Excitation-contraction Coupling Mechanism Research Articles

Functional Significance of Na+/Ca2+ Exchangers Co‐localization with Ryanodine Receptors

Co-localization of Na+/Ca2+ exchangers (NCX) with ryanodine receptors (RyRs) is debated. We incorporate local NCX current in a biophysically detailed model of L-type Ca2+ channels (LCCs) and RyRs and study the effect of NCX on the regulation of Ca2+-induced Ca2+ release and the shape of the action potential. In canine ventricular cells, under pathological conditions, e.g., impaired LCCs, local NCXs become an enhancer of sarcoplasmic reticulum release. Under such conditions incorporation of local NCXs is critical to accurately capture mechanisms of excitation-contraction coupling.

Expression of the tail‐anchored protein SLMAP in developing myocardium and during remodelling after myocardial infarction

The Organization of Excitation-Contraction (E-C) coupling mechanism is temporally regulated during heart remodelling. The molecular components involved in organizing membranes junctions with respect to the contractile apparatus remain elusive. SLMAP (Sarcolemmal Membrane Associated Protein) is a unique molecule shown to localize in cardiac membranes as well as the contractile apparatus and may serve to organizing the E-C coupling mechanism. The aim of this study was to investigate SLMAP expression and localization in developing myocardium and after Myocardial Infarction (MI) to correlate changes with any reorganization of the E-C coupling mechanism. SLMAP was located at the Z-disc, T-tubules and Sarcolemmal Membranes of rat myocardium as assessed by Immunostaining and Western blot analysis. SLMAP expression was significantly increased (p<0.005) from embryonic (E) day 16 to postnatal (P1) and gradually decreased (p<0.01) to low levels at 13 weeks. In a rat heart MI model induced by coronary ligation, SLMAP1 levels significantly increased (4 weeks post-surgery) in Infarct tissue vs. remote left ventricle (LV) (p<0.05) and SHAM (p<0.005). The location of SLMAP and temporal changes in its expression in normal and defective myocardium suggest that SLMAP may serve a role in organizing the E-C coupling apparatus during normal cardiac growth and in remodelling post MI. This work was supported by a grant from HSFO.

EXCITATION–CONTRACTION COUPLING FROM THE 1950s INTO THE NEW MILLENNIUM

1. Excitation-contraction coupling is broadly defined as the process linking the action potential to contraction in striated muscle or, more narrowly, as the process coupling surface membrane depolarization to Ca(2+) release from the sarcoplasmic reticulum. 2. We now know that excitation-contraction coupling depends on a macromolecular protein complex or 'calcium release unit'. The complex extends the extracellular space within the transverse tubule invaginations of the surface membrane, across the transverse tubule membrane into the cytoplasm and then across the sarcoplasmic reticulum membrane and into the lumen of the sarcoplasmic reticulum. 3. The central element of the macromolecular complex is the ryanodine receptor calcium release channel in the sarcoplasmic reticulum membrane. The ryanodine receptor has recruited a surface membrane L-type calcium channel as a 'voltage sensor' to detect the action potential and the calcium-binding protein calsequestrin to detect in the environment within the sarcoplasmic reticulum. Consequently, the calcium release channel is able to respond to surface depolarization in a manner that depends on the Ca(2+) load within the calcium store. 4. The molecular components of the 'calcium release unit' are the same in skeletal and cardiac muscle. However, the mechanism of excitation-contraction coupling is different. The signal from the voltage sensor to ryanodine receptor is chemical in the heart, depending on an influx of external Ca(2+) through the surface calcium channel. In contrast, conformational coupling links the voltage sensor and the ryanodine receptor in skeletal muscle. 5. Our current understanding of this amazingly efficient molecular signal transduction machine has evolved over the past 50 years. None of the proteins had been identified in the 1950s; indeed, there was debate about whether the molecules involved were, in fact, protein. Nevertheless, a multitude of questions about the molecular interactions and structures of the proteins and their interaction sites remain to be answered and provide a challenge for the next 50 years.

