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In vivo studies of transcriptional regulation in cardiac myocytes using direct gene transfer into myocardium
J Mol Cell Cardiol 24 (Supplement
P76
III) (1992)
IN VIVO STUDIES OF TRANSCRIPTIONAL REGULATION IN CARDIAC MYOCYTES USING DIRECT GENE TRANSFER INTO MYOCARDIUM Eliav Barr, Michael S. Parmacek, and Jeffrey M. Leiden, University of Michigan, Ann Arbor, Ml In vitro studies defining transcriptional regulatory elements in primary cardiac myocytes are limited by the rapid loss of differentiated phenotype and finite cell survival in culture. Transgenic animals can be used to map transcriptional regulatory elements in vivo but are expensive and time consuming. We have previously reported that recombinant genes injected directly into the LV wall are taken up and expressed stably in cardiac myocytes. We now demonstrate that this approach can be used to rapidly delineate the transcriptional regulatory elements of cardiac-specific genes in vivo. Direct injections of CAT reporter constructs into B-week-old Sprague-Dawley rat hearts were used to map the transcriptional regulatory sequences of the slow/cardiac Troponin C (cTnC) gene. Using a series of deletion constructs containing sequences from the 5’ flanking region of the murine cTnC gene, we have identified a potent cardiac-specific promoter/enhancer located between bp -124 and +32. This element is able to reproducibly increase CAT transcription by more than 70-fold following injection into the rat ventricle. Further analysis of this element by direct injection of CAT reporter constructs demonstrated that it is composed of a minimal proximal promoter (bp -79 to +32) and a cardiac-specific transcriptional enhancer (bp -124 to -56). Taken together, these results identify a novel cardiac-specific promoter/enhancer in the 5’ flanking region of the cTnC gene and demonstrate the usefulness of direct DNA injection for studies of cardiac-specific transcriptional regulatory elements.
P77 PRIOR HEAT
SHOCK TREATMENT ATTENUATES THE MOLECULAR RESPONSE OF HEARTS TO THE METABOLIC STRESS OF ISOLATION AND PERFUSION. Todd A. D&in and R. William Currie, Anatomy Dept, Dalhousie Univ, Halifax, B3H 4H7, Canada. Induction of the heat shock response has been shown to improve post-isohemic ventricular contractile recovery. However, the precise mechanism of this protection is unknown. We examined the expression of the mBNA encoding for several heat shook proteins during isolated working heart perfusion. Control and 24 hour post-heat shook rat hearts were perfused in the working heart mode for 1, 2, 3, and 4 hours. Total RNA was extracted from hearts, fractionated by gel electrophoresis, transferred to membranes, and probed with 32Pradio-labelled cDNA encoding for the inducible HSP71, the constitutive HSP73, HSP60, HSP90, ubiquitin and actin. Control hearts had a large and progressive increase in mRNA for HSP71 during perfusion in the working heart mode for 1, 2, 3, and 4 hr. Heat shocked hearts had minimal increase. of mRNA for HSP71 even after 4 hr of perfusion. Similarly, control hearts had a progressive increase in mBNA for HSF73 while heat shocked hearts had less increase of mRNA for HSP73 during perfusion for 1, 2, 3, and 4 hr. Actin mRNA was not affected by the length of perfusion in control or heat shocked hearts. Thus, the heat shook pretreatment seems to attenuate the accumulation of mRNA for at least two heat shock proteins during perfusion. Negative regulation of synthesis of hsp transcripts and their respective proteins by the heat shocked hearts may be important in cardiac metabolic demands during ischemia and roperfusion and in post-ischemic ventricular recovery. Supported by New Brunswick Heart and Stroke Foundation.
