Absence of SOCS3 in the cardiomyocyte increases mortality in a gp130-dependent manner accompanied by contractile dysfunction and ventricular arrhythmias

Abstract

Background: Suppressor of cytokine signaling-3 (SOCS3) is a key negative-feedback regulator of the gp130 receptor that provides crucial signaling for cardiac hypertrophy and survival; however, an in vivo role of SOCS3 regulation on cardiac gp130 signaling remains obscure.

Methods and results: We generated cardiac-specific SOCS3 knockout (SOCS3 cKO) mice. These mice showed increased activation of gp130 downstream signaling targets (STAT3, ERK1/2, AKT, and p38) from 15 weeks of age and developed cardiac dysfunction from approximately 25 weeks of age with signs of heart failure. Surprisingly, SOCS3 cKO failing hearts had minimal histological abnormalities with intact myofibril ultrastructure. In addition, Ca(2+) transients were significantly increased in SOCS3 cKO failing hearts compared with wild-type hearts. We also found that Ser23/24 residues of troponin I were hypophosphorylated in SOCS3 cKO hearts before the manifestation of cardiac dysfunction. These data suggested the presence of abnormalities in myofilament Ca(2+) sensitivity in SOCS3 cKO mice. In addition to the contractile dysfunction, we found various ventricular arrhythmias in SOCS3 cKO nonfailing hearts accompanied by a sarcoplasmic reticulum Ca(2+) overload. To determine the contribution of gp130 signaling to the cardiac phenotype that occurs with SOCS3 deficiency, we generated cardiac-specific gp130 and SOCS3 double KO mice. Double KO mice lived significantly longer and had different histological abnormalities when compared with SOCS3 cKO mice, thus demonstrating the importance of gp130 signaling in the SOCS3 cKO cardiac phenotype.

Conclusions: Our results demonstrate an important role of SOCS3 regulation on cardiac gp130 signaling in the pathogenesis of contractile dysfunction and ventricular arrhythmias.

Figure 1

Successful disruption of SOCS3 and unregulated activation of gp130 downstream signaling in the heart. A, After 12 hours of serum starvation, isolated adult cardiomyocytes (8 weeks old) were stimulated with CT-1 (1nM) for the indicated time. The expression of SOCS3, phospho-STAT3 and endogenous STAT3 were estimated by western-blot (n=3 separate experiments). B, Known gp130 downstream signaling in the heart was examined at 8 and 15 weeks of age. The level of expression of each phospho-protein was normalized to the endogenous protein expression level. Expression level was calculated relative to the mean value of wild-type (WT) group (n=6 in each group). cKO; SOCS3 cKO * p<0.05 comparing SOCS3 cKO with WT hearts. Representative western-blot from three independent experiments is shown.

Figure 1

Successful disruption of SOCS3 and unregulated activation of gp130 downstream signaling in the heart. A, After 12 hours of serum starvation, isolated adult cardiomyocytes (8 weeks old) were stimulated with CT-1 (1nM) for the indicated time. The expression of SOCS3, phospho-STAT3 and endogenous STAT3 were estimated by western-blot (n=3 separate experiments). B, Known gp130 downstream signaling in the heart was examined at 8 and 15 weeks of age. The level of expression of each phospho-protein was normalized to the endogenous protein expression level. Expression level was calculated relative to the mean value of wild-type (WT) group (n=6 in each group). cKO; SOCS3 cKO * p<0.05 comparing SOCS3 cKO with WT hearts. Representative western-blot from three independent experiments is shown.

