Héctor F. del Valle

Ischemic preconditioning protection against stunning in conscious diabetic sheep: role of glucose, insulin, sarcolemmal and mitochondrial KATP channels

INTRODUCTION Sarcolemmal and mitochondrial ATP-sensitive potassium (KATP) channels have been postulated to participate in preconditioning protection against infarction and stunning. However, these structures appear to be altered in diabetes and thus, it would be possible that preconditioning does not develop in diabetic hearts. OBJECTIVE The purpose of this study was to know whether early (EP) and late (LP) ischemic preconditioning against stunning develop in conscious diabetic (D) sheep and whether diabetes affects KATP channel function. METHODS Male castrated sheep received alloxan monohydrate (1 g) and were ascribed to three experimental groups: control [DC, 12 min of ischemia (i) followed by 2 h of reperfusion (r)], early preconditioning (DEP, six 5 min i-5 min r periods were performed 45 min before the 12 min i) and late preconditioning (DLP, same as DEP except that the preconditioning stimulus was performed 24 h before the 12 min i). Regional mechanics during reperfusion was evaluated by wall thickening fraction (%WTH) and expressed as percentage of basal values (100%), and KATP channel behavior was indirectly assessed by monophasic action potential duration (MAPD) in relation to its sensitivity to glibenclamide blockade (0.1 and 0.4 mg/kg). The results were compared to those obtained in normal (N) sheep. The effects of sarcolemmal and mitochondrial KATP channel blockade on recovery from stunning were assessed by administration of glibenclamide (0.1 and 0.4 mg/kg) and 5-hydroxydecanoate (5-HD, 5 mg/kg i.v.) and/or diazoxide (10 microg/kg/min over 90 min). Whether acute hyperglycemia (H) in normal animals and insulin (I) treatment in diabetic sheep affected preconditioning protection and KATP channel behavior were also evaluated. RESULTS Results expressed as mean % recovery of %WTH showed that preconditioning protected against stunning in normal sheep (NC=65+/-3.5, NLP=82+/-6**, NEP=76+/-4*, *P<0.05 and **P<0.01 against NC) while this did not occur in diabetic ones, where DLP (58+/-7.6) afforded a similar recovery to DC (54+/-5) and DEP worsened instead of improving mechanical function (37+/-9, P<0.01 against DC). Acute hyperglycemia did not affect preconditioning development (NEPH=72+/-3 and NLPH=80+/-4) and insulin treatment reverted the lack of early and late preconditioning protection in diabetic hearts (DEPI=72+/-4* and DLPI=76+/-3*, P<0.05 against DC). Sarcolemmal KATP channel behavior appeared altered in diabetic hearts as shown by MAPD in normal sheep (276+10 ms) compared to diabetic ones (365+9 ms, P<0.05) and by the sensitivity to glibenclamide [0.1 mg/kg completely blocked KATP channels in diabetic (P<0.05) but not in normal hearts]. Insulin also restored MAPD in diabetic heart. Mitochondrial KATP channels appeared not to account for the reported results in diabetes, since glibenclamide (%WTH=40+/-4, P<0.01 vs. NC), but not 5HD nor diazoxide affected myocardial functional recovery during reperfusion. CONCLUSIONS Sarcolemmal KATP channel dysfunction due to the lack of insulin affords a primary approach to explain the absence of preconditioning protection against stunning in diabetic sheep hearts.

Arteriogenesis Induced by Intramyocardial Vascular Endothelial Growth Factor 165 Gene Transfer in Chronically Ischemic Pigs

Exogenous vascular endothelial growth factor (VEGF) improves tissue perfusion in large animals and humans with chronic myocardial ischemia. Because tissue perfusion is mainly dependent on the arteriolar tree, we hypothesized that the neovascularizing effect of VEGF should include arteriogenesis, an effect not as yet described in large mammalian models of myocardial ischemia. In the present study we investigated the effect of intramyocardial plasmid-mediated human VEGF(165) gene transfer (pVEGF(165)) on the proliferation of vessels with smooth muscle in a pig model of myocardial ischemia. In addition, we assessed the effect of treatment on capillary growth, myocardial perfusion, myocardial function and collateralization. Three weeks after positioning of an Ameroid constrictor (Research Instruments SW, Escondido, CA) in the left circumflex artery, pigs underwent basal perfusion (single-photon emission computed tomography [SPECT] with (99m)Tc-sestamibi) and regional function (echocardiography) studies at rest and under dobutamine stress, and were then randomly assigned to receive transepicardial injection of pVEGF(165) 3.8 mg (n = 8) or placebo (empty plasmid, n = 8). All experimental steps and data analysis were done in a blinded fashion. Five weeks later, pVEGF(165)-treated pigs showed a significantly higher density of small (8-50 microm in diameter) vessels with smooth muscle, higher density of capillaries, and improved myocardial perfusion. These results indicate an arteriogenic effect of VEGF in a large mammalian model of myocardial ischemia and encourage the use of VEGF to promote arteriolar growth in patients with severe coronary artery disease.

