Stromal cell-derived factor-1α signals via the endothelium to protect the heart against ischaemia-reperfusion injury

Fig. 1. Effect of SDF-1α administration prior to reperfusion on infarct size after in vivo ischaemia-reperfusion injury. Phenotypically wild-type mice (CXCR4fl/fl without Cre or tamoxifen) were anaesthetised and the indicated quantities of vehicle or SDF-1α injected prior to 40 min ischaemia and 2 h reperfusion in vivo; (A) Analysis of their respective AAR revealed no statistically significant differences; (B) Infarct size (IS) as a proportion of AAR was analysed using Evans Blue and TTC staining. Statistical significance was assessed using one-way ANOVA and Tukey's multiple comparison test, n = 6–14, ***P < 0.001 vs. 80 μg/kg and control. Data presented as individual hearts with mean ± SEM; (C) Representative scanned transverse heart sections demonstrating Evans Blue area (blue), area at risk (non-blue) and infarct (white). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. Effect of SDF-1α administration prior to reperfusion on infarct size after in vivo ischaemia-reperfusion injury in EC-CXCR4KO mice. EC-CXCR4WT (i.e.: CXCR4fl/fl; Cre+/+) and EC-CXCR4KO (i.e.: CXCR4fl/fl; Cre+/−) mice were injected with tamoxifen as an intraperitoneal bolus daily for 5 consecutive days at a dose of 20 mg/kg. Mice were left for 3 weeks after completion of tamoxifen. Mice were treated with either vehicle or SDF-1α by jugular vein injection and subject to 40 min ischaemia and 2 h reperfusion in vivo; (A) Analysis of their respective AAR revealed no statistically significant differences; (B) Infarct size as a proportion of AAR was analysed using Evans Blue and TTC staining and demonstrated no statistically significant differences, n = 6–8. Statistical significance was assessed using one-way ANOVA and Tukey's multiple comparison test. Data presented as individual hearts with mean ± SEM; (C) Representative heart sections as per Fig. 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. Effect of SDF-1α administration on Phospho-Erk and Phospho-Akt in HUVECs. HUVECs in M199 conditioning media were treated with rhSDF-1α. Further groups were pre-treated 5 mM AMD3100 prior to rhSDF-1α or M199 media alone. After 10 min, cells were washed and analysed by Western blot. Treatment with SDF-1α increased Phospho-Erk relative to tubulin, which was abrogated by pre-treatment with AMD3100. Phospho-Erk and Phospho-Akt were run on the same membrane and normalised to tubulin loading control. Size of standard marker proteins is indicated (kDa). Statistical significance was assessed using repeated measures one-way ANOVA and Tukey's multiple comparison test, n = 4, *P < 0.05, **P < 0.01, *** P < 0.001 vs. control; & P < 0.05 vs. 25 nM SDF-1α. Data presented as mean ± SEM.

Fig. 4. Effect of cardiomyocyte-specific CXCR4 deletion on infarct size prior to in vivo ischaemia-reperfusion injury. CM-CXCR4WT (i.e.: CXCR4fl/fl; Cre+/+), CXCR4fl/+; Cre+/− and CM-CXCR4KO (i.e.: CXCR4fl/fl; Cre+/−) mice were injected with tamoxifen as an intraperitoneal bolus daily for 5 consecutive days at a dose of 20 mg/kg. Mice were left for 3 weeks after completion of tamoxifen. Mice were anaesthetised and subject to 40 min ischaemia and 2 h reperfusion in vivo; (A) Analysis of their respective AAR revealed no statistically significant differences; (B) Infarct size as a proportion of AAR was analysed using Evans Blue and TTC staining and was significantly smaller in CM-CXCR4KO compared to CM-CXCR4WT mice (12.2 ± 2.6% vs. 35.8 ± 4.3%). Statistical significance was assessed using one-way ANOVA and Tukey's multiple comparison test, n = 6, **P < 0.01 vs. CM-CXCR4WT. Data presented as mean ± SEM; (C) Representative heart sections as per Fig. 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Baseline echocardiographic parameters in CM-CXCR4WT and CM-CXCR4KO mice. CM-CXCR4WT (CXCR4fl/fl; Cre+/+) and CM-CXCR4KO (CXCR4fl/fl; Cre+/−) mice were injected with tamoxifen as an intraperitoneal bolus daily for 5 consecutive days at a dose of 20 mg/kg. Mice were left for 3 weeks after completion of tamoxifen. There were no statistically significant differences between CM-CXCR4WT and CM-CXCR4KO groups with respect to (A) fractional shortening, (B) stroke volume, (C) cardiac output or (D) LV dimensions at baseline. Statistical significance was assessed using unpaired t-test for A-C and two-way ANOVA with Bonferroni correction for multiple comparisons in D; n = 5–6, PNS.

Fig. 6. RNAseq analysis comparing mRNA expression in CM-CXCR4WT and CM-CXCR4KO mouse hearts. A) Of ~12K mRNAs detected (green dots), only 37 exhibited a significant difference in expression level (red dots). B) A heat map of genes expressed at significantly different levels. Clustering based on expression patterns demonstrates a clear separation between the CM-CXCR4WT and CM-CXCR4KO hearts (N = 6). C) qRT-PCR confirmed an increase in expression of Coq10b in CM-CXCR4KO hearts, resulting in a lower 2-ΔΔCT, (n = 4, P < 0.05 by t-test, lower number indicates greater transcript numbers). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Supplementary Fig. 1. Mouse primer sequences used in qRT-PCR experiments.

Supplementary Fig. 2. Analysis of whole heart tissue from CM-CXCR4WT and CM-CXCR4KO mice. Western blot analysis demonstrated no difference in CXCR7 (A) or Phospho-Akt / Total-Akt (B) protein expression. QPCR analysis demonstrated no difference in SDF-1α gene expression (C). Statistical significance was assessed using unpaired t-tests, n=4-6. Data presented as mean ± SEM.

Supplementary Fig. 3. Comparison of baseline inflammatory cells between CM-CXCR4WT and CM-CXCR4KO mice. CM-CXCR4WT (CXCR4Fl/Fl; Cre+/+) and CM-CXCR4KO (CXCR4Fl/Fl; Cre+/-) mice were injected with tamoxifen as an intraperitoneal bolus daily for 5 consecutive days at a dose of 20 mg/kg. Mice were left for 3 weeks after completion of tamoxifen; (A-D) 5 μM sections were stained with Ly6G, CD45, CD68 and Toliudine blue for neutrophils, total leukocytes, macrophages and mast cells, respectively. No statistically significant differences between CM-CXCR4WT and CM-CXCR4KO groups were seen at baseline. Statistical significance was assessed using unpaired t-test; n=3-6, P=NS for all comparisons; (E-H) representative sections at x40 magnification with positive staining arrowed.