Figure 2

Heart failure and early mortality in SOCS3 cKO mice. A, Kaplan-Meier survival curves of WT and SOCS3 cKO mice. B, Morphology of SOCS3 cKO failing heart (28 weeks old). Mural thrombus in left atrium (arrow) was frequently observed in SOCS3 cKO mice. Scales: 1 mm. Ventricular weight to tibia length ratio (VW/TL) is shown in the panel on the right. C, Isolated cardiomyocytes were stained with WGA conjugated with Texas Red to visualize the sarcolemma membrane clearly (left panels). Width and length of SOCS3 cKO cardiomyocyte were calculated relative to that of WT cardiomyocytes (right panel). Scale bars: 100 µm. D, Echocardiography at 8, 20, 25 and 28 weeks of age (different groups of mice, n=5 in each group, were used at each time point). Representative M-mode images are shown on the top. LVEDD; Left ventricular end-diastolic dimension, LVESD; LV end-systolic dimension, %FS; percent fractional shortening. E, Hematoxylin-eosin (HE) and Masson-Trichrome (MT) staining of WT non-failing and SOCS3 cKO failing heart sections (28 weeks old, left panels). Scale bars: 100 µm (HE) and 200 µm (MT). Ultrastructure of myofibrils in WT non-failing and SOCS3 cKO failing hearts by electron microscopy (right panels). Scale bars: 1.5 µm. F, A representative TUNEL staining of the SOCS3 cKO failing heart using the spleen tissue from the same animal as a positive control (left panels). All the TUNEL positive cells in the heart were blood cells in capillaries (left panel, arrow). Caspase-3 (Casp3) cleavage was examined by western-blot using anti-casp3 antibody that can detect both cleaved and uncleaved Casp3 (right panel). Scale bar: 200 µm. *p<0.05 comparing SOCS3 cKO with WT.

Figure 2

Heart failure and early mortality in SOCS3 cKO mice. A, Kaplan-Meier survival curves of WT and SOCS3 cKO mice. B, Morphology of SOCS3 cKO failing heart (28 weeks old). Mural thrombus in left atrium (arrow) was frequently observed in SOCS3 cKO mice. Scales: 1 mm. Ventricular weight to tibia length ratio (VW/TL) is shown in the panel on the right. C, Isolated cardiomyocytes were stained with WGA conjugated with Texas Red to visualize the sarcolemma membrane clearly (left panels). Width and length of SOCS3 cKO cardiomyocyte were calculated relative to that of WT cardiomyocytes (right panel). Scale bars: 100 µm. D, Echocardiography at 8, 20, 25 and 28 weeks of age (different groups of mice, n=5 in each group, were used at each time point). Representative M-mode images are shown on the top. LVEDD; Left ventricular end-diastolic dimension, LVESD; LV end-systolic dimension, %FS; percent fractional shortening. E, Hematoxylin-eosin (HE) and Masson-Trichrome (MT) staining of WT non-failing and SOCS3 cKO failing heart sections (28 weeks old, left panels). Scale bars: 100 µm (HE) and 200 µm (MT). Ultrastructure of myofibrils in WT non-failing and SOCS3 cKO failing hearts by electron microscopy (right panels). Scale bars: 1.5 µm. F, A representative TUNEL staining of the SOCS3 cKO failing heart using the spleen tissue from the same animal as a positive control (left panels). All the TUNEL positive cells in the heart were blood cells in capillaries (left panel, arrow). Caspase-3 (Casp3) cleavage was examined by western-blot using anti-casp3 antibody that can detect both cleaved and uncleaved Casp3 (right panel). Scale bar: 200 µm. *p<0.05 comparing SOCS3 cKO with WT.

Figure 2

Heart failure and early mortality in SOCS3 cKO mice. A, Kaplan-Meier survival curves of WT and SOCS3 cKO mice. B, Morphology of SOCS3 cKO failing heart (28 weeks old). Mural thrombus in left atrium (arrow) was frequently observed in SOCS3 cKO mice. Scales: 1 mm. Ventricular weight to tibia length ratio (VW/TL) is shown in the panel on the right. C, Isolated cardiomyocytes were stained with WGA conjugated with Texas Red to visualize the sarcolemma membrane clearly (left panels). Width and length of SOCS3 cKO cardiomyocyte were calculated relative to that of WT cardiomyocytes (right panel). Scale bars: 100 µm. D, Echocardiography at 8, 20, 25 and 28 weeks of age (different groups of mice, n=5 in each group, were used at each time point). Representative M-mode images are shown on the top. LVEDD; Left ventricular end-diastolic dimension, LVESD; LV end-systolic dimension, %FS; percent fractional shortening. E, Hematoxylin-eosin (HE) and Masson-Trichrome (MT) staining of WT non-failing and SOCS3 cKO failing heart sections (28 weeks old, left panels). Scale bars: 100 µm (HE) and 200 µm (MT). Ultrastructure of myofibrils in WT non-failing and SOCS3 cKO failing hearts by electron microscopy (right panels). Scale bars: 1.5 µm. F, A representative TUNEL staining of the SOCS3 cKO failing heart using the spleen tissue from the same animal as a positive control (left panels). All the TUNEL positive cells in the heart were blood cells in capillaries (left panel, arrow). Caspase-3 (Casp3) cleavage was examined by western-blot using anti-casp3 antibody that can detect both cleaved and uncleaved Casp3 (right panel). Scale bar: 200 µm. *p<0.05 comparing SOCS3 cKO with WT.

