Effects of strophanthin K and beta-acetyldigoxin in vitro on energy transformation of myocardial contractile protein system in toxic-allergic lesions of the cardiac muscle

Strophanthin K and beta-acetyldigoxin in vitro in concentration of 10(-6) M in TAM sharply increased the force generated by isolated myocardial contractile protein system (MCPS), and normalized the work performed by the system. This was accompanied by increase of ATP internal energy release (enthalpy) intensity, while a portion of energy, dissipating into heat did not increase proportionally. The mechanical efficiency of contractile process was normalized due to beta-acetyldigoxin, and exceeded the normal level due to strophanthin K effect. Strophanthin K proved a positive effect on quantitative and qualitative economy of MCPS energy utilization, while beta-acetyldigoxin effected, on the whole, extensively.     

Effect of twitch interval duration on the contractile function of subsequent twitches in isolated rat, rabbit, and dog myocardium under physiological conditions

Many studies have shown that a change in stimulation frequency leads to altered contractility of the myocardium. However, it remains unclear what changes occur directly after a change in frequency and which ones are a result of the slow processes that lead to the altered homeostasis, which develops after a change in stimulation frequency. To distinguish the immediate from the slow responses, we assessed contractile function in two species that have distinctively different calcium (Ca(2+))-handling properties using a recently developed, randomized pacing protocol. In isolated dog and rat right ventricular trabeculae, twitch contractions at five different cycle lengths within the physiologic range of each species were randomized around a steady-state frequency. We found, in both species, that the duration of the cycle length just prior to the analyzed twitch (primary) positively correlated with the increased force of the analyzed twitch. In sharp contrast, the cycle lengths, one and two more removed from the analyzed twitch ("secondary" and "tertiary"), displayed a negative correlation with force of the analyzed twitch. In additional experiments, assessment of intracellular Ca(2+) transients in rabbit trabeculae revealed that diastolic Ca(2+) levels were closely correlated to contractile function outcome. The relative contribution of the primary cycle length was different between dog (51%) and rat (71%), whereas in neither species was a significant effect on relaxation time observed. With the use of randomized cycle lengths, we have distinguished the intrinsic response from the signaling-mediated effects of frequency-dependent activation on myofilament properties and Ca(2+) handling.     

Mechanical control of the rising phase of contraction of frog skeletal and cardiac muscle

The effect of shortening on contractile activity was studied in experiments in which shortening during the rising phase of an isotonic contraction was suddenly stopped. At the same muscle length and the same time after stimulation the rise in tension was much faster, if preceded by shortening, than during an isometric contraction, demonstrating an increase in contractile activity. In this experiment the rate of tension rise determined in various phases of contraction was proportional to the rate of isotonic shortening at the same time after stimulation. Therefore, the time course of the isotonic rising phase could be derived from the tension rise after shortening. The rate of isotonic shortening was found to be unrelated to the tension generated at various lengths and to correspond closely to the activation process induced by shortening. The length response explains differences between isotonic and isometric contractions with regard to energy release (Fenn effect) and time relations. These results extend previous work which showed that shortening during later phases of a twitch prolongs, while lengthening abbreviates contraction. Thus the length responses, which have been called shortening activation and lengthening deactivation, control activity throughout an isotonic twitch.     

Activation of phosphorylase in frog muscle as determined by contractile activity

The state of activation of phosphorylation in muscle has been reinvestigated by combining the extraction procedures of Danforth, Helmreich, and Cori with the low-temperature techniques of this laboratory. In resting frog muscle, the phosphorylase-alpha content is usually below detectability. Upon contractile activity in series of twitches, activation of phosphorylase beta to alpha took place, without activation of phosphorylase beta kinase as defined by the assay procedure. Two different experimental designs were used to examine the relation between phosphorylase activation and the myothermally determined energy turnover per twitch, and these showed, identically, that the enzyme activation is proportional to the energy per twitch.     

Histochemical and contractile responses of rat medial gastrocnemius to 2 weeks of complete disuse.

