Deficiency of Senescence Marker Protein 30 Exacerbates Cardiac Injury after Ischemia/Reperfusion

Early myocardial reperfusion is an effective therapy but ischemia/reperfusion (I/R) causes lethal myocardial injury. The aging heart was reported to show greater cardiac damage after I/R injury than that observed in young hearts. Senescence marker protein 30 (SMP30), whose expression decreases with age, plays a role in reducing oxidative stress and apoptosis. However, the impact of SMP30 on myocardial I/R injury remains to be determined. In this study, the left anterior descending coronary artery was occluded for 30 min, followed by reperfusion in wild-type (WT) and SMP30 knockout (KO) mice. After I/R, cardiomyocyte apoptosis and the ratio of infarct area/area at risk were higher, left ventricular fractional shortening was lower, and reactive oxygen species (ROS) generation was enhanced in SMP30 KO mice. Moreover, the previously increased phosphorylation of GSK-3β and Akt was lower in SMP30 KO mice than in WT mice. In cardiomyocytes, silencing of SMP30 expression attenuated Akt and GSK-3β phosphorylation, and increased Bax to Bcl-2 ratio and cardiomyocyte apoptosis induced by hydrogen peroxide. These results suggested that SMP30 deficiency augments myocardial I/R injury through ROS generation and attenuation of Akt activation.


Introduction
Percutaneous coronary intervention has been reported to reduce the mortality in patients with acute coronary infarction, since coronary reperfusion therapy is the most effective treatment for salvaging viable myocardium [1,2]. Ischemia/reperfusion (I/R) involves reactive oxygen species (ROS) generation, intracellular Ca 2+ disturbance, rapid pH restoration, and inflammation [3]; therefore, I/R is accompanied by detrimental manifestations [3], including myocardial necrosis and apoptosis [4,5].

Deficiency of SMP30 Exacerbates Infarct Size Induced by I/R
We compared myocardial damage in WT mice and SMP30 KO mice. After 30 min of ischemia and 24 h of reperfusion, the area at risk (AAR)/LV ratio in SMP30 KO mice was similar to that in WT mice. However, the size of infarction area (IA)/AAR ratio was significantly higher in SMP30 KO than in WT mice ( Figure 1A,B). In addition, serum CPK levels of SMP30 KO mice were significantly higher than those of WT mice 24 h after I/R ( Figure 1C). There was no significant difference in serum CPK levels between sham-operated WT and SMP30 KO mice. These results indicate that SMP30 deficiency enhanced myocardial injury during I/R.

Cardiac Function after I/R Was Exacerbated in SMP30 KO Mice Compared with WT Mice
We performed an echocardiography to assess the cardiac function in SMP30 KO and WT mice. There were no significant changes in echocardiographic parameters between sham-operated SMP30 KO and WT mice (Table 1, Figure 1D). At 24 h after reperfusion, the fractional shortening was significantly lower in SMP30 KO mice than in WT mice as shown in Table 1 and Figure 1E. . † p < 0.05 vs. WT I/R mice; (C) Serum CPK levels 24 h after I/R or sham operation in WT mice and SMP30 KO mice. Graphs show mean ± SE of serum CPK levels (n = 7). I/R, ischemia/reperfusion; AAR, area at risk; IA, infarct area; CPK, creatine phosphokinase. * p < 0.05 vs. sham-operated mice, † p < 0.05 vs. WT I/R mice; (D) Representative M-mode echocardiograms of left ventricles in WT and SMP30 KO mice 24 h after I/R and sham operation; (E) Fractional shortening 24 h after I/R or sham operation in WT mice and SMP30 KO mice. Graphs show mean ± SE of fractional shortening (n = 10 to 15). * p < 0.05 vs. sham-operated mice, † p < 0.05 vs. WT I/R mice.

