Author + information
- Received June 6, 2000
- Revision received September 28, 2000
- Accepted November 3, 2000
- Published online March 1, 2001.
- ↵*Reprint requests and correspondence: Professor Manuel Galiñanes, Division of Cardiac Surgery, Department of Surgery, University Hospitals Leicester, Glenfield Campus, Leicester LE3 9QP, United Kingdom
Presented at the 72nd Annual AHA Scientific Congress, November 7–10, 1999, Atlanta, Georgia.
We investigated the effects of ischemic preconditioning (PC) on diabetic and failing human myocardium and the role of mitochondrial KATPchannels on the response in these diseased tissues.
There is conflicting evidence to suggest that PC is a healthy heart phenomenon.
Right atrial appendages were obtained from seven different groups of patients: nondiabetics, diet-controlled diabetics, noninsulin-dependent diabetics (NIDD) receiving KATPchannel blockers, insulin-dependent diabetics (IDD), and patients with left ventricular ejection fraction (LVEF) >50%, LVEF between 30% and 50% and LVEF <30%. After stabilization, the muscle slices were randomized into five experimental groups (n = 6/group): 1) aerobic control—incubated in oxygenated buffer for 210 min, 2) ischemia alone—90 min ischemia followed by 120 min reoxygenation, 3) preconditioning by 5 min ischemia/5 min reoxygenation before 90 min ischemia/120 min reoxygenation, 4) diazoxide (Mito KATPopener, 0.1 mm)—for 10 min before the 90 min ischemia/120 min reoxygenation and 5) glibenclamide (10 μm)—10 min exposure prior to PC (only in the diabetic patient groups). Creatine kinase leakage into the medium (CK, U/g wet wt) and MTT reduction (OD/mg wet wt), an index of cell viability, were assessed at the end of the experiment.
Ischemia caused similar injury in both normal and diseased tissue. Preconditioning prevented the effects of ischemia in all groups except NIDD, IDD and poor cardiac function (<30%). In the diazoxide-treated groups, protection was mimicked in all groups except the NIDD and IDD groups. Interestingly, glybenclamide abolished protection in nondiabetic and diet-controlled NIDD groups and did not affect NIDD groups receiving KATPchannel blockers or IDD groups.
These results show that failure to precondition the diabetic heart is due to dysfunction of the mitochondrial KATPchannels and that the mechanism of failure in the diabetic heart lies in elements of the signal transduction pathway different from the mitochondrial KATPchannels.
Ischemic preconditioning (PC) falls within a spectrum of adaptive responses to ischemia and represents the ability of the myocardium to adapt to sublethal ischemic stress in the short term so that it is more resistant to a subsequent, potentially injurious period of ischemia. Preconditioning consists of two phases of protection: an early or first window of protection (≤2 h) and a delayed or second window of protection (≥24 h). The underlying mechanism of PC has been extensively investigated; however, the basis of such cardioprotection is not fully elucidated. The most favored hypothesis for the first window of PC suggests that a variety of endogenous ligands such as adenosine, bradykinin, catecholamines and opoids activate receptors linked to protein kinase C (PKC) to initiate an intracellular signal transduction pathway. Protein kinase C may activate a tyrosine kinase, which in turn activates mitogen-activated protein or c-Jun-N-terminal kinases (JNK) kinases leading to phosphorylation of KATPchannels (1). There is convincing evidence in the literature that KATPchannels are involved in the protection of ischemic PC in the human myocardium (2,3), although the exact place of these channels in the signal transduction pathway is still unclear (4–6). Recently we have shown that the mitochondrial, not the sarcolemmal, KATPchannel is responsible for this powerful protective mechanism in the human myocardium (7).
Within the enormous amount of research describing the cellular basis of the PC response, relatively few studies relevant to coronary artery disease in humans have focused on the effect of PC in hearts with concurrent abnormalities. More importantly, even amongst those studies, the conclusions have been conflicting. Clinical studies identify a number of conditions that increase mortality from myocardial infarction; these include heart failure, diabetes, hypertension, aging and hypercholesterolemia (8,9). It is plausible that these conditions interfere with the biochemical pathways underlying the PC response.
