996-10 A Novel Approach to Identifying mRNAs Differentially Expressed Under Hemodynamic Pressure Overload During Sheep Fetal Heart Development

Abstract

The adaptive response to pressure overload is different in neonatal hearts with congenital lesions than in adult hearts with acquired lesions. In adult hearts this response is characterized by an altered pattern of gene expression. Little is known about the changes in gene expression that occur in neonates. We have used a novel approach to examine these changes in expression of messenger RNAs in an in vivo model of fetal lambs heart after induced acute pressure overload. In ewes (which normally carry twin gestationsl the main pulmonary artery of one fetal lamb was banded in utero for one hour to produce an acute pressure overload on the right ventricle (RV) while the twin underwent a sham operation. Total RNA was isolated and reverse transcribed from both the banded and control RV The resulting cDNAs were amplified by the polymerase chain reaction technique using generalized oligonucleotide primers and the products displayed on sequencing gels. Comparative analysis of the differentially expressed cDNA bands from each condition revealed both up-regulation and down-regulation of several cardiac genes in the banded fetal hearts compared with the control lamb hearts. No morphological changes in the cardiac tissues were observed during this same time period. Eighty (300–500bp) differentially expressed cDNAs were identified. Twenty-three of these were analyzed by Northem blot and four were confirmed as being differentially expressed. Two of these were sequenced, identifying the 3’-untranslated regions which had no identifiable homology to the sequences in GENEBANK. This study reports on using an in vivo fetal lamb model to study changes in gene expression induced by hemodynamic pressure overload using a technique that identifies all differentially expressed messages. This is the first study to examine alterations in fetal cardiac gene expression to hemodynamic overload in a large mammalian model.