Rafael Stuani Floriano

Hypertension and aging are associated with endothelial dysfunction in skeletal muscle, potentially compromising exercise capacity. Although high-intensity interval training (HIIT) has been proposed as an effective non-pharmacological strategy for individuals with hypertension, its effects on endothelial-related markers in aging skeletal muscle remain unclear This study investigated the impact of HIIT on endothelial-related gene expression in hypertensive aging rats. Twenty male 12-month-old spontaneously hypertensive rats were assigned to sedentary (n = 10) or trained (n = 10) groups. The training protocol (50 min/day, 5 times/week) was performed for 8 weeks, on a treadmill adapted for rodents. Gene expression of nitric oxide synthase 3 ( NOS3 ), vascular endothelial growth factor ( VEGF ), endothelin-1 ( EDN1 ), and phosphoinositide-3-kinase regulatory subunit 2 ( PIK3R2 ) was assessed by reverse transcription quantitative polymerase chain reaction and by reanalysis of transcriptomic datasets available in the Gene Expression Omnibus (GSE109657 and GSE163356). Eight weeks of HIIT reduced blood pressure and improved physical performance, without altering the expression of NOS3, VEGF, EDN1 , and PIK3R2 in the soleus and diaphragm muscles. These findings are consistent with an independent transcriptomic dataset from vastus lateralis biopsies of healthy subjects performing HIIT protocols (4 times/week for 6 weeks); however, changes were identified in NOS3 , VEGF, and PIK3R2 genes in the vastus lateralis muscle after an HIIT protocol performed 3 times/week for 3 weeks. Collectively, the results indicate that HIIT improves functional capacity and blood pressure regulation in hypertensive aging rats, without concomitant changes in endothelial-related gene expression. These results are comparable with those found in healthy individuals undergoing a similar period of HIIT.

The venom of Bothrops bilineatus, an Amazonian arboreal viper, induces neurotoxicity in mammalian nerve-muscle preparations that is characterized by initial neuromuscular facilitation followed by irreversible blockade. Up until now, the toxins responsible for the neuromuscular excitatory action of this venom have remained unidentified. In this study, we characterized two presynaptically active peptides from B. bilineatus venom using mass spectrometry and electrophysiological analysis at the neuromuscular junction. Fractionation by size-exclusion chromatography yielded eight fractions, with fraction P8 (15 μg/ml) inducing an increase in the twitch amplitude recorded in the mouse phrenic nerve-diaphragm (PND) preparations. Mass spectrometry identified two tripeptides, P8-1 (pEKW) and P8-2 (pENW), in this fraction. Peptide P8-1 was prominently involved in the neuromuscular facilitation and increased the frequency of miniature end-plate potentials (MEPPs) in a manner comparable to the whole fraction (P8). This study provides the first identification of bioactive tripeptides with presynaptic neuromodulatory effects in a Viperidae venom. These findings enhance our understanding of snake venom neurotoxicity and support the potential use of venom-derived peptides as tools for studying synaptic physiology and as templates for novel neuroactive therapeutics.

Snake venom is a complex mixture of bioactive substances known to induce a wide range of toxic effects. We investigated the potential of baru flour (Dipteryx alata) in mitigating the neuromuscular blockade induced by Lachesis muta muta venom. Three pharmacological models (pre-incubation, post-venom, and pre-venom), with n=6 each, were exposed to isolated nerve-muscle preparations and conventional myographic technique. Muscular changes were further examined through light microscopy, and protein composition was analyzed via SDS-PAGE electrophoresis for both venom and baru:venom mixture. A concentration of 1000 µg/mL baru flour was chosen for subsequent assays against the venom (100 µg/mL), which inhibited 62.1 % of the contractile response. Notably, pre-incubation with baru flour resulted in a significant 30.6 % inhibition (p<0.05), while post-venom and pre-venom models showed no significant effects. Myotoxicity (in %) revealed that venom affected 35.3 ± 1.7 to cells, while pre-incubation,19.3 ± 1.9 (p<0.05). Electrophoresis analysis of the mixture containing baru flour and venom revealed a reduction in venom-specific proteins, a finding corroborated by a decrease in protein concentration (in µg/mL) from 6.8 ± 0.001 to 1.4 ± 0.003. Concluding, baru flour possesses significant potential as an inhibitor against paralysis and myotoxicity induced by Lachesis venom, a promising therapeutic application.

