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Circulation Research. 1997;80:665-672

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(Circulation Research. 1997;80:665-672.)
© 1997 American Heart Association, Inc.

Articles

Skeletal Muscle–Specific Myosin Binding Protein-H Is Expressed in Purkinje Fibers of the Cardiac Conduction System

Tatiana Alyonycheva, Leona Cohen-Gould, Christiana Siewert, Donald A. Fischman, , Takashi Mikawa

From the Department of Cell Biology and Anatomy, Cornell University Medical College, New York, NY.

Correspondence to Dr Takashi Mikawa, Department of Cell Biology and Anatomy, Cornell University Medical College, 1300 York Ave, New York, NY 10021. E-mail tmikaw{at}mail.med.cornell.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract Heart contraction is coordinated by conduction of electrical excitation through specialized tissues of the cardiac conduction system. By retroviral single-cell tagging and lineage analyses in the embryonic chicken heart, we have recently demonstrated that a subset of cardiac muscle cells terminally differentiates as cells of the peripheral conduction system (Purkinje fibers) and that this occurs invariably in perivascular regions of developing coronary arteries. Cis regulatory elements that function in transcriptional regulation of cells in the conducting system have been distinguished from those in contractile cardiac muscle cells; eg, 5' regulatory sequences of the desmin gene act as enhancer elements in skeletal muscle and in the conduction system but not in cardiac muscle. We hypothesize that Purkinje fiber differentiation involves a switch of the gene expression program from that characteristic of cardiac muscle to one typical of skeletal muscle. To test this hypothesis, we examined the expression of myosin binding protein-H (MyBP-H) in Purkinje fibers of chicken hearts. This unique myosin binding protein is present in skeletal but not cardiac myocytes. A site-directed polyclonal antibody (AB105) was generated against MyBP-H. Immunohistological analysis of the myocardium mapped the AB105 antigen predominantly to A bands of myofibrils within Purkinje fibers. Western blot analysis of whole extracts from the ventricular wall of adult chicken hearts revealed that the AB105 epitope was restricted to a single protein of {approx}86 kD, the same size as MyBP-H in skeletal muscle. Biochemical properties of the Purkinje fiber 86-kD protein and RNase protection analyses of its mRNA indicate that Purkinje fiber 86-kD protein is indistinguishable from skeletal muscle MyBP-H. The results provide evidence that skeletal muscle MyBP-H is expressed in a subset of cardiac muscle cells that differentiate into Purkinje fibers of the heart.


Key Words: Purkinje fiber • heart conduction system • myosin binding protein-H • myofibril


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Rhythmic contraction of the avian and mammalian heart is coordinated by conduction of electrical excitation through a well-defined tissue, the cardiac conduction system.1 An action potential generated at the sinoatrial node is first guided across the atrial chambers to the atrioventricular node and propagated along the atrioventricular bundle, finally spreading into ventricular muscle via the Purkinje fiber network. Purkinje fibers play a crucial role in transmitting electrical impulses, first to the apex of the heart and secondarily to the remainder of the ventricular myocardium. This ensures synchronous apical-to-basal ejection of blood from both ventricular chambers. In the avian heart, Purkinje fibers are identified as a cellular network in close association with the coronary arterial bed2 and are distinguished from cardiac muscle cells by a characteristic pattern of gene expression.3

Little is known about the molecular mechanisms that regulate the differentiation of this essential tissue, and this has in part been related to controversy about a myogenic or neurogenic origin.3 Using retroviral cell-tagging procedures,4 5 6 7 8 9 we have shown that single contractile myocytes in the tubular heart10 11 12 generate a series of daughter cells, which form a cone-shaped colony traversing the myocardial wall later in development.5 6 Subsequent analyses of cell populations within individual myogenic clones have revealed that a subset of contractile cardiomyocytes differentiate into conducting cells invariably at the perivascular regions of developing coronary arteries.4 These results have provided evidence that direct conversion of contractile myocytes into conducting cells is the mechanism of Purkinje fiber differentiation.

