Chronic B-Type Natriuretic Peptide Therapy Prevents Atrial Electrical Remodeling in a Rabbit Model of Atrial Fibrillation

Abstract

Background:

Atrial fibrillation (AF) is an important and growing clinical problem. Current pharmacological treatments are unsatisfactory. Electrical remodeling has been identified as one of the principal pathophysiological mechanisms that promote AF, but there are no effective therapies to prevent or correct electrical remodeling in patients with AF. In AF, cardiac production and circulating levels of B-type natriuretic peptide (BNP) are increased. However, its functional significance in AF remains to be determined. We assessed the hypotheses that chronic BNP treatment may prevent the altered electrophysiology in AF, and preventing AF-induced activation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) may play a role.

Methods and Results:

Forty-four rabbits were randomly divided into sham, rapid atrial pacing (RAP at 600 beats/min for 3 weeks), RAP/BNP, and sham/BNP groups. Rabbits in the RAP/BNP and sham/BNP groups received subcutaneous BNP (20 μg/kg twice daily) during the 3-week study period. HL-1 cells were subjected to rapid field stimulation for 24 hours in the presence or absence of BNP, KN-93 (a CaMKII inhibitor), or KN-92 (a nonactive analog of KN-93). We compared atrial electrical remodeling-related alterations in the ion channel/function/expression of these animals. We found that only in the RAP group, AF inducibility was significantly increased, atrial effective refractory periods and action potential duration were reduced, and the density of I _{Ca, L} and I _to decreased, while I _K1 increased. The changes in the expressions of Cav1.2, Kv4.3, and Kir2.1 and currents showed a similar trend. In addition, in the RAP group, the activation of CaMKIIδ and phosphorylation of ryanodine receptor 2 and phospholamban significantly increased. Importantly, these changes were prevented in the RAP/BNP group, which were further validated by in vitro studies.

Conclusions:

Chronic BNP therapy prevents atrial electrical remodeling in AF. Inhibition of CaMKII activation plays an important role to its anti-AF efficacy in this model.

Keywords

atrial fibrillation B-type natriuretic peptide electrical remodeling

Introduction

Atrial fibrillation (AF) is the most commonly sustained arrhythmia that is associated with pronounced morbidity, mortality, and socioeconomic burden. Over the years, although many important advances in our understanding and treatment of AF have been made, it remains a serious and growing health problem. Presently available therapeutic approaches have limited efficacy and potential adverse effects.^1

-5

Now it is increasingly recognized that the basic arrhythmia mechanisms of AF are rapid focal ectopic firing and/or reentry. Accumulating evidence has shown that electrical remodeling promotes both for the induction of triggered ectopic activity and for the activation of Ca²⁺-related cell signaling that mediates profibrillatory remodeling. Electrical remodeling entails atrial electrophysiological property changes, including the alterations that occur in ion channels, pumps, and exchangers in atrial cells.^3,5
-7 Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) has been identified as a critical player in promoting these electrophysiological changes. Ca²⁺/calmodulin-dependent protein kinase II modulates several of the pathways involved in AF and atrial electrical remodeling. However, there are currently no CaMKII inhibitors available clinically. There is an urgent need for effective therapies to prevent and to correct electrical remodeling in AF.^5,8 Thus, it may be the best to use some complementary approaches to CaMKII inhibition.

Emerging evidence supports B-type natriuretic peptide (BNP) as a new therapeutic option for the treatment in AF. In AF, cardiac production and circulating levels of BNP are increased. It is well known that BNP produced by both atrial myocytes and ventricular myocytes exhibits a range of actions. In addition to its vasodilating and natriuresis actions, BNP has other cardiac protective effects, such as antifibrotic, antihypertrophic, and renin–angiotensin–aldosterone inhibition.^9

-12 These properties are beneficial in AF. Recombinant human BNP (rhBNP) has been recommended to treat acute heart failure (HF).^13,14 Furthermore, rhBNP therapy has been shown to reduce the new onset of AF in HF patients.^12,15,16 Although the beneficial actions have been well-documented in HF patients, its functional effect in AF is unclear. No studies have been reported about the response after chronic BNP therapy in AF.

With this in mind, we assessed the hypothesis that chronic BNP therapy may prevent atrial electrical remodeling in AF. We extended the hypothesis to include the demonstration of the prevention of AF-associated increased activation of CaMKII with chronic BNP treatment. Accordingly, we examined the antiarrhythmic efficacy and underlying electrophysiological mechanisms of chronic BNP therapy in a well-established rabbit model of AF induced by rapid atrial pacing (RAP).

Materials and Methods

Rapid Atrial Pacing Animal Model

This investigation was approved by the Animal Experimentation Ethics Committee of Harbin Medical University (HMU) and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (no. 85-23).

Healthy adult New Zealand white rabbits (male, 2.5-3.0 kg, aged 4-5 months) were obtained from the Experimental Animal Center of HMU (China). The rabbit model of AF was established as reported previously.^17,18 In the current study, the surface electrocardiogram was checked for every rabbit. The rabbits were in normal sinus rhythm and had no detectable arrhythmia before therapy. The rabbits were anesthetized with ketamine (35 mg/kg) and xylazine (5 mg/kg^; Sigma Aldrich, St Louis, Missouri). A pacemaker (Harbin Polytechnic University, Harbin, China) was inserted into a subcutaneous (SC) pocket on the back of the rabbit. The pacemaker electrode was sutured to the right atrium. The animals were confined for 1 week after the surgery for recovery.

