Cardioprotective effect of Notch signaling on the development of myocardial infarction complicated by diabetes mellitus

Abstract

The present study aimed to elucidate the role of Notch signaling in the development of myocardial infarction (MI) concomitant with diabetes in vivo and in vitro and evaluated the therapeutic effect of the Notch signaling in vitro. Streptozotocin-induced diabetic rats were subjected to 25 min of ischemia and 2 h of reperfusion. Cardiac troponin T (cTnT) and creatine kinase-MB (CK-MB) isoenzyme levels were detected. Infarct size was measured by 2,3,5-triphenyltetrazolium chloride staining. Myocardial apoptosis and fibrosis were examined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and Masson Trichrome staining, respectively. The mRNA and protein levels of Notch signaling components, including Notch1, Notch4, Delta-like 1, Jagged1, Mastermind-like protein 1 and p300, were quantified by reverse transcription-quantitative polymerase chain reaction and western blotting analyses, respectively. H9c2 cells were treated with/without 33 mM high glucose (HG) and/or subjected to hypoxia in the presence/absence of Jagged1. Cell viability and apoptosis were determined by MTT assay and Annexin V-fluorescein isothiocyanate/propidium iodide assay. Levels of the Notch signaling pathway members were examined. The present findings revealed that diabetes elevated CK-MB and cTnT, increased infarct size, induced myocardial apoptosis and inhibited the Notch signaling pathway in vivo after ischemia/reperfusion. Ischemia/reperfusion augmented the severity of MI in diabetic rats. Furthermore, HG reduced cell viability and induced cell apoptosis in H9c2 cells after hypoxia exposure, which was inhibited by Jagged1. We also found that HG inhibited Notch signaling in H9c2 cells after hypoxia, whereas Jagged1 exerted its cardioprotective effect on hypoxic injury (in HG environments or not) by activating the Notch signaling pathway. In conclusion, these findings suggest that diabetes promoted the progression of MI in vivo and in vitro via the inhibition of the Notch signaling pathway. Jagged1 may protect against MI in in vitro models by activating Notch signaling.

Keywords: Jagged1; Notch signaling; cardioprotection; diabetes mellitus; myocardial infarction.

Figure 1.

Determination of myocardial infarction by the levels of cTnT and CK-MB isoenzyme. (A) cTnT levels were measured using a commercial ELISA kit. Results are presented as pg/ml. (B) CK-MB levels were detected using a commercial ELISA kit. Results are presented as ng/ml. Data are expressed as the mean ± standard deviation. *P<0.05 and **P<0.01. cTnT, cardiac troponin T; CK-MB, creatine kinase-MB; STZ, streptozotocin; I/R, ischemia/reperfusion; ns, not significant.

Figure 2.

Measurement of infarct size. Following reperfusion, we excised the hearts from the rats and 2,3,5-triphenyltetrazolium chloride staining was performed to assess infarct size. Data are expressed as the mean ± standard deviation. *P<0.05 and **P<0.01. STZ, streptozotocin; I/R, ischemia/reperfusion; ns, not significant.

Figure 3.

Myocardial apoptosis in the myocardium. Paraffin-embedded rat heart tissues were sectioned and myocardial apoptosis was measured by Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Apoptotic cell rate (%) was calculated. Scale bar, 50 µm. Magnification, ×400. Data are expressed as the mean ± standard deviation. *P<0.05 and **P<0.01. STZ, streptozotocin; I/R, ischemia/reperfusion; ns, not significant.

Figure 4.

Myocardial fibrosis in the myocardium. Heart tissues were embedded and cut into sections (5-µm thick). Myocardial fibrosis was examined by Masson Trichrome staining. Scale bar, 100 µm. Magnification, ×200. STZ, streptozotocin; I/R, ischemia/reperfusion.

Figure 5.

Role of the Notch signaling pathway in DM-MI in vivo. Total RNA was isolated from myocardial tissues and mRNA levels of Notch1, Notch4, DLL1, Jagged1, MAML1 and p300 were determined by reverse transcription-quantitative polymerase chain reaction. β-actin served as an internal control. Data are expressed as the mean ± standard deviation. **P<0.01. STZ, streptozotocin; I/R, ischemia/reperfusion; DM, diabetes mellitus; MI, myocardial infarction; DLL1, Delta-like 1; MAML1, Mastermind-like protein 1; ns, not significant.

Figure 6.

Role of the Notch signaling pathway in DM-MI in vivo. Total protein was extracted from myocardial tissues and Notch1, Notch4, DLL1, Jagged1, MAML1 and p300 protein expression levels were determined by western blotting. β-actin served as a loading control. Data are expressed as the mean ± standard deviation. **P<0.01. STZ, streptozotocin; I/R, ischemia/reperfusion; DM, diabetes mellitus; MI, myocardial infarction; DLL1, Delta-like 1; MAML1, Mastermind-like protein 1; ns, not significant.

Figure 7.

Effect of Jagged1 on H9c2 cell viability and apoptosis. (A) Following the indicated treatments, cell viability was measured by MTT assay. (B) Harvested cells were subjected to Annexin V-FITC/PI assay. Apoptotic cell rate was analyzed using a flow cytometer. Data are expressed as the mean ± standard deviation. *P<0.05 and **P<0.01. FITC, fluorescein isothiocyanate; PI, propidium iodide; HG, high glucose.

Figure 8.

Effect of Jagged1 on the Notch signaling pathway in vitro. (A) Gene expression levels of Notch1, Notch4, DLL1 and Jagged1 were measured by reverse transcription-quantitative polymerase chain reaction. Results were normalized to β-actin expression. (B) Protein expression levels of Notch1, Notch4, DLL1 and Jagged1 were measured by western blotting and normalized to β-actin. Data are expressed as the mean ± standard deviation. *P<0.05 and **P<0.01. HG, high glucose; DLL1, Delta-like 1; ns, not significant.