The Effects Of Protein Kinase C Inhibitors On Cardiotoxicity Induced By Imatinib And Sunitinib

Dysfunction of protein kinases involves in the development of manydiseases, especially for tumor. In recent years, tyrosine kinase inhibitors (TKIs)has become the hot spot in the field of pharmaceutical research and new drugdevelopment. Compared to conventional drugs, TKIs have shown excellenceeffectiveness for a variety of tumors and some TKIs have become first-linedrugs. However, during the past year it has become evident that some of theseagents have associated cardiotoxicity and can, at least in some patients, causeleft ventricular dysfunction and even symptomatic congestive heart failure(CHF) without a prior history of heart disease[12,17,38,45,46]. Therefore,TKI-induced cardiotoxicity becomes a major concern in tumor therapy andnew drug development and to determine the mechanisms of cardiotoxicity isof importance.The adverse cardiac effects have been mentioned in the prescribinginformation for imatinib, sunitinib, nilotinib, dasatinib, etc. Imatinib andsunitinib-related cardiotoxicity has been demonstrated in both in vitro and invivo experiments. Electron micrographs of samples in vitro and in vivo haverevealed mitochondrial abnormalities. However, little is known about themolecular mechanism underlying TKI-induced mitochondrial dysfunction. Itwas found that upregulation of protein kinase C (PKC) isoenzymeexpression levels in the hearts of mice and in cardiomyocytes exposed toimatinib.PKC plays an important role in the transmembrane signaling process.Currently, at least12isoforma of PKC have been identified and are classifiedinto three groups based on their structure and activation mechanisms:(1)conventional PKC (α, βI, βII and γ) that are activated by phosphatidylserine (PS), intracellular Ca2+and diacylglycerol (DAG);(2) novel PKC (δ, ε, η andθ) that are activated by DAG and PS, but not intracellular Ca2+; and (3)atypical PKC (ζ and λ) that regulated by PS, but not activated by intracellularCa2+or DAG. All of the PKC isoforms, including α, βI, βII, δ, ε, ζ and λ havebeen identified in the heart. It has been shown that PKC isoforms in the heartare differentially regulated by various stimuli under physiological andpathological conditions[20,31]. It is known that activated PKCδ can activatemitochondrial acetone dehydrogenase kinase, inhibiting the regeneration ofATP and pyruvate dehydrogenase and thus triggers cell death[12,13,10].Therefore,we assume that over activation of PKC signaling pathway may mediatecardiomyocytes. To test our hypothesis, this study was designed to determinethe effects of PKC inhibitor or PKC inhibitor peptide on cardiotoxicityinduced by imatinib (IM) and sunitinib (SU) in cultured neonatal ratcardiomyocytes (NRVMs).Objective:To observe the effects of PKC inhibitor or PKC inhibitor peptide oncardiotoxicity induced by IM and SU in NRVMs.Methods:The neonatal rat ventricular myocytes (NRVMs) were isolated from1-2days newborn SD rats and incubated with DMEM/F-12medium for three days.Then cells were incubated with different concentrations of IM (2,5,10M) orSU (1,5,10M). The mitochondrial membrane potential (MMP) wasdetermined at24h by using confocal detection kit and Leica laser formicroscopic examination. The intracellular ATP content was detected at48husing ATP detection kit and FLUO star Omega automatic multifunctionalmicroplate. The LDH release was determined at72h with LDH assay kit.NRVMs were co-incubated with both IM/SU and non-selective or selectivePKC inhibitors or peptides to test the effects of PKC inhibition oncardiotoxicity. All of data are presented as means±SEM and were analyzedusing the OriginPro8.6. software. The n represents the repetitive times. The statistical significance of the differences between groups was evaluated usingStudent’s unpaired t test and differences with P<0.05were consideredstatistically significant.Results:(1) Toxic effects of IM and SU in NRVMsNRVMs were incubated with different concentrations of IM (2,5,10M)or SU (1,5,10M). Both IM and SU significantly decreased ATP content andincreased LDH releases in a concentration-dependent manner. Next we testedthe time course of toxic responses. NRVMs were separately incubated with IM(10M) and SU (10M) and ATP content, LDH releases, and MMP weredetermined at24,48and72h. The results showed that both IM and SUmarkedly decreased MMP at24h and reached to50%and30%of the controlrespectively. ATP contents were significantly lowered in IM and SU treatedNRVMs at48h, and much decrease in ATP contents was found at72h. LDHrelease showed a similar time course as ATP content in both IM andSU-treated NRVMs. These results indicated that both IM and SU inducedcardiotoxicity in NRVMs in concentration and time-dependent manner. TheMMP decrease turned out earlier than the change in ATP content and LDHreleases.(2) The effect of Bis-1, a non-selective PKC inhibitor, on the toxicitycaused by IM and SU.Non-selective PKC inhibitor, Bis-1(100nM) alone did not affect MMPvalue, ATP content, and LDH release. In the presence of Bis-1, changes in ATPcontent and LDH release caused by IM and SU were almost reverted to thecontrol level. MMP measurement revealed that, compared with the IM or SUalone, Bis-1significantly elevated MMP value. Scanning electron microscopyobservation showed that, both IM group and SU induced great changes inmitochondrial morphology, manifested by mitochondria swelling or medullarychange. In the presence of Bis-1the cells returned to normal mitochondrial morphology. The results prompted that non-selective PKC inhibitionantagonize the cardiotoxicity induced by IM and SU.(3) The effects of selective PKC inhibitors or inhibit peptides on thetoxicity induced by IM and SU.G6976, a cPKC inhibitor didn’t affect the MMP value, ATP content andrelease of LDH. Co-application of G6976(100nM) with IM or SU didn’tchange toxic parameters. Similarly, highly selectivity of PKCα inhibitorypeptide (500nM) had no significant effect on the changes in above parametersinduced by IM and SU. The results indicated that inhibition of conventionalPKC isoforms didn’t antagonize cardiotoxicity caused by IM and SU. Then,the effectiveness of nPKC inhibitors was tested. It is known that rotterlin isPKCδ selective inhibitor. Rotterlin (500nM) alone didn’t affect the value ofMMP, ATP content and LDH release, but it almost completely antagonized thechanges in MMP, ATP content and LDH release caused by IM and SU.Selective PKCε inhibitory peptide (500nM) produced similar effects asrotterlin. On the contrary, scramble peptide had no any effect on the changesin toxic parameters. The results revealed that selective inhibition of novelPKCδ and PKCε had a significant protective effect against cardiomyocytetoxicity induced by IM and SU.To further confirm the cardioprotection of rotterlin and PKCε inhibitorypeptide, we used transmission electron microscopy to observe submicroscopicstructure of the mitochondria. NRVMs in co-application of rotterlin and IMshow clear mitochondria submicroscopic structure, no obvious vacuoles,swelling or medullary change. In the present of PKCε inhibitory peptide, IMcaused no medullary change although with some mitochondria vacuoles.Rotterlin and PKCε inhibitory peptide showed similar protective effect on thechanges in mitochondria caused by SU.Experimental results suggest that selective inhibition of PKC δ and εisoenzymes, but not cPKC isoform antagonize cardiotoxicity induced by IMand SU. Conclusion:(1) Both IM and SU induce cardiotoxicity in NRVMs in concentrationand time-dependent manner. The MMP decrease turns out earlier than thechange in ATP content and LDH releases.(2) Non-selective PKC inhibition antagonizes the cardiotoxicity inducedby IM and SU.(3) Selective inhibition of PKC δ and ε isoenzymes, but not cPKCisoform antagonize cardiotoxicity induced by IM and SU.