The rabbits were randomized into 4 groups (n = 11 per group, 3 for the isolation of left atrial myocytes and whole-cell patch-clamp recording and 8 for other analyses). (1) Sham group: sham surgery and no pacing; (2) RAP group: RAP at 600 beats/min for 3 weeks; (3) RAP/BNP group: RAP at 600 beats/min and SC administration of rhBNP (Nuodikang Biological Pharmaceutical Company Ltd, Chengdu, China) at a dose of 20 μg/kg twice daily for 3 weeks; and (4) sham/BNP group: sham surgery and SC rhBNP therapy (20 μg/kg, twice daily) for 3 weeks. The SC route of administration of rhBNP was used as reported previously. The rhBNP dose regimen was based on previous studies and our initial dosage testing, which had minimal effect on systemic blood pressure and heart rate of the rabbits. The dosage used in the current study was in the range of therapeutic doses, but slightly lower than that used in the past for the treatment of systolic HF patients.^19

-22

HL-1 Cell Model

To determine that the prevention of AF-induced activation of CaMKII may play a critical role to the antielectrical remodeling by BNP, in the current study, we utilized a well-established HL-1 cell model of AF^17,23

-27 and applied CaMKII inhibitor (KN-93) in this model. This approach, to use KN-93 in the HL-1 cell model of AF, will allow a better comparison and minimizing the off-target effects of KN-93. It has been well-documented that HL-1 cells are derived from mouse atrial tumor (AT-1) cells and can proliferate indefinitely in culture. They retain a highly differentiated cardiac morphological, biochemical, and electrophysiological phenotype reflective of atrial cardiomyocytes.^24,25 Rapid stimulation of HL-1 cells produces electrical remodeling, recapitulating principal phenotypic features of atrial tachycardia remodeling in vivo. HL-1 cells express CaMKIIδ, RyR2, and PLN.^28,29 Ca²⁺/calmodulin-dependent protein kinase II is a serine/threonine protein kinase expressed in many cell types. Given the ubiquitous nature of CaMKII and its multifunctional role in several physiological processes, to avoid the off-target effects of KN-93 by systemic use on the rabbits, we studied the HL-1 cells to further validate the role of CaMKII.^5,30

HL-1 cells were subjected to rapid field stimulation (10 Hz, 5 milliseconds) for 24 hours using a YC-2 stimulator (Chengdu, China). The HL-1 cells were divided into 6 groups: (1) control group: cultured for 24 hours, no rapid field stimulation or other drug interventions; (2) BNP group: cultured for 24 hours with mouse BNP (100 nM, GL Biochem, Shanghai, China) and no rapid field stimulation; (3) AF group: rapid field stimulation for 24 hours as aforementioned; (4) AF/KN-93 group: rapid field stimulation for 24 hours with KN-93 (a CaMKII inhibitor, 1 μM; APExBIO, Houston, Texas) pretreatment 1 hour; (5) AF/KN-92 group: rapid field stimulation for 24 hours with KN-92 (a nonactive analog of KN-93, 1 μM; APExBIO, Houston, Texas) pretreatment for 1 hour; and (6) AF/BNP group: rapid field stimulation for 24 hours with mouse BNP (100 nM; GL Biochem, Shanghai, China) pretreatment for 1 hour.

Determine AF Induction Rate and Atrial Effective Refractory Period

The rabbit atrial electrophysiology study was performed as described previously.^17,18 In these animals, atrial effective refractory periods (AERPs) were measured at 2 basic cycle lengths (150 and 200 milliseconds), which were recorded as AERP_{150 ms} and AERP_{200 ms}, respectively. After the AERPs were measured, AF was induced by applying 10 seconds burst pacing (10 Hz, 2 milliseconds) to the right atrium at 4 times the threshold current. Each rabbit was induced 10 times. Atrial fibrillation induction rate in each group was calculated as (AF times)/(induction times, ie, 80).³¹

Determine N-Terminal Pro-BNP Levels

Blood collections were made before the surgery at the start of the study and after 3 weeks of RAP at the end of the study. Serum N-terminal pro-BNP (NT-proBNP) level was determined by an enzyme-linked immunosorbent assay kit (Shanghai Jianglai Biotech, Shanghai, China) following the manufacturer’s instructions.

N-terminal pro-BNP is the amino terminal cleavage fragment of endogenous BNP and is secreted in equimolar amounts with BNP. Therefore, NT-proBNP concentration can be applied to denote the release of endogenous BNP. We measured serum NT-proBNP in this study because it is not affected by exogenous rhBNP and has higher stability and longer half-life.³²

Cell Isolation

Left atrial myocytes were isolated from the hearts of the rabbits as described previously.³³ After the rabbits were anesthetized, their hearts were quickly excised and mounted on a Langendorff apparatus. Then the hearts were perfused with the following solutions in sequence: buffer A (75 mL Ca²⁺-free Tyrode), buffer B (40 mL Ca²⁺-free Tyrode, 10 µL 200 mM Ca²⁺, 400 µL 100 mg/mL bovine serum albumin [BSA]), buffer C (40 mL Ca²⁺-free Tyrode, 200 µL 200 mM Ca²⁺, 400 µL 100 mg/mL BSA), buffer D (50 mL Ca²⁺-free Tyrode, 12.5 µL 200 mM Ca²⁺, 500 µL 100 mg/mL BSA), buffer E (35 mL buffer D, 31.5 mg collagenase), and buffer F (15 mL buffer D, 13.5 mg collagenase). Then, the left atria were separated from the hearts. The myocardia of left atria were minced and filtered. The freshly isolated left atrial myocytes were gently centrifuged and resuspended in the storage solution for subsequent patch clamp experiments.

