Electronegative LDL-mediated cardiac electrical remodeling in a rat model of chronic kidney disease.

We performed UNx on 6 adult Sprague-Dawley rats to create a rat model of early-stage CKD, and we performed a sham operation on 6 control rats. Eight weeks after the operation, body weight and total cholesterol, triglyceride, LDL, and HDL levels were not significantly different between UNx and sham rats ( Table 1 ). However, blood urea nitrogen (BUN) and creatinine levels were significantly higher in UNx rats than in the sham group ( P  = 0.04 and P  < 0.01, respectively). For surface electrocardiographic characteristics, the RR interval, heart rate, PR interval, P duration, and QRS interval were similar between UNx and sham rats ( Fig. 1a ). However, the QT interval and corrected QT interval (QTc) were prolonged by about 60% in UNx rats (0.082 ± 0.003 and 0.060 ± 0.002, respectively). We used Mitchell’s approach for the QTc analysis, which has been widely used at fast heart rates with consistent results in rodents 17 . To further study the mechanisms involved in QT prolongation in UNx rats, we first compared action potentials between EPI and ENDO cardiomyocytes from sham and UNx rats by using the patch-clamp technique. Cardiomyocytes were paced with 3–5 ms suprathreshold depolarizing stimuli in the current-clamp mode. Figure 1b shows the superimposed recordings of action potentials for UNx and sham rat EPI and ENDO cardiomyocytes. The APD at 50% and 90% repolarization was longer in UNx EPI cardiomyocytes than in sham EPI cardiomyocytes ( P  = 0.048 and P  = 0.008, respectively; Fig. 1c ), but not significantly different between UNx and sham ENDO cardiomyocytes. No significant differences were observed between UNx and sham rat cardiomyocytes in action potential amplitude and resting membrane potential ( Fig. 1d ). The transient outward potassium current ( I to ) of cardiomyocytes isolated from ENDO and EPI regions of sham and UNx rats were recorded in the voltage-clamp mode ( Fig. 2a ). To determine I to , we elicited cardiomyocytes by a 10 ms step to −40 mV from the holding potential of −80 mV (to inactivate sodium channel), followed by a 400-ms depolarization to various potential levels ranging from −60 to 60 mV (applied at 20 mV increments every 1 s). The contamination of the calcium current was prevented by adding 1 mM Co 2+ ( Fig. 2b ). Current-voltage relationships showed that I to density was much larger in EPI than in ENDO cardiomyocytes in both sham and UNx rats, as predicted ( Fig. 2c and d). However, EPI cardiomyocytes isolated from UNx showed a significantly smaller I to at 60, 40, and 20 mV than those isolated from the corresponding layer in sham rats ( P  = 0.041, 0.033, and 0.045, respectively; Fig. 2c ). In ENDO myocytes I to density was similar in both groups ( Fig. 2d ). Figure 2e illustrates plots of the transmural gradient (EPI–ENDO) of I to at different voltages from −60 to +60 mV for sham and UNx myocytes. The gradient was significantly smaller in cardiomyocytes from UNx than from sham animals ( P  = 0.021). We found no significant differences between sham and UNx cardiomyocytes in the I Ca (−8.88 ± 1.02 vs. −9.57 ± 1.11 pA/pF at 10 mV, respectively; P  = 0.649) and the I K1 currents (−38.16 ± 4.45 vs. −33.56 ± 2.51 pA/pF at −140 mV, respectively; P  = 0.081) ( Supplementary Fig. S1 ). In addition, sham LDL- and UNx LDL-treated normal cardiomyocytes had similar I Ca (−18.99 ± 1.86 vs. −18.86 ± 3.41 pA/pF at 10 mV, respectively; P  = 0.947) and I K1 currents (−32.11 ± 1.29 vs. −32.61 ± 2.43 pA/pF at −140 mV, respectively; P  = 0.866) ( Supplementary Fig. S2 ). Moreover, the midpoint of inactivation of I Ca was similar between sham and UNx rats (−16.50 ± 1.90 vs. −16.23 ± 2.41; P  = 0.933) and between sham LDL- and UNx LDL-treated cardiomyocytes (−16.00 ± 1.73 vs. −18.19 ± 2.76; P  = 0.527) ( Supplementary Fig. S3 ). Because the smaller current density seen in EPI cardiomyocytes from UNx rats may be due to altered channel kinetics, we examined the gating properties of I to . To examine the voltage dependence of inactivation of I to , we used different voltages of conditioning pulses (−100 to +20 mV), which either depolarized or hyperpolarized from the holding potential of −80 mV for 1 second to inactivate the channel; with the second pulse, we depolarized to +60 mV for 200 ms from different precondition pulses ( Fig. 3a ). Figure 3b shows representative traces of I to after different preconditioning voltages in EPI cardiomyocytes from sham and UNx rats. I to amplitude normalized to the I to of the most negative conditioning pulse ( I max ) was plotted against the conditioning voltage in Fig. 3c , and the curve was fitted by a Boltzmann model to obtain the midpoint of inactivation. We showed that the curve of UNx EPI cardiomyocytes was left-shifted and that the midpoint of inactivation was more negative than that of the sham group ( P  = 0.0004; Fig. 3d ). For the kinetics of I to recovery from inactivation, we used a typical 2-pulse protocol. Two identical pulses (from a holding potential of −80 mV to the test potential of +60 mV for 200 ms) were elicited in a variable interval from 0 to 200 ms in 20-ms increments ( Fig. 3e ). Representative traces of both sham and UNx EPI cardiomyocytes are provided in Fig. 3f , which showed I to recovered from inactivation at a much slower rate in EPI cardiomyocytes from UNx rats than in those from the sham group. Normalized currents were plotted against the interval between the two pulses, and the data were fitted by a single exponential equation ( Fig. 3g ). The time constant (τ) of UNx EPI cardiomyocytes was significantly increased when compared to that in the sham group ( P  = 0.04; Fig. 3h ). In ENDO cardiomyocytes, the inactivation and recovery curves of sham and UNx were comparable, and there were no significant differences between the two groups (data not shown). To clarify the underlying mechanisms of UNx-induced electrophysiologic remodeling and to determine whether circulating LDL plays an important role, we compared the electronegativity of LDL between sham and UNx rats. The results of agarose gel electrophoresis showed that the LDL from UNx rats was more electronegative than that from healthy controls ( Fig. 4a ). We then incubated normal EPI cardiomyocytes with 100 μg/mL LDL from sham or UNx rats for 12 hours. Figure 4b shows the superimposed recordings of action potentials for UNx LDL- and sham LDL-treated normal EPI and ENDO cardiomyocytes. The APD at 50% and 90% repolarization was longer in UNx LDL-treated than in sham LDL-treated EPI cardiomyocytes ( P  = 0.002 and P  = 0.0001, respectively; Fig. 4c ) but was not significantly different between UNx LDL- and sham LDL-treated ENDO cardiomyocytes. No significant differences were observed in action potential amplitude and resting membrane potential between UNx LDL- and sham LDL-treated EPI and ENDO cardiomyocytes ( Fig. 4d ). The transient outward potassium current ( I to ) of both EPI and ENDO cardiomyocytes treated with sham LDL and UNx LDL was recorded ( Fig. 5a ). For the determination of I to , we elicited cardiomyocytes by a 10 ms step to −40 mV from the holding potential of −80 mV, followed by 400 ms depolarization to various potential levels ranging from −60 to 60 mV (applied at 10 mV increments every 1 s) ( Fig. 5b ). Current-voltage relationships showed that I to density at 60, 50, 40, and 30 mV was