Depressed intracellular calcium transients and contraction in myocytes ...

Depressed intracellular calcium transients and contraction in myocytes from hypertrophied and failing guinea pig hearts FRANCIS M. SIRI, JOHN KRUEGER, CHARLES NORDIN, ZHEN MING, AND RONALD S. ARONSON of Medicine, Albert Einstein College of Medicine, Cardiology Division, Department Bronx, New York 10461

SIRI, FRANCIS M., JOHN KRUEGER, CHARLES NORDIN, ZHEN MING, AND RONALD S. ARONSON. Depressed intraceZlular calcium transients and contraction in myocytes from hypertrophied and failing guinea pig hearts. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H514-H530, 1991.-We investigated the basis for impaired left ventricular function of hearts in which hypertrophy was produced by gradual pressure overload. We measured myoplasmic free calcium concentration ([ Ca”+];) with furaand sarcomere shortening in single myocytes isolated from control hearts and hypertrophied failing hearts. Diastolic [ Ca2+]i was normal, but [Ca”+]; at the peak of contraction was depressed in myocytes from failing hypertrophied hearts. Increasing drive rate from 0.20 Hz to 5.00 Hz increased both diastolic and peak [Ca2+];. Norepinephrine (3 X lo-" M) increased diastolic [Ca2+]; in all cells and tended to normalize peak [ Ca”+]; in myocytes from hypertrophied failing hearts during 5.00 Hz drive. Depressed peak [Ca2+]i in the hypertrophied cells was paralleled by significant decreases in both the velocity and percent of sarcomere shortening, which were measured in cells not loaded with fura-2. Sarcomere length was correlated with estimates of [Ca2+]i in intact cells and with controlled levels of [Ca”‘] in chemically “skinned” myocytes. A plot of sarcomere length against [Ca”‘] gave a single continuous relationship that spanned resting and peak values at all drive rates in both the control and hypertrophied myocytes. Thus heart failure in this model is reflected in impaired myocyte contraction, which is closely related to reduced levels of [Ca”‘]i during systole rather than to depressed myofilament sensitivity to Ca2+. fura-2; myoplasmic free calcium; heart enlargement; ure; sarcomere dynamics; contraction; norepinephrine

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is an adaptive mechanism of the heart to pressure and volume overloads (7, 28, 44). For reasons that are still unclear, the adaptive phase of cardiac hypertrophy can eventually deteriorate into a phase of heart failure. Despite a number of previous studies in which various aspects of excitation and contraction have been investigated, the basis for impaired contractile function of the failing myocardium has not been established. In many models of hypertrophy, myosin adenosinetriphosphatase activity is depressed (52). Although this depression has been correlated with various indexes of contractility, it does not result in decreased tension development in skinned muscle (25, 43) or in isolated papillary muscle (10) and is not necessarily associated

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with either reduced pressure-generating ability or diminished pump function of the intact heart (54). Furthermore, there is presently no evidence linking the impaired function of the hypertrophied, failing left ventricular myocardium to either reduced myofibrillar sensitivity to [Ca2+]; or to a decrease in maximum Ca”+-activated force (25, 37, 43, 49). In virtually all models of hypertrophy induced by pressure overload, the action potential is prolonged (2, 16, 18,23, 24,33,47,51,59). The prolonged action potential, if due to increased Ca2+ current (loa), might enhance contraction by increasing activator Ca”+. In one study in single myocytes isolated from rats with left ventricular hypertrophy induced by renal hypertension, Ica increased markedly and the total free myoplasmic calcium ([Ca”‘];) estimated by calculation increased substantially in hypertrophied cells over control levels (30). A more recent study in myocytes isolated from rats with left ventricular hypertrophy induced by abdominal aortic constriction found that I cay normalized for membrane capacitance, was not significantly different in normal and hypertrophied myocytes, nor was there any difference in the time course of decay (51). In single myocytes isolated from cats with right ventricular hypertrophy, 1oaappeared to be slightly reduced or the same as in normal myocytes and the time course of inactivation of Ica was prolonged (33). The cells used in all the voltage-clamp studies were isolated from hypertrophied hearts without evidence of heart failure. The time course of activation and decay of Ica can influence both the release from and loading of Ca2’ into the sarcoplasmic reticulum (SR) (12, 13). The effect of Ica on the [Ca2+]i transient in intact cells is complex (5) and may be altered in diseased cells. In addition, 1ca is only one of many factors that can influence [Ca2+];. For example, the prolonged duration of the action potential characteristic of hypertrophied cells (2) could increase [Ca2+]; via an electrogenic Na+-Ca2+ exchange (32, 41, 45) independent of 1ca. Two previous studies have described alterations in the time course, but not the peak value, of the [Ca2+]; transient as measured by the fractional luminescence of the photoprotein aequorin in hypertrophied and failing muscles from ferrets and humans (19, 20). Although fractional luminescence was not converted into [Ca2+];, the authors concluded that in the ferrets with experimental pressure-overload hypertrophy, “the prolonged time

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course of tension development, but not the diminished peak isometric tension response, may be related to changes in intracellular calcium handling” (20). Preparations from patients with end-stage heart failure had abnormally prolonged [ Ca”+] i transients and impaired ability to restore diastolic [ Ca2+]i to normal low levels (19). There was, however, no evidence of a consistent decrease in the peak of the [Ca2+]i transient in failing muscles. Several studies have shown that SR function is altered in heart failure. For example, the velocity of Ca2’ transport into isolated SR vesicles is depressed by as much as 50% in some models of heart failure (27, 57). The relationship between this change and depressed contractile function is not clear. Thus the available experimental evidence does not provide any convincing explanation for the reduced contractile performance of hypertrophied muscle that has failed. Therefore, we investigated the cellular basis for depressed contractile performance in a model in which chronic heart failure occurs after a gradually developing left ventricular overload in guinea pigs. We have previously characterized the hemodynamic and contractile abnormalities in the intact heart in this model (54). We have also shown that single left ventricular myocytes isolated from hypertrophied hearts are larger than control cells and maintain the prolongation of action potential duration characteristic of the hypertrophy process (47). In this study, we directly measured intracellular [ Ca2+]i transients in single cells with the Ca2+-sensitive dye fura-2. The physiological relevance of our measurements of [Ca’+]; are further supported by our observations that [Ca2+]; measured with furacorrelates strongly with contraction. Our results show that the peak of the [ Ca2+]; transient is markedly reduced in cells isolated from failing hearts and thus provides an explanation for the depressed contractile performance of cells isolated from the failing myocardium. Preliminary reports of this work have been presented as abstracts (37,

55) . METHODS

Experimental animals and aortic banding procedure. Male guinea pigs of the Hartley strain, initially weighing 225-275 g (2-3 wk of age), were used. Under methohexital sodium anesthesia (30 mg/kg), half of the animals underwent a lateral thoracotomy with placement of a small Tygon band on the ascending aorta. This band was of a size that produced very little left ventricular overload at first but led to a progressive rise in systolic left ventricular pressure as the banded animals grew. Each banded animal was age and weight matched with an unoperated control. With growth to -750 g body wt, most aortic-banded animals had substantial left ventricular hypertrophy, and a high percentage (50-60%) also showed clinical dyspnea and cyanosis by 40 days after banding. We have previously described the details of this model and demonstrated that clinical dyspnea and cyanosis in the aortic-banded animals correlated with pathophysiological alterations consistent with heart failure: high lung weight, right ventricular hypertrophy, and

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depressed capacity of the intact left ventricle to generate pressure (54). Thus we compared data from three groups: control guinea pigs, aortic-band.ed guinea pigs without evidence of congestive heart failure (AC), and aorticbanded guinea pigs with evidence (dyspnea and mild cyanosis) of congestive heart failure (ACF). Because this study extended longer than 40 days, a higher percentage of banded animals progressed to overt heart failure (ACF), leaving a relatively low number of animals in the AC group. This limited our statistical analysis of the AC group, particularly with respect to the subgroups used for testing norepinephrine’s effects and for examining contractile parameters. In these cases only the control and ACF groups are compared statistically. We nevertheless present data from myocytes in the AC group to illustrate the continuum of results. The major differences occurred between the control and ACF groups, and that is the principal focus of this study. Isolation of cells and loading with fura-2. Single myocytes were isolated from the left ventricles of guinea pig hearts by enzymatic dispersion by a method described previously (3, 41, 47). The isolated cells were suspended in 10 ml of a solution of the following composition (in mM): 118 NaCl, 4.8 KCl, 1.2 NaH2P04, 1.2 MgSOJ, 1.0 CaC12, 11 glucose, and 25 Na-N-Z-hydroxyethylpiperazine-2V’-2ethanesulfonic acid. One-half milliliter of the cell suspension was added to a small tube containing I.5 ml of normal Tyrode solution of the following composition (in mM): 137.5 NaCl, 12.0 NaHC03, 1.8 NaH2P04, 4.0 KCl, 1.2 CaC12, 0.5 MgC12, and 5.5 glucose. Five microliters of a 1 mM solution of the acetoxymethyl ester derivative of fura(fura-2/AM) in dimethyl sulfoxide was added, after which the tube was capped and rotated at 10 revolutions/min at room temperature (23°C) for 15-20 min. Evaluation of distribution of cellular fluorescence. Cells were selected for photomultiplier measurements based on their rod-shaped morphology and good striations. It is also important to verify that the fura- dye is uniformly distributed throughout the cell, since under some conditions it can become trapped in intracellular organelles (62) and lead to artifacts in the estimation of [ Ca”+];. Such compartmentalization can be minimized by loading the cells with fura- at room temperature, then gradually raising the perfusate temperature to the level at which they are to be studied (42). To evaluate uniformity of dye distribution in this study, we obtained fluorescent video images of representative unstimulated myocytes using a SIT camera (Dage-MTI, SIT66). Each image was digitized rapidly (l/30 s) at 640 X 480 X 8 bit resolution with a microcomputer (Macintosh II equipped with a video-frame grabber, Data Translation, DT 2255). Noise in individual video frames was reduced by averaging 1681 frames for each image obtained at each wavelength. The signal-averaged image was stored on the hard disk for retrospective image processing. Figure 1, A and B, shows representative fluorescent video images of a resting myocyte isolated from an ACF heart. These images show a fairly uniform distribution of fluorescence, and the lower intensity of the image due to 340 nm excitation (compared with the 380-nm image) is typical of an unstimulated myocyte with low [ Ca2+];.

