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Measurement of sarcomere dynamics simultaneously with auxotonic force in isolated cardiac cells
Abstract
We developed an easy to use and non-invasive method to study sarcomere motion of enzymatically isolated myocytes which can be simultaneously combined with auxotonic force detection, thus being very useful when studying the contractile performance of cardiac cells. This method basically consists in analyzing the periodicity of the cell striation pattern using the Cooley-Tukey fast Fourier transform (FFT) algorithm on a video image of the cell during the course of the experiment. A longitudinal fraction of the cell image is recorded with a CCD TV camera, digitized, then transiently stored on a computer and used to calculate the spectrum corresponding to the distribution of the sarcomere lengths (SL). The method gives a real-time measurement of the most probable value of sarcomere length in one isolated cell with a temporal resolution of 20 ms. When used on a cell attached between two carbon fibers, the auxotonic force developed by the cell upon electrical stimulation can be simultaneously measured together with the SL in various conditions of stretch. Preliminary results have been presented in abstract form (Gannier et al., vol 24, pp. S47, 1992).
... As a result, a number of investi-gators have turned to image analysis as a means of examining local sarcomere behavior. A number of such methods have been proposed, ranging from relatively fast, single-line charge coupled device-based methods to slower video analysis and off-line cine-film-based methods (Goldspink et al., 1970;Kawai and Kuntz, 1973;Krueger et al., 1980;Lieber et al., 1983;deClerck et al., 1984;Roos, 1987;Krueger, 1988; Roos and Taylor, 1988;Roos et al., 1989;Periasamy et al., 1990;Krueger and Denton, 1991;Anazawa et al., 1992;Horowitz et al., 1992;Gannier et al., 1993). In the majority of these methods, sarcomere length assessment is carried out directly in the spatial domain. ...
... However, it should be noted that the Rayleigh criterion provides only an approximate estimate of the actual resolution (cf. Gannier et al., 1993). In terms of digitization effects, to simplify matters, let us assume that the frequency FIGURE 7 Comparison of images and associated spectra of a fiber segment stretched to two different sarcomere lengths. ...
... There are a number of ways in which the computation time can be scaled down. Gannier et al. (1993) employ a method in which they reduce the two-dimensional images into one-dimensional line luminances (by simply summing the images across their columns) and then calculating the associated one-dimensional Fourier transforms. However, striation skewness can significantly influence the luminance profiles derived by such analysis, which in turn would affect the sarcomere length measurements. ...
A new image-analysis-based method is described for assessing sarcomere heterogeneity in skinned rabbit psoas muscle fiber segments. This method consists of off-line, two-dimensional Fourier spectral analysis of video-taped muscle images. Local sarcomere length is assessed by partitioning the muscle images into half and quarter images spanning the original image and analyzing the associated spectra. The spectra are analyzed in two different ways, yielding two measures of sarcomere length. The first measure is obtained by calculating and inverting the centroid frequency of the first-order peak associated with each two-dimensional Fourier spectrum. The second measure is obtained in a similar manner, the only difference being that the two-dimensional spectra are first collapsed into one-dimensional line spectra by summing the pixels perpendicular to the fiber axis. Comparison of the two measures provides a measure of striation skewness that cannot be obtained by other image analysis based methods that determine sarcomere length by analyzing selected line luminance profiles.
... The cells were positioned using a remotely controlled rotatable experimental chamber (Kohl et al., 1994) and illuminated using long-wavelength light. The image formed by this light was directed to a video camera; sarcomere length was then determined along the length of the cell using a fast Fourier transform algorithm on an acquired video image of the cell (Gannier et al., 1993). The sampling frequency of the video image acquisition was 50 Hz. ...
... The cells were positioned using a remotely controlled rotatable experimental chamber ( Kohl et al., 1994) and illuminated using long-wavelength light. The image formed by this light was directed to a video camera; sarcomere length was then determined along the length of the cell using a fast Fourier transform algorithm on an acquired video image of the cell ( Gannier et al., 1993). The sampling frequency of the video image acquisition was 50 Hz. ...
... refer to ordinal location of stimulus: 3, before stretch; 6 and 8, during stretch; 10, after stretch). B: APD at both 90% and 50% repolarization (APD 90 and APD 50 ) increased during stretching (5)(6)(7)(8) and returned close to control duration after strain was released (9)(10)(11)(12)(13)(14). C: resting potential was depolarized during stretching. ...
... The peak amplitude of the stretch-activated depolarization from rest and repolarization from the plateau exhibited a linear relationship to voltage and volume change (58). In single guinea pig cardiac myocytes, a 3% strain did not affect the resting potential but did decrease the APD (10,54). ...
Mechanoelectric transduction can initiate cardiac arrhythmias. To examine the origins of this effect at the cellular level, we made whole cell voltage-clamp recordings from acutely isolated rat ventricular myocytes under controlled strain. Longitudinal stretch elicited noninactivating inward cationic currents that increased the action potential duration. These stretch-activated currents could be blocked by 100 microM Gd(3+) but not by octanol. The current-voltage relationship was nearly linear, with a reversal potential of approximately -6 mV in normal Tyrode solution. Current density varied with sarcomere length (SL) according to I (pA/pF) = 8.3 - 5.0 SL (microm). Repeated attempts to record single channel currents from stretch-activated ion channels failed, in accord with the absence of such data from the literature. The inability to record single channel currents may be a result of channels being located on internal membranes such as the T tubules or, possibly, inactivation of the channels by the mechanics of patch formation.
... It is interesting to consider why the presently described changes in sarcomere strain during LDA have not been previously reported in cardiomyocytes. Indeed, even the concept of nonhomogeneous sarcomere function appears to have been overlooked by some investigators, who have examined sarcomere dynamics with Fourier transforms to assess average changes in Z-line positions 40,41 or simply recorded overall cell length. Furthermore, previous efforts to track Z lines have generally not sought to reduce the noise of the individual sarcomere signals or have used too low frame rates to adequately detect changes in length during the contraction cycle. ...
Background:
Increasing cardiomyocyte contraction during myocardial stretch serves as the basis for the Frank-Starling mechanism in the heart. However, it remains unclear how this phenomenon occurs regionally within cardiomyocytes, at the level of individual sarcomeres. We investigated sarcomere contractile synchrony and how intersarcomere dynamics contribute to increasing contractility during cell lengthening.
Methods:
Sarcomere strain and Ca2+ were simultaneously recorded in isolated left ventricular cardiomyocytes during 1 Hz field stimulation at 37 °C, at resting length and following stepwise stretch.
Results:
We observed that in unstretched rat cardiomyocytes, differential sarcomere deformation occurred during each beat. Specifically, while most sarcomeres shortened during the stimulus, ≈10% to 20% of sarcomeres were stretched or remained stationary. This nonuniform strain was not traced to regional Ca2+ disparities but rather shorter resting lengths and lower force production in systolically stretched sarcomeres. Lengthening of the cell recruited additional shortening sarcomeres, which increased contractile efficiency as less negative, wasted work was performed by stretched sarcomeres. Given the known role of titin in setting sarcomere dimensions, we next hypothesized that modulating titin expression would alter intersarcomere dynamics. Indeed, in cardiomyocytes from mice with titin haploinsufficiency, we observed greater variability in resting sarcomere length, lower recruitment of shortening sarcomeres, and impaired work performance during cell lengthening.
Conclusions:
Graded sarcomere recruitment directs cardiomyocyte work performance, and harmonization of sarcomere strain increases contractility during cell stretch. By setting sarcomere dimensions, titin controls sarcomere recruitment, and its lowered expression in haploinsufficiency mutations impairs cardiomyocyte contractility.
... Furthermore, since hiPSC-CM are spontaneously beating, their morphology and contractile properties are important markers of their health and function. However, while powerful method to measure sarcomere parameters exist (Gannier et al., 1993;Guo and Song, 2014;Pasqualin et al., 2015), due to the novelty of hiPSC-CM model, efficient dedicated analysis tools often lack and need to be developed for quantifying the level of organization of sarcomeres which are the essential blocks for the beating of the monolayers. Measurements of the degree of alignments, their spacing, their concentration in proteins are needed for a good characterization of the cell properties in different culture conditions. ...
Motivation:
Cardiomyocytes derived from stem cells are closely followed, notably since the discovery in 2007 of human induced pluripotent stem cells (hiPSC). Cardiomyocytes (hiPSC-CM) derived from hiPSC are indeed more and more used to study specific cardiac diseases as well as for developing novel applications such as drug safety experiments. Robust dedicated tools to characterize hiPSC-CM are now required. The hiPSC-CM morphology constitutes an important parameter since these cells do not demonstrate the expected rod shape, characteristic of native human cardiomyocytes. Similarly, the presence, the density and the organization of contractile structures would be a valuable parameter to study. Precise measurements of such characteristics would be useful in many situations: for describing pathological conditions, for pharmacological screens or even for studies focused on the hiPSC-CM maturation process.
Results:
For this purpose, we developed a MATLAB based image analysis toolbox, which gives accurate values for cellular morphology parameters as well as for the contractile cell organization.
Implementation:
To demonstrate the power of this automated image analysis, we used a commercial maturation medium intended to promote the maturation status of hiPSC-CM, and compare the parameters with the ones obtained with standard culture medium, and with freshly dissociated mouse cardiomyocytes.
Supplementary information:
Supplementary data are available at Bioinformatics online.
... For a pixel size of 0.40 and 0.45 m, the measurement error lies between 0.006 and 0.007 m. These values are far below the pixel size, since they are obtained with a frequency analysis by FFT and not by a simple metric measurement (7). ...
