Homeobox Protein Hop Functions in the Adult Cardiac Conduction System
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循环研究杂志 2005年第4期
the Division of Cardiology (F.I.), Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, Pa
Division of Cardiovascular Medicine (M.Z., B.H., V.A.F., J.A.E., V.V.P.) and the Department of Radiology (R.Z.), University of Pennsylvania, Philadelphia, Pa
the Department of Pharmacology and Medical Research Center for Gene Regulation (H.K.), Chonnam National University Medical School, Gwangju, South Korea.
Abstract
Hop is an unusual homeobox gene expressed in the embryonic and adult heart. Hop acts downstream of Nkx2eC5 during development, and Nkx2eC5 mutations are associated with cardiac conduction system (CCS) defects. Inactivation of Hop in the mouse is lethal in half of the expected null embryos. Here, we show that Hop is expressed strongly in the adult CCS. HopeC/eC adult mice display conduction defects below the atrioventricular node (AVN) as determined by invasive electrophysiological testing. These defects are associated with decreased expression of connexin40. Our results suggest that Hop functions in the adult CCS and demonstrate conservation of molecular hierarchies between embryonic myocardium and the specialized conduction tissue of the mature heart.
Key Words: Hop conduction connexin40 Nkx2eC5 mouse mutants
Introduction
Synchronized contraction of the atrial and ventricular chambers is essential for normal cardiac function, and the cardiac conduction system (CCS) is required for mediating this delicate interplay. However, little is known about the molecular cascades regulating CCS development and function. Several studies suggest that certain components of the avian CCS are locally derived from bipotential cardiomyogenic cells.1 Endothelin, neuregulin-1, and Nkx2eC5 have each been implicated as contributors to the transition between ventricular and conductive cell lineages in animals.2eC5 Additionally, patients with mutations in Nkx2eC5 exhibit congenital heart defects and conduction system disease, notable for progressive atrioventricular block.6 Complete loss of Nkx2eC5 leads to loss of the atrioventricular node (AVN) anlage,5 and a mouse model in which Nxk2eC5 has been selectively removed from the ventricular myocardium demonstrates hypoplasia and progressive loss of cells from the AVN along with atrioventricular block.4
We and others recently identified Hop, a novel homeodomain protein in the embryonic heart functioning downstream of Nkx2eC5.7,8 Hop encodes a 73 amino acid protein that contains a domain (the 60 amino acid homeodomain) homologous to those seen in homeobox (Hox) transcription factors. Unlike all other known Hox transcription factors, Hop does not directly bind DNA.9 Hop is known to interact with serum response factor (SRF) and interfere with SRF-mediated gene transcription.7,8 Mice lacking Hop (HopeC/eC) are born at 50% of the expected frequency, with loss of embryos between E10.5 and E12.5.7 Hop expression also continues into the postnatal period and transgenic overexpression in adult myocardium leads to cardiac hypertrophy and heart failure.9 In this study, we present evidence that Hop is highly expressed in the adult mouse CCS and is required for proper function during adulthood.
Materials and Methods
Animals
Creation of Hop knockout mice (HopeC/eC) has been recently described.7 Mice were bred on a mixed C57BL/6 and 129sv background (Jackson Laboratories, Bar Harbor, Me). All protocols conformed to the guidelines established by the Association for the Assessment and Accreditation of Laboratory Animal Care and were approved by the University of Pennsylvania Animal Care and Use Committees.
-Galactosidase Activity Staining
Specimens were stained for -galactosidase activity using a previously described protocol,13 both for whole-mounted tissue and individual slides, as noted.
Acetylcholinesterase Activity Staining
Staining for acetylcholinesterase activity was performed using a previously described protocol.14
Connexing40 Protein Quantification
The superior portion of the interventricular septum was isolated from adult littermates (Hop+/+, Hop+/eC, and HopeC/eC). Western blots using anti-connexin40 antibody at 1 mcg/mL (Chemicon) and anti--tubulin antibody at 0.1 e/mL (Sigma) were quantified using ImageJ software (NIH).
Immunohistochemistry
Frozen sections of hearts from Hop+/eC and HopeC/eC mice were stained for connexin40. Hearts were embedded unfixed. Slides were air dried, fixed with 4% paraformaldehyde, washed with PBS, and briefly dehydrated through an ethanol series and then rehydrated before washes with PBST (PBS +0.5% Tween-20). They were then blocked with 10% goat serum in PBST, incubated with anti-Cx40 antibody 1:100 in 5% goat serum in 0.1%PBST, washed in PBS, and incubated with fluorescent secondary antibody at 1:500 (Molecular Probes).
