Spatiotemporal regulation of an Hcn4 enhancer defines a role for Mef2c and HDACs in cardiac electrical patterning

Abstract

Regional differences in cardiomyocyte automaticity permit the sinoatrial node (SAN) to function as the leading cardiac pacemaker and the atrioventricular (AV) junction as a subsidiary pacemaker. The regulatory mechanisms controlling the distribution of automaticity within the heart are not understood. To understand regional variation in cardiac automaticity, we carried out an in vivo analysis of cis-regulatory elements that control expression of the hyperpolarization-activated cyclic-nucleotide gated ion channel 4 (Hcn4). Using transgenic mice, we found that spatial and temporal patterning of Hcn4 expression in the AV conduction system required cis-regulatory elements with multiple conserved fragments. One highly conserved region, which contained a myocyte enhancer factor 2C (Mef2C) binding site previously described in vitro, induced reporter expression specifically in the embryonic non-chamber myocardium and the postnatal AV bundle in a Mef2c-dependent manner in vivo. Inhibition of histone deacetylase (HDAC) activity in cultured transgenic embryos showed expansion of reporter activity to working myocardium. In adult animals, hypertrophy induced by transverse aortic constriction, which causes translocation of HDACs out of the nucleus, resulted in ectopic activation of the Hcn4 enhancer in working myocardium, recapitulating pathological electrical remodeling. These findings reveal mechanisms that control the distribution of automaticity among cardiomyocytes during development and in response to stress.

Figure 1. Expression pattern of Hcn4 mRNA by whole-mount in-situ hybridization

(A) At E8.5, Hcn4 was expressed throughout the heart tube in a caudal-to-cranial gradient. By E9.5 (B,C), expression was less in the atrial chamber, right ventricle (rv), and outflow tract (oft) than in other areas. At E10.5 and E11.5 (D–H), Hcn4 was strongly expressed in the sinus horns (sh) 27 and their interface with the atria, and at lower levels in the atria and ventricles. At E12.5 (I), Hcn4 was expressed highly in the sinoatrial node, the sinus venosus contribution to the atria including the coronary sinus (cs), and at lower levels in the ventricles and right atrium. In the neonatal heart (J,K), Hcn4 was expressed in the SA node, the sinus venosus derivatives, and the AV ring tissue. In (L) the ventricles are shown after clearing. A low-intensity meshwork of staining is visible, consistent with the His-Purkinje system (arrowheads delineate an example of a Purkinje fiber strand in the left ventricle). Abbreviations: ra, right atrium; la, left atrium; v, ventricle; rv, right ventricle; lv, left ventricle; sh, sinus horns; sv, sinus venosus oft, outflow tract; cs, coronary sinus.

Figure 2. Identification and in vivo testing of Hcn4 regulatory elements

(A) Genomic regions containing putative CREs were cloned upstream of hsp68LacZ or LacZ (basal promoter) to generate constructs for pronuclear injection. (B) Three species alignment at the Hcn4 locus from the ECR Browser, with mouse as the base genome against human and opossum. Peaks reflect degree of conservation (scale: 50–100%). Color code: red, upstream non-coding DNA; salmon, intronic DNA; blue, exons. Multiple conserved elements were selected for analysis (1–4) from Intron 1. Locations of previously validated transcription factor binding sites (TFBS) are indicated (NRSF, Mef2, AP-1, Sp1). Examples of founders from each construct injected are shown below the corresponding area on the Hcn4 genomic map. Although the presence of variable extra-cardiac reporter activity among different founders confirmed reporter insertion into permissive loci, none of the founders exhibited consistent reporter activity within the heart. (C) Left, an E11 embryo from an R2R3-LacZ transgenic line (line 17) stained with bluo-gal reporter activity within defined areas of the heart. Right, the cardiac region of line 17 in comparison with embryos from two additional transgenic founders (line 15 and line 22) shows similar cardiac reporter activity despite different genomic integration sites. By whole mount imaging, activity is concentrated at the junction of the sinus venosus and the right atrium, a linear area extending from the sinus venosus at the level of the interatrial groove to the ventricles (arrow), and within the ventricles. Sections from one founder embryo (line 15, bottom left) show reporter activity in the interventricular ring at the crest of the developing interventricular septum (section 1); and between the sinus venosus and atrium (section 2). Abbreviations: sv, sinus venosus; ra, right atrium; la, left atrium; v, ventricles; rv, right ventricle; lv, left ventricle.

