CCRR regulate MYZAP-PKP2-Nav1.5 signaling pathway in atrial fibrillation following myocardial infarction

Figures

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  Figure 1. The expression of CCRR and MYZAP in atrial tissue decreased after MI (A) Flow chart of CCRR transgenic mouse construction. (B) Tissue microarray results in atria of CCRR transgenic mice. (C) Scatterplot of gene changes in CCRR transgenic mice. Compared with WT group, 2395 genes were upregulated, 785 genes were downregulated, and 9789 genes had no significant changes. (D) Pathway analysis revealed changes in 57 genes significantly associated with focal adhesion. (E and F) Compared with WT mice, the expression of MYZAP and Nav1.5 in the atria of cardiac-specific transgenic CCRR overexpression mice was significantly increased (n = 3 mice/group). (G) Flow chart of mouse MI model. (H) CCRR levels were determined by real-time PCR after MI 12 h (n = 8 mice/group). (I and J) Real-time PCR and western blot analyses for the mRNA levels (left panel, n = 9 mice/group) and protein levels (right panel, n = 6 mice/group) of MYZAP, respectively, in the myocardium 12 h after MI surgery in mice 4 weeks after injection of AAV9-CCRR virus. (K–N) Atrial cardiomyocytes were isolated from adult mice at 12 h after MI, and immunofluorescence showed decreased expression of MYZAP and PKP2 (K, Sham n = 15,…

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  Figure 2. CCRR reduces the incidence and duration of AF, and improves atrial conduction in mice with MI (A) Experimental flow chart. AAV-CCRR was injected via tail vein for 4 weeks, acute myocardial infarction model was established for 12 h, and related indexes were detected. (B) Representative diagram of AF at 12 h after MI in mice detected by programmed electrical stimulation. (C–F) The incidence and duration of AF were significantly increased in mice 12 h after MI, which was significantly improved after overexpression of CCRR (Sham n = 13, MI n = 15, +AAV-CCRR n = 12, +AAV-NC n = 10 mice/group). +AAV-CCRR: MI+AAV-CCRR, +AAV-NC: MI+AAV-NC. (G) Optical mapping was used to detect atrial conduction. (H) Atrial conduction velocity was decreased after MI, which was restored after AAV-CCRR. (I) Representative trace of action potential duration. (J–M) APD (J, APD50; K, APD70; L, APD80; M, APD90) in atrial cardiomyocytes was significantly prolonged after MI compared with Sham group. Compared with +AAV-NC group, AAV-CCRR could shorten APD duration (Sham n = 7, MI n = 6, +AAV-CCRR n = 4, +AAV-NC n = 6 mice/group). Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. #p <…

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  Figure 3. CCRR regulates the expression of MYZAP, PKP2 and Nav1.5 (A and B) FISH and immunofluorescence staining were employed to examine co-localization of CCRR with MYZAP. CCRR was stained red, MYZAP was stained green, and nucleus were stained blue (A, n = 30, B, n = 28 visions). A, from mouse atrial tissue. B, from primary cultured atrial cardiomyocytes. Scale bar: 10 μm. (C and D) RIP experiment detected the interaction between CCRR and MYZAP (n = 3 mice/group). (E) CCRR-FD is constructed, the interaction between CCRR-Functional domain and MYZAP is predicted via the website (http://hdock.phys.hust.edu.cn/), and the interaction site is predicted using Pymol. (F) Real-time PCR was used to detect the expression of CCRR in atrial cardiomyocytes (n = 6 mice/group). (G) The expression of MYZAP in atrial tissue was detected by western blot. The expression of MYZAP in MI group was significantly lower than that in Sham group. MYZAP expression was significantly upregulated by AAV-CCRR compared with AAV-NC group (n = 6 mice/group). (H) Real-time PCR results showed that MYZAP expression was decreased, which were reversed by overexpression of CCRR (n = 5 mice/group). (I) Western blot was used to…

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  Figure 4. The aberrant expression of PKP2 and Nav1.5 were induced by the deletion of MYZAP (A) Bioanalysis predicts that MYZAP interacts with PKP2. (B) MYZAP overexpression plasmid was transfected into atrial cardiomyocytes, and the expression efficiency of the plasmid was verified by Western blot (n = 6/group). +MYZAP: Hypoxia+MYZAP, +NC: Hypoxia+NC. (C) Compared with Ctl group, the expression of PKP2 protein in atrial cardiomyocytes was decreased after hypoxia for 12 h. Compared with NC group, the protein expression level of PKP2 was significantly increased by MYZAP overexpression after hypoxia (n = 6/group). (D) Compared with Ctl group, the expression of Nav1.5 protein in atrial cardiomyocytes was decreased after hypoxia for 12 h. Compared with +NC group, the protein expression level of PKP2 was significantly increased in the +MYZAP group (n = 6/group). (E) Representative sodium current in different groups. (F) Sodium current density was detected by patch-clamp. Compared with Ctl group, the sodium current density of atrial cardiomyocytes was significantly decreased after 12 h hypoxia. Compared with +NC group, the density of sodium channel was significantly increased in +MYZAP group…

