Role And Mechanisms Of Visfatin In Cardiomyocyte Hypertrophy And Inflammation

Cardiac hypertrophy is an adaptive response to satisfy the demand ofheart work, it can maintain or increase the cardiac output for a short time.However, cardiac hypertrophy will develop into decompensated hypertrophyand leads to the heart failure. There are some side effects in cardiachypertrophy and heart failure treatment perscription. It is definitely importantand urgent to further explore the primary cause of this disease, and to choose aspecific treatment perscription.Inflammatory cytokines are a group of functional proteins which secretedin cells after stimulated by the changes of extracellular or intracellularenvironment, in which TNF-alpha, TGF-beta, IL-1β, IL-6and IL-10play amajor role. Inflammation plays an important role in the development ofcirculatory diseases. There are reciprocal influence and causation betweeninflammation and hypertension. Atherosclerosis (AS) is an inflammatorydisease, ACS induced by the unstable and cracked atherosclerotic plaque isconsidered to be a response of inflammation. Epidemiologic studies haveconfirmed that inflammation biomarkers, such as C-reactive protein (CRP),matrix metalloproteinases (MMPs) and white interleukin-6(IL-6), are elevatedin the serum of patiens with ACS, and they have been used for predictors ofcardiovascular events. Inflammatory cytokine cascade reaction plays animportant role in the pathophysiological processes of the heart failure, and itcan reduce myocardial contractility and cardiac output. Inflammatorycytokines are elevated in heart failuer, and the level of inflammatory cytokinescan be used to judge the severity of heart failure and prognosis, andanti-inflammatory treatment can reduce the incidence of high-risk patientswith heart failure.Generally, adipose tissue is regarded as a terminally differentiated organ for the energy storage. With the discovery of a number of vasoactivesubstances secreted by the adipose tissue which is known as adipocytokines,such as leptin, resistin, adiponectin, omentin and visfatin, etc., the endocrinefunction of adipose tissue is also gradually being recognized, and adiposetissue has become a consensus of the academia as an endocrine organ.Visfatin, a novel adipocytokine, is widely expressed in a variety of organs,which indicates its versatility. Visfatin level in the plasma is significantlyincreased in rheumatic diseases, obesity, diabetes, acute lung injury, naturaldelivery and the infection-induced preterm or other pathophysiologicalprocesses. It is well known that visfatin plays an important role in thecardiovascular diseases. Visfatin level is significantly elevated in the plasmaof patients with ACS, and it was closely related to the unstable plaques, andstduies have found that the expression of visfatin was significantly increasedin macrophage-riched plaques. Visfatin can promote the mature of vascularsmooth muscle cells and vascular remodeling. Visfatin was originally isolatedfrom the cDNA of activated lymphocytes in peripheral blood, and it canpromote the mature of lymphocytes-B. It has been found that visfatin is animportant pro-inflammatory factor, which plays an important role in a varietyof pathophysiological processes, and it can induce the production ofinflammatory cytokines through the combination with some unconfirmedreceptors or special signal transduction pathways probalblely. In addition,visfatin can increase the level of IL-6in the plasma of mouse.There are still a lot of controversies on the functions and effects of visfatin,and it needs to be further explored on its clinical role. Most of studys on therole of visfatin in the circulatory system are involved in vivo experiments, butthere are still few evidences on vitro experiments, particularly at the cellularlevel. There are no any researches focuses on the expression of visfatin in thecardiac cells as well as its role and mechanisms in the cardiac hypertrophy andthe relationship between visfatin and myocardial inflammatory reaction.The objectives of this study are to clarify the role and molecular biologicalmechanisms of visfatin in cardiac hypertrophy and myocardial inflammatory reaction at cellular level through the cultivation of primary cardiomyocytesfrom neonatal SD rats in vitro. It follows by four parts:Part1The expression of visfatin in cardiomyocytesObjectives: To explore whether or not cardiomyocytes can expressvisfatin.Methods: Ventricular cardiomyocytes were dissociated from the2to3-day old neonatal SD rats. Forty-eight hours after seeding, thecardiomyocytes were cultured with the serum-free culture medium for anothertwenty-four hours. Afterthat, visfatin expression in cardiomyocytes weredetermined by RT-PCR and Western-Blot.Results: The mRNA and protein expression of visfatin were detected incardiomyocytes.Conclusions: Cardiomyocytes can express visfatin.Part2Angiotensin II type-1receptor-JAK/STAT pathway mediates theinduction of visfatin in angiotensin II-induced cardiomyocytehypertrophyObjectives: Investigating the expression and mechanisms of visfatin inangiotensin