Contraction Kinetics Research Articles

Deletion of Enigma Homologue from the Z-disc slows tension development kinetics in mouse myocardium.

Enigma Homologue (ENH) is a component of the Z-disc, a structure that anchors actin filaments in the contractile unit of muscle, the sarcomere. Cardiac-specific ablation of ENH protein expression causes contractile dysfunction that ultimately culminates in dilated cardiomyopathy. However, whether ENH is involved in the regulation of myocardial contractility is unknown. To determine if ENH is required for the mechanical activity of cardiac muscle, we analyze muscle mechanics of isolated trabeculae from the hearts of ENH +/+ and ENH -/- mice. We detected no differences in steady-state mechanical properties but show that when muscle fibers are allowed to relax and then are restretched, the rate at which tension redevelops is depressed in ENH -/- mouse myocardium relative to that in ENH +/+ myocardium. SDS-PAGE analysis demonstrated that the expression of β-myosin heavy chain is increased in ENH -/- mouse myocardium, which could partially, but not completely, account for the depression in tension redevelopment kinetics. Using top-down proteomics analysis, we found that the expression of other thin/thick filament regulatory proteins is unaltered, although the phosphorylation of a cardiac troponin T isoform, cardiac troponin I, and myosin regulatory light chain is decreased in ENH -/- mouse myocardium. Nevertheless, these alterations are very small and thus insufficient to explain slowed tension redevelopment kinetics in ENH -/- mouse myocardium. These data suggest that the ENH protein influences tension redevelopment kinetics in mouse myocardium, possibly by affecting cross-bridge cycling kinetics. Previous studies also indicate that ablation of specific Z-disc proteins in myocardium slows contraction kinetics, which could also be a contributing factor in this study.

Contraction and intracellular calcium transport in epicardial and endocardial ventricular myocytes from streptozotocin-induced diabetic rat

Introduction: Diabetes mellitus (DM) is a global health problem. According to the International Diabetes Federation, 424.9 million people suffered from DM in 2017 and this number is expected to rise to 628.6 million by 2045. Although diabetes can affect every organ in the body, cardiovascular disease is a major cause of death and disability in people with diabetes. Diabetic patients frequently suffer from systolic and diastolic dysfunction. Within the ventricles, the electromechanical properties of cardiac myocytes vary transmurally. Aims and Objectives: The aim of this study was to investigate contraction and Ca2+ transport in epicardial (EPI) and endocardial (ENDO) myocytes from the left ventricle in the streptozotocin (STZ) – induced diabetic rat heart. Materials and Methods: Experiments were performed 5-6 months after STZ treatment. Ventricular myocytes were isolated by enzymic and mechanical dispersal techniques from EPI and ENDO regions of the left ventricle. Contraction and free intracellular Ca2+ concentration [Ca2+]i were measured by video edge detection and fluorescence photometry techniques, respectively. Results: Myocyte length and calculated surface area were smaller in EPI-STZ compared to EPI-CON. Time to peak (TPK) shortening was prolonged in EPI-STZ compared to EPI-CON and in ENDO-STZ compared to ENDO-CON myocytes. Time to half (THALF) relaxation of shortening was prolonged in EPI-STZ compared to EPI-CON. TPK Ca2+ transient was prolonged in EPI-STZ compared to EPI-CON, ENDO-STZ compared to ENDO-CON, ENDO-STZ compared to EPI-STZ and in ENDO-CON compared to EPI-CON myocytes. THALF decay of the Ca2+ transient was prolonged in ENDO-STZ compared to ENDO-CON. Fractional release of Ca2+ was increased in ENDO-STZ compared to ENDO-CON and in ENDO-STZ compared to EPI-STZ. Recovery of the Ca2+ transient was prolonged in ENDO-STZ compared to ENDO-CON. Conclusion: In conclusion the kinetics of contraction and Ca2+ transient and fractional release of Ca2+ from the sarcoplasmic reticulum are altered to different extents in EPI and ENDO myocytes from STZ-induced diabetic rat.

