Contraction-relaxation Cycle Research Articles

Proteomic reference map for sarcopenia research: mass spectrometric identification of key muscle proteins located in the sarcomere, cytoskeleton and the extracellular matrix.

Sarcopenia of old age is characterized by the progressive loss of skeletal muscle mass and concomitant decrease in contractile strength. Age-related skeletal muscle dysfunctions play a key pathophysiological role in the frailty syndrome and can result in a drastically diminished quality of life in the elderly. Here we have used mass spectrometric analysis of the mouse hindlimb musculature to establish the muscle protein constellation at advanced age of a widely used sarcopenic animal model. Proteomic results were further analyzed by systems bioinformatics of voluntary muscles. In this report, the proteomic survey of aged muscles has focused on the expression patterns of proteins involved in the contraction-relaxation cycle, membrane cytoskeletal maintenance and the formation of the extracellular matrix. This includes proteomic markers of the fast versus slow phenotypes of myosin-containing thick filaments and actin-containing thin filaments, as well as proteins that are associated with the non-sarcomeric cytoskeleton and various matrisomal layers. The bioanalytical usefulness of the newly established reference map was demonstrated by the comparative screening of normal versus dystrophic muscles of old age, and findings were verified by immunoblot analysis.

SERCA mediated thermogenesis during contraction-relaxation cycle in rat skeletal muscle in vivo

BACKGROUND: Across the lifespan, body temperature homeostasis is tightly regulated and impacts various physiological phenomena. In skeletal muscle, heat is produced via contraction-dependent (shivering) and independent (nonshivering) mechanisms, regulating the body temperature and muscle physiology including contractile properties. PURPOSE: In this study, we aimed to reveal the intracellular temperature dynamics in skeletal muscle in vivo during the contraction-relaxation cycle. Additionally, we tested the hypothesis that calcium ion (Ca2+) handling mediated by sarcoplasmic reticulum Ca2+ ATPase (SERCA) is involved in the thermogenesis process during contraction-relaxation cycle in skeletal muscle fibers. METHOD: The spinotrapezius muscle of anesthetized adult male Wistar rats (n = 18) was exteriorized and subjected to microinjection of fluorescent probe Cellular Thermoprobe for Fluorescence Ratio (49.3 μM) for intracellular temperature imaging in vivo. The fluorescence ratio (R: 580 nm / 515 nm) was measured in vivo during temperature increases induced by an external heater for thermoprobe calibration. R was also measured following Ca2+ injection (3.9 nL, 2.0 mM), and Ca2+ injection with SERCA inhibition by cyclopiazonic acid (CPA, 100 μM). RESULTS: The fluorescence ratio linearly increased with the increase of the muscle surface temperature from 25 °C to 40 °C (r2 = 0.97, P < 0.01). The intracellular temperature in muscle was increased and sustained following the Ca2+ injection and this thermogenetic response was significantly suppressed with SERCA inhibition (Ca2+: 38.3 ± 1.4 °C vs Ca2++CPA: 28.3 ± 2.8 °C, P < 0.01 at 1 min following injection). Importantly, elevated muscle temperature occurred predominantly in the muscle relaxation phase that followed the muscle shortening: That shortening occurred immediately and transiently after the Ca2+ injection. CONCLUSION: In this investigation, we demonstrated a novel technique for in vivo intracellular temperature imaging of skeletal muscle. Herein we demonstrate that substantial heat is generated concomitantly with SERCA mediated Ca2+ handling and muscle relaxation. This work was supported by JSPS KAKENHI Grant Numbers 20H04074, 19K22800. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

In vivo heat production dynamics during a contraction-relaxation cycle in rat single skeletal muscle fibers

