This review discusses the present knowledge on the oxygen uptake kinetics at the onset of exercise in skeletal muscle and the contribution of a previously developed computer model of oxidative phosphorylation in intact skeletal muscle to the understanding of the factors determining this kinetics on the biochemical level. It has been demonstrated recently that an increase in the total creatine pool [PCr + Cr] and in glycolytic ATP supply lengthen the half-transition time of the VO₂ on-kinetics, while an increase in mitochondria content, in parallel activation of ATP supply and ATP usage, in muscle oxygen concentration, in proton leak, in resting energy demand, in resting cytosolic pH, and in initial alkalization diminish this parameter. It has also been shown that the half-transition time is near-linearly proportional to the absolute difference between the phosphocreatine concentration during work and at rest (ΔPCr). The present review discusses whether the VO₂ on-kinetics on the muscle level is strictly or only approximately exponential. Finally, it is post-ulated that a short transition time of the VO₂ on-kinetics in itself does not need be profitable for the skeletal muscle functioning during exercise, but usually a short transition time is correlated with factors that improve exercise capacity. The transition time is a phenomenological parameter resulting from the biochemical properties of the system and not a physical factor that can cause anything in the system.

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We developed an electromagnetic apparatus to perform a quick change in load in the motility system, using magnetizable beads on which myosin thick filaments from molluscan smooth muscle or green algae, Chara, myosin were attached. The quick change in load to beads (diameter 4.5 μm) was applied in the range of 0–85 pN. The movement of beads was recorded by a video-system and analyzed with special software. When the quick increase in load was applied during the movement of beads under no load, the beads showed a transient movement to the reverse direction before the steady slower movement to the normal direction. When the application of load was stopped, the beads showed a transient fast phase of movement. The change in load-sustaining ability was measured by a double load step. The backward velocity at the second constant test load was smaller when the first preceding step was increased, suggesting that the ability to sustain load was higher with a higher preceding step. These phenomena were observed both in molluscan thick filaments and in Chara myosin, and the time course of the movement of a bead was quite similar to those observed previously in frog single muscle fibers. This suggested that the velocity transients are the intrinsic properties induced by the interaction between actin and myosin, irrespective of the hexagonal lattice structure of filaments, the regular sarcomere structure, and myosin type, namely, that the molecule of myosin itself has the ability to adjust to mechanical circumstances.

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This study examines the role of gap junctional communication in smooth muscle in relation to the frequency of stimulation and the innervation density of the tissue in the generation of neurogenic contractions. Toward this end the effects of heptanol, a gap junctional blocker, on the neurogenic contractions of guinea pig and rat vas deferens at different frequencies of stimulation (single pulse, 5, 10, 20, 40, 60, and 80 Hz) were studied. In both the prostatic and epididymal halves of these tissues, heptanol abolished the neurogenic contractions at the lower frequencies of stimulation. At higher frequencies, contractions were resistant to heptanol action. The effect of heptanol on the neurogenic contractions was found to decrease with increasing stimulation frequency. The neurogenic contractions of rat vas deferens were more resistant to heptanol than those of guinea pig vas deferens. Our data indicate that gap junctional communication is significant in the generation of neurogenic contractions in both guinea pig and rat vas deferens in a frequency-dependent manner, and we discuss the mechanisms underlying these findings.

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There is a well-characterized membrane chloride current (I_Cl,cAMP) in the heart that can be activated by β-adrenergic agonists and is due to expression of the cardiac isoform of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR). We have investigated whether 17β-estradiol (E2) modulates I_Cl,cAMP in single ventricular myocytes. Under whole-cell tight-seal voltage-clamp conditions, I_Cl,cAMP was evoked by exposing cells to 20 nM isoprenaline. On the addition of 30 μM E2, membrane slope conductance, measured at potentials near 0 mV, increased over that induced by isoprenaline alone by 2.46 ± 0.16 (p < 0.001). The effects of E2 were concentration-dependent and described by a Hill Plot with an EC₅₀ of 8.2 μM and a Hill coefficient of 1.63. The application of membrane-impermeant E2 conjugated to bovine serum albumin (E2-BSA) potentiated isoprenaline-evoked I_Cl,cAMP by approximately the same degree as that for the equivalent level of free E2. Cell surface binding was observed with confocal microscopy by using BSA-FITC tagged E2. This binding was inhibited by nonlabeled, nonconjugate E2, the specific E2 antagonist ICI 182,780, and incubation of E2coBSA with a specific anti-E2 antibody (E2885). ICI 182,780 (100 μM) significantly reduced the increase in I_Cl,cAMP evoked by 10 μM E2 to 1.46 ± 0.10 (p < 0.02). The preincubation of myocytes with the NOS inhibitor N-ω-nitro-arginine (L-NNA, 1 mM) reduced the potentiation of I_Cl,cAMP by 30 μM E2, to 1.93 ± 0.06 (p < 0.02), and for 10 μM E2, to 1.32 ± 0.05 (p < 0.002). E2 also increased I_Cl,cAMP evoked by bath application of 0.5 μM Forskolin. These experiments demonstrate that, under our experimental conditions, E2 dramatically increases I_Cl,cAMP in ventricular myocytes by mechanisms involving a contribution by NOS, but that can be only partially accounted for through binding to classical plasma membrane estrogen receptor sites. This potentiation of I_Cl,cAMP by E2 may play a significant role in the observed clinical actions of E2 on the incidence of cardiac arrhythmias and hypertrophy.

