Loss of H3K4 methylation destabilizes gene expression patterns and physiological functions in adult murine cardiomyocytes

Abstract

Histone H3 lysine 4 (H3K4me) methyltransferases and their cofactors are essential for embryonic development and the establishment of gene expression patterns in a cell-specific and heritable manner. However, the importance of such epigenetic marks in maintaining gene expression in adults and in initiating human disease is unclear. Here, we addressed this question using a mouse model in which we could inducibly ablate PAX interacting (with transcription-activation domain) protein 1 (PTIP), a key component of the H3K4me complex, in cardiac cells. Reducing H3K4me3 marks in differentiated cardiomyocytes was sufficient to alter gene expression profiles. One gene regulated by H3K4me3 was Kv channel-interacting protein 2 (Kcnip2), which regulates a cardiac repolarization current that is downregulated in heart failure and functions in arrhythmogenesis. This regulation led to a decreased sodium current and action potential upstroke velocity and significantly prolonged action potential duration (APD). The prolonged APD augmented intracellular calcium and in vivo systolic heart function. Treatment with isoproterenol and caffeine in this mouse model resulted in the generation of premature ventricular beats, a harbinger of lethal ventricular arrhythmias. These results suggest that the maintenance of H3K4me3 marks is necessary for the stability of a transcriptional program in differentiated cells and point to an essential function for H3K4me3 epigenetic marks in cellular homeostasis.

Figure 1. Cardiac-specific PTIP deletion affects H3K4me in mice.

(A) PTIP protein expression (140 kDa) was markedly decreased in the PTIP^– mice injected with tamoxifen as compared with that in the vehicle-injected PTIP^– and tamoxifen-injected PTIP⁺ mice. LV tissue was harvested 30 days after tamoxifen or vehicle injection, and immunoblotting was performed using chicken anti-PTIP (anti-PTIP) normalized to mouse anti–β-tubulin. (B) PTIP^– (n = 3) and PTIP⁺ mice (n = 3/group) were injected with tamoxifen at 8 weeks of age. Thirty days later, LV tissue was harvested, mRNA was isolated, and qPCR was performed for PTIP and normalized to GAPDH using TaqMan primers. Data are mean ± SEM. *P < 0.05 versus PTIP^–. (C) Eight-week-old PTIP^– (n = 5) and PTIP⁺ (n = 3) mice were injected with tamoxifen. Eight months later immunoblotting for H3K4me3 and histone H3 (top) was performed from whole heart chromatin. Immunoblot data were quantified by normalizing H3K4me3 levels to H3 (bottom). Data are mean ± SEM. (D) Whole heart tissue from PTIP⁺ mice was harvested and prepped for chromatin. IP was performed on 20 μg chromatin using chicken anti-PTIP and chicken IgY (control). IP samples were then denatured in SDS, loaded into a 6% SDS gel, and probed with chicken anti-PTIP and rabbit anti-RbbP5 (Bethyl). In the sample immunoprecipitated with chicken anti-PTIP (right lane), a 140-kDa band, consistent with PTIP, and a 70-kDa band, consistent with RbBP5, were detected. These bands were not detected in the sample immunoprecipitated with chicken IgY (middle lane).

Figure 2. Gene expression data in PTIP^– and PTIP⁺ mice.

(A) Eight-week-old PTIP^– (n = 3–5) and PTIP⁺ mice (n = 3–5) were injected with tamoxifen for 5 days. Thirty days later, LV tissue was harvested, and qPCR was performed using TaqMan probes for the listed genes normalized to GAPDH. (B) Eight-week-old PTIP^– (n = 5) and PTIP⁺ mice (n = 5) were injected with tamoxifen for 5 days. Eight months later, LV tissue was harvested, and qPCR was performed using TaqMan probes for the listed genes normalized to GAPDH. *P < 0.05 versus PTIP^–. All data are mean ± SD.

Figure 3. ChIP analysis of H3K4me3, PTIP, and RbBP5.

(A) ChIP analysis was performed to study H3K4me3 enrichment at the Kcnip2 5′ regulatory region. A schematic of the Kcnip2 transcript is shown. Boxes represent exons, and lines connecting boxes represent introns. Black boxes represent coding sequence, whereas white boxes represent untranslated region. Primers were designed that amplify DNA at –500 bp (primer 1), –100 bp (primer 2), and +100 bp (primer 3) relative to the first recognized ATG site (arrow tip). IP was performed with 20 μg chromatin using 2 μg anti-rabbit IgG and 2 μg anti-H3K4me3 antibody in PTIP⁺ (n = 4) and PTIP^– (n = 4) mice. Enrichment for H3K4me3 marks is shown at primer set 1 (left), primer set 2 (middle), and primer set 3 (right). *P < 0.05 versus PTIP^– at the same primer set. ChIP was performed to study whether PTIP and RbBP5 localized to the Kcnip2 5′ regulatory region. Primers were designed that amplify DNA at –500 bp, –100 bp, and +100 bp relative to the first recognized Kcnip2 ATG site. IP was performed with 20 μg chromatin using 4 μg anti-rabbit IgG (white bars), 4 μg rabbit anti-PTIP antibody (gray bars), or 4 μg rabbit anti-RbBP5 (black bars) in PTIP⁺ (n = 5) and PTIP^– (n = 4) mice. Data are represented as percentage input (mean ± SD).*P < 0.05 versus PTIP^–; ^#P < 0.05 versus PTIP⁺ at –100 and +100 bp. (B) Schematic illustrating how the decrease in Kcnip2 transcript expression is a direct result of PTIP deletion and the inability of PTIP^– mice to maintain a KMT2 complex (comp.) and H3K4me3 marks at the 5′ regulatory region of the Kcnip2 gene.

