Ser14 phosphorylation of Bcl-xL mediates compensatory cardiac hypertrophy in male mice

Abstract

The anti-apoptotic function of Bcl-xL in the heart during ischemia/reperfusion is diminished by K-Ras-Mst1-mediated phosphorylation of Ser14, which allows dissociation of Bcl-xL from Bax and promotes cardiomyocyte death. Here we show that Ser14 phosphorylation of Bcl-xL is also promoted by hemodynamic stress in the heart, through the H-Ras-ERK pathway. Bcl-xL Ser14 phosphorylation-resistant knock-in male mice develop less cardiac hypertrophy and exhibit contractile dysfunction and increased mortality during acute pressure overload. Bcl-xL Ser14 phosphorylation enhances the Ca2+ transient by blocking the inhibitory interaction between Bcl-xL and IP3Rs, thereby promoting Ca2+ release and activation of the calcineurin-NFAT pathway, a Ca2+-dependent mechanism that promotes cardiac hypertrophy. These results suggest that phosphorylation of Bcl-xL at Ser14 in response to acute pressure overload plays an essential role in mediating compensatory hypertrophy by inducing the release of Bcl-xL from IP3Rs, alleviating the negative constraint of Bcl-xL upon the IP3R-NFAT pathway.

Fig. 1. Knock-in (KI) mice in which Serine (Ser) 14 of Bcl-xL is replaced with Alanine show worse phenotypes in response to pressure overload.

a Representative immunoblots showing phosphorylation of Bcl-xL at Ser14 in the heart with time course after transverse aortic constriction (TAC). Sham is a heart sample collected one hour after sham surgery. h; hours, and d; days after TAC. Lower panel shows densitometric analysis of relative expression of pBcl-xL (Ser14)/Bcl-xL in the heart. Kruskal-Wallis test with sham as the control. p values are shown in the figure (n = 5). b Kaplan–Meier survival curves. Log-rank (Mantel-Cox) test p = 0.0006. n = 25–36. Lower panel shows the survival curves 1 week after TAC. c Representative pictures of M-mode echocardiography. Yellow lines indicate left ventricular (LV) end-systolic and -diastolic diameters. Vertical scale bar indicates 5 mm and horizontal scale bar indicates 100 ms. d Ejection fraction (EF) with time course after TAC or sham surgery. Two-way ANOVA with Tukey’s multiple comparison test. ****p < 0.0001 compared to +/+ (wild type) control. #p = 0.0183 and ####p < 0.0001 compared to knock-in/+. &&&&p < 0.0001, &&&p = 0.0003, && p = 0.0025, and &p = 0.0141 compared to Sham (upaired t test or Mann–Whitney test, two-sided). n = 6–16 (TAC) and 5 (Sham). e LV end-diastolic pressure at the indicated time points after TAC or sham surgery, evaluated by hemodynamic study. Two-way ANOVA with Tukey’s multiple comparison test. **** p < 0.0001. n = 5. f Lung weight normalized by tibia length at the indicated time points after TAC or sham surgery. Two-way ANOVA with Tukey’s multiple comparison test. ****p < 0.0001, ***p < 0.001, and **p < 0.01. Adjusted p values for (f): 0.0004 (1 W, +/+ vs KI/ + ), 0.0018 (2 W, +/+ vs KI/ + ), 0.0008 (2 W, +/+ vs KI/KI). n = 5–8. N represents biologically independent replicates. Data are mean ± SEM. Source data are provided as a Source Data file.

Fig. 2. Phosphorylation of Bcl-xL at Serine 14 is essential for compensatory hypertrophy in response to pressure overload.

