Detrimental proarrhythmogenic interaction of Ca2+/calmodulin-dependent protein kinase II and NaV1.8 in heart failure

Abstract

An interplay between Ca²⁺/calmodulin-dependent protein kinase IIδc (CaMKIIδc) and late Na⁺ current (I_NaL) is known to induce arrhythmias in the failing heart. Here, we elucidate the role of the sodium channel isoform Na_V1.8 for CaMKIIδc-dependent proarrhythmia. In a CRISPR-Cas9-generated human iPSC-cardiomyocyte homozygous knock-out of Na_V1.8, we demonstrate that Na_V1.8 contributes to I_NaL formation. In addition, we reveal a direct interaction between Na_V1.8 and CaMKIIδc in cardiomyocytes isolated from patients with heart failure (HF). Using specific blockers of Na_V1.8 and CaMKIIδc, we show that Na_V1.8-driven I_NaL is CaMKIIδc-dependent and that Na_V1.8-inhibtion reduces diastolic SR-Ca²⁺ leak in human failing cardiomyocytes. Moreover, increased mortality of CaMKIIδc-overexpressing HF mice is reduced when a Na_V1.8 knock-out is introduced. Cellular and in vivo experiments reveal reduced ventricular arrhythmias without changes in HF progression. Our work therefore identifies a proarrhythmic CaMKIIδc downstream target which may constitute a prognostic and antiarrhythmic strategy.

Fig. 1. CaMKIIδc interacts with Na_V1.8 in human myocardium and isolated cardiomyocytes.

a Co-immunoprecipitation of CaMKIIδc and Na_V1.8 from left ventricular homogenates of human non-failing and failing hearts (NF: n = 7; HF: n = 7). b Co-localization of CaMKIIδc and Na_V1.8 in human failing cardiomyocytes with immunofluorescence staining. Scale bar: 10 µm (staining was performed in cardiomyocytes isolated from five heart failure patients).

Fig. 2. Reduced I_NaL upon Na_V1.8 inhibition in human failing and mouse CaMKIIδc transgenic cardiomyocytes, and in SCN10A knockout iPSC-cardiomyocytes.

a Original traces of I_NaL in WT and CaMKIIδc^+/T mouse ventricular cardiomyocytes elicited using the protocol shown in the inset. b Mean data ± SEM along with individual values shown in the graph plotting WT (n = 10 cells/5 mice) and CaMKIIδc^+/T (untreated: n = 15 cells/7 mice; A-806467: n = 12 cells/7 mice; PF-01247324: n = 12 cells/7 mice). Probability vs. untreated (One-way ANOVA with post hoc Bonferroni’s correction). c Original traces of I_NaL from human failing ventricular cardiomyocytes elicited using the protocol shown in the inset. d Mean data ± SEM along with individual values shown in the graph plotting (untreated: n = 14 cells/8 patients; AIP: n = 8 cells/5 patients; PF-01247324: n = 10 cells/6 patients; AIP + PF-01247324 = 9 cells/5 patients). Probability vs. untreated (One-way ANOVA with post hoc Bonferroni’s correction). e Original traces of I_NaL from human ventricular SCN10A knockout iPSC-cardiomyocytes elicited using the protocol shown in the inset. f Mean data ± SEM along with individual values shown in the graph plotting (control + Iso: n = 19 cells/3 cardiac differentiations; control + Iso + PF: n = 4 cells/3 differentiations; SCN10A knockout (KO) + Iso: n = 11 cells/3 differentiations; KO + Iso + PF-01247324 = 12 cells/3 differentiations). Probability vs. control + Iso (One-way ANOVA with post hoc Bonferroni’s correction).

Fig. 3. Effects Na_V1.8 inhibition on intracellular Ca²⁺ handling.

