Excitation-contraction Coupling Mechanism Research Articles

Pericardial Delivery of Sodium Alginate-Infusible Extracellular Matrix Composite Hydrogel Promotes Angiogenesis and Intercellular Electrical Conduction after Myocardial Infarction.

Injectable extracellular matrix (iECM) is a versatile biological material with beneficial properties such as good degradability, promotion of cell survival, immunomodulation, and facilitation of vascular formation. However, intravenous injection of iECM faces challenges like a short retention time in vivo and low concentration at the lesion site. To address these issues, we prepared a composite hydrogel composed of sodium alginate and iECM and administered it via intrapericardial injection, forming a structure akin to cardiac patches within the pericardium. Compared with intramyocardial injection, intrapericardial injection avoids direct myocardial injury and ectopic tumor formation, offering less invasiveness and better biocompatibility. This study demonstrates that the sodium alginate/infusible extracellular matrix (SA/iECM) composite hydrogel can effectively prolong the local retention time of iECM in the heart, enhance electrical conduction between cardiomyocytes, promote angiogenesis at ischemic myocardial sites, inhibit apoptosis in the infarcted region, mitigate left ventricular remodeling postmyocardial infarction (MI), and improve cardiac function after infarction. Precise coordination of cardiomyocyte contraction and relaxation depends on the rhythmic occurrence of calcium-dependent action potentials. Cardiac dysfunction is partially attributed to the disruption of the excitation-contraction coupling (ECC) mechanism, which is associated with prolonged intracellular Ca2+ transients and alterations in contraction and relaxation Ca2+ levels. Our results show that the SA/iECM composite hydrogel improves electrical conduction, as evidenced by increased Cx43 expression and enhanced intercellular electrical connectivity. This research establishes that intrapericardial injection of a SA/iECM composite hydrogel is a safe and effective treatment modality, providing a theoretical basis for the use of biomaterials in MI therapy.

Emerging biotechnologies for screening electromechanical signals of cardiomyocytes

AbstractCardiac diseases threaten human health and burden the global healthcare system. Cardiomyocytes (CMs) are considered the ideal model for studying the signal transduction and regulation of cardiac systems. Based on the principle of the rhythmical beating process (excitation‒contraction coupling mechanism of CMs), investigating the mechanical and electrophysiological signals offered new hope for cardiac disease detection, prevention, and treatment. Considerable technological success has been achieved in electromechanical signal recording. However, most drug assessment platforms attach importance to high‐throughput and dynamic monitoring of mechanical or electrical signals while overlooking the measuring principles and physiological significance of the signal. In this review, the development of biosensing platforms for CMs, sensing principles, key measured parameters, measurement accuracy, and limitations are discussed. Additionally, various approaches for the stimulation and measurement of CMs in vitro are discussed to further elucidate the response of these cells to external stimuli. Furthermore, disease modeling and drug screening are used as examples to intuitively demonstrate the contribution of in vitro CM measurement platforms to the biomedical field, thereby further illustrating the challenges and prospects of these sensing platforms.

In situ structural insights into the excitation-contraction coupling mechanism of skeletal muscle.

Excitation-contraction coupling (ECC) is a fundamental mechanism in control of skeletal muscle contraction and occurs at triad junctions, where dihydropyridine receptors (DHPRs) on transverse tubules sense excitation signals and then cause calcium release from the sarcoplasmic reticulum via coupling to type 1 ryanodine receptors (RyR1s), inducing the subsequent contraction of muscle filaments. However, the molecular mechanism remains unclear due to the lack of structural details. Here, we explored the architecture of triad junction by cryo-electron tomography, solved the in situ structure of RyR1 in complex with FKBP12 and calmodulin with the resolution of 16.7 Angstrom, and found the intact RyR1-DHPR supercomplex. RyR1s arrange into two rows on the terminal cisternae membrane by forming right-hand corner-to-corner contacts, and tetrads of DHPRs bind to RyR1s in an alternating manner, forming another two rows on the transverse tubule membrane. This unique arrangement is important for synergistic calcium release and provides direct evidence of physical coupling in ECC.

