biochemical aspects of cardiac muscle differentiation · fraction of cardiac muscle tissue slices...

$: Biochemical Aspects of Cardiac Muscle Differentiation · fraction of cardiac muscle tissue slices was determined by incubating slices with [SH]thymidine as described. The slices were$
THE JOURNAL OF BIOLOCKAL. CHEMISTRY Vol. 251, No. 19, Issue of October 10, pp. 6082-6089, 1976

Printed in U.S.A.

Biochemical Aspects of Cardiac Muscle Differentiation

POSSIBLE CONTROL OF DEOXYRIBONUCLEIC ACID SYNTHESIS AND CELL DIFFERENTIATION BY ADRENERGIC INNERVATION AND CYCLIC ADENOSINE 3’:5’-MONOPHOSPHATE*

(Received for publication, December 9, 1975)

WILLIAM C. CLAYCOMB$

From the Department of Biochemistry, Louisiana State University School of Medicine, New Orleans, Louisiana 70112

A single injection of either isoproterenol or N6, 0”-dibutyryl adenosine 3’:5’-monophosphate (dibutyryl cyclic AMP) results in an inhibition in the rate of [3H]thymidine incorporation into DNA of differentiating cardiac muscle of the neonatal rat. This inhibition is not due to substantially altered cellular uptake or catabolism of [SH]thymidine. Inhibition of [‘Hlthymidine incorporation by isoproterenol or dibutyryl cyclic AMP is potentiated by theophylline. Maximal inhibition (95%) is observed 24 h after administration of isoproterenol, and the rate of incorporation returns to a value 80% of control by 72 h. Norepinephrine also inhibits [3H]thymidine incorporation whereas cyclic GMP, N2,02-dibutyryl guanosine 3’:5’-monophosphate (dibutyryl cyclic GMP), and phenylephrine have little effect. Equilib- rium sedimentation analysis of cardiac muscle DNA in neutral and alkaline cesium chloride gradients using bromodeoxyuridine as a density label indicate that isoproterenol and dibutyryl cyclic AMP inhibit [aH]thymidine incorporation into DNA that is replicating semiconservatively.

Administration of isoproterenol or dibutyryl cyclic AMP to neonatal rats inhibits by approximately 60% the incorporation of [9H]thymidine into DNA of tissue slices of cardiac muscle prepared 16 h later. [3H]Thymidine incorporation into DNA of tissue slices is into chains that were growing in uiuo. This incorporation is linear for at least 4 h of incubation and is inhibited by isoproterenol and dibutyryl cyclic AMP. Inhibition is not due to altered cellular uptake of [3H]thymidine nor is it due to a cytotoxic action. Several other compounds which elevate intracellular levels of cyclic AMP (epinephrine, norepinephrine, glucagon, and prostaglandin E,) also inhibit [3H]thymidine incorporation into DNA of cardiac muscle tissue slices. Cyclic GMP, dibutyryl cyclic GMP, sodium butyrate, and phenylephrine have little effect.

Isoproterenol administered together with theophylline to neonatal rats significantly stimulates the incorporation of [3H]phenylalanine into total cardiac muscle protein and into myosin. This enhanced incorporation may be due in part to an increase in the cellular uptake of [SH]phenylalanine.

DNA synthesis decreases progressively in differentiating cardiac muscle of the rat during postnatal development and essentially ceases by the middle of the third week (Claycomb, W. C. (1975) J. Biol. Chem. 250, 3229-3235). In reviewing the literature it was found that this decline in synthetic activity correlates temporally with a progressive increase in tissue concentrations of norepinephrine and cyclic AMP and with the anatomical and physiological development of the adrenergic nerves in this tissue. Because of these facts and data presented in this report it is proposed that cell proliferation and cell differentiation in cardiac muscle may be controlled by adrenergic innervation with norepinephrine and cyclic AMP serving as chemical mediators.

DNA synthesis and hence cell proliferation in terminally this loss of synthetic activity is an almost complete loss in differentiating cardiac muscle of the rat decrease progressively activity of DNA polymerase (Y and thymidine kinase and an following birth and essentially cease by the middle of the third increase in activity of nuclear poly(ADP-ribose) polymerase week of postnatal development (1). Temporally correlated with (l-3). The concentration of cyclic AMP’ increases progres-

sively in cardiac muscle of the rat during late fetal and early *This investigation was supported by a grant-in-aid from the neonatal development (4) and adenylate cyclase activity in

American Heart Association, the Muscular Dystrophy Associations of America, and Grant HL 17269-02 from the National Institutes of ‘The abbreviations used are: cyclic AMP, adenosine 3’:5’-mono- Health. This is Paper IV in a series concerning biochemical aspects of phosphate; dibutyryl cyclic AMP, W,O*‘-dibutyryl adenosine 3’:5’- cardiac muscle cell proliferation and cell differentiation. Papers I to monophosphate; cyclic GMP, guanosine 3’:5’-monophosphate; dibuty- III in this series are Refs. 1-3. ryl cyclic GMP, W,O*-dibutyryl guanosine 3’5.monophosphate;

#Established Investigator of the American Heart Association. BrdUrd, 5.bromo-2’.deoxyuridine.

