Sarcomere Shortening Velocity Research Articles

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System.

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches involve either differentiating myogenic progenitor cells into immature myotubes or the short-term ex vivo culture of isolated individual muscle fibers. A key benefit of ex vivo culture over in vitro is the retention of the complex cellular architecture and contractile characteristics. Here, we detail an experimental protocol for the isolation of intact flexor digitorum brevis muscle fibers from mice and their subsequent ex vivo culture. In this protocol, muscle fibers are embedded in a fibrin-based and basement membrane matrix hydrogel to immobilize the fibers and maintain their contractile function. We then describe methods to assess the muscle fiber contractile function using an optics-based, high-throughput contractility system. The embedded muscle fibers are electrically stimulated to induce contractions, after which their functional properties, such as sarcomere shortening and contractile velocity, are assessed using optics-based quantification. Coupling muscle fiber culture with thissystem allows for high-throughput testing of the effects of pharmacological agents on contractile function and ex vivo studies of genetic muscle disorders. Finally, this protocol can also be adapted to study dynamic cellular processes in muscle fibers using live-cell microscopy.

Live-Cell Imaging of the Contractile Velocity and Transient Intracellular Ca2+ Fluctuations in Human Stem Cell-Derived Cardiomyocytes.

Live-cell imaging techniques are essential for acquiring vital physiological and pathophysiological knowledge to understand and treat heart disease. For live-cell imaging of transient alterations of [Ca2+]i in human cardiomyocytes, we engineered human-induced pluripotent stem cells carrying a genetically-encoded Ca2+-indicator (GECI). To monitor sarcomere shortening and relaxation in cardiomyocytes in real-time, we generated a α-cardiac actinin (ACTN2)-copepod (cop) green fluorescent protein (GFP+)-human-induced pluripotent stem cell line by using the CRISPR-Cas9 and a homology directed recombination approach. The engineered human-induced pluripotent stem cells were differentiated in transgenic GECI-enhanced GFP+-cardiomyocytes and ACTN2-copGFP+-cardiomyocytes, allowing real-time imaging of [Ca2+]i transients and live recordings of the sarcomere shortening velocity of ACTN2-copGFP+-cardiomyocytes. We developed a video analysis software tool to quantify various parameters of sarcoplasmic Ca2+ fluctuations recorded during contraction of cardiomyocytes and to calculate the contraction velocity of cardiomyocytes in the presence and absence of different drugs affecting cardiac function. Our cellular and software tool not only proved the positive and negative inotropic and lusitropic effects of the tested cardioactive drugs but also quantified the expected effects precisely. Our platform will offer a human-relevant in vitro alternative for high-throughput drug screenings, as well as a model to explore the underlying mechanisms of cardiac diseases.

Effects of omecamtiv mecarbil on calcium-transients and contractility in a translational canine myocyte model

Omecamtiv mecarbil (OM) is a selective cardiac myosin activator (myotrope), currently in Phase 3 clinical investigation as a novel treatment for heart failure with reduced ejection fraction. OM increases cardiac contractility by enhancing interaction between myosin and actin in a calcium-independent fashion. This study aims to characterize the mechanism of action by evaluating its simultaneous effect on myocyte contractility and calcium-transients (CTs) in healthy canine ventricular myocytes. Left ventricular myocytes were isolated from canines and loaded with Fura-2 AM. With an IonOptix system, contractility parameters including amplitude and duration of sarcomere shortening, contraction and relaxation velocity, and resting sarcomere length were measured. CT parameters including amplitude at systole and diastole, velocity at systole and diastole, and duration at 50% from peak were simultaneously measured. OM was tested at 0.03, 0.1, 0.3, 1, and 3 µmol\L concentrations to simulate therapeutic human plasma exposure levels. OM and isoproterenol (ISO) demonstrated differential effects on CTs and myocyte contractility. OM increased contractility mainly by prolonging duration of contraction while ISO increased contractility mainly by augmenting the amplitude of contraction. ISO increased the amplitude and velocity of CT, shortened duration of CT concurrent with increasing myocyte contraction, while OM did not change the amplitude, velocity, and duration of CT up to 1 µmol\L. Decreases in relaxation velocity and increases in duration were present only at 3 µmol\L. In this translational myocyte model study, therapeutically relevant concentrations of OM increased contractility but did not alter intracellular CTs, a mechanism of action distinct from traditional calcitropes.

