PROTEOMIC REMODELLING OF PROTEASOME IN RIGHT HEART FAILURE

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Until recently, right heart disease has been relatively understudied and underappreciated. A recent report using in-depth quantitative proteomics have investigated the differential proteome of isolated mouse right and left ventricle [29]. Interestingly they found very subtle change between LV and RV, suggesting that changes in protein expression due to HF could be specifically linked to remodeling of the ventricle. In order to dissect differences particular to RHF hypertrophy, the aim of this study was to search for protein that were differentially expressed. Proteomic expression profiling of RV in RHF had identified 126 differential protein expressions (Supplemental Tables S2 and S3). Among the proteins that are up-regulated in RHF, we found several proteins already reported at the RNA level in RHF mice model [30] such as tropomyosin 1, myosin heavy polypeptide 9, Synpo protein, Reticulon-4, Neural cell adhesion molecule 1. However few reports in the literature have look at down-regulated proteins rather than up-regulated proteins. Interestingly, many proteasome subunit α- and β- proteins quantified in our study were found highly depressed in the RV of RHF rats (Table 1). The cardiac proteasome contains a variable proteasome complex consisting of different proportions of β-subunits [31]. Alternations in proteasome subunit composition affect overall proteasome proteolytic activity, which, in turn, may alter the specificity and selectivity of the proteasome for various substrates under certain conditions. The proteasome subunit α- and β- proteins, Psma1, Psma2, Psma4, Psma5, Psma7, Psmb4, Psmb5 and Psmb6 show low expression levels in RHF as compared to the control group (Table 1). Interestingly, the increased expression of protease inhibitors (Figure 4) in RHF let us to suggest that the balance between proteasome components and protease inhibitors is specifically altered in the RV of RHF. Indeed, Serpina3n belongs to the Serpin family of protease inhibitors. Some of them such as Plasminogen activator inhibitor type-1 (PAI-1) [32] and type-2 (PAI-2) [33] have been shown to interact with proteasome and affect its activity, suggesting that an increase in the expression of protease inhibitors could reflect and confirm a decrease in proteasome activity. By Western blot, the down-regulation of two representative proteins, Psma5 and Psmb5, was confirmed (Figure 3). Thus, the trend towards down-regulation for theses two proteasome proteins confirmed the directionality revealed by our proteomic quantitative analysis. Psma1, Psma2, Psma4, Psma5 and Psma7 (α-subunits) transcript levels were all statistically decreased specifically in the RV of RHF compared to control rats (Figure 4A). In contrast, as expected, these α-subunits were not statistically regulated in LV in RHF rats as compared to control rats (Figure 4B). As well, in the RV, the β-subunits mRNAs (Psmb4, Psmb5 and Psmb6) were also significantly decreased in RHF rats compared to control rats (Figure 4A), whereas in the LV the mRNA levels did not differ significantly between RHF and control rats (Figure 4B). In LHF, this type of alteration has also been reported that the transcript levels of some of the α- and β-subunits of the 20S proteasome were downregulated in failing human hearts compared with non-failing hearts [34, 35], however the pattern of alteration such as the type of subunits seems to be slightly different in the RVH as compared to the LHF. This is consistent with the physiological requirement of the RV as compared to the LV. In a non-disease state, the LV encounters the relatively high pressures of systemic circulation, which requires the formation of a thick myocardium, while RV has only to cope with the low pressure of pulmonary circulation and develops a thin wall. Due to their anatomic differences, any increase in pressure will have a much more severe impact on the RV than on the LV. Comparative microarray based transcriptome analysis of RV and LV remodeling identified distinct responses to pressure-induced hypertrophy [30].

The alterated proteasome proteins identified belong to the ubiquitin–proteasome system (UPS). The UPS is the major non-lysosomal pathway for intracellular degradation of proteins and plays a major role in regulating many cellular processes. The key components of the UPS are the 26S proteasome and ubiquitin [36].The role of ubiquitin in the UPS is to act as a tag for the proteasome to identify how the protein targeted for proteolytic degradation is destroyed. It has been reported, that during the transition of hypertrophy to LHF in patients, there was an increased level of ubiquitination during hypertrophy followed by increase upon the onset of heart failure [37] [38]. In our model RHF, we found that the intensity of several bands of poly-ubiquitinated proteins was only increased in the RV of the RHF rats compared with control rats (Figure 5A and B), whereas no major differences in the ubiquitin signals was observed in the LV between RHF and control rats (Figure 5A and C).

