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Protein quality control disruption by PKCβII in heart failure; rescue by the selective PKCβII inhibitor, βIIV5-3.

Ferreira JC, Boer BN, Grinberg M, Brum PC, Mochly-Rosen D - PLoS ONE (2012)

Bottom Line: Importantly, inhibition of PKCβII, using a selective PKCβII peptide inhibitor (βIIV5-3), improved proteasomal activity and conferred protection in cultured neonatal cardiomyocytes.We also show that sustained inhibition of PKCβII increased proteasomal activity, decreased accumulation of damaged and misfolded proteins and increased animal survival in two rat models of HF.Finally, increased cardiac PKCβII activity and accumulation of misfolded proteins associated with decreased proteasomal function were found also in remodeled and failing human hearts, indicating a potential clinical relevance of our findings.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT
Myocardial remodeling and heart failure (HF) are common sequelae of many forms of cardiovascular disease and a leading cause of mortality worldwide. Accumulation of damaged cardiac proteins in heart failure has been described. However, how protein quality control (PQC) is regulated and its contribution to HF development are not known. Here, we describe a novel role for activated protein kinase C isoform βII (PKCβII) in disrupting PQC. We show that active PKCβII directly phosphorylated the proteasome and inhibited proteasomal activity in vitro and in cultured neonatal cardiomyocytes. Importantly, inhibition of PKCβII, using a selective PKCβII peptide inhibitor (βIIV5-3), improved proteasomal activity and conferred protection in cultured neonatal cardiomyocytes. We also show that sustained inhibition of PKCβII increased proteasomal activity, decreased accumulation of damaged and misfolded proteins and increased animal survival in two rat models of HF. Interestingly, βIIV5-3-mediated protection was blunted by sustained proteasomal inhibition in HF. Finally, increased cardiac PKCβII activity and accumulation of misfolded proteins associated with decreased proteasomal function were found also in remodeled and failing human hearts, indicating a potential clinical relevance of our findings. Together, our data highlights PKCβII as a novel inhibitor of proteasomal function. PQC disruption by increased PKCβII activity in vivo appears to contribute to the pathophysiology of heart failure, suggesting that PKCβII inhibition may benefit patients with heart failure. (218 words).

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Sustained PKCβII inhibition re-establishes protein quality control and improves cardiac function in myocardial infarction-induced model of heart failure.A. Schematic panel of PKCβII treatment in the post-MI heart failure model, representative blots of PKCβII total level and translocation to particulate fraction, and PKCβII activity from left ventricle tissue from 22-week-old myocardial infarction-induced heart failure (10 wks after MI surgery) TAT-treated, βIIV5-3-treated and control (sham) rats (n = 3 per group). B. 20S proteasome subunits (α5/7, β1, β5 and β7) were precipitated from left ventricle tissue from 22-week-old myocardial infarction-induced heart failure (10 wks after MI surgery) TAT-treated, βIIV5-3-treated and control (sham) rats (n = 3 per group), and then probed with PKCβII, PKCε and anti-serine and threonine phosphorylation antibodies. Equal sample loading was verified using α5/7, β1, β5 and β7 proteasome subunits antibody. C. ATP-dependent and -independent cardiac proteasomal activity. D. Representative blots of proteasome 20S, α-β-crystallin, HSP27, caspase-3, cleaved caspase-3 and GAPDH in heart samples from 22 week-old rats (10 wks after MI surgery) (n = 6 per group). Data quantification and statistical details are in supplementary Fig. 4. E. Oxidized protein levels, F. polyubiquitinated protein levels and G. soluble oligomer accumulation in heart samples from control (sham, white bars), TAT-treated (gray bars) and βIIV5-3-treated (green bars) heart failure rats as determined by Western blot (E, F) and slot-blot analysis (G). H. Average fractional shortening data from each group at 16 weeks and 22 weeks. All biochemical analyses were performed in the ventricular remote area. Error bars indicate SEM. *, p<0.05 compared to control (sham) rats. §, p<0.05 compared to βIIV5-3-treated heart failure rats.
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pone-0033175-g003: Sustained PKCβII inhibition re-establishes protein quality control and improves cardiac function in myocardial infarction-induced model of heart failure.A. Schematic panel of PKCβII treatment in the post-MI heart failure model, representative blots of PKCβII total level and translocation to particulate fraction, and PKCβII activity from left ventricle tissue from 22-week-old myocardial infarction-induced heart failure (10 wks after MI surgery) TAT-treated, βIIV5-3-treated and control (sham) rats (n = 3 per group). B. 20S proteasome subunits (α5/7, β1, β5 and β7) were precipitated from left ventricle tissue from 22-week-old myocardial infarction-induced heart failure (10 wks after MI surgery) TAT-treated, βIIV5-3-treated and control (sham) rats (n = 3 per group), and then probed with PKCβII, PKCε and anti-serine and threonine phosphorylation antibodies. Equal sample loading was verified using α5/7, β1, β5 and β7 proteasome subunits antibody. C. ATP-dependent and -independent cardiac proteasomal activity. D. Representative blots of proteasome 20S, α-β-crystallin, HSP27, caspase-3, cleaved caspase-3 and GAPDH in heart samples from 22 week-old rats (10 wks after MI surgery) (n = 6 per group). Data quantification and statistical details are in supplementary Fig. 4. E. Oxidized protein levels, F. polyubiquitinated protein levels and G. soluble oligomer accumulation in heart samples from control (sham, white bars), TAT-treated (gray bars) and βIIV5-3-treated (green bars) heart failure rats as determined by Western blot (E, F) and slot-blot analysis (G). H. Average fractional shortening data from each group at 16 weeks and 22 weeks. All biochemical analyses were performed in the ventricular remote area. Error bars indicate SEM. *, p<0.05 compared to control (sham) rats. §, p<0.05 compared to βIIV5-3-treated heart failure rats.