Adrenergic and adenosinergic regulation of the cardiovascular system in an Antarctic icefish: Insight into central and peripheral determinants of cardiac output

Icefishes characteristically lack the oxygen-binding protein haemoglobin and therefore are especially reliant on cardiovascular regulation to augment oxygen transport when oxygen demand increases, such as during activity and warming. Using both in vivo and in vitro experiments, we evaluated the roles for adrenaline and adenosine, two well-established cardio- and vasoactive molecules, in regulating the cardiovascular system of the blackfin icefish, Chaenocephalus aceratus. Despite increasing cardiac contractility (increasing twitch force and contraction kinetics in isometric myocardial strip preparations) and accelerating heart rate (ƒH), adrenaline (5 nmol kg−1 bolus intra-arterial injection) did not significantly increase cardiac output (Q̇) in vivo because it elicited a large decrease in vascular conductance (Gsys). In contrast, and despite preliminary data suggesting a direct negative inotropic effect of adenosine on isolated atria and little effect on isolated ventricle strips, adenosine (500 nmol kg−1) generated a large increase in Q̇ by increasing Gsys, a change reminiscent of that previously reported during both acute warming and invoked activity. Our data thus illustrate how Q̇ in C. aceratus may be much more dependent on peripheral control of vasomotor tone than direct regulation of the heart.

Regulatory light chain phosphorylation augments length-dependent contraction in PTU-treated rats.

Force production by actin-myosin cross-bridges in cardiac muscle is regulated by thin-filament proteins and sarcomere length (SL) throughout the heartbeat. Prior work has shown that myosin regulatory light chain (RLC), which binds to the neck of myosin heavy chain, increases cardiac contractility when phosphorylated. We recently showed that cross-bridge kinetics slow with increasing SLs, and that RLC phosphorylation amplifies this effect, using skinned rat myocardial strips predominantly composed of the faster α-cardiac myosin heavy chain isoform. In the present study, to assess how RLC phosphorylation influences length-dependent myosin function as myosin motor speed varies, we used a propylthiouracil (PTU) diet to induce >95% expression of the slower β-myosin heavy chain isoform in rat cardiac ventricles. We measured the effect of RLC phosphorylation on Ca2+-activated isometric contraction and myosin cross-bridge kinetics (via stochastic length perturbation analysis) in skinned rat papillary muscle strips at 1.9- and 2.2-µm SL. Maximum tension and Ca2+ sensitivity increased with SL, and RLC phosphorylation augmented this response at 2.2-µm SL. Subtle increases in viscoelastic myocardial stiffness occurred with RLC phosphorylation at 2.2-µm SL, but not at 1.9-µm SL, thereby suggesting that RLC phosphorylation increases β-myosin heavy chain binding or stiffness at longer SLs. The cross-bridge detachment rate slowed as SL increased, providing a potential mechanism for prolonged cross-bridge attachment to augment length-dependent activation of contraction at longer SLs. Length-dependent slowing of β-myosin heavy chain detachment rate was not affected by RLC phosphorylation. Together with our previous studies, these data suggest that both α- and β-myosin heavy chain isoforms show a length-dependent activation response and prolonged myosin attachment as SL increases in rat myocardial strips, and that RLC phosphorylation augments length-dependent activation at longer SLs. In comparing cardiac isoforms, however, we found that β-myosin heavy chain consistently showed greater length-dependent sensitivity than α-myosin heavy chain. Our work suggests that RLC phosphorylation is a vital contributor to the regulation of myocardial contractility in both cardiac myosin heavy chain isoforms.

Clonal Kinetics and Single Cell Transcriptional Profiling of Adoptively Transferred CD19 CAR-T Cells

INTRODUCTION:When introduced into polyclonal T cells, chimeric antigen receptors (CAR) redirect specificity of the engineered T cells to an antigen recognized by the CAR. We conducted a phase I/II clinical trial of treatment of relapsed and refractory CD19-positive B cell malignancies using a defined formulation of CD4+ and CD8+ CD19-specific CAR-T cells (NCT01865617). Little is known about the transcriptional heterogeneity of CAR-T cells in the infused product and their clonal kinetics after adoptive transfer.METHODS:To understand the factors that impact clonal CAR-T cell behavior in vivo, we performed TCRBV sequencing and single cell transcriptional profiling (10X Genomics) on CD8+ CAR-T cells isolated from infused products and the blood of treated patients. TCRBV sequencing was performed on 0.8 to 1.5 million cells from the infused product and 700-65,000 CAR-T cells from blood after CAR-T infusion. For single-cell RNA sequencing (scRNAseq), we obtained paired 5' gene expression and V(D)J data from individual CAR-T cells isolated from infused products and from the blood at the peak of in vivo expansion, after contraction, and at a late time point.RESULTS:High-throughput sequencing of the TCRBV genes revealed that CAR-T cells were polyclonal in the infused products, and during in vivo expansion and contraction, and at late times (≥ 3 months) after adoptive transfer. We evaluated the diversity of the TCRBV repertoire using the Shannon entropy index, and found that clonal diversity was highest in the infused product and declined at later time points after adoptive transfer. Loss of diversity after adoptive transfer was due to both expansion of higher frequency CAR-T cell clones and loss of low-frequency clones. We identified distinct CAR-T cell clones in the infused product and in blood at multiple time points after infusion that exhibited different kinetics of expansion and contraction. To examine the transcriptional programs that regulate the fate of CAR-T cells after infusion, we performed scRNAseq on CD8+ CAR-T cells, and found transcriptional heterogeneity in the infused products, which declined in CD8+ CAR-T cells isolated from patient blood after adoptive transfer. Gene set enrichment analysis showed that the infused products expressed higher levels of genes associated with hypoxia, glycolysis, and proliferation, and lower levels of genes associated with cytotoxicity compared to CAR-T cells isolated after adoptive transfer. In the infused product, genes associated with cytotoxicity were expressed at higher levels in CAR-T cells harboring clonotypes that were subsequently represented at relatively higher levels in vivo after adoptive transfer.CONCLUSIONS:There is transcriptional heterogeneity in the infused product and distinct CAR-T cell clones exhibit different kinetics of expansion and contraction after infusion. A better understanding of the kinetics of clonal expansion of CAR-T cells after adoptive transfer may provide insight into strategies to improve CAR-T cell immunotherapy. DisclosuresTurtle:Nektar Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Caribou Biosciences: Membership on an entity's Board of Directors or advisory committees; Juno/Celgene: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Active Calpain Promotes Fibrin Clot Contraction By Strengthening the Coupling of Fibrin-Bound αIIbβ3 to the Platelet Cytoskeleton

