Properties of a clonal muscle cell line from rat heart☆
Abstract
A clonal cell line exhibiting many of the properties of skeletal muscle has been derived from embryonic BDIX rat heart tissue. Multinucleated myotubes, formed by fusion of mononucleated myoblasts, simultaneously contract and produce regenerative action potentials in response to electrical stimulation or iontophoretic application of acetylcholine. The acetylcholine response is inhibited by 1−3 × 10−7 M d-tubocurarine, 10−7 g/ml α-neurotoxin or 1 × 10−4 M atropine. The specific activities of the enzymes myokinase and creatine phosphokinase (CPK) increase 3-fold and 20-fold, respectively, after myotube formation, but only CPK activity parallels the extent of fusion. Exponentially dividing myoblasts synthesize a predominantly brain-type CPK isoenzyme while fused myotubes synthesize a muscle type CPK isoenzyme. Electron microscopic analysis reveals that the myotubes contain elaborately branched tubular systems and numerous bundles of thick filaments with distinct M-bands. Some of the thick filament bundles are associated with thin filaments and organized into sarcomere-like structures with faint Z-regions, but no distinct Z-bands are observed.
References (39)
- BW Kimes et al.
Exp cell res
(1976) - C Richler et al.
Dev biol
(1970) - A Shainberg et al.
Dev biol
(1971) - D Schubert et al.
Dev biol
(1973) - D Delain et al.
Differentiation
(1973) - DC Turner et al.
Dev biol
(1974) - K Horibata et al.
Exp cell res
(1970) - OH Lowry et al.
J biol chem
(1951) - D Yaffe et al.
Dev biol
(1964) - DM Dawson et al.
Biochem biophys res commun
(1965)
Dev biol
Exp neurology
Exp cell res
J ultrastruct res
Exp cell res
J physiol
J physiol
Cited by (488)
Effects of H<inf>2</inf>S-donor ascorbic acid derivative and ischemia/reperfusion-induced injury in isolated rat hearts
2024, European Journal of Pharmaceutical SciencesHydrogen sulfide (H2S), a gasotransmitter, plays a crucial role in vasorelaxation, anti-inflammatory processes and mitigating myocardial ischemia/reperfusion-induced injury by regulating various signaling processes. We designed a water soluble H2S-releasing ascorbic acid derivative, BM-164, to combine the beneficial cardiovascular and anti-inflammatory effects of H2S with the excellent water solubility and antioxidant properties of ascorbic acid. DPPH antioxidant assay revealed that the antioxidant activity of BM-164 in the presence of a myocardial tissue homogenate (extract) increased continuously over the 120 min test interval due to the continuous release of H2S from BM-164. The cytotoxicity of BM-164 was tested by MTT assay on H9c2 cells, which resulted in no cytotoxic effect at concentrations of 10 to 30 μM. The possible beneficial effects of BM-164 (30 µM) was examined in isolated ‘Langendorff’ rat hearts. The incidence of ventricular fibrillation (VF) was significantly reduced from its control value of 79 % to 31 % in the BM-164 treated group, and the infarct size was also diminished from the control value of 28 % to 14 % in the BM-164 treated group. However, coronary flow (CF) and heart rate (HR) values in the BM-164 treated group did not show significantly different levels in comparison with the drug-free control, although a non-significant recovery in both CF and HR was observed at each time point. We attempted to reveal the mechanism of action of BM-164, focusing on the processes of autophagy and apoptosis. The expression of key autophagic and apoptotic markers in isolated rat hearts were detected by Western blot analysis. All the examined autophagy-related proteins showed increased expression levels in the BM-164 treated group in comparison to the drug-free control and/or ascorbic acid treated groups, while the changes in the expression of apoptotic markers were not obvious. In conclusion, the designed water soluble H2S releasing ascorbic acid derivative, BM-164, showed better cardiac protection against ischemia/reperfusion-induced injury compared to the untreated and ascorbic acid treated hearts, respectively.
Moderate but not severe hypothermia increases intracellular cyclic AMP through preserved production and reduced elimination
2023, CryobiologyRewarming from accidental hypothermia could be complicated by acute cardiac dysfunction but providing supportive pharmacotherapy at low core temperatures is challenging. Several pharmacological strategies aim to improve cardiovascular function by increasing cAMP in cardiomyocytes as well as cAMP and cGMP levels in vascular smooth muscle, but it is not clear what effects temperature has on cellular elimination of cAMP and cGMP. We therefore studied the effects of differential temperatures from normothermia to deep hypothermia (37 °C–20 °C) on cAMP levels in embryonic H9c2 cardiac cells and elimination of cAMP and cGMP by PDE-enzymes and ABC-transporter proteins. Our experiments showed significant elevation of intracellular cAMP in H9c2-cells at 30 °C but not 20 °C. Elimination of both cAMP and cGMP through ABC transport-proteins and PDE-enzymes showed a temperature dependent reduction. Accordingly, the increased cardiomyocyte cAMP-levels during moderate hypothermia appears an effect of preserved production and reduced elimination at 30 °C. This correlates with earlier in vivo findings of a positive inotropic effect of moderate hypothermia.