Effects of omecamtiv mecarbil on calcium-transients and contractility in a translational canine myocyte model.

Omecamtiv mecarbil (OM) is a selective cardiac myosin activator (myotrope), currently in Phase 3 clinical investigation as a novel treatment for heart failure with reduced ejection fraction. OM increases cardiac contractility by enhancing interaction between myosin and actin in a calcium‐independent fashion. This study aims to characterize the mechanism of action by evaluating its simultaneous effect on myocyte contractility and calcium‐transients (CTs) in healthy canine ventricular myocytes. Left ventricular myocytes were isolated from canines and loaded with Fura‐2 AM. With an IonOptix system, contractility parameters including amplitude and duration of sarcomere shortening, contraction and relaxation velocity, and resting sarcomere length were measured. CT parameters including amplitude at systole and diastole, velocity at systole and diastole, and duration at 50% from peak were simultaneously measured. OM was tested at 0.03, 0.1, 0.3, 1, and 3 µmol\\L concentrations to simulate therapeutic human plasma exposure levels. OM and isoproterenol (ISO) demonstrated differential effects on CTs and myocyte contractility. OM increased contractility mainly by prolonging duration of contraction while ISO increased contractility mainly by augmenting the amplitude of contraction. ISO increased the amplitude and velocity of CT, shortened duration of CT concurrent with increasing myocyte contraction, while OM did not change the amplitude, velocity, and duration of CT up to 1 µmol\\L. Decreases in relaxation velocity and increases in duration were present only at 3 µmol\\L. In this translational myocyte model study, therapeutically relevant concentrations of OM increased contractility but did not alter intracellular CTs, a mechanism of action distinct from traditional calcitropes.

Beta-adrenergic activation induces cardiac collapse by aggravating cardiomyocyte contractile dysfunction in bupivacaine intoxication.

In order to determine the role of the adrenergic system in bupivacaine-induced cardiotoxicity, a series of experiments were performed. In an animal experiment, male Sprague-Dawley (SD) rats under chloral hydrate anesthesia received intravenous bupivacaine, followed by an intravenous injection of adrenalin or isoprenalin, and the electrocardiogram (ECG), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), the maximum rate of rise of left ventricular pressure (+dP/dtmax) and the maximum rate of pressure decrease (-dP/dtmax) were continually monitored. In a cellular experiment, freshly isolated adult SD rat ventricular myocytes were perfused with bupivacaine at different concentrations in the presence or absence of isoprenalin, with or without esmolol. The percentage of the sarcomere shortening (bl% peak h), departure velocity (dep v) of sarcomere shortening and time to 50% of the peak speed of myocyte contraction (Tp50) was assessed by a video-based edge-detection system. In an additional experiment, Swiss mice pretreated with saline, isoprenalin, esmolol or dexmedetomidine received bupivacaine to determine the 50% lethal dose (LD50) of bupivacaine. Electron microscopy of myocardial mitochondria was performed to assess damage of these structures. To test mitochondrial reactive oxygen species (ROS) production, freshly isolated SD rat ventricular myocytes were incubated with bupivacaine in the presence of isoprenalin, with or without esmolol. First, our results showed that bupivacaine significantly reduced the LVSP and +dP/dtmax, as well as enhanced the LVEDP and -dP/dtmax (P < 0.05, vs. control, and vs. baseline). Adrenalin and isoprenalin induced a further reduction of LVSP and +dP/dtmax (P < 0.05, vs. before adrenalin or isoprenalin delivery, and vs. control). Second, bupivacaine induced a dose-dependent cardiomyocyte contractile depression. While 5.9 μmol/L or 8.9 μmol/L of bupivacaine resulted in no change, 30.0 μmol/L of bupivacaine prolonged the Tp50 and reduced the bl% peak h and dep v (P < 0.05, vs. control and vs. baseline). Isoprenalin aggravated the bupivacaine-induced cardiomyocyte contractile depression, significantly prolonging the Tp50 (P < 0.05, vs. bupivacaine alone) and reducing the dep v (P < 0.05, vs. bupivacaine alone). Third, esmolol and dexmedetomidine significantly enhanced, while isoprenalin significantly reduced, the LD50 of bupivacaine in mice. Fourth, bupivacaine led to significant mitochondrial swelling, and the extent of myocardial mitochondrial swelling in isoprenalin-pretreated mice was significantly higher than that compared with mice pretreated with saline, as reflected by the higher mitochondrial damage score (P < 0.01). Meanwhile, esmolol pretreatment significantly reduced the mitochondrial damage score (P < 0.01). Fifth, bupivacaine significantly increased the ROS in freshly isolated cardiomyocytes, and added isoprenalin induced a further enhancement of ROS production (P < 0.05, vs. bupivacaine alone). Added esmolol significantly decreased ROS production (P < 0.05, vs. bupivacaine + isoprenalin). Our results suggest that bupivacaine depressed cardiac automaticity, conductivity and contractility, but the predominant effect was contractile dysfunction which resulted from the disruption of mitochondrial energy metabolism. β-adrenergic activation aggravated the cellular metabolism disorder and therefore contractile dysfunction.

