Cardiac calcium dysregulation in mice with chronic kidney disease

Abstract Cardiovascular complications are leading causes of morbidity and mortality in patients with chronic kidney disease (CKD). CKD significantly affects cardiac calcium (Ca2+) regulation, but the underlying mechanisms are not clear. The present study investigated the modulation of Ca2+ homeostasis in CKD mice. Echocardiography revealed impaired fractional shortening (FS) and stroke volume (SV) in CKD mice. Electrocardiography showed that CKD mice exhibited longer QT interval, corrected QT (QTc) prolongation, faster spontaneous activities, shorter action potential duration (APD) and increased ventricle arrhythmogenesis, and ranolazine (10 µmol/L) blocked these effects. Conventional microelectrodes and the Fluo‐3 fluorometric ratio techniques indicated that CKD ventricular cardiomyocytes exhibited higher Ca2+ decay time, Ca2+ sparks, and Ca2+ leakage but lower [Ca2+]i transients and sarcoplasmic reticulum Ca2+ contents. The CaMKII inhibitor KN93 and ranolazine (RAN; late sodium current inhibitor) reversed the deterioration in Ca2+ handling. Western blots revealed that CKD ventricles exhibited higher phosphorylated RyR2 and CaMKII and reduced phosphorylated SERCA2 and SERCA2 and the ratio of PLB‐Thr17 to PLB. In conclusions, the modulation of CaMKII, PLB and late Na+ current in CKD significantly altered cardiac Ca2+ regulation and electrophysiological characteristics. These findings may apply on future clinical therapies.


Abstract
Cardiovascular complications are leading causes of morbidity and mortality in patients with chronic kidney disease (CKD). CKD significantly affects cardiac calcium (Ca 2+ ) regulation, but the underlying mechanisms are not clear. The present study investigated the modulation of Ca 2+ homeostasis in CKD mice. Echocardiography revealed impaired fractional shortening (FS) and stroke volume (SV) in CKD mice.
Electrocardiography showed that CKD mice exhibited longer QT interval, corrected QT (QTc) prolongation, faster spontaneous activities, shorter action potential duration (APD) and increased ventricle arrhythmogenesis, and ranolazine (10 µmol/L) blocked these effects. Conventional microelectrodes and the Fluo-3 fluorometric ratio techniques indicated that CKD ventricular cardiomyocytes exhibited higher Ca 2+ decay time, Ca 2+ sparks, and Ca 2+ leakage but lower [Ca 2+ ] i transients and sarcoplasmic reticulum Ca 2+ contents. The CaMKII inhibitor KN93 and ranolazine (RAN; late sodium current inhibitor) reversed the deterioration in Ca 2+ handling. Western blots revealed that CKD ventricles exhibited higher phosphorylated RyR2 and CaMKII and reduced phosphorylated SERCA2 and SERCA2 and the ratio of PLB-Thr17 to PLB. In conclusions, the modulation of CaMKII, PLB and late Na + current in CKD significantly altered cardiac Ca 2+ regulation and electrophysiological characteristics. These findings may apply on future clinical therapies.

K E Y W O R D S
calcium homeostasis, CaMKII, chronic kidney disease, electrophysiology, heart failure

