No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
A new method of attachment of isolated mammalian ventricular myocytes for tension recording: Length dependence of passive and active tension
Abstract
The study of the Frank-Starling's law in mammalian single cells has been hindered by a lack of an easily performed method of stretching cells. Some authors have succeeded in this but their methods required a great deal of technical expertise and in most cases they have not had much success. We have developed an easy method of stretching mammalian ventricular cells from slack sarcomere length (S.L.) (Lo, 1.77 +/- 0.05 microns) to about 117% of this length. Thin carbon fibers (12 microns in diameter) which can be bound electrochemically to the cell membrane surface have been used. A flexible long fiber of known compliance (80 microns/microN) was attached to one end of the cell and a stiff double fiber (4 microns/microN) to the other end. The cell attachment was relatively easy to perform and successful results were obtained in 80% of the attempts. The displacement of the flexible fiber allows the quantitative measurements of the resting tension in a group of non-stimulated cells and of auxotonic contractions developed upon stimulation in another group of cells. Increasing S.L. from Lo to 105-106% of Lo, an increase in active tension from 0.21 +/- 0.03 mN/mm to 0.26 +/- 0.01 mN/mm (n = 4) could be noticed with a stimulation frequency of 0.5 Hz. An increase in active tension was also observed at 1 Hz. Staircase kinetics were accelerated with stretching; this confirms at the single cell level the hypothesis of an effect of length-dependent activation on the staircase. Eulerian differential stiffness constant was calculated and found to be 13.5 +/- 1.2, a value which is comparable to that described in intact heart. Thus the important stiffness found in the whole heart may be due to intracellular component(s) such as myofilament and/or connectin.
... We further characterized this contractile dysfunction in PMAD by measuring single-cell force and contractility using the two-carbon-fiber technique. 30 Figure 2A showed an adult left ventricular myocyte before and during contraction in response to electrical stimulation. Figure 2B and C showed the stepwise stretch protocol used to assess myocyte function. ...
... Figure 2C showed carbon fiber bending for the same stretched cell. 30 Corresponding to the derivation by regression of load independent values in vivo, the ESLTR and EDLTR are defined in single cells as the slopes of the linear regressions of systolic and diastolic force (proportional to CF bending; see Methods) and plotted vs. sarcomere length ( Figure 2D, E). ...
... Simultaneous measurements of CICR and force generation allowed us to test whether length-dependent activation, a putative mechanism for the Frank-Starling effect, 30 was impaired in PMAD by comparing contraction velocity immediately before and after stretch ( Figure 3E). Care was taken to equalize baseline and stretched sarcomere lengths. ...
Background:
-Survival after sudden cardiac arrest is limited by post-arrest myocardial dysfunction but understanding of this phenomenon is constrained by lack of data from a physiological model of disease. In this study, we established an in vivo model of cardiac arrest and resuscitation, characterized the biology of the associated myocardial dysfunction, and tested novel therapeutic strategies.
Methods:
-We developed rodent models of in vivo post-arrest myocardial dysfunction using extra-corporeal membrane oxygenation (ECMO) resuscitation followed by invasive hemodynamics measurement. In post-arrest isolated cardiomyocytes, we assessed mechanical load and Ca(2+) induced Ca(2+) release (CICR) simultaneously using the micro-carbon-fiber technique and observed reduced function and myofilament calcium sensitivity. We used a novel-designed fiber optic catheter imaging system, and a genetically encoded calcium sensor GCaMP6f, to image CICR in vivo RESULTS: -We found potentiation of CICR in isolated cells from this ECMO model and also in cells isolated from an ischemia-reperfusion Langendorff model perfused with oxygenated blood from an arrested animal, but not when reperfused in saline. We established that CICR potentiation begins in vivo The augmented CICR observed post-arrest was mediated by the activation of Ca(2+)/calmodulin kinase II (CaMKII). Increased phosphorylation of CaMKII, phospholamban and ryanodine receptor 2 (RyR2) was detected in the post-arrest period. Exogenous adrenergic activation in vivo recapitulated Ca(2+) potentiation but was associated with lesser CaMKII activation. Since oxidative stress and aldehydic adduct formation were high post arrest, we tested a small molecule activator of aldehyde dehydrogenase type 2, Alda-1, which reduced oxidative stress, restored calcium and CaMKII homeostasis, and improved cardiac function and post-arrest outcome in vivo CONCLUSIONS: -Cardiac arrest and reperfusion lead to CaMKII activation and calcium long-term potentiation which support cardiomyocyte contractility in the face of impaired post-ischemic myofilament calcium sensitivity. Alda-1 mitigates these effects, normalizes calcium cycling and improves outcome.
... At that time, the most advanced technique was developed by Garnier and Le Guennec. Using carbon fibers, they established an easy to reproduce, yet efficient, method of attaching and stretching a single intact mammalian cardiac cell 25 . Later, Le Guennec's colleagues employed this technique to simultaneously measure electrical, calcium and mechanical properties of a single intact cell at different cell lengths 26 . ...
... This technique has been used to study LDA in intact myocytes 25,27 . To this aim, special attention to the adhesion efficiency must be considered. ...
... This technique uses glass micro-rods coated with the MyoTak to attach single cardiac cells. When compared to the carbon fibers technique 25 , the results obtained with this technique show higher force recordings 32 . This may be due to the low compliance of glass micro-rods along with better signal processing units used with the MyoTak and the stronger adhesion between the cell membrane and the micro-rods. ...
The heart is subject to multiple sources of stress. To maintain its normal function, and successfully overcome these stresses, heart muscle is equipped with fine-tuned regulatory mechanisms. Some of these mechanisms are inherent within the myocardium itself and are known as intrinsic mechanisms. Over a century ago, Otto Frank and Ernest Starling described an intrinsic mechanism by which the heart, even ex vivo, regulates its function on a beat-to-beat basis. According to this phenomenon, the higher the ventricular filling is, the bigger the stroke volume. Thus, the Frank-Starling law establishes a direct relationship between the diastolic and systolic function of the heart. To observe this biophysical phenomenon and to investigate it, technologic development has been a pre-requisite to scientific knowledge. It allowed for example to observe, at the cellular level, a Frank-Starling like mechanism and has been termed: Length Dependent Activation (LDA).
In this review, we summarize some experimental systems that have been developed and are currently still in use to investigate cardiac biophysical properties from the whole heart down to the single myofibril. As a scientific support, investigation of the Frank-Starling mechanism will be used as a case study.
... In our approach, the AFM is used to apply and measure radial forces at the centre of a single cell. CF is a technique complimentary to AFM: two CF, attached to the ends of single isolated cardiomyocytes, are used for mechanical manipulation and measurement in the axial direction [29][30][31]. Used appropriately, CF are known to not damage the cell [5,32]. Each CF constitutes a cantilever with a known spring constant, giving the possibility to apply and record forces. ...
... The CF technique is described in detail elsewhere [5,35]. In short: to hold the fibres, thin glass capillaries were pulled from borosilicate glass tubes (inner diameter: 1.16 mm, outer diameter: 2.0 mm, Harvard Apparatus, Holliston, MA, USA), and a CF inserted into the fine shaft of the pulled capillary (CF supplied by Jean-Yves Le Guennec [29]). The capillary holding the CF was thermally bent by 30-35°so CF can have near-parallel alignment with the bottom of the perfusion chamber (figure 3a). ...
Cardiomyocytes sense and shape their mechanical environment, contributing to its dynamics by their passive and active mechanical properties. While axial forces generated by contracting cardiomyocytes have been amply investigated, the corresponding radial mechanics remain poorly characterized. Our aim is to simultaneously monitor passive and active forces, both axially and radially, in cardiomyocytes freshly isolated from adult mouse ventricles. To do so, we combine a carbon fibre (CF) set-up with a custom-made atomic force microscope (AFM). CF allows us to apply stretch and to record passive and active forces in the axial direction. The AFM, modified for frontal access to fit in CF, is used to characterize radial cell mechanics. We show that stretch increases the radial elastic modulus of cardiomyocytes. We further find that during contraction, cardiomyocytes generate radial forces that are reduced, but not abolished, when cells are forced to contract near isometrically. Radial forces may contribute to ventricular wall thickening during contraction, together with the dynamic re-orientation of cells and sheetlets in the myocardium. This new approach for characterizing cell mechanics allows one to obtain a more detailed picture of the balance of axial and radial mechanics in cardiomyocytes at rest, during stretch, and during contraction.
This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’.
... For instance, from the FE-analysis we can obtain huge data about cardiac motion, especially stress and strain of the heart that are two of the most important determinants of many cardiac physiological and pathophysiological functions. These functions include: the pumping performance of the ventricles; the oxygen demand of the myocardium; the distribution of coronary blood flow; the vulnerability of the regions to ischemia and infarction diseases; and the risk of arrhythmia [2]. ...
... Nonlinear curve fitting and optimization using MATLAB tool box and MARC were conducted to determine model's constants that characterize the nonlinear elastic and viscous behavior of the heart tissues. The generalized strain energy function versus time data being used for large strain viscoelasticty was generated by using our experimental data; and our data mixed with equi-biaxial tension and volumetric compression (existing in [2], [3] ) to determine constants of the myocardium free energy function. However, stress relaxation tests at multiple levels of strains were conducted to generate the relaxation function. ...
... Le Guennec et al. (1990) introduced the etched carbon fiber attachment approach to study the Frank-Starling's law in Fig. 9 Two micropipettes in a chamber. A pneumatic micromanipulator controls the movement of a micropipette along three orthogonal axes. ...
... More recently, by combining the efforts of Le Guennec et al. (1990) and Yasuda et al. (2001), as well as the earlier works of Iwazumi (1987), Garcia-Webb et al. (2007) described the design and development of a modular (and inexpensive) instrument for exploring the mechanics of cardiomyocytes using carbon fiber (see Fig. 10). Their goal was to design a system capable of not only quantifying the mechanical properties of individual cardiomyocytes, but that was also appropriate for use in an instrument array setting. ...
The use of enzymatically isolated cardiac myocytes is ubiquitous in modern cardiovascular research. Parallels established between cardiomyocyte shortening responses and those of intact tissue make the cardiomyocyte an invaluable experimental model of cardiac function. Much of our understanding regarding the fundamental processes underlying heart function is owed to our increasing capabilities in single-cell stimulation and direct or indirect observation, as well as quantitative analysis of such cells. Of the many important mechanisms and functions that can be readily assessed in cardiomyocytes at all stages of development, contractility is the most representative and one of the most revealing. The purpose of this review is to provide a survey of various methodological approaches in the literature used to assess adult and neonatal cardiomyocyte contractility. The various methods employed to evaluate the contractile behavior of enzymatically isolated mammalian cardiac myocytes can be conveniently divided into two general categories—those employing optical (image)-based systems and those that use transducer-based technologies. This survey is by no means complete, but we have made an effort to include the most popular methods in terms of reliability and accessibility. These techniques are in constant evolution and hold great promise for the next generation of breakthrough studies in cell biology for the prevention, treatment, and cure of cardiovascular diseases.
