Figure 7. Scheme depicting a possible mechanism of PKCbII- mediated PQC disruption during heart failure establishment. doi:10.1371/journal.pone.0033175.g007 ventricular function (Fig. 3H) and prolonged the life span of two rat models of HF with etiologies most common to HF in humans (Fig. 4C and 5F). These results support a model in which the actions of PKCbII in hypertrophied and failing hearts involve primarily the inactivation of the proteasome. Further studies investigating both direct and indirect proteasomal regulation by PKCbII during heart failure progression are required. However, we cannot exclude the possibility that PKCbII exerts other effects that contribute to this pathology. Also, the contribution of other proteolytic systems such as autophagic/lysosomal pathways to cardiac protein quality control in HF should be considered. Taken together, our data suggest that PKCbII-mediated impairment of cardiac PQC may be critical, at least in part, for the development of cardiac dysfunction in failing hearts. In addition, re-establishment of proteasomal function and PQC with bIIV5-3 treatment suggests specific PKCbII inhibition may be a valuable therapeutic approach for patients with HF. Methods Ethics statement The animal protocols were approved by the Stanford University Institutional Animal Care and Use Committee (Protocol ID: 14746) and by the Ethical Committee of the School of Physical Education and Sport of the University of Sa˜o Paulo, Brazil (Protocol ID: 2009/13). Human biopsies were taken according to the procedure approved by the Human Ethical Committee in Brazil (Protocol ID: CAPP2409/04/029) and USA (IRB number: 350, protocol ID: 96726). Written informed consent was also PKCbII Inhibition in Heart Failure obtained from all patients undergoing aortic valve replacement surgery. Myocardial infarction-induced heart failure model Myocardial infarction was induced by ligation of the left anterior descending coronary artery (LAD) in Wistar normoten- sive rats at 12 weeks of age, as described [41]. After the LAD surgery, animals were followed up to 10 weeks to establish the time window of proteasome activity and protein quality control. In addition, to determine the effect of sustained PKCbII inhibition on PQC during heart failure, another group of animals with fractional shortening ,25% (MI-HF) was treated between the ages of 16 and 22 weeks with TAT47–57-bIIV5-3 (3 mg/kg/day) or with equimolar concentration of TAT47–57-carrier peptide, using Alzet osmotic pumps, which were replaced every two weeks. The sham-infarcted group was subjected to TAT treatment as a negative control. Echocardiography (Acuson Sequoia, 14-MHz) to evaluate fractional shortening was performed 10 weeks after LAD surgery as well as before and after sustained PKCbII inhibition. In addition, MI-HF rats were treated between the ages of 16 and 22 weeks with a specific proteasome inhibitor (bortezomib, 0.2 mg/ kg, thrice weekly) either alone or together with bIIV5-3. The bortezomib dose was previously shown to produce blood concentrations of bortezomib comparable with those seen in humans [36,42]. All the biochemical analyses were performed in the ventricular remote area. Hypertension-induced model of heart failure Male Dahl rats were fed with an 8% NaCl-containing diet (high salt diet) or with a 0.3% NaCl low salt diet from the age of 6 weeks onward, as described [13]. All peptides were delivered using osmotic pumps implanted subcutaneously and replaced every two weeks, Dahl rats were treated between the ages of 11 and 17 weeks with the selective PKCbI inhibitor peptide, TAT47–57-bIV5-3 (3 mg/kg/day); the selective PKCbII inhibitor peptide, TAT47–57- bIIV5-3 (3 mg/kg/day); the selective PKCe inhibitor peptide, TAT47–57-eV1-2 (3 mg/kg/day); or an equimolar concentration of TAT47–57 carrier peptide alone (1.6 mg/kg/day), as a control. Cell culture Cardiac myocytes were isolated from 1-day-old Sprague- Dawley rat litters, as described [43]. Short interfering RNA was transfected into cardiac myocytes as described [43]. Proteasome phosphorylation assay 1 ug of purified 20S proteasome (PW8720, Enzo Lif Sci, PA) was incubated with 50 ng of recombinant PKCa, PKCbI, PKCbII or PKCe (Cell Signaling, MA) in assay buffer (25 mM Tris-HCl, pH 7.5, 1 mM CaCl2, 20 mM MgCl2, 1 mM DTT, 25 nM ATP) at 37uC for 30 minutes. Proteasome phosphorylation was evaluated using serine/threonine phosphorylation antibody (1:1000) and [c32P] ATP incorporation, as described [44]. Histone phosphorylation was used to check the effectiveness of different PKC isozymes. Kinase assay The kinase assay was performed as described [44]. Proteasome activity ATP-dependent (26S) and -independent (20S) chymotrypsin- like activity of the proteasome was assayed in the total lysate from heart or isolated cardiomyocyte using the fluorogenic peptide Suc- Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (LLVY-AMC, PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e33175