Nitric Oxide Modulates Ca2+ Leak and Arrhythmias via S-Nitrosylation of CaMKII

Abstract

Background: Nitric oxide (NO) has been identified as a signaling molecule generated during β-adrenergic receptor stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca²⁺ release via S-nitrosylation of CaMKIIδ (Ca²⁺/calmodulin kinase II delta) is emerging. NO donors are routinely used clinically for their cardioprotective effects on the heart, but it is unknown how NO donors modulate the proarrhythmic CaMKII to alter cardiac arrhythmia incidence. We test the role of S-nitrosylation of CaMKIIδ at the Cysteine-273 inhibitory site and cysteine-290 activating site in cardiac Ca²⁺ handling and arrhythmogenesis before and during β-adrenergic receptor stimulation.

Methods: We measured Ca²⁺-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at cysteine-273 or cysteine-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 μM), sodium nitroprusside (200 μM), and β-adrenergic agonist isoproterenol (100 nmol/L).

Results: Both WT and CaMKIIδ-KO cardiomyocytes responded to isoproterenol with a full inotropic and lusitropic Ca²⁺ transient response as well as increased Ca²⁺ spark frequency. However, the increase in Ca²⁺ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from isoproterenol-induced Ca²⁺ sparks and waves was mimicked by GSNO pretreatment in WT cardiomyocytes but lost in CaMKIIδ-C273S cardiomyocytes. When GSNO was applied after isoproterenol, this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pretreatment limited isoproterenol-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after isoproterenol sustained or exacerbated arrhythmic events.

Conclusions: We conclude that prior S-nitrosylation of CaMKIIδ at cysteine-273 can limit subsequent β-adrenergic receptor-induced arrhythmias, but that S-nitrosylation at cysteine-290 might worsen or sustain β-adrenergic receptor-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

Keywords: calcium; heart; nitric oxide.

Figure 1.

Cardiomyocyte Ca²⁺ transients and sparks with nitric oxide donor S-nitrosoglutathione (GSNO). A, Nitric oxide (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 wild-type (WT) and CaMKIIδ (Ca²⁺/calmodulin kinase II delta) 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).

Figure 2.

Cardiomyocyte Ca²⁺ transient and spark properties in response to β-adrenergic agonist isoproterenol. A, Experimental protocol used for Ca²⁺ imaging, the gray bars represent when line-scan images were acquired. Representative line-scans during pacing (B) and resultant Ca²⁺ transients (C) from a WT and CaMKIIδ (Ca²⁺/calmodulin kinase II delta)-KO cardiomyocyte stimulated at 0.5 Hz under control conditions and with 100 nmol/L 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).

Figure 3.

Characterization of the CaMKIIδ (Ca²⁺/calmodulin kinase II delta)-C273S knock-in mouse model. Schematic of the CaMKIIδ monomer with the residue positions of various posttranslational 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 (SR Ca²⁺ ATPase; F), RyR (ryanodine receptor type; 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), wild type (WT): n=52, N=7 hearts; CaMKIIδ-C273S: n=50, N=8 hearts.

Figure 4.

Pretreatment with S-nitrosoglutathione (GSNO) enhances Ca²⁺ transient response to isoproterenol in CaMKIIδ (Ca²⁺/calmodulin kinase II delta)-C273S cardiomyocytes. Ca²⁺ transients from wild-type (WT) and CaMKIIδ C273S cardiomyocytes from were recorded at 0.5 Hz under control conditions and with 100 nmol/L ISO or with pretreatment of 150 μM GSNO before 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 pretreated with GSNO (gray 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 pretreatment of GSNO before 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 pretreated with GSNO (gray 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). ɸP<0.05 compared with WT ISO, ^aP=0.0002 vs CaMKIIδ-C273S ISO, ^bP<0.0001 vs WT ISO with GSNO pretreatment.

Figure 5.

Pretreatment with clinical NO donor sodium nitroprusside (SNP) and sGC (soluble guanylyl cyclase) inhibitor elicits similar protection against isoproterenol-induced Ca²⁺ sparks. Ca²⁺ transients (A) and sparks (B) from wild-type (WT) and CaMKIIδ (Ca²⁺/calmodulin kinase II delta)-C273S cardiomyocyte were recorded at the end of a 7-minute incubation with SNP (200 μM) followed by wash-in of 100 nmol/L 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 pretreatment with sGC inhibitor ODQ (10 μM; 20 minutes incubation) with representative line-scans (E and F) and mean data (G and H) plotted for WT (n=13 cells, N=3 hearts) and CaMKIIδ-C273S (n=14 cells, N=4 hearts) cardiomyocytes. ISO indicates isoproterenol.

Figure 6.

Effects of S-nitrosoglutathione (GSNO) after ISO on Ca²⁺ transients and sparks in wild-type (WT) or CaMKIIδ (Ca²⁺/calmodulin kinase II delta)-C273S and -C290A cardiomyocytes. WT, CaMKIIδ-C273S, and -C290A myocytes were stimulated at 0.5 Hz and exposed to 100 nmol/L ISO, then following ISO exposure myocytes were exposed to 150-μM GSNO. Experimental protocol used for Ca²⁺ imaging, the gray 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 washout 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. ISO indicates isoproterenol.

Figure 7.

ECG characteristics for wild-type (WT) and CaMKIIδ (Ca²⁺/calmodulin kinase II delta)-C273S mice. Representative electrocardiograms from a WT (A) and CaMKIIδ-C273S (B) mouse under anesthetic and mean data for waveform amplitudes and durations (C through K). N=13 to 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 (HR) (M) and acute periods of bradycardia (N).

Figure 8.

Pretreatment with S-nitrosoglutathione (GSNO) prevents ISO-induced arrhythmias in Langendorff-perfused hearts from wild-type (WT), but not CaMKIIδ (Ca²⁺/calmodulin kinase II delta)-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 pretreatment before GSNO, and (F) quantity of arrhythmic events and (G) arrhythmia score during GSNO pretreatment before ISO. White bar=buffer, red bar=ISO, blue bar=GSNO. n=5 to 6 hearts per group. ISO indicates isoproterenol.