A Hypertrophic Cardiomyopathy-associated MYBPC3 Mutation Common in Populations of South Asian Descent Causes Contractile Dysfunction

Hypertrophic cardiomyopathy (HCM) results from mutations in genes encoding sarcomeric proteins, most often MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C). A recently discovered HCM-associated 25-base pair deletion in MYBPC3 is inherited in millions worldwide. Although this mutation causes changes in the C10 domain of cMyBP-C (cMyBP-C(C10mut)), which binds to the light meromyosin (LMM) region of the myosin heavy chain, the underlying molecular mechanism causing HCM is unknown. In this study, adenoviral expression of cMyBP-C(C10mut) in cultured adult rat cardiomyocytes was used to investigate protein localization and evaluate contractile function and Ca(2+) transients, compared with wild-type cMyBP-C expression (cMyBP-C(WT)) and controls. Forty-eight hours after infection, 44% of cMyBP-C(WT) and 36% of cMyBP-C(C10mut) protein levels were determined in total lysates, confirming equal expression. Immunofluorescence experiments showed little or no localization of cMyBP-C(C10mut) to the C-zone, whereas cMyBP-C(WT) mostly showed C-zone staining, suggesting that cMyBP-C(C10mut) could not properly integrate in the C-zone of the sarcomere. Subcellular fractionation confirmed that most cMyBP-C(C10mut) resided in the soluble fraction, with reduced presence in the myofilament fraction. Also, cMyBP-C(C10mut) displayed significantly reduced fractional shortening, sarcomere shortening, and relaxation velocities, apparently caused by defects in sarcomere function, because Ca(2+) transients were unaffected. Co-sedimentation and protein cross-linking assays confirmed that C10(mut) causes the loss of C10 domain interaction with myosin LMM. Protein homology modeling studies showed significant structural perturbation in cMyBP-C(C10mut), providing a potential structural basis for the alteration in its mode of interaction with myosin LMM. Therefore, expression of cMyBP-C(C10mut) protein is sufficient to cause contractile dysfunction in vitro.

Cardiac Myofilament Sarcomere Shortening is Faster and Exhibits Greater Shortening-Induced Cooperative Deactivation after PKA Treatment