Skeletal muscle generates heat via contraction-dependent (shivering) and independent (nonshivering) mechanisms. While this thermogenic capacity of skeletal muscle undoubtedly contributes to the body temperature homeostasis of animals and impacts various cellular functions, the intracellular temperature and its dynamics in skeletal muscle in vivo remain elusive. We aimed to determine the intracellular temperature and its changes within skeletal muscle in vivo during contraction and following relaxation. In addition, we tested the hypothesis that sarcoplasmic reticulum Ca2+ ATPase (SERCA) generates heat and increases the myocyte temperature during a transitory Ca2+-induced contraction-relaxation cycle. The intact spinotrapezius muscle of anesthetized adult male Wistar rats (n = 18) was exteriorized and loaded with the fluorescent probe Cellular Thermoprobe for Fluorescence Ratio (49.3 μM) by microinjection over 1 s. The fluorescence ratio (i.e., 580 nm/515 nm) was measured in vivo during 1) temperature increases induced by means of an external heater, and 2) Ca2+ injection (3.9 nL, 2.0 mM). The fluorescence ratio increased as a linear function of muscle surface temperature from 25 °C to 40 °C (r2 = 0.97, P < 0.01). Ca2+ injection (3.9 nL, 2.0 mM) significantly increased myocyte intracellular temperature: An effect that was suppressed by SERCA inhibition with cyclopiazonic acid (CPA, Ca2+: 38.3 ± 1.4 °C vs Ca2++CPA: 28.3 ± 2.8 °C, P < 0.01 at 1 min following injection). While muscle shortening occurred immediately after the Ca2+ injection, the increased muscle temperature was maintained during the relaxation phase. In this investigation, we demonstrated a novel model for measuring the intracellular temperature of skeletal muscle in vivo and further that heat generation occurs concomitant principally with SERCA functioning and muscle relaxation.

Sex differences in muscle contraction-induced limb blood flow limitations.

To determined sex differences in absolute- and %-reductions in blood flow during intermittent muscular contractions as well as relationships between blood flow reductions and time to task failure (TTF). Thirteen males (25 ± 4 years) and 13 females (22 ± 5 years) completed intermittent isometric trapezoidal forearm flexion at 50% maximal voluntary contraction until task failure. Doppler ultrasound was used to measure brachial artery blood flow (BABF) during the 12-s plateau phase and 12-s relaxation phase. Target torque was less in females than males (24 ± 5 vs. 42 ± 7 Nm; p < 0.001); however, TTF was not different between sexes (F: 425 ± 187 vs. M: 401 ± 158s; p = 0.72). Relaxation-phase BABF at end-exercise was less in females than males (435 ± 161 vs. 937 ± 281mL/min; p < 0.001) but contraction-phase BABF was not different (127 ± 46 vs. 190 ± 99mL/min; p = 0.42). Absolute- and %-reductions in BABF by contraction were less in females than males (309 ± 146 vs. 747 ± 210mL/min and 69 ± 10 vs. 80% ± 6%, respectively; both p < 0.01) and were associated with target torque independent of sex (r = 0.78 and 0.56, respectively; both p < 0.01). Absolute BABF reduction per target torque (mL/min/Nm) and TTF were positively associated in males (r = 0.60; p = 0.031) but negatively associated in females (r = -0.61; p = 0.029). This study provides evidence that females incur less proportional reduction in limb blood flow from muscular contraction than males at a matched relative intensity suggesting females may maintain higher levels of muscle oxygen delivery and metabolite removal than males across the contraction-relaxation cycle of intermittent exercise.

Nucleus Mechanosensing in Cardiomyocytes.

Cardiac muscle contraction is distinct from the contraction of other muscle types. The heart continuously undergoes contraction-relaxation cycles throughout an animal's lifespan. It must respond to constantly varying physical and energetic burdens over the short term on a beat-to-beat basis and relies on different mechanisms over the long term. Muscle contractility is based on actin and myosin interactions that are regulated by cytoplasmic calcium ions. Genetic variants of sarcomeric proteins can lead to the pathophysiological development of cardiac dysfunction. The sarcomere is physically connected to other cytoskeletal components. Actin filaments, microtubules and desmin proteins are responsible for these interactions. Therefore, mechanical as well as biochemical signals from sarcomeric contractions are transmitted to and sensed by other parts of the cardiomyocyte, particularly the nucleus which can respond to these stimuli. Proteins anchored to the nuclear envelope display a broad response which remodels the structure of the nucleus. In this review, we examine the central aspects of mechanotransduction in the cardiomyocyte where the transmission of mechanical signals to the nucleus can result in changes in gene expression and nucleus morphology. The correlation of nucleus sensing and dysfunction of sarcomeric proteins may assist the understanding of a wide range of functional responses in the progress of cardiomyopathic diseases.

