Vol. 188, No. 2, 1992 October
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Pages 547-553
30, 1992
REGULATION
OF HUMAN THE
CARDIAC
EFFECT
MYOSIN
HEAVY
CHAIN
GENES:
OF CATECHOLAMINE
J.J. Chen’*2*3, D.L. Wang2, N.L. Shih2, K.H. Hsu2, W.P.Lien* and C.C. Liew2*3 ‘Department of Medicine,The National Taiwan University Hospital, and ZInstitute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 3Laboratory of Molecular Cardiology, Departments of Clinical Biochemistry and Medicine, Centre for Cardiovascular Research, Toronto Hospital, University of Toronto, Toronto Ontario, Canada Received
August
24,
1992
The Y-flanking regions of the cr- and B-cardiac myosin heavy chain (MyHC) genes were excised from the cosmid human genomic clones using Hind III and Xbal for the cr-MyHC gene, and the Hind III and Hind III sites for the B-MyHC gene.
These fragments were linked to
chloramphenicol acetyl transferase (CAT) vector to generate a chimeric fusion gene. These fusion genes were subsequently transfected to neonatal rat cardiac cultured cells to analyze the CAT activity. The ol-MyHC gene is preferentially expressed as compared to the J3-MyHC. In the presence of norepinephrine (NE) the B-MyHC gene is remarkably induced (within 24 hours following the addition of norepinephrine to the cardiocyte culture).
However, the ol-MyHC is
also induced. Specific alpha andrenergic antagonists such as terazosin (Tz) partially suppressed both the (Y- and B-MyHC genes as revealed by the CAT activity.
These findings suggest that
catecholamine does activate the human cardiac MyHC genes but does not differentiate the specific expression of either the cr- or 8-MyHC genes.
0 1992 Academic
Press,
Inc.
The cardiac o- and l3-myosin heavy chain (MyHc) genes are subject to extensive hormonal, developmental as well as physiological regulation in cardiocytes (l-4).
These tandemly linked
genes are separated by a 4.1 kb intergenic region (5,6). Recently, we and other laboratories have characterized the human cardiac genes (7- 11). We have also determined the complete nucleotide sequence of both genes, including 2 kb of the 5’-flanking region of the B-MyHC gene and 4.1 kb of the intergenic region including the 5’-flanking region of the cY-myosin heavy chain gene (12). We wish to further characterize the regulation of the human cardiac MyHC genes in relation to catecholamines.
541
0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
BIOCHEMICAL
188, No. 2, 1992
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
It is well known that catecholamine stimulates increased cardiac contractility and resultant cardiac hypertrophy (13-16).
It has been demonstrated that the activation of cq-adrenergic
receptor by catecholamine norepinephrine has selectively enhanced the transcription of the OMyHC gene as well as its polypeptide (17). However, the intracellular mechanism(s) that mediates via the cq-adrenergic receptor and eventually exerts effects on the MyHC genes remains unknown.
In this report, we generated chimeric constructs containing the 5’-flanking
regions of either the R- or ol-MHC genes linked to the reporter gene of chloramphenicol acetyltransferase (CAT) which provides a useful model to investigate how the 5’ flanking region of the MyHC genes respond to hormonal stimuli. The constructs were transfected into neonatal rat myocytes and the effect of the catecholamine on CAT expression was investigated. MATERIALS
AND METHODS
All chemicals used were of reagent grade. Cardiocytes were derived from 2-3 day old Wistar rat neonates. Hearts were excised and cut into 1 mm* pieces prior to trypsin digestion. Cardiocyte isolation followed the method of Lee et al. (18) with minimal modification which has been used routinely in our laboratory (19). Prior to norepinephrine (NE) treatment, cultured medium was replaced by serum-free medium. Cardiocyte extract was prepared, and the CAT activity was assayed as described by Gorman et al. (20). The extract concentration was determined by the Lowry method (21) prior to the CAT assay.
It is essential to measure the protein concentration of the extract to normalize cellular
extracts obtained from each cell culture plate. The plasmid pSV-B-galactosidase was used in transfection experiments as a control.
l3-galactosidase activity was measured as described by
Rosenthal (22). All gene transfer experiments were initially conducted using Ca*’ precipitation (20) and later experiments were carried out by the liposome transfection method as described previously (23). Using the latter method we achieved a high yield of surviving cardiocytes as well as a high transfection efficiency. transfection.
In brief, cardiocytes were maintained in culture for 48 hours prior to
Ten pg of plasmid containing either the Y-flanking region of the D- or a-MyHC
gene were mixed with either the Ca*+ or lysosome suspension (DOTAP reagent, Boe.herlnger Mannhein,
West Germany) and immediately added to the cardiocyte cell culture.