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DNA Synthesis and Cell Differentiation in Cardiac Muscle

this tissue is higher in the adult than in the newborn (5-7). Since cyclic AMP has been implicated in the control of cell proliferation and cell differentiation (8-17), I decided to examine whether this cyclic nucleotide and the adenylate cyclase system might be involved with the regulation of DNA synthesis and cell differentiation in cardiac muscle. A prelimi-

nary account of some of these observations has been reported

(18).

EXPERIMENTAL PROCEDURE

Materials

[Methyl-‘Hlthymidine (specific activity, 17 Ci/mmol) and NCS solubilizer were purchased from Amersham/Searle; or [3-3H]phenylalanine (specific activity, 16.1 Ci/mmol) from New England Nuclear; [methyl-“Clthymidine (specific activity, 57 mCi/mmol) and cesium chloride (optical grade) from Schwarz/Mann; dl-isoproterenol and l-norepinephrine from Winthrop Laboratories; l-epinephrine, cyclic AMP, dibutyryl cyclic AMP, cyclic GMP, dibutyryl cyclic GMP, butyric acid, and BrdUrd from Sigma; theophylline from Nutritional Biochemicals; pancreatic DNase I and pancreatic RNase from Worth- ington; pronase from Calbiochem and glucagon from Eli Lilly. Prosta- glandin E1 was a gift from Dr. J. Pike of the Upjohn Co., Kalamazoo, Mich.

Timed pregnant rats were obtained from Holtzman (Madison, Wise.) on the 14th day of gestation. They were housed in individual cages and maintained on water and standard laboratory chow ad libitum. Neonatal rats were raised in litters of 10.

Methods

Injection of Neonatal Rats and Assay of DNA Synthesis-Neonatal rats were injected subcutaneously with the specified compound at the time indicated. Control littermates received saline (0.15 M NaCl) and all injections were made in 0.1 ml. One hour before the animals were killed by decapitation they were injected subcutaneously with [SH]thymidine, 2 PCilg body weight, and the rate of DNA synthesis (as determined by [3Hjthymidine incorporation into DNA) was measured as previously described (1).

Assay of DNA Synthesis in Tissue Slices of Cardiac Muscle-Tissue slices of ventricular cardiac muscle (approximately 0.5 mm in thick- ness) were prepared with a Stadie-Riggs tissue slicer. Approximately 150 mg of tissue was incubated at 37” in 5 ml of Eagle’s minimum essential medium containing Hanks’ salt base with [‘Hlthymidine (5 rCi/ml) in a 25-ml Erlenmeyer flask. At the end of the incubation period the tissue was homogenized in 2.0 ml of 0.15 M NaCl and [SH]thymidine incorporation into DNA was measured as previously described (1).

[3H]Thymidine Uptake and Catabolism-Cellular uptake and catabolism of [3H]thymidine in uiuo was determined as previously described (1). Uptake of radioactive thymidine intp the acid-soluble fraction of cardiac muscle tissue slices was determined by incubating slices with [SH]thymidine as described. The slices were then washed twice in 0.15 M NaCl, blotted dry, and homogenized in 2.0 ml of 0.15 M

NaCl. Two milliliters of ice-cold 10% perchloric acid was added and acid-soluble radioactivity was determined as previously described (1). To determine zero time binding of [3H]thymidine tissue slices were added to the incubation medium and then immediately removed, washed, and homogenized as described. Values for the zero time binding were subtracted when expressing the results.

DNA Extraction and Equilibrium Sedimentation in CsCl Density Gradients-Ventricular cardiac muscle was homogenized in 0.1 M Tris/HCl, pH 8.5, containing 0.25 M sucrose and a crude nuclear fraction was obtained by low speed centrifugation (750 x g for 10 min). The nuclear fraction was washed once in the above buffer, suspended in 0.1 M Tris/HCl, pH 8.5, made 1% (w/v) in sodium dodecyl sulfate and DNA was extracted with phenol. DNA precipitated from the aqueous phase with ethanol was dissolved in 0.01 M Tris/HCl, pH 7.5, containing’O.01 M MgCl, and incubated with 100 pg/ml of RNase (previously heated to 90” for 10 min to inactivate DNase) for 1 h at 37” and 100 rg/ml of pronase (predigested for 1 h at 37”) for 1 h at 37”. Sodium dodecyl sulfate wa’s added to 1% (w/v) followed by phenol and DNA was precipitated from the aqueous phase with ethanol. The DNA was dissolved in 0.01 M Tris/HCl, pH 7.0, containing 0.15 M NaCl.

DNA samples (50 to 100 Kg) were adjusted to a mean density of 1.728 g/cm” with solid CsCl and centrifuged ar. 32,000 rpm for 50 to 70 h in a

Beckman SW 50.1 rotor at 20”. Ten-drop fractions were collected by puncturing the bottom of the centrifuge tube with a hollow needle and the density of CsCl in every fifth fraction was determined by measuring the refractive index in a Bausch and Lomb refractometer. Fractions were diluted to 1.0 ml with distilled water and analyzed for absorbance at 260 nm in a Gilford model 2000 spectrophotometer. To determine radioactivity 50 rg of calf thymus DNA was added to each fraction and the DNA was precipitated with 10% trichloroacetic acid, collected on filters, and counted as described (1). Alkaline CsCl gradients were made in 0.05 M K,HPO, (pH 12.5). DNA to be sheared was diluted to 1.0 ml with 0.15 M NaCl, chilled to 0”, and sonicated at a power setting of 20 for 5 min with a Brownwill sonifier (Biosonik IV) equipped with a microtip.