Cardiovascular disease associated with diabetes mellitus is a major cause of death in patients with diabetes (10). In the vast majority of animal studies, diabetic hearts demonstrate a reduced tolerance to anoxia, hypoxia or ischemia (11–13), but studies that have investigated the effect of preconditioning on diabetic hearts have yielded confusing data. Tosaki et al. (14)have shown in the streptozotocin-induced diabetic rat heart that PC does not confer cardiac protection. Their results were opposed to those of Liu et al. (15), who had earlier shown, also in the rat heart, that myocardial infarction is reduced in diabetes and that PC further increases the protection of these hearts. There are very few studies in human diabetic tissue. Cleveland et al. (16)used a functional isolated atrial trabeculae model and showed that PC did not confer any protection of the myocardium on patients taking long-term oral hypoglycemic agents. They hypothesized that long-term inhibition of KATPchannels with these agents may be responsible for the excess cardiovascular mortality associated with diabetes.
Heart failure is common in all forms of heart disease. Mechanical dysfunction of the failing heart is due to many factors, including neurohormonal disturbance, accumulation of extracellular matrix, alteration of excitation-contraction coupling and maladaptation of myocardial energetics (17). Very few studies have investigated the effects of the PC response in the failing myocardium in light of alterations in the cellular metabolic and biochemical pathways associated with heart failure. Cleveland et al. (3)showed in isolated ventricular trabeculae from patients requiring heart transplantation that PC conferred protection. However, more recently Dekker et al. (18)have studied perfused papillary muscles from rabbits in which cardiac failure has been induced by a combination of pressure and volume overloading. The end points used to assess responses to ischemia, namely the time to onset of a rise in the intracellular concentration of Ca2+(cellular uncoupling) and of ischemic contracture, were delayed by PC in normal myocardium but were exaggerated by PC in the failing myocardium.
The aims of the present study were 1) to investigate the effects of PC on the diabetic and failing human myocardium, and 2) to investigate the role of mitochondrial KATPchannels in the responses of these pathological conditions. These studies were carried out in an in vitro model of human right atrial myocardium of simulated ischemia and reoxygenation.
Materials and methods
The right atrial appendage of patients undergoing elective coronary artery surgery or aortic valve replacement was obtained. Patients were excluded if they had enlarged right atriums, atrial arrhythmias or right ventricular failure, or were taking opioid analgesia. Patient characteristics are detailed in Table 1. Local ethical committee approval was obtained for the harvesting technique, and the investigation conforms to the principles outlined in the Declaration of Helsinki. The specimens were collected in oxygenated HEPES buffered solution at 4° to 5°C and immediately sectioned and prepared for study. Briefly, the appendage was mounted onto a ground glass plate with the epicardial surface face down and was then sliced with surgical skin graft blades (Shwann-Morton, Sheffield, United Kingdom) to a thickness of between 300 and 500 μm. The specimen and the slide were kept moist throughout the procedure. Then 30 to 50 mg of muscle were transferred to conical flasks (25 ml Erlenmeyer flasks, Duran, Astell Scientific, Kent, United Kingdom) containing 10 ml of oxygenated buffered solution. Following this, the flasks were placed in a shaking water bath maintained at 37°C. The oxygenation of the incubation medium was maintained by a continuous flow of 95% O2/5% CO2gas mixture to obtain a pO2between 25 and 30 kPa and a pCO2between 6.0 and 6.5 kPa. The pO2, pCO2and pH in the incubation medium were monitored by intermittent analyses of the effluent by using an automated blood gas analyzer (Model 855 Blood Gas System, Chiron Diagnostics, Halstead, Essex, United Kingdom) and the pH was kept between 7.36 and 7.45. For the induction of simulated ischemia, the medium was bubbled with 95% N2/5% CO2(pH 6.8 to 7.0) and D-glucose was removed and the pO2was maintained at 0 kPa. In this preparation, tissue injury and viability were assessed but the atrium was not paced and force development was not measured.
The incubation medium was prepared daily with deionized distilled water and contained: NaCl (118 mmol/l), KCl (4.8 mmol/l), NaHCO3(27.2 mmol/l), KH2PO4(1 mmol/l), MgCl2(1.2 mmol/l), CaCl2(1.25 mmol/l), D-glucose (10 mmol/l) and HEPES (20 mmol/l). During simulated ischemia, the substrate D-glucose was removed and replaced with 2-deoxy glucose (10 mmol/l) to maintain a constant osmolarity. The KATPchannel blocker glibenclamide was made up to a concentration of 10 μmol in 100 ml K-H solution and the KATPopener diazoxide was dissolved in dimethylsulfoxide immediately before being added into the experimental solutions. All reagents were obtained from Sigma Chemical Co. (Poole, Dorset, United Kingdom).
