Regulation of myocardial contraction as revealed by intracellular Ca2+ measurements using aequorin.

Of the ions involved in myocardial function, Ca2+ is the most important. Ca2+ is crucial to the process that allows myocardium to repeatedly contract and relax in a well-organized fashion; it is the process called excitation-contraction coupling. In order, therefore, for accurate comprehension of the physiology of the heart, it is fundamentally important to understand the detailed mechanism by which the intracellular Ca2+ concentration is regulated to elicit excitation-contraction coupling. Aequorin was discovered by Shimomura, Johnson and Saiga in 1962. By taking advantage of the fact that aequorin emits blue light when it binds to Ca2+ within the physiologically relevant concentration range, in the 1970s and 1980s, physiologists microinjected it into myocardial preparations. By doing so, they proved that Ca2+ transients occur upon membrane depolarization, and tension development (i.e., actomyosin interaction) subsequently follows, dramatically advancing the research on cardiac excitation-contraction coupling.

Myosin and tropomyosin-troponin complementarily regulate thermal activation of muscles.

Contraction of striated muscles is initiated by an increase in cytosolic Ca2+ concentration, which is regulated by tropomyosin and troponin acting on actin filaments at the sarcomere level. Namely, Ca2+-binding to troponin C shifts the "on-off" equilibrium of the thin filament state toward the "on" state, promoting actomyosin interaction; likewise, an increase in temperature to within the body temperature range shifts the equilibrium to the on state, even in the absence of Ca2+. Here, we investigated the temperature dependence of sarcomere shortening along isolated fast skeletal myofibrils using optical heating microscopy. Rapid heating (25 to 41.5°C) within 2 s induced reversible sarcomere shortening in relaxing solution. Further, we investigated the temperature-dependence of the sliding velocity of reconstituted fast skeletal or cardiac thin filaments on fast skeletal or ß-cardiac myosin in an in vitro motility assay within the body temperature range. We found that (a) with fast skeletal thin filaments on fast skeletal myosin, the temperature dependence was comparable to that obtained for sarcomere shortening in fast skeletal myofibrils (Q10 ∼8), (b) both types of thin filaments started to slide at lower temperatures on fast skeletal myosin than on ß-cardiac myosin, and (c) cardiac thin filaments slid at lower temperatures compared with fast skeletal thin filaments on either type of myosin. Therefore, the mammalian striated muscle may be fine-tuned to contract efficiently via complementary regulation of myosin and tropomyosin-troponin within the body temperature range, depending on the physiological demands of various circumstances.

Rotational-motion measurement of the sacroiliac joint using upright MRI scanning and intensity-based registration: is there a sex difference?

PURPOSE: The sacroiliac joint (SIJ) has attracted increasing attention as a source of low back and groin pain, but the kinematics of SIJ against standing load and its sex difference remain unclear due to the difficulty of in vivo load study. An upright magnetic resonance imaging (MRI) system can provide in vivo imaging both in the supine and standing positions. The reliability of the mobility of SIJ against the standing load was evaluated and its sex difference was examined in healthy young volunteers using an upright MRI. METHOD: Static (reliability) and kinematic studies were performed. In the static study, a dry bone of pelvic ring embedded in gel form and frozen in the plastic box was used. In the kinematic study, 19 volunteers (10 males, 9 females) with a mean age of 23.9 years were included. The ilium positions for the sacrum in supine and standing positions were measured against the pelvic coordinates to evaluate the mobility of the SIJ. RESULTS: In the static study, the residual error of the rotation of the SIJ study was < 0.2°. In the kinematic study, the mean values of SIJ sagittal rotation from supine to standing position in males and females were - 0.9° ± 0.7° (mean ± standard deviation) and - 1.7° ± 0.8°, respectively. The sex difference was statistically significant (p = 0.04). The sagittal rotation of the SIJ showed a significant correlation with the sacral slope. CONCLUSION: The residual error for measuring the SIJ rotation using the upright MRI was < 0.2°. The young healthy participants showed sex differences in the sagittal rotation of the SIJ against the standing load and the females showed a larger posterior rotation of the ilium against the sacrum from the supine to standing position than the males. Therefore, upright MRI is useful to investigate SIJ motion.

