Excitation-contraction Coupling Pathway Research Articles

Fundamental constraints in synchronous muscle limit superfast motor control in vertebrates.

Superfast muscles (SFMs) are extremely fast synchronous muscles capable of contraction rates up to 250 Hz, enabling precise motor execution at the millisecond time scale. SFM phenotypes have been discovered in most major vertebrate lineages, but it remains unknown whether all SFMs share excitation-contraction coupling pathway adaptations for speed, and if SFMs arose once, or from independent evolutionary events. Here, we demonstrate that to achieve rapid actomyosin crossbridge kinetics bat and songbird SFM express myosin heavy chain genes that are evolutionarily and ontologically distinct. Furthermore, we show that all known SFMs share multiple functional adaptations that minimize excitation-contraction coupling transduction times. Our results suggest that SFM evolved independently in sound-producing organs in ray-finned fish, birds, and mammals, and that SFM phenotypes operate at a maximum operational speed set by fundamental constraints in synchronous muscle. Consequentially, these constraints set a fundamental limit to the maximum speed of fine motor control.

TGF-β1 Evokes Human Airway Smooth Muscle Cell Shortening and Hyperresponsiveness via Smad3.

Transforming growth factor β1 (TGF-β1), a cytokine whose levels are elevated in the airways of patients with asthma, perpetuates airway inflammation and modulates airway structural cell remodeling. However, the role of TGF-β1 in excessive airway narrowing in asthma, or airway hyperresponsiveness (AHR), remains unclear. In this study, we set out to investigate the direct effects of TGF-β1 on human airway smooth muscle (HASM) cell shortening and hyperresponsiveness. The dynamics of AHR and single-cell excitation-contraction coupling were measured in human precision-cut lung slices and in isolated HASM cells using supravital microscopy and magnetic twisting cytometry, respectively. In human precision-cut lung slices, overnight treatment with TGF-β1 significantly augmented basal and carbachol-induced bronchoconstriction. In isolated HASM cells, TGF-β1 increased basal and methacholine-induced cytoskeletal stiffness in a dose- and time-dependent manner. TGF-β1-induced single-cell contraction was corroborated by concomitant increases in myosin light chain and myosin phosphatase target subunit 1 phosphorylation levels, which were attenuated by small interfering RNA-mediated knockdown of Smad3 and pharmacological inhibition of Rho kinase. Strikingly, these physiological effects of TGF-β1 occurred through a RhoA-independent mechanism, with little effect on HASM cell [Ca2+]i levels. Together, our data suggest that TGF-β1 enhances HASM excitation-contraction coupling pathways to induce HASM cell shortening and hyperresponsiveness. These findings reveal a potential link between airway injury-repair responses and bronchial hyperreactivity in asthma, and define TGF-β1 signaling as a potential target to reduce AHR in asthma.

The different role of sex hormones on female cardiovascular physiology and function: not only oestrogens.

Human response to different physiologic stimuli and cardiovascular (CV) adaptation to various pathologies seem to be gender specific. Sex-steroid hormones have been postulated as the major contributors towards these sex-related differences. This review will discuss current evidence on gender differences in CV function and remodelling, and will present the different role of the principal sex-steroid hormones on female heart. Starting from a review of sex hormones synthesis, receptors and CV signalling, we will summarize the current knowledge concerning the role of sex hormones on the regulation of our daily activities throughout the life, via the modulation of autonomic nervous system, excitation-contraction coupling pathway and ion channels activity. Many unresolved questions remain even if oestrogen effects on myocardial remodelling and function have been extensively studied. So this work will focus attention also on the controversial and complex relationship existing between androgens, progesterone and female heart.

Introduction: Myometrial physiology – time to translate?

Introduction: Myometrial physiology – time to translate?

