PKCbII Inhibition in Heart Failure Figure 4. Protein quality control re-establishment by PKCbII inhibition improves survival and cardiac integrity in myocardial infarction-induced model of heart failure. A. bIIV5-3 improved cardiac integrity in the post-MI-induced HF model in rats. Heart samples were collected from 22 week-old control (sham), TAT-treated and bIIV5-3-treated heart failure rats (10 wks after MI surgery). Samples from a non-infarcted area (contractile zone) were processed for electron microscopy analysis. The integrity of cardiac myofibril structure (arrow head) was analyzed in each group (n = 3 per group). N, nucleus; S, sarcomere; SR, sarcoplasmic reticulum; Z, Z disc; M, mitochondria. B. Schematic panel of sustained treatment with TAT, bIIV5-3, bortezomib or bortezomib plus bIIV5-3 in post-myocardial infarction-induced heart failure in rats. C. bIIV5-3 improved survival of rats with post-myocardial infarction-induced heart failure. Note that sustained treatment of heart failure rats with a specific proteasome inhibitor (bortezomib, 0,2 mg/kg, thrice weekly) either alone or combined with bIIV5-3 culminated in 100% mortality as well as abrogated the cardioprotective effects of bIIV5-3 treatment. Control (sham) rats treated with bortezomib did not present increased mortality. Error bars indicate SEM. *, p,0.05 compared to control (sham) rats. 1, p,0.05 compared to bIIV5-3-treated heart failure rats. #, p,0.05 compared to TAT-treated heart failure rats. doi:10.1371/journal.pone.0033175.g004 hearts. Furthermore, decreased proteasomal activity was signifi- cantly correlated with increased accumulation of oxidized proteins in the failing human hearts (R2 = 0.70, p = 0.0001, Fig. 6D). Thus, cardiac dysfunction, decreased UPS activity and PQC inadequacy seem to be common phenomena in developing heart failure, and the progressive PQC disruption paralleled cardiac function decline after myocardial infarction in rats (Fig. 1). Although the data using human-derived tissue samples were similar to those we obtained from the two animal models of HF, there is an inherent variability in disease type and comorbidity-associated factors. Further caveat is the use of pathological specimens from subjects who died of causes other than HF; the cause of death and the timing of sample collection relative to the time of death may introduce additional variable and therefore are not optimal controls. Despite these limitations, the similar findings in humans, in two animal models and in culture support our conclusion regarding the role of PKCbII in regulation of proteasomal function in failing hearts. A number of studies have shown that activation of PKC contributes to a variety of heart diseases by targeting contractile myofilaments, mitochondrial proteins and transcriptional factors [26,27]. Different PKC isozymes have been implicated in HF [10]. Similar to previous findings from failed human hearts [15,23], we found that both aortic stenosis and ischemic-failing human hearts presented a significant increase in total PKCbII and PKCa levels accompanied by their activation. Similarly, we found that of the PKC isozymes expressed in heart, only PKCbII was activated in failing hearts from myocardial infarction- and hypertensive- induced HF rats. Further, we showed that PKCbII isozyme activation directly regulates proteasomal activity and PQC. PKCbII (but not aPKC or PKCbI) activation results in phosphorylation of the purified 20S proteasome in vitro with a reduction in its activity (Fig. 2A). The functional consequence of PKCbII-induced 20S proteasome phosphorylation was also demonstrated in isolated neonatal cardiomyocytes since both PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e33175