Inotropic and Antiarrhythmic Transmural Actions of Ranolazine in a Cellular Model of Type 3 Long QT Syndrome

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^oC) at a range of pacing frequencies showed a minor impact on both cell types, but RANO at 30uM and 35^oC 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^oC. 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_NaLto a similar level in ENDO and EPI cells.

Arrhythmias; Type 3 Long QT Syndrome; ATX-II; Late Sodium current; Ranolazine; Contraction

Resumo

A Ranolazina (RANO), conhecida na clínica como Ranexa, é um fármaco que previne a arritmia cardíaca através da inibição da corrente de sódio tardia (I_NaT). Um gradiente de voltagem transmural do canal Nav1.5 encontra-se na parede ventricular esquerda do coração. Assim, investigamos os efeitos da RANO em cardiomiócitos saudáveis e em modelo celular da Síndrome do QT longo tipo 3 (SQTL tipo 3). Usamos células isoladas do endocárdio (ENDO) e do epicárdio (EPI) e um software de medição com detecção de bordas por vídeo e microscopia de fluorescência para monitorar os transientes de cálcio. A RANO (0,1, 1, 10 e 30 uM, a 25^OC) em uma série de frequências de estimulação teve impacto pouco significativo sobre ambos os tipos de células, mas a RANO (30uM) a 35^OC minimizou o encurtamento dos sarcômeros em ~21% para células do endocárdio. Em seguida, para simular a SQTL tipo 3, as células do ENDO e EPI foram expostas à toxina ATX-II da anêmona do mar, que aumenta a I_NaT. As arritmias celulares induzidas por ATX-II foram suprimidas com o uso da RANO (30 µM) a 35^OC. Com base nesses resultados, podemos concluir que a RANO tem um impacto pouco significativo sobre o encurtamento dos sarcômeros de células saudáveis do ENDO e EPI. Além disso, ela suprime as arritmias induzidas por I_NaT para níveis semelhantes nas células do ENDO e EPI.

Arritmias; Síndrome do QT Longo do Tipo 3; ATX-II; Ranolazina; Corrente Tardia de Sódio; Contração

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_NaLand mimics several aspects of type 3 long QT syndrome (LQT3), a diseased linked to increased I_NaLin 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 (T90Ca²) 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_NaLthan EPI cells. Since I_NaLmodulates [Ca²]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^oC 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^oC, 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 present larger I_NaLthan EPI cells. However, it is also important to note that 30 µM RANO could also block L-type calcium current in cardiomyocytes.⁹

Figure 1
– Inotropic effect of ranolazine (RANO) on sarcomere shortening of ENDO and EPI cardiomyocytes. Representative sarcomere shortening recordings before (black (25ºC) and blue (35ºC)) and after (light gray (25ºC) and red (35ºC)) exposure of ENDO (left) and EPI (right) cardiomyocyte to RANO ((A) 10 and (B) 30 µM). Inotropic effect of 0.1, 1, and 10 µM RANO (C) and 30 µM (D) on sarcomere shortening (upper bars); Normalized time to 50% sarcomere contraction (T50C) (middle bars)) and; normalized time to 50% of sarcomere relaxation (T50R) (bottom bars) Hatched bars represent EPI cells (n = 3–6 cells/concentration). *p < 0.05 comparing before and after RANO exposure.

To better understand the mechanism underlying sarcomere shortening induced by RANO, subsequent experiments were performed at 35^oC. Cardiomyocytes were loaded with Fura 2-AM to monitor calcium oscillation during cell contraction, and cells were exposed to ATX-II to increase I_NaLand induce an LQT3 phenotype3 ( Figure 2 ). ENDO ( Figures 2A, B and C ) and EPI ( Figures 2D, E and F ) cells exposed to ATX-II showed clear calcium disturbances and simultaneous mechanical arrhythmias. RANO (30 µM) strongly attenuated the arrhythmic phenotype induced by ATX-II in both cell groups to a similar extent. To confirm that the arrhythmic phenotype observed in our experiments was truly attributed to I_NaL, cells were exposed to 6 nM ATX-II [ Figure 2A (iv) and Figure 2D (iv) ], following exposure to 10 µM TTX and 6 nM ATX-II [ Figure 2A (v) and Fig 2D (v) ]. The results confirmed that the observed arrhythmic phenotype occurred due to I_NaLaugmentation. Despite the fact that rat ENDO cells present larger sodium currents than EPI cells,⁷ , ¹⁰ the arrhythmic phenotype induced by ATX-II and the extent of antiarrhythmic effects of RANO were similar in both cell groups.

Figure 2
– Action of ranolazine (RANO) in ENDO and EPI cardiomyocytes exposed to ATX-II and paced at 0.2 Hz. Representative traces of calcium transients (upper traces) and cardiomyocyte sarcomere shortening (lower traces) following exposure to Tyrode’s solution (i), 6 nM ATX-II (ii), 6 nM ATX-II + 30 µM RANO (iii), 6 nM ATX-II (iv), and 6 nM ATX-II + 10 µM TTX (iv) in ENDO (A) and EPI (D) cells. Time to 90% of Ca2+ reuptake in ENDO (n = 8 cells) (B) and EPI (n = 6 cells) (E). Time to 90% sarcomere relaxation in ENDO (C) and EPI (F) cells. * p < 0.05 compared to the ATX-II group.

Interestingly, the therapeutic concentration range of RANO is 1–10 µM.¹¹ The apparent discrepancy in RANO potency may be explained by the fact that ATX-II at doses of 1–10 nM induces larger I_NaLin cardiomyocytes than that observed in cardiovascular disease.³ , ⁶

Conclusion

RANO exerted a minor impact on sarcomere shortening of healthy cardiomyocytes and abrogated arrhythmias induced by I_NaLto a similar extent in ENDO and EPI cells.

Referências

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• Study Association
  This article is part of the thesis of master submitted by Victor Martins Miranda, from Universidade Federal de São Paulo .
• Ethics approval and consent to participate
  This study was approved by the Ethics Committee of the CEUA UNIFESP under the protocol number 2435/70816. All the procedures in this study were in accordance with the 1975 Helsinki Declaration, updated in 2013.
• Erratum
  In the Brief Communication “Inotropic and Antiarrhythmic Transmural Actions of Ranolazine in a Cellular Model of Type 3 Long QT Syndrome”, pages 732-735, by authors Victor Martins Miranda, Samuel Santos Beserra, Danilo Roman Campos, consider Danilo Roman-Campos as the correct form for the name of the author Danilo Roman Campos.

• Sources of Funding
  This study was funded by FAPESP nº 2014/09861-1.

Publication Dates

• Publication in this collection
  29 May 2020
• Date of issue
  Apr 2020Apr 2020

History

• Received
  03 Apr 2019
• Reviewed
  19 Aug 2019
• Accepted
  10 Sept 2019