Figure 2

Heart failure and early mortality in SOCS3 cKO mice. A, Kaplan-Meier survival curves of WT and SOCS3 cKO mice. B, Morphology of SOCS3 cKO failing heart (28 weeks old). Mural thrombus in left atrium (arrow) was frequently observed in SOCS3 cKO mice. Scales: 1 mm. Ventricular weight to tibia length ratio (VW/TL) is shown in the panel on the right. C, Isolated cardiomyocytes were stained with WGA conjugated with Texas Red to visualize the sarcolemma membrane clearly (left panels). Width and length of SOCS3 cKO cardiomyocyte were calculated relative to that of WT cardiomyocytes (right panel). Scale bars: 100 µm. D, Echocardiography at 8, 20, 25 and 28 weeks of age (different groups of mice, n=5 in each group, were used at each time point). Representative M-mode images are shown on the top. LVEDD; Left ventricular end-diastolic dimension, LVESD; LV end-systolic dimension, %FS; percent fractional shortening. E, Hematoxylin-eosin (HE) and Masson-Trichrome (MT) staining of WT non-failing and SOCS3 cKO failing heart sections (28 weeks old, left panels). Scale bars: 100 µm (HE) and 200 µm (MT). Ultrastructure of myofibrils in WT non-failing and SOCS3 cKO failing hearts by electron microscopy (right panels). Scale bars: 1.5 µm. F, A representative TUNEL staining of the SOCS3 cKO failing heart using the spleen tissue from the same animal as a positive control (left panels). All the TUNEL positive cells in the heart were blood cells in capillaries (left panel, arrow). Caspase-3 (Casp3) cleavage was examined by western-blot using anti-casp3 antibody that can detect both cleaved and uncleaved Casp3 (right panel). Scale bar: 200 µm. *p<0.05 comparing SOCS3 cKO with WT.

Figure 2

Heart failure and early mortality in SOCS3 cKO mice. A, Kaplan-Meier survival curves of WT and SOCS3 cKO mice. B, Morphology of SOCS3 cKO failing heart (28 weeks old). Mural thrombus in left atrium (arrow) was frequently observed in SOCS3 cKO mice. Scales: 1 mm. Ventricular weight to tibia length ratio (VW/TL) is shown in the panel on the right. C, Isolated cardiomyocytes were stained with WGA conjugated with Texas Red to visualize the sarcolemma membrane clearly (left panels). Width and length of SOCS3 cKO cardiomyocyte were calculated relative to that of WT cardiomyocytes (right panel). Scale bars: 100 µm. D, Echocardiography at 8, 20, 25 and 28 weeks of age (different groups of mice, n=5 in each group, were used at each time point). Representative M-mode images are shown on the top. LVEDD; Left ventricular end-diastolic dimension, LVESD; LV end-systolic dimension, %FS; percent fractional shortening. E, Hematoxylin-eosin (HE) and Masson-Trichrome (MT) staining of WT non-failing and SOCS3 cKO failing heart sections (28 weeks old, left panels). Scale bars: 100 µm (HE) and 200 µm (MT). Ultrastructure of myofibrils in WT non-failing and SOCS3 cKO failing hearts by electron microscopy (right panels). Scale bars: 1.5 µm. F, A representative TUNEL staining of the SOCS3 cKO failing heart using the spleen tissue from the same animal as a positive control (left panels). All the TUNEL positive cells in the heart were blood cells in capillaries (left panel, arrow). Caspase-3 (Casp3) cleavage was examined by western-blot using anti-casp3 antibody that can detect both cleaved and uncleaved Casp3 (right panel). Scale bar: 200 µm. *p<0.05 comparing SOCS3 cKO with WT.