Platelet-driven blood clot contraction reduces and compacts clot volume, promoting hemostasis while restoring blood flow past otherwise obstructive thrombi. Clot contraction is driven by traction forces in the range of tens of pN per platelet that are generated by platelet non-muscle myosin IIa and actin on αIIbβ3 bound to extracellular fibrin fibers. In an analysis of the kinetics of thrombin-induced clot formation and contraction in platelet-rich plasma, we found that clot contraction has two discernable phases: an exponential initiation phase and a second exponential contraction phase. It is noteworthy that Azam et al (Mol Cell Biol21:2213-20, 2001) observed that deleting the µ-calpain gene in mice impairs platelet-mediated clot contraction. In this context, we found that inhibiting calpain activity with ALLM (N-Acetyl-Leu-Leu-Methional), a cell-permeable calpain inhibitor, significantly prolonged the duration of the initiation phase and decreased the kinetic rate constants for both the initiation and contraction phases of thrombin-stimulated platelet-mediated clot contraction, but without affecting the overall extent of contraction. The calpain inhibitor ALLN (N-acetyl-Leu-Leu-Norleu) had the same effect. Thus, these studies imply that activation of platelet calpain facilitates the transmission of traction forces from the platelet cytoskeleton to the αIIbβ3 bound to fibrin fibers. μ-Calpain is calcium-dependent cytosolic neutral cysteine protease that is activated 30-60 seconds after the onset of platelet aggregation stimulated by platelet agonists such as thrombin. To identify the calpain-cleaved protein or proteins involved in clot contraction, we incubated washed human platelets with the thrombin activation peptide TRAP in the presence or absence of calpain inhibitors and identified calpain-cleaved proteins using subtiligase-mediated biotin-labeling of nascent N-termini followed by mass spectrometry. We identified 32 proteins in the platelet cytosol that undergo calpain-mediated cleavage after TRAP stimulation. Many of these are cytoskeletal proteins, most prominently talin which connects integrins to the cytoskeleton and vinculin which is required for myosin-contractility-dependent effects on traction force and adhesion strength. Accordingly, we focused our attention on these two proteins. Talin, a 250 kD protein, is composed of a 45 kD N-terminal head domain attached via a flexible linker to a 200 kD C-terminal rod domain. The rod domain consists of 62 amphipathic α-helices organized into a series of four- and five-helix bundles. Vinculin is a 116 kD protein composed of 5 domains and has an autoinhibited structure in which domains 1-3 bind to domain 5. We detected 8 calpain-mediated cleavages in talin, 2 previously identified cleavage sites in unstructured regions and 6 others in α-helical regions known to interact with other proteins. Four of the latter are located within the first 12 talin helices, a region that contains four vinculin binding sites (VBSs). Likewise, the remaining two cleavage sites are in proximity to a VBS. A VBS is a vinculin-binding α-helix that is buried in a helical bundle due to extensive hydrophobic interactions with other amphipathic helices. Because VBS are packed into the interior of a helical bundle, a structural rearrangement is required to initiate vinculin binding. Using magnetic tweezers and atomic force microscopy, del Rio et al demonstrated that force-induced stretching of single talin rod molecules activates VBSs (Science323:638-41, 2009)), implying that stretching causes the conformational change required to expose buried VBSs. Thus, based on the location of the calpain-mediated cleavages in talin, we hypothesize that by cleaving talin in proximity to a VBS, calpain facilitates vinculin to binding talin, thereby strengthening the coupling of fibrin-bound αIIbβ3 to the platelet cytoskeleton to promote fibrin clot contraction. DisclosuresNo relevant conflicts of interest to declare.

