Changes In Sarcomere Length Research Articles

Sarcomere level mechanics of the fast skeletal muscle of the medaka fish larva.

The medaka fish (Oryzias latipes) is a vertebrate model used in developmental biology and genetics. Here we explore its suitability as a model for investigating the molecular mechanisms of human myopathies caused by mutations in sarcomeric proteins. To this end, the relevant mechanical parameters of the intact skeletal muscle of wild-type medaka are determined using the transparent tail at larval stage 40. Tails were mounted at sarcomere length of 2.1 μm in a thermoregulated trough containing physiological solution. Tetanic contractions were elicited at physiological temperature (10°C-30°C) by electrical stimulation, and sarcomere length changes were recorded with nanometer-microsecond resolution during both isometric and isotonic contractions with a striation follower. The force output has been normalized for the actual fraction of the cross section of the tail occupied by the myofilament lattice, as established with transmission electron microscopy (TEM), and then for the actual density of myofilaments, as established with X-ray diffraction. Under these conditions, the mechanical performance of the contracting muscle of the wild-type larva can be defined at the level of the half-thick filament, where ∼300 myosin motors work in parallel as a collective motor, allowing a detailed comparison with the established performance of the skeletal muscle of different vertebrates. The results of this study point out that the medaka fish larva is a suitable model for the investigation of the genotype/phenotype correlations and therapeutic possibilities in skeletal muscle diseases caused by mutations in sarcomeric proteins.NEW & NOTEWORTHY The suitability of the medaka fish as a model for investigating the molecular mechanisms of human myopathies caused by mutations of sarcomeric proteins is tested by combining structural analysis and sarcomere-level mechanics of the skeletal muscle of the tail of medaka larva. The mechanical performance of the medaka muscle, scaled at the level of the myosin-containing thick filament, together with its reduced genome duplication makes this model unique for investigations of the genotype/phenotype correlations in human myopathies.

Hole behavior captured by analysis of instantaneous amplitude and phase of sarcosynced oscillations reveals wave characteristics of sarcomeric oscillations

In this study, we performed signal analysis based on instantaneous amplitude and phase of sarcomeric oscillations, which are generated by skeletal muscle under constant calcium concentration conditions and in which sarcomeres repeatedly contract and relax autonomously. In addition to the changes in sarcomere length that have been attracting attention, we named the Z-line oscillations that partition sarcomeres sarcosynced oscillations, and analyzed their instantaneous amplitude and phase. As a result, the behavior of pairs of sarcosynced oscillations and sarcomeric oscillations, which are produced when propagating waves propagate in one direction or collide, was clearly visualized. By focusing on the behavior of the hole, which is a dip in the instantaneous amplitude accompanied by a sudden jump in the instantaneous phase in sarcosynced oscillations, we were able to discern the wave characteristics. Transient disruption occurred in the propagating waves even when they traveled in one direction. Its properties were captured by the sarcomeric defect hole (SD hole), a dip in the instantaneous amplitude accompanied by a jump in the instantaneous phase in sarcosynced oscillations. When propagating waves collide, the collision site, its persistence, movement, and disappearance process are captured as sarcomeric collision holes (SC holes) of sarcosynced oscillations. These holes are important indicators for understanding the oscillation properties of sarcomeres. In conclusion, although sarcosynced oscillations and sarcomeric oscillations are closely related, they exhibit different oscillations, and the study of the SD holes and SC holes caused by them will contribute to a detailed understanding of the muscle characteristics of sarcomeres. This finding has important implications for improving our understanding of the efficiency of muscle function and its regulatory mechanisms.

Omecamtiv mecarbil augments cardiomyocyte contractile activity both at resting and systolic Ca2+ levels.

