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Article Open Access

ULK1 activates NCOA4‑mediated ferritinophagy via the Beclin1/VPS34 complex in cardiomyocyte hypertrophy

  • Authors:
    • Qianhui Zhang
    • Meitian Zhang
    • Yongsheng Liu
    • Pilong Shi
    • Hanping Qi
    • Man Jiang
    • Yonggang Cao
    • Hongli Sun
  • View Affiliations / Copyright

    Affiliations: Department of Pharmacology, Harbin Medical University, Daqing, Heilongjiang 163319, P.R. China
    Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 116
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    Published online on: February 12, 2026
       https://doi.org/10.3892/mmr.2026.13826
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Abstract

Cardiac hypertrophy is associated with ferroptosis. Serine/threonine protein kinase ULK1 (ULK1) acts as a key activator of autophagy; however, its exact function in the non‑autophagy pathway remains to be fully elucidated. The present study aimed to decipher the role and mechanisms of ULK1 in ferroptosis and cardiomyocyte hypertrophy. Cell survival, lipid peroxidation, iron metabolism and prostaglandin endoperoxide synthase 2 (Ptgs2) mRNA expression were analyzed to investigate the role of ferroptosis in ULK1‑silenced or ULK1‑overexpressing HL‑1 cells. Immunofluorescence staining, western blot analysis and monomeric red fluorescent protein‑green fluorescent protein‑microtubule‑associated protein 1 light chain 3 puncta formation assays were performed to demonstrate the regulatory effect of ULK1 on autophagy and ferritinophagy‑related proteins. Ferritinophagy activation was assessed in cardiomyocytes using immunofluorescence of nuclear receptor coactivator 4 (NCOA4) and microtubule‑associated protein 1 light chain 3‑II colocalization. ULK1 expression was found to be elevated in both transverse aortic constriction‑induced hypertrophic cardiac tissues and angiotensin II‑treated cardiomyocytes. ULK1 knockdown markedly suppressed cardiomyocyte ferroptosis, whereas ULK1 overexpression facilitated ferroptosis in HL‑1 cells. Meanwhile, the ferroptosis inhibitor ferrostatin‑1 reduced iron accumulation, lipid peroxidation and Ptgs2 mRNA expression. Notably, the autophagy inhibitor 3‑methyladenine mitigated ULK1‑induced ferroptosis. Mechanistically, ULK1‑activated NCOA4‑mediated ferritinophagy was found to be dependent on the Beclin1/PI3K catalytic subunit type 3 complex. Finally, the ULK1 inhibitor SBI‑0206965 ameliorated ferroptosis in cardiomyocytes in vitro. For the first time, to the best of our knowledge, the present study demonstrated that ULK1 modulates NCOA4‑mediated ferritinophagy and ferroptosis in HL‑1 cells. The findings of the present study provide a novel insight into the progression of cardiomyocyte hypertrophy.
View Figures

Figure 1

ULK1 expression is elevated in
hypertrophic cardiac tissues and cardiomyocytes. (A) C57BL/6J mice
at 8-weeks-old were subjected to sham or TAC surgery.
Representative whole heart images (scale bar, 0.5 cm), M-mode
echocardiography, H&E staining (scale bar, 100 µm) and WGA
staining (scale bar, 50 µm) of heart tissue. Assessment of (B)
HW/BW, (C) LVW/BW and (D) HW/TL (n=6). Statistical graphs for (E)
LVEF (%), (F) FS (%), (G) IVS, d (mm) and (H) LVPW, d (mm) (n=6).
(I) Cardiomyocyte cross-sectional area acquired from WGA staining.
Cells were measured from different microscopic fields of 6 samples
in each group. The mRNA levels of (J) BNP and (K)
β-MHC in the hearts from sham or TAC-surgery mice (n=6). The
mRNA levels of (L) BNP and (M) β-MHC in HL-1 cells
treated with Ang II (n=3). (N) Stained cells and (O) quantification
of cell surface area of HL-1 cells treated with Ang II. Scale bar,
50 µm (n=3). Western blot analysis and summarized data
demonstrating (P) Beclin1 and (Q) VPS34 protein expression in HL-1
cells treated with Ang II (n=3). (R) Western blot analysis and
summarized data demonstrating ULK1 protein levels in the hearts
obtained after sham or TAC surgery (n=6). (S) Western blot analysis
and summarized data demonstrating ULK1 protein levels in the
cardiomyocytes stimulated with Ang II (n=6). All data are presented
as mean ± SEM. *P<0.05 and **P<0.01 vs. sham or
control group. ULK1, serine/threonine protein kinase ULK1; TAC,
transverse aortic constriction; HW, heart weight; BW, body weight;
LVW, left ventricular weight; TL, tibia length; LVEF, left
ventricular ejection fraction; FS, fractional shortening; d,
diastole; IVS, interventricular septal thickness; LVPW, left
ventricular posterior wall thickness; CSA, cross-sectional area;
BNP, brain natriuretic peptide; β-MHC, β-myosin heavy
chain; α-SMA, α smooth muscle actin; Ang II, angiotensin II; VPS34,
PI3K catalytic subunit type 3; WGA, wheat germ agglutinin.