We studied the histochemical and in situ contractile changes in a rat ankle extensor, medial gastrocnemius, in which activation of muscle fibres by motoneurones was blocked for 14 days, using the sodium channel blocker tetrodotoxin applied to the sciatic nerve. Muscles were atrophied and showed slower twitch responses, greater fusion at subtetanic frequencies of stimulation, and higher twitch/tetanic ratios. Tetanic force/mm2 of fibre area and fatiguability were unchanged. Type II fibres were more atrophied and showed greater decreases in mitochondrial succinate dehydrogenase activity than type I fibres. The contractile changes resulting from complete disuse do not occur in models in which weight-bearing alone has been removed (space flight, hindlimb suspension), suggesting that the residual motoneurone activity reported in models of weightlessness is sufficient to prevent these responses. Similarly, the finding of a greater type II fibre susceptibility to complete disuse, which differs from the pattern seen in models of weightlessness, suggest that this residual motoneurone activity in the latter influences atrophic responses in a manner that is variable among motor unit types, to produce the reported preferential type I atrophy characteristic of removal of weight-bearing.     

Kinetics and compartmentation of energy metabolism in intact skeletal muscle determined from 18O labeling of metabolite phosphoryls

Analyses of isolated intact diaphragm muscle show that at rest only about 30% of the total cellular Pi is metabolically reactive as indicated by 18O incorporation from [18O]water, whereas up to 90% becomes metabolically active incrementally with contractile frequency. Kinetics of [gamma-18O]ATP appearance show that about 90% of the cellular ATP is metabolically active and suggest slowly and rapidly metabolizing compartments of ATP in resting muscle and only rapidly metabolizing compartments in contracting muscle. Rates of [18O]creatine phosphate [( 18O]CrP) appearance are consistent with creatine kinase-catalyzed phosphoryl exchange functioning in an obligatory phosphoryl shuttle system. In noncontracting muscle, ATP turnover rate was 83 nmol.mg protein-1.min-1, and the P/O ratio was determined to be 3.2. ATP utilization increases in direct proportion to contractile frequency with each contracture consuming the equivalent of 0.96 nmol of ATP.mg protein-1 or 2.5-3.5 molecules of ATP/myosin active site. Basal concentrations of nucleotide polyphosphates are not altered when ATP utilization rates increase during contraction. At high contractile frequencies, decreases in CrP concentration occur, but this accounts for less than 4% of total high energy phosphoryls consumed. If metabolic intermediates are free in the aqueous cellular cytosol, each twitch contracture would result in a decrease in ATP concentration of no more than 2% and increases in ADP and AMP concentrations of less than 20 and 7%, respectively. Thus, changes in metabolite concentration must be highly localized or metabolic regulation can be accomplished by a nonallosteric mechanism.     

Effects of previous activity on the energetics of activation in frog skeletal muscle

Effects of previous activity on the ability of frog skeletal muscle at 0 degrees C to liberate energy associated with contractile activation, i.e., activation heat (AH), have been examined. Earlier work suggests that activation heat amplitude (as measured from muscles stretched to lengths where active force development is nearly abolished) is related to the amount of Ca2+ released upon stimulation. After a twitch, greater than 2 s is required before a second stimulus (AHt) can liberate the same activation heat as a first stimulus (AH infinity), i.e., (AHt)/(AH infinity) = 1 -0.83 e-1.40t, where t is time in seconds. Caffeine introduces a time delay in the recovery of the ability to generate activation heat after a twitch. After a tetanus, the activation heat is depressed to a greater extent at any time than after a twitch. The activation heat elicited by a stimulus 1 s after a tetanus is depressed progressively with respect to tetanus duration up to 3 s. For tetani of 3, 40, and 80 s duration the postetanus activation heat is comparably depressed. The time-course of the recovery of the ability of the muscle to produce activation heat after a tetanus can be described as (AHt)/(AH infinity) = 1 -0.80 e-0.95t - 0.20 e-0.02t. Greater than 90 s is required before the posttetanus activation heat is equal to the pretetanus value. The faster phase of recovery is similar to recovery after the twitch and the slower phase may be associated with the return of calcium to the terminal cisternae from uptake sites in the longitudinal sarcoplasmic reticulum.     