This conversion appears to include changes in the regulation of myofibrillar protein expression: (1) cardiac muscle–specific troponin is absent from the cardiac conduction system13 ; (2) myosin heavy chain characteristic of slow skeletal muscle is found in the A bands of Purkinje fiber myofibrils but not in the working myocytes14 15 ; and (3) a skeletal muscle enhancer element of the desmin gene functions in the cardiac conduction system but not in contractile myocytes.16 Together with the cell lineage data, these studies suggest that Purkinje fiber differentiation involves a switch of myofibrillar protein gene expression from that characteristic of cardiomyocytes to that of skeletal muscle. A clear test of this hypothesis requires a gene that is exclusively expressed in skeletal muscle cells. Unfortunately, most muscle proteins are transiently expressed in both skeletal and cardiac muscle during embryonic stages.17 An important exception is MyBP-H,18 19 20 a member of the MyBP gene family.21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 In contrast to other myofibrillar proteins, the single isoform of MyBP-H, encoded by a single gene, is expressed in skeletal muscle, but the protein appears to be absent from adult cardiac muscle.18 20 25 37

In the present study, we examined the expression of MyBP-H within the peripheral conduction system of the avian heart to determine if expression correlated with skeletal muscle–type myosin. A site-directed polyclonal antibody specific for MyBP-H was used to examine the expression and localization of this protein in embryonic and adult chicken hearts. Within the ventricular myocardium, a single protein of {approx}86 kD, the same size as MyBP-H, was identified in the A bands of Purkinje fiber myofibrils; no reactive antigen was detected in ventricular myocytes. Partial purification of the cardiac 86-kD protein and RNase protection assays of its mRNA revealed that the AB105-reactive protein was indistinguishable from skeletal muscle MyBP-H. These results provide the first evidence for MyBP-H expression in the heart and further suggest that myofibrillar proteins previously thought to be restricted to skeletal muscle are expressed in a subset of cardiac muscle cells that differentiate into conducting Purkinje fibers.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Antibodies
Monoclonal antibodies used in the present study were as follows: ALD58, specific for slow skeletal muscle MyHC38 ; MF1, reactive with fast skeletal muscle MyBP-C31 ; and C315, specific for cardiomyocyte MyBP-C.36 A site-directed polyclonal antibody to the peptide Lys41-Pro55 of adult chicken MyBP-H was generated as follows: from the amino acid sequences of MyBP-H of chicken and human skeletal muscle, deduced from cDNA sequence data,20 25 a peptide (Lys41-Pro55, 1630 D) was chemically synthesized (Multiple Peptide Systems). Approximately 5 mg of purified peptide was coupled through the terminal cysteine thiol to KLH in a 1:1 ratio (wt/wt) with the heterobifunctional cross-linking agent maleimidobenzoyl-N-hydroxysuccinimide ester. The peptide Lys41-Pro55–KLH conjugate was suspended in PBS buffer to a final concentration of 3.1 mg/mL, emulsified with an equal volume of Freund's adjuvant, and injected into several subcutaneous dorsal sites of New Zealand White rabbits (3 to 9 months old). Approximately 0.6 mL (1 mg of conjugate containing 0.5 mg of peptide) was injected per immunization. Blood was collected from the auricular artery, allowed to clot, and then centrifuged. The supernatant containing the antiserum, designated as AB105, was stored frozen at -20°C.

Peptide Lys41-Pro55 (4 mg) was coupled to 1 g of CNBr-activated Sepharose 4B (Pharmacia) as described by the instructions of the manufacturer. AB105 serum (25 mL) was loaded onto the N-terminal peptide affinity column in coupling buffer (0.1 mol/L NaHCO3 [pH 8.3] and 0.5 mol/L NaCl). After removal of unconjugated proteins, antibodies (AB105) were eluted with 1.0 mol/L HOAc and 0.5 mol/L KCl, pH 2.3, neutralized and immediately dialyzed against coupling buffer. All of the chromatography steps were carried out at 4°C. Affinity-purified AB105 (0.5 mL) was then absorbed against chicken liver extract (0.1 g in 1 mL PBS, Sigma Chemical Co) overnight at 4°C, isolated by centrifugation, and stored at -20°C.

