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(Circulation Research. 2002;91:970.)
© 2002 American Heart Association, Inc.

Integrative Physiology

Dissociation of Regional Adaptations to Ischemia and Global Myolysis in an Accelerated Swine Model of Chronic Hibernating Myocardium

Salome A. Thomas, James A. Fallavollita, Gen Suzuki, Marcel Borgers, John M. Canty, Jr

From the VA Western New York Healthcare System (J.A.F., J.M.C.), Buffalo, NY; the Department of Medicine and the Department of Physiology and Biophysics (S.A.T., J.A.F., G.S., J.M.C.), University at Buffalo; and the Department of Molecular Cell Biology (M.B.), Cardiovascular Research Institute Maastricht, the Netherlands.

Correspondence to John M. Canty, Jr, MD, Biomedical Research Building, Room 347, Department of Medicine, University at Buffalo, 3435 Main St, Buffalo, NY 14214. E-mail canty{at}buffalo.edu

	Abstract

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We tested the hypothesis that an acute critical limitation in coronary flow reserve could rapidly recapitulate the physiological, molecular, and morphological phenotype of hibernating myocardium. Chronically instrumented swine were subjected to a partial occlusion to produce acute stunning, followed by reperfusion through a critical stenosis. Stenosis severity was adjusted serially so that hyperemic flow was severely reduced yet always higher than the preocclusion resting level. After 24 hours, resting left anterior descending coronary artery (LAD) wall thickening had decreased from 36.3±4.0% to 25.5±3.7% (P<0.05), whereas resting flow had remained normal (67±6 versus 67±8 mL/min, respectively). Although peak hyperemic flow exceeded the prestenotic value, resting flow (45±10 mL/min) and LAD wall thickening (17.0±5.0%) progressively decreased after 2 weeks, when physiological features of hibernating myocardium had developed. Regional reductions in sarcoplasmic reticulum proteins were present in hibernating myocardium but absent in stunned myocardium evaluated after 24 hours. Histological analysis showed an increase in connective tissue along with myolysis (myofibrillar loss per myocyte >10%) and increased glycogen typical of hibernating myocardium in the LAD region (33±3% of myocytes from animals with hibernating myocardium versus 15±4% of myocytes from sham-instrumented animals, P<0.05). Surprisingly, the frequency of myolysis was similar in normally perfused remote regions from animals with hibernating myocardium (32±7%). We conclude that the regional physiological and molecular characteristics of hibernating myocardium develop rapidly after a critical limitation in flow reserve. In contrast, the global nature of myolysis and increased glycogen content dissociate them from the intrinsic adaptations to ischemia. These may be related to chronic elevations in preload but appear unlikely to contribute to chronic contractile dysfunction.

Key Words: hibernating myocardium • stunned myocardium • myocardial ischemia • myofibrillar loss

	Introduction

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Regions of the heart supplied by chronic stenoses can develop chronic left ventricular (LV) dysfunction in the absence of infarction. Not all segments supplied by a stenosis develop viable dysfunctional myocardium, and there appears to be a critical relation to the physiological severity of a coronary stenosis (ie, coronary flow reserve or the ability to increase flow during vasodilation), which determines whether intrinsic adaptations to regional ischemia develop.¹ Furthermore, there is great physiological and molecular diversity in the adaptive responses that are produced in response to reversible ischemia. At one extreme is hibernating myocardium, characterized by chronic regional reductions in resting flow and function.^2–6 These physiological changes occur in the absence of acute metabolic evidence of ischemia or pathological evidence of infarction. When stenosis severity is less marked, chronically stunned myocardium with normal resting flow develops.^7,8 At the molecular level, chronic stunning can be distinguished from hibernating myocardium by the relative lack of reductions in sarcoplasmic reticulum (SR) Ca²⁺-ATPase and phospholamban, which are characteristic of hibernating myocardium.^9,10 In addition, hibernating myocardium appears to have an increase in the number of myocytes having increased glycogen stores and expressing structural proteins characteristic of dedifferentiation.^11,12