... Axial stretch can be applied to single myocytes using a range of techniques to attach probes for mechanical stimulation. One approach is to use carbon fibres, which adhere to single cells without the need for gluing, tying, or suction [22]. Using this technique, Cooper et al. observed a significant increase in BR of rabbit single SAN cells during 5-10% stretch. ...
Cardiac electrical and mechanical activity are closely interrelated, not only via the chain of events commonly referred to as ‘excitation-contraction coupling’ that links electrical excitation to contraction, but equally via feedback from the heart’s mechanical environment to the origin and spread of cardiac excitation. The latter has been termed mechano-electric coupling and complements excitation-contraction coupling to form an intracardiac electro-mechanical regulatory loop. This chapter will explore the relevance of mechano-electric coupling in the heart by reviewing its pro- and anti-arrhythmic effects on heart rate and rhythm, and the underlying mechanisms that may account for clinical and experimental observations.
... Given the technical challenge to stretch single intact cardiac myocytes, for example, using carbon fibers (Le Guennec et al., 1990), studies at the tissue and organ level are useful and allow the study of interactions between cell types. Purkinje fibres are a known culprit for ventricular arrhythmia (Haissaguerre et al., 2016). ...
... The usage and limitations of various techniques for applying mechanical loading at the single-cell level have been analyzed in a review paper (Chen- Izu and Izu, 2017). One technique is to use a pair of micro-cantilevers (i.e., carbon fibers, glass fibers, etc.) attached to the cell surface to stretch the rod-shaped cardiomyocyte along the long axis (Le Guennec et al., 1990;Alvarez et al., 1999;Calaghan and White, 2004;Iribe et al., 2009;Prosser et al., 2011). Such a one-dimensional stretching method applies longitudinal tension on the cell but no transverse compression or surface traction on lateral surfaces. ...
The heart pumps blood into circulation against vascular resistance and actively regulates the contractile force to compensate for mechanical load changes. Our experimental data show that cardiomyocytes have a mechano-chemo-transduction (MCT) mechanism that increases intracellular Ca2+ transient to enhance contractility in response to increased mechanical load. This study advances the cardiac excitation-Ca2+ signaling-contraction (E-C) coupling model on conceptual and technical fronts. First, we developed analytical and computational models to perform 3-dimensional mechanical analysis of cardiomyocytes contracting in a viscoelastic medium under mechanical load. Next, we proposed an MCT feedback loop in the E-C coupling dynamic system to shift the feedforward paradigm of cardiac E-C coupling to an autoregulation model. Our combined modeling and experimental studies reveal that MCT enables autoregulation of E-C coupling and contractility in single cardiomyocytes, which underlies the heart’s intrinsic autoregulation in compensatory response to load changes in order to maintain the stroke volume and cardiac output.
... This stems largely from the specific challenges of noninjurious cell attachment and performing small-force measurement on intact single cells (Brady, 1991). More recently, technical developments (e.g., involving the use of flexible carbon fibers to hold the cells at opposite ends; Iribe et al., 2007;Le Guennec et al., 1990;Yasuda et al., 2001) have made these measurements more practicable. Despite these advances, however, only a fraction of studies in the literature have systematically made direct comparisons between skinned and intact systems taken from the same species under optimally similar conditions (see the selection listed in Table 1). ...
Myofilaments and their associated proteins, which together constitute the sarcomeres, provide the molecular-level basis for contractile function in all muscle types. In intact muscle, sarcomere-level contraction is strongly coupled to other cellular subsystems, in particular the sarcolemmal membrane. Skinned muscle preparations (where the sarcolemma has been removed or permeabilized) are an experimental system designed to probe contractile mechanisms independently of the sarcolemma. Over the last few decades, experiments performed using permeabilized preparations have been invaluable for clarifying the understanding of contractile mechanisms in both skeletal and cardiac muscle. Today, the technique is increasingly harnessed for preclinical and/or pharmacological studies that seek to understand how interventions will impact intact muscle contraction. In this context, intrinsic functional and structural differences between skinned and intact muscle pose a major interpretational challenge. This review first surveys measurements that highlight these differences in terms of the sarcomere structure, passive and active tension generation, and calcium dependence. We then highlight the main practical challenges and caveats faced by experimentalists seeking to emulate the physiological conditions of intact muscle. Gaining an awareness of these complexities is essential for putting experiments in due perspective.
... With enough stretching, a maximum output volume is eventually reached [8,9]. The length-tension relationship describes that when myofibrils are close to an optimal length, also called the resting length (Lmax), maximum active tension and force is achieved [10,11]. This relationship is much steeper in cardiac than skeletal muscle due to the increase in calcium sensitivity [12][13][14][15]. ...
Little has been reported on the left ventricular myocardial distension (bounce) and its utility to assess cardiac function. The purpose of this study is to determine whether myocardial bounce at end diastole is reproducibly visualized by blinded observers and to determine whether it corresponds to systolic and diastolic function. 144 Consecutive cardiac MR exams between September and December 2017 were selected for analysis. The bounce was graded by two blinded observers, and the change in LV diameter pre and post bounce was measured. The bounce was defined as the rapid change in LV volume that occurs at the end of diastole during atrial contraction just prior to systolic ejection. Inter-reader agreement was summarized using Cohen’s kappa. Spearman’s rank correlation coefficient was used to evaluate associations between bounce grade and cardiac physiology parameters. Overall agreement was good with unweighted kappa = 0.69 (95% CI 0.60–0.79). Bounce grade was significantly correlated with the average change in LV diameter before and after the bounce (Spearman’s rho = 0.76, p < 0.001). Median diameter changes were 0.0, 1.9, and 4.2 mm in grades 0 (no bounce), 1 (small bounce), and 2 (normal), respectively. The bounce lasted 8 to 12 ms in all patients. Bounce grade was significantly correlated with LV EF (Spearman’s rho = 0.43, p < 0.001). Median EF was 44%, 51%, and 58% in grades 0, 1, and 2, respectively. Of the 87 patients who had E/A ratio or E/e′ ratio measured, bounce grade was also significantly correlated with E/A ratio (r = − 0.24, p = 0.034) and E/e′ ratio (r = − 0.24, p = 0.022), with lower grades having higher ratio values on average (Table 4). Of the 15 patients with a bounce grade of 0 by one or both readers and EF ≥ 50%, 8 had E/A ratio measurements and 7 had E/e′ ratio measurements. The E/A ratio values ranged from 1 to 2.7 (median 1.5). The E/e′ ratio values ranged from 4.8 to 9.6 (median 7.7). The simple observation of a normal myocardial bounce during cine loop review of cardiac MR exams was predictive of normal diastolic and systolic cardiac function. Lack of myocardial bounce was highly associated with both systolic and diastolic dysfunction. The subpopulation of patients with loss of myocardial bounce and normal ejection fraction appear to represent patients with early diastolic dysfunction. Further studies with more diastolic dysfunction MRs are needed to examine this relationship. This study suggests changes to the myocardial bounce seen on cardiac MR may be a simple useful tool for detecting cardiac dysfunction. This study is not to replace, but rather aid the clinical diagnosis and management of both diastolic and systolic dysfunction.
... We developed a method that can overcome these limitations by directly measuring the forces generated from single-cell hPSC-CMs. To this end, we modified a technique first used (Le Guennec et al., 1990) for quantifying forces of primary rodent cardiomyocytes. This technique involves the attachment of an intact rodent cardiomyocyte at its distal ends to carbon fibers (Sugiura et al., 2006). ...
Current platforms for studying the mechanical properties of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) as single cells do not measure forces directly, require numerous assumptions, and cannot study cell mechanics at different loading conditions. We present a method for directly measuring the active and passive forces generated by single-cell hPSC-CMs at different stretch levels. Utilizing this technique, single hPSC-CMs exhibited positive length-tension relationship and appropriate inotropic, klinotropic, and lusitropic changes in response to pharmacological treatments (isoproterenol and verapamil). The unique potential of the approach for drug testing and disease modeling was exemplified by doxorubicin and omecamtiv mecarbil drug studies revealing their known actions to suppress (doxorubicin) or augment (omecamtiv mecarbil at low dose) cardiomyocyte contractility, respectively. Finally, mechanistic insights were gained regarding the cellular effects of these drugs as doxorubicin treatment led to cellular mechanical alternans and high doses of omecamtiv mecarbil suppressed contractility and worsened the cellular diastolic properties.
... Furthermore, complex stretching patterns have been proposed to mimic in vivo tissue contraction. 47 Several methods can be used to apply mechanical stimulation to cells: axial stretch using carbon fibers, 48 unidirectional stretch using an elastic substrate, 46 and biaxial stretch of elastic substrate. 49,50 While biaxial stretch is the most physiologically relevant deformation, uniaxial stretch implemented in our prototype remains interesting but can be limited in part by its Poisson contraction. ...
A new open-hardware bioreactor capable of applying electrical field stimulation in conjunction with static or cyclic stretch is presented. Stretch is applied to cells by a specially designed elastomeric membrane with a central seeding region. The main interest of our approach is the fine control of the characteristics of stimulations in regard to timing and amplitude in a simple design based on affordable, easy to find components and 3D printable parts. Our approach opens the way to more complex protocols for electrical and/or mechanical stimulations, which are known important regulators of cardiac phenotypes.
... The mechanical response in isolated cardiomyocyte was measured by a carbon fiber technique that was introduced ~30 years ago (Le Guennec et al., 1990) and that is now extensively used (White et al., 1995;Hongo et al., 1996;Iribe et al., 2007;Prosser et al., 2013;Fowler et al., 2015;Helmes et al., 2016;Yamaguchi et al., 2017). The conventional method of two carbon fibers used in this study allowed sarcomere stretch by ~8%. ...
