Lack of β2‐adrenoceptors aggravates heart failure‐induced skeletal muscle myopathy in mice

Skeletal myopathy is a hallmark of heart failure (HF) and has been associated with a poor prognosis. HF and other chronic degenerative diseases share a common feature of a stressed system: sympathetic hyperactivity. Although beneficial acutely, chronic sympathetic hyperactivity is one of the main triggers of skeletal myopathy in HF. Considering that β₂‐adrenoceptors mediate the activity of sympathetic nervous system in skeletal muscle, we presently evaluated the contribution of β₂‐adrenoceptors for the morphofunctional alterations in skeletal muscle and also for exercise intolerance induced by HF. Male WT and β₂‐adrenoceptor knockout mice on a FVB genetic background (β₂KO) were submitted to myocardial infarction (MI) or SHAM surgery. Ninety days after MI both WT and β₂KO mice presented to cardiac dysfunction and remodelling accompanied by significantly increased norepinephrine and epinephrine plasma levels, exercise intolerance, changes towards more glycolytic fibres and vascular rarefaction in plantaris muscle. However, β₂KO MI mice displayed more pronounced exercise intolerance and skeletal myopathy when compared to WT MI mice. Skeletal muscle atrophy of infarcted β₂KO mice was paralleled by reduced levels of phosphorylated Akt at Ser 473 while increased levels of proteins related with the ubiquitin‐–proteasome system, and increased 26S proteasome activity. Taken together, our results suggest that lack of β₂‐adrenoceptors worsen and/or anticipate the skeletal myopathy observed in HF.     

Autophagy inhibition induces atrophy and myopathy in adult skeletal muscles

Autophagy is required for cellular survival and for the clearance of damaged proteins and altered organelles. Excessive autophagy activation contributes to muscle loss in different catabolic conditions. However, the function of basal autophagy for homeostasis of skeletal muscle was unknown. To clarify this issue we have generated conditional and inducible knockout mice for the critical gene Atg7, to block autophagy specifically in skeletal muscle. Atg7 null muscles reveal an unexpected phenotype which is characterized by muscle atrophy, weakness and features of myofiber degeneration. Morphological, biochemical, and molecular analyses of our autophagy knockout mice show the presence of protein aggregates, abnormal mitochondria, accumulation of membrane bodies, sarcoplasmic reticulum distension, vacuolization, oxidative stress and apoptosis. Moreover, autophagy inhibition does not protect skeletal muscles from atrophy during denervation and fasting, but instead promotes greater muscle loss. In conclusion, autophagy plays a critical role for myofiber maintenance and its activation is crucial to avoid accumulation of toxic proteins and dysfunctional organelles that, in the end, would lead to atrophy and weakness.     

Effects of IGF‐1 isoforms on muscle growth and sarcopenia

The decline in skeletal muscle mass and strength occurring in aging, referred as sarcopenia, is the result of many factors including an imbalance between protein synthesis and degradation, changes in metabolic/hormonal status, and in circulating levels of inflammatory mediators. Thus, factors that increase muscle mass and promote anabolic pathways might be of therapeutic benefit to counteract sarcopenia. Among these, the insulin‐like growth factor‐1 (IGF‐1) has been implicated in many anabolic pathways in skeletal muscle. IGF‐1 exists in different isoforms that might exert different role in skeletal muscle. Here we study the effects of two full propeptides IGF‐1Ea and IGF‐1Eb in skeletal muscle, with the aim to define whether and through which mechanisms their overexpression impacts muscle aging. We report that only IGF‐1Ea expression promotes a pronounced hypertrophic phenotype in young mice, which is maintained in aged mice. Nevertheless, examination of aged transgenic mice revealed that the local expression of either IGF‐1Ea or IGF‐1Eb transgenes was protective against age‐related loss of muscle mass and force. At molecular level, both isoforms activate the autophagy/lysosome system, normally altered during aging, and increase PGC1‐α expression, modulating mitochondrial function, ROS detoxification, and the basal inflammatory state occurring at old age. Moreover, morphological integrity of neuromuscular junctions was maintained and preserved in both MLC/IGF‐1Ea and MLC/IGF‐1Eb mice during aging. These data suggest that IGF‐1 is a promising therapeutic agent in staving off advancing muscle weakness.     

Obese mice incur greater myofiber membrane disruption in response to mechanical load compared with lean mice

Objective:

Obesity is associated with modified transmembrane signaling events in skeletal muscle, such as insulin signaling and glucose transport. The underlying cause of these obesity‐related effects on transmembrane signaling is still unknown. In general, the function of membrane proteins responsible for transmembrane signaling is modulated by the biochemical makeup of the membrane, such as lipid composition, in which they are embedded. Any obesity‐related alterations in membrane composition would also be predicted to modify membrane biomechanical properties and membrane susceptibility to mechanical load‐induced damage. The primary objective of this study was to investigate whether obesity influences myofiber membrane susceptibility to mechanical damage in skeletal muscle.