Increased myocardial contractility by beta-adrenergic stimulation requires precise coordination between cardiac myocyte membrane potential, calcium handling, and myofilament mechanics. From a myofilament standpoint, more stoke volume at a given end-diastolic volume ideally would involve both faster loaded shortening for more ejection coupled with greater shortening-induced cooperative deactivation to assist in relaxation, which would help assure adequate filling at higher heart rates. We tested if myofilaments elicit these characteristics by direct measurement of myocardial sarcomere shortening during load clamps before and after PKA treatment, a downstream beta-adrenergic signaling molecule. Rat skinned ventricular cardiac myocyte preparations (n = 6) were attached between a force transducer and motor, calcium activated to elicit ∼30% maximal force, and then light-to-moderate load clamps were induced by a servo-controlled feedback system. Initial sarcomere length was set at ∼2.25 μm and sarcomere shortening was monitored during the load clamps using an IonOptix SarcLen system at 240 Hz. Interestingly, PKA seemed to result in the aforementioned ideal changes in sarcomere mechanics. First, loaded sarcomere shortening velocities were faster following PKA treatment (control = 0.838 ± 0.198 μm/sec/sarcomere (n= 20) at loads of 0.17 ± 0.06 isometric force (Po); PKA = 1.215 ± 0.071 μm/sec/sarcomere (n = 31) at loads of 0.15 ± 0.07 Po (p < 0.001) (means ± SD)). In addition, PKA appeared to yield greater shortening-induced cooperative deactivation during load clamps by exhibiting greater curvature of sarcomere length traces, which was indexed by the rate constant of sarcomere shortening (kshortening), (Control kshortening = 4.27 ± 1.47; PKA kshortening = 6.93 ± 2.48 (p < 0.001) (means ± SD)). These findings illustrate the fine tuning of the myofilament clock to help match the resetting of the membrane and calcium clocks after beta-adrenergic stimulation.

Mechanical properties of respiratory muscles.

Striated respiratory muscles are necessary for lung ventilation and to maintain the patency of the upper airway. The basic structural and functional properties of respiratory muscles are similar to those of other striated muscles (both skeletal and cardiac). The sarcomere is the fundamental organizational unit of striated muscles and sarcomeric proteins underlie the passive and active mechanical properties of muscle fibers. In this respect, the functional categorization of different fiber types provides a conceptual framework to understand the physiological properties of respiratory muscles. Within the sarcomere, the interaction between the thick and thin filaments at the level of cross-bridges provides the elementary unit of force generation and contraction. Key to an understanding of the unique functional differences across muscle fiber types are differences in cross-bridge recruitment and cycling that relate to the expression of different myosin heavy chain isoforms in the thick filament. The active mechanical properties of muscle fibers are characterized by the relationship between myoplasmic Ca2+ and cross-bridge recruitment, force generation and sarcomere length (also cross-bridge recruitment), external load and shortening velocity (cross-bridge cycling rate), and cross-bridge cycling rate and ATP consumption. Passive mechanical properties are also important reflecting viscoelastic elements within sarcomeres as well as the extracellular matrix. Conditions that affect respiratory muscle performance may have a range of underlying pathophysiological causes, but their manifestations will depend on their impact on these basic elemental structures.

Inotropic and lusitropic effects of calcitonin gene-related peptide in the heart

Previous studies have demonstrated positive-inotropic effects of calcitonin gene-related peptide (CGRP), but the mechanisms remain unclear. Therefore, two experiments were performed to determine the physiological correlates of the positive-inotropic effects of CGRP. Treatments designed to antagonize the effects of physiologically active CGRP₁₋₃₇ included posttreatment with CGRP₈₋₃₇ and pretreatment with LY-294002 (LY, an inhibitor of phosphatidylinositol 3-kinase), 17β-estradiol (E), and progesterone (P) were also used to modulate the effects of CGRP₁₋₃₇. Experiment 1 was in vitro studies on sarcomeres and cells of isolated adult rat cardiac myocytes. CGRP₁₋₃₇, alone and in combination with E and P, decreased sarcomere shortening velocities and increased shortening percentages, effects that were antagonized by CGRP₈₋₃₇, but not by LY. CGRP₁₋₃₇ increased resting intracellular calcium ion concentrations and Ca(2+) influxes, effects that were also antagonized by both CGRP₈₋₃₇ and LY. Experiment 2 was in vivo studies on left ventricular pressure-volume (PV) loops. CGRP₁₋₃₇ increased end-systolic pressure, ejection fraction, and velocities of contraction and relaxation while decreasing stroke volume, cardiac output, stroke work, PV area, and compliance. After partial occlusion of the vena cava, CGRP₁₋₃₇ increased the slope of the end-systolic PV relationship. CGRP₈₋₃₇ and LY attenuated most of the CGRP-induced changes. These findings suggest that CGRP-induced positive-inotropic effects may be increased by treatments with estradiol and progesterone and inhibited by LY. The physiological correlates of CGRP-induced positive inotropy observed in rat sarcomeres, cells, and intact hearts are likely to reveal novel mechanisms of heart failure in humans.

The velocity of cardiac sarcomere shortening: mechanisms and implications.