Effect of age on cardiac excitation–contraction coupling

1. Cardiovascular diseases most commonly occur in the elderly and are a frequent cause of disability or death. However, the effect of age itself on cardiac function is not well understood. 2. Studies in both human and animal hearts indicate that contractile function is unaffected by age while at rest. However, the ability to increase cardiac contractile force during strenuous activities, such as exercise, declines with age. 3. Similar findings have been observed in individual ventricular myocytes isolated from aged hearts. When myocytes are stimulated with beta-adrenoceptor agonists or rapid pacing frequencies, aged cells show a much smaller increase in peak contractions and Ca(2+) transients than young adult cells. In addition, contractions and Ca(2+) transients are prolonged in aged cells compared with younger cells under these conditions. 4. These observations suggest that the age-related decline in cardiac contractile function originates at the cellular level and may reflect modifications in processes involved in excitation-contraction (EC) coupling. 5. Biochemical studies have shown that there are age-related modifications in the expression, regulation and function of a number of proteins essential to EC coupling in the heart. 6. Functional studies indicate that these changes in EC coupling proteins disrupt Ca(2+) homeostasis and contribute to decrease in peak contraction and prolongation of contraction duration observed in myocytes from aged hearts. 7. The present review describes modifications in cardiac contractile function that occur in the ageing heart and evaluates underlying alterations in the EC coupling pathway that may be responsible for this decline in contractile function in ageing.

Signal-Transduction Pathways that Regulate Visceral Smooth Muscle Function III. Coupling of muscarinic receptors to signaling kinases and effector proteins in gastrointestinal smooth muscles

Stimulation of muscarinic M3 and M2 receptors on gastrointestinal smooth muscle elicits contraction via activation of G proteins that are coupled to a diverse set of downstream signaling pathways and effector proteins. Many studies suggest a canonical excitation-contraction coupling pathway that includes activation of phospholipases, production of inositol 1,4,5-trisphosphate and diacylglycerol, release of calcium from the sarcoplasmic reticulum, activation of L-type calcium channels, and activation of nonselective cation channels. These events lead to elevated intracellular calcium concentration, which activates myosin light chain kinase to phosphorylate and activate myosin II thus causing contraction. In addition, muscarinic receptors are coupled to signaling pathways that modulate the effect of activator calcium. The Rho/Rho kinase pathway inhibits myosin light chain phosphatase, one of the key steps in sensitization of the contractile proteins to calcium. Phosphatidylinositol 3-kinases and Src family tyrosine kinases are also activated by muscarinic agonists. Src family tyrosine kinases regulate L-type calcium and nonselective cation channels. Src activation also leads to activation of ERK and p38 MAPKs. ERK MAPKs phosphorylate caldesmon, an actin filament binding protein. P38 MAPKs activate phospholipases and MAPKAP kinase 2/3, which phosphorylate HSP27. HSP27 may regulate cross-bridge function, actin filament formation, and actin filament attachment to the cell membrane. In addition to the well-known role of M3 muscarinic receptors to regulate myoplasmic calcium levels, the integrated effect of muscarinic activation probably also includes signaling pathways that modulate phospholipases, cyclic nucleotides, contractile protein function, and cytoskeletal protein function.

Effects of PP1/PP2A inhibitor calyculin A on the E-C coupling cascade in murine ventricular myocytes.

Calyculin A was used to examine the importance of phosphatases in the modulation of cardiac contractile magnitude in the absence of any neural or humoral stimulation. Protein phosphatase (PP)1 and PP2A activity, twitch contractions, intracellular Ca(2+) concentration ([Ca(2+)](i)) transients, action potentials, membrane currents, and myofilament Ca(2+) sensitivity were measured in isolated mouse ventricular myocytes. Calyculin A (125 nM) inhibited PP1 and PP2A by 50% and 85%, respectively, whereas it doubled the twitch magnitude and increased twitch duration by 50% in field-stimulated cells. Calyculin A-evoked increases in L-type Ca(2+) current (70%) and the resulting [Ca(2+)](i) transient (83%) explain the positive inotropic response. However, increases in twitch and action potential durations did not result from increased myofilament Ca(2+) sensitivity or K(+) current inhibition, respectively. Comparison of the effects of calyculin A and isoproterenol on [Ca(2+)](i) transients and twitch contractions revealed that calyculin A had a much smaller lusitropic effect than the beta-agonist, indicating that calyculin A did not significantly increase sarcoplasmic reticulum Ca(2+) reuptake. Thus while cardiac contractile magnitude is controlled by a steady-state kinase/phosphatase balance, this regulation is not equally operative at all of the steps in the excitation-contraction coupling pathway and may in fact be most important to the regulation of the L-type Ca(2+) channel.

