PKCbII Inhibition in Heart Failure Figure 3. Sustained PKCbII inhibition re-establishes protein quality control and improves cardiac function in myocardial infarction- induced model of heart failure. A. Schematic panel of PKCbII treatment in the post-MI heart failure model, representative blots of PKCbII total level and translocation to particulate fraction, and PKCbII activity from left ventricle tissue from 22-week-old myocardial infarction-induced heart failure (10 wks after MI surgery) TAT-treated, bIIV5-3-treated and control (sham) rats (n = 3 per group). B. 20S proteasome subunits (a5/7, b1, b5 and b7) were precipitated from left ventricle tissue from 22-week-old myocardial infarction-induced heart failure (10 wks after MI surgery) TAT-treated, bIIV5-3-treated and control (sham) rats (n = 3 per group), and then probed with PKCbII, PKCe and anti-serine and threonine phosphorylation antibodies. Equal sample loading was verified using a5/7, b1, b5 and b7 proteasome subunits antibody. C. ATP-dependent and -independent cardiac proteasomal activity. D. Representative blots of proteasome 20S, a-b-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 bIIV5-3-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. 1, p,0.05 compared to bIIV5-3-treated heart failure rats. doi:10.1371/journal.pone.0033175.g003 infarction- and hypertensive-induced heart failure rat models displayed UPS dysfunction-mediated PQC disruption and elevat- ed PKCbII protein activity. We also demonstrated for the first time that PKCbII activation resulted in decreased proteasomal activity and accumulated damaged proteins. Moreover, improved proteasomal function by sustained inhibition of PKCbII, using the highly selective PKCbII inhibitor peptide, bIIV5-3 [16], signifi- cantly improved cardiac PQC, ventricular function and survival of myocardial infarction- and hypertensive-induced heart failure models in rats. Of interest, sustained proteasomal inhibition (by bortezomib treatment) abrogated PKCbII-mediated cardioprotec- tive effects and resulted in elevated mortality in the myocardial infarction-induced rat heart failure model. Thus, PKCbII hyper- activation appears to contribute to UPS dysfunction-mediated PQC disruption and subsequent decreased cardiomyocyte viabil- ity, cardiac function and survival in HF (Fig. 7). UPS-mediated PQC disruption has been involved in several chronic degenerative diseases, including neurodegenerative dis- eases, cancer and cardiac ischemia [24,25,26], where UPS malfunction culminates in accumulation of abnormal protein- mediated cellular dysfunction and apoptosis. These findings are extended to human heart failure, since we demonstrate here a ,50% decrease in proteasomal activity and an ,3-fold increase in both polyubiquitinated and oxidized proteins in human failing PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e33175