| INTRODUC TI ON
Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD). 1 CKD patients are 2-6 times more likely to die of a CVD than progress to dialysis. 2 CVD mortality rate in end-stage renal failure patients is 10-to 30-fold higher than the age-matched general population. 3 The uraemic milieu contains numerous cardiac risk factors that lead to a distinct cardiac pathology termed uraemic cardiomyopathy (UCM). 4 UCM impairs cardiac performance and causes global cardiac dysfunction, which greatly contributes to the high mortality of the CKD population. 5 Unique aspects of UCM include left ventricular hypertrophy (LVH), reduced capillary density, fibrosis, and ventricular remodelling; and LVH is the most prevalent characteristic. 6 One characteristic feature of LVH in UCM is a metabolic remodelling that results in heart failure (HF). The complex pathogenesis of UCM is not clearly understood.
Electrical excitation coupling (EC coupling) in the normal heart involves the interaction of numerous cellular proteins involved in calcium (Ca 2+ ) homeostasis. 7 Increased intracellular Ca 2+ in cardiomyocytes initiates lethal ventricular tachyarrhythmias, including ventricular fibrillation (VF), in various cardiomyopathies, such as myocardial ischaemia or HF. 8,9 Intracellular Ca 2+ overload in cardiomyocytes triggers activity, delayed afterdepolarizations and life-threatening ventricular tachyarrhythmia. 10 One of the major pathological changes in cardiomyopathy is dysregulation of intracellular Ca 2+ homeostasis, which is caused by functional alterations of the proteins involved in Ca 2+ release and uptake across the sarcolemma and the sarcoplasmic reticulum (SR). 11 Decreased SR Ca 2+ ATPase (SERCA) 2a activity was reported in experimental models of CKD in association with the prolongation of diastolic Ca 2+ transients. 12,13 Cyclic adenosine monophosphate (cAMP) in human SR regulates the phosphorylation of phospholamban (PLB) via protein kinase A (PKA), which affects SERCA2 activity and Ca 2+ transport. Phosphodiesterase 3 (PDE3) inhibition potentiates this signalling pathway. 14

| Animal experiments
Chronic kidney disease was induced by way of partial nephrectomy (PNx) in C57BL/6J mice as described formerly. 16 PNx was reached by a procedure to remove the right kidney 2 weeks later after the initial PNx. The animals were sacrificed by CO 2 inhalation 6 months later after the sham operation or PNx. Then, we collected blood and the hearts were dissected for analysis. The concentrations of blood urea nitrogen (BUN) and creatinine of serum (sCr) were detected by using the Jaffe method (Beckman Coulter Synchron LX System; Beckman Coulter Inc).

| Echocardiography
A Philips iE33 ultrasound imaging system with a 7-15 MHz linear array transducer was used to perform echocardiography (Philips Medical Systems). Inhalation of 3% isoflurane was performed to anaesthesia, until animals were sedated and maintained 1% isoflurane during the examination of echocardiography. First of all, it was to obtain 2D left ventricular (LV) short-axis images. M-mode was used to measure the thickness of LV wall and dimension of chamber and ejection fraction (EF) at diastole and systole phases. All measurements of echocardiography were averaged for consecutive 5 cardiac cycles.

| Electrocardiographic measurements
Under isoflurane anaesthesia (5% for induction and 2% for maintenance), electrocardiograms (ECGs) were recorded from standard lead II limb leads via a bio-amplifier (ADInstruments), were connected to a ML845 Powerlab polygraph recorder (ADInstruments) and were continuously displayed throughout the experiment in sham or CKD mice.

| Isolation of single control and uraemic cardiomyocytes
Mice used in this study were anaesthetized by using sodium pentobarbital (100 mg/kg, i.p.). The heart and lungs were removed quickly after a midline thoracotomy was performed. The heart was perfused in a retrograde manner via a dispensing needle (OD,

| Intracellular Ca 2+ regulation
Isolated cardiomyocytes were loaded with 10 μmol/L of fluo-3/AM for 30 min at room temperature (RT). The bath solution was changed to remove excess dye at 35 ± 1°C for 30 minutes. A 488-nm argon ion laser was used to excite Fluo-3. The emission fluorescence was recorded at >515 nm. Cells were scanned repetitively at 3-ms intervals for a total duration of 6 seconds. Fluorescence imaging was per-   total PLB (Thermo), CaMKII and NCX (Swant). A GAPDH antibody was used to normalize protein bands in each blot. ImageJ software was used to quantify relative protein level.

| Statistical analysis
All quantitative data are expressed as the means ± the standard error of the mean (SEM). Statistical significance between different groups was determined by an unpaired t test or one-way analysis of variance (ANOVA) with Tukey's test for multiple comparisons, as appropriate.
A value of P < .05 was considered significant.