Determining sex-driven excitation-contraction coupling mechanisms in young populations

Determining sex-driven excitation-contraction coupling mechanisms in young populations

Two-pore channels (TPCs) acts as a hub for excitation-contraction coupling, metabolism and cardiac hypertrophy signalling

Ca2+ signaling is essential for cardiac contractility and excitability in heart function and remodeling. Intriguingly, little is known about the role of a new family of ion channels, the endo-lysosomal non-selective cation “two-pore channel” (TPCs) in heart function. Here we have used double TPC knock-out mice for the 1 and 2 isoforms of TPCs (Tpcn1/2−/−) and evaluated their cardiac function. Doppler-echocardiography unveils altered left ventricular (LV) systolic function associated with a LV relaxation impairment. In cardiomyocytes isolated from Tpcn1/2−/- mice, we observed a reduction in the contractile function with a decrease in the sarcoplasmic reticulum Ca2+ content and a reduced expression of various key proteins regulating Ca2+ stores, such as calsequestrin. We also found that two main regulators of the energy metabolism, AMP-activated protein kinase and mTOR, were down regulated. We found an increase in the expression of TPC1 and TPC2 in a model of transverse aortic constriction (TAC) mice and in chronically isoproterenol infused WT mice. In this last model, adaptive cardiac hypertrophy was reduced by Tpcn1/2 deletion. Here, we propose a central role for TPCs and lysosomes that could act as a hub integrating information from the excitation-contraction coupling mechanisms, cellular energy metabolism and hypertrophy signaling.

Fasciculation electromechanical latency is prolonged in amyotrophic lateral sclerosis

ObjectiveIn amyotrophic lateral sclerosis (ALS), motor neurons become hyperexcitable and spontaneously discharge electrical impulses causing fasciculations. These can be detected by two noninvasive methods: high-density surface electromyography (HDSEMG) and muscle ultrasonography (MUS). We combined these methods simultaneously to explore the electromechanical properties of fasciculations, seeking a novel biomarker of disease. MethodsTwelve ALS patients and thirteen healthy participants each provided up to 24 minutes of recordings from the right biceps brachii (BB) and gastrocnemius medialis (GM). Two automated algorithms (Surface Potential Quantification Engine and a Gaussian mixture model) were applied to HDSEMG and MUS data to identify correlated electromechanical fasciculation events. ResultsWe identified 4,197 correlated electromechanical fasciculation events. HDSEMG reliably detected electromechanical events up to 30 mm below the skin surface with an inverse correlation between amplitude and depth in ALS muscles. Compared to Healthy-GM muscles (mean = 79.8 ms), electromechanical latency was prolonged in ALS-GM (mean = 108.8 ms; p = 0.0458) and ALS-BB (mean = 112.0 ms; p = 0.0128) muscles. Electromechanical latency did not correlate with disease duration, symptom burden, sum muscle power score or fasciculation frequency. ConclusionsProlonged fasciculation electromechanical latency indicates impairment of the excitation–contraction coupling mechanism, warranting further exploration as a potential novel biomarker of disease in ALS. SignificanceThis study points to an electromechanical defect within the muscles of ALS patients.

GPER limits adverse changes to Ca2+ signalling and arrhythmogenic activity in ovariectomised guinea pig cardiomyocytes.

Background: The increased risk of post-menopausal women developing abnormalities of heart function emphasises the requirement to understand the effect of declining oestrogen levels on cardiac electrophysiology and structure, and investigate possible therapeutic targets, namely the G protein-coupled oestrogen receptor 1 (GPER). Methods: Female guinea pigs underwent sham or ovariectomy (OVx) surgeries. Cardiomyocytes were isolated 150-days post-operatively. Membrane structure was assessed using di-8-ANEPPs staining and scanning ion conductance microscopy. Imunnohistochemistry (IHC) determined the localisation of oestrogen receptors. The effect of GPER activation on excitation-contraction coupling mechanisms were assessed using electrophysiological and fluorescence techniques. Downstream signalling proteins were investigated by western blot. Results: IHC staining confirmed the presence of nuclear oestrogen receptors and GPER, the latter prominently localised to the peri-nuclear region and having a clear striated pattern elsewhere in the cells. Following OVx, GPER expression increased and its activation reduced Ca2+ transient amplitude (by 40%) and sarcomere shortening (by 32%). In these cells, GPER activation reduced abnormal spontaneous Ca2+ activity, shortened action potential duration and limited drug-induced early after-depolarisation formation. Conclusion: In an animal species with comparable steroidogenesis and cardiac physiology to humans, we show the expression and localisation of all three oestrogen receptors in cardiac myocytes. We found that following oestrogen withdrawal, GPER expression increased and its activation limited arrhythmogenic behaviours in this low oestrogen state, indicating a potential cardioprotective role of this receptor in post-menopausal women.

