B-arrestin-2 Signaling Is Important to Preserve Cardiac Function During Aging

Figures

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  Figure 1. Deletion of β-arrestin-2 causes enlargement of left atria and left ventricle in aged mice. Representative B-mode and M-mode long-axis images of (A) left atrium (LA) diameter and short-axis images of (B) left ventricular internal diameter during diastole (LVIDd) in young and aged WT and KO mice. Quantification of (C) LA and (D) LVIDd shows enlargement in both aged WT and KO mice. (E) Both aged WT and KO show increased left ventricular (LV) mass. Young: 3–6months and old: 12–16months. N=5–13. Data presented as mean ± SEM. Outliers were identified with the ROUT method and excluded from analyses. Data analyzed with a two-way ANOVA post-hoc Fisher’s LSD test. *p < 0.05.

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  Figure 2. Deletion of β-arrestin-2 signaling causes impaired systolic and diastolic function in aged mice. (A) All mice show ejection fraction (EF)≥50%. (B) Aortic ejection time is prolonged in aged KO. (C) Circumferential fiber shortening velocity calculated as minor axis shortening/ejection time is slower in aged KO mice. (D) KO mice show decreased peak myocardial contraction velocity, s′. (E) Young and old KO mice show slower peak myocardial relaxation velocity, e′. (F) Aged KO mice show increased peak blood inflow velocity E/e′ ratio. (G) Sample tissue Doppler traces show slower myocardial relaxation velocity (e′) in aged KO mice. (H) Aged KO mice show prolonged isovolumetric relaxation time. Young: 3–6months and old: 12–16months. N=5–13. Data presented as mean ± SEM. Outliers were identified with the ROUT method and excluded from analyses. Data analyzed with a two-way ANOVA post-hoc Fisher’s LSD test. *p < 0.05.

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  Figure 3. Deletion of β-arrestin-2 alters the phosphorylation of myofilament proteins. (A) Pro-Q Diamond staining of phosphorylated myofilament proteins on 15% SDS-PAGE gel. (B) Coomassie staining of total protein of the corresponding bands on the same gel. (C) Quantification of phosphorylated titin, (D) cardiac myosin binding protein-C (cMyBP-C), (E) cardiac troponin T, (F) cardiac tropomyosin (cTm), (G) cardiac troponin I (cTnI), and (H) regulatory light chain (RLC) in arbitrary units (a.u.). Young: 3–6months and old: 12–16months. N=4–5. Data presented as mean ± SEM. Outliers were identified with the ROUT method and excluded from analyses. Data analyzed with a two-way ANOVA post-hoc Fisher’s LSD test. *p < 0.05.

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  Figure 4. Phosphorylation of cMyBP-C at Ser302 increases in aged KO mice. Western blot images showing phosphorylation of cMyBP-C at (A) Ser273, (B) Ser282, and (C) Ser302 with summarized quantification and actin as loading control. Third sample on the KO old group in (A) was excluded from analyses due to loading problems as indicated with vertical black line drew across bands. t-cMyBP-C: total cMyBP-C. Young: 3–6months and old: 12–16months. Data presented as mean ± SEM. N=4–5. Outliers were identified with the ROUT method and excluded from analyses. Data analyzed with a two-way ANOVA post-hoc Fisher’s LSD test. *p < 0.05.

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  Figure 5. Phosphorylation of cMyBP-C at Ser302 is increased in a PKCɛ overexpression mouse model. Western blot images show phosphorylation of cMyBP-C at (A) Ser273, (B) Ser282, and (C) Ser302 with summarized quantification and actin as loading control. t-cMyBP-C: total cMyBP-C. Young: 3–6months and old: 12–16months. Data presented as mean ± SEM. N=3–5. Outliers were identified with the ROUT method and excluded from analyses. Data analyzed with a two-way ANOVA post-hoc Fisher’s LSD test. *p < 0.05.

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  Figure 6. Protein kinase A (PKA) phosphorylation and Cdc42 phosphorylation are increased in aged KO mice. (A) Phosphorylation of catalytic (C)-α subunit at Thr197, total PKA, and actin as loading control was detected by Western blot. Aged KO mice show increased quantification of PKA phosphorylation at Thr197 over total PKA. (B) Representative images of the phosphorylation levels of Cdc42 at Ser71 detected by Western blot. Due to a strong phospho Cdc42 signal that was difficult to strip and both phospho and total antibodies being from rabbit, we used a separate blot to detect total Cdc42 and actin as loading control. Quantification of Cdc42 phosphorylation at Ser71 over total Cdc42 shows increased phosphorylation levels in aged KO mice. Young: 3–6months and old: 12–16months. Data presented as mean ± SEM. N=4–5. Outliers were identified with the ROUT method and excluded from analyses. Data were analyzed with a two-way ANOVA post-hoc Fisher’s LSD test. *p < 0.05.

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  Figure 7. Scheme illustration of proposed β-arrestin-2 signaling in cardiomyocytes. (A) β-arrestin-2 signaling inhibits PKA activation by phosphorylation. Decreased PKA activity results in decreased Cdc42 phosphorylation that promotes its activation. Active Cdc42 may promote RLC phosphorylation through myotonic dystrophy-related Cdc42-binding kinase (MRCK) activation to preserve contractile function in aged mice. (B) β-arrestin-2 also inhibits phospholipase C (activated by AT1R signaling) resulting in the inhibition of PKCɛ signaling. Inhibition of PKCɛ activity results in decreased cMyBP-C phosphorylation at Ser302 that may prevent the heart from failing. AC, adenylate cyclase; MRCK, myotonic dystrophy-related Cdc42-binding kinase; RLC, regulatory light chain; PLC, phospholipase C; PIP2, Phosphatidylinositol 4, 5-bisphosphate; IP3, inositol triphosphate; DAG, diacylglycerol; and cMyBP-C, cardiac myosin binding protein-C. Created with BioRender.com.