Time course of the MAPK and PI3-kinase response within 24 h of skeletal muscle overload.

Knowledge of the molecular mechanisms by which skeletal muscle hypertrophies in response to increased mechanical loading may lead to the discovery of novel treatment strategies for muscle wasting and frailty. To gain insight into potential early signaling mechanisms associated with skeletal muscle hypertrophy, the temporal pattern of mitogen-activated protein kinase (MAPK) phosphorylation and phosphatidylinositol 3-kinase (PI3-kinase) activity during the first 24 h of muscle overload was determined in the rat slow-twitch soleus and fast-twitch plantaris muscles after ablation of the gastrocnemius muscle. p38alpha MAPK phosphorylation was elevated for the entire 24-h overload period in both muscles. In contrast, Erk 2 and p54 JNK phosphorylation were transiently increased by overload, returning to the levels of sham-operated controls by 24 h. PI3-kinase activity was increased by muscle overload only at 12 h of overload and only in the plantaris muscle. In summary, sustained elevation of p38alpha MAPK phosphorylation occurred early in response to muscle overload, identifying this pathway as a potential candidate for mediating early hypertrophic signals in response to skeletal muscle overload.

Differential global gene expression in red and white skeletal muscle.

The differences in gene expression among the fiber types of skeletal muscle have long fascinated scientists, but for the most part, previous experiments have only reported differences of one or two genes at a time. The evolving technology of global mRNA expression analysis was employed to determine the potential differential expression of approximately 3,000 mRNAs between the white quad (white muscle) and the red soleus muscle (mixed red muscle) of female ICR mice (30-35 g). Microarray analysis identified 49 mRNA sequences that were differentially expressed between white and mixed red skeletal muscle, including newly identified differential expressions between muscle types. For example, the current findings increase the number of known, differentially expressed mRNAs for transcription factors/coregulators by nine and signaling proteins by three. The expanding knowledge of the diversity of mRNA expression between white and mixed red muscle suggests that there could be quite a complex regulation of phenotype between muscles of different fiber types.

ANG II is required for optimal overload-induced skeletal muscle hypertrophy.

ANG II mediates the hypertrophic response of overloaded cardiac muscle, likely via the ANG II type 1 (AT(1)) receptor. To examine the potential role of ANG II in overload-induced skeletal muscle hypertrophy, plantaris and/or soleus muscle overload was produced in female Sprague-Dawley rats (225-250 g) by the bilateral surgical ablation of either the synergistic gastrocnemius muscle (experiment 1) or both the gastrocnemius and plantaris muscles (experiment 2). In experiment 1 (n = 10/group), inhibiting endogenous ANG II production by oral administration of an angiotensin-converting enzyme (ACE) inhibitor during a 28-day overloading protocol attenuated plantaris and soleus muscle hypertrophy by 57 and 96%, respectively (as measured by total muscle protein content). ACE inhibition had no effect on nonoverloaded (sham-operated) muscles. With the use of new animals (experiment 2; n = 8/group), locally perfusing overloaded soleus muscles with exogenous ANG II (via osmotic pump) rescued the lost hypertrophic response in ACE-inhibited animals by 71%. Furthermore, orally administering an AT(1) receptor antagonist instead of an ACE inhibitor produced a 48% attenuation of overload-induced hypertrophy that could not be rescued by ANG II perfusion. Thus ANG II may be necessary for optimal overload-induced skeletal muscle hypertrophy, acting at least in part via an AT(1) receptor-dependent pathway.

Waging war on modern chronic diseases: primary prevention through exercise biology.