Design and Methods:

Myofiber membrane damage was compared between 12‐week‐old obese, hypercholesterolemic (B6.V Lep^ob/J) and isogenic, normocholesterolemic control (C57BL6/J) male mice following either normal cage activity or strenuous eccentric exercise (downhill running). Myofiber membrane damage was quantified in perfusion‐fixed frozen sections of the gastrocnemius muscle via sarcoplasmic concentration of either albumin (cage activity experiment) or a fluorescent marker that had been injected immediately before activity (eccentric exercise experiment).

Results:

Obese mice exhibited evidence of increased myofiber membrane damage compared with lean mice after both normal cage activity and eccentric exercise indicating that myofiber membranes of obese mice are more susceptible to mechanical damage in general and that eccentric exercise exacerbates this effect.

Conclusions:

These observations are consistent with the notion that obesity influences the biochemical and biomechanical properties of myofiber membranes.     

Alteration of neurotransmitter phenotype in noradrenergic neurons of transgenic mice.

The normal complement of neurotransmitters in noradrenergic neurons was altered by expressing the structural gene for the enzyme phenylethanolamine-N-methyltransferase (PNMT) under the control of the dopamine-beta-hydroxylase gene promoter in transgenic mice. This resulted in accumulation of large amounts of epinephrine in neurons of the sympathetic nervous system (SNS) and central nervous system (CNS) but did not reduce norepinephrine levels. Adrenalectomy reduced PNMT levels in the SNS and CNS, suggesting that the transgene is positively regulated by adrenal steroids. Epinephrine levels were unaffected by this treatment in the CNS, suggesting that PNMT is not rate limiting for epinephrine synthesis. However, catecholamines were elevated in a sympathetic ganglion and a target tissue of the SNS, perhaps due to up-regulation of tyrosine hydroxylase in response to adrenalectomy. These transgenic mice also reveal a marked difference in the ability of chromaffin cells and neurons to synthesize epinephrine.     

A missense mutant myostatin causes hyperplasia without hypertrophy in the mouse muscle

Myostatin, which is a member of the TGF-beta superfamily, is a negative regulator of skeletal muscle formation. Double-muscled Piedmontese cattle have a C313Y mutation in myostatin and show increased skeletal muscle mass which resulted from an increase of myofiber number (hyperplasia) without that of myofiber size (hypertrophy). To examine whether this mutation in myostatin gene affects muscle development in a dominant negative manner, we generated transgenic mice overexpressing the mutated gene. The transgenic mice exhibited dramatic increases in the skeletal muscle mass resulting from hyperplasia without hypertrophy. In contrast, it has been reported that a myostatin mutated at its cleavage site produces hypertrophy without hyperplasia in the muscle. Thus, these results suggest that (1) the myostatin containing the missense mutation exhibits a dominant negative activity and that (2) there are two types in the dominant negative form of myostatin, causing either hypertrophy or hyperplasia.     

Pharmacological activation of PPARbeta promotes rapid and calcineurin-dependent fiber remodeling and angiogenesis in mouse skeletal muscle

Recent studies have shown that administration of peroxisome proliferator-activated receptor-beta (PPARbeta) agonists enhances fatty acid oxidation in rodent and human skeletal muscle and that muscle-restricted PPARbeta overexpression affects muscle metabolic profile by increasing oxidative myofiber number, which raises the possibility that PPARbeta agonists alter muscle morphology in adult animals. This possibility was examined in this study in which adult mice were treated with a PPARbeta agonist, and the resulting changes in myofiber metabolic phenotype and angiogenesis were quantified in tibialis anterior muscles. The findings indicate a muscle remodeling that is completed within 2 days and is characterized by a 1.63-fold increase in oxidative fiber number and by a 1.55-fold increase in capillary number. These changes were associated with a quick and transient upregulation of myogenic and angiogenic markers. Both myogenic and angiogenic responses were dependent on the calcineurin pathway, as they were blunted by cyclosporine A administration. In conclusion, the data indicate that PPARbeta activation is associated with a calcineurin-dependent effect on muscle morphology that enhances the oxidative phenotype.     

Exercise pressor reflex in humans with end-stage renal disease

Previous work has suggested that end-stage renal disease (ESRD) patients may have an exaggerated sympathetic nervous system (SNS) response during exercise. We hypothesized that ESRD patients have an exaggerated blood pressure (BP) response during moderate static handgrip exercise (SHG 30%) and that the exaggerated BP response is mediated by SNS overactivation, characterized by augmented mechanoreceptor activation and blunted metaboreceptor control, as has been described in other chronic diseases. We measured hemodynamics and muscle sympathetic nerve activity (MSNA) in 13 ESRD and 16 controls during: 1) passive hand movement (PHM; mechanoreceptor isolation); 2) low-level rhythmic handgrip exercise (RHG 20%; central command and mechanoreceptor activation); 3) SHG 30%, followed by posthandgrip circulatory arrest (PHGCA; metaboreceptor activation); and 4) cold pressor test (CPT; nonexercise stimulus). ESRD patients had exaggerated increases in systolic BP during SHG 30%; however, the absolute and relative increase in MSNA was not augmented, excluding SNS overactivation as the cause of the exaggerated BP response. Increase in MSNA was not exaggerated during RHG 20% and PHM, demonstrating that mechanoreceptor activation is not heightened in ESRD. During PHGCA, MSNA remained elevated in controls but decreased rapidly to baseline levels in ESRD, indicative of markedly blunted metaboreceptor control of MSNA. MSNA response to CPT was virtually identical in ESRD and controls, excluding a generalized sympathetic hyporeactivity in ESRD. In conclusion, ESRD patients have an exaggerated increase in SBP during SHG 30% that is not mediated by overactivation of the SNS directed to muscle. SBP responses were also exaggerated during mechanoreceptor activation and metaboreceptor activation, but without concomitant augmentation in MSNA responses. Metaboreceptor control of MSNA was blunted in ESRD, but the overall ability to mount a SNS response was not impaired. Other mechanisms besides SNS overactivation, such as impaired vasodilatation, should be explored to explain the exaggerated exercise pressor reflex in ESRD.     

