B-type natriuretic peptide (BNP) attenuates the L-type calcium current and regulates ventricular myocyte function

Regulatory Peptides 151 (2008) 95–105

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Regulatory Peptides j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / r e g p e p

B-type natriuretic peptide (BNP) attenuates the L-type calcium current and regulates ventricular myocyte function R. Sodi a,b,⁎, E. Dubuis b, A. Shenkin a,b, G. Hart b a b

Department of Clinical Biochemistry & Metabolic Medicine, Royal Liverpool & Broadgreen University Hospital, Prescot street, Liverpool L7 8XP, United Kingdom Division of Clinical Sciences, Faculty of Medicine, University of Liverpool, Liverpool L69 3GA, United Kingdom

A R T I C L E

I N F O

Article history: Received 12 February 2008 Received in revised form 22 May 2008 Accepted 15 June 2008 Available online 20 June 2008 Keywords: B-type natriuretic peptide Ventricular myocytes Action potential Calcium current Calcium transients

A B S T R A C T A fundamental question in physiology is how hormones regulate the functioning of a cell or organ. It was therefore the aim of this study to investigate the effect(s) of BNP-32 on calcium handling by ventricular myocytes obtained from the rat left ventricle. We specifically tested the hypothesis that BNP-32 decreased the L-type calcium current (ICa,L). Perforated patch clamp technique was used to record ICa,L and action potential (AP) in voltage and current clamp mode, respectively. Myocyte shortening was measured using a photodiode array edge-detection system and intracellular calcium transients were measured by fluorescence photometry. Western blotting was used to determine the relative change in the expression of proteins. At the concentrations tested, BNP-32 significantly decreased cell shortening in a dose-dependent manner; increased the phase II slope of the AP by 53.0%; increased the APD50 by 16.9%; reduced the ICa,L amplitude with a 22.9% decrease in the peak amplitude and reduced Ca2+-dependent inactivation; increased the V1/2 activation of the L-type calcium channel by 51.1% and decreased V1/2 inactivation by 31.8%; and, intracellular calcium transient amplitude was significantly decreased by 32.0%, whereas the time to peak amplitude and T1/2 were both significantly increased by 38.7% and 89.4% respectively. Sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) protein expression was reduced by BNP-32. These data suggest that BNP-32 regulates ventricular myocyte function by attenuating ICa,L, altering the AP and reducing SERCA2a activity and/or expression. This study suggests a novel constitutive mechanism for the autocrine action of BNP on the L-type calcium channel in ventricular myocytes. © 2008 Elsevier B.V. All rights reserved.

1. Introduction The cardiac L-type calcium channel current (ICa,L) is an important regulator of myocardial contractility [4]. A number of factors including hormones and neurotransmitters affect the ICa,L to control cardiac output in order to meet the body's demands. One such hormone family are the natriuretic peptides, which are structurally similar and have potent diuretic, natriuretic and vasorelaxant activity [23,24,34]. There are three major members in the natriuretic peptide family that have a role on the cardiovascular system: atrial natriuretic peptide (ANP), brain or B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) [23,24,34]. ANP and BNP are produced mainly in the atrium and ventricle of the mammalian heart respectively and their secretion is triggered by transmural pressure and myocyte stretch [23]. CNP is present in the central nervous system, anterior pituitary, kidney and vascular endothelial cells and has various paracrine functions [2].

⁎ Corresponding author. Department of Clinical Biochemistry & Metabolic Medicine, Royal Liverpool & Broadgreen University Hospital, Prescot street, Liverpool L7 8XP, United Kingdom. Tel.: +44 151 706 4245; fax: +44 151 706 4250. E-mail address: [email protected] (R. Sodi). 0167-0115/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2008.06.006

ANP and BNP are ligands for the natriuretic receptor NPRA (GC-A), whereas CNP binds to NPRB (GC-B) [20,34]. The NPRA receptor has been identified on the rat and human heart [20,23]. Binding of the natriuretic peptides to their receptors activates the particulate guanylyl cyclase system that results in the production of intracellular cyclic guanosine monophosphate (cGMP) [23,24,34]. There is also a clearance receptor (NPRC) that binds all three peptides but lacks the particulate guanylyl cyclase domain [10]. It is well known that natriuretic peptides act through cGMP-dependent mechanisms [9,12,31,34,41,44]; however, there is currently no published evidence to show the effects of BNP per se on the action potential (AP) and ionic channels in ventricular myocytes. It is posited that BNP has an autocrine effect on the heart [9,28], but the specific effects of the bioactive fragment has not yet been clarified. BNP is synthesized as a pre-pro-hormone of 132 amino acids that is processed into a prohormone of 108 amino acids (proBNP) [38]. This is subsequently cleaved into the bioactive 32-amino acid C-terminal fragment (BNP-32) and a 76-amino acid N-terminal fragment (NTproBNP) [38]. A recent study has now shown that there is functional heterogeneity in circulating BNP but reported that BNP-32 is 6- to 8fold more biologically potent than proBNP [25]. There is currently great interest in the diagnostic [23,24,27], prognostic [8] and therapeutic application [39] of BNP in clinical

