Modulation of contractile proteins in embryonic and fetal chick cardiac cells by phorbol ester, gamma-interferon, 5-azacytidine and diacylglycerols

Life Sciences, Vol. 54, pp. 171-183 Printed in the USA

Pergamon Press

M O D U L A T I O N OF C O N T R A C T I L E P R O T E I N S IN E M B R Y O N I C AND F E T A L C H I C K C A R D I A C C E L L S BY P H O R B O L ESTER, G A M M A - I N T E R F E R O N , 5A Z A C Y T I D I N E AND D I A C Y L G L Y C E R O L S . M.A. Muros, A.E. Ar~inega, C. V61ez, C. Melguizo, L. Alvarez, A. Argnega. Basic Cardiovascular Research Section, Department of Morphological Sciences, School of Medicine, University of Granada, E- 18071 Granada, Spain (Received in final form October 29, 1993) Summary We studied changes in the concentration of tropomyosin, actin, desmin and vimentin in cultured myocardiocytes from Hamburger and Hamilton's stages 29 and 39 chick embryos (HH29 and HH39) (1), treated with 12-o-tetradecanoyl-phorbol-13-acetate (TPA), 5-azacytidine (AZA), gamma interferon (INF) and dyacylglycerols (DAG). In embryonic myocardiocytes at HH29, the first three agents modified the intracellular distribution of the thin filament proteins tropomyosin and actin, increasing their cytoplasmic concentration and decreasing their cytoskeletal concentration. The concentration of the intermediate filament proteins desmin and vimentin increased in both subcellular fractions after treatment with these drugs. In fetal myocardiocytes at HH39, total protein content decreased after treatment with these drugs. Cytoplasmic and cytoskeletal concentrations of actin and tropomyosin decreased to different degrees after treatment with TPA, AZA or DAG in HH39 myocardiocytes. TPA, AZA and DAG decreased desmin in the cytoplasmic and cytoskeletal fractions. These findings suggest that the drugs tested alter the normal protein composition in cultured myocardiocytes, and have different effects depending on the developmental stage in which the embryo is treated.

In studies of cellular differentiation, an area of great interest is the possible influence of drugs which, when added to the culture medium, can act as either differentiating or dedifferentiating agents (2,3,4,5). Many studies have documented the complex variety of cellular responses to phorbol esters. Treatment with diacylglycerols (DAG) or 12-o -tetradecanoyl-phorbol-13 acetate (TPA), the most active of the phorbol ester derivatives, leads to a wide variety of responses arising from the interactions between the drug and its cellular receptor (6). In cultures of adult rat myocardiocytes, TPA stimulates DNA synthesis, activating genes involved in terminal cellular differentiation (7). Claycomb and Moses (8) found that TPA led to profound morphological alterations in adult rat myocardial cells, such as chemical rupture of the myofibrils and accumulation of intermediate filaments. These authors noted that in general, DAG caused ultrastructural alterations like those seen after TPA treatment in the same type of cell. The ability of gamma interferon (INF) byha to potentiate differentiation in different cell lines (9), and 5-azacytidine (AZA), an inhibitor of DNA methylation, has also been reported to promote cell differentiation when added to the culture medium (10). Actin, one of the main proteins in muscle tissue, comprises up to 20% of the total protein content in muscle tissue (11). This protein forms part of the thin muscular filaments, providing the mechanochemical foundation for muscle contraction (12). Tropomyosin (TM), another contractile protein, is also a component of the thin filaments, and acts as a regulator of muscle 0024-3205/94 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.

172

Modulation of Contractile Proteins

'7ol. 54, No. 3, 1994

contraction. We recently analyzed the expression of tropomyosin in different stages of normal heart development (13), and the changes in intracellular distribution during myocardial cell culture (14). Adequate expression of the thin filament proteins tropomyosin and actin is fundamental for the correct development of the contractile apparatus in cultured muscle ceils. Desmin and vimentin, the major proteins in the intermediate filaments in the muscle cell (15), are coexpressed during chick embryo cardiogenesis (16). Desmin is thought to be related with maturation of the muscle fiber (17). These proteins are modified in cultured myocardiocytes when chemical agents are added to the culture medium (18). In this study we compared the effects of TPA, DAG, INF y AZA on cultures of embryonic and fetal chick myocardiocytes obtained at appropriate stages of heart development (1). Our basic approach was to quantify thin filament (tropomyosin and actin) and intermediate filament proteins (desmin and vimentin) in the cytoskeletal and cytoplasmic subfractions of developing myocardiocytes. Methods Cell Cultures (CMC): After 7 and 13 days of incubation, hen the embryos had reached stage 29 and 39 of Hamburger and Hamilton respectively (1), the hearts were removed. Myocardiocytes were isolated and cultured as described by Muros et al. (14). Treatment of CMC: On day 6 of culture, cells were treated with medium containing 12-otetradecanoyl-phorbol-13-acetate (TPA) (Sigma) at a concentration of 100 ng/ml in 0.02% dimethylsulfoxide (DMSO). On the same day, some cultures were treated with 1-oleoyl-2-acetylrac-glycerol (DAG-A) or 1,2-dicapryloyl-rac-glycerol (DAG-B) (both Sigma) at a concentration of 50 I.tg/ml in 0.01% ethanol. In addition, other cultures were treated with medium containing gamma-interferon (INF) (1OO U/ml) (kindly supplied by Dr. F. Garrido of the Virgen de las Nieves Hospital in Granada) and 5-azacytidine (AZA) (Sigma) (10 I.tM). After 24 h of incubation the cells were harvested. On the same day, fresh medium only was added to some cultures, which served as controls. Processing CMC Extracts for SDS-PAGE: The CMC were processed for SDS-PAGE according to Lewis et al. (19,20) as described in Muros et al. (14). SDS-PAGE and Densitometric Analysis of CMC Extracts: The proteins in the Triton X-100- and SDS-soluble fractions (15 Ixl, 5 mg/ml) were separated by 12% SDS-PAGE (Mini Protean II Cell, BioRad) at 60 mA for 1 h at room temperature, stained with Coomassie brilliant blue R-25, and destained (21) (all reagents BioRad). The gels were dried on filter paper (Whatman) with a BioRad 583 drier. Once dry, the gels were laid in plastic film, and the protein bands in the different lanes were analyzed by densitometry at 540 nm using a slit width of 4 mm (Beckman Appraise) and an integral computing device. Immunobl0tting: After electrophoretic separation, the proteins were transferred onto nitrocellulose paper (22). The blots were treated with blocking solution (20 mM Tris, 0.9% NaC1, 10% nonfat milk) and reacted with a 1:200 dilution of anti-tropomyosin (Clone TM228, Sigma), actin (Clone 5C5, Sigma) desmin (Clone DE-U-10, Sigma) or vimentin (Clone V9, Sigma) monoclonal antibodies. To visualize specific antigen-antibody reactions we used an AGR-HRP immunoblot enzyme assay kit and protocol from BioRad (Fig. 1). The immunoblots band were analyzed by densitometry at 540 nm using a slit width of 4 mm (Beckman Appraise) and a integral computing device. Statistical Analysis: All data of the densitometric angdysis are means + SEM of four measurements. Student's t test was used to analyze the differences between treated and untreated CMC. A difference at the level P<0.05 was considered statistically significant.