Figure 2

Fer-1 reverses ferroptosis in
ULK1-overexpressing cells. The mRNA levels of (A) BNP and
(B) β-MHC in HL-1 cells. (C) Images of stained cells and (D)
quantification of cell surface area under the indicated
experimental group. Scale bar, 50 µm. (E) Cell viability of HL-1
cells was assessed using a Cell Counting Kit-8 assay. The levels of
(F) Fe2+, (G) MDA and (H) SOD were determined in the
indicated experimental group. (I) The mRNA levels of Ptgs2.
(J) Immunofluorescence staining for 4-HNE. Scale bar, 50 µm. All
data are presented as mean ± SEM. n=3. *P<0.05 and **P<0.01
vs. control group; #P<0.05 and ##P<0.01
vs. ULK1 group. ULK1, serine/threonine protein kinase ULK1; Fer-1,
ferrostatin-1; BNP, brain natriuretic peptide; β-MHC,
β-myosin heavy chain; α-SMA, α smooth muscle actin; MDA,
malondialdehyde; SOD, superoxide dismutase; Ptgs2,
prostaglandin endoperoxide synthase 2; 4-HNE, 4-hydroxynonenal.

Figure 3

Autophagy activation is required for
ULK1-induced ferroptosis in HL-1 cells. Western blot analysis and
summarized data demonstrating (A) Beclin1, (B) LC3-II and (C) p62
protein levels in HL-1 cells treated with 3-MA. (D) HL-1 cells were
infected with mRFP-GFP-LC3-labeled adenoviruses and the formation
of autophagosomes (yellow) and autolysosomes (red) were observed in
indicated group. Scale bar, 50 µm. (E) Red and yellow dots/cell in
indicated group. (F) Cell viability of HL-1 cells was detected
using a Cell Counting Kit-8 assay in the 3-MA group. The contents
of (G) Fe2+, (H) MDA and (I) SOD were determined in 3-MA
group. (J) The mRNA level of Ptgs2. (K) Immunofluorescence
staining for 4-HNE in 3-MA group. Scale bar, 50 µm. All data are
presented as mean ± SEM. n=3. *P<0.05 and **P<0.01 vs.
control group; #P<0.05 and ##P<0.01 vs.
ULK1 group. ULK1, serine/threonine protein kinase ULK1; 3-MA,
3-methyladenine; LC3, microtubule-associated protein 1 light chain
3; mRFP, monomeric red fluorescent protein; MDA, malondialdehyde;
SOD, superoxide dismutase; Ptgs2, prostaglandin endoperoxide
synthase 2; 4-HNE, 4-hydroxynonenal.

Figure 4

Autophagy inhibition significantly
reduces NCOA4-mediated ferritinophagy induced by ULK1 exposure.
Western blot analysis and summarized data demonstrating (A) NCOA4
and (B) FTH1 protein levels in 3-MA group. (C) The colocalization
of NCOA4 (red) and LC3-II (green) in HL-1 cells was determined by
immunofluorescence in 3-MA group. Scale bar, 50 µm. All data are
presented as mean ± SEM. n=3. *P<0.05 and **P<0.01 vs.
control group; #P<0.05 vs. ULK1 group. ULK1,
serine/threonine protein kinase ULK1; NCOA4, nuclear receptor
coactivator 4; FTH1, ferritin heavy chain 1; 3-MA, 3-methyladenine;
LC3, microtubule-associated protein 1 light chain 3.

