Exercise training alters length dependence of contractile properties in rat myocardium.

Myocardial function is enhanced by endurance exercise training, but the cellular mechanisms underlying this improved function remain unclear. Exercise training increases the sensitivity of rat cardiac myocytes to activation by Ca(2+), and this Ca(2+) sensitivity has been shown to be highly dependent on sarcomere length. We tested the hypothesis that exercise training increases this length dependence in cardiac myocytes. Female Sprague-Dawley rats were divided into sedentary control (C) and exercise-trained (T) groups. The T rats underwent 11 wk of progressive treadmill exercise. Heart weight increased by 14% in T compared with C rats, and plantaris muscle citrate synthase activity showed a 39% increase with training. Steady-state tension was determined in permeabilized myocytes by using solutions of various Ca(2+) concentration (pCa), and tension-pCa curves were generated at two different sarcomere lengths for each myocyte (1.9 and 2.3 microm). We found an increased sarcomere length dependence of both maximal tension and pCa(50) (the Ca(2+) concentration giving 50% of maximal tension) in T compared with C myocytes. The DeltapCa(50) between the long and short sarcomere length was 0.084 +/- 0.023 (mean +/- SD) in myocytes from C hearts compared with 0.132 +/- 0.014 in myocytes from T hearts (n = 50 myocytes per group). The Deltamaximal tension was 5.11 +/- 1.42 kN/m(2) in C myocytes and 9.01 +/- 1.28 in T myocytes. We conclude that exercise training increases the length dependence of maximal and submaximal tension in cardiac myocytes, and this change may underlie, at least in part, training-induced enhancement of myocardial function.     

Contributions of stretch activation to length-dependent contraction in murine myocardium

The steep relationship between systolic force production and end diastolic volume (Frank-Starling relationship) in myocardium is a potentially important mechanism by which the work capacity of the heart varies on a beat-to-beat basis, but the molecular basis for the effects of myocardial fiber length on cardiac work are still not well understood. Recent studies have suggested that an intrinsic property of myocardium, stretch activation, contributes to force generation during systolic ejection in myocardium. To examine the role of stretch activation in length dependence of activation we recorded the force responses of murine skinned myocardium to sudden stretches of 1% of muscle length at both short (1.90 microm) and long (2.25 microm) sarcomere lengths (SL). Maximal Ca(2+)-activated force and Ca(2+) sensitivity of force were greater at longer SL, such that more force was produced at a given Ca(2+) concentration. Sudden stretch of myocardium during an otherwise isometric contraction resulted in a concomitant increase in force that quickly decayed to a minimum and was followed by a delayed development of force, i.e., stretch activation, to levels greater than prestretch force. At both maximal and submaximal activations, increased SL significantly reduced the initial rate of force decay following stretch; at submaximal activations (but not at maximal) the rate of delayed force development was accelerated. This combination of mechanical effects of increased SL would be expected to increase force generation during systolic ejection in vivo and prolong the period of ejection. These results suggest that sarcomere length dependence of stretch activation contributes to the steepness of the Frank-Starling relationship in living myocardium.     

Sarcomere mechanics in uniform and non-uniform cardiac muscle: a link between pump function and arrhythmias