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medicine. BNP is elevated in patients with heart failure and correlates with the New York Heart Association (NYHA) classification [27] and is therefore measured in clinical laboratories to aid diagnoses. BNP has also been shown to be prognostic for mortality in patients with heart failure with a 35% increase in the relative risk of death for each 100 pg/ mL increase in its measured plasma level [8]. Recombinant BNP (Nesiritide) has been used in the treatment of acute decompensated heart failure [39] but recent meta-analyses have shown increased mortality rates in its trials [36,37,42]. Thus, there is need to demonstrate the pharmacological effects of BNP on the heart so as to establish the underlying mechanisms attributable to the reported increase in mortality. Importantly, a recent report has suggested that there are alternate forms of BNP in circulation [21], whereas another study showed the paradoxical absence of circulating BNP-32 in severe heart failure [14]. The physiological effects of the various forms of circulating BNP remain to be clarified. Here we specifically explore the autocrine effects of the bioactive fragment (BNP-32) on the site of its release — the ventricles. Our aim was to clarify its effects on ionic conductances, expression of relevant calcium-regulatory proteins, and on measures of ventricular myocyte function. We tested the hypothesis that BNP-32 decreases the L-type calcium current (ICa,L). 2. Materials and methods 2.1. Peptide, solutions and reagents BNP-32 was purchased from Sigma-Aldrich (Gillingham, Dorset, U.K). The molecular weight was listed as 3452.94 and the amino acid sequence is Asn-Ser-Lys-Met-Ala-His-Ser-Ser-Ser-Cys-Phe-Gly-Gln-Lys-Ile-AspArg-Ile-Gly-Ala-Val-Ser-Arg-Leu-Gly-Cys-Asp-Gly-Leu-Arg-Leu-Phe. BNP-32 was dissolved in distilled H2O to obtain a 1.0 mM stock solution, which was stored frozen in 10 μL aliquots and added to freshly-prepared extracellular control solution (ECS) at the required final concentration. Collagenase type I was purchased from Worthington (Lakehead, CO, USA). Fura-2-AM and BAPTA-AM were purchased from Invitrogen Molecular Probes (Eugene, OR, USA). All other chemicals and kits were purchased from Sigma-Aldrich (Gillingham, Dorset, U.K) unless otherwise stated. The HEPES-Tyrode buffer used for isolation of the myocytes contained (mM): NaCl 130, KCl 5.4, MgCl2 1.4, NaH2PO4 0.4, creatine 10, taurine 20, Dglucose 10, HEPES 10; pH 7.3 adjusted with NaOH. Extracellular control solution (ECS) used in all experiments in this study contained (mM): NaCl 140, KCl 5.4, CaCl2 1, MgCl2 1, D-glucose 10, HEPES 10; pH 7.35 adjusted with NaOH. To record action potentials, microelectrodes were filled with intrapipette solution that contained (mM): NaCl 10, KCl 20, K-glutamate 120, HEPES 10, Mg-ATP 5; pH 7.25 adjusted with KOH. To record ICa,L microelectrodes were filled with intrapipette solution containing (mM): NaCl 10, KCl 20, Cs-glutamate 120, HEPES 10; pH 7.3 adjusted with CsOH. Amphotericin-B was dissolved in dimethyl sulfoxide, DMSO (v/v 0.3%) and then added to intracellular solution at a final concentration of 250 μg/mL. BAPTA-AM was dissolved in DMSO (v/v 0.01%) and used at a final working concentration of 10 μM. Fura-2-AM was also dissolved in DMSO (v/v 0.5%) and added to a suspension of cells at a final concentration of 5 μM. 2.2. Isolation of rat ventricular myocytes Male Wistar rats weighing approximately 250–300 g were sacrificed in accordance with the United Kingdom Home Office Guidelines on the Operation of Animals (Schedule 1, Scientific Procedures Act 1986) and the investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The thoracic cavity was opened, the heart removed and mounted onto a modified Langendorff apparatus maintained at 37 °C as previously described [11]. The heart was cleared of blood by

