Micromechanical function of myofibrils isolated from skeletal and cardiac muscles of the zebrafish

The zebrafish is a potentially important and cost-effective model for studies of development, motility, regeneration, and inherited human diseases. The object of our work was to show whether myofibrils isolated from zebrafish striated muscle represent a valid subcellular contractile model. These organelles, which determine contractile function in muscle, were used in a fast kinetic mechanical technique based on an atomic force probe and video microscopy. Mechanical variables measured included rate constants of force development (k(ACT)) after Ca(2+) activation and of force decay (τ(REL)(-1)) during relaxation upon Ca(2+) removal, isometric force at maximal (F(max)) or partial Ca(2+) activations, and force response to an external stretch applied to the relaxed myofibril (F(pass)). Myotomal myofibrils from larvae developed greater active and passive forces, and contracted and relaxed faster than skeletal myofibrils from adult zebrafish, indicating developmental changes in the contractile organelles of the myotomal muscles. Compared with murine cardiac myofibrils, measurements of adult zebrafish ventricular myofibrils show that k(ACT), F(max), Ca(2+) sensitivity of the force, and F(pass) were comparable and τ(REL)(-1) was smaller. These results suggest that cardiac myofibrils from zebrafish, like those from mice, are suitable contractile models to study cardiac function at the sarcomeric level. The results prove the practicability and usefulness of mechanical and kinetic investigations on myofibrils isolated from larval and adult zebrafish muscles. This novel approach for investigating myotomal and myocardial function in zebrafish at the subcellular level, combined with the powerful genetic manipulations that are possible in the zebrafish, will allow the investigation of the functional primary consequences of human disease-related mutations in sarcomeric proteins in the zebrafish model.     

Expression and purification of human cardiac troponin subunits and their functional incorporation into isolated cardiac mouse myofibrils

The three subunits of the human cardiac troponin complex (hcTnC, hcTnI, hcTnT) were overexpressed in E. coli, purified and reconstituted to form the hcTn complex. This complex was then incorporated into subcellular bundles of mouse cardiac myofibrils whereby the native mcTn complex was replaced. On thus exchanged myofibrils, isometric force kinetics following sudden changes in free Ca(2+) concentration were measured using atomic force cantilevers. Following the exchange, the myofibrillar force remained fully Ca(2+) regulated, i.e. myofibrils were completely relaxed at pCa 7.5 and developed the same maximum Ca(2+)-activated isometric force upon increasing the pCa to 4.5 as unexchanged myofibrils. The replacement of endogenous mcTn by wild-type hcTn neither altered the kinetics of Ca(2+)-induced force development of the mouse myofibrils nor the kinetics of force relaxation induced by the sudden, complete removal of Ca(2+). Preparations of functional Tn reconstituted myofibrils provide a promising model to study the role of Tn in kinetic mechanisms of cardiac myofibrillar contraction and relaxation.     

The interaction of Ca2+ with sarcomeric proteins: role in function and dysfunction of the heart

The hallmarks of the normal heartbeat are both rapid onset of contraction and rapid relaxation as well as an inotropic response to both increased end-diastolic volume and increased heart rate. At the microscopic level, Ca(2+) plays a crucial role in normal cardiac contraction. This paper reviews the cycle of Ca(2+) fluxes during the normal heartbeat, which underlie the coupling between excitation and contraction and permit a highly synchronized action of cardiac sarcomeres. Length dependence of the response of the regulatory sarcomeric proteins mediates the Frank-Starling Law of the heart. However, Ca(2+) transport may go astray in heart disease such as in congestive heart failure, and both jeopardize systole and diastole and triggering arrhythmias. The interaction between weak and strong segments in nonuniform cardiac muscle allows partial preservation of force of contraction but may further lead to mechanoelectric feedback or reverse excitation-contraction coupling mediating an early diastolic Ca(2+) transient caused by the rapid force decrease during the relaxation phase. These rapid force changes in nonuniform muscle may cause arrhythmogenic Ca(2+) waves to propagate by the activation of neighboring sarcoplasmic reticulum by diffusing Ca(2+) ions.     