Starling's Law and the well-known end-systolic pressure-volume relationship (ESPVR) of the left ventricle reflect the effect of sarcomere length (SL) on stress (sigma) development and shortening by myocytes in the uniform ventricle. We show here that tetanic contractions of rat cardiac trabeculae exhibit a sigma-SL relationship at saturating [Ca2+] that depends on sarcomere geometry in a manner similar to skeletal sarcomeres and the existence of opposing forces in cardiac muscle shortened below slack length. The sigma-SL-[Ca2+]free relationships (sigma-SL-CaR) at submaximal [Ca2+] in intact and skinned trabeculae were similar, albeit that the sensitivity for Ca2+ of intact muscle was higher. We analyzed the mechanisms underlying the sigma-SL-CaR using a kinetic model where we assumed that the rates of Ca2+ binding by Troponin-C (Tn-C) and/or cross-bridge (XB) cycling are determined by SL, [Ca2+] or stress. We analyzed the correlation between the model results and steady state stress measurements at varied SL and [Ca2+] from skinned rat cardiac trabeculae to test the hypotheses that: (i) the dominant feedback mechanism is SL, stress or [Ca2+]-dependent; and (ii) the feedback mechanism regulates: Tn-C-Ca2+ affinity, XB kinetics or, unitary XB-force. The analysis strongly suggests that feedback of the number of strong XBs to cardiac Tn-C-Ca2+ affinity is the dominant mechanism that regulates XB recruitment. Application of this concept in a mathematical model of twitch-stress accurately reproduced the sigma-SL-CaR and the time course of twitch-stress as well as the time course of intracellular [Ca2+]i. Modeling of the response of the cardiac twitch to rapid stress changes using the above feedback model uniquely predicted the occurrence of [Ca2+]i transients as a result of accelerated Ca2+ dissociation from Tn-C. The above concept has important repercussions for the non-uniformly contracting heart in which arrhythmogenic Ca2+ waves arise from weakened areas in cardiac muscle. These Ca2+ waves can reversibly be induced in muscle with non-uniform excitation contraction coupling (ECC) by the cycle of stretch and release in the border zone between the damaged and intact regions. Stimulus trains induced propagating Ca2+ waves and reversibly induced arrhythmias. We hypothesize that rapid force loss by sarcomeres in the border zone during relaxation causes Ca2+ release from Tn-C and initiates Ca2+ waves propagated by the sarcoplasmic reticulum (SR). These observations suggest the unifying hypothesis that force feedback to Ca2+ binding by Tn-C is responsible for Starling's Law and the ESPVR in uniform myocardium and leads in non-uniform myocardium to a surge of Ca2+ released by the myofilaments during relaxation, which initiates arrhythmogenic propagating Ca2+ release by the SR.     

Exercise training increases the Ca(2+) sensitivity of tension in rat cardiac myocytes.

The heart is known to respond to a program of chronic exercise in ways that enhance cardiac function. However, the cellular mechanisms involved in training-induced improvements in the contractile function of the myocardium are not known. In this study we tested the hypothesis that increased contractility of the myocardium associated with exercise training is due, in part, to increases in the Ca(2+) sensitivity of steady-state tension. Female Sprague-Dawley rats were randomly divided into sedentary control (C) and exercise-trained (T) groups. The T rats underwent 11 wk of progressive treadmill exercise (1 h/day, 5 days/wk, 26 m/min, 20% grade). Evidence of training effect included a 5.9% increase in heart mass, increases in heart weight-to-body weight ratio, and a 60% increase in skeletal muscle citrate synthase activity in T rats compared with C rats. After the training program, cardiac myocytes were isolated from T and C hearts. Myocytes were chemically skinned (i.e., the sarcolemma was removed) and attached to a force transducer, and steady-state tension was determined in solutions of various Ca(2+) concentrations ([Ca(2+)]). Myocytes isolated from the hearts of T rats showed a significantly (P < 0.01) increased sensitivity of tension to [Ca(2+)]. The [Ca(2+)] giving 50% of maximal tension (pCa(50)) was 5.90 +/- 0.033 and 5.82 +/- 0.023 (SD) in T and C myocytes, respectively (n = 70 myocytes/group). This result suggests that exercise training affects the myofibrillar proteins, such that Ca(2+) sensitivity is increased, and that this may be the mechanism that underlies, at least in part, the effect of training to increase myocardial contractility.     

