Inward Rectifier K ⁺ Currents Contribute to the Proarrhythmic Electrical Phenotype of Atria Overexpressing Cyclic Adenosine Monophosphate Response Element Modulator Isoform CREM‐IbΔC‐X

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  Figure 1. Total K+ currents of transgenic atrial myocytes. Experimental animals involved wild‐type mice (WT) and transgenic mice (TG) expressing the repressor isoform of cyclic adenosine monophosphate response element modulator, CREM‐IbΔC‐X, and were distributed according to age in 6‐week‐old (WT6w and TG6w) and 12‐week‐old (WT12w and TG12w) groups. A, Representative K+ currents recorded from atrial myocytes of WT12w and TG12w using the voltage protocol illustrated in the inset. For clarity, current density (measured in picoamperes/picofarads [pA/pF]) traces from −40 mV with +20 mV increments are displayed. B through D, Mean±SE of current–voltage plots of peak total outward K+ current (IKtot) (B), total outward K+ current at the end of the pulse (IKend) (C), and tail K+ current (IKtail) (D). For WT6w, n/N are 54/17 (B and C) and 45/17 (D), for TG6w n/N are 32/18 (B and C) and 31/18 (D), for WT12w n/N are 31/16 (B and C) and 29/15 (D), and for TG12w n/N are 22/14 (B and C) and 17/11 (D). E, Data show mean±SE of relative mRNA levels of the displayed genes normalized to WT6w vs Hprt1 (hypoxanthine phosphoribosyltransferase 1) as the housekeeping gene. Y‐axes scale is Log2. N=8 per group…

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  Figure 2. Transient outward K+ channel expression in transgenic atrial myocytes. Experimental animals involved wild‐type mice (WT) and transgenic mice (TG) expressing the repressor isoform of cyclic adenosine monophosphate response element modulator, CREM‐IbΔC‐X, and were distributed according to age in 6‐week‐old (WT6w and TG6w) and 12‐week‐old (WT12w and TG12w) groups. A, Representative transient outward K+ current (Ito) traces recorded as −40 mV sensitive currents from atrial cardiomyocytes of WT12w and TG12w. For clarity, current density (expressed in picoamperes/picofarads [pA/pF]) traces from −40 mV with +20 mV increments are displayed. B, Mean±SE of current–voltage plots of the peak current density (Ito). For WT6w, n/N are 41/15, for TG6w n/N are 22/14, for WT12w n/N are 35/19, and for TG12w n/N are 20/16. Ca through d, Representative immunoblots and quantification of protein expression of calsequestrin (CSQ; Ca) normalized to Ponceau and of K+ voltage‐gated channel subfamily D member 2 (Kv4.2, Cb) and 3 (Kv4.3, Cc), and K+ channel interacting protein 2 (KChIP2) (Cd) normalized to CSQ. Data show mean±SE of N=7 mice per group, except for Kv4.3, where N=6 as the result of an…

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  Figure 3. Inward rectifier K+ current IK1 expression and function in transgenic atrial myocytes. Experimental animals involved wild‐type mice (WT) and transgenic mice (TG) expressing the repressor isoform of cyclic adenosine monophosphate response element modulator, CREM‐IbΔC‐X, and were distributed according to age in 6‐week‐old (WT6w and TG6w) and 12‐week‐old (WT12w and TG12w) groups. A, Representative traces of inward rectifiers K+channel (IK1) recorded in WT12w and TG12w myocytes before and after application of 10 μmol/L BaCl2 using a ramp protocol (inset, scale bar 0.2 seconds). The traces show current densitiy (expressed in picoamperes/picofarads [pA/pF]) over time. (Aa). Current–voltage plots of IK1 averaged from traces of all cells. For WT6w, n/N are 8/5, for TG6w n/N are 10/6, for WT12w n/N are 13/7, and for TG12w n/N are 9/6 (Ab). B, Data show mean±SE of relative mRNA levels of atrial K+ voltage‐gated channel subfamily J member 2 (Kcnj2) and 4 (Kcnj4) normalized to WT6w vs Hprt1 (hypoxanthine phosphoribosyltransferase 1) as the housekeeping gene; N=8 per group. Y‐axes scale is Log2. Ca through c, Representative immunoblots (Ca) and quantification of K+ inwardly rectifying channel…

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  Figure 4. Acetylcholine‐activated K+ current (IKACh) expression and function in transgenic atrial myocytes. Experimental animals involved wild‐type mice (WT) and transgenic mice (TG) expressing the repressor isoform of cyclic adenosine monophosphate response element modulator, CREM‐IbΔC‐X, and were distributed according to age in 6‐week‐old (WT6w and TG6w) and 12‐week‐old (WT12w and TG12w) groups. A, Representative traces recorded before and after application of 10 μmol/L acetylcholine using the ramp protocol (inset, scale bar 0.2 seconds) in 1 isolated WT12w atrial cardiomyocyte. The acetylcholine‐sensitive current derived by subtraction is IKACh. The traces show current densitiy (expressed in picoamperes/picofarads [pA/pF]) over time. (Aa). Current–voltage plots of IKACh averaged from traces of all cells. n/N for WT6w are 12/6, for TG6w are 13/7, for WT12w are 12/6, and for TG12w are 10/6 (Ab). B, Relative mRNA levels of K+ voltage‐gated channel subfamily J members 3 (Kcnj3) and 5 (Kcnj5) measured in atrial tissue, with Hprt1 (hypoxanthine phosphoribosyltransferase 1) as the housekeeping gene. Data were normalized to WT6w. Y‐axes scale is Log2. Data show mean±SE of 8 per group. Ca…

