Effects of exercise on mitochondrial content and function in aging human skeletal muscle

Skeletal muscle mitochondria are implicated with age-related loss of function and insulin resistance. We examined the effects of exercise on skeletal muscle mitochondria in older (age = 67.3 +/- 0.6 years) men (n = 5) and women (n = 3). Similar increases in (p <.01) cardiolipin (88.2 +/- 9.0 to 130.6 +/- 7.5 microg/mU creatine kinase activity [CK]) and the total mitochondrial DNA (1264 +/- 170 to 1895 +/- 273 copies per diploid of nuclear genome) reflected increased mitochondria content. Succinate oxidase activity, complexes 2-4 of the electron transport chain (ETC), increased from 0.13 +/- 0.02 to 0.20 +/- 0.02 U/mU CK (p <.01). This improvement was more pronounced (p <.05) in subsarcolemmal (127 +/- 48%) compared to intermyofibrillar (56 +/- 12%) mitochondria. NADH oxidase activity, representing total ETC activity, increased from 0.51 +/- 0.09 to 1.00 +/- 0.09 U/mU CK (p <.01). In conclusion, exercise enhances mitochondria ETC activity in older human skeletal muscle, particularly in subsarcolemmal mitochondria, which is likely related to the concomitant increases in mitochondrial biogenesis.     

Effect of heart failure on skeletal muscle myofibrillar protein content, isoform expression and calcium sensitivity

BACKGROUND: Alterations in skeletal muscle with heart failure contribute to exercise intolerance and physical disability. The majority of studies to date have examined abnormalities in skeletal muscle oxidative capacity and mitochondrial function. In contrast, less information is available regarding the effect of heart failure on myofibrillar protein metabolism and function. To address this issue, we examined the effect of heart failure on skeletal muscle myofibrillar protein content, isoform distribution and Ca2+ sensitivity. METHODS: We measured skeletal muscle myosin heavy chain (MHC) and actin protein content and MHC isoform distribution in soleus (SOL), extensor digitorum longus (EDL), plantaris (PL) and diaphragm (DIA) muscles and myofibrillar Ca2+ sensitivity in EDL muscles from Dahl salt-sensitive rats with (high-salt fed: HS; n=10) or without heart failure (low-salt fed: LS; n=8) and assessed the relationship of these variables to markers of disease severity. RESULTS: No differences in muscle mass were found. Similarly, no differences in MHC (mean+/-SE; SOL: 1353+/-29 vs. 1247+/-52; EDL: 1471+/-31 vs. 1441+/-31; PL: 1207+/-66 vs. 1286+/-36; DIA: 1166+/-42 vs. 1239+/-26 AU/microg protein) or actin (EDL: 348+/-13 vs. 358+/-19; PL: 245+/-20 vs. 242+/-9; DIA: 383+/-9 vs. 376+/-17 AU/microg protein) protein content or the actin-to-MHC ratio were observed, with the exception of lower (P<0.01) actin content in the soleus of LS rats (352+/-7 vs. 310+/-8 AU/microg protein). MHC isoform expression (I, IIa, IIx, IIb) did not differ between groups in SOL (I: 89+/-1% vs. 85+/-2%; IIa: 11+/-1% vs. 15+/-2%), EDL (IIx: 43+/-10% vs. 38+/-10%; IIb: 57+/-10% vs. 62+/-10%), PL (I: 6+/-4% vs. 3+/-3%; IIa: 1+/-1% vs. 1+/-1%; IIx: 31+/-3% vs. 26+/-4%; IIb: 62+/-5% vs. 71+/-6%) or DIA (I: 43+/-6% vs. 36+/-6 %; IIa: 9+/-1% vs. 7+/-1%; IIx: 47+/-6% vs. 56+/-7%; IIb: 2+/-1% vs. 1+/-0.5%) muscles. Moreover, heart failure did not affect the Ca2+ sensitivity (i.e., pCa50) of extensor digitorum longus myofilaments (5.68+/-0.11 vs. 5.65+/-0.09). Finally, MHC and actin content, MHC isoform distribution and myofibrillar Ca2+ sensitivity were not related to markers of disease severity. CONCLUSIONS: Our results show that this animal model of heart failure is not characterized by alterations in the quantity or isoform distribution of key skeletal muscle myofibrillar proteins or the Ca2+ sensitivity of isometric force production. These findings suggest that alterations in skeletal muscle myofibrillar protein metabolism do not develop in parallel with myocardial failure in the Dahl salt-sensitive rat.     

