Temporal and spatial variations in structural protein expression during the progression from stunned to hibernating myocardium.

BACKGROUND: Dysfunctional and normally perfused remote regions show equal myolysis and glycogen accumulation in pig hibernating myocardium. We tested the hypothesis that these arose secondary to elevations in preload rather than ischemia. METHODS AND RESULTS: Expression of structural protein (desmin, desmoplakin, titin, cardiotin, alpha-smooth muscle actin, lamin-A/C, and lamin-B2) in viable dysfunctional myocardium was analyzed by immunohistochemistry. We performed blinded analysis of paired dysfunctional left anterior descending coronary artery and normal remote subendocardial samples from stunned (24 hours; n=6), and hibernating (2 weeks; n=6) myocardium versus sham controls pigs (n=7). Within 24 hours, cardiac myocytes globally reexpressed alpha-smooth muscle actin. In stunned myocardium, cardiotin was globally reduced, whereas reductions in desmin were restricted to the dysfunctional region. Alterations progressed with the transition to hibernating myocardium, in which desmin, cardiotin, and titin were globally reduced. A qualitatively similar reorganization of cytoskeletal proteins occurred 3 hours after transient elevation of left ventricular end-diastolic pressure to 33+/-3 mm Hg. CONCLUSIONS: Qualitative cardiomyocyte remodeling similar to that in humans with chronic hibernation occurs rapidly after a critical coronary stenosis is applied, as well as after transient elevations in left ventricular end-diastolic pressure in the absence of ischemia. Thus, reorganization of cytoskeletal proteins in patients with viable dysfunctional myocardium appears to reflect chronic and/or cyclical elevations in preload associated with episodes of spontaneous regional ischemia.

Myocyte apoptosis and reduced SR gene expression precede the transition from chronically stunned to hibernating myocardium.

A systematic transition from chronic stunning to hibernation occurs as coronary flow reserve decreases to a critical level. Hibernating myocardium exhibits apoptosis-induced myocyte loss and a reduction in the expression of the sarcoplasmic reticulum (SR) Ca2+ ATPase but whether similar cellular changes occur in chronic stunning is unknown. Pigs with a chronic left anterior descending coronary artery (LAD) stenosis were studied one (n=9) or two (n=10) months after instrumentation. Anterior hypokinesis with normal levels of resting perfusion developed at each time-point, consistent with chronic stunning. After 1 month, sub-endocardial flow reserve was moderately reduced (adenosine/rest, LAD: 3.60+/-0.91 v Remote: 6.00+/-0.54, P<0.01) with no regional differences in SR protein expression, no increase in apoptosis (32+/-6 v 21+/-5 nuclei/10(6) myocyte nuclei, p-ns) and no regional myocyte loss (1976+/-44 v 1955+/-30 nuclei/mm2, p-ns). After 2 months, sub-endocardial flow reserve in chronically stunned myocardium was severely impaired (LAD: 1.41+/-0.21 v Remote: 5.59+/-0.96, P<0.01). There were small but significant reductions in LAD mRNA and protein levels for the SRCa2+ ATPase and phospholamban whereas calsequestrin was unchanged. In addition, regional myocyte apoptosis increased (127+/-24 v 55+/-9 nuclei/10(6) myocyte nuclei, P<0.01), resulting in the onset of myocyte loss (1293+/-50 v 1394+/-32 nuclei/mm2, P<0.01). Apoptosis-induced myocyte loss and reductions in SR protein expression are not invariably present in viable chronically dysfunctional myocardium. They are induced as the propensity of a region to develop reversible ischemia increases (as reflected by coronary flow reserve). The temporal progression indicates that alterations in SR protein expression and myocyte apoptosis precede the transition from chronically stunned to hibernating myocardium.

Lessons from experimental models of hibernating myocardium.