Length-dependent activation (LDA) of contraction is an important mechanism of proper myocardial function that is often blunted in diseases accompanied by deficient contractility and impaired calcium homeostasis. We evaluated how the extent of LDA is related to the decreased force in healthy rat myocardium under negative inotropic conditions that affect the calcium cycle. The length-dependent effects on auxotonic twitch and Ca-transient were compared in isolated rat ventricular cardiomyocytes at room temperature (“25C”) and near-physiological temperature (“35C”) in normal Tyrode and at 25°C with thapsigargin-depleted sarcoplasmic reticulum (“25C + Thap”). At the slack length, a similar negative inotropy in “35C” and “25C + Thap” was accompanied by totally different changes in Ca-transient amplitude, time-to-peak, and time-to-decline from peak to 50% amplitude. End-systolic/end-diastolic tension-sarcomere length relationships were obtained for each individual cell, and the ratio of their slopes, the dimensionless Frank-Starling Gain index, was 2.32 ± 0.16, 1.78 ± 0.09, and 1.37 ± 0.06 in “25C,” “35C” and “25C + Thap,” respectively (mean ± S.E.M.). Ca-transient diastolic level and amplitude did not differ between “25C” and “35C” at any SL, but in “35C” it developed and declined significantly faster. In contrast, thapsigargin-induced depletion of SERCA2a significantly attenuated and retarded Ca-transient. The relative amount of Ca²⁺ utilized by troponin C, evaluated by the integral magnitude of a short-lived component of Ca-transient decline (“bump”), increased by ~25% per each 0.05 μm increase in SL in all groups. The kinetics of the Ca-TnC dissociation, evaluated by the bump time-to-peak, was significantly faster in “35C” and slower in “25C + Thap” vs. “25C” (respectively, 63.7 ± 5.3 and 253.6 ± 8.3% of the value in “25C,” mean ± S.E.M.). In conclusion, a similar inotropic effect can be observed in rat ventricular myocardium under totally different kinetics of free cytosolic calcium. The extent of LDA is not determined by actual peak systolic tension but is regulated by the level of peak systolic calcium and the kinetics of Ca-transient decline which, in turn, are governed by Ca-TnC dissociation and Ca²⁺ reuptake by the sarcoplasmic reticulum. Altogether, these findings constitute new evidence about the role of the length-dependent modulation of Ca²⁺ homeostasis in the mechanisms of calcium regulation of contraction and mechano-calcium feedback in the myocardium.
... Several methods were developed to study the response of single cardiac cells to applied load and to elucidate the underlying mechano-chemo-transduction mechanism. These methods include stretching a substrate on top of which cells are attached (reviewed in Quinn and Kohl, 2012), using carbon fibers attached to both ends of a cardiac cell (Le Guennec et al., 1990;Cooper et al., 2000;Prosser et al., 2011) and a "cell-in-a-gel" system whereby isolated cardiomyocytes contract against an elastic three dimensional matrix (Jian et al., 2014). Most of these studies used spontaneously beating cardiac cells with no electrical stimulation. ...
Cardiac cells are subjected to mechanical load during each heart-beat. Normal heart load is essential for physiological development and cardiac function. At the same time, excessive load can induce pathologies such as cardiac hypertrophy. While the forces working on the heart as an organ are well-understood, information regarding stretch response at the cellular level is limited. Since cardiac stretch-response depends on the amplitude and pattern of the applied load as well as its timing during the beating cycle, the directionality of load application and its phase relative to action potential generation must be controlled precisely. Here, we design a new experimental setup, which enables high-resolution fluorescence imaging of cultured cardiac cells under cyclic uniaxial mechanical load and electrical stimulation. Cyclic stretch was applied in different phases relative to the electrical stimulus and the effect on cardiac cell beating was monitored. The results show a clear phase-dependent response and provide insight into cardiac response to excessive loading conditions.
... This approach allows attachment of carbon fibers of known stiffness to both ends of a cardiomyocyte. CFbased stretching gives rise to a relatively homogeneous increase in sarcomere length (SL) and allows calculation of passive and active forces from optically monitored changes in CF bending [9]. Adapted from [8]. ...
The technologies to study cardiac cell mechanics in near-physiological conditions are limited. Carbon fiber (CF) technique is a unique tool to study single cardiomyocyte contractility. However, the CF adhesion to a cell is limited and it is difficult to control CF sliding occurred due to inappropriate adhesion. In this study, we present a CF adhesion quality index – a linear coefficient (slope) derived from "end-diastolic cell length - end-diastolic sarcomere length" relationship. Potential applicability of this index is demonstrated on isolated rat and guinea pig ventricular cardiomyocytes. Further improvement of the approach may help to increase the quality of the experimental data obtained by CF technique.
... Due to this, drug effects that depend on, or are modulated by, the mechanical environment may be missed in standard in vitro studies. The 2-carbon fibre (CF) technique (Le Guennec et al., 1990) offers an experimental approach to controlling the mechanical environment of isolated cells. It can be used therefore to explore drug effects under different mechanical loads. ...
The apelin peptide is described as one of the most potent inotropic agents, produced endogenously in a wide range of cells, including cardiomyocytes. Despite positive effects on cardiac contractility in multicellular preparations, as well as indications of cardio-protective actions in several diseases, its effects and mechanisms of action at the cellular level are incompletely understood.
Here, we report apelin effects on dynamic mechanical characteristics of single ventricular cardiomyocytes, isolated from mouse models (control, apelin-deficient [Apelin-KO], apelin-receptor KO mouse [APJ-KO]), and rat. Dynamic changes in maximal velocity of cell shortening and relaxation were monitored. In addition, more traditional indicators of inotropic effects, such as maximum shortening (in mechanically unloaded cells) or peak force development (in auxotonic contracting cells, preloaded using the carbon fibre technique) were studied.
The key finding is that, using Apelin-KO cardiomyocytes exposed to different preloads with the 2-carbon fibre technique, we observe a lowering of the slope of the end-diastolic stress-length relation in response to 10 nM apelin, an effect that is preload-dependent. This suggests a positive lusitropic effect of apelin, which could explain earlier counter-intuitive findings on an apelin-induced increase in contractility occurring without matching rise in oxygen consumption.
... The most common approach to subject a single myocyte to preload is stretching the cell in longitudinal direction with a pair of carbon fibers or glass rods attached at both cell ends. The carbon fiber technique was introduced by Le Guennec et al. in the late 1980s (Le Guennec et al., 1990). The attachment is presumably due to electrostatic forces between the carbon fiber and myocyte surfaces (Yasuda et al., 2001). ...
In cardiac myocytes, calcium (Ca²⁺) signalling is tightly controlled in dedicated microdomains. At the dyad, i.e. the narrow cleft between t-tubules and junctional sarcoplasmic reticulum (SR), many signalling pathways combine to control Ca²⁺-induced Ca²⁺ release during contraction. Local Ca²⁺ gradients also exist in regions where SR and mitochondria are in close contact to regulate energetic demands. Loss of microdomain structures, or dysregulation of local Ca²⁺ fluxes in cardiac disease, is often associated with oxidative stress, contractile dysfunction and arrhythmias. Ca²⁺ signalling at these microdomains is highly mechanosensitive. Recent work has demonstrated that increasing mechanical load triggers rapid local Ca²⁺ releases that are not reflected by changes in global Ca²⁺. Key mechanisms involve rapid mechanotransduction with reactive oxygen species or nitric oxide as primary signalling molecules targeting SR or mitochondria microdomains depending on the nature of the mechanical stimulus. This review summarizes the most recent insights in rapid Ca²⁺ microdomain mechanosensitivity and re-evaluates its (patho)physiological significance in the context of historical data on the macroscopic role of Ca²⁺ in acute force adaptation and mechanically-induced arrhythmias. We distinguish between preload and afterload mediated effects on local Ca²⁺ release, and highlight differences between atrial and ventricular myocytes. Finally, we provide an outlook for further investigation in chronic models of abnormal mechanics (eg post-myocardial infarction, atrial fibrillation), to identify the clinical significance of disturbed Ca²⁺ mechanosensitivity for arrhythmogenesis.
... Few studies have quantified the stretch imparted upon patch-clamped membranes during SAC investigations. 216 At the cell and tissue level, cell or sarcomere length are used as a readout of strain, 217 and occasionally as an input parameter to gauge mechanical modulation of cardiomyocytes. 24,218 In native heart, implanted ultrasonic transducers, 219 echo, and magnetic resonance imaging 220 have all been applied to characterize transmural deformation patterns. ...
Mechanical forces will have been omnipresent since the origin of life, and living organisms have evolved mechanisms to sense, interpret, and respond to mechanical stimuli. The cardiovascular system in general, and the heart in particular, is exposed to constantly changing mechanical signals, including stretch, compression, bending, and shear. The heart adjusts its performance to the mechanical environment, modifying electrical, mechanical, metabolic, and structural properties over a range of time scales. Many of the underlying regulatory processes are encoded intracardially and are, thus, maintained even in heart transplant recipients. Although mechanosensitivity of heart rhythm has been described in the medical literature for over a century, its molecular mechanisms are incompletely understood. Thanks to modern biophysical and molecular technologies, the roles of mechanical forces in cardiac biology are being explored in more detail, and detailed mechanisms of mechanotransduction have started to emerge. Mechano-gated ion channels are cardiac mechanoreceptors. They give rise to mechano-electric feedback, thought to contribute to normal function, disease development, and, potentially, therapeutic interventions. In this review, we focus on acute mechanical effects on cardiac electrophysiology, explore molecular candidates underlying observed responses, and discuss their pharmaceutical regulation. From this, we identify open research questions and highlight emerging technologies that may help in addressing them.
... In this work, divalent cation entry has been explored in isolated ventricular Wild Type (WT) and mdx cardiomyocytes in two different conditions: at rest and during the application of an axial stretch. Ventricular cardiomyocytes have been stretched using a carbon microfibers technique [50,51], which has the advantage to achieve an almost homogenous lengthening of the sarcomeres and to best mimic the effect of diastolic filling in physiological conditions [52]. Moreover, the involvement of other channels, as store-operated channels, in the observed Ca 2+ influx has been investigated with the use of channels blockers. ...
... The carbon fiber technique allows a controlled axial stretch of the cell and was first developed on cardiomyocytes (Le Guennec et al., 1990). Carbon fibers are attached to the cell membrane via electrostatic forces (the same forces that seal the patch-clamp pipette to the membrane). ...
Plants, like other organisms, are facing multiple mechanical constraints generated both in their tissues and by the surrounding environments. They need to sense and adapt to these forces throughout their lifetimes. To do so, different mechanisms devoted to force transduction have emerged. Here we focus on fascinating proteins: the mechanosensitive (MS) channels. Mechanosensing in plants has been described for centuries but the molecular identification of MS channels occurred only recently. This review is aimed at plant biologists and plant biomechanists who want to be introduced to MS channel identity, how they work and what they might do in planta? In this review, electrophysiological properties, regulations, and functions of well-characterized MS channels belonging to bacteria and animals are compared with those of plants. Common and specific properties are discussed. We deduce which tools and concepts from animal and bacterial fields could be helpful for improving our understanding of plant mechanotransduction. MS channels embedded in their plasma membrane are sandwiched between the cell wall and the cytoskeleton. The consequences of this peculiar situation are analyzed and discussed. We also stress how important it is to probe mechanical forces at cellular and subcellular levels in planta in order to reveal the intimate relationship linking the membrane with MS channel activity. Finally we will propose new tracks to help to reveal their physiological functions at tissue and plant levels.
