A Novel Mechanism for Myocardial Stunning Involving Impaired Ca2+ Handling
The mechanism of myocardial stunning has been studied extensively in rodents and is thought to involve a decrease in Ca2+ responsiveness of the myofilaments, degradation of Troponin I (TnI), and no change in Ca2+ handling. We studied the mechanism of stunning in isolated myocytes from chronically instrumented pigs. Myocytes were isolated from the ischemic (stunned) and nonischemic (normal) regions after 90-minute coronary stenosis followed by 60-minute reperfusion. Baseline myocyte contraction was reduced, P<0.01, in stunned myocytes (6.3±0.4%) compared with normal myocytes (8.8±0.4%). The time for 70% relaxation was prolonged, P<0.01, in stunned myocytes (131±8 ms) compared with normal myocytes (105±5 ms). The impaired contractile function was associated with decreased Ca2+ transients (stunned, 0.33±0.04 versus normal, 0.49±0.05, P<0.01). Action potential measurements in stunned myocytes demonstrated a decrease in plateau potential without a change in resting membrane potential. These changes were associated with decreased L-type Ca2+-current density (stunned, −4.8±0.4 versus normal, −6.6±0.4 pA/pF, P<0.01). There were no differences in TnI, sarcoplasmic reticulum Ca2+ ATPase (SERCA2a), and phospholamban protein quantities. However, the fraction of phosphorylated phospholamban monomer was reduced in stunned myocardium. In rats, stunned myocytes demonstrated reduced systolic contraction but actually accelerated relaxation and no change in Ca2+ transients. Thus, mechanisms of stunning in the pig are radically different from the widely held concepts derived from studies in rodents and involve impaired Ca2+ handling and dephosphorylation of phospholamban, but not TnI degradation.
Myocardial stunning, recognized as the reversible reduction of contractile function following an ischemic episode,1,2 is a universal response in mammalian species and is an integral component of myocardial dysfunction observed in patients with acute and chronic ischemic heart disease. The reversible impairment of contractile function after myocardial stunning may play a role in mediating hibernating myocardium.3
The cellular mechanism of myocardial stunning has been studied primarily in rodents and is thought to involve altered Ca2+ responsiveness of the myofilaments, degradation of TnI, and no change in Ca2+ handling.4–8 The same mechanism of stunning is presumed to be active in larger mammalian models, but there are no supporting data. Indeed, recent studies in pigs and dogs have failed to find TnI degradation in stunned myocytes.9–13 In view of this, it becomes important to reexamine Ca2+ handling as a potential mechanism for stunning in large mammalian models.
To facilitate the examination of the mechanism of stunning, it is necessary to have an in vitro model of stunning. Therefore, the first goal of the study was to determine that the myocardial stunning phenotype observed in vivo, ie, impaired contractile function, can be observed in vitro in isolated myocytes from the pig. The second goal was to determine whether the mechanism of contractile and relaxation dysfunction involves impaired intracellular Ca2+ handling, and whether changes in Ca2+-handling proteins such as L-type Ca2+ channel, SERCA2a, or phospholamban are observed in stunned myocytes. The third goal was to confirm that degradation of TnI, the cellular hallmark of stunning in rodents, was not observed in the stunned swine myocytes.
Accordingly, isolated myocytes from chronically instrumented conscious pigs were used for this study14 because (1) the coronary anatomy of this species resembles that found in humans, (2) the conscious animal is more physiological and avoids complicating factors such as anesthesia and recent surgery and cardiac denervation,15 all of which can alter the response to ischemia/reperfusion, (3) the lack of preformed collateral vessels allows more precise regulation of the stenosis and concurrent flow reduction, and (4) myocardial stunning could be induced regionally so that myocytes from stunned and nonischemic regions could be compared in the same heart.