Mechanisms of Excitation-Contraction Coupling in an Integrative Model of the Cardiac Ventricular Myocyte

It is now well established that characteristic properties of excitation-contraction (EC) coupling in cardiac myocytes, such as high gain and graded Ca 2+ release, arise from the interactions that occur between L-type Ca 2+ channels (LCCs) and nearby ryanodine-sensitive Ca 2+ release channels (RyRs) in localized microdomains. Descriptions of Ca 2+-induced Ca 2+ release (CICR) that account for these local mechanisms are lacking from many previous models of the cardiac action potential, and those that do include local control of CICR are able to reconstruct properties of EC coupling, but require computationally demanding stochastic simulations of ∼10 5 individual ion channels. In this study, we generalize a recently developed analytical approach for deriving simplified mechanistic models of CICR to formulate an integrative model of the canine cardiac myocyte which is computationally efficient. The resulting model faithfully reproduces experimentally measured properties of EC coupling and whole cell phenomena. The model is used to study the role of local redundancy in L-type Ca 2+ channel gating and the role of dyad configuration on EC coupling. Simulations suggest that the characteristic steep rise in EC coupling gain observed at hyperpolarized potentials is a result of increased functional coupling between LCCs and RyRs. We also demonstrate mechanisms by which alterations in the early repolarization phase of the action potential, resulting from reduction of the transient outward potassium current, alters properties of EC coupling.

Hypoxic modulation of cardiac -type Ca current: Interaction of reactive oxygen species and β-adrenergic signaling

See article by Hool et al. [5] (pages 624–635) in this issue . Sarcolemmal L -type Ca2+ channels are integral components of the excitation–contraction coupling mechanism in cardiomyocytes. Membrane depolarization to −30 mV opens these channels [1], allowing rapid influx of Ca2+ which peaks within 2–3 ms [2]. This inward Ca2+ current ( I Ca) produces the plateau phase of the cardiomyocyte action potential and triggers a massive release of Ca2+ from the sarcoplasmic reticulum via ryanodine-sensitive Ca2+ channels, arrayed in close proximity to the L -type channels. The resultant cytosolic Ca2+ transient activates crossbridge cycling and mechanical force production by the contractile machinery and inactivates the L -type Ca2+ current [2]. Cardiac L -type channels contain four subunits (α1C, α2, β2, δ) [1,2]. Each of the four homologous domains of the large α1C subunit harbor six membrane-spanning α helices; helices 5 and 6 of the four α1C domains combine to form a Ca2+ pore [2]. L -type Ca2+ current is physiologically regulated by protein kinases that covalently modify the α1C subunit to modulate intrinsic channel gating properties. β-Adrenergic activity increases I Ca via protein kinase A phosphorylation of Ser-1928 of α1C [2] (Fig. 1), which shifts gating behavior of the channels from mode 0 (rarely or never open) to modes 1 (low open probability; channels open only briefly) and 2 (high open probability; prolonged opening of channels) [1,3]. This mechanism contributes importantly to β-adrenergic enhancement of myocardial contractile performance. Protein kinase C may phosphorylate Thr-27 and/or Thr-31 near the amino terminus of α1C, producing either … *Tel.: +1 817 735 2260; fax: +1 817 735 5084. Email address: malletr{at}hsc.unt.edu

Inhibition of In Vitro Contractions of Human Myometrium by Mibefradil, a T-Type Calcium Channel Blocker: Support for a Model Using Excitation-Contraction Coupling, and Autocrine and Paracrine Signaling Mechanisms

