Myocardial functional correlates of cardiac myosin light chain 2 phosphorylation

In vitro and in situ studies have proposed a potentiation of submaximal force production after myosin light chain 2 (P-light chain) phosphorylation in mammalian striated muscle. The purpose of this study was to ascertain the relationship between the augmentation in left ventricular pressure development and cardiac myosin P-light chain phosphorylation at different times during and after submaximal treadmill exercise involving adult female Sprague-Dawley rats. In vivo hemodynamic measurements were monitored with an indwelling high-fidelity solid-state pressure transducer. Exercise heart rate, peak left ventricular (LV) pressure, and rate of LV pressure development/relaxation (LV +/- dP/dt) were significantly elevated compared with a normal sedentary group (P less than 0.001). Peak LV pressure remained significantly elevated throughout 20 min of postexercise recovery (P less than 0.01), and heart rate, LV end-diastolic pressure, and LV +/- dP/dt returned rapidly to preexercise values. Corresponding to these in vivo hemodynamic changes, increased levels of P-light chain phosphorylation were observed during both exercise (16%, P less than 0.01) and subsequent recovery periods (14%, P less than 0.02) compared with the NC group. A quasi-temporal relationship was observed between postexercise peak LV pressure potentiation and P-light chain phosphorylation. These results demonstrate that cardiac myosin P-light chain phosphorylation is associated, in part, with the augmentation of peak LV pressure observed during both exercise and recovery.     

Left ventricular functional capacity in the endurance-trained rodent

Cardiac myosin P-light chain phosphorylation [P-LC(P)] has been proposed to augment myocardial force production. This study was undertaken to examine the potential for cardiac myosin P-LC(P) for both equivalent heart rate and work load in exercising endurance-trained and nontrained rodents. A 10-wk training protocol elicited a significant reduction in submaximal running O2 uptake while enhancing peak O2 uptake (-17 and 10%, respectively, P less than 0.05). Left ventricular functional index during submaximal exercise, obtained with a high-fidelity Millar ultraminiature pressure transducer, indicated that the trained animals were able to maintain peak left ventricular pressure (LVP) in comparison to their sedentary counterparts, even though both heart rate and rate of LVP development were significantly reduced (P less than 0.05). When expressed on the basis of equivalent submaximal heart rate, peak LVP was augmented in the trained animals. Cardiac myosin P-LC(P) was examined under two conditions known to produce disparate responses in trained vs. sedentary animals. For an equivalent work load, we observed parallel increases in P-LC(P) (20%) and systolic pressure (17%) in both groups, even though the trained animals exhibited significantly lower heart rates (P less than 0.05). For an equivalent heart rate, training evoked a significant increase in systolic pressure (26%, P less than 0.05) and caused a slight increase in P-LC(P) relative to the nontrained controls. Cardiac myosin adenosinetriphosphatase was reduced approximately 10% in the trained animals (P less than 0.05), commensurate with a 2.0-fold increase in the V3 (low adenosinetriphosphatase) isomyosin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of autonomic blockade on cardiac function at rest and during upright exercise in humans

The cardiac function was studied by radionuclide cardiography in eight healthy subjects at rest and during submaximal upright exercise before and after autonomic blockade with metoprolol and atropine. At rest the median stroke volume was reduced by 21% during autonomic blockade (P less than 0.01), but cardiac output was maintained by a concomitant increase in heart rate. The systolic blood pressure was reduced from 120 to 105 mmHg (P less than 0.01), and left ventricular ejection fraction was reduced from 61 to 56% (P less than 0.05). After autonomic blockade the heart rate reached during exercise was the same. Stroke volume and cardiac output were maintained through cardiac dilation. The increase in left ventricular end-diastolic volume was 31 vs. 10% during control conditions (P less than 0.01). The systolic blood pressure was reduced from 174 to 135 mmHg (P less than 0.01). Left ventricular ejection fraction was reduced from 75 to 67% (P less than 0.05), but the increase from rest to exercise was preserved. Total peripheral resistance was reduced by 17% (P less than 0.05). These findings suggest that the heart possesses intrinsic mechanisms to maintain cardiac output during submaximal upright exercise. End-diastolic dilation results in a preserved stroke volume despite a reduced contractility.     

