1. Dysfunctional Sarcoplasmic Reticulum Ca 2+ Release Underlies Arrhythmogenic Triggers in Catecholaminergic Polymorphic Ventricular Tachycardia: A Simulation Study in a Human Ventricular Myocyte Model Introduction Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is a highly malignant arrhythmogenic disorder, triggered by exertion or emotional stress 1. Mutations in the Ryanodine Receptor (RyR) and Calsequestrin (CSQN) have been reported in the families affected by the disorder. The bidirectional ventricular tachycardia (VT) that is the phenotypic manifestation of the disease suggests that spontaneous Ca release-mediated delayed afterdepolarizations (DADs) and triggered activity (TA) is the most likely electrophysiological mechanism for arrhythmia initiation in CPVT 2. Although it is known that Sarcoplasmic Reticulum (SR) Ca release is altered by the disease, the molecular mechanisms that lead to the abnormal Ca cycling are unclear. In particular, it has been proposed that mutations in RyR lead to increased sensitivity of RyR openings to cytosolic Ca (Ca cyt ) 3. It has also been suggested that in CPVT mutations, faster recovery from refractoriness of Ca release can lead to premature SR Ca release and arrhythmogenic afterdepolarizations in cardiac myocytes 4. Using a multiscale Ca cycling model 5 that scales processes from single L-type Ca channels (LCCs), RyR openings and Ca sparks to whole-cell Ca cycling and electrophysiology in a human ventricular myocyte, we simulate these CPVT effects and explore their arrhythmogenic consequences during β-adrenergic stimulation (βAS). Interventions that target membrane ionic currents, including the L-type Ca current (I CaL ), Na-Ca exchange current (I NaCa ), or the Ca cycling system (RyR, Sarcoplasmic endoplasmic reticulum Ca ATPAse pump (SERCA)) are explored as potential anti- arrhythmic therapy. Methods A previously published model of Ca cycling in a ventricular myocyte provides the basis for the simulations in this study 5. The model consists of 10,000 diffusion-coupled units (called dyads) in which Ca release occurs stochastically within a dyad. Each unit comprises of the following Ca distribution compartments: myoplasm, network sarcoplasmic reticulum (NSR), junctional sarcoplasmic reticulum (JSR), dyadic space and the submembrane space. The number of LCCs and RyRs in a dyad is 15 and 100 respectively, and their states fluctuate stochastically. RyR kinetics is simulated by a Markov model. All other ionic currents, pumps and exchangers are formulated from the computer model of the human ventricular action potential 6. Modeling Isoproterenol (ISO) Effects Steady-state PKA phosphorylation effects of βAS due to ISO application were simulated using the β-adrenergic cascade model as adapted for human conditions 7. Targets included I CaL, the slow delayed rectifier K current (I Ks ), buffering constant of Troponin C (TnC), Na-K pump current (I NaK ), Fast Na current (I Na ) and Ca uptake via SERCA. CPVT-mutant RyR model The CPVT-mutant RyR model was simulated by (1) Increasing the cytosolic Ca sensitivity of RyR and (2) Increasing the luminal SR Ca sensitivity of RyR, thus simulating slower termination and faster recovery from refractoriness of the SR Ca release process. Computations In order to handle the required large amount of computations, the numerical simulation code was parallelized and optimized to facilitate the effective use of clusters of multicore CPUs. Namit Gaur 1, Xing Cai 1, Yoram Rudy 2 1.Simula Research Laboratory, Oslo, Norway 2. Washington University in Saint Louis, USA Figure 2. Ca handling in wild-type (WT) and CPVT-mutant (MT) under basal (no βAS) conditions. (A) Action potential (AP) (B) Whole-cell cytosolic Ca transient (Ca cyt ) and (C) Whole-cell junctional SR Ca (JSR) is identical between MT and WT at slow pacing rate (cycle length (CL) = 1500 ms). (D) AP (E) Ca cyt and (F) JSR is also identical during a paced beat at faster pacing (CL = 500 ms). After the cessation of pacing, the Ca release is slightly different in the MT. The cell is paced to steady-state. The last paced beat is indicated