Determination of age- and exercise-dependent changes in myoglobin contents in murine skeletal and cardiac muscles

A new method was developed to determine myoglobin (Mb) contents in as least as 5 mg of murine skeletal muscles. The method was a modification of Reynafarje's spectroscopic technique and was based on the Soret absorptions at 416 and 422 nm of the muscle extract. Mb contents in the skeletal and cardiac muscles increased with age and were widely different from muscle to muscle. The contents in most of the 13 muscles examined at the 30th week of age were less than 1.5 mg/g wet muscle, but the cardiac, soleus and gracilis muscles showed exceptionally high values of 2.2-6.0 mg/g. The relative content of one muscle to the other was the same independent of differences in age, strain and sex. There was a positive correlation between the muscle Mb contents and citrate synthase activity (r = 0.930). Young male mice (5 wk-old) were endurance-trained by a gradual load-increment program on treadmill for 10 weeks (5 days/week), but the training had no effects on the Mb contents. No substantial alteration of the contents was also observed in the limbs immobilized by plaster-fixation for 4 weeks.     

Alterations of the Z-band in cardiac and skeletal muscles.

The heart muscle may react to various hypoxic damaging effects (e.g.N2, CO, haemorrhagic shock, electroshock) by identical responses similarly as in the case of skeletal muscle damages. One of the early manifestations of the process is the alteration of the Z-band, which is considered pathological. The alterations of the Z-band region precede the changes of the mitochondrial and sacrotubular systems and might form the morphological basis of functional changes induced by hypoxic effects. The alterations observed are differentiated from the hypertrophy of the Z-band. In the development of the alterations of the Z-band the role of other factors (e.g. calcium metabolism, sacroplasmatic membrane changes) is emphasized.     

Ruthenium-red staining of skeletal and cardiac muscles

Summary The effects of ruthenium red (RR) on amphibian and mammalian skeletal muscles and mammalian myocardium were examined. In skeletal muscle cells, a discrete pattern of staining can be brought about within the lumina of the terminal cisternae (junctional sarcoplasmic reticulum [SR]) by sequential exposure to RR and OsO₄. After prolonged immersion in RR solution, formation of pentalaminar segments (zippering) occurs at various points along the longitudinal (network) SR tubules. Zippering can be elicited in skeletal SR at any stage of preparation prior to postfixation with OsO₄. By means of dispersive X-ray analysis, both ruthenium and osmium were seen to be deposited in skeletal muscle junctional SR, and ruthenium was detected in the myoplasm as well. In skeletal muscles whose T tubules were ruptured by exposure to glycerol, the pattern of SR staining and zippering resulting from ruthenium-osmium treatment was not affected. These findings indicate that RR is capable of passage across the sarcolemma of skeletal muscle and that this passage does not occur solely under conditions in which the plasma membrane is damaged. In contrast, RR does not opacify or modify any region of the SR of cardiac muscle. However, after this treatment, randomly distributed opaque bodies, composed of parallel lamellar structures, appear throughout the myocardial cells. A few of these bodies are associated with lipid droplets, but the rest are of unknown origin. The failure of the SR of cardiac muscle to stain after exposure to ruthenium dye (even though this material enters these cells) suggests that the chemical composition of cardiac SR is significantly different from that of skeletal muscle SR.Supported in part by PHS grant HL-11155 (to N.S.) and American Heart Grant-in-Aid 78-753 (to M.S.F.). The authors are grateful to Drs. David Harder and Lawrence Sellin for their assistance with the preparation of frog skeletal muscle, to Dr. S.K. Jirge for his helpful suggestions and discussions, and particularly to Dr. Kenneth R. Lawless and Ms. Ann Marshall of the Department of Materials Sciences, University of Virginia School of Engineering, and Col. John M. Brady of the United States Army Institute of Dental Research, Walter Reed Army Medical Center, for their help with, and for the use of, the X-ray analysis equipment     

Effects of exercise and food restriction on rat skeletal muscles.

Studies were undertaken to compare the effects of exercise and food restriction on body weight (BW), muscle weight (MW), muscle fiber size, and proportion of muscle fiber types. 20 male Fischer 344 rats were randomly assigned to four equal groups: ad libitum-fed control (AC), ad libitum-fed exercise (AE), food restricted control (RC) and food restricted exercise (RE). From 6 weeks of age, RC and RE rats received 60% of the daily food intake of AC and AE rats, respectively. At 7 months of age, AE and RE rats began 40-50 min of daily treadmill exercise. Running speed increased from 1.2 to 1.6 miles/hour and the grade increased to 15% during the first 2 weeks of training. After 10 weeks of training, rats were weighed, sacrificed, and the soleus (SOL), plantaris (PLN) and extensor digitorum longus (EDL) muscles were removed at in situ rest length, weighed, and quick-frozen. Standard histochemical assays were performed, and muscle fiber cross-sectional area was determined planimetrically. Training had little effect on MW or BW, but food restriction greatly reduced BW. This resulted in greater MW/BW ratio in RC and RE than AC and AE rats, respectively. Exercise also increased SOL muscle fiber area in ad libitum-fed but not food restricted rats resulting in smaller fibers in SOL of RE than AE. No changes in percentage of SOL fiber types occurred with food restriction or exercise. In PLN, the percentage of fast-twitch oxidative fibers of AE and RE was greater than in AC and RC, but there was no effect of food restriction or exercise on fiber area.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased kallikrein expression protects against cardiac ischemia.