Ion channels from chemosensory olfactory neurons

The olfactory epithelium has the ability to respond to a large number of volatile compounds of small molecular weight. Ultimately, such a property lies on a specialized type of neuron, the olfactory receptor cell. In the presence of odorants, the olfactory receptor neuron responds with action potentials whose frequency depends on odorant concentration. The primary events in the process of olfactory transduction are thought to occur at the cilia of olfactory receptor neurons and involve the binding of odorants to receptor molecules followed by the opening of ion channels. A crucial step in understanding olfactory transduction requires identifying the mechanisms that regulate the electrical activity of olfactory cells. In the last couple of years, patch-clamp recording from isolated olfactory cells and reconstitution of olfactory membranes in planar lipid bilayers have begun to shed light on some of these mechanisms. Although the information emerging from such studies is still preliminary, there are already well-defined hypotheses on the molecular events that might underlie the primary events in olfactory transduction. Currently, attention is being focused on the notions that second messengers might be involved in the activation of ion channels in olfactory cilia, and that odorant binding to a receptor molecule might lead directly to the gating of ion channels in chemosensory olfactory membranes. The coming years promise to be exciting ones in the field of olfactory transduction. We have now the necessary tools to be able to confront hypotheses and experimental facts.     

Using NMR Metabolomics to Investigate Tricarboxylic Acid Cycle-dependent Signal Transduction in Staphylococcus epidermidis

Staphylococcus epidermidis is a skin-resident bacterium and a major cause of biomaterial-associated infections. The transition from residing on the skin to residing on an implanted biomaterial is accompanied by regulatory changes that facilitate bacterial survival in the new environment. These regulatory changes are dependent upon the ability of bacteria to “sense” environmental changes. In S. epidermidis, disparate environmental signals can affect synthesis of the biofilm matrix polysaccharide intercellular adhesin (PIA). Previously, we demonstrated that PIA biosynthesis is regulated by tricarboxylic acid (TCA) cycle activity. The observations that very different environmental signals result in a common phenotype (i.e. increased PIA synthesis) and that TCA cycle activity regulates PIA biosynthesis led us to hypothesize that S. epidermidis is “sensing” disparate environmental signals through the modulation of TCA cycle activity. In this study, we used NMR metabolomics to demonstrate that divergent environmental signals are transduced into common metabolomic changes that are “sensed” by metabolite-responsive regulators, such as CcpA, to affect PIA biosynthesis. These data clarify one mechanism by which very different environmental signals cause common phenotypic changes. In addition, due to the frequency of the TCA cycle in diverse genera of bacteria and the intrinsic properties of TCA cycle enzymes, it is likely the TCA cycle acts as a signal transduction pathway in many bacteria.     

Diversity of mechanical responses and their possible underlying mechanisms in the proboscis muscles ofBusycon canaliculatum

Summary Mechanical responses of the radular protractor and retractor, the odontophore retractor and the radular sac muscles ofBusycon canaliculatum were compared. The radular protractor responded to both ACh and high K salines with similar slow, smooth contractures showing no evidence of fast twitch activity. The radular sac, odontophore retractor, and radular retractor muscles responded to low K salines with bursts of fast twitches at a mechanical threshold below that for responses in the radular protractor. With high K salines these three muscles showed inactivation of fast twitch activity and replacement by slow maintained tonic force. With rare exceptions, the ACh responses of all four muscles consisted of slow, maintained tonic contractures with no fast twitch activity, although individual muscles differed in their ACh sensitivity. A scheme is presented to explain the mechanical modus operandi of this complex organ by the co-operative actions of these four physiologically diverse muscles. It is proposed that fast twitch responses depend upon the activity of fast transient Ca channels showing strong voltage sensitivity and ready voltage inactivation. It is proposed that maintained tonic contractures in all the muscles depends upon the activity of slow long-lasting voltage-dependent Ca channels which only open with substantial membrane depolarization. It is suggested that K-induced and ACh-induced responses may activate a similar cellular Ca pool but by different membrane transduction routes.     