Preadaptivity of Non-Contractive Thermogenesis in the Evolution of Warm-Bloodedness in Vertebrates

Most of the heat that is released in the vertebrate body is produced in the muscles during contractive (during movement or trembling) and non-contractive (without muscle activity) thermogenesis. Contractive thermogenesis is characteristic for all vertebrates, but it is not able to constantly maintain a high body temperature in animals. The main idea discussed in this article, and based on a large number of publications in recent years: the main biochemical base of warm-bloodedness in vertebrates is part of the cycle of contraction–relaxation of striated skeletal muscles, in which the act of muscle contraction somehow falls out, and the energy that should have been used for it is dissipated in the form of heat. This non-contractive thermogenesis, which is able to support the regional and general endothermy in vertebrates, can be considered the real biochemical basis of warm-bloodedness. Thus, the presence of skeletal muscles in all vertebrates and the common biochemical foundations of the contraction–relaxation cycle represent a single preadaptive property of the manifestation of non-contractive thermogenesis in all vertebrates, starting with fish, which is the basis for the evolution of warm-bloodedness. Therefore, it is understandable and unsurprising modern data that the first terrestrial vertebrates were most likely animals with high levels of both metabolism and body temperature.

Characterization of a robust mouse model of heart failure with preserved ejection fraction.

Heart failure (HF) is a leading cause of morbidity and mortality particularly in older adults and patients with multiple metabolic comorbidities. Heart failure with preserved ejection fraction (HFpEF) is a clinical syndrome with multisystem organ dysfunction in which patients develop symptoms of HF as a result of high left ventricular (LV) diastolic pressure in the context of normal or near normal LV ejection fraction (LVEF; ≥50%). Challenges to create and reproduce a robust rodent phenotype that recapitulates the multiple comorbidities that exist in this syndrome explain the presence of various animal models that fail to satisfy all the criteria of HFpEF. Using a continuous infusion of angiotensin II and phenylephrine (ANG II/PE), we demonstrate a strong HFpEF phenotype satisfying major clinically relevant manifestations and criteria of this pathology, including exercise intolerance, pulmonary edema, concentric myocardial hypertrophy, diastolic dysfunction, histological signs of microvascular impairment, and fibrosis. Conventional echocardiographic analysis of diastolic dysfunction identified early stages of HFpEF development and speckle tracking echocardiography analysis including the left atrium (LA) identified strain abnormalities indicative of contraction-relaxation cycle impairment. Diastolic dysfunction was validated by retrograde cardiac catheterization and analysis of LV end-diastolic pressure (LVEDP). Among mice that developed HFpEF, two major subgroups were identified with predominantly perivascular fibrosis and interstitial myocardial fibrosis. In addition to major phenotypic criteria of HFpEF that were evident at early stages of this model (3 and 10 days), accompanying RNAseq data demonstrate activation of pathways associated with myocardial metabolic changes, inflammation, activation of extracellular matrix (ECM) deposition, microvascular rarefaction, and pressure- and volume-related myocardial stress.NEW & NOTEWORTHY Heart failure with preserved ejection fraction (HFpEF) is an emerging epidemic affecting up to half of patients with heart failure. Here we used a chronic angiotensin II/phenylephrine (ANG II/PE) infusion model and instituted an updated algorithm for HFpEF assessment. Given the simplicity in generating this model, it may become a useful tool for investigating pathogenic mechanisms, identification of diagnostic markers, and for drug discovery aimed at both prevention and treatment of HFpEF.

Mechanokinetics of the spontaneous contractions of smooth muscles in the stomach and large intestine of rats under chronic effect of ТiО2 nanoparticles

