The mammalian Ste20-like kinase 2 (Mst2) modulates stress-induced cardiac hypertrophy

Abstract

The Hippo signaling pathway has recently moved to center stage in cardiac research because of its key role in cardiomyocyte proliferation and regeneration of the embryonic and newborn heart. However, its role in the adult heart is incompletely understood. We investigate here the role of mammalian Ste20-like kinase 2 (Mst2), one of the central regulators of this pathway. Mst2(-/-) mice showed no alteration in cardiomyocyte proliferation. However, Mst2(-/-) mice exhibited a significant reduction of hypertrophy and fibrosis in response to pressure overload. Consistently, overexpression of MST2 in neonatal rat cardiomyocytes significantly enhanced phenylephrine-induced cellular hypertrophy. Mechanistically, Mst2 positively modulated the prohypertrophic Raf1-ERK1/2 pathway. However, activation of the downstream effectors of the Hippo pathway (Yes-associated protein) was not affected by Mst2 ablation. An initial genetic study in mitral valve prolapse patients revealed an association between a polymorphism in the human MST2 gene and adverse cardiac remodeling. These results reveal a novel role of Mst2 in stress-dependent cardiac hypertrophy and remodeling in the adult mouse and likely human heart.

Keywords: Cardiac Hypertrophy; Cardiovascular Disease; Genetic Polymorphism; Signal Transduction; Transgenic Mice.

FIGURE 1.

Analysis of cardiomyocyte proliferation in Mst2^−/− hearts. A, expression of MST2 was completely ablated in Mst2^−/− hearts as indicated by Western blot. B, immunostaining of heart sections from WT and Mst2^−/− neonates (3 days of age). Sections were stained with anti-α-actinin (red), anti-phospho-HH3 (green), and DAPI (blue) (scale bars, 50 μm). C, no difference in the number of pHH3-positive cells between WT and Mst2^−/− neonates was observed. D, quantification of heart weight/body weight ratio showed that there was no difference in cardiac size between WT and Mst2^−/− neonate hearts. E, immunostaining of heart sections from WT and Mst2^−/− mice stained with anti-α-actinin (red), anti-phospho-HH3 (green), and DAPI (blue) (scale bars, 50 μm). F, heart weight/body weight ratio was not different between WT and Mst2^−/− adult mice. G, immunofluorescence analysis using pHH3 antibody in small intestine sections as positive control of this assay. The results showed that pHH3 staining was able to detect proliferating cells (arrows indicate pHH3-positive cells).

FIGURE 2.

Cardiac expression of MST2 in pathological conditions. A, wild type mice were subjected to TAC to induce pressure overload cardiac hypertrophy. Heart weight/tibia length ratio was significantly increased in the TAC group (n = 5–10 in each group; **, p < 0.01 versus sham). B, Western blot analysis of Mst2 expression in mouse heart lysates following TAC or sham operation. C, analysis of band density showed a significant increase in MST2 level in hypertrophic hearts (n = 5;**, p < 0.01). D, MST2 expression in humans with end-stage heart failure (HF) was detected by Western blot. Quantification of band density showed that MST2 level was elevated in failing hearts (n = 3 in each group; *, p < 0.05). E, immunofluorescence analysis of MST2 expression in cardiomyocytes and cardiofibroblasts isolated from rat neonatal hearts. F, Westen blot and band density quantification of MST2 expression in neonatal rat cardiomyocytes following phenylephrine (PE) treatment (20 μm, 48 h). Con, control.

FIGURE 3.

In vivo model of pressure overload induced hypertrophy in Mst2^−/− mice. A, hearts from WT and Mst2^−/− mice following TAC (2 weeks) or sham operation. B, analysis of HW/tibia length ratio showed significant reduction of hypertrophic response in Mst2^−/− mice (n = 7–8 in each group; *, p < 0.05; **, p < 0.01; ***, p < 0.001). Expression of hypertrophic markers were as follows: BNP (C) and atrial natriuretic peptide (ANP; D) were detected using real time RT-PCR. Values indicate fold change of expression after normalization with GAPDH level (n = 5–7 in each group; *, p < 0.05). Histological sections stained with hematoxylin and eosin (E) and quantification of cardiomyocyte cross-sectional area (F) showed smaller cardiomyocyte size in Mst2^−/− mice after TAC (scale bars, 20 μm; ***, p < 0.001). Masson's trichrome staining (G) and quantification of fibrotic area (H) indicated significantly less fibrosis in Mst2^−/− mice (*, p < 0.05). Expressions of collagen 1 (I) and collagen 3 (J) were detected using real time RT-PCR. Values indicate fold change of expression following normalization with GAPDH level (n = 5–7 in each group; *, p < 0.05).

FIGURE 4.

Echocardiography and hemodynamic analysis. Ventricular wall thickness (average of posterior and interventricular septum wall thickness) (A) and left ventricular end diastolic diameter (B) in Mst2^−/− mice compared with WT after TAC or sham operation (n = 7–8; *, p < 0. 05). C, left ventricular mass/body weight ratio was significantly higher in WT mice (n = 7–8; *, p < 0.05). However, there were no differences in cardiac contractility indices as indicated by D. End systolic pressure volume relationship (ESPVR), ejection fraction (E), dP/dt max (F), and dP/dt min (G). H, left ventricular systolic pressure was significantly elevated following TAC in both genotypes; however, no difference between WT and knock-out was observed basally or after TAC.

FIGURE 5.