Heart failure with reduced ejection fraction (HFrEF) is a disease with high mortality and morbidity. Recent positive inotropic drug developments focused on cardiac myofilaments, that is, direct activators of the myosin molecule and Ca2+ sensitizers for patients with advanced HFrEF. Omecamtiv mecarbil (OM) is the first direct myosin activator with promising results in clinical studies. Here, we aimed to elucidate the cellular mechanisms of the positive inotropic effect of OM in a comparative in vitro investigation where Ca2+ -sensitizing positive inotropic agents with distinct mechanisms of action [EMD 53998 (EMD), which also docks on the myosin molecule, and levosimendan (Levo), which binds to troponin C] were included. Enzymatically isolated canine cardiomyocytes with intact cell membranes were loaded with Fura-2AM, a Ca2+ -sensitive, ratiometric, fluorescent dye. Changes in sarcomere length (SL) and intracellular Ca2+ concentration were recorded in parallel at room temperature, whereas cardiomyocyte contractions were evoked by field stimulation at 0.1Hz in the presence of different OM, EMD, or Levo concentrations. SL was reduced by about 23% or 9% in the presence of 1μM OM or 1μM EMD in the absence of electrical stimulation, whereas 1μM Levo had no effect on resting SL. Fractional sarcomere shortening was increased by 1μM EMD or 1μM Levo to about 152%, but only to about 128% in the presence of 0.03μM OM. At higher OM concentrations, no significant increase in fractional sarcomere shortening could be recorded. Contraction durations largely increased, whereas the kinetics of contractions and relaxations decreased with increasing OM concentrations. One-micromole EMD or 1μM Levo had no effects on contraction durations. One-micromole Levo, but not 1μM EMD, accelerated the kinetics of cardiomyocyte contractions and relaxations. Ca2+ transient amplitudes were unaffected by all treatments. Our data revealed major distinctions between the cellular effects of myofilament targeted agents (OM, EMD, or Levo) depending on their target proteins and binding sites, although they were compatible with the involvement of Ca2+ -sensitizing mechanisms for all three drugs. Significant part of the cardiotonic effect of OM relates to the prolongation of systolic contraction in combination with its Ca2+ -sensitizing effect.

The Novel Cardiac Myosin Activator Danicamtiv Improves Cardiac Systolic Function at the Expense of Diastolic Dysfunction In Vitro and In Vivo: Implications for Clinical Applications.

Recent cardiotropic drug developments have focused on cardiac myofilaments. Danicamtiv, the second direct myosin activator, has achieved encouraging results in preclinical and clinical studies, thus implicating its potential applicability in the treatment of heart failure with reduced ejection fraction (HFrEF). Here, we analyzed the inotropic effects of danicamtiv in detail. To this end, changes in sarcomere length and intracellular Ca2+ levels were monitored in parallel, in enzymatically isolated canine cardiomyocytes, and detailed echocardiographic examinations were performed in anesthetized rats in the absence or presence of danicamtiv. The systolic and diastolic sarcomere lengths decreased; contraction and relaxation kinetics slowed down with increasing danicamtiv concentrations without changes in intracellular Ca2+ transients in vitro. Danicamtiv evoked remarkable increases in left ventricular ejection fraction and fractional shortening, also reflected by changes in systolic strain. Nevertheless, the systolic ejection time was significantly prolonged, the ratio of diastolic to systolic duration was reduced, and signs of diastolic dysfunction were also observed upon danicamtiv treatment in vivo. Taken together, danicamtiv improves cardiac systolic function, but it can also limit diastolic performance, especially at high drug concentrations.

What's all the hype with fibre type? Selective single fibre adaptations with lifelong endurance exercise.

What's all the hype with fibre type? Selective single fibre adaptations with lifelong endurance exercise.

Muscle in Variable Gravity: "I Do Not Know Where I Am, But I Know What to Do".