In this review, we develop a blueprint for exercise biology research in the new millennium. The first part of our plan provides statistics to support the contention that there has been an epidemic emergence of modern chronic diseases in the latter part of the 20th century. The health care costs of these conditions were almost two-thirds of a trillion dollars and affected 90 million Americans in 1990. We estimate that these costs are now approaching $1 trillion and stand to further dramatically increase as the baby boom generation ages. We discuss the reaction of the biomedical establishment to this epidemic, which has primarily been to apply modern technologies to stabilize overt clinical problems (e.g., secondary and tertiary prevention). Because this approach has been largely unsuccessful in reversing the epidemic, we argue that more emphasis must be placed on novel approaches such as primary prevention, which requires attacking the environmental roots of these conditions. In this respect, a strong association exists between the increase in physical inactivity and the emergence of modern chronic diseases in 20th century industrialized societies. Approximately 250,000 deaths per year in the United States are premature due to physical inactivity. Epidemiological data have established that physical inactivity increases the incidence of at least 17 unhealthy conditions, almost all of which are chronic diseases or considered risk factors for chronic diseases. Therefore, as part of this review, we present the concept that the human genome evolved within an environment of high physical activity. Accordingly, we propose that exercise biologists do not study "the effect of physical activity" but in reality study the effect of reintroducing exercise into an unhealthy sedentary population that is genetically programmed to expect physical activity. On the basis of healthy gene function, exercise research should thus be viewed from a nontraditional perspective in that the "control" group should actually be taken from a physically active population and not from a sedentary population with its predisposition to modern chronic diseases. We provide exciting examples of exercise biology research that is elucidating the underlying mechanisms by which physical inactivity may predispose individuals to chronic disease conditions, such as mechanisms contributing to insulin resistance and decreased skeletal muscle lipoprotein lipase activity. Some findings have been surprising and remarkable in that novel signaling mechanisms have been discovered that vary with the type and level of physical activity/inactivity at multiple levels of gene expression. Because this area of research is underfunded despite its high impact, the final part of our blueprint for the next millennium calls for the National Institutes of Health (NIH) to establish a major initiative devoted to the study of the biology of the primary prevention of modern chronic diseases. We justify this in several ways, including the following estimate: if the percentage of all US morbidity and mortality statistics attributed to the combination of physical inactivity and inappropriate diet were applied as a percentage of the NIH's total operating budget, the resulting funds would equal the budgets of two full institutes at the NIH! Furthermore, the fiscal support of studies elucidating the scientific foundation(s) targeted by primary prevention strategies in other public health efforts has resulted in an increased efficacy of the overall prevention effort. We estimate that physical inactivity impacts 80-90% of the 24 integrated review group (IRG) topics proposed by the NIH's Panel on Scientific Boundaries for Review, which is currently directing a major restructuring of the NIH's scientific funding system. Unfortunately, the primary prevention of chronic disease and the investigation of physical activity/inactivity and/or exercise are not mentioned in the almost 200 total subtopics comprising t

Skeletal muscle myostatin mRNA expression is fiber-type specific and increases during hindlimb unloading.

Transgenic mice lacking a functional myostatin (MSTN) gene demonstrate greater skeletal muscle mass resulting from muscle fiber hypertrophy and hyperplasia (McPherron, A. C., A. M. Lawler, and S. -J. Lee. Nature 387: 83-90, 1997). Therefore, we hypothesized that, in normal mice, MSTN may act as a negative regulator of muscle mass. Specifically, we hypothesized that the predominately slow (type I) soleus muscle, which demonstrates greater atrophy than the fast (type II) gastrocnemius-plantaris complex (Gast/PLT), would show more elevation in MSTN mRNA abundance during hindlimb unloading (HU). Surprisingly, MSTN mRNA was not detectable in weight-bearing or HU soleus muscle, which atrophied 42% by the 7th day of HU in female ICR mice. In contrast, MSTN mRNA was present in weight-bearing Gast/PLT muscle and was significantly elevated (67%) at 1 day but not at 3 or 7 days of HU. However, the Gast/PLT muscle had only atrophied 17% by the 7th day of HU. Because the soleus is composed only of type I and IIa fibers, whereas the Gast/PLT expresses type IId/x and IIb in addition to type I and IIa, it was necessary to perform a more careful analysis of the relationship between MSTN mRNA levels and myosin heavy-chain (MHC) isoform expression (as a marker of fiber type). A significant correlation (r = 0.725, P < 0. 0005) was noted between the percentage of MHC isoform IIb expression and MSTN mRNA abundance in several muscles of the mouse hindlimb. These results indicate that MSTN expression is not strongly associated with muscle atrophy induced by HU; however, it is strongly associated with MHC isoform IIb expression in normal muscle.