II (Ang II)-induced cardiomyocyte hypertrophy in vitro by meansof the cultured neonatal rat cardiomyocytes.Methods: After primary culture of2-3day-old Sprague-Dawley (SD) ratcardiomyocytes, cardiomyocytes were pretreated with Ang II. Ang II type-1receptor (AT1-R) antagonist telmisartan and Ang II type-2receptor (AT2-R)antagonist PD123319were used to block effects of Ang II. These inhibitorsused for the AT1-R pathway determination included SP600125, AG490andU0126. Cell viability was examined using the MTT assay. The expression ofvisfatin was examined by means of RT-PCR and western-blot. The expressionof brain natriuretic peptide (BNP) was examined through western-blotanalysis.Results: Ang II treatment induced the increased expression of visfatinand BNP in a dose-and time-dependent manner in cardiomyocytes, andpretreatment with AT1-R antagonist telmisartan completely blocked Ang II-induced visfatin expression increasement. The increased visfatin expressionwas also blocked by the Janus kinase/signal transducer and activator oftranscription (JAK/STAT) pathway inhibitor AG490.Conclusions: Visfatin expression was increased mainly through theAT1-R-JAK/STAT pathyway in the process of Ang II-induced cardiomyocytehypertrophy.Part3Activation of RhoA/ROCK2-NF-kB is required for visfatin-inducedhypertrophic growth of primary rat neonatal ventricularcardiomyocytesObjectives: To observed the effect of visfatin on cardiomyocytehypertrophy induced by Ang II, and to explore weather or not visfatin caninduce cardiomyocyte hypertrophy independlly and its mechanisms.Methods: After primary culture of2-3day-old Sprague-Dawley (SD) ratcardiomyocytes, visfatin antagonist FK866was used to block the effects ofvisfatin in Ang II induced cardiomyocyte hypertrophy. Cardiomyocytes werethen pretreated with recombine visfatin with different concentrations anddifferent times. These inhibitors used for the RhoA/ROCK-NFκB pathwaydetermination included simvastatin, Y27632and SN50. Cardiomyocytehypertrophy was evaluated with the measurement of cardiomyocyte surfacearea and BNP expression. The expression of BNP was examined by means ofreal time-PCR and western-blot. The expression of SIRT1was examinedthrough western-blot analysis.Results: FK866attenuated BNP protein expression in Ang II inducedcardiomyocyte hypertrophy. Cell surface area and BNP expression of thecardiomyocytes pretreat with visfatin increased in a dose and time-dependentmanner, peaking when the concentration of visfatin was100ng/mL after48h.RhoA and ROCK2protein expression in cardiomyocytes pretreat with visfatinincreased in a dose and time-dependent manner, peaking after48h when theconcentration of visfatin was100ng/mL, on the contrary, IκB-α proteinexpression in cardiomyocytes pretreat with visfatin decreased in a dose andtime-dependent manner. The nuclear translocation of p65was significantly stimulated by visfatin in a dose and time-dependent manner, and reaching amaximal at the concentration of100ng/ml at48h. The increased cell surfacearea and BNP expression were blocked by simvastatin,Y27632and SN50.The degradation of IκB-α and the nuclear translocation of p65were blockedby simvastatin and Y27632, but the activation of ROCK2was not blocked bySN50. SIRT1protein expression in cardiomyocytes pretreated with visfatinwas increased in a dose and time-dependent manner, peaking after48h whenthe concentration of visfatin was100ng/mL.Conclusions: SIRT1play an important role in cardiomyocytehypertrophy mediated by RhoA/ROCK2-NFκB pathway induced withvisfatin.Part4RhoA/ROCK2-NFκB pathway mediates visfatin induced myocardial in-flammationObjectives: To explore weather or not visfatin can induce myocardialinflammation and its mechanisms.Methods: During the3rdpart experiment, we also explored TNF-αmRNA expression in cardiomyocytes pretreated with visfatin. TNF-α mRNAexpression was examined by means of real time-PCR. Cell viability wasexamined using the MTT assay.Results: TNF-α mRNA expression in cardiomyocytes pretreated withvisfatin was increased in a dose and time-dependent manner. The OD value ofMTT products of cardiomyocytes pretreated with visfatin reached a maximalwhen the concentration of visfatin was10ng/ml. Afterthat, it decreasedsignificantly in a dose-dependent manner, and lowest when the concentrationof visfatin was100ng/ml. Then cardiomyocytes were pretreated with100ng/mL visfatin for different times, and the OD value of MTT products ofcardiomyocytes decreased significantly after24h. Afterthat, the OD value ofMTT products of cardiomyocytes decreased persistently, but there was nosignificantly difference between24h group and48h and72h group. The ODvalue of MTT products of cardiomyocytes in sirtinol+visfatin group wassignificant lower than control gorup, but significant higher than100ng/ml visfatin treating group.Conclusions: Visfatin can induce myocardial inflammation throughRhoA/ROCK2-NFκB pathway. SIRT1modestly elevated induced by visfatincan attenuate cardiomyocyte damage induced by inflammatory cytokines.Inflammatory cytokines play an important role in visfatin inducedcardiomyocyte hypertrophy.