To investigate the importance of T-type calcium channels in human myometrial contractility and to refine a model of contractility. We performed in vitro muscle bath experiments on human myometrial tissue strips while simultaneously monitoring bioelectrical activity with loose-contact electrodes. Tissue was obtained by myometrial biopsy from term pregnant women at the time of cesarean delivery and studied after 1 to 7 days in organ culture. Tissue strips were exposed to 5 nM oxytocin to obtain regular contractions. Tissue was then exposed to the T-type calcium channel blocker mibefradil (1 microM), the L-type calcium channel blocker nifedipine (5 nM), or the cyclooxygenase (COX) inhibitor indomethacin (30 microM). All study drugs reduced the strength of contractions. Data were analyzed using a model composed of two phases of force production--an electrical phase (E), which encompassed the first 3 seconds of each contraction, and a paracrine phase (P), which continued until the peak of the contraction. For each phase, the relative force reduction was calculated by the ratio of forces measured after and before drug exposure (RE and RP, respectively). For each drug, experiments were performed in at least triplicate. Myometrial contractions and bioelectrical signals were routinely observed following oxytocin exposure. Spike-like bioelectrical signals occurred only at the beginning of each contraction. Nifedipine and indomethacin slightly modulated the bioelectrical signals, but mibefradil appeared to block the spike component. Mibefradil caused a similar reduction of E and P (RE = 58 +/- 10%, RP = 62 +/- 22%). Nifedipine also reduced E and P, although P was reduced more than E (RE = 72 +/- 12%, RP = 38 +/- 8%, P = .006). Indomethacin yielded variable results, with some experiments showing similar RE and RP values, while other experiments showed RP > RE. Mibefradil inhibition of the bioelectrical signal and uterine contractile forces suggest that T-type calcium channels are important in the initiation of each contraction. Some results using indomethacin suggest that for a specific tissue or under some conditions, autocrine stimulation by prostaglandins may be important for recruitment of myocytes in the electrical phase. Stimulation by prostaglandins is likely important in the paracrine phase. Enhanced inhibition by nifedipine in the paracrine phase suggests the mechanism of action of nifedipine tocolytic effect is block of prostaglandin F2alpha (PGF2alpha) paracrine stimulation. Our initial model proposed at least two mechanisms (electrical and nonelectrical) for the recruitment of myocytes. Our modified model suggests that the electrical phase contains a prostaglandin autocrine mechanism in addition to excitation-contraction coupling, and the mechanism in the nonelectrical phase is prostaglandin paracrine signaling.

Central and Peripheral Fatigue of the Knee Extensor Muscles Induced by Electromyostimulation

The main purpose of this study was to characterise neuromuscular fatigue induced by 30 contractions of the knee extensor muscles evoked by electromyostimulation (EMS). Twelve healthy subjects were tested before and after a typical EMS session (frequency: 75 Hz, on-off ratio: 6.25 s on-20 s off) used for quadriceps femoris muscle strengthening. Surface electromyographic (EMG) activity and torque obtained during maximal voluntary and electrically evoked contractions were analysed to distinguish peripheral from central fatigue. Maximal voluntary torque of the knee extensor muscles decreased approximately 20 % (p < 0.001) following EMS. In the same way, peak torque associated to single (p < 0.05) and paired (p < 0.001) stimuli as well as M-wave amplitude (p < 0.05) significantly decreased as a result of EMS. The raw EMG activity of both vastus lateralis and rectus femoris muscle recorded during maximal voluntary isometric contraction significantly decreased after the session (-17.3 and -14.5 %, respectively) whereas no changes were observed when EMG signals were normalised to respective M-wave amplitudes. Similarly, voluntary activation estimated by using the twitch interpolation technique was unchanged following EMS. In conclusion, a typical session of EMS of the knee extensor muscles mainly induced neuromuscular propagation failure while excitation-contraction coupling and neural mechanisms were not significantly affected. It is recommended to interpret surface EMG data together with the corresponding M wave, at least for the knee extensor muscles, in order to distinguish peripheral from central causes of fatigue.

Multiphysics Simulation of Left Ventricular Filling Dynamics Using Fluid-Structure Interaction Finite Element Method

To relate the subcellular molecular events to organ level physiology in heart, we have developed a three-dimensional finite-element-based simulation program incorporating the cellular mechanisms of excitation-contraction coupling and its propagation, and simulated the fluid-structure interaction involved in the contraction and relaxation of the human left ventricle. The FitzHugh-Nagumo model and four-state model representing the cross-bridge kinetics were adopted for cellular model. Both ventricular wall and blood in the cavity were modeled by finite element mesh. An arbitrary Lagrangian Eulerian finite element method with automatic mesh updating has been formulated for large domain changes, and a strong coupling strategy has been taken. Using electrical analog of pulmonary circulation and left atrium as a preload and the windkessel model as an afterload, dynamics of ventricular filling as well as ejection was simulated. We successfully reproduced the biphasic filling flow consisting of early rapid filling and atrial contraction similar to that reported in clinical observation. Furthermore, fluid-structure analysis enabled us to analyze the wave propagation velocity of filling flow. This simulator can be a powerful tool for establishing a link between molecular abnormality and the clinical disorder at the macroscopic level.

