Isometric and Dynamic Contractile Properties of Porcine Skinned Cardiac Myocytes After Stunning
Abstract The purpose of this study was to investigate myofibrillar mechanisms of depressed contractile function associated with myocardial stunning. We first tested whether the degree of stunning was directly related to changes in myofilament Ca2+ sensitivity. Variable degrees and durations of low-flow ischemia were followed by 30 minutes of reperfusion in an open-chest porcine model of regional myocardial stunning (n=27). Ca2+ sensitivity of isometric tension was measured in skinned myocytes obtained from endocardial biopsies taken during control aerobic flow and after 30 minutes of reperfusion. The degree of stunning, as assessed by percent systolic wall thickening, ranged from −3% to 75% of control but did not correlate (r=.11) with changes in pCa50, ie, pCa for half-maximal tension. Only in the group (n=10) with the most severe level of ischemia was there a significant decrease in pCa50 (from 5.97±0.06 in the control condition to 5.86±0.07 after ischemia, P<.05). Less severe levels of ischemia (n=17) resulted in significant stunning (percent systolic wall thickening, 38±4% of control) but no change in pCa50. To investigate the possibility that alterations in myofibrillar cross-bridge kinetics contribute to depressed function in stunning, maximum velocity of shortening (Vo) was measured in postischemic myocytes. Vo in postischemic myocytes was reduced to 56±4% of Vo in control myocytes and was independent both of the degree of stunning (r=.26) and changes in Ca2+ sensitivity. We conclude that the basis of stunning involves decreased cycling rates of myofibrillar cross-bridges and, after more severe ischemia, a reduction in myofilament Ca2+ sensitivity.
Myocardial stunning is a form of reversible contractile dysfunction that occurs after brief periods of ischemia.1 Stunning is known to occur after exercise-induced ischemia,2 coronary angioplasty,3 and cardiopulmonary bypass4 and in patients with unstable angina5 and, to varying degrees, in patients with myocardial infarction after reperfusion.6 7 Although the etiology of stunning is not fully understood, considerable evidence suggests that the formation of oxygen-derived free radicals and elevated cytosolic Ca2+ during ischemia and early reperfusion contributes to the development of stunning.8 9 10 11 How these two factors alter myocardial contractility, which ultimately results in stunning, has been the focus of a number of recent studies. Kusouka et al12 reported that isolated stunned ferret hearts have reduced responsiveness of myofilaments to Ca2+. Both peak Ca2+-activated pressure (measured during tetani after exposure to ryanodine) and developed pressure at a given perfusate Ca2+ were reduced in the stunned heart.12 Such changes in contractility could result from alterations in the amplitude or duration of the Ca2+ transient or changes in the myofilament sensitivity to available Ca2+ or both. Using gated NMR with the Ca2+ indicator 5F-BAPTA, Kusuoka et al13 observed a paradoxical increase in peak intracellular Ca2+ in stunned myocardium compared with control conditions, suggesting that the intracellular Ca2+ transient in stunned myocardium is preserved and that the decrease in developed pressure results from a reduction in myofilament sensitivity to Ca2+. Consistent with this idea, studies in our laboratory using an in vivo open-chest porcine heart model found a decrease in the Ca2+ sensitivity of isometric tension in skinned myocytes from postischemic stunned myocardium.14 A subsequent study using the same model showed that Ca2+ sensitivity of tension is unchanged during ischemia and that the decrease in Ca2+ sensitivity occurs during reperfusion.15 Taken together, these results demonstrate that Ca2+ sensitivity of myocardial tension is reduced after reperfusion, and this may account for the depressed contractile function associated with stunning.
The goal of the present study was to further investigate possible myofibrillar mechanisms of depressed contractile function associated with stunning. Ca2+ sensitivity of isometric tension and the rate of cross-bridge cycling were assessed, since a reduction in either of these properties would likely decrease both tension and the amount of muscle shortening during an individual heartbeat. We first tested the hypothesis that the degree of stunning is directly related to changes in myofilament Ca2+ sensitivity. To examine this idea, we imposed variable degrees and durations of low-flow ischemia followed by 30 minutes of reperfusion in the open-chest porcine model of regional myocardial stunning. Ca2+ sensitivity of isometric tension was measured in skinned myocytes obtained from endocardial biopsies taken during the control period of aerobic flow and after the period of reperfusion. We also tested the hypothesis that the maximum rate of cross-bridge turnover is reduced in stunned myocytes, which was determined by measuring Vo in isolated control and postischemic myocytes.
