In the present study, we showed that human hypertrophied and failing hearts of different etiologies, as well as myocardial infarction- and hypertensive-induced heart failure rat models displayed UPS dysfunction-mediated PQC disruption and elevated PKCβII protein activity. We also demonstrated for the first time that PKCβII activation resulted in decreased proteasomal activity and accumulated damaged proteins. Moreover, improved proteasomal function by sustained inhibition of PKCβII, using the highly selective PKCβII inhibitor peptide, βIIV5-3 
, significantly improved cardiac PQC, ventricular function and survival of myocardial infarction- and hypertensive-induced heart failure models in rats. Of interest, sustained proteasomal inhibition (by bortezomib treatment) abrogated PKCβII-mediated cardioprotective effects and resulted in elevated mortality in the myocardial infarction-induced rat heart failure model. Thus, PKCβII hyper-activation appears to contribute to UPS dysfunction-mediated PQC disruption and subsequent decreased cardiomyocyte viability, cardiac function and survival in HF ().
Scheme depicting a possible mechanism of PKCβII-mediated PQC disruption during heart failure establishment.
UPS-mediated PQC disruption has been involved in several chronic degenerative diseases, including neurodegenerative diseases, cancer and cardiac ischemia 
, where UPS malfunction culminates in accumulation of abnormal protein-mediated cellular dysfunction and apoptosis. These findings are extended to human heart failure, since we demonstrate here a ~50% decrease in proteasomal activity and an ~3-fold increase in both polyubiquitinated and oxidized proteins in human failing hearts. Furthermore, decreased proteasomal activity was significantly correlated with increased accumulation of oxidized proteins in the failing human hearts (R2
0.0001, ). 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 (). 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 PKCβII 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 
. Different PKC isozymes have been implicated in HF 
. Similar to previous findings from failed human hearts 
, we found that both aortic stenosis and ischemic-failing human hearts presented a significant increase in total PKCβII and PKCα levels accompanied by their activation. Similarly, we found that of the PKC isozymes expressed in heart, only PKCβII was activated in failing hearts from myocardial infarction- and hypertensive-induced HF rats. Further, we showed that PKCβII isozyme activation directly regulates proteasomal activity and PQC. PKCβII (but not αPKC or PKCβI) activation results in phosphorylation of the purified 20S proteasome in vitro
with a reduction in its activity (). The functional consequence of PKCβII-induced 20S proteasome phosphorylation was also demonstrated in isolated neonatal cardiomyocytes since both βIIV5-3, a PKCβII-specific inhibitor (but not selective inhibitors for α, βI or PKCε) and PKCβ knockdown using siRNA abrogated PMA-induced proteasomal dysfunction and the accumulation of damaged proteins (). Since proteasomal activity is regulated by multiple factors, such as intracellular ATP levels 
and post-translational modification of the proteasome 
, the in vitro
findings might not reflect proteasomal regulation in vivo
. Thus, we next examined whether PKCβII 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, PKCβII, which is over-activated in these failing rat hearts, co-immunoprecipitated with the 20S proteasome and was found to have decreased activity (). Taken together, in vitro
cell culture and in vivo
data identify PKCβII 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 PKCβII could provide a new pharmacological 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 
. 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 
, cardiotoxic effects were attributed to proteasome inhibition in normal and ischemic hearts 
. 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 
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 
. 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 PKCβII-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 improvements in heart function/survival induced by PKCβII 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 
, which might suggest that aged hearts are more susceptible to UPS dysfunction and PQC disruption.
Considering that proteasomal dysfunction is likely responsible for PQC disruption in HF, therapies that prevent or reverse selectively HF-induced proteasomal dysfunction may be of value for these patients. Our data in two models of HF in rats suggest that sustained inhibition of PKCβII may provide such novel treatment for HF, since selective inhibition of PKCβII with βIIV5-3 appears safe, even after many weeks of treatment 
. We showed in animal models that inhibition of PKCβII with βIIV5-3 treatment re-established cardiac PQC, and not only prevented further deterioration of cardiac function, but actually improved ventricular function () and prolonged the life span of two rat models of HF with etiologies most common to HF in humans ( and ). These results support a model in which the actions of PKCβII in hypertrophied and failing hearts involve primarily the inactivation of the proteasome. Further studies investigating both direct and indirect proteasomal regulation by PKCβII during heart failure progression are required. However, we cannot exclude the possibility that PKCβII 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 PKCβII-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 βIIV5-3 treatment suggests specific PKCβII inhibition may be a valuable therapeutic approach for patients with HF.