Absence of ischemic preconditioning protection in diabetic sheep hearts: Role of sarcolemmal KATP channel dysfunction

Sarcolemmal ATP-sensitive potassium (KATP) channels have been mentioned to participate in preconditioning protection. Since these channels are altered in diabetes, it would be possible that preconditioning does not develop in diabetic (D) hearts. The purpose of this study was to assess whether early (EP) and late (LP) ischemic preconditioning protect diabetic hearts against stunning in a conscious diabetic sheep model and whether diabetes might have altered KATP channel functioning. Sheep received alloxan monohydrate (1 g) and were ascribed to three experimental groups: control (DC, 12 min of ischemia (I) followed by 2 h of reperfusion (R)), early preconditioning (DEP, six 5 min I-5 min R periods were performed before the 12 min I) and late preconditioning (DLP, same as DEP except that the preconditioning stimulus was performed 24 h before the 12 min I). Regional mechanics during reperfusion was evaluated as the percent recovery of wall thickening fraction (%WTH) expressed as percentage of basal values (100%) and KATP behaviour was indirectly assessed by monophasic action potential duration (MAPD) and sensitivity to glibenclamide blockade (0.1 and 0.4 mg/Kg). The results were compared to those obtained in normal (N) sheep. EP and LP protected against stunning in normal sheep (%WTH: NC = 63 ± 3.7, NLP = 80 ± 5**, NEP = 78 ± 3*, *p < 0.05 and **p < 0.01 against NC) whereas contrary results occurred in diabetic ones, where DLP (%WTH = 60 ± 4) afforded a similar recovery to DC (%WTH = 54 ± 5) and DEP surprisingly worsened instead of improving mechanical function (%WTH = 38 ± 6, p < 0.01 against DC). KATP channel behaviour appeared altered in diabetic hearts as shown by MAPD during ischemia in normal sheep (153 ± 9 msec) compared to diabetic ones (128 ±11 msec, p < 0.05) and by the sensitivity to glibenclamide (while 0.4 mg/Kg blocked action potential shortening in normal and diabetic animals, 0.1 mg/Kg completely blocked KATP in diabetic but not in normal hearts, p < 0.05). A sarcolemmal KATP channel dysfunction might afford a primary approach to explain the absence of ischemie preconditioning protection against stunning in diabetic sheep. (Mol Cell Biochem 249: 21–30, 2003)

Cardiomyocyte hyperplasia after plasmid-mediated vascular endothelial growth factor gene transfer in pigs with chronic myocardial ischemia.

For over 40 years it has been proposed that cardiomyocyte hyperplasia may occur in hypertrophic human hearts. While this implies that heart myocytes can undergo cytokinesis, evidence of conventional cell division has been exceptionally reported. Recently, we found that gene transfer of vascular endothelial growth factor (VEGF) displays a mitogenic effect on adult cardiomyocytes. In the present study we searched for cardiomyocyte hyperplasia as evidence of VEGF‐induced cardiomyocyte cytokinesis.

Glibenclamide effects on reperfusion-induced malignant arrhythmias and left ventricular mechanical recovery from stunning in conscious sheep