Multiple indirect lines of evidence point at a cardioprotective role for enhanced bradykinin formation. In particular, the inhibition of angiotensin-converting enzyme, also known as kininase II, can protect against cardiac ischemia, putatively via accumulation of bradykinin. To address whether an increase in kinin formation is sufficient to protect against cardiac ischemia, we studied transgenic rats harboring the human tissue kallikrein gene TGR(hKLK1) under the control of the metallothionein promoter, which drives expression of the transgene in various organs including the heart. We subjected the isolated hearts from transgenic rats and their transgene negative littermates to ex vivo regional cardiac ischemia and reperfusion. During the experiment, the hearts were treated with either vehicle or the specific bradykinin type 2 receptor antagonist HOE 140 (10-9 M). In the transgenic rats, overflow of nucleotide breakdown products upon reperfusion was significantly less (455 +-54 nmol/min/g in transgene negative rats vs. 270+-57 nmol/min/g in the transgenic rats, P.     

Pentacyclic triterpene oleanolic acid protects against cardiac aging through regulation of mitophagy and mitochondrial integrity

Advanced aging exhibits altered cardiac geometry and function involving mitochondrial anomaly. Natural compounds display promises in the regulation of cardiac homeostasis via governance of mitochondrial integrity in aging. This study examined the effect of oleanolic acid (OA), a natural pentacyclic triterpenoid with free radical scavenging and P450 cyclooxygenase-regulating properties, on cardiac aging and mechanisms involved with a focus on mitophagy. Young (4–5 month-old) and old (22–24 month-old) mice were treated with OA for 6 weeks prior to assessment of cardiac function, morphology, ultrastructure, mitochondrial integrity, cell death and autophagy. Our data revealed that OA treatment alleviated aging-induced changes in myocardial remodeling (increased heart weight, chamber size, cardiomyocyte area and interstitial fibrosis), contractile function and intracellular Ca²⁺ handling, apoptosis, necroptosis, inflammation, autophagy and mitophagy (LC3B, p62, TOM20 and FUNDC1 but not BNIP3 and Parkin). OA treatment rescued aging-induced anomalies in mitochondrial ultrastructure (loss of myofilament alignment, swollen mitochondria, increased circularity), mitochondrial biogenesis and O₂^? production without any notable effect at young age. Interestingly, OA-offered benefit against cardiomyocyte aging was nullified by deletion of the mitophagy receptor FUNDC1 using FUNDC1 knockout mice, denoting an obligatory role for FUNDC1 in OA-elicited preservation of mitophagy. OA reconciled aging-induced changes in E3 ligase MARCH5 but not FBXL2, and failed to affect aging-induced rises in IP3R3. Taken together, our data indicated a beneficial role for OA in attenuating cardiac remodeling and contractile dysfunction in aging through a FUNDC1-mediated mechanism.     

Contents of myofibrillar proteins in cardiac, skeletal, and smooth muscles

The in situ contents of myosin, actin, alpha-actinin, tropomyosin, troponin, desmin were estimated in dog cardiac, rabbit skeletal, and chicken smooth muscles. Whole muscle tissues were dissolved with 8 M guanidine hydrochloride and subjected to two-dimensional gel electrophoresis, which is a nonequilibrium pH gradient electrophoresis (Murakami, U. & Uchida, K. (1984) J. Biochem. 95, 1577-1584) with some modification. The amount of protein in a spot on a slab gel was determined by quantification of the extracted dye. Dye binding capacity of individual myofibrillar proteins was determined by using the purified protein. Myosin contents were 82 +/- 7 pmol/mg wet weight in cardiac muscle, 105 +/- 10 pmol/mg wet weight in skeletal muscle, and 45 +/- 4 pmol/mg wet weight in smooth muscle. Actin contents were 339 +/- 15 pmol/mg wet weight in cardiac muscle, 625 +/- 27 pmol/mg wet weight in skeletal muscle, and 742 +/- 13 pmol/mg wet weight in smooth muscle. The subunit stoichiometry of myosin in the three types of muscles was two heavy chains and four light chains, and there was one light chain 2 for every heavy chain. The molar ratio of actin to tropomyosin was 7/1 in the three types of muscles. Striking differences were seen in the molar ratio of myosin to actin: 1.0/4.1 in cardiac muscle, 1.0/6.0 in skeletal muscle, and 1.0/16.5 in smooth muscle.     

