PKCbII Inhibition in Heart Failure Figure 5. PKCbII inhibition repairs protein quality control in hypertension-induced heart failure model in rats. A. Schematic panel of sustained PKCbI, bII or e inhibition in hypertension-induced model of heart failure in rats. Representative blots of PKCbII total level and translocation to particulate fraction. B. ATP-dependent and -independent cardiac proteasomal activity, C. Oxidized proteins (as determined by Western blot) and D. cardiac soluble oligomer accumulation (as determined by slot blot) in heart samples from 17 week-old normotensive rats (white bar), TAT-treated (gray bar), bIV5-3-treated (gray bar), bIIV5-3-treated (green bar) and eV1-2-treated (gray bar) hypertensive rats. E. Average fractional shortening data from each group at the age of 17 weeks-old. F. bIIV5-3 improved survival of rats with hypertension-induced heart failure. Error bars indicate SEM. *, p,0.05 compared to control (sham) rats. 1, p,0.05 compared to bIIV5-3-treated heart failure rats. Data from Fig. b–d were analyzed by one-way analysis of variance (ANOVA) with post-hoc testing by Tukey. Survival was analyzed by the standard Kaplan-Meier analysis with log-rank test. doi:10.1371/journal.pone.0033175.g005 bIIV5-3, a PKCbII-specific inhibitor (but not selective inhibitors for a, bI or PKCe) and PKCb knockdown using siRNA abrogated PMA-induced proteasomal dysfunction and the accumulation of damaged proteins (Fig. 2B–C). Since proteasomal activity is regulated by multiple factors, such as intracellular ATP levels [26] and post-translational modification of the proteasome [28,29,30,31], the in vitro findings might not reflect proteasomal regulation in vivo. Thus, we next examined whether PKCbII activation disrupts cardiac PQC related to UPS dysfunction in myocardial infarction- and hypertensive-induced HF rats. Both HF animal models displayed accumulated misfolded proteins associated with proteasomal dysfunction. Indeed, PKCbII, which is over-activated in these failing rat hearts, co-immunoprecipitated with the 20S proteasome and was found to have decreased activity (Fig. 3B). Taken together, in vitro cell culture and in vivo data identify PKCbII as a key enzyme in down-regulating proteasomal activity, which resulted in disrupting cardiac PQC and worsening HF with increased mortality. Considering this scenario, the usage of selective inhibitors of PKCbII could provide a new pharma- cological tool against PQC disruption in heart failure. The use of proteasome inhibitors for therapeutic purposes has been proposed based on the major role of proteasomes in degrading intracellular proteins involved in uncontrolled cell proliferation and growth [32]. In cardiac disease, both beneficial and detrimental effects were reported for pharmacologically- induced proteasome inhibition. While most studies on pressure- overload hypertrophy have shown that systemic proteasome inhibition prevented or reversed concentric cardiac hypertrophy with no impact on cardiac function [33,34], cardiotoxic effects were attributed to proteasome inhibition in normal and ischemic hearts [26,35,36]. These findings raise important questions regarding the degree of proteasomal inhibition, which inhibitor should be used (reversible or irreversible) and the appropriate therapeutic time window (when and for how long) such an inhibitor should be used. The latter is of particular interest, since long-term use of proteasome inhibitors seem counterintuitive based on UPS dysfunction- mediated PQC disruption reported in chronic cardiac proteinopathies [37,38] and as reported here in human HF. Further, the chronic use of the proteasome inhibitor, bortezomib, for chemotherapy was reported to cause cardiac complications ranging from cardiotoxicity to HF in some cancer patients [39]. Relevant to these observations, we found that sustained bortezomib treatment resulted in 100% mortality in myocardial infarction-induced HF rats (fractional shortening below 25%) and blocked PKCbII-related cardioprotective effects. Since control animals treated with bortezomib did not die, these findings highlight the contribution of intact UPS function to cardiac integrity and strengthen our premise that the improve- ments in heart function/survival induced by PKCbII inhibition is mediated in part by protecting proteasomal function. It is also important to emphasize that most HF patients are elderly and that proteasome activity declines with ageing [40], which might suggest PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e33175