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Lin28a protects against hypoxia/reoxygenation induced cardiomyocytes apoptosis by alleviating mitochondrial dysfunction under high glucose/high fat conditions.

Zhang M, Niu X, Hu J, Yuan Y, Sun S, Wang J, Yu W, Wang C, Sun D, Wang H - PLoS ONE (2014)

Bottom Line: Our results revealed that Lin28a cDNA transfection (overexpression) significantly inhibited cardiomyocyte apoptotic index, improved mitochondria biogenesis, increased ATP production and reduced ROS production as compared with the H/R group in HG/HF conditions.The present results suggest that Lin28a inhibits cardiomyocytes apoptosis by enhancing mitochondrial biogenesis and function under high glucose/high fat conditions.The mechanism responsible for the effects of Lin28a is associated with the PI3K/Akt dependent pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, China.

ABSTRACT

Aim: The aim of the present study was to investigate the role of Lin28a in protecting against hypoxia/reoxygenation (H/R)-induced cardiomyocytes apoptosis under high glucose/high fat (HG/HF) conditions.

Methods: Primary cardiomyocytes which were isolated from neonatal mouse were randomized to be treated with lentivirus carrying Lin28a siRNA, Lin28acDNA 72 h before H/R (9 h/2 h). Cardiomyocytes biomarkers release (LDH and CK), cardiomyocytes apoptosis, mitochondria biogenesis and morphology, intracellular reactive oxygen species (ROS) production, ATP content and inflammatory cytokines levels after H/R injury in high glucose/high fat conditions were compared between groups. The target proteins of Lin28a were examined by western blot analysis.

Results: Our results revealed that Lin28a cDNA transfection (overexpression) significantly inhibited cardiomyocyte apoptotic index, improved mitochondria biogenesis, increased ATP production and reduced ROS production as compared with the H/R group in HG/HF conditions. Lin28a siRNA transfection (knockdown) rendered the cardiomyocytes more susceptible to H/R injury as evidenced by increased apoptotic index, impaired mitochondrial biogenesis, decreased ATP production and increased ROS level. Interestingly, these effects of Lin28a were blocked by pretreatment with the PI3K inhibitor wortmannin. Lin28a overexpression increased, while Lin28a knockdown inhibited IGF1R, Nrf-1, Tfam, p-IRS-1, p-Akt, p-mTOR, p-p70s6k, p-AMPK expression levels after H/R injury in HG/HF conditions. Moreover, pretreatment with wortmannin abolished the effects of Lin28a on the expression levels of p-AKT, p-mTOR, p-p70s6k, p-AMPK.

Conclusions: The present results suggest that Lin28a inhibits cardiomyocytes apoptosis by enhancing mitochondrial biogenesis and function under high glucose/high fat conditions. The mechanism responsible for the effects of Lin28a is associated with the PI3K/Akt dependent pathway.

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Validation of Lin28a overexpression and Lin28a knockdown.Lin28a expression levels with representative gel blots of Lin28a and β-actin (loading control) were shown (A); Lin28a mRNA expression as evaluated by real-time PCR analysis (B); Let7a (microRNA) expression as evaluated by real-time PCR analysis (C). Columns and bars represent mean ± SD. Data were obtained from at least three independent experiments. W, wortmannin.*p<0.05 vs. H/R, #p<0.05 vs. H/R+Lin28a.
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pone-0110580-g001: Validation of Lin28a overexpression and Lin28a knockdown.Lin28a expression levels with representative gel blots of Lin28a and β-actin (loading control) were shown (A); Lin28a mRNA expression as evaluated by real-time PCR analysis (B); Let7a (microRNA) expression as evaluated by real-time PCR analysis (C). Columns and bars represent mean ± SD. Data were obtained from at least three independent experiments. W, wortmannin.*p<0.05 vs. H/R, #p<0.05 vs. H/R+Lin28a.

Mentions: Primary cardiomyocytes were treated with lentivirus carrying Lin28a siRNA, Lin28a cDNA 72 h before H/R (9 h/2 h). HG/HF treatment decreased Lin28a expression levels compared with the control group. H/R group decreased Lin28a expression levels compared with the HG/HF group (Figure 1A, 1B). Lin28a overexpression inhibited, while Lin28a siRNA administration promoted let7a expression as indicated by real-time PCR analysis (Figure 1C). HG/HF treatment increased Let7a expression levels compared with the control group. H/R group increased Lin28a expression levels compared with the HG/HF group (Figure 1A, 1B). There was no differences between H/R+Lin28a group and H/R+Lin28a+W group in Lin28a and Let7a expression levels (Figure 1A–1C). The Ct values were presented in table S1.