Cardiac-Gated Magnetic Resonance Imaging
HopeC/eC and wild-type mice underwent cardiac-gated magnetic resonance imaging (MRI) to compare left ventricular morphology and function, as previously described.18 Left ventricular (LV) wall thickness, mass, and ejection fraction were measured in the short-axis planes.
EPS Preparation and Protocol
Surface ECG and invasive mouse electrophysiology studies were performed as previously described.15,16 Briefly, surface ECG recordings and complete in vivo electrophysiological studies (EPS) were obtained from HopeC/eC adult mice (10 to 14 weeks old) and age-matched Hop+/+ littermates. Each mouse was anesthetized with pentobarbital (0.033 mg/kg IP), and multi-lead ECGs were obtained using 26-gauge subcutaneous electrodes. A jugular vein cutdown was performed and an octapolar 2-French electrode catheter (CIBer mouse-EP; NuMED, Inc) placed in the right atrium and ventricle under electrogram guidance to confirm catheter position. A programmed digital stimulator (DTU-215A, Bloom Associates Ltd) was used to deliver electrical impulses at approximately twice diastolic threshold. Surface ECG and intracardiac electrograms were simultaneously displayed on a multi-channel oscilloscope recorder (Quinton Electrophysiology, Inc) at a digitization rate of 2 kHz and stored on optical media for offline analysis. ECG channels were filtered from 0.5 to 250 Hz and intracardiac electrograms were filtered from 5 to 400 Hz. ECG intervals were measured by two independent observers blinded to the animal’s genotype.
Ambulatory ECG Recordings
Ambulatory ECG recordings were obtained by aseptic, subcutaneous implantation of a 3.5-g wireless radiofrequency telemetry device (Data Sciences International) configured to record a signal analogous to ECG lead I as previously described.17 Following a 72-hour recovery period after surgical instrumentation, ECG recordings were obtained from HopeC/eC and Hop+/+ mice, each placed in a separate cage overlying a receiver.
CCS Volume Calculations
Digital photomicrographs of all sections through the AVN and proximal CCS were obtained with an Axiophot2 microscope (Carl Zeiss) and Nikon DXM1200 digital camera after staining for -galactosidase activity. The structural regions of the CCS were defined according to the method of Rentschler et al10 where stained sections above the bifurcation of the bundle branches, encompassed by atrial tissues, were taken as the AVN and stained tissues distal to the AVN, but proximal to the division of the bundle branches, were taken as the His-bundle. Measurement of the area of interest (the AVN proper and His-bundle) was performed with ImageJ software. The sum of the areas of interest multiplied by the section thickness (6 to 8 e) was used to compute the estimated volume.
Quantitative RT-PCR
Quantitative RT-PCR was performed using SYBR Green according to the manufacturer’s protocol. Total RNA was isolated from whole hearts for real-time RT-PCR with TRIzol (Invitrogen, Inc). PCR was preformed with 5 e蘈 of reverse transcribed cDNA reaction mixture, 400 nmol/L of specific forward and reverse primers and 1xSYBR Green PCR Master Mix (Applied Biosystems, Inc.). The following primer pairs were used: (1) connexin40 (Cx40), 5'-ACAAGCACT-CCACAGTCATCGGC-3' and 5'-AGCAGGACAGCTCGGCCTT-TTC-3'; (2) connexin43 (Cx43), 5'-CCTTGGGGAAGCTGCTGG-ACAA-3' and 5'-CAGCAGGCCACCTCTCATCTTCA-3'; (3) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-GTGGCAAAGTGGATTGTTGCC-3' and 5'-GATGATGACCCG-TTTGGCTCC-3'. Quantification of the reaction product was performed using the MJ Research DNA Engine Opticon 2 real-time detection system. PCR cycle conditions were 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing and extension at 60°C for 1 minute. All reactions were performed in triplicate with and without RT as controls. Cycle threshold values were converted to relative gene expression levels using the 2eCC(t) method.
Statistical Analysis
Continuous variables, such as ECG intervals and cardiac conduction properties, were compared using a two-tailed Student t test. All values are reported as the mean±1SD, unless otherwise noted. A probability value <0.05 was considered significant.