Chronic animal models of viable dysfunctional myocardium are now available that recapitulate most if not all of the physiological findings in humans with hibernating myocardium. These include chronic reductions in resting perfusion and contractile function, critical limitations in coronary flow reserve and increased uptake of 18F-2-deoxyglucose. These changes occur in the absence of infarction or necrosis and are accompanied by regional reductions in sarcoplasmic reticulum calcium-handling proteins and myocyte loss that arise secondary to apoptosis. Longitudinal studies of viable dysfunctional myocardium indicate that a state of chronic stunning with normal resting flow precedes the development of hibernating myocardium but these are distinct entities within a continuum of chronic adaptations to ischemia. This indicates that reductions in resting flow are the result rather than cause of chronic contractile dysfunction. Thus, the original concept proposing an acute prolonged reduction in flow as the initial stimulus producing hibernating myocardium needs to be revised.

Stability of hibernating myocardium in pigs with a chronic left anterior descending coronary artery stenosis: absence of progressive fibrosis in the setting of stable reductions in flow, function and coronary flow reserve.

OBJECTIVES: This study was performed to determine whether hibernating myocardium is adaptive or is destined to undergo progressive irreversible injury. BACKGROUND: Previous studies have suggested that hibernating myocardium eventually results in progressive dysfunction. Since serial studies cannot be performed in humans, the temporal progression of physiologic and structural adaptations was evaluated in pigs with hibernating myocardium. METHODS: Pigs were instrumented with a left anterior descending coronary artery (LAD) stenosis (1.5 mm) and underwent physiologic studies three to five months later to quantify regional function, perfusion and 18F-2-deoxyglucose (FDG) uptake. Viability was confirmed by histology and contractile reserve. RESULTS: Hibernating myocardium was characterized by severe regional dysfunction (centerline score, -1.9+/-0.1), reduced resting subendocardial flow (LAD: 0.85+/-0.03 vs. normal: 1.02+/-0.03 ml/min/g, p < 0.01), critically reduced subendocardial flow reserve (adenosine flow: 1.04+/-0.09 ml/min/g, p = NS vs. rest; epinephrine flow: 0.88+/-0.07 ml/min/g, p = NS vs. rest) and increased FDG uptake (0.022+/-0.002 vs. 0.014+/-0.001 ml/g/min, p < 0.01). Physiologic parameters were not different among animals studied at three (93+/-1 days, n = 27), four (118+/-2 days, n = 26) or five months (150+/-6 days, n = 9). Pathology revealed a small increase in LAD connective tissue (6.4+/-0.4% vs. 4.0+/-0.2%, p < 0.001), with no change over this time frame. CONCLUSIONS: Thus, physiologic and structural features of hibernating myocardium remain constant for at least two months. The absence of functional deterioration or progressive fibrosis suggests that hibernation is adaptive rather than an unstable physiology destined to progress to irreversible injury. The stability of this model appears ideally suited for interventions targeted to improve flow and function in chronically dysfunctional myocardium.

Preload induces troponin I degradation independently of myocardial ischemia.

BACKGROUND: Although global ischemia induces troponin I (TnI) degradation, regional ischemia does not. We hypothesized that this disparity is related to preload-induced proteolysis, which varies as a function of the amount of myocardium at risk of ischemia. METHODS AND RESULTS: Isolated rat hearts were buffer-perfused at controlled levels of preload. Increasing preload to 25 mm Hg in the absence of ischemia produced pronounced TnI degradation (27 kDa versus 31 kDa bands: 16.4 +/- 3.6% versus 4.7 +/- 1.9% in immediately excised controls, P<0.05). TnI degradation could be blocked by preventing the activation of endogenous calpains with 25 micromol/L calpeptin (4.3 +/- 0.6%). This improved function, with left ventricular systolic pressure increasing from 103 +/- 4 mm Hg to 137 +/- 7 mm Hg (P<0.05). Eliminating elevations in preload after global ischemia-induced stunning also prevented TnI degradation. CONCLUSIONS: Calpain-mediated TnI proteolysis can be dissociated from stunning and arises from elevations in preload rather than ischemia. This raises the possibility that ongoing preload-induced TnI degradation could impair myocardial function long-term.

Chronic hibernation and chronic stunning: a continuum.

