© 1997 by European Society of Cardiology
Copyright © 1997, European Society of Cardiology
Acceleration of stiffness in underperfused diabetic rat hearts by glyburide, a KATP channel blocker, and its prevention by levcromakalim and insulin
aDepartment of Pharmacology, School of Medicine, Faculty of Medicine, University of the Ryukyus, 207 Nishihara, Okinawa 903-01, Japan
bResearch Laboratory Center, Faculty of Medicine, University of the Ryukyus, 207 Nishihara, Okinawa 903-01, Japan
* Corresponding author. Tel.: +81 (98) 895-3331, ext. 2276; fax: +81 (98) 895-3293; e-mail: tomo@med.u-ryukyu.ac.jp
Received 7 October 1996; accepted 21 April 1997
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Abstract |
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Object: To clarify the role of the KATP channels in myocardial dysfunction during underperfusion with norepinephrine (NE) in the diabetic heart, particularly the heart treated with sulphonylurea derivatives. Methods: Isolated 6-week streptozotocin-diabetic rat hearts with a balloon in the left ventricle (LV) were paced and perfused with normoxic Krebs-Henseleit solution. Agents were infused for 15–25 min before as well as during 60-min underperfusion (2 ml/min/g heart weight) with 10–6 M NE. Regional myocardial flow distribution was measured using dye microspheres. The effects of ex vivo glyburide (10–6 M, a sulphonylurea anti-diabetic drug and a specific KATP channel inhibitor) on contractile dysfunction and abnormal regional myocardial energy metabolism were examined during underperfusion with NE in the absence or presence of levcromakalim (10–4 M, a selective K+ channel opener) and insulin (2 mU/min/g heart weight). Results: The flow rate was greater in the LV subendocardium than the subepicardium during normal perfusion, and smaller at 60-min underperfusion with NE. The LV diastolic tension and pressure during underperfusion with NE increased more rapidly in the presence of glyburide. At 60-min underperfusion with NE, the diastolic pressure elevation was still higher in the glyburide-treated heart, and decreases in tissue ATP, creatine phosphate (CP), energy charge, phosphorylation potential and CP/inorganic phosphate (Pi) ratio, and increases in AMP, Pi and lactate were more marked in the glyburide-treated heart, particularly in the LV subendocardium. Thus, ex vivo glyburide enhanced the increase in LV stiffness and abnormal myocardial energy metabolism during underperfusion with NE in diabetic hearts. These changes were reduced by levcromakalim to the level during underperfusion with NE without glyburide. Insulin did not prevent the glyburide-induced earlier exacerbation of the increase in LV stiffness during underperfusion with NE, but reduced the detrimental effects 20 min after the onset of underperfusion. Conclusions: KATP channels in the diabetic myocardium probably open during underperfusion with NE, and it helps delay the initiation of the increase in cardiac stiffness. Glyburide may have harmful effects in the ischemic diabetic heart; the myocardial KATP channel blockade during underperfusion with NE enhanced the increase in LV stiffness and abnormal myocardial energy metabolism. The glyburide-induced detrimental effects in the ischemic diabetic heart are prevented by levcromakalim and partly by insulin.
KEYWORDS Rat, heart; Diabetes mellitus; Norepinephrine; Coronary flow; Energy metabolism; Potassium channel, ATP sensitive; Potassium channel opener; Sulfonylureas; Glibenclamide; Levcromakalim; Insulin
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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In 1983, Noma [1]inferred that the ATP-dependent K+ channels (KATP channels) open by a fall in the cytosolic ATP concentration and play a cardioprotective role during myocardial ischemia or hypoxia in non-diabetic hearts [2–5]. Thus, blocking the KATP channels may be detrimental to the ischemic myocardium. In 1970, patients with diabetes mellitus (DM) treated with the oral hypoglycemic drug, tolbutamide, were reported to have a higher mortality than those without treatment [6]. Tolbutamide, a sulphonylurea derivative, blocks the KATP channels in the beta-cells of the pancreas [7]and the myocardial cells [1, 8]. The detrimental effects may be partly caused by the dysfunction of the KATP channel in the myocardium [9].
