Differences in sarcoplasmic reticulum gene expression in myocardium from patients undergoing cardiac surgery. Quantification of steady-state levels of messenger RNA using the reverse transcription-polymerase chain reaction.

Little is known about any alterations in sarcoplasmic reticulum (SR) gene expression associated with cardiac diseases of varying degrees of severity. We assessed, using the reverse transcription-polymerase chain reaction (RT-PCR) technique, SR Ca2+ transport protein gene expression in small tissue samples from failing hearts in patients undergoing cardiac surgery. Total RNA was extracted from 30- to 50-mg samples from the hearts of 13 patients with coronary artery disease, congenital heart disease, or valvular heart disease. We used RT-PCR to synthesize and amplify cDNA encoding cardiac SR Ca(2+)-ATPase, ryanodine receptor (RYR), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The amount of each mRNA in the sample was expressed relative to the amount of GAPDH mRNA. The expression level of each mRNA was correlated with the cardiac functional index. The mRNA levels for Ca(2+)-ATPase and RYR varied between heart samples, but showed a positive correlation with left ventricular ejection fraction. Ca(2+)-ATPase mRNA levels showed in inverse relationship with plasma brain natriuretic peptide. In addition, we isolated partial cDNA encoding a human cardiac RYR. The cDNA consisted of 487 nucleotides, and the nucleotide and deduced amino acid sequences showed 93% and 99% homology, respectively, to those of rabbit cardiac RYR. These results suggest that decreased levels of mRNA for SR Ca2+ transport protein could be related to abnormal cardiac function, regardless of the etiology of the heart disease. RT-PCR provides a rapid and economical way of quantifying the expression of multiple genes in small specimens and may, therefore, aid understanding of the pathophysiology and treatment of heart disease.