Contraction Kinetics Research Articles

B-PO03-019 CARDIAC CONTRACTILITY MODULATION STIMULATION DURING SYSTOLE ENHANCES CONTRACTION AND CALCIUM HANDLING PROPERTIES IN HUMAN-INDUCED PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES

Cardiac contractility modulation (CCM) is a cardiac therapy whereby non-excitatory electrical simulations are delivered to the heart during the absolute refractory period. We previously evaluated effects of CCM and found adult rabbit cardiomyocytes display a transient increase in calcium and contractility. In the present study, we sought to extend these results to human stem-cell cardiomyocytes (hiPSC-CMs). hiPSC-CMs (iCell Cardiomyocytes2, Fujifilm Cellular Dynamic Inc) were studied in monolayer format on Matrigel mattress substrate. Contractility, calcium and electrophysiology were evaluated by image and fluorescence analysis (CellOPTIQ, Clyde Biosciences). Standard clinical CCM pulse parameters were applied with an A-M Systems 4100 pulse generator (Figure 1). Robust CCM response was observed at 11 V/cm (64 mA) for pacing and 22 V/cm (128 mA) for CCM. The first CCM beat displayed a 19.7±0.03 %, (**P<0.01, n=10) contraction amplitude increase that reached steady-state within 5 beats at 8.3±0.16 %. CCM stimulation resulted in faster contraction and relaxation kinetics of 25.9±0.07 % and 18.6±0.07 % respectively. Likewise, calcium amplitude increased by 59.3±0.03% (*P<0.05, n=3). This study provides a comprehensive characterization of the effects of CCM on hiPSC-CMs. These data suggest, CCM exerts its effects by, at a minimum, two calcium centric mechanisms including 1) modulation of L-type calcium channels and 2) increased myofilament calcium sensitivity. These data provide the first study of CCM in hiPSC-CMs and demonstrates the utility of hiPSC-CMs to evaluate safety and effectiveness of cardiac medical devices.

Pathophysiological Effects of Overactive STIM1 on Murine Muscle Function and Structure.

Store-operated Ca2+ entry (SOCE) is a ubiquitous mechanism regulating extracellular Ca2+ entry to control a multitude of Ca2+-dependent signaling pathways and cellular processes. SOCE relies on the concerted activity of the reticular Ca2+ sensor STIM1 and the plasma membrane Ca2+ channel ORAI1, and dysfunctions of these key factors result in human pathologies. STIM1 and ORAI1 gain-of-function (GoF) mutations induce excessive Ca2+ influx through SOCE over-activation, and cause tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK), two overlapping disorders characterized by muscle weakness and additional multi-systemic signs affecting growth, platelets, spleen, skin, and intellectual abilities. In order to investigate the pathophysiological effect of overactive SOCE on muscle function and structure, we combined transcriptomics with morphological and functional studies on a TAM/STRMK mouse model. Muscles from Stim1R304W/+ mice displayed aberrant expression profiles of genes implicated in Ca2+ handling and excitation-contraction coupling (ECC), and in vivo investigations evidenced delayed muscle contraction and relaxation kinetics. We also identified signs of reticular stress and abnormal mitochondrial activity, and histological and respirometric analyses on muscle samples revealed enhanced myofiber degeneration associated with reduced mitochondrial respiration. Taken together, we uncovered a molecular disease signature and deciphered the pathomechanism underlying the functional and structural muscle anomalies characterizing TAM/STRMK.

Asymptomatic and symptomatic SARS-CoV-2 infections elicit polyfunctional antibodies

SummaryMany SARS-CoV-2-infected individuals remain asymptomatic. Little is known about the extent and quality of their antiviral humoral response. Here, we analyze antibody functions in 52 asymptomatic infected individuals, 119 mildly symptomatic, and 21 hospitalized patients with COVID-19. We measure anti-spike immunoglobulin G (IgG), IgA, and IgM levels with the S-Flow assay and map IgG-targeted epitopes with a Luminex assay. We also evaluate neutralization, complement deposition, and antibody-dependent cellular cytotoxicity (ADCC) using replication-competent SARS-CoV-2 or reporter cell systems. We show that COVID-19 sera mediate complement deposition and kill infected cells by ADCC. Sera from asymptomatic individuals neutralize the virus, activate ADCC, and trigger complement deposition. Antibody levels and functions are lower in asymptomatic individuals than they are in symptomatic cases. Antibody functions are correlated, regardless of disease severity. Longitudinal samplings show that antibody functions follow similar kinetics of induction and contraction. Overall, asymptomatic SARS-CoV-2 infection elicits polyfunctional antibodies neutralizing the virus and targeting infected cells.

