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Sarcomeric contraction in cardiomyocytes serves as the basis for the heart’s pump functions. It has generally been considered that in cardiac muscle as well as in skeletal muscle, sarcomeres equally contribute to myofibrillar dynamics in myocytes at varying loads by producing similar levels of active and passive force. In the present study, we expressed α-actinin–AcGFP in Z-disks to analyze dynamic behaviors of sequentially connected individual sarcomeres along a myofibril in a left ventricular (LV) myocyte of the in vivo beating mouse heart. To quantify the magnitude of the contribution of individual sarcomeres to myofibrillar dynamics, we introduced the novel parameter “contribution index” (CI) to measure the synchrony in movements between a sarcomere and a myofibril (from −1 [complete asynchrony] to 1 [complete synchrony]). First, CI varied markedly between sarcomeres, with an average value of ∼0.3 during normal systole. Second, when the movements between adjacent sarcomeres were asynchronous (CI < 0), a sarcomere and the ones next to the adjacent sarcomeres and farther away moved in synchrony (CI > 0) along a myofibril. Third, when difference in LV pressure in diastole and systole (ΔLVP) was lowered to

We investigated the non-inferiority of continuous rectus sheath block to continuous epidural anesthesia for postoperative analgesia of gynecological cancer patients. One hundred ASA-PS 1–2 patients via a median incision up to 5 cm above the navel were randomized into a continuous epidural anesthesia (CEA) group and a continuous rectus sheath block (CRSB) group. Following surgery, they have controlled with intravenous patient-controlled analgesia (IV-PCA) as basal postoperative analgesia. For patients in the CEA group were administered 0.25% levobupivacaine at 5 mg/h. Patients in the CRSB group, catheters were inserted on both sides of the posterior rectus sheath after surgery. They received 0.25% levobupivacaine on both sides at 7.5 mg/h. To determine whether CRSB is non-inferior to CEA in postoperative treatment, pain at rest and movement was assessed using the Numerical Rating Scale (NRS). The non-inferiority margin of NRS difference between CRSB and CEA was set at 1.3 difference in means. The primary outcome was non-inferiority comparisons of NRS at rest/at movement after surgery, while the secondary outcome included the frequency of requesting IV-PCA and rescue drugs. NRS at rest in the CRSB group was not inferior to that in the CEA group. On the other hand, the NRS at movement at 4, 6, 8, 12 h following surgery in the CRSB group was inferior to CEA. There was no difference in the frequency of requesting IV-PCA and rescue drugs. CRSB showed the non-inferiority to CEA for postoperative analgesia at rest, while CRSB was not non-inferior to CEA at movement in gynecological cancer patients. CRSB would be a substitute when CEA is contraindicated as a component of postoperative multimodal analgesia.

Myocardial contraction is initiated by action potential propagation through the conduction system of the heart. It has been thought that connexin 43 in the gap junctions (GJ) within the intercalated disc (ID) provides direct electric connectivity between cardiomyocytes (electronic conduction). However, recent studies challenge this view by providing evidence that the mechanosensitive cardiac sodium channels Nav1.5 localized in perinexii at the GJ edge play an important role in spreading action potentials between neighboring cells (ephaptic conduction). In the present study, we performed real-time confocal imaging of the CellMask-stained ID in the living mouse heart in vivo. We found that the ID structure was not rigid. Instead, we observed marked flexing of the ID during propagation of contraction from cell to cell. The variation in ID length was between ~30 and ~42 &mu, m (i.e., magnitude of change, ~30%). In contrast, tracking of &alpha, actinin-AcGFP revealed a comparatively small change in the lateral dimension of the transitional junction near the ID (i.e., magnitude of change, ~20%). The present findings suggest that, when the heart is at work, mechanostress across the perinexii may activate Nav1.5 by promoting ephaptic conduction in coordination with electronic conduction, and, thereby, efficiently transmitting excitation-contraction coupling between cardiomyocytes.

The present study was conducted to systematically investigate the optimal viral titer as well as the volume of the adenovirus vector (ADV) that expresses α-actinin-AcGFP in the Z-disks of myocytes in the left ventricle (LV) of mice. An injection of 10 μL ADV at viral titers of 2 to 4 × 1011 viral particles per mL (VP/mL) into the LV epicardial surface consistently expressed α-actinin-AcGFP in myocytes in vivo, with the fraction of AcGFP-expressing myocytes at ~10%. Our analysis revealed that SL was ~1.90-2.15 μm upon heart arrest via deep anesthesia. Likewise, we developed a novel fluorescence labeling method of the T-tubular system by treating the LV surface with CellMask Orange (CellMask). We found that the T-tubular distance was ~2.10-2.25 μm, similar to SL, in the healthy heart in vivo. Therefore, the present high-precision visualization method for the Z-disks or the T-tubules is beneficial to unveiling the mechanisms of myocyte contraction in health and disease in vivo.

