Investigating a role for CaMKIIδ in mediating the cardiotoxic effects of anti-cancer tyrosine kinase inhibitors

Error on title page – year of award is 2022.Introduction: Tyrosine kinase inhibitors (TKIs) have dramatically improved cancer treatment but are known to cause cardiotoxicity. The pathophysiological consequences of TKI therapy are likely to manifest across different cell types of the heart, yet there is little understanding of the differential adverse cellular effects. Cardiac fibroblasts (CFs) play a pivotal role in the repair and remodelling of the heart following insult or injury, yet their involvement in anticancer drug induced cardiotoxicity has been largely overlooked. Here, we examine and compare the direct effects of two TKIs, sunitinib malate and imatinib mesylate, on CFs and progenitor cardiac myocytes (PCMs). Methodology: CFs and PCMs were treated with sunitinib and imatinib (0.1-10μM) for 18 hours and then subject to in vitro analyses to determine the role of Ca2+/calmodulin dependent protein kinase II (CaMKII) in the cardiotoxic mechanism of TKIs. Variations in cell phenotype were monitored via brightfield imaging. Changes in cell viability were determined via MTT assays and flow cytometry. Western blot and immunofluorescent imaging were utilised to investigate potential changes in protein expression. Changes in calcium mobility were investigated using a Fluo4-AM intracellular calcium release assay and mitochondrial function was explored using MitoSOX Red live cell imaging and an Oroborous oxygraph O2k respirometer. Results/Discussion: In investigating the cardiotoxic mechanism of anti-cancer TKIs, this project has shown that (i) TKI treatment leads to contractile and non-contractile cellular dysfunction and death, (ii) TKI treatment, particularly sunitinib treatment, increases intracellular [Ca2+] release and cellular oxidative stress in CFs by increasing reactive oxygen species (ROS) production via effects at the level of the mitochondria, correlating with increased oxidation and autonomous activation of CaMKII and (iii) CaMKII inhibition via KN-93 reduces the detrimental effects of sunitinib and imatinib treatment at the level of the mitochondria, but this does not improve CF viability. Interestingly, the study found that TKI treatment had no effect on intracellular [Ca2+] release in PCMs and actually reduced CaMKII phosphorylation, despite previous reports of pathophysiological changes in CMs that are similar to those obtained in CFs here. This study also established that the PCMs used here do not express key CM Ca2+ handling proteins and this makes them unsuitable to investigate the cardiotoxic mechanism of TKIs. Conclusion: These findings highlight a new role for CaMKII in TKI-induced cardiotoxicity, particularly at the level of the mitochondria, and confirm differential off-target toxicity in both contractile and non-contractile cardiac cells, consistent with the differential selectivity of sunitinib and imatinib.Introduction: Tyrosine kinase inhibitors (TKIs) have dramatically improved cancer treatment but are known to cause cardiotoxicity. The pathophysiological consequences of TKI therapy are likely to manifest across different cell types of the heart, yet there is little understanding of the differential adverse cellular effects. Cardiac fibroblasts (CFs) play a pivotal role in the repair and remodelling of the heart following insult or injury, yet their involvement in anticancer drug induced cardiotoxicity has been largely overlooked. Here, we examine and compare the direct effects of two TKIs, sunitinib malate and imatinib mesylate, on CFs and progenitor cardiac myocytes (PCMs). Methodology: CFs and PCMs were treated with sunitinib and imatinib (0.1-10μM) for 18 hours and then subject to in vitro analyses to determine the role of Ca2+/calmodulin dependent protein kinase II (CaMKII) in the cardiotoxic mechanism of TKIs. Variations in cell phenotype were monitored via brightfield imaging. Changes in cell viability were determined via MTT assays and flow cytometry. Western blot and immunofluorescent imaging were utilised to investigate potential changes in protein expression. Changes in calcium mobility were investigated using a Fluo4-AM intracellular calcium release assay and mitochondrial function was explored using MitoSOX Red live cell imaging and an Oroborous oxygraph O2k respirometer. Results/Discussion: In investigating the cardiotoxic mechanism of anti-cancer TKIs, this project has shown that (i) TKI treatment leads to contractile and non-contractile cellular dysfunction and death, (ii) TKI treatment, particularly sunitinib treatment, increases intracellular [Ca2+] release and cellular oxidative stress in CFs by increasing reactive oxygen species (ROS) production via effects at the level of the mitochondria, correlating with increased oxidation and autonomous activation of CaMKII and (iii) CaMKII inhibition via KN-93 reduces the detrimental effects of sunitinib and imatinib treatment at the level of the mitochondria, but this does not improve CF viability. Interestingly, the study found that TKI treatment had no effect on intracellular [Ca2+] release in PCMs and actually reduced CaMKII phosphorylation, despite previous reports of pathophysiological changes in CMs that are similar to those obtained in CFs here. This study also established that the PCMs used here do not express key CM Ca2+ handling proteins and this makes them unsuitable to investigate the cardiotoxic mechanism of TKIs. Conclusion: These findings highlight a new role for CaMKII in TKI-induced cardiotoxicity, particularly at the level of the mitochondria, and confirm differential off-target toxicity in both contractile and non-contractile cardiac cells, consistent with the differential selectivity of sunitinib and imatinib