No abstract

Many small molecule kinase inhibitors (SMKIs) used to fight cancer have been associated with cardiotoxicity in the clinic. Therefore, preventing their failure in clinical development is a priority for preclinical discovery. Our study focused on the integration and concurrent measurement of ATP, apoptosis dynamics and functional cardiac indexes in human stem cell-derived cardiomyocytes (hSC-CMs) to provide further insights into molecular determinants of compromised cardiac function. Ten out of the fourteen tested SMKIs resulted in a biologically relevant decrease in either beating rate or base impedance (cell number index), illustrating cardiotoxicity as one of the major safety liabilities of SMKIs, in particular of those involved in the PI3K–AKT pathway. Pearson's correlation analysis indicated a good correlation between the different read-outs of functional importance. Therefore, measurement of ATP concentrations and apoptosis in vitro could provide important insight into mechanisms of cardiotoxicity. Detailed investigation of the cellular signals facilitated multi-parameter evaluation allowing integrative assessment of cardiomyocyte behavior. The resulting correlation can be used as a tool to highlight changes in cardiac function and potentially to categorize drugs based on their mechanisms of action.

We have previously shown that reducing Na þ influx by a reduction in peak Na þ current (I Na,peak ) in isolated cardiac myocytes leads to a decrease in proarrhythmic Ca 2þ waves. However, I Na,peak inhibition can result in reduced conduction velocity and promote wavebreak leading to enhanced arrhythmia in vivo. Here we test if ranolazine, an inhibitor of late Na þ current (I Na,late ) could also reduce the occurrence of Ca 2þ waves without potentially problematic reductions in I Na,peak . Cells, loaded with fluo-4AM to monitor intracellular Ca 2þ , were stimulated at different pacing rates followed by a one-minute rest period for wave detection. 10mM ranolazine reduced Ca 2þ wave frequency in cells following pacing at 5Hz although less potently than 5mM flecainide (0.1350.02 waves.s À1 (flecainide) vs 0.1650.02 waves.s À1 (ranolazine), P<0.01). Comparable effects were achieved when the concentration of ranolazine was increased to 20mM (0.1450.02 waves.s À1 (flecainide) and 0.1850.03 waves.s À1 (ranolazine) vs 0.2250.03 waves.s À1 (control), P<0.001 and 0.05 respectively) although at this concentration there is a significant reduction in I Na,peak . Ranolazine's ability to reduce wave occurrence was tested further under two conditions in which enhancement of INa,late, would be expected; in the presence of anemone toxin II and in cells from an established model of chronic heart failure (HF). In both conditions wave frequency was increased (e.g. 0.0850.01 waves.s À1 (HF) vs 0.0650.01 waves.s À1 (control), P<0.05) but ranolazine was not effective at reducing frequency (0.1950.04 waves.s À1 (ranolazine 10mM) vs 0.1950.04 waves.s À1 (control), P=0.92). Ranolazine decreased Ca 2þ wave frequency in isolated cardiomyocytes, particularly at concentrations where I Na,peak is reduced. The lack of efficacy in states of enhanced I Na,late suggests specific effects of the drug are less important than off-target effects on I Na,peak . Thus similar dose-limiting side effects as with conventional I Na blockers are expected. Development of in vitro toxicity assays based on the human stem cell-derived cardiomyocytes (SC-CMs) provides an opportunity to assess the specific cardiac safety end-points early in drug development. The cardiac action potential (AP) is an established biomarker for cardiac safety and efficacy in humans and a good detector of proarrhythmic events. However, its utility has been mainly limited by a low throughput of manual patch clamp and a restricted access to healthy human cardiomyocytes. The recent progress in automated electrophysiology combined with availability of human SC-CMs provides an exciting possibility to measure AP in human cells expressing endogenous cardiac ion channels. Electrophysiological properties of human SC-CMs were investigated with special focus on establishment of automated cell-based cardiac safety screens. For the first time assay protocols and procedures were established to measure and characterize both cardiac AP and the main underlying currents in human SC-CMs using automated electr...