Guoliang Jiang

Effects of Heart Rate and Anesthetic Timing on High-Resolution Echocardiographic Assessment Under Isoflurane Anesthesia in Mice

Objective. Anesthesia provides sedation and immobility, facilitating echocardiography in mice, but it influences cardiovascular function and therefore outcomes of measurement. This study aimed to determine the effect of the optimal heart rate (HR) and anesthetic timing on echocardiographic reproducibility under isoflurane anesthesia. Methods. Male C57BL/6J mice underwent high‐resolution echocardiography with relative fixed HRs and anesthetic timing. The same experiment was repeated once again after 1 week. Results. Echocardiography was highly reproducible in repeated measurements under low‐HR (350–400 beats per minute [bpm]) and high‐HR (475–525 bpm) conditions except some M‐mode parameters under low‐HR conditions. With similar anesthetic timing, mice with a high HR had decreased preload indices and increased ejection phase and Doppler indices. Inversely, when the HR was similar, the echocardiographic results of mice under short anesthetic timing showed little difference from the ones under long anesthetic timing. Conclusions. This study shows that echocardiographic assessment is greatly reproducible under a high HR. The HR is more important than anesthetic timing for echocardiographic evaluation in mice.

High density lipoprotein downregulates angiotensin II type 1 receptor and inhibits angiotensin II-induced cardiac hypertrophy

Angiotensin II (AngII) and its type receptor (AT1-R) play important roles in the development of cardiac hypertrophy. Low level of high density lipoprotein (HDL) is also an independent risk factor for cardiac hypertrophy. We therefore investigated in the present study whether HDL inhibits cardiac hypertrophy relatively to inhibition of AngII and AT1-R in both in vitro and in vivo experiments. Stimulation of cultured cardiomyocytes of neonatal rats with AngII for 24 h and infusion of AngII in mice for 2 weeks resulted in marked cardiac hypertrophic responses including increased protein synthesis, enlarged sizes of cardiomyocytes and hearts, upregulated phosphorylation levels of protein kinases and reprogrammed expression of specific genes, all of which were significantly attenuated by the treatment with HDL. Furthermore, AngII-treatment induced upregulation of AT-R expression either in cultured cardiomyocytes or in hearts of mice and HDL significantly suppressed the upregulation of AT1-R. Our results suggest that HDL may abrogate AngII-induced cardiac hypertrophy through downregulation of AT1-R expression.

Src Is Required for Mechanical Stretch-Induced Cardiomyocyte Hypertrophy through Angiotensin II Type 1 Receptor-Dependent β-Arrestin2 Pathways

Angiotensin II (AngII) type 1 receptor (AT1-R) can be activated by mechanical stress (MS) without the involvement of AngII during the development of cardiomyocyte hypertrophy, in which G protein-independent pathways are critically involved. Although β-arrestin2-biased signaling has been speculated, little is known about how AT1-R/β-arrestin2 leads to ERK1/2 activation. Here, we present a novel mechanism by which Src kinase mediates AT1-R/β-arrestin2-dependent ERK1/2 phosphorylation in response to MS. Differing from stimulation by AngII, MS-triggered ERK1/2 phosphorylation is neither suppressed by overexpression of RGS4 (the negative regulator of the G-protein coupling signal) nor by inhibition of Gαq downstream protein kinase C (PKC) with GF109203X. The release of inositol 1,4,5-triphosphate (IP3) is increased by AngII but not by MS. These results collectively suggest that MS-induced ERK1/2 activation through AT1-R might be independent of G-protein coupling. Moreover, either knockdown of β-arrestin2 or overexpression of a dominant negative mutant of β-arrestin2 prevents MS-induced activation of ERK1/2. We further identifies a relationship between Src, a non-receptor tyrosine kinase and β-arrestin2 using analyses of co-immunoprecipitation and immunofluorescence after MS stimulation. Furthermore, MS-, but not AngII-induced ERK1/2 phosphorylation is attenuated by Src inhibition, which also significantly improves pressure overload-induced cardiac hypertrophy and dysfunction in mice lacking AngII. Finally, MS-induced Src activation and hypertrophic response are abolished by candesartan but not by valsartan whereas AngII-induced responses can be abrogated by both blockers. Our results suggest that Src plays a critical role in MS-induced cardiomyocyte hypertrophy through β-arrestin2-associated angiotensin II type 1 receptor signaling.

Olmesartan attenuates cardiac remodeling through DLL4/Notch1 pathway activation in pressure overload mice.

