Zhidan Chen

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.

Association of Stat3 with HSF1 plays a critical role in G-CSF-induced cardio-protection against ischemia/reperfusion injury.

Granulocyte colony-stimulating factor (G-CSF) has been shown to be cardio-protective against ischemia through activating Jak2/Stat3 pathway, however, the mechanism is unclear. Heat shock transcription factor 1 (HSF1), a definite endogenous protective protein in cardiomyocytes, may interact with Stat family under stress conditions. We hypothesized that G-CSF could induce cardio-protection against ischemia/reperfusion (I/R) through association of HSF1 with Stat3. To test the hypothesis, we built cardiac I/R injury model with HSF1 knockout (KO) mice and wild type (WT) mice by occlusion of the left anterior descending (LAD) coronary artery for 30min and subsequent release of the occlusion for 24h. These mice were administered with G-CSF (100μg/kg/day) or vehicle subcutaneously for 3days before surgery. As expected, G-CSF induced significant cardio-protections against I/R injury, characterized by higher ejection fraction (EF%), lower left ventricular end diastolic pressure (LVEDP), increased dp/dt value and decreased infarct area as compared with the vehicle treatment in WT mice. In HSF1-KO mice, however, these cardio-protections induced by G-CSF were greatly attenuated. Inhibition of oxidative stress-induced cardiomyocyte apoptosis by G-CSF also disappeared due to the deficiency of HSF1 in vitro and in vivo. Furthermore, G-CSF increased the phosphorylation and the association of Stat3 with HSF1, which enhanced transcriptional activity of HSF1. Inhibition of either Stat3 or HSF1 by pharmacological agents suppressed G-CSF-induced association of the two proteins and anti-apoptotic effect on cardiomyocytes. Our data suggest that G-CSF stimulates phosphorylation and association of Stat3 with HSF1 and therefore enhances transcriptional activity of HSF1, leading to the cardio-protection against I/R injury.

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.

Urotensin II Protects Cardiomyocytes from Apoptosis Induced by Oxidative Stress through the CSE/H2S Pathway

Plasma urotensin II (UII) has been observed to be raised in patients with acute myocardial infarction; suggesting a possible cardiac protective role for this peptide. However, the molecular mechanism is unclear. Here, we treated cultured cardiomyocytes with H2O2 to induce oxidative stress; observed the effect of UII on H2O2-induced apoptosis and explored potential mechanisms. UII pretreatment significantly reduced the number of apoptotic cardiomyocytes induced by H2O2; and it partly abolished the increase of pro-apoptotic protein Bax and the decrease of anti-apoptotic protein Bcl-2 in cardiomyocytes induced by H2O2. SiRNA targeted to the urotensin II receptor (UT) greatly inhibited these effects. Further analysis revealed that UII increased the production of hydrogen sulfide (H2S) and the level of cystathionine-γ-lyase (CSE) by activating the ERK signaling in H2O2-treated-cardiomyocytes. Si-CSE or ERK inhibitor not only greatly inhibited the increase in CSE level or the phosphorylation of ERK induced by UII but also reversed anti-apoptosis of UII in H2O2-treated-cadiomyocytes. In conclusion, UII rapidly promoted the phosphorylation of ERK and upregulated CSE level and H2S production, which in turn activated ERK signaling to protect cardiomyocytes from apoptosis under oxidative stress. These results suggest that increased plasma UII level may protect cardiomyocytes at the early-phase of acute myocardial infarction in patients.

Knockdown of Nucleosome Assembly Protein 1-Like 1 Induces Mesoderm Formation and Cardiomyogenesis Via Notch Signaling in Murine-Induced Pluripotent Stem Cells