Axial stretch enhances sarcoplasmic reticulum Ca2+ leak and cellular Ca2+ reuptake in guinea pig ventricular myocytes: experiments and models

Cardiac cellular calcium (Ca2+) handling is the well-investigated mediator of excitation-contraction coupling, the process that translates cardiac electrical activation into mechanical events. The reverse--effects of mechanical stimulation on cardiomyocyte Ca2+ handling--are much less well understood, in particular during the inter-beat period, called 'diastole'. We have investigated the effects of diastolic length changes, applied axially using a pair of carbon fibres attached to opposite ends of Guinea pig isolated ventricular myocytes, on the availability of Ca2+ in the main cellular stores (the sarcoplasmic reticulum; SR), by studying the rest-decay of SR Ca2+ content [Ca2+]SR, and the reloading of the SR after prior depletion of Ca2+ from the cell. Cells were loaded with Fura-2 AM (an indicator of the cytosolic 'free' Ca2+ concentration, [Ca2+]i), and pre-conditioned by field-stimulation (2 Hz) at 37 degrees C, while [Ca2+]i transients and sarcomere length (SL) were recorded simultaneously. After reaching a steady state in the behaviour of observed parameters, stimulation was interrupted for between 5 and 60s, while cells were either held at resting length, or stretched (controlled to cause a 10% increase in SL, to aid inter-individual comparison). Thereafter, each cell was returned to its original resting length, followed by swift administration of 10mM of caffeine (in Na+/Ca2+-free solution), which causes the release of Ca2+ from the SR (caffeine), but largely prevents extrusion of Ca2+ from the cytosol to the cell exterior (Na+/Ca2+-free solution). By comparing the [Ca2+]i in cells exposed/not exposed to diastolic stretch of different duration, we assessed the rest-decay dynamics of [Ca2+]SR. To assess SR reloading after initial Ca2+ depletion, the same stretch protocol was implemented after prior emptying of the cell by application of 10mM of caffeine in normal Tyrode solution (which causes Ca2+ to be released from the SR and extruded from the cell via the Na+/Ca2+ exchanger; NCX). Axial stretch enhanced the rate of both rest-decay and reloading of [Ca2+]SR. Application of 40 microM streptomycin, a blocker of stretch-activated ion channels, did not affect the stretch-induced increase in SR reloading. This behaviour was reproduced in a computer simulation study, using a modified version of the 2006 Iribe-Kohl-Noble model of single cardiac myocyte Ca2+ handling, suggesting that stretch increases both Ca2+ leak from the SR and Ca2+ influx via the sarcolemma. This may have important implications for the mobilisation of Ca2+ in stretched cells, and could contribute to the regional 'matching' of individual cardiomyocyte contractility to dynamic, and regionally varying, changes in mechanical loads, such as diastolic pre-load, of cardiac tissue.     

Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments.

The passive tension-sarcomere length relation of rat cardiac muscle was investigated by studying passive (or not activated) single myocytes and trabeculae. The contribution of collagen, titin, microtubules, and intermediate filaments to tension and stiffness was investigated by measuring (1) the effects of KCl/KI extraction on both trabeculae and single myocytes, (2) the effect of trypsin digestion on single myocytes, and (3) the effect of colchicine on single myocytes. It was found that over the working range of sarcomeres in the heart (lengths approximately 1.9-2.2 microns), collagen and titin are the most important contributors to passive tension with titin dominating at the shorter end of the working range and collagen at longer lengths. Microtubules made a modest contribution to passive tension in some cells, but on average their contribution was not significant. Finally, intermediate filaments contributed about 10% to passive tension of trabeculae at sarcomere lengths from approximately 1.9 to 2.1 microns, and their contribution dropped to only a few percent at longer lengths. At physiological sarcomere lengths of the heart, cardiac titin developed much higher tensions (> 20-fold) than did skeletal muscle titin at comparable lengths. This might be related to the finding that cardiac titin has a molecular mass of 2.5 MDa, 0.3-0.5 MDa smaller than titin of mammalian skeletal muscle, which is predicted to result in a much shorter extensible titin segment in the I-band of cardiac muscle. Passive stress plotted versus the strain of the extensible titin segment showed that the stress-strain relationships are similar in cardiac and skeletal muscle. The difference in passive stress between cardiac and skeletal muscle at the sarcomere level predominantly resulted from much higher strains of the I-segment of cardiac titin at a given sarcomere length. By expressing a smaller titin isoform, without changing the properties of the molecule itself, cardiac muscle is able to develop significant levels of passive tension at physiological sarcomere lengths.     