perfusion with HEPES-Tyrode buffer containing 0.75 mM CaCl2 for about 2 min followed by a 4 min perfusion with Ca2+-free HEPESTyrode buffer containing 0.1 mM ethyleneglycol-bis(β-aminoethyl)-N, N,N′,N′-tetraacetic acid (EGTA) and then finally a 6 min perfusion with HEPES-Tyrode buffer containing 50 μM CaCl2, 1 mg/mL type 1 collagenase and 0.05 mg/mL type XIV protease. The left ventricle was then dissected free, sliced into smaller chunks and agitated for 4 min in the HEPES-Tyrode buffer containing the enzymes as described but supplemented with 10 mg/mL bovine serum albumin (BSA). This was followed by filtration through monofilament nylon cloth, then centrifugation and the pelleted myocytes were resuspended in enzyme-free solution containing 0.75 mM CaCl2 and maintained in a petri dish at room temperature until used. This procedure was repeated 5 times to obtain 5 batches of ventricular myocytes from each rat heart. All experiments were undertaken within 6 h of the isolation procedure and only myocytes showing clearly defined striations, and when applicable, sustained contractions in response to field stimulation were used in this study. 2.3. Cell shortening measurement The effect of BNP-32 on unloaded contractions of ventricular myocytes was studied using a linear photodiode array edge-detection system [3]. Briefly, myocytes were pipetted into a small volume chamber mounted on the stage of an inverted light microscope (Nikon, Japan) and allowed to settle on the glass bottom. The myocytes were superfused with ECS at the rate of 2 mL/min and at room temperature (22–25 °C) using a gravity-assisted flow system. Two platinum wires placed opposite each other in the chamber were used to field stimulate the myocytes using an isolated stimulator (Digitimer Ltd., Hertfordshire, UK) at the rate of 0.5 Hz. A myocyte was selected at random and its longitudinal axis was positioned in alignment with the photodiode array detection system. In this study, cell shortening during field stimulation was monitored at both ends of a myocyte. A different cell was tested for each concentration and 6 consecutive contractions in control and after achieving steady state in the presence of BNP-32 (10− 9 to 5.0 × 10− 6 M) were averaged per cell for data analysis. The parameters recorded included cell shortening, time to peak shortening (TPS) and time to 50% decline of shortening (T1/2) as previously described [40]. For myocyte shortening, the change in the magnitude of shortening induced by BNP-32 was normalised to their respective controls, that is, (Scontrol − SBNP-32) / Scontrol [18]. The time course of inhibition was plotted and fitted to an exponential function: y = y0 + Ae− x / τ, where y0 is the maximal amplitude of inhibition and τ is the time constant. The inverse of the time constant was then plotted against BNP-32 concentration and fitted with the following equation: 1 / τ = k+ 1[BNP32] + k− 1, where τ is the time constant of shortening inhibition for the BNP-32 concentration used, k+ 1 is the association rate constant and k− 1 is the dissociation rate constant. KD is estimated by k− 1 / k+ 1. The dose–response data obtained were fitted to the Hill equation: y = 1 / (1 + ([BNP-32] / IC50)n), where IC50 is the BNP-32 concentration required to obtain half of the maximal inhibition and n is the Hill coefficient. Due to the prohibitive cost of BNP-32, we were unable to undertake experiments at higher concentrations to achieve maximal inhibition. Therefore, the calculated KD value was used as an estimate of IC50 in the Hill equation to determine the maximum inhibition of myocyte shortening caused by BNP-32. 2.4. Electrophysiological measurements Myocytes were loaded with 10 μM BAPTA-AM for 10 min to prevent contractions and then allowed to settle on the bottom of the chamber mounted on an inverted microscope (Nikon, Japan). The myocytes were constantly superfused with ECS at approximately 2 mL/min (at room temperature) using a gravity-assisted flow system. Micropipettes

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were fabricated from filamented borosilicate glass type GC150TF-15 (Harvard apparatus, Kent, UK) with a two-stage patch pipette puller (model PP-830, Narishige Scientific Instrument, Narishige, Japan) to give tip resistances of 1–2 MΩ after fire polishing and when filled with intracellular solutions. The micropipettes were filled with the relevant intracellular solution as described in Section 2.1. Voltage and current clamp protocols were generated and recorded using an AXOPATCH 200A amplifier via a CV 202A headstage and the data were digitized with a Digidata-1322A analog–digital converter (all from Molecular Devices Corporation, Sunnyvale, CA, USA). The data was stored on a PC computer using Clampex and analysed with Clampfit (PCLAMP software version 8.2, Molecular Devices Corporation). The perforated patch clamp technique using amphotericin-B as the pore forming agent was used in this study so as to prevent dialysis of the cell's internal constituents [35]. Briefly, the micropipettes were front filled with the relevant intracellular solution as described in Section 2.1 by capillary action and then backfilled with the same solution but containing 250 μg/mL amphotericin-B. As amphotericin-B perforated the patch, access resistance (Ra) decreased to 10–30 MΩ within 5–10 min. In all cases, Ra b 30 MΩ was ascertained and steady state recording was achieved prior to data collection. APs were recorded in current clamp mode and the myocytes were stimulated with 2 ms supra-threshold current pulses at 0.5 Hz with a sampling rate of 25 kHz. The parameters recorded in control ECS and after achieving steady state with ECS containing 10− 6 M BNP-32 included: resting membrane potential (VR), time taken to reach half the AP amplitude (APD50) and slope of phase II of the AP, which was fitted using a linear function. For each cell, 6 APs before and after exposure to BNP-32 were averaged for data analysis. ICa,L was measured in voltage clamp mode using a cesium-based intrapipette solution to suppress K+ currents (as described in Section 2.1) at a holding potential of −40 mV to inactivate any fast Na+ current. Test pulses of 500 ms duration, ranging from −40 to +50 mV, were imposed in 10 mV stepwise increments to obtain current–voltage relations (I–V curves). ICa,L was filtered at 2 kHz with an 8-pole low-pass Bessel filter and sampled at 10 kHz. Six I–V curves per cell, prior to and after superfusion with 5.0 × 10− 7 M BNP-32 was

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averaged for data analyses. In all cases, currents were normalised to cell capacitance in order to account for cell size variability. We also assessed the time course of the ICa,L decay to investigate any changes in Ca2+-dependent inactivation [6]. The decay phase of the ICa,L were fitted with a mono-exponential function for each incremental depolarisation step. The voltage dependence of activation and inactivation were determined using a conventional double-pulse protocol. Prepulses from −40 to 60 mV for 500 ms in 10 mV steps were applied, followed by a 2 ms gap to allow for the resetting of the activation gate. A test pulse was then applied for 200 ms from the holding potential of −40 to 0 mV. The currents recorded during prepulses were corrected for driving force using the equation: GCa,L =ICa,L / (Vm −Vrev), where GCa,L is the peak conductance, ICa,L is the peak calcium current for the test potential and Vm −Vrev gives the apparent reversal potential for Ca2+ [17]. The results were then normalised to the peak current to obtain activation curves. To obtain the inactivation curves, the currents obtained with the test pulse was normalised and plotted against the potentials applied in the first phase. Data for the activation and inactivation curves were fitted with a Boltzmann function: I /Imax = {1 + exp[(Vm −V1/2) /k]}− 1, where I /Imax is the relative current, Vm is the membrane potential, V1/2 is the voltage of half-maximal activation or inactivation and k is the slope factor. 2.5. Intracellular calcium transient recording The myocytes were loaded with the cell permeable intracellular calcium indicator fura-2-AM (5 μM) for 10 min at room temperature in ECS. They were then washed, resuspended in ECS and an aliquot was allowed to settle on the glass bottom of a chamber on the stage of an inverted microscope (Diaphot 300, Nikon, Japan). The myocytes were field stimulated at 0.5 Hz while superfused with ECS or ECS containing 1 μM BNP-32 at 2 mL/min kept at room temperature. Intracellular calcium was measured using a fluorescence photometry system (Optoscan Monochromator, Cairn Research, Kent, UK) through a 40× oil objective. The excitation wavelengths were 340 and 380 nm, whereas the fluorescence emission was detected at 510 nm. The fluorescence signals were digitized on a computer using the Clampex 8 software

