Tension Development and Sarcomere Length in Rat Cardiac Trabeculae

Transcription

1 703 Tension Development and Sarcomere Length in Rat Cardiac Trabeculae Evidence of Length-Dependent Activation HENK E.D.J. TER KEURS, WIM H. RIJNSBURGER, ROB VAN HEUNINGEN, AND MlCHIEL J. NAGELSMIT SUMMARY We studied the influence of inotropic factors on the shape of the relation between tension and sarcomere length. Tension measurements were performed on thin trabeculae dissected from the right ventricle of the rat heart. Sarcomere length was measured by laser diffraction techniques and controlled by a servomotor system. The relations between tension and sarcomere length were derived from contractions at various extracellular calcium concentrations [Ca 2+ ] o. The time course of tension development was dependent on both sarcomere length and [Ca 2+ ] o. At all [Ca 2+ ] o, the tension attained during contraction was zero at sarcomere lengths of pm and maximal at a sarcomere length of 2.35 /im. Neither a summit nor a descending limb was found in the sarcomere length-tension relation. At [Ca 2+ ] o = 0.5 mm, tension increased linearly with sarcomere length, whereas at [Ca 2+ ] o = 2.5 mm, it approached maximal tension exponentially with sarcomere length. The relations between tension and sarcomere length derived from isometric contractions of the muscle and of sarcomeres were identical, and this suggests that shortening of sarcomeres does not contribute significantly to the effect of [Ca 2+ ] o. The relations between tension and sarcomere length obtained at [Ca 2+ ] o = 0.5 mm from contractions 30 seconds after a potentiating burst of stimuli (4 seconds at 4 Hz) were identical to the relation between tension and sarcomere length at [Ca 2+ ] o = 2.5 mm. Our results are consistent with the hypothesis that cardiac muscle length affects contractile performance by its influence on excitation contraction coupling. CircRes 46: , 1980 THE shape of the relation between tension and sarcomere length in cardiac papillary muscle suggests length-dependent activation of the contractile system (Jewell, 1977). However, there are no data on the effect of inotropic interventions on this curve. Only muscle length-tension curves are available, and these give conflicting results (Jewell, 1977; Huntsman and Stewart, 1977; Sonnenblick, 1962; Bodem et al., 1976). It is known that during isometric contractions there is considerable shortening of sarcomeres in the central region of papillary muscle. This happens at the expense of stretch of damaged regions near the clamps holding the ends of the specimen (Krueger and Pollack, 1975; Pollack and Krueger, 1976; Julian et al., 1976; Julian and Sollins, 1975). The damaged regions as well as the normal region may be influenced by inotropic factors. The interpretation of results of isometric contractions of the muscle is therefore ambiguous. The purpose of the present study was to examine the effect of different calcium concentrations on the relations From the Department of Cardiology, Experimental Cardiology Center, Medical Faculty, State University Leiden, The Netherlands. Supported by Grants and from the Netherlands Heart Foundation. Dr. ter Keurs is an Established Investigator of the Netherlands Heart Foundation. Address for reprints: Henk E.D.J. ter Keurs, Department of Cardiology, Medical Faculty, State University Leiden, Rijnsburgerweg 10, Leiden, The Netherlands. Received November 27,1978; accepted for publication January 7,1980 between tension and sarcomere length derived from contractions at constant muscle length and constant sarcomere length. Methods Preparation and Mounting Procedures Twelve-week-old Wistar rats were anesthetized with ether. The heart was rapidly removed and transferred to a dissection chamber. The aorta was cannulated and perfused with oxygenated physiological salt solution at a flow rate of 5 ml/min. The right ventricle and atrium were opened, and the free wall of the right ventricle was exposed. Papillary muscles were removed. Trabeculae attached to the atrioventricular (AV) ring and the free wall of the right ventricle were selected. One suitable trabecula could be found in approximately half of the hearts. Ventricular and atrial tissue was dissected carefully from the AV ring, thus freeing a part of the tricuspid valve, the AV ring, and the trabecula. The part of the ventricular wall to which the trabecula was attached was dissected. The trabecula next was transported to an experimental chamber mounted on the stage of an inverted Zeiss microscope (Fig. 1). The tricuspid valve and the flat part of right ventricle at the base of the trabecula were mounted in spring-loaded stainless steel clips. The specimen was remounted repeatedly such that all

2 704 CIRCULATION RESEARCH VOL. 46, No. 5, MAY 1980 oisnicum miioutfi mem KOTO DEIECIOI FIGURE 1 Diagram of the experimental setup. The muscle and entering laser beam were observed by means of a microscope. Light of the first order of the diffraction pattern was deflected onto two photodetector systems. The light of the first order passed through Fourier lenses between the muscle and the detectors. It was compressed onto the detectors along the length of the bands by a cylindrical lens and a cylindrical mirror, respectively. The positions of the median and mean of the first order intensity distributions are converted to median and mean sarcomere length, respectively, in the detector systems. Force is measured with a capacitive force transducer. Muscle length is measured and controlled by a servomotor system with length transducer. cell strands throughout the cross-section of the muscle had equal lengths at all muscle lengths. Consequently the preparation, which usually had the shape of a ribbon, did not move in the horizontal plane, but bent uniformly downward during passive shortening below the slack length. The dimensions of the trabeculae reported here were as follows: thickness, 80 ± 15 (SD) /an; width, 200 ± 50 /mi; n = 15. They were unbranched and uniformly shaped along nearly their entire length of more than 2.5 mm. Perfusion Solutions Solutions used during dissection and during experiments consisted of the following components in HIM: Na +, 147.4; K +, 5.0; Cl", 98.5; Mg 2 *, 1.2; H 2 P<V, 2.0; SO, 2 ", 1.2; acetate, 23.9; HCO 3 ", 28.0; glucose, The calcium concentration varied between 0.25 and 4.5 HIM, as will be indicated in the results. The solutions were equilibrated with 95% O2 and 5% CO2; the ph in the perfusion chamber was 7.41; Pco 2, 5.3 kpa; Po 2, 80 kpa. The volume of the chamber was 2 ml and the flow-rate through it 2 ml/min. Temperature of the fluid in the chamber was kept constant at 25.0 C. Under these conditions, stable responses were observed for experiments lasting 6 hours and longer. Measurements of Force and Muscle Length Force was measured with a capacitive force transducer connected to a reactance converter (Disa 51 E01). The moving plate of the force transducer was connected through a lightweight plastic arm to the muscle clip. The sensitivity of the transducer was 0.1 V/mN; linearity, 5% up to 10 mn; drift, 0.2 mn/hr; resonant frequency, 460 Hz; compliance, 0.1 jum/mn. Forces measured in different muscles were normalized at a sarcomere length of 2.00 fim and at [Ca 2+ ] o = 2.5 mm. Tension developed by the trabeculae was computed from the force and cross-sectional areas at a sarcomere length of 2.00 (im. Muscle