Biomarkers For In Vitro And In Vivo Detection Of Cardiotoxicity Induced By Sunitinib And Imatinib

Protein tyrosine kinase(PTK)can catalyze the phosphorylation of multiple tyrosine residues substrate and play a role in cellular growth,proliferation,and differentiation and so on.However,the overexpression of protein tyrosine kinase(PTK)leads to abnormality in cellular proliferation and thus involes in tumorous occurrence and development.Tyrosine kinase inhibitors(TKI)with small molecules,such as sunitinib,imatinib interfere with the kinase activity and slow tumour progression are becoming fervent focus of the development of new anti-tumorous drugs.However,these drugs can cause cardiovascular adverse reactions,including hypertension,cardiac arrhythmia and cardiac systolic dysfunction,severe heart failure,which severely affect the life quality and survival rate of patients,thus it should be importantly clinical significance to early detect and prevent the cardiac toxicity of TKIs.In present,sensitive and specific biomarkers are important clinical indicators to diagnose the drug-induced cardio-toxicity(myocardial injury and/or heart failure).It has been verified that elevated levels of topoisomerase2β,myeloperoxidase,myocardial fatty acid binding protein(H-FABP),troponin T or I(cTn T/cTnI)and other circulating markers could predict myocardial injury of drugs,and the N-terminal pro-brain natriuretic peptide(NT-proBNP)is a marker for the clinical diagnosis of ventricular systolic dysfunction/heart failure.Wherase,there are no unanimous conclusions that whether or not these markers can be used to detect cardiac toxicity caused by TKIs.There are studies shown that different types of drugs,such as arsenic trioxide,anthracyline-anti-tumorous drugs,etc.,cause cardiac toxicity with different dynamic characteristics of the release of biomarkers as mentioned above.It has been clinically observed that increase of some biomarkers are notcertainly associated with the decreased ventricular systolic function caused by TKIs.Animal studies have shown that imatinib induced marked myocardial injury without elevation of cTnT,cTnI,and FABP,and Our previous study found that serum cTnI levels were significantly decreased when sunitinib induced cardiac toxicity in mice.Objective: Based on the contradictory experimental results as mentioned above,the present study was intend to observe the dynamic release of H-FABP,HS-cTnI,and NT-proBNP,which are three currently widely studied biomarkers,by incubating isolated cultured neonatal rat cardiomyocytes(NRVMs)in different concentrations of sunitinib or imatinib;meanwhile,this study also was intend to conclude the alteration of these biomarkers at whole animal level,with an expection to find a sensitive marker and experimental evidence to monitor the cardiac toxicity of TKI.Experimental approach:1 Isolation and culture of neonatal rat cardiomyocytes(NRVMs): 1-3days of neonatal SD rats were used to isolate ventricular myocytes.Using differential adherence to remove fibroblasts,and adding 0.1 mM Brdu in culture inhibits the growth of fibroblasts.2 Sunitinib,Imatinib for NRVNs cytotoxicity assay: the cell viability of different concentrations of sunitinib and imatinib incubated with NRVNs was measured by MTS colorimetry;JC-1 fluorescent probe and chemiluminescence method was applied to evaluate the effect of these drugs on mitochondrial function(mitochondrial membrane potential MMP and ATP content)of NRVNs myocardial cells.In addition,the ultrastructural changes of mitochondria were observed using a transmission electron microscope(TEM).3 In parallel,the dynamic release of H-FABP,HS-cTnI,and NT-proBNP were measured by enzyme-linked immunosorbent assay with different concentrations of sunitinib and imatinib-induced cytotoxicity of NRVNs.4 C57 male mice,weighing approximately 25 g,were randomly divided into experimental and control groups.The experimental group received sunitinib(40 mg/kg/d)by gavage,the control group received gavage of thesame volume of normal saline,and the left ventricular ejection fraction(LVEF)was measured by echocardiography one week later.Blood was taken from the eyeballs to prepare serum and the levels of H-FABP,HS-cTnI and NT-pro BNP were determined.Results:1.Cell viability experiments: the growth curve of NRVMs showed that the toxicity of Sunitinib 、 Imatinib on NRVMs for 72 h was concentrationdependent.According to the cytotoxicity classification,the following experiments were performed on mild to moderate toxicity drug concentrations of sunitinib 1,5,and 10 μM,and imatinib 10,20,and 40 μM.2.Effects on mitochondrial function: Sunitinib and imatinib reduced MMPs and ATP production of NRVMs,and the effects were concentration and time-dependent.High concentrations of sunitinib and imatinib showed a significant decrease in MMP 24 h,which was the earliest manifestation of cytotoxicity.3.Effects on ultrastructure of mitochondria: Transmission electron microscopy showed that the ultrastructure of mitochondria induced by sunitinib and imatinib significantly changed,and most of the mitochondria produced vacuolization,swelling or myelination,and changes in sacral breaks.Further structural validation of the toxic effects of sunitinib and imatinib on NRVMs.4.Sunitinib and Imatinib showed concentration-and time-dependent increases in H-FABP,HS-cTnI,and NT-proBNP in vitro cultured cardiomyocytes,indicating that the three biomarkers are sensitive markers of sunitinib,imatinib cytotoxicity.5.Changes in serum markers: One week after sunitinib administration(40mg/kg/d),the LVEF of mice was significantly decreased(LVEF<50%);compared with the control group,the release of H-FABP in the serum of mice decreased.Compared with the control group,the release of H-FABP in mouse serum had a tendency to decrease,and the release of HS-cTnI had a tendency to increase,but there was no significant difference.The release of NT-proBNPwas significantly decreased(P<0.001),which was approximately 0.30 times that of the control group.Conclusion:1.Sunitinib and Imatinib exhibit concentration-and time-dependent damage to cultured cardiomyocytes in vitro and the decline in mitochondrial membrane potential(MMP)is the earliest manifestation of cytotoxicity.2.Sunitinib and Imatinib showed concentration-and time-dependent increases in H-FABP,HS-cTnI,and NT-proBNP in vitro cultured cardiomyocytes,indicating that the three biomarkers are sensitive markers of cytotoxicity induced by sunitinib and imatinib.However,no significant increase in three serum biomarkers was observed when the overall animal dose of sunitinib resulted in reduced cardiac systolic function,however,the release of NT-proBNP decreased.It is suggested that the decrease of myocardial contractile function caused by repeated clinical administration of sunitinib is not entirely caused by the general cytotoxicity of the drug,and may be related to its direct inhibition of contraction-related mechanisms.