Purpose: Fascicle and sarcomere lengths are important predictors of muscle mechanical performance. However, their regulation during stretch-shortening cycle (SSC) activities in usual and challenging conditions is poorly understood. In this study, we aimed to investigate muscle fascicle and sarcomere behavior during drop jumps (a common SSC activity) in conditions of variable gravity.Methods: Fifteen volunteers performed repeated drop jumps in 1 g, hypo-gravity (0 to 1 g), and hyper-gravity (1 to 2 g) during a parabolic flight. Gastrocnemius medialis (GM) electromyographic activity and fascicle length (Lf) were measured at drop-off, ground contact (GC), minimum ankle joint angle (MAJ), and push-off. GM sarcomere number was estimated by dividing Lf, measured by ultrasound at rest, by published data on GM sarcomere length, and measured in vivo at the same joint angle. Changes in sarcomere length were estimated by dividing GM Lf in each jump phase by sarcomere number calculated individually. The sarcomere force-generating capacity in each jump phase was estimated from the sarcomere length-tension relationship previously reported in the literature.Results: The results showed that, regardless of the gravity level, GM sarcomeres operated in the ascending portion of their length-tension relationship in all the jump phases. Interestingly, although in hypo-gravity and hyper-gravity during the braking phase (GC-MAJ) GM fascicles and sarcomeres experienced a stretch (as opposed to the quasi-isometric behavior in 1 g), at MAJ they reached similar lengths as in 1 g, allowing sarcomeres to develop about the 70% of their maximum force.Conclusion: The observed fascicle behavior during drop jumping seems useful for anchoring the tendon, enabling storage of elastic energy and its release in the subsequent push-off phase for effectively re-bouncing in all gravity levels, suggesting that an innate neuromuscular wisdom enables to perform SSC movements also in challenging conditions.

Stretch-induced satellite cell deformation incontracturedmuscles in children with cerebral palsy

Satellite cells (SCs) are quiescent, adult skeletal muscle stem cells responsible for postnatal muscle growth and repair. Children with cerebral palsy (CP) have muscle contractures with reduced SC abundance, extracellular matrix abnormalities and reduced serial sarcomere number resulting in greatly increased in vivo sarcomere length, perhaps due to impaired sarcomere addition, compared to children with typical development (TD). Stretch is a strong activator of SCs that leads to addition of sarcomeres during bone-muscle growth. Mechanical loading and subsequent deformation of intracellular structures can lead to activation and proliferation, perhaps by cytoskeletal transmissions of extracellular mechanical signals to the nuclei. The primary aim of this study was to determine the effect of ex vivo stretch-induced sarcomere length change on SC deformation in children with CP and TD. Muscle biopsies were obtained from twelve children (7 CP, 5 TD) during surgery. Fiber bundles were labeled with fluorescent antibodies for Pax7 (SC), DRAQ5 (nuclei), and alpha-actinin (sarcomere protein). Fibers were stretched using a custom jig and imaged using confocal microscopy. SC nuclear length, height and aspect ratio underwent increased deformation with increasing sarcomere length (p < 0.05) in both groups. Slopes of association for SC nuclear length, aspect ratio and sarcomere lengths were similar between CP and TD. Our results indicate that SC in children with CP undergo similar deformation as TD across sarcomere lengths.

Sarcomere length measurement reliability in single myofibrils

Sarcomere length non-uniformities occur at all structural levels of skeletal muscles and have been associated with important mechanical properties. Changes in sarcomere length non-uniformities in the nano- and sub-nanometer range have been used to explain muscle properties and contractile mechanisms. Typically, these measurements rely on light microscopy with a limited spatial resolution. One critical aspect in sarcomere length determination is the relatively arbitrary choice of intensity thresholds used to delineate sarcomere structures, such as A-bands or Z-lines. In experiments, these structures are typically distorted, intensity profiles vary, and baselines drift, resulting in asymmetric intensity patterns, causing changes in the centroid location of these structures depending on threshold choice, resulting in changes of sarcomere lengths. The purpose of this study was to determine the changes in (half-) sarcomere lengths associated with small changes in the A-band threshold choice. Sarcomere and half-sarcomere length changes for minute variations in A-band threshold were 28 nm (±28 nm) and 18 nm (±22 nm), respectively, and for the entire feasible range of thresholds across A-bands were 123 nm (±88 nm) and 99 nm (±105 nm), respectively. We conclude from these results that (half-) sarcomere lengths in the nanometer range obtained with light microcopy are noise, and the functional implications associated with such data should be discarded. We suggest that a functional resolution for sarcomere length of 100 nm (0.1 µm) is reasonable and 50 nm (0.05 µm) might be possible under ideal conditions.

