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. 2024 Feb 15;27(3):109219.
doi: 10.1016/j.isci.2024.109219. eCollection 2024 Mar 15.

Cardiomyocyte-fibroblast interaction regulates ferroptosis and fibrosis after myocardial injury

Mary E Mohr  1 , Shuang Li  1 , Allison M Trouten  1 , Rebecca A Stairley  1 , Patrick L Roddy  1 , Chun Liu  2   3 , Min Zhang  4 , Henry M Sucov  1   5 , Ge Tao  1
Affiliations

Cardiomyocyte-fibroblast interaction regulates ferroptosis and fibrosis after myocardial injury

Mary E Mohr et al. iScience. .

Abstract

Neonatal mouse hearts have transient renewal capacity, which is lost in juvenile and adult stages. In neonatal mouse hearts, myocardial infarction (MI) causes an initial loss of cardiomyocytes. However, it is unclear which type of regulated cell death (RCD) occurs in stressed cardiomyocytes. In the current studies, we induced MI in neonatal and juvenile mouse hearts and showed that ischemic cardiomyocytes primarily undergo ferroptosis, a non-apoptotic and iron-dependent form of RCD. We demonstrated that cardiac fibroblasts (CFs) protect cardiomyocytes from ferroptosis through paracrine effects and direct cell-cell interaction. CFs show strong resistance to ferroptosis due to high ferritin expression. The fibrogenic activity of CFs, typically considered detrimental to heart function, is negatively regulated by paired-like homeodomain 2 (Pitx2) signaling from cardiomyocytes. In addition, Pitx2 prevents ferroptosis in cardiomyocytes by regulating ferroptotic genes. Understanding the regulatory mechanisms of cardiomyocyte survival and death can identify potentially translatable therapeutic strategies for MI.