Although serial studies of juvenile swine with a chronic stenosis have demonstrated a systematic progression from chronically stunned to hibernating myocardium,^4,8 the extent to which these changes are unique to the growing swine model or are dependent on the physiological severity versus chronicity of a coronary stenosis remains unclear. We hypothesized that the severity of stenosis is the key physiological variable that drives the myocyte adaptation to ischemia, and we also hypothesized that the progression to hibernating myocardium is independent of growth and can occur in mature swine. To critically test these hypotheses, we determined whether we could reproduce the physiological, molecular, and cellular characteristics of chronic hibernating myocardium by acutely restricting vasodilated perfusion. We used a hydraulic stenosis that could be maintained over a period of weeks in chronically instrumented swine. Swine were evaluated at frequent intervals to confirm that the stenosis critically limited peak hyperemic flow. Importantly, we avoided any circumstance in which the stenosis might cause prolonged acute ischemia, as reported in previous models of "short-term hibernation."^13–18 The results demonstrate that the physiological and molecular changes typical of hibernating myocardium can be induced within days after an acute critical stenosis. Although the myolytic phenotype also develops rapidly, the morphological changes occur globally and are dissociated from the regional adaptive responses in hibernating myocardium.

	Materials and Methods

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A total of 23 swine were chronically instrumented with catheters and hydraulic occluders, as previously described¹⁹ and detailed in an online Materials and Methods section (available in the data supplement at http://www.circresaha.org). In brief, we performed a left thoracotomy and inserted catheters into the aorta and left atrium. A micromanometer was placed into the LV apex, and a volumetric flow probe was placed on the proximal left anterior descending coronary artery (LAD). Ultrasonic crystals were inserted to measure wall thickening. Two hydraulic occluders were placed on the LAD. The primary occluder was used to produce a critical LAD stenosis, which was maintained throughout the study. A secondary occluder was used to produce brief LAD occlusions to confirm the attenuation of reactive hyperemia. The chest was closed, the pneumothorax was evacuated, and analgesics were administered until the animals recovered.

Study Protocol
Studies were conducted with the animals under propofol sedation. After assessment of baseline variables, acute stunning was produced by a 15-minute partial occlusion of the LAD using the primary occluder. Subsequently, hearts were reperfused through a critical stenosis that abolished the reactive hyperemic response to a 20-second occlusion yet allowed peak LAD flow to increase above the preocclusion value. Microspheres were injected to assess the transmural distribution of flow at rest, during a transient total LAD occlusion (to assess collateral flow), and after adenosine vasodilation. In 9 animals, we removed hearts for tissue sampling after 24 hours. The remaining studies were continued for 2 weeks. In the latter group, serial measurements of flow, function, and hemodynamics were assessed at 1- to 3-day intervals. When necessary, the primary occluder was readjusted to achieve a critical coronary stenosis.

Myocardial Sampling
At the end of the protocols, animals were anesthetized, and flash-frozen subendocardial samples were obtained from the LAD and normal regions for protein and RNA isolation. We assessed regional changes in the expression of SR Ca²⁺-ATPase, phospholamban, and calsequestrin.^9,19 Samples were also obtained to assess microsphere perfusion in 3 transmural layers of the LV.

Histological Analysis
Connective tissue was assessed by point counting, and infarction was excluded by triphenyltetrazolium chloride (TTC) staining.^4,19 In animals evaluated for 2 weeks, small subendocardial samples were processed for electron microscopy (EM) and light microscopy and analyzed in a blinded fashion (M.B.).²⁰ At least 200 cells per sample were analyzed. Because the space, formerly occupied by sarcomeres, was occupied by glycogen, quantification of myolysis was performed by planimetrically evaluating the percentage of PAS-positive material per cell. If sarcomere depletion was >10%, cells were classified as being affected by myolysis.²⁰ EM was used to confirm the structural alterations, such as sarcomere depletion and glycogen accumulation, demonstrated on light microscopy.

