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MicroRNA-30d regulates cardiomyocyte pyroptosis by directly targeting foxo3a in diabetic cardiomyopathy.

Li X, Du N, Zhang Q, Li J, Chen X, Liu X, Hu Y, Qin W, Shen N, Xu C, Fang Z, Wei Y, Wang R, Du Z, Zhang Y, Lu Y - Cell Death Dis (2014)

Bottom Line: Diabetic cardiomyopathy is a common cardiac condition in patients with diabetes mellitus, which can result in cardiac hypertrophy and subsequent heart failure, associated with pyroptosis, the pro-inflammatory programmed cell death.In an effort to understand the signaling mechanisms underlying the pro-pyroptotic property of mir-30d, we found that forced expression of mir-30d upregulated caspase-1 and pro-inflammatory cytokines IL-1β and IL-18.These findings promoted us to propose a new signaling pathway leading to cardiomyocyte pyroptosis under hyperglycemic conditions: mir-30d↑→foxo3a↓→ ARC↓→caspase-1↑→IL-1β, IL-18↑→pyroptosis↑.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), Harbin Medical University, Harbin 150081, China.

ABSTRACT
Diabetic cardiomyopathy is a common cardiac condition in patients with diabetes mellitus, which can result in cardiac hypertrophy and subsequent heart failure, associated with pyroptosis, the pro-inflammatory programmed cell death. MicroRNAs (miRNAs), small endogenous non-coding RNAs, have been shown to be involved in diabetic cardiomyopathy. However, whether miRNAs regulate pyroptosis in diabetic cardiomyopathy remains unknown. Our study revealed that mir-30d expression was substantially increased in streptozotocin (STZ)-induced diabetic rats and in high-glucose-treated cardiomyocytes as well. Upregulation of mir-30d promoted cardiomyocyte pyroptosis in diabetic cardiomyopathy; conversely, knockdown of mir-30d attenuated it. In an effort to understand the signaling mechanisms underlying the pro-pyroptotic property of mir-30d, we found that forced expression of mir-30d upregulated caspase-1 and pro-inflammatory cytokines IL-1β and IL-18. Moreover, mir-30d directly repressed foxo3a expression and its downstream protein, apoptosis repressor with caspase recruitment domain (ARC). Furthermore, silencing ARC by siRNA mimicked the action of mir-30d: upregulating caspase-1 and inducing pyroptosis. These findings promoted us to propose a new signaling pathway leading to cardiomyocyte pyroptosis under hyperglycemic conditions: mir-30d↑→foxo3a↓→ ARC↓→caspase-1↑→IL-1β, IL-18↑→pyroptosis↑. Therefore, mir-30d may be a promising therapeutic target for the management of diabetic cardiomyopathy.

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Mir-30d upregulates caspase-1 expression through ARC. (a) Relative protein and mRNA levels of ARC in cardiomyocytes treated with different concentrations of glucose (control, 30 and 50 mM). (b) Relative protein and mRNA levels of ARC in the control, mir-30d and NC groups. ARC levels in the HG, HG+AMO-mir-30d and HG+AMO-NC groups in high-glucose-treated cardiomyocytes are shown in the bottom panel. (c) Immunofluorescence images ( × 400) showing the expression of ARC in cardiomyocytes in the control, HG, HG+mir-30d, and HG+AMO-mir-30d groups. Blue: nuclear staining (DAPI); green: ARC staining. (d) Relative protein and mRNA levels of caspase-1 in the control, mir-30d and NC groups. Caspase-1 levels in the HG, HG+AMO-mir-30d and HG+AMO-NC groups in high-glucose-treated cardiomyocytes are shown in the bottom panel. (e) Immunofluorescence images ( × 400) showing expression of caspase-1 in the control, HG, HG+mir-30d, and HG+AMO-mir-30d groups. Blue: nuclear staining (DAPI); green: caspase-1 staining. n=3. *P<0.05 and **P<0.01 versus control; #P<0.05 and ##P<0.01 versus mir-30d; +P<0.05 and ++P<0.01 versus HG; §P <0.05 and §§P<0.01 versus HG+AMO-mir-30d; mean±S.E.M.
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fig5: Mir-30d upregulates caspase-1 expression through ARC. (a) Relative protein and mRNA levels of ARC in cardiomyocytes treated with different concentrations of glucose (control, 30 and 50 mM). (b) Relative protein and mRNA levels of ARC in the control, mir-30d and NC groups. ARC levels in the HG, HG+AMO-mir-30d and HG+AMO-NC groups in high-glucose-treated cardiomyocytes are shown in the bottom panel. (c) Immunofluorescence images ( × 400) showing the expression of ARC in cardiomyocytes in the control, HG, HG+mir-30d, and HG+AMO-mir-30d groups. Blue: nuclear staining (DAPI); green: ARC staining. (d) Relative protein and mRNA levels of caspase-1 in the control, mir-30d and NC groups. Caspase-1 levels in the HG, HG+AMO-mir-30d and HG+AMO-NC groups in high-glucose-treated cardiomyocytes are shown in the bottom panel. (e) Immunofluorescence images ( × 400) showing expression of caspase-1 in the control, HG, HG+mir-30d, and HG+AMO-mir-30d groups. Blue: nuclear staining (DAPI); green: caspase-1 staining. n=3. *P<0.05 and **P<0.01 versus control; #P<0.05 and ##P<0.01 versus mir-30d; +P<0.05 and ++P<0.01 versus HG; §P <0.05 and §§P<0.01 versus HG+AMO-mir-30d; mean±S.E.M.

