Time microscopy of circadian expression of cardiac clock gene mRNA transcription: chronodiagnostic and chrono-therapeutic implications.
OBJECTIVE
Molecular clocks present in organs and individual cells throughout the body are central for the temporal coordination of internal biological processes among themselves and with external environmental cycles. Relationships between circadian clocks and normal vs. abnormal organ physiology can have significant impact relevant to not only cardiovascular health, but also to the general treatment and prognosis of human disease. Chronobiological statistical procedures were applied to previously published circadian clock gene (CG) mRNA expression data which were described macroscopically, in order to establish rhythm probability and point and interval estimates for amplitudes and acrophases for 14 clock and clock-controlled genes in mouse heart. CGs in general and their importance to cardiovascular health, as well as to diagnosis and treatment of human disease, are reviewed.


METHODS
Organs from male Balb/c mice were harvested every 4 h for 24-h on the 3rd day in constant darkness and analyzed by quantitative real-time reverse transcription-polymerase chain reaction for 14 CGs: mPer1, mPer2, mPer3, mCry1, mCry2, mBmal1, mCK1delta, mCK1epsilon, mClock, mDbp, mNpas2, mRev-erbalpha, mRev-erbbeta, and mTim. Relative mRNA levels normalized to corresponding G3-PDH RNA levels were re-expressed as percent of the highest value for each CG and analyzed for circadian time effect by one-way ANOVA and for circadian rhythm characteristics by single cosinor.


RESULTS
12 CGs showed a significant time-effect at p < or = 0.031 by ANOVA and 13 CGs displayed a significant 24-h rhythm at p < or = 0.011 by cosinor analysis. Five CGs (mRev-erbalpha, mDbp, mPer1, mRev-erbbeta, mPer3) reached their maxima late in the presumed resting span, 5 CGs (mPer2, mCry2, mCK1delta, mCK1epsilon, mCry1) reached their peak early in the presumed activity span, while 3 genes (mBmal1, mClock, mNpas2) reached their peak late in the presumed activity span.


CONCLUSIONS
Macroscopic inspection concluded a robust circadian rhythm in 8 CGs, while cosinor analysis detected significance in 13 of 14 CGs (the developmental gene mTim is usually not circadian rhythmic) and computed point and interval estimates for amplitudes and acrophases, useful in making future objective comparisons among organisms and conditions. Information on statistically-determined rhythm characteristics of the molecular clock presents new avenues for diagnosis and therapeutic intervention in conditions where disturbance of circadian CG expression is an important cause of morbidity in chronic illnesses and diseases with a strong circadian component, including coronary vascular disease.
Sothern
R B
RB
The Rhythmometry Lab, College of Biological Sciences, University of Minnesota, 1445 Gortner Avenue, St. Paul, MN 55108, USA. sothe001@umn.edu
Cornélissen
G
G
Yamamoto
T
T
Takumi
T
T
Halberg
F
F
D000818:Animals; D056926:CLOCK Proteins; D002940:Circadian Rhythm / Q000235:genetics*; D005786:Gene Expression Regulation / Q000528:radiation effects; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008853:Microscopy / Q000379:methods; D009206:Myocardium / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic / Q000528:radiation effects
D012333:RNA, Messenger; D015534:Trans-Activators; D014157:Transcription Factors; D056926:CLOCK Proteins; C538943:CLOCK protein, human; C538944:Clock protein, mouse