Materials and Methods
In Vivo Model of Stunned Porcine Heart
A previously described open-chest porcine heart preparation was used,14 but with anesthesia as follows: Adolescent pigs (n=27) weighing 44.0±0.5 kg were initially sedated with a combination of ketamine (1 g), atropine (0.8 mg), and acepromazine (30 mg) given intramuscularly. After 10 to 15 minutes, another injection of ketamine (1 g) was given intramuscularly. An intravenous catheter was placed in a superficial ear vein, and both thiopental sodium (250 mg) and α-chloralose (1.5 g) were infused. Deep anesthesia was maintained by administering α-chloralose (500 mg IV) and morphine sulfate (45 mg SC) every hour. Euthanasia was performed with an overdose of sodium pentobarbital given at the end of the experiment. Anesthesia, surgery, and general care of the animals conformed strictly to the “Guide for the Care and Use of Laboratory Animals” of the National Institutes of Health, and the protocol was approved by the University of Wisconsin Animal Care Committee. The heart was exposed by bilateral thoracotomy with transsternotomy. For LVP measurements, a high-fidelity micromanometer-tipped catheter (Millar Instruments) was inserted retrogradely from the right internal carotid artery into the LV. For some preparations, leads from a cardiac pacer were attached to the right atrial appendage in order to control heart rate throughout the experiment. After treatment with heparin (10 000 U IV), three extracorporeal perfusion circuits were constructed with low-flow perfusion pumps (Sarns, 3M) in order to separately control flow to the right coronary artery, LAD, and circumflex coronary artery perfusion beds. Another small cannula was placed in the anterior cardiac vein to sample blood for venous oxygen saturation. Oxygen consumption in the LAD bed was measured by using the Fick principle. Control aerobic flow to the LAD was set by adjusting the calibrated perfusion pump to a flow rate that yielded a perfusion pressure equivalent to mean aortic pressure. Flow to the circumflex and right coronary beds was constant at aerobic levels throughout the experiment. LAD blood flow and oxygen consumption were normalized to the wet weight of myocardium in the LAD bed. Wet weight was determined by postmortem injection of colored dye at the conclusion of the experiment.
LV transmural wall thickness was measured with ultrasonic crystals positioned midway between the apex and base of the LV in the LAD bed. Regional LV function was assessed by percent systolic wall thickening, which was defined as the change in wall thickness between end systole and end diastole divided by the end-diastolic thickness. End diastole was defined by the onset of positive dP/dt, and end systole occurred just before peak negative LV dP/dt. LVP, LV dP/dt, ECG, coronary perfusion pressures, and LAD wall thickness were continuously recorded on an eight-channel recorder (Gould Brush 200, Gould Inc), averaged over 10 cardiac cycles, and digitized by a microcomputer (Zenith Data Systems) at 10-minute intervals. Systolic LVP×heart rate was calculated to provide an index of myocardial oxygen demand.16
Protocols for the Induction of Myocardial Stunning
The protocols used to induce stunning and obtain myocardium are similar to the protocol previously described14 but differ in the degree of ischemia as detailed below. Myocardium was obtained by transmural needle biopsies (inner diameter, 0.9 mm) taken serially from areas of the ventricle perfused by the LAD. The first biopsy was taken during preischemic control flow. After 10 minutes of control flow, ischemia was induced in the LAD perfusion bed by reducing LAD flow. At the end of the ischemic period, flow to the LAD was returned to preischemic levels, and a biopsy of postischemic stunned myocardium was taken after 30 minutes of reperfusion. In the present study, four different stunning protocols were used. These protocols induced varying degrees of ischemic insult and were accomplished by varying the magnitude and/or duration of reduced LAD flow and, for group 4, by additionally increasing heart rate. LAD flows during ischemia for the four different groups (protocols) were as follows: group 1 (n=7), 40% of control aerobic flow rate for 20 minutes; group 2 (n=7), 20% of control flow for 20 minutes; and groups 3 (n=6) and 4 (n=10), 40% of control flow for 40 minutes. In group 4, myocardial oxygen demand during ischemia was further increased by atrial pacing at ≈150 bpm. Group 4 is the only group comparable in terms of degree of ischemia to the preparations in Reference 1414 .