Theiler's murine encephalomyelitis virus-induced cardiac and skeletal muscle disease.

The DA strain of Theiler's murine encephalomyelitis virus, a member of the cardiovirus genus of picornaviruses, induces a restricted and persistent infection associated with a demyelinating process following intracerebral inoculation of mice; both virus infection and the immune response are believed to contribute to the late white matter disease. We now report that intraperitoneal inoculation with DA produces an acute myositis that progresses to a chronic inflammatory muscle disease in CD-1 mice as well as several inbred mouse strains. Some mouse strains also develop central nervous system white matter disease and a focal myocarditis. Infectious virus in skeletal muscle falls to undetectable levels 3 weeks postinoculation (p.i.), although viral genome persists for at least 12 weeks p.i., the longest period of observation. Severe combined immunodeficient animals have evidence of muscle pathology as long as 5 weeks p.i., suggesting that DA virus is capable of inducing chronic muscle disease in the absence of an immune response. The presence in immunocompetent mice, however, of prominent muscle inflammation in the absence of infectious virus suggests that the immune system also contributes to the pathology. T lymphocytes are the predominant cell type infiltrating the skeletal muscle during the chronic disease. This murine model may further our understanding of virus-induced chronic myositis and help to clarify the pathogenesis of human inflammatory myopathies.     

Localization of talin in skeletal and cardiac muscles

Antibodies to talin and vinculin were used for localization of these proteins in skeletal and cardiac muscles by the indirect immunofluorescence method. We have found that talin is localized in intercalated discs of cardiac muscle and in costameres of skeletal and cardiac muscles. It is suggested that in striated muscles talin and vinculin play an important role in interactions between actin filaments and membranes.     

Low-intensity exercise training during doxorubicin treatment protects against cardiotoxicity.

Doxorubicin (Dox) is a highly effective antineoplastic antibiotic associated with a dose-limiting cardiotoxicity that may result in irreversible cardiomyopathy and heart failure. The purpose of this study was to examine the effects of low-intensity exercise training (LIET) during the course of Dox treatment on cardiac function, myosin heavy chain expression, oxidative stress, and apoptosis activation following treatment. Male Sprague-Dawley rats either remained sedentary or were exercise trained on a motorized treadmill at 15 m/min, 20 min/day, 5 days/wk (Monday through Friday) for 2 wk. During the same 2-wk period, Dox (2.5 mg/kg) or saline was administered intraperitoneally to sedentary and exercised rats 3 days/wk (Monday, Wednesday, Friday) 1-2 h following the exercise training sessions (cumulative Dox dose: 15 mg/kg). Five days following the final injections, hearts were isolated for determination of left ventricular (LV) function, lipid peroxidation, antioxidant enzyme protein expression, 72-kDa heat shock protein expression, caspase-3 activity, and myosin heavy chain isoform expression. Dox treatment significantly impaired LV function and increased caspase-3 activity in sedentary animals (P < 0.05). LIET attenuated the LV dysfunction and apoptotic signal activation induced by Dox treatment and increased glutathione peroxidase expression, but it had no significant effect on lipid peroxidation, protein expression of myosin heavy chain isoforms, 72-kDa heat shock protein, or superoxide dismutase isoforms. In conclusion, our data suggest that LIET applied during chronic Dox treatment protects against cardiac dysfunction following treatment, possibly by enhancing antioxidant defenses and inhibiting apoptosis.     

Trifluoperazine inhibition of contraction in permeabilized skeletal, cardiac and smooth muscles

To gain insights into the mechanism of the central helix of calmodulin and troponin-C in the Ca2(+)-regulation of force development in striated and smooth muscles, the present study was made of the TFP induced inhibition of contraction, and of the uptake of these proteins by skinned fibers. Calmodulin was four-fold more sensitive to TFP than TnC, but the inhibition was found to be identical for skeletal and cardiac muscles despite the differences in their troponin-C isoforms. Also, the results were comparable between fast-twitch fiber, when calmodulin was exchanged for troponin-C to act on TnI, and smooth muscle, where calmodulin acts on myosin light chain kinase. These findings indicate that the inhibition of force by TFP is entirely due to its binding to the hydrophobic sites in the central helix. The uptakes of troponin-C and calmodulin were also different, and this is explained by a TFP-independent domain in troponin-C that binds TnI.     

