In the present study, we reveal that the cytokine TWEAK downregulates PGC1α and mitochondrial OXPHOS gene expression in cardiomyocytes, which contributes to TWEAK-induced cardiac dysfunction. Moreover, we find that TWEAK regulates PGC1α expression via Fn14/TRAF2/NFκB-dependent signaling pathways ().
Graphical illustration summarizing the proposed mechanism underlying TWEAK-induced cardiac dysfunction.
PGC1α is a transcriptional coactivator that is preferentially expressed in tissues with high-energy demand and greater mitochondrial abundance, including the heart 
. PGC1α directly coactivates the transcription factors PPARs and estrogen-related receptors (ERRs) and regulates mitochondrial fatty acid β-oxidation as well as expression of genes related to electron transport chain and oxidative phosphorylation 
. PGC1α also coactivates nuclear respiratory factor (NRF) and the expression of mitochondrial transcription factor A (Tfam), which are essential for mitochondrial DNA replication and transcription 
. Expression levels of PGC1α are intricately linked to the maintenance of the cardiac structure and function. Genetic ablation or overexpression of PGC1α has been demonstrated to result in cardiac dysfunction 
, thereby suggesting that PGC1α is required for maintenance of proper heart function. Interestingly, we observed a reduced expression of PGC1α and OXPHOS genes in response to TWEAK treatment. Importantly, the downregulation of these genes occurred while heart function was still maintained and prior to the development of functional decline, suggesting a temporal association between suppression of PGC1α expression by TWEAK and the development of cardiac dysfunction. In contrast, we have previously found that genetic overexpression of TWEAK does not result in increased cardiac apoptosis, unlike with other cytokines, prior to the development of contractile dysfunction and heart failure 
. The causal relationship between PGC1α downregulation and cardiac dysfunction is supported by our data demonstrating that maintenance of PGC1α expression in cardiomyocytes protected against TWEAK-induced cardiac dysfunction. Notably, high degree of chronic overexpression of PGC1α has been associated with the development of cardiac dysfunction 
and therefore, adenoviral gene expression approaches used in this study aimed to maintain PGC1α in TWEAK-treated cells at control cell levels.
We have previously found that TWEAK-induced cardiac dysfunction requires expression of Fn14 
. Fn14 has a short cytoplasmic tail that contains a TRAF consensus-binding motif 
. The recruitment of TRAFs to the TRAF binding motif leads to activation of signaling cascades and regulates a variety of cellular functions including survival and death, among other functions 
. To date, there are six known structurally related members in TRAF family, of which TRAF1, TRAF2, TRAF3 and TRAF5 are able to bind with Fn14 
. In adult cardiomyocyte, our data have identified TRAF2 as the most abundant TRAF member. TWEAK stimulation was further found to be capable of initiating membrane translocation of TRAF2 in an Fn14-dependent manner. Interestingly, knockdown of TRAF2 with shRNA is sufficient to prevent TWEAK-induced downregulation of PGC1α expression, indicating that TRAF2 is the principal proximal signaling member involved in this event.
A universal characteristic of TWEAK signaling through Fn14 is the activation of NFκB signaling pathway with modulation of numerous downstream target genes 
. NFκB p65 is present in the cytoplasm and is associated with the repressor protein IκBα. Proinflammatory stimuli such as interleukin-1, TNF, and lipopolysaccharides can activate IκB kinase-β that induces IκBα phosphorylation and proteosome-mediated degradation, which results in NFκB phosphorylation, nuclear translocation, and binding to DNA 
. In this study, we demonstrate that TWEAK induces IκBα phosphorylation accompanied by IκBα degradation and resynthesis, as well as NFκB p65 phosphorylation in cardiomyocytes, consistent with previous reports 
. Interestingly, a selective IκB kinase-β inhibitor, SC-514, prevents the downregulation of PGC1α by TWEAK, indicating that TWEAK repression of PGC1α expression requires activation of NFκB signaling pathway. This is consistent with the observation that other cytokines, such as TNFα, suppress PGC1α expression through activation of NFκB 
. It has also been reported that lipopolysaccharide suppresses PGC-1α gene expression at the transcriptional level, and blocking the toll-like receptor-4/NFκB axis prevented the downregulation of PGC-1 coactivator expression by LPS. However, how activation of NFκB suppresses PGC1α gene expression is unclear and remains under investigation.
In summary, our data suggest that Fn14-TRAF2-NFκB-dependent suppression of PGC1α expression plays a crucial role in TWEAK-induced cardiac dysfunction. Modulation of PGC1α expression or antagonism of Fn14-TRAF2-NFκB may serve as candidate therapeutic targets in preventing TWEAK-induced heart failure.