Feeding with HFD for 16 weeks leads to cardiac dysfunction in mice.

<p>(A) Upper panel: Representative images of M mode of echocardiography. Low panel: Feeding mice with HFD for 16 weeks significantly impaired cardiac performance indicated by decreases in EF and FS levels in HFD mice compared to ND mice (n = 5–7 mice, *p<0.05). (B) Left ventricular end diastolic diameter (LVEDD) and left ventricular end diastolic volume (LVEDV) were significantly elevated in HFD mice compared to ND mice at 16 weeks (n = 5–7 mice, *p<0.05). (C and D) Expression of cardiac hypertrophic markers ANP and β-MHC in HFD and ND mice. The levels of ANP and β-MHC were significantly higher in the hearts of HFD mice than that of ND mice at 16 weeks (n = 6 mice, *p<0.05).</p

Knockout of PHD2 reduces HFD-induced NF-κb activation and macrophage infiltration in HFD mice.

<p>(A) NF-κb expression was significantly decreased in the hearts of PHD2KO+HFD mice compared to WT, Cre⁺ + HFD mice at 16 weeks (n = 4 mice, *p<0.05). (B) TLR4 expression was significantly reduced in the hearts of PHD2KO+HFD mice compared to WT, Cre⁺ + HFD mice at 16 weeks (n = 4–5 mice, *p<0.05). (C) MyD88 expression was significantly suppressed in the hearts of PHD2KO+HFD mice compared to WT, Cre⁺ + HFD mice at 16 weeks (n = 4–5 mice, *p<0.05). (D) IRAK-4 expression was significantly inhibited in the hearts of PHD2KO+HFD mice compared to WT, Cre⁺ + HFD mice at 16 weeks (n = 4 mice, *p<0.05). (E and F) The expression of ICAM-1 and TNF-α was significantly reduced in the hearts of PHD2KO+HFD mice compared to WT, Cre⁺ + HFD mice at 16 weeks (n = 6 mice, *p<0.05). (G and H) Inflammatory cell infiltrations were stained with macrophage markers CD45 (green) and CD11b (Red). Nuclei were counterstained with DAPI (blue). Immunohistochemical analysis of macrophage infiltrations (CD45 and CD11b positive cells) showing the number of macrophage (white arrow) was increased in the hearts of WT, Cre⁺ + HFD mice compared to that of WT, Cre⁺ + ND mice. Knockout of PHD2 dramatic reduced the number of macrophage in the hearts of HFD mice.</p

Knockout of PHD2 improves HFD-induced impairment of cardiac function in mice.

<p>(A) Representative images of M mode of echocardiography. (B) Pretreatment of WT, Cre⁺ mice with tamoxifen for 7 days then fed with HFD for 16 weeks resulted in a significant reduction of FS% and EF% compared to WT, Cre⁺ mice fed with ND (n = 7 mice, *p<0.05). Pretreatment of PHD2^f/f-Cre+ mice with tamoxifen for 7 days then fed with HFD for 16 weeks significantly increased FS% and EF% levels compared to WT, Cre⁺ mice fed with HFD (n = 7 mice, *p<0.05). Pretreatment of PHD2^f/−Cre+ mice with tamoxifen for 7 days then fed with HFD for 16 weeks had little effects on FS% and EF% levels compared to WT, Cre⁺ mice fed with HFD (n = 6 mice, p>0.05). (C and D) Left ventricular end diastolic diameter (LVEDD) and left ventricular end diastolic volume (LVEDV) were significantly elevated in WT, Cre⁺ + HFD mice compared to WT, Cre⁺ + ND mice at 16 weeks (n = 5–7 mice, *p<0.05). Knockout of PHD2 (PHD2KO) significantly reduced HFD-induced elevation of LVEDD and LVEDV (n = 7 mice, *p<0.05). (E) Cardiac dp/dt max and dp/dt min levels were significantly improved in PHD2KO + HFD mice compared to WT, Cre⁺ + HFD mice (n = 6 mice, *p<0.05). (F) Apoptotic cells were stained with apoptotic marker TUNEL (green) and nuclei were counterstained with DAPI (blue). Immunohistochemical analysis of apoptotic cells showing apoptotic cells (TUNEL positive cells, white arrow) were increased in the hearts of WT, Cre⁺ + HFD mice compared to that of WT, Cre⁺ + ND mice. Knockout of PHD2 dramatic reduced the number of TUNEL positive cells in the hearts of HFD mice. (G and H) The levels of ANP and β-MHC were significantly reduced in the hearts of PHD2KO + HFD mice compared to WT, Cre⁺ + HFD mice at 16 weeks (n = 4–5 mice, *p<0.05). (I) Cardiomyocytes were stained with H&E. Cardiomyocyte size (area) was significantly reduced in the hearts of PHD2KO + HFD mice compared to WT, Cre⁺ + HFD mice at 16 weeks (40X, n = 3 mice, *p<0.05).</p

Expression of PHDs in the hears of HFD mice.