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A control cell observed at higher magnification showed a cross-striated pattern of fluorescence that correlated well with the original pattern of the striations in the cell (Fig. 1, C and D). Retrospective analysis showed that these striations were evident at either excitation wavelength in three of the nine cells examined. This pattern also appears in the image of the fluorescence ratio, which suggests that it is at least partly due to variations in [Ca*+]i. Even though our method resolved striations in the fluorescent image, we could not detect a discrete, longitudinal component of fluorescence that would correspond to mitochondria. Thus the fluorescence appears to emanate principally from the myofibrillar compartment of the resting cell. Figure 1E shows the bright-field image of a control myocyte that displayed the clearest spatial variations in fluorescence. Figure 1, F and G, is the fluorescent images of that same cell due to excitation at 340 nm and 380 nm, respectively. Figure 1H is the ratio (R) of Fig. 1F to Fig. 1G after background correction for each. R can be related to [Ca*+];, and a discussion of our calibration procedure follows in this methods section and in the

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Here we see gradations in R, exhibiting higher values in the cell’s interior, most notably at one end. The precise explanation for these gradations is not known. However, from a methodological perspective, the points to be stressed are that they cover a range that is small relative to systolic R values, and do not represent compartmentalization into cellular organelles. Occasionally, we observed cells that had areas of punctate fluorescence in the 340 nm image similar to those described by others (60, 62). The greater fluorescence in these areas was not simply due to greater cell thickness and/or fura- dye concentration, because corresponding areas of high intensity were also seen in the image ratio, indicating higher [ Ca2+] i. In eight myocytes, we computed intensity of the ratio image (Fig. 1H) in two ways: by determining in each cell the mean pixel intensity 1) within a boundary encompassing the entire cell area (see Fig. 1, F and G) and 2) along a longitudinal transept excluding the last 10 pm at the cell’s ends (see Fig. 1A). The mean f SD of R values in the area ratios was 1.159 ? 0.240 and in the transept ratios was 1.173 + 0.270. These results of video image analysis are in good agreement with experimental values obtained by photomultiplier detection of fluorescence in restricted regions of resting myocytes in this study (1.120 & 0.160). Thus, despite some detectable variation in R throughout each cell, mean cellular R appears to be relatively constant for the sampling methods we examined. Experimental protocol in cells loaded with fura-2. After being loaded with dye, the cells were transferred to a small perfusion chamber (volume, -1 ml) mounted on the stage of a Nikon Diaphot microscope equipped with quartz optics. The cells were allowed to settle to the bottom of the perfusion chamber, after which they were perfused continuously at 5 ml/min with Tyrode solution gassed with 95% OZ-5% CO2 and kept at 37°C. The cells were driven regularly with an external glass electrode filled with 3% agarose and 3 M NaCl. Stimulation was unipolar relative to a distant electrode in the cell chamber. In general, cells could be stimulated with DC pulses of 5-10 mA lasting 5 ms. The cells were driven at 1 Hz for -5 min, during which baseline [Ca2+]i transients were recorded. Stimulation was interrupted for 5 min, after which the cells were driven at successively higher frequencies of 0.20, 0.33, 1.00, 2.00, and 5.00 Hz. The effect of norepinephrine (3 X 10m6 M) was investigated in a subgroup of the cells. Because of the pharmacological effects of the drug, the experimental protocol was slightly modified as follows: the cells were exposed to norepinephrine for 10 min, after which they were driven at 0.20, 1.00, and 5.00 Hz, [Ca*+]; transients being recorded at each drive rate. Fluorometry. We used a Spex Fluorolog II spectrofluorometer to excite and record fluorescence from cells loaded with fura-2. Experiments were carried out with a x40 oil immersion lens (Nikon CF fluor, NA 1.30). The fura-loaded cells were excited at light wavelengths of 340 and 380 nm, the two wavelengths being alternated by a computer-controlled mechanical chopper. The dwell time and integration time at each wavelength were 1.2 and 3.6 APPENDIX.

FIG. 1. Appearance of fura-2/AM fluorescence in isolated myocytes. A and B: fluorescent images produced by excitation at 340 nm and 380 nm, respectively, in a myocyte isolated from a hypertrophied, failing heart. Gray scales spanning 256 gray levels from zero (darkest) to greatest fluorescence intensity have been added for reference. The thin horizontal line in A denotes a transept along which a profile of cell fluorescence was obtained. C: the striated appearance of the bright field image in a control myocyte. D: the fluorescent image of the striations (averaged for 81 frames) produced with excitation at 340 nm. In D, the image was shifted at a 45” angle and overlaid to enhance the visibility of the display. E-H: control myocyte and the bright field image (E), the background-subtracted fluorescence due to excitation at 340 nm (F) and at 380 nm (G) and the ratio representation of the 340 nm/380 nm images (H). The six densities in H correspond to mean fluorescent image ratios of 1.37 (darkest gray scale), 1.30, 1.25,1.18,1.10, and 0.82. Fluorescence was also integrated over the area of each of the four fluorescent images, as demonstrated in F and G by the dashed border around the cell. Mean + SD of relative fluorescence intensity for each of these images was 91.3 rt 32.7 (A), 131.4 + 52.4 (B), 149.9 + 22.1 (F), and 119.9 f 12.0 (C). F/G = 1.25. Mean density in H, 1.27.

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FIG. 2. Derivation of [Ca”];. Left: changes in the intensity (in cps X 10") of furafluorescence due to excitation at 340 and 380 nm in a control cell driven at 1 Hz. middle: ratio (R) of the 340 and 380 nm signals after correction for background autofluorescence, and R,;,, and R,,,;,, obtained in that same cell. Right: time course of [Ca2+]; (in nM) calculated from RI,IHX, R ,,,,),, and R.

ms, respectively. The fluorescent light emitted by the excited cells was measured at 510 nm by a photomultiplier tube. To exclude any noncellular fluorescence, an aperture limited sampling of emitted light to a circular area, the diameter of which was smaller than the cell’s width. A detailed discussion of the methods used to convert the fluorescence signals to estimates of [Ca”‘]; is giveninthe APPENDIX. Derivation of [CC?+/;. Figure 2 shows how the [Ca2+]i transient was derived from experimental records. Figure 2, left, shows the recorded intensity of fluorescent light due to excitation at 340 and 380 nm. Figure 2, middle, shows R of the background-corrected light signals (left). R,,, and Rminwere obtained as described in the APPENDIX. Figure 2, right, shows the [Ca2+]; transient obtained by substituting the values for R,,,,, R,i,, and R for this cell into a modification of the equation of Grynkiewicz et al. (17) (see APPENDIX). Thus the time-varying Ca”’ transient reflecting [Ca”+]; is calculated from direct experimental measurements. Data collection and analysis. The individual records shown in Fig. 3 were derived in the same manner as shown in Fig. 2. For statistical purposes, we chose to make comparisons of [Ca2+]; for our experimental groups based on values for each heart rather than on values for each cell (Figs. 4 and 5). As can be seen from Fig. 2 (left), the signal-to-noise ratio was best in the original recordings of fluorescence. Therefore, the values of [Ca”+]; in Figs. 4 and 5 and R in Table 2 were calculated from values of fluorescence obtained from the unprocessed records at time points corresponding to end diastole and the peak of the transient. Noise in Fig. 2 was evaluated by estimating the “mean” signal through the use of a 21-point “smoothing” function (Spex Industries: DM3000CM) and then using this to normalize the unfiltered data. The standard deviation of the resulting data was 14.4% for the [Ca2+]; transient, 3.0% for the 340-nm record and 4.4% for the 380-nm record. Rminwas determined in five cells from each heart, and the average of the two lowest values was taken as Rminfor that heart. In 25 of the 26 hearts studied, both R and R,,, were obtained in each of two cells, and these values were averaged. In one heart, R,,, and R were obtained in just one cell. Measurements of sarcomere dynamics. An aliquot of ventricular myocytes was placed in a cell chamber (vol-

ume, 2 ml) through which Tyrode solution was recirculated (4-5 ml/min) from a 250-ml reservoir. Tyrode solution containing (in mM) 137.5 NaCl, 12.0 NaHC03, 1.8 NaH2P04, 4.0 KCl, 2.4 CaC12, 0.5 MgC12, and 5.5 glucose was gassed with 95% O2-5% Co2 and kept at 36.5-38.O”C. The cell chamber was mounted on the stage of an inverted microscope and the cells were viewed with a x40, NA 0.75 WI H off man modulation contrast objective. The condenser was dipped into the experimental solution to eliminate fluctuations in background illumination. Cells were stimulated electrically with an extracellular glass microelectrode filled with Tyrode solution. Stimulation was unipolar, with a platinum wire in the cell chamber serving as the distant electrode. Sarcomere length was measured 530 times/s by frequency modulation (FM) detection of the cell’s striation pattern by an extension of the phase-locked loop method (46). The image of the cell was projected through the third tube of the microscope onto a rapidly scanned, linear array of photodiodes (RL256C-17, EGG Reticon, Sunnyvale, CA) that was aligned with the long axis of the cell. The array scans a path on the cell that is ~4 pm wide and 60 pm long. Because the array is scanned at a constant velocity, there is an inverse relation between the frequency content of its video signal and sarcomere length. The frequency of the video signal was detected with an FM demodulator (i.e., phase-locked loop). The amplitude of the resulting phased array signal, being proportional to striation frequency, was integrated over an adjustable sample interval, to obtain the average length of the sarcomere in a selected region (46). In all cases, the window region sampled was 20-40 pm long and was located where the phase error signal indicated that initial sarcomere length was uniform. The method enables simultaneous measurements of sarcomere length in an entirely separate region of the cell scanned by the photodiode array to verify uniformity of contractile motions within the isolated cell (35). The measurements of sarcomere length were calibrated with a custom-built optical test grating with a maximum deviation of to.015 ,urn (35). Application of the phase-locked loop method to the test grating indicated that the method can resolve known differences in length between continuous striations with a measurement precision of -1%. Because sarcomere length was sampled from a region >20 pm long, the actual precision