significantly smaller in UNx LDL-treated EPI cardiomyocytes than in sham LDL-treated EPI cardiomyocytes ( P  = 0.020, 0.038, 0.038, and 0.045, respectively; Fig. 5c ). In ENDO cardiomyocytes I to density was similar in both groups ( Fig. 5d ). Figure 5e shows the plots of the transmural gradient of I to at different voltages from −60 to +60 mV for sham LDL- and UNx LDL-treated cardiomyocytes. The gradient was significantly smaller in UNx LDL-treated cardiomyocytes than in sham LDL-treated cardiomyocytes ( P  = 0.029). We compared the gating properties of I to between sham LDL- and UNx LDL-treated cardiomyocytes to clarify the mechanism underlying I to downregulation. To examine the voltage dependence of inactivation of I to , we used different voltages of conditioning pulses (−100 to +20 mV), which either depolarized or hyperpolarized from the holding potential of −80 mV for 1 second to bring the membrane to inactivation; with the second pulse, we depolarized to +60 mV for 200 ms from different precondition pulses ( Fig. 6a ). Figure 6b shows representative traces of I to after different preconditioning voltages in sham LDL- and UNx LDL-treated EPI cardiomyocytes. I to amplitude normalized to the I to of the most negative conditioning pulse ( I max ) was plotted against conditioning voltage in Fig. 6c , and the curve was fitted by a Boltzmann model to obtain the midpoint of inactivation. We showed that the curve of UNx LDL-treated EPI cardiomyocytes was left-shifted and that the midpoint of inactivation was more negative than that of the sham LDL group ( P  = 0.002; Fig. 6d ). For the kinetics of I to recovery from inactivation, we used a typical 2-pulse protocol. Two identical pulses (from holding potential of −80 mV to the test potential of +60 mV for 200 ms) were elicited in variable intervals from 0 to 140 ms in 10-ms increments ( Fig. 6e ). Representative traces of both sham LDL- and UNx LDL-treated EPI cardiomyocytes are presented in Fig. 6f , which shows I to recovered from inactivation at a much slower rate in UNx LDL-treated EPI cardiomyocytes than in the sham LDL-treated group. Normalized currents were plotted against the interval between two pulses, and the data were fitted by a single exponential equation ( Fig. 6g ). The time constant (τ) of UNx LDL-treated EPI cardiomyocytes was significantly increased when compared to that of the sham LDL group ( P  = 0.02; Fig. 6h ). Similar to those found in UNx ENDO cardiomyocytes, the inactivation and recovery curves of sham LDL- and UNx LDL-treated cardiomyocytes were comparable, and there were no significant differences between the groups (data not shown). To determine the mechanism underlying changes of I to channel properties in the EPI layer of UNx rats and UNx LDL-treated EPI cardiomyocytes, we examined the expression levels of Kv4.3, Kv4.2, Kv1.4, and KChIP2 proteins in the EPI layer from sham or UNx rats ( Fig. 7a ) and sham LDL- or UNx LDL-treated EPI cardiomyocytes ( Fig. 7b ). In EPI cardiomyocytes from UNx rats, the expression of Kv4.3, Kv4.2, and Kv1.4 proteins was comparable with that in sham myocytes, suggesting that Kv α-subunits did not contribute to the downregulation of I to . However, the expression of β-subunits KChIP2 protein was significantly reduced in UNx EPI cardiomyocytes compared to sham cardiomyocytes ( P  = 0.017). Similarly, 100 μg/mL UNx LDL incubation could reduce KChIP2 protein expression ( P  = 0.0001) but did not change Kv4.3, Kv4.2, and Kv1.4 expression in control EPI cardiomyocytes. There were no significant differences in both α- and β-subunits of I to between ENDO cardiomyocytes from sham and UNx rats or between sham LDL- and UNx LDL-treated ENDO cardiomyocytes (data not shown).

