Abstract
Fibrosis is a pathognomonic feature of structural heart disease and counteracted by distinct cardioprotective mechanisms, e.g. activation of the phosphoinositide 3-kinase (PI3K) / AKT pro-survival pathway. The Cullin-RING E3 ubiquitin ligase 7 (CRL7) was identified as negative regulator of PI3K/AKT signalling in skeletal muscle, but its role in the heart remains to be elucidated. Here, we sought to determine whether CRL7 modulates to cardiac fibrosis following pressure overload and dissect its underlying mechanisms. For inactivation of CRL7, the Cullin 7 (Cul7) gene was deleted in cardiac myocytes (CM) by injection of adeno-associated virus subtype 9 (AAV9) vectors encoding codon improved Cre-recombinase (AAV9-CMV-iCre) in Cul7flox/flox mice. In addition, Myosin Heavy Chain 6 (Myh6; alpha-MHC)-MerCreMer transgenic mice with tamoxifen-induced CM-specific expression of iCre were used as alternate model. After transverse aortic constriction (TAC), causing chronic pressure overload and fibrosis, AAV9-CMV-iCre induced Cul7-/- mice displayed a ~50% reduction of interstitial cardiac fibrosis when compared to Cul7+/+ animals (6.7% vs. 3.4%, p<0.01). Similar results were obtained with Cul7flox/flox Myh6-Mer-Cre-MerTg(1/0) mice which displayed a ~30% reduction of cardiac fibrosis after TAC when compared to Cul7+/+ Myh6-Mer-Cre-MerTg(1/0) controls after TAC surgery (12.4% vs. 8.7%, p<0.05). No hemodynamic alterations were observed. AKTSer473 phosphorylation was increased 3-fold (p<0.01) in Cul7-/- vs. control mice, together with a ~78% (p<0.001) reduction of TUNEL-positive apoptotic cells three weeks after TAC. In addition, CM-specific expression of a dominant-negative CUL71152stop mutant resulted in a 16.3-fold decrease (p<0.001) of in situ end-labelling (ISEL) positive apoptotic cells. Collectively, our data demonstrate that CM-specific ablation of Cul7 restrains myocardial fibrosis and apoptosis upon pressure overload, and introduce CRL7 as a potential target for anti-fibrotic therapeutic strategies of the heart.
Highlights
The Ubiquitin-Proteasome System (UPS) is a selective protein degradation pathway that is involved in the pathogenesis of several cardiac disorders [1, 2]
We previously reported that binding of Large T (LT)-antigen to CUL7 impairs Cullin-RING E3 ubiquitin ligase 7 (CRL7) ubiquitin ligase function, resulting in the accumulation of the CRL7 substrate protein insulin receptor substrate 1 (IRS-1) and upregulation of its downstream signalling pathways phosphoinositide 3-kinase (PI3K)/AKT and MEK/ERK [15]
Cullin 7 (Cul7) mRNA level were reduced by 60.8 ± 0.1% (p
Summary
The Ubiquitin-Proteasome System (UPS) is a selective protein degradation pathway that is involved in the pathogenesis of several cardiac disorders [1, 2]. Central to the UPS is the recognition of a substrate by an E3 ubiquitin ligase, a step pivotal for the ubiquitin-mediated degradation of substrate proteins by the 26S proteasome [3]. Cullin 7 (Cul; formerly known as p193) is a component of the Cullin-RING E3 ubiquitin ligase 7 (CRL7), a multimeric enzyme composed of the RING finger protein ROC1, SKP1-FBXW8 substrate targeting subunit and CUL7 as scaffold protein [5,6,7]. CRL7 was shown to target insulin receptor substrate 1 (IRS-1), a component of the insulin and insulin-like growth factor 1 (IGF1) signalling pathways, for ubiquitin-mediated degradation by the 26S proteasome [6, 8]. Heterozygosity of either Cul or Fbxw resulted in elevated PI3 kinase/AKT activation in skeletal muscle tissue upon insulin stimulation when compared to wild-type controls [9]
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