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FIG. 3. Representative [Ca”‘]; transients. A: 3 sets of [Ca”‘]; transients recorded during stimulation at 1 Hz and 5 Hz in myocytes from a control (C), a nonfailing aortic-banded (AC), and a hypertrophied failing (ACF) heart. In each case, l- and ~-HZ traces are overlapped to facilitate comparison of diastolic levels. B: transients from the same cells after perfusion with 3 x 10m6 M norepinephrine for 10 min. Horizontal lines drawn adjacent to each transient indicate systolic [Ca2+]; in the absence of norepinephrine.

is even better. At this window width, controlled translation of the image gives rise to fluctuations that in all cases are an order of magnitude less than the precision we attribute to measurement of mean sarcomere length in the sampled region. Sarcomere shortening in two separate areas of the cell was recorded initially on a strip chart recorder and was stored on FM tape. The velocity of sarcomere motion was obtained by electronic differentiation of the sarcomere length changes during replay of the prerecorded data. The contractile parameters were measured after a stable mechanical response had been obtained at drive rates ranging from 0.10 to 5.00 Hz or, in some instances, >5 Hz. All measurements were made in dye-free cells. Protocol for measurements of sarcomere dynamics. Sarcomere length was initially measured in cells after a lomin equilibration in the bath at 37°C but before any electrical stimulation. To establish the effect of stimulation, the drive rate was increased in steps of 1 Hz from 1 Hz to over 5 Hz. Typically, contractile behavior approached a steady state l-2 min after the onset of stim-

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ulation at 1 Hz and lo-15 s after the onset of stimulation at higher drive rates. After the first three hearts were investigated, this sequence was followed by a rest period of -30 s, after which the effects of drive rates of 1.00, 0.70,0.50,0.35, and 0.20 Hz were examined in succession. Finally, drive rate was again increased, in increments of 1 Hz, from 1 Hz to the highest drive rate the cell could follow. This protocol generated two values for the contractile response of a cell at each drive rate, and each pair of values was averaged. For statistical analysis of contraction, we compared only those cells that responded over the full range of drive rates (0.20-5.00 Hz) we used in cells in which we measured [Ca’+]i with fura-2. &a-sarcomere length relations. Because [Ca2+]i modulates mechanical interactions between the contractile proteins, sarcomere length should vary with [Ca”‘]; in a predictable way. Therefore, we compared the relationship between sarcomere length and [Ca2+]i measured with fura- in intact control and hypertrophied cells with the sarcomere length measured in response to known changes in [Ca”‘] in permeabilized preparations. The isolated cells were permeabilized (“skinned”) by treatment with a nonionic detergent (0.5-l% Triton X) for -30 min in a test tube immersed in ice. The cells were suspended in a solution containing (in mM), 140 K aspartate, 2 Mg acetate, 5 Na2ATP, 5 K2EGTA, and 5 imidazole (pH, 7.4). An aliquot (50 ~1) of the cell suspension was placed in a chamber containing 1 ml of the solution in which [Ca”‘] was varied by adjusting the ratio of CaKEGTA and K2EGTA (11, 15, 41), keeping total EGTA at 5 mM. The test solutions contained either 2.00 or 6.75 mM Mg acetate, corresponding to calculated free [Mg2+] of 0.10 or 1.80 mM, respectively. K aspartate was lowered to 125 mM to give a total [K’] of 140 mM, corresponding to prior studies in perfused guinea pig myocytes (41). The half ionic strength of the solution was computed to be 0.15-0.16. The calculation of free [Ca”‘] was adjusted retrospectively to account for the residual amount of K,EGTA contained in the added suspension of cells. Sarcomere length was measured by the phase-locked loop method described above, or directly with a filar eyepiece micrometer if contraction bands developed at short sarcomere lengths in the permeabilized cardiac cells (36). Statistical analysis. The effect of drive rate on various parameters was statistically evaluated by analysis of variance with repeated measures. When significant overall effects or interactions were found, appropriate post hoc tests were made. Comparison of three or more groups at a common drive rate was made by one-way analysis of variance followed by the Newman-Keuls test, which was used to determine the significance of specific pairwise comparisons. The significance of differences between control group values and values of the ACF group was evaluated by Student’s t test. Linear regression lines were determined by the method of least squares, and differences with respect to slope and intercept were evaluated by analysis of covariance. RESULTS

Body and tissue weights. Table 1 shows the body and tissue weights of the guinea pigs, which were divided into

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FIG. 4. Systolic and diastolic [Ca2+]i: effects of hypertrophy and drive rate. Data are given as means & SE of estimates of peak systolic (A) and diastolic (B) free [Ca2+]; recorded during stimulation at 0.20, 0.33, 1.00, 2.00, and 5.00 Hz in cells isolated from control (C: n = 13), aortic-banded nonfailing (AC: n = 4) and hypertrophied failing (ACF: n = 9) hearts. Asterisk(s) indicate a significant difference between the mean for the control group and the mean for the ACF group. *P < 0.05, **p < 0.01.

three groups: controls, AC, and ACF. Body weight was not significantly different between the three experimental groups. There was a small increase in heart weight in the AC animals (not significant), whereas heart weight increased markedly and significantly in the ACF animals. Similarly, the percent increase in heart weight from its predicted value was only 14% (not significant) in the AC animals but was 66% in the ACF animals. The cell isolation technique precluded direct measurement of left ventricular mass. However, percent total heart hypertrophy underestimates percent left ventricular hypertrophy to the extent that heart tissue other than the left ventricle fails to hypertrophy. The normal left ventricle comprises slightly 40% of the mass of the whole heart (as dissected for Langendorff perfusion). Thus, in the absence of right ventricular hypertrophy, the left ventricle would have to hypertrophy by ~30% to cause 15% hypertrophy of the whole heart. Our earlier work suggests that the banding procedure causes significant left ventricular hypertrophy even in the nondyspneic animals by the time they reach this body weight (54), but we cannot be certain that this is the case for the AC group in the present study. For this reason, AC refers strictly to the fact that these animals were banded in the usual manner. On the other hand, total heart hypertrophy was highly significant in the ACF group and was most likely accompanied by an even more marked hypertrophy of the left ventricle. Whereas lung weight in the AC animals was similar to that of control animals, lung weight in the ACF animals increased dramatically and significantly compared with both control and AC values. Table 1 shows the same Darameters for those animals whose myocvtes were ex-

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FIG. 5. Systolic and diastolic [Ca2+];: effects of hypertrophy, drive rate, and norepinephrine. Data are given as estimated mean & SE systolic (A ) and diastolic (B) [ Ca2+]i during stimulation at 0.20, 1.00, and 5.00 Hz in cells isolated from control (C: n = 7) and hypertrophied failing (ACF: IZ = 4) hearts before and after perfusion with 3 x lo-” M norepinephrine (NE) for 10 min. Asterisks indicate a significant difference between the mean for the control group and the mean for the ACF group before NE. **P < 0.01. YAdjacent mean is significantly greater than the mean for that group before norepinephrine (P < 0.05). During 0.2 Hz and 1.0 Hz drive, diastolic [ Ca”+]; increased significantly in both groups after norepinephrine (P < 0.05).

posed to norepinephrine. This ACF subgroup (4 animals) had significantly lower body weight and significantly greater heart weight, percent hypertrophy, and lung weight than the control group (7 animals). Representative [Ca2+/i transients before and after norepinephrine. Figure 3A shows the [Ca’+]; transients recorded in representative cells from each of the experimental groups. In each panel, the single transient on the left was obtained during regular stimulation at 1 Hz and the three consecutive transients on the right were obtained from the same cells during regular stimulation at 5 Hz. The most striking feature of these records is the marked depression of the peak of [Ca2+]i at both rates of stimulation in the ACF myocytes Depressed [ Ca2+]i transients in ACF myocytes could reflect a reduced capacity to increase [Ca2+]; after excitation, or a stronger dependence on neurohumoral factors to maintain performance. One such factor is norepinephrine, which is typically elevated in plasma of patients with heart failure (58), and may act on both the sarcolemma and the SR to incre ase [Ca” +]i. TO assess the possibi .I.ity that the depressed SYStolic [Ca2+]; was restorable, we exposed some of the isolated myocytes to norepinephrine. Figure 3B shows records from the same myocytes used in Fig. 3A, obtained after they were perfused with 3 x 10B6 M norepinephrine for 10 min. In each panel, the single [Ca2+]; transients at the left were obtained during regular stimulation at 1 Hz, and the three consecutive transients at the right were obtained, from the same cell, at 5 Hz. Norepinephrine increased the magnitude of the lCa”+l; transients in all three ex-

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3,153*392 3,482+203 4,950+756**t

14s 66t19**-b

4,100+1,134 4,326+234 7,793+1,624**t

7 4

809t102 582t119”

3,207t439 5,115k1,132*

83t35”

3,525+367 6,921&1,736*

g

mg

5%Hypertrophy

Lungs,

mg

P < 0.01.