a Representative line scan images of CaMKIIδc^+/T ventricular cardiomyocytes. b CaSpF data shown as mean ± SEM for wildtype (WT) (untreated: n = 58 cells/4 mice; PF-01247324: n = 41 cells/4 mice; A-806467: n = 41 cells/4 mice) and CaMKIIδc^+/T (untreated: n = 122 cells/8 mice; PF-01247324: n = 105 cells/7 mice; A-806467: n = 101 cells/8 mice). Data were analyzed by one-way ANOVA with post hoc Bonferroni’s correction. c Representative line scan images of human failing ventricular cardiomyocytes. d Data shown as mean ± SEM (untreated: n = 123 cells/14 patients; Autocamtide-2-related inhibitory peptide (AIP): n = 105 cells/15 patients; PF-01247324: n = 59 cells/10 patients; AIP + PF-01247324 = 89 cells/13 patients). Data were analyzed by one-way ANOVA with post hoc Bonferroni’s correction, Probability vs. untreated. e Representative Ca²⁺ transients stimulated at 1 Hz and caffeine-induced Ca²⁺ transients in ventricular cardiomyocytes from CaMKIIδc^+/T under untreated conditions. f Mean data ± SEM show no effect of PF-01347324 treatment on Ca²⁺ transient amplitude at 1.0, 2.0, and 4.0 Hz stimulation (n = 13 cells/5 mice) compared to untreated (n = 10 cells/5 mice). g Ca²⁺-transient decay (90% of Ca²⁺-removal) RT90 was unchanged in PF-01347324-treated cells (n = 11 cells/5 mice) compared to untreated (n = 8 cells/5 mice). Data were presented as mean values ± SEM. h Representative Ca²⁺ transients stimulated at 1 Hz and caffeine-induced Ca²⁺ transients in ventricular cardiomyocytes from CaMKIIδc^+/T treated with PF-01247324. i Diastolic Ca²⁺ after addition of PF-01347324 (n = 13 cells/5 mice) compared to untreated cells (n = 10 cells/5 mice) at different stimulation frequencies was unchanged (one-way ANOVA with post hoc Bonferroni’s correction, Fig. 3f, g, i). Data were presented as mean values ± SEM. j Mean and individual values ± SEM of caffeine-induced Ca²⁺ transients (untreated: n = 7 cells/4 mice, PF-01247324: n = 11 cells/5 mice) did not differ between the groups (Student’s t-test). k Ca²⁺-reuptake into the SR was not affected by inhibition of Na_V1.8 (untreated: n = 7 cells/4 mice, PF-01247324: n = 11 cells/5 mice), analyzed by Student’s t-test. Data were presented as mean values ± SEM.

Fig. 4. Knockout of the Scn10a (Na_V1.8) gene in CaMKIIδc^+/T mice improves survival.

a Survival curve of CaMKIIδc^+/T and SCN10A^−/−/CaMKIIδc^+/T (43 vs. 64 animals, median survival 72 vs. 98 days, blinded analysis). Log-rank (Mantel–Cox test and Gehan–Breslow–Wilcoxon test (two-tailed analysis) were performed to calculate the survival percentage of mice. Probability vs CaMKIIδc^+/T. b Hearts from WT, SCN10A^−/−, CaMKIIδc^+/T, and SCN10A^−/−/CaMKIIδc^+/T mice. c Ratio of heart weight to tibia length as a parameter of cardiac hypertrophy. CaMKIIδc^+/T and SCN10A^−/−/CaMKIIδc^+/T showed a significant increase in this ratio compared to WT and SCN10A^−/− mice. Data were analyzed by one-way ANOVA with post hoc Bonferroni’s correction. (N = hearts studied, WT = 14, SCN10A^−/− = 16, CaMKIIδc^+/T = 13, and SCN10A^−/−/CaMKIIδc^+/T = 23). Data were presented as mean values ± SEM. d Original histological wheat germ agglutinin staining from WT, SCN10A^−/−, CaMKIIδc^+/T, and SCN10A^−/−/CaMKIIδc^+/T mice. Scale bars = 75 µm. Stainings were produced from different sections and three different regions (basal, mid-ventricular, and apical) of each heart studied. e Cardiomyocyte cross-sectional-area (CSA) as a parameter for cellular hypertrophy. CaMKIIδc^+/T and SCN10A^−/−/CaMKIIδc^+/T showed a significant increase in CSA compared to WT and SCN10A^−/− mice. CSA in CaMKIIδc^+/T and SCN10A^−/−/CaMKIIδc^+/T mice did not significantly differ. Data were analyzed by one-way ANOVA with post hoc Bonferroni’s correction. N = hearts studied (>300 cardiomyocytes were studied per heart, from different sections and different regions (basal, mid-ventricular, apical), WT = 5 hearts, SCN10A^−/− = 5 hearts, CaMKIIδc^+/T = 4 hearts, SCN10A^−/−/CaMKIIδc^+/T = 5 hearts. Data were presented as mean values ± SEM. f Original echocardiography recordings from WT, SCN10A^−/−, CaMKIIδc^+/T, and SCN10A^−/−/CaMKIIδc^+/T at M-mode in 12–13- week-old mice. g Echocardiography recordings revealed a decrease in left ventricular ejection fraction (EF) in CaMKIIδc^+/T (six mice) and SCN10A^−/−/CaMKIIδc^+/T (six mice) compared to WT (seven mice) or SCN10A^−/− (eight mice) (p < 0.0001(one-way ANOVA with post hoc Bonferroni’s correction). Data were presented as mean values ± SEM. h Echocardiography recordings revealed a significant increase in left ventricular end-diastolic diameter (LVEDD) in CaMKIIδc^+/T (six mice) and SCN10A^−/−/CaMKIIδc^+/T (six mice) compared to WT (seven mice) or SCN10A^−/− (eight mice) (p < 0.0001). LVEDD was not significantly different in WT vs SCN10A^−/− or CaMKIIδc^+/T vs SCN10A^−/−/CaMKIIδc^+/T (one-way ANOVA with post hoc Bonferroni’s correction). Data were presented as mean values ± SEM.