A classical paper published by Michael Barany almost 50years ago demonstrated a tight correlation between the mechanical parameter of maximal velocity of shortening and the biochemical parameter of myosin ATPase activity in a wide spectrum of species. Here, we review the determinants of muscle dynamics by mechanical load and the relation between sarcomere shortening velocity and cross-bridge dynamics in rat myocardium containing a range of fast and slow myosin. Observations from molecular level to mechanics of the intact human heart suggest that cardiac actin-myosin kinetic properties are matched so as to optimize myocardial strain rate and allow for the maximum rate of hydraulic energy output observed during ejection in the whole ventricle.

Competitive Regulation of Calcium and Zinc Ions in Cardiomyocyte Contraction-Relaxation Function

Zinc (Zn2+) and calcium (Ca2+) ions are divalent cations having common chemical properties leading to their competing for the same regulatory channels and pumps in the intact cardiomyocyte. Diastolic dysfunction may be due in part to elevated diastolic Ca2+ concentration ([Ca2+]). We hypothesized that Zn2+ reduces systolic and enhances diastolic function due to its effects on Ca2+ regulation. We examined the effects of 32µM extracellular zinc ([Zn2+]ext) exposure and intracellular zinc ([Zn2+]int) accumulation on rat cardiomyocyte function. We measured sarcomere dynamics, [Ca2+]int by Fura-2FF and [Zn2+]int by FluoZin-3 under three [Zn2+] conditions: no [Zn2+]ext and low [Zn2+]int; 32µM [Zn2+]ext and low [Zn2+]int; 32µM [Zn2+]ext and high [Zn2+]int. Cardiomyocytes were paced at 2Hz, exposed to 2mM [Ca2+]ext at 37°C. After reaching [Zn2+]int steady-state, 10mM caffeine was rapidly applied to measure sarcoplasmic reticulum (SR) Ca2+ content and Na+-Ca2+ exchanger (NaCaX) efflux rate. Sarcomere shortening velocity and peak shortening were significantly (P<0.05) reduced with [Zn2+]ext exposure in either low or high [Zn2+]int conditions. Interestingly, peak shortening was significantly enhanced with high [Zn2+]int compared to low [Zn2+]int. Diastolic sarcomere length was significantly increased with high [Zn2+]int. Peak [Ca2+]int was significantly reduced under 32μM [Zn2+]ext with high [Zn2+]int, which was consistent with lower SR Ca2+ content detected by caffeine experiment. SR Ca2+ uptake rate by SERCA and NaCaX efflux rate were not affected by [Zn2+]int. All the above changes due to Zn2+ were not observed in control cardiomyocytes without [Zn2+]ext exposure. These findings suggest that Zn2+ competes with Ca2+ for calcium channels (L-type and SR release channels) and thereby reduces contractile function without affecting SERCA or NaCaX. Interestingly, high [Zn2+]int causes a slightly increased contractile function despite the reduction in peak [Ca2+]int, suggesting that [Zn2+]int enhances myofilament contraction by mechanisms yet to be explained.

Comparative Kinetics of the ATPase and Actin Sliding Velocity of Myosin Isoforms

Myosin isoform expression varies according to demand and pathology, and the kinetics of the resultant actomyosin motor protein determine maximal sarcomere shortening velocity. Studies of muscle fibers and isolated myosin isoforms have shown that actin sliding velocity correlates with ATP hydrolysis. We studied this relationship for isoforms of actomyosin complexes and examined ATP hydrolysis and the effect of association and dissociation of myosin with actin. Sliding velocity of actin filaments was measured in motility assays with different myosin isoforms. Actin-dependent ATP hydrolysis rate of isolated myosin sub-fragments interacting with filamentous actin, or cross-linked with actin by the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were measured. ATPase activity and velocity of actin moved by myosin isoforms from rat and canine cardiac and skeletal muscle were measured at 25°, 30° and 35°C. Motility velocity plotted against ATPase activity of different myosin isoforms showed a linear correlation with a slope of 330 ±20 (nm/sec)/(ATP/sec) ( R2 = 0.98); the slope coefficient was 19% of the slope of the relationship for intact muscle described by M. Barany (J Gen Physiol, 1967) across a wide range of temperatures. Activation energy of sliding velocity (92 - 96 kJ/mol) and ATPase rate (83 - 121 kJ/mol) of different myosin isoforms were similar. Cross-linking of actomyosin complexes by EDC increased ATP hydrolysis rate 4-fold above ATPase at saturating [Actin]. This suggests that association/dissociation kinetics are rate-limiting and that ATPase is activated in maximally 25% myosin molecules interacting with actin in solution. The calculated displacement of actin filaments (D = 330 nm) per ATP hydrolyzed under the experimental conditions used here suggests that unloaded cross-bridges may displace actin over a multitude of the minimal steps of 2.7 nm that can be made by myosin along the actin filaments.

