AUTOPHAGY 2016, VOL. 12, NO. 11, 2252–2253 http://dx.doi.org/10.1080/15548627.2016.1215384

AUTOPHAGIC PUNCTUM

Making the connections: Autophagy and post-translational modifications in cardiomyocytes Manish K. Gupta and Jeffrey Robbins Division of Molecular Cardiovascular Biology, The Heart Institute, Cincinnati Children’s Hospital, Cincinnati, OH, USA

ABSTRACT

ARTICLE HISTORY

Cardiac proteins are subject to continuous stress and these intrinsic and extrinsic factors, both physiological and pathological can lead to protein misfolding. If the protein quality control (PQC) pathways are in any way compromised or their activities diminished, intracellular aggregates can form and a proteotoxic environment is generated, which contributes to cardiac disease and heart failure. We studied the role that SUMO post-translational modification plays in a proteotoxic cardiac environment. SUMOylation can have an integral role in controlling flux through the ubiquitin-proteasome system, and expression of the SUMO (small ubiquitin-like modifier) E2 enzyme UBE2I/UBC9 improves cardiac PQC. Our data focus on using gain- and loss-of-function approaches to modify UBE2I levels and measure the effects on cardiomyocyte autophagic flux. UBE2I expression does have an impact on macroautophagy/autophagy as increased SUMOylation results in increased autophagy. We show that increased SUMOylation is cardioprotective and can decrease morbidity in proteotoxic cardiac disease.

Received 14 July 2016 Revised 15 July 2016 Accepted 15 July 2016

We have found that proteotoxicity is an important, common pathway in cardiac disease. Data from our lab as well as others have shown the therapeutic efficacy of maintaining or upregulating protein clearance pathways such as proteasomal-based protein degradation or the clearance of protein aggregates via autophagy in cardiac disease. The sufficiency, in terms of decreasing morbidity, of upregulating autophagic function to reduce proteotoxic accumulations in cardiomyocytes in a proteotoxic environment has been demonstrated. SUMOylation is a reversible protein posttranslational modifier in which a SUMO (small ubiquitin-like modifier) protein is covalently attached to a lysine residue of the target protein. The SUMO system regulates multiple cellular functions including transcription, cell division, protein stability and translocation, signal transduction, chromatin segregation and PQC (Fig. 1). A critical component of this system is the SUMO E2 conjugating enzyme, UBE2I and this protein’s activity can result in increased ubiquitination and a subsequent increase in proteasomal flux of the targeted proteins. However, SUMOylation’s effects on another PQC arm, autophagy, is unexplored and, considering the importance of autophagy in cardiomyocyte aggregate clearance, we set out to to modulate cardiomyocyte SUMO activity and determine the effects on autophagy. The effects of posttranslational protein modification on autophagy are not well understood. We monitored the consequences for autophagy as a result of increased or decreased SUMOylation using the fusion protein GFP-LC3 construct in neonatal rat cardiomyocytes. A hallmark of autophagy is the sequestering of LC3-II to the autophagic vesicle and this

CONTACT Jeffrey Robbins

[email protected]

