Suppression of cardiomyocyte functions by ß-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method

Beta-cardiotoxin (ß-CTX), the three-finger toxin isolated from king cobra (Ophiophagus hannah) venom, possesses ß-blocker activity as indicated by its negative chronotropy and its binding property to both ß-1 and ß-2 adrenergic receptors and has been proposed as a novel ß-blocker candidate. Previously, ß-CTX was isolated and purified by FPLC. Here, we present an alternative method to purify this toxin. In addition, we tested its cytotoxicity against different mammalian muscle cell types and determined the impact on cardiac function in isolated cardiac myocyte so as to provide insights into the pharmacological action of this protein. Methods: ß-CTX was isolated from the crude venom of the Thai king cobra using reverse-phased and cation exchange HPLC. In vitro cellular viability MTT assays were performed on mouse myoblast (C2C12), rat smooth muscle (A7r5), and rat cardiac myoblast (H9c2) cells. Cell shortening and calcium transient dynamics were recorded on isolated rat cardiac myocytes over a range of ß-CTX concentration. Results: Purified ß-CTX was recovered from crude venom (0.53% w/w). MTT assays revealed 50% cytotoxicity on A7r5 cells at 9.41 ± 1.14 µM (n = 3), but no cytotoxicity on C2C12 and H9c2 cells up to 114.09 µM. ß-CTX suppressed the extend of rat cardiac cell shortening in a dose-dependent manner; the half-maximal inhibition concentration was 95.97 ± 50.10 nM (n = 3). In addition, the rates of cell shortening and re-lengthening were decreased in ß-CTX treated myocytes concomitant with a prolongation of the intracellular calcium transient decay, indicating depression of cardiac contractility secondary to altered cardiac calcium homeostasis. Conclusion: We present an alternative purification method for ß-CTX from king cobra venom. We reveal cytotoxicity towards smooth muscle and depression of cardiac contractility by this protein. These data are useful to aid future development of pharmacological agents derived from ß-CTX.(AU)

Suppression of cardiomyocyte functions by ß-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method

Beta-cardiotoxin (ß-CTX), the three-finger toxin isolated from king cobra (Ophiophagus hannah) venom, possesses ß-blocker activity as indicated by its negative chronotropy and its binding property to both ß-1 and ß-2 adrenergic receptors and has been proposed as a novel ß-blocker candidate. Previously, ß-CTX was isolated and purified by FPLC. Here, we present an alternative method to purify this toxin. In addition, we tested its cytotoxicity against different mammalian muscle cell types and determined the impact on cardiac function in isolated cardiac myocyte so as to provide insights into the pharmacological action of this protein. Methods: ß-CTX was isolated from the crude venom of the Thai king cobra using reverse-phased and cation exchange HPLC. In vitro cellular viability MTT assays were performed on mouse myoblast (C2C12), rat smooth muscle (A7r5), and rat cardiac myoblast (H9c2) cells. Cell shortening and calcium transient dynamics were recorded on isolated rat cardiac myocytes over a range of ß-CTX concentration. Results: Purified ß-CTX was recovered from crude venom (0.53% w/w). MTT assays revealed 50% cytotoxicity on A7r5 cells at 9.41 ± 1.14 µM (n = 3), but no cytotoxicity on C2C12 and H9c2 cells up to 114.09 µM. ß-CTX suppressed the extend of rat cardiac cell shortening in a dose-dependent manner; the half-maximal inhibition concentration was 95.97 ± 50.10 nM (n = 3). In addition, the rates of cell shortening and re-lengthening were decreased in ß-CTX treated myocytes concomitant with a prolongation of the intracellular calcium transient decay, indicating depression of cardiac contractility secondary to altered cardiac calcium homeostasis. Conclusion: We present an alternative purification method for ß-CTX from king cobra venom. We reveal cytotoxicity towards smooth muscle and depression of cardiac contractility by this protein. These data are useful to aid future development of pharmacological agents derived from ß-CTX.(AU)