Keywords: Physiology; cardiovascular medicine; cell biology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Ferroptosis is more prevalent in cardiomyocytes than apoptosis and necroptosis in infarcted postnatal hearts LAD-O was performed at either P1 or P7, heart tissue section prepared at 1, 3, or 6 DPMI. (A–G) Infarct zone stained for cCasp3 (magenta), MF20 (green), and DAPI (blue). Arrow: cardiomyocytes positive for cCasp3, ratio quantified in (G). Arrowhead: non-cardiomyocyte positive for cCasp3. (H–N) Infarct zone stained for pMLKL (magenta), cTnT (green), and DAPI (blue). Arrow: cardiomyocytes positive for pMLKL, ratio quantified in (N). Arrowhead: non-cardiomyocyte positive for pMLKL. (O–U) Infarct zone stained for Ptgs2 (magenta), cTnT (green), and DAPI (blue). Arrow: cardiomyocytes positive for Ptgs2, ratio quantified in (U). All bar graphs represent mean ± SD. ∗p < 0.05; NS, not significant by t test. NS in (G), no significance among all groups. #, infarct zone. Scale bar, 25 μm (A–F, H–M, O–T). See also Figures S1 and S2.
Figure 2
Figure 2
Cardiac fibroblasts protect cardiomyocytes from ferroptosis (A) Schematic of high cell density protecting tumor cells from ferroptosis. (B) Schematic showing the change of cardiac cell density during MI. (C) Survival (negative for trypan blue) rates of iCMs cultured at low, mid, or high density after erastin treatment (15 μM, 5 h). (D) Survival rates of HCFs cultured at low, mid, or high density after erastin treatment (15 μM, 5 h). (E and F) PTGS2 (red) and DAPI (blue) were stained and imaged with endogenous TITIN-GFP (green) in iCM after erastin (30 μM) treatment. (G and H) PTGS2 (red), αSMA (gray), and DAPI (blue) were stained and imaged with endogenous TITIN-GFP (green) in co-cultured iCMs and HCFs after erastin (30 μM) treatment. (I) Fold change of PTGS2 fluorescent intensity in iCMs and HCFs. (J–Q) Heart tissue of controls (PostnMCM/+, J, K, N, O) and PostnMCM/+;ROSA-DTA (L, M, P, Q) mice were stained for 4-HNE (magenta, J-M) or Ptgs2 (magenta, N-Q) at 4 DPMI after P1 LAD-O; tamoxifen was administered daily at 1–3 DPMI. Green, MF20; blue, DAPI. #, infarct zone. (R) Ratio of cardiomyocytes positive for Ptgs2 or 4-HNE. (S–V) Heart sections of 2-month-old control (ROSA-DTA, S, T) and Pdgfrα-CreERT2;ROSA-DTA (U, V) mice stained for 4-HNE (red), cTnT (green), Pdgfrα (gray), and DAPI (blue) after tamoxifen administration (see Figure S3). (W) Density of Pdgfrα-positive cells in control and mutant groups. (X) Average 4-HNE intensity in cardiomyocytes from both groups. #, infarct zone. All bar graphs represent mean ± SD. ∗p < 0.05; ∗∗p < 0.01; NS, not significant by t test. NS in (A), no significance among three groups. Scale bar, 50 μm (E–H), 25 μm (J–Q, S–V). See also Figure S3.
Figure 3
Figure 3
Cardiac fibroblasts are resistant to ferroptosis (A–C) HCFs were stained for pSMAD2 [green in (A)], pSMAD3 [green in (B)], and TGFβR2 [green in (C)], with αSMA (magenta) and DAPI (blue). (D) Survival rate of HCFs after treatment with 50, 100, or 200 μM of H2O2, compared with vehicle (H2O). (E) Survival rate of iCM after treatment with 50, 100, or 200 μM of H2O2, compared with vehicle (H2O). (F and G) Brightfield of HCFs after erastin or DMSO treatment. Survival rate quantified in (G). (H) Survival rate of iCMs after erastin treatment at 2, 10, 15, 20, or 30 μM, compared with DMSO control. (I and J) Survival rate of primary mouse cardiac fibroblasts (CFs), prepared from P1 (I) and P7 (J) hearts, after erastin treatment. (K) Survival rate of HEK293 cells after H2O2 or vehicle treatment. (L) Survival rate of HEK293 cells treated with erastin at gradient concentration. (M) Survival rate of HEK293 cells cultured at low, mid, and high density after erastin treatment at 15 or 30 μM, compared with DMSO groups. All bar graphs represent mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; NS, not significant by t test. Scale bar, 100 μm (A–C, E). See also Figure S3.
Figure 4
Figure 4
Fibroblast-derived cytokine and chemokine promote iCM survival after erastin treatment (A) Flowchart of conditioned media and cytokine array experiment. (B) Survival rate of iCMs, cultured in control medium (DMEM), HCF conditioned medium, or H2O2-treated HCF conditioned medium, after erastin treatment, normalized to respective DMSO control groups. (C) Cytokine-chemokine protein array blotting image of conditioned media from vehicle- or H2O2-treated HCFs. (D) Blotting signal intensity of IL-8 and EGF. (E and F) Survival rate of iCMs after erastin treatment in the presence of IL-8 (E) or EGF (F), normalized to DMSO control groups. (G) qPCR of CXCR1 and CXCR2 in total blood cells and purified cardiomyocyte nuclei after P1 LAD-O. (H) Western blot of CXCR1, CXCR2, and α-tubulin in human iCMs after DMSO or erastin treatment. (I) Normalized band (immuno-blotting) intensity of CXCR1 and CXCR2 in (H). All bar graphs represent mean ± SD. ∗p < 0.05; ∗∗p < 0.01; NS, not significant by t test.
