Decreased Myocyte Tension Development and Calcium Responsiveness in Rat Right Ventricular Pressure Overload
Background The contractile dysfunction observed in end-stage myocardial hypertrophy has at its base an abnormality in myocyte function. However, whether depressed contractile function is related to an alteration in contractile protein function is presently unknown.
Methods and Results Contractile force, tension, and calcium responsiveness were measured in single-skinned myocytes isolated from rats with right ventricular hypertrophy (RVH) and control rats. RVH was induced by pulmonary artery constriction for 36 weeks and was associated with significant myocyte hypertrophy. Myocytes were attached to micropipettes that extended from a force transducer and motor. Isometric force was measured over a wide range of calcium concentrations at two sarcomere lengths (SLs). Maximal force was increased in the RVH group: 1.20±0.10 versus 1.62±0.13 mg at SL=2.0 μm and 1.33±0.10 versus 1.84±0.15 mg at SL=2.3 μm (P<.05). Maximal tension, however, was reduced in the RVH group: 24.3±1.91 versus 37.5±2.92 mN/mm2 at SL=2.0 μm and 27.4±1.78 versus 41.8±3.19 mN/mm2 at SL=2.3 μm (P<.01). The concentration of calcium ions required for half-maximal activation was increased in the RVH group: 2.64±0.13 versus 3.47±0.22 μmol/L at SL=2.0 μm and 2.23±0.15 versus 2.86±0.18 μmol/L at SL=2.3 μm (P<.01). The slope of the force-calcium relationship (Hill coefficient) was decreased in the RVH group at SL=2.0 μm (4.3±0.4 versus 3.1±0.2, P=.04) but not at SL=2.3 μm (3.8±0.2 versus 3.6±0.2, P=NS).
Conclusions These results suggest that the depressed cardiac function of end-stage myocardial hypertrophy may be due, in part, to altered contractile protein function.
Chronic ventricular pressure overload inevitably leads to MH. The increase in ventricular mass is initially a seemingly successful adaptation, both by reducing wall stress and by compensating for a reduction in contractility per unit mass.1 Unfortunately the compensatory phase eventually ends, and the underlying myocardial dysfunction becomes clinically overt with the onset of ventricular dilation and heart failure.2 The mechanism that underlies the diminished contractile function seen at end-stage MH has not been completely elucidated. A multitude of studies have indicated that abnormalities in excitation-contraction coupling may play an important role in the development of contractile dysfunction.3 4 5 6 In these studies, tension generation was shown to be depressed in isolated muscle preparations (papillary muscle or cardiac trabeculae) obtained from hearts of animals with experimentally induced pressure overload hypertrophy. Likewise, we have recently observed a similar depression of tension development in RV trabeculae isolated from rats with chronic left ventricular myocardial infarction.7 Interpretation of these experimental observations, however, is complicated by the potential confounding effects of alterations in content and biophysical properties of the extracellular collagen matrix. Increased concentrations of extracellular matrix proteins have been observed in both human end-stage heart failure8 and experimental models of heart failure and MH.4 8 9 Thus, the decrease in contractile tension that is observed in isolated myocardium may be due to a reduction of myofilament density per cross-sectional area or to an alteration in myocardial contractile protein function. Therefore, whether the decrease in contractile tension in MH has at its base a reduction in tension generation at the level of a single myocyte is presently unknown. Accordingly, in the present study we investigated the effect of experimentally induced pressure overload MH on contractile tension development of chemically permeabilized (skinned) single isolated rat myocytes in the absence of confounding influences of extracellular matrix components. Our results suggest that depressed myocardial function in hypertrophy is due, in part, to an alteration of cardiac contractile protein function.