Piezo-bending actuators for isometric or auxotonic contraction analysis of engineered heart tissue.

Engineered heart tissue (EHT) has proven as valuable tool for disease modelling, drug safety screening, and cardiac repair. Especially in combination with the stem cell technology, these in vitro models of the human heart have generated interest not only of basic cardiovascular researchers but also of regulatory authorities responsible for drug safety. A main limitation of 3D-based assays for evaluating cardiotoxicity is their limited throughput. We integrated piezo-bending actuators in a 24-well system for the generation of strip-like rat and human EHT attached to hollow, elastic silicone posts. Muscle contractions of EHTs induced a measurable electrical current in the piezo-bending actuators that could be analysed for contraction amplitude, frequency, and contraction and relaxation kinetics. Compared with the standard video-optical analysis of contractile activity, the new system allows for (a) the analysis of several tissues in parallel, (b) switching between auxotonic and isometric contractions by inserting a stiff metal post in the silicone post opposing the piezo actuator, (c) continuous measurement over days with low data volume (megabyte), (d) automated measurement without the necessity of adjustment of tissue position for video-optical analysis, (e) reduced complexity and costs, (f) high sensitivity of contraction detection, (g) calculation of absolute contraction force, and (h) suitability for variable tissue geometries. The new set-up for contraction analysis based on piezo-bending actuators is a promising new method for the parallel screening of EHT for pharmacological drug effects and other applications of muscle tissue engineering (e.g., skeletal muscle engineering or cardiac repair).

Atrial-like Engineered Heart Tissue: An InVitro Model of the Human Atrium.

SummaryCardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) are under investigation for their suitability as human models in preclinical drug development. Antiarrhythmic drug development focuses on atrial biology for the treatment of atrial fibrillation. Here we used recent retinoic acid-based protocols to generate atrial CMs from hiPSCs and establish right atrial engineered heart tissue (RA-EHT) as a 3D model of human atrium. EHT from standard protocol-derived hiPSC-CMs (Ctrl-EHT) and intact human muscle strips served as comparators. RA-EHT exhibited higher mRNA and protein concentrations of atrial-selective markers, faster contraction kinetics, lower force generation, shorter action potential duration, and higher repolarization fraction than Ctrl-EHTs. In addition, RA-EHTs but not Ctrl-EHTs responded to pharmacological manipulation of atrial-selective potassium currents. RA- and Ctrl-EHTs’ behavior reflected differences between human atrial and ventricular muscle preparations. Taken together, RA-EHT is a model of human atrium that may be useful in preclinical drug screening.

Abstract 16773: Ablation of Cardiac Myosin Binding Protein-C in Human iPSC- Engineered Cardiac Tissue Model Causes Increased Calcium Sensitivity and Accelerated Contractile Kinetics

Hypertrophic cardiomyopathy (HCM) is an inherited cardiac disease that can cause sudden cardiac arrest, diastolic dysfunction and heart failure. Mutations in cardiac myosin binding protein-C (cMyBP-C), a regulator of contractility, are among the most prevalent causes of HCM. cMyBP-C truncation mutations may affect as many as 1 in 1,600 Americans. Though murine models of cMyBP-C ablation have aided in understanding the role of cMyBP-C in health and disease, they are limited by differences in cardiac physiology between mice and humans. iPSC technology now allows researchers to study the effect of HCM mutations in iPSC-derived cardiomyocytes from HCM patients and controls, however differences in genetic background between patients and controls and clonal variation remain confounders. We hypothesized that cMyBP-C ablation in human cardiomyocytes would primarily alter cardiac contractility. CRISPR/Cas9 was used to introduce frame shift mutations in cMyBP-C in the well characterized DF19-9-11T human iPSC line. Human iPSCs were differentiated into cardiomyocytes and then cast into an integrated 3D engineered cardiac tissue (ECT) model for functional testing against non-targeted isogenic controls. Homozygous cMyBP-C ablation significantly accelerated contraction (CT 100 = 219±3 vs. 235±5ms; p = 0.02) and early relaxation (RT 50 = 144±2 vs. 167±4ms; p <0.001), while late relaxation was slowed (RT 50-90 = 127±2 vs. 112±2ms; p <0.001; n=6 for each group). These accelerated contractile kinetics were accompanied by a significantly blunted response to adrenergic stimulation and an increase in Ca 2+ sensitivity. Heterozygous cMyBP-C ablation resulted in acceleration in relaxation kinetics (RT 50 = 148±7 vs. 167±4ms; p = 0.04 and RT 50-90 = 96±4 vs. 112±2ms; p = 0.002), without significantly affecting the rate of contraction. The accelerated kinetics of contraction and early relaxation in cMyBP-C null ECT provides important human evidence that cMyBP-C acts as a brake on cardiac contractility. These data provide strong evidence that the human model system can be used to study the function of cMyBP-C and provide a platform to test therapies that may rectify the contractile defects caused by cMyBP-C haploinsufficiency.

