Function and Mechanism of Trimetazidine in Myocardial Infarction-Induced Myocardial Energy Metabolism Disorder Through the SIRT1-AMPK Pathway

Abstract

Myocardial energy metabolism (MEM) is an important factor of myocardial injury. Trimetazidine (TMZ) provides protection against myocardial ischemia/reperfusion injury. The current study set out to evaluate the effect and mechanism of TMZ on MEM disorder induced by myocardial infarction (MI). Firstly, a MI mouse model was established by coronary artery ligation, which was then treated with different concentrations of TMZ (5, 10, and 20 mg kg^-1 day^-1). The results suggested that TMZ reduced the heart/weight ratio in a concentration-dependent manner. TMZ also reduced the levels of Bax and cleaved caspase-3 and promoted Bcl-2 expression. In addition, TMZ augmented adenosine triphosphate (ATP) production and superoxide dismutase (SOD) activity induced by MI and decreased the levels of lipid peroxide (LPO), free fatty acids (FFA), and nitric oxide (NO) in a concentration-dependent manner (all P < 0.05). Furthermore, an H₂O₂-induced cell injury model was established and treated with different concentrations of TMZ (1, 5, and 10 μM). The results showed that SIRT1 overexpression promoted ATP production and reactive oxygen species (ROS) activity and reduced the levels of LPO, FFA, and NO in H9C2 cardiomyocytes treated with H₂O₂ and TMZ. Silencing SIRT1 suppressed ATP production and ROS activity and increased the levels of LPO, FFA, and NO (all P < 0.05). TMZ activated the SIRT1-AMPK pathway by increasing SIRT1 expression and AMPK phosphorylation. In conclusion, TMZ inhibited MI-induced myocardial apoptosis and MEM disorder by activating the SIRT1-AMPK pathway.

Keywords: SIRT1-AMPK pathway; apoptosis; myocardial energy metabolism disorder; myocardial infarction; trimetazidine.

FIGURE 1

Trimetazidine (TMZ) inhibited myocardial infarction (MI)-induced myocardial apoptosis. (A) MI was detected by triphenyltetrazolium chloride (TTC) staining. Areas in white represent the MI region. (B) Heart weight was measured and the heart/weight ratio calculated. (C) Pathological changes of the myocardial tissue were detected by hematoxylin–eosin (HE) staining. (D) Myocardial apoptosis was detected by transferase-mediated biotin-16-dUTP (TUNEL) staining. (E) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3 (N = 3 per group). Detection was repeated three times. All data were expressed as the mean ± standard deviation. Data were analyzed using one-way one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test. *P < 0.05.

FIGURE 2

Trimetazidine inhibited MI-induced myocardial energy metabolism disorder. (A) ATP content, (B) SOD activity, (C) LPO content, (D) FFA content, and (E) NO content in the myocardium (N = 10 per group). All data were expressed as the mean ± standard deviation. Data were analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons test. *P < 0.05. ATP, adenosine triphosphate; SOD, superoxide dismutase; LPO, lipid peroxide; FFA, free fatty acids; NO, nitric oxide.

FIGURE 3

Trimetazidine inhibited H₂O₂-induced cardiomyocyte apoptosis and energy metabolism disorder. (A) Flow cytometry analysis for apoptosis. (B) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3 expressions. (C) ATP content in H9C2 cardiomyocytes. (D) SOD activity in H9C2 cardiomyocytes. (E–G) Contents of LPO in panel (E), FFA in panel (F), and NO in panel (G) in H9C2 cardiomyocytes. Cell experiment was repeated three times. All data were expressed as the mean ± standard deviation. Data were analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons test. *P < 0.05. ATP, adenosine triphosphate; SOD, superoxide dismutase; LPO, lipid peroxide; FFA, free fatty acids; NO, nitric oxide.

FIGURE 4

Trimetazidine inhibited H₂O₂-induced cardiomyocyte apoptosis and energy metabolism disorder via the SIRT1–AMPK pathway. (A) Western blot analysis of SIRT1, p-AMPK, and t-AMPK. (B) Western blot analysis of SIRT1, p-AMPK, and t-AMPK after overexpressing or silencing SIRT1 in H9C2 cardiomyocytes jointly processed by H₂O₂ and TMZ. (C) Flow cytometry analysis for apoptosis. (D) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3. (E) ATP content in H9C2 cardiomyocytes. (F) SOD activity in H9C2 cardiomyocytes. (G–I) Contents of LPO (G), FFA (H), and NO (I) in H9C2 cardiomyocytes. Cell experiment was repeated three times. All data were expressed as the mean ± standard deviation. Data were analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons test. *P < 0.05. ATP, adenosine triphosphate; SOD, superoxide dismutase; LPO, lipid peroxide; FFA, free fatty acids; NO, nitric oxide.

FIGURE 5

Trimetazidine inhibited MI-induced myocardial energy metabolism via the SIRT1–AMPK pathway in vivo. (A) Western blot analysis of SIRT1, p-AMPK, and t-AMPK. (B) ATP content in H9C2 cardiomyocytes. (C) SOD activity in H9C2 cardiomyocytes. (D–F) Contents of LPO (D), FFA (E), and NO (F) in H9C2 cardiomyocytes (N = 10 per group). All data were expressed as the mean ± standard deviation. Data were analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons test. *P < 0.05. ATP, adenosine triphosphate; SOD, superoxide dismutase; LPO, lipid peroxide; FFA, free fatty acids; NO, nitric oxide.

FIGURE 6

Trimetazidine inhibited MI-induced apoptosis via the SIRT1–AMPK pathway in vivo. (A) MI was detected by TTC staining. Areas in white represent the MI region. (B) Heart weight was measured and the heart/weight ratio calculated. (C) Pathological changes of the myocardial tissue were detected by HE staining. (D) Myocardial apoptosis was detected by TUNEL staining. (E) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3 (N = 10 per group). All data were expressed as the mean ± standard deviation. Data were analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons test. *P < 0.05.