amples. Typically, the effect of norepinephrine was smallest in control cells at 5 Hz and greatest in the AC and ACF cells at 5 Hz. Effects of hypertrophy and drive rate on [Ca2+]; transients. The data in Fig. 4 show peak systolic (4A) and diastolic (4B) [Ca”+]; as a function of drive rate in the three groups of animals (13 control, 4 AC, and 9 ACF). In all three groups, peak systolic [Ca2+]; increased as drive rate was increased. The frequency-dependent increase in peak systolic [Ca2+]; was blunted in AC and ACF myocytes, the relationship being most depressed in the ACF myocytes. Statistical analysis of the data indicated a significant interaction between drive rate and experimental group on peak systolic [Ca2+]; (ANOVA, F test: P < 0.05). Post hoc analysis indicated that the interaction was a consequence of the severely blunted response of the ACF myocytes compared with control myocytes. In contrast to systolic [Ca2+];, diastolic [Ca2+]; responded similarly in all three groups of animals (Fig. 4B), increasing almost linearly with drive rate. Before data for drive rates of 0.20-5 Hz were gathered, cells were driven at 1 Hz for 5 min, followed by a 5-min period of no stimulation. Diastolic [Ca2+]i near the end of the initial period of 1 Hz stimulation was 75 t 33 nM (mean t SD) in the control myocytes and 77 t 21 nM in ACF myocytes. Both of these values decreased significantly by the end of the 5-min period of no stimulation (control: 37 t 14 nM; ACF: 58 t 23 nM, P < 0.01 in each case). The two group means were also significantly different from each other (P < O.Ol), indicating that [Ca2+]; decreased less in ACF myocytes than in control myocytes during the intervening period of rest. Effects of hypertrophy, drive rate, and norepinephrine on [Ca2+]i transients. Figure 5 shows the effects of norepinephrine on systolic (5A) and diastolic (5B) [ Ca”+]; in a subset of hearts (7 control and 4 ACF hearts). There was only one heart from the AC group included in the norepinephrine testing, so statistical analysis was confined to comparison of the control and ACF groups. As was seen in the larger data set (Fig. 4), systolic [Ca2+]; was significantly depressed at all drive rates in the ACF group, before norepinephrine administration (Fig. 5A). Norepinephrine markedly increased the slope of the curve for the ACF group. In fact, after norepinephrine peak systolic [Ca”‘]; at 5 Hz in the ACF group did not differ significantly from that of the control group. Nor-

epinephrine tended to increase peak systolic [Ca”‘]; at each drive rate in control myocytes, but the increase was statistically significant only at 0.20 Hz (P < 0.05). In the ACF group, the norepinephrine-induced rise in systolic [Ca2+]; was not significant at 0.20 Hz but was marked and significant at both 1 and 5 Hz (P < 0.05 in each case). Norepinephrine caused a parallel and significant (P < 0.01) increase in the diastolic [Ca2+];-drive rate curve in both normal and ACF cells (Fig. 5B). Group differences in R,i,, R,,,, and R. The results just described depend to some extent on the values of Rmin and Rmaxfor each group. Therefore, we considered the potential impact of the calibration procedure on our experimental findings. Table 2 summarizes the values we obtained for Rmin,R,,,,, and R in cells isolated from the three experimental groups. Data are tabulated for cells during stimulation at 5 Hz. For the entire series of cells, the values for Rminand R,,, were not significantly different in control, AC, or ACF cells. Nor were the values for Rdiastsignificantly different in cells isolated from the three experimental groups. In contrast, Rsyst . was significantly depressed in ACF cells. Similar results were obtained in the hypertrophied cells that were subsequently treated with norepinephrine (Table 2). In this subset, R min was significantly lower and Rm,, tended to be higher (not significant) in the ACF group compared with controls. Two-way analysis of variance indicated that norepinephrine generally increased both Rdiastand Rsyst(P < 0.05 in each case). Post hoc tests (paired t) showed that the increase in Rsystwas due to a significant increase in J&t in the ACF group but not in the control group. The fact that the unaltered, directly measured values for Rsystwere significantly depressed in ACF cells excludes the possibility that the depressed systolic [Ca”+]; can be attributed to their greater Rm,, value, which in any case did not differ significantly from that of control myocytes. Relation of systolic [Ca”“/i to extracellular Ca2+.In this study, we perfused myocytes with 1.2 mM extracellular [ Ca”+] because this is close to estimated [Ca2+]; in plasma and also because we anticipated that the combination of high drive rate plus norepinephrine would increase [Ca2+]; to the upper level of the physiological range. To confirm that the depression seen in [Ca2+]; transients was not dependent on a specific external Ca2+, we measured [Ca2+]; in normal myocytes (n = 8) and in myocytes

MYOCYTE

2. R,;,

TABLE

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CALCIUM

AND

CONTRACTION

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R,,x- and R values at 5 Hz Group

All animals Control AC ACF NE test group Control ACF Control + NE ACF + NE

n

Rmin

13 4 9

0.88t0.04 0.89t0.07 O.BOkO.09

7 4 7 4

0.88t0.04 0.78&0.04**

R max

R dmsi

R syst

6.25tl.06 7.12t1.19 7.33k1.37

2.00~0.20 2.02kO.16 1.93t0.36

3.91t0.74 3.78t0.48 2.93+0.4o**j-

6.65k1.97 8.46kl.37

2.07t0.32 1.77kO.30 2.49t0.53 2.18t0.51

3.95t0.51 2.70&0.26** 4.13tl.16 4.04+0.91$

Values are means t SD of the ratios of fluorescence intensity (340/380) obtained under conditions of minimum (R,i,) and saturating (R,,,) myoplasmic free calcium, the ratio obtained at end diastole (Rdiast), and the ratio seen at peak systole (R,,,,) in myocytes isolated from control hearts (Control), nonfailing hypertrophied hearts (AC), and failing hypertrophied hearts (ACF). NE, norepinephrine. NE test group includes data obtained in a subgroup both before and after (+ NE) 10 min perfusion with 3 x 10m6 M NE. ** Significant difference between ACF group and Control group, P < 0.01. t Significant difference between ACF group and AC group, P < 0.01. $ Significant increase in mean of ACF group after NE, P < 0.05.

from hypertrophied and failing hearts (n = 6) perfused at 37°C with normal Tyrode solution containing 2.4 mM [Ca”‘] and d riven at 1 and 3 Hz. The means t SD for peak [Ca2+]; at 1 Hz were 600 t 197 for normal myocytes and 303 t 60 nM for ACF myocytes (P < 0.01) and at 3 Hz were 1,277 t 492 and 599 t 138 nM, respectively (P < 0.01). As in the present study, there were no significant differences in diastolic [Ca2+]; between the control and ACF groups at any drive rate. Thus the higher extracellular [Ca”‘] increased systolic [Ca2+]; in both groups during 3 Hz drive, compared with values that would be predicted from Fig. 4. Nevertheless, systolic [Ca2+]; remained depressed in ACF myocytes. [Ca’+]i transient: timingparameters. Table 3 shows the kinetic parameters of [Ca2+]i in myocytes from the three experimental groups. The rate of rise of the [Ca2+]; transient (+d[Ca2+];/dt) was reduced in AC cells, but not enough to be statistically significant. In ACF myocytes, +d[ Ca2+];/dt was significantly depressed compared with values in control myocytes, a result consistent with the TABLE

3. Timing parameters Group

All animals Control AC ACF NE test group Control +NE +NE ACF +NE +NE

Hz

+ d[Ca2’];/dt, nM/ms

13 4 9

1 1 1

26tll 20t8 14&4**

7

1 1 5 5 1 1 5 5

24-t9 56&51 115+78”f$ 126+8O”f$ 10t5** 20t8 23tll* 91+52”r$§

n

4

Rate,

TTP, ms

TR&o, ms

149t47 128k56 177t56

355t74 357254 378t56

129t49 80+29-t 39+12t$ 42+16t$ 198*68 86+37-t 47+12j39+11-j-

356t102 248+59-f 74+17”f$ 83+17”ff 403&68 330t112 99&4**‘f$ 96+25t$

Values are means t SD for the rate of rise in [ Ca2+]; (+ d[Ca2’];/ dt), time to peak [Ca2+]; (TTP) and time to 50% return from peak [ Ca’+]i to end-diastolic [ Ca2+]i (TRE& in isolated myocytes from control guinea pigs (Control), from aortic-banded guinea pigs without heart failure (AC), and from aortic-banded guinea pigs with heart failure; n = sample size. Rate, drive rate of electrical stimulation; NE, norepinephrine (3 x 10e6 M) infused (+NE) during electrical stimulation. *,** Significant difference from corresponding control group mean, * P < 0.05, ** P < 0.01. f-$§ Significant differences from the mean for 1 Hz without NE, 1 Hz + NE, and 5 Hz without NE, respectively (P < 0.05 in all cases).

depressed maximum velocity of sarcomere shortening observed in those cells (see Fig. 7B). Neither the time required to reach the peak of the [Ca2+]; transient (TTP) nor the time required for the transient to decay to 50% of its peak value (TRE& was significantly different in cells from the three experimental groups, although both parameters tended to be longer in ACF cells. Table 3 shows the effects of norepinephrine on the kinetic parameters of the [Ca2+]i transient. During stimulation at 1 Hz in control cells, norepinephrine significantly reduced TTP and TRE50. Norepinephrine also tended to increase +d[Ca2+];/dt but the increase was not statistically significant. Increasing the drive rate to 5 Hz caused a significant reduction in TTP and TRE,, and significantly increased +d[Ca2+]i/dt in the absence of norepinephrine. In control cells driven at 5 Hz, the addition of norepinephrine did not have any additional significant effects on any of the three kinetic parameters. Compared with control myocytes, ACF myocytes driven at 1 Hz had significantly lower +d[ Ca2+]Jdt, which was not significantly increased by norepinephrine. Increasing drive rate to 5 Hz increased +d[Ca2’]i/dt significantly only in the control myocytes and consequently the relative performance of ACF myocytes was even more depressed at 5 Hz than at 1 Hz. However, the combination of 5 Hz drive and norepinephrine increased +d[Ca2’]i/dt substantially in ACF myocytes, to the point that it was no longer significantly less than the value for control myocytes. Both norepinephrine and 5 Hz drive decreased TTP significantly, compared with values at 1 Hz drive, but their combination did not produce any further significant reduction. In ACF myocytes, norepinephrine did not reduce TRE,, significantly below the value seen during 1 Hz drive, whereas increasing drive rate to 5 Hz did. At 5 Hz in the absence of norepinephrine, TRE,, was significantly longer in ACF myocytes than in control myocytes. The combination of 5 Hz drive and norepinephrine did not produce any further decrease in TRE,, in ACF myocytes. Contractile behavior of isolated myocytes. To provide a physiological correlation with the alterations we observed in [Ca2+]; in hypertrophied cells, we measured sarcomere length in unstimulated myocytes (resting sarcomere length), and sarcomere length, sarcomere shortening, and the velocities of sarcomere motion as a func-