Diabetics have an increased incidence of congestive heart failure despite smaller infarct areas following ischemia than non-diabetics [10]; during exercise the same is true even in young men with no evidence of cardiovascular disease [11]. In our previous studies in isolated perfused rat hearts [12–15], we showed the diabetic heart to be more susceptible than the non-diabetic heart to flow reduction and to exhibit contractile dysfunctions and abnormal energy metabolism of the left ventricular (LV) wall readily; norepinephrine (NE) exacerbated this effect, particularly in the diabetic heart [12, 13, 15]. Diabetic hearts were more susceptible to a decrease of total ATP in the tissue during underperfusion with NE; the increase in LV stiffness correlated closely with ATP depletion in the subendocardium [13], and in vivo [12]and ex vivo [14]insulin prevented the injury.
Therefore, it is important to clarify the participation of the KATP channels in cardiac dysfunction during underperfusion in the diabetic heart, particularly the heart treated with sulphonylurea derivatives. In the present study, we examined the effects of glyburide (glibenclamide, a sulphonylurea anti-diabetic drug and a specific KATP channel inhibitor) [16]on the dysfunction during underperfusion with NE in isolated 6-week streptozotocin-induced diabetic rat hearts. We focused on the increase in LV diastolic stiffness, changes in regional myocardial flow distribution and regional abnormal myocardial energy metabolism. We also investigated whether levcromakalim (a selective K+ channel and a KATP channel opener) [17–19]prevents the effects of glyburide. The beneficial metabolic effects of sulphonylurea derivatives in non-insulin-dependent DM are considered to be based on an augmented release of insulin from the beta-cells of the pancreas [20]. Therefore, we examined the effects of glyburide in the absence and presence of ex vivo insulin.
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2 Methods |
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2.1 Animals and treatment
Diabetes mellitus (DM) was induced by a single intravenous (i.v.) injection of 50 mg/kg streptozotocin (Sigma) in 8-week-old male Sprague-Dawley rats, each weighing 250±3 g (n = 42). The diabetic state was assessed by confirming that blood glucose in the evening was >300 mg/dl with the Glucoboy set (E-PG30, Eikenkagaku) 3 days after the injection of streptozotocin. The rats were housed in groups of 2–4 to a cage and received food and water ad libitum and were killed 44±0 days after the injection. The investigation conforms with the principles outlined in the Declaration of Helsinki.
2.2 Heart perfusion and measurement of mechanical performance
As described previously [12–15], the animals were anesthetized with ether and venous blood samples were obtained for measurement of plasma glucose by an enzymatic method with Glu neo (Shinotest).
After thoracotomy, the hearts were rapidly excised and perfused with a Langendorff apparatus. The perfusate was Krebs-Henseleit bicarbonate buffer solution (pH 7.4, 36°C) containing (in mM): glucose 11, NaCl 120, KCl 4.8, CaCl2 1.25, MgSO4 1.2, KH2PO4 1.2 and NaHCO3 25, gassed with 95% O2/5% CO2. Mean coronary perfusion pressure (CPP) was measured through a side tube of the cannula inserted in the aorta. Heart rate (HR) was measured with a cardiotachometer (AT-601G, Nihon Kohden). Coronary flow rate (CFR) was measured by collecting drops of venous effluent from the heart and was gradually increased by a microtube pump (Minipuls 2, Gilson) to provide a CPP of about 55 mmHg. The hearts were then paced at 300 beats/min by an electronic stimulator (SEN-3301, Nihon Kohden) throughout the experiments.
To detect changes in the contractile responses of the left ventricle, the isometric tension along the longitudinal direction of the whole heart [as contractile force (CF)] and the isovolumic pressure [as left ventricular pressure (LVP)] were monitored simultaneously. To monitor CF, a force-displacement transducer (TB-611T and AP-621G, Nihon Kohden) was attached by a thread with a hook to the ventricular apex. To monitor LVP, a fluid-filled balloon connected to a pressure transducer (TP-101T and AP-620G, Nihon Kohden) was placed in the left ventricle. Diastolic force (resting tension) in the CF and the LV diastolic pressure were expressed in terms of resting CF and resting LVP, respectively. The resting CF and resting LVP during the normal perfusion before the infusion of agents were almost adjusted to 1 g and 0 mmHg, respectively, and differences (
g and mmHg) from the normal perfusion levels just before underperfusion were measured to detect changes in LV stiffness. CF and LVP developed from the diastolic to the systolic tension and pressure were measured as developed CF and developed LVP, respectively; the ±dF/dt and the ±dP/dt were derived with differentiators (ED-601G, Nihon Kohden), respectively.