Background. Currently, nano-sized materials of titanium dioxide (TiО2) have wide industrial uses, particularly in the food industry and pharmacology. Therefore, the problem of TiО2 toxicity to living organisms in case of their chronic in vivo intake needs thorough investigation. As nanoparticles enter the internal environment of the body, they spread with the bloodstream to tissues and organs, where they partially accumulate. Thus studying the state of the pacemaker mechanisms of regulation of smooth muscle spontaneous contractions in the stomach and large intestine under chronic exposure to TiО2 nanoparticles is relevant and interesting. The purpose of this research work was to study the spontaneous contractile activity of the gastric and large intestine smooth muscles of rats under chronic (for 6 months) intake of an aqueous suspension of TiО2 nanoparticles. Materials and Methods. Wistar rats were used in the experiments. Rats of the experimental group were daily intragastrically administered an aqueous suspension of ТіО2 nanoparticles for six months at a dose of 0.1 mg/kg. The study of the mechanokinetics of the contraction-relaxation process of muscle preparations was carried out according to the method (Kosterin et al., 2021) with the calculation of the mechanokinetic parameters of the contraction-relaxation cycle: force (Fmax, FC and FR), time (τ0, τC and τR), impulse (Іmax, ІC and ІR) and velocity (VC and VR). Results. A comprehensive mechanokinetic analysis of spontaneous contractions of the antrum and caecum circular smooth muscles was carried out in control and under chronic in vivo exposure to ТіО2 nanocolloids (0.1 mg/kg/day) for 6 months. It was found that the chronic action of ТіО2 nanocolloids significantly inhibits the contractile activity of the antrum smooth muscles accompanied by a decrease in all mechanokinetic parameters of time, force, velocity and impulse. Under the same conditions, inhibition of the spontaneous contractions of the large intestine smooth muscles was observed. However, the time (τ0, τC and τR) and impulse (Іmax, ІC and ІR) parameters increased for these muscles against the background of the decrease in the force and velocity mechanokinetic parameters. Conclusions. Modulation of the mechanokinetic parameters of the spontaneous contractile activity of the stomach and large intestine smooth muscles of rats under chronic intragastric administration of ТіО2 nanocolloids suggests that the functioning of pacemakers changes significantly under these conditions. Since the parameters of the contraction and relaxation phases of both the stomach and the large intestine smooth muscles do not differ by the magnitude of the inhibitory effects, it can be assumed that the effects of ТіО2 are not specific for particular Са2+ transport systems involved in contractile responses.

The mechanism of dwarf open reading frame (DWORF) activation of the sarcoplasmic reticulum calcium pump SERCA.

The sarcoplasmic reticulum calcium pump SERCA plays a critical role in the contraction-relaxation cycle of muscle. In cardiac muscle, SERCA is regulated by the inhibitory transmembrane peptide phospholamban. A new transmembrane peptide regulator, dwarf open reading frame (DWORF), has been reported to displace phospholamban from SERCA. We have shown that DWORF is a direct activator of SERCA, increasing its turnover rate in the absence of phospholamban (1). Measurement of in-cell calcium dynamics supports this observation and demonstrates that DWORF increases SERCA-dependent calcium reuptake, while phospholamban decreases SERCA-dependent calcium reuptake. These functional observations reveal opposing effects of DWORF activation and phospholamban inhibition of SERCA. To gain mechanistic insight into SERCA activation, fluorescence resonance energy transfer experiments revealed that DWORF has a higher affinity for SERCA in the presence of calcium, while phospholamban has a higher affinity for SERCA in the absence of calcium. Molecular modeling and molecular dynamics simulations provide a model for DWORF activation of SERCA, where DWORF modulates the membrane bilayer and stabilizes the conformations of SERCA that predominate during elevated cytosolic calcium. Mutagenesis and structure-function analysis of DWORF reveals three important features required for SERCA activation: Pro15 promotes a helix-linker-helix structure of DWORF; the N-terminal amphipathic helix of DWORF perturbs the membrane bilayer; and the short transmembrane helix of DWORF contributes to hydrophobic mismatch and positions the amphipathic helix at the surface of the membrane bilayer. Our current understanding of the mechanism of SERCA activation by DWORF will be presented.

Tropomyosin R160A is crucial for TnI phosphorylation-induced effects on calcium sensitivity.