Detection of apoptotic cells in Mst2^−/− hearts and in isolated cardiomyocytes overexpressing Mst2. A, examples of TUNEL staining to detect apoptotic cells. Triple staining was performed: TUNEL (green), DAPI (blue), and α-actinin (red). Arrows indicate TUNEL-positive nuclei. B, calculation of TUNEL-positive nuclei revealed that Mst2^−/− mice exhibited a reduced level of apoptosis compared with WT following TAC (n = 4–5; *, p < 0.05). C, immunoblot analysis showing dose-dependent expression of Mst2 in NRCM following infection with adenovirus expressing human Mst2 (Ad-Mst2). Adenovirus expressing LacZ (Ad-LacZ) was used as a control. (MOI, multiplicity of infection). D, representatives TUNEL staining in neonatal rat cardiomyocytes treated with adenovirus expressing Mst2 or LacZ (control). E, quantification of TUNEL-positive nuclei showed more apoptotic cells in cardiomyocytes expressing Mst2 (*, p < 0.05).

FIGURE 6.

Analysis of adult cardiac fibroblast proliferation rate. Adult cardiac fibroblasts were isolated from Mst2^−/− and WT littermates. A, immunostaining with mitosis marker Ki67. Arrows indicate Ki67-positive nuclei. B, quantification of Ki67-positive cells showed that there was no difference between WT and Mst2^−/− cardiac fibroblasts. C, fibroblasts were treated with 1:100 dilution of BrdU-labeling reagent (Invitrogen) for 24 h, and the presence of BrdU-positive cells was determined by immunostaining. D, quantification of BrdU-positive cells supported the Ki67 staining data that there was no difference in the proliferation rate between WT and Mst2^−/− cardiac fibroblasts.

FIGURE 7.

Mst2 regulates Raf1 and ERK1/2 activation. A, immunoprecipitation analysis of lysates from NRCM using Raf1 or control (luc, luciferase) antibody (Ab). Raf1 interacted with Mst2 in NRCM, but no difference in the amount of interacting protein was detected following phenylephrine (PE) stimulation. B, consistently, immunoprecipitation analysis of total heart lysates from WT and Mst2^−/− showed that Mst2 was co-precipitated with Raf1. C, immunoblot analysis to detect phosphorylated/total Raf1 and phosphorylated/total ERK1/2 in NRCM overexpressing LacZ (control), Mst2, or Mst2 kinase-defective mutant (Mst2 K56R). Band density quantification of phospho-Raf1/total Raf1 (D) and phospho-ERK1/2/total ERK1/2 (E) (*, p < 0.05; n = 3–4 independent experiments). E, Western blot analysis showing the expression of truncated mutant Mst2-ΔC. F, immunoprecipitation analysis using Raf1 or control antibody in NRCM overexpressing Mst2-ΔC showed that mutant Mst2-ΔC did not interact with Raf1. G, overexpression of mutant Mst2-ΔC enhanced the level of phospho/total ERK1/2. H, representative immunoblots for the detection of PP2A C subunit in NRCM. I, densitometric analysis showed a trend of higher PP2AC level in NRCM overexpressing Mst2.

FIGURE 8.

Phenylephrine-induced cardiomyocyte hypertrophy model. A, images of NRCM overexpressing Mst2 and control (LacZ) following treatment with 20 μm phenylephrine (PE) for 48 h with or without the addition of ERK1/2 inhibitor FR180204 (50 μm). Cells were stained with α-actinin antibody to specifically visualize cardiomyocytes. Scale bars, 50 μm. B, quantification of cell surface area indicated that Mst2 overexpression significantly enhanced phenylephrine-induced hypertrophic response. (Results were from four independent experiments conducted in triplicate; a minimum of 100 cells were measured per replicate.) (***, p < 0.001). Treatment with FR180204 reduced the hypertrophic response in Mst2-overexpressing cells to the level comparable with control cells with the same treatment. C, using adenovirus expressing a luciferase reporter driven by the rat BNP promoter, we found that Mst2 overexpression increased activation of the BNP promoter both basally and after phenylephrine stimulation (n = 8; **, p < 0.01;***, p < 0.001). Consistently, treatment with FR180204 abolished the difference in hypertrophic response between Mst2-overexpressing myocytes and control cells. D, representative images of NRCM-overexpressing Mst2 K56R mutant and the quantification of cell surface area at basal condition and after stimulation with phenylephrine (***, p < 0.001).

FIGURE 9.

Analysis of Raf1 and ERK1/2 activation during TAC-induced hypertrophy. A, representative Western blots to detect phosphorylated/total Raf1 and phosphorylated/total ERK1/2 in total heart extracts of Mst2^−/− and WT mice following TAC. Quantification of phosphorylated/total Raf1 (B) and ERK1/2 (C) showed that activation of Raf1 and ERK1/2 were decreased in Mst2^−/− mice during pathological hypertrophy (*, p < 0.05; n = 5–7 in each group).

FIGURE 10.

Analysis of Mst1 expression and YAP activation in the hearts during hypertrophy. A, immunoblot analyses to determine the level of MST1 and phosphorylated/total YAP (tYAP). Band density quantification of MST1 normalized to GAPDH level (B) and phosphorylated/total YAP (C) showed that the level of Mst1 expression as well as activation of YAP were not altered in Mst2^−/− mice following TAC (n = 5–7 in each group).

FIGURE 11.

Functional analysis of the human MST2 + 214G/A polymorphism. A, schematic diagram of the luciferase constructs used for the experiment. The 1.2-kb 3′-UTR of the human MST2 gene carrying either +214 G or A allele were inserted downstream of the luciferase-coding region. The expression of Renilla luciferase was used as a control for transfection efficiency. B, analysis using human primary fibroblasts indicated that the 3′-UTR bearing G allele produced significantly higher luciferase (luc) compared with 3′-UTR with A allele. (*, p < 0.05).