Whole-Cell Patch Clamp Recording

Myocyte ion channel responses were recorded with a patch clamp amplifier (HEKA EPC10, Ludwigshafen, Germany). Action potential duration (APD) was recorded at 37°C under current clamp mode. Current densities (pA/pF) were obtained by current amplitudes normalized to cell capacitance in each cell (n = 9 cells from 3 rabbits per group). The amplitude of L-type calcium current (I _{Ca, L}), transient outward potassium current (I _to), and inward rectifier potassium current (I _K1) were measured as described previously.³³

The pipette solution for I _{Ca, L} contained (mM) 1 MgCl₂, 120 CsCl, 10 EGTA, 10 HEPES, 5 phosphocreatine disodium salt, 4 Mg-ATP, and 0.3 Na₂-GTP (pH adjusted to 7.20 with CsOH). The pipette solution for potassium current (I _to and I _K1) and action potential (AP) recordings contained (mM) 0.1 CaCl₂, 140 K-gluconate, 1 MgCl₂, 5 NaCl, 10 HEPES, 2 Mg-ATP, and 1 EGTA (pH adjusted to 7.20 with KOH). The extracellular solution for I _{Ca, L} recordings contained (mM) 140 TEA-Cl, 10 HEPES, 2 MgCl₂, 10 CaCl₂, and 5 glucose (pH adjusted to 7.4 with TEA-OH). The extracellular solution for potassium currents and AP recording contained (mM) 3.5 KCl, 2 mM CaCl₂,140 NaCl, 1 MgCl₂, 10 glucose, 1.25 NaH₂PO₄, and 10 HEPES (pH adjusted to 7.4 with NaOH). To record I _K1 and I _to, both I _{Ca, L} and I _Na were blocked with 0.2 mM CdCl₂ and 30 μM TTX, respectively.

Real-Time Polymerase Chain Reaction

From the left atria of the rabbit, total RNA was extracted using TRIzol reagent (BioTeke, Beijing, China). Using M-MLV reverse transcriptase (BioTeke), total RNA was reverse-transcribed to obtain complementary DNA. The messenger RNA (mRNA) levels of rabbit BNP, Cav1.2 (the subunit of L-type calcium channel), Kv4.3 (the subunit of transient outward potassium channel), and Kir2.1 (the subunit of inward rectifier potassium channel) were determined using SYBR Green I incorporation method on an ABI 7500 fast real-time polymerase chain reaction system (Applied Biosystems, Foster, California). β-Actin was used as an internal control. The primers of related genes used in this study are listed in Table 1.

Table 1.

Primers for Real-Time PCR.

Genes	Sequence
BNP	Forward primer 5′-AGCAGACGACCCTGGAGCCT-3′
	Reverse primer 5′-GAGCCAATCCGGTCCATCCT-3′
Cav1.2	Forward primer 5′-TTCGTTCTCATCCTGCTCAA-3′
	Reverse primer 5′-TATCAACAATGCTACCCACA-3′
Kv4.3	Forward primer 5′-CGACGGACTGAGACCAAACT-3′
	Reverse primer 5′-GCTGGCTATGGAAGGAATGT-3′
Kir2.1	Forward primer 5′-GATGCTGGTTATCTTCTGCC-3′
	Reverse primer 5′-TAGCCTATGGTTGTCTGGGT-3′
β-Actin	Forward primer 5′-CATCTACGAGGGCTACGC-3′
	Reverse primer 5′-TCTCCTTGATGTCCCGCA-3′

Abbreviations: BNP, B-type natriuretic peptide; PCR, polymerase chain reaction.

Western Blot

Protein samples were extracted from the left atria of the rabbits or from HL-1 cells. Western blot analysis was performed as described previously.³⁴ Primary antibodies included p-CaMKIIδ antibody (1:500; Santa Cruz, Dallas, Texas), anti-oxidized-CaMKII (Met281/282) antibody (ox-CaMKIIδ, 1:1000; Merck, Temecula, California), RYR2 (pSer2814) pAb (1:5000; Badrilla, Leeds, UK), and phospho-phospholamban (Thr17) antibody (1:2500; Thermo, Waltham, Massachusetts). Secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit or rabbit anti-mouse immunoglobulin G (1:1000; Zhongshan, Beijing, China). Western blot bands were normalized to β-actin as an internal control.

Statistical Analyses

SPSS 19.0 software was used for statistical analyses. All data are presented as mean ± standard error of the mean, except for AF induction rate as a percentage. Statistical significance between different groups was determined by 1-way analysis of variance (ANOVA) and least significant difference post hoc test. Serum concentrations of NT-proBNP in 4 groups before and after 3 weeks of RAP were compared with 2-factor ANOVA. The AF induction rate was compared with χ² test. A value of P < .05 was considered statistically significant.

Results

Effects of Chronic rhBNP Therapy on AF Inducibility and AERPs After 3 Weeks of RAP

As displayed in Figure 1, the AF induction rate increased significantly in the RAP group compared with the sham group (RAP: 51.3% vs sham: 7.5%). Both AERP_{200 ms} and AERP_{150 ms} were significantly shortened (AERP_{200 ms}:78.3 ± 9.2 milliseconds vs 100.3 ± 16.3 milliseconds; AERP_{150 ms}: 75.3 ± 9.3 milliseconds vs 94.8 ± 15.6 milliseconds). Importantly, in the RAP/BNP group, the AF induction rate significantly decreased to 13.8%, the AERP_{200 ms} and AERP_{150 ms} reached to 95.8 ± 16.7 milliseconds and 92.0 ± 18.1 milliseconds, respectively. There was no significant difference in AF induction rate, AERP_{200 ms}, or AERP_{150 ms} between the RAP/BNP and sham groups (Figure 1A-C).

Figure 1.