Assay of [SH~henylalanine Uptake and Incorpomtion into Protein and Myosin-The incorporation of [‘Hlphenylalanine into ventricular cardiac muscle protein and into myosin during a l-h period was used as a measure of total muscle protein synthesis and myosin synthesis, respectively. Radioactive phenylalanine was chosen since Morgan et al. (19) had determined previously that it was a good amino acid to use to estimate protein and myosin synthesis in cardiac muscle. Rats were injected subcutaneously with [3H]phenylalanine, 1 &i/g body weight. One hour later they were killed by decapitation. Approximately 300 mg of ventricular cardiac muscle was homogenized in 3.0 ml of 0.3 M KC1 containing 0.1 M KH,PO, and 0.05 M K,HPO, at pH 6.5. The homogenate, 0.4 ml, was taken for estimation of DNA and 0.2 ml was taken for estimation of protein. The remainder of the homogenate was divided into two equal portions. Myosin was extracted from one portion by the procedure of Weeds and Hartley (20). The myosin was dissolved in 0.5 M KC1 and an aliquot was taken for estimation of protein; to measure radioactivity the remainder was dissolved in NCS solubilizer and counted in toluene scintillation fluid (1). Myosin synthesis is expressed as [3H]phenylalanine incorporation/mg of protein. The remainder of the original homogenate was used to measure [3H]phenylalanine uptake and incorporation into total muscle protein. Two milliliters of ice-cold 10% trichloroacetic acid was added to the homogenate and it was chilled on ice for 20 to 30 min. Following centrifugation at high speed in a clinical centrifuge, portions of the acid-soluble fraction were counted to estimate cellular uptake of [SH]phenylalanine (1). To measure incorporation into protein the acid-insoluble material was washed twice with ice-cold 5% trichloroacetic acid, extracted at 90” for 20 min in 5% trichloroacetic acid, washed once in 5% trichloroacetic acid, twice with ethanol/ether (3/l), dried under nitrogen, dissolved in NCS solubilizer and radioactivity determined as described (1) Since it had been noted previously that the protein content of these cells increases as they differentiate (3) and to estimate incorporation on a cellular basis, the results are expressed as [3H]phenylalanine incorporation/fig of DNA.

Chemical Assays-DNA was determined using the Burton modifica- tion of the diphenylamine reaction (21) with calf thymus DNA as a standard. Protein was determined by the procedure of Lowry et al. (22) with crystalline bovine serum albumin as a standard.

RESULTS

[3H]Thymidine Incorporation, Uptake, and Catabolism-

Isoproterenol injected into neonatal rats inhibits [‘HIthy- midine incorporation into DNA of cardiac muscle in a concentration-related manner (Fig. 1). This inhibition is potentiated by theophylline (Fig. 2). A synergistic response in the inhibition of DNA synthesis is observed when theophylline and a suboptimal amount of isoproterenol are administered simulta- neously. The time course of the isoproterenol-induced inhibition of DNA synthesis is shown in Fig. 3. The rate of DNA synthesis is inhibited by more than 90%, 24 h after a single injection of isoproterenol together with theophylline. The rate of synthesis returns to 80% of the control value by 72 h. It should be pointed out that isoproterenol is known to be cytotoxic in cardiac tissue of the adult when administered at high concentrations (23, 24). Under the present experimental conditions necrotic lesions in cardiac muscle of neonatal rats were never observed at isoproterenol concentrations less than 50 mg/kg or at isoproterenol concentrations of 25 mg/kg administered together with theophylline at 50 mg/kg.


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6084 DNA Synthesis and Cell Differentiation in Cardiac Muscle

% F- IO 4,‘;” 9:

8

84 6 Y z ,s 4 Z-- =I 2 5:: >- E

0 20 40 60 80 100

P % lSOPROTERENOL]( mq I kg)

FIG. 1. Effect of isoproterenol on the incorporation of [‘H]thymidine into DNA of differentiating cardiac muscle. Rats were injected subcutaneously at approximately 6:06 p.m. on the 3rd day of postnatal development with the indicated amount of isoproterenol. Controls received saline. Fifteen hours later they were injected subcutaneously with [9H]thymidine (2 rCi/g) and incorporation into DNA was measured as described (1). Values are the mean of four determinations. Hearts from two animals were pooled for each determination.

-

0

+i

Theophylline KW/W

0 + 0 t

FIG. 2. Effect of isoproterenol and theophylline on the incorporation of [SH]thymidine into DNA of differentiating cardiac muscle. Eats were injected subcutaneously at approximately 6:00 p.m. on the 6th day of postnatal development with the indicated compound. [3H]Thy- midine incorporation was determined as described in the legend to Fig. 1. Values are the mean + S.E. of five determinations.

Dibutyryl cyclic AMP administered to neonatal rats also inhibits [3H]thymidine incorporation into cardiac muscle DNA

(Fig. 4) and administered together with theophylline inhibits in a concentration-related manner. In experiments carried out thus far the magnitude of inhibition of [‘Hlthymidine incorporation by either isoproterenol (25 mg/kg, together with theophylline, 50 mg/kg) or dibutyryl cyclic AMP (25 mg/kg, together with theophylline, 50 mg/kg) has not been substantially different in neonatal rats between the ages of 1 to 7 days. Therefore the magnitude of inhibition by these compounds apparently is not dependent on the extent of DNA synthesis taking place in the muscle (1).