Accurate measurement of cardiomyocyte contraction is a critical issue for scientists working on cardiac physiology and physiopathology of diseases implying contraction impairment. Cardiomyocytes contraction can be quantified by measuring sarcomere length but few tools are available for this and none is freely distributed. We developed a plug-in (SarcOptiM) for the ImageJ/Fiji image analysis platform developed by the National Institute of Health. SarcOptiM computes sarcomere length via FFT analysis of video frames captured or displayed in ImageJ and thus is not tied to a dedicated video camera. It can work in real time or offline, the latter overcoming rotating motion or displacement related artifacts. SarcOptiM includes a simulator and video generator of cardiomyocyte contraction. Acquisition parameters such as pixel size and camera frame rate were tested with both experimental recordings of rat ventricular cardiomyocytes and synthetic videos. It is freely distributed and its source code is available. It works under Windows, Mac or Linux operating systems. The camera speed is the limiting factor since the algorithm can compute online sarcomere shortening at frame rates above 10 kHz. In conclusion, SarcOptiM is a free and validated user-friendly tool for studying cardiomyocyte contraction in all species including human.
... Comparison of the two provided a measure of striation skewing. Gannier et al. (1993) developed a non-invasive, easy-to-use method to study sarcomere motion in cardiomyocytes. This was based on analyzing the periodicity of the cell striation pattern using the fast Fourier transform (FFT) algorithm on a video image of the cell during the course of the experiment. ...
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.
... g & Parikh. 1993; Tung & Zou. 1995; Brady. 1991; Wellner & Isenberg. 1994). The best methods to date use carbon fibers (White, Le Guennec, Nigretto, Gannier, Argibay, & Garnier. 1993) or sticky glass probes (Sasaki, Mitsuiye, & Noma. 1992; Tung & Parikh. 1993; Palmer, Brady, & Roos. 1996) that adhere to the sarcolemma. The work with carbon fibers (Gannier, Beranengo, Jacquemond, & Garnier. 1993) suggests that, for reasons not understood, the strain is rather uniform ⎯ when cells are stretched, the sarcomere spacing doesn't vary significantly below the attachment site of the probe. Presumably this means that there is significant rigidity of the contractile apparatus across the fiber diameter. ...
... While edge detection can be used to define sarcomere length ~Infantolino et al., 2010!, the longitudinal regularity of muscle is especially suited for analysis by Fourier transform ~Shah & Lieber, 2003!. The one-dimensional fast Fourier transform ~FFT! has been most often used to find the dominant frequency in high-quality images, which corresponds to the sarcomere length ~Gannier et al., 1993; Helmes et al., 1999; Weiwad et al., 2000; Ockleford et al., 2002; Shah & Lieber, 2003!, although the FFT-derived power spectrum also provides information on the relative strength of that frequency and may help determine whether an ROI is acceptable by quantifying how clear repeating structures are in that region. Texture and structural measures calculated from ROIs can be used to sort regions into two or more classifications. ...
Understanding cytoskeletal dynamics in living tissue is prerequisite to understanding mechanisms of injury, mechanotransduction, and mechanical signaling. Real-time visualization is now possible using transfection with plasmids that encode fluorescent cytoskeletal proteins. Using this approach with the muscle-specific intermediate filament protein desmin, we found that a green fluorescent protein-desmin chimeric protein was unevenly distributed throughout the muscle fiber, resulting in some image areas that were saturated as well as others that lacked any signal. Our goal was to analyze the muscle fiber cytoskeletal network quantitatively in an unbiased fashion. To objectively select areas of the muscle fiber that are suitable for analysis, we devised a method that provides objective classification of regions of images of striated cytoskeletal structures into "usable" and "unusable" categories. This method consists of a combination of spatial analysis of the image using Fourier methods along with a boosted neural network that "decides" on the quality of the image based on previous training. We trained the neural network using the expert opinion of three scientists familiar with these types of images. We found that this method was over 300 times faster than manual classification and that it permitted objective and accurate classification of image regions.
... Both ends of skinned myocytes were glued to a thin glass rod with optical glue (NOA 63, Norland products Inc., North Brunswick, NJ, U.S.A.) that was polymerized by UV illumination for 3 min. An SL was determined online at 50 Hz by using a fast Fourier transform algorithm (FFT) on the digitized striation images of the attached cell (Gannier et al., 1993). The system for recording force consisted of a piezoresistive strain gauge (model AE 801, SensoNor a.s., Horten, Norway) with a light, 1-mm diameter, 3-cm long glass rod. ...
SR33805, a potent Ca(2+) channel blocker, increases cardiac myofilament Ca(2+) sensitivity in healthy rat cardiomyocytes. Therefore, the aim of the present study was to evaluate the effects of SR33805 on contractile properties in ischaemic failing hearts after myocardial infarction (MI) in vivo and in vitro at the cellular level.
The effect of SR33805 (10 µM) was tested on the excitation-contraction coupling of cardiomyocytes isolated from rat with end-stage heart failure. Cell shortening and Ca(2+) transients were measured in intact cardiomyocytes, while contractile properties were determined in Triton X-100 permeabilized myocytes. Acute treatment with SR33805 restored the MI-altered cell shortening without affecting the Ca(2+) transient amplitude, suggesting an increase of myofilament Ca(2+) sensitivity in MI myocytes. Indeed, a SR33805-induced sensitization of myofilament activation was found to be associated with a slight increase in myosin light chain-2 phosphorylation and a more significant decrease on troponin I (TnI) phosphorylation. Decreased TnI phosphorylation was related to inhibition of protein kinase A activity by SR33805. Finally, administration of a single intra-peritoneal bolus of SR33805 (20 mg/kg) improved end-systolic strain and fractional shortening of MI hearts.
The present study indicates that treatment with SR33805 improved contractility of ischaemic failing hearts after MI in the rat by selectively modulating the phosphorylation status of sarcomeric regulatory proteins, which then sensitized the myofilaments to Ca(2+). Our results gave a proof of concept that manipulation of the Ca(2+) sensitivity of sarcomeric regulatory proteins can be used to improve contractility of a failing heart.
... The cells were positioned with a manually or remotely controlled (Kohl, Spindler & Le Guennec, 1994) rotatable experimental chamber. Sarcomere length was determined by using a fast Fourier transform algorithm on an acquired video image of the cell (Gannier, Bernengo, Jacquemond & Garnier, 1993 Ijfect of Gd' oni the stiretchindulced increase in [Ca'l]1 ...
The effect of Gd3+ on the delayed rectifier potassium current (IK) in single guinea-pig ventricular myocytes was tested using whole-cell patch-clamp techniques. It was found that Gd3+ blocked 70% of the IK tail current at a concentration of 100 microM. The EC50 was 24 microM. Action potential durations were, however, reduced, consistent with a predominant effect on depolarizing L-type Ca2+ current (Ica.L). In the presence of 5 microM nifedipine Gd3+ prolonged the action potential. Using carbon fibres to stretch cells we observed that 10 microM Gd3+ was not effective in reducing a large stretch-activated increase in resting calcium. Modelling studies using the OXSOFT HEART program suggest that this lack of response is influenced by blockade of repolarizing current but is best reproduced by additional blockade of Ca2+ extrusion via the Na(+)-Ca2+ exchanger. When Gd3+ is used as a blocker of stretch-activated channels its actions upon both Ica.L and IK must therefore be accounted for.
... The best methods to date use carbon fibers (White et al. 1993) or sticky glass probes (Sasaki et al. 1992; Tung and Parikh 1993;) that adhere to the sarcolemma. The work with carbon fibers (Gannier et al. 1993) suggests that, for reasons not understood, the strain is rather uniform -when cells are stretched, the sarcomere spacing doesn't vary significantly below the attachment site of the probe. Presumably this means that there is significant rigidity of the contractile apparatus across the fiber diameter. ...
... Therefore, several investigators have preferred image analysis for the study of local Sl (Periasamy et al., 1990;Roos, 1990;Horowitz and Pollack, 1993). Standard video techniques (Lundblad et al., 1986;Sato et al., 1988;Gannier et al., 1993) allow satisfactory spatial information and Sl measurement along "bers, but are ine!ective during fast twitches. This led us to develop a low-cost, high-speed video system that provides su$cient spatial information (Hajjar and LeH oty, 1996). ...
A low-cost, high-resolution (spatial and temporal) image analysis system was developed to measure sarcomere length (Sl) during fast twitch of isolated striated muscle fibers at different temperatures. Fiber images were examined during twitch with an imaging rate of 220 Hz. To increase temporal resolution beyond 220 Hz, consecutive temporally shifted image sequences (N sequences) were acquired. Individual or average Sl was directly measured from a horizontal profile without spatial-frequency assessment. Measurement precision (E) was determined and expressed as: E(%) = 100xPs/(IsxSl), where Ps is the pixel size and Is the involved sarcomere number. At 18 degrees C during isometric twitch, Sls were measured with 220 Hz temporal and 0.2% spatial resolutions. Sl shortened in the central region (0.21+/-0.12 microm) as tension developed, reaching a maximal shortening of 8.09 + 2.05% (at rest, Sl = 2.59+/-0.05 microm, n = 4) in 32.5+/-1.96 ms. At 30 degrees C, Sl variations were examined with 880 Hz temporal resolution, in which case maximal S1 shortening was reached in 15.74+/-1.99 ms, and then decreased to 5.19+/-1.97% (at rest, S1 = 2.6+/-0.06 microm). The twitch tension developed by the whole fiber was recorded and compared with sarcomere length behavior. Sarcomere length variations in the central region were representative of overall developed tensions at 18 and 30 degrees C.