Both heterozygous and homozygous Serine (S14A) knock-in (KI) mice were used in a and e whereas homozygous mice were used in b–d and f–h. a Heart weight normalized by tibia length at the indicated time points after TAC or sham. Sham: +/+ n = 5, KI/+ n = 7, KI/KI n = 5; 1 W: +/+ n = 5, KI/+ n = 6, KI/KI n = 5; 2 W: +/+ n = 6, KI/+ n = 8, KI/KI n = 5; 4 W: +/+ n = 5, KI/+ n = 7, KI/KI n = 5. b Wheat Germ Agglutinin (WGA) staining of the indicated heart tissues. Scale bar; 100 μm. c Quantitative analysis of relative cardiomyocyte size. n = 5. d Relative NPPA, NPPB, MYH7, and Rcan1.4 gene expressions. NPPA, NPPB, and MYH7: n = 6 (sham) and 9 (TAC). Rcan1.4: n = 4. e Calculated end-diastolic wall stress of the indicated mice 1-week post-TAC (n = 5). f Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of the indicated heart tissues. Arrows indicate TUNEL-positive nuclei. Scale bar; 100 μm. g Quantitative analysis of TUNEL-positive nuclei. n = 5. h Immunoblots showing cleaved caspase 3 and 9 expression levels in the heart after TAC or sham. Immunoblots were repeated at least three times using biologically independent replicates. In all graphs, WT is indicated by +/+ and knock-in (KI) is indicated by KI/+ (heterozygous) or KI/KI (homozygous). n represents biologically independent replicates. Two-way ANOVA with Tukey’s multiple comparison test. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns not significant. Adjusted p values for (a): 0.0015 (1 W, +/+ vs KI/ + ), 0.0001 (1 W, +/+ vs KI/KI), 0.0002 (2 W, +/+ vs KI/+). Adjusted p values for (c): 0.0012 (TAC 1 W, +/+ vs KI/KI), 0.0021 (KI/KI, sham vs TAC 2 W). Adjusted p values for (d): 0.0029 (NPPA: WT, Sham vs TAC), 0.002 (NPPA: TAC, +/+ vs KI/KI), 0.0251 (NPPB: TAC, +/+ vs KI/KI), 0.0004 (NPPB: KI/KI, Sham vs TAC), 0.0491 (MYH7: +/+, Sham vs TAC). Adjusted p values for (e): 0.0061 (WT vs KI/ + ), 0.0001 (WT vs KI/KI). Data are mean ± SEM. Source data are provided as a Source Data file.

Fig. 3. The Ras-MEK-ERK pathway is activated immediately after hypertrophic stimulation, phosphorylating Bcl-xL at Serine 14 and promoting nuclear translocation of NFAT3.

a Heat map of differentially expressed genes in the hearts of WT and Bcl-xL-S14A knock-in (KI) mice 9 hs after TAC or sham surgery. n = 3. b Gene set enrichment analysis plots of the FCεR1, ERK and Integrin pathways enriched after TAC compared to sham in WT mice (Supplementary Table 1). NES, normalized enrichment score. GSEA nominal p value is the statistical significance of the enrichment score by using a phenotype-based permutation test with no adjustment. c Immunoblots showing the phosphorylation status of ERK1/2 in the hearts of WT and KI mice after TAC or sham surgery. Immunoblots were repeated at least three times using biologically independent replicates. d Immunoblots showing the phosphorylation status of Bcl-xL (Ser14) in cardiomyocytes after phenylephrine (PE). e, g Relative expression of pBcl-xL (Ser14)/Bcl-xL. Kruskal–Wallis test with vehicle as the control. p values are shown in the figure (n = 5). f Immunoblots showing the effect of a MEK inhibitor (PD0325901) on PE-induced Bcl-xL-Ser14 phosphorylation in cardiomyocytes. h MS/MS spectrum of a doubly charged ion (m/z 646.81) corresponding to the peptide sequence ⁷ELVVDFLpSYK¹⁶ with a phosphorylation modification at S¹⁴ in Bcl-xL. The observed y- and b-ion series confirmed the peptide sequence and phosphorylation modification site. i, j Immunoblots showing the nuclear and cytosolic localization of NFAT3 in WT and S14A KI adult mouse cardiomyocytes transduced with adenovirus harboring H-Ras or LacZ (i) and its quantification analysis (j, n = 5). Two-way ANOVA with Tukey’s multiple comparison test. ****p < 0.0001, ns not significant. k Immunoprecipitation assay using α-H-Ras antibody with heart lysates from mice subjected to 9 hs of pressure overload. The numbers indicate the ratio of Bcl-xL (upper arrow) to H-Ras (lower arrow) by densitometric analysis. l FLAG-pull down assay using rat neonatal ventricular cardiomyocytes transduced with adenovirus harboring FLAG-Bcl-xL for two days in the presence of PE or vehicle for 20 mins. Immunoblots were repeated at least three times using independently prepared cardiomyocytes. n represents biologically independent replicates. Data are mean ± SEM.

