HBP activation and apoptosis contributed to contractile dysfunction. The heart functional data are consistent with our earlier work and reveal attenuated contractile function without significant alterations to heart rate. Here the 6dP/dt findings implicate the myocardial calcium handling pathway, as diastolic calcium is a key determinant of contractile function and calcium signaling. Since PI treatment decreased and increased myocardial UPS activity and ubiquitination, respectively, this may lead to an accumulation of contractile protein aggregates and impaired cardiac contractility and signaling pathways. For example, protein turnover of AB1010 msds connexin 43, PLB and SERCA-2a are all regulated by the UPS and may explain the higher expression levels found here and before by us. Others have established that altered connexin 43 expression can precede arrhythmias, ventricular fibrillation and incorrect signal propagation in the long-term. Therefore we tentatively suggest that elevated connexin 43 expression in our model may results in detrimental effects on contractile function in the future, especially within the context of HIV-AIDS. We previously identified lower myocardial calcium levels and higher SERCA-2a protein expression with PI treatment, and now report attenuated and elevated calmodulin and pPLB expression levels, respectively. In parallel, we found increased myocardial calcineurin and NFAT3 
expression levels. Of note, others found that cardiac-specific calcineurin overexpression resulted in enhanced pPLB and SERCA-2a expression and diminished phosphorylation and redistribution of connexin 43. This was associated with depressed contractility and cardiac hypertrophy. Here the authors proposed that connexin 43 may be a downstream target of calcineurin and that attenuated connexin 43 levels may be linked to perturbed gap junction assembly and arrhythmogenesis. We propose that a similar scenario may exist in our model and that greater calcineurin activation is linked to elevated connexin 43 expression that may compromise gap junction function. Increased SERCA-2a, connexin 43 and pPLB expression may occur as a result of lower myocardial UPS and have also been implicated as downstream transcriptional targets of calcineurin. Thus, elevated connexin 43 and pPLB expression may represent an adaptive response by PI-treated hearts to improve calcium handling, which may improve cardiac function. Higher calcineurin activation also leads to increased dephosphorylation and translocation of NFAT3 to the nucleus for activation of downstream targets, e.g. PGC-1a and pro-hypertrophic genes. However, since the calcineurin-NFAT3 pathway did not result in cardiac hypertrophy in our model, we are of the opinion that longer-term activation may eventually result in a hypertrophic response. These findings, however, represent a model of altered cardiac physiology and suggest a potential association with PI-induced molecular alterations to key junction and ionic proteins that may precede the onset of contractile dysfunction. Moreover, the metabolic side-effects elicited by PI treatment in our model �C although at a relatively early stage �C may also affect heart function as a downstream target. Thus we do not imply that the protein expression alterations are directly associated with the altered contractility found in our model. Data linking these phenomena are scarce and therefore makes definitive conclusions difficult. Together these findings indicate that perturbed calcium handling may contribute to the TWS119 PI-mediated contractile dysfunction found in our experimental model in the longer term. However, further studies are required to confirm whether this is indeed the case. Since myocardial PGC-1a was upregulated, this implies that PIs exert initial effects at the mitochondrial level. PGC-1a is a welldescribed transcriptional regulator of mitochondrial biogenesis and we propose that higher expression levels may represent an early compensatory response to energetic stress. In agreement with this notion, NRF-1 and mtTFA expression remained unaltered while we previously identified no changes for myocardial ATP levels and AMPKa expression following 8 weeks of PI administration. It is likely that reduced UPS activity in PI-treated hearts may contribute to the increased PGC1a levels here observed. In support, others established that lower UPS-mediated protein turnover in fibroblasts resulted in PGC-1a stabilization and mitochondrial biogenesis, while it can also be rapidly degraded in the nucleus.