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Protein phosphatase 2C-alpha knockdown reduces angiotensin II-mediated skeletal muscle wasting via restoration of mitochondrial recycling and function.

Tabony AM, Yoshida T, Sukhanov S, Delafontaine P - Skelet Muscle (2014)

Bottom Line: This induction was reduced by PP2Cα knockdown, which also increased beclin-1 expression and microtubule-associated protein 1 light chain 3 (LC3)-II conversion in AngII-infused Gas.AngII reduced activating S555 phosphorylation of UNC-51-like kinase 1 (ULK1), a critical regulator of autophagosome formation, and increased inhibitory S757 ULK1 phosphorylation and these effects were prevented by PP2Cα siRNA.These results demonstrate novel effects of AngII on cellular metabolism that are likely critical in mediating the muscle wasting that is a hallmark of CHF.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tulane University Department of Medicine, Heart and Vascular Institute, New Orleans, LA 70112, USA.

ABSTRACT

Background: Circulating angiotensin II (AngII) is elevated in congestive heart failure (CHF), and leads to skeletal muscle wasting, which is strongly associated with poor patient outcomes. We previously found that AngII upregulates protein phosphatase 2C-alpha (PP2Cα) and dephosphorylates AMP-activated protein kinase (AMPK), a critical regulator of cellular metabolism, in skeletal muscle.

Methods: To determine the role of PP2Cα in AngII-induced wasting, gastrocnemius (Gas) muscles of FVB mice were injected with scrambled or PP2Cα siRNA and mice were infused with saline or AngII for 4 days.

Results: Knockdown of PP2Cα reduced AngII wasting, blocked AngII upregulation of PP2Cα, increased p-T172-AMPK, and inhibited AngII-mediated reductions in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (TFAM), in complex IV activity, and in ATP levels. AngII impaired the rate of autophagy as determined by a 2.4-fold increase in p62/SQSTM1 (p62) accumulation. This induction was reduced by PP2Cα knockdown, which also increased beclin-1 expression and microtubule-associated protein 1 light chain 3 (LC3)-II conversion in AngII-infused Gas. AngII reduced activating S555 phosphorylation of UNC-51-like kinase 1 (ULK1), a critical regulator of autophagosome formation, and increased inhibitory S757 ULK1 phosphorylation and these effects were prevented by PP2Cα siRNA.

Conclusions: AngII inhibited AMPK activity and reduced PGC-1α and TFAM expression (thereby inhibiting mitochondrial biogenesis) and impaired ULK1 activation and autophagy (thereby also inhibiting clearance of damaged mitochondria), resulting in mitochondrial dysfunction, decreased ATP, and wasting. Knockdown of PP2Cα normalized AMPK activity, PGC-1α, NRF1, and TFAM levels and blocked AngII inhibition of ULK1, leading to improved mitochondrial biogenesis/recycling/function, energy production, and inhibition of AngII-induced wasting. These results demonstrate novel effects of AngII on cellular metabolism that are likely critical in mediating the muscle wasting that is a hallmark of CHF.

No MeSH data available.


Related in: MedlinePlus

In vivo knockdown of PP2Cα. The indicated siRNAs were injected at five sites in Gas muscles which were then electroporated to induce uptake of the siRNA. The most effective sequence, time point, and dose of siRNA were chosen for subsequent AngII-infusion experiments. (A) Real-time PCR showing PP2Cα mRNA knockdown in Gas using 2.5 μg of four different PP2Cα siRNA target sequences relative to negative control siRNA at day 4 post electroporation. PP2Cα siRNA ‘A’ was used in all time course and dose response experiments. (B) Time-course of PP2Cα mRNA knockdown in Gas with 2.5 μg of scrambled and PP2Cα siRNA. (C) Time-course of PP2Cα protein knockdown in Gas with 2.5 μg of scrambled and PP2Cα siRNA. (D) Time-course of AMPK phosphorylation in Gas with 2.5 μg of scrambled and PP2Cα siRNA. (E) Dose response of PP2Cα mRNA knockdown in Gas at day 7 post electroporation. (F) Dose response of PP2Cα protein knockdown in Gas at day 7 post electroporation. (G) Dose response of AMPK phosphorylation in Gas at day 7 post electroporation. (H) Representative western blots showing PP2Cα protein knockdown and AMPK activation with 5 μg of PP2Cα siRNA at day 7. n =4-8 per group, Mean ± SEM, *P <0.05, **P <0.01, ***P <0.001.
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Figure 1: In vivo knockdown of PP2Cα. The indicated siRNAs were injected at five sites in Gas muscles which were then electroporated to induce uptake of the siRNA. The most effective sequence, time point, and dose of siRNA were chosen for subsequent AngII-infusion experiments. (A) Real-time PCR showing PP2Cα mRNA knockdown in Gas using 2.5 μg of four different PP2Cα siRNA target sequences relative to negative control siRNA at day 4 post electroporation. PP2Cα siRNA ‘A’ was used in all time course and dose response experiments. (B) Time-course of PP2Cα mRNA knockdown in Gas with 2.5 μg of scrambled and PP2Cα siRNA. (C) Time-course of PP2Cα protein knockdown in Gas with 2.5 μg of scrambled and PP2Cα siRNA. (D) Time-course of AMPK phosphorylation in Gas with 2.5 μg of scrambled and PP2Cα siRNA. (E) Dose response of PP2Cα mRNA knockdown in Gas at day 7 post electroporation. (F) Dose response of PP2Cα protein knockdown in Gas at day 7 post electroporation. (G) Dose response of AMPK phosphorylation in Gas at day 7 post electroporation. (H) Representative western blots showing PP2Cα protein knockdown and AMPK activation with 5 μg of PP2Cα siRNA at day 7. n =4-8 per group, Mean ± SEM, *P <0.05, **P <0.01, ***P <0.001.