INTRODUCTION Sulfonylureas have been associated with a high incidence of cardiovascular death in diabetic patients treated with these drugs. Although the evidence on the cardiovascular effects of sulfonylureas is contradictory and scarce, many experiments have shown that the second-generation compound glibenclamide has a protective effect on mechanical function and against generation of malignant arrhythmias. OBJECTIVE The purpose of this study was to assess whether glibenclamide elicits protection on postischemic myocardial functional recovery (stunning) and against reperfusion-induced arrhythmias in a conscious sheep model. METHODS Sheep were divided into three groups: control, glibenclamide (0.4 mg/kg) and vehicle. After a 12-min ischemic period, the heart was reperfused and recordings for index calculation were acquired during 2 h of reperfusion. Percent systolic wall thickening fraction (%WTH), radial diastolic compliance (CR), arrhythmia incidence and Bernauers arrhythmia severity index (ASI) were calculated for each group. RESULTS Glibenclamide infusion had a high proarrhythmic action (ASI: glibenclamide 143, control 54 and vehicle 23; ANOVA P < 0.001 drug vs. control and vehicle) and a detrimental effect on regional systolic (%WTH: glibenclamide 26.9 +/- 6.7, control 65.7 +/- 3.5 and vehicle 68.6 +/- 5.6, ANOVA P < 0.01 drug vs. control and vehicle) and diastolic function (CR: glibenclamide 76.2 +/- 7.8, control 104.7 +/- 4.2 and vehicle 106 +/- 4.9, ANOVA P < 0.05 drug vs. control and vehicle) during reperfusion. CONCLUSIONS Glibenclamide infusion resulted in adverse cardiovascular effects. The combined deleterious effects on reperfusion-induced arrhythmias and on myocardial recovery from stunning could be the cause of the unexplained high mortality in diabetic patients treated with sulfonylurea derivatives. The mechanism involved seems to be the blockade of the cardiac ATP sensitive potassium (K-ATP) channel.

Ischemic shortening of action potential duration as a result of KATP channel opening attenuates myocardial stunning by reducing calcium influx

Action potential duration (APD) shortening due to opening of sarcolemmal ATP-dependent potassium (KATP) channels has been postulated to protect the myocardium against postischemic damage by reducing Ca2+ influx. This hypothesis was assessed, assuming that increased postischemic stunning due to KATP channel inhibition with glibenclamide could be reverted by the addition of the Ca2+ channel blocker diltiazem. Percent wall thickening fraction (% WTh, conscious sheep) and APD (open-chest sheep) were obtained from the following groups: control: 12 min ischemia by anterior descending coronary artery occlusion followed by 2 h reperfusion; glibenclamide: same as control, with glibenclamide (0.4 mg/kg) infused 30 min before ischemia; diltiazem: same as control, with diltiazem (100 μg/kg) administered prior to ischemia; glibenclamide+diltiazem: both drugs infused as in glibenclamide and diltiazem groups. APD was reduced in control ischemia. Conversely, KATP-channel blockade by glibenclamide lengthened APD and increased postischemic stunning (p < 0.01 vs. control); glibenclamide+diltiazem did not shorten APD but enhanced functional recovery (p < 0.01 vs. glibenclamide). Ca2+ channel blockade improvement of increased stunning provoked by KATP channel inhibition supports the hypothesis that APD shortening due to opening of KATP channels protects against postischemic stunning by limiting Ca2+ influx.

ATP-sensitive potassium channels do not have a main role in mediating late preconditioning protection against arrhythmias and stunning in conscious sheep

Objective Although late preconditioning protects against stunning following several short periods of ischemia-reperfusion, it is not clear if it confers protection against stunning and malignant arrhythmias after a sustained reversible ischemia, and whether KATP channels are involved as triggers and/or end effectors of the protective mechanism. The purpose of this work was thus to test these issues in conscious sheep. Methods Five groups were considered: CONT (control): the animals were submitted to 12 min ischemia followed by 2 h reperfusion; SWOP (late preconditioning): on the first day, the animals were preconditioned with 6 periods of 5 min ischemia 5 min reperfusion and 24 h later they were submitted to 12 min ischemia followed by 2 h reperfusion; GLIB: same as CONT with the KATP channel inhibitor glibenclamide (0.4 mg/kg) infused 30 min prior to the 12 min ischemia; SWOPG2: same as SWOP with glibenclamide before the 12 min ischemia; SWOPG1: same as SWOP with glibenclamide prior to the preconditioning stimulus. Results Percent reperfusion recovery of wall thickening fraction (% WTh) showed late preconditioning protection against stunning throughout reperfusion (SWOP vs CONT, p < 0.01). Arrhythmia severity index (ASI) also demonstrated that late preconditioning protects against malignant arrhythmias at the onset of reperfusion (CONT: 4.87 ± 1.62 vs SWOP: 1.39 ± 0.93, p < 0.01). Glibenclamide was unable to prevent preconditioning, both against stunning and arrhythmia incidence, when administered either before the preconditioning stimulus (SWOPG1 vs CONT, p < 0.01) or before the sustained ischemia (SWOPG2 vs GLI, p < 0.01). Conclusions Results indicate that late preconditioning protects against stunning and arrhythmias following a reversible, sustained ischemia in conscious sheep and that KATP channel participation is negligible as triggers and end effectors of both types of protection.

