| ObjectivesRheumatic aortic stenosis is a common disease leading to LV overloading.while the overloading can be lowered through aortic valve replacement therapy.However,not all patients can benefit from ventricular load decreasing therapy.Preliminary study indicated that KLF15 can modulate the process of gluconeogenesis,lipid metabolism of skeletal musle and physiological nitrogen balance in vivo.KLF15 is an important modulator for cardiac lipid metabolism and can also connect endoplasmic reticulum stress and insulin resistance,and even it has close relation with heart failure and arterial aneurysm formation.Pilot study suggested that lipid accumulation in myocardia can result in toxin effect,even can worsen heart function.Base on the studies before,we made a model of coarctation-release above aortic valves in rats to investigate the changes of cardiac lipid metabolism after cardiac pressure overloading and unloading decreasing and also the modulating effect in process of lipid metabolism.Hence,we hope to offer one useful therapy to the patients post LV overloading deceasing procedure in clinical practice.Methods1.Animal groups: Forty-five 5-week-old male SD rats were randomly divided into sham operation group,which was control group included 9 weeks,12 weeks and 15 weeks,ascending aortic constriction group included overload 9 weeks,12 weeks and 15 weeks,unload group,included unload 0 week,3 weeks,6 weeks after overload 9 weeks.Sham group: SD rats underwent sham surgery,which was exposed the ascending aorta and closed the thoracic cavity.2.Build the pressure overload/unload model: we exposed the ascending aorta from the path of the upper corner of the sternum,playing a slipknot after 4-0Prolene plastic string encircles the ascending aorta,the diameter of 1.4mm steel wire was placed inside the knot running parallel to the ascending aorta and tightened up.It produced aortic stenosis after withdrawing the wire immediately.We released the pressure overloading with the same surgical path by cutting the ligature.3.Cardiac echocardiography: Small animal ultrasond machine(Vevo 2100 Imaging system),with a probe frequency 21 MHz,was adopted in the narrowed ascending aorta to evaluate left ventricular pressure 9 weeks,12 weeks and 15 weeks after overload,0 week,3 weeks and 6 weeks after unloading,and at the 9th,12 th,and 15 th week in the control group.The measured parameters of the left ventricle were ejection fraction(EF)and fractional shortening(FS).4.Free Fatty Acid tested: Quantitation Kit(SIGMA-ALDRICH),and we tested free fatty acid(FFA)(nmol/ul/mg)in the rats’ heart according to technical bulletin.The plate was kept away from light during the incubation.For colorimetric assays,the absorbance was measured at 570 nm.5.Histopathological observation: The removed heart by formaldehyde solution fixed and paraffin-embedded sections were used for oil red O staining,microscopy image acquisition system with myocardial pathological changes and save the images.6.RNA isolation and reverse transcriptin PCR:RNA PCR Kit(AMV)Ver3.0 and we tested KLF15 m RNA in the rats’ heart according to technical bulletin.c DNA was diluted in DNase-free water before quantification by real-time PCR.The primers for quantification of m RNA by real-time PCR for KLF15.7.Western blot analysis of KLF15: myocardium was homogenized in a lysis buffer.The protein fraction in cell lysis solution was extracted by using centrifugal machine and was are well preserved in a low temperature environment for Western blot analysis.The blots were visualized with ECL method,and were reprobed with anti-GAPDH for equal loading.Results1.The mortality of TAC model without artificial ventilation is 15%,and 6 rats died during the operation.the mortality of unloading model without artificial ventilation is 11.8%,and 4 rats died during the operation.After unloading 0 week,3 weeks and 6 weeks,respectively,5 rats survived in the control group.Echocardiography confirmed significant narrowing of the ascending aorta.The narrowed site disappeared after unloading,and blood flow was restored to normal state,which was also confirmed by Echocardiography.2.We evaluated changes in ejection fraction(EF)and fractional shortening(FS)in the 5pressure-overloaded and-unloaded heart of rats.EF and FS were decreased from the 9-week to the 15-week pressure-overload rats.Compared to control group,cardiac function is decreased in overload 9w,12 w,15w.EF%:(85.32±3.90)vs(54.32±3.88),(85.45±4.26)vs(47.91±3.64),(82.83±4.93)vs(40.08±4.45),P<0.01;There was no statistical difference in EF and FS between the 0-weekpressure-unloaded and 9-week pressure-overloaded groups.3.Compared to overload 9w,cardiac function is significant increased in unload 3w,6w after overload 9w.EF% :(54.32±3.88)vs(63.28±5.81),(54.32±3.88)vs(63.59±6.48),P<0.05;However,no significant difference was found in EF and FS between the 3-week and 6-week pressure-unloaded groups.4.FFA deposited progressively in the heart from the 9th to 15 th week of pressure overloading[(0.091±0.03)vs(0.159±0.032),(0.088±0.029)vs(0.196±0.024),(0.091±0.031)vs(0.23±0.029),P<0.05 ].The unload groups showed a significant increase in FFA but the control groups did not.3-week and 6-week unloaded groups showed a significant decrease in FFA while the 9-week pressure-overloaded group did not[(0.091±0.03)vs(0.124±0.027),(0.091±0.03)vs(0.125±0.024),P<0.05 ].5.Compared to control group,Lipid deposition in heart increased in unload 0w,3w,6w after overload 9w;compared to overload 9w,Lipid deposition in heart deceased unload 3w,6w after overload 9w,but still higher than control group.6.Compared to control group,the m RNA and protein level of KLF15 significant decreased in unload 0w,3w,6w after overload 9w[m RNA:(0.66±0.09)vs(1.76±0.07),(0.52±0.07)vs(1.78±0.12),(0.51±0.04)vs(1.70±0.13),P<0.01;Protein:(0.99±0.05)vs(1.25±0.04),(0.96±0.05)vs(1.30±0.09),(1.01±0.08)vs(1.30±0.06),P<0.01].Compared to overload 9w,the m RNA and protein level of KLF15 have no Statistical differences in unload 3w,6w after overload 9w.Conclusion1.During cardiac pressure overload,lipid deposited progressively in the heart.The unload groups showed a significant increase in lipid deposition but the control groups did not.2.KLF15 protein expressions were significantly decreased in the pressure-overloaded groups,but not in the control and unloaded groups.Moreover,the KLF15 protein expression levels still decreased in the pressure-unloaded groups,but not in the control groups.3.During cardiac pressure overload-unload,KLF15 maybe reduce the lipid deposition in heart,and involve in cardiac function recovery after unloading. |
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