length was measured by means of a variable mutual inductance displacement transducer (Metrisite) incorporated in a conventional servomotor system (Brush pen motor ). A plastic arm connected the vertical motor axis to the steel clip. The compliance of the arm and motor was 0.8 jiim/mn. Muscle force and length were recorded on a chart recorder (Gould Brush 440) and storage oscilloscope and hard copy unit (Tektronix 5103,613,4631). Sarcomere Length Measurement Microscopy The muscle was contained in a glass-covered chamber, which allowed simultaneous microscopic and laser diffraction measurements. For observation or photography of sarcomeres at high magnification, Kdhlers illumination was used with the aid of fiber optics which served as the light source for the modified Zeiss objective (Zeiss 14612) which was used as condenser. A clear image of the sarcomeres could be obtained with the use of a 40x objective with long-working distance (Zeiss ). In five experiments, sarcomeres were photographed during diastole on Ilford HP4 film using a Zeiss microcamera (C 35). Enlargements of these photographs enabled us to measure sarcomere lengths by dividing the total length of sarcomeres by the number of sarcomeres. Calibrations were obtained from a Zeiss stage micrometer, commercial phase gratings, or transmission gratings (Dijkstra and Landwaard, 1975) that spanned the physiological range of striation spacings. Sarcomere Length and Light Diffraction Figure 1 illustrates the methods used in this study. The trabecula acts as a grating and diffracts

3 LENGTH^DEPENDENT ACTIVATION OF CARDIAC MUSCLE/ter Keurs et al. 705 the incident light of a laser beam into a zero order band and multiple symmetrical higher order band pairs. The spacing (d) between the bands is related. uniquely to sarcomere length (SL), given by: SL = K X/d, where K is constant, and X is the wavelength of the laser light ( pm) (Goodman, 1968). There is a considerable spread of light under the principal orders of diffraction pattern of cardiac tissue (Krueger and Pollack, 1975; Pollack and Krueger, 1976; Nassar et al., 1974) due to scatter by noncontractile elements in the muscle and to nonuniformity of the sarcomere lengths. Consequently, the first order straddles the skirt of the zero order. Sarcomere length was computed from the intensity distribution after correction for the contribution of scattered light under the first order. The intensity distribution of one first order was scanned 4 times per millisecond by a photodiode array (Reticon 256 EC). Each scan generated a profile of intensity along the array. Polaroid photographs of the intensity distribution were taken (Hewlett Packard CRO Camera, Philips CRO PM3110) at rest and at 40-msec intervals during contraction. The intensity distribution of the zero order at both sides of the first order was measured. From this, the zero order intensity under the first order was approximated by exponential interpolation (Iwazumi and Pollack, 1979). Then the position of the median of the first order intensity distribution above the zero order was determined. From this position, the median sarcomere length could be calculated with the use of a calibration curve, obtained with the aid of phase and transmission gratings positioned in the muscle plane. In a later stage of the experiments, sarcomere length was computed electronically with the aid of a computation circuit modified after Iwazumi (Iwazumi and Pollack, 1979; R Van Heuningen, WH Rijnsburger, HEDJ ter Keurs, unpublished observations), which performed the operations described above during each scan of the photodiode array. Calibration of the calculating circuitry was performed with test gratings in every experiment. The calibration error was smaller than 1%. The resolution of the measurement was limited by noise to 5 nm optimally and 20 nm nominally in cardiac trabeculae. The other first order of the diffraction pattern was deflected onto a position-sensitive photodetector (United Detector Technology LSC 4) (Iwazumi and Pollack, 1979). Due to the "lateral photo effect," the difference and sum of detector output currents are related directly to first and zero moment of the distribution of light impinging on the detector, respectively. The zero and first moment of the zero order intensity distribution were calculated with the aid of photodiodes placed at both ends of the detector and a network which simulated a parabolic decay of the zero order light. The curvature of the simulated decay could be adjusted with one parameter. After subtraction of the approximated zero order contribution, the ratios of first and zero moment of the intensity distribution of the first order band were calculated. This yielded the position of the center of gravity of the first order light distribution, which was converted to sarcomere length with the use of the same transfer function as used in the photodiode array system. Calibration of both systems was similar. Frequency response of the lateral photodetector was 23 khz, and the error due to noise and drift typically was 30 nm. Maximal nonunearity of the system was 10 nm. The error due to intensity variations of a factor x5 was less than 1 nm. The difference in sarcomere length computed by the two systems and three observers was less than 3%. Dynamic Control of Sarcomere Length For the purpose of this study, we attempted to eliminate sarcomere length changes by modifying the servomotor system to allow direct feedback control of sarcomere length. Interchange of the muscle length signal for sarcomere length signal could be obtained by potentiometric selection of either one of the signals for feedback. The signal levels were adjusted so that critical damping in any mode of control was obtained prior to switching from one feedback mode to any other. A correction amplifier was used to optimize the open loop frequency characteristic of the system. Characteristics of the performance of system were as follows: step response rise time, 3 msec; frequency response, 3 db, 200 Hz; overshoot, 8%. Experimental Protocol After the muscle had been mounted, a region that did not move longitudinally was selected for diffraction study. The trabeculae usually were stimulated at a rate of 0.2 Hz by two silver electrodes parallel to the muscle. Stimulus intensity was 50% above threshold, and the stimulus duration was 5 msec. The data were first obtained from contractions in a medium containing a calcium concentration of 2.5 mm. The muscle was kept at reference length (sarcomere length, 2.10 jum) for contractions between series of three test contractions. Peak force for relations between tension and sarcomere length was derived from the third contraction of each test series. The results from the first and second contractions were similar. Test lengths were chosen in random order. Tension developed during the test contractions was highly reproducible with this procedure (Jewell and Rovell, 1973). Perfusion then was changed to solutions containing calcium concentrations of 1.5,1.0, 0.5, or 0.25, and occasionally of higher than 2.5 mm. Each series of measurements was performed after tension had attained a constant value i.e., approximately 10 minutes after a change of solution. In between different measurements at test calcium concentrations, control measurements were obtained at [Ca 2+ ] o = 2.5 mm. Data were accepted only if these controls varied less than 10%.