Functional analysis of the SAP30 gene and its relation to muscle development

Entender a maquinaria molecular envolvida em determinadas características de interesse econômico é importante para que informações genéticas possam ser aplicadas junto aos programas de melhoramento animal, visando estabelecer maior acurácia na seleção genômica. Estudos de GWAS identificaram regiões de QTL e CNV localizadas no cromossomo 4 na espécie Gallus gallus, em uma população brasileira de frangos de corte, associadas com características de desenvolvimento muscular, como peso e porcentagem de peito. Buscas por genes candidatos presentes nessas regiões e que pudessem estar participando do desenvolvimento muscular foram realizadas. O gene SAP30 foi identificado como um possível candidato ao desenvolvimento muscular. Diante disso, e com o objetivo de validar sua função, SAP30 foi editado usando CRISPR/Cas9 e teve sua expressão reduzida por meio de siRNA, em cultura de mioblastos in vitro. Após essas análises, análises morfométricas foram realizadas na cultura celular para avaliar as alterações ocorridas no tamanho de área dessas células. Posteriormente, as análises de sequenciamento de RNA, genes diferencialmente expressos e enriquecimento funcional foram realizadas para investigar redes gênicas e processos biológicos que as mutações e/ou redução da expressão do SAP30 estariam ocasionando. Os resultados para ambos os trabalhos mostram o envolvimento do SAP30 na hipertrofia de células musculares. Tanto a redução na expressão do gene, quanto a indução de mutações em sua sequência, fizeram com que os miotubos tivessem maior área nos grupos tratados, quando comparado aos controles. As análises de sequenciamento de RNA revelaram, para ambos, uma importante diferença na expressão de genes relacionados ao desenvolvimento muscular, bem como, genes que atuam em vias de formação e regeneração do músculo. Assim, pela primeira vez, apresentamos o gene SAP30 como um possível regulador de hipertrofia muscular.

Understanding the molecular machinery involved in certain traits of economic interest is important because the genetic information can be applied to animal breeding programs, defining greater accuracy in genomic selection. GWAS studies have identified general QTL and CNV regions on chromosome 4 in Gallus gallus species, in a Brazilian broiler population, associated with muscle development traits such as weight and percentage of breast. Searches for candidate genes present in these regions and that could be participating in muscle development were performed. The SAP30 gene was identified as a possible candidate for muscle development. Therefore, and to validate its function, SAP30 was edited using CRISPR/Cas9 and had its expression reduced by means of siRNA, in myoblast culture in vitro. After these analyses, morphometric analyzes were performed in cell culture to evaluate changes in the area size of these cells. Subsequently, RNA sequencing, differentially expressed genes and functional enrichment analyzes were performed to investigate gene networks and biological processes that mutations and/or reduced expression of SAP30 would be causing. The results for both papers presented the involvement of SAP30 in muscle cell hypertrophy. Both the reduction in gene expression and the induction of mutations in its sequence made the myotubes have a larger area in the treated groups when compared to controls. RNA sequencing analyzes revealed, for both, an important difference in the expression of genes related to muscle development, as well as genes that act in muscle formation and regeneration pathways. So, for the first time, we present the SAP30 gene as a possible regulator of muscle hypertrophy.

The c-jun N-terminal kinases (JNK) / mitogen-activated protein kinase (MAPK) is responsible for the protection of tanshinol (Danshensu) upon H2 O2 -induced L6 rat myoblast cell injury

Background: Promoting L6 rat myoblast cell (L6 RMC) survival in the pro-apoptotic environment is critical to myoblastcell replacement for skeletal muscle degenerative disease therapy. Tanshinol (Danshensu), one of the principal bioactivecomponents in salvia miltiorrhiza bunge, has been used widely in skeletal muscle system (SMS) diseases treatment andserves as an antioxidant to protect myoblast cells against oxidative stress. The present study was undertaken to investigatethe protective effects of Tanshinol on L6 RMC injury induced by hydrogen peroxide (H2O2). After challenge with 100 µMH2O2 for 1h, loss of cell viability and excessive apoptotic cell death were observed in cultured L6 RMC, tanshinol treatmentconferred protective effects against the loss of cellular viability in a concentration-dependent manner.Materials, Methods & Results: L6 rat myoblast cells (L6 RMC) were maintained DMEM containing 4.5 g/L glucose andsupplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin on tissue culturefl asks in a 37°C humidifi ed atmosphere of 95% air and 5% CO2. Cells were subcultured every 2-3 days. The cells werestained with SABC-Cy3 fl uorescence and measured by counting the nuclei. L6 RMC viability was determined by MTTassay in 96-well plates. The cells were counter-stained with H & E staining and Hoechst 33342 fl uorescent staining. Cellcycle phase distribution and apoptosis rates were detected by fl ow cytometry. Western blot were repeated three times, andqualitatively similar results were obtained. L6 RMC were pretreated with Tanshinol 375, 187.5, 93.75 mg/mL for 24 h,followed by treatment of 1000, 500, 100 µM H2O2 for 1h (P < 0.05) with viability by MTT method. Tanshinol 187.5 mg/mL pretreatment was protected in L6 RMC from H2O2-induced apoptosis with H&E staining and Hoechst 33342 fl uorescent...(AU)

