Protein Kinase B2 (PKB2/AKT2) Is Essential for Host Protection in CVB3-Induced Acute Viral Myocarditis

Figures

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  Figure 1. Generation of cardiac-specific AKT2 knockout mice. Protein and RNA were extracted from wild-type (WT) and AKT2 knockout (KO) mouse hearts. (A) Proteins were subjected to Western blot analysis using the indicated antibodies. Total AKT immunoblotting bands indicate the fold changes of total AKT normalized to GAPDH bands. (B) AKT1, -2, and -3 mRNA levels were determined by real-time PCR. (C) Heart sectioned and subjected to immunofluorescence staining. AKT (green) and connexin-43 (red) in WT and AKT2 KO hearts. All data are means ± s.e.m from three independent experiments (scale bar, 100 µm). * p < 0.05, *** p < 0.001 according to a two-tailed Student’s t-test.

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  Figure 2. AKT2 deletion increased mouse mortality and heart virus replication. (A) CVB3 was injected intraperitoneally into WT and KO mice. (B) The survival rate of the mice was recorded on Day 14 post-infection (p.i). (C) The heart and pancreas were lysed on Day 5 p.i. Tissue lysates were subjected to a plaque-forming unit (PFU) assay to measure tissue virus titers. All data are the means ± s.e.m. * p < 0.05 according to a two-tailed Student’s t-test.

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  Figure 3. Myocardial damage increased in AKT2 knockout mice. (A) WT and KO mice were injected with 10% Evans blue dye (EBD) for 18 h before being sacrificed. Hearts were sectioned and subjected to fluorescence microscopy to determine the red area of EBD uptake (arrow). Heart extracts were subjected to Western blot analysis using the indicated antibodies. (B) Molecules indicating muscle damage (TnT, CK-MB, and LDH) were measured in Day 5 p.i mouse sera. (C) Histological findings using hematoxylin and eosin (H&E) staining in sectioned hearts showed inflammatory cell infiltration on Day 14 p.i. (D) Quantification of the inflamed area (%) of the heart (n = 4 each group). All data are the means ± s.e.m. ** p < 0.01, and *** p < 0.001 according to a two-tailed Student’s t-test.

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  Figure 4. AKT2 deletion prolonged PR interval in CVB3-induced myocarditis. (A). Electrocardiogram (ECG) recorded on Day 3 p.i. The PR interval is extended in AKT2 KO mice (blue arrow). (B) ECG pattern of 100 mean beats in both groups of mice. (C) PR interval and QRS duration, measured by LabChart8.0 software. All data are the means ± s.e.m. * p < 0.05 according to a two-tailed Student’s t-test.

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  Figure 5. AKT2 deletion attenuates inflammatory cytokines and Type 1 interferon induction. (A–C) Total RNA was extracted from WT and KO mouse hearts on Days 5 and 14 p.i. and subjected to real-time PCR with the indicated primer sets. All data are the means ± s.e.m from two independent experiments. NS > 0.05, * p < 0.05, ** p < 0.01, and *** p < 0.001 according to a two-tailed Student’s t-test.

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  Figure 6. AKT2 deletion increased TLR-4 expression in isolated adult cardiomyocytes. (A) Adult cardiomyocytes were isolated from the hearts of WT and KO mice. Cell extracts were subjected to Western blot analysis using the indicated antibodies. (B,C) TLR-4 immunoblotting bands indicate the fold changes in TLR-4 normalized to GAPDH bands, and pAKT and pP38 bands normalized to total Akt and P38 bands. All data are the means ± s.e.m. from independent experiments. ** p < 0.01 and *** p < 0.001 according to a two-tailed Student’s t-test.

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  Figure 7. AKT2 deletion attenuated Type 1 interferon transcription during CVB3 infection. Isolated adult myocytes were infected with CVB3, and total RNA was extracted from WT and KO ventricular cardiac myocytes. RNA was subjected to real-time PCR using gene primers relating to inflammatory cytokines, heart damage, and autophagy formation. (A) AKT isoform expression was observed in AKT2 KO cardiac myocytes. (B) Cardiac myocyte damage was indicated by early gene (ANP and MyH7) induction. (C,D) Expression of Type 1 interferon and autophagosome formation genes. Expression of IFN-α, -β, and p62 was not induced by CVB3 infection in cardiac myocytes under AKT2 deletion. All data are the means ± s.e.m. from independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 according to a two-tailed Student’s t-test.