H522

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tion of drive rate. These measurements were made in cells not loaded with furato eliminate the possibility that any contractile differences observed between the control and ACF cells might be due to differential Ca”+ buffering by the fura- dye. Resting sarcomere length was slightly but significantly shorter in ACF myocytes. Average resting sarcomere length was measured by the phase-lock-loop method in 94 myocytes isolated from 8 normal hearts (1.862 t 0.041 pm: mean t SD), in 32 cells from 3 AC group hearts (1.849 t 0.046 ,um), and in 77 cells from 5 ACF group hearts (1.837 t 0.051 ,um). The difference between control myocytes and ACF myocytes was statistically significant (P < 0.05). Resting sarcomere length of intact myocytes was also measured directly from photographs taken from the video monitor in 62 cells isolated from 13 normal hearts (1.834 t 0.064 pm) and in 31 cells from 6 ACF group hearts (1.785 t 0.067 pm). The group difference (normal vs. ACF) using this measurement system was also statistically significant (P < 0.01). Finally, resting sarcomere length was measured in some chemically skinned myocytes to see if the mean for the ACF group remained shorter even in nominally calciumfree solution. Average sarcomere length in 139 chemically skinned myocytes from 5 normal hearts was 1.910 t 0.041 pm. This was significantly longer than either the average for 18 myocytes from an AC heart (1.852 t 0.038 pm) or the average for 10 myocytes from an ACF heart (1.831 t 0.098 pm: P < 0.01 in both cases). Figure 6 shows the effect of stimulation on the initial sarcomere length and its length at the peak of shortening in normal and hypertrophied cells. Figure 6A shows chart recordings of sarcomere length and the velocity of shortening at progressively higher drive rates in an ACF myocyte. After the onset of stimulation at 1 Hz, the initial (diastolic) sarcomere length reached a steady state as shown at the beginning of the upper trace. Up to 2 Hz, increasing drive rate decreased the initial sarcomere length and increased the degree and velocity of shortening. Higher drive rates decreased the extent of shortening and shortening velocity in this myocyte (left panel, Fig. 6A). The decrease in velocity of shortening at high drive rates is not attributable to a change in sarcomere length because sarcomere length at peak velocity of shortening was the same at both low and high drive rates (Fig. 6A, horizontal arrows). Stimulation at 1 Hz immediately after the highest drive rate promptly restored contraction (data not shown), suggesting that depletion of energy stores and/or SR stores of Ca2+ does not explain the decreased contractile performance at high drive rates. Figure 6B summarizes the relationship between sarcomere length and drive rate in 9 cells isolated from four normal hearts and in 21 cells isolated from three ACF hearts. The overall effect of stimulation on initial sarcomere length and length at peak shortening was similar to that shown in Fig. 6A. As with sarcomere length in the unstimulated cells, the initial length of the contracting sarcomere tended to be shorter in the hypertrophied cells than in the control cells, but the difference was not significant in this subgroup. Length at the peak of contraction tended to be longer in the ACF cells than in the control cells (also not significant).

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A 1.83 1.63

0.0

1.0

STIMULUS

2.0

3.0

FREQUENCY

4.0

5.0

(Hz)

FIG. 6. Effect of drive rate on sarcomere shortening in isolated myocytes. A, left: slow chart recording showing the contractile adjustments in a myocyte isolated from hypertrophied failing myocardium. A, right: details of shortening at a near-optimal drive rate (2 Hz); the sarcomere length at the peak velocity of shortening is changed little by stimulation at a high rate (arrows). Note the change of calibration for the velocity trace at the right. B: effects of drive rate on initial sarcomere length (open symbols) and on sarcomere length at the peak of shortening (closed symbols) in control (circles) and ACF (triangles) myocytes. Values represent means t SE for contractile data for 9 myocytes isolated from 4 control hearts, and 21 myocytes from 3 hypertrophied failing hearts, studied over a compete set of drive rates. There were no significant differences between the groups in either the initial sarcomere length or the sarcomere length at the peak of contraction. The reduction of sarcomere shortening at higher drive rates (>3 Hz) is not accompanied by any significant decrease in initial sarcomere length. Lines represent third-order polynomial fits (R” > 0.96).

Thus the initial sarcomere length tended to be shorter and the sarcomere length at the peak of contraction tended to be longer in ACF cells than in normal cells. Even though those differences in sarcomere length were not statistically significant, the fractional shortening computed from these values was significantly lower in ACF cells than in normal cells. Figure 7 shows the fractional shortening and the peak velocity of shortening for the same cells depicted in Fig. 6B. Figure 7A shows that the maximum extent of shortening was depressed in ACF myocytes over the entire range of drive rates (analysis of variance, F test: P < O.Ol), and the depression reached statistical significance at drive rates ~2 Hz (t test: P < 0.05). Accordingly, the maximum fractional shortening at 2 Hz was significantly lower in ACF cells (8.6 t 2.6%, mean t SD) than in control cells (10.8 t 2.1%). As shown in Fig. 7B, a similar depression of the peak velocity of sarcomere shortening occurred in the ACF myocytes (F test: P < 0.05). A comparable analysis showed that the peak velocity of relengthening was also significantly depressed over the range of drive rates in ACF cells (analvsis of variance.

MYOCYTE

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.

0.0

1.0

2.0

STIMULUS

3 a

3.0

4.0

FREQUENCY

5.0

(Hz)

1

2

-

AND

.

3

-

.

CONTRACTION

-

4

AK VELOCITY OF SARCOMERE SHORTENING (pm/s)

0; 0.0

1.0

2.0

STIMULUS

3.0

4.0

FREQUENCY

5.0

(HZ)

FIG. 7. Effect of drive rate on derived measurements of contraction. Circles and triangles denote data (means t SE) from control myocytes (n = 9) and from myocytes isolated from hypertrophied failing hearts (n = 21). These are the same myocytes depicted in Fig. 6. A: effect of drive rate on sarcomere shortening, computed as 100 X (SLdiast where SLdiast and SLsyst are the length of the sarcomere SLyst)/SLliast, before the stimulus and at the peak of shortening, respectively. B: effect of drive rate on the peak velocity of sarcomere shortening. *Significant differences between the group means at a particular drive rate (P < 0.05). Lines are third-order polynomial fits of the data (R” > 0.9). C: relation between shortening and relengthening dynamics in the control and ACF groups. Here, the control group values are depicted by open circles to facilitate identification.

F test: P < 0.01). Figure 7C plots the relationship between the velocity of shortening and the velocity of relengthening in both control and ACF myocytes, and shows that data from the two groups formed a continuum. This suggests that the slower relengthening in the ACF myocytes cannot be attributed to a specific alteration in the relaxation mechanism, per se. The group differences between ACF and control myocytes was a consistent finding for all contractile parameters examined; i.e., the extent and percent of sarcomere shortening and the peak velocities of sarcomere motion, based on 252 measurements of contraction in 38 normal cells, and 323 measurements of contraction in 43 ACF cells driven at rates from 0.1 to 9.0 Hz. The time from the onset to the peak of shortening in the control and ACF cells was 129 t 5.6 ms (mean t SE, n = 34) and 134 t 4.4 (n = 42) ms, respectively, at a drive rate of 1 Hz. These values are somewhat less than those for the TTP of the [Ca’+]; transient (Table 3). When [Ca’+]; and contraction are measured simultaneously in the same cells, the rise in [ Ca2+]; typically precedes cell shortening (47, 55). However, in this study contraction was measured in cells not loaded with fura2 and therefore those cells probably shortened more rapidly than the cells in which [Ca2+]; was measured. In addition, the broader [Ca2+]; transients characteristic of guinea pig myocytes make determination of TTP [Ca2+]; less precise than determination of time to peak shortening. Aside from these considerations, the maximal

-I

5

IN

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+d[Ca”+];/dt at 1 Hz was >4% of peak [Ca”‘]; per ms in both groups, compared with normalized values for peak velocity of shortening, which showed rates ~2% of maximal shortening per millisecond in both groups. Thus, although there is some question about the relative timing of peak [Ca2+]; and peak shortening, [Ca2+]i clearly increased faster than shortening during the initial period after excitation. As drive rate increased, time to peak shortening decreased in both groups, but it decreased to a greater extent in the control group. As a result, at 5 Hz, time to peak shortening in the control group (69.4 t 3.1 ms, n = 26) was significantly shorter than it was in the hypertrophy group (87.9 t 3.6 ms, n = 24; P < 0.05). Relation between [Ca2”/i and sarcomere shortening in intact cells. Although the depressed [Ca’+]i can account for the depressed contractile performance of the ACF myocytes, it is also possible that the sensitivity of the myofibrils to the Ca2’ may be depressed. To investigate that possibility we studied the relationship between [Ca2+]; measured with fura- and sarcomere length in normal and ACF myocytes. This relationship was obtained by plotting the sarcomere length measured in cells not loaded with fura- against [Ca2+]; measured with fura- in other cells from the same heart, at the same drive rate. Figure 8A shows the relationship between [Ca2+]; -

A

1

sE

1.90

?

(3 zi

1.80

w

1.70-

DIAST

SYST 0

ma0 w

AC ACF

O4

Cl

A

:

I

E

-

8

1.60-

2 CJj

1.50!

+ fSEM . 10

. . . . . ..I 100

-

MYOPLASMIC

;

/t 1

.

10

.

. . -..-I

.

100

MYOPLASMIC

.

’

. .-.--I

. .....q

10000

CALCIUM

$ fSEM

k

* * 1 . . . . ..‘I 1000

(nM)

.

.

1000

CALCIUM

- - .--I

10000

(nM)

FIG. 8. Relation between [Ca’+]; and contraction. Individual data points represent the respective means for [Ca’+]; and sarcomere length in 10 cells from a control heart, 5 cells from a hypertrophied heart without heart failure (AC), and 15 cells from a hypertrophied failing heart (ACF). All observations were paired by common drive rate. Drive rates were varied from 0.20 to 5.00 Hz to grade both sarcomere length in diastole and sarcomere length at the peak of shortening. Overlap of the respective data points demonstrates a common time-independent relation between [Ca2+]; and sarcomere length. B: correlation between [Ca’+]; and the peak velocity of unloaded sarcomere shortening in 9 control and 21 ACF myocytes for data pooled from all animals. Data on shortening velocity were those used for Fig. 7B, and data on [Ca2+]i were those used for Fig. 4. The peak velocity of sarcomere shortening is graded by [ Ca”‘];, but it is limited at the highest drive rate (asterisk). All data are means of: SE.

