Loss of H3K4 methylation destabilizes gene expression patterns and physiological functions in adult murine cardiomyocytes (original) (raw)
Generation of an inducible, cardiac-specific PTIP-null mouse. In order to delete the PTIP protein in cardiomyocytes, mice were bred to generate the tamoxifen-inducible Paxip1 deletion strains. Using a floxed (fl), a null (–), and a wild-type (+) Paxip1 allele (11), the Cre-modified estrogen receptor transgene, driven by the cardiac-specific α myosin heavy chain promoter (αMHCMCM), was crossed into the Paxip1fl/+ or Paxip1fl/– genetic background to generate αMHCMCM_Paxip1fl/–_ and αMHCMCM_Paxip1fl/fl_ (designated as PTIP–) or αMHCMCM_Paxip1fl/+_ and αMHCMCM_Paxip1+/+_ (designated as PTIP+) control mice. Thirty days after tamoxifen injection in 8-week-old mice, there was a marked decrease in steady-state PTIP protein levels in the tamoxifen-injected PTIP– mice as compared with that in the vehicle-injected PTIP– and the tamoxifen-injected PTIP+ mice (Figure 1A). After tamoxifen injection, PTIP– mice also have a 7- to 8-fold decrease in PTIP mRNA when compared with that in PTIP+ mice (Figure 1B).
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).
PTIP is part of a histone methyltransferase complex that regulates histone methylation. To assess whether PTIP protein affects H3K4me in ventricular whole heart samples, we examined levels of H3K4me3 by quantitative Western blotting (Figure 1C). Compared with total amounts of histone H3, levels of H3K4me3 were reduced to approximately 53% of controls. We prepared chromatin from control hearts for immunoprecipitation with anti-PTIP antibodies to examine protein-protein interactions directly on DNA. After reverse-crosslinking, proteins were separated on SDS/PAGE gels and probed for RbBP5 (Figure 1D), a component of the KMT2C/D complex (11), to show that PTIP is part of the H3K4me complex in whole heart tissue.
Histone methylation regulates gene expression. We next determined whether PTIP deletion and reduction in H3K4me3 was sufficient to alter gene expression profiles in the adult heart. Total RNA was isolated from cardiac tissue 30 days after tamoxifen injection from PTIP– (n = 3) and PTIP+ (n = 3) mice. The RNAs were analyzed by Affymetrix microarrays to identify changes in gene expression patterns. A total of 221 genes were significantly, altered with 60% of them showing a decrease after PTIP deletion (Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI44641DS1). These results are consistent with the hypothesis that the PTIP/KMT2C/D complex imparts H3K4me3 marks that are associated with active gene expression, at least within a subset of genes. Differential gene expression was confirmed by qPCR for selected genes (Figure 2A). Gene expression analysis was also performed on hearts from mice 8 months after tamoxifen injection (Figure 2B). Many genes that showed a decrease in expression at 1 month continued to exhibit a significant decrease 8 months after tamoxifen. In order to demonstrate that the attenuation in Kcnip2 was due to PTIP deletion and not due to genetic background or the injection of tamoxifen, Kcnip2 gene expression by qPCR was measured in Paxip1fl/+ and Paxip1fl/fl mice, with and without the αMHCMCM transgene and with and without tamoxifen (Supplemental Figure 1).
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.