All studies were conducted in accordance with institutional guidelines for the care and use of laboratory animals. RVH was induced in 4-week-old male Sprague-Dawley rats by pulmonary artery constriction.10 This procedure was performed by the vendor (Zivic-Miller), and the animals were allowed to recover for a few days before being brought to our Animal Resources Center. Thirty-six weeks following surgery was allowed for the development of RVH. During this time the animals received food and water ad libitum. Control animals were treated similarly, but with a sham surgical procedure. In addition, the control group also included two age-matched animals that were not operated on, a procedure that has been used by other investigators.5 6 11
Single isolated myocytes were obtained by using an enzymatic retroperfusion technique.12 In brief, rats were deeply anesthetized with halothane by inhalation to effect, and the heart was rapidly removed. The aorta was then cannulated and immediately connected to a Langendorff retroperfusion system. Next, the heart was perfused for 5 minutes with a Ringer’s solution (see “Solutions” below) that contained 1.0 mmol/L Ca2+ to allow for the washout of blood from the coronary circulation. After this the heart was perfused for 4 minutes with a similar solution but without added Ca2+ (nominally calcium-free Ringer’s solution). This perfusion was followed by 22 minutes of perfusion with an enzymatic Ringer’s solution containing 0.8 mg/mL class I collagenase (Whorthington), 0.38 mg/mL hyaluronidase (Sigma), and 50 μmol/L added Ca2+. Upon completion of this perfusion, the free wall of the RV was dissected from the heart. To ensure that only RV myocytes were obtained and that no part of the left ventricle or the intraventricular septum was included, only the center portion of the RV free wall was dissected. The RV free wall was minced and gently agitated in the enzymatic Ringer’s solution to which 1 mg/mL BSA had been added. Debris were removed by filtering the processed RV free wall through a 300-μm nylon mesh. Cells were then allowed to precipitate by gravitation, and the supernatant was discarded. The cells were resuspended in enzyme-free Ringer’s solution with 50 μmol/L added Ca2+. The cells were then washed two additional times with a Ringer’s solution by using this procedure; the first solution contained 0.5 mmol/L added Ca2+, and the second had 1.0 mmol/L added Ca2+. Rod-shaped cells that were obtained via this procedure were “calcium-tolerant,” intact cells. For the purpose of measuring force, cells were next sedimented and then resuspended in a skinning solution for 6 minutes to chemically permeabilize the myocytes. The skinning solution was composed of standard relaxing solution (see “Solutions” below) to which 1% ultrapure Triton-X100 (Pierce) had been added. Finally, the skinned myocytes were washed twice in relaxing solution to remove the Triton-X100 and stored on ice for <12 hours before data collection.
Experimental Setup and Cell Attachment
Experiments were performed on the stage of an inverted microscope (Olympus). Single myocytes were attached at either end to glass micropipettes with silicone glue (Dow Corning).12 13 14 The stage was modified to allow for temperature control of the superfusate (16±0.2°C) and contained several solution wells into which the attached cell could rapidly be moved to expose the myocyte to solutions containing different concentrations of free calcium ions.15 Fig 1⇓ shows an example of an attached cell. One pipette was mounted on a sensitive force transducer (Cambridge model 403A; ≈300 Hz resonant frequency), and the other pipette was attached to a high-speed motor (Cambridge model 308; ≈1-msec 90% step response). Both pipettes were connected to X-Y-Z manipulators (Newport). Cell length was adjusted by using the motor, which was controlled via computer (Apple PPC 100 mHz) using custom-designed software (Labview, National Instruments). FD and cell length were also recorded by computer for off-line analysis. Developed force was measured during each activating cycle; the zero force level was identified by instituting a quick ramp shortening in cell length just before moving the cell back to the relaxing solution (pCa=9.0). Fig 2A⇓ shows a series of such quick length-release steps recorded at varying levels of activation for a single myocyte.
The attached cell was monitored by video microscopy using a ×40 Hoffman Modulation Contrast objective, a ×10 video adapter tube, and a gray-scale video camera. The image was displayed on a video monitor and sampled via computer for on-line analysis of SL using custom-designed software (Labview) as follows. A region of the image that encompassed most of the attached myocyte was selected, and each horizontal pixel line was transformed by fast Fourier transformation into the spatial frequency domain. The amplitude spectra were averaged, and the peak power of the first-order harmonic in the spatial frequency domain was detected (see Fig 1D⇑ and 1E⇑). This value was then converted into a median SL across the region. The system was calibrated with glass gratings of known spacing; the resolution of the system was ≈10 nm, and acquisition speed was ≈0.5 seconds.
Measurement of Cross-sectional Area
To compare tension generation between cells of different sizes, we normalized total force to myocyte cross-sectional area measured while the cell was placed in relaxing solution at SL=2.0 μm. Cell width was measured directly from the vertical projection of the cell on the video image. Cell thickness was measured from a horizontal projection of the cell from a 45° mirror that was mounted on a hydraulic manipulator (Newport) positioned next to the cell (see Fig 1C⇑). Cross-sectional area was calculated by assuming that the cell had a rectangular shape. Tension development, calculated as force normalized to cross-sectional area, is expressed as mN/mm2.
The perfusate used for cell isolation was a modified Ringer’s solution of the following composition (in mmol/L): Na+ 125, K+ 6.8, Cl− 125.2, Mg2+ 1.2, H2PO4− 2, d-glucose 11, insulin 1.0, and Ca2+ as indicated. pH was adjusted to 7.4 by adding NaOH. For some steps in the cell isolation, collagenase and hyaluronidase were added as solids to this solution.