Background: Notch1 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 + olmsartan + DAPT group (DAPT: &ggr;-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 &mgr;mole·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 (ANP and SAA) expression. Notch1-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 the 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.

Early estimation of left ventricular systolic pressure and prediction of successful aortic constriction in a mouse model of pressure overload by ultrasound biomicroscopy.

Elevation of left ventricular end-systolic pressure (LVESP) and hypertrophic response in mice varies after transverse aorta constriction (TAC). Micromanometric catheterization, conventionally used to select 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). Out of 55 C57BL/6J mice, we randomly selected 45 as TAC group and 10 as controls. UMB 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. With LVESP ≥ 150 mm Hg set as indicator of successful TAC (TAC+) and LVESP < 150 mm Hg 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/l) 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/l correlated best with LVESP at day 14 (R(2) = 0.5740, 0.6549, 0.5208, 0.2274, respectively. p < 0.01 for all). Furthermore, IDb, PVb, and PVr/l 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. UBM can be a noninvasive and effective option for early prediction of successful TAC.

Identification of Amino Acid Residues in Angiotensin II Type 1 Receptor Sensing Mechanical Stretch and Function in Cardiomyocyte Hypertrophy.

Background/Aims: Angiotensin II (AngII) type 1 receptor (AT₁R) could be activated by mechanical stress without the involvement of AngII during the development of cardiac hypertrophy. We aimed to identify sensing sites of AT₁R for activation by mechanical stretch. Methods: We constructed several site-directed mutations of AT₁R (AT₁R^K199Q, AT₁R^L212F, AT₁R^Q257A and AT₁R^C289A), transfected them respectively into COS7 cells or angiotensinogen knockout cardiomyocytes (ATG^−/−-CMs), and observed cellular events after mechanical stretch. Results: AngII-induced phosphorylation of ERKs and Jak2, and redistribution of Gαq11 in AT₁R^WT- COS7 or -ATG^−/−-CMs were dramatically decreased in AT₁R^K199Q- or AT₁R^Q257A- COS7 cells or -ATG^−/−-CMs, while those effects induced by mechanical stretch were greatly suppressed in COS7 cells or ATG^−/−-CMs expressing AT₁R^L212F, AT₁R^Q257A or AT₁R^C289A compared with these cells expressing AT₁R^WT. AngII-induced hypertrophic responses (the increase in hypertrophic genes expression and cross-sectional area) in AT₁R^WT- ATG^−/−-CMs were partly abolished in AT₁R^K199Q-ATG^−/−-CMs or AT₁R^Q257A-ATG^−/−-CMs, while these responses induced by mechanical stretch were greatly inhibited in ATG^−/−-CMs overexpressing AT₁R^L212F, AT₁R^Q257A or AT₁R^C289A. Conclusion: These results indicated that Leu212, Gln257 and Cys289 in AT₁R are not only sensing sites for mechanical stretch but also functional amino residues for activation of the receptor and cardiomyocytes hypertrophy induced by mechanical stretch.

Noninvasive Estimation of Infarct Size in a Mouse Model of Myocardial Infarction by Echocardiographic Coronary Perfusion

Animal models of myocardial infarction (MI) are widely used not only in analyses of 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 the left anterior descending coronary artery.

Cardiomyocyte-Restricted Low Density Lipoprotein Receptor-Related Protein 6 (LRP6) Deletion Leads to Lethal Dilated Cardiomyopathy Partly Through Drp1 Signaling

Low density lipoprotein receptor-related protein 6 (LRP6), a wnt co-receptor, regulates multiple functions in various organs. However, the roles of LRP6 in the adult heart are not well understood. Methods: We observed LRP6 expression in heart with end-stage dilated cardiomyopathy (DCM) by western blot. Tamoxifen-inducible cardiac-specific LRP6 knockout mouse was constructed. Hemodynamic and echocardiographic analyses were performed to these mice. Results: Cardiac LRP6 expression was dramatically decreased in patients with end-stage dilated cardiomyopathy (DCM) compared to control group. Tamoxifen-inducible cardiac-specific LRP6 knockout mice developed acute heart failure and mitochondrial dysfunction with reduced survival. Proteomic analysis suggests the fatty acid metabolism disorder involving peroxisome proliferator-activated receptors (PPARs) signaling in the LRP6 deficient heart. Accumulation of mitochondrial targeting to autophagosomes and lipid droplet were observed in LRP6 deletion hearts. Further analysis revealed cardiac LRP6 deletion suppressed autophagic degradation and fatty acid utilization, coinciding with activation of dynamin-related protein 1 (Drp1) and downregulation of nuclear TFEB (Transcription factor EB). Injection of Mdivi-1, a Drp1 inhibitor, not only promoted nuclear translocation of TFEB, but also partially rescued autophagic degradation, improved PPARs signaling, and attenuated cardiac dysfunction induced by cardiac specific LRP6 deletion. Conclusions: Cardiac LRP6 deficiency greatly suppressed autophagic degradation and fatty acid utilization, and subsequently leads to lethal dilated cardiomyopathy and cardiac dysfunction through activation of Drp1 signaling. It suggests that heart failure progression may be attenuated by therapeutic modulation of LRP6 expression.

