Assessment of extracellular field potential and Ca2+ transient signals for early QT/pro-arrhythmia detection using human induced pluripotent stem cell-derived cardiomyocytes.

Cardiovascular toxicity is a prominent reason for failures in drug development, resulting in the demand for assays that can predict this liability in early drug discovery. We investigated whether iCell® cardiomyocytes have utility as an early QT/TdP screen. Thirty clinical drugs with known QT/TdP outcomes were evaluated blind using label-free microelectrode array (parameters measured were beating period (BP), field potential duration (FPD), fast Na+ amplitude and slope) and live cell, fast kinetic fluorescent Ca2+ transient FLIPR® Tetra (parameters measured were peak count, width, amplitude) systems. Many FPD-altering drugs also altered BP. Correction for BP, using a Log-Log (LL) model, was required to appropriately interpret direct drug effects on FPD. In comparison with human QT effects and when drug activity was to be predicted at top test concentration (TTC), LL-corrected FPD and peak count had poor assay sensitivity and specificity values: 13%/64% and 65%/11%, respectively. If effective free therapeutic plasma concentration (EFTPC) was used instead of TTC, the values were 0%/100% and 6%/100%, respectively. When compared to LL-corrected FPD and peak count, predictive values of uncorrected FPD, BP, width and amplitude were not much different. If pro-arrhythmic risk was to be predicted using Ca2+ transient data, the values were 67%/100% and 78%/53% at EFTPC and TTC, respectively. Thus, iCell® cardiomyocytes have limited value as an integrated QT/TdP assay, highlighting the urgent need for improved experimental alternatives that may offer an accurate integrated cardiomyocyte safety model for supporting the development of new drugs without QT/TdP effects.

Enhanced characterization of contractility in cardiomyocytes during early drug safety assessment.

We sought to investigate whether drug-induced changes in contractility were affected by pacing rates that represent the range of heart rates encountered in vivo. Using the cell geometry measurement system (IonOptix), we paced dog cardiomyocytes at different cycle lengths (CLs) of 2000, 1000, 500, and 333.3 ms, before and after exposure to 13 inotropic drugs. Time course data using vehicle control (0.1% dimethyl sulfoxide (DMSO)) demonstrated stability of the system at all CLs tested. Seven positive inotropes (eg isoproterenol) exerted rate-dependent increases in sarcomere shortening (Sarc. short.; maximal effect at a CL of 333.3 ms [0.1 µM isoproterenol increased Sarc. short. by 41.1% and 145.9% at 2000 and 333.3 ms, respectively]). Omecamtiv mecarbil showed an atypical profile (increased Sarc. short. at 2000 ms [106.9%] and decreased at 333.3 ms [IC(50) = 0.64 µM]). Four negative inotropes (eg flecainide) showed rate-independent inhibition of Sarc. short. (IC(50)s: 3.3 µM [2000 ms] versus 2.3 µM [333.3 ms]). The remaining negative inotropes, verapamil, and BTS (N-benzyl-p-toluene sulphonamide) produced an increase (IC(50)s: 3.9 µM [2000 ms] versus 0.043 µM [333.3ms]) and decrease (IC(50)s: 18.3 µM [2000 ms] versus 34.0 µM [333.3 ms]) in potency, respectively. Negative inotropes (eg flecainide, BTS, and verapamil) decreased the area of the Ca(2+) transient versus Sarc. short. hysteresis loop, although rate dependency was seen with verapamil only. Positive inotropes (eg isoproterenol and levosimendan) induced a rate-dependent increase in the area, however Omecamtiv mecarbil increased and decreased the area at CLs of 2000 and 333.3 ms, respectively. Thus, the use of different pacing rates may improve the detection of inotropes in early drug discovery and illustrate the potential for finger-printing different mechanisms of action.

Preservation of cardiomyocytes from the adult heart.

Cardiomyocytes represent one of the most useful models to conduct cardiac research. A single adult heart yields millions of cardiomyocytes, but these cells do not survive for long after isolation. We aimed to determine whether inhibition of myosin II ATPase that is essential for muscle contraction may preserve fully differentiated adult cardiomyocytes. Using inhibitors of the myosin II ATPase, blebbistatin and N-benzyl-p-toluene sulphonamide (BTS), we preserved freshly isolated fully differentiated adult primary cardiomyocytes that were stored at a refrigerated temperature. Specifically, preserved cardiomyocytes stayed viable for a 2-week period with a stable expression of cardiac genes and retained the expression of key markers characteristic of cardiomyocytes. Furthermore, voltage-clamp, action potential, calcium transient and contractility studies confirmed that the preserved cardiomyocytes are comparable to freshly isolated cells. Long-term exposure of preserved cardiomyocytes to four tyrosine kinase inhibitors, sunitinib malate, dasatinib, sorafenib tosylate and imatinib mesylate, revealed their potential to induce cardiac toxicity that was manifested with a decrease in contractility and induction of cell death, but this toxicity was not observed in acute experiments conducted over the time course amenable to freshly prepared cardiomyocytes. This study introduces the concept that the inhibition of myosin II ATPase safeguards the structure and function of fully differentiated adult cardiomyocytes. The fact that these preserved cardiomyocytes can be used for numerous days after preparation makes them a robust and versatile tool in cardiac research and allows the investigation of long-term exposure to novel drugs on cardiomyocyte function.