Effects Of β₁ And β₂-ARs Overexpression On Contraction And Survival Of Cardiac Myocytes Injured By Isoprenaline And The Primary Analysis Of Their Mechanisms

Evidence has demonstrated thatβ₁ andβ₂-ARs exist at least inhuman and mammalian cardiac myocytes. In normal state, Theβ₁-ARsubtype is the predominant influence on ventricular contraction.Chronicfailure heart in animal models and in humans with enhancedcirculating catecholamine levels is characterized by a severely diminishedcontractile response to sympathetic stimulation. Meanwhile theexpression ofβ₁-AR decreases in these patients. So the contractile abilityof cardiac muscle is reduced, heart rate is increased, the assumption of O₂is increased, and the cardiac output is decreased, leading to a high enddiastolic pressure of left ventricle. According to the pathophysiology, theantagonist ofβ-AR had been used to improve the function of heart bydecreasing the heart rate, reducing blood pressure and the assumption ofO₂. But the use ofβ-AR antagonist didn't increase the contractile functionof cardiac muscle itself. Because theβ₁-AR subtype is down-regulated,theβ₂-AR subtype assumes greater importance in old man, especiallyduring the development of failure heart.β₂-AR maybe a critical subtypein response to synthetic and exogenousβ-ARs agonists. To restore cardiac function, up-regulation ofβ-AR expression isthought as a potential therapeutic strategy. In knockout animals,overexpressions ofβ₁-AR andβ₂-AR had significantly increased thediastolic and systolic contractile function.But it has few reported whether increase ofβ₁ orβ₂-AR expressioncan improve the contractility in ventricular myocytes chronic stimulatedby isoprenaline, which mimics the state of high catecholaminestimulation in vivo, or directly in failure heart myocytes. The presentstudy was performed to determine potential effects of overexpression ofβ₁ orβ₂-AR by adenoviral-mediated gene transfection on contractility inisolated rat ventricular myocytes stimulated by isoprenaline and in failureheart ventricular myocytes of rats, and primarily analysis theirmechanisms. The experiments are include four parts as following:1. Effects of overexpression ofβ₁ orβ₂-AR on contractionamplitude in cardiac myocytesObjects: Myocardial contractility is diminished during chronic activationof cardiacβ-adrenoceptors (β-ARs) in some cardiac diseases. Thepurpose of the study is to investigate whether overexpression ofβ-adrenoceptors can improve contractile amplitude in adult rat ventricularmyocytes chronic stimulated by isoprenaline,Methods: we have adenovirally overexpressedβ₁- andβ₂-adrenoceptor inisolated, cultured adult rat ventricular myocytes in the absence or presence of chronic isoprenaline stimulation. Contraction amplitude ofmyocytes was measured.Results:β₁-adrenoceptor overexpression enhanced the inotropic potencyof isoprenaline in normal cultured cells. The effect was abolished byselectiveβ₂-adrenoceptor antagonist ICI118,551.β₁-adrenoceptoroverexpression decreased the basal and isoprenaline-stimulatedcontraction amplitude in myocytes cultured with isoprenaline.β₂-adrenoceptor overexpression enhanced the basal contraction amplitudein myocytes cultured for 24hr. It also enhanced the basal andisoprenaline-stimulated contraction amplitude in myocytes cultured in thepresence of isoprenaline. Selectiveβ₁-adrenoceptor antagonistCGP20712A downregulated the basal and isoprenaline-stimulatedcontraction amplitude in myocytes overexpressedβ₂-adrenoceptor andcultured with or without isoprenaline. Adenovirally green fluorescentprotein overexpression did not alter the basal and isoprenaline-stimulatedcontraction amplitude.Conclusions: This study demonstrates that overexpression ofβ₁-adrenoceptor aggravate the downregulation of contraction amplitude,but overexpression ofβ₂-adrenoceptor improve the response toisoprenaline in myocytes injured by chronically isoprenaline stimulation. 2. The effects ofβ-adrenoceptors overexpression on cellsurvival are mediated by Bax/Bcl-2 and Gi pathwayin rat cardiac myocytesObjectives: Chronic activation ofβ-adrenoceptors (β-ARs) results incardiac myocytes injury, even death, diminishes the number ofβ-ARs.The purpose of the study is to investigate the effects of overexpression ofβ₁ orβ₂-AR on cardiomyocytes survival injured by isoprenaline (ISO).Methods: We have used an adenoviral vector carrying the sequence forhumanβ₁ orβ₂-AR(Adv.