Influence of inorganic phosphate and energy state on force in skinned cardiac muscle from freshwater turtle and rainbow trout

Inorganic phosphate, which increases in the hypoxic cardiac cell, depresses force development. The cardiac muscle of freshwater turtle maintains a remarkably high contractility during hypoxia; this may involve a low sensitivity to phosphate. Therefore, freshwater turtle and rainbow trout were compared with regard to Ca²⁺-activated force in skinned atrial trabeculae in a bath containing 3 mM ATP buffered by 15 mM creatine phosphate in the presence of creatine kinase. For turtle, an increase in phosphate from 0 mM to either 6 mM or 12 mM reduced maximal force by 50% and 80% respectively, whereas the Ca²⁺ activity eliciting half maximal force (Ca_0.5) was increased by 70% in 6 mM and could not be reliably recorded in 12 mM. For trout, the effects of phosphate were less pronounced. An increase from 0 mM to 12 mM did not affect maximal force significantly, but elevated Ca_0.5 by 70%. Hypoxia increases ADP as creatine phosphate is shifted to creatine, therefore, creatine phosphate was changed from 15 mM to 3 mM and creatine from 0 mM to 12 mM. After these changes, the elevation of phosphate from 0 mM to 12 mM had no significant effects for either turtle or trout. In conclusion, the high performance of turtle cardiac muscle during hypoxia does not involve a low sensitivity of the contractile system to phosphate. In addition, the effect of increased phosphate seems to be offset by a concomitant increase in ADP. Accepted: 28 June 1999     

Sodium-hydrogen exchange and its role in controlling contractility during acidosis in cardiac muscle

Intracellular pH (pH_i) and Na (a_na ⁱ) were recorded in isolated sheep cardiac Purkinje fibres using ion-selective microelectrodes while simultaneously recording twitch tension. A fall of (pH_i) stimulated acid-extrusion via sarcolemmal Na-H exchange but the extrusion was inhibited by reducing extracellular pH (pH_o), indicating an inhibitory effect of external H ions upon the exchanger. Intracellular acidosis can reduce contraction by directly reducing myofibrillar Ca^2– sensitivity. The activation of Na-H exchange at low (pH_i) can offset this direct inhibitory effect of H^– ions since exchange-activation elevates a_na ⁱ which then indirectly elevates Ca_i ²⁺ (via Na-Ca exchange) thus tending to restore tension. This protection of contraction during intracellular acidosis can be removed if extracellular (pH_i) is also allowed to fall since, under these conditions, Na-H exchange is inhibited.     

Oxygen consumption and flight muscle activity during heating in workers and drones of Apis mellifera

Summary Instantaneous oxygen consumption, muscle potential frequency, thoracic and ambient temperature were simultaneously measured during heating in individual workers and drones of honey bees. Relationships between these parameters and effects of thoracic temperature on power input and temperature elevation were studied. Oxygen consumption increased above basal levels only when flight muscles became active. Increasing muscle potential frequencies correlated with elevated oxygen consumption and raised thoracic temperature. The difference between thoracic and ambient temperature and oxygen consumption were linearly related. Oxygen consumption per muscle potential (l O₂ · g _–1 ^thorax · MP^–1) was two-fold higher in drones than in workers. However, oxygen consumption for heating the thorax (l O₂ · g _–1 ^thorax · (T_th-T_a) · °C^–1) was nearly the same in workers and drones. Thoracic temperature affected the amount of oxygen consumed per muscle potential (R₁₀=1.5). Achieved temperature elevation per 100 MP was more temperature sensitive in drones (R₁₀=6–10) than in workers (R₁₀=3.6). Q₁₀ values for oxygen consumption were 3 in workers and 4.5–6 in drones. Muscle potential frequency decreased with a Q₁₀=1.8 in workers and 2.7 in drones. Heating behaviour of workers and drones was different. Drones generated heat less continuously than workers, and showed greater interindividual variability in predilection to heat. However, the maximal difference between ambient and thoracic temperature observed was 22 °C in drones and 14 °C in workers, indicating greater potential for drones.Abbreviations DL dorsal-longitudinal muscle - DV dorsoventral muscle - MP muscle potential - T _a ambient temperature - T _th thoracic temperature     

Role of creatine kinase in force development in chemically skinned rat cardiac muscle