Effect of SMP30 Deficiency on Oxidative Stress and Myocardial Apoptosis Induced by I/R
Because we have previously shown that ROS generation was increased in SMP30 KO mice after angiotensin II or doxorubicin administration, we evaluated the myocardial oxidative stress after I/R by dihydroethidium (DHE), which indicates superoxide production. I/R increased ROS generation in both WT and SMP30 KO mice; however, ROS generation induced in I/R SMP30 KO mice was significantly higher than that in WT mice (Figure 2A,B). Since ROS generation during I/R induced myocardial apoptosis, we performed a TUNEL staining to investigate the extent of apoptosis in the ischemic area after I/R. TUNEL-positive cells were evident in heart sections obtained from both SMP30-KO and WT ischemic areas. There was no significant difference between WT mice and SMP30 KO mice in sham operation. SMP30 KO mice exhibited significantly higher percentages of TUNEL-positive nuclei compared with WT mice, as shown in Figure 2C,D. These results indicated that SMP30 deficiency enhanced ROS generation in the heart and cardiomyocyte apoptosis during I/R.

Effect of SMP30 Deficiency on Oxidative Stress and Myocardial Apoptosis Induced by I/R
Because we have previously shown that ROS generation was increased in SMP30 KO mice after angiotensin II or doxorubicin administration, we evaluated the myocardial oxidative stress after I/R by dihydroethidium (DHE), which indicates superoxide production. I/R increased ROS generation in both WT and SMP30 KO mice; however, ROS generation induced in I/R SMP30 KO mice was significantly higher than that in WT mice (Figure 2A,B). Since ROS generation during I/R induced myocardial apoptosis, we performed a TUNEL staining to investigate the extent of apoptosis in the ischemic area after I/R. TUNEL-positive cells were evident in heart sections obtained from both SMP30-KO and WT ischemic areas. There was no significant difference between WT mice and SMP30 KO mice in sham operation. SMP30 KO mice exhibited significantly higher percentages of TUNEL-positive nuclei compared with WT mice, as shown in Figure 2C,D. These results indicated that SMP30 deficiency enhanced ROS generation in the heart and cardiomyocyte apoptosis during I/R.

Phosphorylation of ERK1/2, Akt, and Glycogen Synthase Kinase-3β (GSK-3β) after I/R
Activation of the reperfusion injury salvage kinase (RISK) pathway contributes to the reduction of I/R injury [26,27]. Therefore, we examined the phosphorylation of ERK1/2, Akt, and GSK-3β before and after I/R. Western blots revealed that ERK1/2, Akt, and GSK-3β phosphorylation were increased 30 min after reperfusion. There was no significant difference in ERK1/2 phosphorylation between WT and SMP30 KO mice; however, phosphorylation of Akt and GSK-3β after I/R was suppressed in SMP30 KO mice compared with WT mice. Since Akt and GSK-3β regulate the

Phosphorylation of ERK1/2, Akt, and Glycogen Synthase Kinase-3β (GSK-3β) after I/R
Activation of the reperfusion injury salvage kinase (RISK) pathway contributes to the reduction of I/R injury [26,27]. Therefore, we examined the phosphorylation of ERK1/2, Akt, and GSK-3β before and after I/R. Western blots revealed that ERK1/2, Akt, and GSK-3β phosphorylation were increased 30 min after reperfusion. There was no significant difference in ERK1/2 phosphorylation between WT and SMP30 KO mice; however, phosphorylation of Akt and GSK-3β after I/R was suppressed in SMP30 KO mice compared with WT mice. Since Akt and GSK-3β regulate the myocardial apoptotic pathway, we evaluated Bax and Bcl-2 expression levels after ischemia/reperfusion. The ratio of Bax to Bcl-2 expression was significantly higher in SMP30 KO mice than in WT mice ( Figure 3A,B). myocardial apoptotic pathway, we evaluated Bax and Bcl-2 expression levels after ischemia/reperfusion. The ratio of Bax to Bcl-2 expression was significantly higher in SMP30 KO mice than in WT mice ( Figure 3A,B). sham-operated mice, and † p < 0.05 vs. WT I/R mice.