... A number of methods for mechanical stimulation have been developed, in a preparation-dependent manner. For single isolated cells, stretch can be achieved by attaching carbon fibres (Le Guennec et al., 1990;Le Guennec et al., 1991) to both ends of a cell and stretching the cell by increasing the distance between the fibres Iribe et al., 2009), potentially even mimicking intra-cardiac force-length loop behaviour (Iribe et al., 2007;Bollensdorff et al., 2011). Single cardiomyocytes can also be mechanically manipulated by a pair of glass pipettes (Tung and Zou, 1995;Zeng et al., 2000), a glass pipette and a glass stylus (Kamkin et al., 2000), or be glued to a pair of glass rods (Prosser et al., 2011). ...
Living cardiac tissue slices, a pseudo two-dimensional (2D) preparation, have received less attention than isolated single cells, cell cultures, or Langendorff-perfused hearts in cardiac biophysics research. This is, in part, due to difficulties associated with sectioning cardiac tissue to obtain live slices. With moderate complexity, native cell-types, and well-preserved cell-cell electrical and mechanical interconnections, cardiac tissue slices have several advantages for studying cardiac electrophysiology. The trans-membrane potential (Vm) has, thus far, mainly been explored using multi-electrode arrays. Here, we combine tissue slices with optical mapping to monitor Vm and intracellular Ca2+ concentration ([Ca2+]i). This combination opens up the possibility of studying the effects of experimental interventions upon action potential (AP) and calcium transient (CaT) dynamics in 2D, and with relatively high spatio-temporal resolution.
... For example, Kamkin applied local stretches to ventricular myocytes by pulling with a glass stylus and patch-pipette (Kamkin et al., 2000), while Zeng and Riemer used a pair of suction pipettes to pull the myocyte from each end (Riemer and Tung, 2003;Zeng et al., 2000). A totally different technique was introduced by Le Guennec (Le Guennec et al., 1990) in which a pair of thin carbon fibers was attached to the myocyte surface, likely because of electrostatic forces between the fibers and the surface (Garnier, 1994). This technique was later modified with the use of graphite-reinforced carbon (GRC; Tsukuba Material Information Laboratory Ltd, Tsukuba, Japan) fiber (Sugiura et al., 2006) and/or biocompatible adhesive (MyoTak; IonOptix, Milton, MA, USA; or World Precision Instruments Inc., Sarasota, FL, USA) (Khairallah et al., 2012;Prosser et al., 2011) to enable firmer attachment between the fibers and the cellular surface, and was used for the dynamic stretching of single cardiomyocytes (Nishimura et al., 2006a(Nishimura et al., , 2006bSeo et al., 2014). ...
Stretch-induced arrhythmias are multi-scale phenomena in which alterations in channel activities and/or calcium handling lead to the organ level derangement of the heart rhythm. To understand how cellular mechano-electric coupling (MEC) leads to stretch-induced arrhythmias at the organ level, we developed stretching devices and optical voltage/calcium measurement techniques optimized to each cardiac level. This review introduces these experimental techniques of (1) optical voltage measurement coupled with a carbon-fiber technique for single isolated cardiomyocytes, (2) optical voltage mapping combined with motion tracking technique for myocardial tissue/whole heart preparations and (3) real-time calcium imaging coupled with a laser optical trap technique for cardiomyocytes. Following the overview of each methodology, results are presented. We conclude that individual MEC in cardiomyocytes can be heterogeneous at the ventricular level, especially when moderate amplitude mechanical stretches are applied to the heart, and that this heterogeneous MEC can evoke focal excitation that develops into re-entrant arrhythmias.
... Currently, little is known about force production in single developing cardiomyocytes, while mature cardiomyocytes have been previously studied (Sivaramakrishnan et al. 2009; Bollensdorff et al. 2011; Curtis and Russell 2011). Their contractile forces have been measured by suspending single, isolated cardiomyocytes between pairs of carbon fibers (Le Guennec et al. 1990; Yasuda et al. 2001; Iribe et al. 2007), glass micropipettes (Brady et al. 1979; Fabiato 1981), or micromachined silicon grippers (Lin et al. 1995) that deflect during contraction; the resulting deflections can be used to calculate applied contractile forces. The two-point carbon fiber technique has been used with displacement feedback to perform both isometric and auxotonic contractile force measurements of adult rat cardiomyocytes (Yasuda et al. 2001; Nishimura et al. 2004; Iribe et al. 2007), leading to the measurement of peak contractile forces in the range of 0.7–12.6 ...
Immature primary and stem cell-derived cardiomyocytes provide useful models for fundamental studies of heart development and cardiac disease, and offer potential for patient specific drug testing and differentiation protocols aimed at cardiac grafts. To assess their potential for augmenting heart function, and to gain insight into cardiac growth and disease, tissue engineers must quantify the contractile forces of these single cells. Currently, axial contractile forces of isolated adult heart cells can only be measured by two-point methods such as carbon fiber techniques, which cannot be applied to neonatal and stem cell-derived heart cells because they are more difficult to handle and lack a persistent shape. Here we present a novel axial technique for measuring the contractile forces of isolated immature cardiomyocytes. We overcome cell manipulation and patterning challenges by using a thermoresponsive sacrificial support layer in conjunction with arrays of widely separated elastomeric microposts. Our approach has the potential to be high-throughput, is functionally analogous to current gold-standard axial force assays for adult heart cells, and prescribes elongated cell shapes without protein patterning. Finally, we calibrate these force posts with piezoresistive cantilevers to dramatically reduce measurement error typical for soft polymer-based force assays. We report quantitative measurements of peak contractile forces up to 146 nN with post stiffness standard error (26 nN) far better than that based on geometry and stiffness estimates alone. The addition of sacrificial layers to future 2D and 3D cell culture platforms will enable improved cell placement and the complex suspension of cells across 3D constructs.
... Techniques used to apply axial stretch to intact isolated cardiomyocytes include distension of agarose gels with embedded cells [39] or strain application to single cells using suction pipettes [6], adhesives [5], or carbon fibres (CF) which can be attached to cells glue-free [31]. The CF technique in particular has been developed to a point where it can be used to prescribe to single cells a stress-strain environment that mimics relevant aspects of in situ dynamics, such as by exposing cells to work-loop-like contractions (see black traces in Fig. 1; see also [23,36]). ...
This paper briefly recapitulates the Frank–Star-ling law of the heart, reviews approaches to establishing diastolic and systolic force–length behaviour in intact isolated cardiomyocytes, and introduces a dimensionless index called 'Frank–Starling Gain', calculated as the ratio of slopes of end-systolic and end-diastolic force–length relations. The benefits and limitations of this index are illustrated on the example of regional differences in Guinea pig intact ventricular cardiomyocyte mechanics. Potential applicability of the Frank–Starling Gain for the comparison of cell contractility changes upon stretch will be discussed in the context of intra-and inter-individual variability of cardiomyocyte properties.
The first of its kind, this comprehensive resource integrates cellular mechanobiology with micro-nano techniques to provide unrivalled in-depth coverage of the field, including state-of-the-art methods, recent advances, and biological discoveries. Structured in two parts, the first part offers detailed analysis of innovative micro-nano techniques including FRET imaging, electron cryo-microscopy, micropost arrays, nanotopography devices, laser ablation, and computational image analysis. The second part of the book provides valuable insights into the most recent technological advances and discoveries in areas such as stem cell, heart, bone, brain, tumor, and fibroblast mechanobiology. Written by a team of leading experts and well-recognised researchers, this is an essential resource for students and researchers in biomedical engineering.
The first of its kind, this comprehensive resource integrates cellular mechanobiology with micro-nano techniques to provide unrivalled in-depth coverage of the field, including state-of-the-art methods, recent advances, and biological discoveries. Structured in two parts, the first part offers detailed analysis of innovative micro-nano techniques including FRET imaging, electron cryo-microscopy, micropost arrays, nanotopography devices, laser ablation, and computational image analysis. The second part of the book provides valuable insights into the most recent technological advances and discoveries in areas such as stem cell, heart, bone, brain, tumor, and fibroblast mechanobiology. Written by a team of leading experts and well-recognised researchers, this is an essential resource for students and researchers in biomedical engineering.
The first of its kind, this comprehensive resource integrates cellular mechanobiology with micro-nano techniques to provide unrivalled in-depth coverage of the field, including state-of-the-art methods, recent advances, and biological discoveries. Structured in two parts, the first part offers detailed analysis of innovative micro-nano techniques including FRET imaging, electron cryo-microscopy, micropost arrays, nanotopography devices, laser ablation, and computational image analysis. The second part of the book provides valuable insights into the most recent technological advances and discoveries in areas such as stem cell, heart, bone, brain, tumor, and fibroblast mechanobiology. Written by a team of leading experts and well-recognised researchers, this is an essential resource for students and researchers in biomedical engineering.
The first of its kind, this comprehensive resource integrates cellular mechanobiology with micro-nano techniques to provide unrivalled in-depth coverage of the field, including state-of-the-art methods, recent advances, and biological discoveries. Structured in two parts, the first part offers detailed analysis of innovative micro-nano techniques including FRET imaging, electron cryo-microscopy, micropost arrays, nanotopography devices, laser ablation, and computational image analysis. The second part of the book provides valuable insights into the most recent technological advances and discoveries in areas such as stem cell, heart, bone, brain, tumor, and fibroblast mechanobiology. Written by a team of leading experts and well-recognised researchers, this is an essential resource for students and researchers in biomedical engineering.
The first of its kind, this comprehensive resource integrates cellular mechanobiology with micro-nano techniques to provide unrivalled in-depth coverage of the field, including state-of-the-art methods, recent advances, and biological discoveries. Structured in two parts, the first part offers detailed analysis of innovative micro-nano techniques including FRET imaging, electron cryo-microscopy, micropost arrays, nanotopography devices, laser ablation, and computational image analysis. The second part of the book provides valuable insights into the most recent technological advances and discoveries in areas such as stem cell, heart, bone, brain, tumor, and fibroblast mechanobiology. Written by a team of leading experts and well-recognised researchers, this is an essential resource for students and researchers in biomedical engineering.
The first of its kind, this comprehensive resource integrates cellular mechanobiology with micro-nano techniques to provide unrivalled in-depth coverage of the field, including state-of-the-art methods, recent advances, and biological discoveries. Structured in two parts, the first part offers detailed analysis of innovative micro-nano techniques including FRET imaging, electron cryo-microscopy, micropost arrays, nanotopography devices, laser ablation, and computational image analysis. The second part of the book provides valuable insights into the most recent technological advances and discoveries in areas such as stem cell, heart, bone, brain, tumor, and fibroblast mechanobiology. Written by a team of leading experts and well-recognised researchers, this is an essential resource for students and researchers in biomedical engineering.
The first of its kind, this comprehensive resource integrates cellular mechanobiology with micro-nano techniques to provide unrivalled in-depth coverage of the field, including state-of-the-art methods, recent advances, and biological discoveries. Structured in two parts, the first part offers detailed analysis of innovative micro-nano techniques including FRET imaging, electron cryo-microscopy, micropost arrays, nanotopography devices, laser ablation, and computational image analysis. The second part of the book provides valuable insights into the most recent technological advances and discoveries in areas such as stem cell, heart, bone, brain, tumor, and fibroblast mechanobiology. Written by a team of leading experts and well-recognised researchers, this is an essential resource for students and researchers in biomedical engineering.