To evaluate the transmural distribution of myolysis, we also quantified myofibrillar volume loss by use of blinded analysis (G.S.) and a point-counting grid. Trichrome-stained sections were used to identify myocytes, and grid intersections that were not stained in the perinuclear area (myolysis) were expressed as a percentage of the intersections that were overlying myofibrils. Myofibrillar loss was analyzed in subendocardial and subepicardial regions of sham-instrumented animals (sham animals) and animals instrumented for 24 hours and 2 weeks (24-hour and 2-week animals, respectively).

Statistical Analysis
Hemodynamics and wall thickness measurements were recorded (Gould model TA11) and digitized (sampling rate, 500 Hz). Values reported are the mean±SEM. Time-dependent changes were evaluated by ANOVA, with post hoc paired t tests comparing data at each time point with the corresponding baseline. Perfusion in stenotic LAD regions was also compared with that in remote left circumflex (LC) myocardium. A value of P<0.05 was considered significant.

	Results

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Animals were in good health at the time of study. Of the 23 animals initially instrumented, 7 were excluded from analysis by experimental design. In 3 animals, we were unable to maintain a stable occlusion, and the lack of a significant reduction in flow reserve prevented the development of depressed LAD function. In 4 others, there was TTC evidence of infarction that averaged 5.5±1.0% of LV mass.

Serial Flow, Function, and Hemodynamics
Figure 1 illustrates representative analog recordings from an animal undergoing serial evaluation for the 2-week period. Hemodynamics, flow, and function at selected time points are summarized in the Table. A 15-minute partial occlusion resulted in acute myocardial stunning, with LAD wall thickening decreasing from 36.3±4.0% to 24.0±3.3% (P<0.01), whereas remote-zone LC function remained normal (29.3±4.0% versus 30.9±4.1%). Although flow was somewhat lower than baseline, peak hyperemic flow after a total occlusion exceeded the initial baseline, confirming that the stenosis was not flow limiting. When the heart was reperfused through a critical stenosis, function did not return to normal. After 24 hours, LAD wall thickening averaged 25.5±3.7% (P<0.05 versus baseline), but LAD flow was normal; these findings are consistent with sustained myocardial stunning. There were no significant differences in hemodynamics, and there was no infarction as assessed by TTC.

View larger version (27K): [in this window] [in a new window]   Figure 1. Analog recordings at selected time points from a representative animal from the 2-week group. Dotted vertical lines depict end diastole and end systole. On day 1, a 15-minute partial LAD occlusion produced acute stunning. When reperfused with a critical limitation in flow reserve, function remained depressed when the animals were reevaluated 24 hours later (day 2), but resting flow was normal, consistent with myocardial stunning. Function continued to be depressed throughout the study, but resting flow became reduced, consistent with a progression to hibernating myocardium (day 17). Throughout the study, heart rate and systemic hemodynamics remained constant, with the exception of an increase in LV end-diastolic pressure. dPLV/dt represents the first derivative of LV pressure.

View this table: [in this window] [in a new window]   Table 1. Hemodynamics, Flow, and Function

Serial measurements of flow and function in animals completing the 2-week study protocol are summarized in Figure 2, and hemodynamic measurements are summarized in the Table. The physiological severity of the stenosis varied little, and initial peak reactive hyperemic flow increased to less than twice the preocclusion baseline flow at all time points. There was a progressive reduction in LAD wall thickening, which was initially associated with normal resting perfusion, consistent with myocardial stunning. After 1 week, resting flow decreased and remained so for the duration of the study. In animals studied after 2 weeks, flow was 45±10 mL/min (P<0.05 versus preocclusion), and wall thickening averaged 17.0±5.0% (P<0.05). The reduction in flow developed despite the fact that hyperemic flow always exceeded the preocclusion value at rest. These findings indicate that an acute critical stenosis caused a rapid progression from stunned to hibernating myocardium within 1 week. Reductions in function preceded reductions in resting flow and occurred in the presence of residual vasodilator reserve.