Effects of aging on atrial and ventricular human myosin

Enzymatic and structural studies of human cardiac myosin from young and old subjects have been investigated to determine possible changes in myosin properties in aging hearts. Human ventricular myosin from old subjects (47-70 years old) has lower actin-activated ATPase activity than and increased alkaline sensitivity as compared to myosin from young subjects (1-132 months old). Ca2+-and K+(EDTA)-ATPase activities, pyrophosphate gel patterns and one-dimensional peptide mapping of heavy chains of ventricular myosin from old subjects are similar to those observed for myosin from young subjects. Atrial myosin from human hearts differs significantly from ventricular myosin in that the Ca2+-, Mg2+- and actin-activated myosin Mg2+-ATPase activities of atrial myosin are significantly higher than those of ventricular myosin. Pyrophosphate gel electrophoresis patterns and peptide mapping of heavy chains of atrial myosin are also different from those of ventricular myosin. Unlike ventricular myosin, atrial myosin from young hearts is similar to that of atrial myosin from old hearts in its enzymatic and structural properties.     

Changes in skeletal muscle protein metabolism and myosin heavy chain isoform messenger ribonucleic acid abundance after treatment of hyperthyroidism

BACKGROUND: Hyperthyroidism causes a hypermetabolic state and skeletal muscle dysfunction, but the underlying mechanism remains incompletely defined. OBJECTIVE: The objective of the study was to determine whether treatment of hyperthyroidism causes changes in amino acid fluxes, synthesis rates of muscle proteins, and expression of muscle myosin heavy chain (MHC) that may impact skeletal muscle function and metabolic rate. METHODS: Eight hyperthyroid patients were studied (TSH 0.008 +/- 0.001 mU/liter) before treatment and at least 9 months after correction of hyperthyroidism (TSH 2.3 +/- 0.4) (P < 0.03). Fluxes of leucine and phenylalanine as well as muscle protein synthesis rates were measured using L[1,2 13C] leucine and L(15N) phenylalanine as tracers. mRNA levels of selected genes were measured in muscle biopsy samples. RESULTS: Treatment decreased resting metabolic rate that paralleled changes in fluxes of leucine and phenylalanine accompanied by improved muscle strength and mass. Synthesis rates of mixed muscle proteins (P = 0.01), sarcoplasmic (P = 0.04), and mitochondrial (P = 0.08) proteins decreased, whereas MHC synthesis was unchanged. Selective increases in mRNA abundance of muscle MHC1 isoform (P = 0.04) and decrease of MHCIIA (P = 0.007) and MHCIIx (P = 024) were observed. Muscle mitochondrial oxidative enzymes and mRNA levels of mitochondrial proteins were unchanged, but uncoupling protein2 and uncoupling protein3 mRNA levels (P = 0.02) decreased. CONCLUSION: Increased amino acid flux, mixed muscle protein synthesis, and synthesis of sarcoplasmic proteins are consistent with the hypermetabolic state in hyperthyroidism. After treatment, MHC synthesis rates were unchanged, but mRNA levels of isoforms of MHC found in slow-twitch and fast-twitch fibers increased and decreased, respectively. These results offer a mechanistic explanation for posttreatment improvement in muscle functions in hyperthyroidism.     

Recombinant growth hormone enhances muscle myosin heavy-chain mRNA accumulation and amino acid accrual in humans.

A potentially lethal complication of trauma, malignancy, and infection is a progressive erosion of muscle protein mass that is not readily reversed by nutritional support. Growth hormone is capable of improving total body nitrogen balance, but its role in myofibrillar protein synthesis in humans is unknown. The acute, in situ muscle protein response to an infusion of methionyl human growth hormone was investigated in the limbs of nutritionally depleted subjects during a period of intravenous refeeding. A 6-hr methionyl growth hormone infusion achieved steady-state serum levels comparable to normal physiologic peaks and was associated with a significant increase in limb amino acid uptake, without a change in body amino acid oxidation. Myosin heavy-chain mRNA levels, measured by quantitative dot blot hybridization, were also significantly elevated after growth hormone administration. The data indicate that methionyl growth hormone can induce intracellular amino acid accrual and increased levels of myofibrillar protein mRNA during hospitalized nutritional support and suggest growth hormone to be a potential therapy of lean body wasting.     

