Selective gene expression in failing human heart. Quantification of steady-state levels of messenger RNA in endomyocardial biopsies using the polymerase chain reaction.

Abstract

BACKGROUND Evaluation of gene expression in failing human heart has been limited by the availability of cardiac tissue. METHODS AND RESULTS We used the polymerase chain reaction (PCR) to assess gene expression in small quantities of failing and nonfailing human heart. PCR is a powerful new molecular biological tool that allows a small quantity of DNA to be amplified as much as 1 million-fold. Total RNA was extracted from 3-5 mg samples of human heart and reverse-transcribed to complementary DNA (cDNA). With selected oligonucleotide primers, we used PCR to amplify cDNAs encoding atrial natriuretic peptide, beta-myosin heavy chain, phospholamban, and cytoskeletal beta-actin. To quantify the relative levels of messenger RNA (mRNA) in human heart, a known amount of a control RNA was present in the reverse transcription and PCR reactions. The amount of mRNA in the sample could therefore be assessed in relation to the amount of control product. The control RNA was transcribed from a synthetic DNA template containing primers complementary to those used to amplify the cDNAs of interest. Atrial natriuretic factor mRNA could not be detected in nonfailing human heart but was abundant in ventricular myocardium from failing human heart. In contrast, steady-state levels of phospholamban mRNA decreased, whereas levels of beta-myosin heavy-chain mRNA were unchanged with heart failure. CONCLUSIONS Alterations in gene expression in the failing human heart appear to be selective. In addition, the present study suggests that PCR provides a rapid and economical way to quantify the expression of multiple genes of interest in endomyocardial biopsy specimens and may therefore be used to advance our understanding of heart muscle disease.