Identification of myocardial viability is of increasing clinical importance in managing patients with coronary artery disease and advanced left ventricular dysfunction. Although viable chronically dysfunctional myocardium is always the result of repetitive episodes of reversible ischemia, there may be multiple mechanisms responsible for the contractile dysfunction. Many patients have contractile dysfunction with normal resting perfusion, as determined by imaging, that is related to chronic myocardial stunning. Viability studies are generally unnecessary because normal resting perfusion would preclude significant fibrosis. The clinical problem arises in evaluating patients with depressed resting flow that can be due to hibernating myocardium or nontransmural infarction. In this circumstance viability studies are required to assess the likelihood of functional recovery after revascularization. Although hibernating myocardium was originally posited to develop in response to prolonged episodes of myocardial ischemia (experimentally termed "short-term hibernation"), subsequent studies have shown that this tenuous balance can only be maintained for a period of several hours before resulting in some degree of subendocardial infarction. More recent experimental studies have demonstrated that there is a progression from chronic stunning with normal flow to hibernating myocardium with reduced resting flow. This presumably arises from repetitive episodes of spontaneous ischemia that increase in frequency as the physiologic significance of a coronary stenosis progresses. Thus in this new paradigm reduced flow is a result, rather than the cause, of the contractile dysfunction. This review summarizes basic and clinical pathophysiologic studies supporting the claim that chronic stunning and hibernation are distinct entities that may represent opposite ends of a continuum of mechanisms in viable chronically dysfunctional myocardium.

Spatial heterogeneity in fasting and insulin-stimulated (18)F-2-deoxyglucose uptake in pigs with hibernating myocardium.

BACKGROUND: Previous studies of hibernating myocardium in the fasting state have shown regionally increased (18)F-2-deoxyglucose (FDG) uptake with a marked transmural gradient. We hypothesized that this adaptation to chronic ischemia might be associated with altered maximal FDG uptake. METHODS AND RESULTS: Pigs were instrumented with a 1.5-mm proximal left anterior descending artery (LAD) stenosis. Studies were conducted 106+/-4 days later on anesthetized animals with complete LAD occlusion and anteroapical dysfunction. In fasting animals (n=9), FDG uptake in dysfunctional LAD regions was 2-fold higher than in normally perfused myocardium (7.9+/-1.2 versus 4. 0+/-0.5 micromol x min(-1) x 100 g(-1), P<0.05), with a pronounced transmural gradient (endocardial/epicardial ratio 2.56+/-0.19 versus 1.25+/-0.03, P<0.05). Euglycemic, hyperinsulinemic clamp (insulin clamp, n=8) produced a 5- to 9-fold increase in FDG uptake, but there was no longer a regional difference in accumulation (LAD, 37. 8+/-4.2 versus normal, 36.4+/-5.1 micromol x min(-1) x 100 g(-1), P=NS) and the transmural distribution was uniform. FDG uptake in the fasting state varied inversely with coronary flow during vasodilation. In contrast, during insulin clamp there was no relation between FDG uptake and vasodilated flow, resulting in a reduced spatial heterogeneity in individual samples (relative dispersion=SD/mean; fasting, 52+/-5% versus insulin, 24+/-2%, P<0.05). CONCLUSIONS: In the fasting state, FDG uptake in pigs with hibernating myocardium was heterogeneous and was increased in dysfunctional regions with a marked transmural gradient and high spatial heterogeneity. In contrast, FDG uptake was more homogeneously distributed during insulin clamp with (1) uptake in dysfunctional myocardium similar to remote normal regions, (2) uniform transmural distribution, and (3) reduced spatial heterogeneity.

Transmural distribution of FDG uptake in stunned myocardium.

Fasting [(18)F]fluoro-2-deoxyglucose (FDG) uptake is increased in viable, chronically dysfunctional myocardium, but the relationship to acute episodes of ischemia remains undefined. To investigate FDG uptake in acute stunning, chronically instrumented pigs (n = 9) and sham controls (n = 8) were studied while in a fasted, closed-chest, anesthetized state. One-hour partial occlusion reduced subendocardial flow from 1.24 +/- 0.14 to 0.35 +/- 0.06 ml. min(-1). g(-1) and wall thickening from 16.8 +/- 2.1 to 3.7 +/- 0.7%. Regional function remained depressed during reperfusion (8.3 +/- 1. 4%) despite the return of flow to resting levels. Triphenyl tetrazolium chloride staining showed no irreversible injury. FDG uptake in stunned myocardium was variably increased and averaged 1. 5-fold higher than that of normal regions, with no consistent transmural variation. Subgroup analysis showed that variability in FDG uptake was related to alterations in insulin levels that varied directly with ischemic risk region.

