Calcium dysregulation increases right ventricular outflow tract arrhythmogenesis in rabbit model of chronic kidney disease

Abstract

Chronic kidney disease (CKD) increases the risk of arrhythmia. The right ventricular outflow tract (RVOT) is a crucial site of ventricular tachycardia (VT) origination. We hypothesize that CKD increases RVOT arrhythmogenesis through its effects on calcium dysregulation. We analysed measurements obtained using conventional microelectrodes, patch clamp, confocal microscopy, western blotting, immunohistochemical examination and lipid peroxidation for both control and CKD (induced by 150 mg/kg neomycin and 500 mg/kg cefazolin daily) rabbit RVOT tissues or cardiomyocytes. The RVOT of CKD rabbits exhibited a short action potential duration, high incidence of tachypacing (20 Hz)-induced sustained VT, and long duration of isoproterenol and tachypacing-induced sustained and non-sustained VT. Tachypacing-induced sustained and non-sustained VT in isoproterenol-treated CKD RVOT tissues were attenuated by KB-R7943 and partially inhibited by KN93 and H89. The CKD RVOT myocytes had high levels of phosphorylated CaMKII and PKA, and an increased expression of tyrosine hydroxylase-positive neural density. The CKD RVOT myocytes exhibited large levels of I_to , I_Kr , NCX and L-type calcium currents, calcium leak and malondialdehyde but low sodium current, SERCA2a activity and SR calcium content. The RVOT in CKD with oxidative stress and autonomic neuron hyperactivity exhibited calcium handling abnormalities, which contributed to the induction of VT.

Keywords: calcium homeostasis; chronic kidney disease; right ventricular outflow tract; ventricular tachycardia.

FIGURE 1

Experimental localizations of the right ventricular outflow tract (RVOT) and the effects of rapid ventricular pacing (RVP, 20 Hz) and isoproterenol (1 μM) in the RVOTs of the chronic kidney disease (CKD) and control tissues. (A) Location of the isolated RVOT tissues, superior to the supraventricular crest and ≤5 mm below the pulmonary valve. RVA and PA denote the right ventricular apex and pulmonary artery respectively. (B) Tracings and average data of RVP‐induced non‐sustained ventricular tachycardia (VT) in the CKD (n = 8) and control (n = 10) RVOT tissues. (C) Tracings of regular pacing (2 Hz) before and after the isoproterenol infusion in the CKD and control RVOT tissues. (D) Means of the incidence, beating rate and duration of RVP‐induced sustained and non‐sustained VT in the isoproterenol‐treated CKD (n = 17) and control (n = 19) RVOT tissues. The tracing indicates examples of RVP‐induced sustained VT in the isoproterenol‐treated CKD and control RVOTs. *p < 0.05, ***p < 0.005 versus control. Error bars indicate SEMs

FIGURE 2

Effects of KB‐R7943 (10 μM), H89 (10 μM), KN93 (1 μM) and KN92 (1 μM) on RVP (20 Hz)‐induced VT (sustained and non‐sustained) in the isoproterenol (1 μM)‐treated CKD and control RVOT tissues. (A) Upper panel shows the tracings of RVP‐induced VT in the isoproterenol‐treated CKD (n = 9) and control (n = 10) RVOT tissues before and after KB‐R7943 (10 μM). Lower panel indicates that KB‐R7943 (10 μM) yielded a lower rate and shorter duration of RVP‐induced VT in the isoproterenol‐treated CKD RVOTs compared with those in the control RVOTs. (B) Upper panel indicates the tracings of RVP‐induced VT in the isoproterenol‐treated CKD (n = 6) and control (n = 7) RVOT tissues before and after H89 (10 μM). Lower panel indicates that H89 (10 μM) yielded a shorter duration of RVP‐induced VT in the isoproterenol‐treated CKD RVOTs compared with that in the control RVOTs. (C) Upper panel indicates the tracings of RVP‐induced VT in the isoproterenol‐treated CKD and control RVOT tissues before and after KN93 (1 μM) and KN92 (1 μM). Lower panel indicates that KN93 (1 μM) resulted in a shorter duration of RVP‐induced VT in the isoproterenol‐treated CKD RVOT tissues compared with that in the control RVOT (both n = 8) tissues. (D) Upper panel presents the tracings of RVP‐induced VT in the isoproterenol‐treated CKD and control RVOT tissues before and after KN92 (1 μM). However, KN92 (1 μM) did not yield a lower beating rate or shorter duration of RVP‐induced VT in the isoproterenol‐treated CKD compared with the control RVOT tissues (both n = 4). *p < 0.05, ***p < 0.005 versus control. Error bars indicate SEMs