Fig. 4. Wild type (WT), but not Bcl-xL-S14A knock-in (KI), cardiomyocytes exhibit increases in Ca²⁺ transient amplitude and sarcoplasmic reticulum (SR) Ca²⁺ content after TAC-induced pressure overload, which are suppressed by IP3R inhibition.

a Hemodynamic stress differentially alters intracellular Ca²⁺ dynamics in WT and KI cardiomyocytes, as shown in the Ca²⁺ transient amplitude, SR Ca²⁺ content, fractional Ca²⁺ release, and T₅₀. Cardiomyocytes were isolated from the indicated mouse hearts 1 day after TAC. WT baseline n = 21; WT TAC n = 18; KI baseline n = 24; KI TAC n = 20 from 3 hearts/group. Two-way ANOVA with Tukey’s multiple comparison test. b Representative traces of Ca²⁺ transient (F/F0) with 2-APB treatment in WT and KI cardiomyocytes 1 day after TAC. Orange dotted lines indicate the level of F/F0 at the time of 2-APB administration and arrows indicate the direction of change in F/F0. c Ca²⁺ transient amplitude before and after 2-APB treatment in cardiomyocytes 1 day after TAC. WT n = 13; KI n = 14 cells from 3 hearts/group. Two-sided paired t test (WT) or Wilcoxon matched-pairs signed rank test (KI). d Quantification of intracellular Ca²⁺ dynamics after 2-APB treatment in WT and KI cardiomyocytes 1 day after TAC. WT n = 13 and KI n = 14 cells (Ca²⁺ transient amplitude, two-sided Mann-Whitney U test); WT n = 9 and KI n = 10 cells (SR Ca²⁺ content and fractional Ca²⁺ release, two-sided unpaired t test); WT and KI n = 13 cells (T₅₀, two-sided Mann-Whitney U test) from 3 hearts/group. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns not significant. Adjusted p values for (a): 0.0216 (Ca²⁺ transient amplitude, WT, Baseline vs TAC), 0.0435 (Ca²⁺ transient amplitude, KI, Baseline vs TAC), 0.0071 (SR Ca²⁺ content, WT, Baseline vs TAC), 0.0007 (SR Ca²⁺ content, TAC, WT vs KI), 0.0423 (Fractional Ca²⁺ release, TAC, WT vs KI), 0.0001 (T₅₀, TAC, WT vs KI). Adjusted p values for (c): 0.0353 (KI, Pre-2APB vs Post-2APB). Adjusted p values for (d): 0.0373 (T₅₀, WT vs KI). Data are mean ± SEM. Source data are provided as a Source Data file.

Fig. 5. Bcl-xL Serine (Ser) 14 phosphorylation disrupts its inhibitory interaction with IP3R, enhancing hypertrophic stimuli-induced calcineurin-NFAT signaling.

a Gene set enrichment analysis plots of calcineurin and calcium regulation pathways enriched in WT compared to knock-in (KI) mouse hearts after 9 hs of TAC. NES, normalized enrichment score. GSEA nominal p value is the statistical significance of the enrichment score by using a phenotype-based permutation test with no adjustment. b The relative NFAT transcriptional activity in response to phenylephrine (PE) with or without 2-APB in cardiomyocytes transfected with adenovirus harboring Bcl-xL-WT or -S14A mutant (n = 6 independently prepared cardiomyocyte preparations/cultures). c Immunoprecipitation assay with anti-IP3R type 2 antibody using WT and KI mouse hearts 1 day after TAC. Repeated three times. d Flag pull-down assay using Flag-Bcl-xL-S14D or -S14A and recombinant GST-IP3R-fragment3, followed by Coomassie Brilliant Blue staining. Right panel shows quantification analysis of immunoprecipitated proteins (GST-IP3R-fragment 3 versus Flag-Bcl-xL-S14D or -S14A) (n = 8 independently prepared cardiomyocyte preparations/cultures, followed by in vitro binding assay). e The relative NFAT transcriptional activity in response to PE with or without synthetic peptide corresponding to the amino acid sequence in IP3R Fragment 3 (n = 6, independently prepared cardiomyocyte preparations/cultures). Two-way ANOVA with Tukey’s multiple comparison test. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns not significant. Adjusted p values for (b): 0.0041 (WT, PE vs PE + 2-APB), 0.001 (S14A, Vehicle vs PE), 0.0003 (S14A, Vehicle vs PE + 2-APB). Adjusted p values for (e): 0.0251 (PE, WT vs S14A), 0.0096 (S14A, Vehicle vs PE), 0.0037 (S14A, PE vs PE+Pep). Data are mean ± SEM. Source data are provided as a Source Data file.