Ne that prevent RyR2 opening by slowing RyR2 activation could be proarrhythmic because they favor the induction of calcium alternans. In line with this, it has been shown experimentally that tetracaine favors the induction of non-uniform beat-to-beat responses at lower stimulation frequencies in human atrial myocytes [25], [36]. Another field 
gaining increasing attention is genetic mutations get ��-Sitosterol ��-D-glucoside linked to abnormal RyR2 function or SR calcium loading [20], [21]. Here, current paradigms are that mutations causing SR overload or calcium leak are likely to be arrhythmogenic by promoting calcium release-induced afterdepolarizations [37]. However, as pointed out above, the present model predicts that mutations that increase RyR2 open probability by increasing RyR2 activation may be antiarrhythmic because they are expected to prevent the induction of calcium alternans. On the other hand, our results also suggest that mutations which decrease RyR2 open probability by reducing RyR2 activation are likely arrhytmogenic because they induce calcium alternans at lower beating rates. In accordance with this prediction, the first mutation in the RyR2 associated with ventricular fibrillation (A4860G), which dramatically reduces RyR2 opening, was recently described and shown to be associated with a strong reduction in luminal calcium activation of the RyR2 [38].Together, this shows that the present model may be useful to understand and predict the relationship between molecular alterations that affect RyR2 refractoriness and rate-dependent beat-to-beat changes in the intracellular calcium transient in isolated cardiomyocytes.ConclusionThe present study has used a well characterized rabbit numerical ventricular myocyte model and a dynamic clamping protocol to systematically investigate how fundamental RyR2 properties such as activation, inactivation, and recovery from inactivation as well as SR calcium loading contribute to determine the frequency dependent induction of cytosolic calcium alternans. This approach allows a mapping of the beat-to-beat response as a function of RyR2 activation and inactivation as well as the identification of domains where SR calcium load and/or RyR2 recovery from inactivation contribute to the induction of calcium alternans. It also allows the identification of transition zones where one predominant mechanism is substituted by another, and a characterization of how the transition zones depend on the stimulation frequency or the RyR2 recovery time. Importantly, the developed clamping protocols can also be used to study the mechanism behind alternans in other cardiac myocyte models. A consequence of our study relevant to the analysis of other cardiac cell types is that even when experimental data shows concurrent alternations in calcium load and the cytosolic calcium transient, this does not necessarily imply that alternation in calcium load is the underlying mechanism.Supporting InformationAppendix S1 Supplemental material with MedChemExpress PLV-2 further information on the modifications of the RyR2 properties in the model by Shannon et al. necessary to obtain cytosolic calcium alternans. It also includes some extra simulations, using an action potential clamp to eliminate potential interference from alternations in action potential amplitude or duration. Finally, it provides a mathematical study of the instability leading to calcium alternans and a more detailed analysis of the post-rest potentiation of the calcium transient. The appendix includes.Ne that prevent RyR2 opening by slowing RyR2 activation could be proarrhythmic because they favor the induction of calcium alternans. In line with this, it has been shown experimentally that tetracaine favors the induction of non-uniform beat-to-beat responses at lower stimulation frequencies in human atrial myocytes [25], [36]. Another field gaining increasing attention is genetic mutations linked to abnormal RyR2 function or SR calcium loading [20], [21]. Here, current paradigms are that mutations causing SR overload or calcium leak are likely to be arrhythmogenic by promoting calcium release-induced afterdepolarizations [37]. However, as pointed out above, the present model predicts that mutations that increase RyR2 open probability by increasing RyR2 activation may be antiarrhythmic because they are expected to prevent the induction of calcium alternans. On the other hand, our results also suggest that mutations which decrease RyR2 open probability by reducing RyR2 activation are likely arrhytmogenic because they induce calcium alternans at lower beating rates. In accordance with this prediction, the first mutation in the RyR2 associated with ventricular fibrillation (A4860G), which dramatically reduces RyR2 opening, was recently described and shown to be associated with a strong reduction in luminal calcium activation of the RyR2 [38].Together, this shows that the present model may be useful to understand and predict the relationship between molecular alterations that affect RyR2 refractoriness and rate-dependent beat-to-beat changes in the intracellular calcium transient in isolated cardiomyocytes.ConclusionThe present study has used a well characterized rabbit numerical ventricular myocyte model and a dynamic clamping protocol to systematically investigate how fundamental RyR2 properties such as activation, inactivation, and recovery from inactivation as well as SR calcium loading contribute to determine the frequency dependent induction of cytosolic calcium alternans. This approach allows a mapping of the beat-to-beat response as a function of RyR2 activation and inactivation as well as the identification of domains where SR calcium load and/or RyR2 recovery from inactivation contribute to the induction of calcium alternans. It also allows the identification of transition zones where one predominant mechanism is substituted by another, and a characterization of how the transition zones depend on the stimulation frequency or the RyR2 recovery time. Importantly, the developed clamping protocols can also be used to study the mechanism behind alternans in other cardiac myocyte 
models. A consequence of our study relevant to the analysis of other cardiac cell types is that even when experimental data shows concurrent alternations in calcium load and the cytosolic calcium transient, this does not necessarily imply that alternation in calcium load is the underlying mechanism.Supporting InformationAppendix S1 Supplemental material with further information on the modifications of the RyR2 properties in the model by Shannon et al. necessary to obtain cytosolic calcium alternans. It also includes some extra simulations, using an action potential clamp to eliminate potential interference from alternations in action potential amplitude or duration. Finally, it provides a mathematical study of the instability leading to calcium alternans and a more detailed analysis of the post-rest potentiation of the calcium transient. The appendix includes.
