Nitric Oxide modulates spontaneous Ca ²⁺ release and ventricular arrhythmias during β-adrenergic signalling through S -nitrosylation of Calcium/Calmodulin dependent kinase II

Figures

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  Figure 1.. Cardiomyocyte Ca2+ transients and sparks with nitric oxide donor GSNO. (A) NO liberated from increasing GSNO concentrations in buffer was measured using a NO electrode. (B) The kinetics of NO release following the addition of 150 μM GSNO to buffer. (C) Western blot of ventricle tissue samples from WT and CaMKIIδ KO mice (n=3 hearts) showing loss of CaMKIIδ protein expression in CaMKIIδ-KO vs. WT hearts (with GAPDH loading controls). (D) WT or CaMKIIδ-KO cardiomyocytes were treated with 150 μM GSNO and paced at 0.5 Hz. There was no change in Ca2+ transient amplitude (E) or decay kinetics (F). In quiescent cardiomyocytes, Ca2+ sparks were measured from line-scan images (G) and there was also no effect of GSNO on Ca2+ spark frequency in either WT or CaMKIIδ-KO cardiomyocytes (H), (WT n=8 cells, N=3 hearts; CaMKIIδ-KO n=8 cells, N=3 hearts).

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  Figure 2.. Cardiomyocyte Ca2+ transient and spark properties in response to β-adrenergic agonist isoproterenol. (A) Experimental protocol used for Ca2+ imaging, the grey bars represent when line-scan images were acquired. Representative line-scans during pacing (B) and resultant Ca2+ transients (C) from a WT and CaMKIIδ-KO cardiomyocyte stimulated at 0.5 Hz under control conditions and with 100 nM ISO. ISO increased Ca2+ transient amplitude (D) and accelerated decay (E) in both WT and CaMKIIδ-KO cardiomyocytes to a similar degree (WT n=8 cells, N=2 hearts; CaMKIIδ-KO n=13 cells, N=4 hearts). (F) Representative line-scans from a WT and CaMKIIδ-KO cardiomyocyte show an increase in the number of Ca2+ sparks following exposure to ISO. Mean data show the ISO-induced increase in Ca2+ spark frequency (CaSpF) was attenuated in the CaMKIIδ-KO cardiomyocytes (G) (WT n=11 cells, N=2 hearts; CaMKIIδ-KO n=13 cells, N=4 heats).

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  Figure 3.. Characterization of the CaMKIIδ-C273S knock-in mouse model. Schematic of the CaMKIIδ monomer with the residue positions of various post-translational modifications within the regulatory domain including S-nitrosylation (NO-), phosphorylation (P), oxidation (O) and O-GlcNAc modification which are known to alter CaMKII activity (A). A mouse model was generated using CRISPR/cas9 causing a single point mutation in the cystine codon at position 273, resulting in replacement with a serine residue which cannot be S-nitrosylated (B). Example of a CaMKIIδ-C273S offspring confirmed with genotyping of ear notch (C). Protein expression in ventricular tissue (N = 5 hearts) was measured using western blots (D) for CaMKIIδ (E), SERCA (F) RyR (H), and normalized to actin or protein load. Ventricular fibrosis was measured in fixed and stained tissue (G) by quantifying collagen content (I). Representative cardiomyocytes are shown in J for measurement of cell length (K) and width (L), WT: n = 52, N = 7 hearts; CaMKIIδ-C273S: n=50, N=8 hearts.

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  Figure 4.. Pre-treatment with GSNO enhances Ca2+ transient response to isoproterenol in CaMKIIδ-C273S cardiomyocytes. Ca2+ transients from WT and CaMKIIδ C273S cardiomyocytes from were recorded at 0.5 Hz under control conditions and with 100 nM ISO or with pre-treatment of 150 μM GSNO prior to ISO exposure. Mean Ca2+ transient amplitude (A) and decay (B) data in response to ISO with control KRH buffer (white bars; WT: n=28 cells, N=14 hearts; CaMKIIδ-C273S: n=24 cells, N=14 hearts) or when pre-treated with GSNO (grey bars; WT: n=18 cells, N=7 hearts; CaMKIIδ-C273S: n=24, N=9 hearts). Representative line-scans from quiescent WT (C) and CaMKIIδ-C273S (D) cardiomyocytes under control conditions and with ISO or with pre-treatment of GSNO prior to ISO exposure. Mean Ca2+ spark frequency (E) data are shown for cardiomyocytes in response to ISO with control buffer (white bars; WT: n=31 cells, N=14 hearts; CaMKIIδ-C273S: n=24 cells, N=9 hearts) or when pre-treated with GSNO (grey bars; WT: n=16 cells, N=7 hearts; CaMKIIδ-C273S: n=25 cells, N=9 hearts). Percentage of cardiomyocytes exhibiting Ca2+ waves in quiescent WT and CaMKIIδ-C273S cardiomyocytes (F). ϕ p<0.05 compared to WT ISO, ap = 0.0002 vs…