Effect of ionophore A23187 on the strength of contraction and transmembrane action potential of guinea pig papillary muscle]

Calcium ionophore A23187 being added at 2.5 mM concentration induced 2--3-fold increase of peak contractile force of isolated guinea pig papillary muscle stimulated with the frequency of 0.2 Hz. The increase of force of contraction is not followed by any change in amplitude and duration of membrane action potential. A23187 decreases testing tension as well as duration of the twitch and increases relaxation rate of the muscle. This phenomenon probably reflects increased capability of sarcoplasmic reticulum in the process of calcium ion accumulation. In the presence of ionophore appearance of shoulder on the rising part of the twitch has been registered. In this case the single twitch being differentiated reveals two positive maxima. Addition of D-600 leads to decrease of action potential duration and eliminates the second part of the twitch, the amplitude and the velocity of the first phase of contraction being without change. It was proposed that the first component of the twitch appearing in the presence of A23187 and insensitive to D-600 corresponds to the release of calcium from some intracellular stores.     

Effect of free fatty acids on the heart: effects of nonanoic acid on contractility and postextrasystolic potentiation in the rat papillary muscle

1. Nonanoic acid causes a depression in the contractile force of rat papillary muscles working isometrically. The effect depends on both the concentration of the fatty acid (0.1-1 mM) and the time of exposure (2-12 min), and shows a nearly complete reversibility. 2. Vmax-values derived from the force-velocity relation as well as the index of relation suggested by Meerson do not change in muscles exposed to nonanoic acid. During paired pulse stimulation with a 400 ms-delay of the second impulse, both the postextrasystolic potentiation and the time to reach a new steady level in the contraction amplitude are significantly increased. The speed of restituting the contraction after a twitch (resitution) is descreased. 4. The results suggest that the action site of nonanoic acid may be the excitation-contraction coupling system (including the action potential) rather than the contractile element or the relaxation of the muscle.     

Grape seed proanthocyanidin extract promotes skeletal muscle fiber type transformation via AMPK signaling pathway

Transformation of skeletal muscle fiber type from fast twitch to slow twitch has significances for sustained contractile and stretchable events, energy homeostasis and antifatigue ability. However, the regulation of skeletal muscle fiber type transformation through nutritional intervention is still not fully spelled out. Grape seed proanthocyanidin extract (GSPE) has been widely reported to play a broader role in many aspects of diseases with its various pharmacological and health-promoting effects. In this study, we found that GSPE significantly improved the fatigue resistance in mice. GSPE up-regulated slow myosin heavy chain (MyHC) and down-regulated fast MyHC, accompanied by increases in activities of succinic dehydrogenase and malate dehydrogenase and by decreased lactate dehydrogenase activity in muscle of mice and in C2C12 myotubes. The AMP-activated protein kinase (AMPK) signaling can be activated by GSPE. Several upstream and downstream factors of AMPK signaling such as liver kinase B1, nuclear respiratory factor 1, calcium calmodulin-dependent protein kinase kinase β, sirtuin1 and peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α) were also up-regulated by GSPE. Specific inhibition of AMPK signaling by AMPK inhibitor compound C or by AMPKα1 siRNA significantly abolished the GSPE-induced the activation of AMPK and the increase of PGC-1α, and attenuated the GSPE-induced increase of slow MyHC and decrease of fast MyHC in C2C12 myotubes. Taken together, we revealed that GSPE promotes skeletal muscle fiber type transformation from fast twitch to slow twitch through AMPK signaling pathway, and this GSPE-induced fiber type transformation may contribute to increased fatigue resistance.     

Maintained contractile reserve in a transgenic mouse model of myocardial stunning

Cardiac excitation-contraction (E-C) coupling is impaired at the myofilament level in the reversible postischemic dysfunction known as "stunned" myocardium. We characterized tension development and calcium cycling in intact isolated trabeculae from transgenic (TG) mice expressing the major proteolytic degradation fragment of troponin I (TnI) found in stunned myocardium (TnI(1-193)) and determined the ATPase activity of myofibrils extracted from TG and non-TG mouse hearts. The phenotype of these mice at baseline recapitulates that of stunning. Here, we address the question of whether contractile reserve is preserved in these mice, as it is in genuine stunned myocardium. During twitch contractions, calcium cycling was normal, whereas tension was greatly reduced, compared with non-TG controls. A decrease in maximum Ca2+-activated tension and Ca2+ desensitization of the myofilaments accounted for this contractile dysfunction. The decrease in maximum tension was paralleled by an equivalent decrease in maximum Ca2+-activated myofibrillar ATPase activity. Exposure to high calcium or isoproterenol recruited a sizable contractile reserve in TG muscles, which was proportionately similar to that in control muscles but scaled downward in amplitude. These results suggest that calcium regulatory pathways and beta-adrenergic signal transduction remain intact in isolated trabeculae from stunned TG mice, further recapitulating key features of genuine stunned myocardium.     

