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Cardiac stem cells (CSCs) transplantation has been recognized to be effective on the treatment of myocardial infarction (MI), but some techniques still need to be developed in the isolation and culture of CSCs, which is the key problem restricting the clinical application of CSCs. This study was focused on the isolation of Lin - (lineage-negative) Sca-1 + (stem cell antigen-1-positive) CSCs from newborn C57BL/6J mice (0-3 d) by mixed enzymatic-explant isolation in combination with immunomagnetic separation. The digesting time, digesting frequency, incubation temperature, stirring speed, centrifugation time and rotational speed were strictly controlled in the experiment. In order to increase the survival rate of CSCs, the medium changing time and manner were optimized in primary CSCs culture. The percentages of Sca-1 + cells in primary and passage cells were detected by flow cytometry and immunofluorescence staining. The results showed that: (1) the proportion of Lin - Sca-1 + cells within the collected cells could be as high as (85.03 ± 5.60)% after isolation and purification; (2) In vitro culture of Lin - Sca-1 + CSCs grew into spheres on the 5 th day, and over the whole bottom of the dish on the 7 th day. The growth curve showed that the cells were in logarithmic growth phase on the 3 rd day; (3) Immunofluorescence staining data showed that the expression of Sca-1, the CSCs membrane-specific marker, was decreased after subculture, and flow cytometry data showed that the percentages of Sca-1 + cells were (71.82 ± 2.63)%, (58.38 ± 3.70)% and (46.19 ± 4.72)% in passage 1 (P1), P3, and P5 CSCs, respectively. The above results suggest that high purity of Lin - Sca-1 + CSCs can be obtained by enzymolysis combined with immunomagnetic separation method. Moreover, the CSCs culture system is stable. In our experiment, the Sca-1 + CSCs isolation and culture method has been successfully established, and it is simple, stable, effective and reliable. The method can provide a stable methodological basis for the treatment of MI by Lin - Sca-1 + CSCs transplantation.

Introduction: Ischemia causes myocardial infarction and arrhythmias. Up-regulation of cardiac CLC-3 chloride channels is important for ischemic preconditioning-induced second-window protection against myocardial infarction. But its consequences in ischemia-induced electrical remodeling are still unknown., Methods: The recently-characterized heart-specific overexpression of human short CLC-3 isoform (hsCLC-3(OE)) mice was used to study the effects of CLC-3 up-regulation on cardiac electrophysiology under ischemia/reperfusion conditions. In vivo surface electrocardiography (ECG) and intracardiac electrophysiology (ICEP) were used to compare the electrophysiological properties of age-matched wild-type (Clcn3(+/+)) and hsCLC-3(OE) mice under control and myocardial ischemia-reperfusion conditions., Results: QT and QTc intervals of hsCLC-3(OE) mice were significantly shorter than those of Clcn3(+/+) mice under control, ischemia and reperfusion conditions. In the ICEP, ventricular effective refractory period (VERP) of hsCLC-3(OE) mice (26.7±1.7ms, n=6) was significantly shorter than that of Clcn3(+/+) mice (36.9±2.8ms, n=8, P<0.05). Under ischemia condition, both VERP (19.8±1.3ms) and atrial effective refractory period (AERP, 34.8±2.5ms) of hsCLC-3(OE) mice were significantly shorter than those of Clcn3(+/+) mice (35.2±3.0ms and 45.8±1.6ms, P<0.01, respectively). Wenckebach atrioventricular nodal block point (AVBP, 91.13±4.08ms) and 2:1 AVBP (71.3±3.8ms) of hsCLC-3(OE) mice were significantly shorter than those of Clcn3(+/+) mice (102.0±2.0ms and 84.1±2.8ms, P<0.05, respectively). However, no differences of ICEP parameters between hsCLC-3(OE) and Clcn3(+/+) mice were observed under reperfusion conditions., Conclusion: Heart-specific overexpression of hsCLC-3 limited the ischemia-induced QT and ERP prolongation and postponed the advancements of Wenckebach and 2:1 AVBP. CLC-3 up-regulation may serve as an important adaptive mechanism against myocardial ischemia., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Anoctamin1 (ANO1) encodes a Ca(2+)-activated chloride (Cl(-)) channel (CaCC) in variety tissues of many species. Whether ANO1 expresses and functions as a CaCC in cardiomyocytes remain unknown. The objective of this study is to characterize the molecular and functional expression of ANO1 in cardiac myocytes and the role of ANO1-encoded CaCCs in ischemia-induced arrhythmias in the heart. Quantitative real-time RT-PCR, immunofluorescence staining assays, and immunohistochemistry identified the molecular expression, location, and distribution of ANO1 in mouse ventricular myocytes (mVMs). Patch-clamp recordings combined with pharmacological analyses found that ANO1 was responsible for a Ca(2+)-activated Cl(-) current (I(Cl.Ca)) in cardiomyocytes. Myocardial ischemia led to a significant increase in the current density of I(Cl.Ca), which was inhibited by a specific ANO1 inhibitor, T16A(inh)-A01, and an antibody targeting at the pore area of ANO1. Moreover, cardiomyocytes isolated from mice with ischemia-induced arrhythmias had an accelerated early phase 1 repolarization of action potentials (APs) and a deeper "spike and dome" compared to control cardiomyocytes from non-ischemia mice. Application of the antibody targeting at ANO1 pore prevented the ischemia-induced early phase 1 repolarization acceleration and caused a much shallower "spike and dome". We conclude that ANO1 encodes CaCC and plays a significant role in the phase 1 repolarization of APs in mVMs. The ischemia-induced increase in ANO1 expression may be responsible for the increased density of I(Cl.Ca) in the ischemic heart and may contribute, at least in part, to ischemia-induced arrhythmias., (© 2014 Wiley Periodicals, Inc.)