KEYWORDS

autophagy; cardiomyopathy; cell death; heart failure; protein misfolding; SUMO

process can be detected by the appearance and quantification of green puncta, visualized by fluorescence microscopy. We were able to quantify a positive, dose-dependent effect of increased UBE2I expression on autophagy and autophagic flux as well and showed that starvation induced both UBE2I expression and increased autophagy in the cardiomyocyte cultures. We then explored the consequences of decreased UBE2I expression, using siRNA to knock down the protein levels in the cells. Decreasing UBE2I results in decreased expression of the autophagy marker protein LC3-II but upregulated SQSTM1/p62, which functions in transporting ubiquitinated protein to the proteasome and phagophore (the precursor to the autophagosome). We also noted that knocking down UBE2I results in decreased BECN1/Beclin 1 as well, a protein that functions in multiple steps in autophagy. Overall, ubiquitination levels were also increased by UBE2I knockdown, pointing to the importance of SUMOylation in modulating this critical posttranslational modification. Finally, the importance of UBE2I was confirmed by simultaneously knocking down UBE2I expression and starving the cells. Under these conditions we were unable to detect any activation of autophagy, emphasizing the importance of UBE2I expression for the autophagic response during starvation. We were also interested in understanding the cellular networks and their interactions in the intact organ and so altered UBE2I levels by transgenic manipulation of cardiomyocyte proteins in the mouse. Cardiomyocyte-specific overexpression of UBE2I (2- to 4-fold relative to wild-type levels) has no effect on cardiomyocyte size, cardiac function or survival into adulthood.

MLC 7020, 240 Albert Sabin Way, Cincinnati, OH 45229-3039, USA

Punctum to: Gupta MK, McLendon PM, Gulick J, James J, Khalili K, Robbins J. UBC9-Mediated SUMOylation Favorably Impacts Cardiac Function in Compromised Hearts. Circulation Research, 2016: 118(12):1894-905. doi: 10.1161/CIRCRESAHA.115.308268. © 2016 Taylor & Francis

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Figure 1. Documented examples of the multiple roles that posttranslational modification via SUMOylation can play.

However, UBE2I overexpression leads to increased levels of SUMOylated proteins and elevated levels of autophagic markers such as LC3-II. We then focused on a specific protein, PIK3C3/Vps34, which is positively regulated via SUMOylation, with this modification playing an instrumental role in autophagosome formation. Increased SUMOylation of PIK3C3 occurs in UBE2I-overexpressing hearts as detected by immunoprecipation affinity isolation experiments, confirming that UBE2I directs SUMOylation to an essential autophagic protein. We subsequently examined UBE2I’s ability to directly mediate autophagy by crossing the UBE2I mice to reporter transgenic animals that express GFP-LC3 in cardiomyocytes so that autophagy could be directly detected and quantified. Puncta were quantified with and without bafilomycin A1 treatment so that autophagic flux could be measured as well. These experiments confirmed that the number and density of green puncta are increased in UBE2I overexpressors. Increasing autophagy can prolong survival in a proteotoxic cardiomyopathy model. Mutations in the chaperone CRYAB (crystallin a B) cause DES/desmin-related cardiomyopathy. We have studied this disease using cardiomyocyte-specific expression of a mutated CRYAB (CRYABR120G). CRYABR120G hearts have significant deficits in autophagy, and restoration of normal autophagic flux is cardioprotective. We crossed UBE2I-overexpressing mice with the CRYABR120G animals and showed that at 5

mo, when heart disease is significant, UBE2I expression is cardioprotective. Hypertrophy and fibrosis are significantly decreased in the double-transgenic animals relative to the CRYABR120G mice, and aggregate accumulation is also decreased. Crossing the double transgenics into a transgenic reporter line that allowed us to titrate autophagy showed that autolysosome formation is increased, confirming that autophagy is upregulated in these animals. Consistent with these data, UBE2I overexpression significantly prolongs mouse survival in the presence of CRYABR120G expression. While these data are intriguing and begin to define some ways in which the cell’s PQC pathways communicate, the data do not establish an unambiguous mechanism for how this is occurring in the cardiomyocyte. As SUMOylation’s roles are so diverse, one would need to establish whether UBE2I can rescue the CRYABR120G phenotype through an autophagy-independent pathway to test the necessity of autophagic upregulation. That is, we have shown that SUMOylation can upregulate autophagy and this is beneficial in a proteotoxic environment. But whether this pathway is necessary for rescue remains subject to future investigation.

Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.

Making the connections: Autophagy and post-translational modifications in cardiomyocytes.

Cardiac proteins are subject to continuous stress and these intrinsic and extrinsic factors, both physiological and pathological can lead to protein m...
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