Fig. 1. Inhibition of ventricular myocyte shortening by BNP-32. Representative fast-time base traces of ventricular myocyte shortening in the presence of control solution and with BNP-32 (left) and slow-time base traces (right) at the stated concentrations. Arrows indicate the start of BNP-32 superfusion. In each case a different cell was used.

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(Molecular Devices Corporation) and expressed as the ratio of the emission at the two excitation wavelengths (F340/380). For each cell, 6 consecutive transients in ECS and after achieving steady state with exposure to 10− 6 M BNP-32 were averaged for data analysis. The parameters recorded included: F340/380; time to peak amplitude (TTP); and time to 50% decline of calcium transients (T1/2) [40]. To calculate the amount of calcium flux, we integrated the area under the Ca2+ transients.

antibodies, the blots were incubated for 2 h at room temperature (RT) with constant rocking. Following further washes, the blots were incubated with mouse anti-IgG antibodies conjugated to horse-radish peroxidase (1:1000, Sigma-Aldrich) for 1 h at RT. For a negative control, the primary antibody was omitted. Visualization of the immunoreactive bands was performed using a sensitive chemiluminescent substrate for detecting horse-radish peroxidase on immunoblots (SuperSignal West Dura Extended Duration Substrate, Pierce, Rockford, IL, USA).

2.6. Western immunoblot analysis 2.7. Statistical analysis Myocytes exposed to 1 μM BNP-32 for 6 h were resuspended in a buffer containing the protease inhibitors: 1 mM iodoacetimide, 1 mM benzithonium chloride, 5.7 mM phenylmethlsulphonyl fluoride, 2 mM EGTA in 1% sodium dodecyl sulphate (SDS) solution. The cells were sonicated on ice, centrifuged and the protein content of the supernatant was determined by the Bicinchoninic acid (BCA) method using BSA for standard curve [43]. Samples containing 100 μg of protein and Laemmli buffer were subjected to SDS-polyacrylamide gel electrophoresis (SDSPAGE) in a 4% polyacrylamide stacking gel at 20 mA current and then in 8% polyacrylamide separating gel at 40 mA until the proteins reached the bottom of the gel (Protogel, National Diagnostics, Hull, UK). The proteins were then transferred on to nitrocellulose membranes (Hybond ECL, Amersham Biosciences, Buckinghamshire, UK) by electroblotting with a 45 mA current for 90 min using the Multiphor II discontinuous blotting system (Pharmacia Biotech, Milton Keynes, UK). To block non-specific binding, the membranes were incubated overnight at 4 °C in 5% non-fat dry milk powder in phosphate buffered saline containing 0.05% Tween20 (PBS/Tween). After washes, the blots were probed with a monoclonal mouse (clone 2A7-A1) anti-SERCA2 ATPase antibody (1:500, SigmaAldrich). A monoclonal mouse (clone 5C5) anti-α-sacromeric actin antibody (1:500, Sigma-Aldrich) was used as a loading control. For both

Results were expressed as mean ± standard error (SEM). N denotes the number of animals, whereas n denotes the number of cells studied. Statistical analyses were undertaken using the paired Student's t test or when the normality test failed using the Wilcoxon matched-pairs sign rank test. For comparison between more than two means, one-way analysis of variance followed by multiple comparisons versus control group was undertaken using the Holm–Sidak method. For data that was not normally distributed, the Kruskal– Wallis test was used followed by Dunn's method for multiple comparisons. Statistical significance was considered when p b 0.05. All statistical analyses were carried out using SigmaStat version 3.0 (Systat Software, London, UK) and figures were prepared using Origin version 6.0 (Microcal Software Inc., Northampton, MA, USA). 3. Results 3.1. Effect of BNP-32 on ventricular myocyte shortening To investigate the effect of the bioactive fragment of BNP (BNP-32) on ventricular myocytes isolated from the rat heart, we first evaluated

Fig. 2. Dose- and time-dependent inhibition of ventricular myocyte shortening by BNP-32. (A) Dose-dependent inhibitory effect of BNP-32 on cell shortening shown as percentage of control. (B) Time course of the effect of BNP-32 (diamond −10− 7, triangle −5 × 10− 7, square − 10− 6 and circle − 5 × 10− 6 M). Changes in the magnitude of shortening was normalised to control shortening. The time course was fitted to an exponential function that yielded a concentration-dependent time constant (τ). (C) Reciprocal of τ plotted versus BNP-32 concentration. The line represents the least-squares fit of the data. (D) Dose–response curve for inhibition of ventricular myocyte shortening by BNP-32. The calculated KD was used as an estimate of the IC50 concentration of BNP-32 in the Hill equation to determine the maximal inhibition of shortening. Data are expressed as mean ± SEM. N = 2–3, n = 3–5.