β₁, Adv.β₂)or Gi to increase the content ofβ₁orβ₂-AR or Gi in isolated adult rat ventricular myocytes, and we haveexamined the cell survival and the expression of Bax and Bcl-2.Results: With use of adenoviral vectors, theβ₁ andβ₂-AR contents ofmyocytes were increased 2.98-fold and 2.87-fold, respectively.Overexpression ofβ₁-AR sharpened the cellular injury of ISO. Ifβ₂-ARactivity was further blocked by addition of selectiveβ₂-AR antagonistICI118,551, the cells were more sensitive to the impairment of Adv.β₁+ISO. Overexpression of Adv.β₂ partially inversed the cytotoxicity of ISOstimulation. The beneficial effects were strengthened by addition ofCGP20712A, aβ₁-AR blocking agent. Western blot analysisdemonstrated that both increasingβ₁-AR and inhibition ofβ₂-ARincreased the ratio of Bax/Bcl-2. Whereas, increasingβ₂-AR andinhibition ofβ₁-AR decreased the ratio of Bax/Bcl-2. Control adenovirus CGP had no effect on cell survival. Overexpression of Gi had no effect onregularly cultured cardiomyocytes. But overexpression of Gi improvedthe survival of cardiomyocytes cultured with isoprenaline. The effectswere abolished by selectiveβ₂-adrenoceptor antagonist ICI118,551 andthe Gi inhibitor pertussis toxin(PTX), respectively.Conclusions: Overexpression of Adv.β₂ and/or inhibition ofβ₁-AR haveprotective effect on adult rat ventricular myocytes chronically stimulatedby ISO. Overexpression of Adv.β₁ and/or inhibition ofβ₂-AR aredeleterious in the same state. The effects ofβ-ARs on cell survival mightbe mediated by Bax/Bcl-2 and Gi signal pathway.3. The Effect of Overexpression ofβ₁-adrenoceptors on thecontraction amplitude in rat failure heart ventricular myocytesObjects: To observe the changes of contractile amplitude in ventricularmyocytes of rat with failure heart after overexpressedβ₁-ARs.Methods: Heart failure model was made with isoprenaline. Then,ventricular myocytes were isolated, cultured and infected withadenoviruses containing the sequence for humanβ₁-adrenoceptor. Thecontents ofβ₁-AR were tested after the cells were infected withadenoviruses contain the sequence for human for 24 hours.Results: The content in failure heart group was lower than that incontrol group. Overexpression ofβ₁-AR markedly increased the content. The isoprenaline-stimulated contraction amplitudes in failure group weresignificantly lower than that in control group. Overexpression ofβ₁-ARimproved the contraction amplitudes. Selectiveβ₁-AR antagonistCGP20712A 300nM descended the isoprenaline-stimulated contractionamplitudes in failure heart myocytes infected with or withoutadenoviruses. Selectiveβ₂-adrenoceptor antagonist ICI118,551 55nMpartially decreased the contraction amplitude infailure heart myocytesinfected with or without adenoviruses.Conclusions: Overexpression ofβ₁-ARs increase the contractionamplitude in failure heart myocytes. The effect is directly related toβ₁-AR.β₂-ARs participate in the isoprenaline-stimulated contractionamplitudes.4. Effects of overexpression ofβ₂-adrenoceptor on contractionin cardiac myocytes isolated from failure hearts of ratsObjects: To investigate the role of overexpression ofβ₂-adrenoceptor oncontraction in cardiac myocytes isolated from failure hearts of rats andprimarily analyses its mechanisms.Methods: Adenovirus containing the sequence for humanβ₂-adrenoceptors were overexpressed in cardiac myocytes. Thecontraction amplitudes induced by isoprenaline stimulation were tested.Results: Overexpression ofβ₂-adrenoceptor increased the content in failure cardiac myocytes. The contraction amplitudes in failure cardiacmyocytes were lower than that in the control(P＜0.01). Overexpression ofβ₂-adrenoceptor improved the contraction of failure cardiac myocytes(P＜0.01, Failure+Adv.β₂group vs Failure group).Selectiveβ₂-adrenoceptor antagonist ICI 118,551 partially reversed theeffects (P＜0.05, Failure+Adv.β₂+ICI group vs Failure+Adv.β₂ group),but the contraction amplitudes in this Failure+Adv.β₂+ICI 118,551group were still higher than that in only failure heart group(P＜0.05).Selectiveβ₁-adrenoceptor antagonist CGP20712A completely inhibitedthe effects of overexpression ofβ₂-adrenoceptor on contraction amplitudein failure cardiac myocytes.Conclusions: Overexpression ofβ₂-adrenoceptors improves thecontraction of cardiac myocytes isolated from failure hearts of rats. Theeffect is related toβ₁-adrenoceptor...