Ischemia in the heart produces rapid deterioration of contractions. Depletion of high-energy phosphates (HEP) plays a central role in the reduced contractile performance of ischemia (1,2). Conflicting results have been reported regarding the sensitivity of the diabetic (DM) heart to ischemic insult. The streptozotocin (STZ)-induced DM heart is reportedly resistant to ischemia/reperfusion injury (3–6), although the precise mechanisms remain unclear. Diabetic animal models show widespread abnormalities of energy metabolism in several types of tissue, including cardiac myocardium and its subcellular organelles (7). Contrary to the reduced HEP level during ischemia, the level is increased during reperfusion. This increase in HEP level may explain the functional recovery of cardiac performance.
These findings raise an assumption that preservation of HEP during ischemia and reperfusion in the STZ-DM heart may be associated with cardioprotection against an ischemic insult. STZ-DM animals are deficient in insulin, which is indispensable for myocardial glucose uptake. Addition of insulin to the perfusate reportedly improved functional recovery during reperfusion after 15 min of no-flow ischemia (8). In contrast, adverse effects of insulin have also been reported (9,10). Furthermore, there have been no reports that described the effects of insulin perfusion on ischemic insult using STZ-DM hearts. The myocardial metabolism of HEP under these conditions also remains to be studied. The pathogenesis of STZ-DM rats may be comparable to that of type 1 diabetic patients, who need to be treated with insulin. Diabetic myocardium is known to have an increased level of glycogen, and insulin also increases the myocardial glycogen content (7). The preischemic content of myocardial glycogen is crucial for the myocardial damage caused by ischemic insult (9–11). Therefore, it is interesting to investigate the effects of insulin perfusion on ischemia/reperfusion injury in STZ-DM heart in comparison with normal heart from the point of clinical view.
The present study assessed cardiac performance in the presence or absence of insulin during reperfusion after low-flow ischemia, using isolated perfused STZ-DM and age-matched control hearts. Myocardial concentrations of adenine nucleotides, creatine phosphate (CP) and glycogen at baseline, at the end of ischemia, and at the end of reperfusion were measured for evaluation of cardiac energy metabolism.
METHODS
Animals
We used mature male Wistar King A rats weighing 250 to 300 g. All animals were housed in a room illuminated daily with a 12:12 h light/dark cycle, with the temperature maintained at 21 ± 1°C, and humidity at 55 ± 5%. STZ (Sigma Co. Ltd., St. Louis, MO, USA) was freshly prepared on the infusion day by dissolving STZ in sterile sodium citrate buffer solution at a final concentration of 0.05 M (pH 4.5). Rats were made diabetic by a bolus injection of STZ (65 mg/kg) into the tail vein (12) under light ether anesthesia. The same volume of buffer without STZ was injected in age-matched controls. At the time of heart isolation 4 weeks after the injection, STZ rats whose plasma glucose concentration was >22.0 mM in a fed state were defined as diabetic. All studies were conducted in accordance with the Oita Medical University Guidelines based on the NIH Guide for the Care and Use of Laboratory Animals.
Isolated heart preparation
Four weeks after injection, rats were heparinized intraperitoneally (i.p.) at a dose of 500 IU/kg and anesthetized with pentobarbital (50 mg/kg, i.p.). Body weight was measured, and a blood sample was taken for plasma glucose and insulin concentrations. The heart was rapidly removed and placed in ice-cold Krebs-Henseleit buffer until cessation of contraction. The aorta was then cannulated and the coronary vasculature perfused in the Langendorff mode at a constant pressure of 70 mmHg. The perfusion medium was Krebs-Henseleit buffer (pH 7.4, in mM 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, 11.0 glucose). The perfusion buffer was bubbled with a 95% O2 and 5% CO2 gas mixture at 36.5°C. Following initiation of Langendorff perfusion, the left atrium was removed and a water-filled latex balloon was cannulated through the mitral orifice into the left ventricle. The heart was then equilibrated for 10 min, followed by the left ventricular end-diastolic pressure (LVEDP) adjusted to 0 to 5 mmHg. The balloon volume was maintained throughout the experiment. LV pressure, coronary perfusion pressure (CPP), and electrocardiogram (ECG) were simultaneously recorded on a polygraph recorder (WS-681G, Nihon Kohden, Tokyo, Japan) and stored in a PCM data recorder (RD-111T, TEAC, Tokyo, Japan) for later analysis.