Vol. 54, No. 3, 1994

Modulation of Contractile Proteins

173

Northern Blot Analysis: Total cellular RNA from control and treated HH29 and HH39 CMC was isolated by the guanidinium isothiocyanate method (23). RNA samples (40 Ixg per lane) were separated by electrophoresis through 1% agarose gels containing 18% formaldehyde. After transfer to nylon membranes (Boehringer Mannheim), RNA blots were prehybridized at 42 ° C for 3-4 h. The blots were hybridized with probe for 18 h at 42°C and washed three times at 68 ° C in 2XSSC, 0.1% SDS, and then twice at 68°C in 0.1XSSC, 0.1% SDS. The filters were incubated with anti-DIG AP conjugate for 30 min (75 mU/ml, anti-digoxigenin-AP-conjugate) (Boehringer Mannheim), then with the substrate AMPPD (10 mg/ml) (3-(2'-spiroadamantane) 4methoxy-4-(3"-phosphoryloxy)-phenyl-l,2 dioxietane) (Boehringer Mannheim) for 15 min at 37 -0C. The blots were exposed to X-ray film for at least 2 h. As the probe we used full-length cDNA for chick heart ~-tropomyosin, a 1050 bp Eco RI insert from the KS plasmid (courtesy of Dr. S. Hughes of the Cancer Research and Development Center in Frederik, Maryland). The DNA was labeled with DIG (digoxigenin- 11-dUTP) (Boehringer Mannheim).

g 7 ,,,,-rot

"

ee--

t__.

4

-,~AC o,,

i-,~1"11

3 1 mmmmP~

"

i I

II

III

IV

V

I

Vl

FIG. 1 A representative selection of immunobloting bands used in densitometric analyses of tropomyosin, actin, desmin and vimentin proteins in cultured myocardial cells treated with TPA, INF or DAG-B. Lane I, molecular weight control; Lane II, actin in Triton X100-soluble cytoplasmic fraction treated with INF; Lane III, Tropomyosin in Triton X100-soluble cytoplasmic fraction treated with TPA; Lane IV, desmin in SDS-soluble cytoskeletal fraction treated with INF; Lane V, vimentin in SDS-soluble cytoskeletal fraction treated with TPA; Lane VI, desmin in SDS-soluble cytoskeletal fraction treated with DAG-B. Results Total proteins: H H 2 9 (Table 1): In control cultures the total protein concentration in cellular homogenate was 1.014 mg/ml (0.790 mg/ml in the citoplasmic (CP) and 0.276 mg/ml in the cytoskeletal (CS) fraction). In cultures treated with AZA, total protein in the cellular homogenate increased 61% with respect to the control value (P<0.001). The increases in the CP and CS fractions were 44% and 17% of their respective control values (both P<0.001). Treatment with INF also significantly raised total protein content in the whole cell homogenate by 52% (p<0.001) in comparision with the control culture. The corresponding increases in the CP and CS fractions were 31% and 16% (both P<0.001). Total protein in cultures treated with TPA increased 25% (P<0.001), with increases in the CP and CS fractions of 6% and 8% respectively (both P<0.001). Treatment with DAG-A and DAG-B led to increases in total protein content due to increases in

174

Modulation of Contractile Proteins

Vol. 54, No. 3, 1994

cytoplasmic protein levels only. A slight rise in total protein (20%) was observed after exposure to DAG-A; this change reflected a 24% increase in CP protein concentration together with a 4% decrease in CS protein. The increase in protein content in the whole cell homogenate of cultures treated with DAG-B was 34%, representing a 16% rise in CP protein and a 10% decrease in CS protein content (Table 1). H H 3 9 : (Table2): In control cultures at HH39, total protein concentration in the cellular homogenate was 1.186 mg/ml. The corresponding concentrations in the cytoplasmic and cytoskeletal fractions were 0.988 and 0.601 mg/ml.