... Frame grabber, acquisition, and analysis software for sarcomere length measurements were obtained from IonOptix (Milton, MA). The algorithm for measuring sarcomere spacing was adapted from Gannier et al. (9). First, the user aligns the cell image horizontally, after which a user-defined rectangular region of interest (ROI) within the myocyte is selected. ...
Isolated permeabilized cardiac myocytes have been used in the study of myofilament calcium sensitivity through measurement of the isometric force-pCa curve. Determining this force-pCa relationship in skinned myocytes is relatively expensive and carries a high degree of variability. We therefore attempted to establish an alternative high-throughput method to measure calcium sensitivity in cardiac myocytes. With the use of commercially available software that allows for precise measurement of sarcomere spacing, we measured sarcomere length changes in unloaded skinned cardiac myocytes over a range of calcium concentrations. With the use of this technique, we were able to accurately detect acute increases or decreases in myofilament calcium sensitivity after exposure to 10 mM caffeine or 5 mM 2,3-butanedione monoxime, respectively. This technique allows for the simple and rapid determination of myofilament calcium sensitivity in cardiac myocytes in a reproducible and inexpensive manner and could be used for high-throughput screening of pharmacological agents and/or transgenic mouse models for changes in myofilament calcium sensitivity.
... Contraction was monitored by following the sarcomere length (SL) as described by Gannier et al. (1993). Brie¯y, the periodicity of the cell striation pattern (A and I bands) was analysed with a Fast Fourier Transform (FFT) of a video image of the cell during the course of the experiment. ...
The effects of 10 m M caffeine (CAF) on intramembrane charge movements (ICM) were studied in isolated guinea‐pig ventricular heart cells with the whole‐cell patch‐clamp technique.
In the presence of CAF, the properties (voltage dependence, maximum Q ON [Q max ], availability with voltage) of Q ON charge activated from −110 mV were barely affected. Following a 100 ms prepulse to −50 mV to decrease the participation of charges originating from Na channels, the voltage dependence of Q ON was shifted by 5 mV (negative component) and by 10 mV (positive component) towards negative potentials, and Q max was depressed by 16.5%.
CAF drastically reduced in a time‐ and voltage‐dependent manner Q OFF on repolarization to −50 mV, the effects being greater at positive potentials.
CAF‐induced Q OFF immobilization could be almost entirely removed by repolarization to voltages as negative as −170 mV. In these conditions, the voltage‐dependence of Q OFF (repolarization to +30 to −170 mV) was shifted by 17 mV (negative component) and 30 mV (positive component) towards negative potentials, suggesting an interconversion into charge 2.
Most of CAF effects were suppressed when the sarcoplasmic reticulum (SR) was not functional or when the cells were loaded with BAPTA‐AM.
We conclude that CAF effects on ICM are likely due to Ca ²⁺ ions released from the SR, and which accumulate in the subsarcolemmal fuzzy spaces in the vicinity of the Ca channels. Because CAF effects were more pronounced on Q OFF than on Q ON the channels have likely to open before Ca ²⁺ ions could affect their gating properties. It is speculated that such an effect on gating charges might contribute to the Ca‐induced inactivation of the Ca current.
British Journal of Pharmacology (2002) 135 , 721–734; doi: 10.1038/sj.bjp.0704520
... Displacement of the fibres during stimulation was used to calculate force developed, which could be normalised to cell cross-sectional area (CSA). Increased sarcomere length (SL) was used as our index of stretch, which was monitored online using a Fourier analysis of the video image of the cell (Gannier et al. 1993; White et al. 1995). The change in tension over the change in SL (dT/dSL) was our index of the slope of the passive and active SL–tension curves (see Cazorla et al. 2000b). ...
Short-term (6 weeks) voluntary wheel running exercise in young female rats that were in an active growth phase resulted in whole-heart hypertrophy and myocyte concentric hypertrophy, when compared to sedentary controls. The cross-sectional area of ventricular myocytes from trained rats was significantly greater than for those isolated from sedentary rats, with the greatest change in morphology seen in sub-endocardial cells. There was no statistically significant effect of training on cell shortening in the absence of external mechanical loading, in [Ca2+](i) transients, or in myofilament Ca2+ sensitivity (assessed during re-lengthening following tetanic stimulation). Under the external mechanical load of carbon fibres, absolute force developed in myocytes from trained rats was significantly greater than in those from sedentary rats. This suggests that increased myocyte cross-sectional area is a major contractile adaptation to exercise in this model. Training did not alter the passive mechanical properties of myocytes or the relative distribution of titin isomers, which was exclusively of the short, N2B form. However, training did increase the steepness of the active tension-sarcomere length relationship, suggesting an exercise-induced modulation of the Frank-Starling mechanism. This effect would be expected to enhance cardiac contractility. Training lengthened the action potential duration of sub-epicardial myocytes, reducing the transmural gradient in action potential duration. This observation may be important in understanding the cellular causes of T-wave abnormalities found in the electrocardiograms of some athletes. Our study shows that voluntary exercise modulates the morphological, mechanical and electrical properties of cardiac myocytes, and that this modulation is dependent upon the regional origin of the myocytes.
... Finally, we have attempted to introduce analytical tools to characterize images in a more systematic fashion , potentially aiding in the development of more accurate structural computational models for muscle mechanics or providing quantitative definitions of muscle pathology. The FFT has been used by muscle biologists to calculate sarcomere lengths (Gannier et al. 1993; Helmes et al. 1999; Weiwad et al. 2000; Ockleford et al. 2002 ). To date, however, relative positions between periodic structures have not been quantified. ...
We describe a novel system that permits simultaneous confocal imaging of protein interactions and measurement of cell mechanical properties during passive loading. A mechanical apparatus was designed to replace the stage of a confocal microscope, enabling cell manipulation, force transduction, and imaging. In addition, image processing algorithms were developed to quantify the degree of connectivity between subcellular structures. Using this system, we examined the interactions among three cellular structures thought to be linked by the muscle's intermediate filament system: Z-disks, nuclei, and the costamere protein complexes located at the muscle cell surface. Fast Fourier transforms (FFTs) and autocorrelations (ACs) were implemented to quantify image periodicity and relative phase shifts among structures. We demonstrated in sample wild-type muscle cells that there was significant connectivity among Z-disks in the same fiber at various sarcomere lengths, as well as between Z-disks and the costamere complexes. This approach can be applied to any cell system in which structural periodicity and mechanical connectivity are of interest.
... The index of stretch used in our experiments was an increase in sarcomere length (SL). This was monitored on-line by a Cooley- Tukey fast Fourier transform of the video image of the myocyte, sampled at 50 Hz (Gannier et al. 1993) and calibrated using a 50 w 2 mm graticule. Sarcomere length was not altered by carbon fibre attachment and was stable following increases due to stretch; this was indicative of the absence of damage-induced changes in [Ca 2+ ] i (see White et al. 1993 White et al. , 1995 Hongo et al. 1996). ...
We tested the hypothesis that both stretch-activated channels (SACs) and intracellular calcium ([Ca(2+)](i)) are important in the electrical response of single guinea-pig ventricular myocytes to axial stretch. Myocytes were attached to carbon fibre transducers and stretched, sarcomere length increased by approximately 9 %, and there was a prolongation of the action potential duration. Streptomycin, a blocker of SACs, had no effect upon the shortening, [Ca(2+)](i) transients or action potentials of electrically stimulated, unstretched myocytes, at a concentration of 50 microM, but at 40 microM, prevented any stretch-induced increase in action potential duration. Under action potential clamp, stretch elicited a current with a linear current-voltage relationship that was inward at membrane potentials negative to its reversal potential of -30 mV, in 10 of 24 cells tested, and was consistent with the activation of non-specific, cationic SACs. This current was not seen in any stretched cells that were exposed to 40 microM streptomycin. However, exposure of cells to 5 microM BAPTA-AM, in order to reduce [Ca(2+)](i) transients, also abolished stretch-induced prolongation of the action potential. We conclude that both SACs and [Ca(2+)](i) are important in the electrical response of cardiac myocytes to stretch, and propose that stretch-induced changes in electrical activity and [Ca(2+)](i) may be linked by inter-dependent mechanisms.
... Both ends of skinned myocytes were glued to a thin glass rod with optical glue (NOA 63, Norland products Inc., North Brunswick, NJ, U.S.A.) that was polymerized by UV illumination for 3 min. An SL was determined online at 50 Hz by using a fast Fourier transform algorithm (FFT) on the digitized striation images of the attached cell (Gannier et al., 1993). The system for recording force consisted of a piezoresistive strain gauge (model AE 801, SensoNor a.s., Horten, Norway) with a light, 1-mm diameter, 3-cm long glass rod. ...
This study examined the effects of SR33805, a fantofarone derivative with reported strong Ca2+ -antagonistic properties, on the contractile properties of intact and skinned rat ventricular myocytes.
On intact cells loaded with the Ca2+-fluorescent indicator Indo-1, the application of low concentrations of SR33805 enhanced the amplitude of unloaded cell shortening and decreased the duration of cell shortening. Amplitude of the Ca2+ transient was also decreased.
These effects were accompanied with a shortening of the action potential and a dose-dependent blockade of L-type calcium current (IC50=2.4 × 10−8M).