Figure 5

NCOA4 knockdown alleviates
ULK1-induced ferroptosis events. (A) Cell viability of HL-1 cells
was assessed using a Cell Counting Kit-8 assay in indicated group.
The levels of (B) Fe2+, (C) MDA and (D) SOD were
determined in each indicated group. (E) The mRNA levels of
Ptgs2. (F) Immunofluorescence staining for 4-HNE in
indicated group. Scale bar, 50 µm. (G) Western blot analysis and
(H) summarized data demonstrating FTH1 protein levels in the
indicated groups. All data are presented as mean ± SEM. n=3.
*P<0.05 and **P<0.01 vs. control group; #P<0.05
and ##P<0.01 vs. ULK1 group. ULK1, serine/threonine
protein kinase ULK1; NCOA4, nuclear receptor coactivator 4; MDA,
malondialdehyde; SOD, superoxide dismutase; siRNA, small
interfering RNA; NC, negative control; Ptgs2, prostaglandin
endoperoxide synthase 2; 4-HNE, 4-hydroxynonenal; FTH1, ferritin
heavy chain 1.

Figure 6

ULK1 activates ferritinophagy
dependent on Beclin1/VPS34 complex. (A) Cell viability of HL-1
cells was assessed using a Cell Counting Kit-8 assay in the
indicated groups. The levels of (B) Fe2+, (C) MDA and
(D) SOD were determined. (E) The mRNA levels of Ptgs2. (F)
Immunofluorescence staining for 4-HNE in indicated groups. Scale
bar, 50 µm. Western blot analysis and summarized data demonstrating
(G) NCOA4 and (H) FTH1 protein levels in indicated group. (I) The
colocalization of NCOA4 (red) and LC3-II (green) in HL-1 cells was
determined using immunofluorescence staining. Scale bar, 50 µm. All
data are presented as mean ± SEM. n=3. *P<0.05 and **P<0.01
vs. control group; #P<0.05 and ##P<0.01
vs. ULK1 group. ULK1, serine/threonine protein kinase ULK1; VPS34,
PI3K catalytic subunit type 3; siRNA, small interfering RNA; NC,
negative control; MDA, malondialdehyde; SOD, superoxide dismutase;
Ptgs2, prostaglandin endoperoxide synthase 2; 4-HNE,
4-hydroxynonenal; FTH1, ferritin heavy chain 1; NCOA4, nuclear
receptor coactivator 4; LC3, microtubule-associated protein 1 light
chain 3.

Figure 7

Beclin1/VPS34 complex is associated
with NCOA4-mediated ferritinophagy. (A) Cell viability of HL-1
cells was assessed using a Cell Counting Kit-8 assay. The levels of
(B) Fe2+, (C) MDA and (D) SOD were determined. (E) The
mRNA levels of Ptgs2. (F) Immunofluorescence staining for
4-HNE in indicated group. Scale bar, 50 µm. Western blot analysis
and summarized data demonstrating (G) NCOA4 and (H) FTH1 protein
levels in the indicated groups. All data are presented as mean ±
SEM. n=3. *P<0.05 and **P<0.01 vs. control group;
#P<0.05 and ##P<0.01 vs. Beclin1 group.
VPS34, PI3K catalytic subunit type 3; NCOA4, nuclear receptor
coactivator 4; siRNA, small interfering RNA; NC, negative control;
MDA, malondialdehyde; SOD, superoxide dismutase; Ptgs2,
prostaglandin endoperoxide synthase 2; 4-HNE, 4-hydroxynonenal;
FTH1, ferritin heavy chain 1.