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The effects of dietary L-arginine supplementation on exercise-induced angiogenesis and VEGF expression were examined in male middle-aged (12 months old) Wistar rats. Exercise training lasted for six weeks at 20 m/min on a 0% gradient for 10–60 min/day. Rats in the L-arginine–treated groups drank water containing 2.5% L-arginine. According to histochemical identification of the capillary profile, in the soleus muscle the capillary-to-fiber (C:F) ratio showed a significantly greater value in the L-arginine–treated training group than in both the sedentary control and training groups. Training with L-arginine significantly increased the C:F ratio in the subendocardium of the left ventricle, whereas training alone did not. In the plantaris muscle, training with or without L-arginine significantly increased the capillary density, but it did not affect the C:F ratio. A Western blot analysis showed that training with L-arginine significantly increased VEGF protein expression by 2.9-fold in the soleus muscle and by 1.7-fold in the left ventricle, but the increase with training alone was insignificant. The tissue endothelial nitric oxide synthase protein levels were significantly increased in both the soleus (by 1.3-fold) and the left ventricle (by 1.4-fold) only after training with L-arginine supplementation. In the plantaris muscle, these protein levels did not change after either training or L-arginine treatment. The present results suggest that in middle-aged rats, L-arginine administration caused additional effects on exercise-induced angiogenesis by presumably promoting VEGF expression in the hind-leg muscle as well as in the left ventricle.

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The Valsalva maneuver is a frequently used physiological test in evaluating the cardiovascular autonomic functions in human. Although a large pool of experimental data has provided substantial insights into different aspects of the mechanisms underlying the cardiovascular regulations during the Valsalva maneuver, so far a complete comprehension of these mechanisms and the interactions among them is unavailable. In the present study, a computational model of the cardiovascular system (CVS) and its interaction with the autonomic nervous system (ANS) was developed for the purpose of quantifying the individual roles of the CVS and the ANS in the hemodynamic regulations during the Valsalva maneuver. A detailed computational compartmental parameter model of the global CVS, a system of mathematical equations representing the autonomic nervous reflex regulatory functions, and an empirical cerebral autoregulation (CA) model formed the main body of the present model. Based on simulations of the Valsalva maneuvers at several typical postures, it was demonstrated that hemodynamic responses to the maneuver were not only determined by the ANS-mediated cardiovascular regulations, but also significantly affected by the postural-change-induced hemodynamic alterations preceding the maneuver. Moreover, the large-magnitude overshoot in cerebral perfusion immediately after the Valsalva maneuver was found to result from a combined effect of the circulatory autonomic functions, the CA, and the cerebral venous blood pressure.

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We have recently reported that the atrioventricular (AV) nodal mechanism functions to cancel fluctuation in the atrial excitation interval during a stair-stepping exercise. However, it remained unknown at which level of heart rate (HR) this mechanism started to operate and whether fluctuation in the interval might influence AV conduction over the following beats. To solve these questions, the variability of PP, RR, and PR intervals and their interrelationships were analyzed throughout ergometer exercise in eight subjects. The variability of the RR interval decreased to 0.7% of the control at 160 beats/min during exercise, much more than the PP interval variability, which decreased to 10%, despite the same shortened average interval. In contrast, the PR interval variability tended to increase by 87% during exercise, but the mean PR interval decreased. A strong inverse relationship between PP and the subsequent change in PR [ΔPR] intervals became evident during exercise, implying that the ΔPR interval canceled fluctuation in the PP interval. However, there was little correlation between the RR and ΔPR intervals and between the PP interval and the next PR intervals in the forthcoming beat. When the slope of the PP–ΔPR relationship, considered as sensitivity of the AV nodal function opposing an alteration in the PP interval, was plotted against the PP interval, the AV nodal function curve was approximated to a sigmoidal curve having a threshold of PP interval near 650 ms and a maximum plateau level of the slope near 1.0. We conclude that when HR exceeds 90–100 beats/min during dynamic exercise, the AV nodal mechanism will function to cancel fluctuation in the PP interval within one beat and keep the RR interval constant.