The heart has an intrinsic ability to increase systolic force in response to a rise in ventricular filling (the Frank–Starling law of the heart). It is widely accepted that the length dependence of myocardial activation underlies the Frank–Starling law of the heart. Recent advances in muscle physiology have enabled the identification of the factors involved in length-dependent activation, viz., titin (connectin)-based interfilament lattice spacing reduction and thin filament “on–off” regulation, with the former triggering length-dependent activation and the latter determining the number of myosin molecules recruited to thin filaments. Patients with a failing heart have demonstrated reduced exercise tolerance at least in part via depression of the Frank–Starling mechanism. Recent studies revealed that various mutations occur in the thin filament regulatory proteins, such as troponin, in the ventricular muscle of failing hearts, which consequently alter the Frank–Starling mechanism. In this article, we review the molecular mechanisms of length-dependent activation, and the influence of troponin mutations on the phenomenon.

Long-term disuse results in atrophy in skeletal muscle, which is characterized by reduced functional capability, impaired locomotor condition, and reduced resistance to fatigue. Here we show how long-term disuse affects contractility and fatigue resistance in single fibers of soleus muscle taken from the hindlimb immobilization model of the rat. We found that long-term disuse results in depression of caffeine-induced transient contractions in saponin-treated single fibers. However, when normalized to maximal Ca2+-activated force, the magnitude of the transient contractions became similar to that in control fibers. Control experiments indicated that the active force depression in disused muscle is not coupled with isoform switching of myosin heavy chain or troponin, or with disruptions of sarcomere structure or excessive internal sarcomere shortening during contraction. In contrast, our electronmicroscopic observation supported our earlier observation that interfilament lattice spacing is expanded after disuse. Then, to investigate the molecular mechanism of the reduced fatigue resistance in disused muscle, we compared the inhibitory effects of inorganic phosphate (Pi) on maximal Ca2+-activated force in control vs. disused fibers. The effect of Pi was more pronounced in disused fibers, and it approached that observed in control fibers after osmotic compression. These results suggest that contractile depression in disuse results from the lowering of myofibrillar force-generating capacity, rather than from defective Ca2+mobilization, and the reduced resistance to fatigue is from an enhanced inhibitory effect of Pi coupled with a decrease in the number of attached cross bridges, presumably due to lattice spacing expansion.

In skeletal muscle, active force production varies as a function of sarcomere length (SL). It has been considered that this SL dependence results simply from a change in the overlap length between the thick and thin filaments. The purpose of this study was to provide a systematic understanding of the SL-dependent increase in Ca2+ sensitivity in skeletal muscle, by investigating how thin filament “on–off” switching and passive force are involved in the regulation. Rabbit psoas muscles were skinned, and active force measurements were taken at various Ca2+ concentrations with single fibers, in the short (2.0 and 2.4 μm) and long (2.4 and 2.8 μm) SL ranges. Despite the same magnitude of SL elongation, the SL-dependent increase in Ca2+ sensitivity was more pronounced in the long SL range. MgADP (3 mM) increased the rate of rise of active force and attenuated SL-dependent Ca2+ activation in both SL ranges. Conversely, inorganic phosphate (Pi, 20 mM) decreased the rate of rise of active force and enhanced SL-dependent Ca2+ activation in both SL ranges. Our analyses revealed that, in the absence and presence of MgADP or Pi, the magnitude of SL-dependent Ca2+ activation was (1) inversely correlated with the rate of rise of active force, and (2) in proportion to passive force. These findings suggest that the SL dependence of active force in skeletal muscle is regulated via thin filament “on–off” switching and titin (connectin)-based interfilament lattice spacing modulation in a coordinated fashion, in addition to the regulation via the filament overlap. Electronic supplementary material The online version of this article (doi:10.1007/s12576-011-0173-8) contains supplementary material, which is available to authorized users.

The basis of the Frank-Starling mechanism of the heart is the intrinsic ability of cardiac muscle to produce greater active force in response to stretch, a phenomenon known as length-dependent activation. A feedback mechanism transmitted from cross-bridge formation to troponin C to enhance Ca(2+) binding has long been proposed to account for length-dependent activation. However, recent advances in muscle physiology research technologies have enabled the identification of other factors involved in length-dependent activation. The striated muscle sarcomere contains a third filament system composed of the giant elastic protein titin, which is responsible for most passive stiffness in the physiological sarcomere length range. Recent studies have revealed a significant coupling of active and passive forces in cardiac muscle, where titin-based passive force promotes cross-bridge recruitment, resulting in greater active force production in response to stretch. More currently, the focus has been placed on the troponin-based "on-off" switching of the thin filament state in the regulation of length-dependent activation. In this review, we discuss how myocardial length-dependent activation is coordinately regulated by sarcomere proteins.

