© 1999 by European Society of Cardiology
Copyright © 1999, European Society of Cardiology
Measure is treasure
Department of Anatomy and Embryology, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
* Corresponding author. Tel.: +31-20-5669111; fax: +31-20-6976177 m.j.vandenhoff{at}amc.uva.nl a.f.moorman{at}amc.uva.nl
Received 26 February 1999; accepted 24 March 1999
See article by Ribadeau-Dumas et al. [1] (pages 426–436) in this issue.
The paper of Ribadeau-Dumas et al. in this issue [1] deals with the interesting, but complex regulation of the SERCA2 gene. This gene encodes the sarcoplasmatic calcium pump, which is a crucial enzyme in the calcium handling of the cardiomyocyte. Insight into the regulation of this gene is therefore of paramount importance. A proper understanding of the regulation of the expression of a gene in adult, developing, experimental or diseased (cardiac) tissue requires precise knowledge of the control at each step from gene to the function of the encoded protein, comprising RNA and protein accumulation (being the product of synthesis and degradation) and biological activity of the studied protein. This list can of course be extended (Fig. 1). If we are to appreciate at which step in this pathway control is predominantly exerted, it is important to quantify as many of the parameters as possible. It is equally important to relate the disparate parameters with one another, to evaluate, for example, whether or not the increase in biological activity is proportional with the increase in the amount of protein, or whether the increase in protein follows a proportionate increase in the encoding mRNA. The vital role of quantification of the distinct parameters for our understanding of the regulation of (cardiac) gene expression is beyond any dispute. The lightheartedness concerning this topic is therefore surprising.
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It is convention to use total RNA (UV-measurement, ethidium bromide staining, 18S or 28S hybridization) or a housekeeping gene (GAPDH-mRNA or EF1
-mRNA) as tissue base if the levels of a specific mRNA are to be evaluated (e.g. by Northern blotting, or RNase-protection assay) and to use total protein as tissue base if levels of a specific protein or activity are to be evaluated. Not only is it often implicitly assumed that the tissue base (total RNA, or total protein) does not change during the conditions studied, it is also often implicitly assumed that the two tissue bases (total protein and total RNA) do not change relative to one another. Comparison of the changes in the levels of a distinct protein with its encoding mRNA without taking into account the possible changes in the respective tissue bases relative to one another, is a comparison of two incomparable quantities. It is conventional wisdom that apples and oranges are not to be compared.
In a single study Ribadeau-Dumas et al. have determined the rate of SERCA2 transcription, SERCA2 mRNA accumulation, SERCA2 protein accumulation and SERCA2 protein activity [1]. In accordance with common practice, they expressed SERCA2 transcription (run on assays) per ‘transcribed’ DNA (total transcriptional activity), SERCA2 mRNA accumulation (Northern blot) per 18S rRNA, SERCA2 protein accumulation (ELISA) and SERCA2 activity per total protein (Table 1). However, direct comparison of the developmental changes of a single parameter, or comparison of the distinct parameters with each other is only allowed if the denominators (i.e. total DNA, total RNA and total protein) do not change with development and also do not change relative to one another. This is, however, not the case. Compared to the embryonic heart, in the adult heart total protein concentration has increased almost 3-fold, total RNA concentration has decreased almost 4-fold, and total DNA concentration has decreased almost 2-fold [2]. As a consequence the ratios protein to RNA, RNA to DNA and protein to DNA have changed as well. Therefore, we have recalculated the data presented by Ribadeau-Dumas et al. [1] taking into account these changing tissue bases, and the results are presented in Table 2. From this table one can appreciate that per ventricular wet weight the SERCA2 mRNA accumulates 2.7-fold, SERCA2 protein 10-fold and SERCA2 activity 34.1-fold in the developmental period studied. These figures differ significantly from conventional representation (see Tables 1 and 2
). Moreover, from a functional rather than a regulatory point of view it seems more appropriate to relate the increase in SERCA2 activity to the increase in cardiac mass, which will match approximately the volume. Recalculation of the developmental change in SERCA2 activity per mg wet weight reveals in a 34-fold increase in activity (Table 2). This number is in agreement with the well-known increase in cardiac performance after birth [3].
