RGFP966 Improved Myocardial Function Recovery In Rat Hearts Suffered From Hypothermia Preservation

BackgroundHeart transplantation is a major strategy to improve the life quality and prolong survival of patients with end-stage heart failure.The number of patients who need to undergo heart transplantation is increasing every year.However,the longest cold ischemic preservation time of donor heart is still within 4-6 hours,which highly limits the number of patients who accepted heart transplant surgery.Therefore,it has greatly practical significance to explore how to alleviate the hypothermic preservation-induced myocardial injury and prolong the preservation time.Histone deacetylase?HDAC?inhibitors have been shown to protect aganist cardiac injury during a variety of cardiovascular diseases.Non-specific HDAC inhibitors could improve myocardial energy metabolism,delay myocardial hypertrophy and fibrosis,and enhance cardiac function by reducing oxidative stress and inflammation in experimental heart failure animals.Therefore,we hypothesized that RGFP966 might be potential for alleviation of hypothermic preservation-induced cardiac dysfunction.ObjectivesThe aims of this study was to investigate whether HDAC3 inhibitor RGFP966 could protect against hypothermic preservation-induced cardiac injury,and to further explore whether Hippo-YAP signaling pathway is involved in the beneficial effect of RGFP966in rat hearts.Methods1)Establishment of Langendorff perfusion model:Isolated rat hearts were fixed onto Langendorff perfusion device.The hearts were perfused with Krebs-Henseleit?KH?solution retrogradly rom aortic root at aconstant pressure.2)Establishment ofheart hypothermic preservation model:Rat hearts were placed in Celsior solution containing RGFP966?0,0.25,0.50 or 0.75?M?for 12 h at 4?.3)Measurement of cardiac function:A balloon was inserted into left ventricle through left atrial appendage,and the other end of the balloon was connected with a pressure transducer and recorder.Cardiac functions before preservation and at 60 min of reperfusion after preservation were measured,which included left ventricular end-diastolic pressure?LVEDP?,left ventricular developed pressure?LVDP?,maximal systolic and diastolic velocity of left ventricular pressure(±d P/dt_max),heart rate?HR?,and coronary flow.4)Western blotting analysis:Total protein or nuclear protein was extracted from myocardial tissue.The expression levels of HDAC3,p-Mst1,Mst1,p-YAP,YAP,cleaved caspase-3,Bcl-2 and Bax were detected.5)RT-PCR:Total RNA was extracted from myocardial tissue.RNA was retranscribed to a c DNA using Prime Script?RT Master Mix?Perfect Real Time?.PCR amplification was performed using ABI 7500 Fast Real-Time PCR system..6)Determination of LDH activity:Coronary flow was collected.LDH activity was determined according to the instructions of the LDH assay kit.7)Detection of SOD activity,CAT activity,MDA content,and GPx activity::Left ventricular myocardium was homogenized and supernatant was collected.The SOD activity,CAT activity,MDA content,and GPx activity were determined by their commercial test kits..8)Immunohistochemistry:Myocardial morphology was observed by immunohistochemistry:left ventricular tissue was fixed and paraffin-embedded.After dewaxed,tissue slices were stained with hematoxylin and eosin.9)TUNEL staining:After dewaxed and hydrated,slices were incubated with TUNEL reaction solution at 37?for 1 h in dark.Then sections were stained with DAB and hematoxylin.The slices were observed under optical microscope and the number of apoptotic cells was counted.Results1)Compared with Celsior group,addition of RGFP966 in Celsior solution could significantly inhibit hypothermic preservation-induced increase of LVEDP,prevent preservation-induced decrease of LVDP,±d P/dt_max,and coronary flow.2)RGFP966 significantly inhibited the hypothermic preservation-induced enhancement of p-Mst1/Mst1 ratio and p-YAP/YAP ratio induced by,prevented the decline of total YAP protein expression,and increased the nuclear YAP protein level.3)Verteporfin,a small molecular inhibitor of YAP-TEAD interaction,partially abolished protective effect of RGFP966 on cardiac function,decreased LDH activity and MDA content of hearts.4)RGFP966 increased SOD,GPx,CAT gene and protein expression,which could be prevented by addition of verteporfin.5)RGFP966 could inhibit hypothermic preservation-induced overexpression of Bax and cleaved caspase-3,enhanced the Bcl-2 m RNA and protein expression,and declined cardiomyocytes apoptosis.The beneficial effects of RGFP966 could be cancelled by supplement with verteporfin.ConclusionTreatment with RGFP966 attenuated hypothermic preservation-induced cardiac dysfunction.The mechanism is involved in inhibition of oxidative stress and apoptosis via inactivation of YAP/TEAD pathway.