Abstract
Myostatin (MSTN), a member of the TGF-β superfamily, negatively regulates muscle growth. MSTN inhibition has been known to cause a double-muscled phenotype in skeletal muscle and fibrosis reduction in the heart. However, the role of MSTN in the cardiac extracellular matrix (ECM) needs more studies in various species of animal models to draw more objective conclusions. The main objective of the present study was to investigate whether loss of MSTN affects the cardiac extracellular matrix in pigs. Three MSTN knockouts (MSTN−/−) and three wild type (WT) male pigs were generated by crossing MSTN ± heterozygous gilts and boars. Cardiac ECM and underlying mechanisms were determined post-mortem. The role of MSTN on collagen expression was investigated by treating cardiac fibroblasts with active MSTN protein in vitro. MSTN protein was detected in WT hearts, while no expression was detected in MSTN−/− hearts. The heart-to-body weight ratio was significantly decreased in MSTN−/− pigs. The morphometric analyses, including picrosirius red staining, immunofluorescent staining, and ultra-structural thickness examination of the endomysium, revealed a significant reduction of connective tissue content in MSTN−/− hearts compared to WT. Hydroxyproline, type I collagen (Col1A), and p-Smad3/Smad3 levels were significantly lower in MSTN−/− hearts in vivo. On the contrary, cardiac fibroblasts treated with exogenous MSTN protein overexpressed Col1A and activated Smad and AKT signaling pathways in vitro. The present study suggests that inhibition of MSTN decreases cardiac extracellular matrix.
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The data supporting the findings of this study are available from the corresponding author upon reasonable request.
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Acknowledgements
The authors thank all the staff at the Longxing Pig Farm (Longjing, China) for their assistance with feeding and careing for the animals.
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This work was supported by the National Natural Science Foundation of China (Nos. 31860297 and 32160820) and the Science and Technology Fund of Yanbian University (Grant No. 2021101).
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All authors contributed to the writing, review, and editing. H.-J. Paek contributed to the conception and design of the study; H.-J. Paek and B.-H. Quan collected the field data and samples; H.-M. Choe and Z.-Y. Li performed experiments and analyzed the data statistically; X.-J. Yin provided supervision and funding support.
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Figure S1.
Cells isolated from MSTN−/− pig heart was stained with primary anti-collagen I antibody for validation of cardiac fibroblast cells (200X. DAPI: 4′,6-diamidino-2-phenylindole). (TIF 3191 kb)
Figure S2
. Cross-sectional area of cardiomyocyte was not changed in MSTN−/− heart (a) Representative images of the laminin immunofluorescent staining of WT and MSTN−/− heart to show the cross sectional-area of cardiomyocyte. (b) Statistical analysis data of the cross-sectional area. (n = 3, p > 0.05). (TIF 4631 kb)
Figure S3.
Electrocardiogram (ECG) of 6-month-old WT and MSTN−/− pig (ECG speed: 25 mm/s). ECG of the heart was recorded with the animals in a supine position using electocardiography machine (Neusoft, China). according to the manufacturer’s insructions. (TIF 1375 kb)
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Paek, HJ., Quan, BH., Choe, HM. et al. Myostatin deficiency decreases cardiac extracellular matrix in pigs. Transgenic Res 31, 553–565 (2022). https://doi.org/10.1007/s11248-022-00322-w
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DOI: https://doi.org/10.1007/s11248-022-00322-w