To characterize electrical changes in early CKD, we used a rat model of early CKD induced by UNx. All animal research was approved by the China Medical University Institutional Animal Care and Use Committee (2016-128), and all procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals by the US National Institutes of Health. Adult male 8-week-old Sprague Dawley rats purchased from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan) were assigned to 1 of 2 groups: the UNx group or the sham group. All rats were anesthetized with 2% isoflurane (Abbott Laboratories, Abbott Park, IL, USA) and supported by a rodent ventilator (New England Medical Instruments, Medway, MA, USA). For UNx, the left kidney was removed immediately following ligation of the left renal artery and vein using a 4/0 silk suture. After 8 weeks, the biochemical parameters of blood samples including total cholesterol, triglyceride, LDL, HDL, BUN, and creatinine levels were measured by the Automated Biochemical Analyzer (SP-4430, Spotchem EZ, Arkray USA, Edina, MN, USA) with different strips. The lead II surface electrocardiogram was recorded 8 weeks after the operation at the sampling rate of 1000 Hz by using PONEMAH real-time acquisition interface P3P Plus coupled to a digital converter (ML-870, ADInstruments, Colorado Springs, CO, USA). Data were analyzed by the software Lab Chart 7 plus (ADInstruments), and all characteristics were measured as the average of five consecutive cycles. The rate-corrected QTc interval was calculated according to the equation: where QT 0 , QTc, y, RR100 are the observed QT, rate-corrected QT interval, value of the exponent, and normalized RR interval, respectively 17 . Left ventricular cardiomyocytes of rats were enzymatically isolated by the Langendorff perfusion method. Hearts were retrogradely perfused with Krebs buffer (in mmole/L): 120 NaCl, 12 glucose, 25 NaHCO 3 , 1.2 KH 2 PO 4 , 1.2 MgSO 4 , and 5.4 KCl; pH was adjusted to 7.4 by using HEPES. After a 5-min equilibration, we added 0.4 mg/mL collagenase (type II, Worthington) for 20 min. Hearts were then dissected from the left ventricular EPI and ENDO layer, leaving a distinct and dividing mid-myocardial layer. Single EPI or ENDO cardiomyocytes were then isolated by digestion with 0.4 mg/mL collagenase and 0.02 mg/mL trypsin (ThermoFisher Scientific, Waltham, MA, USA) in Krebs buffer for 20 min. After filtration, the cardiomyocytes were washed twice with Krebs buffer and stored in Krebs buffer. To examine the direct effect of LDL on control cardiomyocytes, we pretreated the cells with LDL isolated from sham and UNx rats for 12 hours. The whole-cell patch-clamp technique was used to record ionic currents and membrane potential with the Axon CNS 700B amplifier (Molecular Devices, LLC, Sunnyvale, CA, USA) with Digidata 1550 A data acquisition system and pClamp software (Version 10, Molecular Devices). A droplet of cell suspension was placed in a chamber mounted on the stage of an inverted microscope (Eclipse Ti-U, Nikon Corporation, Tokyo, Japan) in bath (extracellular) solution containing (in mM/L) 137 NaCl, 5.4 KCl, 1.8 CaCl 2 , 1.1 MgCl 2 , 6 HEPES, 22 glucose, and 0.33 NaH 2 PO 4 ; pH was adjusted to 7.4 using NaOH at room temperature. Only quiescent rod-shaped cells showing clear-cross striations were studied. The mean capacitance of EPI cardiomyocytes from sham and UNx rats was 198.72 ± 20.57 and 192.79 ± 16.74 pF, respectively, and that of ENDO cardiomyocytes was 182.78 ± 12.38 and 186.89 ± 16.85, respectively (n = 20 per group). Heat-polished glass electrodes (tip resistances about 1.5 MΩ when filled with pipette internal solution) were prepared from borosilicate glass capillaries (outer diameter 1.5 mm) by the Glass Microelectrode Puller (PC-10, Narishige International Inc., East Meadow, NY, USA) and polished by the Microforge (MF-830, Narishige). The internal solution contained (in mmol/L) 120 KCl, 5 MgCl 2 , 5 MgATP, 10 HEPES, and 15 EGTA; the pH was adjusted to 7.2 using KOH at room temperature. Junctional potentials were zeroed before the formation of the membrane-pipette seal in the bath solution. Capacitances of cells were measured by calculating the total charge movement of the capacitative transient in response to a 10 mV hyperpolarizing pulse. I to was defined as the difference between the peak value and the current level at the end of a 400 ms pulse. The cycle length of the stimulation in action potential measurements was 1000 ms. All patch-clamp experiments were performed at room temperature. Rat plasma LDL was isolated by using sequential potassium bromide density-gradient ultracentrifugation (d = 1.030–1.063 g/mL). To confirm the quality and electronegativity of LDL samples, we separated them in 0.7% agarose by using electrophoresis and subjected the delipidated LDL samples to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The left ventricular EPI layer of each UNx and sham rat was homogenized by using a Mini-Beadbeater-1 (BioSpec Products, Inc., Bartlesville, OK, USA) in T-PER Tissue Protein Extraction Reagent. For immunoblotting, polyclonal antibodies against Kv4.2/4.3 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), Kv1.4 (Academy Bio-Medical Company, Houston, TX, USA), KChIP2 (Merck Millipore, Darmstadt, Germany), and β-actin (Sigma Aldrich Corporation, St. Louis, MO, USA) were used. The data are expressed as the mean ± standard error of the mean, and the differences between the 2 groups were determined by using the Mann-Whitney U test. The difference between the mean EPI and ENDO I to current was plotted as the transmural gradient of I to . The transmural gradient of I to between sham and UNx rats was compared by using two-way ANOVA with the post-hoc Bonferroni correction. A P -value < 0.05 was considered statistically significant. The inactivation curve of I to was fitted by using the Boltzmann equation: where I gives the current amplitude and I max its maximum, V m the potential of pulse, V 0.5 the half-maximal inactivation potential, and k the slope factor. The recovery curve of I to was fitted by the single exponential function: where τ is the time constant of decaying component of inactivation.

