The remodelling of skeletal muscle for indefatigable hemodynamic work

Skeletal muscle possesses inherent plasticity of gene expression. Low frequency pulse-train stimulation can remodel the biochemical machinery that confers physiological expression and fatigue resistance approaching that of the myocardium. This fatigue-resistant muscle can generate sufficient force to meet the power requirements for useful cardiac work. This ultimate goal is currently being pursued in models of cardiomyoplasty and muscle-powered cardiac assist devices. In this article, we review the three major subcellular systems subserving canine skeletal muscle transformation and compare them to those of cardiac muscle. The magnitude of the problem of clinical heart failure and the feasibility of fatigue-resistant skeletal muscle joining the therapeutic armamentarium are addressed. The adaptation and transformation of fast-twitch skeletal muscle in response to chronic electrical stimulation augers therapeutic potential as an endogenous, readily available power source for myocardial assistance. The basis mechanisms of skeletal muscle fatigue require elucidation to gain a complete and thorough understanding of how to manipulate this property to provide continuous hemodynamic work.     

High‐fat diet affects skeletal muscle mitochondria comparable to pressure overload‐induced heart failure

In heart failure, high‐fat diet (HFD) may exert beneficial effects on cardiac mitochondria and contractility. Skeletal muscle mitochondrial dysfunction in heart failure is associated with myopathy. However, it is not clear if HFD affects skeletal muscle mitochondria in heart failure as well. To induce heart failure, we used pressure overload (PO) in rats fed normal chow or HFD. Interfibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM) from gastrocnemius were isolated and functionally characterized. With PO heart failure, maximal respiratory capacity was impaired in IFM but increased in SSM of gastrocnemius. Unexpectedly, HFD affected mitochondria comparably to PO. In combination, PO and HFD showed additive effects on mitochondrial subpopulations which were reflected by isolated complex activities. While PO impaired diastolic as well as systolic cardiac function and increased glucose tolerance, HFD did not affect cardiac function but decreased glucose tolerance. We conclude that HFD and PO heart failure have comparable effects leading to more severe impairment of IFM. Glucose tolerance seems not causally related to skeletal muscle mitochondrial dysfunction. The additive effects of HFD and PO may suggest accelerated skeletal muscle mitochondrial dysfunction when heart failure is accompanied with a diet containing high fat.     

A simple ballistocardiographic system for a medical cardiovascular physiology course

Ballistocardiography is an old, noninvasive technique used to record the movements of the body synchronous with the heartbeat due to left ventricular pump activity. Despite the fact that this technique to measure cardiac output has been superseded by more advanced and precise techniques, it is useful for teaching cardiac cycle physiology in an undergraduate practical course because of its noninvasive application in humans, clear physiological and physiopathological analysis, and practical approach to considering cardiac output issues. In the present report, a simple, low cost, easy-to-build ballistocardiography system is implemented together with a theoretical and practical session that includes Newton's laws, cardiac output, cardiac pump activity, anatomy and physiology of the vessel circulation, vectorial composition, and signal transduction, which makes cardiovascular physiology easy to understand and focuses on the study of cardiac output otherwise seen only with the help of computer simulation or echocardiography. The proposed system is able to record body displacement or force as ballistocardiography traces and its changes caused by different physiological factors. The ballistocardiography session was included in our medical physiology course six years ago with very high acceptance and approval rates from the students.     

Role of troponin I isoform switching in determining the pH sensitivity of Ca(2+) regulation in developing rabbit cardiac muscle

Skinned muscle fibers prepared from fetal rabbit heart (28 days of gestation) showed a marked resistance to acidic pH in the Ca(2+) regulation of force generation, compared to the fibers prepared from adult heart. SDS-PAGE and immunoblot analysis showed that the slow skeletal troponin I was predominantly expressed in the fetal cardiac muscle, while the cardiac isoform was predominantly expressed in the adult cardiac muscle. Direct exchange of purified slow skeletal and cardiac troponin I isoforms into these skinned muscle fibers revealed that cardiac troponin I made the Ca(2+) regulation of contraction sensitive to acidic pH just as in the adult fibers, whereas slow skeletal troponin I made the Ca(2+) regulation of contraction resistant to acidic pH just as in the fetal fibers. These results demonstrate that the troponin I isoform switching accounts fully for the change in the pH dependence of Ca(2+) regulation of contraction in developmental cardiac muscle.     

