摘要：目的许多心血管变量存在着近日节律，心肌收缩反应及收缩蛋白基因表达是否存在着相应的周期性改变是值得深入研究。方法在24h内采用直接在大白鼠左心室内插入左心导管记录左室压(LVP)和左室压力微分最大值(dp/dtmax)以及检测比较心肌细胞的α-MHC基因表达改变。结果LVP(P＜0.001)、dp/dtmax(P＜0.001)和α-MHC(P＜0.01)的变化存在着近日节律。通过比较三者近日节律振幅显示LVP的振幅最大，dp/dtmax次之，α-MHC基因表达的节律振幅再次之，表明心肌收缩力的近日节律的变化是由心肌细胞内在和外在作用的结果。结论α-MHC基因表达水平的近日变化是决定着心肌收缩功能的近日节律的因素之一。

中图分类号：R331.31文献标识码：A文章编号：1002-0837(1999)06-0391-06

Circadian Rhythm of Gene Expression of Myocardial

Contractile Protein,Left Ventricular Pressure

and Contractility^*

WANG Zheng-rong¹,WANG Ling¹,WAN Chao-min¹,

Germaine Cornelissen²,Inder Anand³,Franz Halberg²

(1.Biomedical Engineering Department, West China University of Medic al Sciences, Chengdu, 610041, P.R. China；2. Chronobiology Laboratories , University of Minnesota, Minneapolis, MN 55455, USA；3. Cardiology Section, VA Medical Center, Minneapolis, MN 55417,USA)

Abstract： Objective A number of cardiovascular variables exhibit a circ adian rhythm. Whethe r myocardial contractile response and gene expression of the contractile protein also show changes with a similar period was here investigated. Method Circadi an variabilities in the left ventricular developed pressure (LVP) and contractil ity (LV dp/dt max) were measured in 24 Sprague-Dawley r ats by directly left ve ntricular catheterizing and compared with changes in the gene expression of α- myosin heavy chain (α-MHC) in myocytes obtained from the same animals by dot b lottin g analysis. Results A circadian rhythm was seen in the variabili ty of LVP (P＜0.001), LV dp/dt max (P＜0.001) and the bio chemically measured expression of the α- MHC gene (P＜0.01). As compared to the amplitude of the rhythm i n α-MHC gene exp ression, the amplitude of the contractility rhythm was large (P＜ 0.01) and the ci rcadian amplitude of the LVP(P＜0.001) was the largest, represent ing perhaps a co mposite of intracardiac plus any extracardiac contributions. Conclusion One of factors determing the circadian rhythm of myocardial contractile function is α -MHC gene expression level.

Key words:circadian rhythm;gene expression;myocardial contracti on;contractility

Clinical and experimental studies have established that cardiovascular variables like blood pressure, heart rate, cardiac output and peripheral resistance exhib it spontaneous circadian variation in humans and other mammals^［1～5］. Th e geneti c aspects of circadian variation are documented by its persistence in isolation from society^［6］. Like higher frequencies, it is neither societally nor pacing i nduced^［7］.These investigations, including studies of twins^［8，9］ , do not diffe rentiate whether the circadian rhythm relates to the nervous, hormonal or metabo lic systems^［10］ or is intrinsic to cardiac muscle. Spoor and Jackson ^［11］ demon strated a circadian rhythm in the sensitivity of isolated rat atria to acetylcho line. Goshima^［12］ found very high frequency ultradian variation in the contraction of cultured heart muscle cells.

We have reported in a single murine myocardial cell and in two sets of grouped c ells in culture for several days, variations in myocyte beating with anticipated about-daily, half-daily and half-weekly components, which could not be separ ated from trends in view of the brevity of the series^［13］. Features of a presum ably bult-in spectrum of multifrequency rhythms, chaotic-appearing changes and tren ds are part of a broad organized time structure, the chronome^［14］. Beca use myoc ytes in culture undergo rapid fibroblast transformation, it is difficult to be c ertain whether changes in the observed rhythm are related to circadian variation of the myocytes or are the result of fibroblast transformation. In humans, the timing of myocardial infarction, cardiac rhythm disturbances and other morbidit y as well as mortality have been shown to have definite cyclic patterns^{［1 5］}. Wh ether such disturbances are influenced by variability intrinsic to myocytes rema ins to be determined.

Myocardial contractile function is influenced by the state of its contractile pr oteins, myosin and actin. An important component of the mammalian heart's contr actile protein is myosin heavy chian (MHC), which exists in three distinct isofo rms, V1, V2 and V3. The V1 and V3 isoforms are homodimers of the polypeptides en coded by α-MHC and β-MHC, and the V2 isoform is a heterodimer, consisting of α-M HC and β-MHC. The relative amounts of the three isoforms vary depending upon t he developmental stage and disease states. Under usual conditions, α-MHC (or V1) i s the predominant isoform in the adult rat. Hearts with a greater α-MHC gene e xp ression demonstrate a higher contractile response than hearts with a predominant β-MHC isoform.

In this study we tested the hypothesis that the rat heart undergoes a circadian variability in global myocardial contractility and that the latter is associated with similar circadian changes in the expression of contractile proteins.

Method

12 male and 12 female Sprague-Dawley rats, weighing 216±12g (SE), were stud ied.All animals were housed in an experimental animal laboratory under natural conditions of light alternating with darkness. Animals were randomly divided into 6 groups, each containing 2 m ale and 2 female rats and these sub-groups of 4 animals were studied at six test times, 4 hours apart (at 00:00, 04:00, 08:00, 12:00, 16:00 and 20:00, local tim e). After intraperitoneal administration of pentobarbital (30 mg/kg), the abdome n was opened and a polythene LV catheter (id 0.2 mm) was inserted into the animal's LV through the d iaphragm. The left ventricular pressure (LVP) and LV dp/dt were recorded using a pressure transducer (Statham P23Db) and a physiological polygraph system (Optic al Electronic, Japan). Hearts were then removed, atrial and the right ventricle discarded, and the LV was cleared of blood with saline solution and immediately sto red in a freezer at -80℃. For the analysis of α-MHC gene expression, total RN A w as prepared by the single-step method^［16］. Briefly, LV tissue from eac h heart w as homogenized in a Teflon homogenizer with a denaturing solution containing 4M guanidium thiocyanate. The homogenate was mixed sequentially with 2M sodium acet ate (pH4), phenol, and chloroform/isoamyl alcohol. The resulting mixture was cen trifuged at 10000xg, yielding an upper aqueous phase containing total RNA. The total RNA content was measured for absorbent optical density with 260 nm UV-vis ible recording spectrophotometer (UV-280, Shimadzu, Japan).

The oligonucleotide probe of α-MHC was used to test for the α-MHC gene expre ssio n. The sequence of the probe was α-MHC, 5'-TTGTGGGATAGCAACAGCGA-3'. The oligon uc leotide probe was labeled with Digoxigenin-11-dUTP/dATP at the 3'-tailing, using the Digoxigenin labeling kit (S@CPolymer GmbH, Germany) as described in the Dig oxigenin labeling manual. 60 μg of total RNA were dropped through a nylon membr an e and fixed at 80℃ for 30 minutes. The membrane was pre