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ABCC9 knockdown attenuates isoproterenol‑induced myocardial hypertrophy by inhibiting the PI3K/AKT signaling pathway

  • Authors:
    • Qian Peng
    • Rui Chang
    • Linlin Ma
    • Yanfei Li
  • View Affiliations / Copyright

    Affiliations: Graduate School, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, P.R. China, Department of Scientific Research, Shanghai University of Medicine and Health Sciences Affiliated Zhoupu Hospital, Shanghai 201318, P.R. China
    Copyright: © Peng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 60
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    Published online on: December 4, 2025
       https://doi.org/10.3892/mmr.2025.13770
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Abstract

Myocardial hypertrophy (MH) represents an early pathological manifestation that progresses to severe cardiovascular disease (CVD), and its reversal is important for preventing and treating heart failure. Dysregulated expression of ATP‑binding cassette subfamily C member 9 (ABCC9) has been associated with complex CVD pathogenesis, although its precise mechanistic role remains ambiguous. The present study was designed to investigate the protective effects of ABCC9 knockdown against isoproterenol (ISO)‑induced MH and elucidate its underlying molecular mechanisms. AC16 cardiomyocytes were treated with ISO to establish an MH model, in which ABCC9 protein expression was significantly elevated. Fluorescence staining of cardiomyocyte surface area and quantification of MH‑related biomarkers, including atrial natriuretic peptide, brain natriuretic peptide and β‑myosin heavy chain, demonstrated that ABCC9 knockdown effectively attenuated MH and improved cardiac function. Furthermore, western blot analysis and flow cytometry revealed that ABCC9 knockdown not only decreased cardiomyocyte apoptosis but also reduced oxidative stress, as indicated by lower reactive oxygen species levels. Mechanistically, western blotting and mitochondrial membrane potential assays showed that ABCC9 knockdown inhibited the phosphatidylinositol 3‑kinase/protein kinase B (PI3K/AKT) signaling pathway and improved mitochondrial function. Notably, these protective effects were diminished by treatment with the PI3K/AKT activator 740Y‑P. These findings collectively suggest that ABCC9 knockdown protects against MH by inhibiting the PI3K/AKT signaling pathway, thereby alleviating mitochondrial dysfunction and reducing apoptosis and oxidative stress, positioning ABCC9 as a potential therapeutic target for MH treatment.
View Figures

Figure 1

Elevated expression of ABCC9 in the
ISO-induced myocardial hypertrophy model. (A) Cell Counting Kit-8
was used to determine the viability of ISO-induced AC16 cells.
After treating AC16 cardiomyocytes with 10 µM ISO for different
time periods, western blot analysis was performed to assess changes
in ABCC9 protein expression. (B) Representative western blot image
of ABCC9 and (C) semi-quantitative detection of ABCC9 protein
expression levels using ImageJ software. (D) Evaluation of mRNA
expression levels of ANP, BNP and β-MHC in ISO-treated AC16 cells
using reverse transcription-quantitative PCR. After ISO
stimulation, cardiomyocytes were stained with (E) rhodamine
phalloidin and (F) cell surface area was analyzed. After ISO
stimulation, cardiomyocytes were stained with (G) WGA and (H) cell
surface area was analyzed. Fluorescent images of ISO-treated AC16
cells were obtained using a laser confocal microscope, and cell
surface area was quantified using ImageJ software. Scale bar, 20
µm. AC16 cells were treated with ISO (10 µmol/l) for 24 h. n=3;
*P<0.05 and **P<0.01 vs. control. ABCC9, ATP-binding cassette
subfamily C member 9; NC, negative control; ns, not significant;
ISO, isoproterenol; ANP, atrial natriuretic peptide; BNP, brain
natriuretic peptide; β-MHC, β-myosin heavy chain; WGA, wheat germ
agglutinin.

Figure 2

Silencing of ABCC9 attenuates
ISO-induced myocardial hypertrophy in AC16 cells. (A) Western blot
analysis assessed the transfection efficiency of ABCC9 knockdown.
(B) ImageJ was used for semi-quantitative analysis of ABCC9 protein
expression levels. (C) Western blotting and (D) semi-quantitative
analysis confirmed the knockdown efficiency of ABCC9 si-3
transfection in normal cardiomyocytes. (E) Western blot analysis
was used to assess the protein expression levels of ABCC9 in
ISO-treated AC16 cells after ABCC9 knockdown. (F) ImageJ was used
for semi-quantitative analysis of ABCC9 protein expression levels.
(G) Representative western blot images of ANP and BNP. (H) ANP and
(I) BNP protein expression levels were semi-quantified using
ImageJ. Myocardial cells transfected with ABCC9-siRNA for 24 h were
treated with (J) rhodamine-phalloidin and (K) analyzed
quantitatively. Myocardial cells transfected with ABCC9-siRNA for
24 h were treated with (L) WGA staining and (M) analyzed
quantitatively. The ISO-induced surface area of AC16 myocardial
cells was detected using a laser confocal microscope. The cell
surface area was quantified using ImageJ software. Scale bar, 20
µm. AC16 cells were treated with ISO (10 µmol/l) for 24 h. n=3;
*P<0.05 and **P<0.01. NC, negative control; ns, not
significant; ISO, isoproterenol; ABCC9, ATP-binding cassette
subfamily C member 9; ANP, atrial natriuretic peptide; BNP, brain
natriuretic peptide; si, small interfering RNA; WGA, wheat germ
agglutinin; lip, Lipofectamine® 3000 transfection
reagent.