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  Figure 5.. Pre-treatment with clinical NO donor SNP and sGC inhibitor elicits similar protection against isoproterenol induced Ca2+ sparks. Ca2+ transients (A) and sparks (B) from WT and CaMKIIδ-C273S cardiomyocyte were recorded at the end of a 7-minute incubation with SNP (200 μM) followed by wash-in of 100 nM ISO. Mean Ca2+ transient amplitude and spark frequency data are shown in C and D respectively (WT: n=10 cells, N=3 hearts; CaMKIIδ-C273S: n=23 cells, N=6 hearts). The same experiments were performed following a pre-treatment with sGC inhibitor ODQ (10 μM; 20 min incubation) with representative line-scans (E-F) and mean data (G-H) plotted for WT (n=13 cells, N=3 hearts) and CaMKIIδ-C273S (n=14 cells, N=4 hearts) cardiomyocytes.

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  Figure 6.. Effects of GSNO after ISO on Ca2+ transients and sparks in WT or CaMKIIδ-C273S and -C290A cardiomyocytes. WT, CaMKIIδ-C273S and -C290A myocytes were stimulated at 0.5 Hz and exposed to 100 nM ISO, then following ISO exposure myocytes were exposed to 150 μM GSNO. Experimental protocol used for Ca2+ imaging, the grey bars represent when line-scan images were acquired (A). Mean Ca2+ transient amplitude (B) and decay (C) data are shown for cardiomyocytes in response to ISO and wash-out with GSNO (WT: n=20 cells, N=9 hearts; CaMKIIδ C273S: n=7 cells, N=5 hearts; CaMKIIδ-C290A: n=13 cells, N=5 hearts). Representative line-scans from quiescent WT, CaMKIIδ-C273S and -C290A cardiomyocytes under indicated conditions (D). Mean Ca2+ spark frequency data (E) are shown for cardiomyocytes in response to ISO with washout with GSNO (WT: n=18 cells, N=9 hearts; CaMKIIδ C273S: n=8 cells, N=5 hearts; CaMKIIδ-C290A: n=17 cells, N=5 hearts). All data are normalized to baseline control before addition of ISO.

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  Figure 7.. Electrocardiogram characteristics for WT and CaMKIIδ-C273S mice. Representative electrocardiograms from a WT (A) and CaMKIIδ-C273S (B) mouse under anaesthetic and mean data for waveform amplitudes and durations (C-K). N = 13-17 per group. ECG traces for some of the CaMKIIδ-C273S mice showed spontaneous arrhythmic events (L). These events led to variability in total heart rate (M) and acute periods of bradycardia (N).

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  Figure 8.. Whole mouse hearts exposed to ISO and GSNO have increased S-nitrosylation of cardiac proteins. (A) WT mouse hearts undergoing Langendorff-perfusion were treated with ISO and GSNO in varying order, snap-frozen and lysed, followed by a modified biotin switch assay to measure total protein S-nitrosylation. (B) Total cardiac protein S-nitrosylation in each lysate. (C) Treatment with ISO followed by GSNO (red bar) resulted in a significant increase in total cardiac protein S-nitrosylation compared to control (white bar, n=3-4 hearts per group).

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  Figure 9.. Pre-treatment with GSNO prevents ISO-induced arrhythmias in Langendorff-perfused hearts from WT, but not CaMKIIδ-C273S, mice. Arrhythmias were measured in Langendorff-perfused hearts following protocols outlined in Figure 7A. (A) Examples of arrhythmic events observed in the isolated hearts. We measured (B) quantity of arrhythmic events and (C) arrhythmia score during ISO perfusion only, (D) quantity of arrhythmic events and (E) arrhythmia score during ISO pre-treatment before GSNO, and (F) quantity of arrhythmic events and (G) arrhythmia score during GSNO pre-treatment before ISO. White bar = buffer, red bar = ISO, blue bar = GSNO. n = 5-6 hearts per group.