Since isoproterenol and dibutyryl cyclic AMP have been reported to influence the metabolism and transport of various nucleosides (25-28) the effect of these compounds on the uptake and on the catabolism of [‘Hlthymidine was determined (Table I). Neither compound had an appreciable influence cm either the uptake or on the catabolism of radioactive thymidine.

Isopycnic Analysis of DNA Synthesized by Differentiating Cardiac Muscle-These experiments were designed to examine the nature of the synthesis involved with [3H]thymidine

incorporation. DNA of differentiating cardiac muscle was labeled with BrdUrd and [3H]thymidine, extracted, and ana-

L CL. TIME AFTER IPT (HRSI

FIG. 3. Time course of the inhibition of [3H]thymidine incorporation into DNA of differentiating cardiac muscle. Four-day neonatal rats were injected once at approximately 6:00 p.m. with isoproterenol (IPT) (25 mg/kg) together with theophylline (50 mg/kg). [3H]Thymidine incorporation during a l-h pulse was determined at the time intervals indicated on the abscissa. Since the rate of thymidine incorporation is decreasing in cardiac muscle during the time period studied (l), the results are expressed as a per cent of the control value. Each point is the mean of three control and three experimental groups of animals. Further details are given in the legend to Fig. 1 and under “Experi- mental Procedure.”

r 0 20 40 60 80

[DBcAMPl (mglkql

FIG. 4. Effect of dibutyryl cyclic AMP (DBcAMPj and theophylline on the incorporation of [‘Hlthymidine into DNA of differentiating cardiac muscle. Animals were injected and [3H Jthymidine incorporation was measured as described in the legend to Fig. 1. Values are the mean of four determinations. Eats were injected on the 5th day of postnatal development with: 0, saline; 0, dibutyryl cyclic AMP (25 mg/kg); A, theophylline (50 mg/kg); A---A, theophylline (50 mg/kg) together with the indicated amount of dibutyryl cyclic AMP.

lyzed in neutral CsCl equilibrium density gradients. Badioac- tivity incorporated into DNA banded at three different densi- ties (Fig. 5A). The most dense fraction corresponds to double- stranded DNA in which both strands had incorporated BrdUrd; the next less dense fraction corresponds to a hybrid molecule in which one strand is heavy and one strand is light. Radioactivity in the main band DNA corresponds to both strands having incorporated little or no BrdUrd. DNA strands containing [3H]thymidine and little or no BrdUrd were most likely initiated during the last 10-h interval of this experiment when high intracellular concentrations of BrdUrd were not maintained by repeated injections. To confirm that these density shifts were indeed due to BrdUrd incorporation the DNA was analyzed in alkaline CsCl gradients (Fig. 5B). A large shift in the density of single stranded DNA was observed. These results indicate that [3H]thymidine incorporation is into DNA that is being replicated semiconservatively (29, 30). The specific activity of DNA extracted from cardiac muscle prepared from 2-day-old rats that had been injected 15 h previously with 0.15 M NaCl (0.1 ml), isoproterenol (25 mg/kg) together with theophylline (50 mg/kg), or dibutyryl cyclic

AMP (25 mg/kg) together with theophylline (50 mg/kg) was


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TABLE I

Cellular uptake and catabolism of [3H]thymidine by differentiating cardiac muscle of neonutal rats

Rats were injected subcutaneously at approximately 6:00 p.m. on the 4th day of postnatal development with the indicated compound. Fifteen hours later they were injected with [3H]thymidine (2 &i/g) and cellular uptake 15 min later was determined (1). Catabolism of radioactive thymidine to nonaromatic pyrimidine derivatives was determined as previously described (1). Values are the mean * S.E. of three determinations.

Compound tented ?HlThymidine (JH]Th~midine uptake catabolism

Saline Isoproterenol (25 m&g) Isoproterenol (25 mg/kg)

+ theophylline (50 mg/kg) Dibutyryl cyclic AMP (25 mgikg)

+ theophylline (50 mg/kg)

dpmlpg DNA

866 l 63 807 * 23 829 * 59

812 * 56

12 14 17

16

TABLE II

Effect of various compounds on DNA synthesis in differentiating cardiac muscle of neonatal ruts

Rats were injected subcutaneously at approximately 6:00 p.m. on the 3rd or 4th day of postnatal development with the indicated compound or with saline (control) and [‘Hlthymidine incorporation was measured 16 h later as described in the legend to Fig. 1. Values are the mean * S.E. of three to five determinations.

Compound tested [‘H ]Thymldine incorporation

Cyclic AMP (25 mg/kg) Dibutyryl cyclic AMP (25 mg/kg) Cyclic GMP (25 mg/kg) Dibutyryl cyclic GMP (25 mg/kg) Norepinephrine (1 mg/kg) Norepinephrine (10 mg/kg) Phenylephrine (25 mg/kg)

% of control 86 + 6 79 * 8

108 i 9 105 i 8 74 * 7 59 f 9 97 l 7

TABLE III

[“H]Thymidine incorporation into DNA of tissue slices of cardiac muscle prepared from rats of different ages

Approximately 150 mg of tissue was incubated with [3H]thymidine (5 pCi/ml) in Eagle’s minimum essential medium for 1 h. Each value is the mean * S.E. of three determinations. Adults were females that weighed approximately 250 g.

