E3-ligase knock down revealed differential titin degradation by autophagy and the ubiquitin proteasome system

Abstract

The sarcomere protein titin is a major determinant of cardiomyocyte stiffness and ventricular distensibility. The constant mechanical stress on titin requires well-controlled protein quality control, the exact mechanisms of which have not yet been fully elucidated. Here, we analyzed E3-ligases potentially responsible for cardiac titin ubiquitination and specifically studied the involvement of the autophagosomal system in titin degradation. Pharmacological inhibition of autophagy and the proteasome in cultured primary rat cardiomyocytes significantly elevated titin ubiquitination and increased titin degradation. Using in-vitro pull down assays we identified binding of E3-ligases MuRF1-3, CHIP and Fbx32 to several titin domains. Immunofluorescence analysis showed sarcomeric localization of the E3-ligases. siRNA-mediated knock-down of the E3-ligases MuRF-1, -3 and a combination of CHIP/Fbx32 significantly reduced autophagy-related titin ubiquitination, whereas knock-down of MuRF-2 and -3 reduced proteasome-related titin ubiquitination. We demonstrated that the proteasomal and the autophagosomal-lysosomal system participate in degradation of the titin filament. We found that ubiquitination and degradation of titin are partially regulated by E3-ligases of the MuRF family. We further identified CHIP and Fbx32 as E3-ligases involved in titin ubiquitination.

Protein Quality Control at the Sarcomere: Titin Protection and Turnover and Implications for Disease Development

“…Their conclusion of increased autophagy was partially based on findings of increased LC3-II and p62/sequestosome-1 level in a type 1 diabetes model. This observation was challenged by recent findings, showing elevated LC3-II and p62 protein levels also after pharmacological inhibition of autophagy in cultured cardiomyocytes (Müller et al, 2021). Apart from disunity concerning elevated or reduced autophagy, modification of PQC seems to be a general phenomenon in the setting of diabetes.…”

“…Inhibition of MuRF-1 by small molecules improves diastolic function in heart failure with preserved ejection fraction (HFpEF) and attenuates skeletal muscle wasting in cardiac cachexia (Bowen et al, 2017;Adams et al, 2022). Recently, the involvement of MuRF-1, -2, -3, Fbx32 and CHIP in proteasome-and autophagosome dependent titin filament ubiquitination has been demonstrated (Section 4.1 and Figure 2) (Müller et al, 2021).…”

“…Current evidence suggests that both the proteasome ( Kötter et al, 2016 ; Higashikuse et al, 2019 ) and autophagy ( Lange et al, 2005 ; Bogomolovas et al, 2021 ; Müller et al, 2021 ), are involved in the degradation of titin and that the degradation of the different titin domains might occur specifically via one or the other system. This hypothesis was lately supported by experimental in vitro inhibition of the proteasome, which lead to increased proteasome-dependent K48 polyubiquitination, and inhibition of autophagy, causing increased autophagy-dependent K63 polyubiquitination of titin ( Kötter et al, 2016 ; Müller et al, 2021 ).…”

“…The observed titin ubiquitination is at least in part performed by the muscle specific E3-ligases MuRF-1, -2, -3, CHIP and Fbx32/atrogin-1. Importantly, polyubiquitination occurs at full-length titin indicating is fully accessible for ubiquitination while still incorporated into the sarcomere ( Müller et al, 2021 ). This is in accordance with other studies showing ubiquitination of other sarcomere proteins within the sarcomere ( Martin et al, 2021 ).…”

“…This is in accordance with other studies showing ubiquitination of other sarcomere proteins within the sarcomere ( Martin et al, 2021 ). It can therefore be concluded that also for a giant protein like titin, a predigest is not necessarily needed for the ubiquitination reaction itself ( Müller et al, 2021 ). Nevertheless, predigest of titin is important for further processing by autophagy or the proteasome and could be performed by the proteases calpain 1 and/or 3 as well as MMP2 ( Barta et al, 2005 ; Hayashi et al, 2008 ; Azevedo et al, 2014 ).…”