Profound apoptosis-mediated regional myocyte loss and compensatory hypertrophy in pigs with hibernating myocardium.

BACKGROUND: Myocyte apoptosis is seen in ischemic heart disease, but whether it can occur after reversible ischemia or independent of necrosis and replacement fibrosis is unknown. METHODS AND RESULTS: Pigs were instrumented with a stenosis of the left anterior descending coronary artery to chronically reduce coronary flow reserve over a period of 3 months. At this time, there was viable dysfunctional myocardium having the physiological features of hibernating myocardium. Resting subendocardial perfusion was reduced to 0.65+/-0.08 (mean+/-SEM) mL. min(-1). g(-1) in hibernating myocardium of instrumented pigs compared with 0.98+/-0.14 mL. min(-1). g(-1) in myocardium of sham-operated pigs (P<0.05). There was a critical limitation in subendocardial flow during vasodilation to 0.78+/-0.20 mL. min(-1). g(-1) in instrumented pigs versus 3. 24+/-0.50 mL. min(-1). g(-1) in sham-operated pigs (P<0.001). Histology revealed a regional reduction in myocyte nuclear density to 995+/-100 nuclei/mm(2) in hibernating myocardium from the instrumented group versus 1534+/-65 nuclei/mm(2) in myocardium from the sham-operated group (P<0.05), regional myocyte hypertrophy (myocyte volume per nucleus, 14 183+/-2594 in the instrumented group versus 9130+/-1301 microm(3) in the sham group; P<0.05), and minimal increases in connective tissue (5.8+/-0.9% in the instrumented group versus 3.0+/-0.2% in the sham group, P<0.05). Necrosis was not identified, but apoptosis was increased from 30+/-9 myocytes per 10(6) myocyte nuclei in myocardium from the sham group to 220+/-77 myocytes per 10(6) myocyte nuclei in hibernating myocardium (P<0.05). CONCLUSIONS: These findings indicate that reversible ischemia in an area of chronically reduced coronary flow reserve induces regional myocyte loss via an apoptotic mechanism. This may contribute to the progression of chronic coronary disease to heart failure and explain the lack of complete functional recovery after revascularization in hibernating myocardium.

Regional alterations in SR Ca(2+)-ATPase, phospholamban, and HSP-70 expression in chronic hibernating myocardium.

We sought to identify mechanisms for chronic dysfunction in hibernating myocardium. Pigs were instrumented with a left anterior descending artery stenosis for 3 mo. Angiography demonstrated high-grade stenoses and hibernating myocardium with 1) severe anterior hypokinesis (P < 0.001 vs. shams), 2) reduced subendocardial perfusion [0.73 +/- 0.05 (SE) vs. 1.01 +/- 0.06 ml. min(-1). g(-1) in normal, P < 0.001], and 3) critically reduced adenosine flow (1.0 +/- 0.17 vs. 3.84 +/- 0.26 ml. min(-1). g(-1) in normal, P < 0.001). Histology did not reveal necrosis. Northern blot analysis of hibernating myocardium demonstrated regional downregulation in mRNAs for sarcoplasmic reticulum (SR) proteins phospholamban (0.76 +/- 0.08 vs. 1.07 +/- 0.06, P < 0.02) and SR Ca(2+)-ATPase (0.83 +/- 0.06 vs. 1.02 +/- 0.06, P < 0.05) with no change in calsequestrin (1.08 +/- 0.06 vs. 0.96 +/- 0.05, P = not significant). Heat shock protein (HSP)-70 mRNA was regionally induced in hibernating myocardium (2.4 +/- 0.3 vs. 1.0 +/- 0.11, P < 0.01). Directionally similar changes were confirmed by Western blot analysis of respective proteins. Our results indicate that hibernating myocardium exhibits a molecular phenotype that on a regional basis is similar to end-stage ischemic cardiomyopathy. This supports the hypothesis that SR dysfunction from reversible ischemia may be an early defect in the progression of left ventricular dysfunction.

Absence of troponin I degradation or altered sarcoplasmic reticulum uptake protein expression after reversible ischemia in swine.

