Sex-based differences in skeletal muscle kinetics and fiber-type composition.

Previous studies have identified over 3,000 genes that are differentially expressed in male and female skeletal muscle. Here, we review the sex-based differences in skeletal muscle fiber composition, myosin heavy chain expression, contractile function, and the regulation of these physiological differences by thyroid hormone, estrogen, and testosterone. The findings presented lay the basis for the continued work needed to fully understand the skeletal muscle differences between males and females.

Skeletal muscle adaptations to microgravity exposure in the mouse.

To investigate the effects of microgravity on murine skeletal muscle fiber size, muscle contractile protein, and enzymatic activity, female C57BL/6J mice, aged 64 days, were divided into animal enclosure module (AEM) ground control and spaceflight (SF) treatment groups. SF animals were flown on the space shuttle Endeavour (STS-108/UF-1) and subjected to approximately 11 days and 19 h of microgravity. Immunohistochemical analysis of muscle fiber cross-sectional area revealed that, in each of the muscles analyzed, mean muscle fiber cross-sectional area was significantly reduced (P < 0.0001) for all fiber types for SF vs. AEM control. In the soleus, immunohistochemical analysis of myosin heavy chain (MHC) isoform expression revealed a significant increase in the percentage of muscle fibers expressing MHC IIx and MHC IIb (P < 0.05). For the gastrocnemius and plantaris, no significant changes in MHC isoform expression were observed. For the muscles analyzed, no alterations in MHC I or MHC IIa protein expression were observed. Enzymatic analysis of the gastrocnemius revealed a significant decrease in citrate synthase activity in SF vs. AEM control.

Potential limitations of transcription terminators used as transgene insulators in adenoviral vectors.

The presence of adenoviral cis-elements interfering with the activity of tissue-specific promoters has seriously impaired the use of transcriptional targeting adenoviruses for gene therapy purposes. As an approach to overcome this limitation, transcription terminators were previously employed in cultured cells to insulate a transgene promoter from viral activation. To extend these studies in vivo, we have injected into heart and skeletal muscle, adenoviruses containing the human growth hormone terminator and the cardiac-specific alpha-myosin heavy chain promoter (alphaMyHC) driving the chloramphenicol acetyltransferase (CAT) reporter gene. Promoterless CAT constructs were also tested to study interfering viral transcription and terminator activity. Here we demonstrate that the presence of a terminator can produce undesirable effects on the activity of heterologous promoters. Our analysis shows that in particular conditions, a terminator can reduce the tissue specificity of the transgene promoter. By RNAse protection assay performed on cardiac myocytes, we also show that adenoviral elements can direct high levels of autonomous transcription within the E1A enhancer region. This finding supports the model that passive readthrough of the transgene promoter is responsible for loss of selective expression.

Skeletal muscle adaptations in response to voluntary wheel running in myosin heavy chain null mice.

10.1152/ japplphysiol.00832.2001.-To examine the effects of gene inactivation on the plasticity of skeletal muscle, mice null for a specific myosin heavy chain (MHC) isoform were subjected to a voluntary wheel-running paradigm. Despite reduced running performance compared with nontransgenic C57BL/6 mice (NTG), both MHC IIb and MHC IId/x null animals exhibited increased muscle fiber size and muscle oxidative capacity with wheel running. In the MHC IIb null animals, there was no significant change in the percentage of muscle fibers expressing a particular MHC isoform with voluntary wheel running at any time point. In MHC IId/x null mice, wheel running produced a significant increase in the percentage of fibers expressing MHC IIa and MHC I and a significant decrease in the percentage of fibers expressing MHC IIb. Muscle pathology was not affected by wheel running for either MHC null strain. In summary, despite their phenotypes, MHC null mice do engage in voluntary wheel running. Although this wheel-running activity is lessened compared with NTG, there is evidence of distinct patterns of muscle adaptation in both null strains.

Different pathways regulate expression of the skeletal myosin heavy chain genes.