View larger version (23K): [in this window] [in a new window]   Figure 2. Serial measurements of LAD flow and wall thickening in animals in the 2-week study protocol. The initial (triangles) and final (squares) reactive hyperemic (RH) flows were reduced by the stenosis but always exceeded the preocclusion baseline flow at the beginning of the study (dotted line). LAD wall thickening was initially depressed with normal flow, consistent with chronic myocardial stunning. After a critical stenosis was applied for 8 days, resting flow (circles) became reduced. Thus, a critical coronary stenosis caused a rapid progression from stunned to hibernating myocardium with reductions in resting flow occurring in the presence of recruitable flow reserve.

To confirm that the reductions in flow were indicative of reductions in tissue perfusion, we assessed regional flow with microspheres. Figure 3 summarizes serial resting flow measurements in animals evaluated for 2 weeks. Before occlusion, resting flow was similar in the LAD and normal LC regions. Although function was reduced after 24 hours, microsphere flow was similar in LAD and remote regions, which is consistent with stunning. After 2 weeks, there was a reduction in resting LAD flow to 1.03±0.08 versus 1.39±0.09 mL · min^-1 · g^-1 in normal regions (P<0.05). Flow was reduced in each myocardial layer (P<0.05) and was consistent with a progression from stunned to hibernating myocardium.

View larger version (17K): [in this window] [in a new window]   Figure 3. Microsphere measurements of resting flow in animals completing the 2-week protocol. Paired analysis of flow measurements in the LAD and normally perfused remote region were similar before occlusion (day 1) and in stunned myocardium after 24 hours (day 2). At the end of the study, LAD flow was significantly lower than flow in normally perfused myocardium in each myocardial layer. Endo, Mid, and Epi indicate subendocardial, midmyocardial, and subepicardial samples, and FT indicates full thickness.

Figure 4 summarizes transmural variations in adenosine flow in the presence of a LAD stenosis. After application of a critical stenosis with the primary occluder (Figure 4, top), adenosine flow was similar to the initial baseline value (1.34±0.19 versus 1.14±0.07 mL · min^-1 · g^-1, respectively; P=NS), with no significant increase in any transmural layer. After 24 hours (Figure 4, middle), the physiological severity of the stenosis had decreased, but full-thickness vasodilated flow still did not significantly increase above resting flow (2.46±0.83 [adenosine] versus 1.28±0.12 [baseline] mL · min^-1 · g^-1 at rest, P=NS). Subendocardial flow continued to be critically reduced (1.79±0.65 versus 1.28±0.18 mL · min^-1 · g^-1). After 2 weeks, adenosine flow was similar to that after 24 hours (2.25±0.46 [full thickness] versus 1.62±0.43 [subendocardium] mL · min^-1 · g^-1). Flow measurements during a total LAD occlusion at 24 hours (0.10±0.06 mL · min^-1 · g^-1) and 2 weeks (0.25±0.17 mL · min^-1 · g^-1, P=NS versus 24 hours) were severely reduced, indicating little change in coronary collateral flow over time.

View larger version (25K): [in this window] [in a new window]   Figure 4. Microsphere flow at rest (circles), after acute ischemia (diamonds), after adenosine vasodilation in the presence of a critical stenosis (triangles), and after a transient total LAD occlusion (squares). At each time point, compared with flow in normally perfused remote regions, LAD flow during vasodilation was severely reduced. Subendocardial flow was critically reduced and failed to increase above the resting value before occlusion. Flow during a transient LAD occlusion was markedly reduced and indicated little, if any, stimulation of collateral flow in this model.