Do mental events cause neural events analogously to the probability fields of quantum mechanics?

If non-material mental events, such as the intention to carry out an action, are to have an effective action on neural events in the brain, it has to be at the most subtle and plastic level of these events. In the first stage of our enquiry an introduction to conventional synaptic theory leads on to an account of the manner of operation of the ultimate synaptic units. These units are the synaptic boutons that, when excited by an all-or-nothing nerve impulse, deliver the total contents of a single synaptic vesicle, not regularly, but probabilistically. This quantal emission of the synaptic transmitter molecules (about 5000-10 000) is the elementary unit of the transmission process from one neuron to another. In the second stage this refined physiological analysis leads on to an account of the ultrastructure of the synapse, which gives clues as to the manner of its unitary probabilistic operation. The essential feature is that the effective structure of each bouton is a paracrystalline presynaptic vesicular grid with about 50 vesicles, which acts probabilistically in vesicular (quantal) release. In the third stage it is considered how a non-material mental event, such as an intention to move, could influence the subtle probabilistic operations of synaptic boutons. On the biological side, attention is focused on the paracrystalline presynaptic vesicular grids as the targets for non-material mental events. On the physical side, attention is focused on the probabilistic fields of quantum mechanics which carry neither mass nor energy, but which nevertheless can exert effective action at microsites. The new light on the mind-brain problem came from the hypothesis that the non-material mental events, the 'World 2' of Popper, relate to the neural events of the brain (the 'World 1' of matter and energy) by actions in conformity with quantum theory. This hypothesis that mental events act on probabilistic synaptic events in a manner analogous to the probability fields of quantum mechanics seems to open up an immense field of scientific investigation both in quantum physics and in neuroscience.     

Initial force production before sag is enhanced by prior contraction followed by a 3-minute rest period in fast motor units of the rat medial gastrocnemius

Unfused tetanic contractions evoked in fast motor units exhibit extra-efficient force production at the onset of contraction, an effect called “boost”. Boost is diminished in subsequent contractions if there is a short rest period between contractions, but can be re-established with a longer period of rest. We tested the hypothesis that contractile activity and rest could enhance boost-related metrics. Two sets of 3 unfused tetani were evoked 3 min apart in fast fatigable (FF) and fast fatigue-resistant (FR) motor units of the rat medial gastrocnemius. The greatest changes occurred in the first unfused tetanic contractions. Relative to the first contraction in the first set, the first contraction in the second set exhibited higher peak force during boost in a subset of motor units (76% of FF and 48% of FR). Enhanced force during boost was influenced by interaction of slowing of twitch contraction time (up to 20% and 25%, for FF and FR motor units, respectively), half-relaxation time (up to 37% and 49% for FF and FR motor units, respectively), and potentiation of the first twitch (up to 13% and 5% for FF and FR motor units, respectively). Examination of twitches evoked between sets suggested opportunity for greater enhancement of boost with shorter intervening rest periods. The phenomenon of enhanced boost following motor unit activity may interest sports scientists.     

Proteomic profiling of chronic low-frequency stimulated fast muscle

Skeletal muscle fibre transitions occur in many biological processes, in response to alterations in neuromuscular activity, in muscular disorders, during age-induced muscle wasting and in myogenesis. It was therefore of interest to perform a comprehensive proteomic profiling of muscle transformation. Chronic low-frequency stimulation of the rabbit tibialis anterior muscle represents an established model system for studying the response of fast fibres to enhanced neuromuscular activity under conditions of maximum activation. We have conducted a DIGE analysis of unstimulated control specimens versus 14- and 60-day conditioned muscles. A differential expression pattern was observed for 41 protein species with 29 increased and 12 decreased muscle proteins. Identified classes of proteins that are changed during the fast-to-slow transition process belong to the contractile machinery, ion homeostasis, excitation-contraction coupling, capillarization, metabolism and stress response. Results from immunoblotting agreed with the conversion of the metabolic, regulatory and contractile molecular apparatus to support muscle fibres with slower twitch characteristics. Besides confirming established muscle elements as reliable transition markers, this proteomics-based study has established the actin-binding protein cofilin-2 and the endothelial marker transgelin as novel biomarkers for evaluating muscle transformation.     