Fig. 5. Knockout of Scn10a (Na_V1.8) in CaMKIIδc^+/T mice (SCN10A^−/−/CaMKIIδc^+/T) significantly reduces I_NaL and proarrhythmic triggers.

a Original traces of I_NaL in WT, SCN10A^−/−, CaMKIIδc^+/T, and SCN10A^−/−/CaMKIIδc^+/T mouse ventricular cardiomyocytes elicited using the protocol shown in the inset. b Mean data ± SEM along with individual values shown in the graph plotting (WT: n = 7 cells/4 mice, SCN10A^−/− n = 10 cells/5 mice, CaMKIIδc^+/T: n = 9 cells/5 mice; SCN10A^−/−/CaMKIIδc^+/T: n = 4 cells/4 mice), there was a significantly reduced I_NaL in SCN10A^−/−/CaMKIIδc^+/T cardiomyocytes compared to CaMKIIδc^+/T. Data were analyzed by one-way ANOVA with post hoc Bonferroni’s correction. c Original traces of action potentials showing triggered action potentials originating from delayed afterdepolarizations (DADs) in CaMKIIδc^+/T and SCN10A^−/−/CaMKIIδc^+/T cardiomyocytes. d Graph of mean data ± SEM along with individual values showing DADs per minute in WT (n = 10 cells/5 mice), SCN10A^−/− (n = 12 cells/5 mice), CaMKIIδc^+/T (n = 21 cells/5 mice) and SCN10A^−/−/CaMKIIδc^+/T (n = 15 cells/5 mice) cardiomyocytes. There were significantly less events of afterdepolarizations in SCN10A^−/−/CaMKIIδc^+/T compared to CaMKIIδc^+/T cardiomyocytes. Data were analyzed by one-way ANOVA with the post hoc two-stage step-up method of Benjamini, Krieger, and Yekutieli. e Original traces of action potential showing early afterdepolarizations (EADs) in CaMKIIδc^+/T and SCN10A^−/−/CaMKIIδc^+/T cardiomyocytes. f Graph of mean data ± SEM along with individual values showing EADs per minute in WT (n = 10 cells/5 mice), SCN10A^−/− (n = 12 cells/5 mice), CaMKIIδc^+/T (n = 21 cells/5 mice) and SCN10A^−/−/CaMKIIδc^+/T (n = 16 cells/5 mice) cardiomyocytes. There were significantly less events of afterdepolarizations in SCN10A^−/−/CaMKIIδc^+/T compared to CaMKIIδc^+/T cardiomyocytes. Data were analyzed by one-way ANOVA with the post hoc two-stage step-up method of Benjamini, Krieger, and Yekutieli. g Original confocal line scans images of CaMKIIδc^+/T and SCN10A^−/−/CaMKIIδc^+/T cardiomyocytes showing diastolic Ca²⁺ waves. h Percentage of cells exhibiting waves was significantly less in SCN10A^−/−/CaMKIIδc^+/T (n = 74 cells/7 mice) compared to CaMKIIδc^+/T (n = 104 cells/9 mice). Data were analyzed by Chi-square test, two-tailed analysis. i Significantly decreased number of Ca²⁺ waves per minute in SCN10A^−/−/CaMKIIδc^+/T compared to CaMKIIδc^+/T. Data were analyzed by one-way ANOVA with post hoc Bonferroni’s correction. Cells/mice studied, WT: n = 48 cells/5 mice, SCN10A^−/−: n = 52 cells/5 mice, CaMKIIδc^+/T: n = 104 cells/9 mice; SCN10A^−/−/CaMKIIδc^+/T: n = 74 cells/7 mice. Data were presented as mean values ± SEM.

Fig. 6. SCN10A^−/−/CaMKIIδc^+/T exhibit less in vivo arrhythmias compared to CaMKIIδc^+/T mice.

a Original ECG traces from telemetry recordings of 10-week-old CaMKIIδc^+/T and SCN10A^−/−/CaMKIIδc^+/T mice showing ventricular arrhythmias. b Unchanged activity levels in SCN10A^−/−/CaMKIIδc^+/T compared to CaMKIIδc^+/T (CaMKIIδc^+/T (four mice); SCN10A^−/−/CaMKIIδc^+/T (three mice), four individual recordings each). Data were presented as mean values ± SEM, analyzed by unpaired two-tailed Student’s t-test. c Mean values of premature ventricular contractions (PVCs) in SCN10A^−/−/CaMKIIδc^+/T (p = 0.08, Unpaired two-tailed Student’s t-test), CaMKIIδc^+/T (four mice); SCN10A^−/−/CaMKIIδc^+/T (three mice), four individual recordings each. Data were presented as mean values ± SEM. d Reduction of ventricular tachycardia (VT) incidence in SCN10A^−/−/CaMKIIδc^+/T (p < 0.05, Unpaired two-tailed Student’s t-test), CaMKIIδc^+/T (four mice); SCN10A^−/−/CaMKIIδc^+/T (three mice), four individual recordings each. Data were presented as mean values ± SEM.