Ultrastructure of the sarcoplasmic reticulum in cardiac myocytes from Pacific bluefin tuna

Pacific bluefin tuna are active teleost fish with a large capacity for heat conservation and endothermy. They have a high metabolism, and hence the myocardium must be capable of sustaining elevated levels of cardiac output over a wide range of temperatures. To examine the way that the myocardial cells of bluefin tuna respond to their unique cardiac physiology, we have studied the ultrastructure of the internal membrane system and mitochondria of atrial and ventricular myocytes by light and electron microscopy. Our results reveal that cardiomyocytes of juvenile bluefin tuna posses a relatively high content of sarcoplasmic reticulum (SR), together with a large volume of mitochondria within the two (compact and spongy) ventricular compartments and in the atrial myocardium. The mitochondrial structure and distribution in bluefin tuna myocardium follow specific metabolic zonation resulting in a higher volume and lower cristae density in the compact ventricular layer than in atrium and spongy layer. The presence of junctional SR profiles and an extensive network of free SR within cells may ensure a rapid delivery of Ca(2+) to the myofibrils. This, in conjunction with transarcolemmal Ca(2+) entry, might contribute to a faster excitation-contraction-relaxation cycle and thus enhance cardiac performance, cardiac output, and the maintenance of excitability at low temperatures. We propose that the mitochondrial configuration together with the developed SR ultrastructure of bluefin tunas myocardium are important evolutionary steps for the maintenance of high heart rates and endothermy in this teleost fish.     

Expression of active p21-activated kinase-1 induces Ca2+ flux modification with altered regulatory protein phosphorylation in cardiac myocytes

p21-Activated kinase-1 (Pak1) is a serine-threonine kinase that associates with and activates protein phosphatase 2A in adult ventricular myocytes and, thereby, induces increased Ca2+ sensitivity of skinned-fiber tension development mediated by dephosphorylation of myofilament proteins (Ke Y, Wang L, Pyle WG, de Tombe PP, Solaro RJ. Circ Res 94: 194-200, 2004). We test the hypothesis that activation of Pak1 also moderates cardiac contractility through regulation of intracellular Ca2+ fluxes. We found no difference in field-stimulated intracellular Ca2+ concentration ([Ca2+]i) transient amplitude and extent of cell shortening between myocytes expressing constitutively active Pak1 (CA-Pak1) and controls expressing LacZ; however, time to peak shortening was significantly faster and rate of [Ca2+]i decay and time of relengthening were slower. Neither caffeine-releasable sarcoplasmic reticulum (SR) Ca2+ content nor fractional release was different in CA-Pak1 myocytes compared with controls. Isoproterenol application revealed a significantly blunted increase in [Ca2+]i transient amplitude, as well as a slowed rate of [Ca2+]i decay, increased SR Ca2+ content, and increased cell shortening, in CA-Pak1 myocytes. We found no significant change in phospholamban phosphorylation at Ser16 or Thr17 in CA-Pak1 myocytes. Analysis of cardiac troponin I revealed a significant reduction in phosphorylated species that are primarily attributable to Ser(23/24) in CA-Pak1 myocytes. Nonstimulated, spontaneous SR Ca2+ release sparks were significantly smaller in amplitude in CA-Pak1 than LacZ myocytes. Propagation of spontaneous Ca2+ waves resulting from SR Ca2+ overload was significantly slower in CA-Pak1 myocytes. Our data indicate that CA-Pak1 expression has significant effects on ventricular myocyte contractility through altered myofilament Ca2+ sensitivity and modification of the [Ca2+]i transient.     