Animal grouping
STZ-DM and control hearts were perfused with Krebs-Henseleit buffer in the absence (DM-BUF and CON-BUF groups, respectively) or presence of 1 U/L insulin (DM-INS and CON-INS groups, respectively). Cardiac function and released creatine kinase (CK) during the ischemic and reperfusion periods were evaluated in 8 rats in each of the 4 groups. Adenine nucleotides, CP and glycogen were assessed at baseline, after 30 min of ischemia, and after 30 min of reperfusion. Activity of ecto-5´-nucleotidase was assessed in ventricular tissues from the CON-BUF (n = 7) and DM-BUF groups (n = 10) after 30 min of low-flow ischemia.
Ischemia-reperfusion paradigm
During the initial 10 min of constant pressure perfusion, the perfusion flow rate was determined for each heart. The heart was then perfused at a determined perfusion rate using a microtube pump (MP-3, Tokyo-Rikakikai, Tokyo, Japan). During global ischemia, the suspended heart was covered with thermostatic glassware containing 90 to 100% stable vapor to prevent dryness. Normothermic low-flow global ischemia was initiated after a 30 min baseline recording by reducing the perfusion rate to 5% of the baseline flow. In insulin-perfused hearts, insulin perfusion was started 30 min before the initiation of global ischemia and maintained throughout the perfusion period. Reperfusion was carried out for 30 min at the same perfusion rate used during the baseline period.
Hemodynamics and released CK
LV pressure was monitored through a latex balloon attached to a pressure transducer (AP-601G, Nihon Kohden, Tokyo, Japan). LV developed pressure (LVDP) was defined as the difference between the LV systolic and diastolic pressure. Cardiac performance was assessed using the rate pressure product (RPP) calculated as follows: RPP = LVDP x heart rate (HR) (mmHg/min). CPP was monitored as hydraulic pressure measured at the level of aortic cannulation. LVDP, CPP, and ECG were analyzed using a MacLab 8s (AD Instruments, Milford, MA, USA) and a Power Macintosh G4 computer (Apple Computer, Cupertino, CA, USA). The coronary effluent was collected during the 30 min of low-flow ischemia and the initial 1 min after introduction of reperfusion for measurement of CK content (released CK). At the end of the reperfusion period, the wet ventricular weight of each heart was measured. The released CK was expressed as the ratio of CK content to wet ventricular weight.
Myocardial adenine nucleotides and glycogen
The concentrations of adenine nucleotides, CP and glycogen were measured according to the method previously described (13,14). At each time point, 4 animals from each group had isolated hearts prechilled in liquid nitrogen and freeze-clamped with aluminum plates. The hearts were then kept in liquid nitrogen and stored at -80°C until assayed. Frozen tissue was homogenized in 0.6 N ice-cold perchloric acid and centrifuged at 2,000 g for 10 min at 4°C. The supernatant was neutralized with KOH between pH 5.0 and 7.0. The extracts were centrifuged for 10 min after the neutralization to remove the KClO4, and the supernatants were used for the assays. The myocardial contents of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and CP were measured by high performance liquid chromatography (HPLC) (LC-9A liquid chromatograph, Shimadzu, Kyoto, Japan) with a column of STR ODS-M (Shimadzu, Kyoto, Japan). The values are expressed as μmol/g frozen tissue weight. For myocardial glycogen measurement, the homogenate was incubated for 2 h at 40°C with amyloglucosidase to hydrolyze the glycogen. The resulting glucose residue was then measured using an NADP-linked spectrophotometric method using hexokinase and glucose-6-phosphate dehydrogenase (14). Glycogen concentrations were expressed as mg glycogen/g tissue wet weight. Activity of ecto-5´-nucleotidase was assessed as previously described (15). Ventricular tissues were freeze-clamped at the end of 30 min of low-flow ischemia and homogenized for 30 s in 3 ml ice-cold 10 mM HEPES-KOH buffer (pH 7.4, in mM 250 sucrose, 1.0 MgCl2 and 1.0 mercaptoethanol). To prepare a membrane fraction, the homogenate was centrifuged at 1000 g for 10 min at 4°C. The resultant pellet was resuspended in HEPES-KOH buffer and divided into aliquots. The activity of ecto-5´-nucleotidase was determined by an enzymatic assay technique using a commercially available kit (Sigma Chemical Co., St. Louis, MO, USA). Concentrations of protein in each sample were measured by the method described by Bradford (16).