Materials and Methods
In Vivo Studies
A left thoracotomy was performed in domestic swine (weight 22 to 25 kg; Whippo Farms, Darlington, Pa), and the pigs were instrumented to measure global and regional myocardial function, as previously described.3,14 After 1 week of postoperative recovery, myocardial stunning was induced by introducing air into the hydraulic occluder to reduce coronary blood flow by approximately 40% for 90 minutes and followed by 60-minute full reperfusion. Myocyte contractile function was studied in 8 pigs. Seven of these 8 pigs were used for Western blot analysis of TnI, SERCA2a, and phospholamban and for Ca2+ transient studies as well as for analysis of myocyte contractile function. An additional 9 pigs (5 for action potential [AP], 4 for current measurements) were used either for L-type Ca2+ (ICa) currents or for AP. To determine whether there are species differences, we also examined stunned myocyte function from 5 rats subjected to 15 minutes occlusion and 30 minutes reperfusion. Animals used in this study were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, revised 1996).
In Vitro Studies
After completion of in vivo experiments, calcium-tolerant myocytes were isolated at 37°C from subendocardial regions of the left ventricles according to standard procedure. Briefly, cardiac tissue (approximately 15 to 20 g) incorporating the left anterior descending coronary artery (LAD, previously ischemic, ie, stunned, region), and the left circumflex artery (LCX, nonischemic, ie, normal, region) was isolated. Each coronary artery was cannulated, the distal branch was ligated, and the tissue was then perfused with Krebs-Henseleit solution (mmol/L) (130 NaCl, 4.8 KCl, 1.2 MgSO4, 12 HEPES, 2.5 NaHCO3, 1.2 NaH2PO4 · H2O, and 12.5 glucose) containing 75 U/mL each of collagenase 1 and 2 (Worthington), as described previously.16
Measurement of Myocyte Contractile and Relaxation Function and Ca2+ Transients
Myocyte contractile and relaxation function was measured using a video motion edge detector at 1 Hz (35±2°C).16 The chamber was perfused with Krebs-Henseleit solution containing 1.8 mmol/L CaCl2. Myocyte contraction was induced once per second (1 Hz) by platinum field electrodes placed in the cell chamber that were attached to a stimulator. The following contractile properties were calculated from the length data: percent contraction and rate of shortening (−dL/dt). Relaxation properties (+dL/dt: rate of relengthening; TR 70%: the time for 70% relaxation) were also assessed. The effects of 4 mmol/L Ca2+ were also examined in isolated myocytes. Intracellular Ca2+ concentration ([Ca2+]i) in loaded myocytes with 5 μmol/L of Fura 2-AM (Sigma) was measured using the Photoscan dual-beam spectrofluorophotometer (Photon Technology). The Fura-2 fluorescence was calibrated as described previously.17,18
Whole-cell currents and action potentials were measured using patch-clamp techniques at 35±0.5°C, as previously described.19 Action potentials were recorded in Tyrode solution in the absence of intracellular Ca2+ buffer.
Western Blot Analysis for TnI, SERCA2a, and Phospholamban
Subendocardial tissue was obtained from stunned and normal regions for immunoblot analysis. Samples were homogenized in 160 mmol/L Tris, pH 8.0, 6 mol/L urea, with protease, phosphatase, and kinase inhibitors. Myofibrils were also isolated from biopsy samples using a myofibril prep,20 with the exception that cardiac muscle was homogenized in 60 mmol/L KCl, 30 mmol/L imidazole, 2 mmol/L MgCl2, and protease inhibitor cocktail. Equal amounts of protein were dissolved in 2% SDS, 62.5 mmol/L Tris-HCl, pH 6.5, 10% glycerol, 0.05% bromophenol blue, 6 mol/L urea, and 100 mmol/L DDT and then separated by 12.5% SDS-PAGE using the Biorad mini-gel system. Equal loading of samples was confirmed by densitometry of actin-TnI of coomassie-stained gels. Gels were transferred to nitrocellulose using a wet transfer apparatus (Biorad) with 20% methanol, 25 mmol/L Tris, and 192 mmol/L glycine buffer. The primary anti-TnI Mab 8I-7 (Spectral Diagnostics) was used. Primary antibodies were detected using anti-mouse IgG conjugated to alkaline phosphatase (Jackson Immuno Research Labs) and CDP-Star chemiluminescence reagent (NEN-Mandel). All Western blot exposures were in the linear range of detection, and the intensities of the resulting bands were quantified by densitometry (Corel Photo). The primary antibody used, 8I-7, was tested to ensure its ability to detect intact TnI, modified TnI products, and cross-reactivity to other proteins.
