Abnormal p38α mitogen-activated protein kinase signaling in dilated cardiomyopathy caused by lamin A/C gene mutation

Abstract

We previously interrogated the transcriptome in heart tissue from Lmna(H222P/H222P) mice, a mouse model of cardiomyopathy caused by lamin A/C gene (LMNA) mutation, and found that the extracellular signal-regulated kinase 1/2 and Jun N-terminal kinase branches of the mitogen-activated protein (MAP) kinase signaling pathway were abnormally hyperactivated prior to the onset of significant cardiac impairment. We have now used an alternative gene expression analysis tool to reanalyze this transcriptome and identify hyperactivation of a third branch of the MAP kinase cascade, p38α signaling. Biochemical analysis of hearts from Lmna(H222P/H222P) mice showed enhanced p38α activation prior to and after the onset of heart disease as well as in hearts from human subjects with cardiomyopathy caused by LMNA mutations. Treatment of Lmna(H222P/H222P) mice with the p38α inhibitor ARRY-371797 prevented left ventricular dilatation and deterioration of fractional shortening compared with placebo-treated mice but did not block the expression of collagen genes involved in cardiac fibrosis. These results demonstrate that three different branches of the MAP kinase signaling pathway with overlapping consequences are involved in the pathogenesis of cardiomyopathy caused by LMNA mutations. They further suggest that pharmacological inhibition of p38α may be useful in the treatment of this disease.

Figure 1.

Altered expression of genes in the p38α branch of the MAP kinase signaling cascade in hearts from Lmna^H222P/H222P compared with wild-type mice at 10 weeks of age. (A) Representation of genes in the MAP kinase signaling pathway (MAPK signaling pathway) identified by the KEGG visual pathway resource, which includes the ERK1/2 (ERK signaling), JNK (JNK signaling) and p38α (p38 signaling) branches within this GO term. Genes with statistically significant differences (q< 0.05) in expression detected on Affymetrix Mouse Genome 430 2.0 Arrays in hearts of Lmna^H222P/H222P mice compared with wild-type mice are indicated by asterisks; red asterisks indicate genes in the p38α branch. (B) Matrices showing array data of corresponding probe sets corresponding to 10 genes in the in the p38α branch with statistically significant differences (q< 0.05) in expression between wild-type (Lmna^+/+) and Lmna^H222P/H222P mouse hearts. In the matrices, each probe set is visualized as a row of colored squares and each column is a biological replicate (sample form a different mouse heart). Darker color means lower expression and brighter color higher expression. The genes correspond to those indicated with red asterisks in (A) [DDIT3/CHOP10 is the same as GADD153 and MAP2K6/MEK6 the same as MKK6 in (A)]. (C) Validation of differential expression in hearts from Lmna^H222P/H222P mice of four selected genes in the p38α MAP signaling pathway using real-time quantitative RT–PCR. RNA was obtained from hearts of mice at different ages as indicated on the x-axis. White bars show relative RNA expression levels in hearts of Lmna^+/+ mice and black bars in hearts of Lmna^H222P/H222P mice. Values are means ± SEM for n= 3 samples per group. The real-time quantitative RT–PCR was performed in triplicate with the different RNA samples. *P< 0.05, **P< 0.005, ***P< 0.0005.

Figure 2.

p38α signaling is hyperactivated in hearts of mice and humans with cardiomyopathy and lamin A/C gene mutaitons. (A) Immunoblots showing phosphorylated p38α (p-p38α), total p38α, phosphorylated MKK6 (p-MKK6) and total MKK6 in protein extracts of hearts of wild-type (Lmna^+/+) and Lmna^H222P/H222P mice at 8 weeks (left panel) and 16 weeks (right panel) of age. Each lane contains protein extracts from a different mouse. (B) Immunoblot showing p-p38α and total p38α  in protein extracts of cardiomyocytes isolated from hearts of wild-type (Lmna^+/+) and Lmna^H222P/H222P mice at 16 weeks. Each lane contains protein extracts from a different mouse. (C) Immunoblots showing p-p38α and total p38α in protein extracts of heart tissue from two human controls (LMNA^+/+) and three individuals with dilated cardiomyopathy caused by LMNA mutations.

Figure 3.

ARRY-371797 prevents LV dilatation and deterioration of FS in Lmna^H222P/H222P mice. (A) Schematic representation of the treatment protocol of Lmna^H222P/H222P mice with ARRY-371797. (B) Representative immunoblots using antibodies against phosphorylated p38α (p-p38α), total p38α, phosphorylated ERK1/2 (pERK1/2) and total ERK1/2 (ERK1/2) to probe proteins extracted from hearts from Lmna^H222P/H222P mice treated with placebo or ARRY-371797. Each lane contains protein extracts from a different mouse. (C) Representative M-mode transthoracic echocardiographic tracings from 20-week-old male Lmna^H222P/H222P mice treated with placebo or ARRY-371797. (D) Graphs showing mean LVEDD, mean LVESD and FS in 20-week-old male Lmna^H222P/H222P mice treated with placebo (n= 5) or ARRY-371797 (n= 7). Values for each individual mouse receiving placebo (circles) or ARRY-371797 (squares) as well SEMs of means (bars) are shown. *P< 0.05, **P< 0.005.

Figure 4.

ARRY-371797 decreases expression of genes encoding proteins involved in sarcomere organization and natriuretic peptides but not collagens in Lmna^H222P/H222P mice. (A) Relative expression of mRNAs from genes encoding myosin light chain (Mlc-1a) and cardiac-specific actin (Acta2) in Lmna^H222P/H222P mice treated with placebo or ARRY-371797. (B) Relative expression of mRNAs from genes encoding atrial natriuretic peptide A (NppA) and brain natriuretic peptide B (NppB) in Lmna^H222P/H222P mice treated with placebo or ARRY-371797. (C) Relative expression of mRNAs from genes encoding type 1 collagen isoforms (Col1a1 and Col1a2) in Lmna^H222P/H222P mice treated with placebo or ARRY-371797. Real-time quantitative RT–PCR, performed in triplicate on three separate samples, was used to measure mRNAs in hearts from Lmna^H222P/H2222P mice treated with placebo (n= 5) or ARRY-371797 (n= 7). Values shown are means ± SEM. *P< 0.05, **P< 0.005, n.s., not significant.