4 706 CIRCULATION RESEARCH VOL. 46, No. 5, MAY 1980 In a number of experiments, the effects of frequency potentiation on the relation between tension and sarcomere length was studied. The potentiating procedure consisted of giving a burst of 20 stimuli within 4 seconds, and then a test stimulus was delivered to the muscle after a pause, usually of 30 seconds. Muscle length was kept at reference length during the train of 20 stimuli and was changed to test lengths immediately prior to the test stimulus. Results General Properties of Trabeculae The properties of a typical trabecula are illustrated in Figure 2. The thin ribbon shape of the trabeculae made it possible to observe sarcomeres along most of the length of the muscle. The presence of atrial and ventricular remnants precluded observations very close to the AV ring. The ventricular remnants sometimes prevented observation of the insertion into the ventricular wall. The sarcomeres near the AV ring and those located more centrally in the trabeculae always exhibited uniform shortening behavior during contraction, and their length at rest varied only slightly. Uniform shortening (see Fig. 2) was observed along the trabecula to within a distance of about 500 /mi from the insertion of the trabecula into the ventricular myocardium. The latter region exhibited frequent irregular contractions of sarcomeres in individual cells and did not respond to stimuli. The clarity of the crossstriations in this region gradually disappeared during the first 30 minutes after dissection until only an opaque area remained. By then, distinct stretch of the region adjacent to the attached clip was observed, but there was little variation in length of this region from one experiment to another. Regions with similar properties were found invariably in the vicinity of cut branches and also in other areas of obvious damage in trabeculae which were rejected from this study. In earlier experiments, it appeared that such areas developed anew after excessive stretch, especially in the neighborhood of the opaque region. An area in between the opaque area and the uniform region in some trabeculae exhibited short resting sarcomere lengths, little shortening during contraction, and early relaxation with varying degrees of stretch of the relaxing sarcomeres. Resting sarcomere length of 2.16 pm was uniform throughout 50% of the visible sarcomeres in the muscle. Sarcomere length decreased toward both ends of the muscle. The sarcomeres shortened during contraction, on average by 12.4% to 1.86 jum, at the expense of stretch of the regions near the clamps. The resting length of the opaque region was 260 fan; that of the tricuspid valve region was 140 /im. Their total length increased during a contraction by about 200 jun as measured from displacement of natural markers. An area in the central region of the trabeculae, which did not move lon n SARCOMERE LENGTH ms FIGURE 2 A composite microphotograph of a typical trabecula is shown in the middle panel. The trabecula was 2300 pm long and 180 jim wide (at region 5) and 90 fim thick. Sarcomere length recorded at position 1 and 2, 3-6, and 7-10, respectively, together with twitch force records, are shown in the top panel. Length of sarcomeres at rest could also be measured closer to the muscle clamps. Bottom panel shows the microscopically observed resting length of compliant ends (indicated by the bars) and their stretch during contraction (arrows), together with the resting and the active sarcomere length distribution along the trabecula. gitudinally upon contraction, was selected for studying the dependence of tension development on sarcomere length. Records of isometric tension development (see Fig. 2) show a slow rise of tension to a maximum 150 msec after onset of contraction followed by a gradual decay and then a relaxation phase. The sarcomere length changes that occur during contraction in the central region of the muscle consist of abrupt shortening at a high initial velocity. The velocity of shortening gradually decreased during contraction. Shortening persisted at a low rate until after peak force had been attained. Sarcomere lengthening occurred abruptly and usually coincided with an increase in the rate of force relaxation (see Figs. 2 and 4). An increase of SL dispersion along the fibers was found in some muscles during relaxation, but this was less pronounced than in skeletal muscle fibers (Cleworth and Edman, 1972). Stepwise sarcomere shortening was observed regularly, but it was not studied in detail (Pollack et al., 1977). Sarcomere length-muscle length relations for the

5 LENGTH-DEPENDENT ACTIVATION OF CARDIAC MUSCLE/ter Keurs et al. 707 resting and active muscle (Fig. 3) were similar to those of papillary muscle (Krueger and Pollack, 1975; Pollack and Krueger, 1976; Pollack and Huntsman, 1974). Sarcomere length at rest (SLR) was related to muscle length in a linear manner between 0.8 and 0.95 L ma)[. The muscles appeared to buckle below 0.8 L^* at which SLR remained constant at about 1.85 /im. At muscle lengths greater than 0.95 Lmax, SL R hardly increased if the muscle was stretched. We never found SLR to be larger than 2.39 jum, even at muscle lengths exceeding 1.3 L m ax. The amount of shortening of the sarcomeres during contraction varied 7-18% depending on muscle length (see Fig. 3). Maximal shortening was found at SLR range of jum. The central region apparently is much less compliant than the end region at rest, as no length changes of the central region were observed, even though passive FORCE MUSCLE LENGTH J 1.5 irr FIGURE 3 Shows the relations between muscle lengthsarcomere length and force in a representative trabecula. Muscle length is expressed relative to L max (horizontal calibration). Muscle length (lower trace, calibration bar at the right side) was varied between 4.5 mm (0.75 L m ax), at which length the muscle just buckled, and 6.5 mm (1.06 L ma x). The muscle was ribbon-shaped and had a width of 225 [im and a thickness of 90 fim at L max. Sarcomere length (middle trace, calibration indicated from 1.5 to 2.3 fim at the right side) increased linearly from 1.82 to 2.25 fan, with a length increase of the muscle at rest from 0.75 L max to 0.97 L ma *. Sarcomere length increased much less, i.e., from 2.25 to 2.28 nm, if the muscle was further stretched to 1.06 Lmax. Stretch of the damaged ends of the preparation accounted for the difference between stretch of the muscle and total stretch of the sarcomeres. Active force development during twitches, which were elicited at a rate of 0.2 Hz, increases with muscle length to L m<tc. The apparent decrease of actively developed force beyond L m ax occurred while the passive element parallel to the shortening sarcomeres was released. In the following figures, force (or tension), therefore, will be expressed as total peak force minus the passive force which would exist at the same sarcomere length as the sarcomere length which exists at peak force. tension exceeded maximal active tension by a factor of X5 when the muscle was stretched beyond L max. The ventricular end exhibited most of the stretch during lengthening of the whole muscle beyond 0.95 Tension Development and Sarcomere Length It is evident from previous studies and the results described that the interpretation of tension development at various sarcomere