Figure 2

Heart failure and early mortality in SOCS3 cKO mice. A, Kaplan-Meier survival curves of WT and SOCS3 cKO mice. B, Morphology of SOCS3 cKO failing heart (28 weeks old). Mural thrombus in left atrium (arrow) was frequently observed in SOCS3 cKO mice. Scales: 1 mm. Ventricular weight to tibia length ratio (VW/TL) is shown in the panel on the right. C, Isolated cardiomyocytes were stained with WGA conjugated with Texas Red to visualize the sarcolemma membrane clearly (left panels). Width and length of SOCS3 cKO cardiomyocyte were calculated relative to that of WT cardiomyocytes (right panel). Scale bars: 100 µm. D, Echocardiography at 8, 20, 25 and 28 weeks of age (different groups of mice, n=5 in each group, were used at each time point). Representative M-mode images are shown on the top. LVEDD; Left ventricular end-diastolic dimension, LVESD; LV end-systolic dimension, %FS; percent fractional shortening. E, Hematoxylin-eosin (HE) and Masson-Trichrome (MT) staining of WT non-failing and SOCS3 cKO failing heart sections (28 weeks old, left panels). Scale bars: 100 µm (HE) and 200 µm (MT). Ultrastructure of myofibrils in WT non-failing and SOCS3 cKO failing hearts by electron microscopy (right panels). Scale bars: 1.5 µm. F, A representative TUNEL staining of the SOCS3 cKO failing heart using the spleen tissue from the same animal as a positive control (left panels). All the TUNEL positive cells in the heart were blood cells in capillaries (left panel, arrow). Caspase-3 (Casp3) cleavage was examined by western-blot using anti-casp3 antibody that can detect both cleaved and uncleaved Casp3 (right panel). Scale bar: 200 µm. *p<0.05 comparing SOCS3 cKO with WT.

Figure 3

SOCS3 cKO mice developed cardiac dysfunction under acute pressure overload. A, Echocardiography before (Pre TAC) and 10 days after TAC (Post TAC) (8 weeks old, female, n=6 and 7, respectively). The changes in echo data before and after TAC were compared between the two groups using repeated measure ANOVA (*p<0.05). As a control, sham operated mice (n=5 in each group) were also examined at 10 days. PG; pressure gradient between right and left carotid arteries measured 10 days post TAC. LV/TL; LV weight and tibia length ratio. B, Activation of STAT3, ERK1/2 and AKT signaling in the heart 10 days after TAC. **p<0.05 comparing SOCS3 cKO with WT.

Figure 3

SOCS3 cKO mice developed cardiac dysfunction under acute pressure overload. A, Echocardiography before (Pre TAC) and 10 days after TAC (Post TAC) (8 weeks old, female, n=6 and 7, respectively). The changes in echo data before and after TAC were compared between the two groups using repeated measure ANOVA (*p<0.05). As a control, sham operated mice (n=5 in each group) were also examined at 10 days. PG; pressure gradient between right and left carotid arteries measured 10 days post TAC. LV/TL; LV weight and tibia length ratio. B, Activation of STAT3, ERK1/2 and AKT signaling in the heart 10 days after TAC. **p<0.05 comparing SOCS3 cKO with WT.

Figure 4

Increased Ca²⁺ transients and abnormal myofilament Ca²⁺ sensitivity in SOCS3 cKO mice. A, Ca²⁺ transient and sarcomere shortening measurements in SOCS3 cKO failing cardiomyocytes (28 week old). Cardiomyocytes were labeled with Fura-2 and voltage paced at 0.5 Hz. Representative recoding images for WT and SOCS3 cKO were shown on the left. Results for amplitude of [Ca²⁺]_i, time constant of [Ca²⁺]_i decline, max shortening, and time constant of relaxation are shown on the right. Representative recordings of 15 cardiomyocytes from three mice are shown. * p<0.05 comparing SOCS3 cKO with WT cardiomyocytes. B, The relationship between peak [Ca²⁺]_i and %sarcomere shortening. Each dot represents each cardiomyocyte recorded. C, The expression level of SR proteins in SOCS3 cKO failing hearts. A representative western-blot from three independent experiments is shown. * p<0.05 comparing SOCS3 cKO with WT (n=5 in each group). D, Increased triggered activities in SOCS3 cKO failing cardiomyocytes. Cardiomyocytes were voltage paced at 1 Hz. Simultaneous recoding images of Ca²⁺ transient (top) and sarcomere shortening (bottom) are shown. Arrowheads show triggered activity. Short coupling of the triggered activity induced subsequent abnormal automaticity followed by fibrillation (bottom panel).