2.3 Perfusion protocol
Fig. 1 shows the perfusion protocol. The paced diabetic hearts were divided into two groups—normal perfusion and underperfusion with norepinephrine (NE). In the normal perfusion group (n = 7), the hearts were perfused at a flow rate adjusted to provide a CPP of about 55 mmHg (normal perfusion) for 90 min. In the underperfusion with NE group, after 30-min normal perfusion (CFR was 6.30±0.11 ml/min/g heart weight, n = 42), the hearts were exposed to 60-min underperfusion by reducing the flow rate to 2 ml/min/g heart weight, and 5 min after the start of underperfusion the perfusate was changed to that containing 10–6 M NE (Sankyo). The 60-min underperfusion with NE group was divided into 4 subgroups: 0.002% dimethyl sulfoxide (DMSO as a solvent, Sigma) subgroup (n = 6 for measuring tissue substrates and n = 5 for measuring regional flow rate); 10–6 M glyburide subgroup (n = 7 for measuring tissue substrates and n = 6 for measuring regional flow rate); glyburide + 10–4 M levcromakalim subgroup (n = 5); and glyburide + insulin (2 mU/min/g heart weight, Novo, Actrapid) subgroup (n = 6). Insulin was infused 25 min before, and DMSO, glyburide and levcromakalim were infused 15 min before and during underperfusion by an infusion pump (SP-60, Nipro). Glyburide and levcromakalim were dissolved in DMSO. The solutions containing the agents were diluted with Krebs-Henseleit solution and infused at a rate of 0.1 ml/min into the perfusate. The above concentrations of the agents are those during underperfusion. At the end of each perfusion the hearts were quickly frozen in liquid nitrogen for subsequent measurement of tissue substrates.
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2.4 Determination of myocardial energy metabolites
The tissue substrate concentration in the subendocardium and the subepicardium of the left ventricle was measured and analyzed separately. The LV free walls of the frozen hearts were dissected into inner and outer halves, which corresponded to the subendocardial and subepicardial portions, respectively. The solidly frozen tissue was weighed (wet weight).
After a 5-h lyophilization, the dried tissue was again weighed (dry weight). The tissue water content was estimated from the wet and dry weights. The dried tissue was extracted with 0.6 M perchloric acid. The mixture was centrifuged at 12 000xg for 15 min at 2°C, and the supernatant was used to determine tissue metabolites. Creatine phosphate (CP) and inorganic phosphate (Pi) were determined by the method of Fiske and Subbarow, as modified by Furchgott and DeGubareff [21]. ATP was determined by the firefly luminescence method of Strehler, using an ATP monitoring reagent (Pharmacia Biosystems) and a Lumiphotometer (TD-4000, Laboscience). Adenine nucleotides shown in Table 2 were also determined by high-performance liquid chromatography (HPLC, 600E, Waters). Lactate was determined by an enzymatic method with Lactate Test BMY (Boehringer Mannheim). The approximate tissue lactate concentration was estimated from the water and lactate content. Tissue glycogen was determined by the enzymatic method of Keppler and Decker [22]with a starch measuring reagent (‘Starch’, Boehringer Mannheim), as described in our previous paper [15]in detail.
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2.5 Measurement of regional myocardial flow rate
Tissue flow distribution was determined by the dye microspheres method with Dye-trak (Primetech) in the DMSO and the glyburide subgroups. Blue and yellow microspheres (diameter, 15 µm; density, about 50 000/50 µl of 0.01% Tween 80) were injected into the coronary perfusion line just before and after 60-min underperfusion, respectively. The effluences were collected for 5 min after each injection. The hearts were dissected into 6 portions—both atria, the right ventricular free wall, the ventricular septum, the subendocardium, and the subepicardium of the LV free wall, and the apex. The 6 portions were lyophilized and the dried tissues were weighed. Each dried tissue was dissolved in 4 M KOH/20% Tween 80 (Sigma). Multiple dye microspheres in the solutions were collected using a 10-µm pored filter, and the dyes were dissolved in N,N-dimethylformamide (Nacalai) and determined with a UV-VIS recording spectrophotometer (UV-2200A, Shimadzu).