[3H]Thymidine incorporation

Fetal, 19 day Neonatal, 1 day Neonatal, 7 day Neonatal, 14 day Adult

dpmhg DNA

3027 zt 212 2418 + 198 2082 + 257

272 zt 59 64 * 12

3590 dpm/pg of DNA, 685 dpm/pg of DNA, and 650 dpm/pg of DNA, respectively, and banded in neutral CsCl gradients at a density of main band DNA.

Effect of Other Compounds on DNA Synthesis-The effect of several other compounds on [3H]thymidine incorporation into DNA of differentiating cardiac muscle is summarized in Table II. Cyclic AMP, dibutyryl cyclic AMP, and norepineph-

10 20 30 40 FRACTION NUMBER

FIG. 5. Isopycnic analysis of cardiac muscle DNA in neutral and alkaline CsCl gradients. Neonatal rats were injected subcutaneously over a period of 47 h at 4- or 10-h intervals (10-h intervals were between 9:OO p.m. and 7:00 a.m.) beginning on the morning of birth with BrdUrd (250 mg/kg). Ten hours after the last injection of BrdUrd they were injected with [3H]thymidine (10 &i/g) and killed 1.5 h later. DNA was extracted from cardiac muscle and analyzed in neutral (A) or alkaline (B) CsCl density gradients. Further details are given under “Experi- mental Procedure.”

rine inhibit, whereas cyclic GMP, dibutyryl cyclic GMP, and phenylephrine have little effect.

[SH]Thymidine Incorporation into DNA of Tissue Slices of Cardiac Muscle-In order to determine whether cyclic AMP and catecholamines inhibit DNA synthesis in vivo by a direct action on cardiac muscle or by some indirect mechanism, DNA synthesis was studied in tissue slices prepared from differentiating cardiac muscle. Incorporation of [aH]thymidine into DNA of tissue slices of cardiac muscle prepared from rats of different ages is shown in Table III. The rate of incorporation in this in vitro system decreases during development and is approximately 50-fold less in slices prepared from the adult than in slices prepared from the 19-day fetal rat. During the developmental period between 1 and 7 days after birth the decrease in the rate of [3H]thymidine incorporation into DNA of tissue slices is not as large as that observed previously in vivo (1). Experiments were next designed to determine whether [‘Hlthymidine incorporation into DNA of tissue slices is a continuation of the process that is occurring in vivo, i.e. semiconservative replication. If this were the case one would expect that strands initiated in vivo would continue to be elongated in vitro. This was tested by labeling cardiac muscle DNA in vivo with [SH]thymidine and BrdUrd and then tissue slices were prepared from these animals and incubated with [“Clthymidine in vitro. The DNA was extracted and analyzed in CsCl equilibrium density gradients. Both aH- and “C-


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1.600 L J

0.25

1.9w

1.800

I.700

1.m

10 20 30 40 FRACTION NUMBER

- 0.10

I- z - T E 0 s x - 0.05 I

y b 4

E? “- 4 ::

-2 J

FIG. 6. Isopycnic analysis of cardiac muscle DNA labeled in uiuo with [‘Hlthymidine and BrdUrd and in vitro with [‘“Clthymidine. Twenty l-day neonatal rats were injected subcutaneously with YH]- thymidine (10 pCi/g); 2 h later and every l/r hour thereafter for 4 h they were injected with BrdUrd (500 mg/kg). Tissue slices were then pre-. pared and incubated for 2 h in 10Iml of Eagle’s minimum essential medium containing [“Clthymidine (10 rCi/ml). DNA was extracted and analyzed in neutral CsCl equilibrium density gradients (A) or sheared by sonication and analyzed in alkaline CsCl equilibrium density gradients (B). Further details are given under “Experimental Pro- cedure.”

labeled DNA sedimented in ,neutral C&l gradients with the main band DNA (absorbance) (Fig. 6A). This result does not

establish whether [3H]thymidine and [“Clthymidine were incorporated in tandem in the same strand or in separate

strands which were held together by hydrogen bonds. To examine this the DNA was sheared in order to emphasize a

density shift and analyzed in alkaline CsCl gradients (Fig. 6B). The majority of the fragmented DNA containing 3H and “C was shifted towards a heavier density away from the bulk of DNA (absorbance). This indicates that [“Clthymidine incorporation into DNA of tissue slices occurs relatively close if not in tandem to the region where [‘Hlthymidine and BrdUrd were incorporated in vivo and, thus, is an elongation of the growing chains initiated in vivo. This is consistent with

semiconservative DNA replication being continued in tissue slices.’ [3H]Thymidine incorporation in this tissue slice system is linear for at least 4 h of incubation and is inhibited by the

%These experiments do not eliminate the possibility that ‘H and “C were incorporated into separate strands which band together in CsCl gradients after shearing because they contain the same fractional content of BrdUrd. This could occur if the BrdUrd pool size in oitro was large enough to allow the DNA to be labeled during the 2-h incubation period to a density equivalent to that achieved in uiuo. Although remnant BrdUrd pools, chain growth rates, and sizes of DNA frag- ments have not been measured in these experiments, this interpreta- tion seems unlikely, especially since additional experiments using

I 2 3 4 INCUBATIONTIMEfHRSI

FIG. 7. Kinetics of [3H]thymidine transport and incorporation into DNA of tissue slices of differentiating cardiac muscle. Tissue slices were prepared from 5.day neonatal rats and incubated with [SH]thymidine (5 rCi/ml). Further details are given under “Experimental Procedure.” Closed symbols represent [3H]thymidine uptake into the acid-soluble fraction (transport) and open symbols represent [3H]thymidine incorporation into DNA. Circles are control values; triangles are values obtained when slices were incubated with 1 mM dibutyryl cyclic AMP; squares are values obtained when slices were incubated with 1 mM isoproterenol. Each point is the mean of three to five determinations.

