Microtubules Sequester Acetylated YAP in the Cytoplasm and Inhibit Heart Regeneration

Abstract

Background: The Hippo pathway effector YAP (Yes-associated protein) plays an essential role in cardiomyocyte proliferation and heart regeneration. In response to physiological changes, YAP moves in and out of the nucleus. The pathophysiological mechanisms regulating YAP subcellular localization after myocardial infarction remain poorly defined.

Methods: We identified YAP acetylation at site K265 by in vitro acetylation followed by mass spectrometry analysis. We used adeno-associated virus to express YAP-containing mutations that either abolished acetylation (YAP-K265R) or mimicked acetylation (YAP-K265Q) and studied how acetylation regulates YAP subcellular localization in mouse hearts. We generated a cell line with YAP-K265R mutation and investigated the protein-protein interactors by YAP immunoprecipitation followed by mass spectrometry, then validated the YAP interaction in neonatal rat ventricular myocytes. We examined colocalization of YAP and TUBA4A (tubulin α 4A) by superresolution imaging. Furthermore, we developed YAP-K265R and αMHC-MerCreMer (MCM); Yap-loxP/K265R mutant mice to examine the pathophysiological role of YAP acetylation in cardiomyocytes during cardiac regeneration.

Results: We found that YAP is acetylated at K265 by CBP (CREB-binding protein)/P300 (E1A-binding protein P300) and is deacetylated by nicotinamide phosphoribosyltransferase/nicotinamide adenine dinucleotide/sirtuins axis in cardiomyocytes. After myocardial infarction, YAP acetylation is increased, which promotes YAP cytoplasmic localization. Compared with controls, mice that were genetically engineered to express a K265R mutation that prevents YAP K265 acetylation showed improved cardiac regenerative ability and increased YAP nuclear localization. Mechanistically, YAP acetylation facilitates its interaction with TUBA4A, a component of the microtubule network that sequesters acetylated YAP in the cytoplasm. After myocardial infarction, the microtubule network increased in cardiomyocytes, resulting in the accumulation of YAP in the cytoplasm.

Conclusions: After myocardial infarction, decreased sirtuin activity enriches YAP acetylation at K265. The growing TUBA4A network sequesters acetylated YAP within the cytoplasm, which is detrimental to cardiac regeneration.

Keywords: Hippo signaling pathway; NAD+; Sirtuin 1, Sirtuin 2; acetylation; microtubules; myocardial infarction.

Figure 1.. YAP is acetylated on K265.

(A) K265 acetylation site on YAP. (B) Mass spectrometry (MS) was used to identify the K265 YAP acetylation site. (C) In vitro acetylation assay shows that the CBP catalytic domain (CBP-CAT) directly acetylates YAP K265 in the presence of acetyl-CoA. CBP-CAT autoacetylation levels increase in the presence of acetyl-CoA. Acetylated YAPK265 antibodies nonspecifically detect acetylated CBP-CAT. (D) Immunoblot of Ac-YAP in neonatal rat ventricular cardiomyocytes (NRVMs) treated with indicated drugs (n=5). (E) Quantification of D, DMSO-treated samples (controls), paired t-tests were used. (F) IP-Western (top) showing Ac-YAP levels in NRVMs treated with Sirt1 or Sirt2 siRNAs. NRVM protein lysates were incubated with rabbit Ac-YAPK265 antibodies, and Ac-YAP was detected via a mouse YAP antibody. (G) Ac-YAP and YAP levels in postnatal day 0 (P0), P10, and 3-month-old mouse hearts. Ac-YAP/YAP ratios are quantified in H (n = 3) via one-way ANOVA with Bonferroni’s post-hoc test. (I) Ac-YAP/YAP levels in mouse hearts 1 day or 2 days after myocardial infarction (MI). (J) Quantification of I, data were compared via one-way ANOVA with Bonferroni’s post-hoc test (n=5 for sham; n=3 for post-MI groups). Data are expressed as the mean ± SEM.

Figure 2.. Acetylation promotes YAP cytoplasmic localization.

(A) Western blotting reveals P300 knockdown (KD) in NRVMs. (B) Immunofluorescence (IF) staining shows YAP nuclear localization after P300-KD in NRVMs. Scale bar (10 μm). (C) Quantification of B. (D) IF of exogenously expressed mouse YAP (mYAP, Flag (green)) in mouse hearts two weeks after injection of AAV9 virus expressing Flag-tagged YAP, arrowheads (nuclear YAP in CMs), scale bar (10 μm). (E) Quantification of D. (F-I) RNA-scope of YAP target genes Amotl2 and Csf1 in mouse hearts injected with AAV9-mYap-WT, AAV9-mYap-K265R, and AAV9-mYap-K265Q, Scale bar (25 μm). Amotl2 and Csf1 expression is quantified in H, and I. Mann–Whitney test was used in C, one-way ANOVA with Bonferroni’s post-hoc test was used in E, and two-way ANOVA with Tukey’s post-hoc test was used in H and I. Data are shown as the mean ± SEM.

Figure 3.. Heart regeneration in YapK265R mice.

