Age-associated increase in Arg-II levels in mouse heart.

(A) arg-ii mRNA levels of male and female young (3-4 months) and old (20-22 months) wild type (wt) heart tissues analyzed by qRT-PCR. rps12 served as the reference (n=6 to 7 animals per group); (B) Representative histological images of heart interstitial and perivascular fibrosis in young and old wt and arg-ii-/-female mice. Fibrosis is shown by the blue-colored Trichrome Masson’s staining. Scale bar = 50 µm. (n=5 to 7 mice per group); (C) Quantification of total fibrotic area in cardiac tissue (% of total area); (D) Hydroxyproline content of mouse heart from young and old wt and arg-ii-/-female mice. (n=4 mice in each group); (E) Representative confocal images showing immunofluorescence staining of PDGF-Rα (green, fibroblasts marker) in young and old wt and arg-ii-/- heart tissue. DAPI (blue) is used to stain nuclei. Scale bar = 50 µm; (F) Relative PDGF-Rα signal quantification of confocal images (n= 4 per each group). The values shown are mean ± SD. Data are presented as the fold change to the young-wt group, except for panel D. *P ≤ 0.05, **P ≤ 0.01 between the indicated groups. wt, wild-type mice; arg-ii-/-, arg-ii gene knockout mice.

Age-associated elevation in inflammatory cytokines and apoptosis in heart is prevented in female arg-ii-/-mice.

mRNA expression levels of (A) f4/80, (B) mcp-1, (C) il-1β and (D) tnf-α in young and old wt and arg-ii-/- female mouse hearts were analyzed by qRT-PCR. rps12 served as the reference. (n=6-8 mice per group); (E-F) Representative confocal images and relative quantification of apoptotic cardiac cells in young and old wt and arg-ii-/- heart tissue. DAPI (blue) is used to stain nuclei. Scale bar = 50 µm; (G) wheat germ agglutinin (WGA)-Alexa Fluor 488-conjugate was used to stain cell membrane, and separate cardiomyocytes and non-cardiomyocytes apoptotic cells. Cell distinction was based on cell size and shape. Scale bar = 10 µm; (H) graphs showing the quantification of the TUNEL-positive cardiomyocytes (CM) and non-myocytes (NCM) in old wt and arg-ii-/-hearts. The values shown are mean ± SD. Data are expressed as fold change to the young wt group, expect for panel H. *p ≤ 0.05, **p≤ 0.01, ***p ≤ 0.005 and ****p ≤ 0.001 between the indicated groups. wt, wild-type mice; arg-ii-/-, arg-ii gene knockout mice.

Age-related endothelial-to-mesenchymal transition (EndMT) in heart tissue.

(A) Immunoblotting analysis of CD31 (endothelial marker), vimentin (mesenchymal marker) and SNAIL (master regulator of EndMT) in the heart of wt and arg-ii-/-female young and old mice; tubulin and vinculin served as protein loading controls. Molecular weight (kDa) is indicated at the side of the blots. The plot graphs show the quantification of the SNAIL (B), vimentin (C) and CD31 (D) signals on immunoblots (n=6-10 mice in each group); (E) Representative confocal images showing co-localization of CD31 (green) and vimentin (red) in young and old wt and arg-ii-/- heart tissues. DAPI (blue) is used to stain nuclei. Scale bar = 20 µm. *p≤0.05 and **p ≤ 0.01, between the indicated groups. wt, wild-type mice; arg-ii-/-, arg-ii gene knockout mice.

Cellular localization of Arg-II in aging heart of female mice.