Exogenous hydrogen peroxide as a possible messenger for the stimulation effect of magnetized physiological solution on heart contractility

The dual effect of magnetized physiological solution (MPS) on snail heart muscle contractility (muscle relaxation and stimulation of heartbeat) was shown previously. The MPS-induced relaxation of the heart muscle has been explained by activation of cGMP-dependent Ca(2+) effluxes from the muscle; however, the mechanism of the stimulating effect of MPS on heartbeat remains unclear. As in the presence of paramagnetic oxygen molecules, magnetic fields could generate the exogenous reactive oxygen species such as hydrogen peroxide (H(2)O(2)), we hypothesize that H(2)O(2) may play a role as the possible messengers through which the activation effect of MPS on heartbeat is realized. To test this hypothesis, the dose-dependent effects of exogenous H(2)O(2) on heart muscle contractility and (45)Ca uptake were studied. Here we compared the obtained data with the previous results of the effects of MPS on heart muscle contractility and (45)Ca uptake. We found that exogenous H(2)O(2) and MPS have similar effects on Na(+)-K(+) pump-induced transient inhibition of muscle contractility and (45)Ca uptake. The Na(+)-K(+) pump-induced depression of H(2)O(2)-sensitivity of muscle contractility is determined by activation of Ca(2+) efflux from the cell. On the basis of these data we suggest the exogenous H(2)O(2) as a possible messenger through which the stimulation effect of MPS on heart muscle is realized.     

The Rates of Ca2+ Dissociation and Cross-bridge Detachment from Ventricular Myofibrils as Reported by a Fluorescent Cardiac Troponin C

The rate-limiting step of cardiac muscle relaxation has been proposed to reside in the myofilament. Both the rates of cross-bridge detachment and Ca(2+) dissociation from troponin C (TnC) have been hypothesized to rate-limit myofilament inactivation. In this study we used a fluorescent TnC to measure both the rate of Ca(2+) dissociation from TnC and the rate of cross-bridge detachment from several different species of ventricular myofibrils. The fluorescently labeled TnC was sensitive to both Ca(2+) dissociation and cross-bridge detachment at low Ca(2+) (presence of EGTA), allowing for a direct comparison between the two proposed rates of myofilament inactivation. Unlike Ca(2+) dissociation from TnC, cross-bridge detachment varied in myofibrils from different species and was rate-limited by ADP release. At subphysiological temperatures (<20 °C), the rate of Ca(2+) dissociation from TnC was faster than the rate of cross-bridge detachment in the presence of ADP. These results support the hypothesis that cross-bridge detachment rate-limits relaxation. However, Ca(2+) dissociation from TnC was not as temperature-sensitive as cross-bridge detachment. At a near physiological temperature (35 °C) and ADP, the rate of cross-bridge detachment may actually be faster than the rate of Ca(2+) dissociation. This provides evidence that there may not be a simple, single rate-limiting step of myofilament inactivation.     

Characteristics of troponin C binding to the myofibrillar thin filament: extraction of troponin C is not random along the length of the thin filament.