Figure 3. In vivo temporal reporter activity directed by the combined R2-R3 Hcn4 Enhancers

A stable transgenic R2R3-LacZ mouse line was examined at E11.5 (A–C), at E15.5 (D–F) and at the neonatal stage (G–I). At E11.5, a whole mount bluo-gal stained embryo (A) and paraffin sections of bluo-gal stained embryos counterstained with nuclear fast red (B,C) showed reporter activity in the non-chamber embryonic myocardium including the AV canal, the interventricular ring at the crest of the developing interventricular septum, the posterior atrium/interatrial groove and right venous valve. At E15.5 (D–F), expression was confined to the atrial non-chamber myocardium and the developing AV conduction system. By late development, only faint expression was visible in atrial non-chamber myocardium while more robust expression was present within the AV conduction system (G–I). Of note, high-level activity was never noted in the SAN primordium or the perinatal SAN, indicating that R2R3 is not sufficient to direct expression to this tissue. Immunohistochemistry for Cx43, marking chamber myocardium, on bluo-gal stained cryosections (bottom row) showed mutual exclusion of Cx43 and β-gal activity in the ventricles at E11.5 (J,K) and at E15.5 (L,M). Abbreviations: san, sinoatrial node; avc, atrioventricular canal; rvv, right venous valve; pas, primary atrial septum; ra, right atrium; rv, right ventricle; ivs, interventricular septum; avn, atrioventricular node; avb, atrioventricular bundle; av ring, atrioventricular ring; a, common atrium; ivr, interventricular ring.

Figure 4. In vivo expression directed by the R2R3 Hcn4 enhancer in the post-natal AV bundle and its interface with the AV Node

(A–E) A postnatal (P5) R2R3-LacZ heart was stained with bluo-gal, imaged with optical projection tomography in whole-mount, then sectioned and counterstained with hematoxylin-eosin. (A) An image from a 3D reconstruction of optical projection tomograms shows a discrete band of enhancer activity along the anterior-posterior axis of the AV junction. Dashed lines indicate the positions of the coronal sections shown in panels B-E proceeding from anterior-posterior (1–4). The band of reporter activity begins below the annulus fibrosis (af) anteriorly at the crest of the interventricular septum (ivs) just below the aortic valve (ao) (B). As it moves posteriorly (C,D) expression band proceeds superiorly and penetrates the af, eventually terminating within it. In section 4 (E), the solid black line demarcates the area of the compact atrioventricular node (cavn) + inferior nodal extension (ine) and the solid white line demarcates the atrioventricular bundle (avb) + lower nodal cells (lnc) where reporter activity was present. (F) Post-natal hearts were also sectioned and counterstained with trichrome to visualize fibrous tissue (green). A close-up view of the area of bluo-gal is shown to contain abundant insulating collagen fibers (light blue), consistent with its identity as the avb. (G,H) Immunohistochemical analysis of an R2R3-LacZ section from an adult heart at the level of the avb (outlined, dashed line) showed mutual exclusion of β-gal and Cx43, but co-expression of β-gal and Cx40 in the avb. (I) A more posterior section at the level of the cavn (outlined, solid white) showed β-gal expression within the Cx40+ lnc domain (outlined, dashed line) that comprises the interface between the avb and the avn. Abbreviations: A, anterior; P, posterior; Cr, cranial; Ca, caudal; lv, left ventricle; ra, right atrium; rv, right ventricle; tv, tricuspid valve; mv, mitral valve.

Figure 5. Identification of minimal enhancer regions within R2R3 that direct reporter activity in the Hcn4 expression domain

(A) A deletion analysis of R2R3-LacZ revealed that only constructs bearing both deeply conserved sequences could direct robust reporter expression to the atrial and ventricular nonchamber myocardium at E11. (B) Representative 30 examples of E11 founder embryos from each construct is shown after bluo-gal staining and imaging of the heart in whole mount from the right side. A diagram is shown at the top left depicting the heart region. The pattern of full-length R2R3-LacZ is shown for comparison as construct 1. Of the deletion constructs tested that did not contain both conserved sequences (constructs 2–5), only construct 2 had some cardiac activity in atrial non-chamber myocardium in 2/5 embryos (D). In contrast, those with both conserved regions (constructs 6–8, bottom row) did preserve the activity of full length R2R3. The minimal enhancer capable of reconstituting the entire expression pattern of the full-length sequence, construct 8, consisted of the two conserved regulatory elements (light blue) without intervening sequence. Abbreviations: p, promoter; sv, sinus venosus; ra, right atrium; v, ventricle; oft, outflow tract; lb, limb bud; pa, pharyngeal arch; “++” denotes robust and consistent activity, “+” denotes inconsistent or lowlevel activity.

