An optimized Langendorff-free isolating method and electrophysiology studies for adult mouse atrioventricular node cells

Figures

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  Fig.1. Animal surgical procedure. A Chest cavity opened, exposing the heart; B Insert a gavage needle into the aorta and ligate it. C Block the gavage needle opening with gauze. D Preheat the three liquids in advance. E Injection of EDTA buffer into the left ventricle. F Injection of perfusion buffer into the left ventricle. G Injection of collagenase buffer r into the left ventricle. H Digestive endpoint. Animal surgical procedure of the isolation of adult mouse AVN cells

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  Fig. 2. Procedure after complete digestion of the heart

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  Fig. 3. Immunofluorescent imaging confirmed the presence of HCN4-positive cells. A Representative image of isolated adult mouse atrioventricular node cells. B AVN cell was demonstrated by the immunofluorescent staining of HCN4 (red) and anti-nucleic acid stain (blue). A significantly higher fluorescence intensity of HCN4 was observed in AVN cells (smaller cells within the solid frame) compared to ventricular or atrial cells (larger, rod-shaped cells indicated by the arrow). C Single HCN4-positive AVN cell. D Western blot images of HCN4 expression in AVN cells compared to ventricular cells in the same mice (n = 4mice)

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  Fig. 4. Morphological differences between AVN, atrial and ventricular cells. A Representative image of mouse AVN cell. B Representative image of mouse atrial cell. C Representative image of mouse ventricular cell. Scale bars = 200 μm

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  Fig. 5. Isolated AVN cells display normal APs and If current. APs and If current were measured in freshly isolated mouse atrioventricular node cell. A Representative spontaneous action potential raw traces recorded from a single atrioventricular node cell. Scale bars = 500 ms. Figure 5 summarizing AVN action potential parameters recorded from WT mice(n = 11). Rate, beating rate; MDP, maximum diastolic potential; Eth, action potential threshold; DDR, the rate of diastolic depolarization; APA, action potential amplitude; APD50, action potential duration at 50% of the amplitude; APD70 action potential duration at 70% of the amplitude; APD90, action potential duration at 90% of the amplitude; RT, time of repolarization. B Representative example of If from AVN cell isolated from mice. The upper shows the voltage-clamp protocol used. C If IV relationships recorded from WT mouse AVN cells. Representative APs and If current obtained from isolated AVN myocytes

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  Fig. 6. Isolated AVN cells display normal calcium current and sodium current. A Representative T-ca raw trace recorded from a single AVN cell. The upper shows the voltage-clamp protocol used. B T-ca IV relationships recorded from WT mouse AVN cells. C Representative L-ca raw trace recorded from a single AVN cell. The upper shows the voltage-clamp protocol used. D L-ca IV relationships recorded from WT mouse AVN cells. E Representative I-Na raw trace recorded from a single AVN cell. The upper shows the voltage-clamp protocol used. F, I-Na IV relationships recorded from WT mouse AVN cells. Representative Ca2 + and Na + current obtained from isolated AVN cells

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  Fig. 7. Isolated AVN cells display normal calcium transient and sarcomere length. A Representative Ca2+ transient raw trace recorded from a single AVN cell. B Representative raw sarcomere length trace recorded from a single AVN cell. Figure 7 Summarizing AVN Ca2+ parameters recorded from WT mice. Dep v, the maximal contraction rate; Dep vt, the time to maximal contraction rate; Time to peak, the time to contraction peak; Ret V, the maximal rate of sarcomere relaxation; Tau, relaxation time constant (n = 10 cells/group). Figure 7 Summarizing sarcomere length parameters recorded from WT mice. Dep v, the maximal contraction rate; Dep vt, the time to maximal contraction rate; Time to peak, the time to contraction peak; Ret V, the maximal rate of sarcomere relaxation; Tau, relaxation time constant. (n = 10cells/group). Representative Ca2 + transient and sarcomere length parameters obtained from isolated AVN myocytes