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. 2022 Nov;164(5):e207-e226.
doi: 10.1016/j.jtcvs.2021.06.029. Epub 2021 Jun 26.

Inhibition of mitochondrial reactive oxygen species improves coronary endothelial function after cardioplegic hypoxia/reoxygenation

Yi Song  1 , Hang Xing  1 , Yixin He  1 , Zhiqi Zhang  1 , Guangbin Shi  1 , Su Wu  1 , Yuhong Liu  1 , Elizabeth O Harrington  2 , Frank W Sellke  1 , Jun Feng  3
Affiliations

Inhibition of mitochondrial reactive oxygen species improves coronary endothelial function after cardioplegic hypoxia/reoxygenation

Yi Song et al. J Thorac Cardiovasc Surg. 2022 Nov.

Abstract

Objective: Cardioplegic ischemia-reperfusion and diabetes mellitus are correlated with coronary endothelial dysfunction and inactivation of small conductance calcium-activated potassium channels. Increased reactive oxidative species, such as mitochondrial reactive oxidative species, may contribute to oxidative injury. Thus, we hypothesized that inhibition of mitochondrial reactive oxidative species may protect coronary small conductance calcium-activated potassium channels and endothelial function against cardioplegic ischemia-reperfusion-induced injury.

Methods: Small coronary arteries and endothelial cells from the hearts of mice with and without diabetes mellitus were isolated and examined by using a cardioplegic hypoxia and reoxygenation model to determine whether the mitochondria-targeted antioxidant Mito-Tempo could protect against coronary endothelial and small conductance calcium-activated potassium channel dysfunction. The microvessels or mouse heart endothelial cells were treated with or without Mito-Tempo (0-10 μM) 5 minutes before and during cardioplegic hypoxia and reoxygenation. Microvascular function was assessed in vitro by vessel myography. K+ currents of mouse heart endothelial cells were measured by whole-cell patch clamp. The levels of intracellular cytosolic free calcium (Ca2+) concentration, mitochondrial reactive oxidative species, and small conductance calcium-activated potassium protein expression of mouse heart endothelial cells were measured by Rhod-2 fluorescence staining, MitoSox, and Western blotting, respectively.

Results: Cardioplegic hypoxia and reoxygenation significantly attenuated endothelial small conductance calcium-activated potassium channel activity, caused calcium overload, and increased mitochondrial reactive oxidative species of mouse heart endothelial cells in both the nondiabetic and diabetes mellitus groups. In addition, treating mouse heart endothelial cells with Mito-Tempo (10 μM) reduced cardioplegic hypoxia and reoxygenation-induced Ca2+ and mitochondrial reactive oxidative species overload in both the nondiabetic and diabetes mellitus groups, respectively (P < .05). Treatment with Mito-Tempo (10 μM) significantly enhanced coronary relaxation responses to adenosine 5'-diphosphate and NS309 (P < .05), and endothelial small conductance calcium-activated potassium channel currents in both the nondiabetic and diabetes mellitus groups (P < .05).

Conclusions: Administration of Mito-Tempo improves endothelial function and small conductance calcium-activated potassium channel activity, which may contribute to its enhancement of endothelium-dependent vasorelaxation after cardioplegic hypoxia and reoxygenation.

Keywords: cardiac surgery; cardioplegia; cardioplegic arrest; cardioplegic hypoxia and reoxygenation; cardioplegic ischemia and reperfusion; cardiopulmonary bypass; coronary endothelial function; coronary endothelium-dependent vasodilation; coronary microcirculation; diabetes; endothelial function; ion channels; ischemia–reperfusion; mitochondria; mitochondrial reactive oxygen species; potassium channels; reactive oxygen species; small conductance calcium-activated potassium channels.

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Conflict of interest statement

Conflict of interests and any other disclosure: Conflict of interests: No any potential conflicts of interest exist for all authors; Any other disclosure: No any potential conflicts of interest exist for all authors.

