Cardioprotection during diabetes: the role of mitochondrial DNA

Abstract

Background: Diabetes alters mitochondrial bioenergetics and consequently disrupts cardioprotective signaling. The authors investigated whether mitochondrial DNA (mtDNA) modulates anesthetic preconditioning (APC) and cardiac susceptibility to ischemia-reperfusion injury by using two strains of rats, both sharing nuclear genome of type 2 diabetes mellitus (T2DN) rats and having distinct mitochondrial genomes of Wistar and fawn-hooded hypertensive (FHH) rat strains (T2DN(mtWistar) and T2DN(mtFHH), respectively).

Methods: Myocardial infarct size was measured in Wistar, T2DN(mtWistar), and T2DN(mtFHH) rats with or without APC (1.4% isoflurane) in the presence or absence of antioxidant N-acetylcysteine. Flavoprotein fluorescence intensity, a marker of mitochondrial redox state, 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein fluorescence intensity, a marker of reactive oxygen species generation, and mitochondrial permeability transition pore opening were assessed in isolated rat ventricular cardiomyocytes with or without isoflurane (0.5 mmol/l).

Results: Myocardial infarct size was decreased by APC in Wistar and T2DN(mtWistar) rats (to 42 ± 6%, n = 8; and 44 ± 7%, n = 8; of risk area, respectively) compared with their respective controls (60 ± 3%, n = 6; and 59 ± 9%, n = 7), but not in T2DN(mtFHH) rats (60 ± 2%, n = 8). N-acetylcysteine applied during isoflurane treatment restored APC in T2DN(mtFHH) (39 ± 6%, n = 7; and 38 ± 5%, n = 7; 150 and 75 mg/kg N-acetylcysteine, respectively), but abolished protection in control rats (54 ± 8%, n = 6). Similar to the data on infarct size, APC delayed mitochondrial permeability transition pore opening in T2DN(mtWistar) but not in T2DN(mtFHH) cardiomyocytes. Isoflurane increased flavoprotein and 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein fluorescence intensity in all rat strains, with the greatest effect in T2DN(mtFHH) cardiomyocytes.

Conclusion: Differences in the mitochondrial genome modulate isoflurane-induced generation of reactive oxygen species which translates into differential susceptibility to APC and ischemia-reperfusion injury in diabetic rats.

Figure 1

Schematic diagram depicting the experimental protocols used to determine myocardial infarct size in Wistar and type 2 diabetes mellitus (T2DN) rats in vivo. APC = anesthetic preconditioning; MAC = minimum alveolar concentration; NAC = N-acetylcysteine; OCC = coronary artery occlusion.

Figure 2

Myocardial infarct size depicted as a percentage of the area at risk (% of AAR) for infarction in Wistar, type 2 diabetes mellitus (T2DN)^mtWistar and T2DN^mtFHH rats, respectively. Data are expressed as mean ± SD. *P < 0.05 versus respective control without treatment; †P < 0.05 versus respective Wistar control. APC = anesthetic preconditioning; FHH = fawn-hooded hypertensive; mt = mitochondrial; NAC = N-acetylcysteine

Figure 3

Native fluorescence of flavoproteins (FP) depicted as an indicator of mitochondrial redox state in cardiomyocytes. (A) Experimental protocol. (B) Representative confocal microscopy images of FP fluorescence in cardiomyocytes before (Baseline), during (ISO) and after (Washout) the addition of 0.5 mmol/l isoflurane. (C) Data summary of baseline fluorescence intensity indicates no significant difference between the strains (P > 0.05). (D) Summarized data of FP fluorescence during ISO exposure and washout expressed as change from the baseline (100%). Isoflurane induced oxidation of mitochondrial flavoproteins in cardiomyocytes isolated from type 2 diabetes mellitus (T2DN)^mtFHH rats to a larger extend than in Wistar and T2DN^mtWistar cardiomyocytes, respectively. Data are expressed as mean ± SD, n = 6. *P < 0.05 versus baseline; ^#P < 0.05 versus Wistar and versus T2DN^mtWistar. a.u. = arbitrary units; FHH = fawn-hooded hypertensive; ISO = isoflurane; mt = mitochondrial; TYR = Tyrode solution.

Figure 4

ROS generation in cardiomyocytes as evaluated by 5-(and-6)-chloromethyl-2′,7′-dichlorofluorescein (CM-DCF) fluorescence. (A) Experimental protocol. (B) Representative confocal microscopy images of CM-DCF fluorescence in cardiomyocytes before and after addition of 0.5 mmol/l isoflurane (ISO). (C) Quantitative analysis of baseline CM-DCF fluorescence intensity indicates a significant increase in ROS generation in type 2 diabetes mellitus (T2DN) as compared with Wistar strains. (D) Time-dependent changes in CM-DCF fluorescence after exposure to ISO. Data are normalized to baseline and presented as mean ± SD. *P < 0.05 versus baseline; ^§P < 0.05 versus Wistar; ^#P < 0.05 versus T2DN^mtWistar. a.u. = arbitrary units; CM-H2DCFDA = 5-(and-6)-chloromethyl-2′,7′-dichlorofluorescein diacetate acetyl ester; FHH = fawn-hooded hypertensive; mt = mitochondrial; TYR = Tyrodes solution;.

Figure 5

Opening of the mitochondrial permeability transition pore [mPTP: measured with the mitochondrial potentiometric dye tetramethylrhodamine (TMRE; 100 nmol/l)] after photoexcitation-generated oxidative stress and assessed as rapid and complete mitochondrial depolarization in cardiomyocytes. (A) Experimental protocol. (B) Representative confocal microscopy images of TMRE fluorescence from a 50 μm² region of cardiomyocytes showing mPTP opening in individual mitochondria. (C) Arbitrary mPTP opening time expressed as time required to decrease baseline TMRE fluorescence intensity by half (arrows). (D) Anesthetic preconditioning (APC) extended the arbitrary mPTP opening time in Wistar, type 2 diabetes mellitus (T2DN)^mtWistar, but not in T2DN^mtFHH cardiomyocytes. *P < 0.05 versus T2DN^mtFHH. a.u. = arbitrary units; FHH = fawn-hooded hypertensive; ISO = isoflurane; mt = mitochondrial; TYR = Tyrodes solution.