In cultures treated for 24 h with TPA, AZA, INF, DAG-A or DAG-B, total proteins decreased in the homogenate and in both subcellular fractions. Azacytidine decreased total protein by 43% in the homogenate (0.671 mg/ml) in comparision to the control culture (P<0.001). In the cytoplasmic fraction, total protein decreased by 51% (0.478 mg/ml) (P<0.001). A reduction of 47% in total protein concentration was found in the homogenate after treatment with DAG-A (0.625 mg/ml) (P<0.001). In the cytoplasmic fraction, the decrease was 66% (0.333 mg/ml) (P<0.001), and in the cytoskeletal fraction, there was a 45% drop (0.326 mg/ml) (P<0.001). In cultures treated with TPA, the decreases in total protein in the cytoplasmic and cytoskeletal fractions were 3% (0.951 mg/ml) and 13% (0.522 mg/ml) (both P<0.05) respectively. Thin Filament Proteins: H H 2 9 (Table 3): In untreated control cultures, TM content was 0.145 gg/I.tl (11.74% for immunoblotting) in the CP fraction and 0.245 p.g/gl (19.82% for immunoblotting) in the CS fraction. The corresponding values for actin concentration were 0.285 gg/~tl (23.06% for immunoblotting) in CP and 0.535 gg/gl (43.29% for immunoblotting) in CS. For both proteins, the CP/CS ratio was approximately 0.5. All cultures treated with TPA, INF or AZA showed increases in contractile protein concentration (actin, and to a lesser extent TM) in the CP fraction, accompained by a decrease in the CS fraction. Exposure to INF led to a 50% increase in actin in the CP fraction (P<0.001), with a 15% drop in the CS fraction. A significant 44% rise in TM concentration (P<0.001) was noted in the CP fraction of cultures treated with TPA, whereas this drug decreased the CS concentration of TM by 11% in comparision to the control value. This drug also significantly increased CP actin by 43% (P<0.001), and reduced CS actin by 18%. Both A and B forms of DAG decreased the amounts of TM and actin in both subcellular fractions, although the decrease in TM was smaller after treatment with DAG-A. H H 3 9 (Table 4): In control cultures of cells from HH39 embryos, tropomyosin concentration reached 0.235 ~tg/I.tl (19.01% for immunoblotting) in the cytoplasmic fraction, and 0.390 I.tg/I.tl (31.55% for immunoblotting) in the cytoskeletal fraction. Actin concentrations in the two subcellular fractions were 0.640 p_g/~tl (51.68% for immunoblotting) and 0.530 I.tg/I.tl (42.88% for immunoblotting) respectivaly.

Treatment with AZA reduced tropomyosin concentration by 55% in the cytoplasm (P<0.001), and by 65% in the cytoskeleton (P<0.001). Actin was reduced in the two subcellular fractions by 58% and 75% (both P<0.001) respectively. In cells incubated with TPA, tropomyosin concentration decreased by 19% in the cytoplasm (P<0.05), and by 73% in the cytoskeleton (P<0.001). This drug also reduced actin in the two subcellular fractions by 38% and 59% (both P<0.001) respectively. The reductions in tropomyosin concentration caused by INF treatment were 51% in the cytoplasmic fraction, and 61% in he cytoskeletal fraction (both P<0.001). Actin concentration decreased by 37% in the cytoplasm, and increased by 32% (both P<0.001) in the cytoskeleton.

Table 1

CP

0.988 + 0.009

-56%

CP: Cytoplasmic fractions; CT: Cytoskeletal fractions; mf: : relative percentage modification

mf -26%

0.443 4- 0.022 c

mf -4.5%

0.326 4- 0.017 c

mf

0.262 4- 0.013 c

mf 0%

0.612 +, 0.022 ns

mf -13%

0,522 +, 0,017II

0,601 + 0.014

with respect to controls, calculated as protein concentration after treatment x 100/protein concentration of CMC untreated,

mf -62%

0.372 + 0.021 c

raf -66%

0.333 +, 0.010c

m/ -51%

0.478 +- 0.020c

mf -53%

0.458 +_0.018c

mf -3%

0,951 + 0.009a

CS

mg/ml

with respect to controls, calculated as protein concentration aftei" treatment x 100/protein concentration of CMC untreated.

mf -29%

0.838 + 0.010 c

mf -47%

0.625 +, 0.025 c

m/ -43%

0.671 +, 0.007c

CP

mg/ml

CP: Cytoplasmic fractions; CT: Cytoskeletal fractions; mf: : relative percentage modification

DAG-B

0.012c

mf -24%

0.898 +

my -8%

1.085~+ 0.009ns

1.186 + 0.0l I

mg/ml

Homogenate

All data are means + SEM of four measurements. All data were compared with the control

mf -10%

0.249 _+ 0,005 b

raf -4%

DAG-A

AZA

INF

TPA

Control

Drug

cytoskeletal fractions at HH39 sta~e.(Lowry assay)

culture using comparision of the means: ns nonsignificant; a p<0.05; b p<0.01; c p<0,001:

mf 16%

0.923 +_ 0.002 c

mf 2 4 %

17%

0.266 + 0.006 ns

mf

0.321 +_0.003c

mf 16%

0.324 +_ 0,006 c

my 8%

0.300 +_0.006b

0.276 4- 0.005

(INF), 5-azacytidine (AZA), l-oleoyl-2-acetyl-rac-glycerol (DAG-A) y 1,2-dicapryloyl-raeglycerol (DAG-B) on the amount of total protein in CMC homogenate, and in cytoplasmic and

All data are means + SEM of four measurements. All data were compared with the control

mf 34%

1.363 +_ 0.003 c

mf 2 0 %

0.983 +_ 0.003 c

mf 44%

mf 61%

1.224 + 0.005 ns

1.140 + 0.010c

mf 31%

m/ 52%

1.634 +_0.005c

1,035 +_ 0.006 c

1.544 +. 0.006c

CS mg/mI

Table 2 Effect o f treatment with 12-0-tetradecanoyl-phorbol-13-acetate (TPA), gamma-interferon

culture using comparision of the means: ns nonsignificant; a p<0.05; b p<0.01; c p<0.001.