R. Sodi et al. / Regulatory Peptides 151 (2008) 95–105 Table 1 Effect of BNP-32 on ventricular myocyte shortening [BNP-32] (M)

TPS (ms) BNP-32/control (%)

T1/2 (ms)

N

n

10− 9 10− 7 5.0 × 10− 7 10− 6 5.0 × 10− 6

98.7 ± 1.5 97.5 ± 1.9 95.9 ± 3.1 103.9 ± 2.3 96.8 ± 3.7

89.2 ± 3.9 95.5 ± 3.9 102.3 ± 2.6 93.2 ± 4.2 72.8 ± 1.1*

2 3 2 3 3

4 4 4 5 3

TPS — time to peak shortening, T1/2 — time to 50% decline of shortening, N — number of animals and n — number of cells studied. Percentage values are expressed as mean ± SEM and are expressed with respect to control recordings for each case (BNP-32/Control). *Denotes p b 0.05.

its effect on cell shortening. Our data demonstrate that BNP-32 decreased cell shortening in a dose-dependent manner between the concentrations 10− 9 to 5 × 10− 6 M (Figs. 1 and 2A). Fig. 2A shows the dose-dependent inhibitory effect of BNP-32 on ventricular myocyte shortening. At all concentrations tested there was a decrease in cell shortening compared to control, although statistical significance was only achieved at concentrations higher than 10− 7 M (p b 0.001).

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The mean amplitude of cell shortening, expressed as percentage of control, was 93.4 ± 4.2% at 10− 9 M, 75.6 ± 2.8% at 10− 6 M and 57.1 ± 8.4% at 5 × 10− 6 M. In addition, as summarised in Table 1 there was no significant difference observable in TPS or in T1/2 although at higher concentrations there was a tendency to decrease in the latter. The time-dependent inhibitory effect of BNP-32 is shown in Fig. 2B. Due to the variability of myocyte shortening, each recording was normalised to its respective control. As shown in Fig. 2B, the time course was fitted to an exponential function that yielded concentration-dependent time constants (τ). A plot of the reciprocal of τ versus BNP-32 concentration (Fig. 2C) gave an apparent association rate constant (k+ 1) of 0.074 ± 0.005 μM− 1 min− 1 and an apparent dissociation rate constant (k− 1) of 0.071 ± 0.003 μM− 1 min− 1 (n = 5). Thus, the estimated KD (k− 1 / k+ 1) was found to be 0.96 ± 0.06 μM with corresponding Hill coefficient (n) of 0.67 ± 0.13. The maximal inhibition of shortening by BNP-32 extrapolated using the IC50 value of 0.96 μM was calculated to be 51% and was achievable at approximately 5 × 10− 4 M (Fig. 2D). Based on these results, we chose a level close to the calculated KD concentration to undertake subsequent experiments as detailed below.

Fig. 3. Effect of BNP-32 on ventricular myocyte action potential. (A) Representative action potential recording in the absence (hashed line) and presence (solid line) of 10− 6 M BNP-32. Magnification of the phase II slope is shown in the inset. The tangents used to calculate the slopes are also shown. The slope was determined in the plateau phase II of the rat action potential as demarcated by the vertical lines. (B) Comparison of the phase II slopes in the absence and presence of BNP-32. Note that the negative sign implies a downward slope not the absolute value. (C) Comparison of time taken to reach half the AP amplitude (APD50) in the absence and presence of BNP-32. (D) Comparison of the resting membrane potential (VR) in the absence and presence of BNP-32. Mean ± SEM shown in all cases (N = 4, n = 5); ⁎Denotes p b 0.001.

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3.2. Effect of BNP-32 on ventricular myocyte action potential As shown in Fig. 3A, 10 − 6 M BNP-32 prolonged the AP (magnified in the inset) and significantly increased the phase II slope by 53.0% from − 6.6 ± 0.6 mV/ms to − 10.1 ± 0.6 mV/ms (negative indicates a downward slope, Fig. 3B, p b 0.001). The mean APD50 was increased by 16.9% from 5.9 ± 0.3 ms in control to 6.9 ± 0.4 ms in the presence of BNP-32 (Fig. 3C, p b 0.001). However, there was no change in resting membrane potential (VR) with a

mean of − 68.4 ± 1.4 mV for control recording and − 64.1 ± 1.9 mV in the presence of BNP-32 (Fig. 3D, p = 0.222). Based on these findings, we investigated the effects of BNP-32 on ICa,L next. 3.3. Effect of BNP-32 on L-type calcium current (ICa,L) We tested the effect of 5.0 × 10− 7 M BNP-32 on the ICa,L using the perforated patch clamp technique. The mean (±SEM) membrane capacitance of the cells used in the recordings was 199.1 ± 28.0 pF. A

Fig. 4. BNP-32 attenuates the L-type Ca2+current (ICa,L) in ventricular myocytes. (A) Representative time-base tracing with control solution (CS) and with 5.0 × 10− 6 M BNP-32. (B) Representative current traces and recording protocol. (C) Current–voltage relationship of ICa,L recorded under control (open squares) and in the presence of BNP-32 (filled squares). In all cases, currents were normalised to cell capacitance. (D) Time constant of inactivation of ICa,L. The decay phases of the current traces were fitted with mono-exponential functions. Open circle represents control recording; filled circle represents recording in the presence of BNP-32. Mean ± SEM are shown (N = 6, n = 10); ⁎Denotes p b 0.05.