Protein and RNA Changes
Figure 5 summarizes SR protein changes in hearts from swine with hibernating myocardium. Subendocardial samples from the LAD region demonstrated reductions in phospholamban (2.12±0.61 [LAD region] versus 3.52±0.62 [normal region] densitometric units, P=0.06) and SR Ca²⁺-ATPase (7.30±0.49 [LAD region] versus 9.81±0.31 [normal region] densitometric units, P<0.05). In contrast, there were no regional changes in calsequestrin protein, indicating that the reductions in the SR Ca²⁺ uptake proteins were not related to a nonspecific loss of SR. There were no systematic changes in any SR protein levels in samples taken after 24 hours. Nevertheless, Northern analysis after 24 hours demonstrated reductions in mRNA levels for phospholamban (0.17±0.03 [LAD region] versus 0.23±0.03 [normal region] densitometric units, P<0.05) and SR Ca²⁺-ATPase (0.94±0.11 [LAD region] versus 1.13±0.07 [normal region] densitometric units, P<0.05), with no changes in calsequestrin mRNA. Collectively, these findings suggest that spontaneous ischemia arising distal to a critical stenosis initiates a transcriptional cascade that results in a regional downregulation in the expression of selected SR proteins during the progression to a physiological phenotype of hibernating myocardium.

View larger version (46K): [in this window] [in a new window]   Figure 5. Summary of SR protein measurements in LAD and normal regions from animals completing the 2-week protocol. There were reductions in phospholamban and SR Ca2+-ATPase after 2 weeks, but the expression of calsequestrin remained unchanged. Thus, the physiological findings of hibernating myocardium were accompanied by the rapid progression of protein changes previously demonstrated over a more protracted time frame in growing swine with a fixed LAD stenosis.4,21

Histological Analysis
Point counting demonstrated a regional increase in connective tissue similar to that in humans with hibernating myocardium. After 24 hours, connective tissue was 6.0±1.5% in the LAD region versus 2.7±0.4% in normal regions (P=0.06). In the group with hibernating myocardium, connective tissue averaged 10.6±4.7% in the LAD region versus 7.3±5.4% in normal regions.

Figure 6 shows PAS/toluidine blue-stained sections from LAD and normal regions along with corresponding EM from swine with hibernating myocardium versus sham control swine. PAS-positive areas (increased glycogen) corresponded to regions of myofibrillar loss (myolysis) by EM. Interestingly, myolysis was present in hibernating LAD regions and in normally perfused remote regions of swine that were evaluated 2 weeks after instrumentation. These changes stood in marked contrast to the lack of myolysis or PAS positivity in sham hearts. Myolytic myocytes averaged 33±3% in hibernating LAD regions versus 15±4% in sham control regions (P<0. 05). The frequency of myolysis was identical to that in normally perfused nondysfunctional regions of swine with hibernating myocardium, which averaged 32±7% (P=NS versus LAD of 2-week animals).

View larger version (91K): [in this window] [in a new window]   Figure 6. EM (A) and light (B) photomicrographs from swine evaluated 2 weeks after a critical LAD stenosis. The EM photomicrographs demonstrate myofibrillar loss along with increased glycogen granules and small mitochondria in hibernating myocardium (right). These changes were absent in sham animals (left). Interestingly, the EM changes in dysfunctional LAD regions were identical to those in normal remote regions. Panel B shows that the perinuclear myofibrillar loss was replaced by PAS-positive material, consistent with glycogen by light microscopy. Like the EM changes, PAS-positive myocytes were markedly increased in animals with hibernating myocardium compared with sham animals, yet the frequency in dysfunctional LAD regions was similar to corresponding remote regions. Thus, the morphological features typical of hibernating myocardium occurred globally and were not confined to areas of contractile dysfunction.

Figure 7 summarizes myofibrillar volume loss on a transmural basis for 24-hour, 2-week, and sham animals. Subendocardial myofibrillar loss was similar in LAD and remote regions of 2-week animals (9.4±1.5% versus 8.1±0.5%, respectively; P=NS) but was markedly higher than that in sham control animals (2.0±0.2% versus 1.9±0.2%, respectively; both P<0.05 versus 2-week animals). Myofibrillar loss was also increased in subepicardial layers of 2-week animals but was not different between regions or corresponding subendocardial values. Myofibrillar loss had already begun after 24 hours but was intermediate between values of 2-week and sham animals. Thus, these data dissociate myofibrillar loss from the regional physiological and protein changes characteristic of hibernating myocardium.