After the atrium was sectioned, the preparations were allowed to stabilize for 30 min and then randomly allocated to various protocols. In all studies simulated ischemia was induced for 90 min followed by 120 min of reoxygenation. The drugs were applied to the sections for 10 min after the initial 30 min of stabilization and then removed before ischemia. Two studies were performed:
To investigate whether diabetes influences the protective effect of PC, atrial specimens were collected from four groups of patients: nondiabetics, diet-controlled diabetics (DCD), noninsulin-dependent diabetics (NIDD) on long-term oral suplhonylureas and insulin-dependent diabetics (IDD). Preparations from each group were then randomly allocated to various protocols (n = 6/group) shown in Figure 1.
To investigate the effect of cardiac function on the protection induced by PC, atrial specimens were collected from three groups of patients: with normal left ventricular ejection fraction (LVEF >50%); with moderately impaired function (LVEF 30% to 50%) and with severely impaired function (LVEF <30%). Preparations from each group were then randomly allocated to various protocols (n = 6/group) shown in Figure 2.
In both these studies, PC was induced by a single cycle of 5 min ischemia/5 min reoxygenation, a protocol that we have demonstrated provides maximal protection in this model (19). Preliminary studies (data not shown) had demonstrated that increasing the number of cycles of 5 min ischemia/5 min reoxygenation from one to three does not elicit protection beyond that obtained with a single cycle. In the groups receiving diazoxide and glibenclamide, the drugs were used at a concentration of 0.1 mm and 10 μm, respectively. We have shown that these drug concentrations are the minimum effective concentration required to elicit a response and to block PC (7).
Assessment of tissue injury and viability
At the end of each experimental protocol, tissue injury was determined by measuring the leakage of creatine kinase (CK) into the incubation medium and tissue viability by the reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) to blue formazan product.
The activity of CK leakage into the medium during the reperfusion period was assayed by a kinetic ultraviolet method based on the formation of NAD (Sigma Catalogue No. 1340-K) at 37°C and the results expressed as U/g wet wt.
At the end of the experimental time, the tissue was loaded into a Falcon conical tube (15 ml, Becton Dickinson Labware, Cowley, United Kingdom) and 2 ml of phosphate buffer solution (0.05 m) containing MTT (1.25 mg/ml, 3 mm at final concentration) was added, incubated for 30 min at 37°C and then homogenized in 2 ml dimethyl sulfoxide (Homogenizer Ultra-Turrax T25, dispersing tool G8, IKA-Labortechnic, Staufen, Germany) at 9,500 rpm for 1 min. The homogenate was then centrifuged at 1,000 gfor 10 min and 0.2 ml of the supernatant was dispensed into a 98-well flat-bottom microtiter plate (Nunc Brand Products, Roskilde, Denmark). After this, the absorbance was measured on a plate reader (Benchmark, Bio-Rad Laboratories, Hercules, California) at 550 nm and the results expressed as OD/mg wet wt.
All data are presented as mean ± SEM. All values were compared by two-way analysis of variance with application of a post hoc Bonferroni’s test. Statistical significance was assumed at the p < 0.05 level.
All samples entering the studies completed the applied experimental protocol and were included in the analysis.
Effect of PC in diabetes (study 1)
Figure 3Ashows that CK leakage was increased to a similar degree by ischemia alone in all study groups. It also shows that PC completely reversed the effect of ischemia in the nondiabetics and DCD groups, so that mean CK leakage values were similar to those in the aerobic control group, but did not have a protective effect in NIDD and IDDM groups. Interestingly, diazoxide, a specific mito KATPchannel opener, mimicked the protection afforded by PC in nondiabetics and DCD groups but failed to protect the NIDD and IDD groups. As expected, glibenclamide, a nonspecific KATPchannel blocker, abolished the protection of PC in nondiabetics and in DCD and had no effect in the NIDD and IDD groups. The MTT results shown in Figure 3Bare a mirror image of the CK leakage results with PC and diazoxide exhibiting a similar protection in the nondiabetic and DCD groups and no protection in the NIDD and IDD groups. Overall, the results suggest that changes in mito KATPchannels in patients with NIDD and IDD are the most likely cause for the failure to precondition the myocardium. However, the possibility that alterations in signal transduction pathways could also contribute cannot be completely excluded.