The thin filament, consisting of the three-protein complex troponin (TnI, TnT, and TnC) and tropomyosin (Tpm) bound to filamentous actin, acts as the main transducer that relates sarcoplasmic calcium levels to the contraction-relaxation cycles of the myocardium. In the absence of calcium, TnI biases Tpm in a position on actin that blocks myosin binding whereas calcium binding to TnC causes a propagated conformational change in Tn/Tpm that relieves this inhibition. During heart failure and adrenergic stimulation, phosphorylation of the N-terminal region of troponin I (TnI) at residues S23 and S24, has been shown to play a role in heart muscle adaptation through a decrease in calcium sensitivity. Previous modeling studies performed in thin filament models have suggested that positively charged groups on Tpm, namely R160, could interact with the phosphorylated serine residues on TnI. Here we tested the hypothesis that interactions between Tpm-R160 and phosphorylated S23/24 participate in phosphorylation-induced effects on calcium sensitivity (Pavadai et al., 2022). Tpm mutated from wild-type R160 to alanine (Tpm-R160A) and phosphomimetic TnI affecting both serine 23 and 24 (S23/24D) were generated and expressed recombinantly. Using in vitro motility (IVM) assays, we quantified the effects of these mutations alone and in combination. Regulated IVM experiments using thin filaments reconstituted with phosphomimetic S23/24D-TnI and wild-type Tpm revealed a decrease in calcium sensitivity, showing that phosphorylation is successfully mimicked by mutating serine to an aspartic acid,consistent with previous studies on human cardiac tissue. However, when TnI-S23/24D is paired with mutant R160A-Tpm, the phosphorylation-induced changes in calcium sensitivity are diminished. These results are consistent with Tpm-R160 participating in the lusitropy observed when the N-terminal of TnI is phosphorylated.

Role of adenosine triphosphate and protein kinase A in the force-frequency relationship in isolated rat cardiomyocytes

The physiological heart rate of rodents is around 4-6 Hz, although a stimulus frequency of 1 Hz is generally used in isolated cardiomyocytes to study changes in the contraction-relaxation cycle in cardiac muscle physiology and pathophysiology. Our study investigated the contraction parameters in isolated cardiomyocytes at 1, 2 and 4 Hz stimulation, and the roles of ATP and protein kinase A (PKA) in the force-frequency relationship in isolated cardiomyocytes. The contraction of the cell and intracellular Ca2+ changes were recorded simultaneously during cell stimulation by applying pulses of 6-8 V amplitude with frequencies of 1, 2 and 4 Hz. The increase in stimulus frequency caused a significant decrease in the percentage of shortening, relaxation times, slowing of the relaxation rate, and a significant increase in diastolic Ca2+ levels, but had no effect on the contraction rate and Ca2+ transients. Administration of ATP and N6-benzoyladenosine-3'-5'-cyclic monophosphate (6-BNZ-cAMP) caused an increase in contraction amplitude and speed which were proportional to the stimulus frequency but had no effect on the relaxation times. The experimental results show that the force-stimulus frequency has a negative correlation in isolated myocytes and that energy metabolism and the ?-adrenergic system may be responsible for this relationship.

HIV-Tat Exacerbates the Actions of Atazanavir, Efavirenz, and Ritonavir on Cardiac Ryanodine Receptor (RyR2).

The incidence of sudden cardiac death (SCD) in people living with HIV infection (PLWH), especially those with inadequate viral suppression, is high and the reasons for this remain incompletely characterized. The timely opening and closing of type 2 ryanodine receptor (RyR2) is critical for ensuring rhythmic cardiac contraction-relaxation cycles, and the disruption of these processes can elicit Ca2+ waves, ventricular arrhythmias, and SCD. Herein, we show that the HIV protein Tat (HIV-Tat: 0-52 ng/mL) and therapeutic levels of the antiretroviral drugs atazanavir (ATV: 0-25,344 ng/mL), efavirenz (EFV: 0-11,376 ng/mL), and ritonavir (RTV: 0-25,956 ng/mL) bind to and modulate the opening and closing of RyR2. Abacavir (0-14,315 ng/mL), bictegravir (0-22,469 ng/mL), Rilpivirine (0-14,360 ng/mL), and tenofovir disoproxil fumarate (0-18,321 ng/mL) did not alter [3H]ryanodine binding to RyR2. Pretreating RyR2 with low HIV-Tat (14 ng/mL) potentiated the abilities of ATV and RTV to bind to open RyR2 and enhanced their ability to bind to EFV to close RyR2. In silico molecular docking using a Schrodinger Prime protein-protein docking algorithm identified three thermodynamically favored interacting sites for HIV-Tat on RyR2. The most favored site resides between amino acids (AA) 1702-1963; the second favored site resides between AA 467-1465, and the third site resides between AA 201-1816. Collectively, these new data show that HIV-Tat, ATV, EFV, and RTV can bind to and modulate the activity of RyR2 and that HIV-Tat can exacerbate the actions of ATV, EFV, and RTV on RyR2. Whether the modulation of RyR2 by these agents increases the risk of arrhythmias and SCD remains to be explored.

