The E3 ubiquitin ligase WWP2 regulates pro-fibrogenic monocyte infiltration and activity in heart fibrosis.

Non-ischemic cardiomyopathy (NICM) can cause left ventricular dysfunction through interstitial fibrosis, which corresponds to the failure of cardiac tissue remodeling. Recent evidence implicates monocytes/macrophages in the etiopathology of cardiac fibrosis, but giving their heterogeneity and the antagonizing roles of macrophage subtypes in fibrosis, targeting these cells has been challenging. Here we focus on WWP2, an E3 ubiquitin ligase that acts as a positive genetic regulator of human and murine cardiac fibrosis, and show that myeloid specific deletion of WWP2 reduces cardiac fibrosis in hypertension-induced NICM. By using single cell RNA sequencing analysis of immune cells in the same model, we establish the functional heterogeneity of macrophages and define an early pro-fibrogenic phase of NICM that is driven by Ccl5-expressing Ly6chigh monocytes. Among cardiac macrophage subtypes, WWP2 dysfunction primarily affects Ly6chigh monocytes via modulating Ccl5, and consequentially macrophage infiltration and activation, which contributes to reduced myofibroblast trans-differentiation. WWP2 interacts with transcription factor IRF7, promoting its non-degradative mono-ubiquitination, nuclear translocation and transcriptional activity, leading to upregulation of Ccl5 at transcriptional level. We identify a pro-fibrogenic macrophage subtype in non-ischemic cardiomyopathy, and demonstrate that WWP2 is a key regulator of IRF7-mediated Ccl5/Ly6chigh monocyte axis in heart fibrosis.

Inhibition of IL11 Signaling Reduces Aortic Pathology in Murine Marfan Syndrome.

BACKGROUND: Marfan syndrome (MFS) is associated with TGF (transforming growth factor) ß-stimulated ERK (extracellular signal-regulated kinase) activity in vascular smooth muscle cells (VSMCs), which adopt a mixed synthetic/contractile phenotype. In VSMCs, TGFß induces IL (interleukin) 11) that stimulates ERK-dependent secretion of collagens and MMPs (matrix metalloproteinases). Here, we examined the role of IL11 in the MFS aorta. METHODS: We used echocardiography, histology, immunostaining, and biochemical methods to study aortic anatomy, physiology, and molecular endophenotypes in Fbn1C1041G/+ mice, an established murine model of MFS (mMFS). mMFS mice were crossed to an IL11-tagged EGFP (enhanced green fluorescent protein; Il11EGFP/+) reporter strain or to a strain deleted for the IL11 receptor (Il11ra1-/-). In therapeutic studies, mMFS were administered an X209 (neutralizing antibody against IL11RA [IL11 receptor subunit alpha]) or IgG for 20 weeks and imaged longitudinally. RESULTS: IL11 mRNA and protein were elevated in the aortas of mMFS mice, as compared to controls. mMFS mice crossed to Il11EGFP/+ mice had increased IL11 expression in VSMCs, notably in the aortic root and ascending aorta. As compared to the mMFS parental strain, double mutant mMFS:Il11ra1-/- mice had reduced aortic dilatation and exhibited lesser fibrosis, inflammation, elastin breaks, and VSMC loss, which was associated with reduced aortic COL1A1 (collagen type I alpha 1 chain), IL11, MMP2/9, and phospho-ERK expression. To explore therapeutic targeting of IL11 signaling in MFS, we administered either a neutralizing antibody against IL11RA (X209) or an IgG control. After 20 weeks of antibody administration, as compared to IgG, mMFS mice receiving X209 had reduced thoracic and abdominal aortic dilation as well as lesser fibrosis, inflammation, elastin breaks, and VSMC loss. By immunoblotting, X209 was shown to reduce aortic COL1A1, IL11, MMP2/9, and phospho-ERK expression. CONCLUSIONS: In MFS, IL11 is upregulated in aortic VSMCs to cause ERK-related thoracic aortic dilatation, inflammation, and fibrosis. Therapeutic inhibition of IL11, imminent in clinical trials, might be considered as a new approach in MFS.