Development of gelatin-silicon dioxide nanoparticles (GSNPs) encapsulating recombinant human secretory leukocyte protease inhibitor (rhSLPI) for reducing in vitro myocardial ischemia/reperfusion injury
2023, Materials Today: ProceedingsIschemic Heart Disease (IHD) is the main global cause of death. Previous studies indicate that recombinant human secretory leukocyte protease inhibitor (rhSLPI) exhibits a cardioprotection effect against myocardial ischemia and reperfusion (I/R) injury. However, SLPI has a short half-life in vivo due to protease enzymes in circulation. Application of nanoparticle encapsulation could be beneficial for SLPI delivery. Several types of nanoparticles have been used to encapsulate SLPI and applied in some disease models. However, silica nanoparticles for rhSLPI delivery, particularly on myocardial I/R injury, has never been studied. In this study, rhSLPI was encapsulated into Gelatin-Silica dioxide nanoparticles (GSNPs), which will be further used to determine an in vitro cardioprotective effect against simulated ischemia/reperfusion injury in cardiomyocytes. Silica dioxide nanoparticles (SNPs) were fabricated followed by incubation with 0.33 mg/mL of rhSLPI. Finally, SNPs containing rhSLPI were coated with gelatin (GSNPs). The GSNPs and rhSLPI-GSNPs were characterized by particle size, zeta potential, and morphology scanning electron microscope (SEM). The concentration of rhSLPI in rhSLPI-GSNPs was determined by ELISA. Next, cytotoxicity was determined by incubation of GSNPs or rhSLPI-GSNPs with rat cardiac myoblast cell line (H9c2). The results show that SNPs, GSNPs, and rhSLPI-GSNPs are sized 280, 303, and 302 nm, respectively, and have a zeta potential of −27.8, −24.4, and −21.5 mV, respectively. SEM indicated a thin layer of gelatin covering SNPs, and the value of zeta potential of SNPs was changed when they were covered by gelatin, indicating a successful coating process. 20 μL of rhSLPI-GSNPs verified by ELISA showed the concentration of rhSLPI was 50.89 ng/mL. The cytotoxicity showed no significant difference between cardiac cells with or without rhSLPI-GSNPs. In conclusion, the researchers were able to fabricate Gelatin-Silica dioxide nanoparticles (GSNPs) to deliver rhSLPI which was used to study cardiomyocyte subjected to simulated ischemia/reperfusion (sI/R) injury.
Quality marker (Q-marker) serves an important role in promoting the standardization of the quality of traditional Chinese medicine (TCM) prescriptions. However, discovering comprehensive and representative Q-markers from TCM prescriptions composed of multiple components remains difficult.
A three-step-based novel strategy integrating drug metabolism and pharmacokinetics (DMPK) with network pharmacology and bioactivity evaluation was proposed to discover the Q-markers and applied to a research example of Danlou tablet (DLT), a famous TCM prescription with remarkable and reliable clinical effects for coronary heart disease (CHD).
Firstly, the metabolic profile in vivo of DLT was systemically characterized, and the pharmacokinetic (PK) properties of PK markers were then investigated. Secondly, an integrated network of “PK markers – CHD targets – pathways – therapeutic effects” was established to screen out the crucial PK markers of DLT against CHD. Thirdly, the crucial PK markers that could exhibit strong myocardial protection activity in the H9c2 cardiomyocyte model were selected as the candidate Q-markers of DLT. According to the proportion of their Cmax value in vivo, the candidate Q-markers were configured into a composition; the bioactivity was then evaluated to confirm their synergistic effect and justify their usage as Q-markers.