P76
DECREASED EXPRESSING
ISCHEMIA (Ism) INDUCED HUMAN INDUCIBLE HEAT
INJURY IN RAT HEART SHOCK PROTEIN 70 (hHSP7Oi)
DERIYED
H!k2
CELLS
Wolfgang H Dillmann, Shun-Hua Chi, M Richard Sayen, Ruben Mestril. Univ of Calif, San Diego, Dept. of Medicine, San Diego, CA 92103. Ism rapidly increases HSP70i mRNA and protein in tbe heart, but no conclusive evidence exists for a protective role of HSP70 against Ism mediated injury. Embryonal rat heart derived H9c2 cells were stably transfected with plasmids expressing hHSP7Oi and the neomycin resistance (NEO) gene. After selection with a neomycin analog, H9c2 cells expressing hHSP70i (H9c2&ISWOi), cells only expressing the NE0 gene (H9c2/NEO) and native H9c2 cells were submitted to simulated Ism (SI) consisting of hypoxia, glucose-free Hanks medium, and hypotenic medium (70 mM NaCI) for 6 hs, reoxgenation, and 6 hs of recovery under normal conditions (NC). hHSP70i formation in H9c2/HSP70i cells was documented by immunoprecipitation. H9c2/NEO and native H9c2 cells did not synthesis HSP70i protein. Cell viability was scored with a colony survival assay in native H9c2 cells, H9c2/HSP70i cells, and H9c2/NEO cells maintained under NC vs. SI conditions. A significantly higher number @<0.05) of colonies formed in H9c2/HSWOi cells (86&l% of colonies with SI vs. NC) vs. H9c2/NEO (63+9%), or native H9c2 (57~8%). Protein synthesiswas determined by [35S]-metbionine labelling in cells after 4 hs of SI. H9c2/HSWOi cells had significantly lessdepressed protein synthesis@> 0.05)(43&3% depression vs. H9c2HSP70i cells under NC), than H9c2/NEO cells (66*3%) or H9c2 cells (6121%). Conclusion: 1) Stably transfected H9c2 cells expressing HSP70i show increased cell survival and less inhibition of protein synthesis induced by SI; 2) HSP7Oi protein exerts a protective effect against ischemlc injury in rat heart H9c2 cells. 5.30
P76
III) (1992)
IN VIVO STUDIES OF TRANSCRIPTIONAL REGULATION IN CARDIAC MYOCYTES USING DIRECT GENE TRANSFER INTO MYOCARDIUM Eliav Barr, Michael S. Parmacek, and Jeffrey M. Leiden, University of Michigan, Ann Arbor, Ml In vitro studies defining transcriptional regulatory elements in primary cardiac myocytes are limited by the rapid loss of differentiated phenotype and finite cell survival in culture. Transgenic animals can be used to map transcriptional regulatory elements in vivo but are expensive and time consuming. We have previously reported that recombinant genes injected directly into the LV wall are taken up and expressed stably in cardiac myocytes. We now demonstrate that this approach can be used to rapidly delineate the transcriptional regulatory elements of cardiac-specific genes in vivo. Direct injections of CAT reporter constructs into B-week-old Sprague-Dawley rat hearts were used to map the transcriptional regulatory sequences of the slow/cardiac Troponin C (cTnC) gene. Using a series of deletion constructs containing sequences from the 5’ flanking region of the murine cTnC gene, we have identified a potent cardiac-specific promoter/enhancer located between bp -124 and +32. This element is able to reproducibly increase CAT transcription by more than 70-fold following injection into the rat ventricle. Further analysis of this element by direct injection of CAT reporter constructs demonstrated that it is composed of a minimal proximal promoter (bp -79 to +32) and a cardiac-specific transcriptional enhancer (bp -124 to -56). Taken together, these results identify a novel cardiac-specific promoter/enhancer in the 5’ flanking region of the cTnC gene and demonstrate the usefulness of direct DNA injection for studies of cardiac-specific transcriptional regulatory elements.