Loss of Yap/Taz in cardiac fibroblasts attenuates adverse remodelling and improves cardiac function.

AIMS: Fibrosis is associated with all forms of adult cardiac diseases including myocardial infarction (MI). In response to MI, the heart undergoes ventricular remodelling that leads to fibrotic scar due to excessive deposition of extracellular matrix mostly produced by myofibroblasts. The structural and mechanical properties of the fibrotic scar are critical determinants of heart function. Yes-associated protein (Yap) and transcriptional coactivator with PDZ-binding motif (Taz) are the key effectors of the Hippo signalling pathway and are crucial for cardiomyocyte proliferation during cardiac development and regeneration. However, their role in cardiac fibroblasts, regulating post-MI fibrotic and fibroinflammatory response, is not well established. METHODS AND RESULTS: Using mouse model, we demonstrate that Yap/Taz are activated in cardiac fibroblasts after MI and fibroblasts-specific deletion of Yap/Taz using Col1a2Cre(ER)T mice reduces post-MI fibrotic and fibroinflammatory response and improves cardiac function. Consistently, Yap overexpression elevated post-MI fibrotic response. Gene expression profiling shows significant downregulation of several cytokines involved in post-MI cardiac remodelling. Furthermore, Yap/Taz directly regulate the promoter activity of pro-fibrotic cytokine interleukin-33 (IL33) in cardiac fibroblasts. Blocking of IL33 receptor ST2 using the neutralizing antibody abrogates the Yap-induced pro-fibrotic response in cardiac fibroblasts. We demonstrate that the altered fibroinflammatory programme not only affects the nature of cardiac fibroblasts but also the polarization as well as infiltration of macrophages in the infarcted hearts. Furthermore, we demonstrate that Yap/Taz act downstream of both Wnt and TGFß signalling pathways in regulating cardiac fibroblasts activation and fibroinflammatory response. CONCLUSION: We demonstrate that Yap/Taz play an important role in controlling MI-induced cardiac fibrosis by modulating fibroblasts proliferation, transdifferentiation into myofibroblasts, and fibroinflammatory programme.

Cardiac Tissue Factor Regulates Inflammation, Hypertrophy, and Heart Failure in Mouse Model of Type 1 Diabetes.

Patients with diabetes have an increased risk of heart failure (HF). Diabetes is highly prevalent in HF with preserved ejection fraction (HFpEF), which is on the rise worldwide. The role of diabetes in HF is less established, and available treatments for HF are not effective in patients with HFpEF. Tissue factor (TF), a transmembrane receptor, plays an important role in immune cell inflammation and atherothrombosis in diabetes. However, its role in diabetes-induced cardiac inflammation, hypertrophy, and HF has not been studied. In this study, we used wild-type (WT), heterozygous, and low-TF (with 1% human TF) mice to determine the role of TF in type 1 diabetes-induced HF. We found significant upregulation of cardiac TF mRNA and protein levels in diabetic WT hearts compared with nondiabetic controls. WT diabetic hearts also exhibited increased inflammation and cardiac hypertrophy versus controls. However, these changes in cardiac inflammation and hypertrophy were not found in low-TF mice with diabetes compared with their nondiabetic controls. TF deficiency was also associated with improved cardiac function parameters suggestive of HFpEF, which was evident in WT mice with diabetes. The TF regulation of inflammation and cardiac remodeling was further dependent on downstream ERK1/2 and STAT3 pathways. In summary, our study demonstrated an important role of TF in regulating diabetes-induced inflammation, hypertrophy, and remodeling of the heart leading to HFpEF.

Antibody-mediated neutralization of IL11 signalling reduces ERK activation and cardiac fibrosis in a mouse model of severe pressure overload.