For skinned single myocytes, relaxing and activating solutions were used; the compositions of these solutions (Table 1⇓) were calculated by using the methods described by Fabiato and Fabiato.16 We have observed a rather high variability in the force-calcium relation of isolated myocardium when solutions were prepared or mixed each day during the ongoing experiment. We sought to reduce this solution-induced variability in the force-calcium relation to allow for a more accurate comparison between the experimental groups. To this end, solutions of varying free calcium concentrations were prepared, and aliquots were stored at −30°C until used for each individual myocyte. All chemicals were of the highest purity available (Sigma Chemical Co).
Protocol and Data Analysis
The responsiveness of the contractile proteins to calcium ions, ie, the force-calcium relation, was determined as follows. SL was initially set at 2.3 μm. After two or three exposures of the myocyte to the activation solution (ie, maximum activation), SL was readjusted to 2.3 μm. The purpose of the preexposure to activating solution was to allow the cell to set into the silicone glue attachments. Isometric force during maximum activation was measured at the beginning, middle, and end of the experiment to assess rundown of the preparation; the cell was not included in the final data analysis if force declined by >10% in a successive test contraction at the maximum activation. The average rundown was 7.8±1.7% in the control group and 5.9±1.2% in the RVH group (n=28, P=.4). We consistently observed clear striations and sarcomere registration at activation levels up to close to maximum activation in individual myocytes (≈80% FD; see Figs 1⇑ and 2⇑). In addition to force rundown, the cell was also discarded if SL shortened by >10% at that level of activation. In between the test contractions, FD was measured at varying submaximal free calcium concentrations. FD was corrected for rundown by assuming that each contraction between the test contractions at maximum activation contributed equally to the force rundown. The relation between free calcium and FD resembled a sigmoidal function, and the data were fit to a modified Hill equation (Fig 2⇑) where F is FD, Fmax is the force at maximum activation, [Ca2+] is the calcium concentration, EC50 is the concentration of Ca2+ at which F is 50% of Fmax and represents a compound affinity constant (ie, the calcium sensitivity index), and H represents the slope of the F-[Ca2+] relation (the Hill coefficient).
It has been reported that the response of isolated cardiac muscle to a change in SL, ie, the Frank-Starling mechanism, is diminished in human heart failure.17 To examine the response of FD to changes in SL in the setting of RVH, we determined the force-calcium relation at both a high (2.3 μm) and low (2.0 μm) SL. These lengths of the cardiac sarcomere encompass most of the working range in in situ hearts.18
A two-tailed unpaired Student’s t test was used to test for significant differences between group means, ie, between control and RVH animals. Thus, the fit parameters that resulted from the nonlinear fit to the Hill equation were treated statistically as if they were obtained by direct measurement. Statistical analyses were performed by using commercially available software (SYSTAT). Data are presented as mean±SEM; a probability value of <.05 was considered significant.
Effect of Chronic Pulmonary Artery Banding
Six of a total of 14 animals with experimentally induced pulmonary arterial constriction died before inclusion into the study. At the time of the study, 36 weeks after pulmonary artery constriction, the 8 surviving animals displayed pleural effusion and vein and liver engorgement. These observations are compatible with a clinical diagnosis of right heart failure. We were not able in our study protocol to measure RV weight. Therefore, to assess the extent of the effect of chronic pulmonary hypertension on RV hypertrophy, we instead measured myocyte cell dimensions by using light microscopy (×40 Hoffman contrast) in a random population of intact, unattached rod-shaped cells (15 to 20 isolated myocytes) obtained from each animal. A total of 177 cells were thus measured in the control group and 120 cells in the RVH group. For this measurement, freshly isolated cells were placed on a coverslip in a droplet of Ringer’s solution ([Ca2+]o=1.0 mmol/L) at ambient temperature, and cell dimensions were measured directly from the video image by using public domain image-analysis software (NIH Image). Pulmonary artery constriction was associated with a concentric growth of myocytes, as cell width increased 26% in the RVH group (from 30.6±0.5 to 38.5±0.6 μm, P<.001). Cell length remained relatively unchanged (127±1.7 versus 131±1.6 μm, P=.11). Likewise, unstressed SL was not affected by RVH (1.76±0.04 versus 1.81±0.03 μm, n=7, P=.2). Consequently, the cell length/width ratio was significantly decreased by 23% in RVH (from 4.4±0.07 to 3.4±0.07, P<.001). Such changes in cell morphology have been reported in experimentally induced pulmonary constriction and are characteristic of pressure overload hypertrophy.19
Seven myocytes from 6 control rats and seven myocytes from 5 RVH rats were included for force measurement. Table 2⇓ shows the average values of maximum force, MT, EC50, and Hill coefficient that were obtained at the high and low SL in each individual myocyte. The average tension-calcium relation derived from the group data is shown in Fig 3⇓. In addition, Table 2⇓ shows the average cell width, thickness, and cross-sectional area that were obtained at the low SL. Tension in myocytes obtained from control animals at the high SL amounted to ≈42 mN/mm2. This value is comparable to the MT value of 37 mN/mm2 reported by Strang et al12 for single rat cardiac myocytes, and it is also within the range of MT values for isolated rat cardiac trabeculae.7 20 Values for passive tension of myocytes obtained from control animals were 0.9±0.04 and 2.5±0.15 mN/mm2 at SL=2.0 and 2.3 μm, respectively. These values are similar to the passive tension values reported for both intact isolated rat cardiac myocytes21 and trabeculae.7 Passive tension was slightly but significantly (P<.05) lower at both SLs in the RVH group (0.6±0.04 and 1.4±0.07 mN/mm2 at SL=2.0 and 2.3 μm, respectively).