DAG-B

DAG-A

AZA

INF

mf 6%

mf 2 5 %

0.790 +. 0.010

0.842 4- 0.007b

1.014 + 0.0(~6

1.276.+, 0.003c

mg/mI

Control

mg/ml

Homogenate

TPA

Drug

cytoskeletal fractions at HH29 sta~e.(Lowry assay)

glycerol (DAG-B) on the amount of total protein in CMC homogenate, and in cytoplasmic and

(INF), 5-azacytidine (AZA), l-oleoyl-2-acetyl-rac-glycerol (DAG-A) y 1,2-dicapryloyl-rac-

Effect of treatment with 12-0-tetradecanoyl-phorbol-13-acetate (TPA), gamma-interferon

O

o

.o

< .o

17.79 + 0.06 mf -11%

0.175 4- 0.010c 14.17 + 0.06 mf 20%

0.115+0.020b 9.31 ± 0.05 mf -51%

mf -19%

15.38 ± 0.05

0.190.+ 0.015a

* 0.235 :L 0.015ns • * 19.01 +0.04

DAG-B

Actin

mf -.50~

0.315 + 0.007c 25.47+0.05

my -50% 0.225 ± 0.015c 18.20.+0.06 mf -42%

0.315",0.007c 25.47 ± 0.07

mf -39%

0.265+0.010c 21.44 :L 0.06 my -58%

0,400 ± 0.010c 32.36 .i- 0.03 mf -37%

mf -38%

31.96 ±0.03

0,395 ± 0.010e

0.640 + 0.018 51.68 .t: 0.06

CP

0.235 :t. 0.010c 19.01 + 0;05

mf .65%

0.135+0.007c 10.93 ± 0.02

0.150 ± 0.010c 12.14 + 0.04 mf .61%

mf -73%

8.50 ± 0.06

0.105.+ 0,013c

0.390:1:0.010 31.55 + 0.07

CS

quantified in

my -~la

0.320 ± O.OlO c 25.87+0.07

my .22qb

0.410 + 0.015b 33.16 + 0.06

0.130 .t 0,012c 10.53 ± 0.05 my .75%

0,700 + 0,015¢ 56.63 + 0205 my 32%

mf -59%

17.38 ± 0.06

0.215 ± 0.017c

0.530 + 0.021 42.88 :t: 0.0~

CS

comparison of the means: ns nonsignificant, a p<0.05, b p<0.01,c p<0.001. CP: Cytoplasmic fractions; CT: Cytoskelctal fractions.; mr: relative percentage modification with respect to

the bands obtained by immunoblotting. All data were compared with the control culture using

comparison of the means: ns nonsignificant, a p<0.05, b p<0.01, c p<0.001. CP: Cytoplasmic

fractions; CT: Cytoskeletal fractions.; mf: relative percentage modification with respect to controls.

controls.

band, divided by 100. Data designed ** are percent values of protein content calculated frorn the bands obtained by immunoblotting. All data were compared with the control culture using

band, divided by 100. Data designed ** are percent values of protein content calculated from

used for SDS-PAGE (~g/Al) x densitometric value of the tropomyosin- or aetin- containing

mf 0%

* 0.230 ± 0.015ns • * 18.60 + 0.03 my -2%

* 0.105 ± 0.015c 8.50 + 0.04 my -55%

• *

* •*

* •*

0.235 ::L 0.015 19,0l ± 0.04

Tropomyosin

DAG-A

AZA

INF

TPA

* •*

CP

used for SDS-PAGE (pg/p.l) x densitometric value of the tropomyosin- or actin- containing

0.360 ± 0.002c 29.12 + 0.08 mf -33%

0.395 ± 0,002 c 31;95±0.09 mf -27%

mf .29%

0.380:1:0.005 c 30.74 ± 0.07

0.455 .t: 0.007 c 34.79 :t. 0.06 my -15%

mf -18%

0,440 ± 0,007c 35.60 ± 0.08

Control

Drug

p.g/pl from the densitomettic values, according to the formula: amount of protein in the sample

0.275 + 0.007ns 22.24+0.07 mf -3%

mf -20%

0.225 ± 0.003C 18,19±0.06

0.405 + 0.005c 32.76 ± 0.08 mf 42%

0.430 + 0,003c 36.50 .'L0.05 mf 50%

33.17:1:0.04 mf 43%

0.410 ± 0.007 c

0.535 + 0.010 43.29 :t. 0.07

CS

All data are means + SEM of four measurements. Tropomyosin and aetin were

0.225 + 0.002a 18.20.+0.05 mf -8%

0.215 + 0.012a 17.39±0.06 mf -13%

Actin

0.285:1:0.002 23.06 ± 0.05

CP

p.g/pl from the densitometric values, according to the formula: amount of protein in the sample

mf -10%

* 0.I 35 + 0.009a • * 10.92+0.03

mf -4%

* 0.140 +_0.010as • * 11.33+_.0.05

0.210 .-L0.002b 16.99 + 0.03 mf -15%

0.210 + 0.010a 16.99 +_0.03 mf -15%

0,220.± 0,007 a

mf 44%

16.98 ± 0.09

0.245:1:0.007 19.82 +_0.08

0.210.+ 0.002 c

* 0.185 ± 0.002a 14.98 + 0.06 mf 27%

• *

• *

*

• *

*

* 0.145 + 0.002 • * 11.74 +_0.05

CS

All data are means +_. SEM of four measurements. Tropomyosin a n d actin were quantified in

DAG-B

DAG-A

AZA

INF

TPA

Control

Tmpomyosin

cytoskeletal fractions at HH29 staee.

CP

glycerol (DAG-B) on the contractile proteins (tropomyosin and aetin) in cytoplasmic and cytoskcletal fractions at HH39 sraee.

glycerol (DAG-B) on the contractile proteins (tropomyosin and actin) in cytoplasmic and

Drug

(INF), 5-azacytidine (AZA), l-oleoyl-2-acetyl-rac-glycerol (DAG-A) y 1,2-dicapryloyl-rac-

(INF), 5-azacytidinc (AZA), l-oleoyl-2-acetyl-rac-glycerul (DAG-A) y 1,2-dicapryloyl-rac-

Table 4 Effect o f treatment with 12-0-tetradecanoyl-phorbol- 13 acetate (TPA), gamma-interferon

Table 3

Effect of treatment with 12-0-tetradecanoyl-phorbol-13 acetate (TPA), gamma-interferon