Figure 6. Mef2C regulates R2R3 Hcn4 enhancer activity via a conserved Mef2 Site

(A) Top, a conserved binding site for Mef2 was present within R3 as shown by an alignment between homologous regions of mouse, human, and opposum genomic DNA. Mismatches, shown in red, were not predicted to prevent Mef2 binding. Bottom, the Mef2 site in R3 was mutated at sites required for binding (mutations in red). (B) Transient R2R3-LacZ transgenic embryos bearing the Mef2 binding site mutation lost cardiac reporter activity. A founder bearing the unmutated R2R3 is shown for comparison (top left). Non-specific extracardiac ectopic activity confirmed transgene insertion into a permissive locus in the three founder embryos shown that bear the mutated Mef2 site in the context of R2R3.

Figure 7. Mef2C regulates R2R3-LacZ and Hcn4 expression in vivo

E11.5 embryos (A,C) and embryonic hearts (B,D) from Mef2C^+/−;R2R3-LacZ compound heterozygous mice after whole-mount bluo-gal staining. Mef2C^+/− embryos (C,D) had reduced cardiac staining. At E16.5, 31 the AV junction region of Mef2C^+/− hearts (G) had reduced reporter activity compared to WT (E). (F,H) show higher magnification of the AV junction region. Immunohistochemistry for Hcn4 at the AV junction showed a moderate reduction in Hcn4 expression in Mef2C^+/− embryos (J) compared to WT (I) at E16.5. The AV bundle is outlined (dashed line). Abreviations: avb, atrioventricular bundle; ra, right atrium; la, left atrium; rv, right ventricle; lv, left ventricle.

Figure 8. HDAC Activity Regulates Hcn4 R2R3

(A) MEF2C-VP16 was co-transfected with WT R3-Luc or MT R3-Luc. The Class II HDAC inhibitor MC1568 or dimethyl sulfoxide (DMSO) vehicle was added after transfection. Relative luciferase activity (RLA) was normalized to the value obtained with co-transfection of WT R3-Luc and empty pcDNA1 in the absence of MC1568. Means were compared with two-tailed t-tests (*, p<0.05; ***, p<0.001) (B,C) Wholemount bluo-gal staining of E11.5 R2R3-LacZ embryonic hearts that were cultured for 48 hours in 10 µM Trichostatin-A (TSA) or DMSO. (D,E). Immunhistochemistry for Hcn4 in cryosections of the left ventricle of TSA or DMSO exposed cultured E11.5 embryonic hearts. R2R3-LacZ embryonic hearts exposed to TSA showed an expansion of enhancer activity and increased ventricular Hcn4 expression.

Figure 9. Hcn4 R2R3-LacZ activity expands in response to ventricular pressure overload

Hcn4 R2R3-LacZ transgenic animals subjected to transverse aortic constriction (TAC) had 30% greater heart weight to body weight (HW/BW) ratios compared to sham-operated animals (A) and had induction of ANF expression as assessed by quantitative PCR on whole ventricle RNA (B). Whole-mount bluo-gal staining showed no expansion of enhancer activity beyond the AVB/LNC in the sham-operated animals (C,D), whereas animals subjected to TAC showed an increase in enhancer activity throughout the subendocardial myocardium of the left ventricle (lv) where pressure load is the greatest (D,E). By comparison, the right ventricle (rv), which is not pressure-loaded in this experiment, did not show any increase in enhancer activity. Each value 32 is expressed as mean with error bars denoting standard error of the mean. Means were compared with two-tailed t-tests (*, p<0.05; ***, p<0.001). Abreviations: ivs, interventricular septum; avb, atrioventricular bundle.