Effect of PC in patients with contractile cardiac dysfunction (study 2)
Figure 4Aalso shows that ischemia alone resulted in a significant increase in CK leakage similar in all study groups regardless of the severity of cardiac dysfunction. Importantly, PC was significantly protective; decrease of CK leakage was observed in the groups with LVEF ≥30% but not in the group with LVEF <30%. As observed in study 1, diazoxide mimicked the effect of PC on CK leakage in the groups with LVEF ≥30%. However, in contrast with the absence of protection by PC in the group with LVEF <30%, diazoxide reduced CK leakage in the LVEF <30% group to an extent similar to that seen in LVEF ≥30% groups. The results on MTT reduction shown in Figure 4Bwere again a mirror image of the CK leakage, suggesting that the cause for the absence of protection by PC in the LVEF <30% group is probably due to alterations in some element(s) of the signal transduction pathway that exclude the mito KATPchannels.
It should be noted that in these two studies the number of patients in each study group was too small for analysis of the possible influence of different clinical characteristics on the effects of ischemia and preconditioning.
The major findings of this study are that myocardium from patients with diabetes and poor cardiac function is not protected from ischemic PC although the injury induced by ischemia/reperfusion is not exacerbated in these conditions. Furthermore, the demonstration that activation of mito KATPchannels in myocardium from hearts with poor cardiac function mimics the protection induced by preconditioning in the myocardium from hearts with good contractility, but not from patients with diabetes, suggests that the failure to precondition in these conditions is due to alterations of different elements of the signal transduction pathway. Cardiovascular mortality is increased in patients with heart failure (20,21)and diabetes (10)and therefore any myocardial adaptation to ischemia would decrease mortality associated with these diseases. The results of our studies may have important clinical implications and a number of points warrant further discussion.
Preconditioning and diabetes
Insulin regulates the balance of energy substrates available to the heart and also regulates metabolism and myocardial perfusion via actions on various intracellular regulatory proteins and messenger systems (22). It is therefore conceivable that diabetes may affect ischemic injury. However, our results show that the diabetic human myocardium is not more sensitive to lethal ischemic injury than the nondiabetic myocardium under the conditions of the present study. Because epidemiological studies have clearly shown that patients with either IDD or NIDD are more prone to develop myocardial infarction and postinfarction complications (10,23), it may be argued that the cause of cardiac complications in diabetics is not the tolerance of the heart to ischemia. The literature is inconsistent with respect to how susceptible the hearts of diabetic animals are to injury from ischemia/reperfusion, and whereas some studies have observed a greater susceptibility to ischemia/reperfusion injury (15), others have reported no significant effect (24,25). The divergent results may be explained by experimental differences, but if our results in the human atrial myocardium can be confirmed in ventricular myocardium then our attention to reduce cardiac complications in diabetes should be centered in the context of blood components and the vasculature rather than in the own myocardium.
Our studies have demonstrated that PC affords protection of the myocardium from patients with DCD but that this is lost when patients are on long-term hypoglycemics or become insulin dependent. These results are in agreement with those reported by Cleveland et al. (16). These investigators showed that myocardium from patients with diabetes on long-term KATPblockers also does not precondition in a model of myocardial stunning. However, the same authors also suggested that the myocardium from IDD subjects can be preconditioned, although the protective effect obtained was not to the same extent as that in nondiabetics. Again, these results in the human tissue contrast with the reported experimental results. Liu and co-workers (15)were the first to examine preconditioning in experimental streptozotocin-induced diabetic rats and found that diabetic hearts were more resistant to infarction than normal control hearts and that preconditioning conferred additional protection under in vivo experimental conditions. However, a subsequent study examined the effect of streptozotocin-induced diabetes on the response to ischemia/reperfusion and preconditioning in the isolated rat heart at different stages following its induction (26)and showed that the diabetic heart is more resistant to ischemia/reperfusion in the early phase of the diabetes (two weeks after onset); but that this protection is lost by four to six weeks. In addition, they observed that the diabetic heart can be preconditioned after six to eight weeks. It must be mentioned that the disparity in the results observed between the two studies could be explained, at least in part, by the differences in the models used. Thus, for example, in the study by Liu and colleagues the model of diabetes is unique in that diabetes was induced by streptozotocin in the neonate and then animals were allowed to grow into adulthood before any intervention was carried out.
These studies do not provide an explanation for such a disparity of results; however, they suggest that abnormalities of mito KATPchannels may be responsible for the failure to precondition the myocardium of diabetics. Several investigators (27,28), including ourselves (7), have previously demonstrated that mito KATPchannels are involved in the protection of ischemic PC. The findings by Smith et al. (29)that KATPchannels are altered, with a greater outward single-channel current in the ventricular myocardium of diabetic rats, further support this thesis. Clearly, further research is needed to fully elucidate how the alteration of this channel contributes to the failure to precondition the myocardium of diabetics.