First of all, a total of 110 DLT-related xenobiotics (35 prototypes and 75 metabolites) were detected in bio-samples, and the pharmacokinetic properties of 13 PK markers of DLT were successfully characterized, revealing the quality transitivity and traceability from prescription to in vivo. Then, 6 crucial PK markers with three topological features (degree, betweenness, and closeness) greater than the average values in the pharmacology network were screened out as the key components of DLT against CHD. Furthermore, among these 6 crucial PK markers, 5 components (puerarin, alisol A, daidzein, paeoniflorin, and tanshinone IIA) with strong myocardial protection activity were chosen as the candidate Q-markers to constitute a new composition. The composition activated the expression of the PI3K/AKT pathway and exhibited strong myocardial protection activity, and the effective concentrations (nM level) of these components in the composition were significantly lower than their individually effective concentrations (μM level), indicating that there was a certain synergistic effect between them. Hence, the 5 components with multiple properties, including testability, quality transitivity and traceability from prescription to in vivo, effectiveness, and compatibility contribution, were defined as comprehensive and representative Q-markers of DLT.
This study not only presented a novel idea for the revelation of comprehensive and representative Q-markers in quality control research of TCM prescriptions, but also identified the reasonable Q-markers of DLT for the first time to improve the quality control level of DLT.
Evaluation of mitochondrial oxidative toxicity in mammalian cardiomyocytes by determining the highly reproducible and reliable increase in mitochondrial superoxides after exposure to therapeutic drugs
2022, Toxicology in VitroMitochondria are important cytoplasmic elements present in eukaryotic cells, and are involved in converting energy to ATP through oxidative phosphorylation. Mitochondria are vulnerable to reactive oxygen species (ROS), thereby making it imperative to evaluate the toxicity. However, existing methods that evaluate mitochondrial toxicity in cardiomyocytes are limited. In the current study, we aimed to determine a mitochondrial biomarker that measures the toxicity of mitochondria, and subsequently suggest an efficient evaluation system for evaluating mitochondrial-specific oxidative toxicity. To achieve this, AC16 human cardiomyocytes, H9C2 rat cardiomyocytes were exposed to acetaminophen (AP), amiodarone hydrochloride (AMD), doxorubicin hydrochloride (Dox), valproic acid sodium salt (Val), and (Z)-4-hydroxytamoxifen (4-OHT). Mitochondrial oxidative stress was determined by staining the drug-treated cells with MitoSOX™ red fluorescence dye, followed by imaging with a fluorescence microscope. All working concentrations of Dox showed increased levels of red fluorescence in AC16 and H9C2 cells, whereas exposure to Val did not alter the red fluorescence level of both cells. Considering our results, increased MitoSOX™ subsequent to drug exposure is a highly reproducible and reliable method to measure the mitochondrial-specific oxidative toxicity. These results indicate that a screening system using MitoSOX™ has the potential to be applied as a reliable biomarker for determining mitochondrial oxidative toxicity in new drug development.
PKGIα is activated by metal-dependent oxidation in vitro but not in intact cells
2022, Journal of Biological ChemistryType I cGMP-dependent protein kinases (PKGIs) are important components of various signaling pathways and are canonically activated by nitric oxide– and natriuretic peptide–induced cGMP generation. However, some reports have shown that PKGIα can also be activated in vitro by oxidizing agents. Using in vitro kinase assays, here, we found that purified PKGIα stored in PBS with Flag peptide became oxidized and activated even in the absence of oxidizing agent; furthermore, once established, this activation could not be reversed by reduction with DTT. We demonstrate that activation was enhanced by addition of Cu2+ before storage, indicating it was driven by oxidation and mediated by trace metals present during storage. Previous reports suggested that PKGIα Cys43, Cys118, and Cys196 play key roles in oxidation-induced kinase activation; we show that activation was reduced by C118A or C196V mutations, although C43S PKGIα activation was not reduced. In contrast, under the same conditions, purified PKGIβ activity only slightly increased with storage. Using PKGIα/PKGIβ chimeras, we found that residues throughout the PKGIα-specific autoinhibitory loop were responsible for this activation. To explore whether oxidants activate PKGIα in H9c2 and C2C12 cells, we monitored vasodilator-stimulated phosphoprotein phosphorylation downstream of PKGIα. While we observed PKGIα Cys43 crosslinking in response to H2O2 (indicating an oxidizing environment in the cells), we were unable to detect increased vasodilator-stimulated phosphoprotein phosphorylation under these conditions. Taken together, we conclude that while PKGIα can be readily activated by oxidation in vitro, there is currently no direct evidence of oxidation-induced PKGIα activation in vivo.
- ☆
This work was supported by National Institutes of Neurological Diseases and Stroke grant number NS09658 to D. S., National Institutes of Allergy and Infectious Disease Training Grant number AI00430 to M. C., and by postdoctoral fellowships from the National Multiple Sclerosis Society to B. L. B. and from the ACS to B. W. K.