Streptokinase alters myocardial creatine kinase depletion after ischaemia and reperfusion in rabbits.

The efficacy of streptokinase as an intracoronary thrombolytic agent is well-recognized. The effect of streptokinase, distinct from its thrombolytic action, on ischaemic myocardium distal to an area of coronary artery occlusion when reperfusion occurs has not been well-defined. In order to do this, myocardial creatine kinase depletion and the histopathology of infarctions produced in rabbits after 1 h of circumflex coronary artery occlusion and mechanical release of the occlusion were assessed. Streptokinase or saline was infused intravenously for 1 h beginning 0.5 h after occlusion. Rabbits were divided into two time intervals: early (less than 10 h) and late (24 h) after release of coronary artery occlusion. When streptokinase was infused in early infarctions, haemorrhage did not correlate with infarction cross-sectional area or myocardial creatine kinase depletion. However, myocardial creatine kinase depletion was 40% less when streptokinase was infused than when saline was infused, suggesting that streptokinase might limit infarct size. In late infarctions, the degree of haemorrhage, infarction cross-sectional area, and myocardial creatine kinase depletion were similar after reperfusion with streptokinase or saline. By 24 h, the beneficial effect of a single dose of streptokinase given early in the course of occlusion-reperfusion myocardial injury was no longer evident in limiting infarct size.

Thermal dilution assessment of proximal aortic and left ventricular mixing chambers in dogs.

The adequacy of left ventricular mixing and the effect of mixing in the left ventricle and aorta on the measurement of the residual fraction by thermal dilution were assessed in open-chest dogs. Temperature was recorded at seven ventricular and two aortic sites during ventricular injections of iced saline at heart rates of 100, 150 and 200 bpm. The residual fraction, K, was calculated for each beat from the inscribed dilution curves and compared with the actual temperature at each measurement site and heart rate. Temperature was non-uniform throughout the ventricle after saline injections indicating incomplete tracer mixing. Thermal equilibration was slowest at the apex and decreased as heart rate increased. K, however, was similar for all sites in the ventricle and aorta at each heart rate by the third beat after saline injection. Thus, in spite of incomplete mixing, the beat-to-beat washout of indicator quickly stabilized which suggests that a reproducible measurement of functional volume can be obtained by this technique.

Creatine kinase: purification, characterization, and tissue distribution of the MM subtypes.

The MM isozyme of creatine kinase can be separated into four subtypes by isoelectric focusing: MM3 (pI 6.90), MM2 (pI 6.70), MM1 (pI 6.45), and MMx (pI 6.25). Individual subtypes were produced and purified from canine heart and then separated by chromatofocusing. Subtypes could not be distinguished by relative molecular mass or kinetic properties. They differed slightly in amino acid composition. Canine and human necropsy tissues were studied to determine subtype distribution. Heart and skeletal muscle contained the most MM isozyme of creatine kinase and it was predominantly of the MM3 subtype.

Effect of venous retroperfusion on experimental myocardial ischemia in the open-chest pig.

Venous retroperfusion, by arterialization of the coronary sinus or the vein accompanying an ischemic artery, has been suggested as an alternative method of myocardial revascularization in patients with severe coronary disease in whom direct revascularization would be an unacceptable risk or technically impossible. This study was carried out to assess whether or not venous retroperfusion can increase myocardial blood flow in the area of ischemia after sudden occlusion of a normal coronary artery in an animal, the pig, with a coronary vasculature similar to that of humans. It was found that net flow measured with an electromagnetic flowmeter through either an aorta-to-coronary sinus shunt or an aorta-to-left anterior descending coronary vein shunt after occlusion of the left anterior descending artery was at first high but rapidly decreased toward zero flow within 1 hr. Blood flow in the ischemic region measured by the microsphere method 20 min after coronary occlusion did not increase. This disparity between electromagnetic flow and regional flow suggests that there are venous-to-thebesian or venous-to-venous shunts into the systemic and pulmonary circulation through vessels greater than 14 micron. It is unlikely that effective oxygen or metabolite exchange would occur in vessels this size.

