Failure of Isoflurane Cardiac Preconditioning in Obese Type 2 Diabetic Mice Involves Aberrant Regulation of MicroRNA-21, Endothelial Nitric-oxide Synthase, and Mitochondrial Complex I

Abstract

Background: Diabetes impairs the cardioprotective effect of volatile anesthetics, yet the mechanisms are still murky. We examined the regulatory effect of isoflurane on microRNA-21, endothelial nitric-oxide synthase, and mitochondrial respiratory complex I in type 2 diabetic mice.

Methods: Myocardial ischemia/reperfusion injury was produced in obese type 2 diabetic (db/db) and C57BL/6 control mice ex vivo in the presence or absence of isoflurane administered before ischemia. Cardiac microRNA-21 was quantified by real-time quantitative reverse transcriptional-polymerase chain reaction. The dimers and monomers of endothelial nitric-oxide synthase were measured by Western blot analysis. Mitochondrial nicotinamide adenine dinucleotide fluorescence was determined in Langendorff-perfused hearts.

Results: Body weight and fasting blood glucose were greater in db/db than C57BL/6 mice. Isoflurane decreased left ventricular end-diastolic pressure from 35 ± 8 mmHg in control to 23 ± 9 mmHg (P = 0.019, n = 8 mice/group, mean ± SD) and elevated ±dP/dt 2 h after post-ischemic reperfusion in C57BL/6 mice. These beneficial effects of isoflurane were lost in db/db mice. Isoflurane elevated microRNA-21 and the ratio of endothelial nitric-oxide synthase dimers/monomers and decreased mitochondrial nicotinamide adenine dinucleotide levels 5 min after ischemia in C57BL/6 but not db/db mice. MicroRNA-21 knockout blocked these favorable effects of isoflurane, whereas endothelial nitric-oxide synthase knockout had no effect on the expression of microRNA-21 but blocked the inhibitory effect of isoflurane preconditioning on nicotinamide adenine dinucleotide.

Conclusions: Failure of isoflurane cardiac preconditioning in obese type 2 diabetic db/db mice is associated with aberrant regulation of microRNA-21, endothelial nitric-oxide synthase, and mitochondrial respiratory complex I.

Figure 1

Isoflurane preconditioning improved the recovery of cardiac fnction during post-ischemic reperfusion in Langendorff-perfused C57BL/6 mouse hearts but not db/db mouse hearts. A: left ventricular end-diastolic pressure (mean ± SD); B: left ventricular developed pressure; C: the maximum rate of developed pressure rise (+dp/dt); D: the maximum rate of developed presure decreases (−dP/dt). Control, C57BL/6 mouse hearts undergoing ischemia/reperfusion injury: db/db, db/db mouse hearts undergoing ischemia/reperfusion injury; isoflurane, C57BL/6 mouse hearts treated with 1.4% isoflurane before ischemia; db/db+isoflurane, db/db mouse hearts treated with 1.4% isoflurane before ischemia. *P<0.05 versus control groups, ^†P<0.05 versus db/db groups, ^#P<0.05 versus isoflurane groups (n=8 mice/group).