Abstract P1104: Lower Troponin Expression In The Right Ventricle Of Rats Explains Interventricular Differences In E-c Coupling

Despite distinctive functional and anatomical differences, a precise understanding of the cardiac interventricular differences in excitation-contraction (E-C) coupling mechanisms is still lacking. Here, we directly compared rat right and left cardiomyocytes (RVCM and LVCM). Whole-cell patch clamp, IonOptix system and fura-2 fluorimetry were used to measure electrical properties (action potential and ionic currents), single cell contractility and cytosolic Ca 2+ ([Ca 2+ ] i ), respectively. Myofilament proteins were analyzed by immunoblotting. RVCMs showed significantly shorter action potential duration (APD) and higher density of transient outward K + current ( I to ). However, the triggered [Ca 2+ ] i change (Ca 2+ transient) was not different, while the decay rate of the Ca 2+ transient was slower in RVCM. Although the relaxation speed was also slower, the sarcomere shortening amplitude (ΔSL) was smaller in RVCM. SERCA activity was approximately 60% lower in RVCM, partly responsible for the slower decay of the Ca 2+ transient. Immunoblot analysis revealed lower expression of the cardiac troponin complex (cTn) in RVCM, implying a smaller Ca 2+ buffering capacity ( κ S ), which was proved by in situ analysis. The introduction of these new levels of cTn, I to and SERCA into a mathematical model of rat LVCM reproduced the similar Ca 2+ transient, slower Ca 2+ decay, shorter APD and smaller ΔSL of RVCM. Taken together, we show reduced expression of cTn proteins in the RVCMs, which provides an explanation for the inter-ventricular difference in the E-C coupling kinetics.

Abstract P1057: Implementing A Novel FRET-Based Myofilament Activation Biosensor To Interrogate Dynamic Cardiac Sarcomere Performance With Small Molecule And Genetic Manipulations During A Physiological Twitch

The sarcomere is the functional unit of cardiac muscle contraction. Understanding the precise mechanisms regulating this contraction is key to advancing insights into cardiomyopathies, which affect as many as 1 in 500 adults. Previous work has identified many key sarcomeric components and modulators necessary for the activation of contraction through inter-myofilament signaling. Although these studies have provided valuable information, the majority of this work has been done using in vitro protein recombination or permeabilized muscle. While informative, these systems lack significant physiological features of muscle, including load and intact excitation-contraction coupling mechanisms. Therefore, a large gap exists in the ability to monitor the time-sensitive stepwise physiological mechanism of muscle sarcomere activation in an intact system in real time. In order to further advance the study of sarcomere activation in both physiological and disease states, a cardiac specific myofilament-targeted FRET based biosensor has been designed and validated. The sarcomere activation biosensor reports myofilament dynamics during physiological twitch contractions in live cardiac muscle in real time in the form of simultaneous force and fluorescence measurements. This allows FRET-detection of the global conformational changes in TnC, a nexus point able to integrate many sarcomere-activating ligands, to be observed. From these changes in fluorescence the ensemble of events which lead to sarcomere activation and muscle contraction can be tracked. Data show regulatory ligands of myofilament activation in real time during a physiological twitch contraction. Currently, the biosensor is being utilized to investigate changes in sarcomere activation driven through the use of small molecules, including myosin inhibitor Mavacamten and TnC-TnI inhibitor W7, and those seen in MyBP-C knockout mice. The results of these investigations will be discussed.

Molecular interactions of STAC proteins with skeletal muscle dihydropyridine receptor and excitation-contraction coupling.

Excitation‐contraction coupling (ECC) is the physiological process in which an electrical signal originating from the central nervous system is converted into muscle contraction. In skeletal muscle tissue, the key step in the molecular mechanism of ECC initiated by the muscle action potential is the cooperation between two Ca2+ channels, dihydropyridine receptor (DHPR; voltage‐dependent L‐type calcium channel) and ryanodine receptor 1 (RyR1). These two channels were originally postulated to communicate with each other via direct mechanical interactions; however, the molecular details of this cooperation have remained ambiguous. Recently, it has been proposed that one or more supporting proteins are in fact required for communication of DHPR with RyR1 during the ECC process. One such protein that is increasingly believed to play a role in this interaction is the SH3 and cysteine‐rich domain‐containing protein 3 (STAC3), which has been proposed to bind a cytosolic portion of the DHPR α1S subunit known as the II–III loop. In this work, we present direct evidence for an interaction between a small peptide sequence of the II–III loop and several residues within the SH3 domains of STAC3 as well as the neuronal isoform STAC2. Differences in this interaction between STAC3 and STAC2 suggest that STAC3 possesses distinct biophysical features that are potentially important for its physiological interactions with the II–III loop. Therefore, this work demonstrates an isoform‐specific interaction between STAC3 and the II–III loop of DHPR and provides novel insights into a putative molecular mechanism behind this association in the skeletal muscle ECC process.