Interleukin-11 (IL11) is important for fibroblast-to-myofibroblast transformations. Here, we examined the signalling and phenotypic effects of inhibiting IL11 signalling using neutralizing antibodies against IL11 or its cognate receptor (IL11RA) in a mouse model of acute and severe pressure overload. C57BL/6J mice underwent ascending aortic constriction (AAC) surgery and were randomized to anti-IL11, anti-IL11RA, or isotype control antibodies (20 mg/kg, bi-weekly for 2 weeks). AAC surgery induced the expression of IL11, IL11RA and extracellular matrix (ECM) genes that was associated with cardiac hypertrophy and aortic remodelling. Inhibition of IL11 signalling reduced AAC-induced cardiac fibrosis and ECM gene expression as well as ERK1/2 phosphorylation but had no effect on cardiac hypertrophy. STAT3 was phosphorylated in the hearts of AAC-treated mice but this was unrelated to IL11 activity, which we confirmed in mouse cardiac fibroblasts in vitro. These data highlight that blocking IL11 signalling reduces cardiac fibrosis due to severe pressure overload and suggests ERK, but not STAT3, activity as the relevant underlying signalling pathway.

Therapeutic Targeting of Interleukin-11 Signalling Reduces Pressure Overload-Induced Cardiac Fibrosis in Mice.

There are currently no specific treatments for cardiac fibrosis. We tested the efficacy of a neutralising anti-IL11 antibody (X203) to reduce cardiac fibrosis in two preclinical models: transverse aortic constriction (TAC) and chronic angiotensin II infusion (AngII). In the first model, male C57BL/6J mice were subjected to TAC for 2 weeks. In the second model, mice received continuous angiotensin II for 4 weeks via subcutaneous pump. In both models, mice received either 20 mg/kg of X203 or isotype-control antibody twice-weekly, starting 24 h after surgery. Cardiac fibrosis and extracellular matrix gene expression were assessed by RT-qPCR, Western blot, histology and collagen (hydroxyproline) assays. In both models, X203 significantly reduced pro-fibrotic gene expression and myocardial fibrosis (TAC: 51% reduction in total collagen, P < 0.001, 39% in perivascular fibrosis, P < 0.001; AngII: 17% reduction in total collagen, P = 0.04, 83% in perivascular fibrosis, P < 0.001). Pharmacological targeting of IL11 reduces cardiac fibrosis in preclinical models. Figa Graphical Abstract.

YAP/TAZ deficiency reprograms macrophage phenotype and improves infarct healing and cardiac function after myocardial infarction.

Adverse cardiac remodeling after myocardial infarction (MI) causes structural and functional changes in the heart leading to heart failure. The initial post-MI pro-inflammatory response followed by reparative or anti-inflammatory response is essential for minimizing the myocardial damage, healing, and scar formation. Bone marrow-derived macrophages (BMDMs) are recruited to the injured myocardium and are essential for cardiac repair as they can adopt both pro-inflammatory or reparative phenotypes to modulate inflammatory and reparative responses, respectively. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are the key mediators of the Hippo signaling pathway and are essential for cardiac regeneration and repair. However, their functions in macrophage polarization and post-MI inflammation, remodeling, and healing are not well established. Here, we demonstrate that expression of YAP and TAZ is increased in macrophages undergoing pro-inflammatory or reparative phenotype changes. Genetic deletion of YAP/TAZ leads to impaired pro-inflammatory and enhanced reparative response. Consistently, YAP activation enhanced pro-inflammatory and impaired reparative response. We show that YAP/TAZ promote pro-inflammatory response by increasing interleukin 6 (IL6) expression and impede reparative response by decreasing Arginase-I (Arg1) expression through interaction with the histone deacetylase 3 (HDAC3)-nuclear receptor corepressor 1 (NCoR1) repressor complex. These changes in macrophages polarization due to YAP/TAZ deletion results in reduced fibrosis, hypertrophy, and increased angiogenesis, leading to improved cardiac function after MI. Also, YAP activation augmented MI-induced cardiac fibrosis and remodeling. In summary, we identify YAP/TAZ as important regulators of macrophage-mediated pro-inflammatory or reparative responses post-MI.

Interleukin-11 is important for vascular smooth muscle phenotypic switching and aortic inflammation, fibrosis and remodeling in mouse models.

