IL-6/STAT3 signaling in mice with dysfunctional type-2 ryanodine receptor

Figures & data

FIGURE 1. IL-6 and downstream signaling molecule expression in hearts of Ryr2^+/+ and Ryr2^ADA/ADA mice. (A) Immunoblots of heart homogenates from 1-day old and 10-day old Ryr2^+/+ (WT) and Ryr2^ADA/ADA (ADA) mice. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the loading control. (B) Relative protein and phosphorylation levels in 1-day old and 10-day old Ryr2^ADA/ADA compared to Ryr2^+/+ mice. Data are the mean ± SEM of 5–19 samples. *p < 0.05 compared to Ryr2^{+/+, #}p < 0.05 compared to corresponding 1 day sample using one way ANOVA.

FIGURE 2. Nuclear and cytosolic location of STAT3 and pSTAT3-Tyr705. (A) Immunoblots of nuclear (N) and cytosolic (C) fractions from hearts of 10-day old Ryr2^+/+ (WT) and Ryr2^ADA/ADA (ADA) mice. GAPDH and histone 3 (His3) were markers for cytosolic and nuclear fractions, respectively. (B) Protein and phosphorylation levels of Ryr2^ADA/ADA mice were normalized to Ryr2^+/+. Data are the mean ± SEM of 5–6 samples.*p < 0.05 compared to Ryr2^+/+ using t-test.

FIGURE 3. Ryr2^ADA/ADA and Ryr2^ADA/ADA/IL-6^−/− mice survival. Mean lifetimes ± SEM of Ryr2^ADA/ADA and Ryr2^ADA/ADA/IL-6^−/− mice of 16.9 ± 1.3 (n = 9) and 25.1 ± 3.5 (n = 7), respectively, were significantly different (p < 0.05).

Table 1. Body and heart weights and echocardiography of 10-day old Ryr2^+/+ and Ryr2^ADA/ADA mice.

	Ryr2^+/+	Ryr2^ADA/ADA	Ryr2^+/+/Il6^−/−	Ryr2^ADA/ADA/Il6^−/−
	n = 8	n = 7	n = 7	n = 7
HW/BW ratio	0.58 ± 0.08	1.13 ± 0.04	0.56 ± 0.01	0.89 ± 0.02
IVSd	0.68 ± 0.06	0.63 ± 0.03	0.80 ± 0.08	0.70 ± 0.06
IVSs	1.23 ± 0.06	0.80 ± 0.04	1.24 ± 0.07	0.92 ± 0.06^Footnote
LVIDd	1.69 ± 0.06	2.96 ± 0.10	1.37 ± 0.13	2.75 ± 0.14^Footnote
LVIDs	0.54 ± 0.07	2.55 ± 0.09	0.39 ± 0.09	2.24 ± 0.16^Footnote
LVPWd	0.67 ± 0.04	0.57 ± 0.08	0.77 ± 0.06	0.68 ± 0.07
LVPWs	1.05 ± 0.06	0.59 ± 0.07	1.29 ± 0.10	0.71 ± 0.09^Footnote
FS (%)	68.4 ± 3.45	13.85 ± 0.88	73.0 ± 3.9	18.9 ± 2.5^Footnote
EF (%)	94.7 ± 1.6	30.7 ± 1.8	96.3 ± 1.6	40.1 ± 4.6^Footnote
HR (bmp)	580 ± 21	327 ± 30	527 ± 24	340 ± 36^Footnote

BW and HW, body and heart weight, respectively; IVSd, interventricular septum diastolic thickness; IVSs, interventricular septum systolic thickness; LVIDd and LIVDs, left ventricular dimensions at end diastole and systole, respectively; LVPWd and LVPWs, left ventricular posterior wall at end diastole and systole, respectively; FS, fractional shortening; EF, ejection fraction; HR, heart rate; bpm, beats/min. Data are the mean ± SEM of indicated number of mice shown in the Table.

* p < 0.05 compared to Ryr2^+/+ mice

^# p<0.05 compared to Ryr2^ADA/ADA mice

^% p < 0.05 compared to Ryr2^+/+/IL-6^−/− mice, using 2 way ANOVA.