Mammalian skeletal muscles are a mosaic of different fiber types largely defined by differential myosin heavy chain (MyHC) expression. Little is known about the molecular mechanisms regulating expression of the MyHC gene family members in different fiber types. In this work, we identified several cis- and trans-elements that regulate expression of the three adult fast MyHC genes. Despite multiple DNA-binding motifs for well characterized muscle transcription factors upstream of all three fast MyHC genes, expression of MyoD/Myf-5, calcineurin, or NFAT3 had different effects on the three promoters. MyoD or Myf-5 overexpression preferentially activated the IIb promoter, whereas NFAT or activated calcineurin overexpression preferentially activated the IIa promoter. Calcineurin had a 50-100-fold stimulatory effect on the IIa promoter, and the known downstream effectors of calcineurin (myocyte enhancer factor-2 and NFAT) cannot completely account for this activation. Finally, we identified two elements critical for regulating MyHC-IId/x expression: a 130-base pair enhancer element and a CArG-like element that inhibited IId/x promoter activity in vitro. Thus, we have found specific regulatory pathways that are distinct for the three adult fast MyHC genes. These elements are logical candidates for fiber-specific control of skeletal muscle gene expression in vivo.

Low sequence variation in the gene encoding the human beta-myosin heavy chain.

Over 40 different mutations in the cardiac myosin heavy chain gene (MYH7) have been associated with familial hypertrophic cardiomyopathy (FHC), but no study has analyzed variation at this locus within the normal human population. Here we determine the extent and distribution of nucleotide variation in the 5808-bp MYH7 coding sequence in 25 normal individuals without FHC. We identified six single-nucleotide polymorphisms, none of which changes the encoded amino acid. At one of these sites, the frequencies of both alleles are equal; at the other five sites, the frequency of the rarer allele varies from 0.02 to 0.08. The nucleotide diversity (pi) calculated from these data is 1.73x10(-4)+/-0.49x10(-4), which is lower than the nucleotide diversity found in most other human autosomal genes. Substitution analysis of homologous genes between human and rodent also indicates that the MYH7 sequence has evolved at a very slow rate. The rate of both synonymous and nonsynonymous substitutions, especially in the portion of the sequence that encodes the alpha-helical myosin rod, is extremely low. The low level of even silent sequence variation in MYH7 in comparisons between human sequences and between human and rodent sequences may be a consequence of strong selective pressure against mutations that cause cardiomyopathy.

Alterations in cardiac adrenergic signaling and calcium cycling differentially affect the progression of cardiomyopathy.

The medical treatment of chronic heart failure has undergone a dramatic transition in the past decade. Short-term approaches for altering hemodynamics have given way to long-term, reparative strategies, including beta-adrenergic receptor (betaAR) blockade. This was once viewed as counterintuitive, because acute administration causes myocardial depression. Cardiac myocytes from failing hearts show changes in betaAR signaling and excitation-contraction coupling that can impair cardiac contractility, but the role of these abnormalities in the progression of heart failure is controversial. We therefore tested the impact of different manipulations that increase contractility on the progression of cardiac dysfunction in a mouse model of hypertrophic cardiomyopathy. High-level overexpression of the beta(2)AR caused rapidly progressive cardiac failure in this model. In contrast, phospholamban ablation prevented systolic dysfunction and exercise intolerance, but not hypertrophy, in hypertrophic cardiomyopathy mice. Cardiac expression of a peptide inhibitor of the betaAR kinase 1 not only prevented systolic dysfunction and exercise intolerance but also decreased cardiac remodeling and hypertrophic gene expression. These three manipulations of cardiac contractility had distinct effects on disease progression, suggesting that selective modulation of particular aspects of betaAR signaling or excitation-contraction coupling can provide therapeutic benefit.

Cardiac and skeletal muscle adaptations to voluntary wheel running in the mouse.