Sarcomere length of the vastus intermedius with the knee joint angle change

The force–length relation of the skeletal muscles is an important factor influencing the joint torque at a given joint angle. We aimed to clarify the relationship between the resting sarcomere length and knee joint angle in the vastus intermedius (VI) and to compare it with that of the vastus lateralis (VL). The left and right legs were fixed at knee joint angles of 0° and 90°, respectively, in seven cadavers (age at the time of death: 70–91 years). Muscle tissues were dissected by necropsy of the VL and the VI, and electron microscopy images were obtained to calculate the sarcomere length. At knee joint angles of 0° and 90°, the VL sarcomere length was 2.28 ± 0.49 μm and 2.30 ± 0.48 μm, respectively, and the VI sarcomere length was 2.19 ± 0.35 μm and 2.46 ± 0.53 μm, respectively, with a significant difference between the two (p = 0.028). The magnitude of sarcomere length changes with knee joint angle changes was significantly greater for the VI (0.27 ± 0.20 μm) than for the VL (0.02 ± 0.09 μm) (p = 0.009). Thus, knee joint angle changes may affect the passive and active tension produced by the VI more than those produced by the VL.

Effect of Sarcomere Length Change During Isovolumic Phase of Left Ventricle on Hemodynamics

Effect of Sarcomere Length Change During Isovolumic Phase of Left Ventricle on Hemodynamics

Ventricular contraction and relaxation rates during muscle metaboreflex activation in heart failure: are they coupled?

What is the central question of this study? Does the muscle metaboreflex affect the ratio of left ventricular contraction/relaxation rates and does heart failure impact this relationship. What is the main finding and its importance? The effect of muscle metaboreflex activation on the ventricular relaxation rate was significantly attenuated in heart failure. Heart failure attenuates the exercise and muscle metaboreflex-induced changes in the contraction/relaxation ratio. In heart failure, the reduced ability to raise cardiac output during muscle metaboreflex activation may not solely be due to attenuation of ventricular contraction but also alterations in ventricular relaxation and diastolic function. The relationship between contraction and relaxation rates of the left ventricle varies with exercise. In in vitro models, this ratio was shown to be relatively unaltered by changes in sarcomere length, frequency of stimulation, and β-adrenergic stimulation. We investigated whether the ratio of contraction to relaxation rate is maintained in the whole heart during exercise and muscle metaboreflex activation and whether heart failure alters these relationships. We observed that in healthy subjects the ratio of contraction to relaxation increases from rest to exercise as a result of a higher increase in contraction relative to relaxation. During muscle metaboreflex activation the ratio of contraction to relaxation is significantly reduced towards 1.0 due to a large increase in relaxation rate matching contraction rate. In heart failure, contraction and relaxation rates are significantly reduced, and increases during exercise are attenuated. A significant increase in the ratio was observed from rest to exercise although baseline ratio values were significantly reduced close to 1.0 when compared to healthy subjects. There was no significant change observed between exercise and muscle metaboreflex activation nor was the ratio during muscle metaboreflex activation significantly different between heart failure and control. We conclude that heart failure reduces the muscle metaboreflex gain and contraction and relaxation rates. Furthermore, we observed that the ratio of the contraction and relaxation rates during muscle metaboreflex activation is not significantly different between control and heart failure, but significant changes in the ratio in healthy subjects due to increased relaxation rate were abolished in heart failure.

