Acute myocardial infarcts (AMI) present a major health problem and additional salvage of myocardial tissue will lead to an improved outcome of AMIs. Recent evidence indicates that brief episodes of myocardial ischemia markedly increase the expression of heat shock/stress protein (HSP) gene family members, especially HSP70 isoforms. Increased expression of HSP70 exerts a protective effect against ischemia mediated myocardial injury. We would like to test the hypothesis that a protective effect against ischemia mediated injury is exerted by specific HSP70 proteins in cardiac myocytes. We will use embryonal rat heart derived H9c2 myocytes which show a similar response to simulated ischemia (SI) as neonatal myocytes (NM) do.
In Aim I, we will extend these findings and confirm that in both cell types the cognate HSP70 (HSP70c) is expressed and that SI increase the expression of at least two HSP70 inducible genes (HSP70ia and HSP70ib). Genomic and cDNA clones to HSP70ib have been isolated in our laboratory and we are obtaining genomic and cDNA clones to HSP0ia. We will also pursue preliminary findings and determine if specific components of ischemia, like hypoxia and tonicity changes, mediate HSP70 gene expression through separate cis-acting response elements of HSP70ia or HSP70ib genes.
In Aim II, we will confirm and extend our preliminary results indicating that protection against ischemia-mediated injury occurs in stably transfected H9c2 cells over-expression HSP70i. Clonal H9c2 cell lines over-expressing rat HSP70c, HSP70ia and HSP70ib in a permanent or inducible manner will be used to test whether these proteins have a protective effect against ischemia mediated injury. To confirm our results obtained with H9c2 cells, cardiac myocytes will be prepared from transgenic mice recently produced by us which express HSP70 in their hearts. In addition, adult myocytes will be infected with adenoviruses expressing different isoforms of HSP70. These myocytes are submitted to SI and creatine kinase and lactate dehydrogenase release and other parameters related to ischemic injury are determined. We will also determine in NM if ischemic stress tolerance can be induced by sequential application of ischemia-related stresses and if it can be correlated to changes in HSP70 protein. In studies of Aim III, transgeic mice were produced by using DNA constructs containing a 2.7 kb fragment of the rat myosin light chain-2 (MLC-2) regulatory regions and HSP70i coding regions. Other isoforms of HSP70 will also be used to produce transgenic mice. The MLC-2 promoter directs cardiac ventricular myocyte specific expression of different HSP70 isoforms (HSP70c, HSP70ia,HSP70ib). A protective effect against ischemic injury will be assessed by permanent and reversible occlusion of the left coronary artery using microsurgical techniques and quantitation of infarct size as a fraction of the bed at risk in transgenic mice with or without increased levels of HSP70 isoform expression. In addition, papillary muscle from transgenic and control mice are submitted to hypoxia and a potential protective effect on contractile function is evaluated. Confirming our preliminary finding that HSP70 has a protective effect against ischemia mediated injury may lead to new treatment strategies.
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