Effect Of Endogenous Substances Acting On G-protein Coupled Receptor On Function And Morphology Of Cardiac Myocytes

The normal function of the heart relies on a stable internal environment and on fine-tuned regulation of heart function by many endogenous active substances. Many of these substances act on G-protein coupled receptors, initiating cell signalling, and regulating cardiac function with different end mechanism. Among them, the receptors coupled with Gi and Gq proteins, and the substances acting on these receptors play important roles in regulation of cardiac function, and thus are important for physiology and pathophysiology of the heart.Among receptors coupled with Gi protein, receptors for acetylcholine (ACh) and adenosine (Ado) are outstanding. One way these receptors regulate cardiac function is through modulation of inwardly rectifying potassium channels. Activation of G-protein gated inwardly rectifying potassium channel (GIRK, Kir3.1/3.4) by these substances has profound effects on cardiac functions.GIRK (Kir3.1/3.4) channels play important role in atrial electrical activity. GIRK is also named IKACh since it is activated by ACh. Both ACh and Ado can activate IKACh through their respective Gi-coupled receptors such as M2 and A1, and subsequently exert their negative inotropic and chronotropic effects on heart, and for Ado exerts its antiarrhythmic effect. IKACh mainly exists and is active in atrium. It has been debated whether IKACh also exists and plays important role in ventricle. There are some reports indicating existence of GIRK in ventricle but little is known about its function. Mr Yang Jijie from this laboratory recorded inwardly rectifying K currents, possibly IK1 currents, from ventricular epi- as well as endocardial myocytes. He also recorded from these myocytes Ado-activated inwardly rectifying K currents; the Ado-activated currents were inhibited by tertiapin, a specific GIRK blocker, indicating presence of functional GIRK channels. Ion channels, including potassium channels are the basis of cardiac action potential, which can have profound influence on cardiac function through excitation-contraction coupling. Thus, following earlier works from this lab, this study will investigate whether GIRK channels play important role on ventricular functions.Congestive heart failure (CHF) is deleterious heart disease. Under this condition, absolute or relative cardiac output is reduced and tissues are short of demand for blood supply, despite efficient cardiac venous inflow. Angiotension II (AngII), norepinepherin (NE), endothelin (ET) and some other endogenous substances are believed to involve in cardiac hypertrophy and CHF.A common future of the above mentioned endogenous substances is that many of these substances can activate Gq protein pathway, indicating involvement of Gq pathway in cardiac hypertrophy. In fact, published work has shown that activation of Gq pathway can promote hypertrophy of cardiac myocytes from newborn mice; overespression of wild type Gq promotes proliferation of cultured cardiac myocytes; overexpression of a constitutive active form of Gq promotes cardiac myocytes from hypertrophy to necrosis. These findings have been supported by experimental data from transgenic mice with overexpression of Gq. Gq protein activates phospholipase C (PLC) which than hydrolyze membrane PIP2 into DAG and IP3, and the later can activate IP3 receptor and release Ca2+ from store. It is not yet clear about the detailed mechanisms for Gq pathway-induced cardiac hypertrophy, but changes of intracellular Ca2+ should be one major candidate.There are many controversies concerning the relationship between Gq pathway and intracellular Ca2+. AngII and NE and some other endogenous substances can increase Ca2+ oscillations of newborn animal but have no effect on intracellular Ca2+ of adult animal. Thus the second part of this thesis will study effects of AngII and NE on morphology and intracellular Ca2+ of ventricular myocytes from adult SD rat, and will also study effect of overexpressing Gαq (using adenovirus) on morphology of adult cardiac myocytes.1. Effect of acetylcholine and adenosine acting on Gi protein-coupled receptors on the function of cardiac myocytesAim: To express Kir channels in Xenopus oocytes to study inhibition of Ba2+ on Kir channels; to study effects of acetylcholine and adenosine on the function of ventricular cardiomyocytes.Methods:①Transcription of Kir2.1, Ki2.1 and Kir3.1/3.4 channel cRNA in vitro: All cDNA constructs were subcloned into the pGEMHE plasmid vector and all cRNAs were transcribed using RibomaxTM Large Scale RNA Production Systems-T7 Kit.②Preparation of oocytes: Xenopus oocytes were digested with collagenase (2mg/ml) in OR2 solution for 1.5-2 hours, and then repeatedly washed with ND96 solution to remove the collagenase. OR2 solution contains (mM) NaCl 82.5, KCl 2, MgCl2 1, HEPES 5 (pH=7.4).③Microinjection of oocytes: Each oocyte was injected with 50nl of water containing the desired cRNA. cRNA of all Kir channels and of receptor was injected in the range of ~2 ng/ oocyte. Injected oocytes were kept at an incubator with temperatue of 18℃, in ND96 solution. The solution was changed every 12 h. ND96 solution contains (mM): NaCl 96,KCl 1, CaCl2 1.8, MgCl2 1, HEPES 5, pH 7.4.