SMP30 Silencing on ERK1/2, Akt, and GSK-3β Phosphorylation in Cardiomyocytes after H2O2 Stimulation
We found that Akt and GSK-3β phosphorylation were attenuated in SMP30-KO mice; however, it was possible that the potentiated generation of ROS in SMP30-KO mice after I/R (Figure 2A,B) influenced the degree of phosphorylation. To investigate the effect of SMP30, we transfected SMP30 siRNA or nonspecific control siRNA into neonatal rat cardiomyocytes. Expression of SMP30 was knocked down by its siRNA (Figure 4).   We found that Akt and GSK-3β phosphorylation were attenuated in SMP30-KO mice; however, it was possible that the potentiated generation of ROS in SMP30-KO mice after I/R (Figure 2A,B) influenced the degree of phosphorylation. To investigate the effect of SMP30, we transfected SMP30 siRNA or nonspecific control siRNA into neonatal rat cardiomyocytes. Expression of SMP30 was knocked down by its siRNA (Figure 4). myocardial apoptotic pathway, we evaluated Bax and Bcl-2 expression levels after ischemia/reperfusion. The ratio of Bax to Bcl-2 expression was significantly higher in SMP30 KO mice than in WT mice ( Figure 3A,B). sham-operated mice, and † p < 0.05 vs. WT I/R mice.

SMP30 Silencing on ERK1/2, Akt, and GSK-3β Phosphorylation in Cardiomyocytes after H2O2 Stimulation
We found that Akt and GSK-3β phosphorylation were attenuated in SMP30-KO mice; however, it was possible that the potentiated generation of ROS in SMP30-KO mice after I/R (Figure 2A,B) influenced the degree of phosphorylation. To investigate the effect of SMP30, we transfected SMP30 siRNA or nonspecific control siRNA into neonatal rat cardiomyocytes. Expression of SMP30 was knocked down by its siRNA (Figure 4).  To confirm the effect of SMP30 silencing on Akt and GSK-3β phosphorylation, neonatal cardiomyocytes were subjected to hydrogen peroxide (H 2 O 2) stimulation. Phosphorylation of ERK1/2, Akt, and GSK-3β were increased after 1 h of H 2 O 2 stimulation. SMP30 silencing significantly suppressed Akt and GSK-3β phosphorylation, but did not influence ERK1/2 phosphorylation ( Figure 5A,B). Moreover, the ratio of Bax to Bcl-2 expression was significantly higher in SMP30 siRNA-transfected cardiomyocytes 24 h after H 2 O 2 stimulation ( Figure 5A,B). To confirm the effect of SMP30 silencing on Akt and GSK-3β phosphorylation, neonatal cardiomyocytes were subjected to hydrogen peroxide (H2O2) stimulation. Phosphorylation of ERK1/2, Akt, and GSK-3β were increased after 1 h of H2O2 stimulation. SMP30 silencing significantly suppressed Akt and GSK-3β phosphorylation, but did not influence ERK1/2 phosphorylation ( Figure 5A,B). Moreover, the ratio of Bax to Bcl-2 expression was significantly higher in SMP30 siRNA-transfected cardiomyocytes 24 h after H2O2 stimulation ( Figure 5A,B).

Impact of Silencing of SMP30 Expression on Cardiomyocyte Apoptosis
SMP30 silencing inhibited ROS-mediated phosphorylation of Akt and GSK-3β in cardiomyocytes. Therefore, we examined TUNEL staining to confirm the effect of SMP30 deficiency on ROS-mediated cardiomyocyte apoptosis. We observed that H2O2 stimulation increased TUNEL-positive cardiomyocytes. Furthermore, the number of TUNEL-positive nuclei after H2O2 stimulation was significantly higher in SMP30 siRNA than in control siRNA cardiomyocytes ( Figure 6A,B).