Assessing the Potential of Nutraceuticals as Geroprotectors on Muscle Performance and Cognition in Aging Mice.

Aging and frailty are associated with a decline in muscle force generation, which is a direct consequence of reduced muscle quantity and quality. Among the leading contributors to aging is the generation of reactive oxygen species, the byproducts of terminal oxidation. Their negative effects can be moderated via antioxidant supplementation. Krill oil and astaxanthin (AX) are nutraceuticals with a variety of health promoting, geroprotective, anti-inflammatory, anti-diabetic and anti-fatigue effects. In this work, we examined the functional effects of these two nutraceutical agents supplemented via pelleted chow in aging mice by examining in vivo and in vitro skeletal muscle function, along with aspects of intracellular and mitochondrial calcium homeostasis, as well as cognition and spatial memory. AX diet regimen limited weight gain compared to the control group; however, this phenomenon was not accompanied by muscle tissue mass decline. On the other hand, both AX and krill oil supplementation increased force production without altering calcium homeostasis during excitation-contraction coupling mechanism or mitochondrial calcium uptake processes. We also provide evidence of improved spatial memory and learning ability in aging mice because of krill oil supplementation. Taken together, our data favors the application of antioxidant nutraceuticals as geroprotectors to improve cognition and healthy aging by virtue of improved skeletal muscle force production.

Abstract 123: Study Of The Role Of Myosin-binding Protein C In The Structure-function Of Intact Cardiac Muscles Through The Use Of A Sarcomere Activation Biosensor

Cardiomyopathies affect as many as 1 in 500 adults, with hypertrophic cardiomyopathy (HCM) being the most commonly inherited heart disease. Although there are various genetic mutations which cause HCM, 40-50% of mutations identified in patients with this disease are found in the cardiac myosin-binding protein C (MyBP-C) gene. Thus, understanding this protein's role in sarcomere activation is critical for the development of effective therapeutic strategies. Previous work has identified MyBP-C as a key modulator of the sarcomere through inter-myofilament signaling. Although these studies have provided valuable information on the function of MyBP-C protein, the bulk of this work has been done with either in vitro protein recombination or permeabilized muscle. Unfortunately, these systems lack significant physiological features of muscle, such as load and intact excitation-contraction coupling mechanisms. Therefore, a large gap exists in the ability to monitor muscle sarcomere activation in an intact system in real time. In order to further advance the study of sarcomere activation in HCM disease models, specifically those targeting MyBP-C, a cardiac specific myofilament-targeted FRET based biosensor has been designed and validated. This new sarcomere activation biosensor reports real-time myofilament dynamics during physiological twitch contractions in live cardiac muscle. This allows for the in vivo biophysical tracking of FRET-detected conformation changes in TnC, which reflect the ensemble of regulatory events which lead to sarcomere activation. Currently, the biosensor is being utilized to investigate changes in sarcomere activation in MyBP-C knockout mice. We find that intact cardiac muscle under load shows slowed isometric twitch relaxation kinetics in MyBP-C knockout mice versus controls. In concert with thin and thick filament modifying small molecules, this biosensor system will be used to investigate sarcomere-based mechanisms of intact cardiac muscle performance in health and disease.