Abstract 17043: Enhanced Na + Influx Alters Myocyte Mechanics and Diastolic Function

The late Na + current has emerged as a therapeutic target for conditions characterized by depressed diastolic function, but whether enhanced Na + fluxes in cardiomyocytes interfere with cellular and myocardial relaxation remain to be clarified. For this purpose, we employed mice with inducible, cardiac restricted deletion of the non-pore forming beta1 subunit of the Na + channel ( Scn1b -KO), a rodent model associated with enhanced Na + influx in cardiomyocytes. Two-dimensional and Doppler echocardiography and ECGs were employed to evaluate cardiac function in Scn1b -KO and control (Ctrl) animals. Properties of Ca 2+ transient and contractility were established in isolated left ventricular (LV) myocytes obtained by enzymatic digestion. Deletion of the Scn1b gene had no consequences on ejection fraction, whereas LV filling pattern was altered, isovolumic relaxation time prolonged (+30%), and electrical recovery, measured by the QT interval, was protracted (+14%). In a subgroup of animals, acute administration of GS967 (0.5 mg/kg body weight), a specific inhibitor of the late Na + current, partly ameliorated diastolic indices and shortened the QT interval. At the cellular level, kinetics of cell shortening and Ca 2+ transients were delayed in Scn1b -KO myocytes (n=609-693), with respect to Ctrl cells (369-298). Specifically, time to peak Ca 2+ and time to 50% decay of the Ca 2+ transient were increased in Scn1b -KO cells by 5% and 17%, respectively. Similarly, time to peak contraction and time to 50% relaxation were enhanced by 6% and 7% in Scn1b -KO. Interestingly, GS967 (300 nM) accelerated temporal parameters of contraction in Scn1b -KO myocytes by ~8% and led to an attenuation of fractional shortening. These effects were not observed in Ctrl cells. Thus, enhanced Na + influx modulates electromechanical coupling in cardiomyocytes and delays kinetics of contraction and relaxation with consequences on diastolic properties of the heart.

Temperature constrains locomotion and muscle function in two temperate labrids

Winter quiescence in fishes is not uncommon, however understanding the mechanisms that cause dormancy are poorly understood. This study highlights the physiological stress temperature places on locomotor musculature and its consequences on whole organism locomotion. Cunner and tautog experience temperatures ranging from 0 to 25 °C and enter dormancy at ~10 °C. We aimed to address the question: how do winter temperatures affect steady swimming and muscle contraction kinetics in cunner? Fishes were collected and housed at 5, 10, 15, or 20 °C. Gait transition speed and fin beat frequency were measured at each acclimation temperature. Twitch and tetanus kinetics were recorded from the aerobic locomotor muscle, which is responsible for the power stroke during swimming. Fish acclimated to colder temperatures (5, 10 °C) demonstrated lower gait transition speeds than the warm temperature treatments. Similarly, twitch kinetics were slower in muscle acclimated at ≤10 °C. Locomotor muscle from tautog was significantly slower to contract and relax than cunner when tested at 5 and 10 °C. These results suggest that muscle acclimation differs in these closely related labrids from the same habitat. Additionally, these results suggest that cunner locomotor musculature can maintain greater performance at a wider range of temperatures. Cunner occupy more northern latitudes which likely allows for greater performance shifts in response to temperature. However, when temperatures get cold enough muscle function is reduced, perhaps contributing to their overwintering ecology.

The effects of endogenous and exogenous catecholamines on hypoxic cardiac performance in red-bellied piranhas.