Myosin light chain phosphorylation and contractile performance of human skeletal muscle

Twitch tension and maximal unloaded velocity of human knee extensor muscles were studied under conditions of low phosphate content of the phosphorylatable light chains (P-light chains) of myosin and elevated phosphate content, following a 10-s maximal voluntary isometric contraction (MVC). After the MVC, twitch tension was significantly potentiated, with greater potentiation observed at a shorter muscle length (p less than 0.05). The MVC was associated with at least a twofold increase in phosphate content of the fast (LC2F) and two slow (LC2S and LC2S') P-light chains, but this increase was unrelated to muscle length. No significant differences in knee extension velocity were observed between conditions where P-light chains had low or elevated phosphate content. Positive but nonsignificant correlations were noted between the extent of twitch potentiation and phosphate content of individual P-light chains as well as the percentage of type II muscle fibres in vastus lateralis muscle. No significant relationships were determined for myosin light chain kinase activity and either P-light chain phosphorylation or type II fibre percentage. These data suggest that, unlike other mammalian fast muscles, P-light chain phosphorylation of mixed human muscles is not strongly associated with altered contractile performance.     

Contraction in intact pig aortic strips is not always associated with phosphorylation of myosin light chains.

Intact pig aortic strips were incubated in medium containing [32P]P1 and various Ca2+ concentrations. The 32P content of the myosin P-light chain was determined by radioautography after electrophoresis in the presence of sodium dodecyl sulphate. Although treatment of the strips with noradrenaline always caused a rise in tension, this was not necessarily accompanied by increased phosphorylation of the P-light chain. These results indicate that, in aortic smooth muscle, phosphorylation of the P-light chain is not obligatory for contraction.     

A comparative study of the myosin light chain kinases from myoblast and muscle sources. Studies on the kinases from proliferative rat myoblasts in culture, rat thigh muscle, and rabbit skeletal muscle.

Myosin light chain kinases have been isolated from rat thigh and rabbit skeletal muscle and cultured rat myoblasts. From these preparations, two types of kinases can be distinguished: calcium-dependent and calcium-independent. Both types of kinases can phosphorylate isolated P-light chains of myosin from several sources (skeletal muscle, cardiac muscle, and platelet). Data are shown which support the phosphorylation of the same site on the non-muscle P-light chains by both types of kinases. The rates of these reactins are, however, different for the two types of kinases. Kinetic analysis of the myoblast kinase shows differing affinities for various P-light chains (non-muscle greater than cardiac greater than skeletal). In the proliferative rat myoblast, phosphorylation of myosin is a prerequisite for actin activation of the myosin ATPase activity.     

The effect of myosin phosphorylation on the contractile properties of skinned rabbit skeletal muscle fibers

We have studied the effect of myosin P-light chain phosphorylation on the isometric tension generated by skinned fibers from rabbit psoas muscle at 0.6 and 10 microM Ca2+. At the lower Ca2+ concentration, which produced 10-20% of the maximal isometric tension obtained at 10 microM Ca2+, addition of purified myosin light chain resulted in a 50% increase in isometric tension which correlated with an increase in P-light chain phosphorylation from 0.10 to 0.80 mol of phosphate/mol of P-light chain. Addition of a phosphoprotein phosphatase reversed the isometric tension response and dephosphorylated P-light chain. At the higher Ca2+ concentration, P-light chain phosphorylation was found to have little effect on isometric tension. Fibers prepared and stored at -20 degrees C in a buffer containing MgATP, KF, and potassium phosphate incorporated 0.80 mol of phosphate/mol of P-light chain. Addition of phosphoprotein phosphatase to these fibers incubated at 0.6 microM Ca2+ caused a reduction in isometric tension and dephosphorylation of the P-light chain. There was no difference before and after phosphorylation of P-light chain in the normalized force-velocity relationship for fibers at the lower Ca2+ concentration, and the extrapolated maximum shortening velocity was 2.2 fiber lengths/s. Our results suggest that in vertebrate skeletal muscle, P-light chain phosphorylation increases the force level at submaximal Ca2+ concentrations, probably by affecting the interaction between the myosin cross-bridge and the thin filament.     

