Phosphodiesterase 10A Inhibitor Modulates Right Ventricular Outflow Tract Electrophysiological Activities and Calcium Homeostasis via the cGMP/PKG Pathway

Figures

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  FIGURE 1. Effects of TP‐10 on action potentials of the RVA and RVOT tissues. (A) (Upper) Representative action potentials and contractile forces from the RVA tissues treated with TP‐10, and (below) the mean values (N = 9). (B) (Upper) Representative action potentials and contractile forces from RVOT tissues treated with TP‐10, and (below) the mean values (N = 6). APA, action potential amplitude; RMP, resting membrane potential; RVA, right ventricular apex; RVOT, right ventricular outflow tract; APD20, APD50, and APD90, action potential duration at repolarisation levels of 20%, 50%, and 90% of the action potential amplitude, respectively.

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  FIGURE 2. Effects of TP‐10, thapsigargin/TP‐10 or KT5823/TP‐10 cotreatment on ISO challenge‐induced bursting firing in RVOT tissues. (A) (Left) Representative recordings of post‐RVP electric activity in RVOT tissues at baseline, as well as treated with ISO and ISO/TP‐10. (Right) Mean rate and duration of post‐RVP bursting firing at baseline, under ISO treatment, and under ISO/TP‐10 cotreatment (N = 7). (B) (Left) Representative recordings of post‐RVP electric activity in ISO‐challenged, ISO/thapsigargin‐cotreated and ISO/thapsigargin/TP‐10‐cotreated RVOT tissues. (Right) Mean rate and duration of post‐RVP bursting firing in ISO‐challenged, ISO/thapsigargin‐cotreated and ISO/thapsigargin/TP‐10‐cotreated RVOT tissues (N = 7). (C) (Left) Representative recordings of post‐RVP electric activity in ISO‐challenged, ISO/KT5823‐cotreated and ISO/KT5823/TP‐10‐cotreated RVOT tissues. (Right) Mean rate and duration of post‐RVP bursting firing in ISO‐challenged, ISO/KT5823‐cotreated and ISO/KT5823/TP‐10‐cotreated RVOT tissues (N = 6). ISO, isoproterenol; RVOT, right ventricular outflow tract; RVP, rapid ventricular pacing.

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  FIGURE 3. Effect of TP‐10 on Ca2+ transient and SR Ca2+ content. (A) (Upper and middle) Typical traces of steady‐state Ca2+ transient in RVOT cardiomyocytes treated with TP‐10 and baseline control (without TP‐10), as well as (lower) the mean values (baseline group n = 15/4, N = 4; TP‐10 group n = 26/5, N = 4). (B) (Upper and middle) Typical traces of caffeine‐elicited Ca2+ transient in RVOT cardiomyocytes treated with TP‐10 and baseline control (without TP‐10), as well as (lower) the mean values (baseline group n = 13/4, N = 4; TP‐10 group n = 24/5, N = 5). (C) (Upper and middle) Typical recording of SR Ca2+ content determined by integral of inward NCX current induced by fast caffeine application in voltage‐clamped RVOT cardiomyocytes treated with TP‐10 and baseline control (without TP‐10), as well as (lower) the mean values (baseline group n = 10/5, N = 5; TP‐10 group n = 10/5, N = 5). NCX, sodium‐calcium exchanger; RVOT, right ventricular outflow tract; SR, sarcoendoplasmic reticulum.

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  FIGURE 4. Effect of TP‐10 on Ito, ICa,L and NCX current. (A) (Upper and middle) Representative traces of Ito in RVOT cardiomyocytes treated with TP‐10 and baseline control (without TP‐10), as well as (lower) the I–V relationship (n = 8/4, N = 4). (B) (Upper and middle) Representative traces of ICa,L in RVOT cardiomyocytes treated with TP‐10 and baseline control (without TP‐10) as well as (lower) the I–V relationship (n = 9/3; *p < 0.05; ***p < 0.005) (C) (Upper and middle) Representative traces of NCX current in RVOT cardiomyocytes treated with TP‐10 and baseline control (without TP‐10), as well as (lower) the I–V relationship (n = 10/4, N = 4; *p < 0.05; ***p < 0.005). I–V, current–voltage; NCX, sodium‐calcium exchanger; RVOT, right ventricular outflow tract.

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  FIGURE 5. Effect of TP‐10 on INa,L. (Left) Representative tracings of INa,L (TTX‐sensitive current) with the voltage‐clamp protocol shown in the inset above the current traces, and (Right) the mean values of RVOT cardiomyocytes treated with TP‐10 and baseline control (without TP‐10) (baseline group n = 9/3, N = 3; TP‐10 group n = 9/3, N = 3;pA/pF to pA/pV, respectively). RVOT, right ventricular outflow tract; TTX, tetrodotoxin.

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  FIGURE 6. Quantification of Ca2+ regulatory proteins in control and TP‐10‐treated RVOT cardiomyocytes. Representative immunoblot and normalised densitometric protein levels of phosphorylated CaMKII, CaMKII, SERCA2a, PLB, PLB pSer16 and PLB pThr17 in RVOT cardiomyocytes treated with TP‐10 and baseline control (without TP‐10). GAPDH was used as an internal control (baseline group N = 4, TP‐10 group N = 4). We used the same sample from the same animal in the same gel for PLB, phospho‐PLB S16, phospho‐PLB T17 and GAPDH western blotting. Consequently, the representative GAPDH is the same for PLB, phospho‐PLB S16 and phospho‐PLB T17. Additionally, the PLB/GAPDH values used for calculating the ratio of phosphorylated PLB at S16 or T17 are identical. PLB, phospholamban; CaMKII, Ca(2+)/calmodulin‐dependent protein kinase II; RVOT, right ventricular outflow tract; SERCA2a, sarcoendoplasmic reticulum calcium ATPase 2a.

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  FIGURE 7. Proposed anti‐arrhythmia mechanism of TP‐10. TP‐10 enhances the cGMP/PKG pathway, resulting in reduced L‐type Ca2+ current and late Na+ current, as well as increased NCX current and upregulated SERCA2a function. These effects collectively increase SR Ca2+ content and Ca2+ transients, thereby attenuating isoproterenol‐induced intracellular Ca2+ overload that promotes tachyarrhythmia. cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; NCX, sodium‐calcium exchanger; PDE10A, phosphodiesterase 10A; PKA, protein Kinase A; PKG, protein kinase G; PLB, phospholamban; RVOT, right ventricular outflow tract; RyR2, ryanodine receptor 2; SERCA2a, sarcoplasmic reticulum calcium ATPase 2a; SR, sarcoplasmic reticulum.