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Brain-derived neurotrophic factor regulates TRPC3/6 channels and protects against myocardial infarction in rodents.

Hang P, Zhao J, Cai B, Tian S, Huang W, Guo J, Sun C, Li Y, Du Z - Int. J. Biol. Sci. (2015)

Bottom Line: Meanwhile, echocardiography indicated that BDNF significantly improved cardiac function of MI mice.Furthermore, protective role of BDNF against hypoxia-induced apoptosis was reversed by 2-APB and TRPC3/6 siRNAs.BDNF/TrkB alleviated cardiac ischemic injury and inhibited cardiomyocytes apoptosis by regulating TRPC3/6 channels, which provides a novel potential therapeutic candidate for MI.

View Article: PubMed Central - PubMed

Affiliation: 1. Institute of Clinical Pharmacology of the Second Affiliated Hospital (Key Laboratory of Drug Research, Heilongjiang Higher Education Institutions), Harbin Medical University, Harbin 150086, China.

ABSTRACT

Background: Brain-derived neurotrophic factor (BDNF) is associated with coronary artery diseases. However, its role and mechanism in myocardial infarction (MI) is not fully understood.

Methods: Wistar rat and Kunming mouse model of MI were induced by the ligation of left coronary artery. Blood samples were collected from MI rats and patients. Plasma BDNF level, protein expression of BDNF, tropomyosin-related kinase B (TrkB) and its downstream transient receptor potential canonical (TRPC)3/6 channels were examined by enzyme-linked immunosorbent assay and Western blot. Infarct size, cardiac function and cardiomyocyte apoptosis were measured after intra-myocardium injection with recombinant human BDNF. Protective role of BDNF against cardiomyocyte apoptosis was confirmed by BDNF scavenger TrkB-Fc. The regulation of TRPC3/6 channels by BDNF was validated by pretreating with TRPC blocker (2-Aminoethyl diphenylborinate, 2-APB) and TRPC3/6 siRNAs.

Results: Circulating BDNF was significantly enhanced in MI rats and patients. Protein expression of BDNF, TrkB and TRPC3/6 channels were upregulated in MI. 3 days post-MI, BDNF treatment markedly reduced the infarct size and serum lactate dehydrogenase activity. Meanwhile, echocardiography indicated that BDNF significantly improved cardiac function of MI mice. Furthermore, BDNF markedly inhibited cardiomyocyte apoptosis by upregulating Bcl-2 expression and downregulating caspase-3 expression and activity in ischemic myocardium. In neonatal rat ventricular myocytes, cell viability was dramatically increased by BDNF in hypoxia, which was restored by TrkB-Fc. Furthermore, protective role of BDNF against hypoxia-induced apoptosis was reversed by 2-APB and TRPC3/6 siRNAs.

Conclusion: BDNF/TrkB alleviated cardiac ischemic injury and inhibited cardiomyocytes apoptosis by regulating TRPC3/6 channels, which provides a novel potential therapeutic candidate for MI.

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Effect of BDNF and 2-APB on cell vialibity and apoptosis in NRVMs. (A) Effect of BDNF on cell viability in hypoxia. *p<0.05 vs. hypoxia, #p<0.05 vs. 200 ng/ml BDNF, &p<0.05 vs. 200 ng/ml BDNF+Si-TRPC3 or 200 ng/ml BDNF +Si-TRPC6, n = 6 each group. (B) Effect of BDNF (200 ng/ml), 2-APB and TrkB-Fc on cell vialibity in normoxia. (C) mRNA level of TRPC3/TRPC6 in control and TRPC3/TRPC6 siRNA treated cardiomyocytes. (D) Effect of BDNF on cardiomyocytes apoptosis subject to hypoxia by TUNEL staining. Nuclei are stained in blue while apoptotic cells are stained in green, scale bar: 100 μm. (E) TUNEL positive cell (%), *p<0.05 vs. hypoxia, #p<0.05 vs. 200 ng/ml BDNF, &p<0.05 vs. 200 ng/ml BDNF+Si-TRPC3 or 200 ng/ml BDNF +Si-TRPC6, n = 5 each group.
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Figure 7: Effect of BDNF and 2-APB on cell vialibity and apoptosis in NRVMs. (A) Effect of BDNF on cell viability in hypoxia. *p<0.05 vs. hypoxia, #p<0.05 vs. 200 ng/ml BDNF, &p<0.05 vs. 200 ng/ml BDNF+Si-TRPC3 or 200 ng/ml BDNF +Si-TRPC6, n = 6 each group. (B) Effect of BDNF (200 ng/ml), 2-APB and TrkB-Fc on cell vialibity in normoxia. (C) mRNA level of TRPC3/TRPC6 in control and TRPC3/TRPC6 siRNA treated cardiomyocytes. (D) Effect of BDNF on cardiomyocytes apoptosis subject to hypoxia by TUNEL staining. Nuclei are stained in blue while apoptotic cells are stained in green, scale bar: 100 μm. (E) TUNEL positive cell (%), *p<0.05 vs. hypoxia, #p<0.05 vs. 200 ng/ml BDNF, &p<0.05 vs. 200 ng/ml BDNF+Si-TRPC3 or 200 ng/ml BDNF +Si-TRPC6, n = 5 each group.