Acceleration of stretch activation in murine myocardium due to phosphorylation of myosin regulatory light chain

The regulatory light chains (RLCs) of vertebrate muscle myosins bind to the neck region of the heavy chain domain and are thought to play important structural roles in force transmission between the cross-bridge head and thick filament backbone. In vertebrate striated muscles, the RLCs are reversibly phosphorylated by a specific myosin light chain kinase (MLCK), and while phosphorylation has been shown to accelerate the kinetics of force development in skeletal muscle, the effects of RLC phosphorylation in cardiac muscle are not well understood. Here, we assessed the effects of RLC phosphorylation on force, and the kinetics of force development in myocardium was isolated in the presence of 2,3-butanedione monoxime (BDM) to dephosphorylate RLC, subsequently skinned, and then treated with MLCK to phosphorylate RLC. Since RLC phosphorylation may be an important determinant of stretch activation in myocardium, we recorded the force responses of skinned myocardium to sudden stretches of 1% of muscle length both before and after treatment with MLCK. MLCK increased RLC phosphorylation, increased the Ca(2+) sensitivity of isometric force, reduced the steepness of the force-pCa relationship, and increased both Ca(2+)-activated and Ca(2+)-independent force. Sudden stretch of myocardium during an otherwise isometric contraction resulted in a concomitant increase in force that quickly decayed to a minimum and was followed by a delayed redevelopment of force, i.e., stretch activation, to levels greater than pre-stretch force. MLCK had profound effects on the stretch activation responses during maximal and submaximal activations: the amplitude and rate of force decay after stretch were significantly reduced, and the rate of delayed force recovery was accelerated and its amplitude reduced. These data show that RLC phosphorylation increases force and the rate of cross-bridge recruitment in murine myocardium, which would increase power generation in vivo and thereby enhance systolic function.     

Cooperative activation in cardiac muscle: impact of sarcomere length

This study was undertaken to determine the impact of sarcomere length (SL) on the level of cooperative activation of the cardiac myofilament at physiological [Mg2+]. Active force development was measured in skinned rat cardiac trabeculae as a function of free [Ca2+] at five SLs (1.85-2.25 microm; 1 mM free [Mg2+]; 15 degrees C). Only muscle preparations with minimal force rundown during the entire protocol were included in the analysis (average 7.2 +/- 1.7%). Median SL was measured by on-line computer video micrometry and controlled within 0.01 microm. Care was taken to ensure a sufficient number of data points in the steep portion of the [Ca2+]-force relationship at every SL to allow for accurate fit of the data to a modified Hill equation. Multiple linear regression analysis of the fit parameters revealed that both maximum, Ca2+-saturated force and Ca2+ sensitivity were a significant function of SL (P < 0.001), whereas the level of cooperativity did not depend on SL (P = 0.2). Further analysis of the [Ca2+]-force relationships revealed a marked asymmetry that, also, was not affected by SL (P = 0.2-0.6). Finally, we found that the level of cooperativity in isolated skinned myocardium was comparable to that reported for intact, nonskinned myocardium. Our results suggest that an increase in SL induces an increase in the Ca2+ responsiveness of the cardiac sarcomere without affecting the level of cooperativity.     

Effects of antisense oligonucleotides to the cardiac Na+/Ca2+ exchanger on calcium dynamics in cultured cardiac myocytes.

The present study was designed to explore the role of the Na+/Ca2+ exchanger on spontaneous beating of cultured cardiac myocytes. Antisense oligonucleotides (AS) based on the sequence of the cardiac Na+/Ca2+ exchanger were used to decrease expression of this Ca2+ transporting protein in cardiac myocytes. An application of AS (10 microM) caused an increase in beating rate of myocytes within 6-24 h. After 24 h of exposure, AS increased the beating rate from an average rate of 77 beats/min in control and sense-treated myocytes to 103 beats/min. Moreover, myocytes treated for 24 h with 10 microM AS exhibited an increase in diastolic [Ca2+]i levels. The antisense treatment also led to a approximately 20% decrease in expression of Na+/Ca2+ exchanger proteins within 6-24 h. Changes in mRNA levels following AS treatment could not be detected within 3- to 24-h periods. The results of these studies suggest that the Na+/Ca2+ exchanger plays a potentiating role in spontaneous the beating process by regulating [Ca2+]i dynamics and that even a small reduction in the levels of the exchanger protein has marked effects on the handling of [Ca2+]i during the cardiac cycle.     