H524

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measured with furaand sarcomere length in 10 myocytes isolated from a control heart, 5 myocytes from an AC heart, and 15 myocytes from an ACF heart. The relation between [ Ca2+]i and sarcomere length was graded by including both the initial (diastolic) sarcomere length and the length of the sarcomere at the peak of contraction (systolic) evoked at drive rates from 0.2 to 5.0 Hz. The data from the control, AC, and ACF cells superimposed, suggesting a single relationship for all groups. Furthermore, data points obtained in diastole at the highest drive rate overlap data points obtained at the peak of contraction during low drive rates. Thus there appears to be a single relationship between [Ca2+]i and sarcomere length throughout the contraction cycle. Figure 8B illustrates the effect of [Ca2+]i on the velocity of sarcomere shortening that was obtained by averaging all measurements of [ Ca2+]i and contraction for each drive rate. Data on shortening velocity were those used for Fig. 7B, and data on [Ca2+]i were those used for Fig. 4. The speed of sarcomere shortening correlated well with the peak level of [ Ca2+]; and, as was seen in the plot of sarcomere length versus [ Ca2+]; (Fig. 8A ), we observe again that data from the control and ACF cells overlap (Fig. 8B). Direct effect of [Ca”/ on sarcomere shortening. The data in Fig. 8 suggest that [Ca”‘] < 100 nM can modulate sarcomere length in normal, AC, and ACF cells. This result is somewhat surprising, since mechanically skinned small fragments of rat cardiac tissue do not seem to show gradation of sarcomere length at comparably low levels of [ Ca”+] (14). One possible explanation is that the measurement of [ Ca2+]; with furais falsely low. To exclude that possibility, we examined the effect of controlled changes in free [Ca”‘] on sarcomere length in normal myocytes permeabilized by a nonionic detergent. The effects of free [Ca”‘] on sarcomere length of these detergent “skinned” cells was investigated at two levels of free [Mg2+]: 0.10 mM in 317 cells from 2 hearts and 1.80 mM in 408 cells from 3 hearts. These amounts of free [Mg2+] span the range expected for intact myocytes, and correspond to 2.00 and 6.75 mM Mg acetate, respectively, in the perfusate. Free [Ca”‘] was varied from -1 to >700 nM by increasing the proportions of CaKEGTA and KZEGTA. Because control of [Ca2+]; by EGTA may be inaccurate when [Ca”‘] < 10 nM (15), the concentration at the lowest calculated value of pCa = 9 was assumed to be -2 nM. The effect of controlled changes in [Ca”‘] in the skinned cells is shown in Fig. 9. At the value of [Ca”‘] nearest the lowest value of [Ca2+]i measured with fura-2, there was activation of shortening. Sarcomere length shortened in a stable fashion as [Ca”‘] was increased to -500 nM at low [Mg2+] and to -900 nM at high [Mg2+]. The relation between sarcomere length (based on data used for Fig. 6B) and [Ca2+]i measured by fura(based on data used for Fig. 4) in control and ACF myocytes was superimposed on the data obtained in response to controlled [Ca”‘]. Thus Fig. 9 shows three sarcomere length- [ Ca2+] relations: 1) for skinned cells in high [Mg”+], 2) for skinned cells in low [Mg2+], and 3) for intact cells in which [Ca2+]; was measured with fura-2. Each of these relations was fit to a third-order polyno-

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mial expression, where all coefficients of fit (R2) were BO.94. In the skinned cells at a [Ca”‘] of -50 nM, the relation indicated that sarcomere length shortened to 1.835 pm at 0.10 mM [Mg2+] or to 1.865 pm at 1.80 mM

CMg2+1 l

Mean resting [ Ca2+]; for all normal myocytes was 37 t 14 nM and for all ACF myocytes was 58 t 23 nM. Second- and third-order polynomials fit to data for intact control and ACF myocytes (Fig. 8) predict that this difference in resting [Ca2+]i would cause resting sarcomere length of intact ACF myocytes to be 0.006-0.013 pm shorter than that of intact normal myocytes if they differed only in free [Ca”‘]. Direct measurement of sarcomere length in these intact myocytes showed a greater difference (0.025-0.049 ,um), suggesting that differences in [Ca2+]; were not entirely responsible for the shorter sarcomere lengths of unstimulated intact ACF myocytes compared with controls. This interpretation is supported by the fact that sarcomere lengths of chemically skinned ACF myocytes in nominally Ca2+-free solution were 0.079 ,urn shorter than sarcomere lengths of identically treated control myocytes. At [Ca”‘] = 200 nM, the predicted sarcomere lengths in the skinned cells would be 1.68 at low [ Mg”+] and 1.76 at high [Mg2+], i.e., values close to the 1.75 pm found for intact cells. Data at higher levels of [Ca”‘] where numerous skinned cells were observed with very short sarcomere lengths of 1.2-1.3 pm are not included because such cells may have shortened in an unstable fashion (14). Thus at the lower [Mg2+] and with calculated [ Ca”‘] to be >500-700 nM, one finds the shortest steady-state sarcomere lengths that can be observed in these cells. However, the inclusion of measurements of extreme

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shortening at high [Ca”‘] would only serve to increase the agreement between the fitted curves for the skinned cell and the intact cell data at low levels of [Ca”‘]. DISCUSSION

Our results provide the first quantitative description of a specific subcellular abnormality that can account for the reduced mechanical performance of hypertrophied and failing cardiac muscle. Previous studies in hypertrophied and failing cardiac muscle have not provided an adequate explanation for the reduced contractile performance of failing myocardium (28). Biochemical studies have identified alterations in myosin isoenzymes and adenosinetriphosphatase activity, neither of which can easily account for the reduced tension-generating capacity of failing heart muscle (28). Studies in skinned preparations of hypertrophied left ventricular muscle have failed to show reduced sensitivity of the myofilaments to [Ca”‘] (25, 37,43, 49). Alterations in excitation-contraction coupling have been suggested, (9, 29, 31) but there is no direct experimental confirmation of such alterations in hypertrophied or failing myocardium. Our results show that the [Ca2+]; transient is severely and significantly depressed in ACF myocytes. We show further that the depressed [ Ca2+]; transient correlated with depressed mechanical performance in ACF myocytes, supporting a pathophysiological relationship between the depressed [Ca2+]i transient and reduced contraction. The reduced contractility in ACF cells provides an adequate explanation for the depressed function of hypertrophied, failing hearts (53). There is no need to invoke the participation of extracellular factors such as alterations in connective tissue, although such factors might play an additional role in the intact heart. We found that both the fractional shortening and the maximum velocity of shortening were depressed in ACF myocytes. Previous studies have observed decreased maximum velocity of shortening and relengthening in hypertrophied left ventricular preparations from the guinea pig (39) and reduced sarcomere shortening in hypertrophied isolated rabbit muscle (21,26). Our results show explicitly that the ability to shorten is reduced in isolated single cells and that contractile behavior can be directly related to the level of [Ca2+]i. We have also shown that other factors may influence the relation between [ Ca2+];, sarcomere length, and contraction. Specifically, during rapid stimulation, the extent and velocity of sarcomere shortening decreased in both normal and ACF myocytes, despite an increase in the mean value of [Ca2+];. Because the negative inotropic effect occurs at similar sarcomere length (Fig. 6A), it cannot be attributed to increased resistance to shortening due to an internal passive elastic element. Furthermore, stimulation at 1 Hz immediately after the period of rapid stimulation promptly restored contraction, suggesting that neither energy depletion nor depletion of [Ca”‘] from the SR can explain the negative inotropic effect of rapid drive. This dissociation between contractility and [ Ca2+]; suggests that under certain conditions factors other than [ Ca2+] i influence contraction. We also found that the sarcomere length in unstimu-

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lated ACF cells was significantly shorter than in normal myocytes. Other investigators have observed shorter sarcomeres in the myocardial wall (38), in papillary muscles (21, 26) and in isolated myocytes (22; not significant) from hypertrophied hearts. The shorter sarcomere lengths we found in unstimulated intact ACF cells cannot be attributed solely to a higher level of [ Ca2+]i, because the greater resting [Ca2+]; in those cells (58 nM) vs. that of control cells (37 nM) can only account for a difference in sarcomere length of -0.010 pm (see Fig. 8), whereas directly measured sarcomere length of intact ACF myocytes was 0.025-0.049 pm shorter than sarcomere length of intact control myocytes. Furthermore, sarcomere length of chemically skinned ACF myocytes in nominally zero free [Ca”‘] was significantly shorter than that of skinned normal myocytes. Thus the shorter sarcomere length in cells from hypertrophied failing myocardium must be at least partly attributable to some other intracellular mechanism that is independent of any putative mechanical effects of the interstitium. Because the relation between [Ca”‘] and sarcomere length depends on 2 wk +], we cannot exclude the possibi lity that differences in [Mg2+]i between the control and hypertrophi .ed myocytes might influen .ce their respective sarcomere lengths. It is also possib le that subtle structural alterations in the hypertrophied myocytes might cause their sarcomere lengths to be shorter than those of control myocytes. We have shown that the sarcomere length is quite sensitive to small changes in myoplasmic [ Ca”+]; in both normal and ACF cells. Although the meaning of sensitivity to Ca2+ is difficult to establish by measurement of shortening, our results show that sensitivity to activation by Ca”+ is clearly not reduced in cells isolated from hypertrophied, failing myocardium. We do not know the mechanism responsible for the reduced peak systolic [Ca2+]; in ACF myocytes. However, our observations suggest that this depression is at least partially reversible by norepinephrine. Our results imply that cardiac failure is associated specifically with a defect in Ca2’ metabolism in the failing heart. The depressed [ Ca2+]; transient in failing myocytes could be caused by a number of mechanisms: 1) an insufficient amount of transmembrane Ca2’ current to evoke a normal amount of Ca2+ release from the SR, 2) an insufficient store of Ca2+ in the SR, and 3) a defect in the release of Ca2+ from an adequately stocked SR. With regard to the first possibility, Scamps et al. (51) have recently shown that Ica, when corrected for cell size, did not differ significantly between normal and hypertrophied cells. However, those studies were done in cells isolated from hypertrophied but nonfailing hearts. Therefore, a reduced Ica trigger cannot be excluded as a possible contributor to the depressed systolic [Ca2+]i in cells from failing hypertrophied hearts. The ability of the SR to sequester Ca”+ is impaired in the hypertrophied myocardium (27, 57) and is further impaired with the onset of heart failure (57, 63). This defect might lead to insufficient Ca”+ within the SR, which in turn could contribute to the depressed [Ca2+]; transient and contraction (61). Our results are also consistent with the possibility that Ca”+ release from the SR may be impaired in the hypertrophied and failing myo-