Figures

Figure 1.
Figure 1.. Dose-dependent effects of Mito-Tempo (MT) treatment (0-10μM) on the recovery of relaxation responses of mouse small coronary arteries following CP-H/R.
The diabetic (DM) or non-diabetic (ND) microvessels were pre-constricted with the thromboxane A2 analog U46619 to reach a 30%-40% reduction of the baseline diameter. After a stable constriction, microvascular relaxation in response to the SK activator NS309 (0-10μM); (A), ADP (10μM); (B); and (C) SNP(100μM) in the presence or absence of MT (0-10μM) was measured. ND(H/R) = ND treated by hypoxia/re-oxygenation; ND(H/R)+1μM MT = ND(H/R) + 1μM mito-Tempo; ND(H/R)+10μM MT = ND(H/R) + 10 μM mito-Tempo; DM(H/R) = DM treated by hypoxia/re-oxygenation; DM(H/R) +1μM MT = DM(H/R) + 1μM mito-Tempo; DM(H/R) +10μM MT = DM(H/R) + 10μM mito-Tempo; n= 5-6/group; A, @P =0.0002, ND(H/R) vs. DM(H/R); *P= 0.002 , ND(H/R) + 10μM MT vs. ND (H/R) #P = 0.0329, DM(H/R) + 10μM MT vs. DM(H/R); $P <0.0001, ND(H/R) +10μM MT vs. DM(H/R) +10μM MT; B, @P <0.0001, ND(H/R) vs. DM(H/R); *P = 0.0438, ND(H/R) + 10μM MT vs. ND (H/R); #P =0.0293, DM(H/R) + 10μM MT vs. DM(H/R); $P <0.0001, ND(H/R) +10μM MT vs. DM(H/R) +10μM MT; Mean ± SD, Two-way ANOVA with a post hoc Bonferroni test.
Figure 2.
Figure 2.. Hypoxia/re-oxygenation (H/R) altered Ca2+ homeostasis in ND and DM mouse house endothelial cells (MHECs).
MHECs were isolated from diabetic (DM) or non-diabetic (ND) mice. A, Representative fluorescent of intracellular calcium (Ca2+) of MHECs treated with or without MT (10μM). B, Quantitative analysis of intracellular Ca2+ concentration changes of MHECs in the following experimental groups: ND, n=22; ND(H/R), n=16; ND(H/R) + 10μM MT, n=18; DM, n=20; DM(H/R), n=25; DM(H/R) +10μM MT, n=28; *P <0.0001, ND(H/R) vs. ND; @P <0.0001, ND(H/R) + 10μM MT vs. ND(H/R); &P <0.0001, DM vs. ND; † P <0.0001, DM(H/R) vs. DM; $P <0.0001, DM(H/R) +10μM MT vs. DM(H/R). C, Representative traces of the whole cell currents of MHECs with different free calcium concentration. D, The plots shows Apamin+TRAM34-sensitive component of K+ current at +100 mV (0 calcium, n=4; 400nM Ca2+, n=3; 2μM Ca2+, n=3), *P <0.0001, 400nM Ca2+ vs. 0 Ca2+; @P <0.0001, 400nM Ca2+ vs. 2μM Ca2+; Mean ± SD, ND(H/R) = ND treated by hypoxia/re-oxygenation; DM(H/R) = DM treated by hypoxia/re-oxygenation; ND(H/R) +10μM MT = ND(H/R) + 10μM mito-Tempo; DM(H/R) +10μM MT = DM(H/R) + 10μM mito-Tempo; Mean ±SD, One-way ANOVA with a post hoc Dunnett's multiple comparisons test.
Figure 3.
Figure 3.. Mito-Tempo (MT) significantly increases SK channel currents of mouse heart endothelial cells (MHECs) in H/R model from ND and DM mice.
A, Representative traces of the whole cell currents of MHECs treated with or without MT (10μM) at holding potential of −50 mV and test potentials from −100 to +100 mV in 20 mV increments. B, whole-cell I–V relationships sensitive to NS309 in MHECs of ND(H/R) and DM(H/R) with or without MT treatment. C, whole-cell I–V relationships sensitive to TRAM34+Apamin in MHECs of ND(H/R) and DM(H/R) with or without MT treatment. D, Box plots shows NS309-sensitive component of potassium current at +100 mV in ND(H/R) and DM(H/R) MHECs treat with or without MT (n=5/group). *P = 0.0074, ND(H/R) +10μM MT vs. ND(H/R); @P = 0.0162, DM(H/R) +10μM MT vs. DM(H/R). E, Box plots shows TRAM34+Apamin-sensitive component of potassium current at +100 mV in ND(H/R) and DM(H/R) MHECs treat with or without MT (n=5/group). *P =0.0322, ND(H/R) +10μM MT vs. ND(H/R); @P =0.0451, DM(H/R) +10μM MT vs. DM(H/R). ND(H/R) = ND treated by hypoxia/re-oxygenation; DM(H/R) = DM treated by hypoxia/re-oxygenation; ND(H/R) +10μM MT = ND(H/R) + 10μM mito-Tempo; DM(H/R) +10μM MT = DM(H/R) + 10μM mito-Tempo; Mean ± SD, One-way ANOVA with a post hoc Sidak's multiple comparisons test.
Figure 4.
Figure 4.. Mito-Tempo (MT) significantly increases SK channel currents of MHECs in H/R model from ND and DM mice.