| Cardiac structure, functions, and ECGs of sham and CKD mice
As shown in Table 1, significantly increased serum BUN (49.0 ± 12.64 mg/dL vs 18.9 ± 0.51 mg/dL, P < .05) and creatinine levels (1.24 ± 0.36 mg/dL vs 0.45 ± 0.01 mg/dL, P < .05) in CKD mice confirmed the successful induction of experimental renal failure. The heart rate and left ventricular mass in the sham and CKD mice were not significantly different. CKD mice exhibited a greater left atrium diameter to aortic root diameter ratio (LA/AO), left ventricular internal diameter at end-diastole (LVIDd), left ventricular internal diameter at end-systole (LVIDs), end-diastolic volume (EDV) and end-systolic volume (ESV) compared with the sham mice. The fractional shortening (FS, 23.1 ± 2.6% vs 32.4 ± 2.5%, P < .05) and stroke volume (SV, 0.082 ± 0.003 ml vs 0.103 ± 0.004 ml, P < .05) were decreased in CKD mice compared to sham mice. The ECG data showed that CKD mice exhibited longer QT intervals and corrected QT (QTc) prolongation than the sham group ( Figure 1A). However, the RR intervals were similar in sham and CKD groups ( Figure 1A).
The electrophysiological experiments showed that ventricular cardiomyocytes of CKD mice exhibited significantly shorter APD 90 but similar APD 20 , APD 50 and contractile forces as the sham group ( Figure 1B). The heart beating rate was significantly higher in CKD mice ( Figure 1C).

| Effects of CKD on Ca 2+ regulation
CKD ventricular myocytes exhibited lower Ca 2+ transients than sham ventricular myocytes and decreased SR Ca 2+ content (Figure 2A). Ca 2+ sparks are primary Ca 2+ release from the stochastic opening of one or more RyRs in cardiomyocytes, and unusual Ca 2+ spark dynamics are involved in various pathologies, such as heart failure and cardiac arrhythmia. [19][20][21] The incidence and frequency of Ca 2+ sparks increased unevenly in cardiomyocytes of the CKD group ( Figure 2B). The ratio of Ca 2+ leakage increased in CKD ventricular myocytes ( Figure 2C).
Early afterdepolarization (EAD) is defined as interruption of phase 2 or 3 of action potentials prior to full repolarization, and EADs are related to a shortening of action potential durations (APDs). 22 Our study demonstrated a greater occurrence of EAD-like waves in the right ventricles of CKD mice ( Figure 2D).

CKD ventricles exhibited higher phosphorylation of RyR2 and
CaMKII than the sham groups ( Figure 3). The protein levels of p-SERCA2 and SERCA2, and the ratio of PLB-Thr17 to PLB was reduced in CKD right ventricles. However, CKD right ventricles exhibited similar levels of NCX compared with the sham groups.

| Effects of KN93 and RAN treatment on electrophysiological characteristics
The addition of KN93 (CaMKII inhibitor) and RAN (late sodium current inhibitor) to CKD and sham cardiomyocytes produced a TA B L E 1 Summary of heart rate, left ventricular mass, serum blood urea nitrogen (BUN), creatinine and echocardiography measurements significant decrease in Ca 2+ transients in the CKD group but little effect in the sham group ( Figure 4A,4). RAN addition to CKD cardiomyocytes significantly decreased the beating rates, incidence of burst firing and the incidence of EADs ( Figure 4C).

| D ISCUSS I ON
We successfully created an animal model of CKD-inducing heart failure in mice and investigated the role of inflammation on heart failure progression.