Figure 3

ABCC9 knockdown inhibits apoptosis in
cardiomyocytes. (A) Western blot analysis was used to assess
changes in the expression of apoptosis-related proteins Bax, Bcl-2,
caspase-3 and cleaved caspase-3 in AC16 cells. ImageJ was used for
semi-quantitative analysis of the expression levels of (B) Bax, (C)
Bcl-2, (D) caspase-3, (E) cleaved caspase-3 proteins and (F)
cleaved caspase-3/caspase-3 ratio. (G) Representative flow
cytometry images and (H) quantitative analysis of Annexin V-FITC/PI
staining in AC16 cells. AC16 cells were treated with ISO (10
µmol/l) for 24 h. n=3; *P<0.05 and **P<0.01. NC, negative
control; ns, not significant; ISO, isoproterenol; ABCC9,
ATP-binding cassette subfamily C member 9; Bax, Bcl-2 associated X
protein; Bcl-2, B-cell lymphoma 2 protein; si, small interfering
RNA.

Figure 4

ABCC9 knockdown alleviates oxidative
stress and mitochondrial damage in cardiomyocytes. (A)
Representative fluorescent images of DCFH-DA staining and (B)
semi-quantification of intracellular reactive oxidative species
levels in AC16 cells. Fluorescence intensity analysis of (C) JC-1
aggregate and (D) JC-1 monomer on mitochondrial membrane potential.
(E) Mitochondrial membrane potential representative fluorescence
images. Scale bar, 100 µm. AC16 cells were treated with ISO (10
µmol/l) for 24 h. n=3; *P<0.05 and **P<0.01. NC, negative
control; ns, not significant; ABCC9, ATP-binding cassette subfamily
C member 9; ISO, isoproterenol; DCFH-DA,
2′,7′-dichlorodihydrofluorescein diacetate; si, small interfering
RNA.

Figure 5

Effect of 740Y-P and ABCC9 knockdown
on PI3K/AKT signaling pathway proteins. (A) Western blot bands of
PI3K, p-PI3K, AKT and p-AKT in AC16 cells. Semi-quantitative
analysis of (B) p-PI3K and (C) p-AKT protein expression using
ImageJ. (D) Western blotting with 740Y-P showed a reversal of the
protective effect of ABCC9 knockdown on ISO-induced AC16 cells. (E)
Semi-quantitative analysis of p-PI3K and (F) p-AKT protein
expression with 740Y-P treatment. AC16 cells were treated with ISO
(10 µmol/l) for 24 h. n=3; *P<0.05 and **P<0.01. NC, negative
control; ns, not significant; ABCC9, ATP-binding cassette subfamily
C member 9; ISO, isoproterenol; si, small interfering RNA; PI3K,
phosphatidylinositol-3-kinase; AKT, protein kinase B; p-,
phosphorylated.

Figure 6

ABCC9 knockdown attenuates myocardial
hypertrophy by inhibiting PI3K/AKT signaling pathway. (A) Western
blot bands showing the effect of 740Y-P on ANP and BNP protein
expression. (B) Semi-quantitative analysis of ANP and (C) BNP
protein expression. Reverse transcription-quantitative PCR was used
to assess the mRNA levels of the hypertrophic genes (D) ANP, (E)
BNP and (F) β-MHC in 740Y-P treated AC16 cells. After transfection
and 740Y-P pretreatment, AC16 cells were stained with (G) rhodamine
phalloidin and (H) WGA, and fluorescence images of si-ABCC9 and
740Y-P-treated AC16 cells were obtained using a laser confocal
microscope. Quantitative analysis of cell surface area in (I)
rhodamine phalloidin and (J) WGA stained samples using ImageJ
software. Scale bar, 20 µm. AC16 cells were treated with ISO (10
µmol/l) for 24 h. n=3; *P<0.05 and **P<0.01. NC, negative
control; ABCC9, ATP-binding cassette subfamily C member 9; ISO,
isoproterenol; ANP, atrial natriuretic peptide; BNP, brain
natriuretic peptide; β-MHC, β-myosin heavy chain. si, small
interfering RNA; WGA, wheat germ agglutinin.