Water exchange induced by unilateral exercise in active and inactive skeletal muscles.

Water exchange was evaluated in active (E-leg) and inactive skeletal muscles by using (1)H-magnetic resonance imaging. Six healthy subjects performed one-legged plantar flexion exercise at low and high workloads. Magnetic resonance imaging measured calf cross-sectional area (CSA), transverse relaxation time (T2), and apparent diffusion capacity (ADC) at rest and during recovery. After high workload, inactive muscle decreased CSA and T2 by 2.1% (P < 0.05) and 3.1% (P < 0.05), respectively, and left ADC unchanged. E-leg simultaneously increased CSA, T2, and ADC by 4.2% (P < 0.001), 15.5% (P < 0.05), and 12.5% (P < 0.001), respectively. In conclusion, ADC and T2 correlated highly with muscle volume, indicative of extravascular water displacement closely related to muscle activity and perfusion, which was presumably a combined effect of increased intracellular osmoles and hydrostatic forces as driving forces. A distinguishable muscle temperature release was initially detected in the E-leg after high workload, and the ensuing recovery of ADC and T2 indicated delayed interstitial restitution than restitution of the intracellular compartment. Furthermore, absorption of extravascular water was detected in inactive muscles at contralateral high-intensity exercise.     

Effect of exercise on adenosine deaminase activity in rat skeletal muscles.

Adenosine deaminase activity was shown to decrease in each skeletal muscle type (the slow-twitch oxydative, fast-twitch oxydative--glycolytic and fast-twitch glycolytic) at the beginning of exercise of moderate intensity and to return to the control when exercise was continued till exhaustion. 5 min occlusion of the femoral artery had no effect on the enzyme activity in either muscle. The reduction of the enzyme activity at the onset of exercise could result in reduction of adenosine breakdown and thus contribute to vasodilation at this stage of increased contractile activity of the muscles.     

Histidine dipeptide levels in ageing and hypertensive rat skeletal and cardiac muscles.

1. In rat skeletal muscles (longissimus dorsi and quadriceps femoris), carnosine and anserine levels decreased 35-50% during senescence, and were 35-45% lower in hypertensive rats compared to normotensive levels. 2. In rat left ventricular cardiac muscle, although no free carnosine and anserine were detected, the total level of histidine dipeptides declined 22% during senescence and in hypertensive animals decreased 35% compared to normotensive levels. 3. The significance of these changes in relation to the possible antioxidant roles of histidine dipeptides in muscle is discussed.     

HSP75 protects against cardiac hypertrophy and fibrosis

Cardiac hypertrophy, a major determinant of heart failure, is associated with heat shock proteins (HSPs). HSP75 has been reported to protect against environmental stresses; however, its roles in cardiac hypertrophy remain unclear. Here, we generated cardiac-specific inducible HSP75 transgenic mice (TG) and cardiac hypertrophy was developed at 4 weeks after aortic banding in TG mice and wild-type littermates. The results revealed that overexpression of HSP75 prevented cardiac hypertrophy and fibrosis as assessed by heart weight/body weight ratio, heart weight/tibia length ratio, echocardiographic and hemodynamic parameters, cardiomyocyte width, left ventricular collagen volume, and gene expression of hypertrophic markers. Further studies showed that overexpression of HSP75 inhibited the activation of TAK/P38, JNK, and AKT signaling pathways. Thus, HSP75 likely reduces the hypertrophy and fibrosis induced by pressure overload through blocking TAK/P38, JNK, and AKT signaling pathways.     

Developmental changes of cardiac and slow skeletal muscle troponin T expression in chicken cardiac and skeletal muscles

Numerous troponin T (TnT) isoforms are produced by alternative splicing from three genes characteristic of cardiac, fast skeletal, and slow skeletal muscles. Apart from the developmental transition of fast skeletal muscle TnT isoforms, switching of TnT expression during muscle development is poorly understood. In this study, we investigated precisely and comprehensively developmental changes in chicken cardiac and slow skeletal muscle TnT isoforms by two-dimensional gel electrophoresis and immunoblotting with specific antisera. Four major isoforms composed of two each of higher and lower molecular weights were found in cardiac TnT (cTnT). Expression of cTnT changed from high- to low-molecular-weight isoforms during cardiac muscle development. On the other hand, such a transition was not found and only high-molecular-weight isoforms were expressed in the early stages of chicken skeletal muscle development. Two major and three minor isoforms of slow skeletal muscle TnT (sTnT), three of which were newly found in this study, were expressed in chicken skeletal muscles. The major sTnT isoforms were commonly detected throughout development in slow and mixed skeletal muscles, and at developmental stages until hatching-out in fast skeletal muscles. The expression of minor sTnT isoforms varied from muscle to muscle and during development.