Isolation of Myosin and MyBPs
A protein fraction containing both myosin and MyBPs was extracted from ventricular heart muscle and pectoralis muscle of adult chicken and enriched as previously described.31 The fraction was further processed to separate MyBP-C and MyBP-H by FPLC over Q-Sepharose and Affigel-blue columns, according to Okagaki et al.27

Immunobloting
Protein fractions and pieces of fresh pectoralis muscle and heart from adult chickens were solubilized in 2% SDS, 5% 2-mercaptoethanol, 20 mmol/L Tris buffer [pH 6.8], 10% glycerol, and 1% bromphenol blue by boiling for 5 minutes. The samples were centrifuged at 8000g for 10 minutes and separated electorphoretically on a 7.5% polyacrylamide gel. The electrophoresed proteins were detected either by staining with 0.1% Coomassie blue or by immunoblotting according to Nawrotzki et al.39 Briefly, proteins transferred to nitrocellulose membrane were incubated for 1 hour at room temperature with PBS-T buffer (10 mmol/L Na-Pi, [pH 7.5], 0.15 mol/L NaCl, and 0.05% Tween 20) containing 5% nonfat dry milk, washed with the same buffer, and incubated with AB105, MF1, or C315 in PBS-T containing 1% BSA for 1 hour at RT. After washing to remove excess primary antibody, the membranes were soaked with alkaline phosphatase–conjugated secondary antibody for 1 hour at RT, washed three times with PBS-T buffer, and reacted with bromochloroindolyl phosphate/nitro blue tetrazolium for color development. The staining reaction was terminated with a PBS solution containing 20 mmol/L EDTA.

Protein Assays
Protein concentrations were determined according to the method of Bradford,40 with bovine serum albumin used as a standard. Concentrations of the purified MyBP-C and myosin were determined spectrophotometrically using extinction coefficients at 280 nm of 1.09 (mg/mL)-1xcm-141 and 0.58 (mg/mL)-1xcm-1,42 respectively.

Immunofluorescent Staining of the Chicken Hearts
The embryonic and adult chicken hearts were cryoprotected by infusion with 20% (wt/vol) sucrose in PBS overnight at 4°C. They were encapsulated in O.C.T. compound (Tissue Tek, Miles Inc) and frozen by immersion in liquid nitrogen-cooled isopentane. Frozen sections (7 to 10 µm) were mounted on Superfrost-Plus glass slides (Fisher Scientific) and air-dried. Sections were rehydrated and blocked with PBS containing 1% BSA. Sections were incubated with primary antibody overnight at 4°C, followed by three washes with PBS-BSA at RT for 10 minutes. Incubation with fluorescently tagged secondary antibody was performed at RT for 90 minutes, followed by four washes for 5 minutes each with PBS at RT. Coverslips were mounted with 10% 0.1 mol/L Tris (pH 7.6) in glycerol containing 100 mg/mL of DABCO (Sigma) to retard photobleaching. Slides were examined and photographed with a Nikon Microphot microscope equipped with phase-contrast and epifluorescence optics. The primary antibodies used were as follows: AB105, dilution 1:10 in PBS buffer containing 1% of BSA; MF20 and ALD58, undiluted hybridoma supernatants. The secondary antibodies were as follows: anti-rabbit Texas red for AB105, dilution 1:100 in PBS containing 1% BSA; anti-mouse FITC for MF20 and ALD58, dilution 1:500 in PBS with 1% BSA.