After
incubation for five hours, the cell medium was replaced with serum-free medium and cardiocyte culture was continued for 48 hours prior to harvesting of the cells for CAT assays. 548
Vol.
188,
No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Construction of chimeric CAT fusion genes was accomplished using plasmid pBLCAT, (with thymidine kinase promoter) and pBLCAT, (without promoter) as described by Lucknow and Schultz (24). The 5’ flanking regions of the HindIII-Hind111 fragments of the g-myosin heavy chain genes and the Hind III-XbaI fragment of the a-myosin heavy chain gene were put into the pBLCAT, vector. RESULTS
The promoter regions of the OL-and B-MyHC genes were linked to the CAT gene and we examined their expression in cultured cardiocytes, Two experimental approaches were used. In one set of experiments, cardiocytes were transfected with either the 5’-flanking region of the (Y-or B-MyHC genes linked to the CAT vector. Treatment with NE was initiated 24 hours after transfection by liposomes. This allowed the fusion genes to transfect into the cardiocytes prior to hormonal stimulation.
Figure 1 indicates that cardiocytes harbouring either the (Y- or g-
MyHC gene constructs exhibit CAT activity with or without NE treatment. However, the CAT activity which was linked to the B-MyHC promoter was strikingly increased with the treatment of NE. CAT activity was also increased in the NE treated cardiocytes harbouring the a-MyHC gene constructs.
The increases of MyHC gene expression by catecholamine was statistically
significant as shown in Table 1. In the second set of experiments, rat neonatal cardiocytes were transfected by liposomes with either the Q- or B-MyHC gene constructs.
As shown in Figure 2, the CAT activity observed
was similar to that of the first set of experiments. Again, CAT activity driven by the l3-MyHC
Fig. 1. Effects of Norekeuhrine [NE‘)on the Expression of Human Cardiac MyHC Genes. The neonatal rat cardiocytes were treated with Ca2’ precipitation to introduce the CAT containing chimeric construct of either (Y-or B-MyHC genes. The CAT activity was examined 48 hours after transfection. Lanes 1 and 3 are the the control for CY-MyHCand l3-MyHC fusion constructs,respectively. Lanes 2 and 4 represent the NE treated cardiocytes for 24 hours.
549
Vol.
188,
No.
2,
BIOCHEMICAL
1992
TABLE
AND
BIOPHYSICAL
1: Effect of Norepinephrine
COMMUNICATIONS
on CAT Activity P
% INCREASE
CY
RESEARCH
13.1k4.5
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Pages 547-553
30, 1992
REGULATION
OF HUMAN THE
CARDIAC
EFFECT
MYOSIN
HEAVY
CHAIN
GENES:
OF CATECHOLAMINE
J.J. Chen’*2*3, D.L. Wang2, N.L. Shih2, K.H. Hsu2, W.P.Lien* and C.C. Liew2*3 ‘Department of Medicine,The National Taiwan University Hospital, and ZInstitute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 3Laboratory of Molecular Cardiology, Departments of Clinical Biochemistry and Medicine, Centre for Cardiovascular Research, Toronto Hospital, University of Toronto, Toronto Ontario, Canada Received
August
24,
1992
The Y-flanking regions of the cr- and B-cardiac myosin heavy chain (MyHC) genes were excised from the cosmid human genomic clones using Hind III and Xbal for the cr-MyHC gene, and the Hind III and Hind III sites for the B-MyHC gene.
These fragments were linked to
chloramphenicol acetyl transferase (CAT) vector to generate a chimeric fusion gene. These fusion genes were subsequently transfected to neonatal rat cardiac cultured cells to analyze the CAT activity. The ol-MyHC gene is preferentially expressed as compared to the J3-MyHC. In the presence of norepinephrine (NE) the B-MyHC gene is remarkably induced (within 24 hours following the addition of norepinephrine to the cardiocyte culture).
However, the ol-MyHC is
also induced. Specific alpha andrenergic antagonists such as terazosin (Tz) partially suppressed both the (Y- and B-MyHC genes as revealed by the CAT activity.
These findings suggest that
catecholamine does activate the human cardiac MyHC genes but does not differentiate the specific expression of either the cr- or 8-MyHC genes.
0 1992 Academic
Press,
Inc.
The cardiac o- and l3-myosin heavy chain (MyHc) genes are subject to extensive hormonal, developmental as well as physiological regulation in cardiocytes (l-4).
These tandemly linked
genes are separated by a 4.1 kb intergenic region (5,6). Recently, we and other laboratories have characterized the human cardiac genes (7- 11). We have also determined the complete nucleotide sequence of both genes, including 2 kb of the 5’-flanking region of the B-MyHC gene and 4.1 kb of the intergenic region including the 5’-flanking region of the cY-myosin heavy chain gene (12). We wish to further characterize the regulation of the human cardiac MyHC genes in relation to catecholamines.