Gene-specific evidence that PTIP and H3K4me regulate gene expression in differentiated tissue. We next sought to demonstrate that PTIP deletion alters H3K4me3 marks at a specific target gene promoter region in the heart. Thus, we performed ChIP assays using antibodies to H3K4me3 to assess the methylation status at the 5′ regulatory region of the Kcnip2 gene (Figure 3A). The Kcnip2 gene was selected as a target partly because Kcnip2 levels were reduced approximately 5 and 10 fold at 1 and 8 months, respectively, after PTIP deletion. In heart chromatin from PTIP+ mice, H3K4me3 marks were enriched substantially at a DNA region –100 bp relative to the translation start site. A moderate enrichment in H3K4me3 marks was seen at +100 bp relative to the first ATG, and little enrichment was observed at –500 bp. In comparison, the H3K4me3 enrichment at –100 bp and +100 bp was significantly attenuated in the heart chromatin from the PTIP– group. These results show that PTIP deletion does indeed result in an alteration in H3K4me3 marks at the 5′ regulatory region of the Kcnip2 gene. Next, we examined PTIP and RbBP5 localization to the 5′ regulatory region of the Kcnip2 gene (Figure 3A). In chromatin from PTIP+ hearts, both PTIP and RbBP5 were enriched at a DNA region –500 bp relative to the first Kcnip2 ATG. This enrichment was not observed at DNA regions –100 bp and +100 bp relative to the first ATG. However, in chromatin from PTIP– hearts, the PTIP and RbBP5 enrichment observed at –500 bp in PTIP+ mice was significantly reduced. No differences in PTIP and RbBP5 enrichment were observed between heart chromatin from PTIP+ and PTIP– mice at –100 and +100 bp relative to the first ATG, as these levels were closer to background. Thus, we propose that the decrease in Kcnip2 transcript expression is a direct result of PTIP deletion and the inability of PTIP– mice to maintain a KMT2 complex and H3K4me3 marks at the 5′ regulatory region of the Kcnip2 gene (Figure 3B). As a negative control, we performed ChIP assays for H3K4me3 marks at the 5′ regulatory region of the Nppb gene, a gene that shows no change in mRNA expression in PTIP– mice as compared with that in PTIP+ mice (Supplemental Figure 2). We observed no changes in H3K4me3 enrichment at the 5′ regulatory region of the Nppb gene in PTIP– mice when compared with that in PTIP+ mice. These results provide mechanistic evidence that maintaining histone methylation marks is critical for maintaining stable gene expression profiles in a fully differentiated, nondividing cell type.
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.
Kcnip2, Kcnd2, and Kcnd3 protein expression. To determine whether mRNA profiles correspond to protein levels, hearts were harvested from PTIP– and PTIP+ mice 4 weeks after tamoxifen injection, and LVs were prepped for immunoblotting with anti-Kcnip2, anti-Kcnd2, and anti-Kcnd3 antibodies (Figure 4A). These data reveal that in PTIP– mice there was a marked decrease in Kcnip2 protein as compared with that in PTIP+ mice. Blotting for Kcnd3 and Kcnd2 revealed no significant difference in protein expression in PTIP+ and PTIP– mice, despite the fact that Kcnd3 mRNA levels were significantly different in PTIP– mice as compared with those in PTIP+ mice.
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.
EKG and action potential measurements. To investigate the significance of H3K4me marks in the heart, we studied the impact that PTIP deletion and the attenuation of Kcnip2 gene expression had on the electrophysiological phenotype. Initially, EKGs were obtained from PTIP+ and PTIP– mice 4 weeks after tamoxifen injection. As shown in Figure 4B, PTIP– mice had a marked and easily identified depression of the ST segment as compared with PTIP+ mice. In addition, analysis of the EKG also revealed a significant increase in the PR interval in PTIP– mice (n = 6) as compared with that in PTIP+ mice (n = 6) (36.9 ± 2.6 ms. vs. 31.6 ± 4.0 ms., respectively; P < 0.05). There were no significant differences in the heart rate of PTIP– mice when compared with that of PTIP+ mice (666 ± 20 bpm vs. 661 ± 28 bpm, respectively). Next, LV apical myocytes were isolated from PTIP+ and PTIP– mice, and action potentials were recorded. As shown in Figure 4, C and D, the action potential profile was appreciably different in the PTIP– mice as compared with that in PTIP+ mice. Objective measurements of the APD presented in Figure 4, E and F, demonstrated that PTIP– mice had a significantly longer APD, at 30% and 90% repolarization. The resting membrane potential (RMP) was also significantly higher in PTIP– mice (N = 4, n = 10) as compared with that in PTIP+ mice (N = 4, n = 15) (–69.95 ± 1.2 mV vs. –73.55 ± 0.71 mV; P < 0.05).
PTIP– mice have reduced INa. As shown above, EKG data revealed that there was a significant increase in the PR interval in PTIP– mice as compared with that in PTIP+ mice, which might have indicated that PTIP– hearts had a conduction defect in the His-Purkinje system. Furthermore, analysis of ventricular action potentials revealed a decrease in maximum upstroke velocity (dV/dt) (Figure 5A). Taken together, these results suggested that there may be a decrease in the sodium inward current (INa) responsible for phase 0 of the cardiac action potential. Accordingly, we measured INa in LV apical myocytes in PTIP+ and PTIP– mice. As shown in Figure 5B, PTIP deletion resulted in attenuated INa in PTIP– hearts as compared with that in PTIP+ hearts.