Mice with R2509C-RYR1 mutation exhibit dysfunctional Ca2+ dynamics in primary skeletal myocytes.

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum (SR) of the skeletal muscle and plays a critical role in excitation-contraction coupling. Mutations in RYR1 cause severe muscle diseases, such as malignant hyperthermia, a disorder of Ca2+-induced Ca2+ release (CICR) through RYR1 from the SR. We recently reported that volatile anesthetics induce malignant hyperthermia (MH)-like episodes through enhanced CICR in heterozygous R2509C-RYR1 mice. However, the characterization of Ca2+ dynamics has yet to be investigated in skeletal muscle cells from homozygous mice because these animals die in utero. In the present study, we generated primary cultured skeletal myocytes from R2509C-RYR1 mice. No differences in cellular morphology were detected between wild type (WT) and mutant myocytes. Spontaneous Ca2+ transients and cellular contractions occurred in WT and heterozygous myocytes, but not in homozygous myocytes. Electron microscopic observation revealed that the sarcomere length was shortened to ∼1.7 µm in homozygous myocytes, as compared to ∼2.2 and ∼2.3 µm in WT and heterozygous myocytes, respectively. Consistently, the resting intracellular Ca2+ concentration was higher in homozygous myocytes than in WT or heterozygous myocytes, which may be coupled with a reduced Ca2+ concentration in the SR. Finally, using infrared laser-based microheating, we found that heterozygous myocytes showed larger heat-induced Ca2+ transients than WT myocytes. Our findings suggest that the R2509C mutation in RYR1 causes dysfunctional Ca2+ dynamics in a mutant-gene dose-dependent manner in the skeletal muscles, in turn provoking MH-like episodes and embryonic lethality in heterozygous and homozygous mice, respectively.

Effects of omecamtiv mecarbil on the contractile properties of skinned porcine left atrial and ventricular muscles.

Omecamtiv mecarbil (OM) is a novel inotropic agent for heart failure with systolic dysfunction. OM prolongs the actomyosin attachment duration, which enhances thin filament cooperative activation and accordingly promotes the binding of neighboring myosin to actin. In the present study, we investigated the effects of OM on the steady-state contractile properties in skinned porcine left ventricular (PLV) and atrial (PLA) muscles. OM increased Ca2+ sensitivity in a concentration-dependent manner in PLV, by left shifting the mid-point (pCa50) of the force-pCa curve (ΔpCa50) by ∼0.16 and ∼0.33 pCa units at 0.5 and 1.0 µM, respectively. The Ca2+-sensitizing effect was likewise observed in PLA, but less pronounced with ΔpCa50 values of ∼0.08 and ∼0.22 pCa units at 0.5 and 1.0 µM, respectively. The Ca2+-sensitizing effect of OM (1.0 µM) was attenuated under enhanced thin filament cooperative activation in both PLV and PLA; this attenuation occurred directly via treatment with fast skeletal troponin (ΔpCa50: ∼0.16 and ∼0.10 pCa units in PLV and PLA, respectively) and indirectly by increasing the number of strongly bound cross-bridges in the presence of 3 mM MgADP (ΔpCa50: ∼0.21 and ∼0.08 pCa units in PLV and PLA, respectively). It is likely that this attenuation of the Ca2+-sensitizing effect of OM is due to a decrease in the number of "recruitable" cross-bridges that can potentially produce active force. When cross-bridge detachment was accelerated in the presence of 20 mM inorganic phosphate, the Ca2+-sensitizing effect of OM (1.0 µM) was markedly decreased in both types of preparations (ΔpCa50: ∼0.09 and ∼0.03 pCa units in PLV and PLA, respectively). The present findings suggest that the positive inotropy of OM is more markedly exerted in the ventricle than in the atrium, which results from the strongly bound cross-bridge-dependent allosteric activation of thin filaments.