FIGURE 3

Action potential (AP) morphology in the RVOT and apical myocytes from the CKD and control rabbits and transient outward potassium current (I _to) and rapid delayed rectifier potassium current (E‐4031‐sensitive repolarization current, I _kr) in the CKD and control RVOT myocytes. (A) Examples and average data of the APs in the RVOT and apical myocytes from the CKD (both n = 8, from three hearts) and control (n = 10, from four and three hearts respectively) RVOTs. *p < 0.05, **p < 0.01, ***p < 0.005 versus the control or CKD RVOTs. Error bars indicate SEMs. (B) Upper panels show the examples of current traces, I–V relationship and average conductance‐voltage relationship of I _to in the CKD (n = 8, from three hearts) and control (n = 10, from four hearts) RVOT myocytes. Lower panels display the voltage dependence of inactivation and the recovery kinetics of I _to from the CKD (n = 8, from three hearts) and control (n = 10, from three hearts) RVOT myocytes. (C) Examples of the current traces, I–V relationship and average conductance‐voltage relationship of I _Kr in the CKD (n = 9, from three hearts) and control (n = 9, from three hearts) RVOT myocytes. *p < 0.05, **p < 0.01, ***p < 0.005 versus control. Error bars indicate SEMs

FIGURE 4

L‐type calcium (I _Ca‐L), sodium‐calcium exchanger (NCX), late sodium (I _Na‐Late) and sodium (I _Na) currents in the CKD and control RVOT myocytes. (A) Upper panels present examples of the current traces, I–V relationship and average conductance‐voltage relationship of I _Ca‐L in the CKD (n = 10, from four hearts) and control (n = 10, from three hearts) RVOT myocytes. Lower panels present the voltage dependence of inactivation and the recovery kinetics of I _Ca‐L from CKD (n = 10, from three hearts) and control (n = 10, from three hearts) RVOT myocytes. (B) Examples of the current traces and I–V relationship of NCX in the CKD and control RVOT myocytes (both n = 9, from four hearts). (C) Examples of the current traces, I–V relationship and average data of I _Na‐Late in the CKD and control RVOT myocytes (both n = 10, from four hearts). (D) Examples of the current traces, I–V relationship, and average conductance‐voltage relationship of I _Na in the CKD and control RVOT myocytes (both n = 8, from three hearts). *p < 0.05, **p < 0.01, ***p < 0.005 versus control. Error bars indicate SEMs

FIGURE 5

Intracellular calcium homeostasis, sarcoplasmic reticulum (SR) calcium stores and SR Ca²⁺‐ATPase (SERCA2a) activity in the CKD and control RVOT myocytes. (A) Examples of tracings of Ca²⁺ transient and SR Ca²⁺ leak in the CKD (n = 16, from eight hearts) and control (n = 16, from eight hearts) RVOT myocytes. (B) Means of Ca²⁺ transient, SR Ca²⁺ leak, taufast and tauslow of Ca²⁺ transient decay and peak to half decay time of Ca²⁺ transient in the CKD and control RVOT myocytes. (C) Examples of tracings and average data of SR Ca²⁺ content in the CKD (n = 8, from four hearts) and control (n = 11, from five hearts) RVOT myocytes. (D) Means of SERCA2a activity in the CKD and control RVOT myocytes (both n = 6, from three hearts). *p < 0.05, ***p < 0.005 versus control. Error bars indicate SEMs

FIGURE 6

Calcium regulatory protein, histological fibrosis, malondialdehyde (MDA) and autonomic neuron activity in the CKD and control RVOTs. (A) Representative immunoblot findings and mean values of levels of phospholamban (PLB), phosphorylated PLB at threonine 17 (pPLB T17), phosphorylated PLB at serine 16 (PLB‐Ser16), ryanodine receptor channels (RyR), phosphorylated RyR at serine 2808 (pRyR S2808), phosphorylated RyR at serine 2814 (pRyR S2814), mature collagen α1 Type I, phosphorylated Ca²⁺/calmodulin‐dependent protein kinase II (pCaMKII) at Thr 286, protein kinase A (PKA), SERCA2a, CaMKII, connexin 43 and stromal interaction molecule 1 (STIM1) from the CKD and control RVOT myocytes (both n = 8, from four hearts). (B) Image and mean values from Masson's trichrome staining in the CKD and control RVOT tissues (both n = 5). (C) Mean of MDA from the CKD and control RVOT myocytes (both n = 6, from three hearts). (D) Image and mean values of immunohistochemistry staining for tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) in the cardiac ganglia at the CKD and control RVOT tissues (both n = 8). Staining of neurofilament verified the location of nerve component. *p < 0.05, **p < 0.01, ***p < 0.005 versus control. Error bars indicate SEMs