Evidence for mitochondrial thioesterase 1 as a peroxisome proliferator-activated receptor-alpha-regulated gene in cardiac and skeletal muscle

The physiological role of mitochondrial thioesterase 1 (MTE1) is unknown. It was proposed that MTE1 promotes fatty acid (FA) oxidation (FAO) by acting in concert with uncoupling protein (UCP)3. We previously showed that ucp3 is a peroxisome proliferator-activated receptor-alpha (PPAR alpha)-regulated gene, allowing induction when FA availability increases. On the assumption that UCP3 and MTE1 act in partnership to increase FAO, we hypothesized that mte1 is also a PPAR alpha-regulated gene in cardiac and skeletal muscle. Using real-time RT-PCR, we characterized mte1 gene expression in rat heart and soleus muscles. Messenger RNA encoding for mte1 was 3.2-fold higher in heart than in soleus muscle. Cardiac mte1 mRNA exhibited modest diurnal variation, with 1.4-fold higher levels during dark phase. In contrast, skeletal muscle mte1 mRNA remained relatively constant over the course of the day. High-fat feeding, fasting, and streptozotocin-induced diabetes, interventions that increase FA availability, muscle PPAR alpha activity, and muscle FAO rates, increased mte1 mRNA in heart and soleus muscle. Conversely, pressure overload and hypoxia, interventions that decrease cardiac PPAR alpha activity and FAO rates, repressed cardiac mte1 expression. Specific activation of PPAR alpha in vivo through WY-14643 administration rapidly induced mte1 mRNA in cardiac and skeletal muscle. WY-14643 also induced mte1 mRNA in isolated adult rat cardiomyocytes dose dependently. Expression of mte1 was markedly lower in hearts and soleus muscles isolated from PPAR alpha-null mice. Alterations in cardiac and skeletal muscle ucp3 expression mirrored that of mte1 in all models investigated. In conclusion, mte1, like ucp3, is a PPAR alpha-regulated gene in cardiac and skeletal muscle.     

Toad Heart Utilizes Exclusively Slow Skeletal Muscle Troponin T: AN EVOLUTIONARY ADAPTATION WITH POTENTIAL FUNCTIONAL BENEFITS

The three isoforms of vertebrate troponin T (TnT) are normally expressed in a muscle type-specific manner. Here we report an exception that the cardiac muscle of toad (Bufo) expresses exclusively slow skeletal muscle TnT (ssTnT) together with cardiac forms of troponin I and myosin as determined using immunoblotting, cDNA cloning, and/or LC-MS/MS. Using RT-PCR and 3'- and 5'-rapid amplification of cDNA ends on toad cardiac mRNA, we cloned full-length cDNAs encoding two alternatively spliced variants of ssTnT. Expression of the cloned cDNAs in Escherichia coli confirmed that the toad cardiac muscle expresses solely ssTnT, predominantly the low molecular weight variant with the exon 5-encoded NH(2)-terminal segment spliced out. Functional studies were performed in ex vivo working toad hearts and compared with the frog (Rana) hearts. The results showed that toad hearts had higher contractile and relaxation velocities and were able to work against a significantly higher afterload than that of frog hearts. Therefore, the unique evolutionary adaptation of utilizing exclusively ssTnT in toad cardiac muscle corresponded to a fitness value from improving systolic function of the heart. The data demonstrated a physiological importance of the functional diversity of TnT isoforms. The structure-function relationship of TnT may be explored for the development of new treatment of heart failure.     