Figure 7

ABCC9 knockdown attenuates apoptosis
in cardiomyocytes by inhibiting the PI3K/AKT pathway and
alleviating mitochondrial dysfunction. (A) Western blot bands
showing the effect of 740Y-P on the expression of the
apoptosis-related proteins Bax, Bcl-2, caspase-3 and cleaved
caspase-3 in AC16 cells. Semi-quantitative analysis of (B) Bax, (C)
Bcl-2, (D) caspase-3, (E) cleaved caspase-3 proteins expression
levels and (F) cleaved caspase-3/caspase-3 ratio was performed
using ImageJ. (G) Representative flow cytometry images of Annexin
V/PI staining and (H) quantification of apoptosis rate in AC16
cells. AC16 cells were treated with ISO (10 µmol/l) for 24 h. n=3;
*P<0.05 and **P<0.01. NC, negative control; ns, not
significant; ISO, isoproterenol; ABCC9, ATP-binding cassette
subfamily C member 9; Bax, Bcl-2 associated X protein; Bcl-2,
B-cell lymphoma 2 protein; DCFH-DA,
2′,7′-dichlorodihydrofluorescein diacetate; si, small interfering
RNA.

Figure 8

ABCC9 knockdown attenuates oxidative
stress in cardiomyocytes by inhibiting the PI3K/AKT pathway and
alleviating mitochondrial dysfunction. (A) Representative images of
staining for reactive oxygen species and (B) fluorescence intensity
analysis. After treatment with 740Y-P, the fluorescence intensity
of (C) JC-1 aggregate and (D) JC-1 monomer of mitochondrial
membrane potential in cardiomyocytes was analyzed. (E)
Mitochondrial membrane potential representative fluorescence
images. Scale bar, 100 µm. AC16 cells were treated with ISO (10
µmol/l) for 24 h. n=3; *P<0.05 and **P<0.01. NC, negative
control; ISO, isoproterenol; ABCC9, ATP-binding cassette subfamily
C member 9; DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate;
si, small interfering RNA.

Figure 9

Schematic diagram describing the
PI3K/AKT signaling pathway mechanism by which ABCC9 acts on MH. ISO
binds to the cellular β-adrenergic surface receptor of
cardiomyocytes, while ABCC9 is highly expressed in ISO-mediated
cardiomyocytes, which triggers apoptosis and oxidative stress
through activation of the PI3K/AKT pathway and results in
mitochondrial dysfunction, ultimately leading to MH. ISO,
isoproterenol; ABCC9, ATP-binding cassette subfamily C member 9;
ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide;
β-MHC, β-myosin heavy chain; Bax, Bcl-2 associated X protein;
Bcl-2, B-cell lymphoma 2 protein; ROS, reactive oxygen species; P,
phosphate group; MH, myocardial hypertrophy.
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Copy and paste a formatted citation
Spandidos Publications style
Peng Q, Chang R, Ma L and Li Y: ABCC9 knockdown attenuates isoproterenol‑induced myocardial hypertrophy by inhibiting the PI3K/AKT signaling pathway. Mol Med Rep 33: 60, 2026.
APA
Peng, Q., Chang, R., Ma, L., & Li, Y. (2026). ABCC9 knockdown attenuates isoproterenol‑induced myocardial hypertrophy by inhibiting the PI3K/AKT signaling pathway. Molecular Medicine Reports, 33, 60. https://doi.org/10.3892/mmr.2025.13770
MLA
Peng, Q., Chang, R., Ma, L., Li, Y."ABCC9 knockdown attenuates isoproterenol‑induced myocardial hypertrophy by inhibiting the PI3K/AKT signaling pathway". Molecular Medicine Reports 33.2 (2026): 60.
Chicago
Peng, Q., Chang, R., Ma, L., Li, Y."ABCC9 knockdown attenuates isoproterenol‑induced myocardial hypertrophy by inhibiting the PI3K/AKT signaling pathway". Molecular Medicine Reports 33, no. 2 (2026): 60. https://doi.org/10.3892/mmr.2025.13770
Copy and paste a formatted citation
x
Spandidos Publications style
Peng Q, Chang R, Ma L and Li Y: ABCC9 knockdown attenuates isoproterenol‑induced myocardial hypertrophy by inhibiting the PI3K/AKT signaling pathway. Mol Med Rep 33: 60, 2026.
APA
Peng, Q., Chang, R., Ma, L., & Li, Y. (2026). ABCC9 knockdown attenuates isoproterenol‑induced myocardial hypertrophy by inhibiting the PI3K/AKT signaling pathway. Molecular Medicine Reports, 33, 60. https://doi.org/10.3892/mmr.2025.13770
MLA
Peng, Q., Chang, R., Ma, L., Li, Y."ABCC9 knockdown attenuates isoproterenol‑induced myocardial hypertrophy by inhibiting the PI3K/AKT signaling pathway". Molecular Medicine Reports 33.2 (2026): 60.
Chicago
Peng, Q., Chang, R., Ma, L., Li, Y."ABCC9 knockdown attenuates isoproterenol‑induced myocardial hypertrophy by inhibiting the PI3K/AKT signaling pathway". Molecular Medicine Reports 33, no. 2 (2026): 60. https://doi.org/10.3892/mmr.2025.13770
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