Here, we report by immunoblot analysis an accumulation of ubiquitinated proteins (Figure 5) and also a down-regulation of both α- and β-subunits of proteasome at the mRNA (Figure 4) and proteins levels (Table 1 and Figure 3) specifically in RV from RHF rats. Altogether, these results suggest impairment of RV proteasomal activity in RHF rats. The vast majority of proteins in mammalian cells is degraded by 26S  proteasomes [39]. The 26S enzyme consists of the 20S proteasome and one or two 19S regulatory complexes [40]. The 20 S proteasome is composed of two outer α- and two inner β-rings [40]. Each ring contains seven different subunits, and each β-ring contains three proteolytic sites, which differ in their substrate specificities. The “chymotrypsin-like” site cleaves peptide bonds preferentially after hydrophobic residues; the “trypsin-like” site cuts mainly after basic residues, and the third site “caspase-like” cuts preferentially after acidic residues [41]. It has been reported that the association of 20 S proteasomes with the 19 S regulatory complexes to form 26 S proteasomes leads to much higher rates of peptide hydrolysis [42] and confers the ability to degrade ubiquitinated proteins as well as certain non-ubiquitinated polypeptides [43]. Thus the decrease of β subunits proteins (Figure 4) and the increased in ubiquitinated proteins (Figure 5) correlates with the effect on 26S proteasome activities (Figure 6). Recently, a growing body of evidence suggests that alteration of proteasome-mediated protein degradation also contributes to the initiation and/or progression of cardiac diseases [44-46]. Inhibition of the proteasome has been shown to impair the heart function, as indicated by the use of bortezomib, a FDA-approved chemotherapeutic anti-cancer medication, which increases the occurrences of heart failure [47]. It has been also demonstrated that bortezomib induced complications in the stressed mouse heart [48]. Kim et al., have shown that a daily administration of Bortezomib after induction of RHF suppressed substantially RHF [28]. Indeed, in both models hypoxia- and MCT-induced animals Bortezomib inhibits RV hypertrophy and vascular remodeling. This type of inhibitors already used clinically for the treatment of multiple myelanoma, could therefore be beneficial in the development of RHF. However, other studies in rat, murine, and porcine models indicate that proteasome inhibition could be cardioprotective [49]. The effects of proteasome inhibition during myocardial ischaemia on cardiac function have been controversial, as both beneficial and deleterious effects have been reported.  In heart failure with proteasome dysfunction, bortezomib has a detrimental effect. In contrast, during early remodeling and left ventricular hypertrophy, Hedhli et al. [49] as well as Drews et al. [50] observed increased proteasome activities, showing that under these circumstances, Bortezomib was cardioprotective. Thus, the proteasome activities in cardiac disease remain a subject of debate and the specific molecular components, function, and regulation of the cardiac proteasome still remains largely unknown. Analyses of human biopsies of failing hearts support the concept that defective protein degradation contributes to heart failure. However, although most studies are congruent in showing high total levels of ubiquitinated proteins in failing hearts [51], it remains unclear whether this is attributable to impaired proteasome function. Some reports show that the levels and activity of the proteasome are unchanged in failing hearts [52]. In contrast, other reports show that proteasome activity is impaired in failing hearts, possibly as a result of oxidative modifications on proteasome subunits [53].

Here, we correlate the time-frame of RHF development to proteasome activity and found that proteasome activity increase and then drop down below basal level, these results might explained the differences between studies based on the day for which proteasome activity was measured. Based on the time-course of proteasome activity during the development of RHF, we believe that caution should be taken before comparing studies since the activity has two phases, first an increase followed by a drastic decrease at the end-stage of RHF. This model is consistent with Tsukamoto et al. paper in mouse model [54]. Thus, the proteasome activities in cardiac disease remain a subject of debate and the specific molecular components, function, and regulation of the cardiac proteasome still remain largely unknown. Several experimental studies have demonstrated the contribution of UPS dysfunction to the pathogenesis of LHF disease, however, there is no report investigating the role of UPS in the pathogenesis of RHF, until recently. In parallel to our study, Rajagopalan et al. have reported that proteasome activity is decreased in another model of RHF also suggesting that UPS dysfunction contributes to RHF [55]. It has been demonstrated previously that ubiquitinated proteins are accumulated when proteasome is deactivated and also that the inhibition of proteasome chymotrypsin-like activity was linked to an accumulation of ubiquitinated proteins [56]. Thus, depression of these two proteasome activities found in our study (Figure 6 and 7) correlates with the accumulation of ubiquitinated proteins observed in Figure 5.