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Isolation of a translation inhibiting peptide (TIP) from myocardium
J Mol Cell Cardiol 19 (Supplement IV) (1987) 88
R E P R O G R A M M I N G OF G E N E E X P R E S S I O N I N D U C E D BY C A R D I A C H Y P E R T R O P H Y . S. Izumo, B. Nadal-Ginard, V. Mahdavi. Dept. of Cardiology, C h i l d l r e n ' s H o s p i t a l , H a r v a r d M e d i c a l School, Boston, MA.
Cardiac myosins have been shown to undergo isoform transitions in various models of hypertrophy. Using gene-specific cDNA probes, we show here that the m R N A s encoding the fetal (skeletal muscle type) isoforms of ~-actin and sarcomeric tropomyosin are re-expressed in the hypertrophic adult myocardium produced by pressure overload. In addition, the atrial natriuretic factor (ANF) mRNA, so far believed to b e expressed primarily in the atria, was readily detectable in the ventricles of neonates and was induced to markedly high levels in pressure-overloaded adult ventricles. In contrast, cardiac hypertrophy produced by thyroid hormone excess was not associated with induction of ANF gene or fetal contractile protein isogenes. Furthermore, the c-fos and c-myc proto-oncogenes and a major heat shock protein (hsp70) gene are induced in the ventricula[ myocardium within one hour after imposition of pressure ove~load. These results suggest that induction of cellular proto-oncogenes and heat shock (stress) protein genes is an early response to pressure overload, while re-induction of fetal genes is a later event and might be a general adaptative process to work overload in the post-mitotic cardiac cells.
89
SIGNAL PROTEINS DURING CARDIAC OVERLOAD. AN IN VITRO STUDY OF TIlE TRANSLATIONNAL PRODUCTS OF TOTAL RNA J.M. Moalic and B. Swynghedauw. U 127 INSERM, Hopital Lariboisi~re, 41 Bd de la Chapelle, 75010, Paris, France. Cardiac overload induces several changes in the genetic expression of the cardiac cell. These modifications have been analysed in rat 4 and 30 days after abdominal aortic stenosis by using an in vitro heterologous translational system. Total RNA was in vitro translated in a reticulocyte lysate in the presence of 35S methionine. The translational products were analyzed by 2 dimentionnal gel (2D) electrophoresis and autoradiography. (i) During the transition which precedes the compensatory hypertrophy, i.e. 4 days after banding aorta, two distinct and reproducible spots (Mr. 21,O00 and pl 7.7 ; Mr. 19,OOO and pI 6.9) apprears on the 2 D gel electrophoresis and may correspond to "signal proteins" for protein sysnthesis. They were not shown at one month. (it) 30 days after stenosis the only reproducible change observed was the appearance of $-tropomyosin, an isoform normally absent in rat ventricle and which characterizes slow skeletal muscles. Informations concerning other signals will also be presented.
90
ISOLATION OF A TRANSLATION INHIBITING PEPTIDE (TIP) FROMMYOCARDIUM. P.A. Havre and G.L. Hammond. Department of Surgery, Yale Medical School, New Haven, CT 06510. Although the end result of acute a o r t i c banding is increased protein synthesis and hypertrophy, we previously reported that the i n i t i a l stress response is accompanied by a temporary but marked decrease in protein synthesis. We examined the effect of protein extracts obtained from control canine hearts and hearts acutely stressed by a o r t i c banding and heat shock on translational a c t i v i t y using an in v i t r o translation assay. We found that extracts from stressed tissue exhibited approximately 20% greater suppressor a c t i v i t y than those obtained from control tissue. P u r i f i c a t i o n of thi# factor, which we have named TIP, revealed that i t has a Stokes radius of less than 16~ and molecular weight, determined by SDS-PAGE, of 17 KD. I t has an i s o e l e c t r i c point of 7.25 and a high content of ASX, GLX and GLY. Sequencing of amino acids 3-19 has been completed. Treatment of polysome preparations, obtained from control canine hearts, with purif i e d TIP shifted the polysome d i s t r i b u t i o n p r o f i l e to that which we previously reported was c h a r a c t e r i s t i c for the acutely stressed polysome pattern in which monosomes predominated.