Group means data (n = 8/group) of the effects of rapid atrial pacing (RAP) and chronic recombinant human BNP (rhBNP) therapy on AF induction rate (A), AERP_{200 ms} (B) and AERP_{150 ms} (C), and mRNA expression of BNP (D) in left atrial myocytes. Compared with same animals, in the untreated RAP group, the AF induction rate, AERP, and mRNA of BNP in the left atrial myocytes were significantly increased. In contrast, in the rhBNP-treated RAP group, these changes in left atrial myocytes were prevented. *P < .05 versus sham group; ^# P < .05, RAP/BNP versus RAP group. AERP indicates atrial effective refractory period; AF, atrial fibrillation; BNP, B-type natriuretic peptide; mRNA, messenger RNA.

Effects of Chronic rhBNP Therapy on APD, I _{Ca, L}, I _to, and I _K1 of the Left Atrial Myocytes After 3 Weeks of RAP

Figure 2 presents the effects of chronic rhBNP therapy on the APD of the left atrial myocytes obtained from the RAP rabbits. After 3 weeks of RAP, both APD₉₀ and APD₅₀ were shortened significantly compared with the sham group. In the RAP/BNP group, these changes were nearly prevented. Figure 3 exhibits the effects of chronic rhBNP therapy on I _{Ca, L} and the expression of Cav1.2 in the left atrial myocytes obtained from the RAP rabbits. After 3 weeks of RAP, the current density of I _{Ca, L} and the expressions of Cav1.2 were significantly decreased. Figures 4 and 5 show the effects of chronic rhBNP therapy on I _to, I _K1, and the expressions of Kv4.3 and Kir2.1 in the left atrial myocytes obtained from the RAP rabbits. As shown in Figure 4, the current density of I _to significantly decreased and Kv4.3 significantly reduced after 3 weeks of RAP. In contrast, as demonstrated in Figure 5, both the current density of I _K1 and the expressions of Kir2.1 significantly increased after 3 weeks of RAP. Importantly, these changes were inhibited significantly after chronic rhBNP therapy.

Figure 2.

The effects of rapid atrial pacing (RAP) and chronic recombinant human BNP (rhBNP) therapy on the action potential duration (APD) of the left atrial myocytes. (A) Examples of the traces of APD of single atrial myocytes by patch clamp technique; (B and C) group means data of APD₅₀ and APD₉₀ of single atrial myocytes, respectively (n = 9 cells from 3 rabbits per group). It is noted, only in the untreated RAP group, both APD₅₀ and APD₉₀ in the left atrial myocytes were significantly shortened. *P < .05 versus sham group; ^# P < .05, RAP/BNP versus RAP group. BNP indicates B-type natriuretic peptide.

Figure 3.

The effects of rapid atrial pacing (RAP) and chronic recombinant human BNP (rhBNP) therapy on the L-type calcium current (I _{Ca, L}) and the expression of Cav1.2 in the left atrial myocytes. (A) Group means of the I _{Ca, L} density (n = 9 cells from 3 rabbits per group); (B) group means of the expression of Cav1.2 (n = 6/group). *P < .05 versus sham group; ^# P < .05, RAP/BNP versus RAP group. BNP indicates B-type natriuretic peptide.

Figure 4.

The effects of rapid atrial pacing (RAP) and chronic recombinant human BNP (rhBNP) therapy on the transient outward potassium current (I _to) and the expression of Kv4.3 in the left atrial myocytes. (A) Group means of I _to density (n = 9 cells from 3 rabbits per group); (B) group means of the expression of Kv4.3 (n = 6/group). *P < .05, versus sham group; ^# P < .05, RAP/BNP versus RAP group. BNP indicates B-type natriuretic peptide.

Figure 5.

The effects of rapid atrial pacing (RAP) and chronic recombinant human BNP (rhBNP) therapy on the inward rectifier potassium current (I _K1) and the expression of Kir2.1 in the left atrial myocytes. (A) Group means of I _K1 density (n = 9 cells from 3 rabbits per group); (B) group means of the expression of Kir2.1 (n = 6/group). *P < .05 versus sham group; ^# P < .05, RAP/BNP versus RAP group. BNP indicates B-type natriuretic peptide.

Effects of Chronic rhBNP Therapy on the mRNA Expression and Plasma Levels of BNP After 3 Weeks of RAP

As exhibited in Figure 1D and summarized in Table 2, the mRNA expression of BNP increased significantly in the RAP group. The increase was attenuated significantly in the RAP/BNP group. There was no significant difference in NT-proBNP concentration among 4 groups before surgery. However, after 3 weeks of RAP, NT-proBNP concentration increased significantly in the RAP group compared with that in the sham animals or preoperation. The increase was inhibited significantly in the RAP/BNP group.

Table 2.

Serum Concentration of NT-proBNP in 4 Groups Before and After 3 Weeks of Rapid Atrial Pacing (RAP).^a

Groups	Sham	Sham/BNP	RAP	RAP/BNP
Baseline	52.4 ± 5.5	53.9 ± 7.6	51.8 ± 4.2	55.1 ± 4.8
3-Week pacing	54.3 ± 6.2	55.7 ± 9.5	228.8 ± 31.5^b,c	132.4 ± 18.6^b,c,d

Abbreviations: BNP, B-type natriuretic peptide; NT-proBNP, N-terminal pro-BNP; RAP, rapid atrial pacing; SEM, standard error of the mean.

^aValues are presented as pg/mL and as mean ± SEM; n = 8 per group.

^b P < .05 versus baseline.

^c P < .05 versus sham group.

^d P < .05, RAP versus RAP/BNP group.