Figure 4. Effects of cardiac-specific PTIP deletion on Kcnip2, EKG, and APD.

(A) Western blot performed on LV samples from PTIP^– and PTIP⁺ mice 4 weeks after tamoxifen injection. Lanes were probed with antibodies to Kcnip2, Kcnd2, Kcnd3, and β-tubulin. (B) EKGs from a lead I configuration in PTIP⁺ and PTIP^– mice 4 weeks after tamoxifen injection. Arrows point to the ST segment in the PTIP⁺ mice and depressed ST segment in the PTIP^– mice. (C and D) Representative action potential tracings from ventricular myocytes in (C) PTIP⁺ and (D) PTIP^– mice. APD was measured at (E) 30% and (F) 90% repolarization in PTIP⁺ mice (number of animals [N] = 4, number of cells [n] = 15) and PTIP^– mice (N = 4, n = 10). BCL, basic cycle length. *P < 0.05, ^#P < 0.001. Data shown for E and F are mean ± SEM.

Figure 5. PTIP deletion reduces dV/dt and I_Na.

(A) dV/dt measured in PTIP⁺ myocytes (n = 3, n = 6) and PTIP^– myocytes (n = 2, n = 4). (B) I_Na measured in PTIP⁺ myocytes (N = 2, n = 4) and PTIP^– myocytes (N = 2, n = 4). *P < 0.05, **P < 0.01. Data are mean ± SEM.

Figure 6. PTIP deletion reduces I_to.

(A) Superimposed whole cell outward K⁺ current traces recorded from PTIP⁺ and PTIP^– mice. (B) I_to current-voltage relationships for PTIP⁺ myocytes (N = 2, n = 6) and PTIP^– myocytes (N = 2, n = 4) **P < 0.01, ***P < 0.001. Data are mean ± SEM.

Figure 7. PTIP deletion increases Ca²⁺ transients, I_Ca,L, and contractility.

(A) Ca²⁺ transients expressed as change in fluorescence (F) over control fluorescence (Fo) in PTIP⁺ myocytes (N = 2, n = 7 cells) and PTIP^– myocytes (N = 2, n = 6 cells) . Cells were loaded with fluo-4AM and field stimulated at 1 to 6 Hz. (B) I_Ca,L current-voltage relationships for PTIP⁺ myocytes (N = 2, n = 7) and PTIP^– myocytes (N = 2, n = 6). (C) PTIP^– myocytes show significantly higher fractional shortening that PTIP⁺ myocytes at 1- to 6-Hz pacing. *P < 0.05, ***P < 0.001. All data are mean ± SEM.

Figure 8. PTIP^– mice show increased in vivo LV function.

Eight-week-old PTIP⁺ were injected with tamoxifen (TAM; n = 10) or vehicle (VEH; n = 5), and PTIP^– mice were injected with tamoxifen (n = 12) or vehicle (n = 6). Echo was subsequently performed at 3 months, 6 months, and 9 months of age. (A) LV systolic function assessed by ejection fraction. (B) Velocity of circumferential fractional shortening (Vcf_c). *P < 0.05 versus PTIP^– vehicle, PTIP⁺ vehicle, and PTIP⁺ tamoxifen. (C) LV weight normalized to tibial length at 9 months. (D) The percentage fibrosis in paraffin-embedded sections of hearts from tamoxifen-injected 9-month-old PTIP⁺ mice (n = 6) and PTIP^– mice (n = 6) stained with picrosirius red and NB green. LV myocardial fibrosis was measured in each of 20 sections per heart. (E) Sections were also stained with wheat germ agglutinin and DAPI. Myocyte cross-sectional area in the LV free wall was measured in approximately 100 myocytes per heart. Scale bar: 100 μm (D); 50 μm (E). All data are mean ± SD.

Figure 9. PTIP^– mice show increased susceptibility to PVCs.

Twelve-week-old PTIP⁺ mice (n = 4) and PTIP^– mice (n = 3) were anesthetized with Avertin and injected i.p. with isoproterenol (2 mg/kg) and caffeine (180 mg/kg). (A) Continuous EKG recordings were performed. PVCs were counted, and analysis revealed that PTIP^– mice had significantly more PVCs than PTIP⁺ mice. Data are mean ± SD. *P < 0.05 vs. PTIP⁺. (B) Representative example of the pattern of ventricular bigeminy that was observed in all PTIP^– mice.