Antioxidants prevent ethanol-induced contractions of canine cerebral vascular smooth muscle: relation to alcohol-induced brain injury

The present study was designed to test the hypothesis that α-tocopherol (Vit. E) and pyrrolidine dithiocarbamate (PDTC) might exert direct effects on alcohol-induced contractions of canine basilar cerebral arteries. After precontraction of arterial ring segments with ethanol, PDTC (10 −8–10 −6 M) and Vit. E (10 −6–10 −4 M) induced concentration-dependent relaxations of cerebral arteries, compared to untreated controls. The effective concentrations producing approximately 50% of the maximal relaxation responses (EC 50 values) were about 2.48±0.09 × 10 −7 M for PDTC, and 1.87±0.10 × 10 −5 mM for Vit. E, respectively. Preincubation of these arterial rings with EC 50’s of PDTC or Vit. E for 40 min attenuate markedly the contractions produced by alcohol, at concentrations of 1–400 mM. However, both PDTC and Vit.E do not relax equi-potent precontractions induced by either KCl or prostaglandin F 2α (PGF 2 α ) or inhibit their contractions. These data suggest that alcohol-induced contractions of cerebral arteries are mediated via excitation-contraction coupling pathways different from those used by KCl or receptor-mediated agonists such as PGF 2 α . The present results, when viewed in light of other recently published data, suggest that antioxidants may prove useful in the amelioration and treatment of alcohol-induced brain damage and strokes.

Ethanol and cocaine cause additive inhibitory effects on the calcium transients and contraction in single cardiomyocytes.

The heart is a major locus for the toxic actions of cocaine and ethanol, each of which has been shown to interfere with excitation-contraction coupling in cardiac muscle cells. Because these drugs are frequently used in combination, the present study was designed to investigate how they interact to modify the Ca2+ transient and associated contraction in fura2-loaded cardiomyocytes. A high-speed imaging technique using a charge-coupled device as detector and short-term image store was used to measure cytosolic Ca2+ and contraction simultaneously from fluorescence images obtained during the contractile cycle. Ethanol (100 mM) and cocaine (50 microM) caused reversible reductions in Ca2+ transient amplitude of 24.3 +/- 3.0% and 25.1 +/- 3.6%, respectively. Neither agent modified basal Ca2+. Ethanol treatment decreased peak shortening by 44.3 +/- 3.5%, whereas the contractile depression by cocaine was 31.4 +/- 5.3%. The relatively greater effect of ethanol on contraction resulted from a Ca2+-independent component of ethanol action on contractility. When cardiomyocytes were exposed simultaneously to ethanol and cocaine, Ca2+ transient amplitude was reduced by 38.7 +/- 3.0%, and peak contraction was decreased by 55.1 +/- 3.5%. These values represent a significantly greater inhibition than observed with either drug alone (p < 0.02) and are compatible with additive effects of the two drugs acting at distinct loci within the excitation-contraction coupling pathway. Thus, simultaneous use of cocaine and ethanol leads to an enhanced depression of myocardial contractility, which is likely to contribute to the cardiotoxic actions of the combination of these two drugs.

Ketamine, at clinical concentrations, does not alter the function of cardiac sarcoplasmic reticulum calcium release channels.

In the absence of sympathetically mediated stimulation, ketamine depresses myocardial contractility. This results from a decrease in the availability of intracellular Ca2+ for excitation-contraction coupling. Although sites of action other than the Ca2+ release channel of sarcoplasmic reticulum have been implicated, ketamine-induced alterations in Ca2+ efflux from the sarcoplasmic reticulum remain contentious. The purpose of the present study was to identify interactions of ketamine with the calcium release channel using sarcoplasmic reticulum enriched vesicles from porcine left ventricle. Ketamine did not alter [3H]ryanodine binding at concentrations of 1 mM or less, while binding was almost completely inhibited at 10 mM. Gating and conductance of SR Ca2+ channels studied in planar bilayers was not altered by clinical concentrations of ketamine over the range of physiologic cytoplasmic free Ca2+ concentrations. Channel inactivation was observed at 10 mM ketamine, well in excess of clinical concentrations. These findings indicate that clinical concentrations of ketamine do not alter the function of the Ca2+ release channel. Alterations in intracellular Ca2+ homeostasis that result in depression of myocardial contractility must therefore result from effects at other sites along the excitation-contraction coupling pathway.

Development of the excitation-contraction coupling apparatus in skeletal muscle: peripheral and internal calcium release units are formed sequentially.