Abstract 14416: Mechano-arrhythmogenicity is Enhanced in Late Repolarisation During Acute Ischemia by a Calcium- and TRPA1-dependent Mechanism

Introduction: Altered tissue mechanics in acute regional ischemia contribute to arrhythmias by a mechano-sensitive, Ca 2+ -dependent mechanism. This is facilitated by uncoupling of voltage-Ca 2+ dynamics, creating a vulnerable period (VP) in late repolarisation for stretch-induced arrhythmias (‘mechano-arrhythmogenicity’). However, cellular mechanisms driving mechano-arrhythmogenicity in acute ischemia are unknown. Objective: Define cellular mechanisms of mechano-arrhythmogenicity in the VP during acute ischemia in rabbit ventricular myocytes. Methods: Rabbit (♀, NZW) LV myocytes were transiently stretched (8-16% change in sarcomere length, 100 ms) during diastole or the VP in normal Tyrode (NT) or simulated ischemia (SI) solution (hyperkalemia, acidosis, metabolic inhibition). Drugs were used to buffer Ca 2+ (BAPTA), stabilise RyR (dantrolene), block mechano-sensitive TRPA1 channels (HC-030031), or block (DPI) or increase (bi-product of di-4-ANBDQPQ excitation) ROS production. Voltage-Ca 2+ was simultaneously monitored with fluorescent dyes (di-4-ANBDQPQ, Fluo-5F) and a single camera-optical splitter system. Results: SI shortened AP duration (APD NT =384 vs APD SI =219ms; p &lt;0.0001) more than Ca 2+ transient duration (CaTD NT =424 vs CaTD SI =357 ms; p &lt;0.0001) and increased the length of the VP (=CaTD-APD; VP NT =54 vs VP SI =146ms; n =50 cells for N NT =6 and N SI =14 rabbits ; p &lt;0.0001). Mechano-arrhythmogenicity (single ectopy and complex sustained activity) was increased in SI compared to NT, but only for stretch in the VP (7 vs 1% of stretches; n =50, N =6 each ; p &lt;0.005), and arrhythmias in the VP were proportionally more complex than those that occurred with stretch in diastole (100 vs 69%; n =50, N =6; p &lt;0.05). Arrhythmia incidence in the VP during SI was reduced by BAPTA (2% of stretches; p &lt;0.05), HC-030031 (1%; p &lt;0.005), and DPI (2%; p &lt;0.05), while dantrolene had no effect ( n =50, N =6 each). Fluorescence imaging during SI further increased mechano-arrhythmogenicity in both the VP and diastole (29 and 14%; n =42, N =4; p &lt;0.05). Conclusions: Acute ischemia enhances cellular mechano-arrhythmogenicity specifically in the VP through a mechanism involving Ca 2+ , ROS, and TRPA1, suggesting potential targets for anti-arrhythmic therapy.

Abstract 14409: TRPA1 Channels Are a Source of Calcium-driven Cellular Mechano-arrhythmogenicity

Introduction: Pathologic changes in myocardial mechanics and hemodynamic load result in arrhythmias via mechanically-induced changes in electrophysiology or intracellular Ca 2+ (‘mechano-arrhythmogenicity’). While molecular mechanisms driving mechano-arrhythmogenicity are poorly defined, they are associated with disease-related alterations in voltage-Ca 2+ dynamics. Objective: Define mechanisms of mechano-arrhythmogenicity during alterations in voltage-Ca 2+ dynamics in rabbit ventricular myocytes. Methods: Rabbit (♀, NZW) LV myocytes were transiently stretched (8-16% change in sarcomere length, 100ms) during diastole or late repolarisation in control or during K ATP channel activation (pinacidil). Drugs were used to buffer Ca 2+ (BAPTA), stabilise RyR (dantrolene), non-selectively block stretch-activated channels (streptomycin), or specifically block (HC-030031) or activate (AITC) mechano-sensitive TRPA1 channels. Voltage-Ca 2+ dynamics were simultaneously monitored with fluorescent dyes (di-4-ANBDQPQ, Fluo-5F) and a single camera-optical splitter system and diastolic Ca 2+ was measured using Fura Red. Results: Pinacidil caused greater shortening of the AP than Ca 2+ transient (-144±17 vs -74±11ms; n =24 cells, N =7 rabbits; p &lt;0.001) with no change in cell stiffness or contractility. Stretch during pinacidil application caused arrhythmias in both diastole and late repolarisation (8 and 10% of stretches; n =46, N =5), which voltage-Ca 2+ imaging revealed were Ca 2+ -driven. Arrhythmias were reduced with BAPTA (3 and 0% of stretches; p &lt;0.05), streptomycin (4 and 2%; p &lt;0.05), and HC-030031(2 and 1%; p &lt;0.01), while dantrolene had no effect ( n =40, N =5 for each). Stretch in diastole during AITC application also caused arrhythmias (15%; p &lt;0.001), which were blocked by HC-030031 (4%; p &lt;0.001) or BAPTA (3%; p &lt;0.001; n =40, N =5 each). Both AITC and pinacidil caused an increase in diastolic Ca 2+ (112±29 and 78±29% of control; p&lt;0.05), which was reduced by HC-030031 with AITC (26±24%; p &lt;0.05), but not with pinacidil ( n =25, N =5 each). Conclusions: TRPA1 activation increases mechano-arrhythmogenicity via a Ca 2+ -driven mechanism and may represent a novel anti-arrhythmic target in pathologies involving altered cardiac mechanics.

Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity.

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including the heart. While Channelrhodopsin-2 (ChR2) is a common tool to depolarize the membrane potential in cardiomyocytes (CM), potentially eliciting action potentials (AP), an effective tool for reliable silencing of CM activity has been missing. It has been suggested to use anion channelrhodopsins (ACR) for optogenetic inhibition. Here, we describe a protocol to assess the effects of activating the natural ACR GtACR1 from Guillardia theta in cultured rabbit CM. Primary readouts are electrophysiological patch-clamp recordings and optical tracking of CM contractions, both performed while applying different patterns of light stimulation. The protocol includes CM isolation from rabbit heart, seeding and culturing of the cells for up to 4 days, transduction via adenovirus coding for the light-gated chloride channel, preparation of patch-clamp and carbon fiber setups, data collection and analysis. Using the patch-clamp technique in whole-cell configuration allows one to record light-activated currents (in voltage-clamp mode, V-clamp) and AP (current-clamp mode, I-clamp) in real time. In addition to patch-clamp experiments, we conduct contractility measurements for functional assessment of CM activity without disturbing the intracellular milieu. To do so, cells are mechanically preloaded using carbon fibers and contractions are recorded by tracking changes in sarcomere length and carbon fiber distance. Data analysis includes assessment of AP duration from I-clamp recordings, peak currents from V-clamp recordings and force calculation from carbon fiber measurements. The described protocol can be applied to the testing of biophysical effects of different optogenetic actuators on CM activity, a prerequisite for the development of a mechanistic understanding of optogenetic experiments in cardiac tissue and whole hearts.

Effect of Sarcomere Length Change During Isovolumic Relaxation Time on Hemodynamics

Effect of Sarcomere Length Change During Isovolumic Relaxation Time on Hemodynamics

A proposed role for non-junctional transverse tubules in skeletal muscle as flexible segments allowing expansion of the transverse network.

Using a variety of technical approaches, we have detected the presence of continuous triads that cover the entire length of T tubules in the main white body muscles of several small fish. This is in contrast to the discontinuous association of sarcoplasmic reticulum with T tubules in the red muscles from the same fish as well as in all other previously described muscles in a large variety of skeletal muscles. We suggest that continuous triads are permissible only in muscle fibers that are not normally subject to significant changes in sarcomere length during normal in vivo activity, as is the case for white muscles in the trunk of fish.

Nebulin and titin modulate cross-bridge cycling and length-dependent calcium sensitivity.