TABLE IV

Evidence that isoproterenol and dibutyryl cyclic AMP are not cytotoxic

Treatment [3H]Thymidine incorporation

Isoproterenol, 2 h” 88 Isoproterenol, 1 hb 96 Dibutyryl cyclic AMP, 2 h” 91 Dibutyryl cyclic AMP, 1 h* 98

“Tissue slices were prepared from 6.day neonatal rats and incubated for 2 h with the indicated compound (1 mM). The slices were then washed twice by placing them in 0.15 M NaCl and blotting dry. They were then placed in fresh Eagle’s minimum essential medium containing [3H]thymidine (5 &X/ml) and incubated for an additional 2 h. Controls were carried through the same procedure except that they were incubated in the absence of the indicated compound. [‘H]Thymi- dine incorporation into DNA was determined as described under “Experimental Procedure.” Values are the mean of two experiments.

‘Tissue slices were prepared as above and incubated for 1 h with the indicated compound (1 mM). They were then washed in 0.15 M NaCl, placed in fresh Eagle’s minimum essential medium, and incubated for 2 h. [3H]Thymidine (5 &i/ml) was then added and they were incubated for an additional 1 h. Controls were carried through the same procedure except that they were incubated in the absence of the indicated compound. Values are the mean of two experiments.

addition of isoproterenol or dibutyryl cyclic AMP (Fig. 7).

Inhibition by these compounds is not due to altered uptake of [SH]thymidine into these cells (Fig. 7). It is also not due to a

cytotoxic action since the rate of DNA synthesis is restored to control values by removing the slices from medium containing these compounds, washing them in 0.15 M NaCl, and incubating them further in fresh medium (Table IV).

Administration of isoproterenol or dibutyryl cyclic AMP along with theophylline to neonatal rats inhibits by approximately 60% the incorporation of [‘Hlthymidine into DNA of cardiac muscle tissue slices prepared 16 h later (Table V). The effect of several other compounds which elevate the intracellular concentration of cyclic AMP is also summarized in Table

[3H]thymidine to label DNA in uiuo and [“C]dATP and BrdUTP in vitro have shown that chains initiated in uiuo in cardiac muscle continue to be elongated in isolated nuclei.


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V. Norepinephrine and epinephrine added to tissue slices inhibit, whereas phenylephrine has little effect. Prostaglandin E, and glucagon also inhibit [3H]thymidine incorporation in this system. Neither cyclic GMP nor its dibutyryl derivative nor possible catabolic products of cyclic AMP or dibutyryl cyclic AMP, or sodium butyrate, had a significant effect on [3H]thymidine incorporation.

[3H]Phenylalanine Uptake and Incorporation into Total Cardiac Muscle Protein and Into Myosin-In order to determine if isoproterenol influences other aspects of cardiac muscle differentiation its effect on protein and myosin synthesis was examined. It was observed previously that the cellular content of protein in cardiac muscle of the rat remains relatively constant during the first 3 weeks of postnatal differentiation and begins to increase by the 22nd day after birth (3). Incorporation of [SH]phenylalanine into cardiac muscle myosin shows a similar temporal pattern.3 The end of the 3rd week of postnatal development is also the period during which the growth pattern of this tissue changes from one of cell proliferation to one of cell enlargement (1, 31, 32). The increase in the protein content which occurs in these cells after they cease dividing and begin to enlarge is due largely to an increase in the synthesis and accumulation of the contractile proteins. Admin- istration of isoproterenol together with theophylline to 3-day neonatal rats significantly stimulates the incorporation of [3H]phenylalanine into total cardiac muscle protein and into myosin measured 18 h later (Table VI). Preliminary work using identical experimental conditions has shown that by 48 h the cellular protein content in cardiac muscle is increased 20 to 40’?X~.~The enhanced [3H]phenylalanine incorporation observed in these experiments may be due in part to an increase in the cellular uptake of this amino acid (Table VI).