On skinned cardiac cells, the application of a low SR33805 concentration (10−8M) induced a significant increase in maximal Ca2+-activated force at the two-tested sarcomere lengths (SLs), 1.9 and 2.3 μm.
The application of a larger dose of SR33805 (10−6–10−5M) induced a significant leftward shift of the tension–pCa relation that accounts for Ca2+-sensitization of the myofilaments, particularly at 2.3 μm SL.
In conclusion, despite its strong Ca2+-antagonistic properties SR33805 increases cardiac cell contractile activity as a consequence of its Ca2+-sensitizing effects. These effects are attributable to both an increase in the maximal Ca2+-activated force and a length-dependent Ca2+-sensitization.
British Journal of Pharmacology (2003) 139, 99–108. doi:10.1038/sj.bjp.0705221
... One needle was attached to a force transducer (SensoNor, Horten, Norway) and the other to an electromagnetic motor (Aurora Scientific Inc., Aurora, Canada), both connected to joystick-controlled micromanipulators. The average sarcomere length was determined by means of a spatial Fourier transform, as described previously (Gannier et al. 1993; Fan et al. 1997), and adjusted to 2.2 µm in the relaxing solution. Solution exchange was achieved by transferring the myocyte from a small temperature-controlled well (volume 50 µl) containing the relaxing solution to a similar temperature-controlled well containing the activating solution. ...
The effects of the Ca2+-sensitiser levosimendan alone or in combination with beta-adrenergic stimulation on the contractile function were studied in various guinea pig cardiac preparations. Echocardiography in narcotised animals indicated that a maximal dose of levosimendan (50 microg x kg(-1)) increased the left ventricular posterior wall movement velocity during systoles and diastoles by 25 +/- 3% (mean +/- S.E.M.) and 17 +/- 2%, respectively. In Langendorff hearts, a saturating concentration of levosimendan (0.3 micromol x l(-1) for 5 min) increased +dP/dt(max) and dP/dt(max) by 28 +/- 3% and 14 +/- 2%, respectively. Further, the Ca2+-sensitising potential of levosimendan in Triton-skinned cardiomyocytes (EC50: 5 +/- 3 nmol x l(-1)) was illustrated by a maximal increase in the isometric force production by 51 +/- 5% (at pCa 6.2). However, following stimulation by isoproterenol, when the level of troponin I phosphorylation was elevated, no significant additional increase in the contractile parameters could be demonstrated upon levosimendan administration. Moreover, the levosimendan-induced increase in force production in isolated skinned myocytes could be prevented by incubation with the catalytic subunit of protein kinase A (100 U x ml(-1) for 40 min). These data indicate that thin filament-targeted Ca2+-sensitisation by levosimendan is modulated by phosphorylation of the contractile filaments, an effect that should be considered during combination therapy with levosimendan.
Recent advances in stem cell biology have allowed researchers to efficiently produce large numbers of cardiomyocytes from various pluripotent cell sources. Unfortunately, these cells exhibit properties that are characteristics of immature cardiomyocytes such as poor sarcomere organization, limited calcium handling, and reduced cell size and alignment. Specifically, the actin–myosin motor proteins that form sarcomeres within these cardiomyocytes fail to produce large, highly ordered repeating structures that are distinctive for adult myocytes. Instead, these cells produce heterogeneous sarcomeres that vary in thickness, alignment, and level of organization. Additionally, a large number of cardiomyopathies have been linked to mutations in genes encoding for sarcomeric proteins, resulting in disrupted sarcomere organization. This research focuses on a series of algorithms that provide a quantitative analysis technique to characterize the alignment and organization of sarcomere structures within aggregates and single cardiomyocytes. The scanning gradient Fourier transform (SGFT) method incorporates gradient analysis along with fast Fourier transforms to determine regions of sarcomere organization within individual and a population of cells, yielding a quantitative method of determining sarcomere organization and alignment at the sub-cellular scale. The utility of the SGFT technique is also demonstrated for additional applications, such as breast cancer collagen microstructure and neural rosette patterning.
La fibrillation atriale (FA) est le trouble du rythme le plus fréquemment rencontré en clinique. Elle est responsable d’une importante morbi-mortalité et d’une forte majoration durisque d’accident vasculaire cérébral. La FA est réfractaire aux traitements pharmacologiques anti-arythmiques, elle constitue donc un problème de santé publique. À son stade initial, la FA est principalement déclenchée par des foyers d’activité électriques anormaux situés dans le manchon de cardiomyocytes (CM) des veines pulmonaires (VP). Le mécanisme à l’origine de leur génération très mal connu. Les ions calcium (Ca 2+ ) étant fréquemment impliqués dans la génération de signaux électriques arythmogènes, nous avons émis l’hypothèse qu’ils pouvaient être impliqués dans les arythmies générées dans la VP.Les caractéristiques structurelles et fonctionnelles du cycle du Ca 2+ n’étant pas connues dans les CM de VP, nous avons donc exploré et comparé ce cycle à celui des CM d’oreillette gauche (OG) et de ventricule gauche (VG) chez le rat.L’étude de l’architecture de la membrane plasmique – point d’entrée du Ca 2+ dans le CM – a nécessité le développement d’un algorithme d’analyse spécifique pour les CM de VP (TTorg). Contrairement aux CM d’OG et de VG, la longueur du réseau de tubules transverses (TT) est variable dans les CM de VP et son niveau d’organisation est très hétérogène d’un CM à l’autre. Cette organisation hétérogène des TT d’un CM à l’autre implique une grande variabilité de distribution spatiale des canaux calciques (Ca v 1.2) par rapport aux récepteurs de la ryanodine (RyR2) et ainsi une grande hétérogénéité de forme et d’amplitude des transitoires calciques. Ces différents types de CM ne sont pas dispersés aléatoirement au sein de la veine, mais regroupés en « îlots » de CM présentant le même type d’organisation.Dans les CM de VP, la cinétique de recapture du Ca 2+ cytoplasmique dans le réticulum sarcoplasmique par la SERCA est identique à celle des CM d’OG, mais plus rapide que celle des CM de VG. En revanche, dans les CM de VP, la cinétique globale de diminution de la concentration calcique du cytoplasme par les mécanismes sarcolemmaux et les mitochondries est plus rapide que dans les CM d’OG, mais plus lente que dans les CM de VG.Dans les CM de VP, la stimulation des récepteurs β 1 -adrénergiques induit une augmentation de l’amplitude du transitoire calcique identique à celle des CM d’OG mais plus faible que celle des CM de VG. La stimulation des récepteurs α 1 -adrénergique induit une diminution de l’amplitude des transitoires calcique qui est plus importante dans les CM de VP que dans ceux d’OG.L’étude de l’amplitude de contraction des CM isolés a nécessité le développement d’un algorithme d’analyse spécifique pour les CM de VP (SarcOptiM). Cette amplitude est très hétérogène dans les CM de VP, à l’image de l’amplitude de leurs transitoires calciques. Contrairement aux anneaux isolés d’OG, la force de contraction des anneaux de VP n’est augmentée ni par l’augmentation de la concentration du Ca 2+ extracellulaire ni par le Bay K 8644, un ouvreur des canaux calciques de type L. Ces différences de réponses ne sont pas dues à une différence de sensibilité des myofilaments pour le Ca 2+ . Les CM de VP ne semblent donc pas présenter de réserve contractile dans les conditions d’étirement permettant d’obtenir une réponse contractile maximale.L’étude des libérations calciques spontanées dans le cytoplasme des CM a montré une plus grande fréquence des sparks et des waves dans les CM de VP que dans les CM d’OG.En conclusion, la forte hétérogénéité des CM de VP ainsi que la plus grande fréquence des libérations calciques spontanées dans leur cytoplasme pourraient expliquer le fort potentiel arythmogène des VP.
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.
Availability and implementation:
The software is freely distributed under the GNU General Public License. Download and setup instructions are available at http://pccv.univ-tours.fr/ImageJ/SarConfoCal It is provided as a toolset for ImageJ (the open-source program for image analysis provided by the National Institutes of Health). SarConfoCal has been tested under Windows, Mac and Linux operating systems.
Contact:
come.pasqualin@univ-tours.frSupplementary information: Supplementary data are available at Bioinformatics online.
Modeling of protein interactions responsible for cardiac tension development can enhance the understanding of physiological and pathophysiological phenomena of the heart. Principal components of muscular tension development are the proteins actin, myosin, troponin and tropomyosin. The tension is produced by cross-bridge cycling of actin and myosin using adenosine triphosphate as energy source. The cross-bridge cycling is initiated by binding of intracellular calcium to troponin, resulting in configuration changes of tropomyosin.
In this work a hybrid model of protein interactions in cardiac tension development is derived on basis of recent measurements and descriptions on protein level. Dependencies on intracellular calcium concentration, sarcomere stretch and stretch velocity as well as cooperativity mechanisms are incorporated. The model quantifies the tension development by states associated to configurations of the involved proteins. The model enables in conjunction with electrophysiological models of cardiac myocytes the reconstruction of electro-mechanical phenomena. Numerical simulations with the hybrid model were performed, which illustrated the reconstruction of steady state and length switches experiments. The steady state experiments describe the force-cytosolic [Ca²⁺] relationship in intact rat cardiac trabeculae. The length switch experiments provide data on the redevelopment of force after sudden stretch in rabbit right ventricular papillary muscles. Results of the numerical simulations show quantitative agreement with experimental studies.
The hybrid model of cardiac tension development offers interfaces to further models of cardiac electro-mechanics. The hybrid model can be coupled with models of cellular electrophysiology and passive mechanics of myocardium allowing the inclusion of mechano-electrical feedback mechanisms. The hybrid model can be applied to elucidate cooperativity mechanisms, pathophysiological changes and metabolism of tension development.