We investigated the molecular mechanism by which troponin (Tn) regulates the Frank-Starling mechanism of the heart. Quasi-complete reconstitution of thin filaments with rabbit fast skeletal Tn (sTn) attenuated length-dependent activation in skinned porcine left ventricular muscle, to a magnitude similar to that observed in rabbit fast skeletal muscle. The rate of force redevelopment increased upon sTn reconstitution at submaximal levels, coupled with an increase in Ca2+ sensitivity of force, suggesting the acceleration of cross-bridge formation and, accordingly, a reduction in the fraction of resting cross-bridges that can potentially produce additional active force. An increase in titin-based passive force, induced by manipulating the prehistory of stretch, enhanced length-dependent activation, in both control and sTn-reconstituted muscles. Furthermore, reconstitution of rabbit fast skeletal muscle with porcine left ventricular Tn enhanced length-dependent activation, accompanied by a decrease in Ca2+ sensitivity of force. These findings demonstrate that Tn plays an important role in the Frank-Starling mechanism of the heart via on–off switching of the thin filament state, in concert with titin-based regulation.

It has been reported that the Frank-Starling mechanism is coordinately regulated in cardiac muscle via thin filament "on-off" equilibrium and titin-based lattice spacing changes. In the present study, we tested the hypothesis that the deletion mutation ΔK210 in the cardiac troponin T gene shifts the equilibrium toward the "off" state and accordingly attenuate the sarcomere length (SL) dependence of active force production, via reduced cross-bridge formation. Confocal imaging in isolated hearts revealed that the cardiomyocytes were enlarged, especially in the longitudinal direction, in ΔK210 hearts, with striation patterns similar to those in wild type (WT) hearts, suggesting that the number of sarcomeres is increased in cardiomyocytes but the sarcomere length remains unaltered. For analysis of the SL dependence of active force, skinned muscle preparations were obtained from the left ventricle of WT and knock-in (ΔK210) mice. An increase in SL from 1.90 to 2.20μm shifted the mid-point (pCa50) of the force-pCa curve leftward by ~0.21pCa units in WT preparations. In ΔK210 muscles, Ca(2+) sensitivity was lower by ~0.37pCa units, and the SL-dependent shift of pCa50, i.e., ΔpCa50, was less pronounced (~0.11pCa units), with and without protein kinase A treatment. The rate of active force redevelopment was lower in ΔK210 preparations than in WT preparations, showing blunted thin filament cooperative activation. An increase in thin filament cooperative activation upon an increase in the fraction of strongly bound cross-bridges by MgADP increased ΔpCa50 to ~0.21pCa units. The depressed Frank-Starling mechanism in ΔK210 hearts is the result of a reduction in thin filament cooperative activation.

Active force in cardiac muscle is highly dependent on sarcomere length (SL), highlighting the need for real-time SL imaging at high spatial and temporal resolution in vivo. In the present study, we expressed α-actinin-AcGFP in the Z-disks in cardiomyocytes of the left ventricle in adult mice, and applied SL nanometry (see Shintani et al., J Gen Physiol 2014) for the measurement of SL displacement at 100 fps under a fluorescence microscope (combined with a spinning disk confocal unit and an EMCCD camera), simultaneously with the measurements of left ventricular pressure (LVP) and the electrocardiogram (ECG). SL changed between ∼1.93 and ∼1.73 µm during diastole and systole, respectively, and varied by ∼300 nm in both phases, even in the same myocyte. LVP was positively correlated with the SL change between diastole and systole (P

