Cardiotoxicity of Tentacle Extract from the Jellyfish Cyanea capillata and Its Potential Mechanism

IntroductionIn recent years, a significant increase in jellyfish blooms has been observedworldwide in marine ecosystems due to global climate warming, eutrophication and otherfactors. In fact, jellyfish was the most common injuries in marine systems. Jellyfish stingcould produce local symptoms such as severe pain, inflammation and necrosis, andsystemic symptoms such as muscle spasms, respiratory distress, circulatory depression andeven shock, among which acute heart failure was considered to be the leading cause ofdeath by jellyfish stings. Jellyfish venom exhibited a variety of toxicities to multiple organsystems such as cardiovascular, nerve, muscles, liver and kidney, and the cardiovasculartoxicity was considered to be the evaluation standard of jellyfish venom virulence.However, the mechanism underlying the cardiovascular toxicity of jellyfish venom wasstill unclear so far. In this study, we firstly proved that tentacle extract （TE） from thejellyfish Cyanea capillata had direct cardiotoxicity in isolated heart. Then we explored themechanism on cardiotoxicity induced by TE, finding that Ca²⁺-induced Ca²⁺release （CICR）signaling pathway played an important role in acute heart dysfunction by TE, andβ-adrenergic receptor-cAMP-PKA-RyR2-Ca²⁺signaling pathway might be also related tothe acute heart dysfunction by TE.MethodsPart I: Direct cardiotoxicity of TE in isolated heartThe Langendorff-perfused isolated heart model was used to determine the changes ofheart function indexes （HR, Lvdp,±dP/dtmax, CF and Lvedp） and ECG after TEadministration. An automatic biochemical analyzer was employed to measure the changesof heart injury-related enzymes, including lactate dehydrogenase （LDH）, aspartateaminotransferase （AST） and creatine kinase （CK）. And the histopathologic changes ofheart injuries by TE were also observed. After the hearts were pretreated with variousdrugs, including dihydropyridine Ca²⁺channel antagonist nifedipine （0.25μM） orphenylalkylamine Ca²⁺channel antagonist verapamil （0.1μM）, nonselective β-adrenergicreceptor propranolol （1μM） or nonselective α-adrenergic receptor antagonist phentolamine（1μM）, competitive nonselective muscarinic receptor antagonist atropine （1.5μM） or reversible anticholinesterase drug neostigmine （3.3μM）, the changes of heart functionindexes were determined, respectively.Part II: Cardiotoxicity mechanism of TEThe changes of heart function indexes were determined in both whole animal andisolated heart after TE administration. The effects of TE on morphology, viability and heartinjuries related-enzymes in neonatal rat cardiomyocytes were observed. The Ca²⁺determination kit was used to detect the changes of Ca²⁺content by TE in heart tissues, andlaser scanning confocal microscopy was used to detect the changes of intracellular Ca²⁺content and morphology of neonatal rat cardiomyocytes by TE. Pretreated with differentcation channel blockers, the effects of TE on cell viability in neonatal rat cardiomyocyteswere determined, respectively, and the effective antagonist, for example diltiazem, wasselected to further verify its antagonism against TE-induced cardiotoxicity at both isolatedheart and whole animal levels. Afterwards, the role of CICR signaling pathway inTE-induced cardiotoxicity was comprehensively analysized.Subsequently, after neonatal rat cardiomyocytes were treated with different doses ofTE, western blot technique was used to detect the protein quantity of β1-AR, and Elisamethod was used to detect the concentration of cAMP. Elisa method was also used todetect the effect of TE on the concentration of cAMP after neonatal rat cardiomyocytespretreated with β-adrenergic receptor blockers （propranolol, atenolol and esmolol）. Underextracellular Ca²⁺-free condition, the effect of H89on TE-induced cardiomyotoxicity wasdetermined. These experiments mainly aimed to discuss the role of β-adrenergicreceptor-cAMP-PKA-RyR2-Ca²⁺signaling pathway in TE-induced cardiotoxicity.ResultsPart I: Direct cardiotoxicity of TE in isolated heartAfter the isolated hearts were treated with different doses of TE （60,120,180,240μg）, the heart function indexes, including HR, Lvdp,±dP/dtmax and CF, dose-dependentlydecreased, while Lvedp dose-dependently increased. And ECG also showed obvious dose-and time-dependent changes. When the dose of TE was higher than240μg, ECG showedsevere arrhythmia （ventricular fibrillation or high-degree atrioventricular block）, and thecardiac arrest appeared15min later. At the TE dose of180μg, heart injury-relatedenzymes （LDH, AST, CK） significantly increased, and a variety of tissue lesions （wavyfibers, irregular myocyte diameters and interstitial edema） were observed. TE-induced heart injuries were significantly improved by nifedipine and verapamil, and partlyimproved by propranolol and phentolamine, but atropine and neostigmine had no obviouseffect on TE-induced heart injuries.Part II: Cardiotoxicity mechanism of TEAfter TE administration, the heart function indexes showed apparent changes at bothwhole animal and isolated heart levels, and the viability of neonatal rat cardiomyocytesshowed dose-and time-dependent decline. Under both extracellular Ca²⁺-containing （1.27mmol/L） and Ca²⁺-free conditions, TE could cause obvious cytoplasmic Ca²⁺overload inneonatal rat cardiomyocytes, but the cytoplasmic Ca²⁺increased faster （within5min）, Ca²⁺overload peaks arrived earlier （about10min） and the morphological changes were moresevere under the extracellular Ca²⁺-containing condition. Different types of Ca²⁺channelblockers were utilized to antagonize the cardiomyotoxicity of TE, founding that L-typeCa²⁺channel blockers （for example diltiazem） and RyR2antagonist （ryanodine） couldimprove the viability of neonatal rat cardiomyocytes. These results suggested thatTE-induced acute heart dysfunction was related to CICR signaling pathway. Diltiazemcould improve TE-induced acute heart dysfunction in vitro and in vivo, further indicatingthat CICR signaling pathway played an important role in TE-induced cardiotoxicity.The protein quantity of β1-AR in neonatal rat cardiomyocytes showed obviousincrease after TE treatment, and the concentration of cAMP also significantly increased,indicating that the over-activation of β-adrenergic receptor-cAMP-PKA-RyR2-Ca²⁺signaling pathway might be related to TE-induced cardiotoxicity. The increase of cAMPconcentration by TE in neonatal rat cardiomyocytes could be antagonized by β-adrenergicreceptor blockers （propranolol, atenolol and esmolol） and H89could improve the viabilityof neonatal rat cardiomyocytes under extracellular Ca²⁺-free conditions, further indicatingthat the β-adrenergic receptor-cAMP-PKA-RyR2-Ca²⁺signaling pathway might also play arole in TE-induced cardiotoxicity.Conclusion1. The changes of heart function indexes, ECG and heart injury-related enzymescaused by TE in isolated heart suggested that TE could induce direct cardiotoxicity.2. At the whole animal, isolated heart, cellular and molecular levels, we proved thatCICR signaling pathway played an important role in TE-induced acute heart dysfunction. 3. At cellular and molecular level, it was preliminarily proved that β-adrenergicreceptor-cAMP-PKA-RyR2-Ca²⁺signaling pathway might also be related to the acute heartdysfunction by TE.