Figure 4

Increased Ca²⁺ transients and abnormal myofilament Ca²⁺ sensitivity in SOCS3 cKO mice. A, Ca²⁺ transient and sarcomere shortening measurements in SOCS3 cKO failing cardiomyocytes (28 week old). Cardiomyocytes were labeled with Fura-2 and voltage paced at 0.5 Hz. Representative recoding images for WT and SOCS3 cKO were shown on the left. Results for amplitude of [Ca²⁺]_i, time constant of [Ca²⁺]_i decline, max shortening, and time constant of relaxation are shown on the right. Representative recordings of 15 cardiomyocytes from three mice are shown. * p<0.05 comparing SOCS3 cKO with WT cardiomyocytes. B, The relationship between peak [Ca²⁺]_i and %sarcomere shortening. Each dot represents each cardiomyocyte recorded. C, The expression level of SR proteins in SOCS3 cKO failing hearts. A representative western-blot from three independent experiments is shown. * p<0.05 comparing SOCS3 cKO with WT (n=5 in each group). D, Increased triggered activities in SOCS3 cKO failing cardiomyocytes. Cardiomyocytes were voltage paced at 1 Hz. Simultaneous recoding images of Ca²⁺ transient (top) and sarcomere shortening (bottom) are shown. Arrowheads show triggered activity. Short coupling of the triggered activity induced subsequent abnormal automaticity followed by fibrillation (bottom panel).

Figure 4

Increased Ca²⁺ transients and abnormal myofilament Ca²⁺ sensitivity in SOCS3 cKO mice. A, Ca²⁺ transient and sarcomere shortening measurements in SOCS3 cKO failing cardiomyocytes (28 week old). Cardiomyocytes were labeled with Fura-2 and voltage paced at 0.5 Hz. Representative recoding images for WT and SOCS3 cKO were shown on the left. Results for amplitude of [Ca²⁺]_i, time constant of [Ca²⁺]_i decline, max shortening, and time constant of relaxation are shown on the right. Representative recordings of 15 cardiomyocytes from three mice are shown. * p<0.05 comparing SOCS3 cKO with WT cardiomyocytes. B, The relationship between peak [Ca²⁺]_i and %sarcomere shortening. Each dot represents each cardiomyocyte recorded. C, The expression level of SR proteins in SOCS3 cKO failing hearts. A representative western-blot from three independent experiments is shown. * p<0.05 comparing SOCS3 cKO with WT (n=5 in each group). D, Increased triggered activities in SOCS3 cKO failing cardiomyocytes. Cardiomyocytes were voltage paced at 1 Hz. Simultaneous recoding images of Ca²⁺ transient (top) and sarcomere shortening (bottom) are shown. Arrowheads show triggered activity. Short coupling of the triggered activity induced subsequent abnormal automaticity followed by fibrillation (bottom panel).

Figure 4

Increased Ca²⁺ transients and abnormal myofilament Ca²⁺ sensitivity in SOCS3 cKO mice. A, Ca²⁺ transient and sarcomere shortening measurements in SOCS3 cKO failing cardiomyocytes (28 week old). Cardiomyocytes were labeled with Fura-2 and voltage paced at 0.5 Hz. Representative recoding images for WT and SOCS3 cKO were shown on the left. Results for amplitude of [Ca²⁺]_i, time constant of [Ca²⁺]_i decline, max shortening, and time constant of relaxation are shown on the right. Representative recordings of 15 cardiomyocytes from three mice are shown. * p<0.05 comparing SOCS3 cKO with WT cardiomyocytes. B, The relationship between peak [Ca²⁺]_i and %sarcomere shortening. Each dot represents each cardiomyocyte recorded. C, The expression level of SR proteins in SOCS3 cKO failing hearts. A representative western-blot from three independent experiments is shown. * p<0.05 comparing SOCS3 cKO with WT (n=5 in each group). D, Increased triggered activities in SOCS3 cKO failing cardiomyocytes. Cardiomyocytes were voltage paced at 1 Hz. Simultaneous recoding images of Ca²⁺ transient (top) and sarcomere shortening (bottom) are shown. Arrowheads show triggered activity. Short coupling of the triggered activity induced subsequent abnormal automaticity followed by fibrillation (bottom panel).

