Fig 2: Proximal C6 cells induce the internalization and mono-ubiquitination of hBMVEC Fpn.(A) Fpn was immunoprecipitated from lysates of hBMVEC seeded in transwell either alone (lanes 1–2), distal to (lanes 3–4), or proximal to (lanes 5–6) C6 cells. Lysates from hBMVEC cultured under the same co-culture conditions but treated for 24 h with 10 µM fursultiamine were probed for Fpn as well. Fpn-immunoreactive eluates were probed by immunoblots for either Fpn or ubiquitin (Ub) conjugated to Fpn. (B) hBMVEC alone or seeded proximal to C6 cells were fixed and permeabilized (0.1% Tween-20) or not followed by processing for indirect immunofluorescence imaging of Fpn. The images were obtained with a 40X objective. (C) Average fluorescent intensity of hBMVEC Fpn from 4–7 separate fields of view from each condition obtained (from (B)) using the AxioVision software. (D) qPCR of human HAMP within total RNA isolated from hBMVEC seeded in transwell either alone (hBMVEC), distal to (hBMVEC/-/C6), or proximal to (hBMVEC/C6) C6 glioma cells. (E) qPCR of rat HAMP within total RNA isolated from C6 glioma cells grown in transwell either alone (C6), distal to (hBMVEC/-/C6), or proximal to (hBMVEC/C6) hBMVEC. (F) hBMVEC HAMP gene expression relative to C6 glioma cells in each of the three transwell orientations. All hBMVEC HAMP values are relative to C6 glioma HAMP in each orientation (C6 HAMP expression are set to 1 in each orientation). One-way ANOVA statistical analysis was used to determine significance. *P<0.05, **P<0.01, ***P<0.001. Data are represented as means ± S.D. (n = 3, technical replicates).

Fig 3: Interplay between systemic and cardiac iron HAMP/FPN axes.Cardiomyocyte iron content is determined by both systemic iron availability, which is regulated by liver HAMP, and by the cardiac HAMP/FPN axis, which regulates cardiomyocyte iron efflux. In the wild type heart, cardiac HAMP regulates the levels of cardiac FPN and iron release from cardiomyocytes. In this study, we have demonstrated that loss of cardiac HAMP (cardiac Hamp KO) or loss of cardiac HAMP responsiveness (cardiac Slc40a1 C326Y KI) result in cardiomyocyte iron deficiency due to increased cardiomyocyte FPN and iron release. Previously, we also demonstrated that loss of cardiomyocyte FPN caused cardiomyocyte iron overload. In these two sets of conditions, cardiomyocyte iron deficiency and cardiomyocyte iron overload cause cardiac dysfunction. We have also shown that upregulation of cardiac FPN occurs as a result of loss of either systemic HAMP or systemic HAMP responsiveness, and is protective against the otherwise detrimental effects of systemic iron overload.DOI: http://dx.doi.org/10.7554/eLife.19804.022

Fig 4: Effect of Furin inhibitor on iron export in cardiomyocytes.Fe55 efflux measured in primary adult cardiomyocytes from Hamp fl/fl and Hamp fl/fl;Myh6.Cre+ mice following culture in control medium (-CMK) or medium containing Furin inhibitor (+CMK) for 2 hr. n = 3. values are plotted as mean ± SEM. *p=0.027. †p = 0.024.DOI: http://dx.doi.org/10.7554/eLife.19804.023

Fig 5: Strategy for generation of Hampfl/fl;Myh6.Cre+ mice.A targeting vector was designed to introduce a floxed Hamp allele into mouse ES cells, with exons 2 and 3, which encode the majority of the peptide, flanked by LoxP sites. Further breeding with a C57BL/6 Flp recombinase deleter mouse allowed removal of the Neo cassette. Cardiac Hamp knockouts were then generated by crossing homozygous Hampfl/fl animals with mice transgenic for Myh6-Cre recombinase, which is under the control of cardiomyocyte-specific Myosin Alpha Heavy chain six promoter.DOI: http://dx.doi.org/10.7554/eLife.19804.026