Figure 2

Isoflurane preconditioning elevated the expression of microRNA-21 associated with cardioprotective effect in C57BL/6 mice but decreased microRNA-21 in db/db mice. A: alterations in microRNA-21 by isoflurane in C57BL/6 and db/db mice (mean ± SD, n=8 mice/group); B: isoflurane improved +dP/dt (the maximum rate of left ventricular developed pressure increase) in C57BL/6 but not microRNA-21 knockout mice 2 h after reperfusion (reperfusion) (n=9 mice in control, 8 mice in microRNA-21 knockout group, 9 mice in isoflurane group, and 7 mice inmicroRNA-21 knockout+isoflurane group); C: isoflurane improved the values of -dP/dt (the maximum rate of left ventricular developed pressure decrease) in C57BL/6 but not microRNA-21 knockout mice 2 h min after reperfusion (n=9 mice in control, 8 mice in microRNA-21 knockout group, 9 mice in isoflurane group, and 7 mice inmicroRNA-21 knockout+isoflurane group). Control, C57BL/6 mouse hearts undergoing ischemia/reperfusion injury: db/db, db/db mouse hearts undergoing ischemia/reperfusion injury; microRNA-21 knockout, microRNA-21 knockout mouse hearts subjected to ischemia/reperfusion injury; isoflurane, C57BL/6 mouse hearts treated with 1.4% isoflurane before ischemia; db/db+isoflurane, C57BL/6 mouse hearts treated with isoflurane before ischemia; microRNA-21 knockout+isoflurane, microRNA-21 knockout mouse hearts treated with isoflurane prior to ischemia. *P<0.05 versus control groups, ^†P<0.05 versus db/db or microRNA-21 knockout groups, ^#P<0.05 versus isoflurane groups.

Figure 3

Isoflurane preconditioning increased the dimerization of endothelial nitric oxide synthase associated with cardioprotective effect in C57BL/6 but not db/db mice. A: alterations in the ratio of endothelial nitric oxide synthase dimers/monomers by isoflurane preconditioning (mean ± SD, n=5 mice/group); B: Western blot bands showing the expression of endothelial nitric oxide synthase dimers and monomers and glyceraldehyde 3-phosphate dehydrogenase as a loading control in mouse hearts (n=3 hearts/blot); C: isoflurane preconditioning increased +dP/dt in C57BL/6 but not endothelial nitric oxide synthase knockout mice 2 h after post-ischemic reperfusion (reperfusion) (n=9 mice in control and endothelial nitric oxide synthase knockout groups and 8 mice in isoflurane and endotheial nitric oxide synthase knockout+isoflurane groups); D: isoflurane preconditioning elevated the value of −dP/dt in C57BL/6 but not endothelial nitric oxide synthase knockout mice 2 h after reperfusion (reperfusion) (n=9 mice in control and endothelial nitric oxide synthase knockout groups and 8 mice in isoflurane and endotheial nitric oxide synthase knockout+isoflurane groups). Control, C57BL/5 mice subjected to ischemia/reperfusion injury; db/db, db/db mice undergoing ischemia/reperfusion injury; endothelial nitric oxide synthase knockout, endothelial nitric oxide synthase knockout mice undergoing ischemia/reperfusion injury; isoflurane, C57BL/6 mice undergoing ischemia/reperfusion injury; db/db+isoflurane, db/db mice treated with 1.4% isoflurane prior to ischemia/reperfusion injury; endothelial nitric oxide synthase knockout+isoflurane, endothelial nitric oxide synthase knockout mice treated with isoflurane before ischemia/reperfusion injury. *P<0.05 versus control groups, ^†P<0.05 versus db/db or endothelial nitric oxide synthase knockout groups, ^#P<0.05 versus isoflurane groups.

Figure 4

Effects of diabetes and isoflurane treatment on the levels of reduced form of nicotinamide adenine dinucleotide during ischemia and reperfusion. All hearts were stabilized in Langendorff apparatus for 30 minutes (baseline) and perfused with the buffer with or without isoflurane prior to 30 minutes of global ischemia (ischemia) followed by 2 h of reperfusion (reperfusion). Control, C57BL/6 mouse hearts undergoing ischemia/reperfusion injury: db/db, C57BL/6 mouse hearts undergoing ischemia/reperfusion injury; isoflurane, C57BL/6 mouse hearts treated with 1.4% isoflurane before ischemia; db/db+isoflurane, db/db mouse hearts treated with 1.4% isoflurane before ischemia. *P<0.05 versus control groups, ^†P<0.05 versus db/db groups, ^#P<0.05 versus isoflurane groups (n=9 mice in control, 8 mice in db/db group, 9 mice in isoflurane, and 8 mice in db/db+isoflurane group).