Acute effects of static stretching on characteristics of the isokinetic angle – torque relationship, surface electromyography, and mechanomyography

The aims of this study were to examine the acute effects of static stretching on peak torque, work, the joint angle at peak torque, acceleration time, isokinetic range of motion, mechanomyographic amplitude, and electromyographic amplitude of the rectus femoris during maximal concentric isokinetic leg extensions at 1.04 and 5.23 rad · s−1 in men and women. Ten women (mean ± s: age 23.0 ± 2.9 years, stature 1.61 ± 0.12 m, mass 63.3 ± 9.9 kg) and eight men (age 21.4 ± 3.0 years, stature 1.83 ± 0.11 m, mass 83.1 ± 15.2 kg) performed maximal voluntary concentric isokinetic leg extensions at 1.04 and 5.23 rad · s−1. Following the initial isokinetic tests, the dominant leg extensors were stretched using four static stretching exercises. After the stretching, the isokinetic tests were repeated. Peak torque, acceleration time, and electromyographic amplitude decreased (P≤ 0.05) from pre- to post-stretching at 1.04 and 5.23 rad · s−1; there were no changes (P > 0.05) in work, joint angle at peak torque, isokinetic range of motion, or mechanomyographic amplitude. These findings indicate no stretching-related changes in the area under the angle – torque curve (work), but a significant decrease in peak torque, which suggests that static stretching may cause a “flattening” of the angle – torque curve that reduces peak strength but allows for greater force production at other joint angles. These findings, in conjunction with the increased limb acceleration rates (decreased acceleration time) observed in the present study, provide tentative support for the hypothesis that static stretching alters the angle – torque relationship and/or sarcomere shortening velocity.

Dependence of Intramyocardial Pressure and Coronary Flow on Ventricular Loading and Contractility: A Model Study

The phasic coronary arterial inflow during the normal cardiac cycle has been explained with simple (waterfall, intramyocardial pump) models, emphasizing the role of ventricular pressure. To explain changes in isovolumic and low afterload beats, these models were extended with the effect of three-dimensional wall stress, nonlinear characteristics of the coronary bed, and extravascular fluid exchange. With the associated increase in the number of model parameters, a detailed parameter sensitivity analysis has become difficult. Therefore we investigated the primary relations between ventricular pressure and volume, wall stress, intramyocardial pressure and coronary blood flow, with a mathematical model with a limited number of parameters. The model replicates several experimental observations: the phasic character of coronary inflow is virtually independent of maximum ventricular pressure, the amplitude of the coronary flow signal varies about proportionally with cardiac contractility, and intramyocardial pressure in the ventricular wall may exceed ventricular pressure. A parameter sensitivity analysis shows that the normalized amplitude of coronary inflow is mainly determined by contractility, reflected in ventricular pressure and, at low ventricular volumes, radial wall stress. Normalized flow amplitude is less sensitive to myocardial coronary compliance and resistance, and to the relation between active fiber stress, time, and sarcomere shortening velocity.