Results
Hop Expression in the Adult Murine Heart
During embryonic development, Hop is strongly expressed throughout the developing heart.7,8 Insertion of the lacZ gene into the endogenous Hop locus allowed us to detect sites of expression with high sensitivity. Gross examination of hearts from adult HopeC/eC and Hop+/eC mice stained for -galactosidase activity demonstrated strong expression within the cardiac conduction system (Figure 1A through 1F). This pattern of expression appears to overlap with that of CCS-LacZ10 and MinK-LacZ mice,11 which each demarcate regions of the CCS. This CCS-delimited staining pattern was less prominent in newborn mice (Figure 1G and 1H) where there was more prominent staining throughout the myocardium as compared with adults, but more restricted to the endocardium and interventricular septum than during embryogenesis. Additionally, uniform expression of -galactosidase activity was detected in the atria of Hop+/eC and HopeC/eC hearts (Figure 1I and 1J). Staining of adjacent sections with -galactosidase and for AchE activity confirmed expression of Hop within the proximal CCS (Figure 1K through 1M). The hearts of adult HopeC/eC mice were structurally normal and had normal function as assessed by invasive hemodynamic monitoring9 and cardiac-gated MRI (Table 1). Quantitative morphometric analysis of proximal CCS volume in HopeC/eC and Hop+/eC hearts suggested that proximal CCS tissue integrity was maintained in null animals (Table 2).
ECG and Electrophysiologic Analysis of HopeC/eC Mice
With our initial anatomical observations, we sought to determine whether Hop played a functional role in cardiac conduction. ECG analysis revealed a wider QRS-complex, longer QT-interval, and wider p-wave in HopeC/eC mice compared with wild-type mice (Figure 2A through 2D). Resting heart rates and baseline PR-intervals were similar between HopeC/eC and wild-type mice. In addition, ECG recordings in 5 of 10 HopeC/eC mice showed right axis deviation that was not present in any wild-type littermates. Similar findings were seen in unsedated, ambulatory ECG recordings (Table 3) with no evidence of spontaneous arrhythmias in either group.
Intracardiac EPS revealed a longer HV interval in HopeC/eC mice compared with wild-type controls, whereas the AH interval trended toward being longer in HopeC/eC mice but did not reach statistical significance. These results suggest that conduction delay accounting for the widened QRS complex on surface ECGs is due predominantly to defective conduction below the AVN and is consistent with strong expression of Hop in the infra-His region and bundle branches. In addition, the atrial effective refractory period (AERP) was prolonged in HopeC/eC mice (Table 4).
Connexin40 Expression Is Reduced in Adult HopeC/eC Mice
Because cardiac connexin expression is dependent on proper expression of Nkx2eC5 in the heart and Hop functions downstream of Nkx2eC5, we sought to determine whether deletion of Hop might also affect expression of cardiac connexins. RT-PCR analysis showed a significant reduction in Cx40/GAPDH normalized mRNA expression in hearts from HopeC/eC mice versus wild-type littermates (1.64±0.06 versus 2.31±0.13; P<0.01). However, Cx43/GAPDH normalized mRNA was not different in the hearts of HopeC/eC mice compared with littermate controls and similar results were obtained from microarray analysis (data not shown). Further, there was a reduction of connexin40 protein in the proximal CCS of HopeC/eC mice, whereas there was no difference between wild-type and Hop+/eC mice (Figure 3A and 3B). In the atria, we also found reduced connexin40 in HopeC/eC compared with Hop+/eC mice (Figure 3E and 3H). Further, we examined connexin40 protein expression by immunohistochemistry in Hop+/eC and HopeC/eC littermates. Areas of intense -galactosidase activity in adjacent sections were observed in the region of the AVN and proximal CCS, but connexin40 expression was markedly downregulated in HopeC/eC as compared with Hop+/eC mice in all observed sections through the CCS. This difference was most dramatic in the more distal portions of the CCS (Figure 3D and 3G).
Discussion
Disease of the CCS is common and can result from congenital abnormalities or can be acquired later in life. As such, a better understanding of the basic biology and molecular pathways underlying cardiac conduction tissue development, maintenance, and function may have significant impact on the clinical care of patients with conduction disorders.
In this report, we show that Hop is highly expressed in the adult CCS of mice and modulates cardiac conduction system function. Hop is a transcriptional target of Nkx2eC5, which is also highly expressed in the CCS12 and is required for normal development and maintenance of adult cardiac conduction tissue.4,5 Mutations in Nkx2eC5 are associated with proximal conduction system defects in humans and with congenital heart disease.6 Tissue-specific inactivation of Nkx2eC5 in adult mouse hearts leads to progressive loss of conduction tissue and downregulation of Hop.4 Our data suggest that Hop is robustly expressed in the adult CCS. However, unlike Nkx2eC5, Hop does not appear necessary for maintenance or survival of conduction tissue, because the size and integrity of the AVN and His-bundle were normal in HopeC/eC mice.