Participation of KАТР-channels of plasma and mitochondrial membranes in the regulation of mechanokinetics of rat myometrium spontaneous contractions

Background. ATP-sensitive K+ channels of the plasma membrane in the smooth muscles of the uterus are one of the most significant ion channels that regulate the excitability of this tissue both in the non-pregnant state and during pregnancy. MitoKATР-channels ensure regulation of the bioenergetic state of mitochondria (the intensity of mitochondrial respiration and the potential of the inner mitochondrial membrane). Thus far, there is no information on the participation of both types of these channels in the regulation of the mechanokinetics of spontaneous contractions, therefore the aim of this work was to perform a complete mechanokinetic analysis of spontaneous contractions of rat myometrium under conditions of blocking and activation of ATP-sensitive K+-channels of the plasma membrane and blocking of mitoKATP-channels. Materials and Methods. Experiments were performed on female Wistar rats. The spontaneous activity of smooth muscle stripes of longitudinal smooth muscles of uterine horns was registered by the tenzometric method in the isometric mode. The experiments used the activator of ATP-sensitive K+-channels of the plasma membrane diazoxide (50, 100, 150 and 200 μM) and the blocker of these channels glibenclamide (1, 2, 4, 6, 8 and 10 μM), as well as the blocker of ATP-sensitive mitochondrial K+-channels 5-hydroxydecanoate (5-HD, 50 µM). The study of mechanokinetics of the contraction-relaxation process of muscle preparations was carried out according to the method (Kosterin et al., 2021) with the calculation of the mechanokinetic parameters of the contraction-relaxation cycle: force (Fmax, FC, and FR), time (τ0, τC, and τR), impulse (Іmax, ІC, and ІR) and velocity parameters (VC and VR). Results. It was found that both blocking and activation of plasma membrane KATP-channels cause suppression of the amplitude, probably according to different cellular mechanisms of regulation of ion conductivity. Under the influence of glibenclamide, a significant decrease in the frequency and mechanokinetic parameters of spontaneous contractions of the myometrium was observed, which confirms the contribution of plasma membrane KATP-channels to maintaining the excitability of the myometrium of non-pregnant rats. Activation of plasma membrane KATP-channels by diazoxide caused a change in individual mechanokinetic parameters of spontaneous contractions of the myometrium. Under the action of the mitoKATP-channel blocker 5-HD, suppression of the amplitude and modulation of the mechanokinetic parameters of the contraction phase was observed without changes in the kinetics of the relaxation phase of spontaneous contractions. Conclusions. Thus, modulation of the KATP-channels of the plasma membrane and mitochondria is accompanied by the suppression of spontaneous contractions of the myometrium. Both types of KATP-channels are important regulators of myometrial excitability, however, unlike plasma membrane KATP-channels, mitoKATP-channels probably do not modulate the processes of extrusion of Ca2+ from the cytosol.

Age-related changes of myofibrils in white laboratory rats of different ages

The study of isolated preparations of experimental white rats of 14 months and older reveals that the longitudinal orientation of cardiomyocytes is replaced by an angular orientation in older mice. The number and structural integrity of myofibrils occupy one of the leading places in the regulation of the contraction-relaxation cycle and the implementation of the adaptive capabilities of the myocardium in the normal functioning of cardiomyocytes. Our experimental studies aim at describing the quantitative and morphological characteristics of myofibrils in rats of two age groups and analyzing the morphological and functional results. Isolated heart preparations were examined in two age groups of white rats (middle - 8-14 years; elderly - 14 months and more. The number of rats in each group amounted to six) using different histological staining methods. The results obtained show that, compared with middle-aged rats, the development of degenerative changes in myofibrils is observed in old rats, which is manifested in structural inferiority and quantitative reduction. Degenerative changes revealed with age at the intracellular level can be considered an important morphological basis for the formation of functional heart failure and the depletion of adaptive capabilities.

Proteomic profiling of impaired excitation-contraction coupling and abnormal calcium handling in muscular dystrophy.