lengths measured during isometric contractions of the muscle may be affected by shortening of sarcomeres during contraction. Elimination of sarcomere shortening would give unambiguous information on both the time course of a truly isometric contraction and on the relation between tension and sarcomere length. We chose dynamic sarcomere length control to obtain information on the complete time course of tension developed by sarcomeres under isometric conditions. Tension development at constant muscle length and at constant sarcomere length were compared (Fig. 4). The contractions at constant muscle length showed the features already described. Elimination of sarcomere shortening led to a considerably steeper rise of tension during the early phase of tension development. Time-to-peak tension diminished from 150 to msec, and at all sarcomere lengths, the total duration of contraction increased. At sarcomere lengths above 2.15 jum, the tension developed at constant sarcomere length reached a plateau that lasted msec, and tension then declined. A rapid and approximately exponential relaxation followed a slow initial decline. A monophasic relaxation occurred at shorter sarcomere lengths. Possible sources of error in this measurement of tension at constant sarcomere length are: (1) the introduction of length fluctuations as a consequence of the procedure for controlling sarcomere length, and (2) transient initial shortening of sarcomeres and longitudinal movement of the illuminated region during contraction. Figure 5 shows clearly that fluctuations in the sarcomere length signal during feedback control of sarcomere length do occur and cause fluctuations of developed tension. No deactivation of the contractile mechanism appeared to occur, because the average developed tension at a given sarcomere length was constant, even though tension fluctuations up to 15% of total tension could be observed. The optical and mechanical properties of the muscle, together with the upper limit of the frequency response of the servo system, determine the amplitude and duration of a transient initial shortening of the sarcomere. In three muscles, the transient shortening was negligible (i.e., 0.01 jum over 20 msec) (see Fig. 4). We observed somewhat larger transients in four other muscles studied. The effect of a large transient shortening was a depression of the initial rate of rise of tension. The timeto-peak tension, therefore, is probably underesti-

6 708 CIRCULATION RESEARCH VOL. 46, No. 5, MAY 1980 FIGURE 4 Force development (F) during contractions in which either muscle length or sarcomere length (SL) were kept constant. Four similar panels show contractions which started at sarcomere lengths of 1.90 (upper left), 2.00 (lower left), 2.10 (upper right), and 2.20 /tro (lower right). The upper trace in each panel shows sarcomere length measured by the lateral photodetector system during isometric contraction of the sarcomeres. The second trace from top: sarcomere length measured by the photodiode array system. This signal was used for feedback to the servomotor. The signals were shifted slightly to allow comparison. The third trace shows sarcomere length changes during isometric contractions of the muscle which started at the same sarcomere lengths. The lower two traces in each panel show active force development during muscle isometric (bottom trace) and sarcomere isometric contractions (top trace). Calibration of sarcomere length [0.1 fim per division (div)] and force is indicated. Timescale for all panels is 100 msec/div. Cross-sectional area of the fiber was 0.16 mm 2. Control of sarcomere length leads to an increase in the rate of rise of tension; time-to-peak tension decreases from 150 msec to less than 60 msec. Peak tension decays slowly or remains at the plateau level for approximately 200 msec. Relaxation is slower during contractions at constant sarcomere length than during contractions at constant muscle length. The time course of tension during the rising phase is affected by early transient shortening, but tension is influenced very little by small length fluctuations during the contraction (upper left panel). Sarcomere length control during contractions at large sarcomere length frequently leads to after-contractions, as is shown in the lower right panel. mated in this study. Sarcomere length and tension data were obtained from regions that were selected because they did not move longitudinally during contraction. The sarcomere length signal derived from the same region also was used as the feedback signal during sarcomere length control. One would expect that, if one motor arm stretches the muscle such that sarcomere length stays constant, a different pattern of longitudinal translation could occur. It is likely, therefore, that the area which is stationary during isometric contractions of the muscle will translate relative to the laser beam during contractions in which sarcomere length was kept constant. Data for tension development under these circumstances are a valid measure of the ability of sarcomeres to develop tension only if the properties of the sarcomeres in the neighboring regions are identical. We tested the assumption of uniformity by studying tension development during contractions in which sarcomere length was kept constant. In between contractions (see Fig. 5), the region of illumination was altered. We found no change in the time course of tension development when the illuminated region was intentionally translated over 600 jum. It seems reasonable, therefore, to assume uniformity of sarcomere behavior in the central regions of these trabeculae. Calcium, Sarcomere Length, and Tension Development The behavior of the normal and damaged regions described above (Figs. 2 and 3) suggests that it is reasonable to assume that the length of the elements bearing passive tension and sarcomere length are tightly coupled. Therefore, the tension developed by the sarcomeres can be taken as the total tension minus the passive tension borne at the sarcomere length measured at peak tension. Figure 6, A and B, shows the relations between tension and sarcomere length derived from 12 trabeculae at [Ca 2+ ] o = 2.5 and 0.5 mm, respectively. Externally developed tension is zero at a sarcomere length of 1.58 /un and increases with increasing sarcomere length. In Figure 6A, it is obvious that the relation does not exhibit a descending limb. Lowering of the calcium concentration without a change of the rate of stimulation caused a reduction of the rate of rise of tension and of peak tension during a contraction. The resulting relation between sarcomere length and total tension for seven experiments at [Ca 2+ ] o = 0.5 mm, shown in Figure 6B, was approximately linear. It shows the strong length dependence of the effect of a change of the extracellular calcium concentration for isometric contractions of the muscle. The sarcomeres located in the central region of the trabecula appeared to shorten more at low calcium concentrations (see Table 1) than at high calcium concentrations, despite the fact that they generated less tension. The excess shortening occurred early during relaxation. The extent to which the relation between tension and sarcomere length was influenced by the shortening of sarcomeres, which occurs early during fixed end contraction of the muscle, was studied further by comparing the tension developed in this way with tension developed during a contraction at constant sarcomere length. The results of such a comparison in three muscles at [Ca 2+ ] o = 2.5 mm and [Ca 2+ ] o = 0.5 mm are shown in Figure 7. Sarcomere lengths below 1.8 jum (i.e., below the minimum value attainable in resting muscle) were kept

