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. 2023 Aug 22;12(9):1657.
doi: 10.3390/antiox12091657.

Flt3 Activation Mitigates Mitochondrial Fragmentation and Heart Dysfunction through Rebalanced L-OPA1 Processing by Hindering the Interaction between Acetylated p53 and PHB2 in Cardiac Remodeling

Kaina Zhang  1   2 , Yeqing Zheng  1   2 , Gaowa Bao  1   2 , Wenzhuo Ma  1   2 , Bing Han  1   2 , Hongwen Shi  1   2 , Zhenghang Zhao  1   2
Affiliations

Flt3 Activation Mitigates Mitochondrial Fragmentation and Heart Dysfunction through Rebalanced L-OPA1 Processing by Hindering the Interaction between Acetylated p53 and PHB2 in Cardiac Remodeling

Kaina Zhang et al. Antioxidants (Basel). .

Abstract

Recent studies have shown that FMS-like receptor tyrosine kinase 3 (Flt3) has a beneficial effect on cardiac maladaptive remodeling. However, the role and mechanism of Flt3 in mitochondrial dynamic imbalance under cardiac stress remains poorly understood. This study aims to investigate how Flt3 regulates p53-mediated optic atrophy 1 (OPA1) processing and mitochondrial fragmentation to improve cardiac remodeling. Mitochondrial fragmentation in cardiomyocytes was induced by isoprenaline (ISO) and H2O2 challenge, respectively, in vitro. Cardiac remodeling in mice was established by ligating the left anterior descending coronary artery or by chronic ISO challenge, respectively, in vivo. Our results demonstrated that the protein expression of acetylated-p53 (ac-p53) in mitochondria was significantly increased under cell stress conditions, facilitating the dissociation of PHB2-OPA1 complex by binding to prohibitin 2 (PHB2), a molecular chaperone that stabilizes OPA1 in mitochondria. This led to the degradation of the long isoform of OPA1 (L-OPA1) that facilitates mitochondrial fusion and resultant mitochondrial network fragmentation. This effect was abolished by a p53 K371R mutant that failed to bind to PHB2 and impeded the formation of the ac-p53-PHB2 complex. The activation of Flt3 significantly reduced ac-p53 expression in mitochondria via SIRT1, thereby hindering the formation of the ac-p53-PHB2 complex and potentiating the stability of the PHB2-OPA1 complex. This ultimately inhibits L-OPA1 processing and leads to the balancing of mitochondrial dynamics. These findings highlight a novel mechanism by which Flt3 activation mitigates mitochondrial fragmentation and dysfunction through the reduction of L-OPA1 processing by dampening the interaction between ac-p53 and PHB2 in cardiac maladaptive remodeling.