DISCUSSION

Data presented in this paper show that cyclic AMP and several compounds which elevate the intracellular concentration of cyclic AMP inhibit [3H]thymidine incorporation into DNA of differentiating cardiac muscle that is replicating semiconservatively. The mechanism for this inhibition is unclear. Previous studies have shown that isoproterenol and dibutyryl cyclic AMP both inhibit the activity of DNA polymerase (Y in differentiating cardiac muscle (3). This suggests that initiation and continuation of DNA synthesis in this tissue may be controlled by the availability of this DNA polymerizing enzyme. This idea is supported by work which has shown that addition of DNA polymerase to nuclei and chromatin isolated from cardiac muscle in which the cells are withdrawn from the cell cycle and DNA synthesis is repressed, substantially stimulates [3H]dTTP incorporation into DNA (33). Further studies indicate that the number of 3’-OH initiation sites in nuclei and chromatin of cardiac muscle cells does not change as DNA synthesis is restricted during differentiation; this is consistent with initiation sites being available in repressed cells but not being utilized.*

The inhibitory effect of isoproterenol on DNA synthesis in cardiac muscle is in sharp contrast to its action on other mammalian tissues. In rodents, isoproterenol stimulates DNA synthesis and mitosis in the salivary gland (34-37), kidney (38), duodenum (39), urinary bladder epithelium (40), and dorsal prostate gland tubules (41). In several other normal and

3 W. C. Claycomb, unpublished observation. ’ W. C. Claycomb, manuscript in preparation.

TABLE V

Effect of uarious compounds on [3H]thymidine incorporation into

DNA of tissue slices of differentiating cardiac muscle

Tissue slices were prepared from either 5., 6., or ‘I-day neonatal rats and incubated for 2 h. Each compound was added to the incubation medium in 0.1 ml of saline and when necessary the pH of the medium was adjusted to 7.4 with 7.5% NaHCO,. Saline (0.1 ml) was added to controls. Values are the mean + S.E. of three to five determinations.

Addition [3H]Thymidine incorporation

% Dibutyryl cyclic AMP (25 mg/kg)

+ theophylline (50 mg/kg) in uiuo” Isoproterenol (25 mg/kg)

+theophylline (50 mg/kg) in uiuo” Norepinephrine (0.1 mM)

Norepinephrine (0.01 mM)

Norepinephrine (0.001 mM)

Epinephrine (0.1 mM)

Epinephrine (0.01 mM)

Isoproterenol (1 mM)

Isoproterenol (0.1 mM)



Phenylephrine (1 IIIM)

Cyclic AMP (1 mM)

Dibutyryl cyclic AMP (1 mM)

Dibutyryl cyclic AMP (0.1 mM)

Cyclic GMP (1 mM)

Dibutyryl cyclic GMP (1 mM) Sodium butyrate (0.1 mM)

Glucagon (100 pg/ml) Prostaglandin E, (10 pg/ml)

of contml

45 * 8

38 + 9

53 * 5 68 * 5 85 * 7 84 i 5 91 * 4 48 l 8

69 zt 7 75 + 8 89 * 8 95 + 6 64 * 9 52 zt 6 69 + 5 98 i 7 96 + 5 92 * 8 82 i 6 71 * 7

a Tissue slices were prepared from 2-day neonatal rats that had been injected subcutaneously 16 h earlier with the indicated compounds.

TABLE VI

Effect of isoproterenol and theophylline on cellular uptake and incorporation of [SHlphenyylalanine into total muscle protein

and into myosin

Rats were injected subcutaneously at approximately 2:00 p.m. on the 3rd day of postnatal development with isoproterenol (25 mg/kg) together with theophylline (50 mg/kg). Eighteen hours later they were injected subcutaneously with [3H]phenylalanine, 1 MCilg body weight. One hour later they were killed and uptake and incorporation of [3H]phenylalanine into protein and myosin was measured as described under “Experimental Procedure. ” Values are the mean * S.E. of nine individual determinations.

[3H]P~;;kelanine [3?+;n;;$$e [3H]Phenylalanine into total incorporation

muscle protein Into myosin

dpmlpg DNA dpm1F.z DNA dpmlmgprotein

Control 33 i 2 170 i- 8 6726 i 386 Isoproterenol + 59 i 6” 204 zt 14” 7842 i 502”

theophylline

Op <0.05; p values were calculated by Students t test.

tumour tissues isoproterenol has been reported to inhibit DNA synthesis and cell proliferation (8, 42, 43).

Catecholamines are known to be cytotoxic in cardiac muscle of the adult rat when administered at high concentrations (23, 24). Therefore the possibility should be considered that the catecholamine-induced inhibition of DNA synthesis observed in the present studies is due to a nonspecific cytotoxic action.


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Several facts argue against this possibility. First, 48 h after maximal inhibition of [3H]thymidine incorporation by isoproterenol the rate of incorporation was returned to a value 80% of control (Fig. 3). Recovery from almost total inhibition would not be expected to be so rapid if the inhibition were due to tissue damage. Second, cellular uptake and catabolism of [3H]- thymidine might be expected to be altered if cellular damage had occurrred. This was not observed (Table I). Third, isoproterenol does not inhibit protein or myosin synthesis in cardiac muscle under conditions in which DNA synthesis is severely inhibited (Fig. 3 and Table VI). In fact [3H]phenylalanine incorporation into total muscle protein and into myosin is significantly stimulated. Fourth, previous studies have shown that other developmental events which occur during the normal differentiation of cardiac muscle (decrease in the activity of DNA polymerase 01 and thymidine kinase, increase in nuclear poly(ADP-ribose) polymerase activity, and increase in the cellular concentration of NAD+) can also be accelerated by the administration of isoproterenol or dibutyryl cyclic AMP to neonatal rats (3, 44). Finally, the question of cytotoxicity in vitro was tested directly (Table IV).