Discordant interventricular differences in the excitation‐contraction coupling explained by the lower levels of troponin and Ca 2+ buffering in the right ventricle of rats

The left and right ventricles have distinctive functional characteristics as well as the anatomical differences. However, precise understanding of interventricular differences in the excitation-contraction (E-C) coupling mechanisms and the Ca2+ homeostasis is still lacking. Here we compared the results from the right and left cardiomyocytes (RVCMs and LVCMs) of rats by using whole-cell patch clamp and IonOptix measuring cytosolic Ca2+ ([Ca2+]i) and contractility. RVCMs showed significantly shorter action potential duration (APD), with higher density of transient outward K+ current (Ito) while similar L-type Ca2+ current. However, the triggered [Ca2+]ichange (Ca2+ transient) was not different while the decaying rate of Ca2+ transient was slower in RVCMs. More perplexingly, the sarcomere shortening amplitude (DSL) was smaller and the relaxation speed was slower in RVCMs than LVCMs. The analysis of SERCA activity in situ revealed approximately 50% lower level in RVCMs. Interestingly, the immunoblot analysis revealed lower expression of cardiac troponin complex (cTnC, cTnI and cTnT) in RVCMs. The lower Ca2+ binding cTnC implied smaller Ca2+ buffering capacity (κS), which could be responsible for the similar amplitude of Ca2+-transient despite the shorter APD in RVCMs. In situ analysis using known concentration of fura-2 revealed lower κS in the RVCMs. Introduction of the 1.9 fold Ito, 0.5 fold SERCA, and 0.7 fold cTn into the mathematical model of rat LVCM reproduced the similar Ca2+ transient and the slower Ca2+ decay along with the shorter APD of RVCM. Taken together, we firstly show the lower expression of cTn proteins in the RVCMs, which gives a clue to explain the inter-ventricular difference in the E-C coupling kinetics.

Inverse mechano-electrical reconstruction of cardiac excitation wave patterns from mechanical deformation using deep learning.

The inverse mechano-electrical problem in cardiac electrophysiology is the attempt to reconstruct electrical excitation or action potential wave patterns from the heart's mechanical deformation that occurs in response to electrical excitation. Because heart muscle cells contract upon electrical excitation due to the excitation-contraction coupling mechanism, the resulting deformation of the heart should reflect macroscopic action potential wave phenomena. However, whether the relationship between macroscopic electrical and mechanical phenomena is well-defined and unique enough to be utilized for an inverse imaging technique in which mechanical activation mapping is used as a surrogate for electrical mapping has yet to be determined. Here, we provide a numerical proof-of-principle that deep learning can be used to solve the inverse mechano-electrical problem in phenomenological two- and three-dimensional computer simulations of the contracting heart wall, or in elastic excitable media, with muscle fiber anisotropy. We trained a convolutional autoencoder neural network to learn the complex relationship between electrical excitation, active stress, and tissue deformation during both focal or reentrant chaotic wave activity and, consequently, used the network to successfully estimate or reconstruct electrical excitation wave patterns from mechanical deformation in sheets and bulk-shaped tissues, even in the presence of noise and at low spatial resolutions. We demonstrate that even complicated three-dimensional electrical excitation wave phenomena, such as scroll waves and their vortex filaments, can be computed with very high reconstruction accuracies of about 95% from mechanical deformation using autoencoder neural networks, and we provide a comparison with results that were obtained previously with a physics- or knowledge-based approach.

Abstract 15998: Therapeutic Effectiveness Against Heart Failure With Preserved Ejection Fraction by the Activation of the Growth Hormone-releasing Hormone Receptor in a Murine Model

Introduction: Heart failure with preserved ejection fraction (HFpEF) accounts for ~50% of heart failure cases and is characterized by impaired relaxation, ventricular stiffening and fibrosis. Growth hormone releasing hormone-agonists (GHRH-As) reduce fibrosis in rat and swine models of ischemic myocardial injury. However, their therapeutic effect in HFpEF is unknown. Hypothesis: We hypothesized that activation of GHRH receptor signaling prevents pathological remodeling and targets cardio-myocyte (CM) excitation-contraction coupling, ameliorating the compromised myocardial performance associated with HFpEF. Methods: CD1 mice implanted with a mini-osmotic pump (Alzet) to deliver angiotensin-II (Ang-II) for 8 weeks, were randomized at week 4 to receive daily injections of either GHRH-A (MR-356; n=12) or vehicle (n=12). Echocardiographic data and pressure-volume loops were recorded. Biometric measurements and fibrosis were assessed. CMs were isolated and intracellular Ca2 + and sarcomere length acquired. Results: Ang-II-treated mice exhibited functional and morphologic features consistent with HFpEF. CMs exhibited reduced sarcomere length, indicating an inability to relax. The Ca2 + decay rate and sarcomere re-lengthening were delayed. MR-356 treatment reverted the structural remodeling, including fibrosis, improving diastolic function (Figure Top panel). This treatment also abrogated the HFpEF-associated delay in Ca2 + decay and further enhanced sarcomere re-lengthening rate (Figure Bottom panel). Conclusions: Our findings confirm that chronic infusion of Ang-II mediates functional changes in CMs consistent with HFpEF and demonstrate that activation of GHRH receptor signaling pathways targets mechanisms of excitation-contraction coupling, inflammation and fibrosis, thereby preventing HFpEF-associated dysfunctional CM relaxation and myocardial remodeling.