7 LENGTH-DEPENDENT ACTIVATION OF CARDIAC MUSCLE/ter Keurs et al. 709 FIGURE 5 Force and sarcomere length uniformity during contraction. All contractions started at a sarcomere length of 2.15 fim. Sarcomere length measurements were performed in five adjacent regions covering about 750 itm of the length of the trabecula, as shown in the upper panel. The left lower panel shows sarcomere length changes in each region measured with the photodiode array system (upper trace), and force (lower trace) occurring during contraction elicited at constant muscle length. Note that the sarcomere length records superimpose. The right lower panel shows sarcomere length measured in the same regions with the photodiode array system (top trace) and with the lateral photodetector system (middle trace) during contractions at constant sarcomere length. The signal from the lateral photodetector system was selected for feedback; the other system served as verification of the result of feedback. Force is recorded on the bottom trace. The force records superimpose, although the sarcomere length signal which was used for control was obtained from a different region in each contraction. In the middle lower panel, sarcomere length measured with the photodiode array system (top trace) and with the lateral photodetector system (middle trace), during contractions in which sarcomere length was kept constant with the aid of the signal from the lateral photodetector system, is shown together with force on the bottom trace. The first contraction was started while sarcomere length was measured at the outer left position indicated in the muscle diagram; the second, while sarcomere length was measured at the outer right position. During the third contraction, the measuring area was moved from the outer right to the outer left position. It is clear that, in this muscle region, longitudinal translation does not affect the resultant force development. constant after the sarcomeres had shortened from the resting lengths to a preset length that was maintained during contraction. Frequency Potentiation and Tension Development at Low Calcium Concentration It has been shown that rat cardiac muscle is more sensitive to the rate of stimulation at low external calcium concentrations (e.g., 0.5 nw) than at high external calcium concentrations (e.g., equal to or higher than 2.5 mm) (Forester and Mainwood, 1974). The difference in response of the trabeculae to frequency potentiation at low (0.5 mm) and high (2.5 HIM) external calcium concentrations was used to study whether the extracellular calcium concentration has a direct influence on the shape of the relation between tension and sarcomere length, or whether [Ca 2+ ] o exerts its influence through the calcium turnover within the cell. Tension development after frequency potentiation was studied in three muscles. The results are shown in Figure 8. Test contractions were elicited at various times after a conditioning series of 20 stimuli delivered within 4 seconds. Tension of the test contraction at low [Ca 2+ ] o increased rapidly with the duration of the interval between end of the stimulus train and the test contraction. Peak tension of the test contraction was maximal after an interval of 10 seconds and then remained constant for at least 120 seconds. Maximal peak tension of the test contraction attained with this procedure at [Ca 2+ ] o = 0.5 mm was identical to the tension developed at comparable sarcomere length at [Ca 2+ ] o = 2.5 mm. Also, the time course of tension development in maximally potentiated contractions at [Ca 2+ ] 0 = 0.5 mm was identical to those of contractions at [Ca 2+ ] o = 2.5 mm. Peak tension decayed (see Fig. 8, inset) to the control value in a beat-dependent manner during the course of 10 contractions. It was noteworthy that, although developed tension increased with frequency potentiation, the amount of sarcomere shortening in the potentiated

8 710 CIRCULATION RESEARCH VOL. 46, No. 5, MAY A TABLE 1 Sarcomere Shortening and [Ca 2+ J o SL rest {fira) ASL [Ca 2+ ] o = 2.5 (jim ± SD) ASL [Ca 2 *] o = 0.5 (/un ± SD) n 100- mn.mrrt Tension ± ± ± ± ± ± ± ± ± ± ' 8* 5' Mean sarcomere shortening during isometric contractions of a muscle in high external calcium concentration was less than at low external calcium concentration, although developed force was larger than at low [Ca 2+ ] o. *P s 0.05 in paired t-tests " 50- '»/ " * / " 2 A jjm contractions diminished (Fig. 8, inset), indicating that the compliance of the damaged regions had decreased. The relations between tension and sarcomere length obtained from frequency-potentiated contractions both at [Ca 2+ ] o = 2.5 mm and at [Ca 2+ ] o = 0.5 mm are shown in Figure 8. Potentiation.had no effect on developed tension at an external calcium concentration of 2.5 mm as was found previously (Forester and Mainwood, 1974). The relation between tension and sarcomere length obtained from potentiated contractions at [Ca 2+ ] o = 2.5 mm was identical to the relation at [Ca 2+ ] o = 2.5 mm at a stimulus rate of 0.2 Hz. The relation between tension and sarcomere length of potentiated contractions at [Ca 2+ ] o = 0.5 mm is identical to the relations between tension and sarcomere length obtained at [Ca 2+ ] o = 2.5 mm. Discussion Several mechanisms have been proposed to explain the rapid decline of tension with decreasing sarcomere length observed in cardiac muscle (Krueger and Pollack, 1975; Pollack and Krueger, 1976; Julian et al., 1976; Julian and Sollins, 1975). These mechanisms include length-dependent activation (Jewell, 1977; Bodem et al., 1976), internal loads, and opposing forces (Jewell, 1977). Studies of the influence of inotropic interventions on the relation between tension and muscle length undertaken to unravel the contribution of these factors (Allen et al., 1977; Jewell, 1977; Huntsman and Stewart, 1977; Sonnenblick, 1962; Bodem et al., 1976) have not been conclusive so far. This may be due to the fact that the sarcomeres in the central region of cardiac muscle preparations shorten at the expense of stretch of damaged regions near the clamps (Krueger and Pollack, 1975; Pollack and Krueger, 1976; Julian et al., 1976; Julian and Sollins, 1975; Krueger Sarcomere length FIGURE 6 A: TAe relation between the active and passive tension in the muscle and sarcomere length measured during isometric contraction. Active tension is expressed as total tension minus passive tension borne at the sarcomere length which is measured at peak tension. Pooled data from 12 muscles at [Ca 2+ ] o = 2.5 mm. Passive tension data are indicated (small dots). The line drawn through the total tension data fits an exponential relation: T = 255 [1 - exp( SL)] mn/ mm' 2. B: Pooled data for eight muscles at [Ca 2+ ] o = 0.5 mm. Data of these muscles are shown in Figure 7 as well. A linear relation fits the active tension data well. No change of the relation between passive tension (small dots) and length was detected with varied [Ca 2 *] o.

