3ournd of
Molecular
Cardiac
LORIN BioZogical
and Cellular
Cardiology
(1975)
SHORT
COMMUNICATIONS
Hypertrophy, Ribosomal
K. JOHNSON, Sciences Department, (Received
RNA
ROGER
7, 125-133
Aging and Changes in Cardiac Gene Dosage in Man*
W. JOHNSON
University 25 April
AND BERNARD
of Southern California, 1974,
L. STREHLER
Los Angeles,
and accepted 29 June
California
90007
1974)
L. K. JOHNSON, R. W. JOHNSON AND B. L. STRJXHLER. Cardiac Hypertrophy, Aging and Changes in Cardiac Ribosomal RNA Gene Dosage in Man. Journal of Molecular and Cellular Cardiology (1975) 7, 125-133. The dosage of ribosomal RNA genes was determined in left ventricular myocardium obtained from two young individuals (ages 18 and 20 years), and from three older individuals (ages 64, 73 and 75 years), two of whom were diagnosed to have left ventricular hypertrophy. The technique of RNA-DNA molecular hybridization was employed to determine the gene dosage. The results indicate that there is a significant decline in the dosage of ribosomal RNA genes in the myocardium from the older individuals; the average hybridization level (the parameter of gene dosage) was 0.096% (range: 0.092 to 0.100%) for the young myocardium, and 0.068% (range: 0.063 to 0.076%) for the older myocardium. Within the older age group the hybridization level for the one tissue judged to be non-hypertrophic was essentially identical in its dosage to the tissues derived from hypertrophic myocardimn. These results, which are highly consistent with earlier studies on canine tissues, are interpreted to suggest that: (1) a ribosomal RNA gene loss such as that measured in aging myocardium may impose limitations on the rate of RNA synthesis, and thus indirectly cause abnormally low rates of protein synthesis within the myocardium during periods of stress, and (2) that such restrictions on the maximum rate of protein synthesis may account for the decreased ability of aged myocardium to respond to increased work loads, and the reduced contractility characteristic of aging muscle, hypertrophic myocardium and the failing heart. KEY WORDS: Cardiomegaly; Cardiac hyperfunction; ventricular hypertrophy; Ribosomes; Postmitotic cells;
RNA-DNA Congestive
hybridization; heart failure.
Left
1. Introduction Cardiac hypertrophy can be an adaptive physiological response to increased functional demands on the heart. However, when the functional demands are excessive and protracted the hypertrophy exceeds adaptive limits and congestive heart failure ensues. Left ventricular hypertrophy is the most common form occurring in man, and is frequently associated with systemic arterial hypertension. Systemic arterial hypertension, in turn, is commonly a correlate of age-dependent deterioration of the vascular system, often including arteriosclerosis. * This work
was supported
in part
by the Los Angeles
County
Heart
Association,
Grant
No. 482.
126
L.
K. JOHNSON,
R. W.
JOHNSON
AND
B. L.
STREHLER
A variety of biochemical changes have been associated with cardiac hypertrophy, both in experimental animals, and clinically in man [I]. Nearly every important molecular parameter to the cardiac protein synthetic system has been found to change during experimentally induced cardiac hypertrophy [IO], including alterations in the myocardial DNA content and concentration [4]. In our laboratory we have previously found changes in the DNA from postmitotic tissues (myocardium, skeletal muscle and brain) of aging canines [S, 71. In particular, we found that the dosage of ribosomal RNA genes decreases by 30% in postmitotic tissues from aging animals. Such a deficiency is likely to have a limiting effect on rRNA* synthesis, particularly during periods of maximum demand. Impaired protein synthesis due to ribosome overloading has been observed in in vitro protein synthesizing systems derived from hypertrophied myocardium [17], and may be an important factor in the reduced contractility characteristic of the failing ventricle [3]. The present investigation was undertaken in order to determine whether decreases in ribosomal RNA gene dosage in ventricular myocardium are associated with left ventricular hypertrophy and heart failure in man.
2. Materials
and
Methods
Tissue source Samples of hypertrophied and normal human left ventricle were graciously provided by Drs H. Edmonson and G. Lundberg of the Pathology Dept, University of Southern California Medical School, and Dr T. Noguchi of the Los Angeles County Coroner’s Office. All autopsies were performed within 24 h of death. At the time of autopsy all tissues were determined to be non-necrotic, and they were then frozen at -20°C until the DNA extractions were carried out (never more than 4 days later).
DNA
preflaration
All steps were carried out at 0°C except where otherwise indicated. Ten to 15 g samples of frozen left ventricular myocardium were broken into small pieces, and homogenized with 6 volumes of SSC buffer (pH 7.4) in a precooled Waring blender at top speed for 4 min. The homogenate was then passed through four layers of cheesecloth, the filtrate centrifuged at 10000 x g for 5 min. DNA was extracted from this crude pellet using the chloroform extraction procedure of Marmur [9], * Abbreviations: 2 x SSC is twice for rRNA.