The influence of phosphocreatine in the presence or absence of MgATP and MgADP was studied in Triton X-100-treated thin papillary muscles and ventricular strips of the rat heart. The pCa/tension relationships, the pMgATP/tension relationships, and the tension responses to quick length changes were analyzed. The results show three major consequences of the reduction of the phosphocreatine concentration in the presence of millimolar concentrations of the MgATP. (a) The resting tension and the maximal Ca2+-activated tension were increased, and the pCa/tension relationship was shifted toward higher pCa values and its steepness was decreased; these effects were enhanced by the inclusion of MgADP. (b) The time constant of tension recoveries after quick stretches applied during maximal activation was increased, while the extent of these recoveries was decreased. (c) The study of pMgATP/tension relationships in low Ca concentrations showed that the decrease in phosphocreatine induced a shift toward higher MgATP values with no changes in maximal rigor tension or the slope coefficient; these effects were increased by the increase in MgADP and were independent of the preparation diameter. Thus, modifications of the apparent Ca sensitivity and resting and maximal tension when phosphocreatine is decreased seem to be due to an increasing participation of rigor-like or slowly cycling cross-bridges spending more time in the attached state. These results suggest that endogenous creatine kinase is able to ensure maximal efficiency of myosin ATPase by producing a local high MgATP/MgADP ratio.     

Thymidine kinase activity in cardiac muscle during embryonic and postnatal development

Cytoplasmic thymidine kinase from cardiac muscle of the rat has been characterized. It has a pH optimum of 9.0 and a K(m) value for thymidine of 1.6mum. The sedimentation coefficient of this enzyme in sucrose gradients is 4.5S, which represents a molecular weight of approx. 69000. Thymidine kinase prepared from cardiac muscle of foetal, neonatal and adult rats is inhibited by dTTP and dTDP; there is neither inhibition nor stimulation by dTMP, dCTP, dATP, dGTP or cyclic AMP. The activity of thymidine kinase in differentiating cardiac muscle of foetal and neonatal rats declines progressively with development, reaching adult values of almost zero by the fifteenth to seventeenth day of postnatal development. This represents a 70-fold decrease in enzyme activity from 3 days before birth to 17 days after birth. The loss of thymidine kinase activity in differentiating cardiac muscle correlates temporally with the cessation of DNA biosynthesis and the loss of cytoplasmic DNA polymerase activity in this tissue.     

The effects of different types of acidosis and extracellular calcium on the mechanical activity of turtle atria

Summary The effects of acidosis and extracellular calcium were examined at 20°C in the isolated spontaneously contracting atria of the freshwater turtle (Chrysemys picta bellii). The atria were subjected to treatments of lactic acidosis, hypercapnic acidosis or chloride acidosis in the presence of both normal (2.0 mM) and high (10.0 mM) calcium, which simulated levels of acidosis and calcium observed in vivo. In all cases of acidosis, pH was reduced to 6.80 from a control pH of 7.80.All three forms of acidosis significantly depressed the force of atrial contraction. During lactic and chloride acidosis a progressive decrease in contractile force was seen, while during hypercapnic acidosis a spontaneous partial recovery was observed following an initial sharp drop in tension. Hypercapnic acidosis had the most rapid effect on contractility, while chloride had the slowest effect.Elevated levels of calcium during lactic and hypercapnic acidoses significantly moderated the negative inotropic effects of acidosis, although contractile force was still below pre-acid values. During chloride acidosis with increased [Ca], no decline in contractile force was observed compared to the control values. Each of the three types of acidoses caused a significant decrease in the frequency of the spontaneous atrial contractions but this effect was not significantly improved with acidosis plus increased [Ca].Based on the present findings and on related observations of acidosis, it appears that the fresh-water turtle is able to compensate for the negative inotropic effects on the heart of both lactic and hypercapnic acidosis, and these compensations may contribute to its remarkable tolerance to anoxia.     

Hierarchical modeling of force generation in cardiac muscle

Biomechanics and Modeling in Mechanobiology - Performing physiologically relevant simulations of the beating heart in clinical context requires to develop detailed models of the microscale force...     

Phalloidin suppresses force in nebulin-rich lamprey cardiac muscle

We assessed the effect of phalloidin, known to detach nebulin from actin in skeletal myofibrils, on the isometric force of skinned lamprey cardiac muscle, which has nebulin in amounts comparable to that in skeletal muscle. In contrast to mammalian cardiac muscle, which contains much less nebulin and reacts to phalloidin only by an increase in force, the lamprey cardiac muscle responds to phalloidin by a pronounced (～ 50%) reduction in isometric force, thereby resembling the behavior of skeletal muscle. These results support our hypothesis that nebulin detachment from actin underlies phalloidin-induced force loss and suggest a role of actin-nebulin interaction in contractile function.     