(A, B) Echocardiography at 3, 5, and 7 weeks after myocardial infarction (MI) was induced in P9-stage wild-type (WT) and YapK265R mice. WT mice, n = 18, 10 males/8 females; K265R mice, n = 14, 7 males/7 females. (C) Masson’s trichome staining of cardiac fibrosis 8 weeks post-MI. Scale bar = 2 mm. Quantification is shown in (D), n = 12. WT mice, n = 12, 6 males/6 females; K265R mice, n = 12, 5 males/7 females. Two-way ANOVA with Tukey’s post-hoc test was used for A and B, and Mann–Whitney test was used for D. Data are expressed as the mean ± SEM.

Figure 4.. Increased YAP nuclear localization and CM proliferation in YapK265R mice.

(A-C) YAP heart IF six hours after MI or Sham operation. Scale bar (5 μm) (B) Percentage of nuclear YAP in CMs. (C) Percentage of nuclear YAP in non-CMs. n = 4 per group, 2 males/2 females. (D, E) IF staining of EdU-labeled CMs in WT and YapK265R mice three days after MI, scale bar (25 μm), (E) Quantification of D. n = 6, WT-sham, 4 males/2 females; K265R-sham, 3 males/3 females; WT-MI, 3 males/3 females; K265R-MI, 3 males/3 females. (F, G) IF staining of pHH3-labeled CMs in the G2-M phase of the cell cycle in WT and YapK265R mice three days after MI, scale bar (20 μm). (G) Quantification of F. n = 6, WT, 3 males/3 females; K265R, 3 males/3 females. (H, I) IF staining of the midbody protein Aurora B reveals dividing CMs within the border zone of YapK265R mouse hearts, scale bar (10 μm), n = 8 for WT, 4 males/4 females and n = 6 for YapK265R, 3 males/3 females. (I) Quantification of H. Two-way ANOVA with Tukey’s post-hoc test was used for B, C, and E, Mann–Whitney test was used for G and I, and data are expressed as the mean ± SEM.

Figure 5.. Functional recovery after MI in YapK265R mice.

(A) Echo reveals that deleting one Yap allele (αMHC-MCM/+; Yap^flox/+) in CMs does not alter heart function. αMHC-MCM/+; Yap^flox/+, n = 7, 5 males/2 females; αMHC-MCM/+; Yap^flox/K265R, n = 6, 4 males/2 females; (B) Ejection fraction in αMHC-MCM/+; Yap^flox/+ and αMHC-MCM/+; Yap^flox/K265R post-P9 MI mice without TAM treatment. αMHC-MCM/+; Yap^flox/+, n = 15, 6 males/9 females; αMHC-MCM/+; Yap^flox/K265R, n = 6, 2 males/4 females. (C) Fractional shortening and (D) ejection fraction values for post-P9 mice of indicated genotypes at indicated time points. αMHC-MCM/+; Yap^flox/+, n = 12, 6 males/6 females; αMHC-MCM/+; Yap^flox/K265R, n = 13, 8 males/5 females. Data were compared via two-way ANOVA with Bonferroni’s post-hoc test. (E) Masson’s trichome staining of hearts 8 weeks after P9 MI from mice of indicated genotypes, scale bar (2 mm). (F) Fibrosis quantification of E, data were compared via Mann–Whitney test, αMHC-MCM/+; Yap^flox/+ (n= 5, 3 males/2 females) and αMHC-MCM/+; Yap^flox/K265R (n=6, 3 males/3 females), data are expressed as the mean ± SEM.

Figure 6.. Acetylated YAP and TUBA4A interact in the cytoplasm.

(A) IP-MS volcano plot reveals YAP interaction partners in P19 cells expressing either YapK265R or WT YAP. Interaction partners (blue, interactions have p < 0.05 FDR), interaction intensities (fold changes between WT and K265R (-Log2_FC)). (B) Representative YAP interaction partners that are significantly different between WT YAP or YapK265R-expressing P19 cells. (C) YAP IP reveals a decreased interaction between TUBA4A, MYL6, and YAP in YapK265R P19 cells. (D) IP-Western reveals that FK866 treatment increases Ac-YAP levels and the interaction between Ac-YAP and TUBA4A, MYL6, and MYH10 in NRVMs. (E) IF staining of YAP and TUBA4A in mouse CMs 8 weeks after AAV9-Yap-WT or AAV9-Yap-K265R injection. The colocalization efficiency is shown in F and G (n = 20), data were compared via Mann–Whitney test and data are expressed as the mean ± SEM, scale bar (10 μm).

Figure 7.. Microtubule network growth and Ac-YAP sequestration in the CM cytoplasm after MI.

(A, B) Super-resolution images of the detyrosinated microtubule network (A) and TUBA4A (B) in mice CMs two days after P9 sham or MI. (C) TUBA4A density quantification. (D) YAP and TUBA4A colocalization in mice CMs two days after sham or MI. (E) TUBA4A/YAP colocalization efficiency. A, D, scale bar (5 μm); B, scale bar (10 μm). (F, G) Subcellular fractionation reveals YAP localization in the cytosol (C), nucleus (N), and cytoskeleton (P) of heart tissues four days after P9 sham or MI surgery. s.e.: short exposure, l.e.: long exposure. (G) Quantification of F. Fraction normalization: cytoplasm (GAPDH), nuclear (HDAC1), and cytoskeleton (Vimentin). Sham group (n = 3) and MI group (n = 4). (H, I) YAP distribution in WT and K265R mice four days after P9 MI. (I) Quantification of H, n = 4 per group. Data were compared using the Mann–Whitney test for C and E and the two-way ANOVA test with Bonferroni’s post-hoc tests for G and I. Data are expressed as the mean ± SEM.