The study of cardiac function is important issue in physiological and biological sciences and has relevant implications for clinical practice. The interpretation of the whole-heart experiments is complicated by multi-cellular interactions. This work is devoted to the development of biomechanical tests for studying force-length and passive viscoelastic properties of intact single cardiac muscle cells using a glue-free carbon fiber technique. The developed tests allow analyzing the passive force-length, total force-length and active force-length relations in a beating cardiomyocyte by applying the step stretch protocol. The protocols also can be used to investigate in detail the viscoelastic hysteresis applying sawtooth stretch and release commands on a resting cell. The tracking of viscoelastic hysteresis allows one to study not only changes in the passive tension amplitude, but also the delay between the micromanipulator movement and the cell response or energy dissipation specifying viscoelastic properties. Thus, the developed biomechanical tests are powerful research tools to study cardiac biomechanics at the cell level.
Computer-controlled biomechanical experiments on single cells require high accuracy and synchronicity of signals and stability of experimental set-ups. In this paper, we propose a digital micromanipulator network consisting of two micromanipulators to study single cardiac cell mechanics. The micromanipulation system using Ethernet for this network has a large positioning range (20 mm), covering the experimental bath with the cells and nanometer movement resolution (5 nm) for precise experiments. The software for the micromanipulator network was developed in the LabVIEW to provide sufficient synchronicity of micromanipulator movement. The testing of seven consistent steps of two micromanipulators showed that movement was synchronous and maximum loss of synchrony of coordinates was 170 nm. We believe that studies on the changes in single cardiac cell length and force under various mechanical loads can be carried out using the developed network.
This paper reports on a method for evaluating the force-length relationship of adhering human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on the substrate using a measurement system comprising of a micromachined movable plate and a piezoresistive force probe. The cells on the plate are stretched by pushing the movable plate with the piezoresistive cantilever, which is actuated by a piezo stage. The twitch forces and the applied stretch are measured quantitatively with the piezoresistive cantilever. The results demonstrated that the twitch forces of the hiPSC-CMs increased when a stretch was applied. This evaluation method improves the understanding of the intrinsic force-length relationship of hiPSC-CMs at the cellular scale.
The externally homogeneous mechanical activity of the heart results from the integration of the heterogeneous activity of the cardiomyocytes. Therefore, in order to understand the integrated mechanical function of the heart, it is important to study the regional single cell mechanics. Cardiac left ventricular pressure-volume relationship has been commonly used to evaluate the cardiac performance. In physiological contractions, pressure-volume relation curve for one beat forms a rectangular work-loop that is divided into four phases: isovolumic contraction, ejection, isovolumic relaxation, and filling phase. For single cardiomyocytes that constitute the heart, these phases correspond to isometric contraction, quasi-isotonic contraction, isometric relaxation, and quasi-isotonic relaxation in their force-length relationship, respectively. To reproduce these physiological work-loop style contractions in the single cell preparations, various techniques have been developed using amphibian or mammalian cardiomyocytes. The most important procedure in these techniques is holding the cell ends in order to apply mechanical load. Frog cardiomyocytes are thin and compliant; therefore, they are suitable for holding the cell ends to stretch the cells and measure the twitch force. On the other hand, applying these techniques in the mammalian cardiomyocytes has been challenging because of their short and thick shape and stiffness that makes holding cell ends difficult. Recently, carbon fiber technique has been introduced to attach the mammalian cell ends by electric statics. Since then, this technique has been modified by many researchers in order to improve the cell end attachment. To reproduce the work-loop style contractions, the afterload should be controlled using the force or length feedback control in each of the four phases separately. For this purpose, real-time feedback control is ideal; however, the relatively low signal-to-noise ratio of force/length signal in the carbon fiber technique makes real-time feedback control difficult. Instead, adaptive feedback or feed-forward approach enables it. Recently developed new optical force transducer using laser interferometry allowed force/length measurement with low enough noise for real-time feedback control.
Hollow organs (e.g. heart) experience pressure-induced mechanical wall stress sensed by molecular mechano-biosensors, including mechanosensitive ion channels, to translate into intracellular signaling. For direct mechanistic studies, stretch devices to apply defined extensions to cells adhered to elastomeric membranes have stimulated mechanotransduction research. However, most engineered systems only exploit unilateral cellular stretch. In addition, it is often taken for granted that stretch applied by hardware translates 1:1 to the cell membrane. However, the latter crucially depends on the tightness of the cell-substrate junction by focal adhesion complexes and is often not calibrated for. In the heart, (increased) hemodynamic volume/pressure load is associated with (increased) multiaxial wall tension, stretching individual cardiomyocytes in multiple directions. To adequately study cellular models of chronic organ distension on a cellular level, biomedical engineering faces challenges to implement multiaxial cell stretch systems that allow observing cell reactions to stretch during live-cell imaging, and to calibrate for hardware-to-cell membrane stretch translation. Here, we review mechanotransduction, cell stretch technologies from uni-to multiaxial designs in cardio-vascular research, and the importance of the stretch substrate-cell membrane junction. We also present new results using our IsoStretcher to demonstrate mechanosensitivity of Piezo1 in HEK293 cells and stretch-induced Ca²⁺ entry in 3D-hydrogel-embedded cardiomyocytes.
The heart has the ability to adjust to changing mechanical loads. The Frank-Starling law and the Anrep effect describe exquisite intrinsic mechanisms the heart has for autoregulating the force of contraction to maintain cardiac output under preload and afterload. Although these mechanisms have been known for more than a century, their cellular and molecular underpinnings are still debated. How does the cardiac myocyte sense a change in preload or afterload? How does the myocyte adjust its response to compensate for such changes? In cardiac myocytes Ca(2+) is a crucial regulator of contractile force and in this review we compare and contrast recent results from different labs that address two important questions. The "dimensionality" of the mechanical milieu under which experiments are carried out provide important clues to the location of the mechanosensors and the kinds of mechanical forces they can sense and respond to. As a first approximation, sensors inside the myocyte appear to modulate reactive oxygen species (ROS) while sensors on the cell surface appear to also modulate nitric oxide (NO) signalling; both signalling pathways affect Ca(2+) handling. Undoubtedly, further studies will add layers to this simplified picture. Clarifying the intimate links from cellular mechanics to ROS and NO signalling and to Ca(2+) handling will deepen our understanding of the Frank-Starling law and the Anrep effect, and also provide a unified view on how arrhythmias may arise in seemingly disparate diseases that have in common altered myocyte mechanics. This article is protected by copyright. All rights reserved.
The quantitative description of muscle contraction has evolved into two separate foci: Lumped descriptions based on Hill’s contractile element [e.g., (31)] and crossbridge models based on Huxley’s description of a single sarcomere [e.g., (9)]. The earliest quantitative descriptions of muscle are lumped whole muscle models, with the simplest mechanical description being a purely elastic spring. Potential energy is stored when the spring is stretched and shortening occurs when it is released. The idea of muscle elastance can be traced back to Weber (1846) (48), who considered muscle as an elastic material that changes state during activation via conversion of chemical energy. Subsequently, investigators retained the elastic description but ignored metabolic alteration of muscle stiffness. Fick (1891) (12) and later Blix (1893) (2) refuted the purely elastic model on thermody-namic grounds. They found that potential energy stored during stretching was less than the sum of the energy released during shortening as work and heat.
The left-ventricular diastolic pressure—volume relation (LV-DPVR) is determined by intrinsic properties of the chamber as well as by forces that are external to the heart, principally resulting from pericardial and right heart loads. Intrinsic factors can be divided into passive and active components. Passive elements include myocardial viscoelasticity, caused by structural proteins within the sarcomere, extra-cellular matrix proteins, and coronary vascular turgor, and chamber geometric factors such as wall thickness, cavity shape, and filling deformation. Active components of chamber stiffness are principally related to calcium handling and neurohumoral activation. This can manifest as delayed isovolumic relaxation that influences the early portion of the LV-DPVR, or altered diastolic “tone” which influences overall chamber distensibility. Among these factors, the dominant determinants of the LV-DPVR appear to be structural, because the relation is generally unaltered by acute pharmacological or physiological manipulations. External influences on the LV-DPVR are primarily direct ventricular interaction and constraining forces from the pericardium. These external loads are more easily altered by acute interventions and likely underlie many reported upward or downward shifts in the LV-DPVR from various interventions (e.g., vasodilators, channel blockers, exercise, ischemia). External constraining forces contribute a substantial percent (≈35%) of the resting diastolic pressures in humans, and thereby play an important role in determining the net dependence of cardiac output on filling pressure. In this chapter, we review the determinants of the LV-DPVR and focus on the relative influence of intrinsic and extrinsic factors.
The increasing number of animal models available has undoubtedly contributed to the great advances in cardiovascular research. Not with standing the development of these valuable tools, it is important to properly assess myocardial function both in physiological and in pathological conditions as well as in response to certain drugs. It is clear from recent literature that significant technological progresses to evaluate cardiac function has been made over the last 10 years. This progress has substantially overcome many of the difficulties associated with cardiovascular function assessment in disease models. Moreover by means of a reductionist approach it has been possible to dissect the complexities of in vivo heart function using more detailed and sensitive in vitro techniques that are able to exclude confounding factors of the in vivo setting. Thus many new questions are constantly arising and additional experiments and methodologies urge.
Cardiac contractility is the hallmark of cardiac function and is a predictor of healthy or diseased cardiac muscle. Despite advancements over the last two decades, the techniques and tools available to cardiovascular scientists are limited in their utility to accurately and reliably measure the amplitude and frequency of cardiomyocyte contractions. Isometric force measurements in the past have entailed cumbersome attachment of isolated and permeabilized cardiomyocytes to a force transducer followed by measurements of sarcomere lengths under conditions of submaximal and maximal Ca2+ activation. These techniques have the inherent disadvantages of being labor intensive and costly. We have engineered a micro-machined cantilever sensor with an embedded deflection-sensing element that, in preliminary experiments, has demonstrated to reliably measure
cardiac
cell contractions in real-time. Here, we describe this new bioengineering tool with applicability in the cardiovascular research field to effectively and reliably measure
cardiac
cell contractility in a quantitative manner. We measured contractility in both primary neonatal rat heart cardiomyocyte monolayers that demonstrated a beat frequency of 3 Hz as well as human embryonic stem cell-derived cardiomyocytes with a contractile frequency of about 1 Hz. We also employed the β-adrenergic agonist isoproterenol (100 nmol l−1) and observed that our cantilever demonstrated high sensitivity in detecting subtle changes in both chronotropic and inotropic responses of monolayers. This report describes the utility of our micro-device in both basic cardiovascular research as well as in small molecule drug discovery to monitor cardiac
cell contractions.
