Single cell mechanics of rat cardiomyocytes under isometric, unloaded, and physiologically loaded conditions

One of the most salient characteristics of the heart is its ability to adjust work output to external load. To examine whether a single cardiomyocyte preparation retains this property, we measured the contractile function of a single rat cardiomyocyte under a wide range of loading conditions using a force-length measurement system implemented with adaptive control. A pair of carbon fibers was used to clamp the cardiomyocyte, attached to each end under a microscope. One fiber was stiff, serving as a mechanical anchor, while the bending motion of the compliant fiber was monitored for force-length measurement. Furthermore, by controlling the position of the compliant fiber using a piezoelectric translator based on adaptive control, we could change load dynamically during contractions. Under unloaded conditions, maximal shortening velocity was 106 +/- 8.9 microm/s (n = 13 cells), and, under isometric conditions, peak developed force reached 5,720 nN (41.6 +/- 5.6 mN/mm(2); n = 17 cells). When we simulated physiological working conditions consisting of an isometric contraction, followed by shortening and relaxation, the average work output was 828 +/- 123 J/m(3) (n = 20 cells). The top left corners of tension-length loops obtained under all of these conditions approximate a line, analogous to the end-systolic pressure-volume relation of the ventricle. All of the functional characteristics described were analogous to those established by studies using papillary muscle or trabeculae preparations. In conclusion, the present results confirmed the fact that each myocyte forms the functional basis for ventricular function and that single cell mechanics can be a link between subcellular events and ventricular mechanics.     

Approximate model of cooperative activation and crossbridge cycling in cardiac muscle using ordinary differential equations

We develop a point model of the cardiac myofilament (MF) to simulate a wide variety of experimental muscle characterizations including Force-Ca relations and twitches under isometric, isosarcometric, isotonic, and auxotonic conditions. Complex MF behaviors are difficult to model because spatial interactions cannot be directly implemented as ordinary differential equations. We therefore allow phenomenological approximations with careful consideration to the relationships with the underlying biophysical mechanisms. We describe new formulations that avoid mean-field approximations found in most existing MF models. To increase the scope and applicability of the model, we include length- and temperature-dependent effects that play important roles in MF responses. We have also included a representation of passive restoring forces to simulate isolated cell shortening protocols. Possessing both computational efficiency and the ability to simulate a wide variety of muscle responses, the MF representation is well suited for coupling to existing cardiac cell models of electrophysiology and Ca-handling mechanisms. To illustrate this suitability, the MF model is coupled to the Chicago rabbit cardiomyocyte model. The combined model generates realistic appearing action potentials, intracellular Ca transients, and cell shortening signals. The combined model also demonstrates that the feedback effects of force on Ca binding to troponin can modify the cytosolic Ca transient.     

Measuring single cardiac myocyte contractile force via moving a magnetic bead

One of the biggest problems of heart failure is the heart's inability to effectively pump blood to meet the body's demands, which may be caused by disease-induced alterations in contraction properties (such as contractile force and Young's modulus). Thus, it is very important to measure contractile properties at single cardiac myocyte level that can lay the foundation for quantitatively understanding the mechanism of heart failure and understanding molecular alterations in diseased heart cells. In this article, we report a novel single cardiac myocyte contractile force measurement technique based on moving a magnetic bead. The measuring system is mainly composed of 1), a high-power inverted microscope with video output and edge detection; and 2), a moving magnetic bead based magnetic force loading module. The main measurement procedures are as follows: 1), record maximal displacement of single cardiac myocyte during contraction; 2), attach a magnetic bead on one end of the myocyte that will move with myocyte during the contraction; 3), repeat step 1 and record contraction processes under different magnitudes of magnetic force loading by adjusting the magnetic field applied on the magnetic bead; and 4), derive the myocyte contractile force base on the maximal displacement of cell contraction and magnetic loading force. The major advantages of this unique approach are: 1), measuring the force without direct connections to the cell specimen (i.e., "remote sensing", a noninvasive/minimally invasive approach); 2), high sensitivity and large dynamic range (force measurement range: from pico Newton to micro Newton); 3), a convenient and cost-effective approach; and 4), more importantly, it can be used to study the contractile properties of heart cells under different levels of external loading forces by adjusting the magnitude of applied magnetic field, which is very important for studying disease induced alterations in contraction properties. Experimental results demonstrated the feasibility of proposed approach.     