In Vitro Isolation and Mechanical Measurements of Single Cell-Sized Myocytes
The ventricular myocardial biopsy was placed in cold relaxing solution (pCa 9.0) and separated into epicardial and endocardial pieces. The endocardial pieces were mechanically disrupted for 15 seconds by using a tissue homogenizer (Polytron, Kinematica) set at a low speed. This resulted in a suspension containing small clumps of myocytes, single myocyte–sized preparations, and cell fragments. Single cell–sized preparations were selected for mechanical measurements on the basis of size (100 to 150 μm in length and ≈20 μm in width) and uniformity of striation pattern. The preparation was attached with silicone adhesive to micropipettes extending from a force transducer (model 403, Cambridge Technology, Inc) and a piezoelectric translator (Physik Instrumente) by using methods previously described14 (Fig 1⇓). After a 40-minute cure time, the preparation was first bathed for 30 seconds in relaxing solution containing 0.3% ultrapure Triton X-100 (Pierce Chemical Co) to disrupt any remaining sarcoplasmic reticulum and to improve resolution of the striation pattern.
The length of the preparation was adjusted to achieve a sarcomere length of 2.25 to 2.30 μm. Sarcomere length and uniformity, as well as the length and width of the cell preparation, were monitored throughout the experiment by using a video camera and video monitor. Only preparations in which sarcomere shortening was <0.15 μm per sarcomere during maximal activations of these fixed-end preparations were used. Upon maximal activation, there was invariably partial loss of the striation pattern (see Fig 1⇑ for an example). The extent of retention of striation pattern did not differ between control (62±4%) and postischemic myocytes (61±4%). The exact reason for the partial loss of striation pattern during maximal activation is unknown, but it is likely in part due to small branching cell fragments that shorten excessively during activation and obscure underlying sarcomeres. Additionally, changes in shape of myocyte cross section upon activation could contribute to degradation of the images obtained in bright-field interference microscopy, which is used in the present study. Indeed, by changing the plane of focus, striations were usually observed in regions that in other planes of focus were apparently devoid of striations.
Analysis of Ca2+ Sensitivity in Cardiac Myocytes
Tension-pCa relations were characterized at 22°C by first maximally activating the preparation in a solution of pCa 4.5 and, subsequently, transferring the preparation into a series of submaximally activating solutions. At each pCa, a steady tension was allowed to develop, and the preparation was then slackened in order to determine the total tension generated. The preparation was then transferred back into relaxing solution and reextended to its original length. Active tension at each pCa was calculated as the difference between the total tension and passive tension at pCa 9.0, which was measured by slackening the relaxed preparation. To monitor any decline in maximal tension, the preparation was maximally activated at pCa 4.5 after every third or fourth activation. If maximum tension fell below 80% of the initial value, the preparation was not used. Tension per unit cross-sectional area was calculated from cell width, assuming a circular cross section.
Tension at each pCa was expressed as a fraction of maximum tension developed in the same preparation at pCa 4.5. When maximal tension declined between the beginning and end of the experiment, tension at each pCa was expressed relative to the value of maximal Ca2+-activated tension obtained by linear interpolation.14 17 18 Ca2+ sensitivity of isometric tension was assessed from the tension-pCa relation by determining the pCa for half-maximal tension, ie, pCa50. The pCa50 and the slope (ie, the Hill coefficient) of each tension-pCa relation were quantified by Hill plot transformation of the tension-pCa data.19 A single straight line was fit to the transformed data by least-squares regression using the following equation: where Pr is relative tension and nH is the Hill coefficient. By using the constants derived from the Hill analysis, curves were fit by computer to the tension-pCa relation according to the following equation: where P is submaximal tension at a given [Ca2+] of <10−4.5 mol/L and Po is maximal Ca2+-activated tension.