The findings of troponin I (TnI) proteolysis (in isolated rat hearts) and induction of selected sarcoplasmic reticulum (SR) calcium-regulatory genes (after repetitive total coronary occlusions in swine) have given rise to the hypothesis that the time course of functional recovery of stunned myocardium reflects the resynthesis of reversibly damaged proteins. Although stunning occurs after brief total occlusions and prolonged partial occlusions (ie, short-term hibernation), the time course of functional recovery varies from a few hours to several days, suggesting that the severity of protein damage or mechanisms responsible for the dysfunction may differ. To study this, we examined SR gene expression and TnI degradation in stunned myocardium produced by 10-minute total left anterior descending coronary artery (LAD) occlusions (n=4) or 1-hour partial LAD occlusions, in which flow was reduced to approximately 50% of control values for 60 minutes (n=6) in swine. One hour after reperfusion, LAD wall thickening was severely depressed in both models despite normal perfusion and no triphenyltetrazolium chloride evidence of necrosis. Normal myocardium exhibited TnI immunoreactivity at 31 kDa and a weak secondary band at 27 kDa. Irreversible injury or calpain activation in vitro produced a marked increase in the intensity of the 27-kDa band, consistent with TnI degradation. Stunned myocardium demonstrated no change in the 31- or the 27-kDa band, and the percentage of the 27- to 31-kDa band remained constant after 10-minute total occlusions (LAD, 5.9+/-0.9%; normal, 4.9+/-1.6%) and 1-hour partial occlusions (LAD, 8.5+/-1.9%; normal, 7.3+/-1.4%) and in sham controls (LAD, 10.9+/-1.5%; normal, 9.8+/-1.4%). Northern analysis showed no alterations in TnI or SR gene expression, but the stress protein HSP-70 was variably induced. Thus, stunned myocardium occurs without TnI degradation or altered SR gene expression, indicating that additional mechanisms are responsible for the reversible dysfunction after single episodes of regional ischemia in swine.

Differential 18F-2-deoxyglucose uptake in viable dysfunctional myocardium with normal resting perfusion: evidence for chronic stunning in pigs.

BACKGROUND: Viable, chronically dysfunctional myocardium can have normal or reduced resting flow. We previously produced hibernating myocardium with reduced resting flow in pigs with a chronic stenosis and hypothesized that hibernation is preceded by chronic stunning with normal resting perfusion. METHODS AND RESULTS: Pigs instrumented with a proximal left anterior descending coronary artery (LAD) stenosis were studied 1 or 2 months later in the closed-chest anesthetized state. Stenosis severity increased from 74+/-5% at 1 month to 83+/-6% at 2 months and was accompanied by anteroapical hypokinesis (wall motion score, 2.1+/-0.1 at 1 month and 1.5+/-0.3 at 2 months; normal=3). Resting perfusion was similar in normal and dysfunctional regions, but the deposition of 18F-2-deoxyglucose (FDG) varied. At 1 month, subendocardial FDG deposition by excised tissue counting was similar in each region (0. 034+/-0.006 mL. g-1. min-1 LAD region versus 0.032+/-0.004 mL. g-1. min-1 in normal regions, P=NS). At 2 months, subendocardial FDG deposition was increased (0.084+/-0.025 mL. g-1. min-1 LAD region versus 0.042+/-0.017 mL. g-1. min-1 in normal regions, P<0.05). Increases in FDG uptake were inversely related to LAD subendocardial flow reserve during adenosine (3.5+/-0.6 at 1 month versus 1.4+/-0.2 at 2 months, P<0.01). CONCLUSIONS: These data indicate a progression of physiological adaptations in pigs with viable, chronically dysfunctional myocardium. As coronary flow reserve decreases, fasting FDG uptake increases. Flow at rest remains normal, consistent with "chronic stunning," and contrasts with reduced flow and increased FDG characteristic of hibernating myocardium in similarly instrumented pigs after 3 months. This temporal progression of adaptations supports the hypothesis of a transition from a physiological phenotype of stunning to hibernation.

18F-2-deoxyglucose deposition and regional flow in pigs with chronically dysfunctional myocardium. Evidence for transmural variations in chronic hibernating myocardium.

