VEGFB is widely expressed in tissues and cells with a strong metabolic phenotype, including cardiac and skeletal myocytes and endothelial cells. It has two different subtypes: heparin-binding VEGFB167 and diffusible VEGFB186 isoforms. VEGFB167 has a heparin-binding carboxy-terminus structure, allowing it to bind specifically to heparin-like sulfated protein polysaccharide on the surface of the cell membrane. The biological functions of VEGFB include promoting angiogenesis, promoting fatty acid transport and resisting apoptosis. Notably, VEGFB plays an important role in inhibiting apoptosis and promoting survival in some pathophysiological conditions. For instance, Li et al. proposed that revascularization of the ischemic border zone was impaired in VEGFB−/− mice in a model of acute myocardial infarction (MI), whereas VEGFB167 therapy enhanced ischemic myocardial revascularization. Additionally, the expression of cleaved caspase-3 in cardiomyocytes decreased significantly after treatment with adeno-associated virus serotype 9-VEGFB for 4 weeks in mice with heart failure induced by aortic coarctation. VEGFB was proven to inhibit apoptosis in cardiomyocytes treated with hypoxic conditions or epirubicin or in the myocardial infarction area induced by left anterior descending coronary artery ligation. Similarly, VEGFB treatment increased Bcl-2 expression while reducing Bax expression in a hypoxia-reoxygenation-induced H9c2 cardiomyocyte injury model in a dose-dependent manner, mechanistically VEGFB promoted cardiomyocyte survival and inhibited autophagy by activating the PI3K/Akt/Bcl-2/Beclin1 signaling pathway. Lal et al. revealed that incubation with exogenous VEGFB (100ng/ml) for 1 h inhibited cardiomyocyte apoptosis by reducing the Bax-to-Bcl-2 gene expression ratio, which has been used as an index of cell apoptosis. In addition, inhibition of VEGFB through siRNA abolished its protective effects in DOX-treated cardiomyocytes. In this experiment, we treated differentiated C2C12 myotubes co-incubated with 20 ng/ml TNF-α and 100 ng/ml VEGFB for 24 hours. TNF-α is a critical inflammatory cytokine elevated in heart failure and one of the key cytokines inducing skeletal muscle atrophy. We observed that VEGFB prominently inhibited TNF-α-induced apoptosis in C2C12 myotubes by upregulating various survival genes and inhibiting apoptosis-related genes.
Skeletal muscle is a tissue with a high level of expression of VEGFB. Exercise has been authenticated to stimulate the expression of VEGFB in skeletal muscle. Kivelä et al. reported the mRNA level of VEGFB in the lateralis muscle of an exercised leg in healthy men was upregulated at 48 h postexercise, which may be one of the early mechanisms involved in skeletal muscle remodeling after high mechanical loading. Long-term endurance exercise was shown to upregulate VEGFB gene expression in male rats, which may result in disturbance to angiogenesis in muscles. VEGFB mRNA expression increased in healthy skeletal muscle at 6 h postexercise. Birot et al. reported that VEGFB mRNA levels increased by 90%, while the VEGFB protein content increased by 72% in rat muscle fibers at the end of an acute running session. In brief, previous studies have indicated that VEGFB is highly expressed in skeletal muscle and markedly regulated by exercise as well. However, the specific role of VEGFB in skeletal muscle remains unclear. Therefore, our team has been conducting relevant experiments to understand the effect of VEGFB in skeletal muscle during exercise. Our previous studies fond that aerobic exercise promotes VEGFR1, which is a specific receptor of VEGFB expression in obese mice induced by a high-fat diet. Additionally, chronic aerobic exercise can significantly increase the mRNA and protein expression of VEGFB in the skeletal muscle of rats with heart failure. We also investigated whether EPS promoted the expression of VEGFR1 in C2C12 myotubes incubated with oleic acid and fatty acid. In this experiment, we revealed that the mRNA and protein expression levels of VEGFB in mature C2C12 myotubes increased prominently due to muscle contraction induced by EPS, suggesting that exercise can regulate the expression of VEGFB in muscle cells. Taken together, VEGFB is highly expressed in skeletal muscle, and exercise can induce the expression of VEGFB both in vivo and in vitro, which suggests that VEGFB is a novel myokine regulated by muscle contraction.
Exercise has a strong antiapoptotic effec. Caspase-3, one of the key apoptotic regulators, cleaves myofibrillar proteins, thereby providing substrates for the ubiquitin–proteasome system. Moreover, proapoptotic molecules, such as B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), and antiapoptotic molecules (e.g., Bcl-2) are involved in skeletal muscle apoptosis. Aerobic exercise has been confirmed to inhibit the expression of caspase-3 and Bax and increase the expression of Bcl-2 in TBI-induced apoptotic neuronal cells, indicating a strong effect in resisting apoptosis. HIIT is an effective exercise strategy that has been demonstrated to downregulate the protein levels of caspase-3 and Bax and to upregulate the Bcl-2 and Bcl-2 to Bax ratios in gastrocnemius muscle of 4T1 breast cancer-bearing mice . In the gastrocnemius muscle, 8 weeks of treadmill exercise increased the Bcl-2/Bax ratio but decreased the caspase-3 mRNA content to inhibit skeletal muscle apoptosis in aging rats. Long-term aerobic exercise training also prevents aging-induced apoptosis in cardiac muscles by decreasing Bax and cleaved caspase-3 and increasing Bcl-2 protein levels while decreasing the Bax/Bcl-2 ratio. Kwak et al. revealed that aging may target the Bcl-2 pathway of apoptosis in the heart; however, 12 weeks of exercise in aging reduced caspase levels and the Bax/Bcl-2 ratio by lowering Bax protein expression while increasing Bcl-2 levels compared with the age-matched sedentary group.
Electrical pulse stimulation (EPS), an in vitro model for skeletal muscle contraction, has been a major model used for the study of in vitro exercise to understand skeletal muscle and its role in the complex multidirectional crosstalk between different tissues and its implications for metabolic regulation. Nedachi et al. reported that EPS for 24 hr at 1 Hz induced visible contraction of myotubes and exercise-like effects. Horie et al. reported that EPS (40 V, 1 Hz, 2 ms) markedly decreased the ROS/RNS redox potential and cell viability and increased the expression of the apoptosis marker annexin V in C2C12 myotubes. Zhao et al. stimulated C2C12 mouse skeletal muscle cells with EPS, and conditioned medium (CM) was collected after 12 h of EPS at 20 V, 1 Hz, and 2 ms. The phosphorylation of NF-κB, which is a protein associated with cell inflammation and apoptosis that indicates apoptosis in endothelial cells, was significantly reduced by the CM-EPS. In this experiment, EPS (20 V, 1 Hz, 2 ms) for 12 h significantly downregulated apoptosis indicator, including Bax and cleaved caspase-3, but promoted the antiapoptotic index Bcl-2, suggesting that EPS could repress apoptosis in skeletal muscle cells.
It has been confirmed that exercise stimulates skeletal muscle contraction to secrete a great variety of myokines, which may play important roles in the benefit of exercise. VEGFB is a novel myokine regulated by muscle contraction. However, whether it is involved in the antiapoptotic effect of exercise is not known. To understand the role of VEGFB in skeletal muscle apoptosis, we used RNAi to interfere with the expression of VEGFB in differentiated C2C12 myotubes. Both the mRNA and protein levels of VEGFB were decreased in myotubes by siRNA. Our results showed that the EPS-mediated prevention of apoptosis weakened when the expression of VEGFB in myotubes was knocked down, suggesting that VEGFB is involved in exercise resistance to skeletal muscle apoptosis.