Omecamtiv mecarbil evokes diastolic dysfunction and leads to periodic electromechanical alternans

Omecamtiv mecarbil (OM) is a promising novel drug for improving cardiac contractility. We tested the therapeutic range of OM and identified previously unrecognized side effects. The Ca2+ sensitivity of isometric force production (pCa50) and force at low Ca2+ levels increased with OM concentration in human permeabilized cardiomyocytes. OM (1 µM) slowed the kinetics of contractions and relaxations and evoked an oscillation between normal and reduced intracellular Ca2+ transients, action potential lengths and contractions in isolated canine cardiomyocytes. Echocardiographic studies and left ventricular pressure–volume analyses demonstrated concentration-dependent improvements in cardiac systolic function at OM concentrations of 600–1200 µg/kg in rats. Administration of OM at a concentration of 1200 µg/kg was associated with hypotension, while doses of 600–1200 µg/kg were associated with the following aspects of diastolic dysfunction: decreases in E/A ratio and the maximal rate of diastolic pressure decrement (dP/dtmin) and increases in isovolumic relaxation time, left atrial diameter, the isovolumic relaxation constant Tau, left ventricular end-diastolic pressure and the slope of the end-diastolic pressure–volume relationship. Moreover, OM 1200 µg/kg frequently evoked transient electromechanical alternans in the rat in vivo in which normal systoles were followed by smaller contractions (and T-wave amplitudes) without major differences on the QRS complexes. Besides improving systolic function, OM evoked diastolic dysfunction and pulsus alternans. The narrow therapeutic window for OM may necessitate the monitoring of additional clinical safety parameters in clinical application.

Quantitative and qualitative changes in blood cells associated with COVID-19

Aim. To establish the relationship of hematological disorders with the pathogenesis, course and outcomes of COVID-19.&#x0D; Methods. We examined 235 hospitalized patients with moderate and severe forms of acute COVID-19 receiving anticoagulants and immunosuppressive drugs. We studied the full blood cell counts and morphology along with the platelet function by flow cytometry in comparison with the clinical features and synthesis of inflammatory markers. To assess platelet contractility, blood clot contraction (retraction) kinetics was used in combination with scanning electron microscopy of platelets and blood clots.&#x0D; Results. Hemolytic anemia, neutrophilia and lymphopenia were associated with immature erythrocytes and leukocytes, indicating activation of hematopoiesis. Contraction of blood clots in COVID-19 was impaired, especially in severe and lethal cases, as well as in the presence of comorbidities, including myeloproliferative and coronary heart diseases and acute cerebrovascular disease. In male patients, the changes in clot contraction were more pronounced. Suppression of clot contraction correlated directly with anemia and coagulopathy, including a high D-dimer level, which confirms the pathogenetic significance of blood clot contraction in COVID-19. A decrease in platelet contractility was due to moderate thrombocytopenia in combination with chronic platelet activation and secondary platelet dysfunction. The structure and cellular composition of blood clots depended on the extent of contraction; clots with impaired contraction were porous, had a low content of deformed polyhedral erythrocytes (polyhedrocytes) and an even distribution of fibrin.&#x0D; Conclusion. Blood cells undergoing both quantitative and qualitative changes are involved in the pathogenesis of COVID-19; the suppressed platelet-driven contraction of intravital blood clots may be a part of the prothrombotic mechanisms.

