REPOLARISATION ASSAYS

SINGLE VENTRICULAR MYOCYTES

STABILITY OF ACTION POTENTIAL DURATION UNDER CONTROL CONDITIONS

As noted in the ㈠ Guidelines on Safety Pharmacology Studies for Human Pharmaceuticals艃H S7B section 3.2.2.1) <b>action potential duration studies using disaggregated cardiac myocytes provide a powerful approach</b> to elucidate the ionic current mechanism responsible for a change in action potential parameters.

Although this preparation requires a degree of technical expertise we at OCP have over 15 years experience in using such preparations and have the skill and expertise to consistently produce stable recordings of the highest quality.

Figure 1 Stability of action potential duration in a single guinea-pig ventricular myocyte.

Figure 1   shows 10 consecutive action potential records taken from a single left ventricular myocyte under our standard experimental conditions used for the action potential measurements. These data demonstrate that measurements of action potential duration were stable under the conditions of these experiments.

It has been demonstrated that repolarisation assays using single ventricular myocytes provide results that are comparable to other preparations (such as dog Purkinje fibres) for assessing the effects of compounds that cause QT prolongation, including sotalol and dofetilide
(also see www.physoc.org/Proceedings/Abstracts/544P/Files/S156.html).

Ventricular myocytes when paced at a constant rate have been shown to have a natural beat-to-beat variability in APD₉₀ which thought to be due in part to the stochastic variability of major ionic currents that operate during the plateau phase of the action potential.  This intrinsic beat-to-beat variability is reduced by cell-to-cell coupling (Zaniboni et al., 2000). However, data in Figure 1 and Figure 2 show that under the test conditions used by OCP that beat-to-beat variation in action potential duration is small and that the effects of test compounds can be easily detected.

Figure 2   Mean data for the stability of APD₅₀ and APD₉₀.

Figure 2 shows mean 㥭 for measurements of APD₅₀ and APD₉₀ over a 30 s period for n=30 cells. These data demonstrate that measurements of action potential duration were stable under the conditions of these experiments.

Figure 3 shows mean 㥭 for measurements of APD₉₀ over a 3 min period for n=20 cells, the red dashed line represented the mean value of APD₉₀ during this period. 

Beat-to-beat variability can be quantified by calculating the coefficient of variability (CV) which is defined as the percentage of SD/mean APD₉₀.  A recent study derived a CV value of 2.4î¹ for single ventricular myocytes and showed that this value was reduced to 1.5by myocyte coupling  (Zaniboni et al., 2000).  From the data presented in Figure 3 we calculated a CV of 1.38î°· (n=20) showing that in our test conditions beat-to-beat variability of APD₉₀ is less than 2% of the mean. This stability enables us to accurate assess the potential of an NCE to prolong APD₉₀ by as little as 5%.

B

Figure 4 As a working example Figure 3 shows the effect of dofetilide (100 nM) on APD₉₀ recorded in 5 ventricular myocytes (meanå­ is plotted in figure 2 A&B though for the sake of clarity sem is only shown for every 10^th beat i.e. every 10 s).

Dofetilide prolongs APD₉₀ by ~25% which is clearly illustrated in Fig 3B.

References

Zaniboni, M., Pollard, Yang, and. Spitzer. Beat-to-beat repolarisation variability in ventricular myocytes and its suppression by electrical coupling. Am. J. Physiol. Heart Circ. Physiol. 278: H677-H687,2000