Figure 5

MicroRNA-21 knockout blocked the regulatory effects of isoflurane preconditioning on endothelial nitric oxide synthase and mitochontrial nicotinamide adenine dinucleotide in ischemic myocardium. A: microRNA-21 knockout blocked isoflurane-induced increases in the ratio of endothelial nitric oxide synthase dimers/monomers in myocardium 5 min after ischemia (mean ± SD, n=5 hearts/group); B: representativive Western blot bands showing the expression of endothelial nitric oxide synthase dimers (230 KDa) and monomers (130 KDa) and glyceraldehyde 3-phosphate dehydrogenase (37 KDa) as a loading control in myocardium 5 min after ischemia (n=3 hearts/blot); C: microRNA-21 knockout blocked isoflurane-induced decreases in mitochondrial nicotinamide adenine dinucleotide levels 5 min after ischemia (n=9 hearts in control, microRNA-21 knockout, and isoflurane groups and 8 hearts in microRNA-21 knockout+isoflurane group). All hearts were stabilized for 30 minutes in a Langendorff apparatus and subjected to 5 minutes of global ischemia. Control, C57BL/6 mouse hearts underlying 5 minutes of ischemia; microRNA-21 knockout, microRNA-21 knockout hearts subjected to 5 minutes of ischemia; isoflurane, C57BL/6 mouse hearts treated with isoflurane and subsequently underlying 5 minutes of ischemia; microRNA-21 knockout+isoflurane, microRNA-21 knockout hearts treated with isoflurane and subsequently subjected to 5 minutes of ischemia. *P<0.05 versus control groups, ^†P<0.05 versus microRNA-21 knockout groups, ^#P<0.05 versus isoflurane groups. D: endothelial nitric oxide synthase knockout blocked isoflurane-induced decreases in mitochondrial nicotinamide adenine dinucleotide levels 5 minutes after ischemia (n=10 hearts in control, 9 hearts in endothelial nitric oxide synthase knockout and isoflurane groups, and 8 hearts in endothelial nitric oxide synthase knockout+isoflurane group). Control, C57BL/6 mouse hearts underlying 5 minutes of ischemia; endothelial nitric oxide synthase knockout, endothelial nitric oxide synthase knockout hearts subjected to 5 minutes of ischemia; isoflurane, C57BL/6 mouse hearts treated with isoflurane and subsequently subjected to 5 minutes of ischemia; endothelial nitric oxide synthase knockout+isoflurane, endothelial nitric oxide synthase knockout hearts treated with isoflurane and subsequently subjected to 5 minutes of ischemia. *P<0.05 versus control groups, ^†P<0.05 versus endothelial nitric oxide synthase knockout groups, ^#P<0.05 versus isoflurane groups.

Figure 6

Proposed mechanisms responsible for failure of isoflurane preconditioning to protect the heart against ischemia/reperfusion injury in obese type 2 diabetic mice. InC57BL/6 control mice, isoflurane preconditioning up-reglates microRNA-21, leading to the dimerization of endothelial nitric oxide synthase through direct and/or indirect mechanisms. Dimeric endothelial nitric oxide synthase produces nitric oxide, that acts on respiratory chain Complex I [reduced form of nicotinamide adenine dinucleotide (NADH):ubiquinone] in the mitochondria and facilitaes NADH to release electrons. The electrons removed from respiratory chain Complex I are subsequently transferred to coenzyme Q, Complex III, cytochrome c (Cyt c), and Complex IV, which uses the electrons and hydrogen ions to reduce molecular oxygen to water. Type 2 diabetes mellitus with obesity down-regulates microRNA-21 and prevents the up-regulation of microRNA-21 by isoflurane. Moreover, diabetes, obesity, and ischemia together facilitates the transfer of endothelial nitric oxide synthase dimers to monomers, inhibits respiratory chain Complex I, and elevates the production of NADH though multiple pathways including citric acid cycle. These changes elicited by diabetes and obesity contribute to failure of isoflurane cardiac preconditioning.