Troponin C (TnC) is the Ca(2+)-sensing subunit of troponin responsible for initiating the cascade of events resulting in contraction of striated muscle. This protein can be readily extracted from myofibrils with low-ionic-strength EDTA-containing buffers. The properties of TnC extraction have not been characterized at the structural level, nor have the interactions of TnC with the native myofibrillar thin filament been studied. To address these issues, fluorescein-labeled TnC, in conjunction with high-resolution digital fluorescence microscopy, was used to characterize TnC binding to myofibrils and to determine the randomness of TnC extraction. Fluorescein-5-maleimide TnC (F5M TnC) retained biological activity, as evidenced by reconstitution of Ca(2+)-dependent ATPase activity in extracted myofibrils and binding to TnI in a Ca(2+)-sensitive manner. The binding of F5M TnC to highly extracted myofibrils at low Ca2+ was restricted to the overlap region under rigor conditions, and the location of binding was not influenced by F5M TnC concentration. The addition of myosin subfragment 1 to occupy all actin sites resulted in F5M TnC being bound in both the overlap and nonoverlap regions. However, very little F5M TnC was bound to myofibrils under relaxing conditions. These results suggest that strong binding of myosin heads enhances TnC binding. At high Ca2+, the pattern of F5M TnC binding was concentration dependent: binding was restricted to the overlap region at low F5M TnC concentration, whereas the binding propagated into the nonoverlap region at higher levels. Analysis of fluorescence intensity showed the greatest binding of F5M TnC at high Ca2+ with S1, and these conditions were used to characterize partially TnC-extracted myofibrils. Comparison of partially extracted myofibrils showed that low levels of extraction were associated with greater F5M TnC being bound in the nonoverlap region than in the overlap region relative to higher levels of extraction. These results show that TnC extraction is not random along the length of the thin filament, but occurs more readily in the nonoverlap region. This observation, in conjunction with the influence of rigor heads on the pattern of F5M TnC binding, suggests that strong myosin binding to actin stabilizes TnC binding at low Ca2+.     

Ca2+ shuttling between endoplasmic reticulum and mitochondria underlying Ca2+ oscillations

Although many cell functions are regulated by Ca(2+) oscillations induced by a cyclic release of Ca(2+) from intracellular Ca(2+) stores, the pacemaker mechanism of Ca(2+) oscillations remains to be explained. Using green fluorescent protein-based Ca(2+) indicators that are targeted to intracellular Ca(2+) stores, the endoplasmic reticulum (ER) and mitochondria, we found that Ca(2+) shuttles between the ER and mitochondria in phase with Ca(2+) oscillations. Following agonist stimulation, Ca(2+) release from the ER generated the first Ca(2+) oscillation and loaded mitochondria with Ca(2+). Before the second Ca(2+) oscillation, Ca(2+) release from the mitochondria by means of the Na(+)/Ca(2+) exchanger caused a gradual increase in cytoplasmic Ca(2+) concentration, inducing a regenerative ER Ca(2+) release, which generated the peak of Ca(2+) oscillation and partially reloaded the mitochondria. This sequence of events was repeated until mitochondrial Ca(2+) was depleted. Thus, Ca(2+) shuttling between the ER and mitochondria may have a pacemaker role in the generation of Ca(2+) oscillations.     

Accelerated de novo sarcomere assembly by electric pulse stimulation in C2C12 myotubes

The assembly of sarcomeres, the smallest contractile units in striated muscle, is a complex and highly coordinated process that relies on spatio-temporal organization of sarcomeric proteins, a process requiring spontaneous Ca(2+) transients. To investigate the relationship between Ca(2+) transients and sarcomere assembly in C2C12 myotubes, we employed electric pulse stimulation (EPS), which allows the frequency of Ca(2+) transients to be manipulated. We monitored contractile activity as a means of evaluating functional sarcomere establishment using the differential image subtraction (DIS) method. C2C12 myotubes initially displayed no contractility with EPS, due to a lack of sarcomere architecture. However, C2C12 myotubes showed remarkable contractile activity with EPS-induced repetitive Ca(2+) transients (1 Hz) within only 2 h. This activity was concurrent with the development of sarcomere structure. Importantly, the period required for the acquisition of contractile activity in response to excitation was dependent upon the frequency of Ca(2+) oscillations, but a sustained increase in intracellular Ca(2+) (not oscillatory) by high-frequency EPS (10 Hz) was incapable of conferring either contractility or sarcomere assembly on the myotubes. The EPS-facilitated de novo functional sarcomere assembly appeared to require calpain-mediated proteolysis. In addition, modulation of integrin signals, by adding collagen IV or RGD-peptide, significantly affected the EPS-induced development of contractility. Taken together, these observations indicate that the frequency of the Ca(2+) oscillation determines the time required to establish functionally active sarcomere assembly and also suggest that the Ca(2+) oscillatory signal may be decoded through reorganization of the integrin-cytoskeletal protein complex via calpain-mediated proteolysis.     