The effect of standard chow and reduced hexokinase II on growth, cardiac and skeletal muscle hexokinase and low-flow cardiac ischaemia-reperfusion injury

In the present study, we examined whether standard chow (SDS versus Purina 5001; both low fat, high carbohydrate) and reductions in hexokinase (HK) II (wild-type versus HKII(+/-) mice) affect (1) growth parameters, (2) HK levels in cardiac and skeletal muscle and (3) low-flow cardiac ischaemia-reperfusion (IR) injury. Total HK activity and HKI and HKII expressions were determined, and low-flow IR injury was examined in isolated hearts subjected to 40 min 5% low-flow ischaemia and 120 min reperfusion. Standard chow, but not HKII reductions, significantly affected body weight, heart weight and cardiac hypertrophy. Both standard chow and reduced HKII diminished total cardiac and skeletal muscle HK activity. For the heart, the Purina chow-induced decrease in total HK activity was through decreases in HKI expression, whereas for skeletal muscle post-translational mechanisms are suggested. Both standard chow and reduced HKII demonstrated a non-significant trend for affecting cardiac IR damage. However, the low-flow ischaemia model was associated with mild sublethal injury only (～1% cell death). In conclusion, standard chow affects body weight, heart weight and HK activity and HKI expression in the heart, without altering HKII expression. This implicates standard chow as an important factor in genomic, physiological research models and demonstrates that large differences in fat or carbohydrates in the diet are not necessary to affect growth. In a cardiac low-flow IR model, resulting in only mild injury, standard chow or reduced HKII does not significantly affect IR damage.     

Contractile parameters and occurrence of alternans in isolated rat myocardium at supra-physiological stimulation frequency

The cardiac refractory period prevents the heart from tetanic activation that is typically used in noncardiac striated muscle tissue. To what extent the refractory period prevents successive action potentials to activate the excitation-contraction coupling process and contractile machinery at supra-physiological rates, such as those present during ventricular fibrillation, is unknown. Using multicellular trabeculae isolated from rat hearts, we studied amplitude and kinetics of contraction at rates well above the normal in vivo rat heart range. We show that even at twice the maximal heart rate of the rat, little or no mechanical instability is observed; twitch contractions are at steady state, albeit with an elevated active diastolic force. Although the amplitude of contraction increased within in vivo heart rates (positive force-frequency response), at frequencies beyond the maximal heart rate (10-30 Hz) a steady decline of contractile amplitude is observed. Not until 30 Hz do the majority of the isolated muscle preparations show mechanical alternans, where strong and weak beats alternate. Interestingly, unlike striated limb skeletal muscle, fusing of twitch contractions did not cause a continuous increase in peak force: at frequencies of 10 Hz and above, systolic force declines with relatively little elevation in diastolic force. Contractile kinetics continued to accelerate, from 1 Hz up to 30 Hz, whereas the relative speed of contraction and relaxation remained closely coupled, reflected by a singular linear relationship between the maximal and minimal derivative of force (dF/dt). We conclude that cardiac muscle can produce mechanically stable steady-state contractions at supra-physiological pacing rates, while these contractions continue to decline in amplitude and increase in diastolic force past maximal heart rate.     

Myostatin from the heart: local and systemic actions in cardiac failure and muscle wasting

