Protective Effect Of PI3K On Cardiotoxicity Induced By Tyrosine Kinase Inhibitors

Objective: Small molecule tyrosine kinase inhibitors(TKIs)have a marked effectiveness on tumors and thus greatly improves the survival rate of patients.The target of these kinase inhibitors is the ATP binding pockets of tyrosine kinases,which inhibits the activation of the kinase by blocking the binding of ATP and the kinase.However,some TKIs produce cardiotoxicity,which mainly manifested as left ventricular systolic dysfunction,and even heart failure.So far,of more than 30 TKIs in clinical use,many have a “black box warning” in the prescription information.Due to the limited understanding of the exact molecular mechanism of TKIs-induced systolic dysfunction,there is currently no specific prevention strategy.The tyrosine kinases in heart have important physiological effects.TKIs may inhibit the action of the kinases common to the heart and tumors(on-target).At the same time,it may also inhibit or affect the different kinases in heart from those in tumor and thus generate off-targets effect.It is known that the normal contraction of cardiomyocytes depends on the ATP energy supply and the excitation-contraction coupling(ECC).There are many kinases involved in the regulation of the process.Among them,phosphatidylinositol-3 kinase(PI3K)is essential for maintaining the Ca2+ circulation and contraction in cardiomyocytes.We have previously found that up-regulation of PI3 K would significantly protect the systolic dysfunction caused by sunitinib.Sunitinib is known to be a multi-targeted TKIs that mainly inhibits receptor tyrosine kinases.It is still unknown that whether upregulation of PI3 K may protect myocardial dysfunction of contractility caused by other TKIs? To answer this question,the study was designed to further investigate the effect of PI3 K in human induced stem cell-differentiated cardiomyocytes(hi PSC-CMs)on cardiotoxicity induced by sunitinib,imatinib,trametinib,and sorafenib,which have been clinically reported to cause contraction dysfunction.Methods: 1.Full-automatic cardiomyocyte multifunctional detection platform : Cardio Excyte 96 cardiomyocytes real-time monitoring system was used to observe the effect of TKIs on the contraction of hi PSC-CMs.The hi PSC-CMs(Beijing Cell biotechnology Co,LTD)were resuscitated and counted after trypan blue staining,and then plated into the electrode plate at a density of 3×104 cells/well.The impedance amplitude was measured as the contractile parameter of a sheet of hi PSC-CMs.The effect of sunitinib,imatinib,trametinib,and sorafenib on the contraction of hi PSC-CMs was observed and the action of up-regulation of PI3 K on TKIs was then detected through two methods including an increase of PI3 K activity with the upstream substrate PIP3 and overexpression of p110?,the dominant PI3 K subtype in the heart by adenovirus transfection.2.Measurement of toxic biomarkers: q RT-PCR technology was used to detect the m RNA expression of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP),?-myosin heavy chain(?-MHC).Total RNA from hi PSC-CMs was isolated using RNeasy? Mini kit according to the manufacturer's instruction.Total RNA(2 mg)was converted to c DNA using a reverse transcription kit.Every sample was run and analyzed in triplicate and normalized to GAPDH.3.Detection of reactive oxygen species(ROS): ROS levels were measured with the Reactive Oxygen Species Assay Kit according to the manufacturer's instruction,and the fluorescence signal was detected using a confocal microscope.Results: 1.By real-time monitoring of cell contraction we found that four TKIs including sunitinib,imatinib,trametinib,and sorafenib significantly decreased the amplitudes of contractility.Pre-incubation of hi PSC-CMs with PIP3 significantly prevented the contractile inhibition of hi PSC-CMs caused by sunitinib,imatinib,and trametinib.However,it did not show any protection against the inhibition of contraction by sorafenib.2.Four TKIs increased the m RNA levels of ANP,BNP,?-MHC and ROS release in hi PSC-CMs.Whereas,pre-incubation of hi PSC-CMs with PIP3 was protective against the changes of those biomarkers and ROS induced by sunitinib,imatinib,and trametinib.But,PIP3 had not effects on the sorafenib-caused increase of those biomarkers and ROS levels.3.To verify the protective role of PI3 K activation on TKIs-caused cardiotoxicity,we further overexpressed p110?,the dominant PI3 K subtype in the heart.Consistent with the result with PIP3,p110? overexpression effectively protected the hi PSC-CMs against sunitinib,imatinib,and trametinib-induced reduction in contractility.But it did not protect against the inhibition of contraction by sorafenib.Conclusions: This study have shown that activation of PI3 K can significantly protect against the contractile inhibition induced by sunitinib,imatinib,and trametinib,but have no protective effect on sorafenib.The results suggest that up-regulation of the PI3 K kinase pathway may have protective effects on TKIs,but the effect may have selectivity depending on the kinase target.