In vivo, cells are exposed to mechanical forces in many different ways. These forces can strongly influence cell functions or may even lead to diseases. Through their sensing machinery, cells are able to perceive the physical information of the extracellular matrix and translate it into biochemical signals resulting in cellular responses. Here, by virtue of two-component polymer scaffolds made via direct laser writing, we precisely control the cell matrix adhesions regarding their spatial arrangement and size. This leads to highly controlled and uniform cell morphologies, thereby allowing for averaging over the results obtained from several different individual cells, enabling quantitative analysis. We transiently deform these elastic structures by a micromanipulator, which exerts controlled stretching forces on primary fibroblasts grown in these scaffolds on a subcellular level. We find stretch-induced remodeling of both actin cytoskeleton and cell matrix adhesions. The responses to static and periodic stretching are significantly different. The amount of paxillin and phosphorylated focal adhesion kinase increases in cell matrix adhesions at the manipulated pillar after static stretching whereas it decreases after periodic stretching.
From an engineering perspective, many forms of heart disease can be thought of as a reduction in biomaterial performance, in which the biomaterial is the tissue comprising the ventricular wall. In materials science, the structure and properties of a material are recognized to be interconnected with performance. In addition, for most measurements of structure, properties, and performance, some processing is required. Here, we review the current state of knowledge regarding cardiac tissue structure, properties, and performance as well as the processing steps taken to acquire those measurements. Understanding the impact of these factors and their interactions may enhance our understanding of heart function and heart failure. We also review design considerations for cardiac tissue property and performance measurements because, to date, most data on cardiac tissue has been obtained under non-physiological loading conditions. Novel measurement systems that account for these design considerations may improve future experiments and lead to greater insight into cardiac tissue structure, properties, and ultimately performance.
The mechanical behavior of the heart muscle tissues is the central problem in finite element simulation of the heart contraction, excitation propagation and development of an artificial heart. Nonlinear elastic and viscoelastic passive material properties of the left ventricular papillary muscle of a guinea pig heart were determined based on in-vitro precise uniaxial and relaxation tests. The nonlinear elastic behavior was modeled by a hypoelastic model and different hyperelastic strain energy functions such as Ogden and Mooney-Rivlin. Nonlinear least square fitting and constrained optimization were conducted under MATLAB and MSC.MARC in order to obtain the model material parameters. The experimental tensile data was used to get the nonlinear elastic mechanical behavior of the heart muscle. However, stress relaxation data was used to determine the relaxation behavior as well as viscosity of the tissues. Viscohyperelastic behavior was constructed by a multiplicative decomposition of a standard Ogden strain energy function, W, for instantaneous deformation and a relaxation function, R (t), in a Prony series form. The study reveals that hypoelastic and hyperelastic (Ogden) models fit the tissue mechanical behaviors well and can be safely used for heart mechanics simulation. Since the characteristic relaxation time (900 s) of heart muscle tissues is very large compared with the actual time of heart beating cycle (800 ms), the effect of viscosity can be reasonably ignored. The amount and type of experimental data has a strong effect on the Ogden parameters. The in vitro passive mechanical properties are good initial values to start running the biosimulation codes for heart mechanics. However, an optimization algorithm is developed, based on clinical intact heart measurements, to estimate and re-correct the material parameters in order to get the in vivo mechanical properties, needed for very accurate bio-simulation and for the development of new materials for the artificial heart. (C) 2011 Elsevier Ltd. All rights reserved.
Phase-sensitive detection has long been recognized as a mechanism for increasing imaging contrast. The proliferation of quantitative phase contrast techniques and the breadth of emerging applications reflects the potential for achieving subdiffraction- limited resolution of cellular structure and dynamic phenomena with phase. Our laboratory developed spectral domain phase microscopy (SDPM) as a simple, phase-stable tool for studying cell dynamics and structure. As a functional extension of optical coherence tomography (OCT), SDPM inherited the high-resolution depth-sectioning capabilities for which OCT is well known, but adds to this an ability to discriminate sub-coherence length changes in optical pathlength within target samples at discrete axial positions. Early demonstrations of SDPM showed it to be extremely sensitive to thickness changes in biological and non-biological samples; the results of our previous studies investigating cell surface motion in cardiomyocyte contractility, cytoplasmic streaming rates in single-celled organisms, and rheological properties of the cytoskeleton suggest that SDPM can contribute insights of biological relevance. The principal aim of this work is to refine SDPM to enable imaging, interrogation, and quantification of parameters of interest in developing cardiomyocytes. In this manuscript, we report on the technology advances that enable multidimensional SDPM, and the results of new inotropic imaging studies of chick embryo cardiomyocytes.
During partial Ca2+ activation, skinned cardiac cells with sarcoplasmic reticulum destroyed by detergent developed spontaneous tension oscillations consisting of cycles (0.1-1 Hz) of rapid decrease of tension corresponding to the yield of some sarcomeres and slow redevelopment of tension corresponding to the reshortening of these sarcomeres. Such myofilament-generated tension oscillations were never observed during the full activation induced by a saturating [free Ca2+] or during the rigor tension induced by decreasing [MgATP] in the absence of free Ca2+ or when the mean sarcomere length (SL) of the preparation was greater than 3.10 microm during partial Ca2+ activation. A stiff parallel elastic element borne by a structure that could be digested by elastase hindered the study of the SL--active tension diagram in 8-13-microm-wide skinned cells from the rat ventricle, but this study was possible in 2-7-microm-wide myofibril bundles from the frog or dog ventricle. During rigor the tension decreased linearly when SL was increased from 2.35 to 3.80 microm. During full Ca2+ activation the tension decreased by less than 20% when SL was increased from 2.35 to approximately 3.10 microm. During partial Ca2+ activation the tension increased when SL was increased from 2.35 to 3.00 microm. From this observation of an apparent increase in the sensitivity of the myofilaments to Ca2+ induced by increasing SL during partial Ca2+ activation, a model was proposed that describes the tension oscillations and permits the derivation of the maximal velocity of shortening (Vmax). Vmax was increased by increasing [free Ca2+] or decreasing [free Mg2+] but not by increasing SL.
We studied contraction in single voltage-clamped, internally perfused myocytes isolated from guinea pig ventricles. The microscopic appearance of the cell was observed and recorded with a television system, while contractile shortening was measured 1,000 times/s using a linear photodiode array. Uniform, synchronous sarcomere shortening occurred in response to depolarizations that triggered a slow inward current (Isi). Changes in Isi caused by altering the amplitude of the voltage step, the extracellular [Ca2+], or the holding potential were accompanied by immediate parallel changes in the extent and velocity of shortening. In particular, twitch shortening during depolarization was immediately decreased when large voltage steps decreased Isi, and was eliminated by depolarizations that exceeded +75 mV, the apparent reversal potential for Ca2+. In these cases, shortening was associated with the tail current during repolarization. Increases in the amplitude, duration, and the rate of the depolarizing step increased the extent and speed of sarcomere shortening over the course of four to five contractions without a simultaneous parallel increase of Isi. Large prolonged depolarizations caused an asynchronous, nonuniform, oscillatory shortening of the cell and potentiated future twitch contractions. Increases in the duration of the depolarizing step immediately prolonged contraction; otherwise, interventions that altered the extent, velocity, and time course of shortening in intact, nonperfused cells did not affect the time course of the contraction in the internally perfused single cells. Our results provide direct support for the hypothesis that Isi both induces and grades the size of the Ca2+ release from the sarcoplasmic reticulum of intact cardiac muscle. In addition, a separate, depolarization-dependent process unrelated to Isi grades the size of contraction, presumably by modulating Ca2+ accumulation in the intracellular stores, and affects its time course.
Skinned canine cardiac Purkinje cells were stimulated by regularly repeated microinjection-aspiration sequences that were programmed to simulate the fast initial component of the transsarcolemmal Ca2+ current and the subsequent slow component corresponding to noninactivating Ca2+ channels. The simulated fast component triggered a tension transient through Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum (SR). The simulated slow component did not affect the tension transient during which it was first introduced but it potentiated the subsequent transients. The potentiation was not observed when the SR function had been destroyed by detergent. The potentiation decreased progressively when the slow component was separated by an increasing time interval from the fast component. The potentiation was progressive over several beats under conditions that decreased the rate of Ca2+ accumulation into the SR (deletion of calmodulin from the solutions; a decrease of the temperature from 22 to 12 degrees C). In the presence of a slow component, an increase of frequency caused a positive staircase, and the introduction of an extrasystole caused a postextrasystolic potentiation. There was a negative staircase and no postextrasystolic potentiation in the absence of a slow component. These results can be explained by a time- and Ca2+-dependent functional separation of the release and accumulation processes of the SR, rather than by Ca2+ circulation between anatomically distinct loading and release compartments. The fast initial component of transsarcolemmal Ca2+ current would trigger Ca2+ release, whereas the slow component would load the SR with an amount of Ca2+ available for release during the subsequent tension transients.
Intact cardiac cells from the adult rat or rabbit ventricle were isolated by enzymatic digestion with a progressive increase of the [free Ca2+] in the solution. These cells were electrically stimulated in the presence of 2.50 mM free Ca2+, and a twitch of maximum amplitude was elicited by the positive inotropic interventions that were found to be optimum. Then the cells were chemically skinned, and the maximum tension induced by a saturating [free Ca2+] was used as a reference to express the tension developed during the twitch of the intact cells. The myoplasmic [free Ca2+] reached during the twitch was inferred from the tension-pCa curve. In mechanically skinned cells of the same animal species, the myoplasmic [free Ca2+] reached during Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum (SR) was inferred by two methods using (a) the tension-pCa curve and (b) a direct calibration of the transients of aequorin bioluminescence. The induction of a maximum Ca2+ release from the SR required a larger Ca2+ preload of the SR and a higher [free Ca2+] trigger in the rabbit than in the rat skinned cells. However, the results obtained with the two methods of inference of the myoplasmic [free Ca2+] suggest that in both animal species a maximum myoplasmic [free Ca2+] of pCa approximately 5.40 was reached during both the optimum Ca2+-induced release of Ca2+ from the SR of the skinned cells and the optimum twitch of the intact cells. This was much lower than the [free Ca2+] necessary for the full activation of the myofilaments (pCa approximately 4.90).
