Ablation of p21-activated kinase-1 in mice promotes isoproterenol-induced cardiac hypertrophy in association with activation of Erk1/2 and inhibition of protein phosphatase 2A

Abstract

Earlier investigations in our lab indicated an anti-adrenergic effect induced by activation of p21-activated kinase (Pak-1) and protein phosphatase 2A (PP2A). Our objective was to test the hypothesis that Pak-1/PP2A is a signaling cascade controlling stress-induced cardiac growth. We determined the effects of ablation of the Pak-1 gene on the response of the myocardium to chronic stress of isoproterenol (ISO) administration. Wild-type (WT) and Pak-1-knockout (Pak-1-KO) mice were randomized into six groups to receive either ISO, saline (CTRL), or ISO and FR180204, a selective inhibitor of Erk1/2. Echocardiography revealed that hearts of the Pak-1-KO/ISO group had increased LV fractional shortening, reduced LV chamber volume in diastole and systole, increased cardiac hypertrophy, and enhanced transmitral early filling deceleration time, compared to all other groups. The changes were associated with an increase in relative Erk1/2 activation in Pak-1-KO/ISO mice versus all other groups. ISO-induced cardiac hypertrophy and Erk1/2 activation in Pak-1-KO/ISO were attenuated when the selective Erk1/2 inhibitor FR180204 was administered. Immunoprecipitation showed an association between Pak-1, PP2A, and Erk1/2. Cardiac myocytes infected with an adenoviral vector expressing constitutively active Pak-1 showed a repression of Erk1/2 activation. p38 MAPK phosphorylation was decreased in Pak-1-KO/ISO and Pak-1-KO/CTRL mice compared to WT. Levels of phosphorylated PP2A were increased in ISO-treated Pak-1-KO mice, indicating reduced phosphatase activity. Maximum Ca(2+)-activated tension in detergent-extracted bundles of papillary fibers from ISO-treated Pak-1-KO mice was higher than in all other groups. Analysis of cTnI phosphorylation indicated that compared to WT, ISO-induced phosphorylation of cTnI was blunted in Pak-1-KO mice. Active Pak-1 is a natural inhibitor of Erk1/2 and a novel anti-hypertrophic signaling molecule upstream of PP2A.

Figure 1. Cardiac hemodynamics

M-mode echocardiography obtained from the parasternal short axis view at the mid-papillary level (top), and mitral pulsed-wave Doppler recordings showing early (E) and late (atrial-A) ventricular filling velocity (bottom), in WT and Pak-1-KO mice, in the absence (CTRL) and presence of ISO, or during administration of ISO and FR180204. Ablation of Pak-1 in mice promotes ISO-induced cardiac hypertrophy, compared to ISO-treated WT mice, which is prevented when the selective Erk1/2 inhibitor FR180204 is administered.

Figure 2. Gross anatomy and histology of WT and PAK-1-KO hearts

A, Gross anatomy of WT and Pak-1-KO hearts illustrating the increased cardiac mass in ISO-treated Pak-1-KO hearts (middle, right) compared to WT and Pak-1-KO hearts that received ISO or CTRL. ISO-treated Pak-1-KO hearts that received FR180204, a selective inhibitor of Erk1/2, were characterized by LV total mass comparable to WT and Pak-1-KO hearts that received CTRL saline. B, Cross-section at the mid-papillary level of hearts in (A). C, Hematoxylin and eosin stain of myocardial LV tissue shows myocyte hypertrophy in ISO-treated hearts, and attenuated myocyte growth in mice that received FR180204. Data are representative of three separate experiments. Magnification is 40X for all images.

Figure 3. Western immunoblotting analysis of AKT, MAPKs, and PP2A indicate that cardiac hypertrophy is due to enhanced Erk1/2 activation in Pak-1-KO mice

A, AKT phosphorylation at T308 was not significantly different among any of the experimental groups. B-D, Western immunoblotting of Erk1/2 phosphorylation in whole cardiac tissue shows maximal activation in Pak-1-KO/ISO mice vs. all other groups. Erk1 phosphorylation at residues T202 and Y204, and Erk2 phosphorylation at T188 indicate that Erks activation is dependent on ISO stimulation and Pak-1 expression. E, Phosphorylation of JNK1/2/3 MAPK at residues Y185 + Y185 + Y223 was not significantly different among all experimental groups. F-G, Phosphorylation of p38 MAPK is reduced in Pak-1-KO mice, in the presence and in the absence of ISO, compared to ISO-treated and untreated WT mice. H-I, Phosphorylation of PP2A at residue Y307 was increased in treated and untreated Pak-1-KO mice, compared to WT groups. * indicates p < 0.05.

Figure 4. Ca²⁺-dependent isometric tension development of detergent-extracted cardiac fibers

A-B, ISO-treated Pak-1-KO hearts develop a statistically significantly higher maximal Ca²⁺ activated tension compared to all other experimental groups. * indicates p < 0.05.

Figure 5. Analysis of cTnI by 2-D DIGE

A-B, Two-dimensional map of the cTnI in WT and Pak-1-KO mice. C, Total phosphorylation of cTnI increased in WT mice that received ISO vs. untreated WT control. No change in total cTnI phosphorylation was observed in Pak-1-KO mice after ISO treatment. D, Individual analysis of phosphorylation of spot P1 revealed a decrease in phosphorylation in Pak-1-KO mice treated with ISO vs. untreated Pak-1-KO mice. E, Individual analysis of phosphorylation of spot P4 revealed an increase of phosphorylation in Pak-1-KO mice that received ISO vs. untreated Pak-1-KO mice. * indicates p < 0.05.

Figure 6. Proposed mechanism of LV myocardial hypertrophy in a Pak-1-KO mouse model

A, In the presence of Pak-1, ISO stimulation of the β-adrenercic receptor activates protein kinase A (PKA) signaling via activation of heterotrimeric G protein (α, β, and γ) and adenylate cyclase (AC). Also, ISO stimulation of the β-adrenercic receptor activates Erk1/2 signaling pathway via β-arrestin-mediated transactivation of the Epidermal Growth Factor receptor (EGFr). Active PKA activates Pak-1 by promoting Pak-1 autophosphorylation; in turn, active Pak-1 mediates PP2A conformational change and autodephosphorylation leading to PP2A activation. The now active PP2A induces dephosphorylation and reduced activation of Erk1/2, resulting in attenuation of cardiac hypertrophy. B, In the absence of Pak-1, Pak-1/PP2A-mediated inhibition of Erk-1/2 is lost (//), resulting in enhanced, Erk1/2-mediated cardiac hypertrophy. The dotted line proposes a potential PKA-mediated mechanism through which PP2A phosphorylation and inactivation may occur when Pak-1 is absent.