Table 5

0.110+0.002 8.91 + 0.03

*

Control

*

• *

0.385 _+ 0,002 c 3 I.14 + 0.08

mf 220%

0.I 25 4. 0.002 b I0.12 + 0.02

mf 13%

0.205 ± 0.005c 16.59 + 0.05 mf 70%

mf 120%

0.265 + 0.006c 21.44 + 0.07

m/ 133%

0.280 + 0.007c 22.65 + 0.06

0.215.+0.007c 17.404.0.06 mf 79%

0.1204-0.002 9.72 + 0.05

CS

35%

mf 48%

0.275 4. 0.002 c 22.25 + 0.07

mf

0.250 4. 0.005c 20.23 4. 0.07

mf 91%

0.355 + 0.013 c 28.72 + 0.08

mf 102%

0.375 4. 0.002 c 30.34 4. 0.0~

0.280 + 0.005c 22.664-0.07 mf 51%

mf 15%

0.220 4. 0.028c 17.80 4. 0.06

mf 31%

0.250 4. 0.008 c 20.23 4. 0.04

mf 31%

0.250 4. 0.005 c 20.23 4. 0.05

0.490 4. 0.018c 39.65+0.08 mf 157%

0.190+0.007 15.38 4. 0.07

CS

mf 13%

0.215 4. 0.002 b 17.40 4. 0.05

Vimenfin

0.185+0.002 14.98 4- 0.05

CP

Desmin

* •*

0.135 + 0.012 c 10.93+0.03 mf .41%

* 0.165 + 0.013b • * 13.12+0.04 ,n/ -28%

* 0.160+0.013 c • * 12.95 + 0.06 mf -30%

• *

0.400+0.015 c 32.36 +_0.06 mf 73%

0.i 70 4. 0.002 c 13.76+0.06 mf -55%

0.125 4. 0.005c 10.12 ± 0.06 ,,¢"-67%

0.160 ± 0.005c 12.95 + 0.07 mf -58%

0.477+0.012 b 22.65 + 0.06 mf 24%

mf -40%

mf 0% *

18.60+0.05

18.60 .t- 0 . 0 6

•*

0.160 4. 0.022ns 12.95+0.05

mf 10%

mf -34%

m/417%

0.750 + 0.010c 60.67+0.07

0.160 4. 0.015a$ 12.95 4- 0.06 mf 3%

0.305:1:0.017c 24.66 4. 0.05 0~" 106%

mf 68~

39.654-0.08

0.245 4. 0.015 b

15.38 4. 0.06

0.145 + 0.016

CS

0.160 + 0.012 a 12.954-0.05

0.200 + 0.0lSnS 16.174.0.05 mf .iS%

0.240 + 0.010ns 19,41 + 0.05 mf -2%

0.450 4. 0.015c 36.40 4. 0.07 mf 83%

mf 26%

25.07+0.07

0.310 ± 0.015 a

19.82 + 0.05

0.230.+ 0.012 c

0.245 4. 0.033

Vimentin

0.385 + 0.002

CP

31.14 + 0.04

0.230 + 0.008 ns

18.60 .'L 0.02

CS

*

•*

* 0.230 + 0.010

CP

comparison of the means: ns nonsignificant,a p<0.05,b p<0.01 c p<0.001. CP: Cytoplasmic fractions; CT: Cytoskeletal fractions.; mf: relative percentage modification with respect to controls.

of the means:as nonsignificant,a p<0.05 b p<0.01 c p
used for SDS-PAGE (p.g/p.l) x deasitometfic value of the desmin- or vimentin- containing band, divided by 100. Data designed ** are percent values of protein content calculated from the bands obtained by immunoblotting. All data were compared with the control culture using

All data are means _+. SEM of four measurements. Desmin and vimentin were quantified in pg/p.l from the densitometric values, according to the formula: amount of protein in the sample

DAG-B

DAG-A

AZA

INF

TPA

Control

Drug

and cytoskeletal fractions at HH39 sta~e.

grycerol (DAG-B) on the intermediate filament proteins (desmin and vimentin) in cytoplasmic

(INF), 5-azacytidine (AZA), l-oleoyl-2-acetyl-rac-glycerol (DAG-A) y 1,2-dicapryloyl-rac-

Table 6 Effect of treatment with 12-0-tetradecanoyl.phorbol- 13-acetate (TPA), gamma-interferon

fractions; CT: Cytoskeletal fractions.; mr: relative percentage modification with respect to controls.

com~

the bands obtained by immunoblotting. All data were compared with the control culture using

band, divided by 100. Data designed ** are percent values of protein content calculated from

used for SDS-PAGE (p.g/I.tl) x densitometric value of the desmin- or vimentin- containing

All data are means _+ SEM of four measurements. Desmin and vimentin were quantified in ttg/~l from the deasitometric values, according to the formula: amount of protein in the sample

DAG-B

mf -5%

mf 81%

* 0.105 + 0.004ns • * 8.50 4. 0.03

DAG-A

0.200 4. 0.007 c 16.18 ± 0.05

* •*

mf 90%

AZA

• *

*

INF

0.2 I0 + 0.003 c 16.99 -t-0.07

* 0.125 + 0.007a • * 10.12+0.05 mf 13%

TPA

•*

CP

Drug

Desmin

and eytoskeletal fractions at HH29 staee.

glycerol (DAG-B) on the intermediate filament proteins (desmin and vimentin) in cytoplasmic

(INb'), 5-azacytidine (AZA), l-oleoyl-2-acetyl-rac-glycerol (DAG-A) y 1,2-dicapryloyl-rac-