It generally has been thought that the relatively high resting tension characteristic of cardiac tissue resides in structures (collagen, elastin) external to the individual cardiac cells, but the evidence to support this conclusion has been indirect, since the resting tension of intact single cardiac cells has not been determined previously. The purpose of the present investigation was to determine the resting tension (stress)-sarcomere length relationships of single intact frog atrial cells. For tension determinations, a single cell was attached between two poly-L-lysine coated glass beams; one beam served as a compliant calibrated cantilevered force beam, and length changes were imposed on the cell by movement of the other beam. Coventional bright-field light microscope techniques were used to view the cell, the sarcomere pattern within the cell, and the position of the force beam. The resting tension of the intact cell increased from a value of about 10 nN at a sarcomere length of 2.35 microns to a value of about 130 nN at a sarcomere length of 3.45 microns. Lagrangian and Eulerian resting stress-sarcomere length relationships were computed from the resting tension-sarcomere length relationships. The Lagrangian stress increased from a value of about 0.6 mN/mm2 at a sarcomere length of 2.35 microns to a value of about 7 mN/mm2 at a sarcomere length of 3.45 microns. These values of stress are about 8- to 30-fold less than those previously reported for intact frog atrial tissue and indicate that the resting tension of intact frog atrial preparations resides primarily in structures external to the individual cardiac cell.
A number of investigators have succeeded in preparing isolated cardiac cells by enzymatic digestion which tolerate external [Ca2+] in the millimolar range. However, a persistent problem with these preparations is that, unlike in situ adult ventricular fibres, the isolated fibres usually beat spontaneously. This spontaneity suggests persistent ionic leakage not present in situ. A preferable preparation for mechanical and electrical studies would be one which is quiescent but excitable in response to electrical stimulation and which does not undergo contracture with repeated stimulation. We report here a modified method of cardiac fibre isolation and perfusion which leaves the fibre membrane electrically excitable and moderately resistant to mechanical stress so that the attachment of suction micropipettes to the fibre is possible for force measurement and length control. Force generation in single isolated adult rat heart fibres is consistent with in situ contractile force. The negative staircase effect (treppe) characteristic of adult not heart tissue is present with increased frequency of stimulation. Isometric developed tension increases with fibre length as in in situ ventricular tissue.
Length dependence of activation accounts almost entirely for the dependence of tension production on muscle length over the ascending limb of the length tension relation in isolated papillary muscles. If the inotropic state of the muscle is equated with the degree of activation of the contractile system, then muscle length influences inotropic state and a change of muscle length must therefore be regarded as an inotropic intervention. If these results are applicable to the intact heart, then diastolic volume and inotropic state cannot be regarded as independent regulators of cardiac output.
Pure myofibrils were isolated from bovine heart by sucrose layer ultracentrifugation. Cardiac myofibrils thus prepared contained more protein as insoluble stroma than skeletal muscle. The insoluble stroma largely consisted of connectin, an elastic protein of muscle. The connectin content in cardiac myofibrils was about 18% of the total myofibrillar protein and was three times that in skeletal myofibrils. In view of the role of connectin as an elastic component of muscle, the abundance of connectin in cardiac myofibrils may be responsible for keeping myofibrils short at rest. This would account for the more effective tension generation in cardiac muscle on passive stretching due to blood inflow (Stirling's law).
FRANK1 and Starling2 have demonstrated that the systolic pressure developed by the heart decreased as its diastolic volume was altered in either direction from an optimum value. This law has been explained by variations of the number of cross bridges between the thin (actin) and the thick (myosin) filaments which generate force in individual cells3,4 in accordance with the sliding filament theory5.
Analysis of passive right and left ventricular pressure-volume curves for hearts of 72 Syrian golden hamsters studied in vitro showed increases in ventricular weight, volume, and compliance at mid-age. Both ventricles were filled by syringe pumps at a constant rate. Ventricular compliance (dV/dP) was determined by electronic differentiation of the intraventricular pressures and formation of the ratio (dV/dt)/(dP/dt) as a continuous function of intraventricular pressure between 0 and 30 mm Hg. By relating, with justification, the left ventricle to a thin-walled elastic sphere, ventricular elastic moduli, E, for different ages were compared at constant levels of myocardial wall stress, sigma. The elastic modulus E proved to be a linear function of sigma. The slope of the E-sigma plot yielded a stiffness constant, K, for each age group. Body weight, heart weight, end-diastolic volume, and dV/dP all varied by more than 200% up and then down as a function of age, but K was not a significant function of age. These results suggest that the aging heart does not normally undergo substantial alterations in passive properties that affect the muscle cells and fibers themselves, but rather that the observed changes in compliance are primarily attributable to alterations in ventricular size.
The ascending limb of the length-tension diagram was studied in single skinned (sarcolemma-free) cardiac cells of rat ventricle. The free (Ca2+) in the perfusing solutions was buffered with ethyleneglycol-bis(beta-aminoethyl)N,N'-tetraacetic acid (EGTA). Tension was recorded with a photo-diode force transducer (detection limit: 1 mug, compliance 2-3 mum/mg) and was expressed as a function of the sarcomere length (SL) measured during contraction with a high-speed movie camera. When the SL was decreased by a brief (1-2 sec) exposure to high free (Ca2+), a restoring force qas observed upon return of the cell to a relaxing solution. The restoring force was comprised of two components: (1) a rapid elongation to 1.57 identical to 0.06 mum developing a negative tension (relaxing force) as large as 4% of the maximum positive tension, which was observed in both skinned cells and in single myofibrils, and (2) a slow elongation from 1.57 identical to 0.06 to 1.93 identical to 0.14 mum, which was observed in skinned cells but not in single myofibrils. When the SL was kept extremely short by continuous Ca2+ activation for more than 30 sec, a shortening of the A band much below 1.0 mum persisted for several minutes after imposed re-elongation of the sarcomers (delta state). The amplitude of the tonic tension developed by direct activation of the myofilaments in the presence of high total (EGTA) was maximum for 2.20 mum SL and decreased very little when the SL was decreased. However, the decrease of tension became more pronounced at SL shorter than 1.55 mum when polyvinylpyrrolidone was added to the solutions at a concentration reducing the swelling of the myofilament lattice. This finding imposes some caution in applying the results obtained in skinned cardiac cells to intact tissue. The SL-tension diagram of the phasic contractions induced by Ca2+-triggered of Ca2+ in the presence of a slight EGTA buffering was similar to the SL-tension diagram of the intact rat ventricular muscle when the latter was expressed as a function of the active SL. In contrast, the SL-tension diagram of caffeine-induced phasic contractions was similar to that of the tonic tension produced by direct activation of the myofilaments. Decreasing SL results therefore in a partial inhibition of the Ca2+ triggered release process. It was concluded that Starling's Law may correspond to a length-dependence of several mechanisms including Ca2+-triggered release from the SR, interaction between thick and thin filaments and restoring forces.
The purpose of this study was to develop a method for attachment of single isolated cardiac myocytes to a transducer for recording isometric rension development. Cardiac myocytes were isolated from the hearts of the toad,Bufo
marinus or ferrets by enzymatic digestion with collagenase. The method that we used provided a 60–80% yield of Ca++-tolerant cells. A suspension of cells was placed into a superfusion chamber coated with bovine thrombin. Two glass microtools — each attached to a micromanipulator — were brought into proximity with the ends of a single myocyte; one of the microtools was attached to the element of a low-level force transducer. Human fibrinogen was loaded into a fine-tipped glass micropipette mounted on a micromanipulator. Small amounts of fibrinogen were pressure-ejected from the pipette at each junction between the microtool and the end of the myocyte. The fibrin that formed produced a stable attachment of the ends of the myocyte to the microtools. The myocyte could subsequently be stretched, and a length-tension curve recorded. We have used this method to record concentration-dependent tension development in response to the Ca++-ionophore, A23187, and potassium depolarization. Our results indicate that fibrin glue may facilitate the study of the mechanical properties of isolated myocytes.
In isolated myocytes from mammalian ventricles a fast and a slow component in the contractile response to depolarizing voltage clamp steps were identified. The potential dependence of the slow component was identical to the activation curve of iCa. The fast component, however, remained at its maximal amplitude at potentials positive to +10 mV (up to +100 mV), in which potential range iCa declined and eventually disappeared. The results suggest that the slow component may be activated by Ca++ entering through sarcolemmal Ca channels, whereas the fast component depends on Ca release from intracellular sites and may depend on both Cai and voltage.
1. Action potentials, calcium currents (iCa) and cell contraction have been recorded from single guinea-pig myocytes during periods of stimulation from rest. Voltage clamp was carried out using a single microelectrode. Cell contraction was measured optically. All experiments were performed at 18-22 degrees C. 2. An inverse relationship was observed between cell contraction and action potential duration or iCa. Mixed trains of action potentials and voltage clamp pulses preserved this relationship. Long voltage clamp pulses induced negative 'staircases' of iCa and positive 'staircases' of cell contraction. A facilitation of iCa was observed during repetitive stimulation with clamp pulses of 100 ms duration or less and was accompanied by a decrease in cell contraction. 3. The voltage dependence of inward current staircases was found to depend on Ca2+ entry rather than membrane voltage for long voltage clamp pulses and was not affected by 30 mM-TEA or 50 microM-TTX. Current reduction was greatest at 0 mV (P less than 0.05) when iCa was largest. Changes in cell contraction during pulse trains showed a similar voltage dependence. The time constant of current staircases was only mildly voltage dependent. 4. Interference with normal cellular mechanisms for Ca2+ uptake and release by strontium, 1-5 mM-caffeine and 1 microM-ryanodine increased current staircases and could abolish iCa facilitation with short clamp pulses. 5. Variations in the level of Ca2+-dependent inactivation of iCa can explain many features of the changes in iCa during stimulation after rest. Long clamp pulses (or action potentials) may increase cell Ca2+ loading and inhibit iCa. Short clamp pulses reduce available Ca2+ for cell contraction and this may reflect a lowered myoplasmic Ca2+ level which allows facilitation of iCa.
A method is presented that consistently yields a large number of calcium-tolerant myocytes from mammalian, amphibian, and elasmobranch hearts and from mammalian stomach. The use of incubating solutions or cell harvesting techniques was not required. The time needed to isolate cells was shorter than previously reported values. Action potentials recorded from each cell type appear similar in configuration to that of the intact multicellular tissue. The isolated myocytes appear to tolerate long periods of electrophysiological experimentation using the "giga-seal" suction electrode technique of Hamill et al. (Pfluegers Arch. 391: 85-100, 1981). This method is ideally suited for comparative electrophysiological studies, since the procedure for cell isolation was not seriously modified according to the preparation or species used.
Single heart cells were obtained from frog ventricle with an enzymatic dispersion technique. Isometric contractile force was measured in these cells by an ultrasensitive force transducer and compared with that generated by multicellular muscle strips under similar conditions. The shape of the single-cell twitch was qualitatively similar to that obtained in intact tissue; however, the time to peak was generally shorter, and the falling phase was prolonged in the single cell compared with the muscle strip. The single-cell contractile force was measured in response to alterations in stimulus rate, resting length, and extracellular Ca2+ concentration [Ca2+]o or by addition of epinephrine; in all cases, the force response resembled the physiological response seen in the muscle strip. However, at a constant stimulation rate, the steady-state amplitude of the single-cell twitch exhibited beat-to-beat variations under all conditions tested, whereas that of the muscle strip was essentially constant. These results may prove to be useful in assessing the suitability of the single-cell preparation as a model for the intact tissue.