衰老对肌肉组织中过氧化体增殖物激活型受体-γ及葡萄糖转运子-4基础表达水平的影响

目的　观察衰老对肌肉组织中过氧化体增殖物激活型受体 γ(PPARγ)及葡萄糖转运子 4 (GLUT 4 )基础表达水平的影响,探讨其在胰岛素抵抗形成中的意义。方法　用Bergman创立的最小模型技术评价青年鼠(10～12周龄)和老年鼠(2 4月龄)的胰岛素敏感性,采用半定量逆转录聚合酶链反应(RT PCR)法检测肌肉组织中PPARγ1和PPARγ2 mRNA及GLUT 4mRNA的表达水平。结果　与青年鼠组比较,老年鼠组存在明显的胰岛素抵抗[胰岛素抵抗指数:(11.4 9±6 .92 )vs(5 .2 8±1.94 ) ,P <0 .0 5 ;胰岛素敏感指数:- (0 .0 5±0 .0 3)vs - (0 .0 2±0 .0 2 ) ,P <0 .0 5 ]。用RT PCR方法检测到骨骼肌中PPARγ1和PPARγ2 mRNA均有表达,而以PPARγ1mRNA为主要表达形式;与青年鼠组比较,老年鼠组PPARγ1mRNA [(0 .79±0 .0 4 ) vs(0 .5 2±0 .0 3) ,P <0 .0 1]及GLUT 4mRNA[(0 .73±0 .0 4 )vs(0 .6 1±0 .0 3) ,P <0 .0 5 ]的表达水平均明显降低。结论　衰老降低肌肉组织中PPARγmRNA及GLUT 4mRNA的基础表达水平,直接或间接使骨骼肌糖代谢作用受损,可能参与胰岛素抵抗的形成。     

The effect of weight reduction on skeletal muscle UCP2 and UCP3 mRNA expression and UCP3 protein content in Type II diabetic subjects

Aims/hypothesis. The aim of this study was to examine the effect of weight loss on UCP2/UCP3 mRNA expression and UCP3 protein content in subjects with Type II (non-insulin-dependent) diabetes mellitus.¶Methods. We studied seven Type II diabetic subjects who followed a 10-week very low calorie diet. Expression of skeletal muscle UCP2 and UCP3 mRNA was measured using RT-competitive PCR and UCP3 protein content by western blotting, before and after the diet. Total and plasma fatty acid oxidation was measured using infusion of ¹³C labelled palmitate.¶Results. Body weight decreased from 105.5 ± 8.2 kg to 91.6 ± 7.2 kg (p < 0.001), after 10 weeks of diet intervention. Expression of UCP2 and UCP3 mRNA were significantly reduced after 10 weeks of diet (p < 0.05) but UCP3 protein contents were not significantly altered. Notably, the change in UCP3L mRNA expression and UCP3 protein content after the very low calorie diet were negatively associated with changes in body weight (r = – 0.97, p = 0.006 and r = – 0.83, p = 0.043, respectively) and BMI (r = – 0.99, p = 0.0007 and r = – 0.9, p = 0.016, respectively). Furthermore, changes in UCP3L mRNA expression and UCP3 protein content induced by the diet were positively correlated with changes in cytosolic fatty acid-binding protein content (r = 0.93, p = 0.023 and r = 0.84, p = 0.039, respectively). No correlation between diet-induced changes in UCP3 protein and resting energy expenditure or plasma non-esterified fatty acid concentrations were found.¶Conclusion/interpretation. The negative correlation between the change in UCP3 protein content after weight loss and the change in BMI, suggests that the decrease in UCP3 during weight loss could prevent further weight loss. The finding that the change in UCP3 protein content correlates with the change in skeletal muscle fatty acid-binding protein content, suggests a role for UCPs in the handling of lipids as a fuel. [Diabetologia (2000) 43: 1408–1416]     

Effect of aging on expression of angiogenesis-related factors in mouse skeletal muscle

The molecular mechanisms by which capillary supply is maintained with advancing age remain to be elucidated. To help clarify these mechanisms, we investigated the gene expression levels of angiogenesis-related factors in young (2.5-month-old), adult (6-month-old), and old (22-month-old) mice. To assess the capillary supply, the capillary endothelium in frozen transverse sections was identified by staining for alkaline phosphatase. The mRNA levels for angiogenesis-related factors were analyzed using real-time RT-PCR. The capillary supply to individual muscle fibers, assessed as the number of capillaries around a muscle fiber, did not change with advancing age. Real-time RT-PCR analysis showed that (1) the level of mRNA for VEGF was lower in old animals than young animals; (2) the mRNA levels of Flt-1 and neuropilin-1 are lower in old animals than young animals, while that of KDR/Flk-1 remained unchanged with advancing age; and (3) the levels of mRNA for angiopoietin-1 and -2 remained unchanged, while the mRNA for Tie-2 was lower in old animals than young animals. These findings suggest that capillary supply is maintained irrespective of the down-regulation of several angiogenesis-related factors and that old animals possess the minimum levels of maintenance and reparative abilities needed to preserve the capillary supply.     

