models [12,13], disturbed cardiac PQC by decreasing proteasomal activity. Using different PKC-selective regulators [14], we then demonstrated here that the PKCbII-specific peptide inhibitor, bIIV5-3, prevented the decline in PQC in cultured neonatal cardiac myocytes and that sustained PKCbII inhibition substan- tially increased survival and cardiac function in myocardial infarction-induced and hypertension-induced heart failure animal models in rats. The molecular bases of these events were also studied. Results PQC dysfunction parallels heart failure development in an animal model To investigate whether injury-induced progression to heart failure is associated with PQC dysfunction, we evaluated proteasomal activity and accumulation of damaged cardiac proteins in a rat model of myocardial infarction-induced heart failure (Fig. 1A). All measurements were performed in a region remote from the infarcted area in the left ventricle (non-infarcted zone). We found a progressive decline in proteasomal activity during 10 weeks following myocardial infarction that exhibited a tight correlation with the decline in cardiac function (R2 = 0.61, p = 0.0001; Fig. 1B, E, F and H), reaching a deficit of 68% and 66%, respectively, when compared with sham-operated rats. The decreased proteasomal activity correlated with an increased accumulation of cardiac oxidized proteins and soluble oligomers of misfolded proteins in the failing hearts (R2 = 0.81, p = 0.0001, Fig. 1C, D, G and H). Similar to results observed in human HF hearts [8,15], we found that, of the PKC isozymes present in rat heart, only PKCbII was activated in the myocardial infarction- induced failed hearts, as evidenced by its increased association with the cell particulate fraction (Fig. 1I); there was also a 3-fold increase in catalytic activity of PKCbII, as compared with that from control rat hearts (Fig. 1J). PKCbII activation directly down-regulates proteasomal activity We next determined whether PKCbII directly regulates proteasomal activity. Active PKCbII (but not aPKC or PKCbI) phosphorylated and decreased the proteolytic function of the purified 20S proteasome by 55%, in vitro (Fig. 2A, bottom and top panel, respectively). PKCbII activation-mediated proteasomal phosphorylation was essential to decreased proteasomal activity, since inactive PKCbII (added in the absence of its activators, PS/ DG/Ca+2) had no effect on proteasomal function (Fig. 2A). Further, treatment of cultured neonatal cardiomyocytes with 10 nM phorbol ester 12-myristate 13-acetate (PMA, an activator of most PKC isozymes) for 30 min induced oxidized protein accumulation, at least in part, by decreasing the 26S proteasomal ATP-dependent proteolytic activity (40% decrease) (Fig. 2B). Using selective peptide inhibitors for PKC isozymes, we found that only bIIV5-3, a PKCbII-specific peptide inhibitor [14,16,17], [and not selective inhibitors for a, bI or PKCe (aV5-3 and bIV5-3 and eV1-2, respectively), which are also present in cardiac myocytes; [18,19]] prevented this PMA-induced proteasomal inhibition and increased accumulation of damaged proteins (Fig. 2B). Confirming the pharmacological effect of bIIV5-3, PKCb knockdown using siRNA similarly abrogated PMA-induced proteasomal inhibition and oxidized protein accumulation in neonatal cardiomyocytes (Fig. 2C). In contrast, PKCa knockdown did not restore PMA-induced proteasomal activity unless cells PKCbII Inhibition in Heart Failure were pre-treated also with bIIV5-3 peptide (Fig. 2C). In addition, bIIV5-3 treatment prevented damaged protein accumulation and diminished cell death in hydrogen peroxide-treated neonatal cardiomyocytes (Fig. S1) and epoxomicin (a selective proteasome inhibitor) abrogated the cytoprotective effect of bIIV5-3. Sustained PKCbII inhibition and cardiac PQC in the myocardial infarction-induced heart failure model To evaluate the effect of PKCbII on cardiac PQC in heart failure, we further determined whether sustained administration of bIIV5-3 in a myocardial infarction-induced heart failure model in rats (Fig. 3A) affected cardiac PQC, cardiac function and survival. PKCbII (but not PKCe, 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 bIIV5-3 abolished the increased cardiac PKCbII translocation and activity (Fig. 3A), but not the activity of a, bI, d, c and PKCe (Fig. S2), and diminished the co-immunoprecipitation of PKCbII and the 20S proteasome as well as its phosphorylation (Fig. 3B). This bIIV5-3 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 PKCbII inhibition completely suppressed the accumu- lation 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, a-b-crystallin and HSP27, and a two-fold increase in caspase 3 activation, effects that were reversed by the sustained PKCbII inhibition (Fig. 3D and Fig. S3). In addition, chronic PKCbII inhibition abolished the HF-induced increase in the levels of well-known proteasome substrates, IkB and p53 (Fig. S3). Importantly, a six-week treatment with bIIV5-3 of animals with HF resulted in a significant increase of fractional shortening compared to non-treated heart failure animals (2762 vs. 1462%, respectively; Fig. 3H). bIIV5-3 treatment also decreased left ventricular end-diastolic diameter compared to non-treated heart failure animals (8.560.3 vs. 10.260.2 mm, respectively). The improved cardiac function following bIIV5-3 treatment (from 19% before treatment to 27% after 6 weeks of bIIV5-3 treatment) was likely due to improved integrity of cardiac myofibril structure in the treated hearts, as evidenced by electron microscopy (Fig. 4A). Improved integrity of cellular structures was also found in bIIV5-3-treated rats relative to non-treated heart failure animals (Fig. 4A arrows). Finally, PKCbII inhibition strikingly improved HF animal survival, whereas sustained bortezomib treatment (a proteasome inhibitor) abrogated bIIV5-3 therapy- mediated survival improvement in HF rats and accelerated death in rats with HF (Fig. 4B–C), but not in control rats (none of the control animals died after chronic treatment with bortezomib). Further, bortezomib-treated HF rats did not present any sign of drug toxicity, based on anatomopathological analysis. The increased mortality rate of rats with HF in the presence of a proteasome inhibitor, together with our in vitro and cell culture experiments, further support our hypothesis that PKCbII inhibition-associated improvements in heart function/survival is mediated, at least in part, by better proteasomal function. PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e33175