Calcineurin Aβ gene knockdown inhibits transient outward potassium current ion channel remodeling in hypertrophic ventricular myocyte

Figures

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  Figure 1. PE stimulation promoted the BNP mRNA expression in ventricular myocytes which was attenuated by pre-treatment with Ad-CnAβshRNA1. Bar chart showed the quantitative analysis results of BNP mRNA by real-time reverse transcription-polymerase chain reaction. *P < 0.05; n = 3. PE: Phenylephrine. A1: Ad-CnAβshRNA.

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  Figure 2. PE stimulation promoted cell hypertrophy which was attenuated by pre-treatment with Ad-CnAβshRNA1. Color picture showed the ventricular cells stained with crystal violet in four groups. a, b, c, and d are the null, null + PE, A1 + PE, and A1 groups, respectively (×100). The histogram showed the comparative results of cell area in groups. *P < 0.05, **P < 0.01. PE: Phenylephrine. A1: Ad-CnAβshRNA.

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  Figure 3. PE stimulation increased CnAβ protein expression which was attenuated by pre-treatment with Ad-CnAβshRNA1. (a) The representative picture of CnAβ protein detected by Western blotting. (b) The semiquantitative analysis results of CnAβ protein in groups. **P < 0.01, n = 3. CnAβ: Calcineurin Aβ. PE: Phenylephrine. A1: Ad-CnAβshRNA.

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  Figure 4. PE intervention inhibited the protein and mRNA expression of Kv4.2 which was attenuated by pre-treatment with Ad-CnAβshRNA1. (a) The representative picture of Kv4.2 protein detected by western blotting. (b and c) The semiquantitative analysis results of Kv4.2 protein (n = 4) and mRNA (n = 5) in groups, respectively. *P < 0.05, **P < 0.01. PE: Phenylephrine. A1: Ad-CnAβshRNA.

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  Figure 5. Densities of transient outward potassium current (I to) in ventricular myocytes. (a) A typical I to recording from isolated ventricular myocytes in groups. (b) Current–voltage (I–V) curve relations. At a stimulation voltage of +20 to +70 mV, PE intervention significantly decreased I to density (P < 0.05), which was markedly attenuated by pre-treatment with Ad-CnAβshRNA1 (P < 0.05; the cell numbers are 14, 12, 12, and 12 in null, null + PE, A1 + PE and A1 groups, respectively). PE: Phenylephrine. A1: Ad-CnAβshRNA.

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  Figure 6. The activation and inactivation curves of transient outward potassium current (I to). (a) The steady-state activation curve of I to. (b) The semi-activated voltage (V 1/2,act) of I to. There were no significant differences of V 1/2, act among groups (P > 0.05; the cell numbers are 14, 12, 12, and 9 in null, null + PE, A1 + PE, and A1 groups, respectively). (c) The steady-state inactivation curve of I to. (d) The semi-inactivated voltage (V 1/2,inact) of I to. PE stimulation accelerated inactivation of I to, which was inhibited by Ad-CnAβshRNA1 intervention (*P < 0.05; n = 10 in each group). PE: Phenylephrine. A1: Ad-CnAβshRNA.

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  Figure 7. Action potentials of ventricular myocytes. (a) Representative action potentials of cultured ventricular myocytes. (b–d) The analysis results of the APD20, APD50, and APD90 in groups. PE stimulation prolonged the APD, which was inhibited by Ad-CnAβshRNA1 intervention. **P < 0.001, *P < 0.05; the cell numbers are 12, 12, 12, and 11 in null, null + PE, A1 + PE, and A1 groups, respectively. PE: Phenylephrine. A1: Ad-CnAβshRNA.

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  Figure A. Identification of ventricular myocytes. After 48 h of culture, myocardial cells were demonstrated by immunofluorescent staining with α striated muscle sarcomere actin (α-SCA) antibody that is specialized to striated muscle. (a) Cells appearing green were identified as myocardial cells. (c) Staining for fibroblasts was negative. (b and d) The blue was nuclei stained with diamidine phenylindole. Panels (a) and (b), (c), and (d) are the same view, respectively (×200).

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  Figure A2. The results of Ad-CnAβshRNA sequence screening. A1 caused the most obvious decrease in the A subunit β subtype of calcineurin (CnAβ) protein expression after its infection of ventricular myocytes for 48 h. (a) Cells appearing green were identified as cells infected by recombinant adenovirus. (b) The photo taken in bright background at the same view of (a). (c) The semiquantitative analysis results of CnAβ protein. *P < 0.05, **P < 0.01; n = 3. (d) The representative picture of CnAβ protein detected by western blotting. A1: Ad-CnAβshRNA1. A2: Ad-CnAβshRNA2. A3: Ad-CnAβshRNA3.