Phosphorylation of CRYAB induces a condensatopathy to worsen post–myocardial infarction left ventricular remodeling
Moydul Islam, David R. Rawnsley, Xiucui Ma, Walter Navid, Chen Zhao, Xumin Guan, Layla Foroughi, John T. Murphy, Honora Navid, Carla J. Weinheimer, Attila Kovacs, Jessica Nigro, Aaradhya Diwan, Ryan P. Chang, Minu Kumari, Martin E. Young, Babak Razani, Kenneth B. Margulies, Mahmoud Abdellatif, Simon Sedej, Ali Javaheri, Douglas F. Covey, Kartik Mani, Abhinav Diwan
Moydul Islam, David R. Rawnsley, Xiucui Ma, Walter Navid, Chen Zhao, Xumin Guan, Layla Foroughi, John T. Murphy, Honora Navid, Carla J. Weinheimer, Attila Kovacs, Jessica Nigro, Aaradhya Diwan, Ryan P. Chang, Minu Kumari, Martin E. Young, Babak Razani, Kenneth B. Margulies, Mahmoud Abdellatif, Simon Sedej, Ali Javaheri, Douglas F. Covey, Kartik Mani, Abhinav Diwan
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Research Article Cardiology Cell biology

Phosphorylation of CRYAB induces a condensatopathy to worsen post–myocardial infarction left ventricular remodeling

Abstract

Protein aggregates are emerging therapeutic targets in rare monogenic causes of cardiomyopathy and amyloid heart disease, but their role in more prevalent heart-failure syndromes remains mechanistically unexamined. We observed mislocalization of desmin and sarcomeric proteins to aggregates in human myocardium with ischemic cardiomyopathy and in mouse hearts with post–myocardial infarction ventricular remodeling, mimicking findings of autosomal-dominant cardiomyopathy induced by the R120G mutation in the cognate chaperone protein CRYAB. In both syndromes, we demonstrate increased partitioning of CRYAB phosphorylated on serine 59 to NP40-insoluble aggregate-rich biochemical fraction. While CRYAB undergoes phase separation to form condensates, the phosphomimetic mutation of serine 59 to aspartate (S59D) in CRYAB mimics R120G-CRYAB mutants with reduced condensate fluidity, formation of protein aggregates, and increased cell death. Conversely, changing serine to alanine (phosphorylation-deficient mutation) at position 59 (S59A) restored condensate fluidity and reduced both R120G-CRYAB aggregates and cell death. In mice, S59D CRYAB knockin was sufficient to induce desmin mislocalization and myocardial protein aggregates, while S59A CRYAB knockin rescued left ventricular systolic dysfunction after myocardial infarction and preserved desmin localization with reduced myocardial protein aggregates. 25-Hydroxycholesterol attenuated CRYAB serine 59 phosphorylation and rescued post–myocardial infarction adverse remodeling. Thus, targeting CRYAB phosphorylation-induced condensatopathy is an attractive strategy to counter ischemic cardiomyopathy.

Authors

Moydul Islam, David R. Rawnsley, Xiucui Ma, Walter Navid, Chen Zhao, Xumin Guan, Layla Foroughi, John T. Murphy, Honora Navid, Carla J. Weinheimer, Attila Kovacs, Jessica Nigro, Aaradhya Diwan, Ryan P. Chang, Minu Kumari, Martin E. Young, Babak Razani, Kenneth B. Margulies, Mahmoud Abdellatif, Simon Sedej, Ali Javaheri, Douglas F. Covey, Kartik Mani, Abhinav Diwan

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Figure 2

Phosphorylation of CRYAB at S59 makes it aggregate prone and toxic.

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Phosphorylation of CRYAB at S59 makes it aggregate prone and toxic. (A) ...
(A) Immunoblot (A) demonstrating expression of GFP-fusion proteins in HEK293A cells transfected with GFP-tagged WT CRYAB, its phospho-mimetic mutant (S59D), phosphorylation-deficient mutant (S59A), R120G mutant, or the R120G and S59A double-mutant proteins. (B and C) Representative immunofluorescence images (B) for detection of protein aggregates with quantitation (C) of aggregate area per cell. **P < 0.01; ****P < 0.0001 by Tukey’s post hoc test after 1-way ANOVA. Nuclei are blue (DAPI). (D) Cell death in cells treated in A. **P < 0.01; ****P < 0.0001 by Tukey’s post hoc test after 1-way ANOVA. (E) Schematic depicting generation of optoIDR constructs. mCh indicates mCherry fluorophone and Cry2 encodes for Arabidopsis thaliana protein with light-activated phase separation characteristics. (F) Various domains of CRYAB with localization of serine 59 and arginine 120 residues depicted. (G) Representative time-lapse images at t = 0 seconds, 100 seconds, 200 seconds, and 300 seconds after light activation in HEK293A cells transfected with constructs generated with CRYAB WT, its phosphorylation-deficient mutant (S59A), phospho-mimetic mutant (S59D), R120G mutant, or the R120G and S59A double-mutant proteins as the IDR in the optoIDR constructs. Cry2 fused with mCherry without an IDR was used as the negative control, and FUS-N fused with mCherry-Cry2 was studied as positive control. (H) Average number of condensates/cell at t = 0 versus t = 300 seconds in cells treated as in E. **P < 0.01 by Mann-Whitney test. (I) Average area of condensates/cell at t = 300 seconds in cells treated in E. ***P < 0.001; ****P < 0.0001 by Tukey’s post hoc test after 1-way ANOVA.