Ageing affects the differentiation potential of human myoblasts

The ageing process causes a reduction in the regenerative potential of skeletal muscles eventually leading to diminished muscle strength. In this work we investigated if ageing affects the excitation–contraction coupling mechanism in human myotubes derived from human satellite cells, thereby contributing to the loss in muscle strength in the aged. To test this hypothesis, satellite cells from differently aged donors were differentiated in vitro and the maturation of the excitation–contraction mechanism was followed by the videoimaging technique monitoring the efficiency of such a mechanism in generating intracellular calcium transients. Our experiments showed a delay in the establishment of the excitation–contraction coupling mechanism depending on the age of the donor. Remarkably, the effect was reproducible in human satellite cells from a young donor aged in vitro, suggesting that the delayed functional maturation was strictly dependent on the number of satellite cell divisions and independent from the host environment.

Involvement of a Heptad Repeat in the Carboxyl Terminus of the Dihydropyridine Receptor β1a Subunit in the Mechanism of Excitation-Contraction Coupling in Skeletal Muscle

Chimeras consisting of the homologous skeletal dihydropyridine receptor (DHPR) β1a subunit and the heterologous cardiac/brain β2a subunit were used to determine which regions of β1a were responsible for the skeletal-type excitation-contraction (EC) coupling phenotype. Chimeras were transiently transfected in β1 knockout myotubes and then voltage-clamped with simultaneous measurement of confocal fluo-4 fluorescence. All chimeras expressed a similar density of DHPR charge movements, indicating that the membrane density of DHPR voltage sensors was not a confounding factor in these studies. The data indicates that a β1a-specific domain present in the carboxyl terminus, namely the D5 region comprising the last 47 residues ( β1a 478–524), is essential for expression of skeletal-type EC coupling. Furthermore, the location of β1aD5 immediately downstream from conserved domain D4 is also critical. In contrast, chimeras in which β1aD5 was swapped by the D5 region of β2a expressed Ca 2+ transients triggered by the Ca 2+ current, or none at all. A hydrophobic heptad repeat is present in domain D5 of β1a (L478, V485, V492). To determine the role of this motif, residues in the heptad repeat were mutated to alanines. The triple mutant β1a(L478A/V485A/V492A) recovered weak skeletal-type EC coupling (Δ F/ F max = 0.4 ± 0.1 vs. 2.7 ± 0.5 for wild-type β1a). However, a triple mutant with alanine substitutions at positions out of phase with the heptad repeat, β1a(S481A/L488A/S495A), was normal (Δ F/ F max = 2.1 ± 0.4). In summary, the presence of the β1a-specific D5 domain, in its correct position after conserved domain D4, is essential for skeletal-type EC coupling. Furthermore, a heptad repeat in β1aD5 controls the EC coupling activity. The carboxyl terminal heptad repeat of β1a might be involved in protein-protein interactions with ryanodine receptor type 1 required for DHPR to ryanodine receptor type 1 signal transmission.

Dantrolene – A review of its pharmacology, therapeutic use and new developments

Human malignant hyperthermia is a life-threatening genetic sensitivity of skeletal muscles to volatile anaesthetics and depolarizing neuromuscular blocking drugs occurring during or after anaesthesia. The skeletal muscle relaxant dantrolene is the only currently available drug for specific and effective therapy of this syndrome in man. After its introduction, the mortality of malignant hyperthermia decreased from 80% in the 1960s to < 10% today. It was soon discovered that dantrolene depresses the intrinsic mechanisms of excitation-contraction coupling in skeletal muscle. However, its precise mechanism of action and its molecular targets are still incompletely known. Recent studies have identified the ryanodine receptor as a dantrolene-binding site. A direct or indirect inhibition of the ryanodine receptor, the major calcium release channel of the skeletal muscle sarcoplasmic reticulum, is thought to be fundamental in the molecular action of dantrolene in decreasing intracellular calcium concentration. Dantrolene is not only used for the treatment of malignant hyperthermia, but also in the management of neuroleptic malignant syndrome, spasticity and Ecstasy intoxication. The main disadvantage of dantrolene is its poor water solubility, and hence difficulties are experienced in rapidly preparing intravenous solutions in emergency situations. Due to economic considerations, no other similar drugs have been introduced into routine clinical practice.

