Assessment of Mitochondrial Dysfunction and Monoamine Oxidase Contribution to Oxidative Stress in Human Diabetic Hearts

Abstract

Mitochondria-related oxidative stress is a pathomechanism causally linked to coronary heart disease (CHD) and diabetes mellitus (DM). Recently, mitochondrial monoamine oxidases (MAOs) have emerged as novel sources of oxidative stress in the cardiovascular system and experimental diabetes. The present study was purported to assess the mitochondrial impairment and the contribution of MAOs-related oxidative stress to the cardiovascular dysfunction in coronary patients with/without DM. Right atrial appendages were obtained from 75 patients randomized into 3 groups: (1) Control (CTRL), valvular patients without CHD; (2) CHD, patients with confirmed CHD; and (3) CHD-DM, patients with CHD and DM. Mitochondrial respiration was measured by high-resolution respirometry and MAOs expression was evaluated by RT-PCR and immunohistochemistry. Hydrogen peroxide (H2O2) emission was assessed by confocal microscopy and spectrophotometrically. The impairment of mitochondrial respiration was substrate-independent in CHD-DM group. MAOs expression was comparable among the groups, with the predominance of MAO-B isoform but no significant differences regarding oxidative stress were detected by either method. Incubation of atrial samples with MAOs inhibitors significantly reduced the H2O2 in all groups. In conclusion, abnormal mitochondrial respiration occurs in CHD and is more severe in DM and MAOs contribute to oxidative stress in human diseased hearts with/without DM.

Figure 1

Respiratory parameters for CI-supported respiration (n = 20/CTRL group, n = 25/CHD group, and n = 15/CHD-DM group; values are means ± SEM; ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001).

Figure 2

Respiratory parameters for CII-supported respiration (n = 20/CTRL group, n = 25/CHD group, and n = 15/CHD-DM group; values are means ± SEM; ^∗∗ p < 0.01).

Figure 3

Atrial H₂O₂ emission detected with DCF fluorescence production. Fluorescent green positive staining was measured by confocal microscopy in the dichlorofluorescein- (DCF-) treated images. Levels of H₂O₂ detected with DCF fluorescence were similar in the studied groups (n = 10/group). Spectral unmixing was done using Lambda scan acquisition mode, range 490–600 nm, step 10 nm. The resulting composite image shows the real DCF component (red channel) on the autofluorescent background (green channel).

Figure 4

Atrial H₂O₂ emission detected with FOX assay. (a) Levels of H₂O₂ were similar in all groups (n = 5/group). (b1)–(b3) H₂O₂ level in the presence of selegiline and clorgyline (10 μM each) versus their corresponding controls (i.e., not treated atrial samples: NO TM) in each of the studied groups (n = 5/group; values are means ± SEM; ^∗ p < 0.05 versus not treated atrial samples).

Figure 5

MAO mRNA expression in human atrial samples. (a)–(c) RT-PCR (mRNA expression: 2^−ΔΔCt) for MAO-A and MAO-B relative to the housekeeping gene EEF2α in atrial samples from CTRL group (a), CHD group (b), CHD-DM (c), n = 10, ^∗ p < 0.05, and (d) RT-PCR (fold increase) for MAO-A and MAO-B relative to the housekeeping gene EEF2α in atrial samples (n = 10/group).

Figure 6

MAO protein expression in human atrial samples. The immunohistochemistry assay for MAO-A and MAO-B in atrial samples (a) and the corresponding results expressed as a mean score of intensity (b) (n = 10/group).