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Letters to the Editor |
Department of Experimental Medicine and Pathology Section of Anatomic Pathology Policlinico Umberto I 00161 Rome, Italy
To the editor:
We appreciate Dr. Kinugawa’s comments and suggestions, and we definitely agree that more data are needed to evaluate the actual thyroid hormone action in failing myocardium. We also agree that the discrepancies observed between Dr. Kinugawa (1) and our study (2) cannot be explained solely by the use of glyceraldehyde-3-phosphate dehydrogenase in the RNAse protection assays.
The multiplex competitive RT-PCR method used in our study relies on the use of target-specific RNA internal standards, which are reverse transcribed together with the target sequences in each reaction tube, thus allowing an accurate estimate of the target transcript. In our hands, this method allows the quantification of the target gene in increments of one order of magnitude by simple visualization of ethidium bromide-stained gels (3).
For Western blotting, we used the MA1-125 antibody from Affinity
Bioreagents that resulted in a single band of the molecular weight of
Tr
1. We used the antibody on eight diseased hearts, the control, and
a kidney sample, with consistent results. In addition, we performed
histology and immunohistochemistry on left ventricular samples from all
diseased hearts. Histology was performed on the same myocardial samples
from which total RNA was later extracted, to exclude the presence of
extensive myocardial fibrosis, which could potentially affect the
results of TR mRNA quantification. Indeed, in all myocardial samples
examined there was a mild increase of interstitial fibrosis, in absence
of replacement fibrosis. Immunohistochemistry localized TRs mostly in
myocyte nuclei, with a focal positive signal of endothelial nuclei.
The concept of "local thyroid hormone homeostasis" represents the series of events involving thyroid hormone intracellular transport, metabolism by the deiodinases, binding with the receptor isoforms, receptor dimerization and interaction with coactivators and corepressors, which ultimately lead to the transcription of the various target genes. It is well known that the local thyroid hormone homeostasis is a redundant, multistep process, which is, at least in part, relatively unaffected by changes in the serum levels of thyroid hormones.
Finally, we would like to point out that some of the differences observed could be related to the source of "normal" controls. Our control samples have been obtained by needle biopsy of unaffected myocardium from patients with normal thyroid function undergoing elective coronary artery bypass for single vessel disease. Great care was taken in obtaining the samples from left ventricular areas away from the distribution of the affected coronary artery. It is possible that this tissue is indeed not normal. However, as previously stated by Drs. Lowes and Bristow, Dr. Kinugawa’s co-authors, one might argue that normal hearts from brain dead donors, "which are exposed to a number of factors that could alter gene expression" (3) [including a fall in circulating hormone levels (4), increase sympathetic activity, and drugs used to maintain the circulation (5)] may not be the ultimate control as well. We really appreciate for the helpful suggestions and criticisms that we will definitely take in consideration in the design of future studies.
Received July 19, 2001.
References
-myosin heavy chain in hypertrophied, failing ventricular
myocardium. J Clin Invest 100:2315–2324[Medline]
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