Background: We compared the enzymatic, coagulant, and neuromuscular activities of two variants (yellow—CDRy and white—CDRw) of Crotalus durissus ruruima venom with a sample of C. d. terrificus (CDT) venom and examined their neutralization by antivenom against CDT venom. Methods: The venoms were screened for enzymatic and coagulant activities using standard assays, and electrophoretic profiles were compared by SDS-PAGE. Neutralization was assessed by preincubating venoms with crotalic antivenom and assaying the residual activity. Results: SDS-PAGE showed that the venoms had similar electrophoretic profiles, with the main bands being phospholipase A₂ (PLA₂), serine proteinases, L-amino acid oxidase (LAAO), and phosphodiesterase. CDRy venom had the highest proteolytic and LAAO activities, CDRw venom had greater PLA₂ and esterolytic activities at the highest quantity tested, and CDT had greater PLA₂ activity than CDRy. CDRw and CDT venoms had similar proteolytic and LAAO activities, and CDRy and CDT venoms had comparable esterolytic activity. None of the venoms altered the prothrombin time (PT), but all of them decreased the activated partial thromboplastin time (aPPT); this activity was neutralized by antivenom. The minimum coagulant dose potency was CDRw >> CDRy > CDT. All venoms had thrombin-like activity that was attenuated by antivenom. CDRy and CDRw venoms showed α-fibrinogenolytic activity. All venoms partially cleaved the β-chain. CDRy and CDT venoms caused neuromuscular facilitation (enhanced muscle contractions) followed by complete blockade, whereas CDRw venom caused only blockade. Antivenom neutralized the neuromuscular activity to varying degrees. Conclusions: These findings indicate that while CDR and CDT venoms share similarities, they also differ in some enzymatic and biological activities and in neutralization by antivenom. Some of these differences could influence the clinical manifestations of envenomation by C. d. ruruima and their neutralization by the currently used therapeutic antivenom.

Coralsnakes (Micrurus spp.) are the only elapids found throughout the Americas. They are recognized for their highly neurotoxic venom, which is comprised of a wide variety of toxins, including the stable, low-mass toxins known as three-finger toxins (3FTx). Due to difficulties in venom extraction and availability, research on coralsnake venoms is still very limited when compared to that of other Elapidae snakes like cobras, kraits, and mambas. In this study, two previously described 3FTx from the venom of M. corallinus, NXH1 (3SOC1_MICCO), and NXH8 (3NO48_MICCO) were characterized. Using in silico, in vitro, and ex vivo experiments, the biological activities of these toxins were predicted and evaluated. The results showed that only NXH8 was capable of binding to skeletal muscle cells and modulating the activity of nAChRs in nerve–diaphragm preparations. These effects were antagonized by anti-rNXH8 or antielapidic sera. Sequence analysis revealed that the NXH1 toxin possesses eight cysteine residues and four disulfide bonds, while the NXH8 toxin has a primary structure similar to that of non-conventional 3FTx, with an additional disulfide bond on the first loop. These findings add more information related to the structural diversity present within the 3FTx class, while expanding our understanding of the mechanisms of the toxicity of this coralsnake venom and opening new perspectives for developing more effective therapeutic interventions.

Envenomations by snakes represent a neglected health problem in tropical and subtropical countries. In South America, Lachesis muta occasionally causes severe human envenomation, with treatment being conditioned to an unspecific antivenom. In this work, we examined the neutralizing ability of Coutarea hexandra stem bark hydroalcoholic extract (Ch‐E), including the commercial phytochemicals coumarin and quinine, on the neuromuscular blockade induced by L. m. muta venom in mouse phrenic nerve‐diaphragm preparation. Biological assays were performed following conventional myographic technique ex vivo. Ch‐E was phytochemically characterized to detect the presence of coumarin and quinine using analytical methods. Ch‐E and commercial phytochemicals were tested separately or combined under pre‐ and post‐venom incubation protocols. Ch‐E attenuated the venom‐induced neuromuscular blockade only under the pre‐venom incubation protocol. Quinine was not detected in Ch‐E. Commercial coumarin and quinine exhibited a concentration‐dependent counteracting effect on the venom‐induced neuromuscular blockade. The pre‐venom incubation protocol showed to be efficient in attenuating the L. m. muta venom–induced neuromuscular blockade, most likely due to the presence of coumarin derivatives and unknown alkaloids in this extract.

In this work, we examined the action of two South American coralsnake (Micrurus corallinus and Micrurus dumerilii carinicauda) venoms on rat heart function in the absence and presence of treatment with Brazilian coralsnake antivenom (CAV) and varespladib (VPL), a potent phospholipase A₂ inhibitor. Anesthetized male Wistar rats were injected with saline (control) or a single dose of venom (1.5 mg/kg, i.m.) and monitored for alterations in echocardiographic parameters, serum CK-MB levels and cardiac histomorphology, the latter using a combination of fractal dimension and histopathological methods. Neither of the venoms caused cardiac functional alterations 2 h after venom injection; however, M. corallinus venom caused tachycardia 2 h after venom injection, with CAV (given i.p. at an antivenom:venom ratio of 1:1.5, v/w), VPL (0.5 mg/kg, i.p.) and CAV + VPL preventing this increase. Both venoms increased the cardiac lesional score and serum CK-MB levels compared to saline-treated rats, but only the combination of CAV + VPL prevented these alterations, although VPL alone was able to attenuate the increase in CK-MB caused by M. corallinus venom. Micrurus corallinus venom increased the heart fractal dimension measurement, but none of the treatments prevented this alteration. In conclusion, M. corallinus and M. d. carinicauda venoms caused no major cardiac functional alterations at the dose tested, although M. corallinus venom caused transient tachycardia. Both venoms caused some cardiac morphological damage, as indicated by histomorphological analyses and the increase in circulating CK-MB levels. These alterations were consistently attenuated by a combination of CAV and VPL.