Potassium-induced potentiation of subtetanic force in rat skeletal muscles: influences of β2-activation, lactic acid, and temperature

Purpose: Moderate elevations of [K+]o occur during exercise and have been shown to potentiate force during contractions elicited with subtetanic frequencies. Here, we investigated whether lactic acid (reduced chloride conductance), β2-adrenoceptor activation, and increased temperature would influence the potentiating effect of potassium in slow- and fast-twitch muscle. Methods: Isometric contractions were elicited by electrical stimulation at various frequencies in isolated rat soleus and extensor digitorum longus (EDL) muscles incubated at normal (4 mM) or elevated K+, in combination with either salbutamol (5 μM), lactic acid (18.1 mM), 9-AC (25 μM) or increased temperature (30 to 35°C). Results: Elevating [K+] from 4 mM to 7 mM (soleus) and 10 mM (EDL) potentiated isometric twitch and subtetanic force while slightly reducing tetanic. In EDL, salbutamol further augmented twitch force (+27±3 %, P<0.001) and subtetanic force (+22±4 %, P<0.001). In contrast, salbutamol reduced subtetanic force (-28±6 %, P<0.001) in soleus muscles. Lactic acid and 9-AC had no significant effects on isometric force of muscles already exposed to moderate elevations of [K+]o. The potentiating effect of elevated [K+]o was still well maintained at 35°C. Conclusion: Addition of salbutamol exerts a further force-potentiating effect in fast-twitch but not in slow-twitch muscles already potentiated by moderately elevated [K+]o, whilst neither lactic acid, 9-AC nor increased temperature exerts any further augmentation. However, the potentiating effect of elevated [K+]o was still maintained in the presence of these, thus emphasizing the positive influence of moderately elevated [K+]o for contractile performance during exercise.

Adaptation of the Calcium-dependent Tension Development in Ventricular Cardiomyocytes

Today a variety of models describe the physiological behavior of the heart on a cellular level. The intracellular calcium concentration plays an important role, since it is the main driver for the active contraction of the heart. Due to different implementations of the calcium dynamics, simulating cardiac electromechanics can lead to severely different behaviors of the active tension when coupling the same tension model with different electrophysiological models. To handle these variations, we present an optimization tool that adapts the parameters of the most recent, human based tension model. The goal is to generate a physiologically valid tension development when coupled to an electrophysiological cellular model independent of the specifics of that model's calcium transient. In this work, we focus on a ventricular cell model. In order to identify the calcium-sensitive parameters, a sensitivity analysis of the tension model was carried out. In a further step, the cell model was adapted to reproduce the sarcomere length-dependent behavior of troponin C. With a maximum relative deviation of 20.3% per defined characteristic of the tension development, satisfactory results could be obtained for isometric twitch tension. Considering the length-dependent troponin handling, physiological behavior could be reproduced. In conclusion, we propose an algorithm to adapt the tension development model to any calcium transient input to achieve a physiologically valid active contraction on a cellular level. As a proof of concept, the algorithm is successfully applied to one of the most recent human ventricular cell models. This is an important step towards fully coupled electromechanical heart models, which are a valuable tool in personalized health care.

Abstract P459: Mavacamten And Danicamtiv Reverse Respective Contractile Abnormalities In Engineered Heart Tissue Models Of Hypertrophic And Dilated Cardiomyopathy

Missense mutations in alpha-tropomyosin (TPM1) can lead to development of hypertrophic (HCM) or dilated cardiomyopathy (DCM). HCM mutation E62Q and DCM mutation E54K have previously been studied extensively in experimental systems ranging from in vitro biochemical assays to animal models, although some conflicting results have been found. We undertook a detailed multi-scale assessment of these mutants that included atomistic simulations, regulated in vitro motility (IVM) assays, and finally physiologically relevant human engineered heart tissues. In IVM assays, E62Q previously has shown increased Calcium sensitivity. New molecular dynamics data shows mutation-induced changes to tropomyosin dynamics and interactions with actin and troponin. Human engineered heart tissues (EHT) were generated by seeding iPSC-derived cardiomyocytes engineered using CRISPR/CAS9 to express either E62Q or E54K cardiomyopathy mutations. After two weeks in culture, E62Q EHTs showed a drastically hypercontractile twitch force and significantly increased stiffness while displaying little difference in twitch kinetics compared to wild-type isogenic control EHTs. On the other hand, E54K EHTs displayed hypocontractile isometric twitch force with faster kinetics, impaired length-dependent activation and lowered stiffness. Given these contractile abnormalities, we hypothesized that small molecule myosin modulators to appropriately activate or inhibit myosin activity would restore E54K or E62Q EHTs to normal behavior. Accordingly, E62Q EHTs were treated with 0.5μM mavacamten (to remedy hypercontractility) and E54K EHTs with 0.5 μM danicamtiv (to remedy hypocontractility) for 4 days, followed by a 1 day washout period. Upon contractility testing, it was observed that the drugs were able to reverse contractile phenotypes observed in mutant EHTs and restore contractile properties to levels resembling those of the untreated wild type group. The computational, IVM and EHT studies provide clear evidence in support of the hyper- vs. hypo-contractility paradigm as a common axis that distinguishes HCM and DCM TPM1 mutations. Myosin modulators that directly compensate for underlying myofilament aberrations show promising efficacy in human in vitro systems.