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  Figure 5. Cyclic adenosine monophosphate response element modulator (CREM)–dependent gene expression. A and B, Chromatin immunoprecipitation results for selected genes: K+ voltage‐gated channel subfamily D member 2 (Kcnd2) and 3 (Kcnd3), K+ voltage‐gated channel subfamily A member 5 (Kcna5), K+ voltage‐gated channel subfamily H member 2 (Kcnh2), K+ voltage‐gated channel subfamily J member 3 (Kcnj3), 5 (Kcnj5), 2 (Kcnj2) and 4 (Kcnj4), FBJ Murine Osteosarcoma Viral Oncogene Homolog (c‐fos) and small inducible adenosine monophosphate early repressor (smIcer). Bar graphs show mean±SE of genomic DNA (gDNA) enrichment at 6 weeks (A) and 12 weeks (B) of age in transgenic mice (TG) expressing the repressor isoform of cyclic adenosine monophosphate response element modulator, CREM‐IbΔC‐X, vs age‐matched wild‐type mice (WT). N=7–8 for WT6w, N=8 for TG6w, N=7–8 for WT12w, and N=9–10 for TG12w. *P<0.05 TG6w vs WT6w (gray) and TG12w vs WT12w (black) calculated by REST 2.07 software.

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  Figure 6. Left atrial contraction of transgenic mice (TG) expressing the repressor isoform of cyclic adenosine monophosphate response element modulator, CREM‐IbΔC‐X. Force of contraction (FOC) measured in the left atria. Experimental animals involved wild‐type (WT) and TG animals distributed according to age in 6‐week‐old (WT6w and TG6w) and 12‐week‐old (WT12w and TG12w) groups. Data show mean±SE of mice from WT6w (N=12), TG6w (N=10), WT12w (N=49), and TG12w (N=47). **P<0.01 TG6w vs WT6w (gray) and TG12w vs WT12w (black) and # P<0.05 TG12w vs TG6w (black) from the Kruskall–Wallis test with the Dunn post hoc test.

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  Figure 7. Dose‐response curve of left atrial contraction in response to autonomic stimulation. Experimental animals involved wild‐type mice (WT) and transgenic mice (TG) expressing the repressor isoform of cyclic adenosine monophosphate response element modulator, CREM‐IbΔC‐X, and were distributed according to age in 6‐week‐old (WT6w and TG6w) and 12‐week‐old (WT12w and TG12w) groups. A, Force of contraction (FOC) of the left atria was measured in WT12w and TG12w mice under increasing concentrations of carbachol. Data show mean±SE of raw FOC (Aa), normalized to baseline value (Ab), and normalized to minimum (in carbachol 10−5 mol/L) and maximum (before carbachol) FOC (Ac). For WT12w N is 8 and for TG12w N is 8. B, FOC of left atria was measured in WT12w and TG12w mice under increasing concentrations of isoproterenol. Data show mean±SE of raw FOC (Ba), normalized to initial FOC (Bb), and normalized to minimum (before isoproterenol) and maximum (in isoproterenol 10−4 mol/L) FOC (Bc). For WT12w N is 10 and for TG12w N is 10. *P<0.05 TG12w vs WT12w from 2‐way repeated‐measures analysis of variance with Bonferroni post hoc test.

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  Figure 8. The effect of functional antagonism of vagal‐sympathetic stimulation on left atrial contraction of transgenic mice (TG) expressing the repressor isoform of cyclic adenosine monophosphate response element modulator, CREM ‐IbΔC‐X. Experimental animals involved wild‐type (WT) and TG animals distributed according to age in 6‐week‐old (WT6w and TG6w) and 12‐week‐old (WT12w and TG12w) groups. A through C, Force of contraction (FOC) of left atria measured before and under continuous presence of 300 nmol/L carbachol during the application of increasing isoproterenol concentrations. Data show mean±SE of raw FOC (A), FOC normalized to initial FOC (B), and FOC normalized to minimum (300 nmol/L carbachol) and maximum (10−4 mol/L isoproterenol) FOC (C). Mice from WT6w (N=12), TG6w (N=10), WT12w (N=24), and TG12w (N=13) (A and B) and from WT6w (N=6), TG6w (N=10), WT12w (N=14), and TG12w (N=12) were included (C). *P<0.05 TG6w vs WT6w (gray) and TG12w vs WT12w (black) and # P<0.05 WT12w vs WT6w (gray) and TG12w vs TG6w (black) from 2‐way repeated‐measures analysis of variance with Bonferroni post hoc test.