FD at maximum activation as well as cross-sectional area were significantly (P<.05 for both) increased in the RVH group (Table 2⇑). However, because cross-sectional area increased out of proportion to FD, MT was significantly decreased in the RVH group. Thus, RVH was associated with 34% and 35% decreases in MT development at SL=2.3 and 2.0 μm, respectively (P<.01). In addition, the calcium sensitivity index (EC50) was significantly (P<.05) increased by 28% and 31%, respectively, at SL=2.3 and 2.0 μm. Thus, RVH was also associated with a significant decrease in calcium responsiveness of the single isolated cardiac myocytes. Finally, the slope of the tension-pCa relation, as indexed by the Hill coefficient, was significantly reduced in the RVH group at SL=2.0 but not 2.3 μm. The average value of the Hill coefficient in the control group is comparable to values found by investigators using skinned single isolated rat myocytes.22 23 This result implies that RVH is associated with a reduction in the level of cooperative FD at the lower but not at the higher SL.
The average difference between the EC50 obtained at the high and low SLs in each individual myocyte amounted to 0.4±0.07 and 0.6±0.10 μmol/L in the control and RVH groups, respectively (P=.13). Thesevalues are comparable to that obtained by McDonald et al14 in rat cardiac myocytes. This result indicates that the effect of a change in SL on the calcium responsiveness of the contractile apparatus, ie, the Frank-Starling mechanism, was not affected by RVH.
Animals developed pleural effusion, vein engorgement, ascites, and hepatic engorgement by 36 weeks after pulmonary artery constriction. Myocytes that were isolated from the hearts of these animals showed significant changes in morphology, a finding that is consistent with RVH. The experimental model adopted in the present study, therefore, represented pressure overload MH that had progressed to heart failure.24
A number of investigators3 4 5 7 employing multicellular isolated muscle preparations have reported a decrease in active tension development in RVH. However, interpretation of those studies may be limited since MH is accompanied by a significant increase in the content of extracellular matrix proteins.4 8 9 Therefore, the reduction in tension development may be due merely to a reduction in the number of force-generating elements per cross-sectional area of myocardium, without an actual change in intrinsic contractile protein function. The results of the present study show that although maximally activated force (Fmax) was significantly increased in RVH, tension was significantly decreased at the cellular level (Table 2⇑ and Fig 3⇑). The average decrease in active tension reported in intact, multicellular preparations in MH amounts to ≈55%3 4 5 ; in the present study we found an ≈35% reduction in tension at the cellular level. Because the myocyte isolation procedure effectively removes the extracellular collagen matrix proteins, these results indicate that part of the reduced tension development in RVH has at its base an alteration in tension development at the level of the cardiac cell. The mechanism underlying the decrease of MT development of myocytes is presently unknown, but may be due either to a reduced availability of myofilaments in the cardiac cell or to a decreased efficiency of myofilaments to generate force.