Impact of etiology on force and kinetics of left ventricular end-stage failing human myocardium

BackgroundHeart failure (HF) is associated with highly significant morbidity, mortality, and health care costs. Despite the significant advances in therapies and prevention, HF remains associated with poor clinical outcomes. Understanding the contractile force and kinetic changes at the level of cardiac muscle during end-stage HF in consideration of underlying etiology would be beneficial in developing targeted therapies that can help improve cardiac performance. ObjectiveInvestigate the impact of the primary etiology of HF (ischemic or non-ischemic) on left ventricular (LV) human myocardium force and kinetics of contraction and relaxation under near-physiological conditions. Methods and resultsContractile and kinetic parameters were assessed in LV intact trabeculae isolated from control non-failing (NF; n = 58) and end-stage failing ischemic (FI; n = 16) and non-ischemic (FNI; n = 38) human myocardium under baseline conditions, length-dependent activation, frequency-dependent activation, and response to the β-adrenergic stimulation. At baseline, there were no significant differences in contractile force between the three groups; however, kinetics were impaired in failing myocardium with significant slowing down of relaxation kinetics in FNI compared to NF myocardium. Length-dependent activation was preserved and virtually identical in all groups. Frequency-dependent activation was clearly seen in NF myocardium (positive force frequency relationship [FFR]), while significantly impaired in both FI and FNI myocardium (negative FFR). Likewise, β-adrenergic regulation of contraction was significantly impaired in both HF groups. ConclusionsEnd-stage failing myocardium exhibited impaired kinetics under baseline conditions as well as with the three contractile regulatory mechanisms. The pattern of these kinetic impairments in relation to NF myocardium was mainly impacted by etiology with a marked slowing down of kinetics in FNI myocardium. These findings suggest that not only force development, but also kinetics should be considered as a therapeutic target for improving cardiac performance and thus treatment of HF.

Effects of Cardiac Myosin Heavy Chain Isoform Composition on Contraction Kinetics and Calcium Transients in Adult Rat Cardiomyocytes

Myosin is an essential structural and functional protein of the sarcomere. In rat ventricular myocardium two isoforms of myosin heavy chain (MyHC) are expressed, α-MyHC and β-MyHC. The two MyHC isoforms show different cross bridge kinetics. Strikingly, in the ventricular rat myocardium, cardiomyocytes with different MyHC composition are connected and contract without producing functional disbalances.

Disease in a Dish Crispr-Edited HIPSC-CMS to Study the Cellular and Subcellular Mechanisms of HCM

Genetic sequencing enables the pinpointing of specific genetic mutations correlated with disease. However, such associations do not describe mechanistic relationships between specific mutations and clinical phenotypes. To address this, we use human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a model system to probe how mutations in β-myosin heavy chain (MHC) lead to disease. β-MHC, a major biochemical motor protein in heart muscle, is a common site of mutations implicated in hypertrophic cardiomyopathy Utilizing CRISPR editing of the MYH7 gene to introduce the G256E point mutation into the converter domain region of myosin, we have studied the effects that molecular level changes (nm) in protein structure have at the cellular level through simultaneous contractility and calcium handling measurements in hiPSC-CMs. These hiPSC-CMs were subjected to glucose starvation to mature and enrich the population of cardiomyocytes, and cultured on Matrigel-stamped 1500 μm2 island patterns to achieve single cells. We have also analyzed differences at the individual contractile organelle level through precise measures of myofibril contraction and relaxation kinetics using a custom-built apparatus. Our experimental results have been supplemented with molecular dynamics simulations of wild type and mutant β-MHC. Preliminary data from molecular dynamics simulations of β-myosin structure, suggests G256E alters the distributions of intra-molecular contacts made by loop 1 and distorts β-MHC structure near the nucleotide binding pocket as well as sites forming the interacting heads motif. Synthesis of computational, mechanical and structural studies will determine the mechanism of contractile dysfunction associated with β-MHC G256E. Development of these platforms for molecular and cellular level analysis serve as a template for future studies of other myofilament proteins and mutations to connect disease genotype with phenotype.

Climate change and anadromous fish: How does thermal acclimation affect the mechanics of themyotomal muscle of theAtlantic salmon, Salmo salar?