The c-jun N-terminal kinases (JNK) / mitogen-activated protein kinase (MAPK) is responsible for the protection of tanshinol (Danshensu) upon H2 O2 -induced L6 rat myoblast cell injury

Background: Promoting L6 rat myoblast cell (L6 RMC) survival in the pro-apoptotic environment is critical to myoblastcell replacement for skeletal muscle degenerative disease therapy. Tanshinol (Danshensu), one of the principal bioactivecomponents in salvia miltiorrhiza bunge, has been used widely in skeletal muscle system (SMS) diseases treatment andserves as an antioxidant to protect myoblast cells against oxidative stress. The present study was undertaken to investigatethe protective effects of Tanshinol on L6 RMC injury induced by hydrogen peroxide (H2O2). After challenge with 100 µMH2O2 for 1h, loss of cell viability and excessive apoptotic cell death were observed in cultured L6 RMC, tanshinol treatmentconferred protective effects against the loss of cellular viability in a concentration-dependent manner.Materials, Methods & Results: L6 rat myoblast cells (L6 RMC) were maintained DMEM containing 4.5 g/L glucose andsupplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin on tissue culturefl asks in a 37°C humidifi ed atmosphere of 95% air and 5% CO2. Cells were subcultured every 2-3 days. The cells werestained with SABC-Cy3 fl uorescence and measured by counting the nuclei. L6 RMC viability was determined by MTTassay in 96-well plates. The cells were counter-stained with H & E staining and Hoechst 33342 fl uorescent staining. Cellcycle phase distribution and apoptosis rates were detected by fl ow cytometry. Western blot were repeated three times, andqualitatively similar results were obtained. L6 RMC were pretreated with Tanshinol 375, 187.5, 93.75 mg/mL for 24 h,followed by treatment of 1000, 500, 100 µM H2O2 for 1h (P < 0.05) with viability by MTT method. Tanshinol 187.5 mg/mL pretreatment was protected in L6 RMC from H2O2-induced apoptosis with H&E staining and Hoechst 33342 fl uorescent...

ALT-C, a disintegrin-like Cys-rich protein from Bothrops alternatus, increases skeletal myoblast viability

ALT-C, an ECD motif (glutamic acid, cysteine, aspartic acid) disintegrin from Bothrops alternatus snake venom, induces 21 integrin-mediated signaling and neutrophil chemotaxis. In vitro, in human umbilical vein endothelial cells (HUVEC), ALT-C induces cell proliferation, thus showing an interesting potential for tissue regeneration studies. This work aimed to evaluate the influence of ALT-C in myoblast viability and differentiation. Myoblasts were obtained from hind limb muscles of 3 to 4-day old Wistar rats. The cells were incubated with ALT-C at different concentrations and incubation periods were followed by total RNA isolation. cDNA synthesis and real time polymerase chain reaction (PCR) were performed with primers of myoD as well as of both (slow and fast) myosin heavy chain isoforms (MHC). ECD-disintegrin increased myoblast viability in a dose-dependent way, mostly with 50 to 100 nM concentrations, and such effect was more prevalent after 48 hours. No changes in gene expression of both MHC isoforms were observed in ALT-C-treated cells. MyoD expression was not detected, which suggests that myoblasts were in mature stages. Protease activity and cytokine array tested in a medium of 50 nM ALT-C-treated cells after 48 hours were not different from controls. In conclusion, it was shown that myoblats are sensitive to ALT-C indicating an integrin-mediated intracellular signaling that increases cell viability.