SSC, Standard this concentration,
Saline etc.;
Citrate rRNA,
buffer ribosomal
(0.15 M NaCI, RNA; rDNA,
0.15 M sodium citrate), the genes which code
CARDIAC
HYPERTROPHY
IN
127
MAN
with the following modifications, some of which have been previously described in detail [S, 71. For precipitation of the DNA, 1 .O to 1.5 volumes of 95% ethanol were used. Ethanol precipitated threads of DNA were collected by pelleting at 2500 rev/min rather than by spooling in order to avoid the loss of low molecular weight fragments. For establishment of the proper ionic strength during the initial chloroform extraction, 1 M NaCl was used rather than 1 M NaC104 as was originally used in the Marmur Method; treatment of DNA with the former is a milder procedure. Chloroform extractions were carried out at room temperature. The isopropanol precipitation step was omitted. Chloroform extraction was repeated until there was no insoluble protein visible at the interphase plane. After ethanol precipitation an aliquot (usually 2 mg) of the DNA was incubated in SSC with Pronase (nuclease free, B grade, from Calbiochem) at a ratio of 0.5 mg Pronase per mg DNA at 37°C for 2 h. To effect denaturation of the DNA and removal of residual RNA, the DNA was then precipitated with 2 volumes ethanol, pelleted by centrifugation, dissolved in 2 ml 0.1 N NaOH and incubated at 27°C for 30 min. The DNA was then dialized against SSC for a minimum of 3 h. This was followed by one final chloroform extraction for removal of the Pronase. The yield and purity of the preparations were determined by optical density measurements at 260, 280, and 235 nm. Purification was judged to be adequate when the ratios of 260:280 and 260:235 were 2.0 or greater. In order to obtain reproducible hybridization values it was found to be necessary to fractionate the DNA on a Sephadex G-100 column, collecting only the high molecular weight fractions, just prior to use in the hybridization experiments. This step removed residual low molecular weight contaminants.
Tritiated
rRNA prefiaration
Tritiated rRNA for use in the hybridization experiments was prepared from young rat liver after intraperitoneal injection of 5 mCi [5-aH]-erotic acid (specific activity = 5 mCi/mmol, New England Nuclear) for 6 successive days. Liver ribosomes were isolated as described by Moldave and Skogerson [13], and rRNA was obtained by phenol extraction of the ribosomes [12]. Final purification of the rRNA was accomplished by gravity filtration through membrane filters (Millipore, type HAWP, 0.45 k), followed by fractionation on a Sephadex G-100 column. The high molecular weight peak, containing the 18s and 28s components, was used in the hybridization experiments. The same preparation of rRNA was used for all hybridizations; its specific activity was 7134 ct/min/pg.
DIVA-RNA The
hybridization
reactions
were
carried
hybridization out with
both
the RNA
and DNA
in
128
L.
K.
JOHNSON,
R.
W.
JOHNSON
AND
B.
L.
STREHLER
solution [.2.5]. Each reaction was carried out for 8 h at 75°C in 2 ml of 2 x SSC containing 5 Pg/ml sH-RNA and 50 pg/ml DNA. After hybridization 25 pg pancreatic RNAase (Calbiochem, heated to 90°C to destroy DNAase) and 25 units T1 RNAase (Calbiochem) were added to digest non-hybridized RNA (1 h at 30°C). This solution was then applied to a Sephadex G-100 column (0.9 x 50 cm) for separation of the hybrids from the RNA hydrolysis products. 2 x SSC was used for elution. Radioactivity and o.D.~~o were determined in 2 ml fractions. The relative percent hybridization (the parameter of rRNA gene dosage) was calculated from the ratio of radioactivity and o.D.~~o in the high molecular weight elution peak, Control experiments indicated that a small fraction of the rRNA (as monitored by sH ct/min) appeared in the high molecular weight elution peak after subjecting the rRNA to hybridization conditions in the absence of DNA, and after treatment with the RNAases; this background was subtracted before calculation of the percent hybridization values in Table 1.
3. Results Sephadex e&ion projle
of the r&VA-D.NA
hybrid
A typical Sephadex G-100 elution profile of the rRNA-DNA hybrid is shown in Figure 1. The DNA was from human myocardium (tissue no. 73-9047). The same figure shows the radioactivity profile resulting when the same amount of sH-rRNA is subjected to identical hybridization conditions in the absence of DNA, and chromatographed without DNA present; to calculate the percent hybridization the background radioactivity in the high molecular weight region (representing the non-specific ribonuclease-resistant material) was subtracted from the radioactive peak in this region which resulted when RNA-DNA hybrids were chromatographed. It was observed that this background varied when different preparations of RNAase were employed. However, in retesting several DNA samples using RNAase preparations which gave different backgrounds the same difference between the hybrid peak and the respective background resulted. This indicated that the method of determining the percent hybridization is reliable even when variable (but proportionally small) amounts of non-hybridized RNA are present in the hybrid peak. The percent hybridization determined in this manner was the parameter which was considered to be directly proportional to (if not identical with) the rRNA gene dosage.
