Analysis of gene expression patterns in small amounts of human ventricular myocardium by a multiplex RNase protection assay.
End-stage human heart failure is associated with changes in expression of steady-state messenger RNA (mRNA) levels. These changes correspond to alterations in protein levels and myocardial function and may have clinical implications regarding etiology, clinical state, or prognosis. However, analysis of mRNA levels in endomyocardial biopsies can be accomplished only by the quantitative polymerase chain reaction, which is difficult to standardize. The aim of the study was to evaluate whether the RNase protection assay is applicable to measure mRNAs of multiple genes simultaneously in small amounts of ventricular myocardium comparable to myocardial biopsies. Total RNA was prepared from left ventricular myocardium from terminally failing hearts with idiopathic (n=9) or ischemic cardiomyopathy (n=7) and from nonfailing control hearts (n=10). mRNA was measured by an optimized RNase protection assay for the beta1-adrenoceptor, the stimulatory G protein alpha-subunit (Gsalpha), phospholamban, the calcium ATPase of the sarcoplasmic reticulum (SERCA), beta-myosin heavy chain (beta-MHC), and the atrial natriuretic peptide (ANP). We extracted 10.7+/-2.1 microg total RNA from three myocardial biopsies taken in vitro. All of the six genes were measurable in duplicate in a total of 7 microg RNA. mRNAs of beta1-adrenoceptor, phospholamban, and SERCA were lower in failing than in nonfailing myocardium by 50%, 33%, and 42% respectively, whereas beta-MHC and Gsalpha mRNAs were unchanged. mRNA of ANP was expressed at high levels only in the failing myocardium, providing a highly specific and sensitive marker for discriminating nonfailing and failing hearts. A direct comparison with ANP and Gsalpha levels obtained by Northern blot analysis with 7.5 microg total RNA showed a good correlation between the two methods. The RNase protection assay is thus a suitable method for simultaneous measurements of multiple mRNA levels in human myocardial biopsies. Changes in mRNA levels closely reflected those identified by other methods using larger amounts of RNA. Increased myocardial ANP mRNA levels determined by the RNase protection assay may serve as a molecular marker of heart failure.
Mittmann
C
C
Pharmakologisches Institut, Universitäts-Krankenhaus Eppendorf, Hamburg, Germany.
Münstermann
U
U
Weil
J
J
Böhm
M
M
Herzig
S
S
Nienaber
C
C
Eschenhagen
T
T
D000293:Adolescent; D000328:Adult; D015152:Blotting, Northern; D005260:Female; D015870:Gene Expression; D006352:Heart Ventricles / Q000378:metabolism*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009206:Myocardium / Q000378:metabolism*; D012313:RNA / Q000096:biosynthesis*; D012260:Ribonucleases / Q000378:metabolism
D012313:RNA; D012260:Ribonucleases