Figure 5

SR Ca²⁺ overload and ventricular arrhythmias in SOCS3 cKO non-failing hearts. A, Increased Ca²⁺ transients and SR Ca²⁺ content in SOCS3 cKO non-failing cardiomyocytes (20 weeks old). Cardiomyocytes were labeled with Indo-1 and voltage paced at 0.5 Hz. For SR Ca²⁺ content evaluation, cardiomyocytes were treated with 10 mM caffeine after 10 second of resting period following the 0.5 Hz pacing. Representative recordings were shown on the left. Results for amplitude of [Ca²⁺]_i, and time constant of [Ca²⁺]_i decline are shown on the right. * p<0.05 comparing SOCS3 cKO with WT cardiomyocytes (n=15 in each group). B and C, PVCs and ventricular conduction aberrancy that may represent ventricular tachycardia recorded in SOCS3 cKO mice (20 weeks old). In C, arrowheads show occasional PVCs. The beginning and ending of ventricular conduction aberrancy are shown in the box, respectively on the top panel. The ECG in the boxes are enlarged and shown on the bottom panel. D, The increase in SCN5A and p-PKA C, and the decrease in p-TnI found in SOCS3 cKO non-failing hearts. Representative western-blot from three independent experiments is shown with quantification from all samples tested (n=5 in each group). *p<0.05 comparing SOCS3 cKO with WT.

Figure 5

SR Ca²⁺ overload and ventricular arrhythmias in SOCS3 cKO non-failing hearts. A, Increased Ca²⁺ transients and SR Ca²⁺ content in SOCS3 cKO non-failing cardiomyocytes (20 weeks old). Cardiomyocytes were labeled with Indo-1 and voltage paced at 0.5 Hz. For SR Ca²⁺ content evaluation, cardiomyocytes were treated with 10 mM caffeine after 10 second of resting period following the 0.5 Hz pacing. Representative recordings were shown on the left. Results for amplitude of [Ca²⁺]_i, and time constant of [Ca²⁺]_i decline are shown on the right. * p<0.05 comparing SOCS3 cKO with WT cardiomyocytes (n=15 in each group). B and C, PVCs and ventricular conduction aberrancy that may represent ventricular tachycardia recorded in SOCS3 cKO mice (20 weeks old). In C, arrowheads show occasional PVCs. The beginning and ending of ventricular conduction aberrancy are shown in the box, respectively on the top panel. The ECG in the boxes are enlarged and shown on the bottom panel. D, The increase in SCN5A and p-PKA C, and the decrease in p-TnI found in SOCS3 cKO non-failing hearts. Representative western-blot from three independent experiments is shown with quantification from all samples tested (n=5 in each group). *p<0.05 comparing SOCS3 cKO with WT.

Figure 5

SR Ca²⁺ overload and ventricular arrhythmias in SOCS3 cKO non-failing hearts. A, Increased Ca²⁺ transients and SR Ca²⁺ content in SOCS3 cKO non-failing cardiomyocytes (20 weeks old). Cardiomyocytes were labeled with Indo-1 and voltage paced at 0.5 Hz. For SR Ca²⁺ content evaluation, cardiomyocytes were treated with 10 mM caffeine after 10 second of resting period following the 0.5 Hz pacing. Representative recordings were shown on the left. Results for amplitude of [Ca²⁺]_i, and time constant of [Ca²⁺]_i decline are shown on the right. * p<0.05 comparing SOCS3 cKO with WT cardiomyocytes (n=15 in each group). B and C, PVCs and ventricular conduction aberrancy that may represent ventricular tachycardia recorded in SOCS3 cKO mice (20 weeks old). In C, arrowheads show occasional PVCs. The beginning and ending of ventricular conduction aberrancy are shown in the box, respectively on the top panel. The ECG in the boxes are enlarged and shown on the bottom panel. D, The increase in SCN5A and p-PKA C, and the decrease in p-TnI found in SOCS3 cKO non-failing hearts. Representative western-blot from three independent experiments is shown with quantification from all samples tested (n=5 in each group). *p<0.05 comparing SOCS3 cKO with WT.

