Margaret A. Craig

Derivation of Endothelial Cells From Human Embryonic Stem Cells by Directed Differentiation: Analysis of MicroRNA and Angiogenesis In Vitro and In Vivo

Objective—To develop an embryoid body-free directed differentiation protocol for the rapid generation of functional vascular endothelial cells derived from human embryonic stem cells (hESCs) and to assess the system for microRNA regulation and angiogenesis. Methods and Results—The production of defined cell lineages from hESCs is a critical requirement for evaluating their potential in regenerative medicine. We developed a feeder- and serum-free protocol. Directed endothelial differentiation of hESCs revealed rapid loss of pluripotency markers and progressive induction of mRNA and protein expression of vascular markers (including CD31 and vascular endothelial [VE]-cadherin) and angiogenic growth factors (including vascular endothelial growth factor), increased expression of angiogenesis-associated microRNAs (including miR-126 and miR-210), and induction of endothelial cell morphological features. In vitro, differentiated cells produced nitric oxide, migrated across a wound, and formed tubular structures in both the absence and the presence of 3D matrices (Matrigel). In vivo, we showed that cells that differentiated for 10 days before implantation were efficient at the induction of therapeutic neovascularization and that hESC-derived cells were incorporated into the blood-perfused vasculature of recipient mice. Conclusion—The directed differentiation of hESCs is efficient and effective for the differentiation of functional endothelial cells from hESCs.

cGMP signals modulate cAMP levels in a compartment-specific manner to regulate catecholamine-dependent signaling in cardiac myocytes.

Rationale: cAMP and cGMP are intracellular second messengers involved in heart pathophysiology. cGMP can potentially affect cAMP signals via cGMP-regulated phosphodiesterases (PDEs). Objective: To study the effect of cGMP signals on the local cAMP response to catecholamines in specific subcellular compartments. Methods and Results: We used real-time FRET imaging of living rat ventriculocytes expressing targeted cAMP and cGMP biosensors to detect cyclic nucleotides levels in specific locales. We found that the compartmentalized, but not the global, cAMP response to isoproterenol is profoundly affected by cGMP signals. The effect of cGMP is to increase cAMP levels in the compartment where the protein kinase (PK)A-RI isoforms reside but to decrease cAMP in the compartment where the PKA-RII isoforms reside. These opposing effects are determined by the cGMP-regulated PDEs, namely PDE2 and PDE3, with the local activity of these PDEs being critically important. The cGMP-mediated modulation of cAMP also affects the phosphorylation of PKA targets and myocyte contractility. Conclusions: cGMP signals exert opposing effects on local cAMP levels via different PDEs the activity of which is exerted in spatially distinct subcellular domains. Inhibition of PDE2 selectively abolishes the negative effects of cGMP on cAMP and may have therapeutic potential.

Onset of Experimental Severe Cardiac Fibrosis Is Mediated by Overexpression of Angiotensin-Converting Enzyme 2

Angiotensin-converting enzyme (ACE) 2 is a recently identified homologue of ACE. There is great interest in the therapeutic benefit for ACE2 overexpression in the heart. However, the role of ACE2 in the regulation of cardiac structure and function, as well as maintenance of systemic blood pressure, remains poorly understood. In cell culture, ACE2 overexpression led to markedly increased myocyte volume, assessed in primary rabbit myocytes. To assess ACE2 function in vivo, we used a recombinant adeno-associated virus 6 delivery system to provide 11-week overexpression of ACE2 in the myocardium of stroke-prone spontaneously hypertensive rats. ACE2, as well as the ACE inhibitor enalapril, significantly reduced systolic blood pressure. However, in the heart, ACE2 overexpression resulted in cardiac fibrosis, as assessed by histological analysis with concomitant deficits in ejection fraction and fractional shortening measured by echocardiography. Furthermore, global gene expression profiling demonstrated the activation of profibrotic pathways in the heart mediated by ACE2 gene delivery. This study demonstrates that sustained overexpression of ACE2 in the heart in vivo leads to the onset of severe fibrosis.

Acidosis slows electrical conduction through the atrio-ventricular node.