Catecholamines protect the fish heart during hypoxia. However, the humoral adrenergic stress response may only be invoked in extremis. We investigated the hypothesis that endogenous (e.g., neuronal) myocardial catecholamines may also impact cardiac performance during hypoxia in a hypoxia-tolerant tropical fish, the red-bellied piranha (Pygocentrus nattereri). First, we measured endogenous tissue catecholamines and in vitro catecholamine release from piranha myocardium using ultraperformance liquid chromatography. Ventricle homogenates contained detectable levels of both adrenaline (7.27 ng/g) and noradrenaline (14.48 ng/g), but only noradrenaline was released from ventricular tissue incubated in Ringer's solution. Noradrenaline released in this assay was not affected by hypoxia but was promoted by the catecholamine releasing agent tyramine. Our second series of experiments explored cardiac contractile performance in vitro using tyramine, exogenous noradrenaline or adrenaline, and propranolol (a β-adrenoceptor antagonist). In ventricular strip preparations, β-adrenergic blockade with propranolol had no effects on twitch force or contraction kinetics in either normoxia or hypoxia, confirming that spontaneous endogenous catecholamine release did not impact cardiac performance. However, in the absence of propranolol, tyramine mimicked the positive inotropic effect of noradrenaline (10 µM) during hypoxia, although adrenaline was capable of generating larger effects. Our results suggest that, although it is not spontaneously released, inducible endogenous noradrenaline release may have a significant β-adrenoceptor-dependent impact on hypoxic performance in the fish heart.

Force-frequency relationship and early relaxation kinetics are preserved upon sarcoplasmic blockade in human myocardium.

In this study, we investigated the quantitative and qualitative role of the sarcoplasmic reticulum (SR) in the regulation of the force‐frequency relationship (FFR). We blocked the function of SR with cyclopiazonic acid (CPA) and ryanodine and measured twitch kinetics and developed force at various stimulation frequencies in nonfailing and failing intact human right ventricular trabeculae. We found that developed forces are only slightly reduced upon SR blockade, while the positive FFR in nonfailing trabeculae and negative FFR in failing trabeculae were both preserved. The contraction kinetics (dF/dt, dF/dt/F, and time to peak), however, were significantly slower at all frequencies tested. Kinetics of first 50% of relaxation (RT50) was not affected by SR blockade. Kinetics of entire relaxation process (RT90) was overall slower at low frequencies, but not at high frequencies. From our findings, we conclude that the SR is not essential for FFR, and its role in regulation of FFR lies mostly in contraction kinetics. Unlike small rodents, human myocardium contractile function is near‐normal in absence of a functional SR with little changes in contractile force, and with preservation with the main regulation of FFR.

Red muscle function and thermal acclimationto cold in rainbow smelt, Osmerus mordax, and rainbow trout, Oncorhynchus mykiss.

Climate change affects the thermal environment of aquatic organisms. Changes in the thermal environment may affect muscle function in the eurythermal rainbow smelt, Osmerus mordax, and relatively more stenothermal rainbow trout, Oncorhynchus mykiss. Literature suggests that the trout will be more sensitive to changes in environmental temperature, as they experience a more limited range of environmental temperatures. To examine the effects of thermal environment on red muscle function, both the smelt and trout were thermally acclimated to either a warm (12-15°C) or cold (4-5°C) temperature, after which studies of swimming performance and muscle mechanics were performed. The data on swimming performance and maximum muscle shortening velocity in rainbow smelt were previously published. In both species, cold-acclimated (CA) fish swam with a significantly faster maximum aerobic swimming speed than warm-acclimated fish, when tested at a common temperature of 10°C. Similarly, CA smelt and trout had faster red muscle contraction kinetics. However, smelt displayed a greater shift in contractile properties, such as having a significant shift in maximum muscle shortening velocity that was not observed in trout. The smelt red muscle outperformed trout, with twitch and tetanic times of relaxation being significantly faster for CA smelt compared with CA trout, especially when contraction kinetics were tested at 2°C. The smelt shows a greater thermal acclimation response compared with trout, with more robust increases in maximum swimming speed and faster muscle contractile properties. These differences in acclimation response may contribute to understanding how smelt and trout cope with climate change.

Comparative study of hot air and vacuum drying on the drying kinetics and physicochemical properties of chicory roots

The effect of the drying conditions on the retention quality for dried chicory roots (Cichorium intibyus L.) was investigated. Cubes of chicory roots were dried using hot air and vacuum dryers at 60 and 80°C. Two different air velocities (0.2 and 0.7m/s) were used in the hot air dryer, and two vacuum pressures (25 and 50mmHg absolute) were set in the vacuum chamber. An exhaustive three dimensional mathematical model to describe mass transfer during drying of chicory roots of 1cm of side was presented considering a polynomial functionality for the contraction kinetics. Experimental data obtained at laboratory scale were used to validate the proposed model showing good agreement between the experimental and estimated moisture profiles for both drying procedures. Moisture diffusivity was found to increase with the air drying temperature, velocity and vacuum pressure depending on the drying method. However, higher moisture diffusivity coefficients and lower activation energy values were obtained for the vacuum drying method. Samples dried using the vacuum drier at 60°C and 25mmHg presented better retention quality attributes, i.e., better rehydration, lower shrinkage and higher total phenolic content. The proposed mathematical model was able to satisfactorily predict the described behavior.