The X-linked inherited neuromuscular disorder Duchenne muscular dystrophy is characterised by primary abnormalities in the membrane cytoskeletal component dystrophin. The almost complete absence of the Dp427-M isoform of dystrophin in skeletal muscles renders contractile fibres more susceptible to progressive degeneration and a leaky sarcolemma membrane. This in turn results in abnormal calcium homeostasis, enhanced proteolysis and impaired excitation-contraction coupling. Biochemical and mass spectrometry-based proteomic studies of both patient biopsy specimens and genetic animal models of dystrophinopathy have demonstrated significant changes in the concentration and/or physiological function of essential calcium-regulatory proteins in dystrophin-lacking voluntary muscles. Abnormalities include dystrophinopathy-associated changes in voltage sensing receptors, calcium release channels, calcium pumps and calcium binding proteins. This review article provides an overview of the importance of the sarcolemmal dystrophin-glycoprotein complex and the wider dystrophin complexome in skeletal muscle and its linkage to depolarisation-induced calcium-release mechanisms and the excitation-contraction-relaxation cycle. Besides chronic inflammation, fat substitution and reactive myofibrosis, a major pathobiochemical hallmark of X-linked muscular dystrophy is represented by the chronic influx of calcium ions through the damaged plasmalemma in conjunction with abnormal intracellular calcium fluxes and buffering. Impaired calcium handling proteins should therefore be included in an improved biomarker signature of Duchenne muscular dystrophy.

Acoustic Fabrication of Living Cardiomyocyte-based Hybrid Biorobots.

Organized assemblies of cells have demonstrated promise as bioinspired actuators and devices; still, the fabrication of such "biorobots" has predominantly relied on passive assembly methods that reduce design capabilities. To address this, we have developed a strategy for the rapid formation of functional biorobots composed of live cardiomyocytes. We employ tunable acoustic fields to facilitate the efficient aggregation of millions of cells into high-density macroscopic architectures with directed cell orientation and enhanced cell-cell interaction. These biorobots can perform actuation functions both through naturally occurring contraction-relaxation cycles and through external control with chemical and electrical stimuli. We demonstrate that these biorobots can be used to achieve controlled actuation of a soft skeleton and pumping of microparticles. The biocompatible acoustic assembly strategy described here should prove generally useful for cellular manipulation in the context of tissue engineering, soft robotics, and other applications.

Destabilizing tropomyosin-troponin I interactions increases calcium sensitivity

The cardiac thin filament (TF), containing troponin (Tn) and tropomyosin (Tpm) bound to filamentous actin, is the main transducer that relates sarcoplasmic calcium levels to the contraction-relaxation cycles of the myocardium. In the absence of calcium, Tn interactions with Tpm inhibit actomyosin interactions. Calcium binding to TnC induces a shift in Tpm position along thin filaments, allowing for heart muscle contraction. While much of the TF structure has been well characterized, important regions of the communication pathway where Tn triggers Tpm movement remain unresolved.

Data analysis technique influences blood flow kinetics parameter estimates for moderate- and heavy-intensity exercise transitions.

What is the central question of this study? During exercise, there are fluctuations in conduit artery blood flow (BF) caused by both cardiac and muscle contraction-relaxation cycles. What is the optimal method to process Doppler ultrasound-measured BF for the purpose of characterizing the dynamic response of BF during step-transitions in exercise? What is the main finding and its importance? Continuous BF data were processed in relation to either cardiac or muscle contraction-relaxation cycles and computed based on 'binned' or 'rolling' averages over one, two or five consecutive cycles. Kinetics characterization revealed no data processing technique-specific differences in steady-state BF, but variability in the rapidity at which BF attained steady-state (i.e., mean response time) was observed. The overall rate of blood flow (BF) adjustment (i.e., kinetics) from the onset of an exercise transition can be quantified by the mean response time (MRT). However, the BF response profile can be distorted during rhythmic, dynamic exercise consequent to variations caused by the cardiac cycle (HR) and the muscle contraction-relaxation (CR) cycle. We examined the extent to which distortions imposed by HR and CR cycles affected BF kinetics. Eight healthy, young men (27 (4) years; mean (SD)) performed transitions of alternate-leg knee-extension exercise from 3W to either a moderate- (MOD) or heavy-intensity (HVY) power output. Femoral artery BF was continuously measured by Doppler ultrasound and averaged over one, two or five 'binned' (e.g., HR2b, etc.) or 'rolling' (e.g., CR5r, etc.) HR and CR cycles. Among analysis techniques, there were no differences for steady-state BF values at the 3W baseline. In MOD, MRT using contraction-relaxation cycle (CR1) was smaller than most other analysis techniques. For both MOD and HVY, the 95% confidence interval for MRT was generally larger when using HR- compared to CR-related methods, and monoexponential fits based on 'rolling' averages (HR2r, HR5r, CR2r, CR5r) had a poorer ability to estimate the true end-exercise BF in HVY than in MOD. When modelling BF kinetics, we conclude that the CR1 method is a good option because of its ability to accurately estimate the 'data-determined' end-exercise BF value from the 'model-derived' response, maintain a relatively high density of data points during the transition and yield a relatively small 95% CI.