Although both Nkx2eC5 and Hop mutant mice display conduction system disease, the precise location of conduction delay appears different. Although the AH interval was longer in HopeC/eC mice compared with Hop+/+ mice, albeit not significantly, the PR intervals between the two groups were almost identical. This suggests no significant alteration in AVN conduction in the absence of Hop. On the other hand, the QRS duration was significantly different between these two groups, consistent with the longer HV intervals we observed in HopeC/eC mice. Therefore, although it has been shown that Nkx2eC5 mutant mice display predominantly proximal conduction defects,4,5 Hop mutant mice appear to have more distal, infrahisian delay. Our electrophysiological observations are consistent with the fact that connexin40 expression is lower, but still present, in the AVN of HopeC/eC mice and absent from the infrahisian conduction system. These findings are distinct from those found in the absence of Nkx2eC5, where there is hypoplasia of the CCS with loss of specific connexin expressing cells, but preservation of overall connexin40 density in the remnant CCS.5 This may reflect a particular sensitivity of the conduction tissue just below the His-bundle to Hop function or to loss of connexin40 expression.19
In addition, atrial electrophysiological defects (increased p-wave duration and prolonged AERP) were observed in the absence of Hop. Our observed downregulation of connexin40, the major connexin in the atrium, likely contributes to slowed atrial conduction but not to increased refractoriness. Increased refractoriness is predominantly a function of increased action potential duration, which is determined largely by transmembrane ionic fluxes. We are currently investigating the role of Hop in regulating transmembrane ionic currents in the heart. The electrophysiological defects reported in this study are not likely to be secondary to structural heart disease because HopeC/eC mice have normal hemodynamics, as assessed by invasive analysis,9 and have normal cardiac structure and function as assessed by cardiac-gated MRI.
Taken together, these results support the existence of a molecular hierarchy in which Nkx2eC5 functions upstream of Hop with conservation of this molecular program between embryonic myocardium and adult conduction tissue. Hop is likely to mediate some, but not all, downstream functions of Nkx2eC5 in cardiac conduction tissues and suggests that individual segments of the adult CCS are regulated by separate molecular programs. Further characterization of the molecular programs involved with CCS function and maintenance should lead to improved diagnosis and therapy of conduction system disease.
Acknowledgments
This work was supported by grants from the NIH (J.A.E. and V.V.P.). H.K. was supported, in part, by the Korea Science & Engineering Foundation through the Medical Research Center for Gene Regulation at Chonnam National University. We thank Sandy Cho for her assistance with the gated-MRI studies.
References
Cheng G, Litchenberg WH, Cole GJ, Mikawa T, Thompson RP, Gourdie RG. Development of the cardiac conduction system involves recruitment within a multipotent cardiomyogenic lineage. Development. 1999; 126: 5041eC5049.
Gourdie RG, Wei Y, Kim D, Klatt SC, Mikawa T. Endothelin-induced conversion of embryonic heart muscle cells into impulse-conducting Purkinje fibers. Proc Natl Acad Sci U S A. 1998; 95: 6815eC6818.
Rentschler S, Zander J, Meyers K, France D, Levine R, Porter G, Rivkees SA, Morley GE, Fishman GI. Neurogulin-1 promotes formation of the murine cardiac conduction system. Proc Natl Acad Sci U S A. 2002; 99: 10464eC10469.
Pashmforoush M, Lu JT, Chen H, Amand TS, Kondo R, Pradervand S, Evans SM, Clark B, Feramisco JR, Giles WR, Ho SY, Benson DW, Siberbach M, Shou W, Chien KR. Nkx2eC5 pathways and congenital heart disease: loss of ventricular myocyte lineage specification leads to progressive cardiomyopathy and complete heart block. Cell. 2004; 117: 378eC386.
Jay PY, Harris BS, Maguire CT, Buerger A, Wakimoto H, Tanaka M, Kupershmidt S, Roden DM, Schultheiss TM, O’Brien TX, Gourdie RG, Berul CI, Izumo S. Nkx2eC5 mutation causes anatomic hypoplasia of the cardiac conduction system. J Clin Invest. 2004; 113: 1130eC1137.
Schott JJ, Benson DW, Basson CT, Pease W, Silberbach GM, Moak JP, Maron BJ, Seidman CE, Seidman JG. Congenital heart disease caused by mutations in the transcription factor NKX2eC5. Science. 1998; 281: 108eC111.
Chen F, Kook H, Milewski R, Gitler AD, Lu MM, Li J, Nazarian R, Schnepp R, Jen K, Biben C, Runke G, Mackay JP, Novotny J, Schwartz RJ, Harvey RP, Mullins MC, Epstein JA. Hop is an unusual homeobox gene that modulates cardiac development. Cell. 2002; 110: 713eC723.
Shin CH, Liu ZP, Passier R, Zhang CL, Wang DZ, Harris TM, Yamagishi H, Richardson JA, Childs G, Olson EN. Modulation of cardiac growth and development by HOP, an unusual homeodomain protein. Cell. 2002; 110: 725eC735.