(A) Confocal microscopy illustration of immunofluorescence double staining of Arg-II (red) and tropoin-t (TnT, green; cardiomyocytes marker). Scale bar = 25 µm; (B) Bright-field microscopy images of isolated wt cardiomyocytes and primary cardiac fibroblasts. The immunoblot shows the level of Arg-II in both cardiomyocytes and fibroblasts upon exposure to hypoxia (1 % O2) for 24 h. C indicates freshly isolated cardiomyocytes used as control, N and H indicates respectively normoxia and hypoxia conditions, and K indicates kidney tissue extract used as positive control. Tubulin served as protein loading control; (C) mRNA expression levels of arg-ii in cardiomyocytes (card) and non-cardiomyocytes (non-c) cells isolated from old wt and arg-ii-/- female mouse hearts. gapdh served as the reference. (n=3-5 mice per group); (D to G) Representative confocal images of old wt mouse heart showing co-localization of (D) Arg-II (red) and Mac-2 (green, mouse macrophage marker), (E) Arg-II (red) and CD31 (green, endothelial marker), (F) Arg-II (red) and PDGF-Rα (green, fibroblasts marker), and (G) Arg-II and α-smooth muscle actin (α-SMA; green, smooth muscle cell/myofibroblasts marker); (H to M) Representative confocal images of human heart tissue showing co-localization of (H) Arg-II (green) and tropoin-t (red, cardiomyocytes marker), (I) Arg-II (red) and CD31 (green, endothelial marker), (J) Arg-II (red) and CD-68 (green, macrophage marker), (K) Arg-II (red) and vimentin (green, fibroblast marker), and (L-M) Arg-II (red) and α-smooth muscle actin (α-SMA; green, (L) myofibroblasts and (M) smooth muscle cell marker). DAPI (blue) stains cell nuclei. Scale bar = 20 µm. Each experiment was repeated with 3 to 5 animals.

Arg-II ablation reduces Il-1β protein levels in aging heart.

(A) Immunoblotting analysis of Il-1β (pro-inflammatory cytokine) in the heart of old wt and arg-ii-/- female mice; tubulin served as protein loading control. Molecular weight (kDa) is indicated at the side of the blots; (B) The plot graph shows quantification of the Il-1β signals on immunoblots (n=6-7 mice in each group); (C) Representative confocal images showing Il-1β localization in old wt and arg-ii-/- heart tissues. DAPI (blue) is used to stain nuclei. Scale bar = 50 µm; (D) Relative Il-1β signal quantification of confocal images (n= 9 per each group); (E) Representative confocal images showing co-localization of Mac-2 (green, mouse macrophage marker), and Il-1β (red) in wt heart tissues. This experiment was repeated with 3 animals. Scale bar = 10 µm. *p≤0.05 and ***p ≤ 0.005, between the indicated groups. wt, wild-type mice; arg-ii-/-, arg-ii gene knockout mice.

In vitro study of crosstalk between aging splenic macrophages and cardiomyocytes.

(A) Immunoblotting analysis of Arg-II and Il-1β in mouse splenic macrophages isolated from young and old wt and arg-ii-/- female mice; tubulin served as protein loading control. Molecular weight (kDa) is indicated at the side of the blots. The plot graphs show the quantification of Arg-II (B) and Il-1β (C) protein signals on immunoblots (n=3 mice in each group); (D) IL-1β levels in the conditioned medium from young and old wt and arg-ii-/- splenic macrophages was measured by ELISA; (E) Schematic representation of in vitro crosstalk study; (F) Representative confocal images of adult isolated mouse cardiomyocytes stimulated with conditioned media (CM) from young and old, wt and arg-ii-/- splenic cells (24 h incubation). Wheat Germ Agglutinin (WGA)-Alexa Fluor 488-conjugate was used to stain cell membrane, and TUNEL was performed to identify apoptotic cells. DAPI is used to stain nuclei. Interleukin receptor antagonist (ILRa; 50 ng/mL) is used to prevent IL-1β binding to its receptor; (G) Quantification of TUNEL-positive cardiomyocytes (% in respect to total number of cells). Scale bar = 20 µm. *p≤0.05, **p ≤ 0.01, ***p ≤ 0.005 and ****p ≤ 0.001 between the indicated groups. MØ, splenic macrophage; Y, young; O, old; wt, wild-type mice; arg-ii-/-, arg-ii gene knockout mice.

Crosstalk between splenic macrophages and cardiac fibroblasts and cell-autonomous effect of Arg-II in cardiac fibroblasts.