Figure 8

Administration of SBI-0206965
ameliorates ferritinophagy in ULK1-overexpressing cells. Western
blot analysis and summarized data demonstrating (A) ULK1, (B) NCOA4
and (C) FTH1 protein levels in HL-1 cells treated with SBI-0206965.
(D) Cell viability of HL-1 cells was assessed using a Cell Counting
Kit-8 assay. (E) The levels of Fe2+. (F)
Immunofluorescence staining for 4-HNE. Scale bar, 50 µm. (G)
Immunofluorescence staining for GPX4. Scale bar, 50 µm. (H)
Immunofluorescence staining for γ-H2AX. Scale bar, 50 µm. (I) The
colocalization of NCOA4 (red) and LC3-II (green) in HL-1 cells was
determined by immunofluorescence staining in the indicated groups.
Scale bar, 50 µm. All data are presented as mean ± SEM. n=3.
*P<0.05 and **P<0.01 vs. control group; #P<0.05
and ##P<0.01 vs. ULK1 group. ULK1, serine/threonine
protein kinase ULK1; NCOA4, nuclear receptor coactivator 4; SBI,
SBI-0206965; FTH1, ferritin heavy chain 1; 4-HNE, 4-hydroxynonenal;
GPX4, glutathione peroxidase 4; LC3, microtubule-associated protein
1 light chain 3.

Figure 9

Mechanisms of ULK1 activating
NCOA4-mediated ferritinophagy via Beclin1/VPS34 complex in
cardiomyocyte hypertrophy. This figure was created using Figdraw
(www.figdraw.com). ULK1, serine/threonine protein
kinase ULK1; NCOA4, nuclear receptor coactivator 4; VPS34, PI3K
catalytic subunit type 3; LC3, microtubule-associated protein 1
light chain 3; TAC, transverse aortic constriction; Ang II,
angiotensin II; ROS, reactive oxygen species.
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Copy and paste a formatted citation
Spandidos Publications style
Zhang Q, Zhang M, Liu Y, Shi P, Qi H, Jiang M, Cao Y and Sun H: ULK1 activates NCOA4‑mediated ferritinophagy via the Beclin1/VPS34 complex in cardiomyocyte hypertrophy. Mol Med Rep 33: 116, 2026.
APA
Zhang, Q., Zhang, M., Liu, Y., Shi, P., Qi, H., Jiang, M. ... Sun, H. (2026). ULK1 activates NCOA4‑mediated ferritinophagy via the Beclin1/VPS34 complex in cardiomyocyte hypertrophy. Molecular Medicine Reports, 33, 116. https://doi.org/10.3892/mmr.2026.13826
MLA
Zhang, Q., Zhang, M., Liu, Y., Shi, P., Qi, H., Jiang, M., Cao, Y., Sun, H."ULK1 activates NCOA4‑mediated ferritinophagy via the Beclin1/VPS34 complex in cardiomyocyte hypertrophy". Molecular Medicine Reports 33.4 (2026): 116.
Chicago
Zhang, Q., Zhang, M., Liu, Y., Shi, P., Qi, H., Jiang, M., Cao, Y., Sun, H."ULK1 activates NCOA4‑mediated ferritinophagy via the Beclin1/VPS34 complex in cardiomyocyte hypertrophy". Molecular Medicine Reports 33, no. 4 (2026): 116. https://doi.org/10.3892/mmr.2026.13826
Copy and paste a formatted citation
x
Spandidos Publications style
Zhang Q, Zhang M, Liu Y, Shi P, Qi H, Jiang M, Cao Y and Sun H: ULK1 activates NCOA4‑mediated ferritinophagy via the Beclin1/VPS34 complex in cardiomyocyte hypertrophy. Mol Med Rep 33: 116, 2026.
APA
Zhang, Q., Zhang, M., Liu, Y., Shi, P., Qi, H., Jiang, M. ... Sun, H. (2026). ULK1 activates NCOA4‑mediated ferritinophagy via the Beclin1/VPS34 complex in cardiomyocyte hypertrophy. Molecular Medicine Reports, 33, 116. https://doi.org/10.3892/mmr.2026.13826
MLA
Zhang, Q., Zhang, M., Liu, Y., Shi, P., Qi, H., Jiang, M., Cao, Y., Sun, H."ULK1 activates NCOA4‑mediated ferritinophagy via the Beclin1/VPS34 complex in cardiomyocyte hypertrophy". Molecular Medicine Reports 33.4 (2026): 116.
Chicago
Zhang, Q., Zhang, M., Liu, Y., Shi, P., Qi, H., Jiang, M., Cao, Y., Sun, H."ULK1 activates NCOA4‑mediated ferritinophagy via the Beclin1/VPS34 complex in cardiomyocyte hypertrophy". Molecular Medicine Reports 33, no. 4 (2026): 116. https://doi.org/10.3892/mmr.2026.13826
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