Keywords: FMS-like receptor tyrosine kinase 3; OPA1; cardiac remodeling; mitochondrial dynamics imbalance; p53; prohibitins.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Effects of Flt3 activation on the protein expression of L-OPA1, S-OPA1, ac-p53, and PHBs in the mitochondria of ISO or H2O2-treated NRCMs in vitro. (A,B) Representative western blots showing the effects of FL treatment on the expression of L-OPA1, S-OPA1, ac-p53, PHB1, and PHB2 in the mitochondria of ISO or H2O2-treated NRCMs, and the quantitative analysis (n = 3 independent experiments). Data were expressed as mean ± SD, * p < 0.05, ** p < 0.01 vs. CON, # p < 0.05, ## p < 0.01 vs. ISO/H2O2.
Figure 2
Figure 2
Flt3 activation decreases L-OPA1 processing via SIRT1/p53 pathway in vitro. (A,B) Representative western blots showing the effects of FL treatment, p53 inhibitor PFT-α, and SIRT1 inhibitor EX527 on the expression of L-OPA1, S-OPA1, SIRT1, p53, ac-p53, PHB1, and PHB2 in ISO or H2O2-treated NRCMs, and the quantitative analysis (n = 3 independent experiments). β-actin was used as an internal reference of cytoplasmic proteins, while COXIV was that of mitochondrial lysate proteins. Data were expressed as mean ± SD, * p < 0.05, ** p < 0.01 vs. CON, # p < 0.05, ## p < 0.01 vs. ISO/H2O2, & p < 0.05, && p <0.01 vs. ISO/H2O2 + FL.
Figure 3
Figure 3
Flt3 activation restores ISO- or H2O2-induced mitochondrial dynamics imbalance by reducing L-OPA1 processing in vitro (A) Representative fluorescence images of mitochondrial morphology of NRCMs challenged by H2O2, FL, or in combined with PFT-α or EX527 intervention by using Mito-Tracker Green probe (the scale, 20 µm). The figure below shows a zoomed-in view of the red boxed region. (B) Quantitative analysis of fragmented, hyperfused (at least one mitochondrion > 5 µm in length), intermediate (at least one mitochondrion between 5 to 2 µm but none more the 5 µm in length), and rounded (none longer than 2 µm) mitochondria (n = 125 cells/group). Data were expressed as mean ± SD, ** p < 0.01 vs. CON, # p < 0.05, ## p < 0.01 vs. ISO/H2O2, && p < 0.01 vs. ISO/H2O2 + FL.
Figure 4
Figure 4
Flt3 activation inhibits H2O2-induced ROS and apoptosis by improving mitochondrial dynamics disturbance in vitro. (A) Representative photographs and quantification of ROS by DCFH-DA probe in NRCMs pretreated with FL, PFT-α, and EX527 followed by exposure to H2O2, n = 10 random fields (Scale bar, 100 μm). (B) Quantitative data of the Opa1 mRNA expression after treatment with OPA1 siRNA or to negative control (NC). (C) Representative photographs and quantification of ROS by DCFH-DA probe in NRCMs pretreated with FL and siOPA1 followed by exposure to H2O2, n = 10 random fields (Scale bar, 100 μm). (D,E) Representative photographs and quantification of apoptosis by Annexin V-FITC/PI probe in NRCMs exposed to H2O2 and performed subsequent drug intervention, n = 50 individual cells (Scale bar, 500 μm). Data were expressed as mean ± SD, * p < 0.05, ** p < 0.01 vs. CON/NC, ## p < 0.01 vs. ISO/H2O2/siOPA1, && p < 0.01 vs. ISO/H2O2 + FL.
Figure 4
Figure 4
Flt3 activation inhibits H2O2-induced ROS and apoptosis by improving mitochondrial dynamics disturbance in vitro. (A) Representative photographs and quantification of ROS by DCFH-DA probe in NRCMs pretreated with FL, PFT-α, and EX527 followed by exposure to H2O2, n = 10 random fields (Scale bar, 100 μm). (B) Quantitative data of the Opa1 mRNA expression after treatment with OPA1 siRNA or to negative control (NC). (C) Representative photographs and quantification of ROS by DCFH-DA probe in NRCMs pretreated with FL and siOPA1 followed by exposure to H2O2, n = 10 random fields (Scale bar, 100 μm). (D,E) Representative photographs and quantification of apoptosis by Annexin V-FITC/PI probe in NRCMs exposed to H2O2 and performed subsequent drug intervention, n = 50 individual cells (Scale bar, 500 μm). Data were expressed as mean ± SD, * p < 0.05, ** p < 0.01 vs. CON/NC, ## p < 0.01 vs. ISO/H2O2/siOPA1, && p < 0.01 vs. ISO/H2O2 + FL.
Figure 5
Figure 5
The mRNA expression of mitochondrial fusion and fission genes in ISO- or H2O2-induced mitochondrial dynamics dysfunction in NRCMs. Quantitative data of the expression of mitochondrial fission and fusion-related genes (Mfn2, Opa1, Fis1, Drp1) in the different groups of NRCMs evoked by ISO (A) or H2O2 (B) (n = 3 independent experiments). Data were expressed as mean ± SD, * p < 0.05, ** p < 0.01 vs. CON, # p < 0.05, ## p < 0.01 vs. ISO/H2O2, & p < 0.05, && p < 0.01 vs. ISO/H2O2 + FL.
Figure 6
Figure 6