2.6 Statistics
In Figs. 2–6
and Table 1, the analysis of variance (ANOVA) for multiple comparisons [23]was restricted to the underperfusion with NE subgroups. The data for the differences among the subgroups were first assessed statistically by two-way ANOVA, and then at any perfusion period by one-way ANOVA and the Scheffé method as post-hoc test. In Table 1, all the flow data for the differences between the levels just before (at the normal perfusion) and 60 min after the start of underperfusion with NE, and between the LV subendocardium and subepicardium were also assessed statistically by the Student t-test. We focused on the differences between the LV subendocardium and subepicardium to clarify the relationship between the regional flow and myocardial energy metabolism. In Table 2, all the data for the differences between the normal perfusion group and the DMSO-underperfusion with NE subgroup, and between the LV subendocardium and subepicardium were assessed statistically by the Student t-test. The one-way ANOVA and the Scheffé method were restricted to the underperfusion with NE subgroups.
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3 Results |
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3.1 Characterization of diabetic state
The characteristics of the diabetic rats used in the isolated heart experiments were significantly different from those of the non-diabetic rats as reported previously [15]. Body and heart weights were significantly reduced together with a significant increase in plasma glucose: body weight, 246±6 g (–4±6
g, difference from those at the streptozotocin treatment); heart weight, 0.90±0.02 g; plasma glucose, 535±14 mg/dl (n = 42).
3.2 CPP during underperfusion with NE
In the DMSO subgroup, with the start of underperfusion, CPP decreased to about one third of the normal perfusion level (Fig. 2). NE infusion during underperfusion induced a transient moderate increase in CPP; CPP returned to almost the level during underperfusion without NE, and about 25 min after the onset of underperfusion with NE, CPP began to increase gradually. In the glyburide subgroup, about 17 min after the onset of underperfusion CPP began to increase markedly. In the presence of levcromakalim with glyburide, CPP during normal perfusion decreased significantly, and the changes in CPP during underperfusion with NE were similar to those in the DMSO subgroups.
3.3 Contractile dysfunction during underperfusion with NE
Fig. 3 shows the changes in the developed LVP and the peak +dP/dt and –dP/dt. With the start of underperfusion, these values decreased rapidly and NE infusion during underperfusion caused a transient marked increase in them. There were almost no significant differences among the three subgroups of underperfusion with NE in these parameters.
Fig. 4 shows the changes in the developed CF and the peak +dF/dt and –dF/dt. The parameters during 13–20 min of underperfusion with NE were lower in the glyburide subgroup than in the other two subgroups.
3.4 Diastolic state during underperfusion with NE
Fig. 5 shows the changes in resting LVP and resting CF. In the normal perfusion group, neither values rose during the perfusion period. In the DMSO subgroup, the resting CF and the resting LVP began to increase about 15 and 20 min, respectively, after the onset of underperfusion with NE. In the glyburide subgroup, the resting CF and the resting LVP began to increase about 8 and 15 min, respectively, after the onset of underperfusion with NE, the increase being greater than in the DMSO subgroup. In the presence of levcromakalim with glyburide, the changes in both parameters during underperfusion with NE were similar to those in the DMSO subgroup.
Fig. 6 shows the changes in resting LVP and resting CF in the glyburide+insulin subgroup. In the presence of insulin with glyburide, the changes in both values during underperfusion with NE were similar to those in the glyburide subgroup until 17 min after the onset of underperfusion with NE, and to those in the DMSO subgroup thereafter.
3.5 Regional myocardial flow distribution
Table 1 shows the changes in the tissue flow rate. During normal perfusion, the flow rate in the DMSO subgroup was greater in the LV subendocardium than in the LV subepicardium. However, at 60 min of underperfusion with NE, the decrease in flow was more marked in the subendocardium; the flow rate was smaller in the subendocardium than the subepicardium. In the glyburide subgroup, the changes in the flow rate were similar to those in the DMSO subgroup.
