Senescence marker protein-30 (SMP30) deficiency impairs myocardium-induced dilation of coronary arterioles associated with reactive oxygen species

Abstract

Senescence marker protein-30 (SMP30) decreases with aging. Mice with SMP30 deficiency, a model of aging, have a short lifespan with increased oxidant stress. To elucidate SMP30's effect on coronary circulation derived from myocytes, we measured the changes in the diameter of isolated coronary arterioles in wild-type (WT) mice exposed to supernatant collected from isolated paced cardiac myocytes from SMP30 KO or WT mice. Pacing increased hydrogen peroxide in myocytes, and hydrogen peroxide was greater in SMP30 KO myocytes compared to WT myocytes. Antimycin enhanced and FCCP (oxidative phosphorylation uncoupler in mitochondria) decreased superoxide production in both groups. Addition of supernatant from stimulated myocytes, either SMP30 KO or WT, caused vasodilation. The degree of the vasodilation response to supernatant was smaller in SMP30 KO mice compared to WT mice. Administration of catalase to arterioles eliminated vasodilation in myocyte supernatant of WT mice and converted vasodilation to vasoconstriction in myocyte supernatant of SMP30 KO mice. This vasoconstriction was eliminated by olmesartan, an angiotensin II receptor antagonist. Thus, SMP30 deficiency combined with oxidant stress increases angiotensin and hydrogen peroxide release from cardiac myocytes. SMP30 plays an important role in the regulation of coronary vascular tone by myocardium.

Figure A1

SMP30 expression in myocardium of young and old mice. We measured SMP30 expression in myocardium of young (3 months old) and old (12 months old) mice (n = 5 each) by western blotting. Total protein was extracted from the snap-frozen left ventricle using Cell Lysis Buffer (Cell Signaling Technology, Inc., Beverly, MA, USA) with Protease Inhibitor Cocktail (BD Biosciences, San Jose, CA, USA) as previously reported [33]. Protein concentration was determined by protein assay (DC Protein Assay Kit, Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal amounts (20 μg) of the protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 5%–20%) and transferred onto polyvinylidene difluoride membranes (ATTO Co., Tokyo, Japan). The primary antibodies were anti-SMP30 (SHIMA Laboratories Co. Ltd., Tokyo, Japan) and mouse anti-β-actin (Santa Cruz Biotechnology Inc., California, CA, USA). The secondary antibodies were goat anti-rabbit IgG-horseradish peroxidase and goat anti-mouse IgG-horseradish peroxidase (Santa Cruz Biotechnology Inc., California, CA, USA). The signals from immunoreactive bands were visualized by an Amersham ECL system (Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, UK) and quantified using densitometric analysis. SMP30 expression was lower in myocardium of old mice (12 months old) than young mice (3 months old). Data are expressed as the mean ± S.D.

Figure 1

DHE and DCF staining in cardiac myocytes. Representative DHE (A) and DCF (B) staining in cardiac myocytes (Upper panel). Summary data of DHE and DCF staining in cardiac myocytes (Lower panel). The signals of DHE and DCF increased with electrical stimulation. DHE and DCF signals were more potent in SMP30 KO cardiac myocytes compared to WT cardiac myocytes with electrical stimulation. Values are expressed as the mean ± S.E.M. * p < 0.01 vs. non-stimulated, # p < 0.01 vs. WT mice in same staining (n = 12 each).

Figure 1

DHE and DCF staining in cardiac myocytes. Representative DHE (A) and DCF (B) staining in cardiac myocytes (Upper panel). Summary data of DHE and DCF staining in cardiac myocytes (Lower panel). The signals of DHE and DCF increased with electrical stimulation. DHE and DCF signals were more potent in SMP30 KO cardiac myocytes compared to WT cardiac myocytes with electrical stimulation. Values are expressed as the mean ± S.E.M. * p < 0.01 vs. non-stimulated, # p < 0.01 vs. WT mice in same staining (n = 12 each).

Figure 2

Effect of SMP30 deficiency on generation of superoxide and activity of NADPH oxidase in cardiac myocytes under electrical stimulation. Generation of superoxide (A) was measured by HPLC. NADPH oxidase activity was measured by lucigenin luminescence; (B) The levels of superoxide and NADPH oxidase activity were greater in SMP30 KO mice compared to WT mice under electrical stimulation. Values were expressed as the mean ± S.E.M. * p < 0.01 vs. without agents. n = 8, each.

Figure 3

DHE and DCF staining in paced cardiac myocytes with antimycin or FCCP. Representative DHE (A) and DCF staining (B) in cardiac myocytes (Upper panel). Summary data of DHE and DCF staining in cardiac myocytes (Lower panel). The signals of DHE and DCF were enhanced by antimycin (2 μM) and attenuated by FCCP (1 μM). Values are expressed as the mean ± S.E.M. * p < 0.01 vs. without agents (n = 12 each).

Figure 4

The level of H₂O₂ in cardiac myocyte supernatant. The concentration of H₂O₂ in the cardiac myocyte supernatant increased with pacing. H₂O₂ in SMP30 KO myocytes was higher than in WT cardiac myocytes. Values are expressed as the mean ± S.E.M. * p < 0.01 vs. WT mice (n = 12 each).

Figure 5

Catalase activity. Catalase activity was not different between WT and SMP30 KO myocardium. Values are expressed as the mean ± S.E.M (n = 12 each).

Figure 6

SOD activity. SOD activity was not different between WT and SMP30 KO myocardia. Values are expressed as the mean ± S.E.M (n = 12 each).

Figure 7

Vasodilation induced by the supernatant from stimulated cardiac myocytes. The supernatant of electrically stimulated cardiac myocytes dilated WT coronary arterioles dose-dependently. Vasodilation with WT cardiac myocyte supernatant was more potent compared to that with SMP30 KO cardiac myocyte supernatant (A) Administration of olmesartan to coronary arterioles enhanced vasodilation in the SMP30 KO cardiac myocyte supernatant treatment group (500 μL of supernatant). Administration of catalase to coronary arterioles converted vasodilation to vasoconstriction in the SMP30 KO cardiac myocyte supernatant treatment group and eliminated vasodilation in the WT cardiac myocyte supernatant treatment group. Treatment with olmesartan in addition to catalase in the vessel bath eliminated vasoconstriction in the SMP 30 KO cardiac myocyte supernatant treatment group; (B) Values are expressed as the mean ± S.E.M. n = 12 each, * p < 0.01 vs. without agent.

Figure 7

Vasodilation induced by the supernatant from stimulated cardiac myocytes. The supernatant of electrically stimulated cardiac myocytes dilated WT coronary arterioles dose-dependently. Vasodilation with WT cardiac myocyte supernatant was more potent compared to that with SMP30 KO cardiac myocyte supernatant (A) Administration of olmesartan to coronary arterioles enhanced vasodilation in the SMP30 KO cardiac myocyte supernatant treatment group (500 μL of supernatant). Administration of catalase to coronary arterioles converted vasodilation to vasoconstriction in the SMP30 KO cardiac myocyte supernatant treatment group and eliminated vasodilation in the WT cardiac myocyte supernatant treatment group. Treatment with olmesartan in addition to catalase in the vessel bath eliminated vasoconstriction in the SMP 30 KO cardiac myocyte supernatant treatment group; (B) Values are expressed as the mean ± S.E.M. n = 12 each, * p < 0.01 vs. without agent.