View larger version (28K): [in this window] [in a new window]   Figure 7. Quantitative analysis of myofibrillar volume loss in subendocardial (black bars) and subepicardial (gray bars) regions. Myofibrillar loss was nearly 4-fold higher in hibernating myocardium than sham control myocardium. Myofibrillar loss began after 24 hours and increased after 2 weeks. Subepicardial and subendocardial values were similar. Furthermore, measurements in the LAD region subjected to ischemia were similar to those in the normal region. Thus, myofibrillar loss was global and was dissociated from regional differences in function and coronary flow reserve.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
There are several important new findings from the present study. First, hibernating myocardium develops within days after a sudden critical impairment in coronary flow reserve. Reductions in contractile function with normal resting perfusion precede the development of reductions in resting flow and are consistent with a rapid progression from stunned to hibernating myocardium. Second, like the regional physiological changes, regional alterations in SR protein expression also develop in an accelerated fashion and go hand in hand with the progression from stunned to hibernating myocardium. In sharp contrast, the cellular morphological changes of myolysis and increased glycogen that have been ascribed to hibernating myocytes from biopsies of dysfunctional regions in humans actually occur globally. Myofibrillar volume loss is similar in subendocardial and subepicardial layers. Although it is already present after 24 hours, it increases globally during the progression to hibernating myocardium. The dissociation of the myolytic cellular phenotype from the regional physiological and molecular changes indicates that these morphological alterations are unrelated to the adaptive responses characteristic of hibernating myocardium.

Relation to Previous Studies of Chronic Hibernating Myocardium
The present study demonstrates that the time frame over which adaptive responses characteristic of hibernating myocardium develop can be quite rapid and is primarily related to the physiological significance of a coronary stenosis rather than time. This contrasts with the slow progression of these adaptations in juvenile swine instrumented with a fixed LAD stenosis.^4,8,21 In the latter model, the cross-sectional area of the proximal LAD is fixed and progressively limits maximal flow. As LV mass increases with growth, there is a gradual but progressive reduction in maximal flow per gram of tissue that reaches a critical level after 3 months and remains stable thereafter.²¹ Although this model also results in a progression from stunned to hibernating myocardium that is accompanied by regional reductions in selected SR proteins,¹⁰ the adaptive responses develop over a much more protracted time frame. Because domestic swine grow rapidly during the first 3 months, the physiological adaptations could potentially be unique to the juvenile animal. The present study excludes this possibility and indicates that hibernating myocardium can develop over a time frame as short as 1 week. Thus, the temporal progression from stunned to hibernating myocardium is most closely related to the physiological severity of flow impairment.^4,8,9,21 We have also accelerated the development of hibernating myocardium by limiting source collateral flow with an LC stenosis in a 2-vessel chronic stenosis model of hibernating myocardium.⁶ Although speculative, flow reserve is likely an integrative measure of the propensity of a region of the heart to develop spontaneous ischemia, which increases as the ability of the myocardium to increase perfusion above resting values becomes more limited.