Heat-hypersensitive mutants of ryanodine receptor type 1 revealed by microscopic heating.

Thermoregulation is an important aspect of human homeostasis, and high temperatures pose serious stresses for the body. Malignant hyperthermia (MH) is a life-threatening disorder in which body temperature can rise to a lethal level. Here we employ an optically controlled local heat-pulse method to manipulate the temperature in cells with a precision of less than 1 °C and find that the mutants of ryanodine receptor type 1 (RyR1), a key Ca2+ release channel underlying MH, are heat hypersensitive compared with the wild type (WT). We show that the local heat pulses induce an intracellular Ca2+ burst in human embryonic kidney 293 cells overexpressing WT RyR1 and some RyR1 mutants related to MH. Fluorescence Ca2+ imaging using the endoplasmic reticulum-targeted fluorescent probes demonstrates that the Ca2+ burst originates from heat-induced Ca2+ release (HICR) through RyR1-mutant channels because of the channels' heat hypersensitivity. Furthermore, the variation in the heat hypersensitivity of four RyR1 mutants highlights the complexity of MH. HICR likewise occurs in skeletal muscles of MH model mice. We propose that HICR contributes an additional positive feedback to accelerate thermogenesis in patients with MH.

Validity of Freehand 3-D Ultrasound System in Measurement of the 3-D Surface Shape of Shoulder Muscles.

Freehand 3-D ultrasound (3DUS) system is a promising technique for accurately assessing muscle morphology. However, its accuracy has been validated mainly in terms of volume by examining lower limb muscles. This study was aimed at validating 3DUS in the measurements of 3-D surface shape and volume by comparing them with magnetic resonance imaging (MRI) measurements while ensuring the reproducibility of participant posture by focusing on the shoulder muscles. The supraspinatus, infraspinatus and posterior deltoid muscles of 10 healthy men were scanned using 3DUS and MRI while secured by an immobilization support customized for each participant. A 3-D surface model of each muscle was created from the 3DUS and MRI methods, and the agreement between them was assessed. For the muscle volume, the mean difference between the two models was within -0.51 cm3. For the 3-D surface shape, the distances between the closest points of the two models and the Dice similarity coefficient were calculated. The results indicated that the median surface distance was less than 1.12 mm and the Dice similarity coefficient was larger than 0.85. These results suggest that, given the aforementioned error is permitted, 3DUS can be used as an alternative to MRI in measuring volume and surface shape, even for the shoulder muscles.

Artificial intelligence trained with integration of multiparametric MR-US imaging data and fusion biopsy trajectory-proven pathology data for 3D prediction of prostate cancer: A proof-of-concept study.

BACKGROUND: We aimed to develop an artificial intelligence (AI) algorithm that predicts the volume and location of clinically significant cancer (CSCa) using convolutional neural network (CNN) trained with integration of multiparametric MR-US image data and MRI-US fusion prostate biopsy (MRI-US PBx) trajectory-proven pathology data. METHODS: Twenty consecutive patients prospectively underwent MRI-US PBx, followed by robot-assisted radical prostatectomy (RARP). The AI algorithm was trained with the integration of MR-US image data with a MRI-US PBx trajectory-proven pathology. The relationship with the 3D-cancer-mapping of RARP specimens was compared between AI system-suggested 3D-CSCa mapping and an experienced radiologist's suggested 3D-CSCa mapping on MRI alone according to the Prostate Imaging Reporting and Data System (PI-RADS) version 2. The characteristics of detected and undetected tumors at AI were compared in 22,968 image data. The relationships between CSCa volumes and volumes predicted by AI as well as the radiologist's reading based on PI-RADS were analyzed. RESULTS: The concordance of the CSCa center with that in RARP specimens was significantly higher in the AI prediction than the radiologist' reading (83% vs. 54%, p = 0.036). CSCa volumes predicted with AI were more accurate (r = 0.90, p < 0.001) than the radiologist's reading. The limitations include that the elastic fusion technology has its own registration error. CONCLUSIONS: We presented a novel pilot AI algorithm for 3D prediction of PCa. AI was trained by integration of multiparametric MR-US image data and fusion biopsy trajectory-proven pathology data. This deep learning AI model may more precisely predict the 3D mapping of CSCa in its volume and center location than a radiologist's reading based on PI-RADS version 2, and has potential in the planning of focal therapy.