Blots for SERCA2a and phospholamban were incubated for 30 minutes at room temperature with a 1:50 000 dilution of rabbit anti-SERCA2a polyclonal Ab (generous gift from Dr Frank Wuytack, Belgium) or 1:50 000 mouse anti-phospholamban monoclonal Ab (Affinity BioReagents Inc, Golden, CO) in TBS containing 0.1% Tween-20 and 2.5% nonfat milk. Blots for phospholamban phosphorylation were probed as described previously.16
All data are expressed as mean±SE. Comparisons of the data between stunned and normal myocytes were performed by Student’s paired t test with significant differences taken at P<0.05. The myocyte data were averaged for each animal for statistical comparison.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
In Vivo Versus In Vitro Contractile Function
In Figure 1A, the results from preliminary experiments demonstrating the response to 90 minutes of coronary stenosis and reperfusion are shown. Note the sustained, stable myocardial stunning induced by this protocol. We selected the 1-hour reperfusion time for isolating myocytes because the stunning was both severe and sustained. Results from a typical in vivo experiment involving coronary stenosis and reperfusion on wall thickening are shown also in Figure 1B. The myocardial stunning protocols used in this study produced essentially no necrosis in the area at risk, as demonstrated by previous studies.14 After 30-minute reperfusion, anterior wall thickening was reduced from baseline by 44±6% (P<0.05). As shown in Figure 1C, the degree of wall thickening observed in vivo was correlated with myocyte contraction in vitro (r=0.864, P<0.01), indicating that the myocytes were stunned. The 100%/100% point was derived from anterior and posterior wall myocytes from 2 pigs without ischemia. However, the y-intercept is not at the origin, most likely because of the 4 to 6 hours required to prepare the isolated myocytes and complete the measurements as well as the lack of tethering forces in isolated myocytes. Note in panel A of Figure 1 that at 6 hours reperfusion, there is also significant recovery of function. To confirm further the stunning phenotype, which is characterized by impaired responsiveness to Ca2+, we examined the responses to Ca2+ in stunned myocytes from 2 pigs. The increase in percent contraction to Ca2+ (4 mmol/L) was significantly impaired in stunned myocytes (+16±7%, P<0.05), compared with normal myocytes (+47±17%).
Myocyte Contraction and Ca2+ Transients in Stunned Myocytes
Figure 2A shows representative contraction/relaxation and Ca2+-transient recordings in normal and stunned swine myocytes. The stunned myocytes demonstrated impaired contractile function compared with myocytes from the normal region. For example, percent contraction was reduced, P<0.01, in stunned myocytes (6.3±0.4%) compared with normal myocytes (8.8±0.4%), and the rate of contraction (−dL/dt) was also reduced in stunned myocytes (−84±5 μm/sec) compared with normal myocytes (−136±14 μm/sec) (Table 1). Relaxation function was also impaired in response to stunning. For example, the rate of relengthening (+dL/dt) was reduced, P<0.01, in stunned myocytes (stunned, 121±10 versus normal, 175±15 μm/sec), and accordingly, the time for 70% relaxation was prolonged, P<0.01, in stunned myocytes (stunned, 131±8 versus normal, 105±5 ms).
As shown in Figure 2A and Table 1, Ca2+ transients in stunned myocytes were depressed (stunned, 0.33±0.04 versus normal, 0.49±0.05, P<0.05), without a change in levels of diastolic free Ca2+ concentration. The calibrated values for [Ca2+]i demonstrated even greater differences between stunned and normal myocytes. Systolic [Ca2+]i was even more depressed in stunned myocytes (stunned, 372±82 versus normal, 904±284 nmol, P<0.05) but not diastolic [Ca2+]i (stunned, 111±31 versus normal, 188±55 nmol, P=NS).