Bistable dynamics of cardiac cell models coupled by dynamic gap junctions linked to Cardiac Memory

In an earlier study, we suggested that adaptive gap junctions (GJs) might be a basis of cardiac memory, a phenomenon which refers to persistent electrophysiological response of the heart to external pacing. Later, it was also shown that the proposed mechanism of adaptation of GJs is consistent with known electrophysiology of GJs. In the present article, we show that a pair of cardiac cell models coupled by dynamic, voltage-sensitive GJs exhibits bistable behavior under certain conditions. Three kinds of cell pairs are considered: (1) a Noble–Noble cell pair that represents adjacent cells in Purkinje network, (2) a pair of DiFranceso–Noble cells that represents adjacent SA nodal cells, and (3) a model of Noble cell coupled to Luo–Rudy cell model, which represents an interacting pair of a Purkinje fiber and a ventricular myocyte. Bistability is demonstrated in all the three cases. We suggest that this bistability might be an underlying factor behind cardiac memory. Focused analysis of a pair of Noble cell models showed that bistability is obtained only when the properties of GJs “match” with the properties of the pair of cells that is coupled by the GJs. This novel notion of match between GJs and cardiac cell types might give an insight into specialized distributions of various connexin proteins in cardiac tissue.     

Effects of series compliance on twitches superimposed on voluntary contractions.

The activation of skeletal muscle during voluntary isometric contraction has been assessed by measuring the increase in force caused by a superimposed maximal shock to the motor nerve (the twitch-interpolation technique). When the muscle is held isometric, the increase in force with stimulation (superimposed twitch force) decreases with increasing voluntary force, and a line fit through the data can be extrapolated to maximal voluntary force at the zero twitch force axis. In a previous paper we questioned the applicability of this technique in situations where a high series compliance allows the muscle to shorten during the superimposed twitch. To explore effects of series compliance, we measured force of the adductor pollicis during voluntary isometric contractions with noncompliant and compliant loading devices. With the compliant loading device, superimposed twitch force was systematically less than with the noncompliant device, and the plot of superimposed twitch force vs. voluntary force was often concave upward, preventing easy extrapolation to maximal voluntary force. These findings are consistent with force-velocity characteristics of muscle and suggest that twitch-interpolation data must be interpreted with caution when the muscle is not held isometric during the superimposed twitch.     

Dynamic equilibration of airway smooth muscle contraction during physiological loading.

Airway smooth muscle contraction is the central event in acute airway narrowing in asthma. Most studies of isolated muscle have focused on statically equilibrated contractile states that arise from isometric or isotonic contractions. It has recently been established, however, that muscle length is determined by a dynamically equilibrated state of the muscle in which small tidal stretches associated with the ongoing action of breathing act to perturb the binding of myosin to actin. To further investigate this phenomenon, we describe in this report an experimental method for subjecting isolated muscle to a dynamic microenvironment designed to closely approximate that experienced in vivo. Unlike previous methods that used either time-varying length control, force control, or time-invariant auxotonic loads, this method uses transpulmonary pressure as the controlled variable, with both muscle force and muscle length free to adjust as they would in vivo. The method was implemented by using a servo-controlled lever arm to load activated airway smooth muscle strips with transpulmonary pressure fluctuations of increasing amplitude, simulating the action of breathing. The results are not consistent with classical ideas of airway narrowing, which rest on the assumption of a statically equilibrated contractile state; they are consistent, however, with the theory of perturbed equilibria of myosin binding. This experimental method will allow for quantitative experimental evaluation of factors that were previously outside of experimental control, including sensitivity of muscle length to changes of tidal volume, changes of lung volume, shape of the load characteristic, loss of parenchymal support and inflammatory thickening of airway wall compartments.     