Synchrony of sarcomeric movement regulates left ventricular pump function in the in vivo beating mouse heart.

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 <10 mm Hg, diastolic sarcomere length increased. Under depressed conditions, the movements between adjacent sarcomeres were in marked asynchrony (CI, -0.3 to -0.4), and, as a result, average CI was linearly decreased in association with a decrease in ΔLVP. These findings suggest that in the left ventricle of the in vivo beating mouse heart, (1) sarcomeres heterogeneously contribute to myofibrillar dynamics due to an imbalance of active and passive force between neighboring sarcomeres, (2) the force imbalance is pronounced under depressed conditions coupled with a marked increase in passive force and the ensuing tug-of-war between sarcomeres, and (3) sarcomere synchrony via the distal intersarcomere interaction regulates the heart's pump function in coordination with myofibrillar contractility.

Single-cell temperature mapping with fluorescent thermometer nanosheets.

Recent studies using intracellular thermometers have shown that the temperature inside cultured single cells varies heterogeneously on the order of 1°C. However, the reliability of intracellular thermometry has been challenged both experimentally and theoretically because it is, in principle, exceedingly difficult to exclude the effects of nonthermal factors on the thermometers. To accurately measure cellular temperatures from outside of cells, we developed novel thermometry with fluorescent thermometer nanosheets, allowing for noninvasive global temperature mapping of cultured single cells. Various types of cells, i.e., HeLa/HEK293 cells, brown adipocytes, cardiomyocytes, and neurons, were cultured on nanosheets containing the temperature-sensitive fluorescent dye europium (III) thenoyltrifluoroacetonate trihydrate. First, we found that the difference in temperature on the nanosheet between nonexcitable HeLa/HEK293 cells and the culture medium was less than 0.2°C. The expression of mutated type 1 ryanodine receptors (R164C or Y523S) in HEK293 cells that cause Ca2+ leak from the endoplasmic reticulum did not change the cellular temperature greater than 0.1°C. Yet intracellular thermometry detected an increase in temperature of greater than ∼2°C at the endoplasmic reticulum in HeLa cells upon ionomycin-induced intracellular Ca2+ burst; global cellular temperature remained nearly constant within ±0.2°C. When rat neonatal cardiomyocytes or brown adipocytes were stimulated by a mitochondrial uncoupling reagent, the temperature was nearly unchanged within ±0.1°C. In cardiomyocytes, the temperature was stable within ±0.01°C during contractions when electrically stimulated at 2 Hz. Similarly, when rat hippocampal neurons were electrically stimulated at 0.25 Hz, the temperature was stable within ±0.03°C. The present findings with nonexcitable and excitable cells demonstrate that heat produced upon activation in single cells does not uniformly increase cellular temperature on a global basis, but merely forms a local temperature gradient on the order of ∼1°C just proximal to a heat source, such as the endoplasmic/sarcoplasmic reticulum ATPase.

Thermal Activation of Thin Filaments in Striated Muscle.

In skeletal and cardiac muscles, contraction is triggered by an increase in the intracellular Ca2+ concentration. During Ca2+ transients, Ca2+-binding to troponin C shifts the "on-off" equilibrium of the thin filament state toward the "on" sate, promoting actomyosin interaction. Likewise, recent studies have revealed that the thin filament state is under the influence of temperature; viz., an increase in temperature increases active force production. In this short review, we discuss the effects of temperature on the contractile performance of mammalian striated muscle at/around body temperature, focusing especially on the temperature-dependent shift of the "on-off" equilibrium of the thin filament state.