BACKGROUND: Hibernating myocardium in patients with collateral-dependent myocardium is characterized by relative reductions in resting flow and increases in the uptake of 18F-2-deoxyglucose (FDG) in the fasting state. We performed the present study to examine whether these key physiological alterations could be produced in a porcine model of chronic coronary occlusion and to assess whether the adaptations consistent with hibernation varied across the myocardial wall. METHODS AND RESULTS: We chronically instrumented pigs (n = 18) with a fixed occluder on the proximal left anterior descending coronary artery (LAD). Three months later, ventricular function, regional myocardial perfusion, and FDG deposition (by excised tissue counting or positron emission tomography) were assessed in pigs after an over-night fast in the closed-chest anesthetized state. Total LAD occlusion with angiographic collaterals was present in the majority of animals. Left ventriculography showed severe anterior hypokinesis, and resting perfusion was significantly reduced in the hibernating LAD region in comparison with the normal remote regions (subendocardium: 0.80 +/- 0.06 versus 1.07 +/- 0.06 mL.min-1.g-1, P < .001; full-thickness: 0.87 +/- 0.04 versus 0.99 +/- 0.06 mL.min-1.g-1, P < .01). There was a twofold increase in full-thickness fasting FDG uptake in the dysfunctional LAD region (1.8 +/- 0.2 by positron emission tomography versus 1.9 +/- 0.1 by ex vivo counting). Ex vivo tissue counting revealed a pronounced transmural variation in FDG uptake in the hibernating region (LAD/normal), which averaged 2.5 +/- 0.2 in the subendocardium, 1.9 +/- 0.2 in the midmyocardium, and 1.4 +/- 0.1 in the subepicardium. CONCLUSIONS: These results demonstrate that pigs instrumented with a proximal LAD stenosis develop hibernating myocardium characterized by relative reductions in resting function and perfusion in association with increased uptake of FDG in the fasting state. The transmural variations in relative resting flow and FDG uptake suggest that myocardial adaptations consistent with hibernation are most pronounced in the subendocardial layers and vary in relation to local coronary flow reserve.

Detection of coronary artery calcium to differentiate patients with early coronary atherosclerosis from luminally normal arteries.

Patients with angiographic evidence of early coronary atherosclerosis (<50% diameter stenosis) have a poorer prognosis than those with normal arteries and may benefit from more aggressive interventions targeted toward the primary prevention of cardiovascular disease. Using a calcium score of 5, fast computed tomography was able to identify 59% of patients with early atherosclerosis, while excluding 87% of patients with smooth, luminally normal coronary arteries.

Fast computed tomography detection of coronary calcification in the diagnosis of coronary artery disease. Comparison with angiography in patients < 50 years old.

BACKGROUND: The predominant cause of coronary artery calcification is atherosclerosis. Although fast x-ray computed tomography (CT) has been demonstrated to be a sensitive technique to detect coronary calcification, the increasing prevalence of calcification with age has been associated with a low specificity for identifying obstructive atherosclerosis. We hypothesized that the specificity of this test would be improved in a younger patient population, making it more useful in the diagnosis of coronary artery disease. METHODS AND RESULTS: We compared fast CT-detected calcification with coronary angiography in 106 patients under the age of 50 years. Nonenhanced fast CT scans consisting of 20 contiguous 3-mm tomograms of the proximal coronary arteries were obtained during a single breath hold. A positive scan was defined as 4 contiguous voxels (> or = 1 mm2) of density > 130 Hounsfield units in the region of the epicardial coronary arteries. Calcification detected by fast CT had an 85% sensitivity to predict patients with significant coronary artery disease (> or = 50% diameter stenosis), with a specificity of 45%. Although the sensitivity to detect multivessel disease was 94%, the sensitivity to detect single-vessel disease was 75%. Changing the threshold for defining a positive fast CT scan from 4 to 2 contiguous voxels produced a small improvement in sensitivity, to 88%, but reduced specificity to 36%. CONCLUSIONS: Although the specificity to detect angiographically significant coronary disease with fast CT improves in a younger patient population, it continues to be relatively low. In contrast to older patient populations, a small but significant number of patients < 50 years old with angiographically significant coronary artery disease do not have coronary calcification demonstrated by fast CT. Thus, caution should be used in excluding significant coronary artery disease on the basis of a negative fast CT study.