Fig 2: ATV protected cardiac function by reducing myocardial infarction area, macrophage recruitment, apoptosis, fibrosis, and increasing vascular density and survival curve, but ATV’s-offered protective effect on cardiac remodeling completely dampened in SPK1-KO miceWT and SPK1−/− mice were fed ATV (10 mg/kg/day) for 1 week before and after MI. Survival curve was observed between WT MI, WT + ATV MI, SPK1−/− MI, SPK1−/− + ATV MI groups. After MI 7 days, the death rate of SPK1−/− MI group is higher than that of WT MI group (A). ATV treatment could decrease the death rate of WT MI model, but did not overtly lower the death rate of SPK1−/− models (A). *P<0.05, **P<0.01 vs WT sham without ATV group; #P < 0.05, ##P < 0.01 compared with WT MI without ATV treatment group; §P < 0.05, §§P < 0.01 compared with WT MI with ATV treatment group; ※P>0.05 vs SPK1−/− MI without ATV treatment group. The infarction area of SPK1−/− MI group is bigger than that of WT MI group (B). ATV could limit infarction area of WT MI with ATV treatment group (B). But there was no difference in the infarction area in SPK1−/− MI mice with and without ATV treatment (B). *P < 0.05, **P < 0.01 compared with WT MI without ATV treatment group. #P< 0.05, ##P < 0.01 compared with WT MI with ATV treatment group. ATV could inhibit P-AKT2 (C) and NBA1 (D) protein levels in WT and SPK1−/− MI animal model. There is no difference in P-AKT2 and NBA1 protein level between WT MI and SPK1−/− MI with and without ATV treatment. ATV could inhibit F4/80 (E) protein levels, F4/80 density (F), Ly6c density (G), Ly6c mRNA (H), flow cytometric analysis of Ly6c (I), apoptosis density (J), caspase 3 protein expression (K), mRNA levels of ANP (L), TNFα (M), IL-6 (N), collagen 1 (O), collagen 3 (P), collagen staining (Q) in the infarction area of WT MI mice. ATV could increase CD31 density (R) and CD 31 (S) mRNA level in WT MI model. But no difference in F4/80 (E) protein 6levels, F4/80 density (F), Ly6c density (G), Ly6c mRNA (H), flow cytometric analysis of Ly6c (I), apoptosis density (J), caspase 3 protein expression (K), mRNA levels of ANP (L), TNFα (M), IL-6 (N), collagen 1 (O), collagen 3 (P), collagen staining (Q), CD31 density (R), CD31(S) mRNA level were detected in SPK1−/− MI model with and without ATV treatment. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01 compared with WT sham without ATV sham group; #P < 0.05, ##P < 0.01 compared with WT MI without ATV treatment group; §P < 0.05, §§P < 0.01 compared with WT MI with ATV treatment group; †P < 0.05, ††P < 0.01 compared with SPK1−/− MI without ATV treatment group; NS=not significant.

Fig 3: NBA1-siRNA decreased NBA1, SPK1, and P-SPK1 protein expressions, but no effect on AKT2 and P-AKT2 protein expressionFirst, 5×104 ANA-1 cells were infected by NBA1-siRNA infection solution (5×108 TU) for 72h, starved overnight and then stimulated with 100 ng/ml LPS for 2h. Whole cell lysates were prepared after cells were stimulated with LPS. Immunoblots show total AKT2 (A), P-AKT2 (B), NBA1 (C), SPK1 (D), P-SPK1 (E) and GAPDH protein expression levels. Graph shows GAPDH normalized AKT2 (A), P-AKT2 (B), NBA1 (C), SPK1 (D), and P-SPK1 (E) levels. Data are presented as the mean ± SEM; n=3. *P<0.05, **P < 0.01 compared with Con without LPS; #P<0.05, ##P < 0.01 compared Con+LPS group; NS=not significant.

Fig 4: Macrophage migration, levels of AKT2, P-AKT2, NBA1, SPK1, and P-SPK1 in LPS induced murine peritoneal macrophages from WT and AKT2 KO (AKT2−/−) mice(A) Migration of peritoneal macrophages from WT and AKT2−/− mice. Primary cells enriched for macrophages were placed into 6-well plates (1×106 cells/ml) in DMEM plus 10% FBS. After 24 h incubation and starved overnight, cells were plated in the upper well and serum-free RPMI 1640 medium containing 100 ng/ml LPS was added to the bottom well. After 16 h of incubation, cells migrating across the membrane were stained and counted. Four random fields were counted and the number of migrated cells was used as an index for migration. (B-F) Protein expression in LPS induced murine peritoneal macrophages. Whole cell lysates were prepared after cells were stimulated with LPS. Immunoblots show total AKT2 (B), P-AKT2 (C), NBA1 (D), SPK1 (E), P-SPK1 (F), and GAPDH protein expression levels. Graph shows GAPDH normalized AKT2 (B), P-AKT2 (C), NBA1 (D), SPK1 (E), and P-SPK1 (F) levels. Data are presented as the mean ± SEM; n=3. *P < 0.05, **P < 0.01 compared with WT without LPS; #P < 0.05, ##P < 0.01 compared with WT+LPS induced group; §P<0.05, §§P<0.01 vs SPK1−/− without ATV treatment group; NS=not significant.

Fig 5: ATV ameliorated cardiac remodeling by inhibiting P-AKT2/NBA1/SPK1(P-SPK1) related macrophages recruitment in the infarction area after MI for 7 daysMice were fed ATV (10 mg/kg/day) for 1 week before and after MI-injury. We produced MI animal model. Levels of P-AKT2 (A), NBA1 z(B), SPK1 (C), P-SPK1 (D), F4/80 (E) protein, F4/80 density (F) and ANP mRNA (G) increased following MI injury. ATV decreased protein levels of P-AKT2 (A), NBA1 (B), SPK1 (C), P-SPK1 (D), F4/80 (E), F4/80 density (F) and ANP mRNA (G) levels in WT MI animal model. Data are presented as the mean ± SEM; n=3. *P < 0.05, **P < 0.01 compared with Con group; #P < 0.05, ##P < 0.01 compared with ATV treatment group; §P< 0.05, §§P < 0.01 compared with MI group; NS=not significant.