PTIP deletion reduces dV/dt and INa. (A) dV/dt measured in PTIP+ myocytes (n = 3, n = 6) and PTIP– myocytes (n = 2, n = 4). (B) INa 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.
Ito is reduced in PTIP– mice. To determine whether H3K4me marks regulate Ito, myocytes were isolated from the apex of PTIP– and PTIP+ mice, and Ito currents were measured in the presence of tetrodotoxin (TTX). Figure 6A shows representative Ito recordings from PTIP+ and PTIP– mice. Figure 6B displays peak Ito amplitudes as a function of test pulse; PTIP deletion resulted in a significant attenuation of Ito in LV apical myocytes.
PTIP deletion reduces Ito. (A) Superimposed whole cell outward K+ current traces recorded from PTIP+ and PTIP– mice. (B) Ito 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.
Altered calcium handling and contractility in PTIP– mice. Previous work has suggested that altering Ito and prolonging APD can alter [Ca2+]i levels and cardiac contractility (17). Accordingly, we assessed [Ca2+]i, ICa,L, and cellular contractility in apical LV myocytes from PTIP+ and PTIP– mice. First, cells were loaded with fluo-4AM, and calcium transients were recorded at different stimulation frequencies. As shown in Figure 7A, the amplitude of [Ca2+]i transients was significantly larger in PTIP– myocytes than in PTIP+ myocytes at all pacing frequencies. We also measured ICa,L to determine whether the increase in [Ca2+]i was at least partly due to an increase in this inward current. Figure 7B shows peak ICa,L as a function of test pulse. Data revealed a significantly higher ICa,L in PTIP– mice as compared with that in PTIP+ mice. In addition, measurements of myocyte contractility at different stimulation frequencies revealed that PTIP– mice have a higher fractional shortening than PTIP+ mice (Figure 7C). These results suggest that attenuating Ito and prolonging the APD alters [Ca2+]i levels and excitation contraction coupling in cardiomyocytes.
PTIP deletion increases Ca2+ transients, ICa,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) ICa,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.
In vivo assessment of cardiac function. We assessed cardiac structure and function noninvasively over time by echocardiography in isoflurane-anesthetized PTIP– and PTIP+ mice, with and without tamoxifen at 3 months, 6 months, and 9 months. Surprisingly, the echoes revealed significant and sustained improvements in LV systolic functions in the PTIP– mice after tamoxifen injection, as measured by ejection fraction (Figure 8A) and velocity of circumferential fractional shortening (Figure 8B), a load-independent assessment of systolic function (18). Despite the increase in systolic function in the PTIP– hearts, no alterations in echo parameters of chamber size, wall thickness, or heart rate were observed (Supplemental Table 2).
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 (Vcfc). *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.
Pathology. At the end of 9 months, heart tissue was harvested, and histology was performed. The PTIP– mice showed no evidence of gross cardiac hypertrophy, as measured by LV weight to tibia length (Figure 8C), or cellular hypertrophy, as measured by myocyte cross-sectional area (Figure 8E). Myocardial fibrosis was quantified by picrosirius red staining, with no significant differences observed in the 2 groups (Figure 8D).
Susceptibility to ventricular premature beats. To determine whether PTIP deletion and the associated changes in INa and [Ca2+]i predispose to a susceptibility to ventricular arrhythmias, lightly anesthetized 12-week-old PTIP+ and PTIP– mice were injected with isoproterenol and caffeine. PVCs were counted for the first 5 minutes after the initial PVCs, and data were analyzed as PVCs per minute. As shown in Figure 9A, PTIP– mice (n = 3) demonstrated a significantly higher number of PVCs per minute than PTIP+ mice (n = 4). One PTIP+ mouse showed no evidence of PVCs, and the other PTIP+ mice demonstrated intermittent isolated PVCs. In contrast, all PTIP– mice developed a sustained pattern of ventricular bigeminy, in which every other beat was a PVC (Figure 9B).
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.