Impact of Silencing of SMP30 Expression on Cardiomyocyte Apoptosis
SMP30 silencing inhibited ROS-mediated phosphorylation of Akt and GSK-3β in cardiomyocytes. Therefore, we examined TUNEL staining to confirm the effect of SMP30 deficiency on ROS-mediated cardiomyocyte apoptosis.
We observed that H 2 O 2 stimulation increased TUNEL-positive cardiomyocytes. Furthermore, the number of TUNEL-positive nuclei after H 2 O 2 stimulation was significantly higher in SMP30 siRNA than in control siRNA cardiomyocytes ( Figure 6A,B). To confirm the effect of SMP30 silencing on Akt and GSK-3β phosphorylation, neonatal cardiomyocytes were subjected to hydrogen peroxide (H2O2) stimulation. Phosphorylation of ERK1/2, Akt, and GSK-3β were increased after 1 h of H2O2 stimulation. SMP30 silencing significantly suppressed Akt and GSK-3β phosphorylation, but did not influence ERK1/2 phosphorylation ( Figure 5A,B). Moreover, the ratio of Bax to Bcl-2 expression was significantly higher in SMP30 siRNA-transfected cardiomyocytes 24 h after H2O2 stimulation ( Figure 5A,B).

Impact of Silencing of SMP30 Expression on Cardiomyocyte Apoptosis
SMP30 silencing inhibited ROS-mediated phosphorylation of Akt and GSK-3β in cardiomyocytes. Therefore, we examined TUNEL staining to confirm the effect of SMP30 deficiency on ROS-mediated cardiomyocyte apoptosis. We observed that H2O2 stimulation increased TUNEL-positive cardiomyocytes. Furthermore, the number of TUNEL-positive nuclei after H2O2 stimulation was significantly higher in SMP30 siRNA than in control siRNA cardiomyocytes ( Figure 6A,B).

Discussion
The main findings of this study are as follows: (1) SMP30 KO exacerbated infarct size and LV dysfunction after I/R; (2) ROS generation and apoptosis after I/R in SMP30 KO mice were significantly higher than those in WT mice; (3) Downregulation of SMP30 attenuated the phosphorylation of Akt and GSK-3β after I/R or H2O2 stimulation; and (4) SMP30-downregulated cardiomyocytes showed increased susceptibility to H2O2-induced apoptosis.
SMP30 is reported to act as an anti-aging factor and prevents oxidative stress and apoptosis [25,28,29]. Advanced age worsens I/R injury due to abnormalities in mitochondrial function, oxidative stress, and increased susceptibility to apoptosis and necrosis [15,16]. In our study, infarct size after I/R was larger and LVFS was lower in SMP30 KO mice than in WT mice. In addition, SMP30 KO mice were far more susceptible to I/R-induced apoptosis associated with an increase in the Bax/Bcl-2 ratio. Age-associated SMP30 decrease might be related to the susceptibility of aging hearts to I/R injury.
ROS-induced ROS-release phenomenon occurs under excessive oxidative stress such as I/R injury [30]. ROS generation and apoptosis after I/R in SMP30 KO mice were significantly higher than that in WT mice. A previous study reported that catalase and superoxide dismutase activities were not significantly different between WT and SMP30 KO mice, suggesting that SMP30 antioxidant effect may result from suppressing ROS generation rather than scavenging ROS [31]. Previous study reported that ascorbic acid deficiency affected Nrf2 and NQO1 [32]. In this study, there is no significant difference in ascorbic acid between WT and SMP30KO mice. Therefore, Nrf2 and NQO1 might not be related in this study. However, we need to elucidate the role of SMP30 in the expression and activity of Nrf2, NQO1, and SREBP1 in ischemia/reperfusion injury in the heart. SMP30 was reported to be associated with intracellular calcium homeostasis, which regulates ROS production in the mitochondria, through the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) in the kidneys, liver, and brain [33,34]. Therefore, it is also possible that SMP30 knockdown reduces SERCA levels and activity in the heart after I/R. This hypothesis was potentiated by the fact that SMP30 regulated SERCA activity, and that SMP30 KO mice demonstrated increases ROS generation due to calcium overload in the mitochondria [35,36].
The RISK pathway is a signaling cascade involving prosurvival kinases, which contribute to cardioprotection when activated during the time of reperfusion [27,37]. The phosphatidylinositol-3 kinase (PI3K), Akt, and ERK1/2 are involved in the RISK pathway. We found that phosphorylation of Akt and GSK-3β was lower in SMP30 KO mice than in WT mice. Similarly, H2O2-induced phosphorylation of Akt and GSK-3β was also inhibited by silencing of SMP30 expression in cardiomyocytes, suggesting that ROS-mediated activation of Akt and GSK-3β was at least in part SMP30-dependent. In the present study, H2O2 stimulation increased the Bax/Bcl-2 expression ratio and enhanced cardiomyocyte apoptosis by siRNA-mediated SMP30 silencing. Taken together,