Functional equivalence of dihydropyridine receptor a1S and b1a subunits in triggering excitation-contraction coupling in skeletal muscle

Molecular understanding of the mechanism of excitation-contraction (EC) coupling in skeletal muscle has been made possible by cultured myotube models lacking specific dihydropyridine receptor (DHPR) subunits and ryanodine receptor type 1 (RyR1) isoforms. Transient expression of missing cDNAs in mutant myotubes leads to a rapid recovery, within days, of various Ca2+ current and EC coupling phenotypes. These myotube models have thus permitted structure-function analysis of EC coupling domains present in the DHPR controlling the opening of RyR1. The purpose of this brief review is to highlight advances made by this laboratory towards understanding the contribution of domains present in alpha1S and beta1a subunits of the skeletal DHPR to EC coupling signaling. Our main contention is that domains of the alpha1S II-III loop are necessary but not sufficient to recapitulate skeletal-type EC coupling. Rather, the structural unit that controls the EC coupling signal appears to be the alpha1S/beta1a pair.

Developmental changes of intracellular Ca2+ transients in beating rat hearts.

Postnatal maturation of the rat heart is characterized by major changes in the mechanism of excitation-contraction (E-C) coupling. In the neonate, the t tubules and sarcoplasmic reticulum (SR) are not fully developed yet. Consequently, Ca(2+)-induced Ca(2+) release (CICR) does not play a central role in E-C coupling. In the neonate, most of the Ca(2+) that triggers contraction comes through the sarcolemma. In this work, we defined the contribution of the sarcolemmal Ca(2+) entry and the Ca(2+) released from the SR to the Ca(2+) transient during the first 3 wk of postnatal development. To this end, intracellular Ca(2+) transients were measured in whole hearts from neonate rats by using the pulsed local field fluorescence technique. To estimate the contribution of each Ca(2+) flux to the global intracellular Ca(2+) transient, different pharmacological agents were used. Ryanodine was applied to evaluate ryanodine receptor-mediated Ca(2+) release from the SR, nifedipine for dihydropyridine-sensitive L-type Ca(2+) current, Ni(2+) for the current resulting from the reverse-mode Na(+)/Ca(2+) exchange, and mibefradil for the T-type Ca(2+) current. Our results showed that the relative contribution of each Ca(2+) flux changes considerably during the first 3 wk of postnatal development. Early after birth (1-5 days), the sarcolemmal Ca(2+) flux predominates, whereas at 3 wk of age, CICR from the SR is the most important. This transition may reflect the progressive development of the t tube-SR units characteristic of mature myocytes. We have hence directly defined in the whole beating heart the developmental changes of E-C coupling previously evaluated in single (acutely isolated or cultured) cells and multicellular preparations.

Cardiac-type EC-Coupling in Dysgenic Myotubes Restored with Ca 2+ Channel Subunit Isoforms α1C and α1D Does not Correlate with Current Density

Ca 2+-induced Ca 2+-release (CICR)—the mechanism of cardiac excitation-contraction (EC) coupling—also contributes to skeletal muscle contraction; however, its properties are still poorly understood. CICR in skeletal muscle can be induced independently of direct, calcium-independent activation of sarcoplasmic reticulum Ca 2+ release, by reconstituting dysgenic myotubes with the cardiac Ca 2+ channel α 1C (Ca V1.2) subunit. Ca 2+ influx through α 1C provides the trigger for opening the sarcoplasmic reticulum Ca 2+ release channels. Here we show that also the Ca 2+ channel α 1D isoform (Ca V1.3) can restore cardiac-type EC-coupling. GFP- α 1D expressed in dysgenic myotubes is correctly targeted into the triad junctions and generates action potential-induced Ca 2+ transients with the same efficiency as GFP- α 1C despite threefold smaller Ca 2+ currents. In contrast, GFP- α 1A, which generates large currents but is not targeted into triads, rarely restores action potential-induced Ca 2+ transients. Thus, cardiac-type EC-coupling in skeletal myotubes depends primarily on the correct targeting of the voltage-gated Ca 2+ channels and less on their current size. Combined patch-clamp/fluo-4 Ca 2+ recordings revealed that the induction of Ca 2+ transients and their maximal amplitudes are independent of the different current densities of GFP- α 1C and GFP- α 1D. These properties of cardiac-type EC-coupling in dysgenic myotubes are consistent with a CICR mechanism under the control of local Ca 2+ gradients in the triad junctions.