In response to accelerated temperature shifts due to climate change, the survival of many species will require forms of thermal acclimation to their changing environment. We were interested in how climate change will impact a commercially and recreationally important species of fish, Atlantic salmon (Salmo salar). As climate change alters the thermal environment of their natal streams, we asked how their muscle function will be altered by extended exposure to both warm and cold temperatures. We performed a thermal acclimation study of S. salar muscle mechanics of both fast-twitch, or white, and slow-twitch, or red, myotomal muscle bundles to investigate how temperature acclimated Atlantic salmon would respond across a range of different temperatures. Isometric contraction properties, maximum shortening velocity, and oscillatory power output were measured and compared amongst three groups of salmon-warm acclimated (20°C), cold-acclimated (2°C), and those at their rearing temperature (12°C). The Atlantic salmon showed limited thermal acclimation in their contraction kinetics, and some of the shifts in contractile properties that were observed would not be predicted to mitigate the impact of a warming environment. For instance, the maximum shortening velocity at a common test temperature was higher in the warm acclimated group and lower in the cold-acclimated group. In addition, critical swimming speed did not vary with temperature of acclimation when tested at a common temperature (12°C). Our results suggest that Atlantic salmon populations will continue to struggle in response to a warming environment.

Pulsatile contractions promote apoptotic cell extrusion in epithelial tissues

Extrusion is a mechanism used to eliminate unfit, excess, or dying cells from epithelial tissues. The initial events guiding which cells will be selectively extruded from the epithelium are not well understood. Here, we induced damage in a subset of epithelial cells in the developing zebrafish and used time-lapse imaging to examine cell and cytoskeletal dynamics leading to extrusion. We show that cell extrusion is preceded by actomyosin contractions that are pulsatile. Our data show that pulsatile contractions are induced by a junctional to medial re-localization of myosin. Analysis of cell area during contractions revealed that cells pulsing with the longest duration and highest amplitude undergo progressive area loss and extrude. Although pulses were driven by local increases in tension, damage to many cells promoted an overall decrease in the tensile state of the epithelium. We demonstrate that caspase activation leads to sphingosine-1-phosphate enrichment that controls both tissue tension and pulses to dictate areas of extrusion. These data suggest that the kinetics of pulsatile contractions define a key behavioral difference between extruding and non-extruding cells and are predictive of extrusion. Altogether, our study provides mechanistic insight into how localized changes in physical forces are coordinated to remove defective cells for homeostatic maintenance of living epithelial tissues.

Accelerated Spatial Fibrin Growth and Impaired Contraction of Blood Clots in Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease associated with thrombotic complications. To elucidate pathogenic mechanisms, hemostatic disorders in RA were correlated with other laboratory and clinical manifestations. Hemostasis was assessed using relatively new complementary tests, the spatial growth of a plasma clot (Thrombodynamics assay), and contraction of whole blood clots. Platelet functionality was assessed with flow cytometry that quantified the expression of P-selectin and the fibrinogen-binding capacity of platelets before and after activation with a thrombin receptor-activating peptide. Parameters of fibrin clot growth and the kinetics of contraction of blood clots were significantly altered in patients with RA compared to the control group. In Thrombodynamics measurements, an increase in the clot growth rate, size, and optical density of plasma clots altogether indicated chronic hypercoagulability. The rate and extent of blood clot contraction in patients with RA was significantly reduced and associated with platelet dysfunction revealed by an impaired response to activation. Changes in the parameters of clot growth and contraction correlated with the laboratory signs of systemic inflammation, including hyperfibrinogenemia. These results confirm the pathogenic role of hemostatic disorders in RA and support the validity of fibrin clot growth and the blood clot contraction assay as indicators of a (pro)thrombotic state.

Sex-Specific Effects of Daily Tadalafil on Contraction Kinetics of the Diabetic Heart. The RECOGITO Randomized, Double-Blind, Placebo-Controlled Trial

