Establishment Of A Novel Murine Cardiovascular Pathophysiology Core And Olmesartan Attenuates Cardiac Remodeling Through DLL4/Notch1 Pathway Activation In Pressure Overload Mice

Nowadays mouse is a widely used animal model in the cardiovascular research. Murine cardiovascular pathophysiology core is established in many world-renowned institutes, providing high-quality and stable murine models, evaluation and physiopathological data. Correspondingly, some institutions are establishing murine pathophysiology core in China now. On the basis of our prototype of pathophysiology core, we take the lead to introduce and optimize murine Doppler coronary flow imaging and mouse model of aortic regurgitation. We hope to establish a first-class and comprehensive murine cardiovascular pathophysiology core which integrates modeling (including myocardial infarction, transverse aortic constriction, aortic regurgitation), imaging and hemodynamic analysis.Notch signaling pathway has been speculated to exert cardioprotection in injured myocardium, by controlling the cardiac adaptation to stress, enhancing myocyte survival, promoting precursor proliferation and differentiation, attenuating cardiac remodeling and dysfunction. DLL4/Notch1 activation is essential to the survival of injured myocardium and the regeneration of damaged tissue. It controls the process of proliferation, differentiation, migration, apoptosis and regeneration. On the contrary, inhibition of Notch1 signaling pathway exacerbates cardiac hypertrophy, fibrosis, apoptosis, and dysfunction. On the basis of our murine cardiovascular pathophysiology core and our previous researches, we validated olmesartan, a widely used angiotensin Ⅱ type 1 receptor blocker, attuenuates cardiac remodeling and improves cardiac function via DLL4/Notch1 signaling pathway activation in mice with chronic pressure overload.Part Ⅰ Establishment of a Novel Murine Cardiovascular Pathophysiology CoreSection 1. Noninvasive Estimation of Infarct Size in a Mouse Model of Myocardial Infarction by Echocardiographic Coronary PerfusionBackground and Objectives:Animal models of myocardial infarction (MI) are widely used not only in analyses for the mechanisms but also in testing the efficacy of therapeutic strategies for the disease. It is therefore critically important, but almost impossible to exactly evaluate the validity of coronary artery ligation in a mouse model of MI except by anatomic and histologic analyses. We explored a noninvasive method to estimate MI through analyses of coronary perfusion by transthoracic echocardiography in mice before and 1 day after ligation of left anterior descending coronary artery.Methods:Transthoracic echocardiography-based cardiac function, geometry and coronary perfusion, electrocardiographic findings, and serum troponin I levels were examined in C57BL6/J mice subjected to left anterior descending artery ligation. The histological infarct size was confirmed by staining the heart with 2,3,5-triphenyltetrazolium chloride.Results:Among all parameters, the postoperative hyperemic peak diastolic velocity and coronary flow reserve were most correlated with infarct size (R2=0.8028 and 0.5825, respectively; both P< 0.0001). With an infarct size of 30% or greater indicating successful ligation and less than 30% indicating unsuccessful ligation, receiver operating characteristic curve analysis showed that the postoperative hyperemic peak diastolic velocity and coronary flow reserve most effectively indicated the infarct size level with optimal cutoff values of 480.16 mm/s and 1.89, respectively. Furthermore, impaired cardiac function, an eccentrically expanded left ventricle, typical pathologic electrocardiographic findings, and elevated troponin I levels were observed most often in the mice with an impaired hyperemic peak diastolic velocity and coronary flow reserve.Conclusions:The echocardiographic hyperemic peak diastolic velocity and coronary flow reserve can estimate the histologic infarct size in mice with coronary occlusion. with an impaired hyperemic peak diastolic velocity and coronary flow reserve.Section 2. Comparison between Adenosine and Isoflurane for Assessing the Coronary Flow Reserve in Mouse Models of Left Ventricular Pressure and Volume OverloadBackground and Objectives:Adenosine and high-concentration isoflurane are commonly used to induce hyperemia for assessment of coronary flow reserve (CFR) in mice, but high-concentration isoflurane may exacerbate cardiac dysfunction, leading to impaired CFR. However, there is no study be found comparing the effects of adenosine and isoflurane on CFR and corresponding cardiac function.Methods:High-resolution echocardiography and invasive hemodynamic assessment were performed in 20 mice 2 weeks after transverse aortic constriction (TAC), aortic regurgitation (AR) and corresponding