A custom-designed microelectromechanical system (MEMS) force transducer, with a volume less than 1 mm 3 , is being fabricated to measure force development in isolated cardiac muscle cells to elucidate the physiology of muscle contraction. A single heart cell is attached to flexible, hinged polysilicon plates submerged in a nutrient saline solution. As the cell contracts, the plates bend, and the contractile force can be measured based on the known spring constant of the plate. The amount of deflection is measured by piezoresistive, ion-implanted strain gauges placed at the base of the plates. Prototype structures have been fabricated and have been mechanically tested using probes. We have demonstrated that living rat heart cells can be attached to polysilicon using a silicone sealant. Polysilicon is an inert material when exposed to cardiac cells and their saline environment and has no effect on the cells themselves
Sarcomere length of a cardiomyocyte is an important control parameter for physiology studies on a single cell level; Its accurate determination in real-time is essential for performing single cardiomyocyte contraction experiments, for instance. The aim of this work is to develop an efficient and accurate method for estimating a mean sarcomere length of a contracting cardiomyocyte using microscopy images as an input. The novelty in developed method lies in (i) using unbiased measure of similarities to eliminate systematic errors from conventional autocorrelation function (ACF) based methods when applied to region of interest of an image, (ii) using a semi-analytical semi-numerical approach for evaluating the similarity measure to take into account spatial dependence of neighboring image pixels, (iii) and using a detrend algorithm to extract the sarcomere striation pattern content from the microscopy images. The developed sarcomere length estimation procedure has superior computational efficiency and estimation accuracy compared to the conventional ACF and spectral analysis based methods using Fast Fourier Transform. As shown by analyzing synthetic images with the known periodicity, the estimates obtained by the developed method are more accurate at the sub-pixel level than ones obtained using ACF analysis. When applied in practice on rat cardiomyocytes, our method was found to be robust to the choice of the region of interest that may (i) include projections of carbon fibers and nucleus, (ii) have uneven background, and (iii) be slightly disoriented with respect to average direction of sarcomere striation pattern. The developed method is implemented in open-source software.
A custom-designed microelectromechanical force transducer, with a volume of less than 1 mm3, is being developed to quantify forces generated in isolated cardiac muscle cells. A single heart cell will be attached to flexible, hinged polysilicon plates submerged in a nutrient solution. As the cell contracts, the plates will bend, and the contractile force can be measured based on the known spring constant of the plate and the amount of deflection. Prototype structures have been fabricated and have been mechanically tested. We have demonstrated that living rat heart cells can be attached to polysilicon using a commercial silicone sealant. We have also observed that polysilicon is an inert material when exposed to cardiac cells and their saline environment, and has no detectable effect on the cells themselves.
Modeling of mechanisms involved in electrophysiology and tension development of cardiac myocytes can enhance the understanding
of physiological and pathophysiological cardiac phenomena. Interactions of divers components are necessary for cellular electro-mechanics.
Particularly, the interactions between proteins in the cell membrane, sarcoplasmic reticulum and sarcomere are of importance.
In this work hybrid electro-mechanical models of cardiac myocytes were derived on basis of recently developed models as well
as of measurements ranging from protein to multi-cell level. The models quantify dynamically the electrophysiology and tension
development by states, partly associated to configurations of the involved proteins, and the transition between these states.
The models allow the reconstruction of electro-mechanical phenomena. Results of simulations with the hybrid models were performed
illustrating their properties. The models may help to clarify feedback and cooperativity mechanisms, pathophysiological changes
and metabolism of myocytes.
Sarcomere length (SL) is an important determinant and indicator of cardiac mechanical function; however, techniques for measuring SL in living, intact tissue are limited. Here, we present a technique that uses two-photon microscopy to directly image striations of living cells in cardioplegic conditions, both in situ (Langendorff-perfused rat hearts and ventricular tissue slices, stained with the fluorescent marker di-4-ANEPPS) and in vitro (acutely isolated rat ventricular myocytes). Software was developed to extract SL from two-photon fluorescence image sets while accounting for measurement errors associated with motion artifact in raster-scanned images and uncertainty of the cell angle relative to the imaging plane. Monte-Carlo simulations were used to guide analysis of SL measurements by determining error bounds as a function of measurement path length. The mode of the distribution of SL measurements in resting Langendorff-perfused heart is 1.95 mum (n = 167 measurements from N = 11 hearts) after correction for tissue orientation, which was significantly greater than that in isolated cells (1.71 mum, n = 346, N = 9 isolations) or ventricular slice preparations (1.79 mum, n = 79, N = 3 hearts) under our experimental conditions. Furthermore, we find that edema in arrested Langendorff-perfused heart is associated with a mean SL increase; this occurs as a function of time ex vivo and correlates with tissue volume changes determined by magnetic resonance imaging. Our results highlight that the proposed method can be used to monitor SL in living cells and that different experimental models from the same species may display significantly different SL values under otherwise comparable conditions, which has implications for experiment design, as well as comparison and interpretation of data.
1. The effects of mechanical loading and changes of length on the contraction of single guinea-pig ventricular myocytes has been investigated. 2. Cell shortening was monitored during isotonic contractions (in which the cell shortened freely) and after attaching carbon fibres of known compliance to the ends of the cell, so that the cell contracted auxotonically (the cell both shortened and developed force). 3. Mechanically loading the cells decreased the amount of shortening during a contraction and abbreviated the contraction. There were, however, no consistent changes in the action potential or the [Ca2+]i transient (measured with the fluorescent dye fura-2). 4. Increasing stimulation rate increased the size of the contraction and the [Ca2+]i transient in both isotonic and auxotonic conditions. The increase in the size of the contraction induced by an increase in stimulation rate was greater in auxotonic conditions but the increase in the size of the [Ca2+]i transient was not. 5. When cells were stretched, there was a step increase in the size of the contraction and a prolongation of its time course. However, neither the size nor the time course of the accompanying [Ca2+]i transient was significantly altered by this intervention. 6. When a stretch was maintained, a further, slow increase in the size of the contraction occurred during the following 3-11 min, in about half the cells studied. The probability of this slow response occurring was increased if the initial degree of activation of the cell was decreased. 7. These data suggest that the mechanisms underlying the responses to mechanical loading and changes of length are the same in both multicellular and single cell preparations of cardiac muscle.
The Frank-Starling mechanism is one of the most fundamental properties of cardiac muscle for exerting intrinsic control over contractile force on a beat to beat basis. According to this law, the systolic contractile force depends on the preceding diastolic blood loading. In other words stretching the ventricular muscle induces an increase in contractility. In multicellular preparations the mechanisms that underlie the length-force relationship in the myocardium have been the subject of considerable discussion and controversy.1-7
Single rat left ventricular myocytes were attached at both ends using a newly described double-barreled micropipette technique. This attachment procedure enabled the measurement of the active and passive mechanical properties of chemically skinned cells that showed little structural deformation. The force and oscillatory stiffness (100 Hz) of the cells were measured with a high signal-to-noise ratio, and the sarcomere length throughout the entire cell was monitored using image analysis. The passive properties were investigated from the resting sarcomere length to > 3 microns. Analysis of the sarcomere behavior indicated a high level of homogeneity throughout the cell. The attachment method supported the full activation of the cells by increased free Ca2+ (pCa 4.5), which produced 22.3 mN/mm (mean sarcomere length 2.11 microns). A force/pCa relationship was determined, which, when fitted according to the Hill equation, gave parameters of nH = 2.62 and pCa50 = 5.58. The described techniques allow the accurate study of the mechanical properties of single myocytes with increased fidelity and reliability over the preexisting methods.
Isolated rat ventricular myocytes were stretched using carbon fibres to investigate the mechanisms underlying the increase in contraction following stretch. 2. [Ca2+]i and [Na+]i were monitored using the fluorescent indicators fura-2 and sodium-binding benzofuran isophthalate, respectively. The L-type Ca2+ current was recorded simultaneously with contraction using the perforated patch-clamp technique. 3. Mechanical stretch caused an immediate increase in contraction, followed by a slow increase. Contraction was prolonged immediately after the stretch, but did not change during the slow phase. 4. The Ca2+ transient did not change immediately after the stretch. The slow increase in contraction was accompanied by an increase in the amplitude of the Ca2+ transient. However, diastolic [Ca2+]i did not change significantly following stretch. 5. [Na+]i did not change significantly either immediately, or during the slow increase in contraction, after the stretch. 6. The L-type Ca2+ current was not significantly altered either by mechanical loading of the cell with carbon fibres or by stretching the cell. 7. These results suggest that: (1) the rapid increase in contraction following a stretch is due to an increase in myofilament Ca2+ sensitivity rather than to changes in the L-type Ca2+ current or [Na+]i; and (2) a slow increase in the Ca2+ transient underlies the slow increase in contraction in isolated myocytes, but is not caused by either an increase in diastolic [Ca2+]i or a change in [Na+]i (and hence Ca2+ influx via Na(+)-Ca2+ exchange) or a change in myofilament Ca2+ sensitivity.