In this paper, we describe the effects of voluntary cage wheel exercise on mouse cardiac and skeletal muscle. Inbred male C57/Bl6 mice (age 6-8 wk; n = 12) [corrected] ran an average of 4.3 h/24 h, for an average distance of 6.8 km/24 h, and at an average speed of 26.4 m/min. A significant increase in the ratio of heart mass to body mass (mg/g) was evident after 2 wk of voluntary exercise, and cardiac atrial natriuretic factor and brain natriuretic peptide mRNA levels were significantly increased in the ventricles after 4 wk of voluntary exercise. A significant increase in the percentage of fibers expressing myosin heavy chain (MHC) IIa was observed in both the gastrocnemius and the tibialis anterior (TA) by 2 wk, and a significant decrease in the percentage of fibers expressing IIb MHC was evident in both muscles after 4 wk of voluntary exercise. The TA muscle showed a greater increase in the percentage of IIa MHC-expressing fibers than did the gastrocnemius muscle (40 and 20%, respectively, compared with 10% for nonexercised). Finally, the number of oxidative fibers as revealed by NADH-tetrazolium reductase histochemical staining was increased in the TA but not the gastrocnemius after 4 wk of voluntary exercise. All results are relative to age-matched mice housed without access to running wheels. Together these data demonstrate that voluntary exercise in mice results in cardiac and skeletal muscle adaptations consistent with endurance exercise.

Myofibroblasts: molecular crossdressers.

Myofibroblasts are unique mesenchymal cells with properties inherent to both muscle and nonmuscle cells. They are widely distributed in embryos, are essential for the formation of functional adult tissues, and are intimately involved in tissue homeostasis and wound healing. Cytoskeletal protein expression and contractile properties distinguish them from other cell types. Myofibroblasts also express skeletal muscle structural and regulatory proteins, including sarcomeric myosin heavy chain and MyoD. Despite the presence of such myogenic regulatory proteins, these cells do not terminally differentiate into skeletal muscle. This article focuses on the interesting biology of myofibroblasts, their origin, and the molecular mechanisms that allow these cells to maintain a state intermediate between muscle and nonmuscle cells.

Ca(2+) activation of myofilaments from transgenic mouse hearts expressing R92Q mutant cardiac troponin T.

The functional consequences of the R92Q mutation in cardiac troponin T (cTnT), linked to familial hypertrophic cardiomyopathy in humans, are not well understood. We have studied steady- and pre-steady-state mechanical activity of detergent-skinned fiber bundles from a transgenic (TG) mouse model in which 67% of the total cTnT in the heart was replaced by the R92Q mutant cTnT. TG fibers were more sensitive to Ca(2+) than nontransgenic (NTG) fibers [negative logarithm of half maximally activating molar Ca(2+) (pCa(50)) = 5.84 +/- 0.01 and 6.12 +/- 0.01 for NTG and TG fibers, respectively]. The shift in pCa(50) caused by increasing the sarcomere length from 1.9 to 2.3 microm was significantly higher for TG than for NTG fibers (DeltapCa(50) = 0.13 +/- 0.01 and 0.29 +/- 0.02 for NTG and TG fibers, respectively). The relationships between rate of ATP consumption and steady-state isometric tension were linear, and the slopes were the same in NTG and TG fibers. Rate of tension redevelopment was more sensitive to Ca(2+) in TG than in NTG fibers (pCa(50) = 5.71 +/- 0.02 and 6.07 +/- 0.02 for NTG and TG fibers, respectively). We concluded that overall cross-bridge cycling kinetics are not altered by the R92Q mutation but that altered troponin-tropomyosin interactions could be responsible for the increase in myofilament Ca(2+) sensitivity in TG myofilaments.

Gender and aging in a transgenic mouse model of hypertrophic cardiomyopathy.

Mutations in the cardiac myosin heavy chain (MHC) can cause familial hypertrophic cardiomyopathy (FHC). A transgenic mouse model has been developed in which a missense (R403Q) allele and an actin-binding deletion in the alpha-MHC are expressed in the heart. We used an isovolumic left heart preparation to study the contractile characteristics of hearts from transgenic (TG) mice and their wild-type (WT) littermates. Both male and female TG mice developed left ventricular (LV) hypertrophy at 4 mo of age. LV hypertrophy was accompanied by LV diastolic dysfunction, but LV systolic function was normal and supranormal in the young TG females and males, respectively. At 10 mo of age, the females continued to present with LV concentric hypertrophy, whereas the males began to display LV dilation. In female TG mice at 10 mo of age, impaired LV diastolic function persisted without evidence of systolic dysfunction. In contrast, in 10-mo-old male TG mice, LV diastolic function worsened and systolic performance was impaired. Diminished coronary flow was observed in both 10-mo-old TG groups. These types of changes may contribute to the functional decompensation typically seen in hypertrophic cardiomyopathy. Collectively, these results further underscore the potential utility of this transgenic mouse model in elucidating pathogenesis of FHC.