A model of muscular control of upper airway geometry

Some disorders of the upper airway in humans are marked by decreased cross-sectional area and increased airway wall compliance. Based on our observations from studies performed in the isolated upper airway of dogs, we hypothesized that the size, and perhaps the geometry, of the airway was altered by changes in the relative activation levels of various muscle pairs. This could be accomplished either by altering the intensity of the neuromuscular input, or by activating muscle pairs which have different geometric orientation to the airway. We developed an analytic relationship to allow us to vary the stimulus level driving any one of six muscle pairs, each with a different anatomic orientation, to evaluate the relationship between those parameters and upper airway volume. With data generated from bilateral electrical stimulation of upper airway muscles, we described a shape factor which allowed us to predict the maximum force produced at optimal length. These findings were applied to a length/tension curve common to striated muscle to allow us to examine the muscle behavior at lengths other than optimal. The position of each muscle was described in spherical coordinates relative to an elastic cylinder, which represented the isolated, sealed upper airway. These coordinates defined the direction in which the force generated by each muscle pair would be applied. Three compliance constants determined the change in airway dimensions produced by the muscle force. This system and its variables were used to calculate the change in volume of the sealed upper airway chamber resulting from muscle contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Porcine cardiac myocyte power output is increased after chronic exercise training.

Chronic exercise training increases the functional capacity of the heart, perhaps by increased myocyte contractile function, as has been observed in rodent exercise models. We examined whether cardiac myocyte function is enhanced after chronic exercise training in Yucatan miniature swine, whose heart characteristics are similar to humans. Animals were designated as either sedentary (Sed), i.e., cage confined, or exercise trained (Ex), i.e., underwent 16-20 wk of progressive treadmill training. Exercise training efficacy was shown with significantly increased heart weight-to-body weight ratios, skeletal muscle citrate synthase activity, and exercise tolerance. Force-velocity properties were measured by attaching skinned cardiac myocytes between a force transducer and position motor, and shortening velocities were measured over a range of loads during maximal Ca2+ activation. Myocytes (n = 9) from nine Ex pigs had comparable force production but a approximately 30% increase in peak power output compared with myocytes (n = 8) from eight Sed. Interestingly, Ex myofibrillar samples also had higher baseline PKA-induced phosphorylation levels of cardiac troponin I, which may contribute to the increase in power. Overall, these results suggest that enhanced power-generating capacity of porcine cardiac myofibrils contributes to improved cardiac function after chronic exercise training.     

The influence of an elastic tendon on the force producing capabilities of a muscle during dynamic movements

With increasing computer power, computer simulation of human movement has become a popular research tool. However, time to complete simulations can still be long even on powerful computers. One possibility for reducing simulation time, with models of musculo-skeletal system, is to simulate the muscle using a rigid tendon rather than the more realistic compliant tendon. This study examines the effect of tendon elasticity on muscle force output under different dynamic conditions. A single muscle, point mass model was used and simulations were performed varying the mass, the tendon length, the initial position, and the task. For simulations for relatively slow motion, as experienced for example in upper limb reaching motions or rising from a chair, tendon properties had little influence on muscle force, in contrast simulations of an explosive task similar to jumping or throwing tendon had a much larger effect.     

The influence of an elastic tendon on the force producing capabilities of a muscle during dynamic movements

With increasing computer power, computer simulation of human movement has become a popular research tool. However, time to complete simulations can still be long even on powerful computers. One possibility for reducing simulation time, with models of musculo-skeletal system, is to simulate the muscle using a rigid tendon rather than the more realistic compliant tendon. This study examines the effect of tendon elasticity on muscle force output under different dynamic conditions. A single muscle, point mass model was used and simulations were performed varying the mass, the tendon length, the initial position, and the task. For simulations for relatively slow motion, as experienced for example in upper limb reaching motions or rising from a chair, tendon properties had little influence on muscle force, in contrast simulations of an explosive task similar to jumping or throwing tendon had a much larger effect.     