Background: Cardiovascular disease is the leading cause of morbidity and mortality in type 2 diabetes (T2DM) patients. Cyclic GMP-phosphodiesterase type 5 (PDE5) inhibition was shown to counteract maladaptive cardiac changes triggered by diabetes in some studies. Our aim was to assess sex differences in cardiac remodeling after PDE5 inhibition in patients with diabetic cardiomyopathy (DCM). Methods: We performed a 20-week, double-blind, randomized, placebo-controlled trial. Men and women (45–80 years) with controlled T2DM and echocardiographic (ECG) signs of cardiac remodeling were randomly assigned (1:1) to placebo or tadalafil 20 mg once daily. The primary outcome was analyzing the sex-difference in cardiac torsion change, from baseline to 20 weeks. Secondary outcomes included changes in cardiovascular, metabolic, immune and renal function. Efficacy analyses included all patients who received at least one dose of treatment. Findings: Between May 2014 and July 2018, we screened 220 diabetic subjects; 122 met the inclusion criteria and were randomized to tadalafil (n=61) or placebo (n=61). At 20 weeks, the sex-by-treatment interaction documented an improvement in cardiac torsion [-3·40°(-5·96;-0·84, p =0·011)], paralleled by a consensual increase in fiber shortening, in men but not women. Biomarkers of cardiovascular remodeling , titin and hsa-miR-199-5p, improved accordingly. In both sexes, tadalafil reduced TGF-beta and improved albuminuria, renal artery’s resistive index, Klotho , and symmetric-di-methyl-arginine markers . Five months of 20 mg daily tadalafil in patients with DCM was proven safe. The most common adverse reactions were myalgia (9[14·5%]), gastric reflux (6[9·7%]), headache (4[6·4%]) and flushing (2[3·8%]); one SAE occurred in the placebo arm. Interpretation: Continuous PDE5 inhibition could represent a new treatment strategy to target cardiac and renal complications of T2DM, with a different tissue-specific response in men and women. Klotho and hsa-miR-199-5p appear as novel players linking renal disease to DCM progression. Trial Registration: NCT01803828. Funding Statement: Italian Ministry of University and Research, SIR RBSI141LY2. Declaration of Interests: Prof. Isidori reports personal fees from Takeda; non-financial support from Takeda and Ipsen; and grants from Shire and Pfizer outside the submitted work; Prof. Pozzilli reports research contracts and consulting from Eli Lilly, Sanofi, Merck, Merck Sharpt Dohme, Astra Zeneca, Medtronic, Abbott; Dr. Defeudis reports grants from Ibsa, personal fees from Abbott, grants from Novo Nordisk, grants from Kyowa Kirin , personal fees from Roche, outside the submitted work; Prof. Badagliacca reports personal fees from Bayer, personal fees from MSD, personal fees from Dompe, personal fees from GSK, personal fees from Ferrer, personal fees from United Therapeutics, personal fees from Galenica, personal fees from Janssen, outside the submitted work; all others have nothing to disclose. Ethics Approval Statement: The study was performed in the Policlinico Umberto I in Rome (Italy) and approved by the local ethical review board (3083/14), published on public registries (NCT01803828, EUDRACT 2014-000077-39), and conducted under the Declaration of Helsinki and good clinical practice.

Susceptibility to Severe PWSCC (primary water stress corrosion cracking) of LTMA (low temperature mill anneal) Alloy 600

It has been proposed that a primary water stress corrosion cracking (PWSCC) in pressurized water reactor (PWR) is governed by a lattice contraction due to a short range ordering reaction in Alloy 600. This leads researcher to think that the kinetics of lattice contraction may control a susceptibility of PWSCC in Alloy 600. A lattice variation with ordering treatment at 400 &lt;sup&gt;o&lt;/sup&gt;C was systematically investigated using high resolution neutron diffraction(HRPD) in high temperature mill anneal (HTMA), low temperature mill anneal (LTMA), and sensitized (SEN) Alloy 600. The results showed that ordering treatment caused an isotropic lattice contraction due to short range ordering (SRO) reaction. The lattice contractions of (111) plane are saturated to be 0.04% in 4 to 1500 hours at 400 &lt;sup&gt;o&lt;/sup&gt;C according to prior treatment condition. The lattice contraction in the magnitude of 0.03% of (111) plane in LTMA Alloy 600 is faster by 8 times and 66 times than that of SEN and HTMA, respectively. This fact may explain why the LTMA is most susceptible to PWSCC through of kinetics of lattice contraction in Alloy 600. Thus, it is possible to conclude that the susceptibility of Alloy 600 to PWSCC is governed by the kinetics of (111) lattice contraction.