Mutation of the IIB myosin heavy chain gene results in muscle fiber loss and compensatory hypertrophy.

The fast skeletal IIb gene is the source of most myosin heavy chain (MyHC) in adult mouse skeletal muscle. We have examined the effects of a null mutation in the IIb MyHC gene on the growth and morphology of mouse skeletal muscle. Loss in muscle mass of several head and hindlimb muscles correlated with amounts of IIb MyHC expressed in that muscle in wild types. Decreased mass was accompanied by decreases in mean fiber number, and immunological and ultrastructural studies revealed fiber pathology. However, mean cross-sectional area was increased in all fiber types, suggesting compensatory hypertrophy. Loss of muscle and body mass was not attributable to impaired chewing, and decreased food intake as a softer diet did not prevent the decrease in body mass. Thus loss of the major MyHC isoform produces fiber loss and fiber pathology reminiscent of muscle disease.

Postnatal myosin heavy chain isoform expression in normal mice and mice null for IIb or IId myosin heavy chains.

The patterns of myosin heavy chain (MyHC) isoform expression in the embryo and in the adult mouse are reasonably well characterized and quite distinct. However, little is known about the transition between these two states, which involves major decreases and increases in the expression of several MyHC genes. In the present study, the expression of seven sarcomeric MyHCs was analyzed in the hindlimb muscles of wild-type mice and in mice null for the MyHC IIb or IId/x genes at several time points from 1 day of postnatal life (dpn) to 20 dpn. In early postnatal life, the developmental isoforms (embryonic and perinatal) comprise >90% of the total MyHC expression, while three adult fast isoforms (IIa, IIb, and IId) comprise <1% of the total MyHC protein. However, between 5 and 20 dpn their expression increases to comprise >90% of the total MyHC. Expression of each of the three adult fast isoforms occurs in a spatially and temporally distinct manner. We also show that alpha MyHC, which is almost exclusively expressed in the heart, is expressed in scattered fibers in all hindlimb muscles during postnatal development. Surprisingly, the timing and localization of expression of the MyHC isoforms is unchanged in IIb and IId/x null mice, although the magnitude of expression is altered for some isoforms. Together these data provide a comprehensive overview of the postnatal expression pattern of the sarcomeric MyHC isoforms in the mouse hindlimb.

Progression from hypertrophic to dilated cardiomyopathy in mice that express a mutant myosin transgene.

A mouse model of hypertrophic cardiomyopathy (HCM) was created by expression of a cardiac alpha-myosin transgene including the R(403)Q mutation and a deletion of a segment of the actin-binding domain. HCM mice show early histopathology and hypertrophy, with progressive hypertrophy in females and ventricular dilation in older males. To test the hypothesis that dilated cardiomyopathy (DCM) is part of the pathological spectrum of HCM, we studied chamber morphology, exercise tolerance, hemodynamics, isolated heart function, adrenergic sensitivity, and embryonic gene expression in 8- to 11-mo-old male transgenic animals. Significantly impaired exercise tolerance and both systolic and diastolic dysfunction were seen in vivo. Contraction and relaxation parameters of isolated hearts were also decreased, and lusitropic responsiveness to the beta-adrenergic agonist isoproterenol was modestly reduced. Myocardial levels of the G protein-coupled beta-adrenergic receptor kinase 1 (beta-ARK1) were increased by more than twofold over controls, and total beta-ARK1 activity was also significantly elevated. Induction of fetal gene expression was also observed in transgenic hearts. We conclude that transgenic male animals have undergone cardiac decompensation resulting in a DCM phenotype. This supports the idea that HCM and DCM may be part of a pathological continuum rather than independent diseases.

Inactivation of myosin heavy chain genes in the mouse: diverse and unexpected phenotypes.