Effects of Chronic BNP Therapy on Activation of CaMKII in the Rabbit and HL-1 Cells Model of AF

Figure 6 displays the effects of chronic rhBNP therapy on the activation of CaMKII signaling pathway in the left atrial myocytes obtained from the RAP rabbits. As demonstrated, the RAP group showed increased phosphorylation and oxidation of CaMKII_δ, phosphorylation of RyR2, and PLN compared with the sham group, while BNP treatment attenuated this increase. These results were further verified by the findings from HL-1 cells. As exhibited in Figure 7, in the AF group, HL-1 cells, p-CaMKII_δ, ox-CaMKII_δ, p-RyR2, and p-PLN increased significantly compared with the control group. KN-93 is a CaMKII inhibitor, which decreases CaMKII-mediated phosphorylation of RyR2 and PLN under stress conditions. B-type natriuretic peptide effectively attenuated the upregulation of p-CaMKII_δ, ox-CaMKII_δ, p-RyR2, and p-PLN in HL-1 cells under rapid field stimulation similar to KN-93, while KN-92 treatment did not show any obvious effects.

Figure 6.

The effects of rapid atrial pacing (RAP) and chronic recombinant human BNP (rhBNP) therapy on the activation of CaMKII signaling pathway in the left atrial myocytes. (A) Representative bands and p-CaMKII_δ expression (phosphorylation of CaMKII_δ at Thr287) obtained from 4 group rabbits; (B) representative bands and quantitative analyses of ox-CaMKII_δ (oxidation of CaMKII_δ at Met281/282); (C) representative bands and quantitative analyses of p-PLN (phosphorylation of PLN at Thr17); (D) representative bands and quantitative analyses of p-RyR2 (phosphorylation of at Ser2814). *P < .05 versus sham group; ^# P < .05, RAP/BNP versus RAP group, n = 6/group. BNP indicates B-type natriuretic peptide; CaMKII, Ca²⁺/calmodulin-dependent protein kinase II.

Figure 7.

The exogenous BNP administration on the activation of CaMKII signaling pathway in the HL-1 cells model of AF. HL-1 cells were subjected to rapid field stimulation for 24 hours in the presence or absence of BNP, KN-93 (a CaMKII inhibitor), or KN-92 (a nonactive analog of KN-93). (A) Representative bands and quantitative analyses of p-CaMKII_δ (phosphorylation of CaMKII_δ at Thr287); (B) representative bands and quantitative analyses of ox-CaMKII_δ (oxidation of CaMKII_δ at Met281/282); (C) representative bands and quantitative analyses of p-PLN (phosphorylation of PLN at Thr17); (D) representative bands and quantitative analyses of p-RyR2 (phosphorylation of RyR2 at Ser2814). *P < .05 versus control (ctrl) group; ^# P < .05 versus AF group, n = 5/group. AF indicates atrial fibrillation; BNP, B-type natriuretic peptide; CaMKII, Ca²⁺/calmodulin-dependent protein kinase II.

Discussion

In the present study, we showed, for the first time, chronic rhBNP therapy in a rabbit model of AF results in the prevention of atrial electrical remodeling, CaMKII activation, and increased expression and secretion of endogenous BNP. The prevalence and burden of AF worldwide call for an urgent increase in investment in basic and translational research.^1
-3 The current investigation provides evidence supporting BNP as an effective therapeutic option for the treatment in AF. This study has significant translational value and high public health importance.

Atrial Electrical Remodeling, CaMKII Activation, and Chronic BNP Therapy

Previously, few clinical studied in patients with AF reported antiarrhythmic efficacy after BNP administration. Intravenous administration of rhBNP in patients with acute HF reduced the new onset of AF. Earlier studies showed that perioperative nesiritide (rhBNP) or carperitide (recombinant human ANP) infusion reduced the occurrence of postoperative AF in patients undergoing coronary artery bypass grafting during hospital stay. In another study, nesiritide therapy caused a significantly reduced new-onset AF in patients with acute decompensated HF during a 90-day follow-up period.^12,15,16 However, no previous AF studies have assessed the effects after BNP administration on the alterations of electrical remodeling.

Conceptually, AF induction requires a vulnerable substrate and a trigger that acts on the substrate to initiate the arrhythmia. It is increasingly recognized that principal electrophysiological mechanisms contributing to AF include electrical remodeling, structural remodeling, autonomic remodeling, and Ca²⁺-handling abnormalities. Abbreviations of refractories mainly due to reduction in APD provide substrate for reentry. Increases in delayed afterpolarization (DAD) activity due to Ca²⁺-handling abnormalities promote local ectopic firing Ca²⁺-handling abnormalities.^3,5,35,36 In the current investigation, consistent with previous experimental and human studies, the untreated RAP animals showed characteristics typical of altered electrophysiology, including in the current density and in ion channel mRNA or protein expressions. We found that in the left atrial myocytes isolated from the untreated RAP animals, atrial electrical remodeling was characterized mainly by the significantly shortening of APD and AERP and the changes of the atrial ion current, which were predominantly mediated via the changes in the expression of the ion channels. The shortening of APD/AERP would be expected to reduce the excitation wavelength given by conduction velocity (CV) × AERP. The additional substrate would be abnormal Ca²⁺ handling, which could generate DADs (and therefore triggered activity) and contribute to reentrant substrates.^35,36

Decreased I _{Ca, L} is the principal component of electrical remodeling, which is mainly due to the reduced expression of the subunit of I _{Ca, L} (Cav1.2) to prevent potentially cytotoxic Ca²⁺ overload. Both the reduced I _{Ca, L} and the increased I _K1 may account for the reduced APD. The increased expression of Kir2.1 subunit results in I _K1 increase, while the downregulation of the Kv4.3 subunit causes a decrease in I _to.^7,30,37
-39 Importantly, we found that in the left atrial myocytes isolated from the RAP/BNP animals, the AF-related APD shortening and the loss of AERP with associated characteristical changes on the downregulation of I _{Ca, L}, I _to and the upregulation of I _K1, as well as the altered expression of atrial ion channels genes (Cav1.2, Kv4.3, and Kir2.1), were all absent. There were no significances in these measurements between sham and RAP/BNP animals, thus demonstrating atrial electrical remodeling was prevented by chronic BNP therapy.