How to cite this article : Lee, A.-S. et al . Electronegative LDL-Mediated Cardiac Electrical Remodeling in a Rat Model of Chronic Kidney Disease. Sci. Rep. 7 , 40676; doi: 10.1038/srep40676 (2017). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

In rats with UNx-induced CKD, we have demonstrated electrocardiographic QTc prolongation in vivo that is mediated by a predominant lengthening of the APD in EPI cardiomyocytes. The differential reduction of I to currents between EPI and ENDO cardiomyocytes in UNx rats, along with the resultant disruption of the physiological transmural gradient of I to , may contribute to the APD phenotype. The electrophysiological remodeling with substantial I to reduction and alteration of transmural I to gradient in epicardial cardiomyocytes was reproduced in UNx-LDL treated cardiomyocytes. The reduction of I to is not due to altered expression levels of Kv4.3, Kv4.2, or Kv1.4 proteins, but rather it is related to downregulation of the Kv channels regulator KChIP2. Taken together, the predominant downregulation of KChIP2 protein and I to currents in EPI cardiomyocytes led to alteration of the transmural I to gradient, which may create a vulnerable substrate for generating life-threatening ventricular tachyarrhythmias in CKD. The progression of CKD has been associated with a significant delay of cardiac repolarization manifested by QTc prolongation, independent of other risk factors or structural heart diseases in humans 18 . The prolongation of QTc can occur even in patients with early-stage CKD (stage 2) 19 . The abnormalities in cardiac repolarization may thus predispose susceptible CKD patients to fatal ventricular tachyarrhythmias. In support of the human data, in a rat model of CKD with a defect in the samcystin gene (Cy/+), Hsueh et al . 10 showed that the APD at 80% of repolarization was longer in CKD rats than in normal rats. Pacing cycle length thresholds to induce calcium transient alternans or APD alternans were also longer in CKD rats. This electrophysiological remodeling led to increased vulnerability to ventricular arrhythmia characterized by the spontaneous occurrence of premature ventricular complexes and a higher frequency of VF induction. Although this model of genetic defect–induced CKD has shown some remodeling abnormalities including altered repolarization reserve and calcium homeostasis by optical mapping technique, the underlying mechanism remains unclear. In contrast to the study by Hsueh et al ., we used a standard UNx-induced CKD model to examine CKD-related cardiac electrophysiological remodeling by the patch-clamp technique. In rodents, APD has been determined mostly by the density of the Ca 2+ -independent I to current 20 . However, I to is not uniformly distributed among the whole ventricle. Rather, I to is more prominent in the sub-epicardial region than in the sub-endocardial tissue in rodent hearts, which causes the baseline APD to be shorter in the EPI region than in the ENDO region 21 . During some pathological conditions, such as cardiac hypertrophy, the regional difference of I to reduction can reverse the gradient in APD and ultimately contribute to the increased susceptibility to ventricular arrhythmias 22 23 . In this early-stage CKD model, we demonstrated that the overall electrocardiographic QTc was prolonged in CKD rats compared to sham-operated rats; this prolongation was mediated by an extensive lengthening of APD in EPI cardiomyocytes compared with ENDO cardiomyocytes and was secondary to a substantial reduction of I to in the EPI region. The finding that modulation of I to leads to prolongation or abbreviation of APD was concordant with the observation by Li et al . 24 , who showed that ablation of cold-inducible, RNA-binding protein–mediated I to amplification shortened the QTc interval in rats. In the current study, the changes in electrophysiological properties in CKD rats occurred independently of ventricular fibrosis because fibrosis levels were similar in CKD and control rats, as shown by histologic staining and examination of early fibrosis markers 15 . This electrophysiological remodeling in CKD rats also occurred regardless of cellular hypertrophy based on the finding of similar cellular capacitance in isolated single ventricular cardiomyocytes by the patch-clamp technique (200.69 ± 9.62 in sham vs. 190.37 ± 12.07 pA/pF in UNx). Moreover, Yu et al . found that the renin-angiotensin-aldosterone system (RAAS) could alter the transmural gradient of I to between EPI and