Transforming growth factor beta-1 (TGFß1) is a major driver of vascular smooth muscle cell (VSMC) phenotypic switching, an important pathobiology in arterial disease. We performed RNA-sequencing of TGFß1-stimulated human aortic or arterial VSMCs which revealed large and consistent upregulation of Interleukin 11 (IL11). IL11 has an unknown function in VSMCs, which highly express the IL11 receptor alpha, suggestive of an autocrine loop. In vitro, IL11 activated ERK signaling, but inhibited STAT3 activity, and caused VSMC phenotypic switching to a similar extent as TGFß1 or angiotensin II (ANGII) stimulation. Genetic or therapeutic inhibition of IL11 signaling reduced TGFß1- or ANGII-induced VSMC phenotypic switching, placing IL11 activity downstream of these factors. Aortas of mice with Myh11-driven IL11 expression were remodeled and had reduced contractile but increased matrix and inflammatory genes expression. In two models of arterial pressure loading, IL11 was upregulated in the aorta and neutralizing IL11 antibodies reduced remodeling along with matrix and pro-inflammatory gene expression. These data show that IL11 plays an important role in VSMC phenotype switching, vascular inflammation and aortic pathobiology.

Prdm16 Deficiency Leads to Age-Dependent Cardiac Hypertrophy, Adverse Remodeling, Mitochondrial Dysfunction, and Heart Failure.

Hypertrophic cardiomyopathy (HCM) is a well-established risk factor for cardiovascular mortality worldwide. Although hypertrophy is traditionally regarded as an adaptive response to physiological or pathological stress, prolonged hypertrophy can lead to heart failure. Here we demonstrate that Prdm16 is dispensable for cardiac development. However, it is required in the adult heart to preserve mitochondrial function and inhibit hypertrophy with advanced age. Cardiac-specific deletion of Prdm16 results in cardiac hypertrophy, excessive ventricular fibrosis, mitochondrial dysfunction, and impaired metabolic flexibility, leading to heart failure. We demonstrate that Prdm16 and euchromatic histone-lysine N-methyltransferase factors (Ehmts) act together to reduce expression of fetal genes reactivated in pathological hypertrophy by inhibiting the functions of the pro-hypertrophic transcription factor Myc. Although young Prdm16 knockout mice show normal cardiac function, they are predisposed to develop heart failure in response to metabolic stress. Our study demonstrates that Prdm16 protects the heart against age-dependent cardiac hypertrophy and heart failure.

Author Correction: WWP2 regulates pathological cardiac fibrosis by modulating SMAD2 signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

WWP2 regulates pathological cardiac fibrosis by modulating SMAD2 signaling.

Cardiac fibrosis is a final common pathology in inherited and acquired heart diseases that causes cardiac electrical and pump failure. Here, we use systems genetics to identify a pro-fibrotic gene network in the diseased heart and show that this network is regulated by the E3 ubiquitin ligase WWP2, specifically by the WWP2-N terminal isoform. Importantly, the WWP2-regulated pro-fibrotic gene network is conserved across different cardiac diseases characterized by fibrosis: human and murine dilated cardiomyopathy and repaired tetralogy of Fallot. Transgenic mice lacking the N-terminal region of the WWP2 protein show improved cardiac function and reduced myocardial fibrosis in response to pressure overload or myocardial infarction. In primary cardiac fibroblasts, WWP2 positively regulates the expression of pro-fibrotic markers and extracellular matrix genes. TGFß1 stimulation promotes nuclear translocation of the WWP2 isoforms containing the N-terminal region and their interaction with SMAD2. WWP2 mediates the TGFß1-induced nucleocytoplasmic shuttling and transcriptional activity of SMAD2.

Semaphorin 3E/PlexinD1 signaling is required for cardiac ventricular compaction.