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cardium, although there is no direct evidence for this. Our study calls into question the concept that chronic cardiac failure is associated with a chronic state of “Ca’+ overload,” if one defines that state as elevated myoplasmic [Ca”‘]; during diastole. Indeed, our data show that the peak of the [Ca2+]; transient is depressed while diastolic [ Ca2+]; is no higher than normal in myocytes isolated from failing hearts. Thus Ca2+ overload may not be a chronic stable state, but rather it may represent an agonal condition in which no further compensation is possible, [ Ca2+] i rises, and severe cardiac dysfunction ensues. On the other hand, contractile stresses and neurohumoral changes in the hypertrophied failing myocardium may influence [ Ca2+]i in vivo. For example, circulating norepinephrine is elevated in heart failure (58), and our data suggest, that this would tend to restore systolic [ Ca2+]; but also raise diastolic [ Ca2+];. Because norepinephrine reportedly decreases sensitivity of the myofilaments to [Ca2+]; (l), it is not clear that its net effect on contraction would be beneficial, and the sustained elevation in diastolic [Ca2+]; might be deleterious to the failing heart. Regardless of the effects of in vivo factors, our studies show that the impaired function of hypertrophied, failing hearts has a basis in weaker myocyte contraction that is associated with a markedly diminished [ Ca2+]i transient. APPENDIX

In this APPENDIX we discuss certain issues regarding conversion of original fluorescence records to estimates of [Ca”+];. Much of the discussion relates to a proposed method for determining B, a coefficient reflecting the properties of the dye at the excitation wavelength chosen, which will be incorrectly estimated by conventional methods when there is appreciable dye loss or change in cell shape. We also compare our measurements of fluorescence ratio and [Ca”+]i to those of other studies. Conversion formula. The time-varying fluorescence ratio during the contraction, R, was converted to [Ca2+]; according to the formula of Grynkiewicz et al. (17) [Ca”‘]i

= B X &(R

- R,i,)/(R,,,

- R)

in which &, the Ca2+-furaapparent dissociation constant at 37OC, is 224 nM (17) and R, R,i,, and R,,, are, respectively, the background-corrected ratios observed during contraction and the ratios obtained under conditions of calcium desaturation and calcium saturation. All of these factors are considered in further detail below. Determination of fluorescence ratio (‘R). Background light counts were obtained in five dye-free cells from each animal. The background counts obtained at each wavelength were averaged and subtracted from the corresponding 340- and 380nm spectra obtained in cells loaded with fura-2. Then, the ratio of emitted light due to excitation at 340 nm, divided by that due to excitation at 380 nm (each corrected for background fluorescence) was computed. Determination of R,i,. In preliminary tests to determine the value for R of cells under the condition of minimal [Ca2+]; (R,i,), we first loaded cells with fura- at room temperature, placed them in a perfusion chamber with normal Tyrode solution at 37OC, and recorded R before and after varying periods of perfusion with Ca”+ -free Tyrode solution + 2 mM EGTA, or with Ca2+-free Tyrode solution + 2 mM EGTA + 1 PM Ca2+ ionophore (4-bromo A23187, Molecular Probes, Eugene, OR). We observed that regardless of the perfusion medium, the

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lowest values were seen in quiescent cells, freshly loaded with fura-2, within the first 5 min of perfusion at 37°C. Because neither EGTA nor its combination with the ionophore drove R lower than the value seen before they were added, their use was subsequently discontinued. The rationale for using our procedure in determining Rmin is as follows: 1) [Ca2+]; must be lowest under the conditions that produce the lowest R; 2) R measured in quiescent cells was stable at a higher level after a brief period of 1 Hz drive (R,i,: 0.86 & 0.07; R of quiescent cells after 1 Hz drive: 1.12 & 0.16 means t SD; P < 0.01); and 3) R measured in diastole increased with increasing drive rate but returned to near its original value with resumption of low drive rate. Thus the Rmin estimate seems reasonable, since it remains lower than any subsequent resting or diastolic R values seen in equilibrated cells. Determination of R,,,. To estimate R in cells exposed to saturating [ Ca2+] i (R,,,), we initially used a procedure in which the myocytes were exposed to metabolic inhibitors, followed by exposure to an ionophore in high [ Ca2+] (40). This procedure produced R,,, estimates that were considerably lower than those obtained by causing the cells to go into contracture with an intense pulse of current. Use of an ionophore does not necessarily guarantee that the intracellular dye will be exposed to saturating levels of [Ca”+]; (17, 50), and it has the added disadvantage of requiring long incubation periods during which photobleaching and other forms of dye breakdown and metabolism can occur. For all of these reasons, we chose to determine R,,,,, by the following method. At the end of the experiment, a brief but intense pulse of current was applied to each cell to cause contracture. The fluorescence ratio obtained immediately after induction of the contracture was taken as R,,,. Determination of B. B is the ratio of the proportionality coefficient for the Ca2+-free dye (SfsgO) to the proportionality coefficient for the Ca”+-saturated dye (Sb& during excitation at 380 nm

If dye concentration and properties and cell shape do not change between the Rmin and R,,, estimates, B can be determined from the ratio of fluorescences due to excitation at 380 nm in Ca2+-unsaturated and Ca2+-saturated conditions, respectively. However, because dye leakage, photobleaching, and change of cell shape may occur in living cells during the calibration procedure, it is preferable to use a formula that does not rest on these assumptions. Our approach was based on our observation that the sum of photon counts at 340 nm + 380 nm remained relatively constant, despite changes in R, in the guinea pig cells we studied. Figure 10 illustrates this point. Figure 1OA shows the individual fluorescence records due to excitation at 340 nm (upper) and at 380 nm (lower) during contraction of a normal myocyte driven at 1 Hz. Background autofluorescence due to excitation at 340 nm and 380 nm was 0.85 X 10” and 0.46 X IO’ counts/s, respectively. For these contractions, the background-corrected ratio was 1.18 in diastole and 3.04 in systole, representing 39% of the maximum possible change in R for this cell (R,,,, - Rmin = 4.77). Above both of the phasic traces is their sum, which shows a barely perceptible increase during contraction. Figure IOB shows records from this same myocyte during 2 Hz drive. Here the ratios in diastole and systole are 1.44 and 3.53, respectively, representing 44% of the maximum possible change in R. These contractions are accompanied by slightly greater increases in the sum of fluorescences. The ratio of total background-corrected fluorescence in systole (FZJO+ F380)systto total background-corrected fluorescence in diastole (F340 + Fsgo)diastwi 11 be 1.00 (0% change) if there is no change in total fluorescence with contraction. Our experimental data show that this ratio increases, but only slightly, as

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1 Hz DRIVE

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340

nm +

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.

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60

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IN R: % OF (R,,,

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FIG. 11. Effect of increasing R on total fluorescence in isolated myocytes. The abscissa indicates the increase in R during a single beat of the maximum possible increase in R (R syst - Rdiast) as a percentage for that cell (RIrlax - R,i,). The ordinate shows corresponding values for the percent increase in total background-corrected fluorescence during systole (F, ‘j4() + F3&yst over total background-corrected fluoresFenye.

during

diastole

+ F:s%))diast-

cF340

Data

were

Obtained

during

153

individual beats of 26 myocytes from 13 control hearts (C), 8 myocytes from 4 nonfailing hearts of aortic-banded animals (AC), and 17 myocytes from 8 hypertrophied failing hearts (ACF) driven at 1.00, 2.00, 1’1 p p$iir ci’,q. and 5.00 Hz. The solid line is the least-squares regression line for all points, with a correlation coefficient of 0.25 (P < 0.0001) and has the equation Y = 0.067X + 2.2.

. &h+b’

4-o I

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“J&$q~+il 380

’41; ,I’kiI/“fI /(Rmin + 1) If the ratio in the second equation is not precisely 1.00, the observed ratio will be the coefficient of B in the last equation. For example, we estimate that in these myocytes, the fluorescence sum (340 nm + 380 nm) increases by 8.9% from Rmin to R max. In this case the second equation would be written (Sb340 + Sb&/(Sf340

+ Sfs,,) = 1.089

Then (1.089)S

= (Rmax + l)/(Rmi,

+ 1)

and B = (Rmax + l)/(Rmi,

+ 1)(1.089)

Because this correction was small and similar in the groups being compared, we did not apply it to our calculations, but we note that [Ca’+]; is overestimated by 8-9% by assuming no change in the fluorescence sum. Although the value of 1.089 derived here is considerably less than the value obtained in vitro (-1.33; derived from Ref. 16, Fig. 3), estimates of Rmax are likewise much lower in vivo than in vitro (6-7 vs. 30-40, respectively), underscoring the importance of making the in vivo measurements. Problems related to estimation of B can, in theory, be circumvented by using the isosbestic point as the second excitation wavelength (-360 nm). In practice, the wavelength chosen may not be precisely isosbestic for a particular experiment (4). Furthermore, use of the isosbestic point reduces the dynamic range of the calcium signal. Thus the derivation used here provides a means of estimating B when it cannot be