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We addressed the question how Ca²⁺ transients affect gap junction conductance (G_j) during action potential (AP) propagation by constructing a dynamic gap junction model coupled with a cardiac cell model. The kinetics of the Ca²⁺ gate was determined based on published experimental findings that the Hill coefficient for the [Ca²⁺]_i–G_j relationship ranges from 3 to 4, indicating multiple ion bindings. It is also suggested that the closure of the Ca²⁺ gate follows a single exponential time course. After adjusting the model parameters, a two-state (open-closed) model, assuming simultaneous ion bindings, well described both the single exponential decay and the [Ca²⁺]_i–G_j relationship. Using this gap junction model, 30 cardiac cell models were electrically connected in a one-dimensional cable. However, G_j decreased in a cumulative manner by the repetitive Ca²⁺ transients, and a conduction block was observed. We found that a reopening of the Ca²⁺ gate is possible only by assuming a sequential ion binding with one rate limiting step in a multistate model. In this model, the gating time constant (τ) has a bell-shaped dependence on [Ca²⁺]_i, with a peak around the half-maximal concentration of [Ca²⁺]_i. Here we propose a five-state model including four open states and one closed state, which allows normal AP propagation; namely, the G_j is decreased ∼15% by a single Ca²⁺ transient, but well recovers to the control level during diastole. Under the Ca²⁺-overload condition, however, the conduction velocity is indeed decreased as demonstrated experimentally. This new gap junction model may also be useful in simulations of the ventricular arrhythmia.

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A chronic administration of isoproterenol hydrochloride (60 mg/kg body weight; 30 days) alters the collagen metabolism in denervated gastrocnemius muscle of mice. Hydroxyproline assay for collagen showed an increase in collagen content by 47%, 44%, and 61% in innervated gastrocnemius + drug, denervated control, and denervated + drug, respectively, in gastrocnemius muscles after 30 days of drug administration. Collagen proliferation is β-agonist (isoproterenol) specific confirmed with the simultaneous administration of β-antagonist propranolol (100 mg/kg body weight; 30 days). Van Gieson staining showed heavy collagen proliferation in the epimysium region of the muscle section and adventitia of blood vessels and some specialized regions. However, denervated gastrocnemius muscle represented a heavy collagen proliferation in the endomysium region, which also is probably responsible for extensive collagen proliferation in denervated muscle after drug administration. The SDS-PAGE of pepsin-soluble collagen revealed five bands from origin to the point of migration, γ, β₁, β₂, α₁, and α₂. The SDS-PAGE of CNBr-treated pepsin-insoluble collagen pointed toward the more prominent remodeling of collagen metabolism in the β-agonist-induced denervated gastrocnemius muscle after drug administration. From the present study, we can conclude that β-agonist, isoproterenol hydrochloride, augments collagen proliferation in innervated as well as in denervated gastrocnemius muscle.

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Background: Hypoxia-inducible factor (HIF)-1α regulates the transcription of lines of genes, including vascular endothelial growth factor (VEGF), a major gene responsible for angiogenesis. Several recent studies have demonstrated that a nonhypoxic pathway via nitric oxide (NO) is involved in the activation of HIF-1α. However, there is no direct evidence demonstrating the release of angiogenic factors by cardiomyocytes through the nonhypoxic induction pathway of HIF-1α in the heart. Therefore we assessed the effects of an NO donor, S-Nitroso-N-acetylpenicillamine (SNAP) on the induction of VEGF via HIF-1α under normoxia, using primary cultured rat cardiomyocytes (PRCMs). Methods and Results: PRCMs treated with acetylcholine (ACh) or SNAP exhibited a significant production of NO. SNAP activated the induction of HIF-1α protein expression in PRCMs during normoxia. Phosphatidylinositol 3-kinase (PI3K)–dependent Akt phosphorylation was induced by SNAP and was completely blocked by wortmannin, a PI3K inhibitor, and N^G-nitro-L-arginine methyl ester (L-NAME), a NO synthase inhibitor. The SNAP treatment also increased VEGF protein expression in PRCMs. Furthermore, conditioned medium derived from SNAP-treated cardiomyocytes phosphorylated the VEGF type–2 receptor (Flk-1) of human umbilical vein endothelial cells (a fourfold increase compared to the control group, p < 0.001, n = 5) and accelerated angiogenesis. Conclusion: Our results suggest that cardiomyocytes produce VEGF through a nonhypoxic HIF-1α induction pathway activated by NO, resulting in angiogenesis.