Length-dependence of isometric twitch tension potentiation and myosin phosphorylation in mouse skeletal muscle

The effect of changes in muscle length on post-tetanic isometric twitch tension potentiation and myosin P-light chain phosphorylation-was studied at 23°C in the mouse extensor digitorum longus muscle. The length-tension relationship was determined for the same muscles after a 30 min period of quiescence and between 30 s and 3 min after a 1.5 s tetanus at L₀. Isometric twitch tension is increased at all muscle lengths after the tetanus; however, the fractional increase in twitch tension rises from 0.2 at L₀ to a maximum of 0.3 at 1.2 L_0. The fractional increase in twitch tension measured at any fixed muscle length is constant between 30 s and 3 min post-tetanus. P-light chain phosphorylation remains constant between 30 s and 3 min post-tetanus followed by a slow decline to basal values. Under fixed length conditions, there is linear relationship between the relative magnitude of the twitch tension and the extent of P-light chain phosphor-ylation. Net myosin phosphorylalion measured after a 1.5 s tetanus at 1.23 L₀ is 35% less than that obtained under the same conditions at L_0. Thus, contraction-induced phosphorylation of P-light chain decreases with increased muscle length and post-tetanic potentiation at a constant level of P-light chain phosphorylation increases with increasing muscle length. These observations may be consistent with alterations in the sarcoplasmic Ca²⁺ ion transient as the muscle is lengthened.     

Different phosphorylated forms of myosin in contracting tracheal smooth muscle

Calmodulin-dependent myosin light chain kinase phosphorylates two light chain subunits on each myosin molecule. We have developed a method for measuring nonphosphorylated, monophosphorylated, and diphosphorylated forms of myosin in smooth muscle. Four protein bands were separated in tissue extracts by nondenaturing polyacrylamide gel electrophoresis in the presence of pyrophosphate. Immunoblots demonstrated that three forms (designated M, MP, and MP2) reacted with rabbit antisera prepared against the purified phosphorylatable light chain (P-light chain) from bovine tracheal smooth muscle. Evidence was obtained that M, MP, and MP2 represented nonphosphorylated, monophosphorylated, and diphosphorylated myosin, respectively, and that the other protein band was probably filamin. The formation of different phosphorylated forms of myosin was measured in bovine trachealis strips neurally stimulated from 1.0 to 3.5 s and quick-frozen. There was no detectable MP or MP2 in unstimulated muscles; the extent of P-light chain phosphorylation measured directly was 0.02 +/- 0.01 mol of phosphate/mol of P-light chain. After 2.5-s stimulation, maximal values of 0.63 +/- 0.06 mol of phosphate/mol of P-light chain and 0.40 +/- 0.06 MP2/myosintotal were obtained. During continuous neural stimulation from 1.0 to 3.5 s, the relationship between the extent of P-light chain phosphorylation (measured directly or calculated) and the relative amount of MP2 is consistent with a random phosphorylation process.     

Effect of cardiac denervation on cardiorespiratory responses to exercise in goats