Redox potentials often differ dramatically for homologous proteins that have identical redox centers. For two types of iron-sulfur proteins, the rubredoxins and the high-potential iron-sulfur proteins (HiPIPs), no structural explanations for these differences have been found. We calculated the classical electrostatic potential at the redox site using static crystal structures of four rubredoxins and four HiPIPs to identify important structural determinants of their redox potentials. The contributions from just the backbone and polar side chains are shown to explain major features of the experimental redox potentials. For instance, in the rubredoxins, the presence of Val 44 versus Ala 44 causes a backbone shift that explains a approximately 50 mV lower redox potential in one of the four rubredoxins. This result is consistent with experimental redox potentials of five additional rubredoxins with known sequence. Also, we attribute the unusually lower redox potentials of two of the HiPIPs studied to a less positive electrostatic environment around their redox sites. Finally, molecular dynamics simulations of solvent around static rubredoxin crystal structures indicate that water alone is a major factor in dampening the contribution of charged side chains, in accord with experiments showing that mutations of surface charges produce relatively little effect on redox potentials.
We studied active and passive properties of intact isolated guinea-pig ventricular myocytes in auxotonic conditions. Cells were attached using carbon fibres. The passive properties of the myocytes, in the presence of the stretch-activated channel blocker streptomycin sulphate, could be separated into two groups: stiff cells (stiffness slope = 2.88 +/- 0.93 nN/micron3, n = 63 cells) and compliant cells (stiffness slope = 0.91 +/- 0.35 nN/micron3, n = 52 cells). The study and the localization of the different kind of cells indicated that endocardium is mainly constituted of stiff cells (80%) while the epicardium contained more compliant cells (60%). When a longitudinal strain was applied to compliant cells, an increase in resting tension, diastolic sarcomere length and active tension were observed. On the other hand, in stiff cells, it induced an increase in resting tension and active tension with little change of diastolic sarcomere length. In both kinds of cells, strain had no effect on Ca2+ transient amplitude and shape. Plotting active tension v diastolic sarcomere length also clearly showed two separated populations of cells, corresponding to stiff and compliant cells. The results of the two groups of cells when plotting active tension v resting tension could not be distinguished. We conclude that resting tension is an important factor in the modulation of active tension by stretch in addition to interfilament lattice spacing or sarcomere length.
Cardiac sarcomere stiffness was investigated during diastole in eighteen trabeculae dissected from the right ventricle of rat heart. The trabeculae were stimulated at 0.5 Hz, in a modified Krebs–Henseleit solution (pH, 7.4; 25 °C). Sarcomere length (SL) was measured using high resolution (±2 nm) laser diffraction techniques. Force ( F ) was measured with a silicon strain gauge.
SL increased exponentially (amplitude, 25 ± 9 nm; n = 5 ) throughout diastole. This increase occurred even at slack SL, showing that this phenomenon was due to an internal expansion. The majority of the muscles showed discrete spontaneous fluctuations of SL (amplitude < 20 nm) starting ∼l s after the end of the twitch.
The intracellular free Ca ²⁺ concentration ([Ca ²⁺ ] i ) was measured from the fluorescence of microinjected fura‐2 salt in seven trabeculae under the same experimental conditions. [Ca ²⁺ ] i continuously declined (from 240 to 90 n m ) during diastole following a monoexponential time course (time constant, 210–325 ms).
The stiffness of the sarcomere was evaluated at 10, 30, 50, 70 and 90% of diastole using bursts (30 ms) of 500 Hz sinusoidal perturbations of muscle length (amplitude of SL oscillations < 30 nm). At 1 m m external Ca ²⁺ concentration ([Ca ²⁺ ] o ), the average stiffness modulus (Mod) increased from 9.3 ± 0.6 to 12 ± 0.6 mN mm ⁻² μm ¹ ( n = 18 ; P < 0.05 ), while the average phase shift (Ф) between F and SL signals decreased from 84 ± 3 to 73 ± 4 deg ( n = 18 ; P < 0.05 ) between 10 and 90% during diastole. The increase in Mod and the decrease in Ф reversed when spontaneous activity occurred. When [Ca ²⁺ ] 0 was raised to 2 m m , the stiffness time course reversed approximately 450 ms earlier, simultaneously with the occurrence of spontaneous activity.
Our results show that diastole is only an apparent steady state and suggest that the structural system responsible for the viscoelastic properties of the sarcomere is regulated by [Ca ²⁺ ] i in the submicromolar range. Different possible origins of the dynamic changes in viscoelasticity during diastole are discussed.
We have investigated the effect of the venom of a Chilean tarantula, Phrixotrichus spatulatus, on cell contraction, intracellular [Ca2+] ([Ca2+]i), and the L-type Ca2+ current (ICa,L) in cells isolated from the ventricles of guinea pig hearts. Whole-cell voltage clamp techniques were used to monitor ICa,L. The action potential was recorded using whole cell current clamp. [Ca2+]i was monitored using the fluorescent indicator indo-1. The venom of P. spatulatus decreased ICa,L in a dilution-dependent manner, with half-maximal inhibition at a dilution of 1.1/10(4) (v/v). At a dilution of 1/10(4), this inhibition occurred at all potentials so that the current-voltage relationship of ICa,L was depressed. However, inhibition of ICa,L by the venom was relieved by positive potentials, the venom being less effective following pulses to positive potentials. The venom reduced the duration of the action potential (at 50% repolarization) by between 26 and 43%. The venom also decreased the amplitude of the [Ca2+]i transient and cell contraction. It is concluded that the venom of P. spatulatus is a potent, voltage-dependent inhibitor of ICa,L; this inhibition of ICa,L may account for the effects of the venom on action potential duration, [Ca2+]i, and contraction.
Type I and II collagen (native-type) fibrils, positively stained with uranyl acetate, present typical periodic (D = 67 nm) cross-striation patterns. Although the two patterns are similar, the distributions of charged amino acids along the type I and II collagen molecules are different. After optical diffraction analysis or computer image processing of electron micrographs, different Fourier transforms were obtained from type I and II collagen fibrils, either as native fibrils or after in vitro reconstitution from purified molecules. With tissues such as tendon and cartilage, better results were obtained after mild trypsin treatment, which allowed better isolation and staining of the collagen fibrils. The main difference observed in the Fourier transforms was the presence in type II collagen fibrils of a strong tenth-order peak (corresponding to the tenth harmonic of the fundamental frequency). In order to discriminate between the two collagens, we measured the ratio (R) of the areas under the ninth- and tenth-order peaks. In trypsin treated tissues, the distributions of these ratios were clearly separated: below 1.0 for type II collagen fibrils and above 1.5 for type I collagen fibrils. This method appears to be suitable for rapid typing of type I and II collagen fibrils and might be useful for determining the exact composition of fibrils in tissues, such as intervertebral discs, that contain these both types of collagen.
Diastolic dysfunction is an important cause of congestive heart failure; however, the basic mechanisms causing diastolic congestive heart failure are not fully understood, especially the role of the cardiac muscle cell, or cardiocyte, in this process. Before the role of the cardiocyte in this pathophysiology can be defined, methods for measuring cardiocyte constitutive properties must be developed and validated. Thus this study was designed to evaluate a new method to characterize cardiocyte constitutive properties, the gel stretch method. Cardiocytes were isolated enzymatically from normal feline hearts and embedded in a 2% agarose gel containing HEPES-Krebs buffer and laminin. This gel was cast in a shape that allowed it to be placed in a stretching device. The ends of the gel were held between a movable roller and fixed plates that acted as mandibles. Distance between the right and left mandibles was increased using a stepper motor system. The force applied to the gel was measured by a force transducer. The resultant cardiocyte strain was determined by imaging the cells with a microscope, capturing the images with a CCD camera, and measuring cardiocyte and sarcomere length changes. Cardiocyte stress was characterized with a finite-element method. These measurements of cardiocyte stress and strain were used to determine cardiocyte stiffness. Two variables affecting cardiocyte stiffness were measured, the passive elastic spring and viscous damping. The passive spring was assessed by increasing the force on the gel at 1 g/min, modeling the resultant stress vs. strain relationship as an exponential [sigma = A/k(ekepsilon - 1)]. In normal cardiocytes, A = 23.0 kN/m2 and k = 16. Viscous damping was assessed by examining the loop area between the stress vs. strain relationship during 1 g/min increases and decreases in force. Normal cardiocytes had a finite loop area = 1.39 kN/m2, indicating the presence of viscous damping. Thus the gel stretch method provided accurate measurements of cardiocyte constitutive properties. These measurements have allowed the first quantitative assessment of passive elastic spring properties and viscous damping in normal mammalian cardiocytes.
The intrinsic cellular mechanisms by which length regulates myocardial contraction, the basis of the Frank-Starling relation, are uncertain. The aim of this work was to test the hypothesis that passive force, possibly via titin, participates in the modulation of Ca2+ sensitivity of cardiac contractile proteins induced by stretch. Titin degradation by a mild trypsin digestion modulated passive force induced by increasing from 1.9 to 2.3 microm sarcomere length in skinned rat cardiac cells. Force-pCa curves were established at these two sarcomere lengths after various durations of trypsin application that induced different passive force levels. They allowed us to evaluate myofilament Ca2+ sensitivity by the pCa of half-maximal activation (pCa50). In control conditions, stretching cells from 1.9 to 2.3 microm induced a leftward shift of pCa50 (DeltapCa50) of 0.39+/-0.03 pCa units (mean+/-SEM, n=8 cells), reflecting an increase in Ca2+ sensitivity of the contractile machinery. Passive force measured every 2 min decreased exponentially after the beginning of the trypsin application (t1/2 approximately 12 min). The first 30% decrease of passive force did not affect the stretch-induced variation in Ca2+ sensitivity. Then, with further decrease in passive force, DeltapCa50 decreased. At the lowest passive force investigated 20% of initial passive force, DeltapCa50 decreased by approximately 55%. These effects were not accompanied by a significant modification of either maximal activated force at pCa 4.5 solution or pCa50 at 1.9 microm sarcomere length. This indicates that there was no major functional alteration of the contractile machinery during the protocol as also suggested by contractile and regulatory protein electrophoresis on 2.5-12% gradient and 15% SDS-PAGE gels. Thus, besides modulation induced by the reduced lattice spacing during enhanced heart refilling, Ca2+ sensitivity of the cardiac contractile machinery may be controlled at least partially by internal passive load, which is known to be largely attributable to titin.