The effect of tissue storage The use of human tissue for biochemical variable due to the comparatively long
studies frequently interval between
involves an additional death and the time at
1. Ribosomal
Cardiorespiratory arrest bronchopneumonia, hypoxemia chronic brain syndrome
Pulmonary infarction, shock pulmonary thromboli cerebral vascular accident organic heart disease
Cardiopulmonary arrest, shock hepatic failure, cirrhosis chronic active viral hepatitis chronic lymphocytic leukemia
Gunshot wound, no other pathology
Bronchopneumonia due to thermal burns
86856
86872
73-9047
73-10818
Cause of death and other conditions
20
18
73
64
75
Age (w-4
360
340
415
700
260
Heart weight (9)
RNA gene dosage in human left ventricular
86891
Tissue identification no.
TABLE
dimensions not determined, non-hypetrophic
dimensions not determined, non-hypertrophic
7.5 x 1.3 hypertrophic
10.4 x 1.8 hypertrophic
6.1 x 1.1 non-hypertrophic
Left ventricular length and thickness (cm) and diagnosis _-_.--
myocardium
0.094
0.092
0.073
0.071
0.065
Sample I
0.097
0.100
0.076
0.063
0.063
Sample II
0.099
0.068
Sample III
O,&Hybridization (RNA/DNA) x 100
130
L.
K. JOHNSON,
R. W.
JOHNSON
Fraction
AND
B. L. STREHLER
no
FIGURE 1. Typical Sephadex G-100 chromatographic separation of sH-rRNA-DNA hybrids. The DNA was from myocardial tissue no. 73-9047. Two ml fractions were eluted with 2 x SSC at a rate of 0.5 ml/min. DNA based on o.~.sso, (0-O); ct/min sH-rRNA in the hybrid complex, (-0); CPM sH-rRNA after being subjected to hybridization conditions in the absence of
DNA, (n----n
).
which the tissue is available for processing. In order to evaluate the possible effect of such varying intervals on our experimental results, preliminary tests were carried out using rabbit heart muscle. Carcasses were stored at a temperature and time period typical for cadaver storage between death and autopsy. Three rabbits were killed by cervical dislocation followed by decapitation. The heart from one rabbit was immediately removed and frozen. The other two were first stored at room temperature for 1 h. Then, one of these was stored for an additional 4 h, and the other for an additional 24 h, at 5°C. After storage these hearts also were removed and frozen. Myocardial DNA was extracted and hybridization experiments were carried out as described for human myocardium. No significant differences in the hybridization level were found between the different animals; the hybridization level (pg sH-rRNA/pg DNA x 100) for the tissue frozen immediately after death (the control) was 0.036%, the tissue stored for 5 h was 0.041%, and the tissue stored for 25 h was 0.038%.
CARDIAC
HYPERTROPHY
IN MAN
131
Ribosomal RNA gene dosage in human left ventricular myocardium
The results of the hybridization experiments with DNA from left ventricular myocardium are shown in Table 1. Two or three hybridizations were carried out with DNA from each tissue.Within the advanced age group (ages64, 73 and 75 years), the values fell in a narrow range with overlap between values for different tissues; there were no significant differences between the hypertrophic and the nonhypertrophic tissues.However, a marked difference was found between the tissues from the young individuals (ages18 and 20 years) and the tissuesfrom the advanced age group; the average hybridization level of the older group was 0.068% (range : 0.063% to 0.076x), while the average value for the young individual was 0.096% (range : 0.092 to 0.100%).
4. Discussion
Previous rRNA-DNA hybridization studies in which DNA from human tissues were used are of interest for comparison to the values obtained in this study. However, the rRNA gene dosagein human myocardial DNA, which would be of primary interest here for purposes of comparison, has not been determined previously. In cultured human kidney cells [a], and in HeLa cells [5J the hybridization levels were 0.025% to 0.045‘& and 0.025% respectively. These values are somewhat lower than the values determined here for myocardium; the differences may be due to the aneuploidy which is a feature of cells grown in culture for extended periods, and of cells derived from cancerous tissue (the HeLa cells). The lower values may also be attributable to the differences in hybridization conditions employed by theseother authors. Most of the previous studies on the biochemical events associatedwith cardiac hypertrophy have necessarily involved experimentally produced overloading of the heart in lower mammals; overloading and the consequent hypertrophy is usually produced by impeding cardiac outflow by banding of the aorta (experimental stenosis). Some of the findings of such studies may bear on the results obtained in the present investigation. One of the early events in experimentally induced hypertrophy is an increasein the total DNA content of the heart, both by increase in polyploid frequency in muscle cells, and by mitosis of non-muscular connective tissuecells [a]. The duplication of DNA in mammalian cells is known to be an asynchronous process: different portions of the genome are replicated in a defined sequence during the S-phase [I4]. Thus, our results could reflect an incomplete replication of the myocardial DNA during hypertrophy (and/or aging), in which the processis terminated before complete duplication of the rDNA. Some support for such an interpretation derives from observations in synchronous