<p>(A) Western blot analysis demonstrating that PHD2 expression was significantly upregulated in the hearts of HFD mice compared to normal chow diet (ND) mice (n = 6 mice, *p<0.05). (B and C) Western blot analysis of PHD1 and PHD3 expression showing that there was no significantly difference in PHD1 and PHD3 expression between HFD and normal diet (ND) mice (n = 6 mice, p>0.05). NS =  Not significant.</p

Knockout of PHD2 does not rescue HFD-induced reduction of HIF-1α expression in HFD mice.

<p>(A) Western blot analysis confirming that PHD2 expression was significantly reduced in PHD2^f/f-Cre+ mice treated with tamoxifen for 7 days and fed with HFD for 16 weeks compared to WT, Cre⁺ + HFD mice (n = 4–5 mice, *p<0.05). (B–F) Western blot analysis showing that expression of PHD1 and PHD3 was not significantly altered in PHD2KO + HFD mice compared to WT, Cre⁺ + HFD mice (n = 4–5 mice, NS). The expression of HIF-1α did not changed in the hearts of PHD2KO + HFD mice compared to WT, Cre⁺ + HFD mice (n = 4–5 mice, NS). VEGF and angiopoietins/Tie-2 system were not altered in PHD2KO + HFD mice compared to WT, Cre⁺ + HFD mice (n = 4–5 mice, NS).</p

Conditional knockout of PHD2 in HFD mice improves glucose tolerance and cardiac function.

<p>(A) Conditional knockout of PHD2 in HFD mice significantly reduced body weight growth compared to WT, Cre⁺−HFD mice (n = 10 mice, *p<0.05). (B) Conditional knockout of PHD2 in normal chow diet mice significantly improved glucose tolerance compared to WT, Cre⁺ mice fed with ND (n = 7–10 mice, *p<0.05). (C) Conditional knockout of PHD2 in HFD mice significantly enhanced glucose tolerance compared to WT, Cre⁺−HFD mice (n = 10 mice, *p<0.05). (D and E) Conditional knockout of PHD2 in HFD mice significantly increased EF% and FS% compared to WT, Cre⁺−HFD mice (n = 5–7 mice, *p<0.05).</p

Expression of HIF-1α, NF-κb and MyD88 in the hears of HFD mice.

<p>(A and B) Western blot analysis revealing that HIF-1α expression was significantly decreased in the hearts of HFD mice compared to normal diet (ND) mice (n = 6 mice, *p<0.05). (B) NF-κb expression was significantly upregulated in the hearts of HFD mice compared to normal diet (ND) mice (n = 6 mice, *p<0.05). (C) MyD88 expression was significantly increased in the hearts of HFD mice compared to normal diet (ND) mice (n = 6 mice, *p<0.05).</p

Ang-1 increases number of SMA⁺ cells in the db/db mouse BM and hearts.

<p><i>A.</i> Representative images showing that BM cells differentiate into vascular smooth muscle-like (SMA⁺) cells in Ad-β-gal treated db/db mice subjected to MI for 24 hours (left panel) and in the Ad-Ang-1treated db/db mice (right panel). <i>B.</i> Quantitative analysis showing a significant increase in number of SMA⁺ cells after myocardial ischemia for 24 hours following Ad-Ang-1 compared to Ad-β-gal in db/db mice subjected to MI. All data represent mean ± SD (n = 3 mice); *p<0.05. <i>C.</i> Representative images of myocardial VSMC in infarcted areas in db/db+IS+Ad-β-gal and db/db+IS+Ad-Ang-l mice stained by Ang-1⁺/SMA⁺ cells. Ang-1 was stained with monoclonal anti-angiopoietin-1 linked to FITC (green, 40X). VSMCs were stained with smooth muscle actin (SMA, Red, 40X) and nuclei were stained with DAPI counterstaining (blue, 40x). Merged image showed that over-expressed Ang-1 co-localized with VSMC. Top and middle panel: representative images showing that systemic delivery of Ad-Ang-1 led to a dramatic increase in the number of Ang-1⁺/SMA⁺ cells in db/db mouse subjected to myocardial ischemia for 24 hours compared to Ad-β-gal in db/db mice. Bottom panel: representative images showing the newly formed arteriole (Ang-1⁺/ SMA⁺) in the Ad-Ang-1 treated db/db mouse heart after ischemia for 14 days.</p