Kook H, Lepore JJ, Gitler AD, Lu MM, Wing-Man Yung W, Mackay J, Zhou R, Ferrari V, Gruber P, Epstein JA. Cardiac hypertrophy and histone deacetylase-dependent transcriptional repression mediated by the atypical homeodomain protein Hop. J Clin Invest. 2003; 112: 863eC871.
Rentschler S, Vaidya DM, Tamaddon H, Degenhardt K, Sassoon D, Morley GE, Jalife J, Fishman GI. Visualization and functional characterization of the developing murine cardiac conduction system. Development. 2001; 128: 1785eC1792.
Kupershmidt S, Yang T, Anderson ME, Wessels A, Niswender KD, Magnuson MA, Roden DM. Replacement by homologous recombination of the minK gene with lacZ reveals restriction of minK expression to the mouse cardiac conduction system. Circ Res. 1999; 84: 146eC152.
Thomas PS, Kasahara H, Edmonson AM, Izumo S, Yacoub MH, Barton PJ, Gourdie RG. Elevated expression of Nkx-2.5 in developing myocardial conduction cells. Anat Rec. 2001; 263: 307eC313.
Brown CB, Feiner L, Lu MM, Li J, Ma X, Webber AL, Jia L, Raper JA, Epstein JA. PlexinA2 and semaphorin signaling during cardiac neural crest development. Development. 2001; 128: 3071eC3080.
El-Badawi A, Schenk EA. Histochemical methods for separate, consecutive and simultaneous demonstration of acetylcholinesterase and norepinephrine in cryostat sections. J Histochem Cytochem. 1967; 15: 580eC588.
Berul CI, Aronovitz MJ, Wang PJ, Mendelsohn ME. In vivo cardiac electrophysiology studies in the mouse. Circulation. 1996; 94: 2641eC2648.
Patel VV, Arad M, Moskowitz IPG, Maguire CT, Branco D, Seidman JG, Seidman CE, Berul CI. Electrophysiological characterization and postnatal development of ventricular preexcitation in a mouse familial WPW model. J Am Coll Cardiol. 2003; 42: 942eC951.
Kramer K, van Acker SA, Voss HP, Grimbergen JA, van der Vijgh WJ, Bast A. Use of telemetry to record electrocardiogram and heart rate in freely moving mice. J Pharmacol Toxicol Methods. 1993; 30: 209eC215.
Zhou R, Pickup S, Glickson J, Scott C, Ferarri VA. Assessment of global and regional myocardial function in the mouse using cine and tagged MRI. Magn Reson Med. 2003; 49: 760eC764.
VanderBrink BA, Sellitto C, Saba S, Link MS, Zhu W, Homoud MK, Estes NA3rd, Paul DL, Wang PJ. Connexin40-deficient mice exhibit atrioventricular nodal and infra-Hisian conduction abnormalities. J Cardiovasc Electrophysiol. 2000; 11: 1270eC1276.(Fraz A. Ismat, Maozhen Zh)
Division of Cardiovascular Medicine (M.Z., B.H., V.A.F., J.A.E., V.V.P.) and the Department of Radiology (R.Z.), University of Pennsylvania, Philadelphia, Pa
the Department of Pharmacology and Medical Research Center for Gene Regulation (H.K.), Chonnam National University Medical School, Gwangju, South Korea.
Abstract
Hop is an unusual homeobox gene expressed in the embryonic and adult heart. Hop acts downstream of Nkx2eC5 during development, and Nkx2eC5 mutations are associated with cardiac conduction system (CCS) defects. Inactivation of Hop in the mouse is lethal in half of the expected null embryos. Here, we show that Hop is expressed strongly in the adult CCS. HopeC/eC adult mice display conduction defects below the atrioventricular node (AVN) as determined by invasive electrophysiological testing. These defects are associated with decreased expression of connexin40. Our results suggest that Hop functions in the adult CCS and demonstrate conservation of molecular hierarchies between embryonic myocardium and the specialized conduction tissue of the mature heart.