Fast and slow skeletal myosin binding protein-C and aging

Aging is associated with skeletal muscle strength decline and cardiac diastolic dysfunction. The structural arrangements of the sarcomeric proteins, such as myosin binding protein-C (MyBP-C) are shown to be pivotal in the pathogenesis of diastolic dysfunction. Yet, the role of fast (fMyBP-C) and slow (sMyBP-C) skeletal muscle MyBP-C remains to be elucidated. Herein, we aimed to characterize MyBP-C and its paralogs in the fast tibialis anterior (TA) muscle from adult and old mice. Immunoreactivity preparations showed that the relative abundance of the fMyBP-C paralog was greater in the TA of both adult and old, but no differences were noted between groups. We further found that the expression level of cardiac myosin binding protein-C (cMyBP-C), an important modulator of cardiac output, was lowered by age. Standard SDS-PAGE along with Pro-Q Diamond phosphoprotein staining did not identify age-related changes in phosphorylated MyBP-C proteins from TA and cardiac muscles; however, it revealed that MyBP-C paralogs in fast skeletal and cardiac muscle were highly phosphorylated. Mass spectrometry further identified glycogen phosphorylase, desmin, actin, troponin T, and myosin regulatory light chain 2 as phosphorylated myofilament proteins in both ages. MyBP-C protein-bound carbonyls were determined using anti-DNP immunostaining and found the carbonyl level of fMyBP-C, sMyBP-C, and cMyBP-C to be similar between old and adult animals. In summary, our data showed some differences regarding the MyBP-C paralog expression and identified an age-related reduction of cMyBP-C expression. Future studies are needed to elucidate which are the age-driven post-translational modifications in the MyBP-C paralogs.

     

Alcohol increases c-myc mRNA and protein in skeletal and cardiac muscle

The pathogenic mechanisms responsible for alcohol-induced muscle disease are unknown, although it is possible that increased proto-oncogene expression may be the causative process. Therefore, we investigated the responses of skeletal muscle c-myc protein and mRNA to a standard acute ethanol dosage regimen (75 mmol/kg/body weight [BW]) for 2.5 to 24 hours. Comparative studies were made on the heart. Acute ethanol administration in vivo led to an increase in c-myc proto-oncogene mRNA in rat skeletal and cardiac muscle. The changes in c-myc mRNA were mirrored by increases in the c-myc protein as demonstrated by immunohistochemistry. The changes in the c-myc protein were localized to the myonuclei, with no corresponding changes seen in the interstitial cell nuclei. This is the first report of altered proto-oncogene expression in muscle in response to ethanol. Increased c-myc mRNA and protein may reflect adaptive changes, a stress response, or another uncharacterized cellular adaptation.     

Diagnostic implications of elevated levels of smooth-muscle myosin heavy-chain protein in acute aortic dissection. The smooth muscle myosin heavy chain study

BACKGROUND: A rapid 30-minute assay of circulating smooth-muscle myosin heavy-chain protein has been developed as a biochemical diagnostic tool for aortic dissection. OBJECTIVE: To determine the sensitivity and specificity of this assay. DESIGN: Cross-sectional study. SETTING: 8 major cardiovascular centers in Japan. PATIENTS: 95 patients with acute aortic dissection, 48 patients with acute myocardial infarction, and 131 healthy volunteers. MEASUREMENTS: Levels of circulating smooth-muscle myosin heavy-chain protein. RESULTS: Patients with acute aortic dissection who presented within 3 hours after onset had elevated levels of circulating smooth-muscle myosin heavy-chain protein. In these patients, the assay had a sensitivity of 90.9%, a specificity of 98% compared with healthy volunteers, and a specificity of 83% compared with patients who had acute myocardial infarction; the clinical decision limit was 2.5 microgram/L. All patients with proximal lesions had elevated levels of smooth-muscle myosin heavy-chain protein, and only patients with distal lesions had decreased levels (<2.5 microgram/L). CONCLUSIONS: Levels of smooth-muscle myosin heavy-chain protein can be used to diagnose aortic dissection soon after symptom onset. The assay had the greatest diagnostic value in patients with proximal lesions.     