Left ventricular noncompaction (LVNC) is one of the most common forms of genetic cardiomyopathy characterized by excessive trabeculation and impaired myocardial compaction during fetal development. Patients with LVNC are at higher risk of developing left/right ventricular failure or both. Although the key regulators for cardiac chamber development are well studied, the role of semaphorin (Sema)/plexin signaling in this process remains poorly understood. In this article, we demonstrate that genetic deletion of Plxnd1, a class-3 Sema receptor in endothelial cells, leads to severe cardiac chamber defects. They were characterized by excessive trabeculation and noncompaction similar to patients with LVNC. Loss of Plxnd1 results in decreased expression of extracellular matrix proteolytic genes, leading to excessive deposition of cardiac jelly. We demonstrate that Plxnd1 deficiency is associated with an increase in Notch1 expression and its downstream target genes. In addition, inhibition of the Notch signaling pathway partially rescues the excessive trabeculation and noncompaction phenotype present in Plxnd1 mutants. Furthermore, we demonstrate that Semaphorin 3E (Sema3E), one of PlexinD1's known ligands, is expressed in the developing heart and is required for myocardial compaction. Collectively, our study uncovers what we believe to be a previously undescribed role of the Sema3E/PlexinD1 signaling pathway in myocardial trabeculation and the compaction process.

Targeting Mitochondrial Fission Using Mdivi-1 in A Clinically Relevant Large Animal Model of Acute Myocardial Infarction: A Pilot Study.

Background: New treatments are needed to reduce myocardial infarct size (MI) and prevent heart failure (HF) following acute myocardial infarction (AMI), which are the leading causes of death and disability worldwide. Studies in rodent AMI models showed that genetic and pharmacological inhibition of mitochondrial fission, induced by acute ischemia and reperfusion, reduced MI size. Whether targeting mitochondrial fission at the onset of reperfusion is also cardioprotective in a clinically-relevant large animal AMI model remains to be determined. Methods: Adult pigs (30-40 kg) were subjected to closed-chest 90-min left anterior descending artery ischemia followed by 72 h of reperfusion and were randomized to receive an intracoronary bolus of either mdivi-1 (1.2 mg/kg, a small molecule inhibitor of the mitochondrial fission protein, Drp1) or vehicle control, 10-min prior to reperfusion. The left ventricular (LV) size and function were both assessed by transthoracic echocardiography prior to AMI and after 72 h of reperfusion. MI size and the area-at-risk (AAR) were determined using dual staining with Tetrazolium and Evans blue. Heart samples were collected for histological determination of fibrosis and for electron microscopic analysis of mitochondrial morphology. Results: A total of 14 pigs underwent the treatment protocols (eight control and six mdivi-1). Administration of mdivi-1 immediately prior to the onset of reperfusion did not reduce MI size (MI size as % of AAR: Control 49.2 ± 8.6 vs. mdivi-1 50.5 ± 11.4; p = 0.815) or preserve LV systolic function (LV ejection fraction %: Control 67.5 ± 0.4 vs. mdivi-1 59.6 ± 0.6; p = 0.420), when compared to vehicle control. Similarly, there were no differences in mitochondrial morphology or myocardial fibrosis between mdivi-1 and vehicle control groups. Conclusion: Our pilot study has shown that treatment with mdivi-1 (1.2 mg/kg) at the onset of reperfusion did not reduce MI size or preserve LV function in the clinically-relevant closed-chest pig AMI model. A larger study, testing different doses of mdivi-1 or using a more specific Drp1 inhibitor are required to confirm these findings.

Construction of a vascularized hydrogel for cardiac tissue formation in a porcine model.