The voltage-sensitive release mechanism of excitation contraction coupling in rabbit cardiac muscle is explained by calcium-induced calcium release.

The putative voltage-sensitive release mechanism (VSRM) was investigated in rabbit cardiac myocytes at 37°C with high resistance microelectrodes to minimize intracellular dialysis. When the holding potential was adjusted from −40 to −60 mV, the putative VSRM was expected to operate alongside CICR. Under these conditions however, we did not observe a plateau at positive potentials of the cell shortening versus voltage relationship. The threshold for cell shortening changed by −10 mV, but this resulted from a similar change of the threshold for activation of inward current. Cell shortening under conditions where the putative VSRM was expected to operate was blocked in a dose dependent way by nifedipine and CdCl2 and blocked completely by NiCl2. “Tail contractions” persisted in the presence of nifedipine and CdCl2 but were blocked completely by NiCl2. Block of early outward current by 4-aminopyridine and 4-acetoamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid (SITS) demonstrated persisting inward current during test depolarizations despite the presence of nifedipine and CdCl2. Inward current did not persist in the presence of NiCl2. A tonic component of cell shortening that was prominent during depolarizations to positive potentials under conditions selective for the putative VSRM was sensitive to rapidly applied changes in superfusate [Na+] and to the outward Na+/Ca2+ exchange current blocking drug KB-R7943. This component of cell shortening was thought to be the result of Na+/Ca2+ exchange–mediated excitation contraction coupling. Cell shortening recorded under conditions selective for the putative VSRM was increased by the enhanced state of phosphorylation induced by isoprenaline (1 μM) and by enhancing sarcoplasmic reticulum Ca2+ content by manipulation of the conditioning steps. Under these conditions, cell shortening at positive test depolarizations was converted from tonic to phasic. We conclude that the putative VSRM is explained by CICR with the Ca2+ “trigger” supplied by unblocked L-type Ca2+ channels and Na+/Ca2+ exchange.

Normal and pathological excitation-contraction coupling in the heart -- an overview.

This issue of The Journal of Physiology includes a series of review articles arising from a symposium held at the joint meeting of the UK, German and Scandinavian Physiological Societies. The articles focus on different aspects of the cellular control of contraction. The basic mechanism of cardiac excitation-contraction coupling ('calcium-induced calcium release') is now reasonably well-established. Calcium enters the cell from the extracellular fluid via the voltage-dependent L-type Ca(2+) channel. This results in a 'trigger' increase of [Ca(2+)](i) in the space between the sarcolemma and sarcoplasmic reticulum (SR) and this leads to the opening of the SR Ca(2+) release channel or 'ryanodine receptor' (RyR). As exemplified by the papers from the symposium, much current work is focused on how this mechanism is modified in different circumstances. These include autonomic modulation, but also pathological conditions such as cardiac hypertrophy and failure, a recurrent theme in several of these papers.

Truncation of the Carboxyl Terminus of the DihydropyridineReceptor β1a Subunit Promotes Ca 2+ Dependent Excitation-Contraction Coupling in Skeletal Myotubes