Figure 5

SR Ca²⁺ overload and ventricular arrhythmias in SOCS3 cKO non-failing hearts. A, Increased Ca²⁺ transients and SR Ca²⁺ content in SOCS3 cKO non-failing cardiomyocytes (20 weeks old). Cardiomyocytes were labeled with Indo-1 and voltage paced at 0.5 Hz. For SR Ca²⁺ content evaluation, cardiomyocytes were treated with 10 mM caffeine after 10 second of resting period following the 0.5 Hz pacing. Representative recordings were shown on the left. Results for amplitude of [Ca²⁺]_i, and time constant of [Ca²⁺]_i decline are shown on the right. * p<0.05 comparing SOCS3 cKO with WT cardiomyocytes (n=15 in each group). B and C, PVCs and ventricular conduction aberrancy that may represent ventricular tachycardia recorded in SOCS3 cKO mice (20 weeks old). In C, arrowheads show occasional PVCs. The beginning and ending of ventricular conduction aberrancy are shown in the box, respectively on the top panel. The ECG in the boxes are enlarged and shown on the bottom panel. D, The increase in SCN5A and p-PKA C, and the decrease in p-TnI found in SOCS3 cKO non-failing hearts. Representative western-blot from three independent experiments is shown with quantification from all samples tested (n=5 in each group). *p<0.05 comparing SOCS3 cKO with WT.

Figure 6

Unregulated gp130-STAT3 is required for the increase in SCN5A mRNA in SOCS3 cKO cardiomyocytes. A, SCN5A mRNA expression levels in isolated WT or SOCS3 cKO cardiomyocytes were examined by quantitative PCR at indicated hours post CT-1 (1 nM) stimulation (n=5 in each group). *p<0.05 B, Inhibition of STAT3 activation significantly decreased SCN5A mRNA level in SOCS3 cKO cardiomyocytes. Dominant-negative STAT3 (dnSTAT3) was expressed in SOCS3 cKO cardiomyocytes with adenovirus vector (Ad-dnSTAT3) infection for 12 hours. Adenovirus expressing lacZ (Ad-lacZ) was used as a negative control. Then, the cardiomyocytes were stimulated with CT-1, and the activation of STAT3 (left panel) and SCN5A mRNA (right panel) levels were examined at 4 hours post CT-1 stimulation by western-blot and quantitative PCR, respectively (n=5 in each group). *, **, ***, **** p<0.05 (1-way ANOVA using a Tukey-Kramer post hoc test).

Figure 6

Unregulated gp130-STAT3 is required for the increase in SCN5A mRNA in SOCS3 cKO cardiomyocytes. A, SCN5A mRNA expression levels in isolated WT or SOCS3 cKO cardiomyocytes were examined by quantitative PCR at indicated hours post CT-1 (1 nM) stimulation (n=5 in each group). *p<0.05 B, Inhibition of STAT3 activation significantly decreased SCN5A mRNA level in SOCS3 cKO cardiomyocytes. Dominant-negative STAT3 (dnSTAT3) was expressed in SOCS3 cKO cardiomyocytes with adenovirus vector (Ad-dnSTAT3) infection for 12 hours. Adenovirus expressing lacZ (Ad-lacZ) was used as a negative control. Then, the cardiomyocytes were stimulated with CT-1, and the activation of STAT3 (left panel) and SCN5A mRNA (right panel) levels were examined at 4 hours post CT-1 stimulation by western-blot and quantitative PCR, respectively (n=5 in each group). *, **, ***, **** p<0.05 (1-way ANOVA using a Tukey-Kramer post hoc test).