Altered single cell force-velocity and power properties in exercise-trained rat myocardium.

Myocardial function is enhanced by endurance exercise training, but the cellular mechanisms underlying this improved function remain unclear. The ability of the myocardium to perform external work is a critical aspect of ventricular function, but previous studies of myocardial adaptation to exercise training have been limited to measurements of isometric tension or unloaded shortening velocity, conditions in which work output is zero. We measured force-velocity properties in single permeabilized myocyte preparations to determine the effect of exercise training on loaded shortening and power output. Female Sprague-Dawley rats were divided into sedentary control (C) and exercise trained (T) groups. T rats underwent 11 wk of progressive treadmill exercise. Myocytes were isolated from T and C hearts, chemically skinned, and attached to a force transducer. Shortening velocity was determined during loaded contractions at 15 degrees C by using a force-clamp technique. Power output was calculated by multiplying force times velocity values. We found that unloaded shortening velocity was not significantly different in T vs. C myocytes (T = 1.43 muscle lengths/s, n = 46 myocytes; C = 1.12 muscle lengths/s, n = 43 myocytes). Training increased the velocity of loaded shortening and increased peak power output (peak power = 0.16 P/P(o) x muscle length/s for T myocytes; peak power = 0.10 P/P(o) x muscle length/s for C myocytes, where P/P(o) is relative tension). We found no effect of training on myosin heavy chain isoform content. These results suggest that training alters power output properties of single cardiac myocytes and that this adaptation may improve the work capacity of the myocardium.     

Plakoglobin is essential for myocardial compliance but dispensable for myofibril insertion into adherens junctions

Plakoglobin (gamma-catenin), a member of the armadillo family of proteins, is a constituent of the cytoplasmic plaque of cardiac junctions and is involved in anchorage of cytoskeletal filaments to specific cadherins. Its genetic inactivation leads to an embryonic lethal phenotype due to heart dysfunction related to an impairment in the architecture of intercalated discs and in the stability of the heart tissue. To elucidate the functional consequences of the loss of plakoglobin for myofibrillar function, we monitored passive stress-strain relationship and contractility parameters of demembranated embryonic fibers. Heart fibers obtained from plakoglobin-deficient embryonic mice were significantly less compliant than were fibers from wild-type embryos. This difference was especially pronounced at lower fiber extension levels: at 120% of slack length, compliance was 2.5-fold lower in plakoglobin-deficient mice than in the corresponding wild-type group. Contractile paramenters (force per cross-section; Ca2+ sensitivity of isometric force and shortening velocity at near-zero load) were comparable in all experimental groups. Therefore, we suggest that plakoglobin is important for cardiac compliance but not necessary for the attachment of the myofibrillar apparatus to adherens junctions. Thus, we conclude that the loss of function of desmosomes and the profound disarrangement of junctional components in plakoglobin null embryos is associated with a decreased passive compliance, which may explain the ventricular rupture and consequent pericardial tamponade in embryos lacking plakoglobin.     

The effect of altered temperature on Ca2(+)-sensitive force in permeabilized myocardium and skeletal muscle. Evidence for force dependence of thin filament activation