Acidosis affects the mechanical and electrical activity of mammalian hearts but comparatively little is known about its effects on the function of the atrio-ventricular node (AVN). In this study, the electrical activity of the epicardial surface of the left ventricle of isolated Langendorff-perfused rabbit hearts was examined using optical methods. Perfusion with hypercapnic Tyrodes solution (20% CO2, pH 6.7) increased the time of earliest activation (Tact) from 100.5 ± 7.9 to 166.1 ± 7.2 ms (n = 8) at a pacing cycle length (PCL) of 300 ms (37°C). Tact increased at shorter PCL, and the hypercapnic solution prolonged Tact further: at 150 ms PCL, Tact was prolonged from 131.0 ± 5.2 to 174.9 ± 16.3 ms. 2:1 AVN block was common at shorter cycle lengths. Atrial and ventricular conduction times were not significantly affected by the hypercapnic solution suggesting that the increased delay originated in the AVN. Isolated right atrial preparations were superfused with Tyrodes solutions at pH 7.4 (control), 6.8 and 6.3. Low pH prolonged the atrial-Hisian (AH) interval, the AVN effective and functional refractory periods and Wenckebach cycle length significantly. Complete AVN block occurred in 6 out of 9 preparations. Optical imaging of conduction at the AV junction revealed increased conduction delay in the region of the AVN, with less marked effects in atrial and ventricular tissue. Thus acidosis can dramatically prolong the AVN delay, and in combination with short cycle lengths, this can cause partial or complete AVN block and is therefore implicated in the development of brady-arrhythmias in conditions of local or systemic acidosis.

Dysregulation of cadherins in the intercalated disc of the spontaneously hypertensive stroke-prone rat

The structural integrity of cardiac cells is maintained by the Ca2+-dependent homophilic cell–cell adhesion of cadherins. N-cadherin is responsible for this adhesion under normal physiological conditions. The role of cadherins in adverse cardiac pathology is less clear. We studied the hearts of the stroke-prone spontaneously hypertensive (SHRSP) rat as a genetic model of cardiac hypertrophy and compared them to Wistar–Kyoto control animals. Western blotting of protein homogenates from 12-week old SHRSP animals indicated that similar levels of β, γ-, and α-catenin and T, N and R-cadherin were expressed in the control and SHRSP animals. However, dramatically higher levels of E-cadherin were detected in SHRSP animals compared to controls at 6, 12 and 18 weeks of age. This was confirmed by quantitative Taqman PCR and immunohistochemistry. E-cadherin was located at the intercalated disc of the myocytes in co-localisation with connexin 43. Adenoviral overexpression of E-cadherin in rat H9c2 cells and primary rabbit myocytes resulted in a significant reduction in myocyte cell diameter and breadth. E-cadherin overexpression resulted in re-localisation of β-catenin to the cell surface particularly to cell–cell junctions. Subsequent immunohistochemistry of the hearts of WKY and SHRSP animals also revealed increased levels of β-catenin in the intercalated disc in the SHRSP compared to WKY. Therefore, remodelling of the intercalated disc in the hearts of SHRSP animals may contribute to the altered function observed in these animals.

Electrophysiological characterization of iCell2 hiPSC derived cardiomyocytes: the new generation of CDI hiPSC-CMs

Abstract Most of the efforts in cardiac safety pharmacology are orientated towards the improvement of early stage drug screening. In this regard, the creation of in vitro human ventricular electrophysiology has been accelerated by the commercial availability hiPSC-CMs. Many studies have validated iCell cardiomycytes (Cellular Dynamics Inc.) as a robust model to carry out predictive cardiotoxicity, but the requirement of at least 10 days in vitro (DIV), increases the handling and associated risks. The electrophysiological characterization of 2nd generation of this cell type (iCell2 cardiomyocytes) with accelerated maturation processes that allows their use after 4-7 DIV is presented. Cellular electrophysiology was examined using CellOPTIQTM (Clyde Biosicences Ltd.). The cells are cultured in 96-well glass bottom plates. The electrical activity is recorded on cell monolayers loaded with di-4ANEPPS (transient incubation in serum-free media). Baseline electrical recordings are registered after 7 DIV using the CellOPTIQTM system (10kHz, 15s). A dosedependent APD shortening was evident in iCell2 following treatment with the L-type Ca2+ channel blocker nifedipine. A dose-dependent prolongation of APD is shown upon E4031 hERG blockade, with EADs evident following treatment with the highest concentration of E4031 (0.1μM). In summary, the electrophysiological characteristics of iCell2 cell line are easily recorded using the CellOPTIQTM system and are suitable for use in cardiotoxicology screening. Methodology iCell Cardiomyocytes were purchased from Cellular Dynamics International (USA) and were seeded in a fibronectin coated 96 well plate (50,000 cells/well) and cultured for 7 days. Following maturation, the cells were transferred to serum free media and transiently exposed to voltage sensitive dye (Di-4-ANEPPS). Measurements were obtained before and after drug treatment by exciting the cells with a 470nm LED. The emitted fluorescence was recorded at 10KHz from regions of iCell2 cardiomyocytes for periods up to 15s on the CellOPTIQTM electrophysiology platform (Clyde Biosciences Ltd). The records were subsequently analysed off-line using proprietary software (Clyde Biosciences Ltd). A Dunnett’s test was completed to test for statistical significance of all data (*p<0.05; **p<0.01; ***p<0.001).