Studies on Components of RNA in Cardiac Muscle with Emphasis on the Rapidly Labeled 4 to 18 S Fraction

Abstract

Studies on RNA from cardiac muscle of rats were performed by labeling the RNA with 32 P, extracting it with phenol, and purifying it prior to fractionating and analyzing it on a sucrose density gradient. The major components of RNA found had sedimentation values of 28, 18, and 4 S. Early 32 P-labeIed RNA ranged in size from 4 to > 45 S. Characterization of RNA from 4 to 18 S showed it to be more rapidly labeled and to have a higher turnover rate than 28 S RNA, to be located, at least in part, in the microsomes, and to have a base composition lower in guanine and cytosine content than 28 S RNA. Labeling of 4 to 18 S and 28 S RNA was equally inhibited by actinomycin. The possibility that 4 to 18 S RNA may represent a portion of messenger RNA in cardiac muscle is considered.ADDITIONAL KEY WORDS 32 P-labeIed RNA phenol extraction actinomycin subcellular particles sucrose-density-gradient analysis fractionation of RNA base analysis of RNA messenger RNA turnover rate of RNA• The messenger hypothesis postulates that RNA molecules transfer information from DNA to ribosomes (1). Studies of bacteria have demonstrated species of RNA thought to function in this capacity: the important characteristics are rapidity of labeb'ng by RNA precursor molecules, decreased stability relative to that of ribosomal RNA, a base composition that resembles that of DNA, heterogeneity of molecular size, a capacity for hybrid formation with homologus DNA, localization in polysomes, and a stimulatory effect on amino-acid incorporation in vitro (2-11).A heterogeneous, rapidly labeled RNA has been demonstrated in a number of mammalian tissues (12-27) as well as in germinating plants (28) and yeast cells (29). No comparable material has been characterized in striated muscle although its existence in this This investigation was supported by grants (5T1 HE 5391 and 2R01 HE 06924) from the U. S. Public Health Service.Accepted for publication July 16, 1966. tissue has been suggested (30). The heart is more suitable than skeletal muscle for the study of RNA synthesis because considerably more cardiac muscle RNA can be labeled in vivo (Fig. 1). In this communication, we wish to record the existence of a heterogeneous, rapidly labeled RNA fraction in cardiac muscle of the rat and to delineate some of its pertinent properties. Methods Animals.Male albino rats, weighing 90 to 120 g except when otherwise noted, were used; they were maintained on Purina chow ad lib. until they were killed.Materials. Phenol, 8-hydroxyquinoline, bentonite, and diphenylamine (special indicator grade) were purchased from the Fisher Scientific Company. Sodium dodecyl sulfate and yeast RNA (type XI) were obtained from the Sigma Chemical Company.3 -P was obtained from the New England Nuclear Corporation.Labeling of RNA. The tail veins of rats lightly anesthetized with ether were injected with carrierfree H R 3 -PO 4 diluted in 0.5 to 1.0 ml of 0.9S NaCl. Animals were killed by decapitation at varying times after injection of the label. The heart, and sometimes the skelet...

Study of the Myocardial Nucleic Acids and Proteins Metabolism in Experimentally Induced Cardiac Hypertrophy and Heart Failure

“…Generally, m-RNA has been considered to be very close to DNA in its base composition. 32) Therefore it is likely that DNA and m-RNA take almost the same tendency in the left ventricular failure. According to the messenger hypothesis, in-RNA transfer the information from DNA to ribosomes.…”

Experimental Cardiac Hypertrophy and the Synthesis of Poly(A) Containing RNA and of Myocardial Proteins in the Rat: The Effect of Digitoxin Treatment

“…1 demonstrates the labelling pattern of cardiac RNA two days after the operation. RNA is separated into three peaks on a 5 to 20% sucrose density gradient, which have been shown to correspond in sedimentation characteristics (28S, 1 8 s and 4s) to the RNA species from liver (Posner et al 1966). Any marked changes could not be seen in the labelling pattern of RNA extracted from the hearts of non-digitalized and digitalized animals with and without constriction of aorta.…”

“…Extraction andpurification of RNA from heart. For sucrose density gradient analysis, RNA was extracted by the method of Posner and Fanburg (1966). The tissue was minced with scissors in ice-cold 20 mM…”

“…The aqueous phase was re-extracted with phenol, and after adding one tenth volume of 20% sodium acetate (pH 5.2) RNA was precipitated with 2.5 volumes of 95% ethanol at -20°C for 4 h. DNA was removed from the precipitate by a treatment with 20 ,ug of RNAase free DNAase (Sigma Chemical Co) for 30 min at room temperature in 2 ml of 20 mM Tris-HCI (pH 7.2) containing 20 mM MgSOI. After adding ice-cold water and SDS to a final concentration of 0.5 %, the mixture was deproteinized with phenol, and RNA was precipitated from the aqueous phase with sodium acetate and ethanol Degraded DNA was removed from the precipitate by 2 precipitations with ethanol (final concentration 2596) at high salt concentration (Posner et al 1966) and the final precipitate was dissolved in 2 % sodium acetate (pH 5.2).…”

Protein Synthesis in Work-Overloaded Myocardium.

“…Of interest is the expansion of the uridylic acid pool (43) possibly the result of the observed increase in uridylate kinase activity (44). Analysis of pulselabeled newly synthesized RNA revealed equal incorporation of label into ribosomal and t-RNA with no evidence for the presence of high specific activity m-RNA (43,45). However changes in the types of m-RNA synthesized are suggested by finding changes in the labeling pattern of sarcoplasmic proteins separated by isoelectric focusing 2 days after aortic constriction (27).…”