteriolar vasoconstriction and fatigue during tetanic contractions of rat skeletal muscle are inhibited by α-receptor blockade

Voluntary muscle contractions activate sympathetic efferent pathways. Using a fatiguing electrical stimulation protocol designed specifically to enhance sympathetically-mediated vasoconstrictor tone, we explored the temporal profile and mechanistic bases of the evoked vasoconstrictor response and its role in muscle fatigue. Spinotrapezius muscles of Wistar rats were exteriorized and stimulated tetanically (100 Hz, 6–8 V, stimulus duration 700 ms) every 3 s for 2.5 min. The extent and time course of diameter changes in arterioles (1A and 2A) and venules (1V and 2V) were determined after each of 10 discrete sets of muscle stimulation at 5-min intervals. At first, to compare the effect of stimulation parameters in this preparation, stimulations were performed with rectangular pulses of either 0.2- or 4-ms pulse duration. As expected the 0.2-ms pulse stimulation did not affect arteriolar diameter or muscle fatigability. In contrast, during and following 4-ms pulse stimulations, there was a surprising arteriolar vasoconstriction rather than the expected vasodilation. Arteriolar (but not venular) vasoconstriction (reduced arteriolar diameter by 38.6 ± 2.6% in the 10th set) increased progressively with muscle fatigue (to 29 ± 12% of initial tension in the 10th set) for the 4-ms pulse condition. Superfusion with the selective α1-adrenergic receptor antagonist prazosin (1 μM) and/or α2-adrenergic receptor antagonist rauwolscine (10 μM) abolished both the arteriolar vasoconstriction and significantly reduced fatigue (i.e., % initial tension, α1: 46.8 ± 10.3%; α2: 39.0 ± 5.8%; α1 + α2: 48.7 ± 16.3% in the 10th set; all P < 0.05 vs. control). We conclude that sequential bouts of contractions induce a progressively greater degree of α-adrenergic receptor-induced arteriolar (but not venular) vasoconstriction which contributes significantly to fatigue in this model.     

Charge movement and calcium currents in skeletal muscle fibers are enhanced by GTPγS

G-proteins play several regulatory roles in the cell. They can modulate ionic channels directly or in association with second messengers. In skeletal muscle, G-proteins modulate the activity of calcium channels either by acting directly on the channel and/or through a cAMP-dependent phosphorylating mechanism¹⁹. The activation of G-proteins by GTPS can also induce force generation in skinned fibers⁷. In this paper we studied the effect of GTP-S on charge movement and calcium currents (I_Ca) in rat and frog skeletal muscle, using the Vaseline gap technique. We observed an increase in both charge movement and I_Ca after the intracellular addition of 10–100 M GTPS. GDPS did not have any effect. Addition of protein kinase A catalytic subunit increased the I_Ca, probably through a phosphorylation process, but did not modify the charge movement. This suggests that protein kinase A and GTPS are acting on different sites of the channel. It can be speculated that G-proteins may have a regulatory role in the excitationcontraction coupling mechanism by a direct effect on charge movement.     

Lymphotoxin-α and galectin-2 SNPs are not associated with myocardial infarction in two different German populations

Recent data provided strong evidence for the association of single nucleotide polymorphisms (SNPs) in the lymphotoxin-alpha (LTA) and galectin-2 (LGALS2) genes with myocardial infarction (MI) in a Japanese population. For populations of other genetic background, the relevance of these polymorphisms in the pathogenesis of MI remains controversial. We aimed to define the role of LTA and LGALS2 SNPs in two German MI populations with markedly different ascertainment strategies. Two different MI populations were studied. In the first population, MI patients were ascertained by a strong family history of MI (n = 1214). Controls were unrelated disease-free participants of the study (n = 1080). The second population included patients suffering from sporadic (nonfamilial) MI from the German KORA register (n = 607). The control group consisted of participants of the WHO MONICA survey in Germany (n = 1492). TaqMan assays were used to determine the genotypes of 4 SNPs in the LTA genomic region and 1 SNP in the LGALS2 gene. Single SNPs in both genomic regions as well as haplotypes in the LTA genomic region were tested for association in various models of inheritance. No association with MI could be found for any of the examined SNPs in the LTA genomic region and LGALS2 gene, or for haplotypes spanning the LTA genomic region. In two MI populations of European descent with markedly different ascertainment strategies, we were not able to identify a significant association of SNPs in the LTA genomic region or the LGALS2 gene with MI. These variants are unlikely to play a significant role in populations of European origin.