9 LENGTH-DEPENDENT ACTIVATION OF CARDIAC MUSCLE/ter Keurs et al _2 mn.mm' TENSION Sarcomere length FIGURE 7 The relations between tension and sarcomere length at [Ca 2+ ] o = 2.5 m.m (circles) and 0.5 mm (triangles) obtained from three muscles. The relations between active tension and length from contractions in which sarcomere length was held constant (filled symbols) did not differ from the sarcomere length-total tension relations derived from isometric contractions of the muscle (unfilled symbols) during which 8-14% shortening occurred. and Strobeck, 1978; Pollack et al., 1977) and inotropic interventions influence both the central sarcomeres and the damaged regions. The experiments on trabeculae in this study show shortening of sarcomeres in a uniform central region of the specimen (Figs. 2 and 4) at the expense of damaged regions near the clamps. Elimination of sarcomere shortening revealed two features of the mechanisms involved in contraction: (1) tension development consists of a rapid rise of tension to a maximum attained within 60 msec followed by a tension plateau which precedes a gradual decline of tension; (2) the relation between peak tension and sarcomere length depends on the instantaneous sarcomere length (Fig. 7), and on the inotropic state of the muscle. Three earlier studies of tension development during contractions in which the length of the contractile element was held constant are well known. Brady (1971) used an ingenious technique in which the relation between length and tension of the series elastic element was measured first. Contractile element length was kept constant during subsequent 16 2b 2.4 pm * SARCOMERE LENGTH FIGURE 8 Relation between passive and active tension and sarcomere length from isometric contractions of the muscle at [Ca 2+ ] o = 2.5 and 0.5 mm at low rate of stimulation and after frequency potentiation in two muscles. The relations were derived from measurements in the following order: high [Ca 2 *] o and a low rate of stimulation (unfilled circles), high [Ca 2+ ] o after frequency potentiation (filled squares), low [Ca 2+ ] o and a low rate of stimulation (unfilled triangles), low [Ca 2+ ] o following frequency potentiation (asterisks), high [Ca 2+ J o and a low rate of stimulation following the series at low [Ca 2+ ] o (unfilled squares). The response to frequency potentiation at low [Ca 2 *] o is illustrated in the inset. Upper trace: force (F) 0.1 mn/div, negative deflections are event markers; lower trace: sarcomere length (SL) 0.02 [im/div, the bar indicates 2.00 nm, upward deflections indicate shortening; time base: 20 sec/div. A series of 20 stimuli is delivered in 4 seconds followed by a 30- second period of rest. The next contraction starts at SL = 2.15 iim and develops 220% of the force at low rate of stimulation; shortening is 0.08 \an less than control. The second stimulus is again delivered after an interval of 30 seconds and is followed by stimuli at intervals of 5 seconds. Tension decays to control value in about 10 beats. The relation between tension and sarcomere length following potentiation at low [Ca*] o (asterisks) is identical to the relations at high [Ca 2+ ] o. contractions by conversion of the measured tension to series elastic element length changes imposed on the muscle by a computer-controlled muscle puller. Julian et al. (1976) used the spot follower technique (Gordon et al., 1966) to control central segment length of rabbit papillary muscle dynamically, and

10 712 CIRCULATION RESEARCH VOL. 46, No. 5, MAY 1980 Pollack and Krueger (1976) compensated for sarcomere shortening by applying a carefully adjusted stretch to a rat papillary muscle during the initial phase of the contraction. The absence of sarcomere length changes was verified simultaneously by means of diffraction techniques. The results obtained in these three studies, although on different species, all showed a rapid rise of tension followed by a quasi-plateau, as observed in our experiments. The relation between tension and sarcomere length at [Ca 2+ ] o = 2.5 mm in this study shows an intercept with the sarcomere length axis at about 1.60 /tin and a near exponential increase of tension with sarcomere length toward a maximal tension at 2.3 /xm. The relation, which is obtained from contractions at constant muscle length, is similar to that obtained from contractions during which small random movements occur while sarcomere length is held constant (Fig. 7). It corresponds closely to that obtained previously (Krueger and Pollack, 1975) in rat papillary muscle. Maximal active tension is slightly higher than found in studies on adult rat papillary muscle and twice that in papillary muscle of very young rats (Julian and Sollins, 1975). We found considerably higher values for the average minimal sarcomere lengths at rest than Julian and Sollins (1975) (1.83 /im vs /im), and during contraction (1.58 vs. 1.4 jum). The difference possibly could be due to differences in method; Julian and Sollins found that it was necessary to repetitively stimulate the muscle at short length to make the muscle stay taut at short lengths, whereas, in this study, only three test contractions were used, and these yielded identical results at short lengths. Another cause of difference could be the fact that we studied adult rats, whereas Julian and Sollins used 16-day-old rats, although it has been suggested (Uchino and Tsuboi, 1970) that rat hearts reach functional maturity in the first week of life. The relation between tension and sarcomere length at high [Ca 2+ ] o is similar to the relation between relative tension and sarcomere length obtained in skinned rat heart cells that were activated by calcium-induced calcium release from the sarcoplasmic reticulum (Fabiato and Fabiato, 1975). The relations differ only at the short sarcomere lengths (i.e., below 1.65 [im). The relations between sarcomere length and tension observed by us contrast with the observation on skinned rat heart cells that tension developed following direct activation is nearly independent of sarcomere length between 1.6 and 2.3 jtun (Fabiato and Fabiato, 1975, 1976). A decrease of [Ca 2+ ] o at a low stimulus rate leads to decreased activation at all sarcomere lengths (Figs. 6 and 7). The effect of varied external calcium concentrations and frequency potentiation on maximal developed tension depends on sarcomere length (Figs. 6-8). This effect has been observed previously (Jewell, 1977; Huntsman and Stewart, 1977; Lakatta and