Isolated single cardiac myocytes are becoming an increasingly popular experimental preparation. To date, however, the mechanical properties of these isolated adult mammalian cardiac myocyte preparations have not been thoroughly evaluated. The aims of this research were to 1) define the basic contractile properties of externally unloaded single feline ventriculocytes, 2) determine their response to standard inotropic stimuli, and 3) determine whether contractile properties vary as a function of cell size. Cell contractions were elicited by field stimulation (1 Hz) and recorded with the use of a photodiode array technique. In the 121 myocytes studied (2 mM Ca2+; 37 degrees C) the maximum extent of shortening averaged 7.5 +/- 0.2% resting cell length (L). The maximum rates of shortening and relengthening were 92 +/- 2.7 and 103 +/- 2.0% L/s, respectively. Elevating the extracellular Ca2+ and paired pulse stimulation increased the magnitude of twitch contractions as well as the rates of shortening and relengthening. The relationship between cell size and contractile performance was assessed in this research either by comparing the contractile properties of large and small myocytes or by plotting specific contractile parameters as a function of myocyte surface area. The results of this research support the idea that single feline ventricular myocytes retain normal contractile capabilities following isolation and respond to inotropic maneuvers in a similar fashion to that observed in multicellular preparations. In addition the present experiments showed that the contractile properties of the myocytes isolated from normal feline hearts are not cell size dependent.
Laser diffraction patterns were investigated from enzymatically isolated, unattached myocardial cells of guinea-pigs and mice. Experiments were performed at 2.5 mM Ca2+ and room temperature. The mean sarcomere length of resting guinea-pig and mouse myocardial cells amounted to about 1.83 micron and 1.75 micron, respectively. When paced with alternating intervals by field stimulation carefully selected ventricular cells showed transient phenomena. (1) The staircase following a rested state contraction was positive in the case of guinea-pig and negative in the case of mouse myocardial cells; (2) The rested state as compared to the steady state sarcomere shortening of guinea-pig and mouse cardiac myocytes amounted to 35% and 600%, respectively; (3) The interval strength curve of guinea-pig myocardial cells passed through a maximum which was 0.26 +/- 0.06 micron (mean +/- S.D.) at a pacing interval of 2 s whereas myocardial cells of mice showed a rise of shortening with increasing intervals reaching a maximum at the rested state (0.24 +/- 0.08 micron). Results were similar to those obtained from multicellular preparations. We conclude therefore, dynamic properties of multicellular preparations are nicely reflected at the sarcomere level.
Myocytes were prepared from enzymatically digested adult rat hearts and attached to concentric double-barreled suction micropipettes. Myocyte stiffness was calculated as the ratio of the oscillatory tension-to-strain amplitude, where the strain was produced by an applied 5-Hz perturbation. Stiffness, as a function of cell length, was measured in relaxing solution (pCa = 9) as the control solution, 0.5% Triton X-100 detergent, 0.47 M KCl, and 0.6 M KI. Ultrastructure of unattached cells in each solution is illustrated with electron micrographs. The dependence of cell stiffness on cell length was described by an exponential relation with a length constant that increased slightly in detergent, whereas the stiffness at control length appeared to fall. The major fall in absolute stiffness occurred with myosin extraction in KCl. Both the stiffness at control length and the slope of the ln stiffness-to-length relation declined with the disappearance of the A band. A further, but smaller, decline of stiffness occurred with KI extraction of the thin filaments. A highly compliant "ghost" remained after KI extraction but the stiffness-to-length relation was still measurable. The fall in stiffness with myosin extraction is discussed in relation to cytoskeletal filaments (titin, nebulin, and intermediate filaments.
A high-resolution laser diffraction system suitable for studying the basic mechanical properties of small contractile single cells has been developed. This method was used to establish the mechanical behavior of 95 ventricular cells isolated from adult guinea pig hearts. During contraction, the sarcomere length shortened from 1828 +/- 43 nm (mean +/- SD) to 1518 +/- 99 nm. The maximal velocities were 1.98 +/- 0.64 micron/s for shortening and 1.93 +/- 0.54 micron/s for re-lengthening. The twitch duration from 20% shortening to 80% re-lengthening was 622 +/- 120 ms.
A novel ultrasensitive force transducer suitable for measuring isometric forces generated by single spindle-shaped muscle cells is described. The transducer components are mechanically and electrically simple, consisting of a low power He-Ne laser, a pair of step index, glass optic fibers, and a photodiode detector circuit. To test the operation of the transducer, enzymatically isolated frog ventricular heart cells were used, having peak contractile forces on the order of 100 nN. The transducer presents a number of advantages over existing designs and has a resolution better than 2 nN. As such, it is suitable for excitation-contraction studies in single muscle cells.
The relation between muscle length or sarcomere length and developed tension for lengths up to the optimal for contraction (Lmax) is much steeper in cardiac muscle than in skeletal muscle. The steepness of the cardiac length--tension relation arises because the degree of activation of the cardiac myofibrils by calcium increases as muscle length is increased. Two processes contribute to this length-dependence of activation: (i) the calcium sensitivity of the myofibrils increases with muscle length and (ii) the amount of calcium supplied to the myofibrils during systole increases with muscle length. Of these two, the change in calcium sensitivity is the most clearly defined and is responsible for a large part of the rapid change in developed tension when muscle length is altered. It is likely that this change in calcium sensitivity is due to a change in the affinity of troponin for calcium but the underlying mechanism has not been identified. There is good evidence that changes in the calcium supply to the myofibrils can account for the slow changes in tension that follow an alteration in length; there may also be rapid changes in calcium supply but this is less clearly established at present.
Single cardiac myocytes and skeletal myocyte fragments, devoid of interstitial collagen but with intact glycocalyx, were prepared by mechanical disaggregation of hamster ventricular myocardium and caudal gracilis muscle, respectively. Passive stiffness was studied by examining the sarcomere length-tension relationship over the approximate Eulerian stress range of 0-20 mN/mm2 for cardiac myocytes and 0-120 mN/mm2 for skeletal myocytes. Creep and stress-relaxation became apparent only when cells were stretched to sarcomere lengths close to, or exceeding, 2.2 micron for the cardiac myocytes, and 2.7 micron for the skeletal myocytes. Stress-relaxation and creep occurred simultaneously, suggesting that the sarcomere is at least one of the structural components responsible for viscoelasticity. The differential strain stiffness constant was calculated from the regression of natural stress [Ln(mN/mm2)] against differential strain [(L-Lo)/Lo] and found to be 7.48 +/- 1.73 for the ventricular myocytes and 5.77 +/- 0.87 for the skeletal myocyte fragments. The natural strain stiffness constant was obtained from the regression of natural stress against natural strain [Ln(L/Lo)]. The natural strain stiffness constant was 30-50% higher than the differential strain constant. The high correlation coefficients obtained for both regressions indicate that the length-tension relationships for these isolated cardiac and skeletal myocytes can be very closely fitted to the single exponential function, sigma = C X exp[K(epsilon)]. The length-tension curves obtained for the skeletal myocyte fragments are qualitatively and quantitatively similar to those obtained by others with intact skeletal muscle. The cardiac myocyte length-tension curves are qualitatively, but not quantitatively, similar to those obtained with cardiac muscle. Isolated ventricular myocytes are stiffer than similarly isolated skeletal myocytes. These findings suggest that cellular structures contribute to myocardial stiffness in the hamster.
Cardiac muscle fragments with disrupted sarcolemmas were obtained by homogenizing the ventricles of guinea pig hearts. Force and frequency of the spontaneous contractions of these fragments were measured under control conditions and after adding 2-[(2-methoxy-4-methylsulfinyl)phenyl]-1H-imidazo[4,5-b]pyridine (AR-L 115 BS), caffeine or dihydro-ouabain. The effects of AR-L 115 BS and caffeine were very similar. Probably AR-L 115 BS, like caffeine, releases calcium from the sarcoplasmic reticulum and inhibits the reuptake of calcium.
1. Quiescent cat papillary muscles were stimulated to contract regularly at L max , the length at which force production is optimal, and at 0·85 L max . The resulting increase in force production (rate staircase) at each length was characterized as an exponential function.
2. When stimulated from quiescence at 24 min ⁻¹ in a bathing fluid [Ca ²⁺ ] of 2·5 m M , sixteen of twenty‐three muscles exhibited biexponential increases in force production at both lengths. The coefficients of the exponential function at L max were 1·5‐2 times greater than their counterparts at the shorter length, and this length difference was highly significant. When the force staircases were normalized to the peak developed force attained at each length, the number of beats to attain 25, 50, 75, and 98% of peak force at 0·85 L max was approximately twice that required at L max .
3. At a given length the force staircase (1) exhibited a dependency on the number of beats rather than on stimulation frequency over a range of 12‐60 min ⁻¹ , (2) was accelerated by increasing the bathing fluid [Ca ²⁺ ] from 1·0 to 5·0 m M , (3) was accelerated in the presence of isoproterenol, and (4) was retarded in the presence of DL ‐verapamil. Over the entire range of bathing fluid [Ca ²⁺ ] and at all stimulation frequencies 24 min ⁻¹ and above, more beats were required to complete a given level of the normalized staircase at 0·85 L max than at L max . There was no length difference in the presence of verapamil. These data suggest that transsarcolemmal Ca ²⁺ influx is an important determinant of the kinetics of the force staircase, and the length dependence of the latter indicates that muscle length is an important determinant of transsarcolemmal Ca ²⁺ influx.
4. This conclusion was strengthened by the results of additional studies in which the [Ca ²⁺ ] of the bathing fluid was abruptly increased from 1·0 to 5·0 m M with the muscle beating in the steady state. The resulting increase in force production (Ca ²⁺ staircase) was described by a monoexponential function with a greater coefficient at L max than 0·85 L max ; when normalized to the peak force difference in the two [Ca ²⁺ ] at each length, a given level of the staircase was achieved in significantly fewer beats at L max than at the shorter length.
5. The data provide a mechanism which, in part, explains the length dependence of excitation‐contraction coupling in cardiac muscle with intact sarcolemmae.
Mechanical properties of isolated guinea-pig ventricular cardiocytes subjected to a mechanical load.
- Argibay JA
- Garnier D
- Le Guennec J-Y
- Nigretto J-M
- Peineau N
Connectin, an elastic protein of muscle: characterisation and function.
- Maruyama K
- Matsubara S
- Natori R
- Nonomura Y
- Kimura S
- Ohasi K
- Marajami F
- Handa S
- Eguchi G
Contractile force measured in unskinned isolated adult rat heart fibers.
- Brady AJ
- Tan ST
- Ricchuiti NV