ABC | Volume 114, Nº4, April 2020

Brief Communication Inotropic and Antiarrhythmic Transmural Actions of Ranolazine in a Cellular Model of Type 3 Long QT Syndrome Victor Martins Miranda, 1 Samuel Santos Beserra, 1 Danilo Roman-Campos 1 Universidade Federal de São Paulo, Departamento de Biofísica, Edifício de Ciências Biomédicas, 1 São Paulo, SP - Brazil Mailing Address: Danilo Roman Campos • Universidade Federal de São Paulo – Biofísica - Rua Botucatu, 862. Postal Code 04023-062, São Paulo, SP – Brazil E-mail: drcbio@gmail.com Manuscript received April 03, 2019, revised manuscript August 19, 2019, accepted September 10, 2019 DOI: https://doi.org/10.36660/abc.20190220 Keywords Arrhythmias, Type 3 Long QT Syndrome, ATX-II, Late Sodium current, Ranolazine, Contraction. Abstract Ranolazine (RANO) prevents cardiac arrhythmia by blocking the late sodium current (I NaL ). A transmural gradient of Nav1.5 is found in the left ventricular wall of the heart. Thus, we investigated the effects of RANO in healthy cardiomyocytes and in a cellular model of type 3 long QT syndrome (LQT3). We used isolated endocardium (ENDO) and epicardium (EPI) cells and a video edge detection system and fluorescence microscopy to monitor calcium transients. RANO (0.1, 1, 10 and 30 uM, at 25 o C) at a range of pacing frequencies showed a minor impact on both cell types, but RANO at 30uM and 35 o C for ENDO cells attenuated sarcomere shortening by~21%. Next, to mimic LQT3, we exposed ENDO and EPI cells to anemone toxin II (ATX-II), which augments I NaL . Cellular arrhythmias induced by ATX-II were abrogated by RANO (30 µM) at 35 o C. Based on our results we can conclude that RANO has a minor impact on sarcomere shortening of healthy ENDO and EPI cells and it abrogates arrhythmias induced by I NaL to a similar level in ENDO and EPI cells. Introduction Arrhythmia in cardiovascular diseases is one of the leading causes of death worldwide. 1 The antiarrhythmic action of RANO is attributed to reduction in the slow inactivating component of cardiac inward current through Nav1.5, known as the late sodium current (I NaL ). 2 Despite major advances in the understanding of molecular mechanisms underlying RANO action, whether RANO exhibits a transmural action in heart muscle cells remains uncertain. Therefore, in the present study our hypothesis is that RANO has transmural action on healthy field-stimulated endocardium (ENDO) and epicardium (EPI) cells and also on arrhythmias and calcium disturbance induced by anemone toxin II (ATX-II), 3 which increases I NaL and mimics several aspects of type 3 long QT syndrome (LQT3), a diseased linked to increased I NaL in heart cells. 2 Methods Animals Male Wistar rats (160–250 g; 5–7-week old) were used in the experiments. All experimental procedures were performed in accordance with institutional guidelines, and the study was approved by the local ethical review committee. Cardiomyocytes were isolated as previously described. 4 Sarcomere shortening and calcium transient Experiments were conducted as previously described by our group. 5 Cells were perfused with RANO (Alomone, Israel) at 0.1, 1, 10, or 30 µM from a 10 mM stock solution. Data were normalized as the function of sarcomere contraction before RANO exposure. To access the antiarrhythmic effect of RANO following exposure to 6 nM ATX-II (Alomone, Israel), the times to 90% sarcomere relaxation (T90R) and calcium reuptake (T90Ca 2+ ) were recorded as arrhythmic indexes. In addition, 10 mM tetrodotoxin (TTX) (Alomone, Israel) was used to confirm that the observed phenotype was indeed due to I NaL . Statistical analysis All results are expressed as mean ± standard error of the mean. Significant differences were determined using two- sample t-test or one-way ANOVA with repeated measures, followed by Tukey’s post hoc test. P < 0.05 was considered significant. Cardiomyocytes from at least two distinct hearts were used in each experiment. Results and discussion Previous studies have shown that healthy cardiomyocytes exhibit I NaL . 6 Moreover, a gradient of sodium current has been recorded in the left ventricular wall, and it has been reported to be larger in ENDO cells than in EPI cells. 7 Thus, we hypothesized that ENDO cells present larger I NaL than EPI cells. Since I NaL modulates [Ca 2+ ]i in cardiomyocytes, 8 RANO would be able to attenuate contraction in both cell groups, although with greater potency in ENDO cells than in EPI cells. To test this hypothesis, cells were perfused at 25 o C with RANO; however, RANO could not attenuate sarcomere shortening in ENDO and EPI cardiomyocytes (Figures 1 A and C). A similar trend was observed when cardiomyocytes were exposed to 30 µM RANO and paced at 0.2 Hz. When ENDO and EPI cells were exposed to 30 µM RANO and paced at 0.2 Hz using a superfusion solution at 35 o C, cell shortening was attenuated in ENDO cells by ~21% (p < 0.05) but not in EPI cells (Figures 1B and D). Thus, corroborating the previous findings, our results suggest that healthy ENDO cells indeed 732