The development of calcium release units and of transverse tubules has been studied in skeletal muscle fibres from embryonal and newborn chicken. Three constituents of calcium release units: the tetrads, the feet and an internal protein directly associated with junctional surface of the sarcoplasmic reticulum are visualized by various electron microscope techniques. Evidence in the literature indicates that the three components correspond to the voltage sensors, the sarcoplasmic reticulum calcium release channels and the calcium binding protein calsequestrin respectively. We recognize two stages at which important events in membrane morphogenesis occur. The first stage coincides with early myofibrillogenesis (starting at approximately embryonal day E5.5), and it involves the assembly of calcium release units at the periphery of the muscle fibre in which feet and the internal protein are identified. Groups of tetrads also are present at very early stages and their disposition indicates a relation to the feet of peripheral couplings. Thus three major components of the excitation-contraction coupling pathway are in place as soon as myofibrils develop. The density of groups of tetrads in the surface membrane of primary and secondary fibres is similar, despite differences in developmental stages. The second stage involves the formation of a complex transverse tubule network and of internal sarcoplasmic reticulum-transverse tubule junctions, while peripheral couplings disappear. This stage starts abruptly (between E15 and E16) and simultaneously in primary and secondary fibres. It coincides with the myotube-to-myofibre transition. The two stages are separated by a relatively long intervening period (between E9 and E16). During the latter part of this period some primitive transverse tubules appear, and form junctions with the sarcoplasmic reticulum, but they remain strictly located at the periphery of the fibre and are not numerous. Finally, after the second stage there is a prolonged (up to 4 weeks) period of maturation, during which density of free sarcoplasmic reticulum increases, triads acquire a location at the A-I junction and fibre type differences appear. We conclude that a system for calcium uptake, storage and release exists at the periphery of the myotube during early myogenesis. The complexity of the system and its ability to deliver calcium through the entire fibre develop in parallel to the formation of myofibrils.

The role of calcium in the control of vascular tone as assessed by the Ca2+ indicator aequorin

An overview is given of the current understanding of the mechanism of contraction of vascular smooth muscle. The regulation of vascular tone by intracellular ionized calcium levels appears to occur primarily through calcium-dependent phosphorylation of the myosin light chains by the enzyme myosin light chain kinase. Evidence is presented that additional relatively calcium-independent processes also exist and contribute to the regulation of vascular tone. A scheme is presented whereby vasoconstriction may occur in the absence of any change in cytoplasmic ionized calcium levels. The multiplicity of excitation-contraction coupling pathways in vascular smooth muscle predicts a multitude of rational therapeutic approaches to vascular pathologies.

Effects of changes of pH on the contractile function of cardiac muscle

It has been known for over 100 years that acidosis decreases the contractility of cardiac muscle. However, the mechanisms underlying this decrease are complicated because acidosis affects every step in the excitation-contraction coupling pathway, including both the delivery of Ca2+ to the myofilaments and the response of the myofilaments to Ca2+. Acidosis has diverse effects on Ca2+ delivery. Actions that may diminish Ca2+ delivery include 1) inhibition of the Ca2+ current, 2) reduction of Ca2+ release from the sarcoplasmic reticulum, and 3) shortening of the action potential, when such shortening occurs. Conversely, Ca2+ delivery may be increased by the prolongation of the action potential that is sometimes observed and by the rise of diastolic Ca2+ that occurs during acidosis. This rise, which will increase the uptake and subsequent release of Ca2+ by the sarcoplasmic reticulum, may be due to 1) stimulation of Na+ entry via Na(+)-Ca2+ exchange; 2) direct inhibition of Na(+)-Ca2+ exchange; 3) mitochondrial release of Ca2+; and 4) displacement of Ca2+ from cytoplasmic buffer sites by H+. Acidosis inhibits myofibrillar responsiveness to Ca2+ by decreasing the sensitivity of the contractile proteins to Ca2+, probably by decreasing the binding of Ca2+ to troponin C, and by decreasing maximum force, possibly by a direct action on the cross bridges. Thus the final amount of force developed by heart muscle during acidosis is the complex sum of these changes.

Muscle-type MM creatine kinase is specifically bound to sarcoplasmic reticulum and can support Ca2+ uptake and regulate local ATP/ADP ratios.