Analysis of Unloaded Shortening Velocity
Vo was measured in cardiac myocytes by using the slack test as described previously.20 21 Myocytes were maximally activated at pCa 4.5, and once tension reached a steady level, the myocytes were rapidly released (<1 millisecond) to various slack lengths. Upon release, tension fell to zero, and the myocyte immediately began to shorten under zero load (ie, at Vo) while taking up the imposed slack. For each release, the duration of unloaded shortening was measured as the time between the release and the point at which tension began to redevelop. The time point of tension redevelopment was determined from the intersection of two lines that were fitted by eye through the zero tension baseline and the initial phase of tension redevelopment. We found that a straight line fit through the baseline and a curvilinear fit through the redevelopment phase provided the most repeatable and accurate estimation of the time point at which force consistently increased above baseline. The length of release was plotted against the duration of unloaded shortening, and Vo, expressed as myocyte lengths per second, was calculated by dividing the slope of a fitted straight line by the length of the myocyte preparation. Representative force traces in response to the imposition of various slack lengths and a corresponding slack test plot are shown for a postischemic myocyte in Fig 2⇓.
Relaxing and activating solutions contained (mmol/L) MgATP 4.0, free Mg2+ 1.0, imidazole 20.0, EGTA 7.0, and creatine phosphate 14.5, along with sufficient KCl to adjust the ionic strength to 180.0 mmol/L. Free Ca2+ concentration was varied between 10−9 mol/L (relaxing solution) and 10−4.5 mol/L (maximally activating solution) and is expressed as pCa(−log[Ca2+]). The computer program of Fabiato22 was used to calculate the final concentration of each metal, ligand, and metal-ligand complex. The apparent stability constant for Ca2+-EGTA was corrected for temperature, pH, and ionic strength.22
A one-way ANOVA was performed to determine whether ischemia and/or reperfusion had significant effects on in vivo measurements of hemodynamics, metabolism, and regional function. When significant interactions were found, a Tukey’s test was used as a post hoc test to determine the level of significance of difference between mean values. Paired t tests were used to test whether contractile properties (passive tension, maximal Ca2+-activated tension normalized to cross-sectional area of the preparation, pCa50, the Hill coefficient, and Vo) of stunned myocytes were significantly different from those of control myocytes, and P<.05 was taken as indicating significance. All values are reported as mean±SEM.
Ca2+ sensitivity of isometric tension in myocytes from control and postischemic myocardium subjected to stunning is shown in Fig 3⇓ for the four experimental groups (n=27). Only in the protocol with the most severe ischemia (n=10) was there a significant reduction in Ca2+ sensitivity (group 4; see Fig 3⇓). Because of this finding, the data from the protocols that had no significant effect on Ca2+ sensitivity of tension (groups 1, 2, and 3) were pooled (n=17) and reported as summary data in Tables 1⇓ and 2⇓. In all four protocols, heart rate, LVP, and the LVP×heart rate product were maintained constant throughout preischemic control, ischemic, and postischemic conditions (Table 1⇓). Both heart rate and the product of systolic LVP×heart rate were significantly greater in group 4 compared with groups 1, 2, and 3 during all three stages (Table 1⇓). Table 2⇓ presents regional LAD blood flow, perfusion pressure, myocardial oxygen consumption, and normalized %Th. Reduced LAD blood flow resulted in ischemia with a significant decrease in perfusion pressure and regional myocardial oxygen consumption. The time course of regional myocardial function, as assessed by %Th, for groups 1, 2, and 3 combined and for group 4 is shown in Fig 4⇓. Regional myocardial function in the LAD perfusion bed was significantly more depressed during ischemia induced by protocol 4; eg, after 40 minutes of ischemia, regional function was reduced to −13±11% in group 4 versus 13±3% in group 3 (P<.05). During the postischemic period, regional function returned to 38±4% of control in groups 1, 2, and 3 combined and to 26±7% of control in group 4, which was not significantly different.