The purpose of this study was to determine whether intact cardiac innervation and a normal cardiovascular (CV) response are required for a normal ventilatory (VE) response to mild and moderate treadmill exercise in awake goats. Accordingly, we measured CV and respiratory responses to two levels of exercise in seven normal (N) and six cardiac-denervated (CD) goats. Evidence of surgical CD included 1) absence of a cardiac response during surgery when the left thoracic cardiac nerves, thoracic vagi, and right and left stellate ganglia were electrically stimulated, 2) total and 80% attenuation of baroreflex changes in heart rate (HR) when arterial blood pressure was raised or lowered, respectively, by infusion of vasoactive agents in awake goats, and 3) attenuation of the CV responses to exercise. At each level of exercise in the CD goats, the HR response was significantly reduced relative to the response observed before CD (P less than 0.05) and the recovery HR response was delayed. Cardiac index increased in a work rate-dependent manner in N and CD goats but was significantly lower in the CD animals (P less than 0.05). Hypotension was consistently observed during exercise following CD. There was no effect of CD on steady-state VE at any metabolic rate or on the VE-O2 uptake relationship (P greater than 0.05). The rest-to-work and work-to-work transition responses of arterial PCO2 were similar between N and CD goats, but there was a tendency toward greater hypocapnia at the exercise onset in CD goats at the highest work rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of training on biochemical and functional properties of rodent neonatal heart

This study was undertaken to determine biochemical and functional (in vivo) adaptations of the rodent neonatal heart in response to a training program of endurance running. Ten day-old rats were progressively trained on a treadmill (final intensity, 21 m/min, 30% grade, 1 h/day) until 75 days of age. The training program induced 14, 57, and 24% increases in relative heart mass, skeletal muscle citrate synthase activity, and whole-body maximal O2 uptake, respectively (P less than 0.05). Cardiac myosin (ATPase) and Ca2+-regulated myofibril ATPase were both reduced by approximately 15% in trained vs. sedentary animals (P less than 0.05). In the majority of trained hearts examined, the myosin isozyme profile reflected an estimated 14 +/- 3% shift toward the V3 or low ATPase isozyme. Left ventricular functional indices during submaximal exercise, derived from a fluid-filled indwelling cannula, indicated that the trained animals maintained similar left ventricular (LV) systolic pressure, LV + the time derivative of pressure, and systemic arterial mean blood pressure compared with their sedentary counterparts. These functional parameters were maintained even though the trained animals performed with lower submaximal exercise heart rate. These findings suggest that maximal exercise capacity can be enhanced in neonatal rats even though the biochemical potential for ATP degradation in the cardiac contractile system is lowered. We speculate that the trend to maintain the myosin isozyme pattern further in the direction of the V3 isozyme in the trained neonatal rat heart may reflect a means to economize cross-bridge cycling while maintaining normal levels of ventricle performance at a given submaximal work load.     

Cardiac biochemical and functional adaptations to exercise in sympathectomized neonatal rats

This study was undertaken to examine the influence of guanethidine monosulfate-induced sympathectomy on exercise-induced adaptations of cardiac contractile protein and on acute hemodynamic responses to exercise involving female neonatal rats. Four groups of rats were studied: 1) normal sedentary (NS), 2) normal trained (NT), 3) sympathectomized sedentary (SS), and 4) sympathectomized trained (ST). The 9-wk running program, which began at 20 days of age, induced increases in whole-body maximal O2 consumption and skeletal-muscle citrate synthase activity in both NT and ST groups compared with NS (P less than 0.05). Submaximal exercise tests demonstrated circulatory adaptations for NT, SS, and ST groups compared with NC; however, the ST group demonstrated the greatest degree of altered cardiac function (decreased heart rate, left ventricular pressure, and contractility index) during exercise. Also, significant reductions in both myosin- and Ca2+-regulated myofibril adenosinetriphosphatase (ATPase) activity and increases in the relative content of the low ATPase myosin isozyme, V3, occurred in the hearts of the two trained groups (P less than 0.05). These findings suggest that chronic exercise involving normal and sympathectomized neonatal rats improves cardiac function without compromising maximal exercise capacity. Also, the exercise-related adaptation involving myosin isozyme shifts are exaggerated when involvement of the sympathetic nervous system is reduced during training.     

Myosin light-chain phosphatase.