We investigated the contribution of the carboxyl terminus region of the β1a subunit of the skeletal dihydropyridine receptor (DHPR) to the mechanism of excitation-contraction (EC) coupling. cDNA-transfected β1 KO myotubes were voltage clamped, and Ca 2+ transients were analyzed by confocal fluo-4 fluorescence. A chimera with an amino terminus half of β2a and a carboxyl terminus half of β1a ( β2a 1-287/ β1a 325-524) recapitulates skeletal-type EC coupling quantitatively and was used to generate truncated variants lacking 7 to 60 residues from the β1a-specific carboxyl terminus (Δ7, Δ21, Δ29, Δ35, and Δ60). Ca 2+ transients recovered by the control chimera have a sigmoidal Ca 2+ fluorescence (Δ F/ F) versus voltage curve with saturation at potentials more positive than +30 mV, independent of external Ca 2+ and stimulus duration. In contrast, the amplitude of Ca 2+ transients expressed by the truncated variants varied with the duration of the pulse, and for Δ29, Δ35, and Δ60, also varied with external Ca 2+ concentration. For Δ7 and Δ21, a 50-ms depolarization produced a sigmoidal Δ F/ F versus voltage curve with a lower than control maximum fluorescence. Moreover, for Δ29, Δ35, and Δ60, a 200-ms depolarization increased the maximum fluorescence and changed the shape of the Δ F/ F versus voltage curve, from sigmoidal to bell-shaped, with a maximum at ∼+30 mV. The change in voltage dependence, together with the external Ca 2+ dependence and additional controls with ryanodine, indicated a loss of skeletal-type EC coupling and the emergence of an EC coupling component triggered by the Ca 2+ current. Analyses of d(Δ F/ F)/ dt showed that the rate of cytosolic Ca 2+ increase during the Ca 2+ transient was fivefold faster for the control chimera than for the severely truncated variants (Δ29, Δ35, and Δ60) and was consistent with the kinetics of the DHPR Ca 2+ current. In summary, absence of the β1a-specific carboxyl terminus (last 29 to 60 residues of the control chimera) results in a loss of the fast component of the Ca 2+ transient, bending of the Δ F/ F versus voltage curve, and emergence of EC coupling triggered by the Ca 2+ current. The studies underscore the essential role of the carboxyl terminus region of the DHPR β1a subunit in fast voltage dependent EC coupling in skeletal myotubes.

148 左心室ポンプ機能と心室壁繊維方向に関する検討

In this study, a 3D finite element based simulation program incorporating the propagation of excitation and excitation-contraction coupling mechanisms developed by us was modified to reproduce the more realistic ventricular wall structure. FE model of LV consists of six layers of muscle bundles and the fiber direction of the inner-most layer was longitudinal. Although the detailed modeling did not influence the global pump function of the LV appreciably, we could successfully identify the functional significance of longitudinal fibers as pointed out by clinical observations. Furthermore, we also changed the heart structure drastically to elucidate the functional significance of the fiber structure. The results suggest that the double-helical structure the heart has developed through the evolutionary process can be an optimal design for a blood pump.

Optical bioimaging: from living tissue to a single molecule: atrio-ventricular difference in myocardial excitation-contraction coupling--sequential versus simultaneous activation of SR Ca2+ release units.

Rapid-scanning cofocal microscopy has been applied to the analysis of early phase Ca(2+) transients in ventricular and atrial cardiomyocytes. On electrical stimulation of ventricular myocytes, Ca(2+) concentration begins to rise earliest at the Z-line level and becomes uniform throughout the cytoplasm within about 10 ms after the onset of the action potential; transsarcolemmal Ca(2+) influx triggers Ca(2+) release from release sites on the junctional sarcoplasmic reticulum (SR) coupled to T-tubules at the Z-line throughout the cytoplasm. In atrial myocytes lacking the T-tubular network, transsarcolemmal Ca(2+) influx during an action potential triggers SR Ca(2+) release only at subsarcolemmal region. SR Ca(2+) release then spreads towards the central region of the cell through a propagated Ca(2+)-induced-Ca(2+) release mechanism. The atrio-ventricular difference in excitation-contraction coupling mechanisms underlies some of the atrio-ventricular difference in response to physiological and pharmacological stimuli.

Finite Element Analysis on the Relationship between Left Ventricular Pump Function and Fiber Structure within the Wall

In this study, a 3D finite element based simulation program incorporating the propagation of excitation and excitation-contraction coupling mechanisms developed by us was modified to reproduce the more realistic ventricular wall structure. FE model of LV consists of six layers of muscle bundles and the fiber direction of the inner-most layer was longitudinal. Although the detailed modeling did not influence the global pump function of the LV appreciably, we could successfully identify the functional significance of longitudinal fibers as pointed out by clinical observations. Furthermore, we also changed the heart structure drastically to elucidate the functional significance of the fiber structure. The results suggest that the double-helical structure the heart has developed through the evolutionary process can be an optimal design for a blood pump.