Cross-bridge versus thin filament contributions to the level and rate of force development in cardiac muscle

In striated muscle thin filament activation is initiated by Ca(2+) binding to troponin C and augmented by strong myosin binding to actin (cross-bridge formation). Several lines of evidence have led us to hypothesize that thin filament properties may limit the level and rate of force development in cardiac muscle at all levels of Ca(2+) activation. As a test of this hypothesis we varied the cross-bridge contribution to thin filament activation by substituting 2 deoxy-ATP (dATP; a strong cross-bridge augmenter) for ATP as the contractile substrate and compared steady-state force and stiffness, and the rate of force redevelopment (k(tr)) in demembranated rat cardiac trabeculae as [Ca(2+)] was varied. We also tested whether thin filament dynamics limits force development kinetics during maximal Ca(2+) activation by comparing the rate of force development (k(Ca)) after a step increase in [Ca(2+)] with photorelease of Ca(2+) from NP-EGTA to maximal k(tr), where Ca(2+) binding to thin filaments should be in (near) equilibrium during force redevelopment. dATP enhanced steady-state force and stiffness at all levels of Ca(2+) activation. At similar submaximal levels of steady-state force there was no increase in k(tr) with dATP, but k(tr) was enhanced at higher Ca(2+) concentrations, resulting in an extension (not elevation) of the k(tr)-force relationship. Interestingly, we found that maximal k(tr) was faster than k(Ca), and that dATP increased both by a similar amount. Our data suggest the dynamics of Ca(2+)-mediated thin filament activation limits the rate that force develops in rat cardiac muscle, even at saturating levels of Ca(2+).     

Electrical and mechanical activity in heart tissue of flounder and rainbow trout during acidosis

1. Twitch force and voltage across the sarcolemma were measured in heart tissue of flounder and rainbow trout. 2. For the trout heart, hypercapnia was followed by a loss of force and an action potential prolongation. 3. This was also observed for the flounder heart, but only initially. 4. About 5 min after the onset of hypercapnia, an increase in force and a shortening of the action potential occurred in the flounder heart. 5. After about 30 min of hypercapnia a decrease in force and a prolongation of the action potential slowly appeared. 6. These results can be interpreted in terms of a species-dependent effect of acidosis on the cellular Ca2+ handling and the influence of intracellular Ca2+ on the action potential.     

Cytoplasmic acidosis induces multiple conductance states in ATP-sensitive potassium channels of cardiac myocytes

We studied the effect of cytoplasmic acidosis on the ionic conducting states of ATP-sensitive potassium channels in heart ventricular cells of guinea pigs and rabbits by using a patch-clamp technique with inside-out patch configuration. Under normal conditions (pH 7.4), the channel alternated between a closed state and a main open state in the absence of nucleotides on the cytoplasmic side. As internal pH was reduced below 6.5, the single channel current manifested distinct subconductance levels. The probability of the appearance of these subconductance levels was pH dependent with a greater probability of subconductance states at lower pH. A variance-mean amplitude analysis technique revealed two subconductance levels approximately equally spaced between the main open level and the closed level (63 and 33%). A current-voltage plot of the two subconductance levels and the main level showed that they had similar reversal potentials and rectification properties. An intrinsic flickering gating property characteristic of these ATP-sensitive channels was found unchanged in the 63% subconductance state, suggesting that this subconductance state and the main conductance state share similar ion pore properties (including ion selection and block) and similar gating mechanisms. The appearance of the subconductance states decreased as ionic strength was increased, and the subconductance states were also slightly voltage dependent, suggesting an electrostatic interaction between the protons and the negative surface charge in the vicinity of the binding sites, which may be close to the inner entrance of the ion pore. Proteolytic modification of the channel on the cytoplasmic side with trypsin did not abolish the subconductance levels. External acidosis did not induce subconductance levels. These results suggest that protons bound to the negatively charged group at the inner entrance of the channel ion pore may induce conformational changes, leading to partially reduced conductance states.     

MicroRNAs in skeletal and cardiac muscle development

MicroRNAs (miRNAs) are a recently discovered class of small non-coding RNAs, which are approximately 22 nucleotides in length. miRNAs negatively regulate gene expression by translational repression and target mRNA degradation. It has become clear that miRNAs are involved in many biological processes, including development, differentiation, proliferation, and apoptosis. Interestingly, many miRNAs are expressed in a tissue-specific manner and several miRNAs are specifically expressed in cardiac and skeletal muscles. In this review, we focus on those miRNAs that have been shown to be involved in muscle development. Compelling evidences have demonstrated that muscle miRNAs play an important role in the regulation of muscle proliferation and differentiation processes. However, it appears that miRNAs are not essential for early myogenesis and muscle specification. Importantly, dysregulation of miRNAs has been linked to muscle-related diseases, such as cardiac hypertrophy. A mutation resulting in a gain-of-function miRNA target site in the myostatin gene leads to down regulation of the targeted protein in Texel sheep. miRNAs therefore are a new class of regulators of muscle biology and they might become novel therapeutic targets in muscle-related human diseases.