Replacing cardiac tissues lost to myocardial infarction remains a therapeutic goal for regenerative therapy in recovering cardiac function. We assessed the feasibility of constructing a macrosized human cardiac tissue construct using pluripotent stem cell-derived cardiomyocytes or control fibroblasts infused fibrin/collagen hydrogel and performed ectopic implantation in peripheral vascular system of a porcine model for 3 weeks. Finally, an optimized vascularized cardiac construct was explanted and grafted onto porcine myocardium for 2 weeks. Myocardial-grafted human cardiac constructs showed a nascent tissue-like organization with aligned cardiomyocytes within the remodelled collagen matrix. Nevertheless, no significant changes in intraconstruct density of cardiomyocytes were observed in the myocardial-grafted constructs (human embryonic stem cell [hESC]-derived cardiomyocyte [n = 4]: 70.5 ± 22.8 troponin I+ cardiomyocytes/high power field [HPF]) as compared to peripherally implanted constructs (hESC-derived cardiomyocyte [n = 4]: 59.0 ± 19.6 troponin I+ cardiomyocytes/HPF; human induced pluripotent stem cell-derived cardiomyocyte [n = 3]: 50.9 ± 8.5 troponin I+ cardiomyocytes/HPF, p = ns). However, the myocardial-grafted constructs showed an increased in neovascularization (194.4 ± 24.7 microvessels/mm2 tissue, p < .05), microvascular maturation (82.8 ± 24.7 mature microvessels/mm2 , p < .05), and tissue-like formation whereas the peripherally implanted constructs of hESC-derived cardiomyocyte (168.3 ± 98.2 microvessels/mm2 tissue and 68.1 ± 33.4 mature microvessels/mm2 ) and human induced pluripotent stem cell-derived cardiomyocyte (86.8 ± 57.4 microvessels/mm2 tissue and 22.0 ± 32.7 mature microvessels/mm2 ) were not significantly different in vascularized response when compared to the control human fibroblasts (n = 3) constructs (65.6 ± 34.1 microvessels/mm2 tissue and 30.7 ± 20.7 mature microvessels/mm2 ). We presented results on technical feasibility and challenges of grafting vascularized centimetre-sized human cardiac construct that may spur novel approaches in cardiac tissue replacement strategy.

Titin-truncating variants affect heart function in disease cohorts and the general population.

Titin-truncating variants (TTNtv) commonly cause dilated cardiomyopathy (DCM). TTNtv are also encountered in ∼1% of the general population, where they may be silent, perhaps reflecting allelic factors. To better understand TTNtv, we integrated TTN allelic series, cardiac imaging and genomic data in humans and studied rat models with disparate TTNtv. In patients with DCM, TTNtv throughout titin were significantly associated with DCM. Ribosomal profiling in rat showed the translational footprint of premature stop codons in Ttn, TTNtv-position-independent nonsense-mediated degradation of the mutant allele and a signature of perturbed cardiac metabolism. Heart physiology in rats with TTNtv was unremarkable at baseline but became impaired during cardiac stress. In healthy humans, machine-learning-based analysis of high-resolution cardiac imaging showed TTNtv to be associated with eccentric cardiac remodeling. These data show that TTNtv have molecular and physiological effects on the heart across species, with a continuum of expressivity in health and disease.

iPSC-derived human cardiac progenitor cells improve ventricular remodelling via angiogenesis and interstitial networking of infarcted myocardium.

We investigate the effects of myocardial transplantation of human induced pluripotent stem cell (iPSC)-derived progenitors and cardiomyocytes into acutely infarcted myocardium in severe combined immune deficiency mice. A total of 2 × 10(5) progenitors, cardiomyocytes or cell-free saline were injected into peri-infarcted anterior free wall. Sham-operated animals received no injection. Myocardial function was assessed at 2-week and 4-week post-infarction by using echocardiography and pressure-volume catheterization. Early myocardial remodelling was observed at 2-week with echocardiography derived stroke volume (SV) in saline (20.45 ± 7.36 µl, P < 0.05) and cardiomyocyte (19.52 ± 3.97 µl, P < 0.05) groups, but not in progenitor group (25.65 ± 3.61 µl), significantly deteriorated as compared to sham control group (28.41 ± 4.41 µl). Consistently, pressure-volume haemodynamic measurements showed worsening chamber dilation in saline (EDV: 23.24 ± 5.01 µl, P < 0.05; ESV: 17.08 ± 5.82 µl, P < 0.05) and cardiomyocyte (EDV: 26.45 ± 5.69 µl, P < 0.05; ESV: 18.03 ± 6.58 µl, P < 0.05) groups by 4-week post-infarction as compared to control (EDV: 15.26 ± 2.96 µl; ESV: 8.41 ± 2.94 µl). In contrast, cardiac progenitors (EDV: 20.09 ± 7.76 µl; ESV: 13.98 ± 6.74 µl) persistently protected chamber geometry against negative cardiac remodelling. Similarly, as compared to sham control (54.64 ± 11.37%), LV ejection fraction was preserved in progenitor group from 2-(38.68 ± 7.34%) to 4-week (39.56 ± 13.26%) while cardiomyocyte (36.52 ± 11.39%, P < 0.05) and saline (35.34 ± 11.86%, P < 0.05) groups deteriorated early at 2-week. Improvements of myocardial function in the progenitor group corresponded to increased vascularization (16.12 ± 1.49/mm(2) to 25.48 ± 2.08/mm(2) myocardial tissue, P < 0.05) and coincided with augmented networking of cardiac telocytes in the interstitial space of infarcted zone.