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representative slow-time base tracing and current traces under control and in the presence of BNP-32 are shown in Fig. 4A and B respectively. We observed a decrease in the current amplitude upon the application of BNP-32 over all potentials tested although a statistically significant difference was only achieved between −30 and 0 mV (Fig. 4C). The peak amplitude at 0 mV was reduced by 22.9% (control = −5.16 ± 0.33; +BNP-32 = −3.98 ± 0.34 pA/pF; p = 0.023). The time course of the current decay was also significantly decreased, that is, increased time constants between 0 and 40 mV (Fig. 4D) suggesting reduced Ca2+-dependent inactivation in the presence of BNP-32. Thus, our findings show that BNP-32 attenuates both the amplitude and time course of ICa,L. 3.4. Effect of BNP-32 on activation and inactivation of ICa,L Fig. 5A shows example recordings of current generated with the conventional double-pulse protocol used to ascertain the

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voltage dependence of ICa,L activation and inactivation. We found that there was a significant shift in the activation curve to more positive potentials with corresponding V1/2 values of − 14.0 ± 0.64 mV for control recordings and − 6.85 ± 0.68 mV for recordings in the presence of 10− 6 M BNP-32 (51.1% increase, Fig. 5B and C, p b 0.001). The respective slopes of the curves in ECS and in the presence of BNP-32 were 5.25 ± 0.42 and 5.28 ± 0.36 and were not significantly different (p = 0.960). In addition, there was also a significant decrease in inactivation with V1/2 values of − 15.1 ± 0.74 mV for control recordings and − 19.9 ± 0.80 mV for recordings in the presence of BNP-32 (31.8% decrease, Fig. 5B and D, p b 0.001). The respective slope values were 3.14 ± 0.38 and 3.76 ± 0.47 and were not significantly different (p = 0.327). These data demonstrate that BNP-32 affects the voltage-dependent activation and inactivation of ICa,L. Overall, there was a decrease in the window current in the presence of BNP-32 (shaded region, Fig. 5B) compared to control (hatched region, Fig. 5B).

Fig. 5. Effect of BNP-32 on steady state activation and inactivation of ICa.L. (A) Representative recordings under control and with 10− 6 M BNP-32. (B) Activation (squares) and inactivation (circles) curves under control conditions (open symbols with hashed lines) and in the presence of BNP-32 (closed symbols with solid lines). The hatched and shaded regions show the window currents in control and with BNP-32 respectively. Data are means ± SEM fitted with a Boltzmann function. (C) Comparisons of V1/2 activation and, (D) V1/2 inactivation with and without BNP-32. Mean ± SEM shown (N = 4, n = 8); ⁎Denotes statistical significance.

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Fig. 6. Effect of BNP-32 on calcium transients. (A) Representative calcium transient tracings from the same cell under control, with 10− 6 M BNP-32 and washout with control solution. (B) Fluorescence ratio at 340/380 (R.U = ratio units). (C) Time to peak amplitude (TTP). (D) Time to 50% decline of calcium transients (T1/2). (E) Cumulative area under the Ca2+ transient over time under control, in the presence of 10− 6 M BNP-32 and after washout. The line shows the mean and the shading shows the SEM. Mean ± SEM shown in all cases (N = 3, n = 5); ⁎Denote p b 0.001 versus control.

3.5. Effect of BNP-32 on ventricular myocyte calcium transients

3.6. Effect of BNP-32 on SERCA2a protein expression

Fig. 6A shows traces of Ca2+ transients recorded from a fieldstimulated left ventricular myocyte that was superfused with ECS, then exposed to 10− 6 M BNP-32 and finally washed with ECS, respectively. Our data show that BNP-32 significantly decreased the amplitude of the calcium transients by 32.0%, which was fully reversible on washing (Fig. 6B, p b 0.001). TTP was significantly increased by 38.7% and was also fully reversible (Fig. 6C, p = 0.034). T1/2 increased significantly by 89.4% (Fig. 6D, p b 0.001) and was apparently irreversible. The cumulative area under the Ca 2+ transients showed that the intracellular calcium flux was reduced under BNP-32 exposure but increased upon washout to slightly more than control levels (Fig. 6E). These findings suggest that BNP32 alters the intracellular Ca2+ homeostatic mechanisms, which might involve some or all the calcium-regulatory proteins. We therefore investigated the effect of BNP-32 on the expression of the heart-specific isoform of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) protein.

As shown in Fig. 7, there was an apparent reduction in the relative expression of SERCA2a in three independent experiments (C, D and E) compared to control (A) even after correcting for loading using αactin.

Fig. 7. Effect of BNP-32 on SERCA2a protein expression. Representative Western immunoblot shown. (A) Control. (B) Negative control for SERCA2A. (C–E) 3 independent experiments where cells were incubated in 1 (μM BNP-32 for 6 h. α-actin was used as a loading control.