Myogenic stage, sarcomere length, and protease activity modulate localization of muscle-specific calpain

p94/calpain 3 is a Ca(2+)-binding intracellular protease predominantly expressed in skeletal muscles. p94 binds to the N2A and M-line regions of connectin/titin and localizes in the Z-bands. Genetic evidence showing that compromised p94 proteolytic activity leads to muscular dystrophy (limb-girdle muscular dystrophy type 2A) indicates the importance of p94 function in myofibrils. Here we show that a series of p94 splice variants is expressed immediately after muscle differentiation and differentially change localization during myofibrillogenesis. We found that the endogenous N-terminal (but not C-terminal) domain of p94 was not only localized in the Z-bands but also directly bound to sarcomeric alpha-actinin. These data suggest the incorporation of proteolytic N-terminal fragments of p94 into the Z-bands. In myofibrils localization of exogenously expressed p94 shifted from the M-line to N2A as the sarcomere lengthens beyond approximately 2.6 and 2.8 microm for wild-type and proteaseinactive p94, respectively. These data demonstrate for the first time that p94 proteolytic activity is involved in responses to muscle conditions, which may explain why p94 inactivation causes limb-girdle muscular dystrophy.     

Myofibrillar Ca2+-stimulated Mg2+-ATPase from chronically ischemic canine heart

Functional properties of myofibrils from chronically ischemic canine myocardium were evaluated. Ischemia was produced by tight stenosis of left anterior descending artery (LAD), followed by 40 min acute ischemia with prior preconditioning. Animals of the first group were sacrificed after 8 weeks. In the second group, angioplasty of LAD was performed after 8 weeks of ischemia and animals were kept alive for other 4 weeks. Control animals were sham operated. Activity and kinetic parameters of myofibrillar Ca2+-stimulated Mg2+-ATPase were measured in myofibrils isolated from anterior and posterior parts of all hearts. We did not find any differences in maximal velocity (Vmax), half-maximal activation constant for calcium (K(Ca2+)50) and cooperativity coefficient (n(hill)) of myofibrils from different experimental groups as compared to controls, either at pH 7, pH 6.5 (acidosis) or pH 7.5 (alkalosis). K(Ca2+)50 increased in medium simulated acidosis (12.6-33.5 times) and n(hill) decreased significantly in all groups as compared with values obtained at pH 7. These results indicate that activity and Ca2+-sensitivity of myofibrillar Mg2+-ATPase remain unchanged despite deteriorated heart function 8 weeks after LAD obstruction. Experiments have confirmed that Ca2+-stimulated-ATPase from canine heart myofibrils responded to pH decrease by a decreased sensitivity to Ca2+ and a decreased cooperativity. However, sensitivity of the enzyme to the pH changes is unaltered by 8 weeks of chronic ischemia.     

Role of NHE1 in calcium signaling and cell proliferation in human CNS pericytes

The central nervous system (CNS) pericytes play an important role in brain microcirculation. Na(+)/H(+) exchanger isoform 1 (NHE1) has been suggested to regulate the proliferation of nonvascular cells through the regulation of intracellular pH, Na(+), and cell volume; however, the relationship between NHE1 and intracellular Ca(2+), an essential signal of cell growth, is still not known. The aim of the present study was to elucidate the role of NHE1 in Ca(2+) signaling and the proliferation of human CNS pericytes. The intracellular Ca(2+) concentration was measured by fura 2 in cultured human CNS pericytes. The cells showed spontaneous Ca(2+) oscillation under quasi-physiological ionic conditions. A decrease in extracellular pH or Na(+) evoked a transient Ca(2+) rise followed by Ca(2+) oscillation, whereas an increase in pH or Na(+) did not induce the Ca(2+) responses. The Ca(2+) oscillation was inhibited by an inhibitor of NHE in a dose-dependent manner and by knockdown of NHE1 by using RNA interference. The Ca(2+) oscillation was completely abolished by thapsigargin. The proliferation of pericytes was attenuated by inhibition of NHE1. These results demonstrate that NHE1 regulates Ca(2+) signaling via the modulation of Ca(2+) release from the endoplasmic reticulum, thus contributing to the regulation of proliferation in CNS pericytes.     