Preconditioning and the failing heart
Left ventricular hypertrophy and LV chamber dilation are among the compensatory mechanisms of the failing heart. There is experimental evidence that the hypertrophied myocardium is at greater risk from ischemia/reperfusion injury, and it is generally believed that the failing heart is less tolerant to such injury. Our results, however, have shown for the first time that the effects of ischemia/reoxygenation are similar in the failing and nonfailing myocardium. In addition, we have also demonstrated for the first time that the myocardium from hearts exhibiting a LVEF <30% cannot be preconditioned. Our results contrast with those of Cleveland et al. (3)showing that the isolated ventricular trabeculae obtained from patients undergoing cardiac transplantation can be preconditioned. Again, the explanation for this difference cannot be found in the reported experimental studies, and whereas some investigators have shown protection of the failing heart by PC (3), others have observed no effect (30)or even further tissue damage (18). The diversity of results is not entirely surprising because of the lack of uniformity of experimental design and the degree of heart failure we have shown in this study.
The protection observed with diazoxide in the myocardium from hearts with LVEF <30% was commensurate with the protection induced by PC in the myocardium from nonfailing hearts. This supports the thesis that the failure to precondition the failing human heart is not due to an alteration in the response of the mitochondrial KATPchannel but is caused by abnormalities in other elements of the preconditioning signaling pathway. Considerable evidence indicates that PKC is intimately involved in ischemic PC (31), and the failure to precondition the failing heart may be due to the chronic activation of PKC observed in this condition (32). There are several PKC isoforms, some of which have been involved with PC, and in future studies the type of isoforms and their expression in the myocardium of the failing heart should be investigated. Indeed, if specific PKC isoforms are proved to be responsible for the failure to precondition, then their manipulation could become a therapeutic intervention to reduce myocardial injury in ischemia/reperfusion of the failing heart.
Possible limitations of the study and clinical implications
A potential limitation of our study was the use of atrial tissue as opposed to ventricular myocardium, and therefore any extrapolation must be conducted with caution; however, Yellon and colleagues have suggested that PC exerts identical protection in atrial and ventricular myocardium (2). The present study also used atrial tissue to characterize the effects of ischemia and reperfusion in the failing and diabetic human myocardium. However, atrial and ventricular myocardium possess characteristics of their own that may influence susceptibility to ischemia/reperfusion injury, and as a consequence results from one may not be applicable to the other. Thus the reported differences in the distribution of potassium channels (33,34), which contribute to the characteristic differences between atrial and ventricular action potentials, may determine a different response to ischemia/reperfusion. Undoubtedly, KATPchannels are present in both atrium and ventricle (33), although their density in both tissues is unknown. It must also be mentioned that the preparation is superfused (“simulated ischemia”) as opposed to being arterially perfused, and simulated ischemia is achieved by removal of oxygen and blocking glycolytic ATP production with 2-deoxyglucose. This results in metabolic conditions within the myocardium that may be different from those that occur in the myocardium during clinical ischemia.
Another limitation might be that right atrial appendages were obtained from patients subjected to various medical treatments (e.g., nitrates, beta-blockers, calcium antagonists), which may have influenced ischemia/reperfusion injury and the protection induced by PC. However, it should be emphasized that all medication was stopped the day before surgery when specimens were taken for the study, and that significant effect of the medication was unlikely because all preparations responded to ischemia/reperfusion with a similar degree of injury. The preparation used in this study was not electrically stimulated (i.e., nonbeating) and therefore one should be cautious when extrapolating to the in vivo situation.
Preconditioning is a potent protective intervention whose use has been advocated in clinical situations such as angioplasty and cardiac surgery. The results of our studies have obvious clinical implications in that PC cannot be beneficial to patients with NIDD or IDD and those with cardiac failure. The results also show that in the failing heart a degree of protection similar to that seen with PC can be obtained by the administration of a selective opener of mito KATPchannels, an intervention that is not effective in diabetics.
We thank Professor David Jones for his help with statistical analysis of the data.
☆ This study was supported in part by a grant from the University of Leicester.
- creatine kinase
- diet-controlled diabetes
- insulin-dependent diabetes
- ATP-dependent potassium channels
- left ventricular ejection fraction
- 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide
- noninsulin-dependent diabetes
- protein kinase C
- Received June 6, 2000.
- Revision received September 28, 2000.
- Accepted November 3, 2000.
- American College of Cardiology
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