Statistics
Data are expressed as the mean ± SEM. Statistical evaluations between groups for serial changes in the hemodynamic parameters, and for myocardial adenine nucleotides, CP, and glycogen concentrations were carried out by two-way analysis of variance (ANOVA) followed by the Bonferroni/Dunn test. Comparisons between groups with respect to body weight, ventricular weight, the ratio of ventricle to body weight, plasma glucose and insulin concentrations, and the ratio of released CK to ventricular weight were analyzed by one-way ANOVA followed by the Bonferroni/Dunn test. Analysis of myocardial adenine nucleotides, CP and glycogen concentrations in each period were also based on one-way ANOVA followed by the Bonferroni/Dunn test. A p value < 0.05 was defined as significant.
RESULTS
Basic characteristics of animals
The basic characteristics of DM-BUF and DM-INS groups and their age-matched CON-BUF and CON-INS groups are summarized in Table 1. Both body weight and ventricular wet weight were lower in both DM groups than in their appropriate controls (p < 0.01 for each). The ratio of ventricular wet weight to body weight was, however, higher in both DM groups than in their appropriate controls (p < 0.01 for each). Significantly elevated glucose and lowered insulin concentrations in plasma were shown in both DM groups, confirming that STZ was sufficient to cause rats to be diabetic.
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TABLE 1: Baseline characteristics of experimental groups
Cardiac performance during ischemia/reperfusion
Figure 1 shows the serial changes in RPP, LVDP, HR, and CPP at baseline, and during low-flow ischemia and reperfusion in the CON-BUF, CON-INS, DM-BUF, and DM-INS groups. At baseline, RPPs in the DM-BUF and DM-INS groups were lower than those in the corresponding controls. Insulin per se exerted no significant effect on RPP. Throughout the low-flow ischemia, RPPs decreased to near zero in all 4 groups. With reperfusion, however, RPP in each group showed progressive recovery. Recovery of RPP was greater in the DM-BUF group than in the CON-BUF group (p < 0.05). Insulin perfusion promoted the recovery of RPP in both the diabetic and control groups (p < 0.05 and p < 0.01, respectively). This depended completely on the lack of change in HR. CPPs in all 4 groups did not differ significantly throughout the experiment.
FIG. 1.:
Serial changes in rate-pressure product (RPP), left ventricular developed pressure (LVDP), heart rate (HR), and coronary perfusion pressure (CPP) in the streptozotocin (STZ)-induced diabetic heart, and the effects of insulin perfusion on these parameters of cardiac function. In this and succeeding figures, CON-BUF is the STZ-free control group without insulin. CON-INS is the STZ-free control group with insulin. DM-BUF is the STZ-treated diabetic group without insulin. DM-INS is the STZ-treated diabetic group with insulin. *p < 0.05, **p < 0.01 vs corresponding non-DM control groups. †p < 0.05, ††p < 0.01 vs corresponding non-insulin perfused groups.
Released CK
Figure 2 illustrates the ratio of released CK to ventricular weight throughout low-flow ischemia and the initial 1 min into the reperfusion period. During ischemia, STZ-induced DM (DM-BUF group) lowered the ratio compared with the CON-BUF group (p < 0.01). Addition of insulin to the perfusate in controls (CON-INS) also reduced the ratio compared with the CON-BUF group (p < 0.05). The reduction of the ratio in the DM-INS group compared with DM-BUF group did not reach statistical significance. STZ-induced DM (DM-BUF group) also reduced the ratio during the initial 1 min of reperfusion when compared with CON-BUF group (p < 0.05). A reduced ratio was similarly noted in the comparison between the CON-INS and DM-INS groups (p < 0.05).