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4. Discussion 4.1. Findings of this study Here we show that BNP-32 inhibited cell shortening in a dosedependent manner, altered the AP, decreased the Ca2+ current through the ICa,L and decreased the window current. The time course of the ICa,L decay was also significantly decreased in the presence of BNP-32. BNP32 also modified the channel's activation and inactivation kinetics but the lack of differences in the slope implies that there is no difference in the response of the channel to voltage change despite changes in the threshold for activation and inactivation, which were at more positive and negative potentials respectively. Thus, it would appear that BNP-32 alters the sensitivity of the channel but not the response of the channel once activated or inactivated. We also showed that BNP-32 decreased the amplitude and area under the curve of Ca2+ transients and increased both the TTP and T1/2. However, on washout the area under the curve and T1/2 were both increased to above control levels. We found that the expression of SERCA2a was reduced after exposure to BNP-32. 4.2. Proposed mechanisms Based on our results, we propose that BNP-32 alters Ca2+ handling by the left ventricular myocytes. By binding to its receptors (NPRA), BNP-32 activates the particulate guanylyl cyclase system (pGC) causing the generation of cGMP [20,34], which it has been shown inhibits the ICa,L via phosphorylation [41]. The ICa,L serves 3 functions: first, the amplitude and time course of its decay determines the shape of the plateau of the action potential; second, it acts as a trigger to release Ca2+ from the sarcoplasmic reticulum (SR); and third, it plays a role as a Ca2+ loading mechanism. Thus, the decreased activation of the Ca2+ channel by BNP-32 may cause decreased influx of Ca2+, attenuating the Ca2+-induced-Ca2+ release (CICR) mechanism causing the decreased release of Ca2+ via the Ryanodine-2 receptors (RyR2) [5]. This would result in decreased intracellular Ca2+ available for myofilament contractility and would explain the decreased cell shortening and reduction in the amplitude of the Ca2+ transients as well as the delayed TTP in Ca2+ transients observed in this study (Figs. 1, 2 and 6) suggesting a negative inotropic effect of BNP-32 on left ventricular myocytes. We also found that the time course of the ICa,L decay was significantly decreased (Fig. 4D) suggesting reduced Ca2+-dependent inactivation in the presence of BNP-32. This is also consistent with the reduced Ca2+ transient observed on exposure to BNP-32 (Fig. 6A and B). Thus, our findings show that BNP-32 attenuates both the amplitude and time course of ICa,L. Furthermore, we have shown that on exposure to BNP-32 the T1/2 of the Ca2+ transient was increased (Fig. 6D) suggesting a possible lusitropic effect. This may be explained by the reduction and/or delay in the rate of Ca2+ re-sequestration by the SR via the SERCA2a or a decrease in the expression of the protein as shown in this study (Fig. 7). The finding that BNP-32 decreases the area under the Ca2+ transient is consistent with the decrease in the Ca2+-dependent inactivation of ICa,L. We also observed that on washout, there was an increase in Ca2+ flux, as assessed by the area under the Ca2+ transient, and in the T1/2. Further work is required to elucidate the basis for this reversibility. The attenuation of the ICa,L (Fig. 4) by BNP-32 would explain the modification of the phase II slope of the AP. The prolongation of the APD50 with the decrease of ICa,L may appear paradoxical; however, previous studies have shown that Ca2+-dependent inactivation of ICa,L is a major determinant of action potential duration [1,6]. As shown here, the reduction in the Ca2+ transient amplitude would result in reduced Ca2+-dependent inactivation of ICa,L and would therefore lead to more Ca2+ entry and hence a prolongation of the action potential duration. Alternatively, these changes may also be due to the involvement of K+ channels such as IK1 although this was beyond the scope of the present study.

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The decrease in the window current demonstrates the modulatory role of BNP-32 on the AP. The window current represents the amount of Ca2+ influx through the Ca2+ channels, which has been shown to be associated with the maintenance of the plateau phase of the AP and is linearly linked to the amplitude of the Ca2+ transient [5]. Therefore, the data in this study showing a decrease in the window current is consistent with the decrease in both the amplitude of the Ca2+ transient and ICa,L. Together with the decrease in the Ca2+-dependent inactivation of ICa,L these data suggest that BNP-32 modulates the duration and shape of the plateau phase (phase II) of the AP. Interestingly, previous studies have shown that the AP duration is prolonged in hypertrophy [22], a clinical scenario where BNP-32 is usually elevated [8,23,24,27,30]. It is important to note the difference in the time scale of the effects of BNP-32 on cell function and protein expression. In the former, all effects occur within minutes whereas in the latter, cells were incubated in BNP-32 for 6 h for changes on protein expression to be seen (Fig. 7). We did not see any changes in protein expression at shorter periods of incubation with BNP-32 (data not shown). Although only speculative, our results suggest that an increase in BNP concentration in heart tissues is likely to exert a dual effect on myocardial function. In the acute phase, it is acting on mechanisms regulating myocyte contractility. On chronic exposure, structural modifications, including changes in the expression of regulatory proteins such as SERCA2a becomes possible. Further studies are needed to answer this question fully. It appears then that BNP-32 has an autocrine effect on left ventricular myocytes by virtue of its action on the cell from which it is secreted. This might be a local constitutive mechanism for the cell to fine-tune the expression and release of various factors including BNP-32 itself in the face of increased ventricular stretch and volume overload. Moreover, it has been shown that BNP levels are increased in heart failure, ventricular hypertrophy, hypertension, valvular heart disease and in acute coronary syndromes [7]. Experimental studies have also shown that there is activation of the BNP gene in mainly the ventricles followed by increased synthesis and release into circulation in patients with heart failure [26,30]. Therefore, in pathological states whether this constitutive mechanism is altered remains an important area for further investigation. Our findings are consistent with a previous report, which showed that BNP (10− 9 to 10− 7 M) decreased myocyte contractility in a dosedependent manner and increased cGMP expression in ventricular myocytes isolated from the rabbit [46]. This report even showed that ANP and CNP had a similar negative inotropic effect. The inhibition of cGMP with KT5823 caused a reversal of this response, demonstrating that the effect was mediated via a cGMP-signalling pathway. Our results are also partly consistent with another recent study [16] that showed no significant effect in time to peak shortening (TPS) and time to 50% decline of shortening (T1/2) in ventricular myocytes but a significant effect in amplitude of shortening at 10− 8 M. In contrast, whereas we showed a significant effect in the amplitude, time to peak amplitude (TTP) and time to 50% decline of Ca2+ transients (T1/2) on exposure to BNP-32, this study [16] did not. This discrepancy may be due to the differences in experimental protocol and cell isolation procedures. Furthermore, this study [16] did not test the effect of a higher concentration of BNP (10− 6 M) on calcium transients as undertaken in the present study. It must be noted that the role of natriuretic peptides in the regulation of cardiac contractility remains controversial with reports of increases, decreases or no change in myocyte function [9]. For example, CNP has been shown to have a negative inotropic effect in the Sabra rat [32] but a positive inotropic effect in mice [45]. Therefore, there also appears to be a speciesspecific effect of the natriuretic peptides in regulating cardiomyocyte functionality. There is a paucity of data regarding the effect of BNP-32. 4.3. Clinical implications One of the pathophysiological hallmarks of heart failure is impaired Ca2+ homeostasis that results in contractile dysfunction, arrhythmogenesis [15] and the expression and release of BNP by the ventricular