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We hypothesized that a freely paced 10,000 m running race would induce a smaller physiological strain (heart rate and oxygen uptake) compared with one performed at the same average speed but with an imposed constant pace. Furthermore, we analyzed the scaling properties with a wavelet transform algorithm computed log₂ (wavelet transform energy) vs. log₂ (scale) to get slope a, which is the scaling exponent, a measure of the irregularity of a time series. HR was sampled beat by beat and VO₂ breath by breath. The enforced constant pace run elicited a significantly higher mean VO₂ value (53 ± 4 vs. 48 ± 5 ml kg⁻¹ min⁻¹, P < 0.001), HR (169 ± 13 vs. 165 ± 14 bpm, P < 0.01), and blood lactate concentration (6.6 ± 0.9 vs. 7.5 ± 1 mM, P < 0.001) than the freely paced run. HR and VO₂ signals showed a scaling behavior, which means that the signals have a similar irregularity (a self-similarity) whatever the scale of analysis may be, in both constant and free-paced 10,000 m runs. The scaling exponent was not significantly different according to the type of run (free vs. constant, P > 0.05) and the signal (HR vs. VO₂, P > 0.05). The higher metabolic cost of constant vs. free paced run did not affect the self-similarity of HR and VO₂ in either run. The HR signal only kept its scaling behavior only with a distance run, no matter the type of run (free or constant).The results suggest that the larger degree of pace variation in freely paced races may be an intentionally chosen strategy designed to minimize the physiological strain during severe exercise and to prevent a premature termination of effort, even if the variability of the heart rate and VO₂ are comparable in an enforced constant vs. a freely paced run and if HR keeps the same variability until the arrival.

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Na⁺,K⁺-ATPases are composed of one α and one β subunit; four α and three β isoforms have been found to date. We elucidated which α and β subunits were present in the ventricular myocytes of rat and guinea-pig and what roles the Na⁺,K⁺-ATPase isozymes play in cardiac contraction. The presence of the α1, α2, and α3 subunits and the β1 and β2 subunits in rat and guinea-pig hearts were confirmed at the protein or mRNA level. Immunocytochemistry showed a patchy presence of α1 in the transverse tubules and surface sarcolemma, whereas α2 was distributed continuously in the transverse tubules alone. The α3 isoform was expressed prominently in the guinea-pig intercalated disc and slightly in the rat. On the other hand, the β1 isoform was located in the transverse tubules and surface sarcolemma, whereas the β2 was mainly located in the intercalated disc. The immunocytochemistry and immunoprecipitation findings indicated that the α1 and α2 form heterodimers with β1 and the α3 with β2 in ventricular myocytes. The application of low concentrations of ouabain enhanced the amplitudes of twitch without a change in resting tension in rat and guinea-pig ventricular stripts, whereas that of high concentrations resulted in a decrease in twitch with an increase in the resting tension. We thus conclude that the α2β1 and α3β2 isozymes are selectively located in the transverse tubules and intercalated disc of the ventricular myocytes, respectively, and the α2β1 is involved in the regulation of the Ca²⁺ contents in the SR.

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The effects of p-CPA (para-chlorophenylalanine) pretreatment was studied on the sleep-wake parameters and patterns of behavioral activities in an animal model of acute immobilization stress. For the experiments, young male Charles Foster rats were divided into three groups, subjected to (i) acute immobilization stress for four hours on specially designed wooden boards, (ii) a similar model of acute immobilization stress after pretreatment of p-CPA (injected through i.p. route), and (iii) control rats (p-CPA untreated and unstressed). Three channels of electrographic signals, i.e., EEG (electroencephalogram), EOG (electrooculogram), and EMG (electromyogram) were recorded continuously for four hours for all three groups of rats to analyze the changes in sleep-wake stages. The assessment of behavior was performed just after the stress on separate groups of rats in Open-Field (OF) and Elevated Plus-Maze (EPM) apparatuses. The significant changes in total sleep time (P < 0.05), total time for rapid eye movement sleep (P < 0.01), and total time in wakefulness (P < 0.01) following acute immobilization stress were found reversed in the p-CPA (a serotonin inhibitor) pretreated group of rats. Simultaneously, the results of the present work also revealed that the changes in grooming behavior (P < 0.05) in OF and the total time spent on the center of EPM (P < 0.05) were observed altered in p-CPA pretreated group of rats.

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