Effect of treatment with 12-0-tetradecanoyl-phorbol-13-acetate (TPA), gamma-interferon

o

o=

o

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Both contractile proteins were decreased in both subcellular fractions after treatment with either DAG-A or DAG-B. Intermediate Filament Proteins: H H 2 9 (Table 5): In control cultures, CP and CS desmin concentrations were 0.110 gg/l.tl (8.91% for immunoblotting) and 0.120 gg/I.tl (9.72% for immunoblotting) respectively. The corresponding control values for CP and CS vimentin were 0.185 gg/gl (14.98% for immunoblotting) and 0.190 (15.38% for immunoblotting). For both intermediate filament proteins, the CP/CS ratio was approximately 1. Treatment with TPA, INF, AZA or DAG-B increased desmin and vimentin concentrations in both the CP and the CS fractions of treated cells (Table 5). Desmin increased by 90% in the CS fraction and 133% in the CP fraction (P<0.001 in both subfractions) after INF treatment. Vimentin concentration in the CP and CS fractions increased by 51% and 157% respectively in cultures treated with TPA (P<0.001). Exposure of cultured myocardiocytes to DAG-B led to increases of 220% and 13% in the CS and CP concentrations of desmin?, (P<0.001 in comparison to control values in both subfractions). H H 3 9 (Table 6): In untreated control cultures at HH39, the concentration of desmin reached 0.230 l.tg/I.tl (18.60% for immunoblotting) in the CP fraction, and 0.385 I.tg/gl (31.14% for immunobloting) in the CS fraction. The corresponding values for vimentin were 0.245 I.tg/gl (19.82% for immunoblotting) and 0.145 ~tg/gl (15.38 for immunoblotting).

Treatment of cultured myocardial cells with TPA, AZA, DAG-A or DAG-B decreased desmin concentrations to different degrees in both subcellular fractions. In contrast, treatment with INF increased desmin by 73% and 24% in the CP and CS fractions (P<0.001 in both cases). Vimentin increased in the CS fraction, but decreased in the CP fraction, after cell cultures were treated with AZA, DAG-A or DAG-B. The greatest increase in the cytoskeleton (417%) was caused by DAG-A (P<0.001). The drugs TPA and INF increased vimentin concentrations by 26% (P<0.05) and 68% (P<0.01) respectively (TPA), and by 83% and 106% (INF) (P<0.001 in both cases), in the cytoskeletal and cytoplasmic fractions. g-Tropomyosin mRNA Levels in Control and Treated CMC: To determine whether posttranscriptional repression of c~-TM occurred in control or treated myocardiocytes at I-IH29 or HH39, a cDNA probe was used to measure the c~-TM total RNA. Myocardiocyte cultures from untreated HH29 and HH39 embryos expressed cardiac 0ttropomyosin (Fig. 2). The amount of total RNA was similar in all lanes, as show by measurements in samples incubated with ethidium bromide and visualized under ultraviolet light. Levels of a - T M mRNA were higher in HH29 myocardiocytes than in control cells after 24 h of treatment with TPA, INF or AZA; however, DAG led to slightly lower mRNA concentrations in treated than in untreated cells. All treatments led to higher a - T M mRNA levels in HH39 myocardiocytes in comparison with control cultures (Fig. 2). Discussion The differentiating and dedifferentiating effects of a number of drugs have recently been studied in many cell lines. For example, Alvarez et al. (2), Pedrinaci et al., (5) and Rainer and Feinberg (3) have investigated the dedifferentiating effects of TPA, AZA, and DAG in tumoral and embryonic cell lines. Interferon, on the other hand, has often been considered a cell differentiating agent (4). The effects of TPA and other dedifferentiating drugs on the concentration of total proteins have been analyzed in cell cultures of different species (8). We found increases in HH29 of the

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A

.. TM

B

TM

FIG.2 Northern blot analysis of ~-tropomyosin mRNA in control and treated myocardiocytes. Lane A, HH29; Lane B, HH39. RNA (40 ~tg/lane) was blotted after electrophoresis and probed as indicated in Methods. Control: untreated myocardiocytes; TPA: treated with 100 ng/ml 12-o-tetradecanoylphorbol-13-acetate in 0.02% dimethylsulfoxide (DMSO); AZA: treated with 10 ~M 5-azacytidine; DAG-A y B: treated with 1-oleoyl-2-acetyl-racglycerol or 1,2-dicapryloyl-rac-grycerol at a concentration of 50 ~g/ml in 0.01% ethanol. INF: treated with 100 U/ml of gamma interferon.

concentration of total protein in the cellular homogenate, a finding in line with the observations of Allo et al. (2) in rat myocardiocytes. Claycomb and Moses (8) reported similar increases in protein content in adult rat cardiac cells treated with TPA or DAG, along with identical morphological alterations. In contrast, the concentration of total proteins in CMC at HH39 decreases when TPA, AZA, INF, DAG-A or DAG-B is added to the culture medium. This effect is the opposite of that found in more immature myocardiocytes at HH29, in which the addition of these modulators significantly increased total protein in comparison with untreated control cultures. We also analyzed the subcellular modifications in total protein concentrations after the addition of these drugs to the culture medium. In chick embryo myocardiocytes at HH29 TPA increased protein concentration by similar amounts in both fractions (6% in CP and 8% in CS). Treatment with AZA or INF led to slightly larger increases in the CP than in the CS fraction (44% after AZA, 31% after INF). The addition of DAG-A or DAG-B to the culture medium led to increases in cytoplasmic concentrations of proteins andto decreases in cytoskeletal levels. These findings suggest that DAG have dedifferentiating effects in chick embryo myocardiocytes, as cellular differentiation is accompained by increased protein content in mainly the CS subfraction (25). Holterz et al., (26) noted that TPA inhibited the synthesis and stimulated the break-down of the muscle-specific proteins myosin, 0~-actin, troponin and TM in skeletal muscle cell cultures.