Riboprobes
A MyBP-H cDNA (2061 bp)20 was fragmented sequentially as follows: nucleotides 1 to 467 by EcoRI–Sph I digestion, nucleotides 467 to 972 by Sph I–BamHI digestion, nucleotides 972 to 1114 by BamHI-HindII digestion, nucleotides 500 to 1176 by Ava I–Ava I digestion, nucleotides 1176 to 1631 by Ava I–Ava I digestion, and nucleotides 1631 to 2061 by Sma I–EcoRI digestion. For convenience, these cDNA fragments were designated as H1-467, H467-972, H972-1114, H500-1176, H1176-1631, and H1631-2061, respectively. Each fragment was inserted into the pGEM4 multiple cloning site flanked by T7 and SP6 promoters (Promega). Each construct was linearized by endonuclease digestion of a restriction site either 5' or 3' of the insert and then reacted with T7 or SP6 RNA polymerase for 40 minutes at 37°C in the presence [{alpha}-32P]UTP (800 Ci/mmol) using a MAXIscript kit (Ambion). Probes to the 777 to 972 sequence (H777-972) were obtained by linearizing a plasmid encoding H467-972 with Bgl II digestion. Probes to the 777 to 1176 sequence (H777-1176) were obtained by linearizing a plasmid encoding H500-1176 with Bgl II. The DNA template was digested with 4 U of DNase I for 45 to 60 minutes at 37°C. The transcribed sense and antisense riboprobes were purified on an 8 mol/L urea–5% polyacrylamide gel before use. Specific activities of the riboprobes were 4x109 to 4x1010 cpm/µg.

RNase Protection Assay
The radiolabled riboprobes (0.05 to 0.3 ng) were hybridized with total RNA43 of pectoralis, ALD, PLD, heart, and liver at 45°C for 12 to 24 hours. Depending on the levels of MyBP-H mRNA in each tissue, the following amounts of total RNA were used: 0.4 µg (pectoralis), 10 µg (ALD), 80 µg (PLD and heart), and 20 µg (liver). Unhybridized riboprobe was digested with a mixture of RNases A and T1 (the RPA II kit, Ambion) for 30 minutes at 14°C or 37°C, according to the manufacturer's instructions. Protected probes were displayed by electrophoresis on a denaturing gel containing 5% polyacrylamide/8 mol/L urea followed by autoradiography with Cronex film (DuPont) exposed at -80°C for 12 to 72 hours.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Detection of MyBP-H in Heart
To detect chicken MyBP-H, a myofibrillar protein expressed in fast and slow skeletal muscle cells,20 we prepared a site-directed polyclonal antibody (AB105) against 15 amino acids (KEGHAPTPKEEHAPP) at its N-terminal region. The sequence was selected because it lacks homology with primary sequences in other members of the MyBP family.27 44 In addition, we assumed that the hydrophilic nature of this peptide would provide greater accessibility of the epitope to reactive antibodies. Specificity of the antibody to the Lys41-Pro55 peptide was demonstrated by immunoblot analysis of tissue extracts of chicken skeletal and cardiac (ventricular) muscle (Fig 1Down). MF1 monoclonal antibody, specific for skeletal muscle MyBP-C,31 and C315, specific for cardiac MyBP-C,36 were used as controls for the immunoreactions. In skeletal muscle extracts, AB105 recognized MyBP-H, primarily as a single band with an Mr of {approx}86 kD (Fig 1Down, lane 4). No significant cross-reactivity of AB105 to MyBP-C ({approx}140 kD), M protein ({approx}165 kD), or myomesin ({approx}185 kD) occurred. In heart extracts, a faint but reproducible protein of {approx}86 kD cross-reacted with AB105, although the signal was significantly weaker than that in skeletal muscle extracts (Fig 1Down, lane 8). The cross-reactive cardiac antigen exhibited a mobility identical to that of skeletal muscle MyBP-H when samples were coelectrophoresed (data not shown). A comparison of Western blot staining intensities suggested that the 86-kD protein in heart is {approx}200 times less abundant than MyBP-H in skeletal muscle. Because of its low concentration, we questioned whether the 86-kD protein was restricted to a small subpopulation of heart cells or expressed at a low level throughout the myocardium.