3.6 Regional myocardial energy metabolism during underperfusion with NE
Table 2 shows the changes in the regional myocardial energy metabolism in the LV free wall. During normal perfusion, the level of phosphorylation potential, the energy charge and tissue content of high-energy phosphate compounds were similar in the subendocardium and the subepicardium; the tissue lactate concentration and CP/Pi ratio were slightly higher, and the levels of Pi and water were slightly lower in the subendocardium. After a 60-min underperfusion with NE, all the metabolic parameters except ADP and subendocardial water significantly changed in both layers in the DMSO subgroup (P<0.01); the metabolic changes were more marked in the subendocardium.
After a 60-min underperfusion with NE, the decreases in ATP and energy charge, the increase in AMP in both the subendo- and subepicardium, and the decrease in CP in the subendocardium were more marked in the glyburide subgroup than in the DMSO subgroup. In the presence of levcromakalim with glyburide, the changes during underperfusion with NE were similar to those in the DMSO subgroup. In the presence of insulin with glyburide, the changes in ATP and CP during underperfusion with NE were also similar to those in the DMSO subgroup. The tissue content of ADP did not change and the total adenine nucleotides decreased similarly in both layers of both DMSO and glyburide subgroups; the tissue levels of both substrates increased slightly in the presence of levcromakalim with glyburide.
After a 60-min underperfusion with NE, the increases in lactate and Pi in both layers were more marked in the glyburide subgroup than in the DMSO subgroup. In the presence of levcromakalim or insulin with glyburide, the changes resembled those in the DMSO subgroup, but the lactate concentration in the subendocardium in the glyburide+levcromakalim subgroup was still greater than that in the DMSO subgroup.
After a 60-min underperfusion with NE, the glycogen levels were markedly low and the content in the subendocardium and the subepicardium was similar in the three subgroups.
After a 60-min underperfusion with NE, the phosphorylation potential and CP/Pi ratio, which reflects the state of myocardial oxidative metabolism, were markedly decreased, particularly in the subendocardium. The changes were more marked in the glyburide subgroup. In the presence of levcromakalim or insulin with glyburide, the changes were similar to those in the DMSO subgroup.
After a 60-min underperfusion with NE, the water content did not change in the subendocardium and decreased in the subepicardium similarly in the three subgroups, and there was no significant difference between the layers.
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4 Discussion |
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Glyburide, a sulphonylurea derivative, is used for treatment of non-insulin-dependent DM, because it augments the release of insulin from the beta-cells of the pancreas [20]. The beneficial effect is probably caused by blocking the KATP channels in the beta-cells [7]. However, concomitant blockade of the KATP channels in the diabetic myocardium may be harmful to the diabetic heart, particularly during ischemia, if opening of the KATP channels has a protective effect on the diabetic ischemic heart as on the non-diabetic ischemic heart [9].
The present study provides evidence that glyburide enhances diabetic myocardial damage during underperfusion with NE. Glyburide during underperfusion with NE accelerated the start of increase in LV diastolic stiffness and the abnormal myocardial energy metabolism in both LV layers of the isolated diabetic rat heart. The glyburide-induced deleterious effects were prevented by levcromakalim and partly by insulin.
4.1 Increased LV stiffness in isolated underperfused diabetic rat hearts
When hearts isolated from diabetic rats were perfused with a NE-containing fluid at a low flow rate, an increase in LV stiffness was accompanied by a decrease in the amount of water contained in the myocardial tissue. This indicates that the observed increase in the LV diastolic stiffness is attributable to myocardial contracture rather than to increased LV lumen volume, myocardial edema or the erectile effect, as we proposed previously [12, 15].
4.2 Regional myocardial flow distribution in isolated underperfused diabetic rat heart
The present findings indicate that the regional myocardial flow distribution during normal perfusion is greater in the LV inner layer (subendocardium) than in the outer layer (subepicardium) of the isolated diabetic rat heart.