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Claycomb and Moses (8) showed that TPA completely disrupted myofibrillar organization in adult rat myocardiocytes. These observations are in consonance with the decrease we found in TM and actin in the CS subfraction. Tropomyosin and actin decreased (P<0.05) in this fraction, and increased in the CP fraction, after the addition of TPA, AZA or INF to the culture medium; these changes altered of the normal pattem of subcellular distribution of these thin filament proteins with respect to control cells. The CP/CS ratio also reflected these modifications, changing from approximately 0.5 in untreated cells to approximately 1 in cells exposed to TPA, AZA or INF, indicating that the relative proportions of these proteins in the two subfractions became nearly equal in response to treatment. Our findings illustrate changes in the intracellular compartmentalization of TM and actin, rather than modifications in the actual amounts of these proteins. This may account for the observation, by Schliwa et al., (27) of a rapid redistribution of actin (and vinculin) in BSC-1 epithelial cells treated with TPA. Manjarrez et al., (28) have described dissociation of myosin from the cytoskeleton toward the cytplasm in human epithelial cells treated with TPA. In general, the increases in tropomyosin are less marked than the changes in actin in both the CP and CS fractions. The addition of DAG to the cultures decreased the amounts of actin and tropomyosin in both subcellular compartments, although the changes in tropomyosin were slightly smaller than the modifications in actin. Treatment of fetal chick myocardiocytes at HH39 with TPA decreased the concentration of the thin filament proteins tropomyosin and actin in both the CP and CS subfractions, suggesting that this drug alters the myofibril at the level of the thin filaments. Our results differ from those of Lin et al. (29), who found that phorbol esters did not alter myofibril integrity in chick myocardiocytes. Other studies in different cell lines have reported TPA-caused alterations in specific muscle proteins. Manjarrez et al. (28) observed dissociation of cytoskeletal myosin in the cytosol of human epithelial cells, and Sell et al. (30) described changes in the organization of actin filaments in response to phorbol ester treatment of cultures of muscle cells from rat aorta. The different effects at HH29 and HH39 suggest that the response of embryonic chick myocardiocytes to this drug depends on the degree of differentiation reached before treatment. These findings are compatible with those of Lin et al., (29), who noted that different phenotypical responses to TPA were dependent on the differentiation program of the cell prior to treatment. Deamond and Bruce (31) reported an inverse correlation between the age of the animal from which the cells were obtained and the morphological and proliferative response of fibroblasts in vitro to TPA. In our cultures of embryonic myocardiocytes, actin and tropomyosin were more markedly altered in the more mature HH39 cells, decreasing in both the cytoplasm and cytoskeleton. The decrease in tropomyosin and actin concentrations after treatment with AZA at HH39 suggests that this drug, like phorbol esters, is able to disorganize myocardiocyte thin filaments. In this connection, Alvarez et al. (2) found that AZA caused phenotypical alterations in MeWo melanoma ceils, and Rainier and Feinberg (3) showed treatment with this drug to induce transformation in many rodent cells lines and primary epithelial cells. Adequate expression of thin filament proteins 0t-tropomyosin and acfin is fundamental for the correct development of the contractile apparatus of cultured myocardiocytes.Because of the close relatioship between these two proteins, and because the changes in ~-TM concentrations were less marked than the modifications of actin levels in cells from both HH29 and HH39, regardless of the drug added to culture, we tested whether the levels of ot-TM mRNA also changed under different experimental conditions, ie, whether post-transcriptional of this protein occurred. Our results conf'u'med that the modifications found with SDS-PAGE and densitometry in cellular ¢zTM after treatment with AZA, TPA, INF and DAG corresponded to changes in mRNA, thus ruling out post-transcriptional repression. Treatment of embryonic myocardiocytes at HH29 with TPA increased ~-TM mRNA in comparison with the concentration in control cells, a finding in consonance with the observations of Shubeita et al., (32) who found that treatment with this drug increased the expression of the gene for myosin ligth chain in myocardiocytes of newborn

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rats. When we analized tx-tropomyosin expression in HH39 myocardiocytes treated with TPA, and found levels of total RNA transcription for this protein to be lower than in untreated control cells.This suggested that TPA acted at the transcriptional level, decreasing the expression of the tx-tropomyosin gene in fetal myocardiocytes. Our results are compatible with the findings of Claycomb et al. (7), who found this drug to activate genes involved in cellular proliferation (c-myc, c-abl, and c-src), and to inactivate genes involved in cellular differentiation (MHC, M-CK) in adult rat myocardiocytes. Northern blot analyses showed that treatment with DAG-A or DAG-B reduced cardiac cz-tropomyosin total RNA in HH 39 and HH29. Claycomb and Moses (8) found that these chemicals induced morphological and ultrastmctural changes similar to those observed after TPA treatment in cultured myocardiocytes. Mature myocardiocytes treated with INF or AZA contained lower levels of cardiac oc-tropomyosin total RNA in comparison with control cells. If we consider tx-tropomyosin as a marker of phenotypical differentiation in cultured muscle cells (33), INF apparently did not show, in embryonic myocardiocytes, the differentiative ability it has displayed in other cell lines. Our findings are similar to those of Aboud et al. (34), who found that INF treatment did not activate expression of the myosin heavy chain gene (MHC) in fibrosarcoma cell lines. In addition, gamma interferon has also been found to inhibit in vitro differentiation of mouse 2T2-LI cells in adipocytes, and of human monocytes in macrophages (35). Treatment with TPA, AZA, INF or DAG (A or B) increased the concentration of the intermediate filament proteins desmin and vimentin in both subcellular fractions at HH 29, leading to the accumulation of these proteins in embryonic chick myocardiocytes. These findings are in agreement with the observations of Holtzer et al. (26), who reported the accumulation of intermediate filament proteins in other types of cells treated with TPA. Claycomb and Moses (8) have reported that TPA induced a reactivation of DNA synthesis in adult rat myocardiocytes, along with a reappearance of the embryonic pattern of development. In the present study vimentin, one of the predominant intermediate filament proteins in the early stages of heart development (16), was especially affected by TPA, increasing by 51% in the CP and 157% in the CS fraction of treated cells. These changes may reflect a return to a less differentiated state in response to TPA. The most significant effects of treatment with INF or AZA were noted in the concentration of desrnin, both in the cytoplasm (increases of 90% and 81% respectively) and in the cytoskeleton (increases of 133% and 120%). The accumulation of desmin, a protein that characterizes more advanced stages of chick heart development (16), suggests that these drugs exert a differentiating effect when added to the cell culture. Other authors have noted similar effects in both mature and embryonic cells: Campbell et al. (36) reported an increase in the synthesis of HLA A, B and C proteins in human pancreatic beta cells treated with INF, and Ballard et al., (37) noted enhanced production of pulmonary surfactant protein by embryonic lung cells exposed to this drug. Our findings with regard to intermediate filament proteins in chick myocardiocytes at HH39 differed from those in more immature (HH29) cells, in that there was no significant accumulation of desmin or vimentin. In contrast, desmin in both subcellular fractions of HH39 cells decreased after treatment with TPA, AZA, DAG-A or DAG-B. That the effects of these drugs on HH39 cells were the opposite of those found In HH29 is in consonance with the hypothesis that the effects of these substances depend on the degree of differentiation reached by the cells prior to treatment (31, 38). In summary, the drugs TPA, AZA, INF, DAG-A and DAG-B significantly modified the concentration of contractile proteins in cultured embryonic chick myocardiocytes, affecting thin filament (actin and tropomyosin) and intermediate filament proteins (desmin and vimentin). Treatment of HH29 myocardiocytes with TPA, AZA or INF increased TM and actin concentrations in the cytoplasmic fraction, and decreased these proteins in the cytoskeletal