Discussion
The main findings of this study are as follows: (1) SMP30 KO exacerbated infarct size and LV dysfunction after I/R; (2) ROS generation and apoptosis after I/R in SMP30 KO mice were significantly higher than those in WT mice; (3) Downregulation of SMP30 attenuated the phosphorylation of Akt and GSK-3β after I/R or H 2 O 2 stimulation; and (4) SMP30-downregulated cardiomyocytes showed increased susceptibility to H 2 O 2-induced apoptosis.
SMP30 is reported to act as an anti-aging factor and prevents oxidative stress and apoptosis [25,28,29]. Advanced age worsens I/R injury due to abnormalities in mitochondrial function, oxidative stress, and increased susceptibility to apoptosis and necrosis [15,16]. In our study, infarct size after I/R was larger and LVFS was lower in SMP30 KO mice than in WT mice. In addition, SMP30 KO mice were far more susceptible to I/R-induced apoptosis associated with an increase in the Bax/Bcl-2 ratio. Age-associated SMP30 decrease might be related to the susceptibility of aging hearts to I/R injury.
ROS-induced ROS-release phenomenon occurs under excessive oxidative stress such as I/R injury [30]. ROS generation and apoptosis after I/R in SMP30 KO mice were significantly higher than that in WT mice. A previous study reported that catalase and superoxide dismutase activities were not significantly different between WT and SMP30 KO mice, suggesting that SMP30 antioxidant effect may result from suppressing ROS generation rather than scavenging ROS [31]. Previous study reported that ascorbic acid deficiency affected Nrf2 and NQO1 [32]. In this study, there is no significant difference in ascorbic acid between WT and SMP30KO mice. Therefore, Nrf2 and NQO1 might not be related in this study. However, we need to elucidate the role of SMP30 in the expression and activity of Nrf2, NQO1, and SREBP1 in ischemia/reperfusion injury in the heart. SMP30 was reported to be associated with intracellular calcium homeostasis, which regulates ROS production in the mitochondria, through the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) in the kidneys, liver, and brain [33,34]. Therefore, it is also possible that SMP30 knockdown reduces SERCA levels and activity in the heart after I/R. This hypothesis was potentiated by the fact that SMP30 regulated SERCA activity, and that SMP30 KO mice demonstrated increases ROS generation due to calcium overload in the mitochondria [35,36].
The RISK pathway is a signaling cascade involving prosurvival kinases, which contribute to cardioprotection when activated during the time of reperfusion [27,37]. The phosphatidylinositol-3 kinase (PI3K), Akt, and ERK1/2 are involved in the RISK pathway. We found that phosphorylation of Akt and GSK-3β was lower in SMP30 KO mice than in WT mice. Similarly, H 2 O 2 -induced phosphorylation of Akt and GSK-3β was also inhibited by silencing of SMP30 expression in cardiomyocytes, suggesting that ROS-mediated activation of Akt and GSK-3β was at least in part SMP30-dependent. In the present study, H 2 O 2 stimulation increased the Bax/Bcl-2 expression ratio and enhanced cardiomyocyte apoptosis by siRNA-mediated SMP30 silencing. Taken together, decreased phosphorylation of GSK-3β in SMP30-silenced cardiomyocytes also contributed to exacerbate cardiomyocyte apoptosis.
Based on the previous studies and our data, we suspect that SMP30 decreased expression in the heart of elderly patients is the cause of higher risk and worsened clinical outcomes during myocardial reperfusion. Lower expression of SMP30 in elderly patients induces increased ROS generation and attenuates AKT and GSK3β, both of which are associated with cardiomyocyte apoptosis. Therefore, preservation of SMP30 expression in aging heart is one of the possible therapeutic targets to inhibit cardiac dysfunction after myocardial infarction in elderly patients. To confirm this hypothesis, we need to evaluate cardiac function, cardiomyocyte apoptosis, and infarct size after ischemia/reperfusion in cardiac-specific overexpression of SMP30 mice in the future study.
There are several limitations in this study. We showed that deficiency of SMP30 exacerbates myocardial I/R injury; however, we did not show that endogenous or exogenous SMP30 has a protective role in myocardial I/R injury. To confirm this point, we might need to evaluate in a future study the cardioprotective role of SMP30 overexpression after I/R and H 2 O 2 stimulation.