Force kinetics and individual sarcomere dynamics in cardiac myofibrils after rapid ca(2+) changes

Kinetics of force development and relaxation after rapid application and removal of Ca(2+) were measured by atomic force cantilevers on subcellular bundles of myofibrils prepared from guinea pig left ventricles. Changes in the structure of individual sarcomeres were simultaneously recorded by video microscopy. Upon Ca(2+) application, force developed with an exponential rate constant k(ACT) almost identical to k(TR), the rate constant of force redevelopment measured during steady-state Ca(2+) activation; this indicates that k(ACT) reflects isometric cross-bridge turnover kinetics. The kinetics of force relaxation after sudden Ca(2+) removal were markedly biphasic. An initial slow linear decline (rate constant k(LIN)) lasting for a time t(LIN) was abruptly followed by an ~20 times faster exponential decay (rate constant k(REL)). k(LIN) is similar to k(TR) measured at low activating [Ca(2+)], indicating that k(LIN) reflects isometric cross-bridge turnover kinetics under relaxed-like conditions (see also. Biophys. J. 83:2142-2151). Video microscopy revealed the following: invariably at t(LIN) a single sarcomere suddenly lengthened and returned to a relaxed-type structure. Originating from this sarcomere, structural relaxation propagated from one sarcomere to the next. Propagated sarcomeric relaxation, along with effects of stretch and P(i) on relaxation kinetics, supports an intersarcomeric chemomechanical coupling mechanism for rapid striated muscle relaxation in which cross-bridges conserve chemical energy by strain-induced rebinding of P(i).     

Blockage of spontaneous Ca2+ oscillation causes cell death in intraerythrocitic Plasmodium falciparum

Malaria remains one of the world's most important infectious diseases and is responsible for enormous mortality and morbidity. Resistance to antimalarial drugs is a challenging problem in malaria control. Clinical malaria is associated with the proliferation and development of Plasmodium parasites in human erythrocytes. Especially, the development into the mature forms (trophozoite and schizont) of Plasmodium falciparum (P. falciparum) causes severe malaria symptoms due to a distinctive property, sequestration which is not shared by any other human malaria. Ca(2+) is well known to be a highly versatile intracellular messenger that regulates many different cellular processes. Cytosolic Ca(2+) increases evoked by extracellular stimuli are often observed in the form of oscillating Ca(2+) spikes (Ca(2+) oscillation) in eukaryotic cells. However, in lower eukaryotic and plant cells the physiological roles and the molecular mechanisms of Ca(2+) oscillation are poorly understood. Here, we showed the observation of the inositol 1,4,5-trisphospate (IP(3))-dependent spontaneous Ca(2+) oscillation in P. falciparum without any exogenous extracellular stimulation by using live cell fluorescence Ca(2+) imaging. Intraerythrocytic P. falciparum exhibited stage-specific Ca(2+) oscillations in ring form and trophozoite stages which were blocked by IP(3) receptor inhibitor, 2-aminoethyl diphenylborinate (2-APB). Analyses of parasitaemia and parasite size and electron micrograph of 2-APB-treated P. falciparum revealed that 2-APB severely obstructed the intraerythrocytic maturation, resulting in cell death of the parasites. Furthermore, we confirmed the similar lethal effect of 2-APB on the chloroquine-resistant strain of P. falciparum. To our best knowledge, we for the first time showed the existence of the spontaneous Ca(2+) oscillation in Plasmodium species and clearly demonstrated that IP(3)-dependent spontaneous Ca(2+) oscillation in P. falciparum is critical for the development of the blood stage of the parasites. Our results provide a novel concept that IP(3)/Ca(2+) signaling pathway in the intraerythrocytic malaria parasites is a promising target for antimalarial drug development.     

