The PTIP-Associated Histone Methyltransferase Complex Prevents Stress-Induced Maladaptive Cardiac Remodeling

<div><p>Pressure overload induces stress-induced signaling pathways and a coordinated transcriptional response that begets concentric cardiac hypertrophy. Although concentric hypertrophy initially attenuates wall stress and maintains cardiac function, continued stress can result in maladaptive cardiac remodeling. Cardiac remodeling is orchestrated by transcription factors that act within the context of an epigenetic landscape. Since the epigenetic landscape serves as a molecular link between environmental factors (stress) and cellular phenotype (disease), defining the role of the epigenome in the development and progression of cardiac remodeling could lead to new therapeutic approaches. In this study, we hypothesized that the epigenetic landscape is important in the development of cardiac hypertrophy and the progression to maladaptive remodeling. To demonstrate the importance of the epigenome in HF, we targeted the PTIP-associated histone methyltransferase complex in adult cardiac myocytes. This complex imparts histone H3 lysine 4 (H3K4) methylation marks at actively expressed genes. We subjected PTIP null (PTIP-) mice to 2 weeks of transverse aortic constriction, a stress that induces concentric hypertrophy in control mice (PTIP+). PTIP- mice have a maladaptive response to 2wk of transverse aortic constriction (TAC)-induced pressure overload characterized by cardiac dilatation, decreased LV function, cardiac fibrosis, and increased cell death. PTIP deletion resulted in altered stress-induced gene expression profiles including blunted expression of ADRA1A, ADRA1B, JUN, ATP2A2, ATP1A2, SCN4B, and CACNA1G. These results suggest that H3K4 methylation patterns and the complexes that regulate them, specifically the PTIP-associated HMT, are necessary for the adaptive response to TAC.</p></div

Attenuated expression of cardiac genes in PTIP- mice after sham or TAC.

<p>qPCR was performed to define the expression of gene expression array-identified genes in PTIP- hearts. These studies were performed 3d after TAC. PTIP deletion resulted in attenuated expression of the calcium handling genes ATP1A2, ATP2A2, JUN, the alpha-adrenergic receptors ADRA1A and ADRA1B, SCN4B, and CACNA1G after TAC. Data shown are means ± SD and normalized to GAPDH.</p

PTIP deletion attenuates H3K4me3 marks.

<p>Immunoblot analysis for PTIP and H3K4me3 was performed to determine the impact of PTIP deletion and TAC on global H3K4me3 levels and PTIP expression. As shown in panel A, PTIP deletion results in a significant attenuation in global H3K4me3 marks when normalized to total histone H3 levels. TAC had no significant impact on PTIP levels or H3K4me3 levels. To determine whether PTIP regulates H3K4me3 levels at the promoter region of specific genes, ChIP-qPCR was performed to assess H3K4me3 enrichment at the promoter region of ADRA1A and SCN4B in PTIP- and PTIP+ mice (panel B). ChIP was performed with an anti-H3K4me3 antibody (black) and a non-specific Rabbit IgG antibody (white). Data reveal a significant decrease enrichment of H3K4me3 marks in PTIP- hearts (n = 6) as compared to PTIP+ hearts (n = 6) at the Adra1a and SCN4b promoter. The amplified ChIP-qPCR products were run on a gel with a 2% input (panel B, below). Data shown are means ± SEM.</p

Increase in cell death after TAC in PTIP- hearts.

<p>Left ventricles were stained for DAPI and TUNEL. DAPI stain for nuclei is shown in blue for PTIP+ TAC (panel A) and PTIP- TAC (panel D). TUNEL positive nuclei are stained in green for PTIP+ TAC (panel B) and PTIP- TAC (panel E). Overlay of the 2 figures shows TUNEL positive cardiomyocyte nuclei (arrowheads) in PTIP+ TAC hearts (panel C) and PTIP- hearts as well as TUNEL positive non-cardiomyocyte nuclei (arrows). TUNEL staining was quantified by counting 4000 nuclei per heart (panel G). Data shown are means ± SEM.</p

LV chamber dilation and depressed cardiac function in PTIP- TAC hearts.

<p>Echo performed on PTIP+ hearts after sham (n = 9) and TAC (n = 10) revealed a significant increase in anterior wall thickness (IVSd; panel B) after TAC without any significant change in LV end diastolic diameter (LVEDD; panel A) or LV ejection fraction (LVEF; panel C). PTIP- hearts after TAC (n = 12) also demonstrated an increase in anterior wall thickness compared with PTIP- sham mice (n = 10). However, PTIP- TAC hearts reveal a significant increase in LVEDD (panel A) and a significant decrease in LVEF when compared to PTIP- sham and PTIP+ TAC hearts. Representative 2D and M-mode images are shown for PTIP+ TAC (panel D) and PTIP- TAC (panel E). Data shown are means ± SEM.</p

Expression of PTIP and fetal genes after TAC.

<p>PTIP expression was significantly attenuated in PTIP- hearts both with and without TAC. Expression of fetal genes after 2wk TAC revealed a significant increase in ANF and β-MHC and a blunted expression of ACTA1 2wk after TAC in PTIP- hearts as compared to PTIP+ TAC hearts. Data shown are means ± SD. Data shown are means ± SD and normalized to GAPDH.</p

Maladaptive remodeling in PTIP- TAC hearts.

<p>PTIP+ and PTIP- hearts were harvested 2wk after sham (<b>A</b> and <b>B</b>) or TAC (<b>C</b> and <b>D</b>). H&E staining of ventricular cross-sections in PTIP+ (<b>A</b>) and PTIP- (<b>B</b>) after sham surgery. After TAC, PTIP+ hearts demonstrate thickened LV walls and preserved chamber size (<b>C</b>) in contrast with PTIP- hearts that demonstrate thinner LV walls and a dilated chamber (<b>D</b>). After TAC PTIP- (n = 15) and PTIP+ (n = 11) hearts demonstrate a similar increase in heart weight/body weight ratio after TAC (<b>E</b>) when compared to PTIP+ sham (n = 8) and PTIP- sham (n = 6). Data shown are means ± SEM.</p

Myocyte cross-sectional area after TAC.

<p>FITC-conjugated WGA staining performed on PTIP+ sham (n = 5; panel <b>A</b>) and PTIP- sham (n = 5; panel <b>B</b>) hearts reveals no significant differences at baseline in myocyte sectional area (CSA). PTIP+ TAC (n = 5; panel <b>C</b>) and PTIP- TAC (n = 7; panel <b>D</b>) both demonstrate that TAC induces an increase in myocyte CSA. The increase in myocyte CSA in PTIP- hearts is similar to that observed in PTIP+ hearts subjected to TAC (panel <b>E</b>). Magnification 400x. At least 200 myocytes from the left ventricle were measured. Data shown are means ± SEM.</p