by arrow. Figure 4. Mechanisms underlying EADs, DADs and TA in a CPVT-mutant (MT) with βAS. (A) V m (B) Simulated line scan image of Ca cyt. (C) Whole-cell Ca cyt (D) JSR Ca (F) SR Ca release (J rel ) (G) I NaCa and (H) I CaL White star * indicates location of initiation of spontaneous Ca wave. Summary In this study we have simulated the functional consequences of suggested mechanisms (increased RyR cytoslic Ca 2+ sensitivity and shortened refractoriness of SR Ca 2+ release) and factors that underlie CPVT and how they can lead to formation of cellular arrhythmogenic triggers. We demonstrated that increased cytosolic Ca sensitivity of RyR and shortened refractoriness of SR Ca release can underlie arrhythmogenesis in CPVT. The mechanism involves spontaneous formation of Ca sparks and waves which result in EADs, DADs and TA. In an attempt to determine possible therapeutic targets for the prevention of CPVT, we studied the functional consequences of block of Ca handling proteins: I CaL, RyR, SERCA and I NaCa. The results identify I CaL inhibition as a potentially effective target for the prevention of CPVT. References Acknowledgments Figure 5. Effects of βAS phosphorylation of selective targets in a CPVT-mutant myocyte. (A and E) Membrane potential (V m ) (B and F) Simulated line scan image of cytosolic Ca (Ca cyt ). (C and G) Whole-cell Ca cyt and (D and H) JSR Ca. 1.Priori and Chen (2011)5. Gaur and Rudy (2011) 2.Cerrone et al (2009)6. O’Hara et al (2011) 3.Velasco et al (2009)7. O’Hara and Rudy (2012) 4.Gyorke S (2009) The authors acknowledge support by the NIH-NHLBI Grants R01- HL033343 and R01-HL049054 (to Y.R.). Y.R. is the Fred Saigh Distinguished Professor at Washington University in St. Louis. The numerical simulations used the NOTUR facilities of Norway, through project NN2849K. Research funding from American Heart Association and Simula Research Laboratory is gratefully acknowledged. Figure 7. Effects of SERCA block (left panels) or I CaL block (right panels) on CPVT-mutant under βAS. 50% block was used in these simulations. (A and E) Membrane potential (V m ) (B and F) Simulated line scan image of Ca cyt (C and G) Whole-cell Ca cyt and (D and H) JSR Ca. Block of the SERCA pump abolished DADs and TA, but EADs still occurred. Block of I CaL abolished all arrhythmogenic triggers: EADs, DADs and TA. Both interventions prevented formation of propagating Ca waves. CPVT-Mutant RyR has Higher Open Probability Compared to Wild-type Figure 1. Increased RyR open probability (P o ) in CPVT-mutant compared to wild- type. (A) Markov model of RyR. (B) P o of CPVT-mutant and wild-type RyR channel as a function of Ca cyt (at fixed Ca SR = 1 mM). (C) P o of CPVT-mutant and wild-type RyR channel as a function of Ca SR (at fixed Ca cyt = 10 µM). Ca 2+ handling in CPVT-mutant during basal (non βAS) conditions Arrhythmogenic triggers occur in CPVT-mutant during βAS conditions Figure 3. Ca handling in wild-type (WT) and CPVT-mutant (MT) under βAS conditions. (A) Action potential (AP) (B) Whole-cell cytosolic Ca transient (Ca cyt ) and (C) Junctional SR Ca (JSR) at slow pacing rate (cycle length (CL = 1500 ms)). Early afterdepolarization (EAD; indicated by +) occurs only in the MT but not the WT. (D) AP (E) Ca cyt and (F) JSR at faster pacing rate (CL = 500 ms). EAD, delayed afterdepolarization (DAD; indicated by #) and triggered activity (TA; indicated by *) occur in the mutant but not the wild-type. Abnormal SR Ca 2+ release underlies arrhythmogenic triggers in the CPVT-mutant Effect of βAS on I CaL underlies arrhythmogenic triggers in CPVT-mutant Effects of RyR block or I NaCa block on CPVT-mutant under βAS Figure 6. Effects of RyR block or I NaCa block on CPVT-mutant under βAS. (A and E) V m. (B and F) Simulated line-scan image of Ca cyt. (C and G) Whole-cell Ca cyt and (D and H) JSR Ca. Block of RyR did not abolish cellular arrhythmogenic events in the form of DADs (indicated by #), EADs (indicated by +) and TA (indicated by *). Block of I NaCa abolished EADs and TA, but DADs and Ca waves still occurred. Effects of SERCA block or I CaL block on CPVT- mutant under βAS