Effects of thyroid hormone on alpha-actin and myosin heavy chain gene expression in cardiac and skeletal muscles of the rat: measurement of mRNA content using synthetic oligonucleotide probes

Effects of thyroid hormone on alpha-actin and myosin heavy chain gene expression were compared in ventricle, soleus, and extensor digitorum longus muscles of hypothyroid rats. Changes in mRNA content were analyzed using synthetic oligonucleotide probes complementary to the unique 3' untranslated regions of four striated myosin heavy chain mRNAs and cardiac and skeletal muscle alpha-actin mRNAs. The results indicate that daily treatment with 3,5,3'-triiodo-L-thyronine (2 micrograms/100 g body weight) increased alpha-myosin heavy chain mRNA content in heart muscle by 500% and decreased beta-myosin heavy chain mRNA by 65% within 48 hours. beta-mRNA in extensor digitorum longus was decreased by 60% at 48 hours while in soleus, beta-mRNA levels were not affected by 9 weeks of treatment. Fast IIa mRNA was present in small amounts in hypothyroid soleus and increased by 150% and 200% after 7 and 9 weeks of thyroid hormone administration, respectively. Fast IIb mRNA also was found in hypothyroid soleus and a small increase (60%) was observed after 1 day of treatment. In extensor digitorum longus, Fast IIb mRNA increased by 200% and Fast IIa mRNA decreased by 50% after 1 week of treatment. When larger daily doses of thyroid hormone (15 micrograms/100 g body weight) were administered, similar changes in mRNA levels were observed, except that beta-mRNA content of soleus muscle was decreased slightly (25%). Expression of the cardiac form of alpha-actin was induced transiently in ventricle, but the skeletal form of alpha-actin mRNA in soleus and extensor digitorum longus did not change significantly after thyroid hormone treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum levels of smooth muscle heavy-chain myosin in patients with ectopic pregnancy

STUDY OBJECTIVE: Previous studies have suggested that serum markers of smooth muscle destruction have utility in predicting ectopic pregnancy. Our goal was to determine whether a novel marker of muscle destruction, smooth muscle heavy-chain myosin (SMHC), is elevated in the serum of patients with ectopic pregnancy. METHODS: We conducted a prospective cohort study, with consecutive enrollment, of all women in the first trimester of pregnancy who presented to our urban emergency department with complaints of lower abdominal pain with or without vaginal bleeding. Patients were excluded if there was a history of recent surgery or major trauma. Means were compared using 2-tailed Student's t test with P values less than.05 set for significance. Data analysis included calculation of receiver operating characteristic (ROC), 95% confidence intervals (CIs), and a regression model. RESULTS: A total of 175 patients were enrolled; ectopic pregnancy was diagnosed in 29, and 146 had other diagnoses. Patients with ectopic pregnancy had a mean serum SMHC concentration of 2.53 ng/dL (95% CI 1.84 to 3.22), whereas those in the non-ectopic pregnancy group had a mean concentration of 1.41 ng/dL (95% CI 1.23 to 1.60; P <.0001). ROC analysis demonstrated an area under the curve of 0.72 (95% CI 0.65 to 0.79). Regression analysis to examine confounders in each group analyzed the effects of race, maternal age, estimated gestational age, and serum levels of human chorionic gonadotropin beta-subunit. Our analysis identified only a positive correlation between estimated gestational age and SMHC in the non-ectopic pregnancy group. CONCLUSION: There is a statistically significant elevation of serum SMHC levels in tubal pregnancy, although our data suggest that the assay has limited clinical utility as a lone marker for ectopic pregnancy. Further investigation is needed to determine whether the assay has a role as an adjunct in the evaluation of suspected ectopic pregnancy.     

The effect of aging on anaerobic and aerobic enzyme activities in human skeletal muscle

The effect of aging on metabolic enzyme activity remains controversial, possibly due to physical activity differences. We examined the effect of aging on the enzyme activity for anaerobic and aerobic pathways in nonweight-bearing human skeletal muscle from relatively sedentary males. The muscle obliquus internus abdominis was analyzed for anaerobic (creatine kinase, adenylate kinase, and lactate dehydrogenase) and aerobic (2-oxoglutarate dehydrogenase and carnitine palmitoyltransferase) enzyme activities in two groups: middle-aged (29-54 years) and older (61-74 years) adults. All enzyme activities were lower in older versus middle-aged adults when results were expressed as muscle wet weight (p <.05). When activity was expressed relative to the protein content, only lactate dehydrogenase remained significantly lower in older versus middle-aged adults (p <.001). In conclusion, some of the reduction in muscle performance in older adults may be due to lower activity of the anaerobic and aerobic enzymes as well as protein content, not solely due to a decrease in physical activity.