The characteristics of isolated skinned myocytes that were analyzed for Ca2+ sensitivity of tension are presented in Table 3⇓. There were no significant changes in passive or active tension per cross-sectional area after ischemia and reperfusion. The Ca2+ sensitivity of tension for each individual myocyte preparation is presented in Table 4⇓. For all groups (n=20), there was no direct relation between the change in pCa50 and the reduction of thickening associated with stunning [%Th, 36.6+23.6(ΔpCa50); r=.11]. Only in group 4, which had the most severe degree of ischemia, as evidenced by the greatest reduction in regional wall thickening during ischemia, was Ca2+ sensitivity of tension significantly shifted to higher [Ca2+].
The characteristics of isolated myocytes in which Vo was measured did not differ in terms of myocyte dimensional characteristics or passive and active tensions (Table 5⇓). Fig 5⇓ shows representative slack plots of a control and postischemic myocyte isolated from the same preparation. Vo determined from the slope of the line fitted to the length change versus time data was 38% less in postischemic myocyte preparations. Mean Vo for postischemic myocytes was reduced to 56±4% compared with control values. Vo values for individual myocytes and means for each group are presented in Table 4⇑. There was no direct relation between Vo (percent of paired control value) and %Th with stunning [%Th=21.9+0.36(%Vo); r=.26]. However, in all three groups in which Vo was measured, ranging from the least (group 1) to the most severe ischemia (group 4), there was a significant decrease in Vo with stunning. As described earlier in other myocytes, there was no significant change in Ca2+ sensitivity of tension in groups 1, 2 or 3, but there was a significant decrease in Ca2+ sensitivity in group 4.
Further, we tested whether the observed decrease in Vo was an artifact related to prior biopsy per se or elapsed time per se. By use of group 4 protocol for LAD bed stunning, Vo was measured in single myocyte-sized preparations from serial endocardial biopsy samples obtained from the aerobically perfused circumflex bed at control (time=0) and after 30 minutes of LAD bed reperfusion (time=80 minutes). Mean Vo was 8% greater in myocytes from the second biopsy compared with myocytes from the first biopsy (n=6, P=NS). From these data, we conclude that a prior biopsy and elapsed time per se have no effect on Vo and that the above observed change in Vo in the LAD bed resulted from ischemia/reperfusion.
In the present study, myocardial stunning was induced in vivo in a large animal model from which myocardium was obtained both before and after stunning for in vitro measurements of myofibrillar contractile properties. The major findings of the present study are as follows: (1) Vo is depressed in stunned myocardium, and this depression is independent of alterations in Ca2+ sensitivity of tension. (2) Changes in Ca2+ sensitivity of tension in stunned myocytes depend on the degree of ischemia, in that Ca2+ sensitivity is reduced only when the degree of ischemia is more severe. These observations suggest that the basis for stunning involves fundamental changes in the kinetics of interaction of myofibrillar proteins and, in more severe ischemia, reductions in Ca2+ sensitivity of isometric tension. However, the present study does not rule out other potential mechanisms that do not directly involve the myofibrillar proteins, such as changes in extracellular matrix, coronary microvasculature, Ca2+ availability, spacing between myofilaments, and cytoskeletal abnormalities, which may also contribute to stunning. Also, since for technical reasons our mechanical measurements on isolated myocytes were performed at lower temperatures (22°C) than the in vivo measurements of %Th (obtained at body temperature), the contributions of these changes in myofibrillar contractile properties to stunning in vivo cannot be known for certain.