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myocardium [26,30]. In addition, the reduced expression and function of SERCA is a major mechanism of defective Ca2+ cycling [29]. Recently, it was shown that BNP decreased myocardial SERCA2a expression and also abolished preload-dependent up-regulation of SERCA2a in muscle strips isolated from the rabbit heart [19]. A previous report has also shown an inverse relationship between SERCA mRNA and plasma BNP concentration in tissue samples from failing hearts in patients undergoing cardiac surgery [33]. Although it is generally considered that BNP expression is a beneficial compensatory mechanism via its vasodilating, natriuretic, diuretic and antihypertrophic effects, it might be possible that BNP-32 also plays a pivotal role in the pathogenesis of heart failure by virtue of its inhibitory effect on SERCA expression and function. In this respect, we indirectly showed that BNP-32 might have a negative effect on SERCA2a function as suggested by the increased T1/2 of the Ca2+ transients (Fig. 6D) and directly by the relative decrease in its expression (Fig. 7). The role of altered ICa,L in disease states remains unclear [15]. In this study we showed that the application of exogenous BNP-32 attenuated the ICa,L. Thus, it is possible that in heart failure where the level of circulating BNP-32 is markedly elevated there is a similar inhibition of the ICa,L and consequently changes in the AP. Future studies may wish to address this hypothesis. Intravenous BNP (nesiritide) is used in patients with acute decompensated heart failure due to its vasodilatory effects [39]. However, recent meta-analyses of clinical trials evaluating the effects of recombinant BNP in the treatment of acute decompensated heart failure have shown increased mortality in patients [36,37,42]. It is possible that in a subset of patients, the infusion of BNP-32 is causing harm rather than benefit by suppressing the expression and/or activity of SERCA2a [19,33] as well as other Ca2+ regulatory proteins and, altering the AP. Further work is required to clarify this idea. This study also reaffirms the notion of the heart not only as a muscular pump but also as an endocrine organ that secretes hormones such as BNP-32, which exerts autocrine effects on the heart itself and endocrine effects on target end-organs such as the kidney [9,28]. It has been noted that instead of the increased natriuretic effect expected with increased levels of BNP, patients with heart failure paradoxically display fluid and salt retention and may have disabling oedema [13]. This leads to 2 very important questions: is heart failure a state of BNP deficiency or BNP resistance and when does BNP contribute to the transition from beneficial hypertrophy to overt heart failure? And what is the role of BNP-32 on Ca 2+ signalling in this context? 4.4. Strengths and limitations of the present study The use of myocytes isolated from the left ventricles assured that the cells were homogenous and from a location where BNP is usually expressed and released in pathological states [7]. We had high yields of myocytes (N80%) that were rod shaped, clearly striated and that contracted strongly upon field stimulation. We also compared the effect of BNP-32 and control solutions on the same cell in a continuous before and after fashion, which eliminated cell-to-cell variability and therefore enabled the clear demonstration of relative effects. It could be argued that the concentrations of BNP-32 tested in this study were supra-physiological; however, our initial experiments on cell shortening were undertaken to determine an optimal range of concentrations to test. Moreover, the levels tested might indeed be supraphysiological in circulating plasma but similar to concentrations in local interstitia surrounding the ventricular myocyte. To the best of our knowledge, there is no information available on local concentrations of BNP released into the myocardial interstitium. 4.5. Conclusion In conclusion, we show for the first time the effects of the bioactive fragment of BNP (BNP-32) on the AP and ICa,L of ventricular myocytes.

We have demonstrated that BNP-32 has a negative inotropic effect, attenuates the ICa,L, alters the AP and reduces Ca2+-dependent inactivation. BNP-32 also alters the handling of intracellular calcium and decreases the expression of SERCA2a. This study reveals a novel constitutive mechanism for the autocrine action of BNP-32 on the Ltype calcium channel in ventricular myocytes.

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