Abstract 13508: Left Ventricular Contractile and Kinetic Changes in Failing Human Myocardium Due to Anthracycline Toxicity

Introduction: Anthracyclines are used effectively to treat many cancers. However, cumulative and irreversible cardiotoxicity can limit anthracyclines’ clinical benefit. Mechanisms of cardiotoxicity have been assessed in murine models, but no studies directly assess human heart contractility. Our objective was to assess left ventricular contractile force, kinetics of contraction and relaxation, and frequency-dependent activation in anthracycline-induced failing human myocardium. Methods: From 2009-2019, we assessed live tissue-level contractile forces and kinetics in isolated left ventricular intact trabeculae from failing and non-failing human hearts. After the trabeculae were transferred to and stabilized in a custom setup, baseline contractile force and kinetic parameters were assessed at 1 Hz (normal resting in vivo heart rate), followed by frequency-dependent activation (0.5-3.0 Hz) under near-physiological conditions. Retrospectively, we analyzed muscles from three cohorts of individuals: (1) with non-ischemic cardiomyopathy due to anthracycline toxicity (NICM-AC; n =14), (2) with NICM and history of cancer without anthracycline or known cardiotoxic treatments (NICM; n =14), and (3) with non-failing myocardium and no history of cancer (NF; n =14). Results: At stimulation of 1 Hz, active developed force (Fdev) of NICM-AC trabeculae was significantly lower than NF and NICM trabeculae. NICM-AC trabeculae exhibited prolonged 90% relaxation time (RT90), significantly slower maximal rate of force decay (-dF/dt), and slower maximal kinetic rate of relaxation (-dF/dt/Fdev). In addition, frequency-dependent activation and relaxation were markedly impaired in both failing groups. Conclusions: Human myocardium failing due to anthracycline toxicity had significantly decreased force and slower relaxation kinetics compared to non-ischemic failing myocardium. With increase in stimulation frequency, anthracycline treated myocardium exhibited impaired activation and relaxation kinetics. These findings suggest that cardio-protection strategies should aim to improve not only contractile force, but also kinetics of relaxation.

A Tenon's capsule/bulbar conjunctiva interface biomimetic to model fibrosis and local drug delivery.

Glaucoma filtration surgery is one of the most effective methods for lowering intraocular pressure in glaucoma. The surgery efficiently reduces intra-ocular pressure but the most common cause of failure is scarring at the incision site. This occurs in the conjunctiva/Tenon's capsule layer overlying the scleral coat of the eye. Currently used antimetabolite treatments to prevent post-surgical scarring are non-selective and are associated with potentially blinding side effects. Developing new treatments to target scarring requires both a better understanding of wound healing and scarring in the conjunctiva, and new means of delivering anti-scarring drugs locally and sustainably. By combining plastic compression of collagen gels with a soft collagen-based layer, we have developed a physiologically relevant model of the sub-epithelial bulbar conjunctiva/Tenon's capsule interface, which allows a more holistic approach to the understanding of subconjunctival tissue behaviour and local drug delivery. The biomimetic tissue hosts both primary human conjunctival fibroblasts and an immune component in the form of macrophages, morphologically and structurally mimicking the mechanical proprieties and contraction kinetics of ex vivo porcine conjunctiva. We show that our model is suitable for the screening of drugs targeting scarring and/or inflammation, and amenable to the study of local drug delivery devices that can be inserted in between the two layers of the biomimetic. We propose that this multicellular-bilayer engineered tissue will be useful to study complex biological aspects of scarring and fibrosis, including the role of inflammation, with potentially significant implications for the management of scarring following glaucoma filtration surgery and other anterior ocular segment scarring conditions. Crucially, it uniquely allows the evaluation of new means of local drug delivery within a physiologically relevant tissue mimetic, mimicking intraoperative drug delivery in vivo.

Muscle Twitch Kinetics Are Dependent on Muscle Group, Disease State, and Age in Duchenne Muscular Dystrophy Mouse Models.

Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by the lack of functional dystrophin protein. In muscular dystrophy preclinical research, it is pertinent to analyze the force of the muscles affected by the disease to assess pathology and potential effectiveness of therapeutic interventions. Although muscles function at sub-maximal levels in vivo, maximal tetanic contractions are most commonly used to assess and report muscle function in muscular dystrophy studies. At submaximal activation, the kinetics of contraction and relaxation are heavily impacted by the kinetics of the single twitch. However, maximal tetanic force is often the main, if not sole, outcome measured in most studies, while contractile kinetics are rarely reported. To investigate the effect of muscle disease on twitch contraction kinetics, isolated diaphragm and extensor digitorum longus (EDL) muscles of 10-, 20-week, “het” (dystrophin deficient and utrophin haplo-insufficient), and 52-week mdx (dystrophin deficient) mice were analyzed and compared to wild-type controls. We observed that twitch contractile kinetics are dependent on muscle type, age, and disease state. Specific findings include that diaphragm from wildtype mice has a greater time to 50% relaxation (RT50) than time to peak tension (TTP) compared to the het and mdx dystrophic models, where there is a similar TTP compared to RT50. Diaphragm twitch kinetics remain virtually unchanged with age, while the EDL from het and mdx mice initially has a greater RT50 than TTP, but the TTP increases with age. The difference between EDL contractile kinetics of dystrophic and wildtype mice is more prominent at young age. Differences in kinetics yielded greater statistical significance compared to previously published force measurements, thus, using kinetics as an outcome parameter could potentially allow for use of smaller experimental groups in future study designs. Although this study focused on DMD models, our findings may be applicable to other skeletal muscle conditions and diseases.

Kinetic Analysis of Label-Free Microscale Collagen Gel Contraction Using Machine Learning-Aided Image Analysis.

Pulmonary fibrosis is a deadly lung disease, wherein normal lung tissue is progressively replaced with fibrotic scar tissue. An aspect of this process can be recreated in vitro by embedding fibroblasts into a collagen matrix and providing a fibrotic stimulus. This work expands upon a previously described method to print microscale cell-laden collagen gels and combines it with live cell imaging and automated image analysis to enable high-throughput analysis of the kinetics of cell-mediated contraction of this collagen matrix. The image analysis method utilizes a plugin for FIJI, built around Waikato Environment for Knowledge Analysis (WEKA) Segmentation. After cross-validation of this automated image analysis with manual shape tracing, the assay was applied to primary human lung fibroblasts including cells isolated from idiopathic pulmonary fibrosis patients. In the absence of any exogenous stimuli, the analysis showed significantly faster and more extensive contraction of the diseased cells compared to the healthy ones. Upon stimulation with transforming growth factor beta 1 (TGF-β1), fibroblasts from the healthy donor showed significantly more contraction throughout the observation period while differences in the response of diseased cells was subtle and could only be detected during a smaller window of time. Finally, dose-response curves for the inhibition of collagen gel contraction were determined for 3 small molecules including the only 2 FDA-approved drugs for idiopathic pulmonary fibrosis.

An Important Role for DNMT3A-Mediated DNA Methylation in Cardiomyocyte Metabolism and Contractility.

DNA methylation acts as a mechanism of gene transcription regulation. It has recently gained attention as a possible therapeutic target in cardiac hypertrophy and heart failure. However, its exact role in cardiomyocytes remains controversial. Thus, we knocked out the main de novo DNA methyltransferase in cardiomyocytes, DNMT3A, in human induced pluripotent stem cells. Functional consequences of DNA methylation-deficiency under control and stress conditions were then assessed in human engineered heart tissue from knockout human induced pluripotent stem cell-derived cardiomyocytes. DNMT3A was knocked out in human induced pluripotent stem cells by CRISPR/Cas9gene editing. Fibrin-based engineered heart tissue was generated from knockout and control human induced pluripotent stem cell-derived cardiomyocytes. Development and baseline contractility were analyzed by video-optical recording. Engineered heart tissue was subjected to different stress protocols, including serum starvation, serum variation, and restrictive feeding. Molecular, histological, and ultrastructural analyses were performed afterward. Knockout of DNMT3A in human cardiomyocytes had three main consequences for cardiomyocyte morphology and function: (1) Gene expression changes of contractile proteins such as higher atrial gene expression and lower MYH7/MYH6 ratio correlated with different contraction kinetics in knockout versus wild-type; (2) Aberrant activation of the glucose/lipid metabolism regulator peroxisome proliferator-activated receptor gamma was associated with accumulation of lipid vacuoles within knockout cardiomyocytes; (3) Hypoxia-inducible factor 1α protein instability was associated with impaired glucose metabolism and lower glycolytic enzyme expression, rendering knockout-engineered heart tissue sensitive to metabolic stress such as serum withdrawal and restrictive feeding. The results suggest an important role of DNA methylation in the normal homeostasis of cardiomyocytes and during cardiac stress, which could make it an interesting target for cardiac therapy.