541
0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
BIOCHEMICAL
188, No. 2, 1992
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
It is well known that catecholamine stimulates increased cardiac contractility and resultant cardiac hypertrophy (13-16).
It has been demonstrated that the activation of cq-adrenergic
receptor by catecholamine norepinephrine has selectively enhanced the transcription of the OMyHC gene as well as its polypeptide (17). However, the intracellular mechanism(s) that mediates via the cq-adrenergic receptor and eventually exerts effects on the MyHC genes remains unknown.
In this report, we generated chimeric constructs containing the 5’-flanking
regions of either the R- or ol-MHC genes linked to the reporter gene of chloramphenicol acetyltransferase (CAT) which provides a useful model to investigate how the 5’ flanking region of the MyHC genes respond to hormonal stimuli. The constructs were transfected into neonatal rat myocytes and the effect of the catecholamine on CAT expression was investigated. MATERIALS
AND METHODS
All chemicals used were of reagent grade. Cardiocytes were derived from 2-3 day old Wistar rat neonates. Hearts were excised and cut into 1 mm* pieces prior to trypsin digestion. Cardiocyte isolation followed the method of Lee et al. (18) with minimal modification which has been used routinely in our laboratory (19). Prior to norepinephrine (NE) treatment, cultured medium was replaced by serum-free medium. Cardiocyte extract was prepared, and the CAT activity was assayed as described by Gorman et al. (20). The extract concentration was determined by the Lowry method (21) prior to the CAT assay.
It is essential to measure the protein concentration of the extract to normalize cellular
extracts obtained from each cell culture plate. The plasmid pSV-B-galactosidase was used in transfection experiments as a control.
l3-galactosidase activity was measured as described by
Rosenthal (22). All gene transfer experiments were initially conducted using Ca*’ precipitation (20) and later experiments were carried out by the liposome transfection method as described previously (23). Using the latter method we achieved a high yield of surviving cardiocytes as well as a high transfection efficiency. transfection.
In brief, cardiocytes were maintained in culture for 48 hours prior to
Ten pg of plasmid containing either the Y-flanking region of the D- or a-MyHC
gene were mixed with either the Ca*+ or lysosome suspension (DOTAP reagent, Boe.herlnger Mannhein,
West Germany) and immediately added to the cardiocyte cell culture.
After
incubation for five hours, the cell medium was replaced with serum-free medium and cardiocyte culture was continued for 48 hours prior to harvesting of the cells for CAT assays. 548
Vol.
188,
No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Construction of chimeric CAT fusion genes was accomplished using plasmid pBLCAT, (with thymidine kinase promoter) and pBLCAT, (without promoter) as described by Lucknow and Schultz (24). The 5’ flanking regions of the HindIII-Hind111 fragments of the g-myosin heavy chain genes and the Hind III-XbaI fragment of the a-myosin heavy chain gene were put into the pBLCAT, vector. RESULTS
The promoter regions of the OL-and B-MyHC genes were linked to the CAT gene and we examined their expression in cultured cardiocytes, Two experimental approaches were used. In one set of experiments, cardiocytes were transfected with either the 5’-flanking region of the (Y-or B-MyHC genes linked to the CAT vector. Treatment with NE was initiated 24 hours after transfection by liposomes. This allowed the fusion genes to transfect into the cardiocytes prior to hormonal stimulation.
Figure 1 indicates that cardiocytes harbouring either the (Y- or g-
MyHC gene constructs exhibit CAT activity with or without NE treatment. However, the CAT activity which was linked to the B-MyHC promoter was strikingly increased with the treatment of NE. CAT activity was also increased in the NE treated cardiocytes harbouring the a-MyHC gene constructs.
The increases of MyHC gene expression by catecholamine was statistically
significant as shown in Table 1. In the second set of experiments, rat neonatal cardiocytes were transfected by liposomes with either the Q- or B-MyHC gene constructs.
As shown in Figure 2, the CAT activity observed
was similar to that of the first set of experiments. Again, CAT activity driven by the l3-MyHC
Fig. 1. Effects of Norekeuhrine [NE‘)on the Expression of Human Cardiac MyHC Genes. The neonatal rat cardiocytes were treated with Ca2’ precipitation to introduce the CAT containing chimeric construct of either (Y-or B-MyHC genes. The CAT activity was examined 48 hours after transfection. Lanes 1 and 3 are the the control for CY-MyHCand l3-MyHC fusion constructs,respectively. Lanes 2 and 4 represent the NE treated cardiocytes for 24 hours.
549
Vol.
188,
No.
2,
BIOCHEMICAL
1992
TABLE
AND
BIOPHYSICAL
1: Effect of Norepinephrine
COMMUNICATIONS
on CAT Activity P
% INCREASE
CY
RESEARCH
13.1k4.5