FIG. 2.:
Changes in the ratio of released creatine kinase (CK) to ventricular weight in the diabetic heart and the effects of insulin perfusion during the low-flow ischemic period (A) and during the initial 1 min of reperfusion (B). *p < 0.01 vs the corresponding non-DM control groups. †p < 0.05 vs the corresponding non-insulin perfused groups.
Adenine nucleotides, CP, and ecto-5´-nucleotidase
Figure 3 shows myocardial concentrations of adenine nucleotides and CP at baseline, and at the end of both low-flow ischemia and reperfusion. ATP concentrations in CON-BUF, CON-INS, and DM-INS groups decreased from the baseline period to the end of the ischemic period (p < 0.01 for each). The reduced values recovered at the end of reperfusion in the DM-BUF and DM-INS groups. At the end of reperfusion, insulin increased ATP concentrations in the DM-INS group compared with the DM-BUF group (p < 0.05). ADP was not affected by STZ-induced DM or insulin throughout the experimental period.
FIG. 3.:
Myocardial concentrations of adenine nucleotides and creatine phosphate (CP) in the diabetic and control heart and the effects of insulin perfusion at baseline, end of ischemia, and end of reperfusion. ATP, adenosine triphosphate. ADP, adenosine diphosphate. AMP, adenosine monophosphate. The differences between time points and groups were analyzed. *p < 0.05, **p < 0.01 vs baseline period in corresponding non-DM control groups. †p < 0.05, ††p < 0.01 vs ischemic period. #p < 0.05, ##p < 0.01 vs the corresponding non-diabetic control groups. ¶p < 0.05, ¶¶p < 0.01 vs the non-insulin perfused groups.
In contrast to ATP, myocardial AMP concentrations in both DM and control groups increased following low-flow ischemia compared with baseline, and this reached statistical significance in the CON-INS group (p < 0.01). The elevation of AMP was suppressed in DM-BUF and DM-INS groups compared with their controls (p < 0.05 and p < 0.01, respectively). At the end of reperfusion, AMP in the DM and controls was lower than in the ischemic period (p < 0.05 in CON-BUF and DM-INS groups, p < 0.01 in CON-INS group). AMP concentrations returned to baseline level and showed no significant differences among the 4 groups in the reperfusion period. To exclude the possibility that the decrease in AMP may be caused by activation of ecto-5´-nucleotidase, enzyme activities in the CON-BUF and the DM-BUF groups in ventricular tissue was measured at the end of ischemia. The activity was not significantly different between these 2 groups (5.3 ± 1.2 vs. 5.6 ± 1.2 U/L, respectively).
Myocardial CP concentrations at baseline were increased by insulin perfusion in both control and DM hearts (p < 0.05 and p < 0.01, respectively). Akin to changes in ATP, the CP concentrations in all 4 groups (except for DM-BUF) were lower at the end of ischemia (p < 0.01) and then increased at the end of reperfusion in all 4 groups (p < 0.01). Myocardial CP was increased by STZ-DM or insulin in the reperfusion period, as shown in comparison between the CON-BUF and DM-BUF groups (p < 0.05), between the CON-INS and DM-INS groups (p < 0.01), between the CON-BUF and CON-INS groups (p < 0.05), and between the DM-BUF and the DM-INS groups (p < 0.01).
Glycogen
At baseline, the myocardial glycogen in DM-BUF group tended to be higher than that in CON-BUF group. However, the difference did not reach statistical significance. Myocardial glycogen at baseline was increased by insulin perfusion in both control and DM hearts (p < 0.01 for each). Glycogen was decreased in all 4 groups (p < 0.01) at the end of ischemia with no significant difference among the 4 groups. At the reperfusion period, glycogen levels were slightly increased in all 4 groups compared with those at the end of ischemia, which did not reach statistical significance.