Large-Scale Contractility Measurements Reveal Large Atrioventricular and Subtle Interventricular Differences in Cultured Unloaded Rat Cardiomyocytes.

The chambers of the heart fulfill different hemodynamic functions, which are reflected in their structural and contractile properties. While the atria are highly elastic to allow filling from the venous system, the ventricles need to be able to produce sufficiently high pressures to eject blood into the circulation. The right ventricle (RV) pumps into the low pressure pulmonary circulation, while the left ventricle (LV) needs to overcome the high pressure of the systemic circulation. It is incompletely understood whether these differences can be explained by the contractile differences at the level of the individual cardiomyocytes of the chambers. We addressed this by isolating cardiomyocytes from atria, RV, LV, and interventricular septum (IVS) of five healthy wild-type rats. Using a high-throughput contractility set-up, we measured contractile function of 2,043 cells after overnight culture. Compared to ventricular cardiomyocytes, atrial cells showed a twofold lower contraction amplitude and 1.4- to 1.7-fold slower kinetics of contraction and relaxation. The interventricular differences in contractile function were much smaller; RV cells displayed 12–13% less fractional shortening and 5–9% slower contraction and 3–15% slower relaxation kinetics relative to their LV and IVS counterparts. Aided by a large dataset, we established relationships between contractile parameters and found contraction velocity, fractional shortening and relaxation velocity to be highly correlated. In conclusion, our findings are in line with contractile differences observed at the atrioventricular level, but can only partly explain the interventricular differences that exist at the organ level.

Recovery From Eccentric Injury Is Maintained In Aging Sarcopenic Muscle

PURPOSE: The progressive loss of muscle function and mass with age, known as sarcopenia, is a leading cause for falls and reduced quality of life in the elderly. Eccentric contractions have been known to induce muscle damage, and it has been suggested that eccentric induced muscle damage (EIMD) contributes to sarcopenia. More recently, however, eccentric training has gained attention as means to improve muscle function in the elderly. These differing views highlight the need for clarification on how EIMD affects sarcopenic muscle performance, particularly force frequency, fatigue, and kinetics of muscle contraction. METHODS: Male CB6F1 mice were used at 7-9 mo (adult) and 29-31 mo (old) of age. In vivo measurement of plantaflexor function was assessed in anesthetized mice using an Aurora Scientific 305C servomotor by stimulating the tibial nerve. Force-frequency and fatigue were assessed at baseline and 3 days following EIMD. EIMD was induced by 20 eccentric contractions of the plantarflexors by forcing dorsiflexion during a maximal contraction. RESULTS: At baseline old mice had lower force (12 ± 1.8 vs 15.2 ± 0.8 mN-m) and were more fatigue resistant compared to adult mice. During the EIMD protocol, old mice lost significantly more force compared to adult mice (34.8% ±11.5 vs 26.3% ± 4.2 respectively). Surprisingly, old mice recovered most of their force (96.4% ± 2.0 vs 90.6 % ± 6.5 adult) and contraction kinetics 3 days after EIMD. Rates of muscle fatigue did not significantly change 3 days after EIMD compared to baseline in either age group. CONCLUSIONS: The plantarflexors of old mice were more susceptible to injury by maximal eccentric contractions compared to adult. However, old mice did not differ from adult mice in their ability to recover muscle function 3 days after EIMD. The impaired regenerative capacity often reported for sarcopenic muscle was not evident under these conditions. The similar recovery in adult and old mice could be due to nature of the EIMD protocol, the muscles tested, and the use of muscle performance rather than molecular and structural markers of damage. These results indicate that mild EIMD may not have a long-term deleterious effect on sarcopenic muscle performance. Further research is needed to determine if EIMD can improve function of sarcopenic muscle.