Possible involvement of ATP-purinoceptor signalling in the intercellular synchronization of intracellular Ca2+ oscillation in cultured cardiac myocytes

Isolated and cultured neonatal cardiac myocytes contract spontaneously and cyclically. The contraction rhythms of two isolated cardiac myocytes, each of which beats at different frequencies at first, become synchronized after the establishment of mutual contacts, suggesting that mutual entrainment occurs due to electrical and/or mechanical interactions between two myocytes. The intracellular concentration of free Ca(2+) also changes rhythmically in association with the rhythmic contraction of myocytes (Ca(2+) oscillation), and such a Ca(2+) oscillation was also synchronized among cultured cardiac myocytes. In this study, we investigated whether intercellular communication other than via gap junctions was involved in the intercellular synchronization of intracellular Ca(2+) oscillation in spontaneously beating cultured cardiac myocytes. Treatment with either blockers of gap junction channels or an un-coupler of E-C coupling did not affect the intercellular synchronization of Ca(2+) oscillation. In contrast, treatment with a blocker of P2 purinoceptors resulted in the asynchronization of Ca(2+) oscillatory rhythms among cardiac myocytes. The present study suggested that the extracellular ATP-purinoceptor system was responsible for the intercellular synchronization of Ca(2+) oscillation among cardiac myocytes.     

Extracellular ATP induces spikes in cytosolic free Ca(2+) but not in NADPH oxidase activity in neutrophils

In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca(2+) concentration, we simultaneously measured Ca(2+) oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast damped Ca(2+) spikes with a period of 15s and slower irregular spikes with a period greater than 50s. Spikes in Ca(2+) occurred in the absence of Ca(2+) influx, but the amplitude was damped by inhibition of Ca(2+) influx. Using the oxidation of hydroethidine as a cytosolic marker of oxidant production, we show that the generation of reactive oxygen species by neutrophils adherent to glass was accelerated by ATP. The step-up in NADPH oxidase activity followed the first elevation of cytosolic Ca(2+) but, despite subsequent spikes in Ca(2+) concentration, no oscillations in oxidase activity could be detected. ATP induced spikes in Ca(2+) in a very reproducible way and we propose that the Ca(2+) signal is an on-switch for oxidase activity, but the activity is apparently not directly correlated with spiking activity in cytosolic Ca(2+).     

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.     

Dynamic simulation of the effect of calcium-release activated calcium channel on cytoplasmic Ca2+ oscillation

A mathematical model is proposed to illustrate the activation of STIM1 (stromal interaction molecule 1) protein, the assembly and activation of calcium-release activated calcium (CRAC) channels in T cells. In combination with De Young-Keizer-Li-Rinzel model, we successfully reproduce a sustained Ca(2+) oscillation in cytoplasm. Our results reveal that Ca(2+) oscillation dynamics in cytoplasm can be significantly affected by the way how the Orai1 CRAC channel are assembled and activated. A low sustained Ca(2+) influx is observed through the CRAC channels across the plasma membrane. In particular, our model shows that a tetrameric channel complex can effectively regulate the total quantity of the channels and the ratio of the active channels to the total channels, and a period of Ca(2+) oscillation about 29 s is in agreement with published experimental data. The bifurcation analyses illustrate the different dynamic properties between our mixed Ca(2+) feedback model and the single positive or negative feedback models.     

Replacement of troponin components in myofibrils.