The study was aimed at determining both passive and Ca(2+)-activated forces of single skinned rat cardiac cells. Particular attention was paid to the descending limb of the active length-tension curve while the sarcomeric order of stretched cells was investigated before and during contraction. To analyse sarcomere length and sarcomere-length inhomogeneity, a fast Fourier transform (FFT) was employed. The fundamental frequency in the FFT spectrum is a measure of sarcomere length. The full-width-half-maximum of the first-order line is a measure of sarcomere-length inhomogeneity. In relaxing buffer, the sarcomere-length inhomogeneity of skinned cells increased linearly with mean sarcomere length. Upon Ca(2+)-dependent activation of skinned cells contracting isometrically, mean sarcomere length decreased slightly and inhomogeneity increased; both effects were greater at higher Ca(2+)concentrations. Maximum activation was reached at sarcomere lengths between 2.2 and 2.4 microm, whereas the descending limb of the active length-tension curve approached zero force already at approximately 2.8 microm. This steep force decline could not be explained by overly inhomogeneous sarcomere lengths in very long, contracting cells. Rather, the results of mechanical measurements on single cardiac myofibrils implied that high stretching is accompanied by irreversible structural alterations within cardiac sarcomeres, most likely thick-filament disarray and disruption of binding sites between myosin and titin due to changes in titin's tertiary structure. Loss of a regular thick-filament organization may then impair active force generation. We conclude that the descending limb of the cardiac length-tension curve is determined both by the degree of actin-myosin overlap and by the intrinsic properties of titin filaments.
In vivo the sub-epicardial myocardium (EPI) and sub-endocardial myocardium (ENDO) operate over different ranges of sarcomere length (SL). However, it has not been previously shown whether EPI and ENDO work upon different ranges of the same or differing length-tension curves. We have compared the SL-tension relationship of intact, single ventricular EPI and ENDO myocytes from rat and ferret hearts. Cells were attached to carbon fibres of known compliance in order to stretch them and to record force at rest (passive tension) and during contractions (active tension). In both species, ENDO cells were significantly stiffer (i.e. had steeper SL-passive tension relationships) than EPI cells. Ferret ENDO cells had significantly steeper SL-active tension relationships than EPI cells; rat cells tended to behave similarly but no significant regional differences in active properties were observed. There were no inter-species differences in the active and passive properties of EPI cells, but ferret ENDO cells displayed significantly steeper passive and active SL-tension relationships than rat ENDO. We conclude that in vivo, ferret EPI and ENDO myocytes will function over different ranges of different SL-tension curves. There is a close relationship between SL and active tension (the Frank-Starling law of the heart), and our observations suggest that regional differences in the response to ventricular dilation will depend on both the change in SL and differing regional slopes of the SL-active tension curves.
The F-actin disrupter cytochalasin D depresses cardiac contractility, an effect previously ascribed to the interaction of cytochalasin D with cytoskeletal actin. We have investigated the possibility that this negative inotropic effect is due to the interaction of cytochalasin D with sarcomeric actin of the thin filament. Confocal images of Triton X-100-skinned myocytes incubated with a fluorescent conjugate of cytochalasin D revealed a longitudinally striated pattern of binding, consistent with a myofibrillar rather than cytoskeletal structure.Tension-pCa relationships were determined at sarcomere lengths (SLs) of 2.0 and 2.3 [mu]m following 2 min incubation with 1 [mu]M cytochalasin D. Cytochalasin D significantly reduced the pCa for half-maximal activation (pCa50) at both SLs. The shift in pCa50 was significantly greater at a SL of 2.3 [mu]m compared with that at a SL of 2.0 [mu]m. Cytochalasin D had no effect on the Hill co-efficient at either SL. Cytochalasin D significantly reduced the maximum tension at both SLs. We suggest that the length-dependent decrease in myofilament Ca2+ sensitivity in response to cytochalasin D is due to a decrease in the affinity of troponin C for Ca2+. Cytochalasin D has been used for many years as the agent of choice for disruption of cytoskeletal actin. However, we have demonstrated for the first time an interaction of cytochalasin D with sarcomeric actin of the thin filament, which can account for the effects of cytochalasin D on cardiac contractility.
We simulated mechanisms that increase Ca2+ transients with two models: the Luo-Rudy II model for guinea pig (GP) ventricle (GP model) representing long action potential (AP) myocytes and the rat atrial (RA) model exemplifying myocytes with short APs. The interventions were activation of stretch-gated cationic channels, increase of intracellular Na+ concentration ([Na+]i), simulated bet-adrenoceptor stimulation, and Ca2+ accumulation into the sarcoplasmic reticulum (SR). In the RA model, interventions caused an increase of AP duration. In the GP model, AP duration decreased except in the simulated beta-stimulation where it lengthened APs as in the RA model. We conclude that the changes in the APs are significantly contributed by the increase of the Ca2+ transient itself. The AP duration is controlled differently in cardiac myocytes with short and long AP durations. With short APs, an increase of the Ca2+ transient promotes an inward current via Na+/Ca2+-exchanger lengthening the AP. This effect is similar regardless of the mechanism causing the increase of the Ca2+ transient. With long APs the Ca2+ transient increase decreases the AP duration via inactivation of the L-type Ca2+ current. However, L-type current increase (as with beta-stimulation) increases the AP duration despite the simultaneous Ca2+ transient augmentation. The results explain the dispersion of AP changes in myocytes with short and long APs during interventions increasing the Ca2+ transients.
Evidence exists for a specific diabetic cardiomyopathy independent of concurrent vascular disease. Our aim was to test the hypothesis that a change in the microtubular cytoskeleton may contribute to cardiac dysfunction in type-1 diabetes. Resting sarcomere length and characteristics of unloaded shortening were measured in ventricular myocytes from rats 2 months after injection of streptozotocin (STZ). Microtubular density and organisation were assessed using immunofluorescence confocal microscopy and the effects of microtubule disruption by colchicine on shortening and microtubules were examined. Diabetic myocytes showed a significant reduction in resting sarcomere length and a 30% increase in time to peak shortening. The microtubule disruptor colchicine (10 micromol/l) had no effect on the amplitude or kinetics of shortening in myocytes from control or diabetic rats. Cardiac microtubular density and organisation were similar in control and diabetic animals, yet although colchicine significantly reduced microtubule density in control myocytes, microtubules in diabetic myocytes were resistant to its effects. These observations of an increase in microtubular stability in STZ-diabetes of 2 months duration imply a disruption to the normal balance between populations of dynamic and drug-stable microtubules. Such disruption has been observed in other pathological conditions and may contribute to diabetic cardiomyopathy.
The article concentrates on the concepts of mechanosensitive ion channels that are present in practically all cells of an organism. Considered are kinetic scheme and activation principles of mechanic-sensitive ion channels. The forces affecting those channels are discussed in detail. The qualities of the channels in lipid monolayer, bilayer and real cell membrane are under consideration. Discussed are various models that analyze possibilities of channel opening depending on the membrane tension. Under discussion are the data received from studying single channels, currents in whole-cell configuration and cloned channels built into bilayer, liposomes and membrane blebs. Problems of transmitting mechanic energy to the channel through the bilayer and through the cytoskeleton are investigated. Inhibitors and activators of mechanosensitive ion channels are mentioned and their effects are considered. The functional classification of mechanosensitive ion channels is given. Described are cation SACs, potassium SACs, Ca(2+)-sensitive and Ca(2+)-insensitive SACs, anion SACs, nonselective SACs and SICs. It is proved that mechanosensitive ion channels can produce considerable currents enough to change the cell electrogenesis.
We have observed the dynamics of sarcomere shortening and the diffracting action of single, functionally intact, unattached cardiac muscle cells enzymatically isolated from the ventricular tissue of adult rats. Sarcomere length was measured either (a) continuously by a light diffraction method or (b) by direct inspection of the cell's striated image as recorded on videotape or by cinemicroscopy (120--400 frames/s). At physiological levels of added CaCl2 (0.5--2.0 mM), many cells were quiescent (i.e., they did not beat spontaneously) and contracted in response to electrical stimulation (less than or equal to 1.0-ms pulse width). Sarcomere length in the quiescent, unstimulated cells (1.93 +/- 0.10 [SD] micrometers), at peak shortening (1.57 +/- 0.13 micrometers, n = 49), and the maximum velocity of sarcomere shortening and relengthening were comparable to previous observations in intact heart muscle preparations. The dispersion of light diffracted by the cell remained narrow, and individual striations remained distinct and laterally well registered throughout the shortening-relengthening cycle. In contrast, appreciable nonuniformity and internal buckling were seen at sarcomere lengths < 1.8 micrometers when the resting cell, embedded in gelatin, was longitudinally compressed These results indicate (a) that shortening and relengthening is characterized by uniform activation between myofibrils within the cardiac cell and (b) that physiologically significant relengthening forces in living heart muscle originate at the level of the cell rather than in extracellular connections. First-order diffracted light intensity, extremely variable during sarcomere shortening, was always greatest during midrelaxation preceding the onset of a very slow and uniform phase of sarcomere relengthening.