Mentions: We then validated the protective effect of BDNF on NRVMs and found that the cell viability was dose-dependently increased by BDNF from 100 ng/ml to 400 ng/ml (Fig. 7A). Thus, 200 ng/ml was chosen for the following study. In addition, we found that TrkB-Fc successfully decreased cell viability and increased TUNEL-positive cells (Fig. 7A, D). Thus, these evidence confirmed the cytoprotection effect of BDNF against cardiomyocytes apoptosis. To examine the role of TRPC3/6 channels in this process, we tested cell viability after treating with TRPC channel block, 2-APB. We found that cell viability was significantly recovered by 2-APB (Fig. 7A). And we also examined the effect BDNF, 2-APB and TrkB-Fc on cell viability under normoxia and found that all of them did not alter the viability of cardiomyocytes (Fig. 7B). Furthermore, because 2-APB is not a specific inhibitor for TRPC3/6, TRPC3/6 siRNAs were used to confirm the role of TRPC3/6 channels in cardioprotective role of BDNF. The efficiency of TRPC3/TRPC6 siRNAs was validated by Real-time RT-PCR analysis (Fig. 7C). We found that cell viability was reduced while apoptotic cardiomyocytes increased by TRPC3/TRPC6 siRNAs (Fig. 7A, D). Taken together, these data supported that TRPC3/6 channels were required in the protective role of BDNF against cardiomyocyte apoptosis.


Brain-derived neurotrophic factor regulates TRPC3/6 channels and protects against myocardial infarction in rodents.

Hang P, Zhao J, Cai B, Tian S, Huang W, Guo J, Sun C, Li Y, Du Z - Int. J. Biol. Sci. (2015)

Effect of BDNF and 2-APB on cell vialibity and apoptosis in NRVMs. (A) Effect of BDNF on cell viability in hypoxia. *p<0.05 vs. hypoxia, #p<0.05 vs. 200 ng/ml BDNF, &p<0.05 vs. 200 ng/ml BDNF+Si-TRPC3 or 200 ng/ml BDNF +Si-TRPC6, n = 6 each group. (B) Effect of BDNF (200 ng/ml), 2-APB and TrkB-Fc on cell vialibity in normoxia. (C) mRNA level of TRPC3/TRPC6 in control and TRPC3/TRPC6 siRNA treated cardiomyocytes. (D) Effect of BDNF on cardiomyocytes apoptosis subject to hypoxia by TUNEL staining. Nuclei are stained in blue while apoptotic cells are stained in green, scale bar: 100 μm. (E) TUNEL positive cell (%), *p<0.05 vs. hypoxia, #p<0.05 vs. 200 ng/ml BDNF, &p<0.05 vs. 200 ng/ml BDNF+Si-TRPC3 or 200 ng/ml BDNF +Si-TRPC6, n = 5 each group.
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Figure 7: Effect of BDNF and 2-APB on cell vialibity and apoptosis in NRVMs. (A) Effect of BDNF on cell viability in hypoxia. *p<0.05 vs. hypoxia, #p<0.05 vs. 200 ng/ml BDNF, &p<0.05 vs. 200 ng/ml BDNF+Si-TRPC3 or 200 ng/ml BDNF +Si-TRPC6, n = 6 each group. (B) Effect of BDNF (200 ng/ml), 2-APB and TrkB-Fc on cell vialibity in normoxia. (C) mRNA level of TRPC3/TRPC6 in control and TRPC3/TRPC6 siRNA treated cardiomyocytes. (D) Effect of BDNF on cardiomyocytes apoptosis subject to hypoxia by TUNEL staining. Nuclei are stained in blue while apoptotic cells are stained in green, scale bar: 100 μm. (E) TUNEL positive cell (%), *p<0.05 vs. hypoxia, #p<0.05 vs. 200 ng/ml BDNF, &p<0.05 vs. 200 ng/ml BDNF+Si-TRPC3 or 200 ng/ml BDNF +Si-TRPC6, n = 5 each group.
Mentions: We then validated the protective effect of BDNF on NRVMs and found that the cell viability was dose-dependently increased by BDNF from 100 ng/ml to 400 ng/ml (Fig. 7A). Thus, 200 ng/ml was chosen for the following study. In addition, we found that TrkB-Fc successfully decreased cell viability and increased TUNEL-positive cells (Fig. 7A, D). Thus, these evidence confirmed the cytoprotection effect of BDNF against cardiomyocytes apoptosis. To examine the role of TRPC3/6 channels in this process, we tested cell viability after treating with TRPC channel block, 2-APB. We found that cell viability was significantly recovered by 2-APB (Fig. 7A). And we also examined the effect BDNF, 2-APB and TrkB-Fc on cell viability under normoxia and found that all of them did not alter the viability of cardiomyocytes (Fig. 7B). Furthermore, because 2-APB is not a specific inhibitor for TRPC3/6, TRPC3/6 siRNAs were used to confirm the role of TRPC3/6 channels in cardioprotective role of BDNF. The efficiency of TRPC3/TRPC6 siRNAs was validated by Real-time RT-PCR analysis (Fig. 7C). We found that cell viability was reduced while apoptotic cardiomyocytes increased by TRPC3/TRPC6 siRNAs (Fig. 7A, D). Taken together, these data supported that TRPC3/6 channels were required in the protective role of BDNF against cardiomyocyte apoptosis.