DISCUSSION
The core findings in the current study are as follows: DM rat hearts showed greater functional recovery than their age-matched controls during reperfusion after low-flow ischemia. The DM rat hearts also showed higher level of myocardial CP concentration than controls at reperfusion period. Insulin perfusion resulted in an increase of myocardial glycogen at baseline and myocardial CP concentration at the reperfusion period.
HEP metabolism in DM hearts
In a previous study that investigated HEP metabolism in the STZ -DM heart, CP concentrations in the DM heart were not significantly affected either at baseline or at the end of reperfusion (6). Functional recovery of contractile performance at the reperfusion period did not differ significantly between groups. Once ischemic preconditioning was produced, however, the myocardial damage assessed by released CK was lower in the DM heart than in controls (6). When compared with the study (6), the cardioprotective effects of STZ-induced DM state appear more remarkable in our study. One explanation for the discrepancy is the experimental protocol, particularly ischemic severity. Perfusion flow was totally blocked during ischemia (6), while low perfusion flow was permitted in the current study. In another report (3), Tani and Neely demonstrated that the resistance to ischemia in STZ or alloxan-induced diabetic hearts was not related to higher tissue levels of HEP during reperfusion but was associated with a reduced Ca2+ uptake. However, the authors carried out no-flow ischemia for 30–60 min. Under such a severe ischemic condition, a crucial intracellular Ca2+ overload must occur. It can be postulated that low-flow ischemia in the current study did not cause such a severe intracellular Ca2+ overload. Therefore, higher tissue levels of HEP during reperfusion may play a more predominant role for functional recovery. In alloxan-DM rats, Allison et al. (17) reported that basal myocardial CP and ATP were reduced by 58 and 45%, respectively, compared with controls, whereas those were similar between STZ-DM and control rats in the current study. The marked reduction in oxidative HEP production in the alloxan-DM hearts in the report (17) can be explained by the more progressive and severe diabetic state, i.e., the plasma glucose concentration of alloxan-DM rats was about 7 times higher than that of controls (17), whereas the difference between our 2 groups was about 3 times. In fact, insulin administration normalized myocardial HEP production in alloxan-DM hearts (17).
In the current study, the increase in AMP during ischemic period was suppressed in DM group compared with control group, despite the lack of difference in myocardial ATP content. The activity of ecto-5´-nucleotidase was not significantly different between the groups. Although the mechanism of suppression of increase in AMP in DM heart during ischemia is not clear, our results indicate that activation of this enzyme and subsequent increase in adenosine are not involved in cardioprotection in STZ-induced diabetic heart.
Cardioprotective effects of insulin
Insulin translocates glucose transporter 4 (GLUT 4) from the intracellular compartment to the plasma membrane, which subsequently increases myocardial glucose uptake (18,19). In the current study, insulin increased baseline CP concentrations in both the control and DM groups, but these increases were markedly reduced compared with the corresponding control levels at the end of ischemia. At the end of the reperfusion period, however, insulin augmented the increase in CP concentrations more in the DM groups over baseline values. Although the mechanism for this overshoot of CP concentration in diabetic heart remains unclear, it was documented that insulin and ischemia had additive effects of on myocardial GLUT1 and GLUT 4 translocation in vivo (19). In insulin-deficient STZ-DM heart, the glucose uptake through GLUT4 is suppressed (20). Therefore, the effects of insulin and ischemia on activation of GLUT4 and subsequent increase in glucose uptake may well be more remarkable in STZ-DM hearts at reperfusion.
With respect to the effects of insulin on ischemia reperfusion injury, addition of insulin to the perfusate was shown to improve functional recovery at the reperfusion period following no-flow ischemia using isolated hearts from the normal rats (8). The authors concluded that an early increase in myocardial glucose uptake accelerated by insulin at reperfusion was critical for cardioprotection. In the current study, insulin perfusion caused increase in preischemic content of myocardial glycogen in both DM and control hearts. Recently, Cross et al. (11) reported that preischemic higher glycogen content is associated with cardioprotection if glycogen may not be fully depleted at the end of ischemia, because glycolysis can be maintained and the detrimental effects of concomitant proton production can be avoided. Consistently, glycogen was not fully depleted at the end of ischemia as shown in Figure 4 in the current study. Thus, the insulin-induced increase of glycogen content at preischemic period may be associated with cardioprotection, although the relationship between the preischemic high glycogen content and the high CP concentration at reperfusion period remains to be studied.