ENDO cardiomyocytes by changing channel kinetics 25 . In our CKD model, the levels of renin, angiotensin, and aldosterone were not significantly different between UNx and sham rats 15 . This finding indicates that the electrophysiological remodeling seen in UNx rats was not mediated by modulation of the RAAS. There are species-specific differences in voltage-gated K + channel expression that are responsible for the timely control of cardiac repolarization. Downregulation of these currents results in prolongation of the APD and a decrease in phase 1 repolarization 26 . Significant levels of mRNA encoding the rapidly inactivating K + channel α-subunits Kv1.4, Kv4.2, and Kv4.3 proteins have been seen in rat ventricle 27 . In addition, previous studies have identified three classes of distinctive cytoplasmic auxiliary proteins, including KChIPs, Kv β-subunits, and KChAP. These proteins not only incorporate with specific Kv α-subunits to generate the native I to current, but also regulate functional expression of these channels in vitro 28 29 30 . Previous in vitro studies have examined the modulatory effects of the KChIP2 protein on the gating kinetics of Kv4 channels in heterologous expression systems. An et al . 28 showed that KChIP proteins may co-localize with Kv4 a-subunits in transiently transfected COS-1 cells. The effects of KChIP1, 2 and 3 may augment A-type currents of Kv4 channels via a variety of mechanisms including increased surface channel density, shifting the activation V 1/2 to more negative potentials, and slower inactivation and faster recovery from inactivation in native cells. Schultz et al . 31 have recorded whole-cell currents from oocytes injected with porcine Kv4.2 cRNA alone or co-injected with porcine Kv4.2 and porcine KChIP2 cRNA. They found that steady-state inactivation was only marginally, not significantly, affected by KChIP2, whereas the recovery from inactivation was significantly accelerated by co-expressing Kv4.2 with KChIP2. The reduction in I to density with the change in channel kinetics in cardiomyocytes from UNx rats and UNx LDL-treated cardiomyocytes suggests that the molecular level of I to is altered. Previous studies have shown that the differential expression of both KChIP2 and Kv4.2 proteins across the ventricular wall may determine the transmural I to gradient in mouse, rat, canine, and human hearts 32 33 34 . In a rat model of type 2 diabetes, Sato et al . 34 found an ENDO-predominant prolongation of the APD via a reduction of steady-state I to induced by type 2 diabetes, in which downregulation of Kv4.2 and KChIP2 proteins may be involved. By using the rat model of early CKD, we discovered an EPI-predominant lengthening of the APD via a reduction of I to currents in EPI cardiomyocytes secondary to downregulation of KChIP2 without alteration of Kv4.2, Kv4.3, and Kv1.4 protein expression. The reduction of I to currents caused by KChIP2 deficiency may be attributed to shifting the voltage dependence of inactivation to more hyperpolarized potentials and slower recovery from inactivation as shown in the current and previous studies 28 29 . Importantly, downregulation of KChIP2 protein abolishes the transmural gradient of I to leading to increased heterogeneity of repolarization; this may in turn cause unidirectional conduction block, creating a substrate for the reentry circuit and thus increasing susceptibility to malignant ventricular tachyarrhythmias 32 . It is generally thought that during the course of CKD, the kidney releases mediators that trigger maladaptive myocardial remodeling, ultimately leading to adverse cardiovascular outcomes. In addition to the well-known inflammatory mediators 35 , we recently discovered that dyslipidemia may be an important upstream mediator of diastolic left ventricular dysfunction in the early stages of CKD 15 . LDL abnormality is a primary etiology of cardiovascular disease caused by dyslipidemia 36 . However, extensive lowering of plasma LDL does not prevent cardiovascular events in all patients. This prompted us to identify a culprit pathogenic LDL subfraction. Our previous study has shown that increased LDL electronegativity in CKD disturbs intracellular calcium homeostasis resulting in cardiac diastolic dysfunction 15 . We have also found that electronegative LDL increases coronary artery disease risk in patients with uremia who are on maintenance hemodialysis 37 . In the current study, we demonstrate that the QT interval was prolonged in UNx rats when compared to the sham