Jewell, 1977; Bodem et al., 1976) in the relation between tension and muscle length and is consistent with the hypothesis that activation depends on length (Jewell, 1977). Other hypotheses are that the relation is determined by the effect of opposing forces or by the effects of the external calcium concentration on the damaged ends. An influence of varying [Ca 2+ ] o and frequency potentiation on the properties of the damaged ends could be inferred from the sarcomere length changes in this study (Table 1; Fig. 8). Similar changes of compliance of the damaged ends with lowering of [Ca 2+ ] o or with frequency potentiation may have been of importance in the variability of the relation between tension and muscle length found previously (Bodem et al., 1976). However, the direct measurements of the change of shape of the relation between tension and sarcomere length in our studies obviously eliminate the possibility that the result seen could be caused solely by the effect of external calcium on the damaged ends. Passive restoring forces obviously must be present in cardiac cells shorter than slack length as the sarcomeres elongate from 1.6 /un length, which exists during active contraction, back to resting length of 1.85 jum without an externally applied force. It is not likely, however, that these forces, which can only play a role below that sarcomere length, depend on the external calcium concentration and thereby change the shape of the relation between tension and sarcomere length (Jewell, 1977). Activation of the contractile apparatus results from binding of calcium by troponin (Ebashi et al., 1969). The activator calcium is released into the sarcoplasm by the sarcolemma and sarcoplasmic reticulum as a result of the action potential (Reuter, 1975; Langer, 1973). The contribution of calcium from the sarcolemma probably is small in rat (Mainwood and Lee, 1969). Changes of activation resulting from some inotropic interventions are thought to be mediated by phosphorylation of proteins involved in calcium storage and release or by phosphorylation of the contractile proteins, e.g., troponin (Drummond and Severson, 1979). Length dependence of calcium binding (England, 1976), calcium release (Ridgway and Gordon, 1975) or calcium storage, or modification of any of these by phosphorylation of the proteins involved, could underlie length dependence of activation. Evidence of changes of phosphorylation as a result of frequency potentiation or changes of Ca 2+ has not been found however (Ezrailson et al., 1977). The calcium release process has been studied in detail in skinned rat cardiac muscle preparations by Fabiato and Fabiato (1976). Their results suggest that the release of calcium by the sarcoplasmic reticulum may contribute to length dependence of activation. Although no conclusive evidence can be obtained from these experiments, our observations do suggest that both calcium release from the sarco-

11 LENGTH-DEPENDENT ACTIVATION OF CARDIAC MUSCLE/ ter Keurs et al. 713 plasmic reticulum and calcium binding by the myofilaments underlie length dependence of activation. The contribution of calcium influx through the sarcolemma during the action potential is less likely, as may be concluded from the effect of frequency potentiation (see Fig. 8). The large observed difference in shape of the relation between potentiated tension and sarcomere length at low [Ca 2+ ] o and the control relation at low [Ca + ] o suggests that [Ca 2+ ] o, which influences the calcium influx, does not affect the shape of the relation directly. This is in agreement with the conclusion that the action potential in rat ventricle mainly serves as a trigger of contraction (Mainwood and Lee, 1969). It is likely that the positive inotropic effects of an increase of [Ca 2+ ] o, and of frequency potentiation, result from enhanced loading of intracallular release sites. This also is suggested by the time course of increase of tension following the period of stimulation at a high rate and the long time-constant of decay of the potentiated tension (Allen et al., 1976; Lakatta and Jewell, 1977). The influence of length on the loading process as a possible contributor to length-dependent activation is unlikely, as potentiation in our experiments (Fig. 8) was elicited at a fixed reference length during the stimulus series, and the muscle length was not changed until just before the test contraction. The constant shape of the relation at [Ca 2+ ] o higher than 2.5 mm and at low [Ca 2+ ] o after frequency potentiation suggests that one of the steps of the activation process has been saturated. Saturation probably takes place in intracellular release sites as direct maximal activation of skinned heart cells causes tension development which is independent of sarcomere length between 1.6 and 2.3 jum (Fabiato and Fabiato, 1975, 1976). The relation between peak tension and instantaneous sarcomere length is not influenced by shortening which occurs early during contraction (cf. Fig. 7; Pollack and Krueger, 1976). This observation suggests that length-dependent release of calcium is not the only process underlying lengthdependent activation, as tension development then would depend on initial sarcomere length, i.e., the length at which calcium release starts, even if release rapidly decreases as a result of subsequent shortening. Calcium binding by the myofilaments, on the other hand, has been suggested to be a rapidly reversible process (Gordon and Ridgway, 1978), such that rapid changes of activation with initial sarcomere shortening readily could be accounted for. If calcium binding by the filaments depends more strongly on sarcomere length than the calcium release process, tension development could increase with stretch of sarcomeres, with little or no evidence of an increase of the amount of calcium which is released. This would be in agreement with the observation that tension in frog trabeculae increases, whereas the free calcium concentration decreases on stretch of the muscle (Allen and Blinks, 1978). Acknowledgments We thank H. ter Keurs, Sr., who contributed greatly to the design of the experimental apparatus. The assistance of I. de Goede van Hoorn in preparing the manuscript is gratefully acknowledged. References Allen DG, Blinks JR (1978) Calcium transients in aequorininjected frog cardiac muscle. Nature 273: Allen DG, Jewell BR, Wood EH (1976) Studies of the contractility of mammalian myocardium at low rates of stimulation. J Physiol (Lond) 254: 1-17 Allen DG, Jewell BR, Murray JW (1977) The contribution of activation processes to the length-tension relation of cardiac muscle. Nature 248: Bodem R, Skelton CL, Sonnenblick EH (1976) Inactivation of contraction as a determinant of the length-active tension relation in heart muscle of the cat. Res Exp Med 168: 1-13 Brady AJ (1971) A measurement of the active state in heart muscle. Cardiovasc Res 5 (suppl I): Cleworth DR, Edman KAP (1972) Changes in sarcomere length during isometric tension development in frog skeletal muscle. J Physiol (Lond) 227: 1-27 Dijkstra JH, Landwaard LJ (1975) Holographic construction of open structure dispersion transmission gratings. Optics Commun 15: Drummond DI, Severson DL (1979) Cyclic nucleotides and cardiac function. Circ Res 44: Ebashi S, Endo M, Ohtsuki I (1969) Control of muscle contraction. 