The Ca(2+)-sensitive ATPase activity of rabbit skeletal myofibrils was desensitized by treatment with excess troponin T and was found to be activated irrespective of the Ca2+ concentrations. A SDS-gel electrophoretic study showed that both troponin C and troponin I were removed from the myofibrils on treatment with troponin T. The Ca(2+)- and Sr(2+)- sensitivities of the ATPase of troponin T-treated myofibrils reconstituted with troponin C. I were the same as in the intact myofibrils. The Ca(2+)-activated ATPase of rabbit skeletal myofibrils was also desensitized on treatment with chicken breast troponin T or its 26K fragment. The SDS-gel electrophoretic study revealed that troponin T, in addition to troponin C and troponin I, was also removed from the myofibrils and, instead, chicken breast troponin T or its 26K fragment was incorporated into the myofibrils. The Ca(2+)- sensitivity of myofibrils treated with chicken breast troponin T or its 26K fragment was then regained on reconstitution with troponin C.I. These findings indicate that the change in composition of myofibrils on treatment with troponin T or its 26K fragment is due to the selective replacement of the troponin C.I.T complex in the myofibrils as a whole with troponin T or its 26K fragment.     

Spontaneous oscillation of tension and sarcomere length in skeletal myofibrils. Microscopic measurement and analysis.

We have devised a simple method for measuring tension development of single myofibrils by micromanipulation with a pair of glass micro-needles. The tension was estimated from the deflection of a flexible needle under an inverted phase-contrast microscope equipped with an image processor, so that the tension development is always accompanied by the shortening of the myofibril (auxotonic condition) in the present setup. The advantage of this method is that the measurement of tension (1/30 s for time resolution and about 0.05 micrograms for accuracy of tension measurement; 0.05 microns as a spatial resolution for displacement of the micro-needle) and the observation of sarcomere structure are possible at the same time, and the technique to hold myofibrils, even single myofibrils, is very simple. This method has been applied to study the tension development of glycerinated skeletal myofibrils under the condition where spontaneous oscillation of sarcomeres is induced, i.e., the coexistence of MgATP, MgADP and inorganic phosphate without free Ca2+. Under this condition, we found that the tension of myofibrils spontaneously oscillates accompanied by the oscillation of sarcomere length with a main period of a few seconds; the period was lengthened and shortened with stretch and release of myofibrils. A possible mechanism of the oscillation is discussed.     

alpha-Adrenoceptor stimulation-mediated negative inotropism and enhanced Na(+)/Ca(2+) exchange in mouse ventricle

Mechanisms underlying the negative inotropic response to alpha-adrenoceptor stimulation in adult mouse ventricular myocardium were studied. In isolated ventricular tissue, phenylephrine (PE), in the presence of propranolol, decreased contractile force by approximately 40% of basal value. The negative inotropic response was similarly observed under low extracellular Ca(2+) concentration ([Ca(2+)](o)) conditions but was significantly smaller under high-[Ca(2+)](o) conditions and was not observed under low-[Na(+)](o) conditions. The negative inotropic response was not affected by nicardipine, ryanodine, ouabain, or dimethylamiloride (DMA), inhibitors of L-type Ca(2+) channel, Ca(2+) release channel, Na(+)-K(+) pump, or Na(+)/H(+) exchanger, respectively. KB-R7943, an inhibitor of Na(+)/Ca(2+) exchanger, suppressed the negative inotropic response mediated by PE. PE reduced the magnitude of postrest contractions. PE caused a decrease in duration of the late plateau phase of action potential and a slight increase in resting membrane potential; time courses of these effects were similar to that of the negative inotropic effect. In whole cell voltage-clamped myocytes, PE increased the L-type Ca(2+) and Na(+)/Ca(2+) exchanger currents but had no effect on the inwardly rectifying K(+), transient outward K(+), or Na(+)-K(+)-pump currents. These results suggest that the sustained negative inotropic response to alpha-adrenoceptor stimulation of adult mouse ventricular myocardium is mediated by enhancement of Ca(2+) efflux through the Na(+)/Ca(2+) exchanger.