Cytotoxicity and morphological analysis of cell death induced by Bothrops venoms from the northeast of Argentina

Bothrops snake venoms have been proved toxic to a variety of cell types, in both in vivo and in vitro models. Studies on the pharmacological actions of Bothrops venoms from Argentina are relatively scarce and the direct action of the crude venoms has not been assessed using cell culture models. In this work, we investigated the cytotoxicity of crude venoms from B. alternatus and B. diporus in a skeletal muscle (C2C12) cell line, which is commonly used as a model for studying the myotoxic action of snake venom. Both venoms (1.25-50 µg/mL) induced an early and significant decrease in cell viability. The cytotoxic concentration 50 (CC50), determined three hours after exposure, revealed that B. diporus venom was significantly more cytotoxic (CC50: 2 µg/mL) than B. alternatus (CC50: 5.8 µg/mL). To investigate the cell death mechanism involved, myoblast cells were examined by phase contrast microscopy and after acridine orange and ethidium bromide fluorescence staining, respectively. Our data clearly demonstrated that an apoptotic mechanism mediated this cell line destruction. The current study aimed to provide new information on the cytotoxicity mechanisms of Argentine Bothrops snake venoms on a skeletal muscle cell line.

Biochemical and morphological analysis of cell death induced by Egyptian cobra (Naja haje) venom on cultured cells

We investigated the in vitro process of cell death caused by Egyptian cobra venom on primary human embryonic kidney (293T) and mouse myoblast (C2C12) cell lines. The aim of these studies was to provide further information about triggering cell death, and suggest methods for eliminating unwanted cells, such as tumour cells. Both cell lines were treated with 10, 20, and 50 m g/ml of Egyptian cobra (Naja haje) venom in serum free media (SFM) and incubated for 8 hours. Total activities of the lactate dehydrogenase (LDH) and creatine kinase (CK) released in the culture during venom incubation were used as an indicator of the venom in vitro cytotoxicity. Cell injury was morphologically recognized and apoptosis determined by a Fluorescing Apoptosis Detection System and confirmed by staining nuclear DNA with DAPI. Our data clearly demonstrated marked cytotoxic effects and acute cell injury for both cell lines. Release of LDH and CK into the culture media induced by the venom correlates well with the morphological changes and extent of cell death. Mostly, these consequences were time and dose-dependent in both cell lines. The results obtained from this study indicated that cobra venom cause cell death by two different mechanisms: necrosis and induction of apoptosis. The apoptotic mechanism, accompanied by cell necrosis, mediated cell destruction of both tested cell lines; however, necrosis was predominant in the C2C12 cell line while apoptosis, in 293T cells. This unusual form of cell death induced by cobra venom may represent a combination of apoptosis and necrosis within the same cell. This is a first-hand investigation showing the apoptotic effects of N. haje venom at the cellular level. However, the contribution of the apoptotic pathway may be dependent on concentration and/or time of exposure to snake venom.

Biochemical and morphological analysis of cell death induced by Egyptian cobra (Naja haje) venom on cultured cells

We investigated the in vitro process of cell death caused by Egyptian cobra venom on primary human embryonic kidney (293T) and mouse myoblast (C2C12) cell lines. The aim of these studies was to provide further information about triggering cell death, and suggest methods for eliminating unwanted cells, such as tumour cells. Both cell lines were treated with 10, 20, and 50 m g/ml of Egyptian cobra (Naja haje) venom in serum free media (SFM) and incubated for 8 hours. Total activities of the lactate dehydrogenase (LDH) and creatine kinase (CK) released in the culture during venom incubation were used as an indicator of the venom in vitro cytotoxicity. Cell injury was morphologically recognized and apoptosis determined by a Fluorescing Apoptosis Detection System and confirmed by staining nuclear DNA with DAPI. Our data clearly demonstrated marked cytotoxic effects and acute cell injury for both cell lines. Release of LDH and CK into the culture media induced by the venom correlates well with the morphological changes and extent of cell death. Mostly, these consequences were time and dose-dependent in both cell lines. The results obtained from this study indicated that cobra venom cause cell death by two different mechanisms: necrosis and induction of apoptosis. The apoptotic mechanism, accompanied by cell necrosis, mediated cell destruction of both tested cell lines; however, necrosis was predominant in the C2C12 cell line while apoptosis, in 293T cells. This unusual form of cell death induced by cobra venom may represent a combination of apoptosis and necrosis within the same cell. This is a first-hand investigation showing the apoptotic effects of N. haje venom at the cellular level. However, the contribution of the apoptotic pathway may be dependent on concentration and/or time of exposure to snake venom.(AU)