Coronary artery spasm related to thiol oxidation and senescence marker protein-30 in aging

Abstract

Background: Senescence marker protein-30 (SMP30) decreases with aging, and SMP30 knockout (KO) mice show a short life with increased oxidant stress.

Aims: We assessed the effect of oxidant stress with SMP30 deficiency in coronary artery spasm and clarify its underlying mechanisms.

Results: We measured vascular responses to acetylcholine (ACh) and sodium nitroprusside (SNP) of isolated coronary arteries from SMP30 KO and wild-type (WT) mice. In SMP30 KO mice, ACh-induced vasoconstriction occurred, which was changed to vasodilation by dithiothreitol (DTT), a thiol-reducing agent. However, Nω-nitro-L-arginine-methyl ester, nitric oxide (NO) synthase inhibitor, or tetrahydrobiopterin did not change the ACh response. In isolated coronary arteries of WT mice, ACh-induced vasodilation occurred. Inhibition of glutathione reductase by 1, 3-bis(2-chloroethyl)-1-nitrosourea decreased ACh-induced vasodilation (n=10, p<0.01), which was restored by DTT. To evaluate the thiol oxidation, we measured the fluorescence of monochlorobimane (MCB) in coronary arteries, which covalently labels the total. The fluorescence level to MCB decreased in SMP30 KO mice, but with DTT treatment restored to a level comparable to that of WT mice. The reduced glutathione and total thiol levels were also low in the aorta of SMP30 KO mice compared with those of WT mice. Administration of ACh into the aortic sinus in vivo of SMP30 KO mice induced coronary artery spasm.

Innovation: The thiol redox state is a key regulator of endothelial NO synthase activity, and thiol oxidation was associated with endothelial dysfunction in the SMP30 deficiency model.

Conclusion: These results suggest that chronic thiol oxidation by oxidant stress is a trigger of coronary artery spasm, resulting in impaired endothelium-dependent vasodilation.

FIG. 1.

Nitric oxide (NO) generation in aorta. Representative electrochemical detection tracing of NO concentration in isolated aortas from the senescence marker protein-30 (SMP30) knockout (KO) and wild type (WT) mice in an organ chamber. NO concentration is directly proportional to the current (pA) by a free-radical analyzer (Apollo 4000). ACh (1 μM)-stimulated NO release was attenuated in SMP30 KO mice vs. WT mice (p<0.01). ACh indicates acetylcholine administration.

FIG. 2.

Isolated coronary artery responses to Ach (1×10⁻¹⁰–1×10⁻⁵ M ) in SMP30 KO and WT mice in an organ chamber. ACh-induced vasoconstriction appeared in the SMP30 KO mice. The NOS inhibitor, N^ω-nitro-l-arginine-methyl ester (L-NAME) (0.3 mM), did not further enhance this response. In the WT mice, ACh-induced vasodilation appeared, blunted with L-NAME (A). BH4 (1 μM) did not change ACh-induced vasoconstriction in the SMP30 KO mice (B). ACh-induced vasoconstriction in the SMP30 KO mice changed to vasodilation with the thiol-reducing agent, dithiothreitol (DTT) (0.1 μM) (C). Inhibition of glutathione reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (80 μM) decreased ACh-induced vasodilation, which was restored by DTT in the WT mice (D). Values were expressed as means±S.E.M., n=10, each, *p<0.01.

FIG. 3.

Sodium nitroprusside (SNP) response in isolated coronary arteries from SMP30 KO and WT mice in an organ chamber. SNP (1×10⁻¹⁰–1×10⁻⁵ M) induced vasodilation dose dependently, which was comparable between the SMP30 and WT mice. Values were expressed as means±S.E.M., n=10, each.

FIG. 4.

Hydrogen peroxide (H₂O₂) generation in the aorta. H₂O₂ production determined by the Amplex-Red assay in the aortic tissue of WT and SMP30 KO mice. SMP30 KO mice had a more than threefold increase in H₂O₂ levels. Values were expressed as means±S.E.M., n=10, each, *p<0.01.

FIG. 5.

Plasma and aorta asymmetric dimethylarginine (ADMA) levels. Plasma ADMA was higher in SMP30 KO mice than WT mice (A). Aorta ADMA was similarly higher in SMP30 KO mice (B). Values were expressed as means±S.E.M., n=10, each, *p<0.01.

FIG. 6.

Aortic tissue total thiols and glutathione levels. In aortic tissue, the levels of total thiols (A) and of reduced glutathione (GSH) (B) decreased in SMP30 KO mice compared to those in WT mice. In contrast, the level of oxidized glutathione (GSSG) increased in SMP30 KO mice (C). Deficiency of SMP30 led to a decrease in the ratio of reduced to oxidized glutathione (GSH/GSSG) (D). The values were expressed as means±S.E.M., n=10, each, *p<0.01.

FIG. 7.

DHE staining in coronary arteries. Production of superoxide anion radical was measured by staining with 10 μM dihydroethidium (DHE) in the isolated coronary arteries. Upper panel shows representative DHE, and the lower panel shows summary data of DHE fluorescent intensity (in arbitrary units) in the coronary arteries. The signal of DHE was potent with SMP30 deficiency, which was attenuated with apocynin (0.3 mM). The values were expressed as means±S.E.M. *p<0.01 vs. without any treatment in WT mice, ^#p<0.01, vs. without treatment in SMP30 KO mice, n=10, each.

FIG. 8.

Oxidation of thiols in coronary arteries. We investigated the modification of thiol oxidation with ACh (1 μM) or DTT (0.1 μM) in the isolated coronary arteries. Total or extracellular thiols were labeled with either monochlorobimane (MCB) (20 μM, A) or monobromotrimethyl ammoniobimane (MBB) (20 μM, B), respectively. Upper panels show fluorescent images of total reduced or extracellular reduced free thiol groups, and the lower panels show the summary data of fluorescent intensity (in arbitrary units) in isolated coronary arteries. A decrease in fluorescence indicates thiol oxidation with less binding of either fluorochrome. Fluorescence from MCB or MBB was decreased (increased thiol oxidation) in SMP30 KO mice compared to those of the WT mice. Ach decreased the MCB signal in the coronary arteries of the WT mice. Fluorescence to either indicator increased after administration of DTT (0.1 μM). Decrease of the fluorescent intensity was greater in MCB than in MBB of the SMP30 KO mice, indicating the effect on intracellular thiol oxidation. The values were expressed as means±S.E.M., n=10, each. *p<0.01, **p<0.05 versus without any treatment of WT mice. ^#p<0.01, ^##p<0.05 versus without any treatment of SMP30 KO mice.

FIG. 9.

Ach-induced coronary artery spasm. Intra-aortic sinus administration of ACh (2 μg) to the SMP30 KO mice induced transient ST-T segment elevation and reciprocal ST-T segment depression in electrocardiogram (A). In the WT mice, ACh-induced ST-T segment change did not appear (B). Before, after 1, 2, and 5 min; before, after 1-, 2-, and 5-min intra-aortic sinus administration of ACh, respectively.