The critical nature of coronary flow reserve reduction required to produce hibernating myocardium likely explains why not all studies have demonstrated a progression to hibernating myocardium distal to a chronic stenosis. For example, circumflex ameroid models (rapidly progressing stenoses) in dogs usually have normal resting function, with a well-developed collateral circulation, yet hibernating myocardium can develop when source collateral flow is limited.^2,22 Bolukoglu et al²³ produced a near-critical stenosis in swine, but flow reserve was not continuously monitored, and hyperemic flow at the end of the study was not lower than control. Although regional function was depressed in this model, resting perfusion was normal, consistent with chronic myocardial stunning.²⁴ Contractile function in the absence of infarction in swine ameroid occluder models is equally as variable, with most studies reporting normal resting function^25–27 and 1 study reporting evidence of chronic myocardial stunning.⁷ The variability of function in circumflex ameroid models probably reflects several factors. First, there is considerable variability in the time course of ameroid occlusion, with some studies even demonstrating a lack of total occlusion.⁷ Second, when vasodilated flow was measured, there was only a modest difference in maximal perfusion between collateral-dependent and normal regions,^28,29 which probably reflects the increase in perfusion from extracardiac collaterals. Although this limits infarction, it does not lead to the degree of spontaneous ischemia necessary to produce chronic myocardial dysfunction. Thus, variability in coronary flow reserve is likely the primary cause of different intrinsic adaptive responses of the myocardium to ischemia.

Relation to Previous Studies of Short-Term Hibernation
Previous investigators have attempted to use animal models of prolonged acute ischemia to produce viable dysfunctional myocardium, a phenomenon that has been termed short-term hibernation.³⁰ Matsuzaki et al¹³ showed that the myocardium could tolerate several hours of moderate ischemia without the development of infarction. Other laboratories have demonstrated that this ability to match flow and function at a reduced level prevents further high-energy phosphate depletion with a resolution of lactate release.^14,15,18,31 The original concept of hibernating myocardium proposed that such a precarious low-flow state could be maintained indefinitely without leading to irreversible injury.³² However, animal studies have demonstrated that irreversible injury ultimately develops in these models. For example, Chen et al¹⁶ found TTC evidence of infarction in the majority of animals subjected to short-term hibernation for a period that exceeded 24 hours, with rates of subendocardial myocyte apoptosis approaching 10%. Kudej et al¹⁷ demonstrated myocyte necrosis after >90 minutes of moderate ischemia in conscious swine. After 24 hours, patchy necrosis involved up to 40% of the subendocardium. Schulz et al³³ found necrosis to be infrequent after 12 hours but common after 24 hours of moderate ischemia in anesthetized swine. Thus, although the ability of the heart to tolerate moderate ischemia is better than the ability of the heart to tolerate total coronary occlusion, it is still time-limited.

Our findings differ from studies evaluating short-term hibernation in several important respects. First, we adjusted the physiological significance of the stenosis to allow flow during vasodilation to exceed the preocclusion resting flow. This strategy avoided uncertainty in interpreting whether reductions in resting flow and function were simply consequences of acute ischemia. In this regard, the present study clearly shows that dysfunction with normal resting flow (myocardial stunning) preceded the development of hibernating myocardium and confirms a similar progression demonstrated in other animal models.^2,8,22

Reductions in SR Proteins in Viable Dysfunctional Myocardium
Our results demonstrate that a critical coronary stenosis initiates transcriptional changes that downregulate the expression of SR Ca²⁺-ATPase and phospholamban within 24 hours. The regional reductions in mRNA contrast with the lack of change in expression that we¹⁹ and others^34–37 have demonstrated after single episodes of reversible ischemia. However, they are remarkably similar to the progression that we have reported in growing swine with a fixed LAD stenosis leading to hibernating myocardium.^9,10 Reductions in mRNA and protein levels for SR Ca²⁺-ATPase and phospholamban began in chronically stunned myocardium after 2 months and decreased further with the progression to hibernating myocardium after 3 months. A similar acceleration in this phenotypic pattern was also seen in swine instrumented with 2-vessel stenoses.⁶ The consistency of these observations in 3 different models of a chronic stenosis supports the notion that regional reductions in SR Ca²⁺-ATPase and phospholamban are at least partly responsible for altered contraction in hibernating myocardium. Although speculative, these changes may lead to an altered calcium responsiveness similar to that in acutely ischemic myocardium.³⁸

