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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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Antiapoptotic effect of Lin28a overexpression on cardiomyocytes after H/R injury in HG/HF conditions.Representative images of TUNEL-stained primary neonatal cardiomyocytes after H/R stress in HG/HF conditions (A). Apoptosis of the primary cardiomyocytes determined by Cy3-annexinV/PI double staining and flow cytometry (n = 3). Region B2: late apoptotic cells (Cy3/PI, where Cy3 is cyanine-3 and PI is propidium iodide); Region B3: vital cells; Region B4: early apoptotic cells (C). Apoptotic index is expressed as the percentage of TUNEL-positive myocytes (in red) over total nuclei determined by DAPI staining(B). Apoptotic rate is expressed as the percentage of late apoptotic cells and early apoptotic cells(D). Protein expression with representative gel blots of Caspase-3, Cleaved Caspase-3, Bcl-2, Bax and β-actin (loading control) (E). Caspase-3(F); Cleaved Caspase-3(G); Bcl-2(H); Bax(I); Bax/Bcl-2 ratio(J). The columns and error bars represent means and SD. Data were obtained from at least three independent experiments.*P<0.05 vs. H/R, #P<0.05 vs. H/R+Lin28a.
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pone-0110580-g002: Antiapoptotic effect of Lin28a overexpression on cardiomyocytes after H/R injury in HG/HF conditions.Representative images of TUNEL-stained primary neonatal cardiomyocytes after H/R stress in HG/HF conditions (A). Apoptosis of the primary cardiomyocytes determined by Cy3-annexinV/PI double staining and flow cytometry (n = 3). Region B2: late apoptotic cells (Cy3/PI, where Cy3 is cyanine-3 and PI is propidium iodide); Region B3: vital cells; Region B4: early apoptotic cells (C). Apoptotic index is expressed as the percentage of TUNEL-positive myocytes (in red) over total nuclei determined by DAPI staining(B). Apoptotic rate is expressed as the percentage of late apoptotic cells and early apoptotic cells(D). Protein expression with representative gel blots of Caspase-3, Cleaved Caspase-3, Bcl-2, Bax and β-actin (loading control) (E). Caspase-3(F); Cleaved Caspase-3(G); Bcl-2(H); Bax(I); Bax/Bcl-2 ratio(J). The columns and error bars represent means and SD. Data were obtained from at least three independent experiments.*P<0.05 vs. H/R, #P<0.05 vs. H/R+Lin28a.

Mentions: As is shown in representative TUNEL images (Figure 2A), TUNEL-positive myocytes were less observed in Lin28a overexpression group as compared with the H/R group. Concomitantly, Cleaved Caspase-3, Caspase-3 and Bax protein levels evaluated by western blot were down-regulated by Lin28a overexpression. The Bcl-2 protein level evaluated by western blot was up-regulated by Lin28a overexpression. Furthermore, Lin28a overexpression decreased Bax/Bcl-2 ratio (Figure 2E–2J). Cardiomyocytes were likewise treated and analyzed by flow cytometry. The results also showed that Lin28a overexpression inhibited cellular apoptosis (Figure 2C). Lentivirus carrying Lin28a siRNA transduced cardiomyocytes showed a trend of cardiomyocytes.


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)

Antiapoptotic effect of Lin28a overexpression on cardiomyocytes after H/R injury in HG/HF conditions.Representative images of TUNEL-stained primary neonatal cardiomyocytes after H/R stress in HG/HF conditions (A). Apoptosis of the primary cardiomyocytes determined by Cy3-annexinV/PI double staining and flow cytometry (n = 3). Region B2: late apoptotic cells (Cy3/PI, where Cy3 is cyanine-3 and PI is propidium iodide); Region B3: vital cells; Region B4: early apoptotic cells (C). Apoptotic index is expressed as the percentage of TUNEL-positive myocytes (in red) over total nuclei determined by DAPI staining(B). Apoptotic rate is expressed as the percentage of late apoptotic cells and early apoptotic cells(D). Protein expression with representative gel blots of Caspase-3, Cleaved Caspase-3, Bcl-2, Bax and β-actin (loading control) (E). Caspase-3(F); Cleaved Caspase-3(G); Bcl-2(H); Bax(I); Bax/Bcl-2 ratio(J). The columns and error bars represent means and SD. Data were obtained from at least three independent experiments.*P<0.05 vs. H/R, #P<0.05 vs. H/R+Lin28a.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110580-g002: Antiapoptotic effect of Lin28a overexpression on cardiomyocytes after H/R injury in HG/HF conditions.Representative images of TUNEL-stained primary neonatal cardiomyocytes after H/R stress in HG/HF conditions (A). Apoptosis of the primary cardiomyocytes determined by Cy3-annexinV/PI double staining and flow cytometry (n = 3). Region B2: late apoptotic cells (Cy3/PI, where Cy3 is cyanine-3 and PI is propidium iodide); Region B3: vital cells; Region B4: early apoptotic cells (C). Apoptotic index is expressed as the percentage of TUNEL-positive myocytes (in red) over total nuclei determined by DAPI staining(B). Apoptotic rate is expressed as the percentage of late apoptotic cells and early apoptotic cells(D). Protein expression with representative gel blots of Caspase-3, Cleaved Caspase-3, Bcl-2, Bax and β-actin (loading control) (E). Caspase-3(F); Cleaved Caspase-3(G); Bcl-2(H); Bax(I); Bax/Bcl-2 ratio(J). The columns and error bars represent means and SD. Data were obtained from at least three independent experiments.*P<0.05 vs. H/R, #P<0.05 vs. H/R+Lin28a.
Mentions: As is shown in representative TUNEL images (Figure 2A), TUNEL-positive myocytes were less observed in Lin28a overexpression group as compared with the H/R group. Concomitantly, Cleaved Caspase-3, Caspase-3 and Bax protein levels evaluated by western blot were down-regulated by Lin28a overexpression. The Bcl-2 protein level evaluated by western blot was up-regulated by Lin28a overexpression. Furthermore, Lin28a overexpression decreased Bax/Bcl-2 ratio (Figure 2E–2J). Cardiomyocytes were likewise treated and analyzed by flow cytometry. The results also showed that Lin28a overexpression inhibited cellular apoptosis (Figure 2C). Lentivirus carrying Lin28a siRNA transduced cardiomyocytes showed a trend of cardiomyocytes.

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