Long-Chain Acylcarnitines and Cardiac Excitation-Contraction Coupling: Links to Arrhythmias.

A growing number of metabolomic studies have associated high circulating levels of the amphiphilic fatty acid metabolites, long-chain acylcarnitines (LCACs), with cardiovascular disease (CVD) risk. These studies show that plasma LCAC levels can be correlated with the stage and severity of CVD and with indices of cardiac hypertrophy and ventricular function. Complementing these recent clinical associations is an extensive body of basic research that stems mostly from the twentieth century. These works, performed in cardiomyocyte and multicellular preparations from animal and cell models, highlight stereotypical derangements in cardiac electrophysiology induced by exogenous LCAC treatment that promote arrhythmic muscle behavior. In many cases, this is coupled with acute inotropic modulation; however, whether LCACs increase or decrease contractility is inconclusive. Linked to the electromechanical alterations induced by LCAC exposure is an array of effects on cardiac excitation-contraction coupling mechanisms that overload the cardiomyocyte cytosol with Na+ and Ca2+ ions. The aim of this review is to revisit this age-old literature and collate it with recent findings to provide a pathophysiological context for the growing body of metabolomic association studies that link circulating LCACs with CVD.

Overlapping Mechanisms of Exertional Heat Stroke and Malignant Hyperthermia: Evidence vs. Conjecture.

Exertional heat stroke (EHS) and malignant hyperthermia (MH) are life-threatening conditions, triggered by different environmental stimuli that share several clinical symptoms and pathophysiological features. EHS manifests during physical activity normally, but not always, in hot and humid environments. MH manifests during exposure to haloalkane anesthetics or succinylcholine, which leads to a rapid, unregulated release of calcium (Ca2+) within the skeletal muscles inducing a positive-feedback loop within the excitation-contraction coupling mechanism that culminates in heat stroke-like symptoms, if not rapidly recognized and treated. Rare cases of awake MH, independent of anesthesia exposure, occur during exercise and heat stress. It has been suggested that EHS and MH are mediated by similar mechanisms, including mutations in Ca2+ regulatory channels within the skeletal muscle. Rapid cooling, which is the most effective treatment for EHS, is ineffective as an MH treatment; rather, a ryanodine receptor antagonist drug, dantrolene sodium (DS), is administered to the victim to prevent further muscle contractions and hyperthermia. Whether DS can be an effective treatment for EHS victims remains uncertain. In the last decade, multiple reports have suggested a number of mechanistic links between EHS and MH. Here, we discuss aspects related to the pathophysiology, incidence, diagnosis and treatment. Furthermore, we present evidence regarding potential overlapping mechanisms between EHS and MH and explore current knowledge to establish what is supported by evidence or a lack thereof (i.e. conjecture).

Activation of endogenous protein phosphatase 1 enhances the calcium sensitivity of the ryanodine receptor type 2 in murine ventricular cardiomyocytes.