Myosin heavy chain (MyHC) is a critical component of the cellular contractile apparatus. The mammalian genome contains two nonmuscle, two smooth muscle, and eight striated muscle isoforms of MyHC. Within each class of genes, there is extremely high sequence homology among different MyHC isoforms, raising the question of whether these isoforms are functionally redundant or whether they perform unique roles in cell function. Recently, strains of mice null for four different MyHC isoforms have been generated. Mice null for the nonmuscle II-B isoform experience significant prenatal lethality and surviving animals have several cardiac abnormalities [Tullio et al. (1997) Proc Natl Acad Sci USA 94:12407-12412]. Mice homozygous null for alpha cardiac MyHC are embryonic lethal, while heterozygous mice are viable but also have numerous cardiac defects [Jones et al. (1996) J Clin Invest 98:1906-1917]. Mice null for IIb or IId adult skeletal MyHC are viable but have skeletal muscle abnormalities compared to wild type mice, despite compensation of a neighboring MyHC gene [Acakpo-Satchivi et al. (1997) J Cell Biol 139:1219-1229]. Both IIb and IId null mice show significant decreases in body mass. Mean muscle mass is also significantly decreased in both null strains but the extent and the pattern of affected muscles differs between the two strains. Both strains show evidence of skeletal muscle pathology but again the pattern and extent differ between the two strains. Finally, both adult skeletal strains demonstrate distinct impairments in contractile function when compared to wild type. Together these observations support the hypothesis that the different isoforms of MyHC are functionally unique and cannot substitute for one another.

Modulation of vascular endothelial gene expression by physical training in patients with chronic heart failure.

BACKGROUND: Abnormalities of the skeletal muscle vasculature, such as endothelial dysfunction and reduced microvascular density, can be reversed by physical training in patients with chronic heart failure. The molecular mechanisms that mediate the beneficial effects of physical training on the vascular endothelium are unknown. METHODS: Endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) gene expression in the skeletal muscle, peak oxygen consumption (VO2) and calf peak reactive hyperemia were measured before and after 12 weeks of supervised physical training in 10 patients with chronic heart failure. Five patients with heart failure of similar severity who did not participate in the training program served as controls. RESULTS: The effects of physical training on eNOS and VEGF gene expression were heterogeneous. eNOS gene expression increased 3-4 fold in 4 patients while it remained constant in 6 patients. VEGF gene expression increased significantly in all patients who were not treated with beta-adrenergic blockade and remained constant in all patients who were treated with beta-adrenergic blockade. In contrast, physical training increased peak VO2 and calf peak reactive hyperemia in all patients. Mean peak VO2 increased from 13.13 +/- 2.21 to 16.19 +/- 2.69 ml/kg/min (p < 0.001) and calf peak reactive hyperemia increased from 19.7 +/- 2.3 to 29.6 +/- 4.0 ml*min(-1)*100 ml(-1) (p < 0.001). CONCLUSIONS: A supervised program of physical training that consistently enhanced peak VO2 and vascular reactivity in patients with chronic heart failure increased or left eNOS and VEGF gene expression unchanged in skeletal muscle. Changes in vascular endothelial gene expression may contribute to the benefits of training on vascular endothelial function but are not solely responsible for these benefits.

Animal models of hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is an autosomal-dominant disease that is both clinically and genetically heterogeneous. Disease-causing mutations have been found in eight genes encoding structural components of the thick and thin filament systems of the cardiac myocyte; it has therefore been coined a disease of the sarcomere. How each mutation leads to the diverse clinical phenotypes is still obscure, and research in this area is very active. Many approaches have been used to characterize the pathogenesis of the disease. Biochemical characterization of mutant alleles expressed in vitro has shed some insight into the functional deficits of several mutant alleles of myosin heavy chain, troponin-T, and alpha-tropomyosin. Transgenic animal models for FHC have been created to gain further insight into the pathogenesis of this disease. Most of these models have been made in mice; however, recently a transgenic rabbit model has been created. In addition, there are several natural-occurring forms of FHC in animals that will be interesting to explore. The discovery of additional responsible genes and the elucidation of the molecular mechanisms of pathogenesis through the use of animal models promise improved and early diagnosis and the potential for developing specific, mutation-, or mechanism-based therapeutics.