Animal Protocol
SMP30 KO (C57BL/6 background) mice were established as previously reported [28]. Vitamin C water (1.5 g/L) was provided for the SMP30 KO mice, as described previously [17,24], because SMP30 is a gluconolactonase, a pivotal enzyme for ascorbic acid (vitamin C) synthesis [38]. Mice were housed in a pathogen-free facility with a 12:12 h light-dark cycle and were given free access to water and standard rodent chow. All experimental procedures were performed according to the animal welfare regulations of Yamagata University School of Medicine, and the study protocol was approved by the Animal Subjects Committee of Yamagata University School of Medicine (No. 21045, 16 March 2009). The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

Echocardiography
The cardiac function was evaluated by two-dimensional echocardiography with the use of an FF Sonic 8900 (Fukudadenshi Co., Tokyo, Japan) equipped with a 13-MHz phase-array transducer, at 24 h after I/R or sham operation. Mice were anesthetized with intraperitoneal administration of pentobarbital (35 mg/kg) so as not to compromise respiration and hemodynamic conditions. The internal dimensions of the left ventricle at end-diastole (LVEDD) and at end-systole (LVESD) were measured and averaged from three cardiac cycles. LV fractional shortening (% FS) was calculated as [(LVEDD´LVESD)/LVEDD]ˆ100 [26].

Heart Surgery of I/R Model
SMP30 KO and WT mice (age 10-12 weeks old) were anesthetized with an intraperitoneal administration of pentobarbital (100 mg/kg), orally intubated, and ventilated with a rodent respirator (Harvard Apparatus, Holliston, MA, USA). Inducing ischemia reperfusion and evaluating infarct size were performed as we previously described [26], using 5% Evans blue (Sigma Chemical Co., St. Louis, MO, USA) and 1% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma Chemical Co.). Rrisk area and infarct area were measured using a Scion imaging system (Scion, Frederick, MD, USA) as we previously described [26]. Serum creatine phosphokinase (CPK) levels were measured in blood samples from mice 24 h after reperfusion. Serum CPK assay was performed with DRIKEM and CPK kits (FUJIFILM, Tokyo, Japan).