An efficient method for the calculation of the interactions of a 2' factorial ex- periment was introduced by Yates and is widely known by his name. The generaliza- tion to 3' was given by Box et al. (1). Good (2) generalized these methods and gave elegant algorithms for which one class of applications is the calculation of Fourier series. In their full generality, Good's methods are applicable to certain problems in which one must multiply an N-vector by an N X N matrix which can be factored into m sparse matrices, where m is proportional to log N. This results inma procedure requiring a number of operations proportional to N log N rather than N2. These methods are applied here to the calculation of complex Fourier series. They are useful in situations where the number of data points is, or can be chosen to be, a highly composite number. The algorithm is here derived and presented in a rather different form. Attention is given to the choice of N. It is also shown how special advantage can be obtained in the use of a binary computer with N = 2' and how the entire calculation can be performed within the array of N data storage locations used for the given Fourier coefficients. Consider the problem of calculating the complex Fourier series N-1 (1) X(j) = EA(k)-Wjk, j = 0 1, * ,N- 1, k=0
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.
We describe an extension of the method of Myers et al. (1982) to measure with high precision the uniformity of contractile motions that occur between sarcomeres in the isolated cardiac muscle cell (guinea pig and rat). The image of the striations, observed with modulation contrast microscopy, was detected by a linear array of photodiodes. Sarcomere length was measured greater than 500/s from the frequency of the array's video signal at two selectable regions of the cell. A precision test grating demonstrated that method resolves known differences in the spacing between two contiguous striations to +/- 0.01 micron and that the effects of image translation and microscopic resolution are minor. The distribution of striation spacing appears to be discrete in isolated segments of the cell, and patches of fairly uniform length can be identified that are laterally contiguous. When electrically triggered, contraction is synchronous and the sarcomeres shorten and relengthen smoothly. The contrast between the striations is transiently enhanced during relengthening, an indication that the contracting cell can not be treated as a simple grating. Pauses that occur during late in relengthening (and transient contractile alternans) are characterized by very synchronized activity. These forms of irregular contractile behavior are not explained by desynchronization of a mechanism of release of intracellular calcium. A companion article describes application of the technique to study the nonuniform motions that occur between sarcomeres.
Single guinea-pig ventricular myocytes were loaded with the fluorescent Ca2+ indicator Indo-1 AM and stretched by carbon fibres. Stretching increased resting tension. Sarcomere lengths were increased by 2-18%. It was observed that a stretch increased resting [Ca2+]i in seven out of eight cells. The change in [Ca2+]i increased with the size of the stretch and returned to pre-stretch levels on return to resting cell length. These observations suggest a means by which changes in resting muscle length can modify the contractile state of cardiac muscle.
A wide variety of techniques have been developed to monitor the mechanical responses of isolated cardiac myocytes. The most successful are those that measure shortening in unattached cells. Because of their relative ease of implementation, edge-detector methods of following cell displacement have become most widespread. Laser diffraction techniques have been applied to the single heart cells, and sophisticated sarcomere imaging systems capable of 2-ms time resolution of shortening responses have also been developed. Active force has been recorded in intact single cells from frog atria; however, the compliance of the force transducers was relatively higher (approximately 5% Lo). (There is an obvious trade-off between transducer sensitivity, which affects noise and drift and compliance.) Some success has been reported with the use of intact rat myocytes supported by suction micropipettes and in guinea pig ventricular myocytes adhering to poly-L-lysine-coated glass beams. With the rat preparation, contractile stress was comparable to that of ventricular muscle, but few cells survived the attachment. In guinea pig myocytes, contractile stress in electrically induced twitches was only approximately 10% of the active stress developed by mammalian trabeculae or papillary muscles at the same temperature (35 degrees C), but, as with the frog atrial transducer, the compliance of the supporting beams was relatively high. Sarcomere uniformity has not been evaluated in these intact preparations. For attachment to the relatively short mammalian cardiac myocytes, the more promising methods that better preserve sarcomere uniformity include double-barreled micropipettes coated with a barnacle adhesive; however, for nonsubmersible transducers, a continuing limitation is the problem of solution surface stability. Unfortunately, the more severe limitation to effective attachment to intact cells is still the extreme sensitivity of the sarcolemma to mechanical stress. The challenge remains to develop an attachment to the intercalated disk such that cell stress can be transferred to the supporting transducers along the normal stress-bearing cellular interface. The ultrastructural and passive mechanical data strongly indicate that although the extracellular collagen limits the extension of cardiac muscle beyond the peak of the active length-tension relation, there is also a substantial cellular component of resistance to extension. Furthermore, this cellular component is related to the cytoskeleton rather than to membranous elements in the cell. The more likely candidates for the longitudinal resting stress-bearing element are titin (connectin) and desmin.(ABSTRACT TRUNCATED AT 400 WORDS)
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 describe the construction and use of a setup that allows the rapid exchange of the solution surrounding an isolated guinea pig heart cell while simultaneously measuring the isometric force and membrane potential (Em). Cells were stably attached, by means of poly-L-lysine, to a force transducer which was adapted from one previously used for a study of frog atrial cells [N. Shepherd and F. Kavaler.Am. J. Physiol. 251 (Cell Physiol. 20): C653-C661, 1986]. The modified transducer is simple to construct and use and can be readily added to existing patch-clamp setups. The strength of attachment of a cell to the transducer exceeded the strength of the gigaseal in all of the experiments. The membrane potential was measured by means of patch electrodes and a high-impedance voltage follower. Rapidly changing extracellular K concentration [( K]o) from 5.4 to 10.8 mM caused a positive change of Em by 16.5 +/- 1.4 mV with a half-time (t1/2) of 27 +/- 4 ms. Replacing calcium in the perfusate by magnesium instantly abolished the contraction and shortened the action potential. Twitch tension returned stepwise to the control value on return of calcium to the perfusate. Our initial observations show that the patch electrode can be used successfully in conjunction with the isometric force transducer and rapid extracellular solution changes for studies of excitation and contraction coupling in isolated mammalian heart cells.
A microcomputer-based image processing system that was developed to obtain contractile properties of single heart cells is described. Cell images were videotaped while action potentials were recorded and were digitized and processed to measure sarcomere length during cell shortening. This system makes it possible to study action potentials and their associated contractions simultaneously.
To measure the mechanical activity of enzymatically isolated mammalian myocytes the principle of laser light diffraction was used. Since the viability of isolated cardiac myocytes showed a marked dependence on the laser power used, an opto-electronic system with improved light sensitivity and low susceptibility to optical noise was developed. The high sensitivity was achieved by a novel approach in the detection of diffraction patterns, that provides a significant reduction of the amount of laser power required. This improvement rendered possible the application of laser diffraction during extended experiments including pharmacological interventions. The static performance of the system, as assessed by means of calibration gratings, showed a resolution in the order of 5 nm for small changes in sarcomere length in the range from 1.2 microns to 2.0 microns. Examples of measurements on resting and contracting cells are presented, and the limitations of the application of the system to biological specimens are discussed.
Isolated cardiac muscle cells enzymatically digested with collagenase and hyaluronidase from whole rat myocardium demonstrate the characteristics of an intact membrane in that they tolerate millimolar concentrations of free Ca2+ and exhibit phasic contractions with electrical excitation. These isolated cells maintain their characteristic A-I band striation patterns when at rest or during contraction. An apparatus has been developed to directly image these cells with phase-contrast micrography onto a 1,728-element charge-coupled device photodiode array for rapid data storage in a digital computer. The digitized striation pattern profile was analyzed for individual and average sarcomere spacing. In isotonic media the average resting sarcomere length ranged from 1.77 to 1.91 micrometers in 13 cells, with a mean length of 1.83 +/- 0.12 micrometers. Electrically stimulated phasic contractions in three cells demonstrated a synchronous 20% decrease in sarcomere spacing to a mean of 1.51 micrometers. Striation spacing decreased under hypertonic stress but elongated only up to 1.93 micrometers in hypotonic solutions, suggesting that some internal elastic constraint exists that limits elongation of the cell.
Behaviour of sarcomere length was analysed in different regions of single cardiac cells (n = 249) of the ventricle, both at rest (n = 144) and during twitch contractions (n = 57). At rest, regional distribution of sarcomere length proved to be uniform. In the leaky cell (n = 48), resting sarcomere length was not affected over longer periods of time (up to 2 h), nor by lowering the ATP concentration (from 5 mM to 2.5 mM and 500 microM), nor by increasing free calcium within subactivating ranges (5, 20, 60 microM). No statistical differences could be detected between resting cell dimensions and sarcomere length between cells isolated from left and right ventricle (n = 64), nor between cells from epicardial or endocardial layers (n = 80). During twitch contraction in the intact unloaded cardiac cell (n = 32), sarcomere lengths in different regions were analysed every 20 ms and behaved synchronously, presenting arguments for uniformity during the myocardial contraction-relaxation cycle in the free-lying intact cardiac cell.
A new technique providing real-time high-speed measurements of sarcomere length from on-line analysis of the striation image has been developed. This method of measurement is not susceptible to the problems of interpretation encountered in laser diffraction. Sarcomere shortening patterns were obtained, using this method, from single toe fibres of Rana pipiens, and stepwise phenomena similar to those previously reported from laser diffraction were observed. The distribution of step size showed several peaks, the most prominent corresponding to 5.7 nm per half sarcomere.