Loss of Sirt3 reduces c-kit⁺/Sca1⁺ cell in post MI mice.

<p><b>A</b>. Western blot analysis revealing that Sirt3 expression was significantly reduced in post-MI mice. BMC treatment significantly increased Sirt3 expression compared to post-MI mice. Loss of Sirt3 blunted BMC-mediated upregulation of Sirt3. n = 6 mice, *p<0.05. <b>B</b>. Immunofluorescence images showing co-localization of Sca1 and c-kit in the border zone of ischemic mouse hearts. Sca1 was stained with mouse Sca1 antibody (green, 10X). c-kit was stained with rabbit c-kit antibody (red, 10X) and nuclei were stained with DAPI (blue, 10X). <b>C</b>. Quantitative analysis of Sca1⁺/c-kit⁺ cells demonstrating that the number of Sca1⁺/c-kit⁺ cells was increased at 14 days of post-MI mice. BMCs significantly increased Sca1⁺/c-kit⁺ cells compared to saline treatment. Treatment with Sirt3KO-BMC had a significant lower number Sca1⁺/c-kit⁺ cells in infracted heart compared to the BMC treated mice. All data represent mean ± SD; n = 5, *p<0.05. ND =  not detected.</p

Ang-1 attenuates myocardial/endothelial apoptosis, and reduces cardiac fibrosis and hypertrophy in db/db mouse.

<p><i>A.</i> TUNEL-stained heart sections from sham control db/db mouse, db/db mouse treated with Ad-β-gal or Ad-Ang-1 at 24 hours and 14 days of MI. Myocardial and endothelial apoptotic cells in the infarcted area of the ischemic hearts were identified by TUNEL staining (green) and TUNEL/vWF (red) positive staining, and total nuclei by DAPI counterstaining (blue). <i>B and C.</i> Quantitative analysis of apoptotic cells in sham control db/db mouse and infarcted area of db/db mouse treated with Ad-β-gal or Ad-Ang-1 at 24 hours and 14 days after ischemia. TUNEL or TUNEL/vWF positive cells are expressed as the total number of per mm². Apoptotic cells were significantly decreased in Ad-Ang-1 compared to Ad-β-gal mice. All data represent mean ± SD (n = 4 mice); *p<0.05. <i>D.</i> Representative images and quantitative analysis of apoptotic cells in the remote area of infarction of db/db mouse at 24 hours after ischemia. Apoptotic cells were significantly decreased in Ad-Ang-1 treated db/db mouse hearts (n = 5) compared to Ad-β-gal treated db/db mice (n = 7). All data represent mean ± SD; sham control n = 5, *p<0.05. <i>E.</i> Representative images of TUNEL and SMA-stained heart sections from sham control db/db mouse, db/db mouse treated with Ad-β-gal or Ad-Ang-1 at 24 hours and 14 days of MI. Myocardial and SMCs apoptotic cells in the infarcted area of the ischemic hearts were identified by TUNEL (green) and SMA (red) positive staining, and total nuclei by DAPI counterstaining (blue). <i>F.</i> Representative images of cardiac fibrosis formation and quantitative analysis of cardiac fibrosis area in db/db mice treated with Ad-β-gal or Ad-Ang-1 stained by Masson’s trichrome. Ad-Ang-1 significantly attenuated area of cardiac fibrosis in db/db mice compared to Ad-β-gal mice. All data represent mean ± SD (n = 5 mice); *p<0.05. <i>G.</i> Heart weight/body weight (HW/BW) ratio in db/db and Ad-Ang-1-treated db/db mouse hearts at 14 days after MI. Treatment with Ad-Ang-1 significantly attenuated cardiac hypertrophy in db/db mouse hearts (n = 5) compared to Ad-β-gal mice (n = 4). All data represent mean ± SD; *p<0.05.</p