Considerable experimental evidence suggests that oxygen-derived free radicals and transient intracellular Ca2+ overload during ischemia and early reperfusion act to produce myocardial injury that results in stunning (for review, see Reference 88 ). Our focus has been directed toward understanding the link between these events and the molecular changes that are the immediate cause of stunning. From the work of others on isolated whole-heart preparations and our work on isolated myocytes, one such effect appears to be a reduction in myofilament responsiveness to Ca2+, which can take any of three forms: a decrease in maximal Ca2+-activated force, a decrease in Ca2+ sensitivity of force, or a decrease of both.9 Regarding maximal Ca2+-activated force in whole hearts, Kusuoka et al12 and Carrozza et al23 observed a reduction in the maximal Ca2+-activated pressure (measured during tetani after exposure to ryanodine) in isolated stunned ferret hearts. On the other hand, results from whole hearts concerning the Ca2+ sensitivity of myofilaments are variable. Kusuoka et al found a shift in the relation between relative developed pressure and perfusate [Ca2+] to higher concentrations of Ca2+, suggesting that stunned myofilaments are less sensitive to Ca2+. On the other hand, Carrozza et al reported no significant change with stunning in the relation between relative pressure and intracellular [Ca2+] measured by use of aequorin. Studies from our laboratory, including the present one, using isolated skinned myocytes found (1) no change in maximum Ca2+-activated tension with stunning and (2) a reduction in Ca2+ sensitivity of tension.14 15 In a recent study, Dietrich et al24 found no change in either maximum isometric tension or Ca2+ sensitivity of tension in skinned trabeculae isolated from stunned rat hearts. The reasons for the discrepancy in Ca2+-sensitivity results are unclear but may involve differences in stunning models and/or differences between ventricular myocytes and trabeculae in their susceptibility to stunning.
The basis for the discrepancy in reported effects on maximal tension-generating capacity between the whole stunned heart and isolated stunned myocytes is not presently known. One possible explanation for the discrepancy is the use of different models of ischemia to induce stunning. The no-flow model of ischemia used in the isolated heart studies could result in a greater accumulation of factors that injure the myocardium (such as oxygen-derived free radicals) and therefore result in depressed peak tension. Along these lines, MacFarlane and Miller25 recently reported that myofilaments exposed to superoxide anion, a radical known to accumulate during ischemia and early reperfusion, exhibited a reduction in maximum Ca2+-activated force. It is plausible that this or related metabolites may be washed out during low-flow ischemia and thus are less of a factor in our experiments. A second possibility is that saturating [Ca2+] and thus maximum tension cannot be obtained in isolated hearts even in the presence of ryanodine. This would mean that the measurements of peak tension (pressure) in isolated hearts were obtained at submaximal [Ca2+] and were therefore not measurements of maximum tension. If myofilament Ca2+ sensitivity were depressed, the stunned heart would generate less tension at the same [Ca2+]i. Hence, the observed reduction in maximal tension might be a consequence of reduced myofilament Ca2+ sensitivity and not maximum tension-generating capacity. In addition, normalizing tension or pressure to a peak submaximal value would artifactually steepen the tension-pCa relation and mask any decreases in Ca2+ sensitivity. A third possibility is that the large variability in maximal Ca2+-activated tension found in isolated myocytes makes it difficult to detect a similar magnitude of change as has been found in isolated hearts. This problem may be especially difficult in our experiments, since myocytes cannot serve as their own controls in tension measurements, whereas isolated hearts can. Yet another possibility to explain the discrepancy in maximal tension between living and skinned preparations is that factors other than changes in the myofilaments (eg, Ca2+ delivery or reuptake by the sarcoplasmic reticulum) are compromised in the stunned heart. Such factors are obviously eliminated in our isolated myocyte preparations.
Previously, we reported a decrease in myofilament Ca2+ sensitivity in permeabilized stunned myocytes from the porcine low-flow ischemia model of stunning.14 15 It is important to point out that the degree of ischemia in our previous studies,14 15 based on the product of LVP×heart rate, was similar to that in group 4 in the present study but more severe than that of groups 1, 2, or 3. It seems reasonable to conclude that this decrease in Ca2+ sensitivity could explain, in part, the reduced contractility of stunned myocardium. A likely consequence of such a mechanism is that the degree of stunning should be inversely related to Ca2+ sensitivity of isometric tension. We tested this hypothesis by measuring the change in Ca2+ sensitivity of tension in skinned myocytes after varying degrees and durations of low-flow ischemia followed by 30 minutes of aerobic reperfusion. The three lesser degrees ischemia (groups 1, 2, and 3) used in the present study did not yield a statistically significant shift in average Ca2+ sensitivity of tension, despite the induction of significant stunning. Only when ischemia was more severe (group 4) did the mean Ca2+ sensitivity of tension become depressed. Thus, reduced Ca2+ sensitivity appears to be a characteristic only of more severe bouts of ischemia. However, since we only tested one pair of myocytes per preparation, the present study does not exclude the possibility that stunning has a heterogeneous effect on Ca2+ sensitivity of tension in individual myocytes when the degree of stunning is relatively less severe, as assessed from measures of regional myocardial function. In such a case, reduced Ca2+ sensitivity in some myocytes could significantly depress regional function. One functional consequence of reduced Ca2+ sensitivity would be that at a given submaximal level of [Ca2+], tension-generating capacity would be less, and the velocity of myocardial shortening against a given load would be reduced. Reduced velocity would slow segmental shortening of myocardium, and slowly shortening myocytes might also become a mechanical load on neighboring myocytes and slow their shortening as well. If the reduction in Ca2+ sensitivity of myocytes became more homogeneous as the ischemia was made more severe, one might expect the degree of regional stunning to increase further.