3D Reconstruction of Chick Embryo Vascular Geometries Using Non-invasive High-Frequency Ultrasound for Computational Fluid Dynamics Studies.

Recent animal studies have provided evidence that prenatal blood flow fluid mechanics may play a role in the pathogenesis of congenital cardiovascular malformations. To further these researches, it is important to have an imaging technique for small animal embryos with sufficient resolution to support computational fluid dynamics studies, and that is also non-invasive and non-destructive to allow for subject-specific, longitudinal studies. In the current study, we developed such a technique, based on ultrasound biomicroscopy scans on chick embryos. Our technique included a motion cancelation algorithm to negate embryonic body motion, a temporal averaging algorithm to differentiate blood spaces from tissue spaces, and 3D reconstruction of blood volumes in the embryo. The accuracy of the reconstructed models was validated with direct stereoscopic measurements. A computational fluid dynamics simulation was performed to model fluid flow in the generated construct of a Hamburger-Hamilton (HH) stage 27 embryo. Simulation results showed that there were divergent streamlines and a low shear region at the carotid duct, which may be linked to the carotid duct's eventual regression and disappearance by HH stage 34. We show that our technique has sufficient resolution to produce accurate geometries for computational fluid dynamics simulations to quantify embryonic cardiovascular fluid mechanics.

Comparative myocardial deformation in 3 myocardial layers in mice by speckle tracking echocardiography.

BACKGROUND: Speckle tracking echocardiography (STE) using dedicated high-resolution ultrasound is a relatively new technique that is useful in assessing myocardial deformation in 3 myocardial layers in small animals. However, comparative studies of STE parameters acquired from murine are limited. METHODS: A high-resolution rodent ultrasound machine (VSI Vevo 2100) and a clinically validated ultrasound machine (GE Vivid 7) were used to consecutively acquire echocardiography images from standardized parasternal long axis and short axis at midpapillary muscle level from 13 BALB/c mice. Speckle tracking strain (longitudinal, circumferential, and radial) from endocardial, myocardial, and epicardial layers was analyzed using vendor-specific offline analysis software. RESULTS: Intersystem differences were not statistically significant in the global peak longitudinal strain (-16.8 ± 1.7% versus -18.7 ± 3.1%) and radial strain (46.8 ± 14.2% versus 41.0 ± 9.5%), except in the global peak circumferential strain (-16.9 ± 3.1% versus 27.0 ± 5.2%, P < 0.05). This was corroborated by Bland Altman analysis that revealed a weak agreement in circumferential strain (mean bias ± 1.96 SD of -10.12 ± 6.06%) between endocardium and midmyocardium. However, a good agreement was observed in longitudinal strain between midmyocardium/endocardium (mean bias ± 1.96 SD of -1.88 ± 3.93%) and between midmyocardium/epicardium (mean bias ± 1.96 SD of 3.63 ± 3.91%). Radial strain (mean bias ± 1.96 SD of -5.84 ± 17.70%) had wide limits of agreement between the two systems that indicated an increased variability. CONCLUSIONS: Our study shows that there is good reproducibility and agreement in longitudinal deformation of the 3 myocardial layers between the two ultrasound systems. Directional deformation gradients at endocardium, myocardium, and epicardium observed in mice were consistent to those reported in human subjects, thus attesting the clinical relevance of STE findings in murine cardiovascular disease models.

iPSC-derived human mesenchymal stem cells improve myocardial strain of infarcted myocardium.