Various mutations in the structural proteins nebulin and titin that are present in human disease are known to affect the contractility of striated muscle. Loss of nebulin is associated with reduced actin filament length and impairment of myosin binding to actin, whereas titin is thought to regulate muscle passive elasticity and is likely involved in length-dependent activation. Here, we sought to assess the modulation of muscle function by these sarcomeric proteins by using the computational platform muscle simulation code (MUSICO) to quantitatively separate the effects of structural changes, kinetics of cross-bridge cycling, and calcium sensitivity of the thin filaments. The simulations show that variation in thin filament length cannot by itself account for experimental observations of the contractility in nebulin-deficient muscle, but instead must be accompanied by a decreased myosin binding rate. Additionally, to match the observed calcium sensitivity, the rate of TnI detachment from actin needed to be increased. Simulations for cardiac muscle provided quantitative estimates of the effects of different titin-based passive elasticities on muscle force and activation in response to changes in sarcomere length and interfilament lattice spacing. Predicted force-pCa relations showed a decrease in both active tension and sensitivity to calcium with a decrease in passive tension and sarcomere length. We conclude that this behavior is caused by partial redistribution of the muscle load between active muscle force and titin-dependent passive force, and also by redistribution of stretch along the thin filament, which together modulate the release of TnI from actin. These data help advance understanding of how nebulin and titin mutations affect muscle function.

Thyroid Hormone Deficiency Adversely Affects Cardiomyocyte T‐tubule Structure and Function

Thyroid hormone deficiency causes adverse changes in cardiomyocyte shape leading to increased wall stress, chamber dilatation and in the long‐term can result in heart failure. We hypothesized that thyroid hormones regulate cardiac contractile function in part by maintaining T‐tubule (TT) structures and efficient excitation‐contraction (EC)‐coupling.MethodsFemale Sprague‐Dawley rats (aged 12 wks) were surgically thyroidectomized (Tx) or were maintained as euthyroid (Eu) controls (6 rats/group). The study was terminated after 12 mo. at which time echocardiographic and hemodynamic measurements were recorded. Ventricular myocytes were isolated by collagenase digestion and plated onto laminin‐coated glass coverslips for TT analysis or were analyzed immediately for contractile and calcium dynamic using videobased edge detection of sarcomere length changes coupled to a fluorescence imaging system (IonOptix). Live cells were labeled with wheat germ agglutinin‐coupled to Alexa‐Fluor (WGA‐488) to image the sarcolemma and TT by confocal microscopy. Multiple z‐stack images were captured per cell and TT periodicity was analyzed using an algorithm to calculate periodicity based on the peak amplitude in the Fourier spectrum of the image at the TT frequency, or TT power. This software program is available as TTorg plugin on ImageJ.ResultsEchocardiography and in vivo hemodynamic measurements showed significantly reduced systolic and diastolic function in Tx vs euthyroid rats. In response to 1 Hz field stimulation at 4V, isolated cardiomyocyte contractile cycles showed significant dysfunction in Tx vs Eu cardiomyocytes: slower max contraction velocity (+Vmax), −2.047±0.30 vs −3.503±0.50 um/sec, Tx vs Eu, respectively, p&lt;0.001; slower max relaxation velocity (‐Vmax), 1.369±0.27 vs 2.44±0.73 um/sec, p&lt;0.01; longer time to peak contraction, 0.279±0.03 vs 0.182±0.03 sec, p&lt;0.001. Myocytes were loaded with Fura2/AM (1uM) and intracellular calcium measured by fluorescence intensity ratio at 340/380 nm. Ca2+ transients showed slower kinetics in Tx vs Eu cardiomyocytes: time to Ca2+ peak, 0.076±0.01 vs 0.0516±0.003 sec, Tx vs Eu, respectively, p&lt;0.0001; time to Ca2+ max return velocity, 0.261 0.03 vs 0.184 0.005, Tx vs Eu, respectively, p&lt;0.0001. TTorg analysis indicated significant disorganization of T‐tubules in Tx cardiomyocytes compared to Eu (TT power = 61±2 vs 80±2, Tx vs Eu, respectively, p&lt;0.00001; arbitrary units with higher values indicate increased periodicity). In conclusion, TH‐deficiency disrupts T‐tubule structures causing cardiomyocyte contractile dysfunction and slower calcium kinetics indicating altered EC‐coupling. Therefore maintaining normal thyroid function is necessary for optimal heart health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Diverse relaxation rates exist among rat cardiomyocytes isolated from a single myocardial region.