Effect of sympathectomy on the heart pump function in rats during postnatal ontogenesis

The influence of guanetidine sympathectomy (30 mg/kg) on the heart pump function in rats during 3 weeks in postnatal ontogenesis has been investigated. Sympathectomy restrains age-dependent establishment of stroke volume, cardiac output and heart rate. The adaptation effects of regular physical training do not develop in the animals with sympathectomy, i.e. heart rate does not decrease and stroke volume does not increase. The initial stage of adaptation of the sympathectomized animals to physical training is accompanied by decrease in stroke volume and remarkable increase in heart rate which indicates the reduction of contractile activity in the myocardium.     

Neuroendocrine properties of intrinsic cardiac adrenergic cells in fetal rat heart

Intrinsic cardiac adrenergic (ICA) cells in developing rat heart constitute a novel adrenergic signaling system involved in cardiac regulation. Regulatory mechanisms of ICA cells remain to be defined. Immunohistochemical study of fetal rat hearts demonstrated ICA cells with catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT). The mRNA of TH and PNMP was also detected in fetal rat hearts before sympathetic innervation. Immunoreactivity of norepinephrine transporter (NET) was localized to ICA cells in rat heart tissue and primary cell culture. For the functional study, the activity of intracellular Ca2+ concentration ([Ca2+]i) transients was quantified by a ratio fluorescent spectrometer in cultured ICA cells and myocytes. ICA cells generated spontaneous [Ca2+]i transients that were eliminated by tetrodotoxin or Ca(2+)-free solutions and showed greatly reduced amplitude with the addition of L-type Ca2+ channel blocker nifedipine. [3H]norepinephrine studies demonstrate release and uptake of norepinephrine. Functional interaction between catecholamines produced by the ICA cells and cocultured myocytes was evident by the effect of the beta-adrenergic blocker atenolol eliciting a dose-dependent reduction in the amplitude and frequency of [Ca2+]i transients of beating myocytes. Hypoxia inhibited [Ca2+]i transient activity of ICA cells, which subsequently produced a reoxygenation-mediated rebound augmentation of [Ca2+]i transients. We conclude that ICA cells are capable of catecholamine synthesis, release, and uptake. They generate spontaneous [Ca2+]i transient activity that can be regulated by oxygen tension. ICA cells may provide an alternative adrenergic supply to maintain cardiac contractile and pacemaker function at rest and during stress in the absence of sympathetic innervation.     

Cardiac natriuretic peptides: a physiological lineage of cardioprotective hormones?

Vertebrate hearts from fish to mammals secrete peptide hormones with profound natriuretic, diuretic, and vasodilatory activity; however, the specific role of these cardiac natriuretic peptides (NPs) in homeostasis is unclear. NPs have been suggested to be involved in salt excretion in saltwater teleosts, whereas they are proposed to be more important in volume regulation in mammals. In this review, we consider an alternative (or perhaps complementary) function of NPs to protect the heart. This hypothesis is based on a number of observations. First, evidence for NPs, or NP-like activity has been found in all vertebrate hearts thus far examined, from osmoconforming saltwater hagfish to euryhaline freshwater and saltwater teleosts to terrestrial mammals. Thus the presence of cardiac NPs appears to be independent of environmental conditions that may variously affect salt and water balance. Second, cardiac stretch is a universal, and one of the most powerful, NP secretagogues. Furthermore, stretch-induced NP release in euryhaline teleosts appears relatively independent of ambient salinity. Third, excessive cardiac stretch that increases end-diastolic volume (EDV) can compromise the mechanical ability of the heart by decreasing actin-myosin interaction (length-tension) or through Laplace effects whereby as EDV increases, the wall tension necessary to maintain a constant pressure must also increase. Excessive cardiac stretch can be produced by factors that decrease cardiac emptying (i.e., increased arterial pressure), or by factors that increase cardiac filling (i.e., increased blood volume, increased venous tone, or decreased venous compliance). Fourth, the major physiological actions of cardiac NPs enhance cardiac emptying and decrease cardiac filling. In fish, NPs promote cardiac emptying by decreasing gill vascular resistance, thereby lowering ventral aortic pressure. In mammals a similar effect is achieved through pulmonary vasodilation. NPs also decrease cardiac filling by decreasing blood volume and increasing venous compliance, the latter producing a rapid fall in central venous pressure. Fifth, the presence of NP clearance receptors in the gill and lung (between the heart and systemic circulation) suggest that these tissues may be exposed to considerably higher NP titers than are systemic tissues. Thus, a decrease in outflow resistance immediately downstream from the heart may be the first response to increased cardiac distension. Because the physiology of cardiac NPs is basically the same in fish and mammals, we propose that the cardioprotective effects of NPs have been well preserved throughout the course of vertebrate evolution. It is also likely that the cardioprotective role of NPs was one of the most primordial homeostatic activities of these peptides in the earliest vertebrates.     