Key Words: Hop conduction connexin40 Nkx2eC5 mouse mutants
Introduction
Synchronized contraction of the atrial and ventricular chambers is essential for normal cardiac function, and the cardiac conduction system (CCS) is required for mediating this delicate interplay. However, little is known about the molecular cascades regulating CCS development and function. Several studies suggest that certain components of the avian CCS are locally derived from bipotential cardiomyogenic cells.1 Endothelin, neuregulin-1, and Nkx2eC5 have each been implicated as contributors to the transition between ventricular and conductive cell lineages in animals.2eC5 Additionally, patients with mutations in Nkx2eC5 exhibit congenital heart defects and conduction system disease, notable for progressive atrioventricular block.6 Complete loss of Nkx2eC5 leads to loss of the atrioventricular node (AVN) anlage,5 and a mouse model in which Nxk2eC5 has been selectively removed from the ventricular myocardium demonstrates hypoplasia and progressive loss of cells from the AVN along with atrioventricular block.4
We and others recently identified Hop, a novel homeodomain protein in the embryonic heart functioning downstream of Nkx2eC5.7,8 Hop encodes a 73 amino acid protein that contains a domain (the 60 amino acid homeodomain) homologous to those seen in homeobox (Hox) transcription factors. Unlike all other known Hox transcription factors, Hop does not directly bind DNA.9 Hop is known to interact with serum response factor (SRF) and interfere with SRF-mediated gene transcription.7,8 Mice lacking Hop (HopeC/eC) are born at 50% of the expected frequency, with loss of embryos between E10.5 and E12.5.7 Hop expression also continues into the postnatal period and transgenic overexpression in adult myocardium leads to cardiac hypertrophy and heart failure.9 In this study, we present evidence that Hop is highly expressed in the adult mouse CCS and is required for proper function during adulthood.
Materials and Methods
Animals
Creation of Hop knockout mice (HopeC/eC) has been recently described.7 Mice were bred on a mixed C57BL/6 and 129sv background (Jackson Laboratories, Bar Harbor, Me). All protocols conformed to the guidelines established by the Association for the Assessment and Accreditation of Laboratory Animal Care and were approved by the University of Pennsylvania Animal Care and Use Committees.
-Galactosidase Activity Staining
Specimens were stained for -galactosidase activity using a previously described protocol,13 both for whole-mounted tissue and individual slides, as noted.
Acetylcholinesterase Activity Staining
Staining for acetylcholinesterase activity was performed using a previously described protocol.14
Connexing40 Protein Quantification
The superior portion of the interventricular septum was isolated from adult littermates (Hop+/+, Hop+/eC, and HopeC/eC). Western blots using anti-connexin40 antibody at 1 mcg/mL (Chemicon) and anti--tubulin antibody at 0.1 e/mL (Sigma) were quantified using ImageJ software (NIH).
Immunohistochemistry
Frozen sections of hearts from Hop+/eC and HopeC/eC mice were stained for connexin40. Hearts were embedded unfixed. Slides were air dried, fixed with 4% paraformaldehyde, washed with PBS, and briefly dehydrated through an ethanol series and then rehydrated before washes with PBST (PBS +0.5% Tween-20). They were then blocked with 10% goat serum in PBST, incubated with anti-Cx40 antibody 1:100 in 5% goat serum in 0.1%PBST, washed in PBS, and incubated with fluorescent secondary antibody at 1:500 (Molecular Probes).
Cardiac-Gated Magnetic Resonance Imaging
HopeC/eC and wild-type mice underwent cardiac-gated magnetic resonance imaging (MRI) to compare left ventricular morphology and function, as previously described.18 Left ventricular (LV) wall thickness, mass, and ejection fraction were measured in the short-axis planes.
EPS Preparation and Protocol
Surface ECG and invasive mouse electrophysiology studies were performed as previously described.15,16 Briefly, surface ECG recordings and complete in vivo electrophysiological studies (EPS) were obtained from HopeC/eC adult mice (10 to 14 weeks old) and age-matched Hop+/+ littermates. Each mouse was anesthetized with pentobarbital (0.033 mg/kg IP), and multi-lead ECGs were obtained using 26-gauge subcutaneous electrodes. A jugular vein cutdown was performed and an octapolar 2-French electrode catheter (CIBer mouse-EP; NuMED, Inc) placed in the right atrium and ventricle under electrogram guidance to confirm catheter position. A programmed digital stimulator (DTU-215A, Bloom Associates Ltd) was used to deliver electrical impulses at approximately twice diastolic threshold. Surface ECG and intracardiac electrograms were simultaneously displayed on a multi-channel oscilloscope recorder (Quinton Electrophysiology, Inc) at a digitization rate of 2 kHz and stored on optical media for offline analysis. ECG channels were filtered from 0.5 to 250 Hz and intracardiac electrograms were filtered from 5 to 400 Hz. ECG intervals were measured by two independent observers blinded to the animal’s genotype.
Ambulatory ECG Recordings
Ambulatory ECG recordings were obtained by aseptic, subcutaneous implantation of a 3.5-g wireless radiofrequency telemetry device (Data Sciences International) configured to record a signal analogous to ECG lead I as previously described.17 Following a 72-hour recovery period after surgical instrumentation, ECG recordings were obtained from HopeC/eC and Hop+/+ mice, each placed in a separate cage overlying a receiver.