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An unexpected and remarkable finding of Ribadeau-Dumas et al. [1] is the observation that the rate of transcription of the SERCA2 gene does not change significantly in the same developmental period (between 17 days of development and 20 days after birth). This conclusion holds only true if the total transcriptional activity does not change with development, as the authors have shown [1]. The important implication of this finding is that all developmental changes in the expression of the SERCA2 gene should be due to post-transcriptional regulation. The significance of this can only be fully exploited if it is compared with the SERCA2 mRNA, protein and activity data. This, in turn, requires a common tissue base that in this case should be DNA, as transcription rates are expressed, of necessity, per ‘transcribed’ DNA (total transcriptional activity). Hardly any differences are observed when DNA is used as tissue base rather than wet weight, because the DNA concentration does not change significantly between 17 days of development and 20 days after birth [2]. Comparison of Tables 1 and 2
immediately reveals that the developmental increase in SERCA2 mRNA per DNA is only 2.4-fold compared to the 4.9-fold increase when expressed per 18S ribosomal RNA. Furthermore, the increase in SERCA2 protein and activity are each about 3-fold higher when expressed per DNA rather than per protein. As a consequence SERCA2 activity has increased more than 30-fold with development. This number may even be underestimated, as with development the contribution of cardiomyocyte DNA relative to total cardiac DNA decreases about one third [4–6]. Moreover, since the denominator of the values for SERCA2 mRNA, protein and activity are now the same, it is also allowed to calculate the ratio SERCA2 protein per SERCA2 mRNA and SERCA2 activity per SERCA2 protein. This permits further evaluation of the levels of control. Based on these additional figures (Table 2) and because the rate of SERCA2 transcription does not change with development, the 2.4-fold increase in SERCA2 mRNA per DNA is most probably due to a change in the stability of the SERCA2 mRNA. Secondly, because with development the ratio of SERCA2 protein to SERCA2 mRNA increases 3.8-fold, either the translational efficiency of SERCA2 mRNA and/or the stability of SERCA2 protein has increased. Thirdly, the finding that the ratio SERCA2 activity to SERCA2 protein increases 3.4-fold indicates that the molecular specific activity of the SERCA2 protein has increased with development.
All together these data demonstrate a considerable post-transcriptional control of SERCA2 gene expression, with cardiac development. This may apply for other genes as well, as with development the heart traverses from a biosynthetic phase fashioned for hyperplastic growth, toward a mature phase optimally constructed for force production. Measure, taken the tissue base into account, may well turn out to be a treasure.
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Acknowledgements |
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M.J.B. van den Hoff is supported by the Netherlands Heart Foundation (Grant no.: M96.002).
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References |
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 Top References |
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- Ribadeau-Dumas A., Boateng S., Schwartz K., Boheler K.R. Sarco(Endo)plasmic reticulum Ca2+-ATPase (SERCA2) gene products are regulated post-transcriptionally during rat cardiac development. Cardiovasc Res (1999) 43:426–436.
[Abstract/Free Full Text] - van den Hoff M.J.B., Lekanne Deprez R.H., Monteiro M., et al. Developmental Changes in rat cardiac DNA, RNA and protein tissue base: Implications for the interpretation of changes in gene expression. J Mol Cell Cardiol (1997) 29:629–639.[CrossRef][Web of Science][Medline]
- Nakanishi T., Sgushi M., Takao A. Development of the myocardial contractile system. Experientia (1988) 44:936–944.[CrossRef][Web of Science][Medline]
- Canale E.D., Campbell G.R., Smolich J.J., Campbell J.H., eds. Handbook of microscopic anatomy: cardiac muscle. (1986) II/7. Berlin: Springer.
- Cluzeaut F., Maurer-Schultze B. Proliferation of cardiomyocytes and interstitial cells in the cardiac muscle of the mouse during pre- and postnatal development. Cell Tissue Kinet (1986) 19:267–274.[Web of Science][Medline]
- Li F., Wang X., Capasso J.M., Gerdes A.M. Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development. J Mol Cell Cardiol (1996) 28:1737–1746.[CrossRef][Web of Science][Medline]
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