(50, 51). These parameters are used to judge the functional maturation of an adrenergic neuron (52, 53). The norepinephrine content of a tissue is also held to reflect the density of the adrenergic neurons and it has been observed that the density of

A point that should be noted about the tissue slice experiments or any experiment carried out with in vitro models such as cell and organ cultures is that catabolism of exogenous hormones and drugs can be extensive. In addition, the cellular permeability to these compounds may be greatly reduced. Therefore it may in some instances be necessary to add apparently unphysiological concentrations of these agents in order to observe a physiological response.

The rates of DNA synthesis and cell proliferation in a variety of normal, transformed, and tumor cells are inversely related or directly related, respectively, to the intracellular concentration of cyclic AMP or cyclic GMP (8-17, 43). It has been proposed that this reciprocal relationship is involved in some manner with the control of several biological processes including cell proliferation (45). A reciprocal relationship between the intracellular concentrations of these cyclic nucleotides and the rate of DNA synthesis and the degree of differentiation is observed in cardiac muscle of the rat. The rate of DNA synthesis and the intracellular concentration of cyclic GMP decrease progressively (1, 46) as the intracellular concentration of cyclic AMP increases progressively (4) during late fetal and early postnatal development. In this context, it is of interest that a similar development-related reciprocal relationship between the activ- ities of cyclic GMP-dependent and cyclic AMP-dependent protein kinases in cardiac muscle of the guinea pig has recently been noted (47). The relationship between cyclic AMP and cyclic GMP in the control of cell proliferation and cell differentiation in cardiac muscle is not clear. It would appear from studies carried out thus far that the presence of cyclic AMP rather than the absence of cyclic GMP is more important in the repression of DNA synthesis in this tissue.

The terminally differentiated ventricular cardiac muscle cell of the adult mammal exists in a repressed state in that it has apparently irreversibly lost the ability to replicate its DNA, undergo mitosis, and proliferate (1, 48, 49). Little is known about the mechanisms responsible for this loss of DNA synthetic activity. It is known that in the rat DNA synthesis essentially ceases by the middle of the third week of postnatal development (1). It is also known that cardiac muscle of the rat manifests a progressive development-dependent increase in endogenous stores of norepinephrine and in the ability to take up, accumulate, and not metabolize exogenous norepinephrine

the adrenergic neurons in the heart increases with development (52-56). A few single axons are observed at birth; innervation increases continually thereafter, attaining the adult pattern by the 22nd day of postnatal development. Therefore, in cardiac muscle of the rat the cessation of DNA synthesis and cell proliferation and the rise in the intracellular concentration of cyclic AMP are correlated temporally with the anatomical and physiological development of the adrenergic nerves. Because of these observations and data presented in this paper it is proposed that cell proliferation and cell differentiation in cardiac muscle may ultimately be controlled by adrenergic innervation with norepinephrine and cyclic AMP serving as chemical mediators. The rise in the intracellular concentration of cyclic AMP during development could result from stimulation of adenylate cyclase by norepinephrine. Cho- linergic innervation and acetylcholine or the lack thereof may be involved in some manner with the fall in the concentration of cyclic GMP that is observed in differentiating cardiac muscle possibly via an action on guanylate cyclase or other enzyme systems. It should be emphasized that at the present time neuronal control of terminal differentiation of cardiac muscle serves as a working hypothesis. A precedent does exist, however, for this hypothesis in the neuronal regulation of enzyme concentration by enzyme induction and for innervation regulating the genes concerned with the synthesis of contractile proteins in skeletal muscle (57-62). One level of growth control during development of higher organisms may be ex- erted in certain tissues by the autonomic nervous system in- fluencing cellular proliferation.

A catecholamine-sensitive adenylate cyclase system capable of generating cyclic AMP when stimulated has been reported to be present in cardiac muscle of the fetal rat and mouse (63, 64). This is prior to development of the inotropic and chrono- tropic responses to the neurotransmitter which may appear when the sinus node is innervated. The presence of an adenylate cyclase system prior to innervation means that one possible direct test of the hypothesis put forth in this paper would be to completely prevent functional adrenergic innervation during development. This could possibly be achieved by chemical or immunological sympathectomy (53).

AclznoLuledgment-I thank Susan Bryde for her conscien- tious technical assistance.

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W C Claycomband cyclic adenosine 3':5'-monophosphate.

deoxyribonucleic acid synthesis and cell differentiation by adrenergic innervation Biochemical aspects of cardiac muscle differentiation. Possible control of

1976, 251:6082-6089.J. Biol. Chem.

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Additions and Corrections

vol. 251 (1976) 5888-5894

Poly(A)-rich ribonucleoprotein complexes from HeLa cell messenger RNA.

Valerie M. Kish and Thoru Pederson

Page 5894, In the last sentence of the Discussion,

References (17, 35) should read (35, 36).

Ref. 36, which was omitted by the authors, is:

36. Auerbach, S., and Pederson, T. (1975) Biochim. Biophys. Acta 395, 388-391

Vol. 251 (1976) 6082-6089

Biochemical aspects of cardiac muscle differentiation. Possi- ble control of deoxyribonucleic acid synthesis and cell differentiation by adrenergic innervation and cyclic adenosine 3’:5’-monophosphate.

William C. Claycomb

Page 6082, Footnote to title:

Line 3 should read:

and in part by Grant HL 17269-02 from the National Insti- tutes of Health while the author was at the Baylor College of Medicine, Houston, Texas.

We suggest that subscribers photocopy these corrections and insert the photocopies at the appropriate places where the article to be corrected originally appeared. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract.) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.

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