Thyroid status and beta-agonistic effects on cytosolic calcium concentrations in single rat cardiac myocytes activated by electrical stimulation or high-K+ depolarization.

The effects of the thyroid status on the cytosolic free Ca2+ concentration ([Ca2+]i) in single cardiomyocytes were studied at rest and during contraction. The mean resting [Ca2+]i increased significantly from the hypothyroid (45 +/- 4 nM) through the euthyroid (69 +/- 12 nM) to the hyperthyroid condition (80 +/- 11 nM) at extracellular Ca2+ concentrations ([Ca2+]o) up to 2.5 mM. At [Ca2+]o above 2.5 mM the differences in [Ca2+]i between the groups became less. The amplitude of the Ca2+ transients became higher in all groups with increasing [Ca2+]o (1, 2.5 and 5 mM), and was highest at all [Ca2+]o in hyperthyroid myocytes. The beta-agonist isoprenaline elevated peak [Ca2+]i during contraction and increased the rate of the decay of the Ca2+ transients to a greater extent in hypothyroid myocytes than in hyperthyroid myocytes. Depolarization with high [K+]o induced a large but transient [Ca2+]i overshoot in hypothyroid myocytes, but not in hyperthyroid myocytes, before a new elevated steady-state [Ca2+]i was reached, which was not different between the groups. When isoprenaline was added to K+ o-depolarized myocytes after a steady state was reached, a significantly larger extra increase in [Ca2+]i was measured in the hypothyroid group (28%) compared with the hyperthyroid group (8%). It is concluded that in cardiac tissue exposed to increasing amounts of thyroid hormones (1) [Ca2+]i increases at rest and during contraction in cardiomyocytes and (2) interventions which favour Ca2+ entry into the cytosol [( Ca2+]o elevation, high [K+]o, beta-agonists) tend to have less impact on Ca2+ homoeostasis.     

Signal-transducing function of Na+-K+-ATPase is essential for ouabain's effect on [Ca2+]i in rat cardiac myocytes

We showed before that Na+-K+-ATPase is also a signal transducer in neonatal rat cardiac myocytes. Binding of ouabain to the enzyme activates multiple signal pathways that regulate cell growth. The aims of this work were to extend such studies to adult cardiac myocytes and to determine whether the signal-transducing function of Na+/K+-ATPase regulates the well-known effects of ouabain on intracellular Ca2+ concentration ([Ca2+]i). In adult myocytes, ouabain activated protein tyrosine phosphorylation and p42/44 mitogen-activated protein kinases (MAPKs), increased production of reactive oxygen species (ROS), and raised both systolic and diastolic [Ca2+]i. Pretreatment of myocytes with several Src kinase inhibitors, or overexpression of a dominant negative Ras, antagonized ouabain-induced activation of MAPKs and increases in [Ca2+]i. Treatment with PD-98059 (a MAPK kinase inhibitor) or overexpression of a dominant negative MAPK kinase 1 also ablated the effect of ouabain on MAPKs and [Ca2+]i. N-acetyl-cysteine, which blocks the effect of ouabain on ROS, did not prevent the ouabain-induced rise in [Ca2+]i. Clearly, the activation of the Ras/MAPK cascade, but not ROS generation, is necessary for ouabain-induced increases in [Ca2+]i in rat cardiac myocytes.     