The interaction between PHBs and ac-p53 in ISO-stimulated NRCMs. (A,B) Representative western blot images showing the expression of OPA1, ac-p53, PHB1, and PHB2 in ISO-treated NRCMs. Cell lysates were immunoprecipitated with IgG (control; lane 3) or PHB1/PHB2 antibody. Protein-protein interaction was determined by western immunoprecipitation. Representative western blot images and the quantitative analysis showed that ISO significantly increased the interaction of ac-p53 and PHB2 but not PHB1 in NRCMs (n = 3 independent experiments). Data were expressed as mean ± SD, * p < 0.05, ** p < 0.01 vs. CON.
Figure 7
Figure 7
Acetylated p53 is necessary for the ISO- or H2O2-induced dissociation of OPA1 from the OPA1-PHB2 complex in cardiomyocytes. (A) H9c2 cells were transfected with wild-type p53 (WTp53), lysine 371 mutant p53 (p53 K371R) plasmid. The overexpression was confirmed by the p53 antibody in the whole cell lysates. Protein−protein interaction was determined by western immunoprecipitation. Representative western blot images and the quantitative analysis showed that p53 K371R significantly enhanced the interaction of OPA1 and PHB2 and decreased that of PHB2 and ac-p53 (n = 3 independent experiments). (B) Representative western blots for the expression of p53 K371R plasmids. Plasmids were transfected into H9c2 cells and then treated with ISO or H2O2 for testing the changes of p53, ac-p53, and PHB2. Data were expressed as mean ± SD, ** p < 0.01 vs. NC, ## p < 0.01 vs. WTp53.
Figure 8
Figure 8
Flt3 activation enhances ISO- or H2O2-induced reduction of OPA1 and PHB2 colocalization within NRCM mitochondria. (A,B) Cultured NRCMs in the different groups were contained with anti-OPA1 antibody (green), anti-PHB2 antibody (red), and DAPI (Scale bar, 50 μm). Representative fluorescence images were shown, and the colocalization of OPA1-PHB2 based on Spearman’s correlation in the merged images was analyzed (n = 30 cells from 3 independent experiments). Data were expressed as mean ± SD, ** p < 0.01 vs. CON, # p < 0.05, ## p < 0.01 vs. ISO/H2O2, && p < 0.01 vs. ISO/H2O2 + FL.
Figure 9
Figure 9
Flt3 activation improves the cardiac dysfunction of mice induced by LAD. (A) Representative images of electrocardiographic (ECG) recordings in the four groups at 28 days post-MI (n = 5). (B) Representative M-mode echocardiography images of mice in the different groups and bar graphs showing quantification of left ventricular internal diastolic diameter (LVIDd), left ventricular internal systolic diameter (LVIDs), left ventricular ejection fraction (LVEF%), left ventricular fractional shortening (LVFS%), left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV) (n = 5). Data were expressed as mean ± SD, * p < 0.05 vs. sham, # p < 0.05, ## p < 0.01 vs. MI.
Figure 10
Figure 10
Flt3 activation ameliorates LAD-induced cardiac remodeling in mice. (A) Representative images of hematoxylin and eosin staining of the transection of the mouse hearts showing the gross cardiac morphology (Scale bar, 1 mm in the upper, 500 μm in the bottom) and quantitative analysis of relative infarct size (n = 5). (B) Representative images and quantitative analysis of Masson’s trichrome intensity calculated from the Masson-stained heart (n = 5). The figures below show the zoomed-in view of the black boxed region. Data were expressed as mean ± SD, ** p < 0.01 vs. sham, ## p < 0.01 vs. MI.
Figure 11
Figure 11
Flt3 activation reduces L-OPA1 processing by hindering interaction between p53 and PHBs in mitochondria in LAD- or ISO-induced cardiac remodeling in vivo. (A) Representative western blots and quantitative analysis of L-OPA1, S-OPA1, SIRT1, p53, ac-p53, PHB1, and PHB2 in heart tissues of mice (n = 3 independent experiments). (B) Representative western blot images showing the expression of OPA1, ac-p53, PHB1, and PHB2 in mitochondria of MI and MI + FL mouse hearts. Mitochondria fraction was separated and immunoprecipitated with IgG (control; lane 4) or PHB2 antibody. Protein–protein interaction was determined by western immunoprecipitation. Representative western blot images and the quantitative analysis showing that FL significantly decreased the interaction of ac-p53 and PHB2 and increased the interaction of OPA1 and PHB2 in mitochondria compared with MI (n = 3 independent experiments). (C) Representative western blot images showing the expression of OPA1, ac-p53, PHB1, and PHB2 in mitochondria in ISO and ISO + FL mouse hearts. Data were expressed as mean ± SD, * p < 0.05, ** p < 0.01 vs. sham/CON, # p < 0.05, ## p < 0.01 vs. MI/ISO.
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