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assumed to be 1.00, and there is reason to suspect that total fluorescence is changing during the time interval between the R,,,,, and Rmin estimates (i.e., because of dye loss or cell shape change). Effect of fura- loading on peak systolic [Ca”+];. An important issue is the extent to which fura-2, which binds at least some Ca”+, influences the [Ca2+]; transient itself, and whether the hypertrophied myocytes might be more sensitive to this effect. To address that issue, we examined the effect of different levels of loading with fura- on peak systolic [Ca”‘]i measured during regular stimulation at 1 Hz. The amount of dye loaded into the cells was varied by altering the concentration of fura- and the duration of incubation with the dye. Figure 12 shows the effect of the degree of fura loading, as estimated by the sum of fluorescence intensity due to excitation at 340 nm + 380 nm, on peak systolic [ Ca”+]; in a series of 40 cells from 2 normal hearts and 60 cells from 2 hypertrophied, failing hearts. These cells were loaded to varying degrees with fura-2, perfused in normal Tyrode solution at 37°C and driven at 1 Hz. Although measuring fluorescence at the isosbestic point is the standard method for following changes in dye concentration, we have shown that the sum of fluorescence due to 340 and 380 nm excitation is also relatively constant during contraction and is therefore primarily dependent on dye concentration. Because we were not using the isosbestic point as one of our excitation wavelengths, this was the most practical way to relate the peaks of the calcium transients to simultaneous relative dye concentrations. The regression lines show that as the level of fura loading increased, peak [Ca2+]; tended to decline in both normal and hypertrophied cells. However, the fall in [ Ca2+]; with higher levels of dye loading was less marked in hypertrophied than in normal cells. Therefore, if anything, peak [Ca2+]i is less depressed by fura- in hypertrophied than in normal cells. Furthermore, the mean level of fura- loading in the hypertrophied cells (7.1 t 3.5 x lo5 counts/ s; mean t SD) was lower than in normal cells (9.3 t 5.2 x IO5 counts/s). Therefore, it is unlikely that the depressed [Ca2+]; transient in hypertrophied myocytes is attributable merely to loading with fura-2. Comparison to other calibration methods and [Ca2+]i estimates. Direct comparison of Rmin and R,,, estimates can only be made when the same cell type, perfusion and stimulation conditions, spectrofluorometric system, and excitation wavelengths are employed. Li et al. (40) studied isolated rat left

5 TOTAL

COUNTS

10

15 (340nm

20

25

+ 380nm)

x lo5

FIG. 12. Effect of furaon peak [Ca2+];. This plot shows the relationship between relative furadye concentration and peak systolic [Cay+]; during 1 Hz stimulation of myocytes from normal hearts (C; 40 myocytes from 2 hearts) and myocytes from hypertrophied, failing hearts (ACF; 60 myocytes from 2 hearts). Regression lines for the control group (solid line) and the ACF group (dotted line) had slight, insignificant downward trends, and their slopes did not differ significantly.

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ventricular myocytes with a furasystem similar to ours. In that study, metabolic inhibitors were added to the perfusate in conjunction with an ionophore and either low (nominally calcium-free) or high calcium concentrations to obtain estimates of Rnin and ha,, respectively. Their Rmin values averaged 0.68 t 0.07 (SD) and their R,,, values averaged 5.24 to.90 in the intact cells. These values corresponded well with those obtained in the supernate from digitonin-lysed preparations, which were 0.70 t 0.04 and 5.44 t 1.36, respectively. The Rmin and R,,, estimates reported here for guinea pig left ventricular myocytes (0.88 t 0.04 and 6.25 t1.06 respectively) are somewhat higher. However, we have also examined rat left ventricular myocytes in the same manner, and in those cells Rmin was 0.76 t 0.04 differences in these and ha, was 5.44 t 1.36. Interspecies values are to be expected. An important requirement for any calibration method is that it produce an estimate of R,,, that exceeds the highest ratio attainable by the myocyte under physiological conditions. In this study, the highest ratios were seen with ~-HZ drive rate in the presence of norepinephrine, and these ratios never exceeded R,,, determined by our procedure. Grynkiewicz et al. (17) obtained an R,,, of 35.1 in a buffer system using fura- and excitation wavelengths of 340 and 380 nm. We have also found R,,, to be in this range for our buffer system. Reading data from Fig. 3 of that study, one could predict that if 10% of the dye were converted to a Ca2+insensitive form having the same spectral characteristics as the unsaturated Ca2+-sensitive dye, relative fluorescence at 340 nm during Ca2+ saturation would be 31.6 (rather than 35.1), while relative fluorescence at 380 nm would be 3.2 (rather than 1.0) and the 340 nm/380 nm ratio would be 9.9 (rather than 35.1). Similarly, this analysis would predict that conversion of 15% of the dye to a Ca2+ -insensitive form would change relative fluorescence at 340 nm to 29.8, and at 380 nm to 4.4, thus lowering the R,,, estimate to 6.8. This analysis suggests that a small amount of Ca2’-insensitive fluorescence can have a major impact on R,,, estimates in myocytes. Differences in viscosity between calibration solutions and myoplasm are another source of error in determining Rmin and R,,,, and Poenie (50) has proposed some corrections for this error. Other undefined constituents of the myoplasm may also influence the sensitivity of cell fluorescence to [Ca’+];. Insofar as our calibration procedure is based entirely on measurements in cells, the net effect of all of these influences should be reflected in our determinations of R-nin, Rnaxy and B, and are thus taken into account in our [ Ca”+]; estimates. With respect to absolute values of estimated [Ca’+];, once again it is essential to consider differences in the measurement conditions before making comparisons. Estimates of resting [ Ca2+]; in mammalian ventricular tissue by calcium-sensitive microelectrodes range from 250 to 290 nM (6). However, these values are considered to be the upper limit of “true” resting [Ca2+]i since cell membranes may not seal perfectly after impalement, thereby allowing leakage of Ca”+ into the cytosol from the perfusate. Barcenas-Ruiz and Wier (4) used fura- to measure [Ca2+]; in guinea pig ventricular myocytes perfused at 37°C and driven externally at 1 Hz. R,,, in that study was determined by cell lysis after mechanical disruption of the sarcolemma, a method similar to ours. Their representative transient showed a diastolic [Ca2+]; value of 100 nM and a systolic [Ca”‘]i value of 600 nM. That transient resembles those we report here for normal guinea pig left ventricular myocytes driven at 1 Hz (diastolic: 85 * 24 nM; systolic: 594 t 180 nM). Estimates of resting [Ca”+]i by Callewaert et al. (8) in fura-2loaded voltage-clamped myocytes (100 nM), and by Williford et al. (62) in fura-2-loaded guinea pig myocytes (73 nM) are also consistent with values reported in this study. Although estimates of absolute [Ca2+]; are affected by a

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number of assumptions and methodological considerations, conversion of the ratio values also serves to adjust for differences in fluorescence sensitivity of the different groups of cells, thus providing a more valid comparison. In a study such as the present one, this function of the calibration procedure is of greater importance than the determination of absolute [Ca”‘];, per se. NOTE

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13.

PROOF

Recent observations on single ventricular myocytes have shown depressed [Ca”+]; transients in a renovascular hypertensive rat model [Moore, R. L., R. V. Yelamarty, H. Misawa, R. C. Scaduto, Jr., D. G. Pawlush, M. Elensky, and J. Y. Cheung. Altered Ca2+ dynamics in single cardiac myocytes from renovascular hypertensive rats. Am. J. Physiol. 260 (Cell Physiol. 29): C327-C337,1991.] and depressed myocyte shortening and relengthening in a model of right ventricular pressure overload in the cat (Mann, D. L., Y. Urabe, R. L. Kent, S. Vinciguerra, and G. Cooper IV. Cellular versus myocardial basis for the contractile dysfunction of hypertrophied myocardium. Circ. Res. 68: 402-415, 1991.)

14.

15.

16.

17.

18. We thank Dr. Regina Talingdan and Dr. Hong Zhang for excellent technical assistance, and also Joanne Pawlowski for assistance in typing this manuscript. We also greatly appreciate the encouragement, support, and critique we received from Dr. Edmund H. Sonnenblick. We are especially grateful for the generous and continued support of the Abraham J. Blumkin fund. This work was supported in part by National Heart, Lung, and Blood Institute Grants PO1 HL-37412, HL21325, and HL-32688 and a New York Heart Association grant-in-aid. Address for reprint requests: F. M. Siri, Albert Einstein College of Medicine, Cardiology Division, Forchheimer G-42, 1300 Morris Park Ave., Bronx, NY 10461. Received

8 August

1990; accepted

in final

form

21 March

1991.

19.

20.

21.

22.

REFERENCES 1. ALLEN, D. G., AND S. KURIHARA. Calcium transients in mammalian ventricular muscle. Eur. Heart J. 1, Suppl. A: 5-15, 1980. 2. ARONSON, R. S. Characteristics of action potentials of hypertrophied myocardium from rats with renal hypertension. Circ. Res. 47: 443-454, 1980. 3. ARONSON, R. S., AND C. NORDIN. Arrythmogenic interaction between low potassium and ouabain in isolated guinea pig ventricular myocytes. J. Physiol. Lord 400: 113-134, 1988. 4. BARCENAS-RUIZ, L., AND W. G. WIER. Voltage dependence of intracellular [Ca2+]; transients in guinea pig ventricular myocytes. Circ. Res. 61: 148-154, 1987. 5. BEUCKELMANN, D. J., AND W. G. WIER. Mechanism of release of calcium from sarcoplasmic reticulum of guinea pig cardiac cells. J. Physiol. Lond. 405: 233-255, 1988. 6. BLINKS, J. R., W. G. WIER, P. HESS, AND F. G. PRENDERGAST. Measurement of Ca2+ concentrations in living cells. Prog. Biophys. MOL. Biol. 40: l-114, 1982. 7. BRAUNWALD, E., J. R. Ross, JR., AND E. H. SONNENBLICK. Mechanisms of Contraction of the Normal and Failing Heart. Boston, MA: Little, Brown, 1976, p. 309-356. G., L. CLEEMAN, AND M. MORAD. Epinephrine 8. CALLEWAERT, enhances Ca2’ current-regulated Ca2’ release and Ca2+ reuptake in rat ventricular myocytes. Proc. Natl. Acad. Sci. USA 85: 20092013,1988. 9. CAPASSO, J. M., R. S. ARONSON, AND E. H. SONNENBLICK. Reversible alterations in excitation-contraction coupling during myocardial hypertrophy in rat papillary muscle. Circ. Res. 51: 189-195, 1982. 10. CAPASSO, J. M., J. E. STROBECK, A. MALHOTRA, J. SCHEUER, AND E. H. SONNENBLICK. Contractile behavior of rat myocardium after reversal of hypertensive hypertrophy. Am. J. Physiol. 242 (Heart Circ. PhysioL. 11): H882-H889, 1982. 11. FABIATO, A. Myoplasmic free calcium concentration reached during the twitch of an intact isolated cardiac cell and during calcium-

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44. 45. 46.

47.

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