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  		Figure 1. Western blot analysis of epitope(s) to the site-directed polyclonal antibody, AB105. Total extracts (15 µg per lane) from pectoralis (lanes 1 to 4) and heart (lanes 5 to 8) were separated by SDS-PAGE, transferred to the nitrocellulose membrane, and stained with monoclonal antibody MF1 (lanes 2 and 6), monoclonal antibody C315 (lanes 3 and 7), or AB105 (lanes 4 and 8). Lanes 1 and 5 represent protein composition. Arrow indicates AB105-positive bands.

The AB105-reactive antigen in the embryonic and adult chicken hearts was localized by staining serial frozen sections of the ventricular wall with immunofluorescence (Fig 2Down). Purkinje fibers were identified with a monoclonal antibody (ALD58) specific for slow skeletal muscle–type MyHC.4 14 15 In the chicken, Purkinje fibers are known to be localized exclusively in perivascular regions of the coronary arteries.2 4 The AB105-positive signal in ventricular myocardium was mainly confined to cells juxtaposed to the coronary arteries (Fig 2ADown and 2BDown). These AB105-positive cells were invariably costained with ALD58 (Fig 2CDown and 2DDown). At higher magnification (Fig 2EDown), it could be seen that AB105 stained the A bands of myofibrils within differentiated Purkinje fibers but was not detected in adjacent ventricular myocytes.



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  		Figure 2. Immunolocalization of the 86-kD protein to Purkinje fibers of the ventricular myocardial wall. Frozen sections (10 µm) of E18 and adult hearts were stained with AB105 to MyBP-H (A through C and E) and ALD58 to slow-type myosin heavy chain (D). Perivascular localization of the AB105-positive Purkinje fibers (arrows) can be seen in embryonic (A) and adult (B) hearts. Double immunolabeling of a section with AB105 (C) and ALD58 (D) demonstrates that the 86-kD protein is preferentially localized to the Purkinje fibers (arrows). Under high-power view, the AB105 epitope (E) was often found in the A band of myofibrils (arrows). Asterisk indicates lumen of coronary artery; m, ventricular myocytes. Bars=100 µm (A through D) and 25 µm (E).

Biochemical Comparison of Cardiac 86-kD Protein With Skeletal Muscle MyBP-H
Since Purkinje fibers express an A band–associated protein with antigenicity and molecular size comparable to those of skeletal MyBP-H,18 19 24 we compared the purification properties of the two 86-kD proteins in skeletal and cardiac muscles.18 19 27 Purification followed four steps27 : (1) extraction of a crude myosin-containing fraction from muscle with a high salt buffer, (2) precipitation of a myosin-MyBP complex at low ionic strength, (3) separation of the MyBPs from myosin by anion exchange chromatography, and (4) separation of MyBP-H from other MyBPs, such as MyBP-C with Affigel blue chromatography. Cardiac and skeletal 86-kD protein isolation was monitored by immunoblotting all purification fractions.