Real-Time In Vivo Imaging of Mouse Left Ventricle Reveals Fluctuating Movements of the Intercalated Discs.

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 µm (i.e., magnitude of change, ~30%). In contrast, tracking of α-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.

Microscopic heat pulses activate cardiac thin filaments.

During the excitation-contraction coupling of the heart, sarcomeres are activated via thin filament structural changes (i.e., from the "off" state to the "on" state) in response to a release of Ca2+ from the sarcoplasmic reticulum. This process involves chemical reactions that are highly dependent on ambient temperature; for example, catalytic activity of the actomyosin ATPase rises with increasing temperature. Here, we investigate the effects of rapid heating by focused infrared (IR) laser irradiation on the sliding of thin filaments reconstituted with human α-tropomyosin and bovine ventricular troponin in an in vitro motility assay. We perform high-precision analyses measuring temperature by the fluorescence intensity of rhodamine-phalloidin-labeled F-actin coupled with a fluorescent thermosensor sheet containing the temperature-sensitive dye Europium (III) thenoyltrifluoroacetonate trihydrate. This approach enables a shift in temperature from 25°C to ∼46°C within 0.2 s. We find that in the absence of Ca2+ and presence of ATP, IR laser irradiation elicits sliding movements of reconstituted thin filaments with a sliding velocity that increases as a function of temperature. The heating-induced acceleration of thin filament sliding likewise occurs in the presence of Ca2+ and ATP; however, the temperature dependence is more than twofold less pronounced. These findings could indicate that in the mammalian heart, the on-off equilibrium of the cardiac thin filament state is partially shifted toward the on state in diastole at physiological body temperature, enabling rapid and efficient myocardial dynamics in systole.

Optimization of Fluorescent Labeling for In Vivo Nanoimaging of Sarcomeres in the Mouse Heart.

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.

Sarcomeric Auto-Oscillations in Single Myofibrils From the Heart of Patients With Dilated Cardiomyopathy.

BACKGROUND: Left ventricular wall motion is depressed in patients with dilated cardiomyopathy (DCM). However, whether or not the depressed left ventricular wall motion is caused by impairment of sarcomere dynamics remains to be fully clarified. METHODS AND RESULTS: We analyzed the mechanical properties of single sarcomere dynamics during sarcomeric auto-oscillations (calcium spontaneous oscillatory contractions [Ca-SPOC]) that occurred at partial activation under the isometric condition in myofibrils from donor hearts and from patients with severe DCM (New York Heart Association classification III-IV). Ca-SPOC reproducibly occurred in the presence of 1 µmol/L free Ca2+ in both nonfailing and DCM myofibrils, and sarcomeres exhibited a saw-tooth waveform along single myofibrils composed of quick lengthening and slow shortening. The period of Ca-SPOC was longer in DCM myofibrils than in nonfailing myofibrils, in association with prolonged shortening time. Lengthening time was similar in both groups. Then, we performed Tn (troponin) exchange in myofibrils with a DCM-causing homozygous mutation (K36Q) in cTnI (cardiac TnI). On exchange with the Tn complex from healthy porcine ventricles, period, shortening time, and shortening velocity in cTnI-K36Q myofibrils became similar to those in Tn-reconstituted nonfailing myofibrils. Protein kinase A abbreviated period in both Tn-reconstituted nonfailing and cTnI-K36Q myofibrils, demonstrating acceleration of cross-bridge kinetics. CONCLUSIONS: Sarcomere dynamics was found to be depressed under loaded conditions in DCM myofibrils because of impairment of thick-thin filament sliding. Thus, microscopic analysis of Ca-SPOC in human cardiac myofibrils is beneficial to systematically unveil the kinetic properties of single sarcomeres in various types of heart disease.