Dissociation of Myolysis and Regional Adaptations in Hibernating Myocardium
Our results indicate that myolysis develops rapidly after a critical limitation in flow reserve and is consistent with the acute myofibrillar disassembly and increased glycogen reported after short-term hibernation by Sherman et al.³⁶ Like acute myofibrillar disassembly, myofibrillar loss and increased glycogen content occurred globally. Quantitative analysis demonstrated similar values in hibernating as well as normally perfused remote regions of dysfunctional hearts. Differences in regional function were not the result of myofibrillar loss because both subendocardial and subepicardial values were similar in dysfunctional and normal remote regions. Thus, the pathological changes described in humans with hibernating myocardium do not appear to reflect the chronicity of LV dysfunction nor do they appear to arise as a direct consequence of ischemia.

A limitation of most clinical studies has been that the biopsy material has been obtained from dysfunctional regions and that an internal control sample from remote myocardium has not been analyzed. Interestingly, Gunning et al³⁹ have recently reported global myolysis in humans with viable dysfunctional myocardium, depressed ejection fraction, and symptomatic heart failure. The fact that myolysis occurs globally in ischemic cardiomyopathy raises the possibility that these morphological changes reflect a response to chronic elevations in preload or stretch. In this regard, we found that LV end-diastolic pressure progressively increased from 16 to 24 mm Hg at the end of the study (Table). This possibility is also supported by the similarity of the changes in LV myocytes to those in atrial myocytes from models of atrial fibrillation.⁴⁰ Interestingly, conversion of the rhythm failed to reverse the myolytic changes in atrial muscle and was associated with chronic increases in atrial filling pressure. Thus, although these cells may revert to a fetal phenotype, a response to global stress rather than repetitive regional ischemia is indicated.

Methodological Limitations
Our experimental approach was quite similar to clinical circumstances in which acute plaque rupture results in a period of prolonged ischemia with reperfusion through a critical stenosis. It is also possible that the transition to hibernating myocardium could occur without a period of brief partial coronary occlusion preceding the stenosis. Nevertheless, studies in 2 additional animals suggest that the time frame for this progression appears slightly longer than when preceded by a period of acute stunning (please see the online data supplement, available at http://www.circresaha.org).

All of our measurements were obtained during propofol sedation. Hemodynamics were similar to those of conscious swine,¹⁷ and heart rates were similar to average values recorded by Holter monitoring of unrestrained swine (authors’ unpublished data, 2002). Although speculative, spontaneous increases in myocardial oxygen demand likely led to episodic ischemia when the animals were unmonitored. Alternatively, ischemia could have arisen from cyclical platelet aggregation and transient total or prolonged partial coronary occlusion. Telemetry of a sensitive index of subendocardial ischemia such as regional function will be required to document the frequency or extent of ischemia necessary to produce chronic regional dysfunction.

In summary, the physiological and molecular changes of hibernating myocardium can develop quite rapidly, in contrast to the several-month time frame required in growing swine with a chronic LAD stenosis. This suggests that the myocyte has remarkable plasticity and can adapt to intrinsic stimuli such as ischemia over a time frame that promotes cell survival at the expense of reduced contractile function. The dissociation between regional physiological and molecular changes with global increases in glycogen and myofibrillar loss indicates that the latter changes are not responsible for the adaptive responses unique to hibernating myocardium. Likewise, myolysis is unlikely to limit functional recovery after revascularization. The rapidity at which hibernating myocardium can develop supports the view that intrinsic adaptations to ischemia can preserve myocyte viability after acute coronary syndromes.

	Acknowledgments

 
This study was supported by the Department of Veterans Affairs, the American Heart Association, the Albert and Elizabeth Rekate Fund, and the National Heart, Lung, and Blood Institute (grants HL-55324 and HL-61610). The technical assistance of Rebeccah Young, Anne Coe, Deana Gretka, Amy Johnson, and Frederick Thoné is greatly appreciated.

Received May 3, 2002; revision received September 23, 2002; accepted September 25, 2002.

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