Mentions: Four days after electroporation, PP2Cα siRNAs B and C both significantly reduced expression of PP2Cα mRNA in Gas by approximately 37% (P <0.05), while siRNA A proved to be the most effective with a 57% reduction (P <0.001) in mRNA compared to control, and siRNA D failed to reduce expression of PP2Cα (Figure 1A). A total of 2.5 μg of PP2Cα siRNA A significantly reduced gene expression from 1 to 7 days post electroporation, with a peak suppression from baseline of 75% measured at day 3, and returning to normal by day 11 (Figure 1B). There was a non-statistically significant trend for transient suppression of PP2Cα mRNA expression with scrambled siRNA/electroporation, which returned to normal between days 3 and 5 (Figure 1B). Similarly, PP2Cα protein tended to be reduced by PP2Cα siRNA A throughout the time course, with a significant reduction of 58% at day 7 (Figure 1C), while AMPK phosphorylation was significantly increased at days 1, 5, and 7 (Figure 1D). The dose response performed at day 7 indicated that 5 μg of siRNA was the optimal dose, with a 51% reduction in expression compared to 31% with 2.5 μg, while scrambled siRNA did not reduce expression at all (Figure 1E). PP2Cα protein expression was significantly reduced at all doses of PP2Cα siRNA (Figure 1F), and there was a concomitant 2.3-fold increase in AMPK phosphorylation with 5 μg of PP2Cα siRNA (Figure 1G).


Protein phosphatase 2C-alpha knockdown reduces angiotensin II-mediated skeletal muscle wasting via restoration of mitochondrial recycling and function.

Tabony AM, Yoshida T, Sukhanov S, Delafontaine P - Skelet Muscle (2014)

In vivo knockdown of PP2Cα. The indicated siRNAs were injected at five sites in Gas muscles which were then electroporated to induce uptake of the siRNA. The most effective sequence, time point, and dose of siRNA were chosen for subsequent AngII-infusion experiments. (A) Real-time PCR showing PP2Cα mRNA knockdown in Gas using 2.5 μg of four different PP2Cα siRNA target sequences relative to negative control siRNA at day 4 post electroporation. PP2Cα siRNA ‘A’ was used in all time course and dose response experiments. (B) Time-course of PP2Cα mRNA knockdown in Gas with 2.5 μg of scrambled and PP2Cα siRNA. (C) Time-course of PP2Cα protein knockdown in Gas with 2.5 μg of scrambled and PP2Cα siRNA. (D) Time-course of AMPK phosphorylation in Gas with 2.5 μg of scrambled and PP2Cα siRNA. (E) Dose response of PP2Cα mRNA knockdown in Gas at day 7 post electroporation. (F) Dose response of PP2Cα protein knockdown in Gas at day 7 post electroporation. (G) Dose response of AMPK phosphorylation in Gas at day 7 post electroporation. (H) Representative western blots showing PP2Cα protein knockdown and AMPK activation with 5 μg of PP2Cα siRNA at day 7. n =4-8 per group, Mean ± SEM, *P <0.05, **P <0.01, ***P <0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4306116&req=5