| Ca 2+ handling in cardiomyocytes of CKD mice
Ca 2+ is a major ionic modulator of the heart, and it plays an important role in the excitation-contraction coupling process.
Ca 2+ -induced Ca 2+ release (CICR) describes a biological process whereby Ca 2+ activates the release of further ionic Ca 2+ from SR stores via the RyR to increase [Ca 2+ ] i and aid in the binding of Ca 2+ to myofilaments in the initiation of cardiac contraction. 23 Our results showed that the ventricular myocytes of CKD mice exhibited decreased Ca 2+ transients, prolonged transient decay, F I G U R E 1 A, ECGs showed prolongation of QT interval and QTc, but not RR interval, in CKD mice. B, APD 20 , APD 50 and contractile force were similar in both groups, but APD 90 was significantly shorter in CKD mice. C, The heart beats of CKD mice were significantly higher F I G U R E 2 A, CKD ventricular myocytes exhibit lower Ca 2+ transients and decreased SR Ca 2+ content. B, The incidence and frequency of Ca 2+ sparks increased unevenly in cardiomyocytes of CKD mice. C, The ratio of Ca 2+ leakage increased in CKD mice. D, More EADs were observed in CKD mice increased Ca 2+ leak and decreased SR Ca 2+ content, which decreased fractional shortening and was arrhythmogenic with more EADs.
CaMKII plays multiple roles in cellular ionic regulation. CaMKII inhibition diminishes cardiac arrhythmias in vitro and in vivo, and transgenic CaMKII-overexpressing mice exhibit an increased incidence of cardiac RyR, which leads to early afterdepolarizations. 26 Ca 2+ sparks represent a major release of Ca 2+ in cardiomyocytes during excitation-contraction coupling. 19 Ca 2+ sparks are stochastic activations of a cluster of RyR2s that are organized into a Ca 2+ -release unit. 27 Diabetes mellitus rats exhibit a higher incidence and frequency of Ca 2+ sparks in cardiomyocytes, 28 which leads to changes in Ca 2+ handling and myocardial dysfunction. We also found an increased frequency and incidence of Ca 2+ sparks in CKD mice. Thus, the increased Ca 2+ leakage from the SR may produce a Ca 2+ deficiency, which leads to myocardial dysfunction in CKD cardiomyopathy. These findings suggest that increased CaMKII in CKD ventricles was arrhythmogenic, and the increased SR Ca 2+ leak was a crucial mechanism, which is consistent with our findings.
A review from Harvath and Bers found that the increased late sodium current also contributed to Ca 2+ modulation to cause heart failure, which was associated with increased ROS. 29 Our data showed that RAN treatment abrogated CKD-affected Ca 2+ transient, decay time and AP, which suggests that a deterioration of Na + regulation also modulated intracellular Ca 2+ handling in CKD.

| ECG changes in CKD
CKD patients exhibit a high incidence of cardiovascular complications that are characterized by complex alterations in the mechanical and electrical properties of the heart. 30 Previous studies of CKD patients revealed prolongation of QT and QTc intervals. 31 Our study found that the CKD mice exhibited prolonged QT and QTc intervals, but there was no effect on RR interval duration. QT records the sum of millions of individual APDs from both right and left ventricle, and could conceal within itself a number of short APDs juxtaposed to long ones. In our study, CKD shortened APD 90 but prolonged QT and QTc intervals. We only recorded APDs from right ventricle, but not from left ventricle. It is possible that the prolongation of QT and QTc intervals may be contributed mostly by prolonged APDs from left ventricle. Therefore, the discrepant effects of CKD on APDs between right and left ventricle may increase interventricular dispersion of APDs, which could increase the genesis of micro-reentry circuits. 32 Moreover, prolonged QT interval may result in ventricular arrhythmia due to triggered activity of EAD, 33 and we found more EADs in our CKD mice.

| CON CLUS IONS
Our findings suggest that CKD induces uraemic cardiomyopathy, which exhibits alterations in cardiac Ca 2+ regulation and electrophysiological characteristics ( Figure 5). These alterations are associated with the regulation of CaMKII, PLB and late Na + current. The antagonists KN93 and RAN may eliminate the observed effects.

ACK N OWLED G EM ENTS
This study was supported by grants from the Ministry of Science and TSGH-C108-026).

CO N FLI C T O F I NTE R E S T
All authors declare that they have no competing interests.

F I G U R E 5
The proposed mechanism of calcium dysregulation in uraemic cardiomyopathy. The underlying mechanisms were associated with the regulation of CaMKII, PLB and late Na current

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.