132
L. KJOHNSON,
R. W.JOHNSON
AND
B. L. STREHLER
cultures of Chinese hamster cells in which there is an initial decrement in the rDNA dosage during the S-phase, due to delayed replication of these genes [2]. We observed a reduced level of rRNA gene dosage in the myocardium of all the aged individuals regardless of whether the myocardium was in a state of hypertrophy. These results are in complete accord with our previously published studies [S, 71 on canine tissues in which we also found a 30% reduction in dosage of rRNA genes in postmitotic tissues (brain, skeletal muscle, and myocardium) of the aged animals; it is particularly interesting that dogs, which had lived a corresponding percentage of their average lifespan displayed a similar decline in gene dosage in the same postmitotic tissues. The biological significance of such a deficiency in myocardium may be inferred from studies of mutants of the toad Xenopus laevis which have a deficiency of rRNA. Mutants with partial deletions which have 35% of the normal diploid amount of rDNA synthesize rRNA at 50% of the normal rate; these mutants however, die at an early stage of embryonic development [11]. A similar reduction in rRNA synthesis as well as other physiological and anatomical defects are also observed in rDNA deficient “bobbed” mutants of the fruit fly Drosophila melanogaster [16]. Based on these studies there appears to be a critical dosage of rDNA, below which there results insufficient rRNA synthesis to support a level of protein synthesis compatible with normal cellular metabolism. Our primary objective was to elucidate the biochemical factors that limit cardiac hypertrophy and heart failure in man. Our findings are likely to have a direct bearing on the reduced ability of the aging heart to respond effectively to increased work loads imposed by other aged-related deteriorative processes such as arteriosclerosis and hypertension. The normal physiological response of the heart to the stress of an increased load is growth and increased output. The growth response requires high levels of protein synthesis, which in turn is directly dependent on the rate of rRNA synthesis and the availability of ribosomes. It follows that a 30% decrement in the dosage of rDNA as we observed would correspondingly decrease the maximum rate of protein synthesis thereby interfering with adaptive growth. The increased incidence of heart failure in the older age categories may be traceable to such a decrement in protein synthesis.
REFERENCES N. R. (Ed.) Cardiac Hypertrophy. New York: Academic Press (1971). F., GIACOMONI, D. & ZITO-BIGNAMI, R. On the duplication of ribosomal RNA cistrons in Chinese hamster cells. European Journal of Biochemistry 11, 419-523 (1969). 3. BING, 0. H. L., MATSUSHITA, S., FANBURG, B. L. & LEVINE, H. J. Mechanical properties of rat cardiac muscle during experimental hypertrophy. In Cardiac Hypertrojhy, N. R. Alpert, Ed., pp. 361-396. New York: Academic Press (1971). 4. GROVE, D., NAIR, K. G. & ZAK, R. Biochemical correlates of cardiac hypertrophy, III changes in DNA content; the relative contribution of polyploidy and mitotic activity. Circulation Research 25, 463472 (1969). 1.
2.
ALPERT, AMALDI,
CARDIAC 5.
6. 7. 8. 9. 10. I 1.
12.
13.
14. 15. 16.
17.
HYPERTROPHYIN
MAN
133
JEANTEIJR, P. & ATTARDI, G. Relationship between HeLa cell ribosomal RNA and its precursors studied by high resolution RNA-DNA hybridization. Journal of Molecular Biology 45, 305-324 (1969). JOHNSON, R., CHRISP, C. & STREHLER, B. Selective loss of ribosomal RNA genes during the aging of postmitotic tissues. Mechanisms of Aging and Development 1, 183-198 (1972). JOHNSON, R. & STREHLER, B. Loss of genes coding for ribosomal RNA in ageing brain cells. Nature 240, 412 (1972). KOCH, J. & CRUCEANU, A. Hormone induced gene amplification in somatic cells. Hoppe-Seyler’s 3ourrud of Physiological Chemistry 352, 137-139 (197 1). MARMUR, J. A procedure for the extraction of DNA from micro-organisms. Journal of Molecular Biology 3, 208-2 18 (1961). MEERSON, F. Z. The myocardium in hyperfunction, hypertrophy and heart failure. Circulation Research 25 (Supplement II), 82-146 (1969). MILLER, L. & KNOWLAND, J. The number and activity of rRNA genes in Xenopus laevis carrying partial deletions in both nucleolar organizers. Biochemical Genetics 6, 65-73 (1972). MOLDAVE, K. Preparation of RNA from mammalian ribosomes. In Methods in Enzymology Vol. 12A. S. P. Colowick and N. D. Kaplan, Eds. pp. 607-608. New York: Academic Press ( 1967). MOLDAVE, K. & SKOGERSON, L. Purification of mammalian ribosomes. In Methods in Enzymology Vol. 12A. S. P. Colowick and N. D. Kaplan, Eds. pp. 478480. New York: Academic Press (1967). MUELLER, C. G. & KAJIWARA, K. Early and late replicating DNA complexes in HeLa nuclei. Biochimica et biofihysica acta 114, 108-l 15 (1966). NYGARD, A. P. & HALL, B. D. A method for detection of RNA-DNA complexes. Biochmical and Biophysical Research Communications 12, 98-104 (1963). RITOSSA, F. M., ATWOOD, K. C., LINDSEY, D. & SPIEGELMAN, S. On the chromosomal distribution of DNA complimentary to ribosomal and soluble RN.4. Proceedings of the National Cancer Institute Monograbh 23, &9-474 (1966). SCHREIEER, S. S., ORATZ, M. & ROTHSCHILD, M. A. Initiation of protein synthesis in the acutely overloaded perfused heart. In Cardiac Hypertrothy, N. R. Alpert, Ed. pp. 215-246. New York: Academic Press (1971).