CARDIOMYOCYTE FUNCTION AFTER BURN INJURY AND LIPOPOLYSACCHARIDE EXPOSURE: SINGLE-CELL CONTRACTION ANALYSIS AND CYTOKINE SECRETION PROFILE

A component of multiorgan dysfunction in burned patients is heart failure. Burn trauma induces cytokine synthesis of interleukin (IL) 1beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) which can negatively impact cardiac function. Infectious complications are common following severe burn injury. We hypothesized that burn injury and lipopolysaccharide (LPS) exposure independently influence peak cardiomyocyte contraction and cytokine secretion. Rats underwent a full-thickness 30% total body surface area scald or sham burn. At 1, 6, 12, and 24 h after burn, cardiomyocytes were isolated and incubated with increasing LPS doses. Peak sarcomere shortening and contractile velocity parameters were recorded using a variable-rate video camera with sarcomere length detection software. Supernatants were assayed for IL-1beta, IL-6, and TNF-alpha by ELISA. Peak sarcomere shortening was decreased in the burn group at 1, 6, 12, and 24 h after burn. IL-1beta, IL-6, and TNF-alpha levels were increased in cardiomyocytes isolated 1 h after burn compared with sham controls, but returned to sham levels at 6, 12, and 24 h after burn. LPS exposure caused dose-dependent decreases in sarcomere shortening in sham and burn animals. LPS exposure did not produce increased cardiomyocyte cytokine expression. Burn injury diminished peak sarcomere shortening. Whereas exposure to LPS did not have an effect on cardiomyocyte cytokine expression, LPS significantly inhibited sarcomere shortening in a dose-dependent fashion. Combined burn and LPS exposure inhibited sarcomere shortening more than each alone. These results demonstrate that LPS exposure and burn injury independently decrease peak cardiac shortening. These decreases did not directly correlate with the levels of cytokines released in response to each stressor.

Systolic modeling of the left ventricle as a mechatronic system: determination of myocardial fiber's sarcomere contractile characteristics and new performance indices.

In this paper, the left ventricle (LV) is modeled as a cylinder with myocardial fibers located helically within its wall. A fiber is modeled into myocardial structural units (MSUs); the core entity of each MSU is the sarcomeric contractile element. The relationship between the sarcomere unit's contractile force and shortening velocity is expressed in terms of the LV model's wall stress and deformation, and hence in terms of the monitored LV pressure and volume. Then, the LV systolic performance is investigated in terms of a mechatronic (excitation-contraction) model of the sarcomere unit located within the LV cylindrical model wall. The governing equation of dynamics of the LV myocardial structural unit (MSU) is developed, involving the parameters of the series-elastic element (SE), the viscous element (VE) and the contractile element (CE). We then relate the MSU's force and displacement variables (in terms of SE, VE and CE parameters) to the LV pressure and volume, using the patient's catheterization-ventriculogram data. We thereby evaluate the MSU elements' parameters. We then determine the sarcomere (CE) 'force vs. shortening-velocity' characteristics as well as the power generated by the sarcomere (or CE) element. These are deemed to be important LV functional indices. When our computed sarcomeric peak-power is compared against the traditional LV contractility indices (by linear regression), a high degree of correlation is obtained. We have provided herein, a LV systolic-phase (cylindrical geometry) model whose wall contains the myocardial fibers having sarcomere units. We have expressed the LV myocardial sarcomere's CE (force vs. shortening-velocity) characteristics in terms of the LV pressure-volume data. These CE properties express the intrinsic performance capacity of the LV. Hence, indices containing these properties are deemed to reflect LV performance. In this regard, our new LV contractility index correlates very well with the traditional LV contractility index dP/dt(max).

Auto-oscillations of Skinned Myocardium Correlating with Heartbeat

Skinned myocardium (or myofibrils) exhibits auto-oscillations of sarcomere length and developed force called SPOC (SPontaneousOscillatoryContraction) under partial activation conditions. In SPOC, each sarcomere repeats the cycle of slow shortening and rapid lengthening, and the lengthening phase propagates sequentially to the adjacent sarcomeres in waves (SPOC wave). In this study, we analyzed the sarcomeric oscillation in SPOC in skinned myocardium of various animal species (rat, rabbit, dog, pig, and cow) with different heart rates. The period of oscillation, the sarcomere shortening velocity, and the velocity of SPOC wave, strongly correlated with the resting heart rate of the animal species. The shortening velocity in particular was proportional to the resting heart rate. We then examined the motile activity of each cardiac myosin by an in vitro motility assay. The sliding velocity of actin filaments, which is an index of the motile activity of myosin, also correlated with the resting heart rate but the relationship was not proportional. As a result, the ratio of sarcomere shortening velocity in SPOC to the sliding velocity of actin filaments was not constant but became higher with a higher heart rate. This suggests that the sarcomere shortening velocity in SPOC is modulated by some additional factors besides the motile activity of myosin, resulting in the proportional relationship between the shortening velocity of the sarcomere and the resting heart rate.