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San Francisco, McGraw Hill, pp Gordon AM, Ridgway EB (1978) Calcium transients and relaxation in single muscle fibers. Eur J Cardiol 7 (suppl): Gordon AM, Huxley AF, Julian FJ (1966) The variation in isometric tension with sarcomere length in vertebrate muscle fibres. J Physiol (Lond) 184: Huntsman LL, Stewart DK (1977) Length dependent calcium inotropism in cat papillary muscle. Circ Res 40: Iwazumi T, Pollack GH (1979) On-line measurement of sarcomere length from diffraction patterns in muscle. IEEE Trans Biomed Eng 26: Jewell BR (1977) A reexamination of the influence of muscle length on myocardial performance. Circ Res 40: Jewell BR, Rovell JM (1973) Influence of previous mechanical events on the contractility of isolated cat papillary muscle. J Physiol (Lond) 235: Julian FJ, Sollins MR (1975) Sarcomere length-tension relation in living rat papillary muscle. 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12 714 CIRCULATION RESEARCH VOL. 46, No. 5, MAY 1980 Krueger JW, Pollack GH (1975) Myocardial sarcomere dynamics during isometric contraction. J Physiol (Lond) 251: Krueger JW, Strobeck JE (1978) Sarcomere relaxation in intact cardiac muscle. Eur J Cardiol 7 (suppl): Lakatta EG, Jewell BR (1977) Length-dependent activation. Its effects on the length-tension relation in cat ventricular muscle. Circ Res 40: Langer GA (1973) Heart: Excitation-contraction coupling. Annu Rev Physiol 35: Mainwood GW, Lee SL (1969) Rat heart papillary muscles: Action potentials and mechanical responses to paired stimuli. Science 166: Nassar R, Manring A, Johnson EA (1974) Light diffraction of cardiac muscle: Sarcomere motion during contraction. In The Physiological Basis of Starling's Law of the Heart, edited by R Porter, DW Fitzsimons. North Holland, Elsevier Excerpta Medica, pp Pollack GH, Huntsman LL (1974) Sarcomere length-active force relations in living mammalian cardiac muscle. Am J Physiol 227: Pollack GH, Krueger JW (1976) Sarcomere dynamics in intact cardiac muscle. Eur J Cardiol 4 (suppl): Pollack GH, Iwazumi T, Keurs HEDJ ter, Shibata EF (1977) Sarcomere shortening in striated muscle occurs in stepwise fashion. Nature 268: Reuter H (1975) Inward calcium current and activation of contraction in mammalian myocardial fibers. In Recent Advances in Studies on Cardiac Structure and Metabolism, vol 5. Basic Functions on Cations in Myocardial Activity, edited by A Fleckenstein, NS Dhalla. Baltimore, University Park Press, pp Ridgway EB, Gordon AM (1975) Muscle activation: Effects of small length changes on calcium release in single fibers. Science 189: Sonnenblick EH (1962) Force-velocity relations in mammalian heart muscle. Am J Physiol 202: Uchino J, Tsuboi KK (1970) Actin accumulation in developing rat muscle. Am J Physiol 219: Prostaglandins and Potassium Relaxation in Vascular Smooth Muscle of the Rat The Role of Na-K ATPase WARREN E. LOCKETTE, R. CLINTON WEBB, AND DAVID F. BOHR SUMMARY We explored the hypothesis that postaglandin-induced vasodilation is caused by activation of the electrogenic sodium-potassium pump which results in membrane hyperpolarization and relaxation of vascular smooth muscle. Helical strips of rat tail artery relax in response to potassium after norepinephrine-induced contractions in physiological salt solution containing a low-potassium concentration. The amplitude of this potassium-induced relaxation is used as an index of sodiumpotassium ATPase activity. It was observed that PGAi, PGE 2, and PGF 2o (10~ 6 g/ml) significantly enhanced the magnitude of potassium-induced relaxation. PGA 2 and PGEi (10~ 6 g/ml) had no significant effect. PGE 2 caused relaxation of contractions induced by either 25 mm KC1 or norepinephrine (10~ 9 g/ ml), and these relaxations were inhibited by 10~ 4 M ouabain. Indomethacin (5.3 x 10"" g/ml) and meclofenamate (10~ 6 g/ml) reduced the magnitude of potassium-induced relaxation by more than 30% of control. PGF 2a (10~ s g/ml) reversed the inhibition of potassium relaxation by meclofenamate. These observations suggest that prostaglandins induce vascular smooth muscle relaxation by stimulation of the sodium pump and that endogenous prostaglandins normally potentiate potassium relaxation. Circ Res 46: , 1980 PROSTAGLANDINS augment tissue blood flow by a direct vasodilator action on vascular smooth muscle (Conway and Halton, 1975; Hedqvist, 1972; Messina et al., 1976; Strong and Bohr, 1974). However, the mechanisms by which prostaglandins induce vasodilation are not known. We have tested the hypothesis that prostaglandin-induced vasodi- From the Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan. These studies were supported by National Heart, Lung, and Blood Institute Grant HL W.E. Lockette is a Medical Student Research Fellow of the Michigan Heart Association. R.C. Webb is a Postdoctoral Research Fellow of the Michigan Heart Association. Original manuscript received June 15, 1979; accepted for publication January 4, lation may be a manifestation of an increase in sodium-potassium adenosine triphosphatase (Na-K ATPase) activity. As a result of the activity of Na- K ATPase, the electrogenic pump in vascular smooth muscle cells maintains high intracellular potassium and low intracellular sodium concentrations. Since more sodium is moved out of the cell than potassium is moved into the cell, the electrogenic pump hyperpolarizes the cell membrane; hyperpolarization causes relaxation of vascular smooth muscle (Anderson, 1976). We hypothesize that this hyperpolarization is augmented by some prostaglandins. Recent observations (Anderson, 1976; Haddy, 1975; Toda, 1974; Webb and Bohr, 1978) have