How does chronic BNP administration prevent AF-induced electrical remodeling? In the current investigation, the prevention of AF-induced activation of CaMKII might play a critical role in the antielectrical remodeling by chronic BNP.

Excessive CaMKII activation has been implicated in AF triggering and reentry mechanisms through its downstream effects on ion channels and proarrhythmic remodeling that results from cell death, fibrosis, and hypertrophy. It has been well-documented that CaMKII_δ activation can be promoted by atrial tachycardia, oxidative stress, and sympathetic hyperactivity and results in phosphorylation of ion channels and Ca²⁺-cycling proteins, including I _{Ca, L}, RyR2, and PLN, thereby facilitating cell membrane hyperexcitability and afterdepolarizations and promotes the development of AF and atrial electrical remodeling.^4,5,30,40 Previous studies on total CaMKII_δ protein expression in AF were not conclusive; some studies reported an increase in total CaMKII_δ protein expression in AF, while others reported no change in expression.^5,40,41 In the current study, we analyzed the phosphorylation and oxidation of CaMKII_δ instead of total CaMKII_δ protein expression. We observed that in untreated RAP animals, RAP for 3 weeks promoted autophosphorylation and oxidation of CaMKII_δ with the phosphorylation of RyR2 (phosphorylation of Ser2814) and PLN (phosphorylation of Thr17). Similar results were obtained from the in vitro HL-1 cell model of AF. These findings are consistent with previous reports and demonstrate that CaMKII_δ activation is an important molecular mechanism of atrial electrical remodeling in AF. Of importance, we further found that these alterations were prevented by a 3-week rhBNP therapy in the RAP/BNP animals. We further validated the effects of exogenous BNP on the activation of CaMKII signaling pathway in HL-1 cells. Previous reports showed that rapid stimulation of HL-1 cells produced electrical remodeling, recapitulating principal phenotypic features of atrial tachycardia remodeling in vivo.^23

-26 Ca²⁺/calmodulin-dependent protein kinase II is a nodal proarrhythmic signal. The changes of p-CaMKII, ox-CaMKII, p-PLN, and pRyR2 in HL-1 cells were similar to that of the rabbits. It is noted that KN-93 exerted similar effects to BNP in terms of expression of p-CaMKII, ox-CaMKII, p-PLN, and pRyR2.

Many previous studies have demonstrated that inhibiting CaMKII activation and/or elimination of CaMKII-dependent RyR2/PLN phosphorylation is an important molecular mechanism that protects animals from developing AF. Also, in fibrillating human atrial cells, CaMKII inhibition appears to be able to correct proarrhythmic, defective intracellular Ca² ⁺ homeostasis. However, there are currently no CaMKII inhibitors available clinically.^5,30,42,43

B-Type Natriuretic Peptide Activation and Chronic BNP Therapy

Both BNP and NT-proBNP are applied as diagnosis, management, and prognostic tool for HF. It is now also clear that elevated BNP levels are closely associated with AF. Plasma BNP levels are increased in patients with paroxysmal, persistent and permanent AF, and normal left ventricle function. It has been shown that both BNP and NT-proBNP are significantly elevated in patients with lone AF. Both BNP and NT-proBNP levels decrease significantly after catheter ablation of AF.^9,10,44,45 In agreement with the clinical observations, in the current study, we found that the mRNA expression and the release of endogenous BNP were significantly increased in the untreated RAP rabbits. Moreover, we further found that the elevated BNP and NT-proBNP were significantly reduced and nearly restored to the levels in the sham rabbits. Our current findings not only support the diagnostic and prognostic value of BNP as a biomarker in AF but also provide new evidence that BNP is an effective therapeutic choice in the management of AF.

It should be realized that BNP has properties that are beneficial in AF and are important for the prevention of CaMKII activation. It is well known that BNP is released from both atrial myocytes and ventricular myocytes. In addition to its effects of diuresis, natriuresis, vasodilation, antihypertrophy, and antifibrosis, BNP has been found to suppress sympathetic tone and the renin–angiotensin system, inhibiting endothelin and vasopressin and imporving cardiac β-adrenergic regulation. It is reported that BNP can act in a paracrine and endocrine manner in many cardiovascular diseases.^9,11,12,46 Although little is known about the electrophysiological effects of BNP, it has been speculated that BNP may have similar properties to ANP, which has been shown to affect the sodium, calcium, and potassium channels and prevent the shortening of the AERP and monophasic APD in a canine RAP model.⁴⁷ Importantly, cardiac production and plasma levels of BNP are increased in AF, which may alter left ventricular and atrial functional responses to BNP and suggesting that it is released as a cytoprotective mechanism. However, the changes of BNP-induced cardiac response in AF are not clear. Moreover, no previous studies have examined antiarrhythmic efficacy after BNP therapy in AF. Further studies are needed to improve the understanding of their therapeutic values.

Study Limitations

First, our observations were obtained from an RAP rabbit model. Although this RAP model mimics “lone AF,” it cannot fully represent the complex clinical spectrum of human AF. Second, in the current investigation, we focused on study that systemically assessed electrical remodeling with and without chronic BNP therapy in AF model. We showed that BNP to protect against AF was due to its effects on the prevention and/or correction of electrical remodeling in fibrillating atrial myocytes. However, it is possible that the improvements in AF-related structure remodeling and intracellular calcium-handling remodeling by BNP might also contribute to our current observations. Histology examination was not performed in the current study. The absence of histology is another limitation. Third, altered intracellular Ca²⁺ dynamics are characteristically observed in cardiomyocytes from AF. The current study did not determine [Ca²⁺]i transients. More insights will be gained by confocal microscopy assessments. It should be assessed in the future.