A Useful Device to Measure Kinetics of Platelet Contraction.

Platelet contraction provides a minimally invasive source for physiologic information. In this article, we describe a device that directly measures the kinetics of platelet contraction. Whole blood is injected between acrylic plates and an adherent clot forms. The bottom plate is fixed, and the top plate is attached to a wire cantilever. Platelet contraction drives deflection of the wire cantilever which is captured by a camera. Force generated by the clot with time is derived using beam equations. Force derivations were verified using a microelectromechanical (MEMS) force sensor. Kinetics of clot contraction were defined, including maximum contraction force (FMAX), lift-off time (TLIFTOFF), and contraction rate (CR). Metrics were compared with optical aggregometry and thromboelastography. FMAX correlates with optical aggregometry maximal amplitude with a Spearman's rho of 0.7904 and p = 0.0195 and thromboelastography maximal amplitude with a Spearman's rho of 0.8857 and p = 0.0188. Lift-off time correlates with optical aggregometry lag time with a Spearman's rho of 0.9048 and p = 0.002. This preliminary study demonstrates the repeatability of a useful platelet contraction device and its correlation with thromboelastography and optical aggregometry, the gold standard platelet function test.

Structure before function: myosin binding protein-C slow is a structural protein with regulatory properties.

Myosin binding protein-C slow (sMyBP-C) comprises a family of accessory proteins in skeletal muscles that bind both myosin and actin filaments. Herein, we examined the role of sMyBP-C in adult skeletal muscles using in vivo gene transfer and clustered regularly interspaced short palindromic repeats technology to knock down all known sMyBP-C variants. Our findings, confirmed in two different skeletal muscles, demonstrated efficient knockdown (KD) of sMyBP-C (>70%) resulting in notably decreased levels of thick, but not thin, filament proteins ranging from ∼50% for slow and fast myosin to ∼20% for myomesin. Consistent with this, A bands were selectively distorted, and sarcomere length was significantly reduced. Contrary to earlier in vitro studies showing that addition of recombinant sMyBP-C slows down the formation of actomyosin crossbridges, our work demonstrates that KD of sMyBP-C in intact myofibers results in decreased contraction and relaxation kinetics under no-load conditions. Similarly, KD muscles develop markedly reduced twitch and tetanic force and contraction velocity. Taken together, our results show that sMyBP-C is essential for the regular organization and maintenance of myosin filaments into A bands and that its structural role precedes its ability to regulate actomyosin crossbridges.-Geist, J., Ward, C. W., Kontrogianni-Konstantopoulos, A. Structure before function: myosin binding protein-C slow is a structural protein with regulatory properties.

Abstract 379: Platelet-driven Contraction of Venous Thrombi Modulates Their Obstructiveness and Embologenicity

Contraction (retraction) of the blood clot is a part of the clotting process driven by activated platelets attached to fibrin. The aim of this work was to reveal the pathogenic importance of contraction of clots and thrombi in venous thromboembolism (VTE). We investigated the kinetics of clot contraction in clots made from the blood of 55 patients with VTE not receiving antiplatelet and anticoagulant medications. In addition, we studied the ultrastructure of ex vivo venous thrombi, as well as the morphology and functionality of isolated platelets. Thrombi from VTE patients contained compressed polyhedral erythrocytes, a marker for clot contraction in vivo . The extent and rate of contraction of in vitro clots were reduced by 2-fold in clots from the blood of VTE patients compared to healthy controls. The contraction of clots from the blood of patients with pulmonary embolism was significantly impaired compared to that of those with isolated venous thrombosis, suggesting that less compacted thrombi may be prone to embolization. The reduced ability of clots to contract correlated with continuous platelet activation followed by their partial refractoriness to stimulation. Morphologically, 75% of platelets from VTE patients were spontaneously partially activated (with filopodia) compared to only 21% of those from healthy controls. At the same time, platelets from VTE patients showed a 1.4-fold reduction in activation markers expressed in response to chemical activation when compared to platelets from healthy individuals. The results obtained suggest that the impaired contraction of thrombi resulting from platelet dysfunction is an underappreciated pathogenic mechanism in VTE that may regulate the obstructiveness and embologenicity of venous thrombi. Furthermore, an assay for clot contraction may have diagnostic and prognostic value for venous thromboembolism. The work was supported by the Program for Competitive Growth at Kazan Federal University.