group. Incubation of normal cardiomyocytes with UNx LDL, which is more electronegative, reproduced a similar electrophysiological phenotype to that seen in UNx rat cardiomyocytes. Importantly, we have shown for the first time that electronegative LDL may underlie the electrophysiological remodeling via downregulation of KChIP2 protein expression in EPI cardiomyocytes with resultant parallel changes of the I to current and altered channel kinetics in CKD. The disruption of the normal transmural gradient of I to via downregulation of KChIP2 protein expression in combination with altered SERCA2a-regulated calcium homeostasis 15 may further promote susceptibility to ventricular tachyarrhythmias in CKD. In previous studies, we have shown that the electronegativity of LDL is increased in metabolic disorders and cardiovascular diseases, such as hypercholesterolemia, diabetes, metabolic syndrome, hypertension, myocardial infarction, and stroke 38 39 40 41 42 43 44 . However, in our UNx model, the rats did not show arterial hypertension, myocardial fibrosis, hyperglycemia, or gross structural heart disease 15 . Therefore, the increased LDL electronegativity in UNx-induced CKD may be a primary mediator of the electrophysiological remodeling rather than it being mediated secondarily through a common downstream effector pathway of general cardiovascular/metabolic disorders associated with CKD. Furthermore, the direct effects of small particles on ion channels usually occur within minutes of exposure. In the present study, none of the ion currents was altered within 10 minutes in UNx LDL-treated cardiomyocytes compared to controls; this finding indicates that it is unlikely that the effect of CKD-electronegative LDL on I to /APD is caused by a direct interaction of LDL on the ion channels, but rather it is caused by other indirect pathways. We have previously shown that the effect of electronegative LDL on vascular endothelial cells is mediated by the lectin-like oxidized LDL receptor (LOX-1). This could also be how electronegative LDL exerts its effects on cardiomyocytes, resulting in electrophysiological changes in CKD. The downstream signaling is still under investigation, but epigenetic regulation may be a possible mechanism 39 . This study was undertaken to assess electrophysiological remodeling in early-stage CKD, which developed 8 weeks after UNx. We do not know if the changes in electrophysiological properties will remain constant or become adapted or maladapted in a more chronic scenario. However, the current UNx model is representative of an early, mild form of renal dysfunction; longer observation periods may show fibrosis and hypertrophy of the hearts, which may overtake the effects of early CKD on electrophysiological remodeling. Another limitation of our study is the significant difference in cardiac electrophysiology between rats and humans; rats have a higher heart rate, a more rapid repolarization, and a predominance of I to over other repolarizing currents compared with humans. Further studies in larger mammal models are necessary to confirm our findings. Furthermore, because only limited amounts of LDL can be isolated from animals, we examined the effects of electronegative LDL on electrophysiological remodeling by incubating cardiomyocytes with UNx LDL instead of by directly injecting LDL into animals. Lastly, although we have identified a disruption in the normal transmural gradient of I to in the current study and an altered intracellular calcium homeostasis in our previous study (which may sufficiently form a vulnerable ventricular substrate to generate ventricular tachyarrhythmias 10 15 32 ), we did not perform a programmed electrical stimulation study to test the inducibility of ventricular tachyarrhythmias in UNx rats. Indeed, further studies are necessary to combine multiple modalities, including patch-clamp techniques, optical mapping, inserted loop recorders for arrhythmias detection, and programmed electrical stimulation, to investigate the electrophysiological substrate and susceptibility to ventricular arrhythmias in CKD.

The results of our study indicate that CKD is associated with EPI-predominant prolongation of the APD and disruption of the physiological transmural gradient of I to via downregulation of KChIP2 protein expression in the EPI region, which together may promote susceptibility to ventricular tachyarrhythmias. Electronegative LDL may underlie the downregulation of KChIP2 and the resultant electrophysiological remodeling in CKD.