Conclusions

Chronic BNP therapy prevents atrial electrical remodeling in a rabbit model of AF induced by RAP. The inhibition of the activation of CaMKII may have contributed to the anti-atrial electrical remodeling effects of BNP. It suggests that chronic BNP may be a useful therapeutic choice for AF.

Footnotes

Author Contributions

Hongyan Zhao contributed to acquisition, analysis and interpretation, and drafted the manuscript. Tiankai Li contributed to analysis and drafted the manuscript. Guangzhong Liu, Li Zhang, Guangnan Li, Jia Yu, Qi Lou, Rui He, Chengchuang Zhan, Luyifei Li, Wen Yang, and Yanxiang Zang contributed to acquisition. Cheping Cheng and Weimin Li contributed to conception and design, and drafted the manuscript. All authors critically revised article, gave final approval. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by grants from Fundamental Research Funds for the Provincial Universities (2017LCZX08), Heilongjiang Postdoctoral Fund (LBH-Z17226), the Health Commission of Heilongjiang Province (2014-376), the National Institutes of Health grant (R01AG049770), and National Natural Science Foundation of China (81270252 and 81700305).

ORCID iD

Weimin Li

References

Calvo

Filgueiras-Rama

Jalife

. Mechanisms and drug development in atrial fibrillation. Pharmacol Rev. 2018;70(3):505–525.

Staerk

Sherer

Benjamin

Helm

. Atrial fibrillation: epidemiology, pathophysiology, and clinical outcomes. Circ Res. 2017;120(9):1501–1517.

Poudel

Cui

Sharma

Tian

Paudel

. Atrial fibrillation: recent advances in understanding the role of microRNAs in atrial remodeling with an electrophysiological overview. Cardiology. 2015;131(1):58–67.

Heijman

Voigt

Wehrens

Dobrev

. Calcium dysregulation in atrial fibrillation: the role of CaMKII. Front Pharmacol. 2014;5:30.

Mesubi

Anderson

. Atrial remodelling in atrial fibrillation: CaMKII as a nodal proarrhythmic signal. Cardiovasc Res. 2016;109(4):542–557.

Nakatani

Nishida

Sakabe

, et al. Tranilast prevents atrial remodeling and development of atrial fibrillation in a canine model of atrial tachycardia and left ventricular dysfunction. J Am Coll Cardiol. 2013;61(5):582–588.

Nattel

Harada

. Atrial remodeling and atrial fibrillation: recent advances and translational perspectives. J Am Coll Cardiol. 2014;63(22):2335–2345.

Hund

Mohler

. Role of CaMKII in cardiac arrhythmias. Trends Cardiovasc Med. 2015;25(5):392–397.

Ping

Zhu

Luo

. Brain natriuretic peptide and its biochemical, analytical, and clinical issues in heart failure: a narrative review. Front Physiol. 2018;9:692.

10.

Inohara

Kim

Pieper

, et al. B-type natriuretic peptide, disease progression and clinical outcomes in atrial fibrillation. Heart. 2019;105(5):370–377.

11.

Kerkela

Ulvila

Magga

. Natriuretic peptides in the regulation of cardiovascular physiology and metabolic events. J Am Heart Assoc. 2015;4(10):e002423.

12.

Lenz

Foral

Malesker

Hunter

Hilleman

. Impact of nesiritide on health care resource utilization and complications in patients with decompensated heart failure. Pharmacotherapy. 2004;24(9):1137–1146.

13.

Ponikowski

Voors

Anker

, et al. 2016 ESC guidelines for the diagnosis and treatment of acute and chronic heart failure: the Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure of the European Society of Cardiology (ESC) Developed with the special contribution of the Heart Failure Association (HFA) of the ESC. Eur Heart J. 2016;37(27):2129–2200.

14.

Yancy

Jessup

Bozkurt

, et al. 2013 ACCF/AHA Guideline for the Management of Heart Failure: executive summary: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Circulation. 2013;128(16):1810–1852.

15.

Mentzer

Jr Oz

Sladen

, et al. Effects of perioperative nesiritide in patients with left ventricular dysfunction undergoing cardiac surgery: the NAPA Trial. J Am Coll Cardiol. 2007;49(6):716–726.

16.

Sezai

Iida

Yoshitake

, et al. Carperitide and atrial fibrillation after coronary bypass grafting: the Nihon University Working Group Study of low-dose HANP infusion therapy during cardiac surgery trial for postoperative atrial fibrillation. Circ Arrhythm Electrophysiol. 2015;8(3):546–553.

17.

Liu

Hou

Yuan

, et al. Fenofibrate inhibits atrial metabolic remodelling in atrial fibrillation through PPAR-alpha/sirtuin 1/PGC-1alpha pathway. Br J Pharmacol. 2016;173(6):1095–1109.

18.

Dong

Zhao

Zhang

, et al. β3-Adrenoceptor impairs mitochondrial biogenesis and energy metabolism during rapid atrial pacing-induced atrial fibrillation. J Cardiovasc Pharmacol Ther. 2016;21(1):114–126.

19.

Clemens

Almirez

Baudouin

Grossbard

Protter

. Human brain natriuretic peptide reduces blood pressure in normotensive and acute norepinephrine-induced hypertensive rabbits. Am J Hypertens. 1997;10(6):654–661.

20.

McKie

Schirger

Benike

, et al. Chronic subcutaneous brain natriuretic peptide therapy in asymptomatic systolic heart failure. Eur J Heart Fail. 2016;18(4):433–441.

21.

