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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.

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 Kruppel-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. Developmental Dynamics 237:725–735, 2008. © 2008 Wiley-Liss, Inc.

Pulmonary hypertension induces right ventricular (RV) overload, which is transmitted to cardiomyocytes via integrins that activate intracellular messengers, including focal adhesion kinase (FAK) and neuronal nitric oxide synthase (NOS1). We investigated whether RV hypertrophy (RVH) and RV failure (RVF) were associated with activation of FAK, NOS1, and matrix metalloproteinases (MMPs). Rats were treated without (RVC) or with a low dose of monocrotaline (30mg/kg) to induce RVH, and with a high dose (80mg/kg) to induce RVF. After approximately 30 days, RV function was determined using a combined pressure-conductance catheter. After sacrifice, FAK, NOS1, their phosphorylated forms (FAK-P and NOS1-P), MMP-2, and MMP-9 were quantified in RV myocardium by immunohistochemistry. In RVH and RVF, RV weight/ body weight increased by 36% and 109%, whereas RV ejection fraction decreased by 23% and 57% compared to RVC, respectively. FAK-P and FAK-P/FAK were highest in RVH (2.87+/-0.12 and 2.52+/-0.23 fold compared to RVC, respectively) and slightly elevated in RVF (1.76+/-0.17 and 1.15+/-0.13 fold compared to RVC, respectively). NOS1-P and NOS1-P/NOS1 were increased in RVH (1.63+/-0.12 and 3.06+/-0.80 fold compared to RVC, respectively) and RVF (2.16+/-0.03 and 3.30+/-0.38 fold compared to RVC, respectively). MMP-2 was highest in RVH and intermediate in RVF (3.50+/-0.12 and 1.84+/-0.22 fold compared to RVC, respectively). MMP-9 was elevated in RVH and RVF (2.39+/-0.35 and 2.92+/-0.68 fold compared to RVC, respectively). Activation of FAK in RVH points to an integrin-dependent hypertrophic response of the myocardium. Activation of NOS1 in failing RV suggests a role of excessive NO in the development of failure and activation of MMPs leading to ventricular remodeling.

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 β-glycerophosphate, 8 mmol/l CaCl2, 10 mmol/l sodium pyruvate, 1 μmol/l insulin, 50 μg/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 β-glycerophosphate, CaCl2, and ascorbic acid (all P

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.

Purpose We characterized the release kinetics of cardiac troponin I and T in relation to lactate dehydrogenase (LDH) from cardiomyocytes before and after the transition from reversible to irreversible cell damage. Methods Cardiomyocytes were exposed to mild metabolic inhibition (1 mmol/L sodium azide) to induce a necrotic cell death process that is characterized by a reversible (0–12 h) and irreversible phase (12–30 h). At various time intervals cells and media were collected and analyzed for LDH activity, intact cTnI and cTnT, and their degradation products. Results During the first 12 h of metabolic inhibition, cell viability was unchanged with no release of intact cTnI and cTnT nor their degradation products. Between 12 and 30 h of azide treatment, cardiomyocytes showed progressive cell death accompanied by release of intact cTnI (29 kDa), intact cTnT (39 kDa), four cTnI degradation products of 26, 20, 17 and 12 kDa, and three cTnT degradation products of 37, 27 and 14 kDa. Possibly due to degradation, there is progressive loss of cTnI and cTnT protein that is obviously undetected by the antibodies used. Conclusions Metabolic inhibition of cardiomyocytes induces a parallel release of intact cTnI and cTnT and their degradation products, starting only after onset of irreversible cardiomyocyte damage.

Elevated cardiac troponin-I (cTnI) levels have been demonstrated in serum of patients without acute coronary syndromes, potentially via a stretch-related process. We hypothesize that this cTnI release from viable cardiomyocytes is mediated by stimulation of stretch-responsive integrins. Cultured cardiomyocytes were treated with (1) Gly-Arg-Gly-Asp-Ser (GRGDS, n = 22) to stimulate integrins, (2) Ser-Asp-Gly-Arg-Gly (SDGRG, n = 8) that does not stimulate integrins, or (3) phosphate-buffered saline (control, n = 38). Cells and media were analyzed for intact cTnI, cTnI degradation products, and matrix metalloproteinase (MMP)-2. Cell viability was examined by assay of lactate dehydrogenase (LDH) activity and by nuclear staining with propidium iodide. GRGDS-induced integrin stimulation caused increased release of intact cTnI (9.6 +/- 3.0%) as compared to SDGRG-treated cardiomyocytes (4.5 +/- 0.8%, p0.001) and control (3.0 +/- 3.4%, p0.001). LDH release from GRGDS-treated cardiomyocytes (15.9 +/- 3.8%) equalled that from controls (15.2 +/- 2.3%, p = n.s.), indicating that the GRGDS-induced release of cTnI is not due to cell necrosis. This result was confirmed by nuclear staining with propidium iodide. Integrin stimulation increased the intracellular and extracellular MMP2 activity as compared to controls (both p0.05). However, despite the ability of active MMP2 to degrade cTnI in vitro, integrin stimulation in cardiomyocytes was not associated with cTnI degradation. The present study demonstrates that intact cTnI can be released from viable cardiomyocytes by stimulation of stretch-responsive integrins.