A significant proportion of heart failure patients develop skeletal muscle wasting and cardiac cachexia, which is associated with a very poor prognosis. Recently, myostatin, a cytokine from the transforming growth factor-β (TGF-β) family and a known strong inhibitor of skeletal muscle growth, has been identified as a direct mediator of skeletal muscle atrophy in mice with heart failure. Myostatin is mainly expressed in skeletal muscle, although basal expression is also detectable in heart and adipose tissue. During pathological loading of the heart, the myocardium produces and secretes myostatin into the circulation where it inhibits skeletal muscle growth. Thus, genetic elimination of myostatin from the heart reduces skeletal muscle atrophy in mice with heart failure, whereas transgenic overexpression of myostatin in the heart is capable of inducing muscle wasting. In addition to its endocrine action on skeletal muscle, cardiac myostatin production also modestly inhibits cardiomyocyte growth under certain circumstances, as well as induces cardiac fibrosis and alterations in ventricular function. Interestingly, heart failure patients show elevated myostatin levels in their serum. To therapeutically influence skeletal muscle wasting, direct inhibition of myostatin was shown to positively impact skeletal muscle mass in heart failure, suggesting a promising strategy for the treatment of cardiac cachexia in the future.     

Myocardial dysfunction in an animal model of cancer cachexia

AimsFatigue is a common occurrence in cancer patients regardless of tumor type or anti-tumor therapies and is an especially problematic symptom in persons with incurable tumor disease. In rodents, tumor-induced fatigue is associated with a progressive loss of skeletal muscle mass and increased expression of biomarkers of muscle protein degradation. The purpose of the present study was to determine if muscle wasting and expression of biomarkers of muscle protein degradation occur in the hearts of tumor-bearing mice, and if these effects of tumor growth are associated with changes in cardiac function.Main methodsThe colon26 adenocarcinoma cell line was implanted into female CD2F1 mice and skeletal muscle wasting, in vivo heart function, in vitro cardiomyocyte function, and biomarkers of muscle protein degradation were determined.Key findingsExpression of biomarkers of protein degradation were increased in both the gastrocnemius and heart muscle of tumor-bearing mice and caused systolic dysfunction in vivo. Cardiomyocyte function was significantly depressed during both cellular contraction and relaxation.SignificanceThese results suggest that heart muscle is directly affected by tumor growth, with myocardial function more severely compromised at the cellular level than what is observed using echocardiography.     

Porcine cardiac myocyte power output is increased after chronic exercise training.

Chronic exercise training increases the functional capacity of the heart, perhaps by increased myocyte contractile function, as has been observed in rodent exercise models. We examined whether cardiac myocyte function is enhanced after chronic exercise training in Yucatan miniature swine, whose heart characteristics are similar to humans. Animals were designated as either sedentary (Sed), i.e., cage confined, or exercise trained (Ex), i.e., underwent 16-20 wk of progressive treadmill training. Exercise training efficacy was shown with significantly increased heart weight-to-body weight ratios, skeletal muscle citrate synthase activity, and exercise tolerance. Force-velocity properties were measured by attaching skinned cardiac myocytes between a force transducer and position motor, and shortening velocities were measured over a range of loads during maximal Ca2+ activation. Myocytes (n = 9) from nine Ex pigs had comparable force production but a approximately 30% increase in peak power output compared with myocytes (n = 8) from eight Sed. Interestingly, Ex myofibrillar samples also had higher baseline PKA-induced phosphorylation levels of cardiac troponin I, which may contribute to the increase in power. Overall, these results suggest that enhanced power-generating capacity of porcine cardiac myofibrils contributes to improved cardiac function after chronic exercise training.     

Neuregulin receptor ErbB2 localization at T-tubule in cardiac and skeletal muscle.

Previous studies have indicated that ErbB receptors for neuregulins play an important role in cardiac development and muscle spindle formation during embryogenesis; however, little is known about their functions in adulthood. Recent reports indicate that breast cancer therapy with humanized monoclonal ErbB2 antibody induces cardiomyopathy, suggesting that ErbB2 serves as a crucial signaling receptor, even in the adult heart. Here, we examine ErbB2 expression and localization in both cardiac and skeletal muscle of adult mice via immunoblot and immunohistochemistry. ErbB2 was detected as a band approximately 185 kD molecular mass in each cardiac and skeletal muscle extraction. Confocal images of double labeling showed that ErbB2 was colocalized with caveolin-3 in cardiac muscle and with dihydropyridine receptor in skeletal muscle, suggesting that ErbB2 was localized at the T-tubule. In addition, immunoelectron micrographs clearly demonstrated that ErbB2 was located at the T-tubule in both types of muscle. Taken together, the results of the present study suggest that neuregulin-ErbB2 signaling plays a role in the physiological function of cardiac and skeletal muscle, even in adulthood.     

Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments.

The passive tension-sarcomere length relation of rat cardiac muscle was investigated by studying passive (or not activated) single myocytes and trabeculae. The contribution of collagen, titin, microtubules, and intermediate filaments to tension and stiffness was investigated by measuring (1) the effects of KCl/KI extraction on both trabeculae and single myocytes, (2) the effect of trypsin digestion on single myocytes, and (3) the effect of colchicine on single myocytes. It was found that over the working range of sarcomeres in the heart (lengths approximately 1.9-2.2 microns), collagen and titin are the most important contributors to passive tension with titin dominating at the shorter end of the working range and collagen at longer lengths. Microtubules made a modest contribution to passive tension in some cells, but on average their contribution was not significant. Finally, intermediate filaments contributed about 10% to passive tension of trabeculae at sarcomere lengths from approximately 1.9 to 2.1 microns, and their contribution dropped to only a few percent at longer lengths. At physiological sarcomere lengths of the heart, cardiac titin developed much higher tensions (> 20-fold) than did skeletal muscle titin at comparable lengths. This might be related to the finding that cardiac titin has a molecular mass of 2.5 MDa, 0.3-0.5 MDa smaller than titin of mammalian skeletal muscle, which is predicted to result in a much shorter extensible titin segment in the I-band of cardiac muscle. Passive stress plotted versus the strain of the extensible titin segment showed that the stress-strain relationships are similar in cardiac and skeletal muscle. The difference in passive stress between cardiac and skeletal muscle at the sarcomere level predominantly resulted from much higher strains of the I-segment of cardiac titin at a given sarcomere length. By expressing a smaller titin isoform, without changing the properties of the molecule itself, cardiac muscle is able to develop significant levels of passive tension at physiological sarcomere lengths.     

MRP1/GS‐X pump ATPase expression: is this the explanation for the cytoprotection of the heart against oxidative stress‐induced redox imbalance in comparison to skeletal muscle cells?

Striated muscle activity is always accompanied by oxidative stress (OxStress): the more intense muscle work and/or its duration, the more a redox imbalance may be attained. In spite of cardiac muscle functioning continuously, it is well known that the heart does not suffer from OxStress-induced damage over a broad physiological range. Although the expression of antioxidant enzymes may be of importance in defending heart muscle against OxStress, a series of combined antioxidant therapeutic approaches have proved to be mostly ineffective in avoiding cellular injury. Hence, additional mechanisms may be involved in heart cytoprotection other than antioxidant enzyme activities. The strong cardiotoxic effect of doxorubicin-induced cancer chemotherapy shed light on the possible role for multidrug resistance-associated proteins (MRP) in this context. Muscle activity-induced 'physiological' OxStress enhances the production of glutathione disulfide (GSSG) thus increasing the ratio of GSSG to glutathione (GSH) content inside the cells, which, in turn, leads to redox imbalance. Since MRP1 gene product (a GS-X pump ATPase) is a physiological GSSG transporter, adult Wistar rats were tested for MRP1 expression and activity in the heart and skeletal muscle (gastrocnemius), in as much as the latter is known to be extremely sensitive to muscle activity-induced OxS. MRP1 expression was completely absent in skeletal muscle. In contrast, the heart showed an exercise training-dependent induction of MRP1 protein expression which was further augmented (2.4-fold) as trained rats were challenged with a session of acute exercise. On the other hand, inducible expression of the 70-kDa heat shock protein (HSP70), a universal marker of cellular stress, was completely absent in the heart of sedentary and acutely exercised rats, whereas skeletal muscle showed a conspicuous exercise-dependent HSP70 expression, which decreased by 45% with exercise training. This effect was paralleled by a 58% decrease in GSH content in skeletal muscle which was even higher (an 80%-fall) after training thus leading to a marked redox imbalance ([GSSG]/[GSH] raised up to 38-fold). In the heart, GSH contents and [GSSG]/[GSH] ratio remained virtually unchanged even after exercise challenges, while GS-X pump activity was found to be 20% higher in the heart related to skeletal muscle. These findings suggest that an intrinsic higher capacity to express the MRP1/GS-X pump may dictate the redox status in the heart muscle thus protecting myocardium by preventing GSSG accumulation in cardiomyocytes as compared to skeletal muscle fibres.     