Highly purified fractions of sarcoplasmic reticulum (SR) were prepared from chicken pectoralis muscles (Saito, A., Seiler, S., Chu, A., and Fleischer, S. (1984) J. Cell Biol. 99, 875-885) and analyzed for the presence of creatine kinase (CK). Vesicles derived from longitudinal SR contained 0.703 +/- 0.428 IU of CK/mg of (SR) protein. Immunogold localization of muscle-type MM-CK on ultrathin cryosections of muscle, after removal of soluble CK, revealed relatively strong in situ labeling of M-CK remaining bound to the M band as well as to the SR membranes. In addition, purified SR vesicles were also labeled by anti-M-CK antibodies, and the peripheral labeling was similar to that observed with anti-Ca2(+)-ATPase antibodies. Only some particulate CK enzyme was released from isolated SR membranes by EDTA/low salt buffer, and CK was resistant to extraction by 0.6 M KCl. Thus, some of the MM-CK present in muscle displays strong associative behavior to the SR membranes. The SR-bound CK was sufficient to support, in the presence of phosphocreatine plus ADP, a significant portion of the maximal in vitro Ca2+ uptake rate. The ATP regeneration potential of SR-bound CK was similar to the rate of Ca2(+)-stimulated ATP hydrolysis of isolated SR vesicles. Thus, CK bound to SR may be physiologically relevant in vivo for regeneration of ATP used by the Ca2(+)-ATPase, as well as for regulation of local ATP/ADP ratios in the proximity of the Ca2+ pump and of other ATP-requiring reactions in the excitation-contraction coupling pathway.

The effect of acidosis on excitation-contraction coupling in isolated ferret heart muscle.

The contractile response to acidosis is the final product of a number of different changes in the excitation-contraction coupling pathway: (i) Cai increases and subsequently decreases during acidosis; (ii) the action potential becomes longer; (iii) the sensitivity of the contractile proteins to Ca2+ decreases. The increase of Cai and the lengthening of the action potential may help to maintain contractile function, although this advantage may be offset if spontaneous Ca2+ release from the s.r. occurs, secondary to the increase of Cai. The recovery of force shown in figure 1 occurs at a time when the calcium transient is decreasing, and therefore represents an increasing sensitivity of the contractile proteins to Cai, probably due to a recovery of intracellular pH (6), although it is also possible that a disappearance of spontaneous Ca2+ releases from the s.r. may be contributing [2].

Pathophysiologic role of calcium in the development of vascular smooth muscle tone

Recent information indicates that the intracellular ionized calcium concentration [Ca 2+]i plays a regulatory role not only in determining the magnitude of vascular tone but also in regulating growth of vascular tissue. Studies on living vascular smooth muscle cells using the calcium indicator aequorin have revealed that the relation between [Ca 2+]i and contraction of the vascular smooth muscle cell is complex. More than 1 intracellular kinase may be involved, leading to the coexistence of multiple excitation-contraction coupling pathways. However, it appears that all of these pathways may be calcium-dependent. It is not yet known whether the cause of human essential hypertension involves an elevated [Ca 2+]i in the vascular smooth muscle cell. However, evidence is presented supporting the concept that a decreased [Ca 2+]i in the hypertensive smooth muscle cell will lead to a decrease in vascular tone and total peripheral resistance, and possibly also antagonize the growth response of the vascular smooth muscle cell associated with the secondary effects of hypertension.

Alterations of excitation-contraction coupling by platelet-derived growth factor in enzymatically isolated and cultured vascular smooth muscle cells

We studied stimulus-specific alterations of the excitation-contraction coupling pathway in freshly isolated contractile and subcultured non-contractile vascular smooth muscle cells. Using the calcium indicator aequorin, we detected physiological increases in cytoplasmic free calcium [( Ca2+]i) in subcultured smooth muscle cells subjected to angiotensin or 33 mM potassium depolarization. These increases were qualitatively identical to those previously measured in intact vascular strips. Platelet-derived growth factor (PDGF) induced a slow, sustained [Ca2+]i increase when applied to the subcultured smooth muscle cells at low picomolar concentrations. Freshly isolated, contractile vascular smooth muscle cells, prepared by a novel technique, exhibited a slow shortening of 20% of resting length in response to PDGF. PDGF also markedly potentiated smooth muscle cell shortening in response to an ED50 dose of phenylephrine. This effect was PDGF concentration dependent. The time course of shortening induced by PDGF alone was consistent with the time course of the PDGF-induced [Ca2+]i increase in the cultured smooth muscle cells. These data suggest that agonists which induce [Ca2+]i changes in contractile smooth muscle cells may retain this ability with respect to cultured smooth muscle cells. PDGF, a peptide mitogen for proliferative smooth muscle cells, may also serve to modulate vascular tone by modestly raising [Ca2+]i in contractile smooth muscle cell and, therefore, sensitizing the cells to alpha adrenergic agonists.