Opto-electronic feedback control of membrane potential for real-time control of action potentials.

To unlock new research possibilities by acquiring control of action potential (AP) morphologies in excitable cells, we developed an opto-electronic feedback loop-based system integrating cellular electrophysiology, real-time computing, and optogenetic approaches and applied it to monolayers of heart muscle cells. This allowed accurate restoration and preservation of cardiac AP morphologies in the presence of electrical perturbations of different origin in an unsupervised, self-regulatory manner, without any prior knowledge of the disturbance. Moreover, arbitrary AP waveforms could be enforced onto these cells. Collectively, these results set the stage for the refinement and application of opto-electronic control systems to enable in-depth investigation into the regulatory role of membrane potential in health and disease.

Effects on cardiac function, remodeling and inflammation following myocardial ischemia-reperfusion injury or unreperfused myocardial infarction in hypercholesterolemic APOE*3-Leiden mice.

Many novel therapies to treat myocardial infarction (MI), yielding promising results in animal models, nowadays failed in clinical trials for several reasons. The most used animal MI model is based on permanent ligation of the left anterior descending (LAD) coronary artery in healthy mice resulting in transmural MI, while in clinical practice reperfusion is usually accomplished by primary percutaneous coronary interventions (PCI) limiting myocardial damage and inducing myocardial ischemia-reperfusion (MI-R) injury. To evaluate a more similar murine MI model we compared MI-R injury to unreperfused MI in hypercholesterolemic apolipoprotein (APO)E*3-Leiden mice regarding effects on cardiac function, left ventricular (LV) remodeling and inflammation. Both MI-R and MI resulted in significant LV dilation and impaired cardiac function after 3 weeks. Although LV dilation, displayed by end-diastolic (EDV) and end-systolic volumes (ESV), and infarct size (IS) were restricted following MI-R compared to MI (respectively by 27.6% for EDV, 39.5% ESV, 36.0% IS), cardiac function was not preserved. LV-wall thinning was limited with non-transmural LV fibrosis in the MI-R group (66.7%). Two days after inducing myocardial ischemia, local leucocyte infiltration in the infarct area was decreased following MI-R compared to MI (36.6%), whereas systemic circulating monocytes were increased in both groups compared to sham (130.0% following MI-R and 120.0% after MI). Both MI-R and MI models against the background of a hypercholesterolemic phenotype appear validated experimental models, however reduced infarct size, restricted LV remodeling as well as a different distributed inflammatory response following MI-R resemble the contemporary clinical outcome regarding primary PCI more accurately which potentially provides better predictive value of experimental therapies in successive clinical trials.

Allogenic stem cell therapy improves right ventricular function by improving lung pathology in rats with pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a chronic lung disease that leads to right ventricular (RV) hypertrophy (RVH), remodeling, and failure. We tested treatment with bone marrow-derived mesenchymal stem cells (MSCs) obtained from donor rats with monocrotaline (MCT)-induced PAH to recipient rats with MCT-induced PAH on pulmonary artery pressure, lung pathology, and RV function. This model was chosen to mimic autologous MSC therapy. On day 1, PAH was induced by MCT (60 mg/kg) in 20 female Wistar rats. On day 14, rats were treated with 10(6) MSCs intravenously (MCT + MSC) or with saline (MCT60). MSCs were obtained from donor rats with PAH at 28 days after MCT. A control group received saline on days 1 and 14. On day 28, the RV function of recipient rats was assessed, followed by isolation of the lungs and heart. RVH was quantified by the weight ratio of the RV/(left ventricle + interventricular septum). MCT induced an increase of RV peak pressure (from 27 + or - 5 to 42 +/- 17 mmHg) and RVH (from 0.25 + or - 0.04 to 0.47 + or - 0.12), depressed the RV ejection fraction (from 56 + or - 11 to 43 + or - 6%), and increased lung weight (from 0.96 + or - 0.15 to 1.66 + or - 0.32 g), including thickening of the arteriolar walls and alveolar septa. MSC treatment attenuated PAH (31 + or - 4 mmHg) and RVH (0.32 + or - 0.07), normalized the RV ejection fraction (52 + or - 5%), reduced lung weight (1.16 + or - 0.24 g), and inhibited the thickening of the arterioles and alveolar septa. We conclude that the application of MSCs from donor rats with PAH reduces RV pressure overload, RV dysfunction, and lung pathology in recipient rats with PAH. These results suggest that autologous MSC therapy may alleviate cardiac and pulmonary symptoms in PAH patients.

Primary cilia sensitize endothelial cells for fluid shear stress.

Primary cilia are mechanosensors for fluid shear stress, and are involved in a number of syndromes and congenital anomalies. We identified endothelial cilia in areas of low shear stress in the embryonic heart. The objective of the present study was to demonstrate the role of primary cilia in mechanosensing. Ciliated embryonic endothelial cells were cultured from the heart, and non-ciliated cells from the arteries. Non-ciliated cells that were subjected to fluid shear stress showed significantly less induction of the shear marker Krüppel-Like Factor-2, as compared to ciliated cells. In addition, ciliated cells from which the cilia were chemically removed show a similar decrease in flow response. This shows that primary cilia sensitize endothelial cells for fluid shear stress. In addition, we targeted and stabilized the connection of the cilium to the cytoplasm by treatment with Colchicine and Taxol/Paclitaxel, respectively, and show that microtubular integrity is essential to sense shear stress.

Modulation of calcification of vascular smooth muscle cells in culture by calcium antagonists, statins, and their combination.

BACKGROUND: Vascular calcification is an organized process in which vascular smooth muscle cells (VSMCs) are implicated primarily. The purpose of the present study was to assess the effects of calcium antagonists and statins on VSMC calcification in vitro. METHODS: VSMC calcification was stimulated by incubation in growth medium supplemented with 10 mmol/l beta-glycerophosphate, 8 mmol/l CaCl(2), 10 mmol/l sodium pyruvate, 1 micromol/l insulin, 50 microg/ml ascorbic acid, and 100 nmol/l dexamethasone (calcification medium). Calcification, proliferation, and apoptosis of VSMCs were quantified. RESULTS: Calcium deposition was stimulated dose-dependently by beta-glycerophosphate, CaCl(2), and ascorbic acid (all P < 0.01). Addition of amlodipine (0.01-1 micromol/l) to the calcification medium did not affect VSMC calcification. However, atorvastatin (2-50 micromol/l) stimulated calcium deposition dose-dependently. Combining treatments stimulated calcification to a degree similar to that observed with atorvastatin alone. Both atorvastatin and amlodipine inhibited VSMC proliferation at the highest concentration used. Only atorvastatin (50 micromol/l) induced considerable apoptosis of VSMCs. CONCLUSION: In vitro calcification of VSMCs is not affected by amlodipine, but is stimulated by atorvastatin at concentrations > or =10 micromol/l, which could contribute to the plaque-stabilizing effect reported for statins.