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  Figure 5. Overexpression of PKP2 improves cardiac function and reduces the incidence of AF (A) Experimental flow chart. AAV-PKP2 virus was injected via tail vein for 4 weeks, MI model was established for 12 h, and related indexes were detected. (B–D) Representative echocardiographic images and statistical data of EF (%), FS (%). NC: the empty AAV9 vector as a negative control for PKP2 overexpression (Sham, MI n = 8, +AAV-PKP2 n = 6, +AAV-NC n = 5 mice/group). +AAV-PKP2: MI+AAV-PKP2, +NC: MI+AAV-NC. (E) LVIDd was detected by echocardiography. (F) LVIDs was detected by echocardiography. (G) Representative AF at 12 h after MI in mice detected by programmed electrical stimulation. (H–K) Compared with Sham group, the incidence and duration of AF in mice were significantly increased after MI 12 h, which was significantly improved after injection of +AAV-PKP2 (Sham, MI n = 7 mice/group, +AAV-PKP2, +AAV-NC n = 6 mice/group). Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. #p < 0.05, and ###p < 0.001. &&p < 0.01, and &&&p < 0.001 (One-way ANOVA).

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  Figure 6. PKP2 is an upstream regulator of TLR2/TLR4/Nav1.5 (A–C) The protein expressions of TLR2, TLR4 and Nav1.5 were detected by western blot. Compared with Sham group, the expressions of TLR2 and TLR4 were upregulated and the expression of Nav1.5 was downregulated in MI group. Compared with AAV-NC group, overexpression of AAV-PKP2 decreased the protein expression of TLR2 and TLR4, and restored the protein expression of Nav1.5 (A n = 7 mice/group, B n = 6 mice/group, C n = 5 mice/group). (D and F) The expression level of TLR2 in atrial tissue of mice overexpressing AAV-PKP2 was detected by immunofluorescence. (Sham, MI n = 21, +AAV-PKP2 n = 13; +AAV-NC n = 19 visions). Red represents TLR2, green represents α-actinin, and blue represents the nucleus. Scale bar = 10 μm. (E and G) The expression level of TLR4 in atrial tissue of mice overexpressing AAV-PKP2 was detected by immunofluorescence. (Sham n = 41, MI n = 21, +AAV-PKP2 n = 37, +AAV-NC n = 41 visions). Red represents TLR4, green represents α-actinin, and blue represents the nucleus. Scale bar = 10 μm. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. ##p < 0.01, and ###p < 0.001. &&p < 0.01 and &&&p < 0.001…

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  Figure 7. Knockdown of PKP2 enhances inflammation after MI via TLR2/TLR4/Nav1.5 pathway (A and B) Western blot (A) and Real-time PCR (B) were used to detect the changes of protein and mRNA expression of PKP2 in atrial cardiomyocytes after transfection of si-PKP2 sequence (A n = 6/group, B n = 8/group). (C–F) The expression of TLR2 and TLR4 protein (C and E) and mRNA (D and F) increased after PKP2 knockdown (C and E n = 6/group, D and F n = 8/group). (G–J) The mRNA expression levels of IL-6, IL-1β, TNF-α and MCP1 increased after PKP2 knockdown in atrial cardiomyocytes (n = 8/group). (K) CCK8 detected a significant decrease in cell viability after PKP2 knockdown (n = 6/group). (L and M) Patch-clamp results showed that the loss of PKP2 resulted in a significant decrease in sodium current density (∗∗p < 0.01 vs. Ctl, ##p < 0.01 vs. NC, n = 6/group). (N and O) Voltage-dependent steady-state activation (N) and inactivation (O) of Na+ channel were not changed by PKP2 knockdown. (N, n = 6/group, O, n = 8/group). Data are presented as mean ± SEM. ∗∗p < 0.01, and ∗∗∗p < 0.001. #p < 0.05, ##p < 0.01, and ###p < 0.001 (One-way ANOVA).

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  Figure 8. PKP2 alleviates inflammatory response after MI by regulating the TLR2/TLR4/Nav1.5 pathway (A and B) Changes of TLR2 protein and mRNA expression after PKP2 overexpression in atrial myocytes (A n = 5/group; B n = 4/group). +PKP2: Hypoxia+PKP2, +NC: Hypoxia+NC. (C and D) Changes of TLR4 protein and mRNA expression after PKP2 overexpression in atrial cardiomyocytes (C n = 3/group; D Ctl n = 3; Hypoxia, +PKP2, +NC n = 4/group). (E) Co-IP results verified the protein interaction between PKP2 and TLR2, TLR4 (n = 3/group). (F–I) After PKP2 overexpression, the mRNA expression levels of IL-6, IL-1β, TNF-α and MCP1 were significantly decreased (n = 8/group). (J) Cell viability decreased after hypoxia, and PKP2 overexpression increased cell viability compared with NC group (n = 8/group). (K and L) Sodium current density was detexted by patch-clamp. Compared with Ctl group, the sodium current density of atrial cardiomyocytes was significantly decreased after 12 h hypoxia. Compared with the NC group, the density of sodium channel was significantly increased in the PKP2 group (∗∗∗p < 0.001 vs. Ctl, ###p < 0.001 vs. Hypoxia, &&&p < 0.001 vs. NC, n = 8/group). (M and N) Voltage-dependent…