To clarify the unique features of the stunned swine myocytes, we also examined 5 rats, which also demonstrated depressed contractile function (percent contraction: stunned, 7.8±0.4% versus normal, 9.1±0.4%). However, in contrast to the data in pigs, stunned rat myocytes actually demonstrated accelerated relaxation (TR 70%: stunned, 68±4 versus normal, 114±12 ms) and no change in Ca2+ transients (P=NS). These features are shown clearly in Figure 2B and Table 2.
Action Potential Changes and L-Type Ca2+ Currents
These experiments were performed at both room and body temperatures. Because the data were comparable, only those at body temperature will be reported. There was no significant change in myocyte size as estimated by the cell membrane capacitance in stunned myocytes (83.5±3.3 pF, n=8) compared with normal myocytes (84.1±2.9 pF, n=6). In addition, the resting membrane potential was comparable in stunned myocytes (−73.7±1.1 mV, n=5) and normal myocytes (−73.9±1.0 mV, n=5), suggesting that myocytes from stunned myocardium were not systematically damaged by the cell isolation procedure.
The action potentials recorded from stunned myocytes showed a negative shift in plateau potential compared with control myocytes (Figure 3A). The overshoot potential was significantly lower in stunned myocytes (42.9±2.2 mV, P<0.01) than that recorded in normal myocytes (52.4±1.8 mV). The action potential duration quantified at 30% and 50% repolarization (APD30 and APD50) was significantly shorter in stunned myocytes (Figure 3B).
When stimulation frequency was increased from 0.2 to 1.0 Hz, the action potential duration became shorter in both control and stunned myocytes, typical of rate adaptation observed in cardiac myocytes (Figure 3A). Interestingly, however, adaptation to faster pacing in stunned myocytes was significantly diminished. The APD50 was reduced by 32±4% (n=5) in normal myocytes when stimulation was increased to 1.0 Hz but only by 12±3% (n=5) in stunned myocytes (Figure 3B). In mammalian cardiac tissue, it has been demonstrated that ICa contributes to rate-dependent abbreviation of APD at rapid rates and that decreased rate-dependent changes in APD are associated with decreased ICa amplitude.21 We therefore examined ICa. Figure 3D shows the I-V relationships for ICa. Stunned myocytes had significantly reduced ICa density (−4.8±0.4 pA/pF, n=4) compared with normal myocytes (−6.6±0.4 pA/pF, n=4, P<0.01). These data suggest that reduced ICa density is at least partially responsible for altered rate-dependent APD changes and also for reduced myocyte contraction observed in our model of stunning.
Immunoblotting of TnI, SERCA2a, and Phospholamban Protein in Pig
To determine whether degradation of TnI contributes to the impaired contractile function in the pig model of stunning, Western blots of TnI were analyzed in tissues from stunned and normal regions. Figure 4 shows an immunoblot of TnI from 8 animals (7, normal and stunned; 1, sham control). Note that neither was the TnI protein degraded, nor were there covalent complexes, in the stunned tissue. Even with overexposure of the intact TnI signal, minimal or no TnI degradation and covalent complexes were detected. These results indicate that the impaired contractile function induced by myocardial stunning was not associated with TnI degradation and covalent modification in pigs. Overexposure of the Western blots of the control and stunned myocardium (Figure 4), as well as purified myofibrils, showed little, if any, degradation of TnI. TnI degradation, when quantified within the linear range of the Western blot signal, was less than 4% of total TnI and was identical whether the sample was from control or stunned myocardium. This lack of TnI proteolysis is not due to the inability of porcine TnI to degrade because porcine myocardium left at room temperature for 2 hours, or treated with calpain in the presence of calcium, caused specific degradation of TnI (Figure 4).