The effect of changes in temperature on the calcium sensitivity of tension development was examined in permeabilized cellular preparations of rat ventricle and rabbit psoas muscle. Maximum force and Ca2+ sensitivity of force development increased with temperature in both muscle types. Cardiac muscle was more sensitive to changes in temperature than skeletal muscle in the range 10-15 degrees C. It was postulated that the level of thin filament activation may be decreased by cooling. To investigate this possibility, troponin C (TnC) was partially extracted from both muscle types, thus decreasing the level of thin filament activation independent of temperature and, at least in skeletal muscle fibers, decreasing cooperative activation of the thin filament as well. TnC extraction from cardiac muscle reduced the calcium sensitivity of tension less than did extraction of TnC from skeletal muscle. In skeletal muscle the midpoint shift of the tension-pCa curve with altered temperature was greater after TnC extraction than in control fibers. Calcium sensitivity of tension development was proportional to the maximum tension generated in cardiac or skeletal muscle under all conditions studied. Based on these results, we conclude that (a) maximum tension-generating capability and calcium sensitivity of tension development are related, perhaps causally, in fast skeletal and cardiac muscles, and (b) thin filament activation is less cooperative in cardiac muscle than in skeletal muscle, which explains the differential sensitivity of the two fiber types to temperature and TnC extraction. Reducing thin filament cooperativity in skeletal muscle by TnC extraction results in a response to temperature similar to that of control cardiac cells. This study provides evidence that force levels in striated muscle influence the calcium binding affinity of TnC.     

Diversion of blood flow from noncompliant to compliant vasculature in awake dogs: mechanical impact on right atrial pressure

The distribution of cardiac output between compliant vasculature (e.g., splanchnic organs and skin) and noncompliant vasculature (e.g., skeletal muscle) is proposed to constitute an important determinant of the amount of blood available to the heart (central blood volume and pressure). The aim here was to directly test the hypothesis that diversion of blood flow from a relatively noncompliant vasculature (muscle) to compliant vasculature (splanchnic organs and skin) acts to reduce right atrial pressure. The approach was to inflate an occluder cuff on the terminal aorta for 30 s in one of two modes of ventricular pacing in five awake dogs with atrioventricular block and autonomic blockade. In one trial, cardiac output was maintained constant, meaning cuff inflation caused a portion of terminal aortic flow (a noncompliant circulation) to be diverted to the splanchnic and skin circulations (compliant circulations). In the other trial, arterial pressure was maintained constant, meaning blood flow to these other regions did not change. The response of right atrial pressure (corrected for differences in arterial pressure between the two trials) fit our hypothesis, being lower when blood flow was diverted to compliant regions. We conclude that a small (4% of cardiac output) diversion of blood flow from a noncompliant region to a compliant region reduces right atrial pressure by 0.7 mmHg.     

Model for left ventricular contraction combining the force length velocity relationship with the time varying elastance theory

A model for the contraction of the left ventricle (LV) is developed for a spheroidal geometry. The classical force-length-velocity relationship for a single muscle fiber is assumed. The linear maximum pressure volume relationship (maximum elastance), a measure of muscle contractility, is further extended into a time-varying function. This is achieved by utilizing a mechanical activation function, assumed as half a sinusoidal wave, to describe the time-dependent isometric stress for the activated cardiac muscle. This, in turn, results in the time-varying elastance function and represents the instantaneous activity of the muscle contractile proteins. The model is tested for a set of boundary conditions that determine preload, afterload, and the inherent properties of the muscle, i.e., the contractility. The computed results of the isovolumic contraction, auxotonic contraction, and isovolumic relaxation are in agreement with the expected behavior of the LV. The relations between the simulated variations on preload, afterload, and contractility, and the set of performance indexes of the LV, are presented and discussed.     

The force-velocity relationship for microtubule sliding in demembranated sperm flagella of the sea urchin

We studied the relationship between the force and velocity of microtubule sliding in demembranated sperm flagella of the sea urchin, Hemicentrotus pulcherrimus, under auxotonic conditions following a quick release of the tension between sliding microtubules. The shape of the force-velocity curve was independent of the concentration of Mg-ATP over the range of 3.7 to 350 microM and appeared either linear or was the reverse of the hyperbolic curve seen for muscle. The power, calculated as the product of velocity and force, passed through a peak at c. 0.7 Fmax (the maximal isometric force). Thus, the maximal power is attained at a larger relative load than in muscle. The sliding velocity at 0.1 Fmax showed a hyperbolic dependence on Mg-ATP concentration, with a Km of 210 microM and a Vmax of 19 micron.sec-1. The maximal force did not significantly change over the Mg-ATP concentration range of 3.7 to 350 microM. These results are discussed in terms of a crossbridge model similar to the one originally proposed by Huxley. It is suggested that the dynein crossbridge cycle is characterized by a relatively rapid rate of attachment and a relatively slow rate of detachment.     