Prior studies have shown variation in the functional properties of cardiomyocytes isolated from different regions of the left ventricular myocardium. We found that these region-dependent variations vanish below a tissue volume of ∼7 mm3 in the adult rat myocardium, revealing a fixed level of intrinsic relaxation rate heterogeneity that is independent of tissue volume. Within these microscopically varying cell populations, fast-relaxing cells were shown to have elevated phosphorylated troponin I compared to slow-relaxing cells. Relaxation rate was also correlated with cardiomyocyte length, in that slow-relaxing cells were longer than fast-relaxing cells. These results show a new relationship between cardiomyocyte morphology and myofilament relaxation, and suggest that functional diversity among individual myocytes at the microscale may contribute to bulk relaxation of the myocardium. The mean contractility and calcium handling properties of cardiomyocytes isolated from different regions of the ventricular myocardium are known to vary significantly. We designed experiments to quantify the variance in contractile properties among cells within the same myocardial region. Longitudinal strips of myocardial tissue were excised from the epicardial left ventricular free walls of adult Sprague-Dawley rats and then treated with collagenase to isolate individual myocytes. Cardiomyocytes were characterized by measuring sarcomere length changes and calcium transients during electrical pacing. Variance of the time from peak sarcomere shortening to 50% re-lengthening (RT50 ) was assessed in each cell population. Isolating cells from progressively shorter strips allowed an estimate of the myocardial volume below which regional variation vanished and only microscale heterogeneity remained (∼7 mm3 ). The SD of RT50 within this myocardial volume was 28% of the mean. In a series of follow-up experiments, RT50 was shown to correlate significantly with resting myocyte length, suggesting a connection between cell morphology and intrinsic relaxation behaviour. To explore the mechanistic basis of varying RT50 , a novel single-cell aspirator was employed to collect small batches of cardiomyocytes grouped according to their relaxation rates (fast or slow). Western blot analysis of the two groups revealed significantly elevated troponin I phosphorylation in fast-relaxing cells. Our observations suggest that cell-to-cell heterogeneity of active contractile properties is substantial, with implications for how we understand myocardial relaxation and design drug therapies intended to alter relaxation rate.

Microendoscopy reveals positive correlation in multiscale length changes and variable sarcomere lengths across different regions of human muscle.

Sarcomere length is a key physiological parameter that affects muscle force output; however, our understanding of the scaling of human muscle from sarcomere to whole muscle is based primarily on cadaveric data. The aims of this study were to explore the in vivo relationship between passive fascicle length and passive sarcomere length at different muscle-tendon unit lengths and determine whether sarcomere and fascicle length relationships are the same in different regions of muscle. A microendoscopy needle probe capable of in vivo sarcomere imaging was inserted into a proximal location of the human tibialis anterior muscle at three different ankle positions [5° dorsiflexion, 5° plantar flexion (PF), and 15° PF] and one distal location at a constant ankle position (5° PF distal). Ultrasound imaging of tibialis anterior fascicles, centered on the location of the needle probe, was performed for each condition to estimate fascicle length. Sarcomere length and fascicle length increased with increasing muscle-tendon unit length, although the correlation between sarcomere length change and muscle fascicle length change was only moderate (r2 = 0.45). Passive sarcomere length was longer at the distal imaging site than the proximal site (P = 0.01). When sarcomere number was estimated from sarcomere length and fascicle length, there were fewer sarcomeres in the fibers of distal location than the proximal location (P = 0.01). These data demonstrate that fascicle length changes are representative of sarcomere length changes, although significant variability in sarcomere length exists within a muscle and sarcomere number per fiber is region-dependent.NEW & NOTEWORTHY Sarcomere and fascicle lengths were measured in vivo from human muscle to examine the relationship between the different scales of organization. Changes in fascicle length were moderately related to sarcomere length changes; however, sarcomere length and number per fiber varied from proximal to distal regions of the muscle. Differences in average sarcomere operating lengths across the muscle suggest potentially different stresses or strains experienced within different regions of muscle.