Increased protein phosphatase 1 (PP-1) activity has been found in end stage human heart failure. Although PP-1 has been extensively studied, a detailed understanding of its role in the excitation-contraction coupling mechanism, in normal and diseased hearts, remains elusive. The present study investigates the functional effect of the PP-1 activity on local Ca2+ release events in ventricular cardiomyocytes, by using an activating peptide (PDP3) for the stimulation of the endogenous PP-1 protein. We report that acute de-phosphorylation may increase the sensitivity of RyR2 channels to Ca2+ in situ, and that the RyR2-serine2808 phosphorylation site may mediate such a process. Our approach unmasks the functional importance of PP-1 in the regulation of RyR2 activity, suggesting a potential role in the generation of a pathophysiological sarcoplasmic reticulum Ca2+ leak in the diseased heart. Changes in cardiac ryanodine receptor (RyR2) phosphorylation are considered to be important regulatory and disease related post-translational protein modifications. The extent of RyR2 phosphorylation is mainly determined by the balance of the activities of protein kinases and phosphatases, respectively. Increased protein phosphatase-1 (PP-1) activity has been observed in heart failure, although the regulatory role of this enzyme on intracellular Ca2+ handling remains poorly understood. To determine the physiological and pathophysiological significance of increased PP-1 activity, we investigated how the PP-1 catalytic subunit (PP-1c) alters Ca2+ sparks in permeabilized cardiomyocytes and we also applied a PP-1-disrupting peptide (PDP3) to specifically activate endogenous PP-1, including the one anchored on the RyR2 macromolecular complex. We compared wild-type and transgenic mice in which the usually highly phosphorylated site RyR2-S2808 has been ablated to investigate its involvement in RyR2 modulation (S2808A+/+ ). In wild-type myocytes, PP-1 increased Ca2+ spark frequency by two-fold, followed by depletion of the sarcoplasmic reticulum Ca2+ store. Similarly, PDP3 transiently increased spark frequency and decreased sarcoplasmic reticulum Ca2+ load. RyR2 Ca2+ sensitivity, which was assessed by Ca2+ spark recovery analysis, was increased in the presence of PDP3 compared to a negative control peptide. S2808A+/+ cardiomyocytes did not respond to both PP-1c and PDP3 treatment. Our results suggest an increased Ca2+ sensitivity of RyR2 upon de-phosphorylation by PP-1. Furthermore, we have confirmed the S2808 site as a target for PP-1 and as a potential link between RyR2s modulation and the cellular response.

Astaxanthin Improves Tetanic Force Without Altering Skeletal Muscle Excitation-Contraction Coupling in Mice

Astaxanthin (AX) is a powerful natural anti-oxidant that protects against the risk of oxidative damage from free radicals and reactive oxygen species. Up to date, there are no studies concerning how and if AX feeding affects the excitation-contraction coupling (ECC) mechanism in skeletal muscle. 8 young adult C57Bl6 mice were divided into two groups: 1) control and 2) AX supplemented for 4 weeks (AstaReal1010, 0.05% w/w added in standard rodent chow). Following the feeding the AX group gained significantly less weight on average (0.16±0.33g vs 1.22±0.31g). When examining the in vitro force of the extensor digitorum longus (EDL) muscle the amplitude of single twitches (5 V, 100 ms) was not altered, but the amplitude of tetani (200 Hz, 200 ms) was significantly increased in the AX group. We also measured the activity-dependent mitochondrial Ca2+ uptake in rhod-2 [SzP1] loaded flexor digitorum brevis (FDB) fibers at rest and during tetanic stimulation. The average (±SE) time course of mitochondrial normalized fluorescence was significantly decreased in the AX group at rest (0.28±0.01 vs 0.12±0.01), after the 1st tetanus (0.3±0.01 vs 0.13±0.01) and after the 5th tetanus (0.39±0.08 vs 0.14±0.02) (p<0.05), respectively. Lastly, we studied the voltage activation of calcium transients in single FDB fibers under whole cell voltage clamp. The voltage dependence of calcium release (fitted with the Boltzmann function) was not statistically different between the two groups (V1/2: -12.4±2.3 mV, k=6.7±0.5 for Ctrl, n=12 vs. V1/2: -14.9±2.7 mV, k=8.1±0.8 for AX, n=17). We conclude that AX supplementation increases the in vitro force of the EDL muscles without affecting the ECC mechanism in skeletal muscle. AX feeding seem to exert a protecting effect on the mitochondria as the Ca2+ uptake was significantly less in the AX group.

Physiological functions and Ca2+ signal transduction pathway exploring during molting of silkworm, Bombyx mori

Abstract Calcium ions are an important second messagers in the life of organisms, such as in silkworm growth and their developmental processes; these ions can trigger various effects, including the muscle excitation-contraction coupling mechanism, secretion of endocrine and exocrine glands, neuronal activities, midgut nutrients metabolisms, sensory system, enzymatic systems, and acceleration of metabolism activities. This paper reviewed the ecdysis process and mechanisms, where calcium plays an important role in larvae-to-larvae ecdysis and larvae-to-pupae and pupae-to-moth metamorphosis of silkworms; the paper focuses on the advanced achievements in the field of calcium actions during larvae-to-larvae and larvae-to-pupae changes. Calcium ion channels are shown to be the action mechanism of phospholipase C in the G protein cascade of the calcium signal transduction pathway, and the activators and inhibitors of these calcium channels are further discussed.