Rationale: Tachycardia-induced atrial fibrosis is a hallmark of structural remodeling of atrial fibrillation (AF). The molecular mechanisms underlying the AF-induced atrial fibrosis remain unclear., Objective: To determine the role of angiotensin II (Ang II)/Ang II type 1 (AT(1)) receptor-coupled transforming growth factor (TGF)-β(1)/Smad signaling pathway in the AF-induced atrial fibrosis., Methods and Results: Rapid atrial pacing (1000 ppm) was applied to the left atrium of rabbit heart to induce atrial fibrillation and fibrosis. Quantitative PCR and Western blot analysis revealed that rapid atrial pacing caused a marked increase in the expression of Ang II, TGF-β(1), phosphorylated Smad2/3 (P-Smad2/3), Arkadia, and hydroxyproline synthesis. However, the expression of Smad7, a key endogenous antagonist of the TGF-β(1)/Smad-mediated fibrosis, was significantly decreased. These changes were dose-dependently reversed by AT(1) receptor antagonist losartan, implicating the involvement of AF-induced release of Ang II and activation of AT(1) receptor-specific pathway. In the adult rabbit cardiac fibroblasts, Ang II increased the expression of TGF-β(1), P-Smad2/3, Smad4, Arkadia, and collagen I synthesis and significantly reduced Smad7 expression. These effects of Ang II were reversed by losartan but not by the AT(2) antagonist (PD123319). In addition, extracellular signal-regulated kinase inhibitor and anti-TGF-β(1) antibody also blocked the Ang II-induced downregulation of Smad7. Silencing of Smad7 gene by small interfering RNA abolished the antagonism of losartan on the fibrogenic effects of Ang II on cardiac fibroblasts, whereas overexpression of Smad7 blocked Ang II-induced increase in collagen I synthesis., Conclusions: Ang II/AT(1) receptor-specific activation of Arkadia-mediated poly-ubiquitination and degradation of Smad7 may decrease the inhibitory feedback regulation of TGF-β(1)/Smad signaling and serves as a key mechanism for AF-induced atrial fibrosis.

Activation of volume regulated chloride channels (VRCCs) has been shown to be cardioprotective in ischemic preconditioning (IPC) of isolated hearts but the underlying molecular mechanisms remain unclear. Recent independent studies support that ClC-3, a ClC voltage-gated chloride channel, may function as a key component of the VRCCs. Thus, ClC-3 knockout (Clcn3(-/-)) mice and their age-matched heterozygous (Clcn3(+/-)) and wild-type (Clcn3(+/+)) littermates were used to test whether activation of VRCCs contributes to cardioprotection in early and/or second-window IPC. Targeted disruption of ClC-3 gene caused a decrease in the body weight but no changes in heart/body weight ratio. Telemetry ECG and echocardiography revealed no differences in ECG and cardiac function under resting conditions among all groups. Under treadmill stress (10 m/min for 10 min), the Clcn3(-/-) mice had significant slower heart rate (648±12 bpm) than Clcn3(+/+) littermates (737±19 bpm, n=6, P<0.05). Ex vivo IPC in the isolated working-heart preparations protected cardiac function during reperfusion and significantly decreased apoptosis and infarct size in all groups. In vivo early IPC significantly reduced infarct size in all groups including Clcn3(-/-) mice (22.7±3.7% vs control 40.1±4.3%, n=22, P=0.004). Second-window IPC significantly reduced apoptosis and infarction in Clcn3(+/+) (22.9±3.2% vs 45.7±5.4%, n=22, P<0.001) and Clcn3(+/-) mice (27.5±4.1% vs 42.2±5.7%, n=15, P<0.05) but not in Clcn3(-/-) littermates (39.8±4.9% vs 41.5±8.2%, n=13, P>0.05). Impaired cell volume regulation of the Clcn3(-/-) myocytes may contribute to the failure of cardioprotection by second-window IPC. These results strongly support that activation of VRCCs may play an important cardioprotective role in second-window IPC., (Copyright © 2011 S. Karger AG, Basel.)