After initial extraction of entire skeletal or cardiac muscle, the cardiac 86-kD protein was detected only in those fractions containing myosin (Fig 3Down) and was probably bound to myosin. The 86-kD protein could then be fractionated from myosin by anion exchange over Q-Sepharose (Fig 4Down). Two protein peaks were obtained: the first contained MyBP-H with MyBP-C, and the second contained myosin.27 SDS-PAGE analysis of each skeletal muscle fraction confirmed that MyBP-H was recovered in the first peak with MyBP-C (Fig 4ADown insert). Identical elution profiles were obtained when cardiac extracts were fractionated on the same Q-Sepharose column (Fig 4BDown). MyBP-C at {approx}155 kD was the major component of the first peak (Fig 4BDown insert). As expected from its low concentration in the heart, cardiac 86-kD protein could not be detected by either Coomassie blue or silver staining of the first peak. However, the band was visualized by Western blots of fractions 10 to 20 with AB106 (Fig 5ADown). Fractions containing the 86-kD protein were pooled and applied to an FPLC–Affigel blue column. Again, similar elution profiles were obtained for both skeletal and cardiac proteins (Fig 6Down). Skeletal muscle MyBP-H was eluted in fractions 20 to 32 (Fig 6ADown insert), and AB105 identified the cardiac 86-kD protein in fractions 22 to 30 (Fig 5BDown). Although the 86-kD protein was a minor component of these fractions (Fig 6BDown), the elution profile was indistinguishable from skeletal MyBP-H. In summary, cardiac 86-kD protein exhibited purification properties identical to those of skeletal muscle MyBP-H. Because the molecular size, antigenicity, myofibrillar localization, and purification properties all indicate that the Purkinje fiber 86-kD protein is closely related or identical to MyBP-H of skeletal muscle, we designate the cardiac 86-kD protein as "Purkinje fiber MyBP-H."



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  		Figure 3. Distribution of the 86-kD protein encoding an AB105 epitope during subpurification of myosin-containing fraction from the heart. Each lane contained 20 µg of protein. Lanes are as follows: 1 and 3, myosin extracted with high salt concentration followed by precipitation of myosin complex at low ionic strength; 2 and 4, supernatant, containing myosin after precipitation of actomyosin. Protein composition was examined with Coomassie blue staining (lanes 1 and 2); the 86-kD protein was detected with AB105 (lanes 3 and 4).



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  		Figure 4. Q-Sepharose column chromatography of the myosin-containing fraction from pectoralis (A) and heart (B). Approximately 100 mg of protein was applied to the Q-Sepharose fast-flow column (16x300 mm) and eluted by a linear gradient of KCl (0 to 0.3 mol/L KCl), as previously described.27 Inserts show the protein composition of fractions displayed by SDS-PAGE after staining with Coomassie blue. The number below each lane corresponds to the column fractions. OD indicates optical density.



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  		Figure 5. Distribution of AB105 epitope(s) during column purification. All fractions obtained during Q-Sepharose (A) and Affigel blue (B) column chromatography were surveyed with AB105. The fraction numbers are identical to those in Figs 4Up and 6Up.



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  		Figure 6. Affigel blue column chromatography of pectoralis (A) and cardiac (B) MyBPs. Approximately 8 to 10 mg of protein was applied to the Affigel blue column (10x100 mm) and eluted by a linear gradient of KCl (0.3 to 1 mol/L KCl). Inserts show the protein composition in fractions eluted from the Affigel blue column. The number below each lane corresponds to the column fractions. OD indicates optical density.