The present study also investigated whether Vo was altered in stunned myocytes. This parameter is an index of the maximum rate of cross-bridge cycling, which is thought to be limited by the rate of cross-bridge detachment,26 and is related to myofibrillar ATPase activity.27 Vo was significantly reduced after all stunning protocols, an effect that was independent of alterations in Ca2+ sensitivity. Interestingly, the magnitude of shift in Vo was similar regardless of the degree of ischemic insult. The functional consequences of a reduction in Vo of stunned myocytes are illustrated in Fig 6⇓. Since it is assumed that the curvature of the force-velocity relation is unchanged by stunning, the velocity of shortening against a given load would be lower in stunned myocardium (point A versus point B). In addition, if the myocyte had reduced sensitivity to Ca2+, the tension-generating capacity and thus the velocity and extent of shortening during a Ca2+ transient at a given load would be further reduced (point B to point C). These shifts to progressively more depressed force-velocity curves are consistent with the significant reduction in ventricular function observed during a given Ca2+ transient in stunned myocardium and would explain progressively greater stunning with increased ischemia.
The basis for reduction in Vo is unknown. Since Vo appears to be limited by ADP release from actomyosin,28 a reduction in Ca2+-dependent myofibrillar ATPase activity should accompany stunning. However, the rate of myofibrillar ATP hydrolysis was unaffected in stunned rabbit hearts.29 Possible changes in myofibrillar ATPase activity in the porcine model of stunning remain to be investigated. One potential mechanism for reduced Vo in stunning is reduced association of MLC2 with myosin, which has previously been suggested to account for some alterations in mechanical properties in failing myocardium.30 In this regard, extraction of MLC2 caused a reduction in the maximal actin-activated cardiac myosin ATPase rate,31 and mechanical studies found that partial extraction of MLC2 significantly reduced Vo in skinned skeletal fibers.32 Again, the apparent lack of effect of stunning on ATPase activity in some models of stunning suggests that MLC2 deficiency is not a common mechanism of stunning. Whether MLC2 concentrations are affected by stunning in our porcine model and whether reduced MLC2 content alters Vo either by enzymatic and/or mechanical means are currently areas of investigation in our laboratory. Other plausible mechanisms for a reduced Vo and myofibrillar Ca2+ sensitivity are covalent modifications (eg, phosphorylation state) of myosin and/or regulatory proteins or damage to contractile proteins by free radicals or by Ca2+-activated proteases. The latter mechanisms are consistent with the time course of replacement of damaged proteins with newly synthesized proteins and may explain why stunning is reversed over several days to weeks.
Selected Abbreviations and Acronyms
|%Th||=||percent systolic wall thickening|
|LAD||=||left anterior descending coronary artery|
|LV||=||left ventricle, left ventricular|
|MLC2||=||myosin regulatory light chain|
|NMR||=||nuclear magnetic resonance|
|Vo||=||maximum velocity of shortening|
This study was supported by National Institutes of Health grants R01 HL-49375 (Dr Miller) and HL-08755 (Dr McDonald) and the Rennebohm Foundation of Wisconsin (Dr Miller). The authors thank Larry Whitesell, Alice Eggleston, and Dr Stephen Nellis for technical assistance in performing the studies.
Presented in part at the 66th Scientific Sessions of the American Heart Association, November 8, 1993, Atlanta, Ga.
- Received December 7, 1994.
- Accepted August 8, 1995.
- © 1995 American Heart Association, Inc.
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