We investigated global and regional effects of myocardial transplantation of human induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) in infarcted myocardium. Acute myocardial infarction (MI) was induced by ligation of left coronary artery of severe combined immunodeficient mice before 2 × 10(5) iMSCs or cell-free saline were injected into peri-infarcted anterior free wall. Sham-operated animals received no injection. Global and regional myocardial function was assessed serially at 1-week and 8-week by segmental strain analysis by using two dimensional (2D) speckle tracking echocardiography. Early myocardial remodelling was observed at 1-week and persisted to 8-week with global contractility of ejection fraction and fractional area change in saline- (32.96 ± 14.23%; 21.50 ± 10.07%) and iMSC-injected (32.95 ± 10.31%; 21.00 ± 7.11%) groups significantly depressed as compared to sham control (51.17 ± 11.69%, P < 0.05; 34.86 ± 9.82%, P < 0.05). However, myocardial dilatation was observed in saline-injected animals (4.40 ± 0.62 mm, P < 0.05), but not iMSCs (4.29 ± 0.57 mm), when compared to sham control (3.74 ± 0.32 mm). Furthermore, strain analysis showed significant improved basal anterior wall strain (28.86 ± 8.16%, P < 0.05) in the iMSC group, but not saline-injected (15.81 ± 13.92%), when compared to sham control (22.18 ± 4.13%). This was corroborated by multi-segments deterioration of radial strain only in saline-injected (21.50 ± 5.31%, P < 0.05), but not iMSC (25.67 ± 12.53%), when compared to sham control (34.88 ± 5.77%). Improvements of the myocardial strain coincided with the presence of interconnecting telocytes in interstitial space of the infarcted anterior segment of the heart. Our results show that localized injection of iMSCs alleviates ventricular remodelling, sustains global and regional myocardial strain by paracrine-driven effect on neoangiogenesis and myocardial deformation/compliance via parenchymal and interstitial cell interactions in the infarcted myocardium.

Role of tumor necrosis factor-α in the pathogenesis of atrial fibrosis and development of an arrhythmogenic substrate.

BACKGROUND: Although tumor necrosis factor-α (TNF-α) levels are increased in patients with atrial fibrillation (AF), its role in the pathogenesis of AF is unclear. We investigated whether direct delivery of TNF-α could induce atrial fibrosis. METHODS AND RESULTS: TNF-α (4 µg/kg) was injected into the tail vein of 20 male Swiss albino mice (TNF group) and saline into 20 control mice (CON group). The dose was carefully chosen to avoid any significant decrease in left ventricular (LV) function. Animals were killed after 16 weeks and their atria examined for fibrosis. We found increased atrial fibrosis in the TNF group compared with the CON group [372.8±21.5 arbitrary units (a.u.) vs. 56.9±6.5 a.u., respectively, mean±SEM; P<0.0001] and decreased connexin-40 immunofluorescence [7.5±0.4 a.u vs. 40.4±1.9 a.u, respectively; P<0.0001]. Transforming growth factor-ß [TGF-ß: 95.6±1.8 a.u vs. 29.4±5.8 a.u; P<0.001], α-smooth muscle actin (α-SMA: 97.9±13.0 a.u vs. 50.1±18.5 a.u; P<0.05] and matrix metalloproteinase 2 (MMP-2)/GAPDH levels [157.3±26.4 a.u vs. 105.8±13.3 a.u; P<0.05] were also increased in the TNF group. CONCLUSIONS: TNF-α is involved in the pathogenesis of atrial fibrosis and altered connexin-40 expression in mice through the TGF-ß signaling pathway, activation of myofibroblasts and increased secretion of MMPs. Collectively, these changes may contribute to the arrhythmogenic substrate and development of AF.