Lin28a protects against hypoxia/reoxygenation induced cardiomyocytes apoptosis by alleviating mitochondrial dysfunction under high glucose/high fat conditions.

Zhang M, Niu X, Hu J, Yuan Y, Sun S, Wang J, Yu W, Wang C, Sun D, Wang H - PLoS ONE (2014)

Validation of Lin28a overexpression and Lin28a knockdown.Lin28a expression levels with representative gel blots of Lin28a and β-actin (loading control) were shown (A); Lin28a mRNA expression as evaluated by real-time PCR analysis (B); Let7a (microRNA) expression as evaluated by real-time PCR analysis (C). Columns and bars represent mean ± SD. Data were obtained from at least three independent experiments. W, wortmannin.*p<0.05 vs. H/R, #p<0.05 vs. H/R+Lin28a.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196990&req=5

pone-0110580-g001: Validation of Lin28a overexpression and Lin28a knockdown.Lin28a expression levels with representative gel blots of Lin28a and β-actin (loading control) were shown (A); Lin28a mRNA expression as evaluated by real-time PCR analysis (B); Let7a (microRNA) expression as evaluated by real-time PCR analysis (C). Columns and bars represent mean ± SD. Data were obtained from at least three independent experiments. W, wortmannin.*p<0.05 vs. H/R, #p<0.05 vs. H/R+Lin28a.
Mentions: Primary cardiomyocytes were treated with lentivirus carrying Lin28a siRNA, Lin28a cDNA 72 h before H/R (9 h/2 h). HG/HF treatment decreased Lin28a expression levels compared with the control group. H/R group decreased Lin28a expression levels compared with the HG/HF group (Figure 1A, 1B). Lin28a overexpression inhibited, while Lin28a siRNA administration promoted let7a expression as indicated by real-time PCR analysis (Figure 1C). HG/HF treatment increased Let7a expression levels compared with the control group. H/R group increased Lin28a expression levels compared with the HG/HF group (Figure 1A, 1B). There was no differences between H/R+Lin28a group and H/R+Lin28a+W group in Lin28a and Let7a expression levels (Figure 1A–1C). The Ct values were presented in table S1.

Bottom Line: Our results revealed that Lin28a cDNA transfection (overexpression) significantly inhibited cardiomyocyte apoptotic index, improved mitochondria biogenesis, increased ATP production and reduced ROS production as compared with the H/R group in HG/HF conditions.The present results suggest that Lin28a inhibits cardiomyocytes apoptosis by enhancing mitochondrial biogenesis and function under high glucose/high fat conditions.The mechanism responsible for the effects of Lin28a is associated with the PI3K/Akt dependent pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, China.

ABSTRACT

Aim: The aim of the present study was to investigate the role of Lin28a in protecting against hypoxia/reoxygenation (H/R)-induced cardiomyocytes apoptosis under high glucose/high fat (HG/HF) conditions.

Methods: Primary cardiomyocytes which were isolated from neonatal mouse were randomized to be treated with lentivirus carrying Lin28a siRNA, Lin28acDNA 72 h before H/R (9 h/2 h). Cardiomyocytes biomarkers release (LDH and CK), cardiomyocytes apoptosis, mitochondria biogenesis and morphology, intracellular reactive oxygen species (ROS) production, ATP content and inflammatory cytokines levels after H/R injury in high glucose/high fat conditions were compared between groups. The target proteins of Lin28a were examined by western blot analysis.

Results: Our results revealed that Lin28a cDNA transfection (overexpression) significantly inhibited cardiomyocyte apoptotic index, improved mitochondria biogenesis, increased ATP production and reduced ROS production as compared with the H/R group in HG/HF conditions. Lin28a siRNA transfection (knockdown) rendered the cardiomyocytes more susceptible to H/R injury as evidenced by increased apoptotic index, impaired mitochondrial biogenesis, decreased ATP production and increased ROS level. Interestingly, these effects of Lin28a were blocked by pretreatment with the PI3K inhibitor wortmannin. Lin28a overexpression increased, while Lin28a knockdown inhibited IGF1R, Nrf-1, Tfam, p-IRS-1, p-Akt, p-mTOR, p-p70s6k, p-AMPK expression levels after H/R injury in HG/HF conditions. Moreover, pretreatment with wortmannin abolished the effects of Lin28a on the expression levels of p-AKT, p-mTOR, p-p70s6k, p-AMPK.

Conclusions: The present results suggest that Lin28a inhibits cardiomyocytes apoptosis by enhancing mitochondrial biogenesis and function under high glucose/high fat conditions. The mechanism responsible for the effects of Lin28a is associated with the PI3K/Akt dependent pathway.

Show MeSH
Related in: MedlinePlus