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. 2024 Nov 16:17:8905-8919.
doi: 10.2147/JIR.S493037. eCollection 2024.

Diltiazem Hydrochloride Protects Against Myocardial Ischemia/Reperfusion Injury in a BNIP3L/NIX-Mediated Mitophagy Manner

Xing Zhou #  1 , Quan Lu #  2 , Qiu Wang #  2 , Wenxin Chu  2 , Jianhao Huang  2 , Jinming Yu  2 , Yuechou Nong  2 , Wensheng Lu #  2
Affiliations

Diltiazem Hydrochloride Protects Against Myocardial Ischemia/Reperfusion Injury in a BNIP3L/NIX-Mediated Mitophagy Manner

Xing Zhou et al. J Inflamm Res. .

Abstract

Background: Mitochondrial calcium uptake-induced mitophagy may play an essential role in myocardial ischemia/reperfusion (MI/R) injury. Diltiazem hydrochloride (DIL), a traditional calcium channel blocker, can alleviate MI/R injury by blocking calcium overload. However, whether the protective mechanism of DIL involves mitophagy remains elusive. This study aimed to clarify the underlying molecular mechanism by which DIL ameliorates MI/R injury by downregulating mitophagy in vivo and in vitro.

Methods: Thirty rats were randomized into three groups: the sham, MI/R, and MI/R+DIL (1 mg/kg) groups (n = 10/per group). MI/R injury was induced by ligating the left anterior descending (LAD) artery for 30 min followed by 60 min of reperfusion in vivo. H9C2 cells were selected to establish an oxygen-glucose deprivation/recovery (OGD/R) model to simulate MI/R injury in vitro. The potential mechanism by which DIL alleviates MI/R injury was analyzed based on tissue morphology, mitophagy-related gene transcription, and protein expression.

Results: According to histological and immunohistochemical evaluations, DIL significantly alleviated myocardial damage in vivo. Moreover, DIL significantly increased cell viability, attenuated OGD/R-induced apoptosis, and inhibited mitochondrial autophagy in vitro. Mechanistically, DIL attenuated mitochondrial autophagy through the upregulation of dual-specificity protein phosphatase 1 (DUSP1) and the downregulation of c-Jun N-terminal kinase (JNK) and Bcl2 interacting protein 3-like (BNIP3L, also known as NIX) expression.

Conclusion: Diltiazem hydrochloride protects against myocardial ischemia/reperfusion injury in a BNIP3L/NIX-mediated mitophagy manner in vivo and in vitro.

Keywords: calcium channel blockers; diltiazem hydrochloride; mitophagy; myocardial infarction; myocardial ischemia/reperfusion injury.