The effect of altered temperature on Ca2(+)-sensitive force in permeabilized myocardium and skeletal muscle. Evidence for force dependence of thin filament activation

The effect of changes in temperature on the calcium sensitivity of tension development was examined in permeabilized cellular preparations of rat ventricle and rabbit psoas muscle. Maximum force and Ca2+ sensitivity of force development increased with temperature in both muscle types. Cardiac muscle was more sensitive to changes in temperature than skeletal muscle in the range 10-15 degrees C. It was postulated that the level of thin filament activation may be decreased by cooling. To investigate this possibility, troponin C (TnC) was partially extracted from both muscle types, thus decreasing the level of thin filament activation independent of temperature and, at least in skeletal muscle fibers, decreasing cooperative activation of the thin filament as well. TnC extraction from cardiac muscle reduced the calcium sensitivity of tension less than did extraction of TnC from skeletal muscle. In skeletal muscle the midpoint shift of the tension-pCa curve with altered temperature was greater after TnC extraction than in control fibers. Calcium sensitivity of tension development was proportional to the maximum tension generated in cardiac or skeletal muscle under all conditions studied. Based on these results, we conclude that (a) maximum tension-generating capability and calcium sensitivity of tension development are related, perhaps causally, in fast skeletal and cardiac muscles, and (b) thin filament activation is less cooperative in cardiac muscle than in skeletal muscle, which explains the differential sensitivity of the two fiber types to temperature and TnC extraction. Reducing thin filament cooperativity in skeletal muscle by TnC extraction results in a response to temperature similar to that of control cardiac cells. This study provides evidence that force levels in striated muscle influence the calcium binding affinity of TnC.     

Immunohistochemical analysis of C-protein isoforms in cardiac and skeletal muscle of the axolotl,Ambystoma mexicanum

Of the several proteins located within sarcomeric A-bands, C-protein, a myosin binding protein (MyBP) is thought to regulate and stabilize thick filaments during assembly. This paper reports the characterization of C-protein isoforms in juvenile and adult axolotls, Ambystoma mexicanum, by means of immunofluorescent microscopy and Western blot analyses. C-protein and myosin are found specifically within the A-bands, whereas tropomyosin and -actin are detected in the I-bands of axolotl myofibrils. The MF1 antibody prepared against the fast skeletal muscle isoform of chicken C-protein specifically recognizes a cardiac isoform (Axcard1) in juvenile and adult axolotls but does not label axolotl skeletal muscle. The ALD66 antibody, which reacts with the C-protein slow isoform in chicken, localizes only in skeletal muscle of the axolotl. This slow axolotl isoform (Ax_slow) displays a heterogeneous distribution in fibers of dorsalis trunci skeletal muscle. The C₃₁₅ antibody against the chicken C-protein cardiac isoform identifies a second axolotl cardiac isoform (Ax_card2), which is present also in axolotl skeletal muscle. No C-protein was detected in smooth muscle of the juvenile and adult axolotl with these antibodies.This work was supported by NIH grants HL-32184 and HL-37702 and a grant-in-aid from the American Heart Association to L.F.L.