Molecular
Cardiac
LORIN BioZogical
and Cellular
Cardiology
(1975)
SHORT
COMMUNICATIONS
Hypertrophy, Ribosomal
K. JOHNSON, Sciences Department, (Received
RNA
ROGER
7, 125-133
Aging and Changes in Cardiac Gene Dosage in Man*
W. JOHNSON
University 25 April
AND BERNARD
of Southern California, 1974,
L. STREHLER
Los Angeles,
and accepted 29 June
California
90007
1974)
L. K. JOHNSON, R. W. JOHNSON AND B. L. STRJXHLER. Cardiac Hypertrophy, Aging and Changes in Cardiac Ribosomal RNA Gene Dosage in Man. Journal of Molecular and Cellular Cardiology (1975) 7, 125-133. The dosage of ribosomal RNA genes was determined in left ventricular myocardium obtained from two young individuals (ages 18 and 20 years), and from three older individuals (ages 64, 73 and 75 years), two of whom were diagnosed to have left ventricular hypertrophy. The technique of RNA-DNA molecular hybridization was employed to determine the gene dosage. The results indicate that there is a significant decline in the dosage of ribosomal RNA genes in the myocardium from the older individuals; the average hybridization level (the parameter of gene dosage) was 0.096% (range: 0.092 to 0.100%) for the young myocardium, and 0.068% (range: 0.063 to 0.076%) for the older myocardium. Within the older age group the hybridization level for the one tissue judged to be non-hypertrophic was essentially identical in its dosage to the tissues derived from hypertrophic myocardimn. These results, which are highly consistent with earlier studies on canine tissues, are interpreted to suggest that: (1) a ribosomal RNA gene loss such as that measured in aging myocardium may impose limitations on the rate of RNA synthesis, and thus indirectly cause abnormally low rates of protein synthesis within the myocardium during periods of stress, and (2) that such restrictions on the maximum rate of protein synthesis may account for the decreased ability of aged myocardium to respond to increased work loads, and the reduced contractility characteristic of aging muscle, hypertrophic myocardium and the failing heart. KEY WORDS: Cardiomegaly; Cardiac hyperfunction; ventricular hypertrophy; Ribosomes; Postmitotic cells;
RNA-DNA Congestive
hybridization; heart failure.
Left
1. Introduction Cardiac hypertrophy can be an adaptive physiological response to increased functional demands on the heart. However, when the functional demands are excessive and protracted the hypertrophy exceeds adaptive limits and congestive heart failure ensues. Left ventricular hypertrophy is the most common form occurring in man, and is frequently associated with systemic arterial hypertension. Systemic arterial hypertension, in turn, is commonly a correlate of age-dependent deterioration of the vascular system, often including arteriosclerosis. * This work
was supported
in part
by the Los Angeles
County
Heart
Association,
Grant
No. 482.
126
L.
K. JOHNSON,
R. W.
JOHNSON
AND
B. L.
STREHLER
A variety of biochemical changes have been associated with cardiac hypertrophy, both in experimental animals, and clinically in man [I]. Nearly every important molecular parameter to the cardiac protein synthetic system has been found to change during experimentally induced cardiac hypertrophy [IO], including alterations in the myocardial DNA content and concentration [4]. In our laboratory we have previously found changes in the DNA from postmitotic tissues (myocardium, skeletal muscle and brain) of aging canines [S, 71. In particular, we found that the dosage of ribosomal RNA genes decreases by 30% in postmitotic tissues from aging animals. Such a deficiency is likely to have a limiting effect on rRNA* synthesis, particularly during periods of maximum demand. Impaired protein synthesis due to ribosome overloading has been observed in in vitro protein synthesizing systems derived from hypertrophied myocardium [17], and may be an important factor in the reduced contractility characteristic of the failing ventricle [3]. The present investigation was undertaken in order to determine whether decreases in ribosomal RNA gene dosage in ventricular myocardium are associated with left ventricular hypertrophy and heart failure in man.
2. Materials
and
Methods
Tissue source Samples of hypertrophied and normal human left ventricle were graciously provided by Drs H. Edmonson and G. Lundberg of the Pathology Dept, University of Southern California Medical School, and Dr T. Noguchi of the Los Angeles County Coroner’s Office. All autopsies were performed within 24 h of death. At the time of autopsy all tissues were determined to be non-necrotic, and they were then frozen at -20°C until the DNA extractions were carried out (never more than 4 days later).
DNA
preflaration
All steps were carried out at 0°C except where otherwise indicated. Ten to 15 g samples of frozen left ventricular myocardium were broken into small pieces, and homogenized with 6 volumes of SSC buffer (pH 7.4) in a precooled Waring blender at top speed for 4 min. The homogenate was then passed through four layers of cheesecloth, the filtrate centrifuged at 10000 x g for 5 min. DNA was extracted from this crude pellet using the chloroform extraction procedure of Marmur [9], * Abbreviations: 2 x SSC is twice for rRNA.