Mentions: ARC, an antiapoptotic gene, is known to be a transcriptional target of foxo3a;31 we therefore assessed the effect of mir-30d on the expression of ARC. Treatment of cardiomyocytes with high glucose (30 and 50 mmol/l) significantly inhibited ARC at both protein and mRNA levels (Figure 5a). Moreover, mir-30d overexpression mimicked high-glucose-induced downregulation of ARC in cardiomyocytes under normal glycemic environment, and knockdown of mir-30d restored ARC levels in cardiomyocytes under hyperglycemic conditions, as verified by western blotting analysis, real-time PCR methods and immunofluorescence staining (Figures 5b and c).


MicroRNA-30d regulates cardiomyocyte pyroptosis by directly targeting foxo3a in diabetic cardiomyopathy.

Li X, Du N, Zhang Q, Li J, Chen X, Liu X, Hu Y, Qin W, Shen N, Xu C, Fang Z, Wei Y, Wang R, Du Z, Zhang Y, Lu Y - Cell Death Dis (2014)

Mir-30d upregulates caspase-1 expression through ARC. (a) Relative protein and mRNA levels of ARC in cardiomyocytes treated with different concentrations of glucose (control, 30 and 50 mM). (b) Relative protein and mRNA levels of ARC in the control, mir-30d and NC groups. ARC levels in the HG, HG+AMO-mir-30d and HG+AMO-NC groups in high-glucose-treated cardiomyocytes are shown in the bottom panel. (c) Immunofluorescence images ( × 400) showing the expression of ARC in cardiomyocytes in the control, HG, HG+mir-30d, and HG+AMO-mir-30d groups. Blue: nuclear staining (DAPI); green: ARC staining. (d) Relative protein and mRNA levels of caspase-1 in the control, mir-30d and NC groups. Caspase-1 levels in the HG, HG+AMO-mir-30d and HG+AMO-NC groups in high-glucose-treated cardiomyocytes are shown in the bottom panel. (e) Immunofluorescence images ( × 400) showing expression of caspase-1 in the control, HG, HG+mir-30d, and HG+AMO-mir-30d groups. Blue: nuclear staining (DAPI); green: caspase-1 staining. n=3. *P<0.05 and **P<0.01 versus control; #P<0.05 and ##P<0.01 versus mir-30d; +P<0.05 and ++P<0.01 versus HG; §P <0.05 and §§P<0.01 versus HG+AMO-mir-30d; mean±S.E.M.
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fig5: Mir-30d upregulates caspase-1 expression through ARC. (a) Relative protein and mRNA levels of ARC in cardiomyocytes treated with different concentrations of glucose (control, 30 and 50 mM). (b) Relative protein and mRNA levels of ARC in the control, mir-30d and NC groups. ARC levels in the HG, HG+AMO-mir-30d and HG+AMO-NC groups in high-glucose-treated cardiomyocytes are shown in the bottom panel. (c) Immunofluorescence images ( × 400) showing the expression of ARC in cardiomyocytes in the control, HG, HG+mir-30d, and HG+AMO-mir-30d groups. Blue: nuclear staining (DAPI); green: ARC staining. (d) Relative protein and mRNA levels of caspase-1 in the control, mir-30d and NC groups. Caspase-1 levels in the HG, HG+AMO-mir-30d and HG+AMO-NC groups in high-glucose-treated cardiomyocytes are shown in the bottom panel. (e) Immunofluorescence images ( × 400) showing expression of caspase-1 in the control, HG, HG+mir-30d, and HG+AMO-mir-30d groups. Blue: nuclear staining (DAPI); green: caspase-1 staining. n=3. *P<0.05 and **P<0.01 versus control; #P<0.05 and ##P<0.01 versus mir-30d; +P<0.05 and ++P<0.01 versus HG; §P <0.05 and §§P<0.01 versus HG+AMO-mir-30d; mean±S.E.M.