CCS Volume Calculations
Digital photomicrographs of all sections through the AVN and proximal CCS were obtained with an Axiophot2 microscope (Carl Zeiss) and Nikon DXM1200 digital camera after staining for -galactosidase activity. The structural regions of the CCS were defined according to the method of Rentschler et al10 where stained sections above the bifurcation of the bundle branches, encompassed by atrial tissues, were taken as the AVN and stained tissues distal to the AVN, but proximal to the division of the bundle branches, were taken as the His-bundle. Measurement of the area of interest (the AVN proper and His-bundle) was performed with ImageJ software. The sum of the areas of interest multiplied by the section thickness (6 to 8 e) was used to compute the estimated volume.
Quantitative RT-PCR
Quantitative RT-PCR was performed using SYBR Green according to the manufacturer’s protocol. Total RNA was isolated from whole hearts for real-time RT-PCR with TRIzol (Invitrogen, Inc). PCR was preformed with 5 e蘈 of reverse transcribed cDNA reaction mixture, 400 nmol/L of specific forward and reverse primers and 1xSYBR Green PCR Master Mix (Applied Biosystems, Inc.). The following primer pairs were used: (1) connexin40 (Cx40), 5'-ACAAGCACT-CCACAGTCATCGGC-3' and 5'-AGCAGGACAGCTCGGCCTT-TTC-3'; (2) connexin43 (Cx43), 5'-CCTTGGGGAAGCTGCTGG-ACAA-3' and 5'-CAGCAGGCCACCTCTCATCTTCA-3'; (3) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-GTGGCAAAGTGGATTGTTGCC-3' and 5'-GATGATGACCCG-TTTGGCTCC-3'. Quantification of the reaction product was performed using the MJ Research DNA Engine Opticon 2 real-time detection system. PCR cycle conditions were 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing and extension at 60°C for 1 minute. All reactions were performed in triplicate with and without RT as controls. Cycle threshold values were converted to relative gene expression levels using the 2eCC(t) method.
Statistical Analysis
Continuous variables, such as ECG intervals and cardiac conduction properties, were compared using a two-tailed Student t test. All values are reported as the mean±1SD, unless otherwise noted. A probability value <0.05 was considered significant.
Results
Hop Expression in the Adult Murine Heart
During embryonic development, Hop is strongly expressed throughout the developing heart.7,8 Insertion of the lacZ gene into the endogenous Hop locus allowed us to detect sites of expression with high sensitivity. Gross examination of hearts from adult HopeC/eC and Hop+/eC mice stained for -galactosidase activity demonstrated strong expression within the cardiac conduction system (Figure 1A through 1F). This pattern of expression appears to overlap with that of CCS-LacZ10 and MinK-LacZ mice,11 which each demarcate regions of the CCS. This CCS-delimited staining pattern was less prominent in newborn mice (Figure 1G and 1H) where there was more prominent staining throughout the myocardium as compared with adults, but more restricted to the endocardium and interventricular septum than during embryogenesis. Additionally, uniform expression of -galactosidase activity was detected in the atria of Hop+/eC and HopeC/eC hearts (Figure 1I and 1J). Staining of adjacent sections with -galactosidase and for AchE activity confirmed expression of Hop within the proximal CCS (Figure 1K through 1M). The hearts of adult HopeC/eC mice were structurally normal and had normal function as assessed by invasive hemodynamic monitoring9 and cardiac-gated MRI (Table 1). Quantitative morphometric analysis of proximal CCS volume in HopeC/eC and Hop+/eC hearts suggested that proximal CCS tissue integrity was maintained in null animals (Table 2).
ECG and Electrophysiologic Analysis of HopeC/eC Mice
With our initial anatomical observations, we sought to determine whether Hop played a functional role in cardiac conduction. ECG analysis revealed a wider QRS-complex, longer QT-interval, and wider p-wave in HopeC/eC mice compared with wild-type mice (Figure 2A through 2D). Resting heart rates and baseline PR-intervals were similar between HopeC/eC and wild-type mice. In addition, ECG recordings in 5 of 10 HopeC/eC mice showed right axis deviation that was not present in any wild-type littermates. Similar findings were seen in unsedated, ambulatory ECG recordings (Table 3) with no evidence of spontaneous arrhythmias in either group.
Intracardiac EPS revealed a longer HV interval in HopeC/eC mice compared with wild-type controls, whereas the AH interval trended toward being longer in HopeC/eC mice but did not reach statistical significance. These results suggest that conduction delay accounting for the widened QRS complex on surface ECGs is due predominantly to defective conduction below the AVN and is consistent with strong expression of Hop in the infra-His region and bundle branches. In addition, the atrial effective refractory period (AERP) was prolonged in HopeC/eC mice (Table 4).