FIGURE 4. Expression of IL-6 downstream signaling molecules in mice targeted for Ryr2^ADA and IL-6^−/−. (A) Immunoblots of heart homogenates from 10-day old Ryr2^+/+ (WT) and Ryr2^ADA/ADA (ADA) mice with and without IL-6. GAPDH was the loading control. (B) Protein levels and phosphorylation ratios of Ryr2^ADA/ADA mice were normalized to Ryr2^+/+. Data are the mean ± SEM of 6–14 determinations. p < 0.05 compared with Ryr2^{+/+, #}p < 0.05 compared with Ryr2^ADA/ADA, using one way ANOVA.

FIGURE 5. Survival data from Ryr2^ADA/ADA mice treated with STAT3 inhibitor NSC74859. Mean lifetimes ± SEM of Ryr2^ADA/ADA mice treated without (Control) and with NSC74859 (NSC) of13.9 ± 0.5 (n = 13) and 18.4 ± 1.8 (n = 13), respectively, were significantly different (p < 0.05).

Table 2. Body and heart weights and echocardiography of 10-day old Ryr2^+/+ and Ryr2^ADA/ADA mice with and without NSC74859.

	Control		NSC74859
	Ryr2^+/+	Ryr2^ADA/ADA	Ryr2^+/+	Ryr2^ADA/ADA
	n = 7	n = 6	n = 7	n = 8
HW/BW ratio	0.58 ± 0.01	1.03 ± 0.03	0.57 ± 0.01	0.94 ± 0.02
IVSd	1.01 ± 0. 06	0.75 ± 0.08	0.89 ± 0.7	0.57 ± 0.06
IVSs	1.50 ± 0. 05	0.88 ± 0.09	1.37 ± 0.06	0.78 ± 0.07
LVIDd	1.55 ± 0.14	3.52 ± 0.20	1.52 ± 0.10	3.14 ± 0.13
LVIDs	0.50 ± 0.11	3.16 ± 0.20	0.42 ± 0.07	2.58 ± 0.13
LVPWd	0.96 ± 0.15	0.68 ± 0.06	0.86 ± 0.08	0.74 ± 0.06
LVPWs	1.28 ± 0.09	0.79 ± 0.07	1.24 ± 0.07	0.85 ± 0.09
FS (%)	68.9 ± 3.6	10.2 ± 2.4	73.0 ± 3.7	17.2 ± 1.7
EF (%)	95.4 ± 1.7	22.6 ± 12.6	96.4 ± 1.2	36.7 ± 3.4
HR (bmp)	514 ± 35	340 ± 31	598 ± 24	329 ± 32

Abbreviations are as in Table 1. Data are the mean ± SEM of indicated number of mice.

* p < 0.05 compared to wild-type mice without NSC74859

^# p < 0.05 compared to Ryr2^ADA/ADA mice without NSC74859

^% p < 0.05 compared to wild-type with NSC74859, using 2 way ANOVA

^and p < 0.05 compared to Ryr2^ADA/ADA mice without NSC74859, as determined by t-test.

FIGURE 6. IL-6 and downstream signaling molecule levels in 10-day old mice treated with or without STAT3 inhibitor NSC74859. (A) Immunoblots of heart homogenates from 10-day old Ryr2^+/+ (WT) and Ryr2^ADA/ADA (ADA) mice treated with or without NSC74859 (NSC). GAPDH was the loading control. (B) Protein levels and phosphorylation ratios of Ryr2^ADA/ADA mice were normalized to Ryr2^+/+ Control. Data are the mean ± SEM of 4–10 samples. *p < 0.05 compared to Ryr2^+/+ mice without NSC74859, ^#p < 0.05 compared to Ryr2^ADA/ADA mice without NSC74859, using one way ANOVA.

FIGURE 7. c-Fos, c-Myc, ANP and BNP mRNA levels in hearts of Ryr2^+/+ and Ryr2^ADA/ADA mice treated with or without STAT3 inhibitor NSC74859. mRNA levels were measured by quantitative RT-PCR and normalized to levels in hearts of Ryr2^+/+ mice not treated with the inhibitor (Ryr2^+/+ Control). Data are the mean ± SEM of 5–6 samples. *p < 0.05 compared to Ryr2^+/+ mice without NSC74859, ^#p < 0.05 compared with Ryr2^ADA/ADA mice without NSC74859, ^%p < 0.05 compared with Ryr2^+/+ mice with NSC74859, using one way ANOVA.