1. A method for the isolation of a new enzyme, myosin light-chain phosphatase, from rabbit white skeletal muscle by using a Sepharose-phosphorylated myosin light-chain affinity column is described. 2. The enzyme migrated as a single component on electrophoresis in sodium dodecyl sulphate/polyacrylamide gel at pH7.0, with apparent mol.wt. 70000. 3. The enzyme was highly specific for the phosphorylated P-light chain of myosin, had pH optima at 6.5 and 8.0 and was not inhibited by NaF. 4. A Ca2+-sensitive 'ATPase' (adenosine triphosphatase) system consisting of myosin light-chain kinase, myosin light-chain phosphatase and the P-light chain is described. 5. Evidence is presented for a phosphoryl exchange between Pi, phosphorylated P-light chain and myosin light-chain phosphatase. 6. Heavy meromyosin prepared by chymotryptic digestion can be phosphorylated by myosin light-chain kinase. 7. The ATPase activities of myosin and heavy meromyosin, in the presence and absence of F-actin, were not significantly changed (+/- 10%) by phosphorylation of the P-light chain.     

Effect of central hypervolemia on cardiac performance during exercise

To investigate the effect of different levels of central blood volume on cardiac performance during exercise, M-mode echocardiography was utilized to determine left ventricular size and performance during cycling exercise in the upright posture (UP), supine posture (SP), and head-out water immersion (WI). At submaximal work loads requiring a mean O2 consumption (Vo2) of 1.2 1/min and 1.5 1/min, mean left ventricular end-diastolic and end-systolic dimensions were significantly greater (P less than 0.05) with WI than UP. In the SP during exercise, left ventricular dimensions were intermediate between UP and WI. Heart rate did not differ significantly among the three conditions at rest and at submaximal exercise up to a mean Vo2 of 1.8 1/min. However, at a mean Vo2 of 2.4 1/min, heart rate in the UP was significantly greater than WI (P less than 0.01) and the SP (P less than 0.05). Maximal Vo2 did not differ statistically in the three conditions. These data indicate that a change in central blood volume results in alterations in left ventricular end-diastolic and end-systolic dimensions during moderate levels of exercise and a change in heart rate at heavy levels of exercise.     

Phosphorylation of myosin light chains in perfused rat heart. Effect of adrenaline and increased cytoplasmic calcium ions.

1. A method was developed for the isolation of essentially pure myosin light chains from perfused rat heart. The phosphorylation of the P-light chains was estimated by hydrolysis and measurement of phosphate released, by electrophoresis in 8 M-urea and by 32P incorporation in perfusion with [32P]Pi. 2. In control perfusions there was 0.5-0.6 mol of phosphate/mol of P-light chain. This was not changed by perfusion with 5 microM-adrenaline for 10-40s. Perfusion for 1 min with medium containing 7.5 mM-CaCl2, or for 30s with medium containing 118 mM-KCl, also did not change the phosphorylation of P-light chains. 3. It is concluded that phosphorylation of P-light chains is not important in mediating the action of inotropic agents in the heart.     

Cardiac myosin light chain kinase is necessary for myosin regulatory light chain phosphorylation and cardiac performance in vivo

In contrast to studies on skeletal and smooth muscles, the identity of kinases in the heart that are important physiologically for direct phosphorylation of myosin regulatory light chain (RLC) is not known. A Ca(2+)/calmodulin-activated myosin light chain kinase is expressed only in cardiac muscle (cMLCK), similar to the tissue-specific expression of skeletal muscle MLCK and in contrast to the ubiquitous expression of smooth muscle MLCK. We have ablated cMLCK expression in male mice to provide insights into its role in RLC phosphorylation in normally contracting myocardium. The extent of RLC phosphorylation was dependent on the extent of cMLCK expression in both ventricular and atrial muscles. Attenuation of RLC phosphorylation led to ventricular myocyte hypertrophy with histological evidence of necrosis and fibrosis. Echocardiography showed increases in left ventricular mass as well as end-diastolic and end-systolic dimensions. Cardiac performance measured as fractional shortening decreased proportionally with decreased cMLCK expression culminating in heart failure in the setting of no RLC phosphorylation. Hearts from female mice showed similar responses with loss of cMLCK associated with diminished RLC phosphorylation and cardiac hypertrophy. Isoproterenol infusion elicited hypertrophic cardiac responses in wild type mice. In mice lacking cMLCK, the hypertrophic hearts showed no additional increases in size with the isoproterenol treatment, suggesting a lack of RLC phosphorylation blunted the stress response. Thus, cMLCK appears to be the predominant protein kinase that maintains basal RLC phosphorylation that is required for normal physiological cardiac performance in vivo.     