Mentions: To evaluate the effect of PKCβII on cardiac PQC in heart failure, we further determined whether sustained administration of βIIV5-3 in a myocardial infarction-induced heart failure model in rats (Fig. 3A) affected cardiac PQC, cardiac function and survival. PKCβII (but not PKCε, an abundant isozyme in the heart) co-immunoprecipitated with the proteasome and decreased its activity in these failing hearts (Fig. 3B and C). After the establishment of HF (4 weeks after myocardial infarction; MI), a subsequent six-week treatment with βIIV5-3 abolished the increased cardiac PKCβII translocation and activity (Fig. 3A), but not the activity of α, βI, δ, γ and PKCε (Fig. S2), and diminished the co-immunoprecipitation of PKCβII and the 20S proteasome as well as its phosphorylation (Fig. 3B). This βIIV5-3 treatment resulted also in a two-fold increase in both ATP-dependent (26S) and -independent (20S) cardiac proteasomal activity back to control levels (Fig. 3C). There were no changes in the protein levels of cardiac proteasome subunits in failing hearts regardless of the treatment (Fig. 3D and Fig. S3). However, sustained PKCβII inhibition completely suppressed the accumulation of cardiac oxidized proteins, polyubiquitinated proteins and soluble oligomers of misfolded proteins in these rat samples (Fig. 3E–G). The increased abnormal protein accumulation in failed non-treated hearts was accompanied by an ∼50% increase in the levels of the small chaperones, α-β-crystallin and HSP27, and a two-fold increase in caspase 3 activation, effects that were reversed by the sustained PKCβII inhibition (Fig. 3D and Fig. S3). In addition, chronic PKCβII inhibition abolished the HF-induced increase in the levels of well-known proteasome substrates, IkB and p53 (Fig. S3).


Protein quality control disruption by PKCβII in heart failure; rescue by the selective PKCβII inhibitor, βIIV5-3.

Ferreira JC, Boer BN, Grinberg M, Brum PC, Mochly-Rosen D - PLoS ONE (2012)

Sustained PKCβII inhibition re-establishes protein quality control and improves cardiac function in myocardial infarction-induced model of heart failure.A. Schematic panel of PKCβII treatment in the post-MI heart failure model, representative blots of PKCβII total level and translocation to particulate fraction, and PKCβII activity from left ventricle tissue from 22-week-old myocardial infarction-induced heart failure (10 wks after MI surgery) TAT-treated, βIIV5-3-treated and control (sham) rats (n = 3 per group). B. 20S proteasome subunits (α5/7, β1, β5 and β7) were precipitated from left ventricle tissue from 22-week-old myocardial infarction-induced heart failure (10 wks after MI surgery) TAT-treated, βIIV5-3-treated and control (sham) rats (n = 3 per group), and then probed with PKCβII, PKCε and anti-serine and threonine phosphorylation antibodies. Equal sample loading was verified using α5/7, β1, β5 and β7 proteasome subunits antibody. C. ATP-dependent and -independent cardiac proteasomal activity. D. Representative blots of proteasome 20S, α-β-crystallin, HSP27, caspase-3, cleaved caspase-3 and GAPDH in heart samples from 22 week-old rats (10 wks after MI surgery) (n = 6 per group). Data quantification and statistical details are in supplementary Fig. 4. E. Oxidized protein levels, F. polyubiquitinated protein levels and G. soluble oligomer accumulation in heart samples from control (sham, white bars), TAT-treated (gray bars) and βIIV5-3-treated (green bars) heart failure rats as determined by Western blot (E, F) and slot-blot analysis (G). H. Average fractional shortening data from each group at 16 weeks and 22 weeks. All biochemical analyses were performed in the ventricular remote area. Error bars indicate SEM. *, p<0.05 compared to control (sham) rats. §, p<0.05 compared to βIIV5-3-treated heart failure rats.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316563&req=5