Expression of the MDR-1 Gene-encoded P-glycoprotein in Cardiomyocytes of Conscious Sheep Undergoing Acute Myocardial Ischemia Followed by Reperfusion

We have recently reported that in chronic myocardial ischemia, adult mammalian cardiomyocytes express P-glycoprotein (P-gp). We now investigate if P-gp is also expressed in acute regional ischemia followed by reperfusion. Adult conscious sheep underwent 12-min occlusion of the mid-left anterior descending artery (inflatable cuff). Successful ischemia-reperfusion was confirmed by monitoring percent systolic left ventricular anterior wall thickening (sonomicrometry) during the whole is chemic period and every 10 min over 2 hr following cuff deflation. At 3, 24, and 48 hr after reperfusion, P-gp expression was investigated by immunohistochemistry and Western blot and MDR-1 mRNA by RT-PCR. Cardiomyocytes in the occluded artery territory (but not those in remote areas) consistently expressed P-gp at their sarcolemma. Whereas at 3 and 24 hr P-gp was mainly observed in the T tubules, at 48 hr it predominated in intercalated discs and gap junctions. RT-PCR and Western blot revealed higher expression in ischemic than in control myocardium. We conclude that in adult sheep with acute myocardial ischemia, the MDR-1 gene-encoded P-gp is expressed at the sarcolemma of the cardiomyocytes from 3 hr up to at least 48 hr after reperfusion.

Left ventricular regional systolic and diastolic function in conscious sheep undergoing ischemic preconditioning

OBJECTIVE Late preconditioning diastolic protection and cardiac function optimization by the combined effects of late and early preconditioning have not been studied in conscious animals. This study assessed in fully conscious sheep: (1) whether 24 h after a reversible ischemia, a new ischemic episode results in lesser systo-diastolic dysfunction (late preconditioning protection) and (2) whether the addition of early preconditioning (brief episodes of ischemia-reperfusion before the subsequent sustained ischemia) on the second day of late preconditioning optimized the second window of protection. METHODS Three protocols were assessed: (a) late preconditioning, 9 min ischemia and 2 h reperfusion was done on two consecutive days; (b) early plus late preconditioning, as in protocol (a) except that on day 2 the heart underwent three periods of 3 min ischemia-6 min reperfusion prior to the sustained 9 min ischemia; (c) early preconditioning, the same protocol as in (b) except that day 2 was separated 1 week from day 1. RESULTS Late preconditioning decreased regional radial diastolic stiffness from 147 +/- 26% (day 1) to 96 +/- 14% (day 2), at 2 h of reperfusion (mean +/- SEM, p < 0.05), but did not protect against systolic stunning (thickening fraction and regional stroke work). Early plus late preconditioning did not improve late preconditioning findings. Early preconditioning alone did not protect either systolic or diastolic functions. CONCLUSION In conscious sheep, there is diastolic but not systolic mechanical protection with late preconditioning. Diastolic protection is not enhanced by the addition of early preconditioning.

Nitroglycerin induces late preconditioning against arrhythmias but not stunning in conscious sheep

Objectives. Nitroglycerin, a nitric oxide donor, induces late preconditioning against stunning by short ischemia-reperfusion periods. The study purpose was to assess similar nitroglycerin protection against stunning and arrhythmias produced by prolonged reversible ischemia. Design. Four groups of conscious sheep were studied, control: 12 minutes ischemia and 2 hour reperfusion; late preconditioning: six periods of 5 min ischemia-5 min reperfusion 24 h before 12 min ischemia and late preconditioning with 120 µg/kg and 600 µg/kg nitroglycerin administered instead of the ischemia-reperfusion periods. Results. Although late preconditioning protected against stunning (mean postischemic recovery of wall thickening fraction, control (n=10): 54.8±3.2, late preconditioning (n=9): 74.4±3.0, p<0.01), nitroglycerin 120 µg/kg (n=6) did not reproduce mechanical protection (50.1±3.8), even with a higher concentration of 600 µg/kg (59.1±3.7, n=4). However, nitroglycerin decreased arrhythmia severity index (control: 2.3±0.6, late preconditioning: 0.5±0.4, nitroglycerin 120 µg/kg: 1±0.4 and 600 µg/kg: 0.1±0.1 (p<0.05 vs. control). Conclusions. Nitroglycerin only has a limited late preconditioning protective effect in conscious animals submitted to a reversible prolonged ischemia since it protects against arrhythmias but not against stunning.