Bottom Line: Meanwhile, echocardiography indicated that BDNF significantly improved cardiac function of MI mice.Furthermore, protective role of BDNF against hypoxia-induced apoptosis was reversed by 2-APB and TRPC3/6 siRNAs.BDNF/TrkB alleviated cardiac ischemic injury and inhibited cardiomyocytes apoptosis by regulating TRPC3/6 channels, which provides a novel potential therapeutic candidate for MI.

View Article: PubMed Central - PubMed

Affiliation: 1. Institute of Clinical Pharmacology of the Second Affiliated Hospital (Key Laboratory of Drug Research, Heilongjiang Higher Education Institutions), Harbin Medical University, Harbin 150086, China.

ABSTRACT

Background: Brain-derived neurotrophic factor (BDNF) is associated with coronary artery diseases. However, its role and mechanism in myocardial infarction (MI) is not fully understood.

Methods: Wistar rat and Kunming mouse model of MI were induced by the ligation of left coronary artery. Blood samples were collected from MI rats and patients. Plasma BDNF level, protein expression of BDNF, tropomyosin-related kinase B (TrkB) and its downstream transient receptor potential canonical (TRPC)3/6 channels were examined by enzyme-linked immunosorbent assay and Western blot. Infarct size, cardiac function and cardiomyocyte apoptosis were measured after intra-myocardium injection with recombinant human BDNF. Protective role of BDNF against cardiomyocyte apoptosis was confirmed by BDNF scavenger TrkB-Fc. The regulation of TRPC3/6 channels by BDNF was validated by pretreating with TRPC blocker (2-Aminoethyl diphenylborinate, 2-APB) and TRPC3/6 siRNAs.

Results: Circulating BDNF was significantly enhanced in MI rats and patients. Protein expression of BDNF, TrkB and TRPC3/6 channels were upregulated in MI. 3 days post-MI, BDNF treatment markedly reduced the infarct size and serum lactate dehydrogenase activity. Meanwhile, echocardiography indicated that BDNF significantly improved cardiac function of MI mice. Furthermore, BDNF markedly inhibited cardiomyocyte apoptosis by upregulating Bcl-2 expression and downregulating caspase-3 expression and activity in ischemic myocardium. In neonatal rat ventricular myocytes, cell viability was dramatically increased by BDNF in hypoxia, which was restored by TrkB-Fc. Furthermore, protective role of BDNF against hypoxia-induced apoptosis was reversed by 2-APB and TRPC3/6 siRNAs.

Conclusion: BDNF/TrkB alleviated cardiac ischemic injury and inhibited cardiomyocytes apoptosis by regulating TRPC3/6 channels, which provides a novel potential therapeutic candidate for MI.

Show MeSH
Related in: MedlinePlus