Creatine kinase MM isoenzyme subforms in myocardium, cardiac lymph and blood after coronary artery occlusion in dogs.

A time-varying pattern of creatine kinase MM (CK-MM) isoenzyme subforms has been found in the blood of patients after acute myocardial infarction, but the site of enzyme modification has not been identified. Therefore, we studied the CK-MM subform patterns in myocardium, cardiac lymph and blood of dogs after coronary artery occlusion. In five conscious dogs, serial blood samples were taken for 72 h after occlusion of the left anterior descending coronary artery. Samples of non-infarcted and infarcted myocardium were taken after 72 h. In five other anaesthetised, open-chest dogs, cardiac lymph and blood samples were taken for 6 h after coronary artery occlusion. CK-MM subforms were quantitated by an isoelectric focusing method. Before coronary occlusion, 64% of the total CK activity in blood appeared as the anodal subform CK-MM 1 (pI 6.3); 20% and 9% as the cathodal subforms CK-MM 2 (pI 6.6) and CK-MM 3 (pI 6.9), respectively. However, after 2 h of coronary occlusion CK-MM 2 and CK-MM 3 were increased (38% and 17% of total activity respectively) compared with CK-MM 1. Between 4 h and 10 h, CK-MM 2 and CK-MM 3 decreased as CK-MM 1 increased restoring the control relative activities of subforms. In contrast to the subform changes in blood, CK-MM 3 was the predominant subform in both non-infarcted and infarcted myocardium after 72 h of coronary occlusion and in cardiac lymph during 6 h of coronary occlusion.(ABSTRACT TRUNCATED AT 250 WORDS)

High-speed eight-channel thermocouple amplifier.

An eight-channel thermocouple amplifier system was constructed to measure rapid changes in temperature of only 1 or 2 degrees C simultaneously from many sites within the heart. The system has a response time of less than 5 ms, a resolution of less than 0.025 degrees C, and a voltage gain of approximately 2,500.

Postextrasystolic pulsus alternans and heart rate.

Postextrasystolic (PES) pulsus alternans, an alternation in systolic blood pressure above and then below base line for several beats after a premature ventricular depolarization was noted many years ago in patients with heart failure. We noted in acute animals studies that the occurrence of PES alternans was related to heart rate. We studied five open-chest dogs suppressing intrinsic heart rate by vagal stimulation and pacing the right ventricle over a range of 100-200 beats/min introducing an extrastimulus at 50% of the paced rate. We found that the pressure of the second PES beat was augmented up to 120 beats/min and then began to decrease approximately 10 Torr for each 10-beats/min increase in rate. The physiological variables most closely related to the occurrence of PES alternans were the duration of systole of the first PES (strong) beat and the rate of rapid diastolic filling of the second PES (weak) beat. Postextrasystolic and possibly sustained pulsus alternans is an inevitable consequence of heart rate and of the relationship between ejection and diastolic filling. The details of that relationship and the effects of various diseases determine whether alternans is observed or not.

Serum creatine kinase MM isoenzyme sub-bands after acute myocardial infarction in man.

Using an isoelectric-focusing (IEF) method developed to quantitate MM isoenzyme-creatine kinase (CK) sub-band activity, we identified a reproducible time-varying pattern of these sub-bands in the serum of eight patients with acute myocardial infarction (MI). Our observations are consistent with the view that MM3-CK (the M2-CK dimer, the pure gene product) is converted intravascularly to MM2-CK, and then to MM1-CK (the M1-CK dimer, the pure postsynthetic sub-band). The MM3-CK reaches a peak first, 16 hours after infarction, followed by MM2-CK, and then by MM1-CK. The MM3-CK is the dominant sub-band in normal myocardium; there is much less MM2-CK and virtually no MM1-CK. The MM3-CK sub-band peak may indicate the time at which enzyme ceases to be released from the injured myocardium. The ratio MM3-CK:MM1-CK rises within 6 hours after onset of chest pain from a baseline of 0.38 and peaks 10 hours after MI. The peak ratio was between 1.1 and 4.2, and the value correlated with the time when total CK activity peaked after MI. The 10-fold change in the MM3:MM1 ratio after MI, as well as the early period at which this ratio peaks (10 hours), makes this an earlier and more sensitive indicator of enzyme release.