Figure 5
Figure 5
Cardiac fibroblasts interact with cardiomyocytes through gap junctions to share free iron (A and B) Wild-type mouse heart tissue stained for Fth1 [magenta, (A)] or Ftl [magenta, (B)], with cTnT (green) and DAPI (blue) at 1DPMI after P7 LAD-O. Arrows: non-cardiomyocytes positive for Fth1 (A) or Ftl (B). (C and D) Mouse heart tissue stained for Fth1 [red, (C)] or Ftl [red, (D)], with Pdgfrα (gray), cTnT (green), and DAPI (blue) at 1 DPMI after P7 LAD-O. Arrows: cells positive for Pdgfrα and Fth1 (C) or Ftl (D). (E and F) Mouse heart tissue stained for Fth1 [green, (E)] or Ftl [green, (F)], with Pdgfrα (red), MF20 (gray), and DAPI (blue) at 6 DPMI after P7 LAD-O. Arrows: cells positive for Pdgfrα and Fth1 (E) or Ftl (F). (G) Diagram of cardiomyocyte-fibroblast interaction after MI. (H and I) Mouse heart section stained for Cx45 [green, (H)] or Cx43 [green, (I)], with Pdgfrα (red), MF20 (gray), and DAPI (blue) after P7 LAD-O. Arrows: potential locations of gap junctions between cardiomyocytes and fibroblasts. (J–L) Co-cultured iCM and HCF stained for VIMENTIN (VIM, gray), free Fe2+ (red), DAPI (blue), and imaged with TITIN-GFP (green) after DMSO (J) or erastin (15 μM) (K, L) treatment. Asterisks: HCFs with accumulation of Fe2+. (M) siRNA knockdown of CX43 and CX45 simultaneously in iCM-HCF co-culture; Fe2+ fluorescent intensity ratio of iCMs over HCFs was quantified after erastin or DMSO treatment. LV, left ventricle. All bar graphs represent mean ± SD. ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 by t test. Scale bar, 75 μm (A, B, E, F, H), 25 μm (C, D, I, J–L). See also Figures S4 and S5.
Figure 6
Figure 6
Pitx2 negatively regulates fibrosis by downregulating Tsp1 expression after MI (A and B) PostnMCM/+;ROSA-mTmG heart sections imaged for endogenous GFP at six days after P1 (A) or P7 (B) LAD-O or sham procedure. Tamoxifen administrated daily at 1, 2, 3, and 5 days after surgery. (C) GFP-positive cell ratio over total nuclei number in sham and LAD-O groups. (D and E) PostnMCM/+;ROSA-mTmG heart sections stained for Tsp1 (red) and DAPI (blue) at 6 DPMI after P1 (D) or P7 (E) LAD-O. (F) Normalized band (immuno-blotting) intensity of myocardial Tsp1 at 4 DPMI after P1 or P7 LAD-O. (G and H) Control (Mhccre-Ert) (G) and Pitx2-iCKO (Mhccre-Ert;Pitx2f/f). (H) Heart tissue stained for Tsp1 (green) and DAPI (blue) at 3 DPMI after P1 LAD-O. Tamoxifen was given at P0–P2. (I) Normalized band intensity of Tsp1 in control and Pitx2-iCKO left ventricle at 3 DPMI after P1 LAD-O. (J and K) Control (Mhccre-Ert) (J) and Pitx2-OE (Mhccre-Ert;Pitx2gof). (K) Hearts were stained for Tsp1 (green) and DAPI (blue) at 3 DPMI after P7 LAD-O. Tamoxifen was given at P6–P8. (L) Normalized band intensity of Tsp1 in control and Pitx2-OE left ventricle at 3 DPMI after P7 LAD-O. (M and O) Control (Mhccre-Ert) (M) and Pitx2-OE (Mhccre-Ert;Pitx2gof). (N) Hearts were stained for pSmad3 (green) and DAPI (blue) at 3 DPMI after P7 LAD-O. Tamoxifen was given at P6–P8. Number of pSmad3-positive cells was quantified in (O). All bar graphs represent mean ± SD. ∗p < 0.05; ∗∗p < 0.01; NS, not significant by t test. LV, left ventricle. Scale bar, 100 μm (A, B, D, E, G, H, J, K, M, N). See also Figure S6.
Figure 7
Figure 7
Pitx2 prevents ferroptosis in cardiomyocytes by regulating ferroptotic genes (A) Heatmap shows transcript level of ferroptotic genes in Pitx2-CKO (MCKcre;Pitx2f/f) ventricles and controls (Pitx2f/f) at five days after apex resection (DPR) or sham. (B) Heatmap shows transcript level of ferroptotic genes in human iCMs after siRNA knockdown of PITX2, with scramble controls. (C) qPCR of ferroptotic targets in iCMs after vehicle or erastin treatment, with PITX2-expressing or control lentivirus transduction. (D) ChIP-Seq showing Pitx2 binding region near ferroptotic gene loci in regenerating neonatal ventricles. (E) qPCR of PITX2 in AC16 cells treated with vehicle or erastin. (F–H) Heart tissue of control (F) and Pitx2-OE (G) were stained for 4HNE (magenta), MF20 (green), and DAPI (blue) at three days after P7 LAD-O. Tamoxifen was administrated daily from P6–P8. Number of 4-HNE-positive cardiomyocytes was quantified in (H). (I and J) Heart tissue of control (I) and Pitx2-OE (J) were stained for Ptgs2 (magenta), MF20 (green), and DAPI (blue) at three days after P7 LAD-O. (K and L) Western blot of Ptgs2 in control and Pitx2-OE left ventricles at three days after sham or LAD-O at P7. Ptgs2 band intensity quantified in (L). (M) Working model of cardiomyocytes interacting with fibroblasts to regulate ferroptosis after MI. All bar graphs represent mean ± SD. ∗p < 0.05; ∗∗p < 0.01; NS, not significant by t test. Scale bar, 75 μm (F, G), 25 μm (I, J).
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