Western Blot Analysis
Samples were lysed in ice-cold lysis buffer and proteins were extracted as previously reported [39,40]. Protein concentrations were determined by protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal amounts of proteins were subjected to 10% or 14% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Immunoreactive bands were detected by using an ECL kit (Amersham Biosciences, Piscataway, NJ, USA). Membranes were incubated with the following primary antibodies: anti-phospho ERK1/2, anti-total ERK1/2, anti-phospho S473-Akt, total-Akt, anti-phospho GSK-3β, GSK-3β, anti-Bax, anti-Bcl-2, β-tubulin (Cell Signaling Technology, Inc., Danvers, MA, USA), and anti-SMP30 antibodies (SHIMA Laboratories Co., Ltd., Tokyo, Japan). To quantify the protein levels, the same membranes were reprobed with total proteins. The relative amount of phosphorylated proteins vs. total proteins was used for phosphorylation kinase activity. Ubiquitously expressed β-tubulin was used as a loading control.

Detection of Apoptosis
Twenty-four hours after I/R, the hearts were excised, fixed with a 10% solution of formalin in phosphate-buffered saline, embedded in paraffin, and serially cut from the apex to the base. Samples were stained by 4 1 ,6-diamidino-2-phenulindole and TdT-mediated dUTP nick end-labeling (TUNEL) according to the manufacturer instructions. The percentage of TUNEL-positive cells was determined by counting 10 random fields per section under a microscope (BX50, Olympus, Tokyo, Japan). TUNEL staining was performed with a commercially available kit for the detection of end-labeled DNA according to the manufacturer instructions (Roche Applied Science, Tokyo, Japan).
In the in vitro study, cultured cardiomyocytes were fixed with 4% paraformaldehyde 24 h after H 2 O 2 stimulation. These samples were stained with a TUNEL kit, and a 4 1 ,6-diamidino-2-phenulindole (DAPI) staining was performed to normalize the results to the cell number. Approximately 400-600 nuclei from random fields were analyzed for each sample [41].

Assessment of Superoxide Generation
Thirty minutes after reperfusion, the heart tissues were embedded in optimum cutting temperature compound and sectioned at 3-µm thickness. Sections were incubated with 10 µM dihydroethidium (Sigma-Aldrich Co., St. Louis, MO, USA) at 37˝C for 30 min [17,42]. The mean dihydroethidium fluorescence intensity of the myocardium was quantified in 10 randomly selected fields in each section with the NIH Image J software under a microscope (BX50, Olympus, Tokyo, Japan).

Cultured Neonatal Rat Cardiomyocytes
Promptly after euthanasia by decapitation, hearts were collected from 1-to 2-day-old neonatal rat pups, and primary cultures of neonatal rat cardiomyocytes were performed as described previously [43,44]. Cardiomyocytes were kept in fetal bovine serum-supplemented Dulbecco's modified Eagle medium (DMEM). SMP30 small interfering RNA (siRNA) was purchased from Thermo Scientific Dharmacon (Lafayette, CO, USA). Two days after cell seeding in culture medium, SMP30 siRNA was transfected into cardiomyocytes using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer instructions [39]. After serum starvation for 4 h, cardiomyocytes were stimulated with H 2 O 2 (200 µM) for 1 and 24 h.

Statistical Analysis
All values are reported as mean˘standard error (SE) in figures and mean˘standard deviation (SD) in the table. Differences between groups were evaluated by analysis of variance (ANOVA) with Bonferroni test. A probability value less than 0.05 was considered statistically significant. The statistical analysis was performed with a standard statistical program package (JMP version 8; SAS Institute, Inc., Cary, NC, USA).

Conclusions
In this study, we found that SMP30 deficiency exacerbated myocardial I/R injury through an increase in oxidative stress and cardiomyocyte apoptosis. Moreover, a lower SMP30 expression suppressed Akt and GSK-3β phosphorylation and increased cardiomyocyte apoptosis after H 2 O 2 stimulation. Therefore, a decreased SMP30 expression in the heart of elderly patients might be one of the causes of a poor prognosis after myocardial infarction.