④Current recording: Whole cell currents were recorded using two-electrodes voltage clamp method using Gene clamp 500B amplifier. Inhibition of Ba2+ on Kir2.1, Kir2.3 and Kir3.1/3.4 was studied.⑤Preparation of ventricular myocytes: Heart from mice was isolated and perfused with digestive solution in a Langendorff system. Digestive solution contains: CaCl2 0.05 mmol/L, BSA 1mg/ml, Taurine20 mmol/L, type II colagenase 0.5mg/ml.⑥Measurement of cardiomyocyte function: the contractility of ventricular myocytes was measured by video-based motion edge-detection system. The cells were stimulated with paramters of 15V, 1Hz and bandth of of 4ms.Results:①Analysis of linearized plasmid DNA by agarose electrophoresis: A proper restriction enzyme was chosen to cut the circular plasmid. The circular plasmid DNA showed more than one bands in the electrophoresis, while the linearized DNA showed only one band.②Analysis of in vitro transcribed cRNA:?cRNA of all Kir channels were transcribed and showed clear RNA bands on the eletrophoresis.③Expression of Kir channels and inhibition by Ba2+: currents from Kir2.1, Kir2.3 and Kir3.1/3.4 channel were recorded using two-electrodes voltage clamp method at 2~3 days after microinjection of oocytes with RNA. These currents showed clear inwardly rectification. Ba2+ inhibited these currents effectively.However, no different sensitivity of Ba2+ was found for inhibition of three Kir channels.④Effect of acetylcholine and adenosine on contractility of ventricular myocytes: Effects of acetylcholine and adenosine on percentage of shortening, maximal systolic velocity, maximal diastolic velocity of ventricular myocytes were measured. No significant effects of acetycholine and adenosine were found on these parameters of cell functions.Conclusion: Kir channels can be expressed by microinjecting the in vitro transcribed RNA into Xenopus oocytes. Kir channel currents can be studied by two-electrodes voltage clamp 2~3 days after the cRNA microinjection. Ba2+ blocks Kir currents effectively, but has no different sensitivity distinguishing different Kir channels. In ventricular cardiomyocytes, acetylcholine and adenosine did not significantly affect cell contractility. Thus GIRK channels may not play an important role in function of ventricular cardiomyocytes.2. Effect of Ang II acting on Gq protein-coupled receptors on morphology and intracellular Ca2+ of adult rat cariomyocytes.Aim: To establish method of adult cardiomyocyte culture; to study effect of Ang II on cardiomyocyte hypertrophy and intracellular Ca2+; to study effect of Gαq overexpression on cardiomyocyte hypertrophy.Methods:①Adult ventricular myocyte culture: Adult ventricular myocytes of rat were isolated in a sterile condition and cultured in M199 medium without albumin.②Overexpression of Gαq protein: Adenovirus was used to carry Gαq gene and to transfect the cardiomyocytes. Overexpression of Gαq protein was verified by using Western blot.③Observation of cell morphology and changes in intracellular calcium: Surface area of culture cardiomyocytes were measured under control or after treatment with Ang II or overexpression of Gαq.④Intracellular Ca2+ of cultured cardiomyocytes was measured by using laser scanning confocal microscope (LSCM) to observe calcium fluorescence from Rhod-2AM.Results:①Morphology of cultured adult ventricular cardiomyotes. Most of the isolated adult rat ventricular cardiomyotes have a rod-like shape, with clear cell structure. These futures did not change significantly during 3 day culturing. Furthmore, although some cells begin to show some morphologic changes after 3 days culturing, other cells maintain their morphologic characteristics even after 5 days culturing.②Angiotensin II (1μM) did not affect size of the cultured cardiomyocytes within 48 h of incubation but significanly increased visible surfacce area of the cardiomyocytes after 72 h incubation.③Gαq overexpression did not affect the size of the cultured cardiomyocytes; however, Gαq overexpression plus Ang II incubation significantly increased visible surfacce area of the cardiomyocytes after 48 h incubation.④Acute application of Ang II did not affect intracellular Ca2+ concentration.Conclusion: Ventricular cardiomyotes of adult rat can be maintained in good stable morphological characters for at least three days, and most of cells keep similar charactreistics after threes day up to one week. Ang II has a hypertrophic effect on adult cardiomyocytes after three days incubation, and this effect of Ang II can be enhanced by overexpression of Gαq. Ang II did not has an acute effect on intracellular Ca2+ of ventricular cardiomyotes of adult rat. SUMMARY1. Ba2+ blocks Kir currents expresssed in Xenopus oocytes effectively, but has no different sensitivity distinguishing different Kir channels.2. In ventricular cardiomyocytes, acetylcholine and adenosine did not significantly affect cell contractility. Thus GIRK channels may not play an important role in function of ventricular cardiomyocytes.3. Ventricular cardiomyotes of adult rat can be maintained in good stable morphological characters for three to seven days.4. Ang II has a hypertrophic effect on adult cardiomyocytes, which can be enhanced by overexpression of Gαq.5. Ang II did not have an acute effect on intracellular Ca2+ of ventricular cardiomyotes of adult rat.