Chen

Glockner

Schirger

Cataliotti

Redfield

Burnett

. Novel protein therapeutics for systolic heart failure: chronic subcutaneous B-type natriuretic peptide. J Am Coll Cardiol. 2012;60(22):2305–2312.

22.

Clemens

Almirez

Baudouin

Mischak

Grossbard

Protter

. Pharmacokinetics and biological actions of subcutaneously administered human brain natriuretic peptide. J Pharmacol Exp Ther. 1998;287(1):67–71.

23.

Yang

Shen

Rottman

Wikswo

Murray

. Rapid stimulation causes electrical remodeling in cultured atrial myocytes. J Mol Cell Cardiol. 2005;38(2):299–308.

24.

Claycomb

Lanson

Stallworth

, et al. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci U S A. 1998;95(6):2979–2984.

25.

White

Constantin

Claycomb

. Cardiac physiology at the cellular level: use of cultured HL-1 cardiomyocytes for studies of cardiac muscle cell structure and function. Am J Physiol Heart Circ Physiol. 2004;286(3): H823–H829.

26.

Brundel

Shiroshita-Takeshita

, et al. Induction of heat shock response protects the heart against atrial fibrillation. Circ Res. 2006;99(12):1394–1402.

27.

Yan

Kong

Zhang

, et al. c-Jun N-terminal kinase activation contributes to reduced connexin43 and development of atrial arrhythmias. Cardiovasc Res. 2013;97(3):589–597.

28.

Chen

Kao

, et al. Colchicine modulates calcium homeostasis and electrical property of HL-1 cells. J Cell Mol Med. 2016;20(6):1182–1190.

29.

Gao

Yan

, et al. Transcriptional regulation of stress kinase JNK2 in pro-arrhythmic CaMKIIdelta expression in the aged atrium. Cardiovasc Res. 2018;114(5):737–746.

30.

Mustroph

Neef

Maier

. CaMKII as a target for arrhythmia suppression. Pharmacol Ther. 2017;176:22–31.

31.

Zhao

, et al. Effects of spironolactone on atrial structural remodelling in a canine model of atrial fibrillation produced by prolonged atrial pacing. Br J Pharmacol. 2010;159(8):1584–1594.

32.

Troughton

Michael Felker

Januzzi

Jr . Natriuretic peptide-guided heart failure management. Eur Heart J. 2014;35(1):16–24.

33.

Laszlo

Bentz

Konior

, et al. Effects of selective mineralocorticoid receptor antagonism on atrial ion currents and early ionic tachycardia-induced electrical remodelling in rabbits. Naunyn Schmiedebergs Arch Pharmacol. 2010;382(4):347–356.

34.

Viatchenko-Karpinski

Kornyeyev

El-Bizri

, et al. Intracellular Na⁺ overload causes oxidation of CaMKII and leads to Ca²⁺ mishandling in isolated ventricular myocytes. J Mol Cell Cardiol. 2014;76:247–256.

35.

Tse

Lai

Lee

Yan

Wong

. Electrophysiological mechanisms of gastrointestinal arrhythmogenesis: lessons from the heart. Front Physiol. 2016;7:230.

36.

Schotten

Verheule

Kirchhof

Goette

. Pathophysiological mechanisms of atrial fibrillation: a translational appraisal. Physiol Rev. 2011;91(1):265–325.

37.

Nattel

Burstein

Dobrev

. Atrial remodeling and atrial fibrillation: mechanisms and implications. Circ Arrhythm Electrophysiol. 2008;1(1):62–73.

38.

Yeh

Xiao

, et al. Cellular signaling underlying atrial tachycardia remodeling of L-type calcium current. Circ Res. 2008;103(8):845–854.

39.

Bosch

Scherer

Rüb

, et al. Molecular mechanisms of early electrical remodeling: transcriptional downregulation of ion channel subunits reduces I_{Ca, L} and I_to in rapid atrial pacing in rabbits. J Am Coll Cardiol. 2003;41(5):858–869.

40.

Purohit

Rokita

Guan

, et al. Oxidized Ca(2+)/calmodulin-dependent protein kinase II triggers atrial fibrillation. Circulation. 2013;128(16):1748–1757.

41.

Fischer

Herting

Mason

, et al. Late INa increases diastolic SR-Ca2+-leak in atrial myocardium by activating PKA and CaMKII. Cardiovasc Res. 2015;107(1):184–196.

42.

Wang

, et al. Inhibition of CaMKII phosphorylation of RyR2 prevents induction of atrial fibrillation in FKBP12.6 knockout mice. Circ Res. 2012;110(3):465–470.

43.

Voigt

Wang

, et al. Enhanced sarcoplasmic reticulum Ca²⁺ leak and increased Na⁺-Ca²⁺ exchanger function underlie delayed afterdepolarizations in patients with chronic atrial fibrillation. Circulation. 2012;125(17):2059–2070.

44.

Maries

Manitiu

. Diagnostic and prognostic values of B-type natriuretic peptides (BNP) and N-terminal fragment brain natriuretic peptides (NT-pro-BNP). Cardiovasc J Afr. 2013;24(7):286–289.

45.

Yamada

Murakami

Okada

, et al. Plasma atrial natriuretic peptide and brain natriuretic peptide levels after radiofrequency catheter ablation of atrial fibrillation. Am J Cardiol. 2006;97(12):1741–1744.

46.

Goetze

Friis-Hansen

Rehfeld

Nilsson

Svendsen

. Atrial secretion of B-type natriuretic peptide. Eur Heart J. 2006;27(14):1648–1650.

47.

Yoshida

Niwano

, et al. Atrial natriuretic peptide (ANP) suppresses acute atrial electrical remodeling in the canine rapid atrial stimulation model. Int J Cardiol. 2008;123(2):147–154.