Biomechanical properties of endothelial glycocalyx: An imperfect pendulum

Endothelial glycocalyx plays a crucial role in hemodynamics in health and disease, yet studying it is met by multiple technical hindrances. We attempted to outline our views on some biomechanical properties of endothelial glycocalyx, which are potentially amenable to mathematical modeling. We start with the null-hypothesis ascribing to glycocalyx the properties of a pendulum and reject this hypothesis on the grounds of multiple obstacles for pendulum behavior, such as rich decoration with flexible negatively charged side-chains, variable length and density, fluid fixation to the plasma membrane. We next analyze the current views on membrane attachments to the cortical actin web, its pulsatile contraction-relaxation cycles which rebound to the changes in tension of the plasma membrane. Based on this, we consider the outside-in signaling, the basis for mechanotransduction, and the dampening action of the inside-out signaling. The aperiodic oscillatory motions of glycocalyx and cortical actin web underlie our prediction of two functional pacemakers. We next advance an idea that the glycocalyx, plasma membrane, and cortical actin web represent a structure-functional unit and propose the concept of tensegrity model. Finally, we present our recent data suggesting that erythrocytes are gliding or hovering and rotating over the surface of intact glycocalyx, whereas the rotational and hovering components of their passage along the capillaries are lost when glycocalyx of either is degraded. These insights into the mechanics of endothelial glycocalyx motions may be of value in crosspollination between biomechanics, physiology, and pathophysiology for deeper appreciation of its rich untapped resources in health and pharmacotherapy in disease.

REDUCED PERIPHERAL AND INSPIRATORY MUSCLE ENDURANCE IN PATIENTS WITH LIVER CIRRHOSIS: A CROSS-SECTIONAL STUDY.

Liver cirrhosis (LC) causes several musculoskeletal changes. To test the hypothesis that the peripheral and inspiratory muscle endurance are reduced in patients with liver cirrhosis. Twenty-one patients with LC (LC group; 61±14 years) and 18 age-matched subjects (control group; 56±17 years) had accepted to participate in this cross-sectional observational study. To assess peripheral muscle endurance, all volunteers performed a rhythmic handgrip exercise at 45% of their maximum voluntary contraction. A metronome was used to control the contraction-relaxation cycles at 60/min. The inspiratory muscle endurance was assessed using PowerBreath®. Participants underwent inspiratory muscle exercise at 60% of their maximal inspiratory muscle strength. The time until failure characterized the muscle endurance for both handgrip and inspiratory muscle exercises. Additionally, the quality of life of the participants was assessed. The muscle endurance was lower in the LC group when compared to the control group for both handgrip (67 vs 130 s, P<0.001) and inspiratory muscle exercises (40 vs 114 s, P<0.001). The peripheral muscle endurance was directly correlated with the total quality of life score (r=0.439, P=0.01) and to the following domains: fatigue (r=0.378 e P=0.030), activity (r=0.583, P=0.001), systemic symptoms (r=0.316, P=0.073) and preoccupation (r=0.370, P=0.034). The inspiratory muscle endurance was inversely correlated with the total quality of life score (r=0.573, P=0.001) and the following domains: fatigue (r=0.503, P=0.002), activity (r=0.464, P=0.004), systemic symptoms (r=0.472, P=0.004), abdominal symptoms (r=0.461, P=0.005), emotional function (r=0.387, P=0.02) and preoccupation (r=0.519, P=0.001). Both peripheral and inspiratory muscle endurance were lower in LC patients when compared to the control group.