sham-operation. CFR was calculated as the ratio of hyperemic to basal peak diastolic velocity (CFRpdv) or diastolic velocity-time integral (CFRdvti).Results:In the sham-operated mice, no differences were observed between the effects of adenosine and isoflurane on CFR, left ventricular systolic function (left ventricular ejection fraction and fractional shortening), left ventricular end-systolic pressure, maximal contraction and relaxation velocity (+dp/dt and-dp/dt), left ventricular developed pressure (△LVP) or +dp/dt (△dp/dt). But adenosine-derived results were significantly higher than isoflurane-derived ones in both the TAC and the AR groups. Moreover, CFRpdv or CFRdvti was positively correlated with both LVEF and LVFS.Conclusions: Compared with adenosine-derived CFR, isoflurane-derived CFR may be underestimated in the TAC and the AR mice, which is probably associated with suppressed cardiac function.Section 3. Early Estimation of Left Ventricular Systolic Pressure and Prediction of Successful Aortic Constriction in a Mouse Model of Pressure Overload by Ultrasound BiomicroscopyBackground and Objectives:Elevation of left ventricular end-systolic pressure (LVESP) and hypertrophic response in mice varies after transverse aortic constriction (TAC). Micromanometric catheterization, conventionally used to select the mice with successful TAC, is invasive and nonreusable. We aimed to establish noninvasive imaging protocols for early estimation of successful TAC by ultrasound biomicroscopy (UBM).Methods:Out of 55 C57BL/6J mice, we randomly selected 45 as TAC group and 10 as controls. UBM was performed before TAC and, at day 3 and day 14 after TAC. In all mice, LVESP was measured with a Millar conductance catheter at day 14.Results:With LVESP≥150 mmHg set as indicator of successful TAC (TAC+) and LVESP< 150 mmHg as unsuccessful (TAC-), receiver operating characteristic curve analysis demonstrated that postoperative inner diameter at aortic banding site (IDb), peak flow velocity at aortic banding site (PVb) and peak flow velocity of right/left common carotid artery (PVr/I) at day 3 served as most effective predictors for LVESP at day 14 (area under curve=0.9016,0.9143, 0.8254, respectively. P<0.01 for all). Among all UBM parameters at day 3, IDb, PVb, right common carotid artery peak flow velocity (PVr) and PVr/I correlated best with LVESP at day 14 (R2=0.5740,0.6549,0.5208,0.2274, respectively. P<0.01 for all). Furthermore, IDb, PVb, and PVr/I at day 3 most effectively predict long-term cardiac hypertrophy, using the cut-off values of 0.45 mm, 2698.00 mm/s,3.08, respectively.Conclusions:UBM can be a noninvasive and effective option for early prediction of successful TAC. predict long-term cardiac hypertrophy, using the cut-off values of 0.45 mm, 2698.00 mm/s,3.08, respectively.Part Ⅱ Olmesartan Attenuates Cardiac Remodeling Through DLL4/Notch1 Pathway Activation in Pressure Overload MiceBackground and Objectives:Notch 1 signaling controls the cardiac adaptation to stress. We therefore aimed to validate whether olmesartan, a widely used angiotensin Ⅱ type 1 receptor blocker, ameliorates cardiac remodeling and dysfunction via delta-like ligand 4 (DLL4)/Notch1 pathway in mice with chronic pressure overload.Methods:Cardiac pressure overload was produced by transverse aortic constriction (TAC). A total of 35 wide-type C57BL/6J mice were randomly divided into sham group, TAC group, TAC+ olmesartan group and TAC+olmesartan+DAPT group (DAPT:γ-secretase inhibitor, Notch signaling inhibitor). Saline (10 ml·kg-1·d-1) or the same volume of olmesartan liquor (3 mg·kg-1·d-1) was administered by gavage, and DAPT (10 μmol·kg-1·d-1) by peritoneal injection. After 28 days of treatment, cardiac hemodynamics, echocardiography, and histology were evaluated, followed by quantitative polymerase chain reaction of fetal gene (atrial natriuretic peptide and skeletal-a actin, ANP and SAA) expression. Notch 1-related proteins and ERK1/2 were examined by western blot, and the serum level of angiotensin Ⅱ was determined by means of enzyme-linked immunosorbent assay kits.Results:Persistent pressure overload induced left ventricular hypertrophy, dysfunction, fibrosis, and microcirculation dysfunction, together with upregulation of angiotensin Ⅱ, ERK1/2 and fetal gene expression. By the activation of DLL4/Notch1, olmesartan decreased left ventricular hypertrophy and fibrosis, preserved cardiac function, and improved capillary density and coronary perfusion. All these curative effects were suppressed by pharmacological blockade of Notch signaling with DAPT.Conclusions:Our findings identify a heretofore unknown pharmacological mechanism that olmesartan improves cardiac remodeling and function via DLL4/Notch1 pathway activation in mice with chronic pressure overload, which may present a new therapeutic target for hypertension.