FIG. 4.:
Myocardial glycogen concentrations in the diabetic and control heart and the effects of insulin perfusion at baseline, end of ischemia, and end of reperfusion. The differences between time points and groups were analyzed. *p < 0.05, **p < 0.01 vs preischemic period. ¶¶p < 0.01 vs the non-insulin perfused groups.
The low-flow ischemia as applied in the present study may promote cardioprotection for the following reasons. First, insulin-induced myocardial glucose uptake which accelerates HEP synthesis is preserved during ischemia. Second, accumulation of metabolites of glycogenolysis can be dissipated by blood flow. The beneficial effects of insulin under low-flow ischemia are in agreement with previous reports using a canine model (21,22). Intravenous and intracoronary insulin infusion markedly increase regional glucose uptake and improve contractile dysfunction, leaving myocardial oxygen consumption unaffected even with a reduction in coronary perfusion pressure (21). Using the same open-chest canine model, another study showed that insulin did not increase ATP or CP in the myocardium with reduced coronary perfusion pressure (22). These observations were attributable to increased myocardial energy demand, which leads to activation of enzymes such as sarcoplasmic reticulum Ca2+ ATPase. Consequently, this mechanism offsets the net increase in glycolytic ATP and CP production. It is most likely that the cardioprotective effects of insulin are mainly caused by lesser myocardial damage due to low-flow ischemia that enables a reserve of insulin-induced glucose uptake to prevent accumulation of glycogenolysis-produced metabolites. Subsequently, production of HEP is enhanced during reperfusion.
Cardioprotective mechanisms of STZ-DM hearts
It remains unclear what mechanisms are involved in the effects of a DM state on cardioprotection. Reduced activities of the Na+/H+ pump and Na+/Ca2+ exchanger were demonstrated in STZ-DM hearts (23–25), indicating less accumulation of Na+ and Ca2+ in the myocardium during ischemia and reperfusion. Indeed, lowered activities of the Na+/H+ pump and Na+/Ca2+ exchanger were found to promote cardioprotection against an ischemic insult in STZ-DM hearts (26). Inhibition of Ca2+ overload may lessen myocardial damage with ischemia/reperfusion and increase net HEP resynthesis at reperfusion.
Activity of protein kinase C (PKC) is increased in STZ-DM hearts (27,28). The mitochondrial ATP-sensitive potassium channel (mitoKATP channel) is downstream from PKC (29,30). There is evidence that the mitoKATP channel plays an important role in cardioprotection against ischemia and reperfusion injury (31). Activation of the mitoKATP channel reduces Ca2+ overload and increases ATP synthesis (32).
Implications
The present study demonstrated beneficial effects of insulin on ischemic insult in the STZ-DM hearts. In the long-term results from Diabetes and Insulin-Glucose Infusion in Acute Myocardial Infarction (DIGAMI) study (33), intensive insulin treatment reduced long-term mortality despite high admission blood glucose and hemoglobin A1c. Our observations may explain, at least in a part, the benefits of insulin infusion recognized in DIGAMI study.
Limitations
There are several limitations to the current study. First, cardiac performance at the baseline period assessed by RPP was significantly lower in DM hearts than in controls. Lowered RPP may permit DM hearts to require less energy and promote cardioprotective effects. Second, glucose was exclusively added to the perfusate as an energy substrate. Under physiologic conditions, however, free fatty acid is a more important energy substrate for myocardial energy metabolism, particularly in the myocardium (7). Third, we measured total myocardial HEP and discussed its role in cardiac performance. According to the compartmentation theory of ATP (34), however, there need be no change in total ATP, whereas the ATP available for transport across the sarcolemma may be crucial. Finally, the STZ-DM is an animal model of type 1 DM with a deficit in insulin secretion. However, patients with type 2 DM make up more than 90% of all DM patients. Further studies will be needed to examine whether an animal model of type 2 DM exhibits similar cardioprotective.
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