SSC, Standard this concentration,
Saline etc.;
Citrate rRNA,
buffer ribosomal
(0.15 M NaCI, RNA; rDNA,
0.15 M sodium citrate), the genes which code
CARDIAC
HYPERTROPHY
IN
127
MAN
with the following modifications, some of which have been previously described in detail [S, 71. For precipitation of the DNA, 1 .O to 1.5 volumes of 95% ethanol were used. Ethanol precipitated threads of DNA were collected by pelleting at 2500 rev/min rather than by spooling in order to avoid the loss of low molecular weight fragments. For establishment of the proper ionic strength during the initial chloroform extraction, 1 M NaCl was used rather than 1 M NaC104 as was originally used in the Marmur Method; treatment of DNA with the former is a milder procedure. Chloroform extractions were carried out at room temperature. The isopropanol precipitation step was omitted. Chloroform extraction was repeated until there was no insoluble protein visible at the interphase plane. After ethanol precipitation an aliquot (usually 2 mg) of the DNA was incubated in SSC with Pronase (nuclease free, B grade, from Calbiochem) at a ratio of 0.5 mg Pronase per mg DNA at 37°C for 2 h. To effect denaturation of the DNA and removal of residual RNA, the DNA was then precipitated with 2 volumes ethanol, pelleted by centrifugation, dissolved in 2 ml 0.1 N NaOH and incubated at 27°C for 30 min. The DNA was then dialized against SSC for a minimum of 3 h. This was followed by one final chloroform extraction for removal of the Pronase. The yield and purity of the preparations were determined by optical density measurements at 260, 280, and 235 nm. Purification was judged to be adequate when the ratios of 260:280 and 260:235 were 2.0 or greater. In order to obtain reproducible hybridization values it was found to be necessary to fractionate the DNA on a Sephadex G-100 column, collecting only the high molecular weight fractions, just prior to use in the hybridization experiments. This step removed residual low molecular weight contaminants.
Tritiated
rRNA prefiaration
Tritiated rRNA for use in the hybridization experiments was prepared from young rat liver after intraperitoneal injection of 5 mCi [5-aH]-erotic acid (specific activity = 5 mCi/mmol, New England Nuclear) for 6 successive days. Liver ribosomes were isolated as described by Moldave and Skogerson [13], and rRNA was obtained by phenol extraction of the ribosomes [12]. Final purification of the rRNA was accomplished by gravity filtration through membrane filters (Millipore, type HAWP, 0.45 k), followed by fractionation on a Sephadex G-100 column. The high molecular weight peak, containing the 18s and 28s components, was used in the hybridization experiments. The same preparation of rRNA was used for all hybridizations; its specific activity was 7134 ct/min/pg.
DIVA-RNA The
hybridization
reactions
were
carried
hybridization out with
both
the RNA
and DNA
in
128
L.
K.
JOHNSON,
R.
W.
JOHNSON
AND
B.
L.
STREHLER
solution [.2.5]. Each reaction was carried out for 8 h at 75°C in 2 ml of 2 x SSC containing 5 Pg/ml sH-RNA and 50 pg/ml DNA. After hybridization 25 pg pancreatic RNAase (Calbiochem, heated to 90°C to destroy DNAase) and 25 units T1 RNAase (Calbiochem) were added to digest non-hybridized RNA (1 h at 30°C). This solution was then applied to a Sephadex G-100 column (0.9 x 50 cm) for separation of the hybrids from the RNA hydrolysis products. 2 x SSC was used for elution. Radioactivity and o.D.~~o were determined in 2 ml fractions. The relative percent hybridization (the parameter of rRNA gene dosage) was calculated from the ratio of radioactivity and o.D.~~o in the high molecular weight elution peak, Control experiments indicated that a small fraction of the rRNA (as monitored by sH ct/min) appeared in the high molecular weight elution peak after subjecting the rRNA to hybridization conditions in the absence of DNA, and after treatment with the RNAases; this background was subtracted before calculation of the percent hybridization values in Table 1.
3. Results Sephadex e&ion projle
of the r&VA-D.NA
hybrid
A typical Sephadex G-100 elution profile of the rRNA-DNA hybrid is shown in Figure 1. The DNA was from human myocardium (tissue no. 73-9047). The same figure shows the radioactivity profile resulting when the same amount of sH-rRNA is subjected to identical hybridization conditions in the absence of DNA, and chromatographed without DNA present; to calculate the percent hybridization the background radioactivity in the high molecular weight region (representing the non-specific ribonuclease-resistant material) was subtracted from the radioactive peak in this region which resulted when RNA-DNA hybrids were chromatographed. It was observed that this background varied when different preparations of RNAase were employed. However, in retesting several DNA samples using RNAase preparations which gave different backgrounds the same difference between the hybrid peak and the respective background resulted. This indicated that the method of determining the percent hybridization is reliable even when variable (but proportionally small) amounts of non-hybridized RNA are present in the hybrid peak. The percent hybridization determined in this manner was the parameter which was considered to be directly proportional to (if not identical with) the rRNA gene dosage.