RNase Protection Analysis of the Transcripts Encoding Purkinje Fiber MyBP-H
Although biochemical characterizations of Purkinje fiber MyBP-H were indistinguishable from skeletal muscle MyBP-H, they did not prove identity. In our hands, peptide mapping analyses and partial amino acid sequencing of Purkinje fiber MyBP-H were unsuccessful because of the limited amounts of this protein in the heart (data not shown). To examine the relationship between Purkinje fiber MyBP-H and skeletal muscle MyBP-H in greater detail, Purkinje fiber MyBP-H mRNA was compared with skeletal muscle MyBP-H cDNA by RNase protection assays. Seven pairs of riboprobes (sense and antisense orientations) covering the complete length of skeletal MyBP-H mRNA were generated to scan both coding and flanking regions of Purkinje fiber MyBP-H mRNA. In the first set of experiments, total mRNA from the heart was mixed with the antisense riboprobe to H1631-2061 encoding 441 nucleotides; 431 nucleotides were derived from coding and flanking sequences of the 3' region of MyBP-H mRNA, and 10 nucleotides were derived from the pGEM-4 multiple cloning site. Subsequent RNase digestion of the mixture revealed that heart contains an RNA population that hybridizes to the entire 431-nucleotide sequence derived from MyBP-H (Fig 7ADown). Identical protection profiles were obtained by hybridization with RNA from pectoralis, PLD, and ALD skeletal muscles, but not with liver RNA (Fig 7ADown). In contrast, no significant protection was observed when sense riboprobe to H1631-2061 was mixed with these RNAs. The results clearly demonstrate that mRNA encoding Purkinje fiber MyBP-H and skeletal muscle MyBP-H are both transcripts of the same gene. However, since the MyBP-H gene of mouse and human consists of 11 exons (D.A. Fischman, unpublished data, 1996), these data did not rule out the possibility that Purkinje fiber MyBP-H may be an alternatively spliced variant of the skeletal MyBP-H gene. To examine this possibility, sequence homology was compared over the entire length of the MyBP-H cDNA (Fig 7BDown through 7G). In all cases, the entire region encoding the MyBP-H sequence within the antisense probes was protected from RNase digestion by heart and skeletal muscle RNA and not by liver RNA. No protection of sense probes was observed (data not shown). Since transcripts of Purkinje fiber MyBP-H preserve the same sequence over the entire length of skeletal muscle MyBP-H cDNA, it is highly unlikely that Purkinje fiber MyBP-H mRNA is an alternatively spliced variant. Sequence identity of mRNA, together with biochemical homologies at the expressed protein, supports the conclusion that Purkinje fiber MyBP-H is the same gene product as that expressed in skeletal muscle cells.



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  		Figure 7. RNase protection assay of heart RNA with MyBP-H riboprobes. A, Antisense (lanes 1 to 6) and antisense (lanes 7 to 12) transcripts from H1631-2061 were hybridized with total RNAs of pectoralis (lanes 2 and 7), heart (lanes 3 and 8), ALD (lanes 4 and 9), PLD (lanes 5 and 10), and liver (lanes 6 and 11). Lanes 1 and 12 are undigested antisense (431 nt from H1631-2061+10 nt from pGEM4) and sense (431 nt from H1631-2061+48 nt from pGEM4) riboprobes, respectively. B through G, Riboprobes were hybridized with RNAs from pectoralis (lane 1), heart (lane 2), and liver (lane 3), respectively. Transcripts are as follows: B, antisense transcripts (from H1176-1631 with 48 nt of pGEM4); C, antisense transcripts (from H1176-777 with 50 nt or pGEM4); D, antisense transcripts (from H972-1114 with 31 nt of pGEM4); E, antisense transcripts (from H777-972 with 31 nt of pGEM4); F, antisense transcripts (from H467-972 with 17 nt of pGEM4); and G, antisense transcripts (from H1-467 with 17 nt of pGEM4). The size of undigested probes is presented in lane 4 of each panel. Numbers indicate the size of markers.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Our retroviral cell lineage studies in embryonic chicken hearts have revealed that a subset of contractile myocytes juxtaposing the developing coronary arteries terminally differentiate into Purkinje fibers.4 Thus, conversion from a contractile to a conducting phenotype is the mechanism for generating Purkinje fibers in the avian cardiomyocyte lineage. The present study was performed to further an understanding of the molecular events associated with differentiation of Purkinje fibers. It addressed the expression of A-band proteins in a subpopulation of cardiac myocytes that terminally differentiate from contracting to conducting fibers. By using a site-directed antibody to skeletal muscle MyBP-H, we demonstrate that {approx}86-kD protein, the same size as MyBP-H, is coexpressed with a slow-type isoform of skeletal muscle myosin in Purkinje fibers of the chicken heart. Antigenicity, molecular size, biochemical properties, and myofibrillar distribution of the Purkinje fiber {approx}86-kD protein identify it as skeletal MyBP-H, previously thought to occur only in skeletal muscle.