A quantitative analysis of cardiac myocyte relaxation: a simulation study

The determinants of relaxation in cardiac muscle are poorly understood, yet compromised relaxation accompanies various pathologies and impaired pump function. In this study, we develop a model of active contraction to elucidate the relative importance of the [Ca2+]i transient magnitude, the unbinding of Ca2+ from troponin C (TnC), and the length-dependence of tension and Ca2+ sensitivity on relaxation. Using the framework proposed by one of our researchers, we extensively reviewed experimental literature, to quantitatively characterize the binding of Ca2+ to TnC, the kinetics of tropomyosin, the availability of binding sites, and the kinetics of crossbridge binding after perturbations in sarcomere length. Model parameters were determined from multiple experimental results and modalities (skinned and intact preparations) and model results were validated against data from length step, caged Ca2+, isometric twitches, and the half-time to relaxation with increasing sarcomere length experiments. A factorial analysis found that the [Ca2+]i transient and the unbinding of Ca2+ from TnC were the primary determinants of relaxation, with a fivefold greater effect than that of length-dependent maximum tension and twice the effect of tension-dependent binding of Ca2+ to TnC and length-dependent Ca2+ sensitivity. The affects of the [Ca2+]i transient and the unbinding rate of Ca2+ from TnC were tightly coupled with the effect of increasing either factor, depending on the reference [Ca2+]i transient and unbinding rate.     

Myosin light chain kinase mediates sarcomere organization during cardiac hypertrophy in vitro

During the development of hypertrophy, cardiac myocytes increase organization of the sarcomere, a highly ordered contractile unit in striated muscle cells. Several hypertrophic agonists, such as angiotensin II, phenylephrine, and endothelin-1, have been shown to promote the sarcomere organization. However, the signaling pathway, which links extracellular stimuli to sarcomere organization, has not been clearly demonstrated. Here, we demonstrate that myosin light chain kinase specifically mediates agonist-induced sarcomere organization during early hypertrophic response. Acute administration of a hypertrophic agonist, phenylephrine, or angiotensin II, causes phosphorylation of myosin light chain 2v both in cultured cardiac myocytes and in the adult heart in vivo. We also show that both sarcomere organization and myosin light chain 2v phosphorylation are dependent on the activation of Ca2+/calmodulin pathway, a known activator of myosin light chain kinase. These results define a new and specific role of myosin light chain kinase in cardiac myocytes, which may provide a rapid adaptive mechanism in response to hypertrophic stimuli.     

Ontogeny of neurohormonal regulation of the cardiovascular system in the crayfish Procambarus clarkii

Adult crayfish have a neurogenic heart which is modulated via inputs from the central nervous system and neurohormones, which act on the cardiac ganglion or directly on the myocardium. This study investigates the ontogeny of cardiac regulation by exploring the temporal sequence of cardiac sensitivity to injections of cardioactive neurohormones (proctolin, serotonin and octopamine) and the neurotransmitter gamma-aminobutyric acid. The cardiac response (delta in heart rate, stroke volume, or in cardiac output) to each neurohormone at each developmental stage was assessed. The observed response elicited by each cardioactive drug was stage dependent and changed as the animals progressed from embryonic through larval and juvenile periods. During early developmental stages, octopamine, serotonin, and proctolin (10(-9)-10(-3) M) did not result in a modulation of stroke volume, yet in later developmental stages they caused significant increases in stroke volume, at comparable concentrations. Early developmental stages are capable of regulating cardiac function, however, the mechanisms appear to be quite different from those used by adults. Evidence is also provided to support the hypothesis that cardiac function is initiated prior to the establishment of an adult-like regulatory system.