The effect of tissue storage The use of human tissue for biochemical variable due to the comparatively long
studies frequently interval between
involves an additional death and the time at
1. Ribosomal
Cardiorespiratory arrest bronchopneumonia, hypoxemia chronic brain syndrome
Pulmonary infarction, shock pulmonary thromboli cerebral vascular accident organic heart disease
Cardiopulmonary arrest, shock hepatic failure, cirrhosis chronic active viral hepatitis chronic lymphocytic leukemia
Gunshot wound, no other pathology
Bronchopneumonia due to thermal burns
86856
86872
73-9047
73-10818
Cause of death and other conditions
20
18
73
64
75
Age (w-4
360
340
415
700
260
Heart weight (9)
RNA gene dosage in human left ventricular
86891
Tissue identification no.
TABLE
dimensions not determined, non-hypetrophic
dimensions not determined, non-hypertrophic
7.5 x 1.3 hypertrophic
10.4 x 1.8 hypertrophic
6.1 x 1.1 non-hypertrophic
Left ventricular length and thickness (cm) and diagnosis _-_.--
myocardium
0.094
0.092
0.073
0.071
0.065
Sample I
0.097
0.100
0.076
0.063
0.063
Sample II
0.099
0.068
Sample III
O,&Hybridization (RNA/DNA) x 100
130
L.
K. JOHNSON,
R. W.
JOHNSON
Fraction
AND
B. L. STREHLER
no
FIGURE 1. Typical Sephadex G-100 chromatographic separation of sH-rRNA-DNA hybrids. The DNA was from myocardial tissue no. 73-9047. Two ml fractions were eluted with 2 x SSC at a rate of 0.5 ml/min. DNA based on o.~.sso, (0-O); ct/min sH-rRNA in the hybrid complex, (-0); CPM sH-rRNA after being subjected to hybridization conditions in the absence of
DNA, (n----n
).
which the tissue is available for processing. In order to evaluate the possible effect of such varying intervals on our experimental results, preliminary tests were carried out using rabbit heart muscle. Carcasses were stored at a temperature and time period typical for cadaver storage between death and autopsy. Three rabbits were killed by cervical dislocation followed by decapitation. The heart from one rabbit was immediately removed and frozen. The other two were first stored at room temperature for 1 h. Then, one of these was stored for an additional 4 h, and the other for an additional 24 h, at 5°C. After storage these hearts also were removed and frozen. Myocardial DNA was extracted and hybridization experiments were carried out as described for human myocardium. No significant differences in the hybridization level were found between the different animals; the hybridization level (pg sH-rRNA/pg DNA x 100) for the tissue frozen immediately after death (the control) was 0.036%, the tissue stored for 5 h was 0.041%, and the tissue stored for 25 h was 0.038%.
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HYPERTROPHY
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131
Ribosomal RNA gene dosage in human left ventricular myocardium
The results of the hybridization experiments with DNA from left ventricular myocardium are shown in Table 1. Two or three hybridizations were carried out with DNA from each tissue.Within the advanced age group (ages64, 73 and 75 years), the values fell in a narrow range with overlap between values for different tissues; there were no significant differences between the hypertrophic and the nonhypertrophic tissues.However, a marked difference was found between the tissues from the young individuals (ages18 and 20 years) and the tissuesfrom the advanced age group; the average hybridization level of the older group was 0.068% (range : 0.063% to 0.076x), while the average value for the young individual was 0.096% (range : 0.092 to 0.100%).
4. Discussion
Previous rRNA-DNA hybridization studies in which DNA from human tissues were used are of interest for comparison to the values obtained in this study. However, the rRNA gene dosagein human myocardial DNA, which would be of primary interest here for purposes of comparison, has not been determined previously. In cultured human kidney cells [a], and in HeLa cells [5J the hybridization levels were 0.025% to 0.045‘& and 0.025% respectively. These values are somewhat lower than the values determined here for myocardium; the differences may be due to the aneuploidy which is a feature of cells grown in culture for extended periods, and of cells derived from cancerous tissue (the HeLa cells). The lower values may also be attributable to the differences in hybridization conditions employed by theseother authors. Most of the previous studies on the biochemical events associatedwith cardiac hypertrophy have necessarily involved experimentally produced overloading of the heart in lower mammals; overloading and the consequent hypertrophy is usually produced by impeding cardiac outflow by banding of the aorta (experimental stenosis). Some of the findings of such studies may bear on the results obtained in the present investigation. One of the early events in experimentally induced hypertrophy is an increasein the total DNA content of the heart, both by increase in polyploid frequency in muscle cells, and by mitosis of non-muscular connective tissuecells [a]. The duplication of DNA in mammalian cells is known to be an asynchronous process: different portions of the genome are replicated in a defined sequence during the S-phase [I4]. Thus, our results could reflect an incomplete replication of the myocardial DNA during hypertrophy (and/or aging), in which the processis terminated before complete duplication of the rDNA. Some support for such an interpretation derives from observations in synchronous