Because of its low concentration within the myocardium, MyBP-H gene transcript has been undetected by Northern hybridization of cardiac mRNAs.20 This is consistent with biochemical fractionation of cardiac MyBPs in which only cardiac-type MyBP-C is a major component in the MyBP fractions and MyBP-H could only be detected by immunoblots. By RNase protection assays, which provide a higher sensitivity and resolution than previous Northern analyses, the present study proves that heart expresses an mRNA species that hybridizes to the entire length of skeletal muscle MyBP-H cDNA. It is likely that MyBP-H expressed in conducting cells is the same gene product as MyBP-H expressed in skeletal muscle.20 25 Our preliminary studies have shown colocalization of ALD58 and AB105 epitopes in atrial cells of internodal tracts (not shown). It remains to be tested whether the AB105 epitope in the internodal tract is MyBP-H expressed in the ventricular conducting cells.

Among the markers used to distinguish Purkinje fibers from contractile myocytes in the chicken heart, slow-type skeletal muscle myosin heavy chain has been the only myofibrillar protein gene known to be induced during conversion of contractile myocytes into conducting cells.4 15 The present study identifies MyBP-H as a new Purkinje fiber marker. In the mammalian heart, cardiac-type troponin I is absent from conduction cells in which slow skeletal muscle troponin I is expressed13 : slow skeletal muscle troponin I is expressed in all embryonic rat cardiomyocytes and persists only in adult conduction tissue cells. Furthermore, transgenic mice carrying a reporter gene fused to a portion of the cis element of the desmin gene exhibits restricted expression of the transgene in cardiac conduction and skeletal muscle fibers.16 These data suggest that skeletal muscle and Purkinje fibers may share the regulatory mechanism for transcription of some, if not all, myofibrillar proteins. It is known that expression of most myofibrillar proteins in skeletal muscle is mediated by members of the MyoD gene family acting in complex with E12, E47, MEF-2, SP-1, and/or other protein factors and bind muscle gene E boxes to regulate transcription.17 These associated factors, capable of dimerizing with MyoD, are expressed in cardiac muscle. Ectopic expression of MyoD in hearts of transgenic mice activates skeletal muscle–specific genes.45 Although expression of the MyoD gene family in heart could potentially upregulate skeletal muscle–type myosin and MyBP-H in cardiomyocytes that differentiate into Purkinje fibers, no members of the MyoD gene family have been found in the developing or adult heart.17 46 47 48 49 50 Identification of cis-element sequences in the MyBP-H gene shared by both skeletal muscle and Purkinje fibers would substantially improve our understanding of gene regulatory mechanisms during formation of the cardiac conduction system.

As in skeletal muscle, expression of the myofibrillar proteins in cardiac muscle is susceptible to extracellular signals such as neural elements,51 growth hormones,52 53 and retinoic acid.54 In earlier studies, we have shown that Purkinje fiber differentiation occurs coordinately with coronary vessel formation and invariably at perivascular boundary of developing arteries.4 7 Normal branching of coronary arteries is regulated by neural crest–derived cardiac ganglion cells55 56 57 58 59 that are closely associated with the vessels. We suggest that a local signal(s) from these nonmuscle tissues may be involved in recruiting Purkinje fibers from contractile myocytes.4 Studies are presently focused on the potential role of these migratory vasculogenic or neurogenic cells in triggering the expression of skeletal muscle–type myosin and MyBP-H during Purkinje fiber formation.


*    Selected Abbreviations and Acronyms
 
FPLC = fast-performance liquid chromatography
KLH = keyhole limpet hemocyanin
MyBP = myosin binding protein
RT = room temperature


*    Acknowledgments
 
This study was supported in part by grants from the American Heart Association (New York Affiliate), the National Institutes of Health (HL-54128, HL-45458, and AR-32148), the Mathers Foundation, Dr Charles Coffrin, and the Tolly Vinik Grant Program. Dr Mikawa is an Irma T. Hirschl Scholar.

Received September 30, 1996; accepted February 19, 1997.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Viragh Sz, Challice CE. The development of the conduction system in the mouse embryo heart: differentiation of the atrioventricular conduction system. Dev Biol. 1982;89:25-40.[Medline] [Order article via Infotrieve]
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