Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Mar:128:187-197.
doi: 10.1016/j.yjmcc.2019.02.002. Epub 2019 Feb 7.

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

Daniel I Bromage  1 , Stasa Taferner  1 , Zhenhe He  1 , Oliver J Ziff  1 , Derek M Yellon  2 , Sean M Davidson  1
Affiliations

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

Daniel I Bromage et al. J Mol Cell Cardiol. 2019 Mar.

Abstract

Aims: The chemokine stromal derived factor-1α (SDF-1α) is known to protect the heart acutely from ischaemia-reperfusion injury via its cognate receptor, CXCR4. However, the timing and cellular location of this effect, remains controversial.

Methods and results: Wild type male and female mice were subjected to 40 min LAD territory ischaemia in vivo and injected with either saline (control) or SDF-1α prior to 2 h reperfusion. Infarct size as a proportion of area at risk was assessed histologically using Evans blue and triphenyltetrazolium chloride. Our results confirm the cardioprotective effect of exogenous SDF-1α in mouse ischaemia-reperfusion injury and, for the first time, show protection when SDF-1α is delivered just prior to reperfusion, which has important therapeutic implications. The role of cell type was examined using the same in vivo ischaemia-reperfusion protocol in cardiomyocyte- and endothelial-specific CXCR4-null mice, and by Western blot analysis of endothelial cells treated in vitro. These experiments demonstrated that the acute infarct-sparing effect is mediated by endothelial cells, possibly via the signalling kinases Erk1/2 and PI3K/Akt. Unexpectedly, cardiomyocyte-specific deletion of CXCR4 was found to be cardioprotective per se. RNAseq analysis indicated altered expression of the mitochondrial protein co-enzyme Q10b in these mice.

Conclusions: Administration of SDF-1α is cardioprotective when administered prior to reperfusion and may, therefore, have clinical utility. SDF-1α-CXCR4-mediated cardioprotection from ischaemia-reperfusion injury is contingent on the cellular location of CXCR4 activation. Specifically, cardioprotection is mediated by endothelial signalling, while cardiomyocyte-specific deletion of CXCR4 has an infarct-sparing effect per se.

Keywords: CXCR4; Cardiomyocyte; Cardioprotection; Endothelial; Ischaemia-reperfusion injury; SDF-1α.

PubMed Disclaimer

Figures

Fig. 1
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
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
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
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
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, Pformula imageNS.
Fig. 6
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
Supplementary Fig. 1
Mouse primer sequences used in qRT-PCR experiments.
Supplementary Fig. 2
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
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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Gibson C.M. NRMI and current treatment patterns for ST-elevation myocardial infarction. Am. Heart J. 2004;148(5 Suppl):S29–S33. - PubMed
    1. Keeley E.C., Boura J.A., Grines C.L. Primary angioplasty versus intravenous thrombolytic therapy for acute myocardial infarction: a quantitative review of 23 randomised trials. Lancet. 2003;361(9351):13–20. - PubMed
    1. Lonborg J., Vejlstrup N., Kelbaek H., Holmvang L., Jorgensen E., Helqvist S., Saunamaki K., Ahtarovski K.A., Botker H.E., Kim W.Y., Clemmensen P., Engstrom T. Final infarct size measured by cardiovascular magnetic resonance in patients with ST elevation myocardial infarction predicts long-term clinical outcome: an observational study. Eur. Heart J. Cardiovasc. Imaging. 2013;14(4):387–395. - PubMed
    1. Chen J., Hsieh A.F., Dharmarajan K., Masoudi F.A., Krumholz H.M. National trends in heart failure hospitalization after acute myocardial infarction for Medicare beneficiaries: 1998-2010. Circulation. 2013;128(24):2577–2584. - PMC - PubMed
    1. Koudstaal S., Pujades-Rodriguez M., Denaxas S., Gho J.M., Shah A.D., Yu N., Patel R.S., Gale C.P., Hoes A.W., Cleland J.G., Asselbergs F.W., Hemingway H. Prognostic burden of heart failure recorded in primary care, acute hospital admissions, or both: a population-based linked electronic health record cohort study in 2.1 million people. Eur. J. Heart Fail. 2017;19(9):1119–1127. - PMC - PubMed

Publication types