When the distribution of low flow rates was examined 60 min after the start of underperfusion with a NE-containing fluid, the flow rate in the inner layer of the LV free wall was significantly lower than that in its outer layer. This reflects the abnormal regional myocardial energy metabolism very well. Regional blood flow rates as determined using multi-colored microspheres are reported to be well consistent with those measured using radioactive microspheres [24]. The distribution of flow rates during perfusion of the isolated heart, determined in this experiment using dye microspheres, does not contradict that determined in situ in non-diabetic animals [25].
In the isolated diabetic rat heart, underperfusion with NE caused a more marked flow decrease in the subendocardium than in the subepicardium in both the presence and absence of glyburide. Glyburide reportedly reduced the coronary blood flow of the non-diabetic heart; however, the coronary vasculature retains the capacity to dilate in response to oxygen demand produced by exercise when KATP channels are blocked [26]. In the present study, the flow rate through the inner layers of the LV myocardium, measured 60 min after the start of underperfusion with NE, was similarly low in the glyburide-treated and untreated DMSO subgroup. The lack of a significant difference is probably because the left ventricular stiffness is markedly elevated even in the absence of glyburide after 60 min of underperfusion with NE.
4.3 Regional myocardial energy metabolism in isolated underperfused diabetic rat heart
As we have described in detail previously [12, 13, 15], underperfusion of hearts isolated from diabetic rats, using a NE-containing fluid as a perfusate, resulted in a marked increase of LV stiffness and in abnormal myocardial energy metabolism. Changes such as an increase in myocardial AMP, Pi and lactate levels, suppression of oxidative energy production and a significant decrease in high-energy phosphates and total adenine nucleotides were more marked in the LV inner layer than the outer layer.
In the present study, abnormalities of regional myocardial energy metabolism reflect the distribution of regional flow rates very well. An increase in LV stiffness and abnormalities of myocardial energy metabolism probably adversely affect the heart and thus precipitate heart failure.
4.4 Involvement of the KATP channels in myocardial dysfunction during underperfusion with NE of diabetic rat heart; the glyburide-induced deleterious effects
In the present study, glyburide treatment resulted in exacerbation of abnormal regional myocardial energy metabolism and an enhanced increase of LV stiffness during underperfusion with NE in the diabetic heart. That is, the increase in LV stiffness during underperfusion with NE appeared earlier and was greater in the glyburide-treated subgroup. Abnormal myocardial energy metabolism in both LV inner and outer layers was exacerbated significantly by treatment with glyburide.
It is suggested that in the non-diabetic heart, myocardial ischemia opens the KATP channels in the vascular smooth muscle and myocardial cells, due to a decrease in the cytosolic ATP concentration [1, 16, 27], an increase in the extracellular adenosine level [28]and the release of an acetylcholine-mediated, endothelium-derived hyperpolarizing factor [29], which leads to protection of the ischemic derangement of myocardium [9]. The activation of KATP channels is thought to lead to an increase in regional coronary flow due to vasodilation, to a decrease in myocardial contraction [19, 27]and to preservation of high-energy phosphates [30]. The preconditioning effect of myocardial ischemia is also thought to be attributable to a similar mechanism, because the beneficial effects of preconditioning disappear if the KATP channels are blocked [3–5]. Inhibiting the KATP channels probably elicits the role of the channels during myocardial ischemia in the diabetic heart. The present results imply the protective effects of opening the KATP channels during underperfusion with NE in diabetic hearts, because the damage during underperfusion with NE was enhanced with the KATP channel inhibitor, glyburide.
Glyburide has been reported to block the KATP channels in cardiac cells in the non-diabetic heart, [8, 16], to suppress the effects of adenosine and acetylcholine in inducing negative chronotropic and inotropic responses [31, 32], and to enhance myocardial damage caused by ischemia/reperfusion [2]. A decrease in outward K+ current induced by blocking the KATP channels may cause depolarization of myocardial cells, as observed in the beta-cells of the pancreas [7, 20], resulting in an influx of Ca2+ via the opening of voltage-dependent Ca2+ channels on the myocardial cell membrane [9]. The exacerbation of abnormal myocardial energy metabolism in the diabetic heart in the presence of glyburide, observed in the present study, may represent the total of the above-mentioned effects plus the adverse effect of myocardial cell calcium accumulation induced by the blockade of the KATP channels (i.e., an increase in the myocardial tension at diastole and suppressed oxidative energy production).