降钙素基因相关肽对不同程度冠脉狭窄时的心脏功能的影响

本实验观察了冠脉内注射降钙素基因相关肽（ＣＧＲＰ０．３μｇ／ｋｇ）对正常及不同程度冠脉狭窄犬的心功能的影响。结果表明正常犬冠脉内注射ＣＧＲＰ后,平均动脉压（ＭＡＰ）下降１．２ｋＰａ（Ｐ＜０．０５）,同时,心率（ＨＲ）、心输出量（ＣＯ）、左室收缩压峰值（ＬＶＳＰ）均不同程度增加;左室舒张末压（ＬＶＥＤＰ）轻度降低。在中度狭窄３０ｍｉｎ后,冠脉内注射ＣＧＲＰ对ＨＲ、ＭＡＰ无明显影响;而重度狭窄后注射ＣＧＲＰ,ＭＡＰ由狭窄时降低逐渐增高,ＨＲ由增快而变慢。ＣＯ、ＬＶＳＰ均显著增高,ＬＶＥＤＰ降低,此作用较冠脉狭窄前更为明显。提示ＣＧＲＰ扩张冠脉动脉,增加冠脉血流量和心排血量,增强心肌收缩力,对缺血心脏功能有保护作用。     

Alterations in autonomic function and cerebral hemodynamics to orthostatic challenge following a mountain marathon.

We examined potential mechanisms (autonomic function, hypotension, and cerebral hypoperfusion) responsible for orthostatic intolerance following prolonged exercise. Autonomic function and cerebral hemodynamics were monitored in seven athletes pre-, post- (<4 h), and 48 h following a mountain marathon [42.2 km; cumulative gain approximately 1,000 m; approximately 15 degrees C; completion time, 261 +/- 27 (SD) min]. In each condition, middle cerebral artery blood velocity (MCAv), blood pressure (BP), heart rate (HR), and cardiac output (Modelflow) were measured continuously before and during a 6-min stand. Measurements of HR and BP variability and time-domain analysis were used as an index of sympathovagal balance and baroreflex sensitivity (BRS). Cerebral autoregulation was assessed using transfer-function gain and phase shift in BP and MCAv. Hypotension was evident following the marathon during supine rest and on standing despite increased sympathetic and reduced parasympathetic control, and elevations in HR and cardiac output. On standing, following the marathon, there was less elevation in normalized low-frequency HR variability (P < 0.05), indicating attenuated sympathetic activation. MCAv was maintained while supine but reduced during orthostasis postmarathon [-10.4 +/- 9.8% pre- vs. -15.4 +/- 9.9% postmarathon (%change from supine); P < 0.05]; such reductions were related to an attenuation in BRS (r = 0.81; P < 0.05). Cerebral autoregulation was unchanged following the marathon. These findings indicate that following prolonged exercise, hypotension and postural reductions in autonomic function or baroreflex control, or both, rather than a compromise in cerebral autoregulation, may place the brain at risk of hypoperfusion. Such changes may be critical factors in collapse following prolonged exercise.     

Selective phosphorylation of myosin light chain in intact skeletal muscle

The phosphorylation of myosin light chain was quantitated in fast and slow chicken skeletal muscles and in frog sartorius and semitendinosus muscles. The phosphate content of light chain was determined either as moles [³²P]phosphate per mole of light chain in ³²P-labeled muscles or as percentage phosphorylated light chain of the total P-light chain, measured by densitometry after separating the phospho and dephospho forms of P-light chain with two-dimensional gel electrophoresis. Both methods revealed that the percentage of total P-light chain which was phosphorylated did not exceed 50% either in maximally tetanized or caffeine-contracted skeletal muscle. This suggests that one of the two P-light chains is selectively phosphorylated in skeletal muscle.