(A) Collagen production measured as hydroxyproline content in mouse wt fibroblasts treated with conditioned media (CM) from old, wt and arg-ii-/- splenic cells (96 h incubation) (n=3 mice in each group). The values shown are mean ± SD. mRNA expression levels of (B) fibronectin, (C) collagen-IIIα (col-IIIα) and (D) tgf-β1 in wt fibroblasts treated with conditioned media (CM) from old wt and arg-ii-/- splenic cells (96 h incubation) were analyzed by qRT-PCR. gapdh served as the reference. (n=4 mice per group); (E) Hydroxyproline content in fibroblasts treated with CM from old wt splenic cells (96 h incubation). ILRa (50 ng/mL) is used to prevent IL-1β binding to its receptor (n=4 independent experiments). (F) Immunoblotting analysis of Arg-II and vimentin in human cardiac fibroblasts (HCFs) upon arg-ii gene overexpression. GAPDH served as protein loading control; (G) qRT-PCR analysis of mRNA expression levels arg-ii in HCF cells; (H) The plot graph shows the quantification of the vimentin signals on immunoblots shown in panel F. (n=3 independent experiments); (I) qRT-PCR analysis of mRNA expression levels of collagen-IIIα in HCF cells. gapdh served as the reference. (n=6 independent experiments); (J) Representative confocal images of human cardiac fibroblasts (HCF) upon transfection with rAd-CMV-Con/Arg-II for 48 h. MitoSOX (Red) is used to stain mitochondrial ROS (mtROS). TEMPO (10 μmol/L) is used to prevent mtROS generation. DAPI (blue) stains cell nuclei. Scale bar = 50 µm. (K) Mitosox signal quantification (n=3 independent experiments); (L) qRT-PCR analysis of mRNA expression levels of col-IIIα in HCF cells treated as indicated. gapdh served as the reference. (n=5 independent experiments). Data are expressed as fold change to respective control group. *p≤0.05, **p ≤ 0.01, ***p ≤ 0.005 and ****p ≤ 0.001 between the indicated groups. MØ, splenic macrophage; Con, control.

Aging wt macrophages induces EndMT in HUVEC.

(A) Immunoblotting analysis of VE-Cadherin (endothelial marker), N-Cadherin and vimentin (both mesenchymal markers), and Arg-II in HUVEC cells upon incubation with conditioned media (CM) from young and old wt and arg-ii-/-splenic cells (4 days incubation); GAPDH served as protein loading control. Molecular weight (kDa) is indicated at the side of the blots. The plot graphs show the quantification of Arg-II (B), N-Cadherin (C), vimentin (D), and VE-Cadherin (E) protein levels (n=3 independent experiments); (F) Immunoblotting analysis of VE-Cadherin, N-Cadherin, vimentin and Arg-II in HUVEC cells upon incubation with CM from old wt splenic cells (4 days incubation); Interleukin receptor antagonist (IL-Ra; 50 ng/mL) is used to prevent IL-1β effect. GAPDH served as protein loading control. Molecular weight (kDa) is indicated at the side of the blots. The plot graphs show the quantification of Arg-II (G), N-Cadherin (H), vimentin (I), and VE-Cadherin (J) protein signals on immunoblots (n=4 independent experiments); Data are expressed as fold change to respective control group. *p≤0.05, **p ≤ 0.01 and ***p ≤ 0.005 between the indicated groups. MØ, splenic macrophage; wt, wild-type mice; arg-ii-/-, arg-ii gene knockout mice.

Arg-II ablation improves heart function recovery from global ischemia/reperfusion injury (I/R-I).

(A) Protocol applied for ex vivo Langendorff-heart functional assessment of old wt and age-matched arg-ii-/- female mice. After baseline recordings, 20 minutes global ischemia is followed by 30 minutes of reperfusion; (B) Ex vivo Langendorff-heart assessment of old female wt (black line) and arg-ii-/- (red line) hearts. The graphs show the functional recovery of the left ventricular developed pressure (LVDP), left ventricular end diastolic pressure (LVED) and maximal rate of contraction (dP/dtmax) and relaxation (dP/dtmin). The data are expressed as % of recovery in respect to baseline values and represent the mean ± SD of data from 5 mice per group; (C) Representative sections of wt and arg-ii-/-female hearts stained with 2,3,5-TTC and (D) relative quantification of infarcted areas. *p≤0.05 and **p ≤ 0.01 between the indicated groups. wt, wild-type mice; arg-ii-/-, arg-ii gene knockout mice.

Schematic illustration of the role of Arg-II and underlying mechanisms in cardiac aging.