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. 2022 Mar;15(3):e010636.
doi: 10.1161/CIRCEP.121.010636. Epub 2022 Feb 25.

Natriuretic Peptide Oligomers Cause Proarrhythmic Metabolic and Electrophysiological Effects in Atrial Myocytes

Zhenjiang Yang #  1 , Tuerdi Subati #  1 , Kyungsoo Kim  1 , Matthew B Murphy  1 , Owen P Dougherty  1 , Isis L Christopher  1 , Joseph C Van Amburg  1 , Kaylen K Woodall  1 , Joey V Barnett  1 , Katherine T Murray  1
Affiliations

Natriuretic Peptide Oligomers Cause Proarrhythmic Metabolic and Electrophysiological Effects in Atrial Myocytes

Zhenjiang Yang et al. Circ Arrhythm Electrophysiol. 2022 Mar.

Abstract

Background: With aging, the human atrium invariably develops amyloid composed of ANP (atrial natriuretic peptide) and BNP (B-type natriuretic peptide). Preamyloid oligomers are the primary cytotoxic species in amyloidosis, and they accumulate in the atrium during human hypertension and a murine hypertensive model of atrial fibrillation susceptibility. We tested the hypothesis that preamyloid oligomers derived from natriuretic peptides cause cytotoxic and electrophysiological effects in atrial cells that promote arrhythmia susceptibility and that oligomer formation is enhanced for a mutant form of ANP linked to familial atrial fibrillation.

Methods: Oligomerization was assessed by Western blot analysis. Bioenergic profiling was performed using the Seahorse platform. Mitochondrial dynamics were investigated with immunostaining and gene expression quantitated using quantitative reverse transcription polymerase chain reaction. Action potentials and ionic currents were recorded using patch-clamp methods and intracellular calcium measured using Fura-2.

Results: Oligomer formation was markedly accelerated for mutant ANP (mutANP) compared with WT (wild type) ANP. Oligomers derived from ANP, BNP, and mutANP suppressed mitochondrial function in atrial HL-1 cardiomyocytes, associated with increased superoxide generation and reduced biogenesis, while monomers had no effects. In hypertensive mice, atrial cardiomyocytes displayed reduced action potential duration and maximal dV/dT of phase 0, with an elevated resting membrane potential, compared with normotensive mice. Similar changes were observed when atrial cells were exposed to oligomers. mutANP monomers produced similar electrophysiological effects as mutANP oligomers, likely due to accelerated oligomer formation, while ANP and BNP monomers did not. Oligomers decreased Na+ current, inward rectifier K+ current, and L-type Ca++ current, while increasing sustained and transient outward K+ currents, to account for these effects.

Conclusions: These findings provide compelling evidence that natriuretic peptide oligomers are novel mediators of atrial arrhythmia susceptibility. Moreover, the accelerated oligomerization by mutANP supports a role for these mediators in the pathophysiology of this mutation in atrial fibrillation.

Keywords: atrial fibrillation; atrial natriuretic factor; calcium; electrophysiology; natriuretic peptide, brain.