Protein levels of SERCA2a, a key protein involved in relaxation, were unchanged in the stunned region when examined by Western blot analysis (Figure 5A). Likewise, phospholamban, a key phosphorylation-dependent modulator of SERCA2a activity, exhibited no change in protein levels (Figure 5B). When Western blots were probed with antibodies specific for the phosphorylated form of phospholamban (Ser16), there was a dramatic reduction in the level of phosphorylated phospholamban in stunned myocardium (Figure 5C). Such a decrease in phosphorylated-phospholamban should result in a substantial decrease in SERCA activity and a subsequent slowing of relaxation.
The first finding of the present investigation is that the myocardial stunning phenotype observed in vivo was also present in isolated myocytes in vitro from a large mammalian model (pig). Figure 1 confirms the marked, prolonged stunning in vivo that is induced by a 90-minute coronary stenosis. The myocytes exhibit the stunned phenotype both in terms of depressed contraction and relaxation (Figure 2) and insensitivity to Ca2+. Secondly, the mechanism(s) of impaired function in myocardial stunning in pigs differs radically from that in rodent models of stunning (Figure 2 and Tables 1and 2⇑), ie, there is (1) impaired relaxation as well as contraction, (2) impaired Ca2+ handling, (3) dephosphorylation of phospholamban, and (4) no TnI degradation. Using our techniques, we recapitulated the previously reported rodent phenotype that showed no change in Fura-2 signaling and more rapid relaxation of stunned myocytes (Figure 2 and Table 2).4,5
The experimental preparation in the present study avoids the confounding effects of anesthesia, recent surgical manipulation, absence of normal cardiac innervation,15 and substrate deficiencies that can occur in buffer-perfused Langendorff preparations. Second, we induced regional myocardial stunning (anterior wall), and thus the nonischemic posterior wall serves as a paired control in the same heart. Third, we investigated mechanisms using isolated myocytes after in vivo studies, which reveal intrinsic functional changes that could not be ascribed to the extracellular matrix. An additional unique feature was to examine the myocytes selectively from the subendocardial region, as there is a disparity in blood flow reduction between the subepicardium and subendocardium in the presence of coronary stenosis.3,14 Furthermore, this model of stunning induced by partial coronary stenosis is particularly relevant to the clinical setting.
Reduction of contractile function, ie, myocardial stunning, after a brief period of ischemia is an almost universal observation across mammalian species. However, mechanisms for contractile dysfunction in stunned myocardium are largely derived from rodent models using isolated heart preparations, which indicate that the principal lesion resides at the level of the contractile proteins.4–8 The impaired function has not been considered to be associated with any change in intracellular Ca2+ transients, although myocardial stunning is associated with impaired responsiveness to Ca2+ both in vitro4,5 and in vivo.22 The latter was confirmed in our study in isolated myocytes.
However, in contrast to the results reported in rodents,4–8 the present investigation demonstrated that the impaired contractile function in stunned swine myocytes was associated with impaired Ca2+ handling. Specifically, we observed reduced Fura-2 Ca2+ transients (Figure 2), diminished action potential plateau (Figure 3), and reduced L-type Ca2+ current density (Figure 3). Importantly, whereas most prior studies on Ca2+ channel function have been performed at room temperature, we observed similar findings at both room and body temperature.
The underlying evolutionary rationale for the observed species difference for intracellular Ca2+ handling remains uncertain. One explanation could be that the rat and most other rodent hearts must contract and relax at physiological frequencies that would be considered pathological tachycardia in large animals. Therefore, in the rodent, approximately 90% of the activator Ca2+ for contraction comes from intracellular stores from sarcoplasmic reticulum (SR).23 In contrast, pigs and most other large animals derive more of their activator Ca2+ from extracellular Ca2+ entry from the L-type Ca2+ channel. As a result, continued pacing at higher frequencies leads to a negative “staircase” in the rat ventricle, which is due to a combination of a small decrease in SR Ca2+ content and a decrease in fractional release at higher frequency, ie, incomplete mechanical restitution.23 In contrast, large mammalian cardiac muscles show a positive staircase, which may be associated with an increase in L-type Ca2+ channel current, diastolic Ca2+ concentration, and Ca2+ availability. In addition, the inactivation of the rat L-type Ca2+ channel is more rapid than that of the pig, possibly because of the large SR Ca2+ release in rodents.19,23 These properties suggest that these animal models need to be different in their ability to handle Ca2+ load and rapid heart rates, and consequently could explain differences in mechanisms for myocardial stunning.