Stretching Your Energetic Budget: How Tendon Compliance Affects the Metabolic Cost of Running

Muscles attach to bones via tendons that stretch and recoil, affecting muscle force generation and metabolic energy consumption. In this study, we investigated the effect of tendon compliance on the metabolic cost of running using a full-body musculoskeletal model with a detailed model of muscle energetics. We performed muscle-driven simulations of running at 2–5 m/s with tendon force–strain curves that produced between 1 and 10% strain when the muscles were developing maximum isometric force. We computed the average metabolic power consumed by each muscle when running at each speed and with each tendon compliance. Average whole-body metabolic power consumption increased as running speed increased, regardless of tendon compliance, and was lowest at each speed when tendon strain reached 2–3% as muscles were developing maximum isometric force. When running at 2 m/s, the soleus muscle consumed less metabolic power at high tendon compliance because the strain of the tendon allowed the muscle fibers to operate nearly isometrically during stance. In contrast, the medial and lateral gastrocnemii consumed less metabolic power at low tendon compliance because less compliant tendons allowed the muscle fibers to operate closer to their optimal lengths during stance. The software and simulations used in this study are freely available at simtk.org and enable examination of muscle energetics with unprecedented detail.     

Simultaneous orientation and cellular force measurements in adult cardiac myocytes using three-dimensional polymeric microstructures

A number of techniques have been developed to monitor contractile function in isolated cardiac myocytes. While invaluable observations have been gained from these methodologies in understanding the contractile processes of the heart, they are invariably limited by their in vitro conditions. The present challenge is to develop innovative assays to mimic the in vivo milieu so as to allow a more physiological assessment of cardiac myocyte contractile forces. Here we demonstrate the use of a silicone elastomer, poly(dimethylsiloxane) (PDMS), to simultaneously orient adult cardiac myocytes in primary culture and measure the cellular forces in a three-dimensional substrate. The realignment of adult cardiac myocytes in long-term culture (7 days) was achieved due to directional reassembly of the myofibrils along the parallel polymeric sidewalls. The cellular mechanical forces were recorded in situ by observing the deformation of the micropillars embedded in the substrate. By coupling the cellular mechanical force measurements with on-chip cell orientation, this novel assay is expected to provide a means of a more physiological assessment of single cardiac myocyte contractile function and may facilitate the future development of in vitro assembled functional cardiac tissue.     

Characterization of the inotropic response induced by stimulation of beta-adrenergic and H1 histaminergic receptors in guinea pig left atria.

The inotropic response induced by beta-adrenergic and H1 histaminergic receptor stimulation was characterized in guinea pig left atria by obtaining dose-response relationships for isoproterenol and histamine under various experimental conditions. Conditions (hypothermia, high frequencies of stimulation, and large extracellular calcium concentrations) which enhanced the ability of cardiac muscle to develop force also increased the sensitivity of the left atrium to isoproterenol while decreasing its efficacy. On the other hand, conditions which enhanced the ability of cardiac muscle to develop force depressed the efficacy of histamine to such an extent that the sensitivity to histamine was also decreased. In addition, conditions which markedly depressed the ability of cardiac muscle to develop force also decreased the efficacy and sensitivity to histamine. The data indicate that while beta-adrenoceptor stimulation results in an inotropic response under all conditions studied, stimulation of H1 histaminergic receptors results in an inotropic response only within a narrow range of experimental conditions.