russell viper bite effect, Bites by Russell's vipers,Bilateral blindness following Russell's viper bite
Abstract
Local tissue damage following snakebite envenoming remains a poorly researched area. To develop better strategies to treat snakebites, it is critical to understand the mechanisms through which venom toxins induce envenomation effects including local tissue damage. Here, we demonstrate how the venoms of two medically important Indian snakes (Russell's viper and cobra) affect human skeletal muscle using a cultured human myoblast cell line. The data suggest that both venoms affect the viability of myoblasts. Russell’s viper venom reduced the total number of cells, their migration, and the area of focal adhesions. It also suppressed myogenic differentiation and induced muscle atrophy. While cobra venom decreased the viability, it did not largely affect cell migration and focal adhesions. Cobra venom affected the formation of myotubes and induced atrophy.
Cobra venom-induced atrophy could not be reversed by small molecule inhibitors such as varespladib (a phospholipase A2 inhibitor) and prinomastat (a metalloprotease inhibitor), and soluble activin type IIb receptor (a molecule used to promote regeneration of skeletal muscle), although the antivenom (raised against the Indian ‘Big Four’ snakes) has attenuated the effects. However, all these molecules rescued the myotubes from Russell’s viper venom-induced atrophy. This study demonstrates key steps in the muscle regeneration process that are affected by both Indian Russell’s viper and cobra venoms and offers insights into the potential causes of clinical features displayed in envenomed victims. Further research is required to investigate the molecular mechanisms of venom-induced myotoxicity under in vivo settings and develop better therapies for snakebite-induced muscle damage.
Introduction
Skeletal muscle damage around the bite site with the loss of muscle mass and function is one of the major complications associated with snakebite envenoming (SBE)1. This negatively impacts the quality of life for victims and is a significant contributor to SBE-induced permanent disabilities2. Snake venom metalloproteases (SVMPs), phospholipase A2 (PLA2), and three-finger toxins (3FTX) are the most recognised mediators of local myonecrosis found in snake venoms3,4. SVMPs mainly act through the degradation of the extracellular matrix (ECM) around the skeletal muscle, damaging blood vessels and leading to serious implications on muscle regeneration5. PLA2s cause the degradation of skeletal muscle fibres through their characteristic disruptions in the cell membrane6. Similarly, 3FTXs induce myonecrosis through pore formation on cell membranes leading to the release of intracellular contents, including biomarkers such as creatine kinase and lactate dehydrogenase5. Skeletal muscle has a robust ability to regenerate through the orchestration of various cellular activities following damage.
The key to this regenerative process is the resident muscle stem cells called satellite cells. These mononucleated cells are found in a quiescent state in an undamaged muscle7. However, following damage, they undergo several changes including proliferation, migration to the site of damage, fusion and hypertrophy, all of which are required to replace the lost muscle fibres8. Perturbations in any of these processes result in attenuated or improper regeneration.
The current understanding of the pathogenesis of SBE-induced myonecrosis points out that the individual and/or synergistic actions of venom toxins directly or indirectly contribute to this process9. Notably, the differential expression of key toxins in elapid (e.g. cobra) and viper (e.g. Russell’s viper) venoms exhibit varying patterns in clinical profiles and muscle damage2. Elapid venoms are generally shown to possess high levels of membranolytic PLA2s and 3FTXs, while viper venoms are rich in SVMPs, and PLA2s10. As a result, the incidence of long-term musculoskeletal disorders in victims of elapid bites is relatively low compared to viper bites, in which the process of innate muscle regeneration is significantly impeded2. The underlying molecular mechanisms resulting in poor skeletal muscle regeneration following exposure to viper and some elapid venoms are poorly understood11. Given that muscle regeneration is underpinned by several phases, it is important to identify which step is affected by a particular venom to understand the basis of the musculoskeletal disorders exhibited by victims.
Currently, antibody (antivenom)-based therapy is the only effective method for neutralising the venom toxicity12. While antivenoms are critical in saving lives, they have serious limitations, such as batch variation, high production cost and importantly, low efficacy in preventing venom-induced local envenomation effects including muscle damage13.
Several alternative methods have been studied in recent years to develop the next generation of antivenoms14. The repurposing of small molecule inhibitors such as PLA2 inhibitor, varespladib has shown the potential to revolutionise SBE treatments15. However, the pharmacological assessment of small molecule inhibitors to date has mainly focused on the neutralisation of venom-induced lethality and systemic effects16. Clinical trials are currently ongoing to determine the efficacy of one (varespladib) of these repurposed broad-spectrum therapeutics for the SBE17.