Force‐frequency Relationship and Early Relaxation Kinetics Are Preserved Upon SR Blockade in Human Myocardium

Cardiomyocytes utilize calcium to activate their myofilaments and shorten, which at the organ level results in blood being pumped out of the heart and into the body. Sarcoplasmic reticulum (SR) is an organelle in cardiomyocytes that stores calcium ions and releases them during systole, when myofilament needs to be activated and sequesters them during diastole, when myofilament needs to be deactivated. Therefore, it is believed to play an integral role in the calcium handling of cardiomyocytes with potential implications in the force‐frequency relationship, as intracellular calcium is increased at higher heart rates. However, the SR is believed to be less important in calcium handling in larger mammals, which have sodium calcium exchangers (NCX) that can extrude calcium out of cardiomyocytes. We hypothesized that when the SR is blocked in human trabeculae, the relaxation function would not be affected and that force‐frequency relationship would be preserved. We isolated intact human right ventricular trabeculae from non‐failing and failing human hearts and measured various contractile and relaxation parameters upon SR blockade. We found that force‐frequency relationship is preserved even when the SR is blocked. We also found that early relaxation speed is not affected when the SR is blocked, probably because of NCX compensating for the lack of SR function. Moreover, we found that the contraction kinetics are slower upon SR blockade, potentially because L‐type calcium channel, which have slower calcium‐release kinetics than the SR, compensates for the lack of SR calcium releaseSupport or Funding InformationR01 HL113084 to Paul ML JanssenAHA 16PRE33410549 to Jae‐Hoon ChungThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rapamycin Treatment Promotes Myofibril Relaxation Kinetics and Reduces Myocardial Stiffness to Improve Diastolic Function in Old Murine Hearts

At least half of heart failure patients have normal systolic function (heart failure with preserved ejection fraction, HFpEF) and there is no current approved therapy that improves mortality in this patient population. Aging is associated with a decline in diastolic function and is a very strong risk factor for HFpEF, which is especially prevalent in aged women. We previously showed that late‐life 10‐week rapamycin treatment can reverse established cardiac aging, including a reversal of diastolic dysfunction, in old mice. Rapamycin induces mitochondrial remodeling within first 2 weeks of treatment to rejuvenate energy metabolism in old hearts, but the time course of improved diastolic function is much slower. This indicates that additional undefined mechanisms are responsible for the reversal of diastolic dysfunction induced by rapamycin. The objective of this study is to determine the mechanisms of rapamycin‐induced reversal of diastolic dysfunction in old mice.Proteomic analysis revealed that 10‐week rapamycin treatment can partially rescue the age‐related changes in abundance of proteins in actin cytoskeleton and calcium handling pathways. The effects of rapamycin on protein expression of these key pathways regulating contraction and relaxation kinetics suggest that rapamycin may promote relaxation kinetics of the aged myocardium. To determine the effects of aging and rapamycin treatment on biomechanical properties of the myocardium, we measured contractile and relaxation properties of multicellular ventricular trabeculae preparations and isolated myofibrils. Analysis of the passive length‐tension relationship of demembranated trabeculae showed a substantial increase in the slope of the length‐tension curve with aging, indicating an age‐related increase in myocardial stiffness. This age‐related increase in myocardial stiffness is significantly reduced by rapamycin treatment. Trichrome staining revealed similar myocardial collagen contents in old control and rapamycin‐treated mice, suggesting the reduced stiffness is not due to reduced fibrosis. Old myocardial tissues also exhibit increased Ca2+ sensitivity, as denoted by a left‐shift of the isometric force‐pCa curve, which is partially restored by rapamycin treatment. In particular, the Ca2+ concentration required to elicit half maximum force (pCa50) decreased from pCa 5.59±0.08 for young controls to 5.75±0.14 for old controls, and rapamycin treatment restored it to 5.65±0.14. For myofibril relaxation kinetics, myofibrils from rapamycin treated mice displayed increased rate of the fast phase of relaxation (kREL, fast) compared to old control at both maximal (pCa 4.0) and submaximal (pCa 5.6) Ca2+ levels.In summary, these results suggest that rapamycin treatment alters the kinetics of myofibril relaxation and reverses the age‐related increase in myocardial stiffness and Ca2+ sensitivity. These biomechanical changes likely contribute to the reversal of diastolic dysfunction in old murine hearts.Support or Funding InformationNIH K99 AG051735 and Glenn/AFAR Postdoctoral Fellowship for Translational Research on Aging to Y.A.C., NIH K08HL128826 to F.M‐H., and NIH R01 AG038550 to P.S.R.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.