Shark liver oil consumption decreases contractility in EDL muscle of trained rats

Introduction: Professional and recreational athletes make daily use of nutritional supplements to improve physical performance. Polyunsaturated fatty acids (PUFAs) have been used in this sense. N-3 PUFA, particularly eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are involved in important physiological functions and the benefits of supplementation are demonstrated in several types of users. Shark liver oil (SLO) is a natural source of n-3 PUFA. Objective: To evaluate the effect of supplementation with SLO on contractility of skeletal muscles with different metabolic characteristics, soleus and extensor digitorum longus (EDL) from rats submitted to eight weeks of interval training of progressive intensity on a motorized treadmill. In the supplemented group, animals were supplemented with SLO (1 g/kg) five times a week for eight weeks. Method: Contractile parameters as maximum isometric twitch force (Tmax), maximum speed of force development (+dF/dt), maximum speed of force decrease (-dF/dt), maximum tetanic force (Fmax) and resistance to fatigue were analyzed in isolated muscle. Results: Compared to the control group, EDL muscles from the supplemented group reduced Tmax at the first (10.82 ± 0.89 vs 14.30 ± 0.67 mN/mm2. p < 0.01) and second minutes of experimentation (9.85 ± 0.63 vs 13.12 ± 0.70 mN/mm2. p < 0.01). However, it increased resistance to fatigue (22.80 ± 0.97 vs 18.60 ± 0.51 seconds. p = 0.005). Conclusion: No difference was observed in the soleus muscle.

Severity of Myasthenia Gravis Influences the Relationship between Train-of-four Ratio and Twitch Tension and Run-down of Rat Endplate Potentials

Background Train-of-four ratio (TOFR) is often used to evaluate muscle relaxation caused by neuromuscular-blocking agents (NMBAs). However, it is unknown whether TOFR reliably correlates with the first twitch tension (T1) in patients with myasthenia gravis (MG). By using rat models of experimental autoimmune MG (EAMG), the authors verified the hypothesis that the severity of MG influences the relationship between TOFR and T1. Methods EAMG rats were divided into sham, moderate MG, and severe MG groups. Isometric twitch tension of the hemidiaphragm was elicited by phrenic nerve stimulation with and without use of the NMBA rocuronium to measure TOFR and T1, and run-down of endplate potentials was estimated in the three groups. Changes around the neuromuscular junction in EAMG rats were investigated by observation of electron micrographs. Results With similar attenuation of T1, TOFR was significantly (n = 6) different among the three groups in the presence of 50% inhibitory concentrations of rocuronium (IC50). Run-down in the sham group was significantly (n = 8) greater with exposure to IC50, whereas that in the severe MG group was statistically insignificant. Width of the primary synaptic cleft in the severe MG group was significantly (n = 80) greater than that in the other groups. Conclusions Severity of MG influences the relationship between TOFR and T1, together with changes in run-down of endplate potentials and those around the neuromuscular junction in rats. TOFR may, therefore, not be an accurate indicator of recovery from NMBAs in MG patients.

R4496C RyR2 mutation impairs atrial and ventricular contractility

Ryanodine receptor (RyR2) is the major Ca2+ channel of the cardiac sarcoplasmic reticulum (SR) and plays a crucial role in the generation of myocardial force. Changes in RyR2 gating properties and resulting increases in its open probability (Po) are associated with Ca2+ leakage from the SR and arrhythmias; however, the effects of RyR2 dysfunction on myocardial contractility are unknown. Here, we investigated the possibility that a RyR2 mutation associated with catecholaminergic polymorphic ventricular tachycardia, R4496C, affects the contractile function of atrial and ventricular myocardium. We measured isometric twitch tension in left ventricular and atrial trabeculae from wild-type mice and heterozygous transgenic mice carrying the R4496C RyR2 mutation and found that twitch force was comparable under baseline conditions (30°C, 2 mM [Ca2+]o, 1 Hz). However, the positive inotropic responses to high stimulation frequency, 0.1 µM isoproterenol, and 5 mM [Ca2+]o were decreased in R4496C trabeculae, as was post-rest potentiation. We investigated the mechanisms underlying inotropic insufficiency in R4496C muscles in single ventricular myocytes. Under baseline conditions, the amplitude of the Ca2+ transient was normal, despite the reduced SR Ca2+ content. Under inotropic challenge, however, R4496C myocytes were unable to boost the amplitude of Ca2+ transients because they are incapable of properly increasing the amount of Ca2+ stored in the SR because of a larger SR Ca2+ leakage. Recovery of force in response to premature stimuli was faster in R4496C myocardium, despite the unchanged rates of recovery of L-type Ca2+ channel current (ICa-L) and SR Ca2+ content in single myocytes. A faster recovery from inactivation of the mutant R4496C channels could explain this behavior. In conclusion, changes in RyR2 channel gating associated with the R4496C mutation could be directly responsible for the alterations in both ventricular and atrial contractility. The increased RyR2 Po and fractional Ca2+ release from the SR induced by the R4496C mutation preserves baseline contractility despite a slight decrease in SR Ca2+ content, but cannot compensate for the inability to increase SR Ca2+ content during inotropic challenge.