The notion of altered myofilament function is further supported by the decrease in calcium responsiveness that was observed in the present study. This finding conflicts with reports that have indicated no changes in calcium responsiveness in RVH.5 25 The reasons for this discrepancy are not entirely clear, but may be related to the fact that other studies have investigated stable myocardial hypertrophy, while in the present model hypertrophy had progressed to heart failure.24 Furthermore, we eliminated the variation in the force-calcium relation between cells that is induced by variations in the “skinned fiber” solutions (see “Methods”). The mechanisms underlying the decrease in calcium responsiveness with MH are not clear. We have shown7 that a shift of isomyosin in itself does not affect the force-calcium relation in rat myocardium. Hence, it is unlikely that an alteration in isomyosin synthesis could cause the reduced maximally activated FD and decreased calcium responsiveness sensitivity. It has been reported that human dilated cardiomyopathy is associated with an alteration in troponin T isoform expression26 and reduced myocardial content of myosin light chain 2.27 Hence, these abnormalities in contractile protein components could potentially be responsible for the altered calcium responsiveness of the cardiac sarcomere.28 Whether such changes in contractile protein composition occur in RVH cannot be determined from the present study. Regardless of the underlying mechanism, however, decreased MT development and calcium responsiveness would explain, in part, the reduction in myocardial contractile function observed in decompensated MH. We cannot exclude the possibility, however, that other factors, such as an alteration in myocyte calcium handling5 29 or matrix change8 24 may also play a role. Wolff et al13 report an increased calcium responsiveness of permeabilized canine myocytes in chronic pacing-induced heart failure, which is in contrast to the present findings. The reason for this discrepancy is unclear, but may be related to differences in the experimental models, ie, pacing-induced canine heart failure versus rat RVH.
Schwinger et al17 report that the effect of changes in SL on myofibrillar calcium sensitivity is attenuated in human heart failure. Our results do not support this conclusion, since the effect of changes in SL on the calcium sensitivity index (EC50) was preserved in RVH. The differences in our findings may be related to differences in the pathophysiology of dilated cardiomyopathy versus pressure overload hypertrophy, species difference, and differences in the methodological approaches, ie, isolated myocytes versus papillary muscle preparations. Our findings suggest that any structural contractile protein abnormalities in ventricular hypertrophy spare the moieties responsible for modulation of length-dependent activation but affect those that modulate overall calcium sensitivity and MT generation.
Several experimental limitations need to be considered. First, the level of contractile protein phosphorylation of the skinned myocytes may be different from the in situ condition. Therefore, it is not possible to directly extrapolate the findings of the present study to contractile function of myocardium under physiological conditions. Second, sarcomere shortening during activation can potentially arise due to the cell attachment method that was employed in the present study.12 14 22 23 As a result, SL may have varied at different levels of contractile activation. Myocytes in which excessive sarcomere shortening occurred were excluded from the present study, and although we attempted to minimize the effect of uncontrolled sarcomere shortening (see “Methods”), it could not altogether be avoided. Likewise, it should be noted that some inhomogeneity in SL may have been present in the cells. The measured values for SL in relaxing solution were averages obtained from most of the cell between the attachment sites. Nevertheless, these factors should not affect the major conclusion of the present study, since myocytes from both the control and RVH groups were affected equally. Third, some of the cells that were included in the present study were derived from the same animal (one heart in the control group provided two cells; two hearts in the RVH group provided two cells). To assess the effect of this potential confounding factor, we averaged the data from those pairs of cells to obtain single measurement values. Analyses of this data set (control n=6, RVH n=5), however, resulted in similar average results and, furthermore, did not alter the statistical outcome of the study. Finally, tension development was calculated as force per cross-sectional area assuming a rectangular shape of the cell in both groups. However, a change in cell geometry in RVH would affect the calculation of tension. To assess the possible effect of this variable, we recalculated tension development in the RVH group based on an ellipsoid shape. This maximum possible change in geometry reduced the difference in MT between the groups to ≈25% (P=.1). Nevertheless, since this is the extreme of the possible shape change between the two groups, we do not consider it likely that this factor alone would be responsible for the observed changes.
In conclusion, in the present study we investigated the effect of MH on myofibrillar contractile function in rat myocardium. MH was associated with a marked depression of maximally activated tension development and a reduction in calcium responsiveness. These results suggest that diminished contractile function in MH is due, in part, to an alteration in contractile protein function.
Selected Abbreviations and Acronyms
|RVH||=||right ventricular hypertrophy|
The current study was supported in part by grants from the National Institutes of Health (HL-52322 to Dr de Tombe and HL-03255 to Dr Wannenburg), the Whitaker Foundation (Dr de Tombe), the American Heart Association National Center (94-006380 and 95001050 to Dr de Tombe and 95-012390 to Dr Wannenburg), and the AHA North Carolina Affiliate (NC-94-GS-42 to Dr Wannenburg). Dr de Tombe is an Established Investigator of the American Heart Association. We thank Dr Kevin Strang for helping us to establish the single myocyte technique in our laboratory.
- Received October 10, 1996.
- Revision received December 2, 1996.
- Accepted December 9, 1996.
- Copyright © 1997 by American Heart Association
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