Active force in cardiac muscle is highly dependent on sarcomere length (SL), which is known as the Frank-Starling mechanism of the heart. Indeed, a change of merely ∼100 nm in SL causes a dramatic change in cardiac contractile performance under partial activation states, highlighting the need for real-time SL imaging at high spatial and temporal resolution in vivo. In the present study, we expressed AcGFP at sarcomeric Z-disks (α-actinin) by means of an adenovirus vector system in adult mice, and applied nanometry for the measurement of SL displacement (i.e., SL nanometry) at 100 fps in the myocytes in the center of the left ventricle in anesthetized open-chest mice under a fluorescence microscope (combined with a spinning disk confocal unit and an EMCCD camera). First, we found that SL changed in the lengths of ∼1.92 and ∼1.63 μm during diastole and systole, respectively, and varied by ∼300 nm in both phases even in the same myocyte. Second, we found that sarcomere contraction started to occur at the T-wave endpoint on the electrocardiogram, followed by a rapid increase in left ventricular pressure (LVP). Similarly a linear relationship existed between the magnitude of the change in SL and that in LVP, demonstrating for the first time direct evidence of the Frank-Starling mechanism at the sarcomere level in vivo. Finally, we reconstructed the Z-sectioning images (at Z = 1 μm) of single sarcomeres by using the piezoelectric actuator, and successfully observed sarcomeric motions during the entire cardiac cycle. The present experimental system has a broad range of application possibilities for unveiling single sarcomere dynamics during excitation-contraction coupling in cardiomyocytes in the beating heart in vivo. At the meeting, we will discuss the organization of sarcomeric contraction in the beating heart in vivo.

Titin is the largest protein in mammals; it forms an elastic filament along the myofibril of cardiac and skeletal muscles. Novel studies employing the recently available varied technologies have revealed the molecular mechanisms by which titin generates passive force in the sarcomere in response to external stretch. Changes in titin stiffness occur during heart disease via a shift in the expression ratio of the two main titin isoforms, called N2B (stiff type) and N2BA (compliant type) titins. Protein kinase (PK)A, PKG and PKC phosphorylate the cardiac specific I-band titin segment, resulting in an acute decrease (by PKA and PKG) or increase (by PKC) in passive force. It has also been discovered that titin performs roles that go beyond passive force generation, by enhancing or terminating active force production, thereby adjusting the Frank-Starling mechanism of the heart. Therefore, titin is a self-adjustable and multi-functional spring that is indispensable for proper heart functions. Here, we discuss how titin regulates the passive and active properties of cardiac muscle in normal physiological conditions as well as in chronic heart disease.

In the present study, we developed an experimental model for real-time imaging of intracellular Ca2+ in ventricular myocytes in the heart. First, the heart isolated from the adult mouse was mounted on the stage of a microscope with a confocal scanning unit, combined with an objective lens [20× (40×), numerical aperture 1.0 (0.8)]. Laser-excited fluorescence was detected by the EMCCD camera, and the signals were analyzed by using the ImageJ software. Ca2+ waves were clearly observed at the cellular level in the isolated heart. Interestingly, randomly occurring Ca2+ waves and/or transients became synchronized by electric stimulation (∼5 Hz). Next, we conducted the isolated heart experiments at various temperatures. We found that 1) higher temperatures (∼37°C) reduced the efficiency of loading Ca2+ indicator into the cardiomyocytes and 2) the fluorescence intensity of Ca2+ waves were less pronounced at ∼37°C than at ∼25°C. Therefore, temperature control is highly important to allow for intracellular Ca2+ imaging in the heart in vivo. Intracellular Ca2+ imaging in the heart will greatly enhance our understanding of the excitation-contraction coupling in health and disease.

It is well known that protein kinase A (PKA) decreases Ca2+ sensitivity in cardiac muscle via phosphorylation of troponin I (TnI). In the present study, we directly tested whether PKA-based phosphorylation of cardiac TnI universally modulates Ca2+ sensitivity regardless of the type of muscle, by taking advantage of our Tn exchange technique (Terui et al., J Gen. Physiol. 131;275-283:2008). Troponins were extracted from porcine ventricular and rabbit fast skeletal muscles (Ca2+ sensitivity: former < latter). Without Tn exchange, PKA decreased Ca2+ sensitivity in cardiac (porcine ventricular) muscle, associated with enhanced phosphorylation of TnI. Reconstitution of cardiac muscle with the skeletal Tn complex (sTn) not only increased Ca2+ sensitivity but abolished the PKA effect, suggesting that phosphorylation of TnI, but not of myosin-binding protein C, is primarily responsible for the PKA-based reduction in Ca2+ sensitivity. Reconstitution of rabbit psoas muscle with the cardiac Tn complex (cTn) decreased Ca2+ sensitivity, as previously reported by us (Terui et al., J Gen. Physiol. 131;275-283:2008). PKA decreased Ca2+ sensitivity in cTn-reconstituted skeletal muscle, and subsequent exchange for sTn restored Ca2+ sensitivity to the original level. A similar result was obtained when skeletal muscle was reconstituted with the hybrid Tn complex (i.e., cTnI-cTnC-sTnT), suggesting that the troponin I-C complex, but not TnT, is essential for PKA-based modulation of Ca2+ sensitivity. These findings support the notion that PKA-based phosphorylation of TnI universally modulates Ca2+ sensitivity regardless of the type of muscle.