pone-0033175-g003: Sustained PKCβII inhibition re-establishes protein quality control and improves cardiac function in myocardial infarction-induced model of heart failure.A. Schematic panel of PKCβII treatment in the post-MI heart failure model, representative blots of PKCβII total level and translocation to particulate fraction, and PKCβII activity from left ventricle tissue from 22-week-old myocardial infarction-induced heart failure (10 wks after MI surgery) TAT-treated, βIIV5-3-treated and control (sham) rats (n = 3 per group). B. 20S proteasome subunits (α5/7, β1, β5 and β7) were precipitated from left ventricle tissue from 22-week-old myocardial infarction-induced heart failure (10 wks after MI surgery) TAT-treated, βIIV5-3-treated and control (sham) rats (n = 3 per group), and then probed with PKCβII, PKCε and anti-serine and threonine phosphorylation antibodies. Equal sample loading was verified using α5/7, β1, β5 and β7 proteasome subunits antibody. C. ATP-dependent and -independent cardiac proteasomal activity. D. Representative blots of proteasome 20S, α-β-crystallin, HSP27, caspase-3, cleaved caspase-3 and GAPDH in heart samples from 22 week-old rats (10 wks after MI surgery) (n = 6 per group). Data quantification and statistical details are in supplementary Fig. 4. E. Oxidized protein levels, F. polyubiquitinated protein levels and G. soluble oligomer accumulation in heart samples from control (sham, white bars), TAT-treated (gray bars) and βIIV5-3-treated (green bars) heart failure rats as determined by Western blot (E, F) and slot-blot analysis (G). H. Average fractional shortening data from each group at 16 weeks and 22 weeks. All biochemical analyses were performed in the ventricular remote area. Error bars indicate SEM. *, p<0.05 compared to control (sham) rats. §, p<0.05 compared to βIIV5-3-treated heart failure rats.
Mentions: To evaluate the effect of PKCβII on cardiac PQC in heart failure, we further determined whether sustained administration of βIIV5-3 in a myocardial infarction-induced heart failure model in rats (Fig. 3A) affected cardiac PQC, cardiac function and survival. PKCβII (but not PKCε, an abundant isozyme in the heart) co-immunoprecipitated with the proteasome and decreased its activity in these failing hearts (Fig. 3B and C). After the establishment of HF (4 weeks after myocardial infarction; MI), a subsequent six-week treatment with βIIV5-3 abolished the increased cardiac PKCβII translocation and activity (Fig. 3A), but not the activity of α, βI, δ, γ and PKCε (Fig. S2), and diminished the co-immunoprecipitation of PKCβII and the 20S proteasome as well as its phosphorylation (Fig. 3B). This βIIV5-3 treatment resulted also in a two-fold increase in both ATP-dependent (26S) and -independent (20S) cardiac proteasomal activity back to control levels (Fig. 3C). There were no changes in the protein levels of cardiac proteasome subunits in failing hearts regardless of the treatment (Fig. 3D and Fig. S3). However, sustained PKCβII inhibition completely suppressed the accumulation of cardiac oxidized proteins, polyubiquitinated proteins and soluble oligomers of misfolded proteins in these rat samples (Fig. 3E–G). The increased abnormal protein accumulation in failed non-treated hearts was accompanied by an ∼50% increase in the levels of the small chaperones, α-β-crystallin and HSP27, and a two-fold increase in caspase 3 activation, effects that were reversed by the sustained PKCβII inhibition (Fig. 3D and Fig. S3). In addition, chronic PKCβII inhibition abolished the HF-induced increase in the levels of well-known proteasome substrates, IkB and p53 (Fig. S3).

Bottom Line: Importantly, inhibition of PKCβII, using a selective PKCβII peptide inhibitor (βIIV5-3), improved proteasomal activity and conferred protection in cultured neonatal cardiomyocytes.We also show that sustained inhibition of PKCβII increased proteasomal activity, decreased accumulation of damaged and misfolded proteins and increased animal survival in two rat models of HF.Finally, increased cardiac PKCβII activity and accumulation of misfolded proteins associated with decreased proteasomal function were found also in remodeled and failing human hearts, indicating a potential clinical relevance of our findings.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT
Myocardial remodeling and heart failure (HF) are common sequelae of many forms of cardiovascular disease and a leading cause of mortality worldwide. Accumulation of damaged cardiac proteins in heart failure has been described. However, how protein quality control (PQC) is regulated and its contribution to HF development are not known. Here, we describe a novel role for activated protein kinase C isoform βII (PKCβII) in disrupting PQC. We show that active PKCβII directly phosphorylated the proteasome and inhibited proteasomal activity in vitro and in cultured neonatal cardiomyocytes. Importantly, inhibition of PKCβII, using a selective PKCβII peptide inhibitor (βIIV5-3), improved proteasomal activity and conferred protection in cultured neonatal cardiomyocytes. We also show that sustained inhibition of PKCβII increased proteasomal activity, decreased accumulation of damaged and misfolded proteins and increased animal survival in two rat models of HF. Interestingly, βIIV5-3-mediated protection was blunted by sustained proteasomal inhibition in HF. Finally, increased cardiac PKCβII activity and accumulation of misfolded proteins associated with decreased proteasomal function were found also in remodeled and failing human hearts, indicating a potential clinical relevance of our findings. Together, our data highlights PKCβII as a novel inhibitor of proteasomal function. PQC disruption by increased PKCβII activity in vivo appears to contribute to the pathophysiology of heart failure, suggesting that PKCβII inhibition may benefit patients with heart failure. (218 words).

Show MeSH
Related in: MedlinePlus