PKCbII Inhibition in Heart Failure Figure 2. PKCbII activation inhibits proteasomal activity in vitro and disrupts protein quality control in cultured neonatal cardiomyocytes. A. Proteolytic activity (upper panel) and phosphorylation of purified proteasome 20S (middle and lower panels) by different recombinant PKC isozymes (n = 3 per group). Purified 20S proteasome (1 ug) was individually incubated with recombinant PKCa, PKCbI, PKCbII or PKCe (50 ng) at 37uC for 30 minutes. Proteasome phosphorylation was evaluated using serine/threonine phosphorylation antibody (1:1000) and [c32P] ATP incorporation. Histone phosphorylation was used to check the effectiveness of different PKC isozymes. Error bars indicate SEM. *, p,0.05 compared to other groups. B. Oxidized protein levels (upper panel) and ATP-dependent proteasomal activity (lower panel) in cultured neonatal cardiomyocytes. Cells were stimulated with PMA (a non-specific PKC activator) and the effect of PKC isozyme-specific peptide inhibitors (aV5-3, an aPKC-specific inhibitor; bIV5-3, a bIPKC-specific inhibitor; bIIV5-3, a bIIPKC-specific inhibitor [16]; and eV1-2, an ePKC-specific inhibitor [46]; or epoxomicin were determined. Error bars indicate SEM. *, p,0.05 compared to control-TAT and bIIV5-3-treated groups. Representative blots of oxidized proteins and GAPDH. Oxidized proteins were normalized against GAPDH. C. Oxidized protein levels (upper panel) and ATP-dependent proteasomal activity (lower panel) in cultured neonatal cardiomyocytes. Cultured neonatal cardiomyocytes were PKCb or PKCa down-regulated (siRNA) plus/minus bIIV5-3 and challenged with phorbol ester (PMA). NC, negative control for siRNA. Representative blots of PKCbII and PKCa showed siRNA effectiveness. Oxidized proteins were normalized against GAPDH. Error bars indicate SEM. *, p,0.05 compared to control (non-treated cells), PKCb down-regulated treated cells and PKCa down-regulated treated cells plus bIIV5-3. doi:10.1371/journal.pone.0033175.g002 PQC in left ventricular remodeling and heart failure in humans To determine the extent of PQC disruption in cardiac remodeling/failure, we used heart biopsies from seven patients with aortic stenosis-induced left ventricular remodeling who underwent aortic valve replacement. Heart biopsies from four patients with ischemic cardiomyopathy-induced HF and from autopsy specimens of 13 non-failing human hearts as controls were also examined (Table S1). Despite preserved systolic function, all patients with aortic stenosis displayed heart failure signs and symptoms, presenting functional class III–IV of the New York Heart Association [21]. Both ATP-dependent (26S) and - independent (20S) proteasomal activities were lower by about 50% in aortic stenosis and ischemic failing hearts as compared with controls (Fig. 6A) and the levels of oxidized and poly- ubiquitinated cardiac proteins were two to three-fold higher in these patients as compared with control subjects (Fig. 6B–C). There was a negative correlation between proteasomal function and oxidized cardiac protein accumulation in failing human hearts (R2 = 0.70, p = 0.001; Fig. 6D). There is an obvious caveat of using autopsied hearts as a control. Nevertheless, when we examined PQC, an ATP-dependent function, we found a better PQC in the autopsied samples of control subjects relative to the biopsy samples from remodeled and failing human hearts, suggesting an impaired PQC in failing human hearts. Because protein kinase C (PKC) isozymes have been implicated in HF [11,22] we determined whether specific PKC isozymes play a role in proteasomal activity and PQC in this disease. Similar to previous reports [15,23], both aortic stenosis and ischemic failing human hearts had a five-fold increase in total PKCbII levels, a two-fold increase in PKCa and no changes in the levels of e and PKCbI relative to controls (Fig. 6E). Elevated PKCbII and PKCa protein levels were accompanied by their activation in failing hearts, as evidenced by their increased association with the cell particulate fraction (Fig. 6F). Discussion In the present study, we showed that human hypertrophied and failing hearts of different etiologies, as well as myocardial PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e33175