132
L. KJOHNSON,
R. W.JOHNSON
AND
B. L. STREHLER
cultures of Chinese hamster cells in which there is an initial decrement in the rDNA dosage during the S-phase, due to delayed replication of these genes [2]. We observed a reduced level of rRNA gene dosage in the myocardium of all the aged individuals regardless of whether the myocardium was in a state of hypertrophy. These results are in complete accord with our previously published studies [S, 71 on canine tissues in which we also found a 30% reduction in dosage of rRNA genes in postmitotic tissues (brain, skeletal muscle, and myocardium) of the aged animals; it is particularly interesting that dogs, which had lived a corresponding percentage of their average lifespan displayed a similar decline in gene dosage in the same postmitotic tissues. The biological significance of such a deficiency in myocardium may be inferred from studies of mutants of the toad Xenopus laevis which have a deficiency of rRNA. Mutants with partial deletions which have 35% of the normal diploid amount of rDNA synthesize rRNA at 50% of the normal rate; these mutants however, die at an early stage of embryonic development [11]. A similar reduction in rRNA synthesis as well as other physiological and anatomical defects are also observed in rDNA deficient “bobbed” mutants of the fruit fly Drosophila melanogaster [16]. Based on these studies there appears to be a critical dosage of rDNA, below which there results insufficient rRNA synthesis to support a level of protein synthesis compatible with normal cellular metabolism. Our primary objective was to elucidate the biochemical factors that limit cardiac hypertrophy and heart failure in man. Our findings are likely to have a direct bearing on the reduced ability of the aging heart to respond effectively to increased work loads imposed by other aged-related deteriorative processes such as arteriosclerosis and hypertension. The normal physiological response of the heart to the stress of an increased load is growth and increased output. The growth response requires high levels of protein synthesis, which in turn is directly dependent on the rate of rRNA synthesis and the availability of ribosomes. It follows that a 30% decrement in the dosage of rDNA as we observed would correspondingly decrease the maximum rate of protein synthesis thereby interfering with adaptive growth. The increased incidence of heart failure in the older age categories may be traceable to such a decrement in protein synthesis.
REFERENCES N. R. (Ed.) Cardiac Hypertrophy. New York: Academic Press (1971). F., GIACOMONI, D. & ZITO-BIGNAMI, R. On the duplication of ribosomal RNA cistrons in Chinese hamster cells. European Journal of Biochemistry 11, 419-523 (1969). 3. BING, 0. H. L., MATSUSHITA, S., FANBURG, B. L. & LEVINE, H. J. Mechanical properties of rat cardiac muscle during experimental hypertrophy. In Cardiac Hypertrojhy, N. R. Alpert, Ed., pp. 361-396. New York: Academic Press (1971). 4. GROVE, D., NAIR, K. G. & ZAK, R. Biochemical correlates of cardiac hypertrophy, III changes in DNA content; the relative contribution of polyploidy and mitotic activity. Circulation Research 25, 463472 (1969). 1.
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JEANTEIJR, P. & ATTARDI, G. Relationship between HeLa cell ribosomal RNA and its precursors studied by high resolution RNA-DNA hybridization. Journal of Molecular Biology 45, 305-324 (1969). JOHNSON, R., CHRISP, C. & STREHLER, B. Selective loss of ribosomal RNA genes during the aging of postmitotic tissues. Mechanisms of Aging and Development 1, 183-198 (1972). JOHNSON, R. & STREHLER, B. Loss of genes coding for ribosomal RNA in ageing brain cells. Nature 240, 412 (1972). KOCH, J. & CRUCEANU, A. Hormone induced gene amplification in somatic cells. Hoppe-Seyler’s 3ourrud of Physiological Chemistry 352, 137-139 (197 1). MARMUR, J. A procedure for the extraction of DNA from micro-organisms. Journal of Molecular Biology 3, 208-2 18 (1961). MEERSON, F. Z. The myocardium in hyperfunction, hypertrophy and heart failure. Circulation Research 25 (Supplement II), 82-146 (1969). MILLER, L. & KNOWLAND, J. The number and activity of rRNA genes in Xenopus laevis carrying partial deletions in both nucleolar organizers. Biochemical Genetics 6, 65-73 (1972). MOLDAVE, K. Preparation of RNA from mammalian ribosomes. In Methods in Enzymology Vol. 12A. S. P. Colowick and N. D. Kaplan, Eds. pp. 607-608. New York: Academic Press ( 1967). MOLDAVE, K. & SKOGERSON, L. Purification of mammalian ribosomes. In Methods in Enzymology Vol. 12A. S. P. Colowick and N. D. Kaplan, Eds. pp. 478480. New York: Academic Press (1967). MUELLER, C. G. & KAJIWARA, K. Early and late replicating DNA complexes in HeLa nuclei. Biochimica et biofihysica acta 114, 108-l 15 (1966). NYGARD, A. P. & HALL, B. D. A method for detection of RNA-DNA complexes. Biochmical and Biophysical Research Communications 12, 98-104 (1963). RITOSSA, F. M., ATWOOD, K. C., LINDSEY, D. & SPIEGELMAN, S. On the chromosomal distribution of DNA complimentary to ribosomal and soluble RN.4. Proceedings of the National Cancer Institute Monograbh 23, &9-474 (1966). SCHREIEER, S. S., ORATZ, M. & ROTHSCHILD, M. A. Initiation of protein synthesis in the acutely overloaded perfused heart. In Cardiac Hypertrothy, N. R. Alpert, Ed. pp. 215-246. New York: Academic Press (1971).