Mentions: ARC, an antiapoptotic gene, is known to be a transcriptional target of foxo3a;31 we therefore assessed the effect of mir-30d on the expression of ARC. Treatment of cardiomyocytes with high glucose (30 and 50 mmol/l) significantly inhibited ARC at both protein and mRNA levels (Figure 5a). Moreover, mir-30d overexpression mimicked high-glucose-induced downregulation of ARC in cardiomyocytes under normal glycemic environment, and knockdown of mir-30d restored ARC levels in cardiomyocytes under hyperglycemic conditions, as verified by western blotting analysis, real-time PCR methods and immunofluorescence staining (Figures 5b and c).

Bottom Line: Diabetic cardiomyopathy is a common cardiac condition in patients with diabetes mellitus, which can result in cardiac hypertrophy and subsequent heart failure, associated with pyroptosis, the pro-inflammatory programmed cell death.In an effort to understand the signaling mechanisms underlying the pro-pyroptotic property of mir-30d, we found that forced expression of mir-30d upregulated caspase-1 and pro-inflammatory cytokines IL-1β and IL-18.These findings promoted us to propose a new signaling pathway leading to cardiomyocyte pyroptosis under hyperglycemic conditions: mir-30d↑→foxo3a↓→ ARC↓→caspase-1↑→IL-1β, IL-18↑→pyroptosis↑.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), Harbin Medical University, Harbin 150081, China.

ABSTRACT
Diabetic cardiomyopathy is a common cardiac condition in patients with diabetes mellitus, which can result in cardiac hypertrophy and subsequent heart failure, associated with pyroptosis, the pro-inflammatory programmed cell death. MicroRNAs (miRNAs), small endogenous non-coding RNAs, have been shown to be involved in diabetic cardiomyopathy. However, whether miRNAs regulate pyroptosis in diabetic cardiomyopathy remains unknown. Our study revealed that mir-30d expression was substantially increased in streptozotocin (STZ)-induced diabetic rats and in high-glucose-treated cardiomyocytes as well. Upregulation of mir-30d promoted cardiomyocyte pyroptosis in diabetic cardiomyopathy; conversely, knockdown of mir-30d attenuated it. In an effort to understand the signaling mechanisms underlying the pro-pyroptotic property of mir-30d, we found that forced expression of mir-30d upregulated caspase-1 and pro-inflammatory cytokines IL-1β and IL-18. Moreover, mir-30d directly repressed foxo3a expression and its downstream protein, apoptosis repressor with caspase recruitment domain (ARC). Furthermore, silencing ARC by siRNA mimicked the action of mir-30d: upregulating caspase-1 and inducing pyroptosis. These findings promoted us to propose a new signaling pathway leading to cardiomyocyte pyroptosis under hyperglycemic conditions: mir-30d↑→foxo3a↓→ ARC↓→caspase-1↑→IL-1β, IL-18↑→pyroptosis↑. Therefore, mir-30d may be a promising therapeutic target for the management of diabetic cardiomyopathy.

Show MeSH
Related in: MedlinePlus