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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 is upregulated and foxo3a is downregulated in high-glucose-treated cardiomyocytes and diabetic rats. (a and b) Relative mRNA levels of mir-129, mir-106, mir-26a, mir-20, mir-197, mir-17, mir-27 and mir-30d in cardiomyocytes under normal and high-glucose (50 mM) conditions. (c and d) Mir-30d levels measured by real-time PCR both in cardiomyocytes treated with different concentrations of glucose (control, 30 and 50 mM) and in the hearts of control and DM rats. (e) Immunofluorescence results ( × 400) indicating the expression of foxo3a in normal glucose (control) and high-glucose-induced cardiomyocytes. Blue: nuclear staining (DAPI); red: foxo3a staining; violet: merged images. (f) Expression of foxo3a at protein and mRNA levels in cardiomyocytes treated with different concentrations of glucose. (g) Relative protein and mRNA levels of foxo3a in the hearts of control and DM rats. (h) Foxo3a expression of consecutive sections of hearts in control and DM rats assessed by immunohistochemical staining. Scale bar: 20 μm. n=3. *P<0.05 & **P<0.01 versus control; mean±S.E.M.
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fig3: Mir-30d is upregulated and foxo3a is downregulated in high-glucose-treated cardiomyocytes and diabetic rats. (a and b) Relative mRNA levels of mir-129, mir-106, mir-26a, mir-20, mir-197, mir-17, mir-27 and mir-30d in cardiomyocytes under normal and high-glucose (50 mM) conditions. (c and d) Mir-30d levels measured by real-time PCR both in cardiomyocytes treated with different concentrations of glucose (control, 30 and 50 mM) and in the hearts of control and DM rats. (e) Immunofluorescence results ( × 400) indicating the expression of foxo3a in normal glucose (control) and high-glucose-induced cardiomyocytes. Blue: nuclear staining (DAPI); red: foxo3a staining; violet: merged images. (f) Expression of foxo3a at protein and mRNA levels in cardiomyocytes treated with different concentrations of glucose. (g) Relative protein and mRNA levels of foxo3a in the hearts of control and DM rats. (h) Foxo3a expression of consecutive sections of hearts in control and DM rats assessed by immunohistochemical staining. Scale bar: 20 μm. n=3. *P<0.05 & **P<0.01 versus control; mean±S.E.M.

Mentions: We further investigated the possible involvement of miRNAs in high-glucose-treated cardiomyocytes to mimic hyperglycemia. We began by measuring the levels of several miRNAs reportedly associated with cardiovascular diseases, including mir-129, mir-106, mir-26a, mir-20, mir-197, mir-17, mir-27 and mir-30d,24, 25, 26, 27, 28, 29 in cardiomyocytes under both normal and high-glucose conditions. The results showed that mir-30d demonstrated the most pronounced deregulation in the high-glucose environment (Figures 3a and b), and high-glucose treatment increased mir-30d expression in a dose-dependent manner (Figure 3c). We subsequently determined whether mir-30d was involved in the pathogenesis of diabetic cardiomyopathy in rats. Overexpression of mir-30d was consistently observed in the DM rats compared with the control animals (Figure 3d). Moreover, we observed a significant downregulation of foxo3a, a transcription factor having a critical role in the regulation of cell death in cardiomyocytes,30 in high-glucose-treated cardiomyocytes, as determined by immunofluorescence staining, real-time PCR and western blotting analysis (Figures 3e and f). Similar downregulation of foxo3a was also observed in DM rats (Figures 3g and h).


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 is upregulated and foxo3a is downregulated in high-glucose-treated cardiomyocytes and diabetic rats. (a and b) Relative mRNA levels of mir-129, mir-106, mir-26a, mir-20, mir-197, mir-17, mir-27 and mir-30d in cardiomyocytes under normal and high-glucose (50 mM) conditions. (c and d) Mir-30d levels measured by real-time PCR both in cardiomyocytes treated with different concentrations of glucose (control, 30 and 50 mM) and in the hearts of control and DM rats. (e) Immunofluorescence results ( × 400) indicating the expression of foxo3a in normal glucose (control) and high-glucose-induced cardiomyocytes. Blue: nuclear staining (DAPI); red: foxo3a staining; violet: merged images. (f) Expression of foxo3a at protein and mRNA levels in cardiomyocytes treated with different concentrations of glucose. (g) Relative protein and mRNA levels of foxo3a in the hearts of control and DM rats. (h) Foxo3a expression of consecutive sections of hearts in control and DM rats assessed by immunohistochemical staining. Scale bar: 20 μm. n=3. *P<0.05 & **P<0.01 versus control; mean±S.E.M.
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fig3: Mir-30d is upregulated and foxo3a is downregulated in high-glucose-treated cardiomyocytes and diabetic rats. (a and b) Relative mRNA levels of mir-129, mir-106, mir-26a, mir-20, mir-197, mir-17, mir-27 and mir-30d in cardiomyocytes under normal and high-glucose (50 mM) conditions. (c and d) Mir-30d levels measured by real-time PCR both in cardiomyocytes treated with different concentrations of glucose (control, 30 and 50 mM) and in the hearts of control and DM rats. (e) Immunofluorescence results ( × 400) indicating the expression of foxo3a in normal glucose (control) and high-glucose-induced cardiomyocytes. Blue: nuclear staining (DAPI); red: foxo3a staining; violet: merged images. (f) Expression of foxo3a at protein and mRNA levels in cardiomyocytes treated with different concentrations of glucose. (g) Relative protein and mRNA levels of foxo3a in the hearts of control and DM rats. (h) Foxo3a expression of consecutive sections of hearts in control and DM rats assessed by immunohistochemical staining. Scale bar: 20 μm. n=3. *P<0.05 & **P<0.01 versus control; mean±S.E.M.
Mentions: We further investigated the possible involvement of miRNAs in high-glucose-treated cardiomyocytes to mimic hyperglycemia. We began by measuring the levels of several miRNAs reportedly associated with cardiovascular diseases, including mir-129, mir-106, mir-26a, mir-20, mir-197, mir-17, mir-27 and mir-30d,24, 25, 26, 27, 28, 29 in cardiomyocytes under both normal and high-glucose conditions. The results showed that mir-30d demonstrated the most pronounced deregulation in the high-glucose environment (Figures 3a and b), and high-glucose treatment increased mir-30d expression in a dose-dependent manner (Figure 3c). We subsequently determined whether mir-30d was involved in the pathogenesis of diabetic cardiomyopathy in rats. Overexpression of mir-30d was consistently observed in the DM rats compared with the control animals (Figure 3d). Moreover, we observed a significant downregulation of foxo3a, a transcription factor having a critical role in the regulation of cell death in cardiomyocytes,30 in high-glucose-treated cardiomyocytes, as determined by immunofluorescence staining, real-time PCR and western blotting analysis (Figures 3e and f). Similar downregulation of foxo3a was also observed in DM rats (Figures 3g and h).

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