Combination Treatment With Antihypertensive Agents Enhances the Effect of Qiliqiangxin on Chronic Pressure Overload–induced Cardiac Hypertrophy and Remodeling in Male Mice

Abstract: We previously showed that Qiliqiangxin (QL) capsules could ameliorate cardiac hypertrophy and remodeling in a mouse model of pressure overload. Here, we compared the effects of QL alone with those of QL combined with the following 3 types of antihypertensive drugs on cardiac remodeling and dysfunction induced by pressure overload for 4 weeks in mice: an angiotensin II type 1 receptor (AT1-R) blocker (ARB), an angiotensin-converting enzyme inhibitor (ACEI), and a &bgr;-adrenergic receptor (&bgr;-AR) blocker (BB). Adult male mice (C57B/L6) were subjected to either transverse aortic constriction or sham operation for 4 weeks, and the drugs (or saline) were orally administered through gastric tubes. Cardiac function and remodeling were evaluated through echocardiography, catheterization, histology, and analysis of hypertrophic gene expression. Cardiomyocyte apoptosis and autophagy, AT1-R and &bgr;1-AR expression, and cell proliferation–related molecules were also examined. Although pressure overload–induced cardiac remodeling and dysfunction, hypertrophic gene reprogramming, AT1-R and &bgr;1-AR expression, and ERK phosphorylation were significantly attenuated by QL alone, QL + ARB, QL + ACEI, and QL + BB, the attenuation was stronger in the combination treatment groups. Moreover, apoptosis was reduced to a larger extent by each combination treatment than by QL alone, whereas autophagy was more strongly attenuated by either QL + ARB or QL + ACEI. None of the treatments significantly upregulated ErbB2 or ErbB4 phosphorylation, and none significantly downregulated C/EBP&bgr; expression. Therefore, the effects of QL on chronic pressure overload–induced cardiac remodeling may be significantly increased when QL is combined with an ARB, an ACEI, or a BB.

Ryanodine Receptor Type 2 Plays a Role in the Development of Cardiac Fibrosis under Mechanical Stretch Through TGFβ-1

Ryanodine receptor type 2 (RyR-2), the main Ca2+ release channel from sarcoplasmic reticulum in cardiomyocytes, plays a vital role in the regulation ofmyocardial contractile function and cardiac hypertrophy. However, the role of RyR-2 in cardiac fibrosis during the development of cardiac hypertrophy remains unclear.In this study, we examined whether RyR-2 regulates TGFβ1, which is secreted from cardiomyocytes and exerts on cardiac fibrosis using cultured cardiomyocytes and cardiac fibroblasts of neonatal rats. The expression of RyR-2 was found only in cardiomyocytesbut not in cardiac fibroblasts. Mechanical stretch induced upregulation of TGFβ1 in cardiomyocytes and RyR-2 knockdown significantly suppressed the upregulation of TGFβ1 expression. The transcript levels of collagen genes were also decreased in fibroblasts compare with wild type, although the expression of both two kinds was higher than those in stationary cardiomyocytes (non-stretch). With the inhibition of the TGFβ1-neutralizing antibody, the expression of collagen genes has no significant difference between the mechanically stretched cardiomyocytes and non-stretchedones. These results indicate that RyR-2 regulated TGFβ1 expression in mechanically stretched cardiomyocytes and TGFβ1 promoted collagen formation of cardiac fibroblasts by a paracrine mechanism.RyR-2 in mechanical stretch could promote the development of cardiac fibrosis involving TGFβ1-dependent paracrine mechanism. Our findings provided more insight into comprehensively understanding the molecular role of RyR-2 in regulating cardiac fibrosis.