Abstract 37: The Positive Inotropic S100a1 Prevents Arrhythmogenic Sarcoplasmic Reticulum Ca2+ Leak And Ventricular Arrhythmias

S100A1 has emerged as a key factor in the control of cardiomyocyte (CM) contractile performance. Improved sarcoplasmic reticulum (SR) function with enhanced Ca2+ resequestration appears critical for its cAMP-independent inotropic effects but raises concerns about potential diastolic SR Ca2+ leakage that might trigger fatal arrhythmias. Thus, the goal of this study was to determine the impact of S100A1 on ryanodine receptor 2 (RyR2)-mediated SR Ca2+ leakage in vitro and in vivo. S100A1 association with the RyR2 was significantly diminished (-50%) in failing cardiomyocytes and hearts with S100A1 downregulation, as shown by co-immunofluorescence, co-immunoprecipitation and proximity ligation assay. Adenoviral-mediated S100A1 overexpression (3-4 fold vs. GFP-control) in quiescent NCs (normal CMs) and FCs (failing CMs) decreased SR Ca2+-frequency (-50 and −40% respectively) and protected from β-AR-triggered diastolic Ca2+-waves (-62 and −58% respectively) in electrically stimulated (2Hz) CMs as assessed by epifluorescent and confocal Ca2+ imaging. In multicellular rat engineered heart tissue (EHT), S100A1-overexpression (6-8 fold vs. GFP-control) protected from Ca2+-triggered after-contractions (ACs) (-50%) with preserved enhancement of isometric twitch force (TF, +40%) at 2Hz. S100A1-mediated rescue of contractile failure of endothelin-1-treated EHT (-50% decrease in TF) was associated with protection from Ca2+-triggered ACs. In mice with post-ischemic heart failure, AAV9-mediated therapeutic administration of S100A1 enhanced S100A1/RyR2 association and prevented epinephrine-induced VTs (70% in MI group vs. 30% in MI-S100A1 group). Mechanistically, S100A1-overexpression changed neither PKA/CaMKII RyR2 phosphorylation pattern nor binding of accessory proteins like FKBP12.6, calmodulin or sorcin to RyR2 but enhanced S100A1/RyR2 stoichiometry. Our data provide evidence that S100A1 interaction with the RyR2 can beneficially modulate and reverse diastolic RyR2 function dysfunction. S100A1 appears to convey a rather unique molecular profile combining cAMP-independent inotropy with protection against Ca2+-triggered arrhythmias.

Potentiation in mouse lumbrical muscle without myosin light chain phosphorylation: Is resting calcium responsible?

The increase in isometric twitch force observed in fast-twitch rodent muscles during or after activity, known universally as potentiation, is normally associated with myosin regulatory light chain (RLC) phosphorylation. Interestingly, fast muscles from mice devoid of detectable skeletal myosin light chain kinase (skMLCK) retain a reduced ability to potentiate twitch force, indicating the presence of a secondary origin for this characteristic feature of the fast muscle phenotype. The purpose of this study was to assess changes in intracellular cytosolic free Ca2+ concentration ([Ca2+]i) after a potentiating stimulus in mouse lumbrical muscle (37°C). Lumbricals were loaded with the Ca2+-sensitive fluorescent indicators fura-2 or furaptra to detect changes in resting and peak, respectively, intracellular Ca2+ levels caused by 2.5 s of 20-Hz stimulation. Although this protocol produced an immediate increase in twitch force of 17 ± 3% (all data are n = 10) (P &lt; 0.01), this potentiation dissipated quickly and was absent 30 s afterward. Fura-2 fluorescence signals at rest were increased by 11.1 ± 1.3% (P &lt; 0.01) during potentiation, indicating a significant increase in resting [Ca2+]i. Interestingly, furaptra signals showed no change to either the amplitude or the duration of the intracellular Ca2+ transients (ICTs) that triggered potentiated twitches during this time (P &lt; 0.50). Immunofluorescence work showed that 77% of lumbrical fibers expressed myosin heavy chain isoform IIx and/or IIb, but with low expression of skMLCK and high expression of myosin phosphatase targeting subunit 2. As a result, lumbrical muscles displayed no detectable RLC phosphorylation either at rest or after stimulation. We conclude that stimulation-induced elevations in resting [Ca2+]i, in the absence of change in the ICT, are responsible for a small-magnitude, short-lived potentiation of isometric twitch force. If operative in other fast-twitch muscles, this mechanism may complement the potentiating influence of myosin RLC phosphorylation.