Myocardial blood flow during acute isovolumic anemia and treadmill exercise in dogs.

We examined whether submaximal treadmill exercise during acute isovolumic anemia altered the distribution of myocardial blood flow and thus caused subendocardial ischemia in unsedated dogs. Myocardial blood flow (determined by the microsphere method) and left ventricular function (indicated by pressures, volumes, and contractile indices) were measured in five dogs at rest and during exercise before and after equal volume exchange of blood and 6% dextran 70, which lowered hematocrit from 36 +/- 4 to 18 +/- 2% (SD). Total myocardial blood flow increased from 175 +/- 65 to 296 +/- 151 ml . min-1 . 100 g-1 (69%) during exercise before anemia and from 329 +/- 61 to 599 +/- 126 (82%) during exercise after anemia. The distribution of blood flow to the left and right ventricles and interventricular septum as well as the subendothelial-to-subepicardial blood flow ratio in these areas did not change during exercise either before or after anemia. Left ventricular function was not impaired during exercise after anemia. We conclude that subendocardial ischemia does not occur when dogs exercise during acute isovolumic anemia.

Clearance of serum creatine kinase activity.

There is confusion regarding the calculation of serum creatine kinase (CK) activity clearance from an elimination rate constant and a distribution volume derived from one- and two-compartment serum enzyme elimination models. We measured serum CK activity clearance from hepatic enzyme extraction and compared this direct measurement with clearance calculated assuming one- and two-compartment elimination models after bolus and constant infusions of a purified preparation of the MM isoenzyme of CK (MM-CK) activity. Although serum CK activity appears to confer on the body the characteristics of first-order disposition from two compartments, clearance is estimated equally well by one-, two-, and noncompartmental models of disposition and is essentially identical to the hepatic clearance of CK activity. As long as the rate constant and distribution volume are compatible and appropriate for a given elimination model, clearance, the product of the two, will be properly estimated regardless of the model chosen.

Acute vasodilator therapy increases renal clearance of digoxin in patients with congestive heart failure.

We studied the effect of vasodilator therapy on renal digoxin clearance in patients with chronic congestive heart failure. Intravenous administration of nitroprusside or hydralazine to eight patients with severe heart failure produced the expected increase in cardiac output and a decrease in central circulatory pressure. Renal clearance of sodium para-aminohippurate and estimated renal blood flow increased without a change in glomerular filtration rate. Total renal clearance of digoxin increased by 50% during vasodilator therapy. Thus, acute administration of vasodilator increases renal digoxin clearance without changing glomerular filtration rate, suggesting an increase in tubular secretion of digoxin. Long-term vasodilator therapy may alter the maintenance dosage of digoxin required for optimal treatment of patients in congestive heart failure.

Continuous positive-pressure ventilation does not alter ventricular pressure-volume relationship.

To determine whether alterations in the mechanical properties (i.e., stiffening) of the right and left ventricles contribute to the decrease in right and left ventricular end-diastolic volumes during continuous positive-pressure ventilation (CPPV), we studied six dogs anesthetized with chloralose urethane and ventilated with a volume ventilator. We varied ventricular volumes by withdrawing or infusing blood. Pressure-volume curves, constructed by plotting transmural ventricular end-diastolic pressures against ventricular end-diastolic volumes, did not change during CPPV (12 cmH2O positive end-expiratory pressure) compared to intermittent positive-pressure ventilation (IPPV, 0 cmH2O end-expiratory pressure). We conclude that decreased ventricular end-diastolic volumes during CPPV result primarily from a decrease in venous return. Alterations in the mechanical properties of the ventricles do not play a significant role in this response.

A new method for quantification of aortic regurgitation during cardiac catheterization.

Clinical methods of estimating aortic insufficiency are unsatisfactory. We developed a radioisotope method of quantitating aortic regurgitant volume. The method is easily performed, convenient, and can be carried out repetitively. The mathematical derivation of the method is presented and a mechanical model is used to verify assumptions. The potential utility and possible difficulties of applying the method clinically are discussed.