There are other major species differences between rodents and large mammalian models with respect to responses to ischemia. For example, the protective effect of ischemic preconditioning on reperfusion arrhythmias is well established in rats, ie, preconditioning dramatically limits reperfusion-induced arrhythmias after short periods of ischemia in the rat heart.24 However, in the pig, ischemic preconditioning has been demonstrated to enhance arrhythmogenesis and to accelerate the development of ventricular fibrillation during subsequent ischemic periods.25 These species differences might be explained by the observed differences in Ca2+ channel function observed in the present study.
Numerous studies in rodents demonstrated TnI proteolysis, covalent modification, or changes in phosphorylation of TnI in the pathogenesis of stunned myocardium.4,6–8 There is also one supportive study from human myocardium as well.26 However, those data were collected from two patients undergoing coronary bypass, where minor amounts of myocardial infarction cannot be excluded. In contrast, studies in large mammalian models of stunning found no significant changes in TnI degradation.9–13 This discrepancy may be due to the more profound ischemia (>20 minutes total ischemia) in rodent models, which could produce irreversible injury,27 or to differences in in vivo versus isolated hearts, or, as noted above, to species differences. This does not preclude that defects in other contractile proteins are not involved in the mechanism of stunning in pigs. Indeed, our preliminary data suggest that TnT and TnC may be altered.28 However, the only contractile protein culprit identified so far in rat models, TnI, was not found to be degraded in stunned porcine myocytes in the current study or in prior studies using the swine model.9–13 The quantity of TnI degradation observed in the normal and stunned myocardium is minimal, and there is no difference between the two tissue types. Interestingly, this is not due to the porcine TnI being resistant to proteolysis (Figure 5) but rather to a true difference in the mechanism between rat and porcine stunning.
Interestingly, relaxation of stunned myocardium also appears to be directionally opposite in rodents and large mammals. Specifically, we observed accelerated relaxation of stunned rodent myocytes (Figure 2), which has been observed previously in rodent preparations.4 In contrast, relaxation in isolated myocytes from the pig was significantly prolonged, which is consistently observed in vivo in large mammalian models of stunning29,30 including studies in humans.31,32 These differences in relaxation may be due to phosphorylation of phospholamban, which is reduced in the swine model of stunning (Figure 5), and although SERCA2a and total phospholamban was not altered, consistent with prior studies by Luss et al.9,11 Thus, it is possible to speculate that differences in phospholamban phosphorylation, Ca2+ handling, and potential alterations in other proteins may explain myocardial stunning in large mammals. In view of the above findings, it is interesting to speculate that sympathetic agonists may improve function in stunned myocardium from large mammals. Indeed, it has been shown that sympathomimetic amines can also improve function in the canine model of stunning.33
There are several similarities between the mechanisms for myocardial stunning elucidated in the present investigation and alterations in myocyte function in heart failure.34–36 In both cases, there is impaired contraction, relaxation, and Ca2+ handling. However, the effects of stunning on SERCA2a and phospholamban noted above are quite different from those observed in heart failure,35 indicating that the mechanisms must diverge at the protein level.
In conclusion, this study demonstrates that mechanisms of myocardial stunning in large mammalian models are radically different from our current concepts derived from rodent models. These differences are of potentially profound importance in the management and treatment of patients suffering from ischemic heart disease and heart failure.
This study was supported in part by US Public Health Services Grants HL59139, HL33107, HL33065, HL69020, HL62442, HL65182, HL65183, HL61476, HL59526, RR16592, GM54169, and AG14121; NSF Grant No. DBI-9873173 and AHA Grant No. 0030125N.
Original received February 21, 2001; revision received August 31, 2001; accepted August 31, 2001.
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