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Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
The effects of DIL on histological and immunohistochemical analyses of cardiac muscular tissue in rats with MI/R injury. (A) Hematoxylin and eosin (HE) staining of cardiac muscular tissue in the three groups. Original magnification: ×100, scale bar = 100 μm (upper row); ×200, scale bar = 50 μm (lower row). (B) Representative images of immunohistochemical (IHC) staining of DUSP1, JNK, and BNIP3L/NIX in the three groups. (C–E) Comparison of the average IOD values of DUSP1, JNK, and BNIP3L/NIX expression in myocardial tissue among the three groups determined by IHC analysis. **P < 0.01 relative to the sham group, ##P < 0.01 relative to the MI/R group.
Figure 2
Figure 2
Effect of DIL intervention on cell viability and the apoptosis rate. (A) Morphological characteristics and growth status of H9C2 cells in the control, OGD/R, and OGD/R + DIL groups (×200). (B) Viability of H9C2 cells treated with different concentrations of DIL for different durations in the control, OGD/R, and OGD/R + DIL groups. (C and D) The apoptosis rates of H9C2 cells in the control, OGD/R, and OGD/R + DIL groups were assessed by Annexin V-FITC/PI staining. **P < 0.01 relative to the control group, ##P < 0.01 relative to the OGD/R group.
Figure 3
Figure 3
Mt-Keima showing normal mitochondria (green) and mitochondria in autophagosomes (red) in H9C2 cells. H9C2 cells stably expressing mt-Keima were generated via lentivirus infection in the control, OGD/R, and OGD/R + DIL groups. The mt-Keima fluorescent protein was excited at 440 nm and 586 nm under a confocal laser scanning microscope, and fluorescence emission was detected in the three groups. Scale bar, 100 μm.
Figure 4
Figure 4
The mitochondrial autophagosomes were observed under a transmission electron microscope (TEM). The morphology of mitochondria and autophagosomes in the control, OGD/R and OGD/R + DIL groups in vitro was observed via transmission electron microscopy (TEM). Original magnification: ×10000, scale bar = 2 μm (upper row); ×30000, scale bar = 500 nm (lower row).
Figure 5
Figure 5
RT‒qPCR analysis of the mRNA levels of Beclin 1, DUSP1, JNK, and BNIP3L/NIX in the control, OGD/R, and OGD/R + DIL groups. Compared to those in the control group, the expression levels of Beclin 1 (A), JNK (C) and BNIP3L/NIX (D) were significantly greater in the OGD/R group, but only DUSP1 expression (B) was significantly lower (all P < 0.01). Remarkably, this effect could be reversed by DIL intervention (all P < 0.01). **P < 0.01 relative to the control group; ##P < 0.01 relative to the OGD/R group.
Figure 6
Figure 6
Western blot analyses of the Beclin 1, LC3B, DUSP1, JNK, and BNIP3L/NIX proteins in the control, OGD/R, and OGD/R + DIL groups. (A) The electrophoresis results of mitophagy-related proteins from the Western blot. The analyses indicated significantly increased protein expression of Beclin 1 (B), LC3B (C), JNK (E), and BNIP3L/NIX (F) and decreased DUSP1 protein expression (D) in the OGD/R group compared to the control group. DIL intervention reversed these changes (all P < 0.05). *P < 0.05, **P < 0.01 relative to the control group; #P < 0.05, ##P < 0.01 relative to the OGD/R group.
Figure 7
Figure 7
RT‒qPCR analysis of the mRNA levels of the DUSP1, JNK, and BNIP3L/NIX genes after treatment with DUSP1, JNK, and BNIP3L/NIX inhibitors or activators in the different groups. (A) The DUSP1 expression level was significantly decreased in the OGD/R+BCI (an inhibitor of DUSP1) group (P < 0.01), and these changes were reversed by DIL in the OGD/R+BCI+DIL group (P < 0.01). (B) Ani (an activator of JNK) significantly upregulated JNK expression in the OGD/R+Ani group (P < 0.01). However, DIL and SP (an inhibitor of JNK) significantly downregulated JNK expression in the OGD/R+DIL and OGD/R+SP groups compared with that in the OGD/R group (P < 0.01). Unexpectedly, compared with that in the OGD/R+Ani group, the JNK expression level did not increase further under DIL intervention but obviously decreased in the OGD/R+Ani+DIL group (P < 0.01), which indicated that DIL-induced DUSP1 partly inhibited JNK expression. (C) MAC (an activator of BNIP3L/NIX) significantly upregulated BNIP3L/NIX expression in the OGD/R+MAC group. Compared with that in the OGD/R+DIL or OGD/R+BAF (an inhibitor of BNIP3L/NIX) group, BNIP3L/NIX expression in the OGD/R+DIL or OGD/R+BAF group was significantly lower in the DIL and BAF groups (P < 0.01). Moreover, compared with that in the OGD/R+MAC group, the BNIP3L/NIX expression level did not increase further under DIL intervention but obviously decreased in the OGD/R+ MAC+DIL group (P < 0.01), which indicated that DIL-induced DUSP1 partly inhibited BNIP3L/NIX expression. The data are presented as the means ± SDs of three independent experiments performed in duplicate. *P < 0.05, **P < 0.01.
Figure 8
Figure 8
Western blot analyses of DUSP1, JNK, and BNIP3L/NIX after treatment with DUSP1, JNK and BNIP3L/NIX inhibitors or activators in the different groups. (A and B) Compared with that in the OGD/R group, the DUSP1 gene expression level was significantly upregulated in the OGD/R+DIL and OGD/R+CTB (an activator of DUSP1) groups (P < 0.01). Moreover, the DUSP1 expression level was significantly decreased in the OGD/R+BCI (an inhibitor of DUSP1) group (P < 0.01), and these changes were reversed by DIL in the OGD/R+BCI+DIL group (P < 0.01). (C and D) Ani (an activator of JNK) significantly upregulated JNK expression in the OGD/R+Ani group (P < 0.01). However, DIL and SP (an inhibitor of JNK) significantly downregulated JNK expression in the OGD/R+DIL and OGD/R+SP groups compared with that in the OGD/R group (P < 0.01). Unexpectedly, compared with that in the OGD/R+Ani group, the JNK expression level did not increase further under DIL intervention but obviously decreased in the OGD/R+Ani+DIL group (P < 0.01), which indicated that DIL-induced DUSP1 partly inhibited JNK expression. (E and F) MAC (an activator of BNIP3L/NIX) significantly upregulated BNIP3L/NIX expression in the OGD/R+MAC group. Compared with that in the OGD/R+DIL or OGD/R+BAF (an inhibitor of BNIP3L/NIX) group, BNIP3L/NIX expression in the OGD/R+DIL or OGD/R+BAF group was significantly lower in the DIL and BAF groups (P < 0.01). Moreover, compared with that in the OGD/R+MAC group, the BNIP3L/NIX expression level did not increase further under DIL intervention but obviously decreased in the OGD/R+ MAC+DIL group (P < 0.01), which indicated that DIL-induced DUSP1 partly inhibited BNIP3L/NIX expression. The data are presented as the means ± SDs of three independent experiments performed in duplicate. *P < 0.05, **P < 0.01.
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