SVMP inhibitors such as prinomastat are also being widely studied to determine their effects in neutralising venom-induced complications. However, the impact of these small molecule inhibitors and other potential molecules such as regenerative medicine approaches that safeguard muscle has not been studied in sufficient detail to determine their efficacy in neutralising venom-induced muscle damage using robust cell-based assays and in vivo animal models. From a translational perspective, the AB1190,
a human-immortalised myoblast cell line, emerges as an attractive research tool for screening a range of therapeutic molecules and pathological studies relating to skeletal muscle damage18. They can be cultured in vitro as undifferentiated cells in growth media and then differentiated into multinucleated myotubes. This cell line acts as a robust system for dissecting the mechanisms of myogenesis and its modulation by pharmacological agents or biologically active compounds19. Here, we evaluated the impact of the venoms of two medically important Indian snakes, Russell’s viper (Daboia russelii) and cobra (Naja naja) on myoblasts as their bites are known to induce significant muscle damage in humans.
Moreover, we determined the effects of varespladib, prinomastat and soluble activin type IIB-receptor [sActRIIB, a molecule that has been widely used to promote muscle growth under diverse pathological settings20 as well as a polyvalent antivenom (raised against the venoms of Russell’s viper, cobra, krait, and saw-scaled viper) to mitigate the muscle damage induced by these venoms.
Methods
Materials used in this study
Lyophilized venoms of D. russelii (lot number: 0120) and N. naja (lot number: 0120) were obtained from Kentucky Reptile Zoo, USA. These are pooled venoms collected from multiple specimens of the same species. The venoms were dissolved in phosphate-buffered saline (PBS) to prepare stock solutions and further diluted at required concentrations in a culture medium before use. The antivenom raised against the venoms of the Indian ‘Big Four’ snakes (Russell’s viper, cobra, krait, and saw-scaled viper) was obtained from Bharat Serums and Vaccines Limited, India (Batch Number: A05317068). Small molecule inhibitors, varespladib and prinomastat were obtained from Sigma Aldrich, UK. Recombinantly expressed soluble activin type IIB-receptor (sActRIIB) was a kind gift from Dr Ritvos, Helsinki, Finland.
Cell culture
The AB1190 is an immortalised myoblast cell line developed by Dr Bigot at INSERM, France using muscle cells isolated from the paravertebral muscle of a disease-free 16-year-old male. This cell line was used as an in vitro tool in this study to determine the impact of venoms. AB1190 myoblasts were routinely maintained in 75 cm2 flasks in skeletal muscle cell growth medium (GM) with the supplied supplement mix (Product Number: C-23160, PromoCell, Germany), and by incubating in a humidified atmosphere of 5% CO2 at 37 °C. The medium was refreshed every other day. The enzymatic disaggregation was performed using 3 mL of recombinant trypsin, TrypLE™ (ThermoFisher Scientific, UK) for sub-culturing to avoid high confluence and contact inhibition or transformation, or for seeding new plates. All our experimental studies were performed in passages 6–12 of AB1190 cells. No significant changes in any parameters including doubling times were noticed during these passages.
Cell viability assay
The CellTiter96® AQueous one solution Cell Proliferation Assay kit that includes 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Promega, UK) was used in AB1190 cells to determine the effects of venoms on myoblast viability. The cells were seeded at a density of 10,000 cells (the use of more than 10,000 cells results in high confluence and random myotube formation at 24 h, hence, we used only 10,000 cells in this assay) per well of a 96-well plate in 100 µL of growth medium overnight at 37 °C and 5% CO2 for the cells to adhere. These undifferentiated myoblasts were then exposed to different venom concentrations dissolved in GM for 24 h. A control group was added with only fresh GM. All experiments were performed in triplicates. Following incubation with venoms, 10 µL of MTS reagent was added to each well. The 96-well plate was then incubated for two hours at 37 °C and 5% CO2. Cell viability was estimated based on the absorbance of the formazan (developed from MTS reagent) product, which was measured at 490 nm using a spectrofluorometer (SpectraMax® ID3, Molecular Devices, UK). In addition, the cell morphology was observed and imaged using a digital cell imager through a 10X objective (EVOS XL, ThermoFisher Scientific, UK).