In the present ex vivo study, the glyburide level which caused marked exacerbation of myocardial dysfunction during underperfusion with NE was 10–6 M, which causes specific blockade of KATP channels and is similar to the plasma glyburide levels seen in clinical cases [20]. Previously [33], we found that the glyburide level causing marked exacerbation of the underperfusion with NE injury was lower in the non-diabetic heart (10–7 M) than in the diabetic heart. In addition, the marked deleterious effects of glyburide at these levels were observed in the presence of 10–6 M NE in both non-diabetic and diabetic hearts. These findings imply that the deleterious effect of glyburide on the underperfused diabetic heart varies with the severity of underlying diabetes mellitus and the activity of the sympathetic nervous system.
On the other hand, some investigators have reported that the glyburide-induced blockade of KATP channels led to a favorable outcome in the non-diabetic heart (i.e., a decrease in the frequency of ischemia-induced ventricular fibrillation) [34]. In the present study, this effect of glyburide could not be assessed because the heart was paced electrically throughout the experiments. Other limitations of the study are that the possible influences of blood components and reflex reaction in in-situ experiments are excluded because in ex vivo experiments the heart is innervated insufficiently and is perfused with Krebs-Henseleit solution containing 11 mM glucose instead of blood.
4.5 Preventive effects of levcromakalim on the glyburide-induced deleterious effects
Levcromakalim, a selective K+ channel opener and a KATP channel opener, suppressed the glyburide-induced exacerbation of myocardial dysfunction during underperfusion with NE in the isolated diabetic rat heart, the severity of myocardial dysfunction being reduced to a level comparable to that seen in the glyburide-untreated heart. Levcromakalim opens the KATP channels of blood vessels [18]and myocardium [17, 19]in non-diabetic animals. This action of levcromakalim is probably involved in the suppression of glyburide-induced exacerbation of myocardial dysfunction during underperfusion with NE in the diabetic heart. This supports the view that the earlier appearance of myocardial dysfunction during underperfusion with NE in the presence of glyburide treatment is due to its action of inhibiting the opening of the KATP channels. The present study also indicates that levcromakalim preserves the adenine nucleotide level in ischemic myocardium of the diabetic heart.
These findings suggest that in the diabetic heart the KATP channels open soon after the onset of ischemia and thus protect the myocardium. Glyburide seems to inhibit this response of the KATP channels and thus enhances the onset and severity of ischemia-caused myocardial injury. Levcromakalim may suppress such actions of glyburide.
4.6 Preventive effects of insulin on the glyburide-induced deleterious effects
In our previous study [12, 14], in vivo and ex vivo insulin reduced the injury associated with underperfusion with a NE-containing fluid in the isolated diabetic heart. In the present study, the glyburide-induced earlier exacerbation of myocardial dysfunction during underperfusion with NE was not reduced by ex vivo insulin. However, 20 min after the start of underperfusion, the elevation of LV stiffness and the abnormal myocardial energy metabolism in the heart treated with ex vivo insulin were reduced to the levels observed in the glyburide-untreated DMSO subgroup. These findings indicate that in the diabetic heart, insulin cannot inhibit the earlier and more severe exacerbation of myocardial dysfunction during underperfusion with NE caused by the KATP channel blocking action of glyburide. Once myocardial glycogen has been consumed almost completely during long-term underperfusion with a NE-containing fluid [15], insulin may protect the myocardium, although incompletely, probably through promotion of glucose utilization [35].
The effects of levcromakalim and insulin strongly suggest that in the diabetic heart the KATP channels open soon after the onset of ischemia and thus protect the myocardium, and that the glyburide-induced earlier exacerbation of myocardial dysfunction during underperfusion with NE is due to its effect in suppressing the opening of the KATP channels. Furthermore, these findings partly explain why treatment with sulphonylurea leads to detrimental effects in some diabetics [6].
Time for primary review 29 days.
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Acknowledgements |
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We are grateful to Dr. Eikoh Uchima for determining adenine nucleotides by HPLC, and to Yamanouchi, Japan, and SmithKline Beecham, UK, for providing glyburide and levcromakalim, respectively.
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