Connexin40 Expression Is Reduced in Adult HopeC/eC Mice
Because cardiac connexin expression is dependent on proper expression of Nkx2eC5 in the heart and Hop functions downstream of Nkx2eC5, we sought to determine whether deletion of Hop might also affect expression of cardiac connexins. RT-PCR analysis showed a significant reduction in Cx40/GAPDH normalized mRNA expression in hearts from HopeC/eC mice versus wild-type littermates (1.64±0.06 versus 2.31±0.13; P<0.01). However, Cx43/GAPDH normalized mRNA was not different in the hearts of HopeC/eC mice compared with littermate controls and similar results were obtained from microarray analysis (data not shown). Further, there was a reduction of connexin40 protein in the proximal CCS of HopeC/eC mice, whereas there was no difference between wild-type and Hop+/eC mice (Figure 3A and 3B). In the atria, we also found reduced connexin40 in HopeC/eC compared with Hop+/eC mice (Figure 3E and 3H). Further, we examined connexin40 protein expression by immunohistochemistry in Hop+/eC and HopeC/eC littermates. Areas of intense -galactosidase activity in adjacent sections were observed in the region of the AVN and proximal CCS, but connexin40 expression was markedly downregulated in HopeC/eC as compared with Hop+/eC mice in all observed sections through the CCS. This difference was most dramatic in the more distal portions of the CCS (Figure 3D and 3G).
Discussion
Disease of the CCS is common and can result from congenital abnormalities or can be acquired later in life. As such, a better understanding of the basic biology and molecular pathways underlying cardiac conduction tissue development, maintenance, and function may have significant impact on the clinical care of patients with conduction disorders.
In this report, we show that Hop is highly expressed in the adult CCS of mice and modulates cardiac conduction system function. Hop is a transcriptional target of Nkx2eC5, which is also highly expressed in the CCS12 and is required for normal development and maintenance of adult cardiac conduction tissue.4,5 Mutations in Nkx2eC5 are associated with proximal conduction system defects in humans and with congenital heart disease.6 Tissue-specific inactivation of Nkx2eC5 in adult mouse hearts leads to progressive loss of conduction tissue and downregulation of Hop.4 Our data suggest that Hop is robustly expressed in the adult CCS. However, unlike Nkx2eC5, Hop does not appear necessary for maintenance or survival of conduction tissue, because the size and integrity of the AVN and His-bundle were normal in HopeC/eC mice.
Although both Nkx2eC5 and Hop mutant mice display conduction system disease, the precise location of conduction delay appears different. Although the AH interval was longer in HopeC/eC mice compared with Hop+/+ mice, albeit not significantly, the PR intervals between the two groups were almost identical. This suggests no significant alteration in AVN conduction in the absence of Hop. On the other hand, the QRS duration was significantly different between these two groups, consistent with the longer HV intervals we observed in HopeC/eC mice. Therefore, although it has been shown that Nkx2eC5 mutant mice display predominantly proximal conduction defects,4,5 Hop mutant mice appear to have more distal, infrahisian delay. Our electrophysiological observations are consistent with the fact that connexin40 expression is lower, but still present, in the AVN of HopeC/eC mice and absent from the infrahisian conduction system. These findings are distinct from those found in the absence of Nkx2eC5, where there is hypoplasia of the CCS with loss of specific connexin expressing cells, but preservation of overall connexin40 density in the remnant CCS.5 This may reflect a particular sensitivity of the conduction tissue just below the His-bundle to Hop function or to loss of connexin40 expression.19
In addition, atrial electrophysiological defects (increased p-wave duration and prolonged AERP) were observed in the absence of Hop. Our observed downregulation of connexin40, the major connexin in the atrium, likely contributes to slowed atrial conduction but not to increased refractoriness. Increased refractoriness is predominantly a function of increased action potential duration, which is determined largely by transmembrane ionic fluxes. We are currently investigating the role of Hop in regulating transmembrane ionic currents in the heart. The electrophysiological defects reported in this study are not likely to be secondary to structural heart disease because HopeC/eC mice have normal hemodynamics, as assessed by invasive analysis,9 and have normal cardiac structure and function as assessed by cardiac-gated MRI.
Taken together, these results support the existence of a molecular hierarchy in which Nkx2eC5 functions upstream of Hop with conservation of this molecular program between embryonic myocardium and adult conduction tissue. Hop is likely to mediate some, but not all, downstream functions of Nkx2eC5 in cardiac conduction tissues and suggests that individual segments of the adult CCS are regulated by separate molecular programs. Further characterization of the molecular programs involved with CCS function and maintenance should lead to improved diagnosis and therapy of conduction system disease.
Acknowledgments
This work was supported by grants from the NIH (J.A.E. and V.V.P.). H.K. was supported, in part, by the Korea Science & Engineering Foundation through the Medical Research Center for Gene Regulation at Chonnam National University. We thank Sandy Cho for her assistance with the gated-MRI studies.
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