Gencefe Client Article | Novel Approach to the Treatment of Doxorubicin (DOX)-Induced Cardiomyopathy

The following article is transferred from GENCEFE BIOTECH （Disease Research Newsletter

Doxorubicin (DOX), a widely used anthracycline drug, is among the first-line chemotherapy agents for various hematologic malignancies and solid tumors [1]. However, many patients undergoing long-term DOX treatment experience progressive and irreversible cardiac toxicity, with limited effective treatment options and relatively poor prognosis for DOX-induced cardiotoxicity [2]. Therefore, identifying new treatment targets and gaining a better understanding of potential molecular mechanisms are crucial for the management of patients with DOX-induced cardiac toxicity.

In September 2023, Professor Ren Jun and Professor Zhang Yingmei's team at Zhongshan Hospital affiliated with Fudan University published an article titled "PHB2 ameliorates Doxorubicin-induced cardiomyopathy through interaction with NDUFV2 and restoration of mitochondrial complex I function" in the journal "Redox Biology" [3]. The research identified a novel role of prohibitin 2 (PHB2) in mitochondrial dynamics and energy metabolism. Forced overexpression of PHB2 may be considered a promising therapeutic approach for patients with DOX-induced cardiomyopathy.

In this study, siRNA and shRNA designed and synthesized by GENCEFE were employed, along with wild-type and mutant plasmids of PHB2 and NDUFV2.

Research Results:

1. Cardiac-specific deficiency of PHB2 exacerbates DOX-induced cardiomyopathy in mice; Cardiac overexpression of PHB2 alleviates DOX-induced cardiomyopathy

Analysis of differentially expressed proteins in cardiac lysates of mice treated with DOX and the control group revealed that mitochondria, inner mitochondrial membrane (IMM), and mitochondrial ribosomes were the most significantly differentially expressed cellular components. Under the influence of DOX, the protein expression of PHB2 was markedly downregulated, while mRNA levels remained unchanged. Subsequent validation using a cardiac-specific PHB2 knockout mouse model confirmed that cardiac-specific PHB2 knockout worsened DOX-induced cardiomyopathy both in vivo and in vitro.

To assess the functional gain of PHB2 in DOX-induced cardiomyopathy, AAV9-mediated transfection of PHB2 into mice was performed [4]. Three weeks after injection, multiple indicators demonstrated that cardiac overexpression of PHB2 using AAV9 mitigated cardiac dysfunction induced by DOX. (Figure 1)

2. PHB2 deficiency induces mitochondrial fragmentation in the hearts of mice through impairing mitochondrial OXPHOS, and exacerbates mitochondrial dysfunction in cardiomyocytes induced by DOX.

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Previous studies have indicated that PHB2 primarily regulates mitochondrial dynamics by modulating the processing and stability of the mitochondrial fusion protein OPA1 [5-7]. This study further evaluated the mitochondrial morphology and function of heart-specific PHB2 knockout mice. Both in vivo and in vitro experimental data suggest that PHB2 deficiency intensifies DOX-induced mitochondrial fragmentation.

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Quantitative proteomics with TMT labeling of heart lysates from PHB2flox/flox-DOX and PHB2CKO-DOX mice identified 1275 differentially expressed proteins. KEGG analysis revealed that mitochondrial metabolic pathways, such as oxidative phosphorylation, the citric acid cycle, fatty acid degradation, and glycolysis, were among the most enriched pathways (Figure 2A). The study focused on mitochondrial respiratory electron transport chain (ETC) and oxidative phosphorylation. Protein analysis showed a significant decrease in ETC subunit protein levels in the hearts of PHB2CKO-DOX mice compared to PHB2flox/flox-DOX mice (Figure 2C). Validation through RT-qPCR and immunoblot confirmed a decrease in nuclear-encoded OXPHOS subunits at both mRNA and protein levels in heart lysates of PHB2CKO-DOX mice. This demonstrates that PHB2 deficiency impairs the protein expression and function of mitochondrial complex I in the hearts of mice under DOX-induced stress."

PHB2 expression in primary cardiomyocytes was modulated by infecting with adenovirus carrying shRNA targeting PHB2. Multiple experiments indicate that the action of DOX leads to various mitochondrial dysfunctions in cardiac cells, and these effects are exacerbated in the case of PHB2 silencing/downregulation (Figure 3). The data above suggest that PHB2 deficiency intensifies DOX-induced mitochondrial dysfunction and in vitro cytotoxicity.

3. PHB interacts with NDUFV2 to regulate its expression levels; Overexpression of PHB2 alleviates mitochondrial dysfunction induced by DOX in cardiac cells through NDUFV2.

Among the identified 1275 differentially expressed proteins, six candidate proteins located in the mitochondria were selected, and further analysis revealed NDUFV2 as the top-ranked candidate. A series of experiments demonstrated the interaction between PHB2 and NDUFV2, with PHB2 deficiency leading to downregulation of NDUFV2.

?Manipulation of PHB2 and NDUFV2 expression in cardiomyocytes was achieved using adenovirus vectors encoding PHB2 or targeting NDUFV2 (Figure 7A). Experimental results indicate that overexpression of PHB2 can improve/reverse DOX-induced mitochondrial dysfunction. However, in most cases, downregulation/silencing of NDUFV2 weakens/eliminates this effect (Figure 4).

4. PHB2 regulates the expression of NDUFV2 by promoting its stability.

Silencing of PHB2 significantly accelerates the degradation of NDUFV2 in cardiac cells treated with DOX, suggesting a potential role of PHB2 in the stability of NDUFV2 (Figure 5A and B). PHB1 and PHB2 form oligomeric ring structures in the inner mitochondrial membrane (IMM) in coordination with the m-AAA protease AFG3L2, inhibiting its proteolytic activity [8-9]. Experiments confirm that PHB2 deficiency allows AFG3L2 to interact with NDUFV2 (Figure 5C). Depletion of AFG3L2 rescues the decreased protein levels of NDUFV2 in PHB2-deficient cardiac cells, indicating the necessity of AFG3L2 for NDUFV2 degradation (Figure 5D and E). These results suggest that PHB2 stabilizes NDUFV2 by inhibiting the proteolytic activity of AFG3L2, limiting its interaction with the substrate NDUFV2.

DOX is a widely used anticancer drug in clinical treatment for solid tumors and hematological malignancies, but its use is restricted to some extent due to cardiotoxicity. This study emphasizes that overexpression of PHB2 promotes mitochondrial bioenergetic metabolism by stabilizing NDUFV2, making it a potential therapeutic target for DOX-induced cardiomyopathy. The findings of this study align with previous perspectives on the role of PHB2 in cardiovascular diseases [10] and further support PHB2 as a crucial regulator of mitochondrial homeostasis.

?References:

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