A-D, Time course of the whole-cell current density evoked at +100mV from MHECs using patch clamp. NS309 was added to the bath to activated IK/SK channels, followed by bath application of Apamin and TRAM34 to block them. E, Box plots shows NS309-sensitive component of potassium current at +100 mV in ND(H/R) and DM(H/R) MHECs treat with or without MT (n=3/group); *P =0.0486, ND(H/R)+10μM MT vs. ND(H/R); @P =0.0082, DM(H/R)+10μMT vs. DM(H/R). F, Box plots shows Apamin+TRAM34-sensitive component of potassium current at +100 mV in ND(H/R) and DM(H/R) MHECs treat with or without MT (n=3/group). *P =0.0233, ND(H/R) +10μM MT vs. ND(H/R); @P =0.0203, DM(H/R) +MT vs. DM(H/R); ND(H/R) = ND treated by hypoxia/re-oxygenation; ND(H/R) +10μM MT = ND(H/R) + 10μM mito-Tempo; DM(H/R) = DM treated by hypoxia/re-oxygenation; DM(H/R) +10μM MT = DM(H/R) + 10 μM mito-Tempo; Mean ± SD, One-way ANOVA with Student's t-test.
Figure 5.
Figure 5.. H/R and MT treatment failed to effect SK3 and SK4 protein expression in the mouse heart endothelial cells (MHECs).
A and B, Immunoblot intensity of small conductance calcium activated potassium channels (SK), SK3 (A), SK4 (B) with a GAPDH loading control, C and D, graphs showing densitometric analysis of immunoband intensity of SK3 (C) and SK4(D) protein expression in the experimental groups. ND = non-diabetes, DM- diabetes; ND (H/R) = ND cells treated by hypoxia/re-oxygenation; DM (H/R) = DM cells treated by hypoxia/re-oxygenation; ND (H/R) + 10μM MT = ND(H/R) cells treated with 10μM mito-Tempo; DM (H/R) + 10 μM MT = DM (H/R) cells treated with 10μM mito-Tempo; n =3/group. Mean ± SD, One-way ANOVA and a post hoc Bonferroni test.
Figure 6.
Figure 6.. The effects of mito-Tempo on the production of mitochondrial ROS (mROS) induced by hypoxia/re-oxygenation in ND and DM MHECs.
Mitochondria localization was visualized using MitoTracker and mROS production was monitored with MitoSOX following exposure to mito-Tempo induced by H/R in ND (A) and DM (B) MHECs. C, Box plots summarizing the data analysis of mROS in the experimental groups, n =5/group. *P <0.0001, ND(H/R) vs. ND; @P <0.0001, ND(H/R) +MT vs. ND(H/R); &P <0.0001, DM vs. ND; †P <0.0001, DM(H/R) vs. DM; $P <0.0001, DM(H/R) +10μM MT vs. DM(H/R). ND = ND cells under normoxic condition; DM = DM cells under normoxic condition; ND(H/R) = ND treated by hypoxia/re-oxygenation; DM(H/R) = DM cells under hypoxia/re-oxygenation; ND(H/R) +10μM MT = ND(H/R) + 10μM mito-Tempo; DM(H/R)+10 μM MT = DM(H/R)+mito-Tempo. Mean ± SD, One-way ANOVA and a post hoc Bonferroni test.
Figure 7:
Figure 7:
Research summary concerning that treatment with the mROS inhibitor, Mito-Tempo (MT) increases endothelial SK-currents and NO signaling pathways, resulting in improving coronary endothelial function, EDH and coronary relaxation in the setting of cardioplegic hypoxia and re-oxygenation (CP-H/R) in mice with or without diabetes (DM); EDH = endothelium-dependent hyperpolarizing; LAD = left anterior descending artery; ND (H/R) = ND treated by hypoxia/re-oxygenation, ND(H/R) + 1μM MT = ND(H/R) + 1μM mito-Tempo; ND(H/R) + 10μM MT = ND(H/R) + 10μM mito-Tempo; DM (H/R) = DM treated by hypoxia/re-oxygenation; DM(H/R) + 1μM MT = DM + 1μM mito-Tempo; DM(H/R) + 10μM MT = DM + 10μM mito-Tempo; CP-H/R = cardioplegic hypoxia/reoxygenation; EDH = endothelial-derived hyperpolarization; LAD = left anterior descending artery; ROS; = reactive oxygen species; mROS = mitochondrial ROS; NO = nitric oxide; SK channels = small conductance calcium-activated potassium channels. @P =0.0002, ND(H/R) vs. DM(H/R); *P= 0.002 , ND(H/R) + 10μM MT vs. ND (H/R); #P = 0.0329, DM(H/R) + 10μM MT vs. DM(H/R); $P <0.0001, ND(H/R) +10μM MT vs. DM(H/R) +10μM MT; n =5-6/group, Mean ± SD, 2-Way-ANOVA repeated measurement.
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