P77 PRIOR HEAT
SHOCK TREATMENT ATTENUATES THE MOLECULAR RESPONSE OF HEARTS TO THE METABOLIC STRESS OF ISOLATION AND PERFUSION. Todd A. D&in and R. William Currie, Anatomy Dept, Dalhousie Univ, Halifax, B3H 4H7, Canada. Induction of the heat shock response has been shown to improve post-isohemic ventricular contractile recovery. However, the precise mechanism of this protection is unknown. We examined the expression of the mBNA encoding for several heat shook proteins during isolated working heart perfusion. Control and 24 hour post-heat shook rat hearts were perfused in the working heart mode for 1, 2, 3, and 4 hours. Total RNA was extracted from hearts, fractionated by gel electrophoresis, transferred to membranes, and probed with 32Pradio-labelled cDNA encoding for the inducible HSP71, the constitutive HSP73, HSP60, HSP90, ubiquitin and actin. Control hearts had a large and progressive increase in mRNA for HSP71 during perfusion in the working heart mode for 1, 2, 3, and 4 hr. Heat shocked hearts had minimal increase. of mRNA for HSP71 even after 4 hr of perfusion. Similarly, control hearts had a progressive increase in mBNA for HSF73 while heat shocked hearts had less increase of mRNA for HSP73 during perfusion for 1, 2, 3, and 4 hr. Actin mRNA was not affected by the length of perfusion in control or heat shocked hearts. Thus, the heat shook pretreatment seems to attenuate the accumulation of mRNA for at least two heat shock proteins during perfusion. Negative regulation of synthesis of hsp transcripts and their respective proteins by the heat shocked hearts may be important in cardiac metabolic demands during ischemia and roperfusion and in post-ischemic ventricular recovery. Supported by New Brunswick Heart and Stroke Foundation.
P76
DECREASED EXPRESSING
ISCHEMIA (Ism) INDUCED HUMAN INDUCIBLE HEAT
INJURY IN RAT HEART SHOCK PROTEIN 70 (hHSP7Oi)
DERIYED
H!k2
CELLS
Wolfgang H Dillmann, Shun-Hua Chi, M Richard Sayen, Ruben Mestril. Univ of Calif, San Diego, Dept. of Medicine, San Diego, CA 92103. Ism rapidly increases HSP70i mRNA and protein in tbe heart, but no conclusive evidence exists for a protective role of HSP70 against Ism mediated injury. Embryonal rat heart derived H9c2 cells were stably transfected with plasmids expressing hHSP7Oi and the neomycin resistance (NEO) gene. After selection with a neomycin analog, H9c2 cells expressing hHSP70i (H9c2&ISWOi), cells only expressing the NE0 gene (H9c2/NEO) and native H9c2 cells were submitted to simulated Ism (SI) consisting of hypoxia, glucose-free Hanks medium, and hypotenic medium (70 mM NaCI) for 6 hs, reoxgenation, and 6 hs of recovery under normal conditions (NC). hHSP70i formation in H9c2/HSP70i cells was documented by immunoprecipitation. H9c2/NEO and native H9c2 cells did not synthesis HSP70i protein. Cell viability was scored with a colony survival assay in native H9c2 cells, H9c2/HSP70i cells, and H9c2/NEO cells maintained under NC vs. SI conditions. A significantly higher number @<0.05) of colonies formed in H9c2/HSWOi cells (86&l% of colonies with SI vs. NC) vs. H9c2/NEO (63+9%), or native H9c2 (57~8%). Protein synthesiswas determined by [35S]-metbionine labelling in cells after 4 hs of SI. H9c2/HSWOi cells had significantly lessdepressed protein synthesis@> 0.05)(43&3% depression vs. H9c2HSP70i cells under NC), than H9c2/NEO cells (66*3%) or H9c2 cells (6121%). Conclusion: 1) Stably transfected H9c2 cells expressing HSP70i show increased cell survival and less inhibition of protein synthesis induced by SI; 2) HSP7Oi protein exerts a protective effect against ischemlc injury in rat heart H9c2 cells. 5.30