BNIP3 Downregulation Ameliorates Muscle Atrophy in Cancer Cachexia

Abstract

Background and aims: Cancer cachexia is a complex syndrome affecting most cancer patients and is directly responsible for about 20% of cancer-related deaths. Previous studies showed muscle proteolysis hyper-activation and mitophagy induction in tumor-bearing animals. While basal mitophagy is required for maintaining muscle mass and quality, excessive mitophagy promotes uncontrolled protein degradation, muscle loss and impaired function. BNIP3, a key mitophagy-related protein, is significantly increased in the muscles of both mice and human cancer hosts. This study aimed to define the potential of mitigating mitophagy via BNIP3 downregulation in preserving mitochondrial integrity, counteracting skeletal muscle loss in experimental cancer cachexia.

Methods: Two in vivo gene delivery methods were performed to knock down muscle BNIP3: electroporation of a BNIP3-specific shRNA expression vector or adenovirus injection.

Results: The electroporation effectively reduced muscle BNIP3 in healthy mice but was ineffective in C26 tumor-bearing mice. In contrast, adenovirus-mediated BNIP3 knockdown successfully decreased BNIP3 levels also in tumor hosts. Although BNIP3 knockdown did not impact overall on body or muscle mass, it improved muscle fiber size in C26-bearing miceh2, suggesting partial prevention of muscle atrophy. Mitochondrial respiratory chain complexes (OxPhos) and TOM20 protein levels were consistently rescued, indicating improvements in mitochondrial mass, while H₂O₂ levels were unchanged among the groups, suggesting that BNIP3 downregulation does not impair the endogenous control of oxidative balance.

Conclusions: These findings suggest that a fine balance between mitochondrial disposal and biogenesis is fundamental for preserving muscle homeostasis and highlight a potential role for BNIP3 modulation against cancer-induced muscle wasting.

Keywords: BNIP3; cancer cachexia; mitochondria; mitophagy; muscle wasting.

Figure A1

Total representative immunoblotting showing the expression of BNIP3 (A,E,G), LC3 I/II (C) in skeletal muscle protein extracts. Densitometric analysis is normalized for the corresponding vinculin content and expressed as fold of C SCR (B,D,F) and C26 SCR (H). Representative images at 10× magnification of GFP, H&E and SDH staining performed on tibialis anterior cryostatic section (I). Red boxes highlight the representative immunoblotting presented in the main text. Data are means ± SD of 6 mice in the control group and 11 mice in the C26 group. Statistical significance: ** p < 0.01, **** p < 0.0001 vs. C SCR.

Figure A2

Representative immunoblotting showing the expression of GFP (A) and BNIP3 (B) in GSN, TOM20 (D) and complexes of mitochondrial respiratory chain (F) in TA skeletal muscle. Densitometric analysis is normalized for the corresponding vinculin content and expressed as fold change related to C SCR (C,E,G). Red boxes highlight the representative immunoblotting presented in the main text. Data are means ± SD of 6–8 mice in the control group and 9–10 mice in the C26 group. Statistical significance: * p < 0.05, **** p < 0.0001 vs. C SCR and ### p< 0.001 vs. C26 SCR.

Figure A3

Representative images of subsarcolemmal mitochondria of gastrocnemius muscles obtained with transmission electron microscopy (A); quantification of area (B), circularity (C) and aspect-ratio (D) parameters of mitochondria. White circles correspond to mean values/animal (n = 3), whereas small grey circles correspond to every individual measurement. Statistical analysis: two-tailed Student’s t-test.

Figure 1

Mitophagy induction in C26 tumor-bearing mice. Representative immunoblotting showing the expression of BNIP3 and LC3-I/II in the skeletal muscle (A,C). Densitometric analysis is normalized for the corresponding vinculin content and expressed as fold change related to C SCR (B,D). Body weight (E), GSN (F) and TA (G) weight correlation to BNIP3 protein expression. Data are means ± SD of 6 mice in the control group and 10 mice in the C26 group. Statistical significance: ** p < 0.01 vs. C SCR.

Figure 2

Silencing BNIP3 through electroporation does not prevent its overexpression in the skeletal muscle of tumor-bearing mice. Scheme of experimental protocol and timeline (A). Representative immunoblotting showing the expression of BNIP3 in TA muscle (B,D). Densitometric analysis is normalized for the corresponding vinculin content and expressed as fold of C SCR (C) and C26 SCR (E), respectively. Differences in tibialis anterior (TA) and gastrocnemius (GSN) muscles (F) expressed in milligrams and normalized for 10 g of body weight. TA weight expressed in mg (G). Data are means ± SD of 6 mice in the control group and 11 mice in the C26 group. Statistical significance: ** p < 0.01, **** p < 0.0001 vs. C SCR.3.3. Adenovirus-Mediated BNIP3 Silencing Improves Muscle Atrophy, Preserving Mitochondrial Homeostasis and Oxidative Capacity.

Figure 3

Adenovirus-mediated BNIP3 silencing prevents BNIP3 overexpression in the skeletal muscle of C26 hosts. Scheme of experimental protocol and timeline (A). Representative immunoblotting showing the expression of GFP (B) and BNIP3 (C) in GSN skeletal muscle protein extracts. Densitometric analysis is normalized for the corresponding vinculin content and expressed as fold of C SCR (D). Tibialis anterior (TA) and gastrocnemius (GSN) muscles (E,F), weight expressed in mg and normalized for 10 g of body weight. Data are means ± SD of 6–8 mice in the control group and 9–10 mice in the C26 group. Statistical significance: * p < 0.05, **** p < 0.0001 vs. C SCR and ### p < 0.001 vs. C26 SCR.

Figure 4

BNIP3 silencing via adenovirus improves muscle atrophy while preserving mitochondrial homeostasis and oxidative capacity. Representative images at 10× magnification of H&E staining performed on tibialis anterior cryostatic sections (A). Myofiber cross-sectional area (CSA) of TA muscles expressed as % relative to the C SCR group (B). Representative immunoblotting showing the expression of complexes of mitochondrial respiratory chain (C) and TOM20 (E) in TA skeletal muscle protein extracts. Densitometric analysis is normalized for the corresponding vinculin content and expressed as fold of C SCR (D,F). Evaluation of H2O2 levels in GSN protein extracts (G) expressed as pmol per milligram of protein. Data are means ± SD of 6–8 mice in the control group and 9–10 mice in the C26 group. Statistical significance: * p < 0.05, **** p < 0.0001 vs. C SCR and #### p < 0.0001 vs. C26 SCR.