Figure 1: In vivo knockdown of PP2Cα. The indicated siRNAs were injected at five sites in Gas muscles which were then electroporated to induce uptake of the siRNA. The most effective sequence, time point, and dose of siRNA were chosen for subsequent AngII-infusion experiments. (A) Real-time PCR showing PP2Cα mRNA knockdown in Gas using 2.5 μg of four different PP2Cα siRNA target sequences relative to negative control siRNA at day 4 post electroporation. PP2Cα siRNA ‘A’ was used in all time course and dose response experiments. (B) Time-course of PP2Cα mRNA knockdown in Gas with 2.5 μg of scrambled and PP2Cα siRNA. (C) Time-course of PP2Cα protein knockdown in Gas with 2.5 μg of scrambled and PP2Cα siRNA. (D) Time-course of AMPK phosphorylation in Gas with 2.5 μg of scrambled and PP2Cα siRNA. (E) Dose response of PP2Cα mRNA knockdown in Gas at day 7 post electroporation. (F) Dose response of PP2Cα protein knockdown in Gas at day 7 post electroporation. (G) Dose response of AMPK phosphorylation in Gas at day 7 post electroporation. (H) Representative western blots showing PP2Cα protein knockdown and AMPK activation with 5 μg of PP2Cα siRNA at day 7. n =4-8 per group, Mean ± SEM, *P <0.05, **P <0.01, ***P <0.001.
Mentions: Four days after electroporation, PP2Cα siRNAs B and C both significantly reduced expression of PP2Cα mRNA in Gas by approximately 37% (P <0.05), while siRNA A proved to be the most effective with a 57% reduction (P <0.001) in mRNA compared to control, and siRNA D failed to reduce expression of PP2Cα (Figure 1A). A total of 2.5 μg of PP2Cα siRNA A significantly reduced gene expression from 1 to 7 days post electroporation, with a peak suppression from baseline of 75% measured at day 3, and returning to normal by day 11 (Figure 1B). There was a non-statistically significant trend for transient suppression of PP2Cα mRNA expression with scrambled siRNA/electroporation, which returned to normal between days 3 and 5 (Figure 1B). Similarly, PP2Cα protein tended to be reduced by PP2Cα siRNA A throughout the time course, with a significant reduction of 58% at day 7 (Figure 1C), while AMPK phosphorylation was significantly increased at days 1, 5, and 7 (Figure 1D). The dose response performed at day 7 indicated that 5 μg of siRNA was the optimal dose, with a 51% reduction in expression compared to 31% with 2.5 μg, while scrambled siRNA did not reduce expression at all (Figure 1E). PP2Cα protein expression was significantly reduced at all doses of PP2Cα siRNA (Figure 1F), and there was a concomitant 2.3-fold increase in AMPK phosphorylation with 5 μg of PP2Cα siRNA (Figure 1G).

Bottom Line: This induction was reduced by PP2Cα knockdown, which also increased beclin-1 expression and microtubule-associated protein 1 light chain 3 (LC3)-II conversion in AngII-infused Gas.AngII reduced activating S555 phosphorylation of UNC-51-like kinase 1 (ULK1), a critical regulator of autophagosome formation, and increased inhibitory S757 ULK1 phosphorylation and these effects were prevented by PP2Cα siRNA.These results demonstrate novel effects of AngII on cellular metabolism that are likely critical in mediating the muscle wasting that is a hallmark of CHF.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tulane University Department of Medicine, Heart and Vascular Institute, New Orleans, LA 70112, USA.

ABSTRACT

Background: Circulating angiotensin II (AngII) is elevated in congestive heart failure (CHF), and leads to skeletal muscle wasting, which is strongly associated with poor patient outcomes. We previously found that AngII upregulates protein phosphatase 2C-alpha (PP2Cα) and dephosphorylates AMP-activated protein kinase (AMPK), a critical regulator of cellular metabolism, in skeletal muscle.

Methods: To determine the role of PP2Cα in AngII-induced wasting, gastrocnemius (Gas) muscles of FVB mice were injected with scrambled or PP2Cα siRNA and mice were infused with saline or AngII for 4 days.

Results: Knockdown of PP2Cα reduced AngII wasting, blocked AngII upregulation of PP2Cα, increased p-T172-AMPK, and inhibited AngII-mediated reductions in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (TFAM), in complex IV activity, and in ATP levels. AngII impaired the rate of autophagy as determined by a 2.4-fold increase in p62/SQSTM1 (p62) accumulation. This induction was reduced by PP2Cα knockdown, which also increased beclin-1 expression and microtubule-associated protein 1 light chain 3 (LC3)-II conversion in AngII-infused Gas. AngII reduced activating S555 phosphorylation of UNC-51-like kinase 1 (ULK1), a critical regulator of autophagosome formation, and increased inhibitory S757 ULK1 phosphorylation and these effects were prevented by PP2Cα siRNA.

Conclusions: AngII inhibited AMPK activity and reduced PGC-1α and TFAM expression (thereby inhibiting mitochondrial biogenesis) and impaired ULK1 activation and autophagy (thereby also inhibiting clearance of damaged mitochondria), resulting in mitochondrial dysfunction, decreased ATP, and wasting. Knockdown of PP2Cα normalized AMPK activity, PGC-1α, NRF1, and TFAM levels and blocked AngII inhibition of ULK1, leading to improved mitochondrial biogenesis/recycling/function, energy production, and inhibition of AngII-induced wasting. These results demonstrate novel effects of AngII on cellular metabolism that are likely critical in mediating the muscle wasting that is a hallmark of CHF.

No MeSH data available.


Related in: MedlinePlus