Low efficiency of cardiomyocyte differentiation from induced pluripotent stem cells (iPSCs) hinders the clinical application of iPSC technology for cardiac repair strategy. Recently, we screened out nucleosome assembly protein 1‐like 1 (Nap1l1), which was downregulated during the differentiation of P19CL6 cells into cardiomyocytes. Here, we attempted to study the role of Nap1l1 in cardiomyogenesis of iPSC. Nap1l1 was downregulated during the differentiation of iPSC. Knockdown of Nap1l1 dramatically enhanced the differentiation of iPSC into functional cardiomyocytes while overexpression of Nap1l1 sharply lowered the differentiation. Moreover, although Nap1l1‐knockdown had little effect on endoderm differentiation, the Nap1l1 modulation significantly accelerated mesoderm development. Re‐expressing Nap1l1 in Nap1l1‐knockdown‐iPSC rescued the effects of Nap1l1. Inducibly overexpressing Nap1l1 at early stage of differentiation greatly inhibited mesoderm induction and cardiogenesis of iPSC. However, mesoderm stem cells (Flk‐1‐positive cells) originated from Nap1l1‐knockdown‐ or ‐overexpression‐iPSC showed no difference in further cardiomyocyte differentiation compared with that of control‐iPSC. Further study revealed that Nap1l1‐overexpression increased γ‐secretase activity and the expression of Notch intracellular domain (NICD) and downstream genes during the differentiation of iPSC. γ‐Secretase inhibitor DAPT (N‐[N‐(3,5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycinet‐butyl ester) greatly suppressed the production of NICD and abolished the inhibitory effects of Nap1l1‐overexpression on mesoderm induction and cardiogenesis. These findings demonstrate that downregulation of Nap1l1 significantly enhances mesodermal induction and subsequent cardiogenesis of murine iPSC via inhibition of γ‐secretase‐regulated Notch signaling, which would facilitate the application of iPSC in heart diseases. Stem Cells 2014;32:1759–1773

Nucleosome Assembly Protein 1-Like 1 (Nap1l1) Regulates the Proliferation of Murine Induced Pluripotent Stem Cells

Background/Aims: To investigate whether nucleosome assembly protein 1-like 1 (Nap1l1) regulates the proliferation of induced pluripotent stem cells (iPSC) and the potential mechanisms. Methods: Nap1l1-knockdown-iPSC and Nap1l1-overexpression-iPSC were constructed by transfection of lentiviral particles. The proliferation of iPSC was detected by MTT analysis, and cell cycle was analyzed by flow cytometry. Results: Nap1l1 overexpression promoted iPSC proliferation and induced G2/M transition compared to their control iPSC while Nap1l1-knockdown-iPSC dramatically displayed the reduced proliferation and accumulated G2/M phase cells. Further analysis showed that Nap1l1 overexpression in iPSC increased the expression of cyclin B1, downregulated the expression of p21 and p27, while knockdown of Nap1l1 showed the opposite effects. In addition, overexpression of Nap1l1 promoted the phosphorylation of AKT and ERK in iPSC, while knockdown of Nap1l1 inhibited the effects. However, these effects displayed in Nap1l1-overexpression-iPSC were greatly suppressed by the inhibition of AKT or ERK signaling. Conclusions: The results indicate that Nap1l1 promotes the proliferation of iPSC attributable to G2/M transition caused by downregulation of p27 and p21, and upregulation of cyclin B1, the activation of AKT or ERK is involved in the process. The present study has revealed a novel molecular mechanism involved in the proliferation of iPSC.

Urotensin II inhibited the proliferation of cardiac side population cells in mice during pressure overload by JNK-LRP6 signalling

Cardiac side population cells (CSPs) are promising cell resource for the regeneration in diseased heart as intrinsic cardiac stem cells. However, the relative low ratio of CSPs in the heart limited the ability of CSPs to repair heart and improve cardiac function effectively under pathophysiological condition. Which factors limiting the proliferation of CSPs in diseased heart are unclear. Here, we show that urotensin II (UII) regulates the proliferation of CSPs by c‐Jun N‐terminal kinase (JNK) and low density lipoprotein receptor‐related protein 6 (LRP6) signalling during pressure overload. Pressure overload greatly upregulated UII level in plasma, UII receptor (UT) antagonist, urantide, promoted CSPs proliferation and improved cardiac dysfunction during chronic pressure overload. In cultured CSPs subjected to mechanical stretch (MS), UII significantly inhibited the proliferation by UT. Nanofluidic proteomic immunoassay showed that it is the JNK activation, but not the extracellular signal‐regulated kinase signalling, that involved in the UII‐inhibited‐ proliferation of CSPs during pressure overload. Further analysis in vitro indicated UII‐induced‐phospho‐JNK regulates phosphorylation of LRP6 in cultured CSPs after MS, which is important in the inhibitory effect of UII on the CSPs during pressure overload. In conclusion, UII inhibited the proliferation of CSPs by JNK/LRP6 signalling during pressure overload. Pharmacological inhibition of UII promotes CSPs proliferation in mice, offering a possible therapeutic approach for cardiac failure induced by pressure overload.

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.

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.