Tropomyosin metabolism in muscles and its blood levels during physical exertion

Physical exercise induces phasic changes in the tropomyosin content and metabolism in muscles and its concentration in blood. The intensive catabolic processes (decrease of 14C-leicin inclusion and time of half-life) of muscle tropomyosin and its appearance in blood were shown 2-24 hours after the exercise. Intensive anabolic processes of muscle tropomyosin were found at the late period of rest (72-144 h). These results reveal the biochemical mechanism of muscle adaptation to physical exercise. Data on the tropomyosin content in blood permit recommending tropomyosin for development of the diagnostic test of functional condition of the skeletal muscle.     

Alpha2-AMPK activity is not essential for an increase in fatty acid oxidation during low-intensity exercise

A single bout of exercise increases glucose uptake and fatty acid oxidation in skeletal muscle, with a corresponding activation of AMP-activated protein kinase (AMPK). While the exercise-induced increase in glucose uptake is partly due to activation of AMPK, it is unclear whether the increase of fatty acid oxidation is dependent on activation of AMPK. To examine this, transgenic mice were produced expressing a dominant-negative (DN) mutant of alpha(1)-AMPK (alpha(1)-AMPK-DN) in skeletal muscle and subjected to treadmill running. alpha(1)-AMPK-DN mice exhibited a 50% reduction in alpha(1)-AMPK activity and almost complete loss of alpha(2)-AMPK activity in skeletal muscle compared with wild-type littermates (WT). The fasting-induced decrease in respiratory quotient (RQ) ratio and reduced body weight were similar in both groups. In contrast with WT mice, alpha(1)-AMPK-DN mice could not perform high-intensity (30 m/min) treadmill exercise, although their response to low-intensity (10 m/min) treadmill exercise was not compromised. Changes in oxygen consumption and the RQ ratio during sedentary and low-intensity exercise were not different between alpha(1)-AMPK-DN and WT. Importantly, at low-intensity exercise, increased fatty acid oxidation in response to exercise in soleus (type I, slow twitch muscle) or extensor digitorum longus muscle (type II, fast twitch muscle) was not impaired in alpha(1)-AMPK-DN mice, indicating that alpha(1)-AMPK-DN mice utilize fatty acid in the same manner as WT mice during low-intensity exercise. These findings suggest that an increased alpha(2)-AMPK activity is not essential for increased skeletal muscle fatty acid oxidation during endurance exercise.     

The α₇β₁-integrin increases muscle hypertrophy following multiple bouts of eccentric exercise

Mechanical stimuli increase skeletal muscle growth in a mammalian target of rapamycin (mTOR)- and p70(S6K)-dependent manner. It has been proposed that costameric proteins at Z bands may sense and transfer tension to these initiators of protein translation, but few candidates have been identified. The purpose of this study was to determine whether a role exists for the α(7)-integrin in the activation of hypertrophic signaling and growth following eccentric exercise training. Five-week-old, wild-type (WT) and α(7)BX2-integrin transgenic (α(7)Tg) mice were randomly assigned to one of two groups: 1) sedentary (SED), or 2) exercise training (EX). Exercise training consisted of downhill running 3 sessions/wk for 4 wk (-20°, 17 m/min, 30 min). Downhill running was used to induce physiological mechanical strain. Twenty-four hours following the final training session, maximal isometric hindlimb plantar flexor force was measured. Gastrocnemius-soleus complexes were collected for further analysis of signaling changes, which included AKT, mTOR and p70(S6K), and muscle growth. Despite increased p70(S6K) activity in WT/EX, no significant changes in cross-sectional area or force were observed in WT/EX compared with WT/SED. AKT, mTOR, and p70(S6K) activation was higher, and whole muscle hypertrophy, relative muscle weight, myofibrillar protein, and force were significantly elevated in α(7)Tg/EX compared with α(7)Tg/SED. A marked increase in average myofiber cross-sectional area was observed in α(7)Tg/EX compared with all groups. Our findings demonstrate that the α(7)β(1)-integrin sensitizes skeletal muscle to mechanical strain and subsequent growth. Thus the α(7)β(1)-integrin may represent a novel molecular therapy for the treatment of disuse muscle atrophy.