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Figures

Figure 1.
Figure 1.. Oligomerization of mutANP is markedly accelerated to generate cytotoxic oligomers that promote mitochondrial dysfunction in atrial cardiomyocytes.
A. ANP and mutANP peptides (10μM) were oligomerized at RT for indicated times followed by Western blot analysis. In parallel, peptides were co-incubated with 15-E2-IsoLG to promote oligomerization as positive controls. B. Cellular ATP levels were measured following incubation with mutANP monomer or oligomers (30μM monomers incubated at RT for 24hr and diluted to a concentration equivalent to 0.45μM monomers, or 0.45μM(m)). N=4 independent experiments; *P<0.05 vs. control, one-way ANOVA followed by Dunnet’s post hoc test. C. Seahorse bioenergetic profiling is illustrated for the mitochondrial stress test using atrial HL-1 cells cultured in the absence (control or CTL) or presence of mutANP oligomers (diluted to 0.45, 1.5, or 4.5μM(m)) for 24hr. Oxygen consumption rate (OCR) was analyzed following sequential injection of respiratory chain inhibitors at indicated time points. D. Bioenergetic parameters were measured following incubation without (CTL) or with mutANP oligomers (OCR values corrected for the number of Hoechst positive nuclei). N=6-7 independent experiments; *P<0.05 and †P<0.01 vs. control, one-way ANOVA with Dunnett’s post-hoc test.
Figure 2.
Figure 2.. Oligomers derived from BNP and ANP also disrupt mitochondrial function.
Bioenergetic profiling results are displayed for atrial cells cultured in the presence or absence of ANP (panels A and B) or BNP (panels C and D) oligomers for 24hr, using the same format as in Figure 1C and 1D. N=6-7 (panel B) or 3-5 (panel D) independent experiments; *P<0.05, †P<0.01, and ‡P<0.001 vs. control, one-way ANOVA with Dunnett’s post-hoc test.
Figure 3.
Figure 3.. Changes in mitochondrial dynamics, ROS generation and mitochondrial biogenesis following treatment with natriuretic peptide monomers or oligomers.
Treatment of atrial HL-1 cardiomyocytes with mutANP (B) and BNP oligomers (C) but not ANP oligomers (A) caused a substantial reduction in TOMM20 immunostaining (boxed region in upper panel [scale bar, 20μm] shown in inset in lower panel [scale bar, 5μm]). D. Exposure of cardiomyocytes to mutANP and BNP oligomers caused intracellular O2•− generation (scale bar, 50μm). E-G. mRNA levels are illustrated for genes encoding PGC-1α and NRF1 (normalized for control [CTL] values) following atrial cell treatment with monomers or oligomers prepared from ANP (E), mutANP (F) and BNP (D). N=4-6 biological replicates for each treatment (3 technical replicates each); *P<0.05 vs. control, Mann-Whitney test.
Figure 4.
Figure 4.. Oligomers, and mutANP monomers, cause proarrhythmic electrophysiologic effects, while monomers do not.
A. Mouse atrial action potentials are shown for a control cell and following acute bath exposure to monomers or oligomers (oligo) of WT and mutANP. Dashed line represents resting membrane potential (RMP) for the control cell. B. Experimental data are displayed for RMP, action potential duration at 90% repolarization (APD90), and maximal dV/dT of Phase 0 (Vmax) for control (CTL) and post-peptide exposure (500nM; ANP monomers and oligomers n=9, 9; mutANP monomers and oligomers n=8, 9). *P<0.05 and †P<0.01 vs. control), Paired Samples Wilcoxon signed rank test or Mann-Whitney test. C. and D. Similar data are shown following exposure to BNP monomers or oligomers (500nM) using the same format as panels A and B (BNP monomers and oligomers n=12, 10). *P<0.05 and †P<0.01 vs. control, Paired Samples Wilcoxon signed rank test.
Figure 5.
Figure 5.. Transient outward (ITo) and rapidly activating, sustained (Isus) currents are increased by mutANP oligomers.
A. K+ currents elicited are displayed under control conditions and following exposure to mutANP oligomers (500nM; voltage clamp protocol shown in inset). B. Summary data are illustrated for the current-voltage relationship on the left and conductance-voltage plot on the right (n=16; P values shown on the Figure). C. Similar data are displayed for Isus currents, with current-voltage curves in panel D for steady-state (left) and tail (right) currents (n=8).
Figure 6.
Figure 6.. Oligomers derived from mutANP suppress L-type Ca++ and Na+ currents, with rightward shifts in the voltage dependence of channel activation.
Using a format similar to Figure 5, data are illustrated for the effects of mutANP oligomers on ICa,L (A and B) and INa (C and D).
Figure 7.
Figure 7.. MutANP oligomers reduce inward rectifier currents, with no effect on intracellular Ca++ concentration or SR content.
A. Data are shown for the effects of mutANP oligomers on IK1. B. In atrial HL-1 cardiomyocytes, extracellular exposure to mutANP oligomers (1μM) did not alter baseline Ca++ concentration, SR content, or the Ca++ transient decay rate (tau) after caffeine administration, when compared to control cells (n=13, 7 cells, respectively).
Figure 8.
Figure 8.. Mechanism(s) of increased AF susceptibility mediated by natriuretic peptide oligomers.
Hypertension-related oxidative stress and mutANP accelerate PAO formation, leading to proarrhythmic electrical remodeling and mitochondrial dysfunction. Modulation of ion channel function underlies action potential changes, while reduced mitochondrial biogenesis/ATP production and increased ROS further promote the AF substrate. Created with BioRender.com.
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