The efficiency of muscle contraction

When a muscle contracts and shortens against a load, it performs work. The performance of work is fuelled by the expenditure of metabolic energy, more properly quantified as enthalpy (i.e., heat plus work). The ratio of work performed to enthalpy produced provides one measure of efficiency. However, if the primary interest is in the efficiency of the actomyosin cross-bridges, then the metabolic overheads associated with basal metabolism and excitation-contraction coupling, together with those of subsequent metabolic recovery process, must be subtracted from the total heat and work observed. By comparing the cross-bridge work component of the remainder to the Gibbs free energy of hydrolysis of ATP, a measure of thermodynamic efficiency is achieved. We describe and quantify this partitioning process, providing estimates of the efficiencies of selected steps, while discussing the errors that can arise in the process of quantification. The dependence of efficiency on animal species, fibre-type, temperature, and contractile velocity is considered. The effect of contractile velocity on energetics is further examined using a two-state, Huxley-style, mathematical model of cross-bridge cycling that incorporates filament compliance. Simulations suggest only a modest effect of filament compliance on peak efficiency, but progressively larger gains (vis-à-vis the rigid filament case) as contractile velocity approaches Vmax. This effect is attributed primarily to a reduction in the component of energy loss arising from detachment of cross-bridge heads at non-zero strain.     

Quantal release of ATP in mouse cortex

Transient currents occur at rest in cortical neurones that reflect the quantal release of transmitters such as glutamate and gamma-aminobutyric acid (GABA). We found a bimodal amplitude distribution for spontaneously occurring inward currents recorded from mouse pyramidal neurones in situ, in acutely isolated brain slices superfused with picrotoxin. Larger events were blocked by glutamate receptor (AMPA, kainate) antagonists; smaller events were partially inhibited by P2X receptor antagonists suramin and PPADS. The decay of the larger events was selectively prolonged by cyclothiazide. Stimulation of single intracortical axons elicited quantal glutamate-mediated currents and also quantal currents with amplitudes corresponding to the smaller spontaneous inward currents. It is likely that the lower amplitude spontaneous events reflect packaged ATP release. This occurs with a lower probability than that of glutamate, and evokes unitary currents about half the amplitude of those mediated through AMPA receptors. Furthermore, the packets of ATP appear to be released from vesicle in a subset of glutamate-containing terminals.     

The effect of audiogenic seizures on regional CNS energy reserves, glycolysis and citric acid cycle flux

Selected energy reserves, glycolytic intermediates and citric acid cycle intermediates were measured in the cerebral cortex, thalamus, brain stem, cerebellum and spinal cord of susceptible mice during audiogenic seizures. Changes in energy reserves (ATP, phosphocreatine and glucose) differed strikingly in extent and temporal pattern from region to region. The audiogenic seizure produced a transient, large decrease in thalamic energy reserves during the early, pretonic phase of the seizure. Less extensive decreases were observed in brain stem and spinal cord; but in these latter regions the changes persisted throughout the pretonic and tonic phases of the seizures. In cerebellum there was a biphasic decrease in energy reserves; a small decrease was observed immediately after the sound stimulus and a second much greater decrease was observed during the tonic phase of the seizure. No change in energy reserves was observed in cerebral cortex. Changes in glycolytic intermediates (glucose 6-phosphate, fructose diphosphate, pyruvate and lactate) also varied from region to region in response to the decreases in energy reserves. In contrast, changes in the two citric acid cycle intermediates, α-oxoglutarate and malate, were essentially the same in all regions studied. α-Oxoglutarate decreased during the tonic phase of the seizure and rose during recovery. Malate remained at control levels throughout the seizure and then slowly increased. These findings are interpreted as indicating regional variations in nueronal activity during audiogenic seizures. During the period when clinical seizure activity is apparent neuronal activity increases in the subcortical regions. This is reflected by an increase in energy utilization and an increase in glycolytic flux in these areas. However, a concomitant increase in citric acid cycle flux does not seem to occur during this period. Citric acid cycle flux does appear to increase after the seizure is over.