Normal myocardial function in severe right ventricular volume overload hypertrophy.

Severe left ventricular volume overloading causes myocardial and cellular contractile dysfunction. Whether this is also true for severe right ventricular volume overloading was unknown. We therefore created severe tricuspid regurgitation percutaneously in seven dogs and then observed them for 3.5-4.0 yr. All five surviving operated dogs had severe tricuspid regurgitation and right heart failure, including massive ascites, but they did not have left heart failure. Right ventricular cardiocytes were isolated from these and from normal dogs, and sarcomere mechanics were assessed via laser diffraction. Right ventricular cardiocytes from the tricuspid regurgitation dogs were 20% longer than control cells, but neither the extent (0.171 +/- 0.005 microm) nor the velocity (2.92 +/- 0.12 microm/s) of sarcomere shortening differed from controls (0.179 +/- 0.005 microm and 3.09 +/- 0.11 microm/s, respectively). Thus, despite massive tricuspid regurgitation causing overt right heart failure, intrinsic right ventricular contractile function was normal. This finding for the severely volume-overloaded right ventricle stands in distinct contrast to our finding for the left ventricle severely volume overloaded by mitral regurgitation, wherein intrinsic contractile function is depressed.

The Effect of Sarcomere Shortening Velocity on Force Generation, Analysis, and Verification of Models for Crossbridge Dynamics

The study tests the hypothesis that the transition rate (G) of the cardiac cross-bridge (XB) from the strong force generating state to the weak state is a linear function of the sarcomere shortening velocity (V(SL)). Force (F) was measured with a strain gauge in six trabeculae from the rat right ventricle in K-H solution [(Ca]0 = 1.5 mM, 25 degrees C). Sarcomere length (SL) was measured with laser diffraction techniques. Twitch F at constant SL and the F response to shortening at constant V(SL) (0-8 microm/s; deltaSL 50-100 nm) were measured at varied times during the twitch. The F response to shortening consisted of an initial fast exponential decline (tau = 2 ms), followed by a slow decrease of F. The instantaneous difference (deltaF) between the isometric F (F(M)) and F during the slow phase depended on the duration of shortening (deltat), the instantaneous F(M) and V(SL). deltaF = G1 x F(M) x deltat x V(SL) x (1 -V(SL)/V(MAX)), where V(MAX) is the unloaded V(SL) and G1 was 6.15+/-2.12 microm(-1) (mean +/- s.d.; n=6). DeltaF/F(M) was independent of the time onset of shortening. The linear interrelation between deltaF and V(SL) is consistent with the suggested feedback, whereby XB kinetics depends on V(SL). This feedback provides a more universal description of the interrelation between shortening and force, as well as the observed linear relation between energy consumption and the mechanical energy output.

Protein kinase A does not alter unloaded velocity of sarcomere shortening in skinned rat cardiac trabeculae.

Whether beta-adrenergic stimulation affects the cross-bridge cycling rate independently of its effect on Ca2+ handling by the cardiac myocyte is still unknown. An increase in cross-bridge cycling rate may result in increased unloaded velocity of sarcomere shortening (V0). To test this hypothesis directly, skinned rat cardiac trabeculae were attached between a silicon strain gauge (approximately 3.5 kHz resonant frequency) and a fast displacement motor. V0 was measured by a modified "Edman slack test" during a single maximal activation using seven to eight sarcomere-length step releases (measured by laser diffraction) ranging between 0.12 and 0.20 micron (15.0 +/- 0.1 degrees C). beta-Adrenergic stimulation was mimicked by exposing the trabeculae to the catalytic subunit of protein kinase A (PKA). Treatment with PKA (3 micrograms/ml; 45 min) caused a significant (P < 0.01) increase (41 +/- 13%) in the Ca2+ concentration required for half-maximal steady-state tension development. Neither maximum tension nor V0 was affected by treatment with PKA, suggesting that beta-adrenergic stimulation does not affect the rate-limiting step of cross-bridge cycling during unloaded shortening in myocardium.