S.30
R E P R O G R A M M I N G OF G E N E E X P R E S S I O N I N D U C E D BY C A R D I A C H Y P E R T R O P H Y . S. Izumo, B. Nadal-Ginard, V. Mahdavi. Dept. of Cardiology, C h i l d l r e n ' s H o s p i t a l , H a r v a r d M e d i c a l School, Boston, MA.
Cardiac myosins have been shown to undergo isoform transitions in various models of hypertrophy. Using gene-specific cDNA probes, we show here that the m R N A s encoding the fetal (skeletal muscle type) isoforms of ~-actin and sarcomeric tropomyosin are re-expressed in the hypertrophic adult myocardium produced by pressure overload. In addition, the atrial natriuretic factor (ANF) mRNA, so far believed to b e expressed primarily in the atria, was readily detectable in the ventricles of neonates and was induced to markedly high levels in pressure-overloaded adult ventricles. In contrast, cardiac hypertrophy produced by thyroid hormone excess was not associated with induction of ANF gene or fetal contractile protein isogenes. Furthermore, the c-fos and c-myc proto-oncogenes and a major heat shock protein (hsp70) gene are induced in the ventricula[ myocardium within one hour after imposition of pressure ove~load. These results suggest that induction of cellular proto-oncogenes and heat shock (stress) protein genes is an early response to pressure overload, while re-induction of fetal genes is a later event and might be a general adaptative process to work overload in the post-mitotic cardiac cells.
89
SIGNAL PROTEINS DURING CARDIAC OVERLOAD. AN IN VITRO STUDY OF TIlE TRANSLATIONNAL PRODUCTS OF TOTAL RNA J.M. Moalic and B. Swynghedauw. U 127 INSERM, Hopital Lariboisi~re, 41 Bd de la Chapelle, 75010, Paris, France. Cardiac overload induces several changes in the genetic expression of the cardiac cell. These modifications have been analysed in rat 4 and 30 days after abdominal aortic stenosis by using an in vitro heterologous translational system. Total RNA was in vitro translated in a reticulocyte lysate in the presence of 35S methionine. The translational products were analyzed by 2 dimentionnal gel (2D) electrophoresis and autoradiography. (i) During the transition which precedes the compensatory hypertrophy, i.e. 4 days after banding aorta, two distinct and reproducible spots (Mr. 21,O00 and pl 7.7 ; Mr. 19,OOO and pI 6.9) apprears on the 2 D gel electrophoresis and may correspond to "signal proteins" for protein sysnthesis. They were not shown at one month. (it) 30 days after stenosis the only reproducible change observed was the appearance of $-tropomyosin, an isoform normally absent in rat ventricle and which characterizes slow skeletal muscles. Informations concerning other signals will also be presented.
90
ISOLATION OF A TRANSLATION INHIBITING PEPTIDE (TIP) FROMMYOCARDIUM. P.A. Havre and G.L. Hammond. Department of Surgery, Yale Medical School, New Haven, CT 06510. Although the end result of acute a o r t i c banding is increased protein synthesis and hypertrophy, we previously reported that the i n i t i a l stress response is accompanied by a temporary but marked decrease in protein synthesis. We examined the effect of protein extracts obtained from control canine hearts and hearts acutely stressed by a o r t i c banding and heat shock on translational a c t i v i t y using an in v i t r o translation assay. We found that extracts from stressed tissue exhibited approximately 20% greater suppressor a c t i v i t y than those obtained from control tissue. P u r i f i c a t i o n of thi# factor, which we have named TIP, revealed that i t has a Stokes radius of less than 16~ and molecular weight, determined by SDS-PAGE, of 17 KD. I t has an i s o e l e c t r i c point of 7.25 and a high content of ASX, GLX and GLY. Sequencing of amino acids 3-19 has been completed. Treatment of polysome preparations, obtained from control canine hearts, with purif i e d TIP shifted the polysome d i s t r i b u t i o n p r o f i l e to that which we previously reported was c h a r a c t e r i s t i c for the acutely stressed polysome pattern in which monosomes predominated.
S.30