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fraction. However, treatment with DAG decreased both proteins in both subcellular fractions of HH29 myocardiocytes. The effects of the drugs differed depending on the developmental stage of the cells at the time of harvest. In HH39 myocardiocytes, decreases in the concentration of thin filament proteins were accompanied by reduced levels of transcription of the gene for cardiac oHropomyosin. The drugs also had different effects on the concentration of intermediate filament proteins in HH29 and HH39 myocardiocytes. Acknowledgments This study was partially supported by the Fondo de Investigaciones Sanitarias through Proyect no. 90/0849. We thank Ms Karen Shashok for the translating the original manuscript into English. References 1. V. HAMBURGER and H.L. HAMILTON, J. Morphol. 15 245-260 (1951). 2. E. ALVAREZ, B.E. ELLIOT, A.N. HOUGHTON and R.S. KERBE, Cancer Res. 48 24402445 (1988). 3. S. RAINER and A.P. FEINBERG, Proc. Natl. Acad. Sci. 85 6384-6388 (1988) 4. B.Y. RUBIN and S.L. GUPTA, Proc. Natl. Acad. Sci. 10 5928-5932 (1980). 5. S. PREDINACI, C. HUELIN, M. PATARROYO, F. RUIZ-CABELLO and F. GARRIDO, Hybridoma 8 13-19 (1989). 6. A. GESCHER, Biochem. Pharm. 34 2587-2592 (1985). 7. W.C. CLAYCOMB and R.L. MOSES, Dev. Biol. 127 257-265 (1988). 8. W.C. CLAYCOMB, Biology of isolated adult cardiac myocytes, W.A. Clark, p.281, Elsevier, New York (1988). 9. C. DE GIOVANI, P. NANNI, G. NICOLLETI, C. CECCARELLI, K. SCOTLANDI, L. LANDUZZI and P.L. LOLLINI, Anticancer Res.9 1943-1949 (1989). 10. B.T. NIXON and H. GREEN, Proc. Natl. Acad. Sci. 85 3429-3432 (1988). 11. E.D. KORN, Physiol. Rev. 62 672-737 (1982). 12. E. EISENBERG and T.L. HILL, Science 22__27999-1006 (1985). 13. J. PRADOS, J.E. FERNANDEZ, F. GARRIDO, L. ALVAREZ, R. HIDALGO and M.A. MUROS, Anat. Rec. 234 302-309 (1992). 14. M.A. MUROS, A.E. ARANEGA, C. VELEZ, F.J. GONZALEZ, J.E. FERNANDEZ, L. ALVAREZ and A. ARANEGA, Cell. Biol. Int. Rep. 16 451-463 (1992). 15. E. LAZARIDES, Nature 283 249-256 (1980). 16. C. VELEZ, M.A. MUROS, A.E. ARANEGA, J.E. FERNANDEZ, F.J. GONZALEZ, L. ALVAREZ and A. ARANEGA, Act. Anat. 139 226-233 (1990). 17. G.S. BENNET, J.M. CROOP and J.J. OTTO, Motility in Cell Function New York, p.324, Academic press, New York (1985). 18. F.J. GONZALEZ, A.E. ARANEGA, A. LINARES, J.E. FERNANDEZ, M.A. MUROS, C. VELEZ, L. ALVAREZ and A. ARANEGA, Life Sci.48 1091-1099 (1991). 19. W. LEWIS, N.L. PERILLO and B. GONZALEZ, J. Lab.Clin. Med. 112 43-51 (1988). 20. W. LEWIS and B. GONZALEZ, J. Lab. Clin. Med. 10____967-74 9 (1987). 21. J. LEIVA, J. MENDOZA, J.M. NAVARRO, J.C. PLATA and M. DE LA ROSA, Enf. Infec. Microbiol. Clin. 8 15-18 (1990). 22. H. TOWBIN, T. STAEHELIN and J. GORDON, Proc. Natl. Acad. Sci. 76 4250-4254 (1979). 23. J.M. GHIRGWIN, A.E. PRZYBYLA, R.J. MAC-DONALD and W.J. RUTTER, Biochem. 18 5294-5299 (1979). 24. S.N. ALLO, P.J. MCDERMOTT, L.L. CARL and H.E. MORGAN, J. Biol. Chem. 266 2203-2209 (1991). 25. C. MELGUIZO, J. PRADOS, C. VELEZ, A.E. ARANEGA, L. ALVAREZ and A. ARANEGA, Histochem J. (submitted) (1993). 26. H. HOLTZER, S. FORRY-SCHAUDIES, G. ANTIN, G. DUBYAK and V. NACHMIAS, Adv. Exp. Med. Biol. 182 179-192 (1985). 27. M. SCHLIWA, T. NAKAMURA, K.R. PORTER and U. EUTENEUER, J. Cell. Biol. 99

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