Figure 7

Role of gp130 activation in the development of mortality and cardiomyopathy in SOCS3 cKO mice. A, gp130 signaling activation was examined in gp130&SOCS3 cDKO (cDKO) hearts at 8 and 15 weeks of age. Representative western-blot from three independent experiments is shown (left panel). The activation of STAT3, ERK1/2, AKT and p38 signaling in SOCS3 cKO mice was abolished in the double KO mice (right panels). Expression level was calculated relative to the mean value of wild-type (WT) group (n=6 in each group). B, gp130&SOCS3 cDKO survived significantly longer than SOCS3 cKO (Logrank test p<0.0001). There was no significant difference in the survival rate between gp130&SOCS3 cDKO and gp130 cKO. C, Histological differences between SOCS3 cKO and gp130&SOCS3 cDKO failing hearts. The transverse section of gp130 cKO (35 weeks old), gp130&SOCS3 cDKO (35 weeks old) and SOCS3 cKO (28 weeks old) failing hearts are shown on the top panels. HE and MT staining of the heart tissue from gp130 cKO and gp130&SOCS3 cDKO are shown on the left lower panel. Unlike SOCS3 cKO, significant fibrosis can be observed in both groups. Scale bar: 200 µm. Heart weight and tibia length ratio (HW/TL) is shown on the right lower panel. Total five hearts in each group were examined. Error bars are ±SEM. *,** p<0.05

Figure 7

Role of gp130 activation in the development of mortality and cardiomyopathy in SOCS3 cKO mice. A, gp130 signaling activation was examined in gp130&SOCS3 cDKO (cDKO) hearts at 8 and 15 weeks of age. Representative western-blot from three independent experiments is shown (left panel). The activation of STAT3, ERK1/2, AKT and p38 signaling in SOCS3 cKO mice was abolished in the double KO mice (right panels). Expression level was calculated relative to the mean value of wild-type (WT) group (n=6 in each group). B, gp130&SOCS3 cDKO survived significantly longer than SOCS3 cKO (Logrank test p<0.0001). There was no significant difference in the survival rate between gp130&SOCS3 cDKO and gp130 cKO. C, Histological differences between SOCS3 cKO and gp130&SOCS3 cDKO failing hearts. The transverse section of gp130 cKO (35 weeks old), gp130&SOCS3 cDKO (35 weeks old) and SOCS3 cKO (28 weeks old) failing hearts are shown on the top panels. HE and MT staining of the heart tissue from gp130 cKO and gp130&SOCS3 cDKO are shown on the left lower panel. Unlike SOCS3 cKO, significant fibrosis can be observed in both groups. Scale bar: 200 µm. Heart weight and tibia length ratio (HW/TL) is shown on the right lower panel. Total five hearts in each group were examined. Error bars are ±SEM. *,** p<0.05

Figure 7

Role of gp130 activation in the development of mortality and cardiomyopathy in SOCS3 cKO mice. A, gp130 signaling activation was examined in gp130&SOCS3 cDKO (cDKO) hearts at 8 and 15 weeks of age. Representative western-blot from three independent experiments is shown (left panel). The activation of STAT3, ERK1/2, AKT and p38 signaling in SOCS3 cKO mice was abolished in the double KO mice (right panels). Expression level was calculated relative to the mean value of wild-type (WT) group (n=6 in each group). B, gp130&SOCS3 cDKO survived significantly longer than SOCS3 cKO (Logrank test p<0.0001). There was no significant difference in the survival rate between gp130&SOCS3 cDKO and gp130 cKO. C, Histological differences between SOCS3 cKO and gp130&SOCS3 cDKO failing hearts. The transverse section of gp130 cKO (35 weeks old), gp130&SOCS3 cDKO (35 weeks old) and SOCS3 cKO (28 weeks old) failing hearts are shown on the top panels. HE and MT staining of the heart tissue from gp130 cKO and gp130&SOCS3 cDKO are shown on the left lower panel. Unlike SOCS3 cKO, significant fibrosis can be observed in both groups. Scale bar: 200 µm. Heart weight and tibia length ratio (HW/TL) is shown on the right lower panel. Total five hearts in each group were examined. Error bars are ±SEM. *,** p<0.05

Figure 8

SOCS3 regulation on gp130 signaling is crucial for the maintenance of cardiomyocyte homeostasis. Under SOCS3 regulation, gp130 signaling stimulates a cardiomyocyte survival signaling cascade. Absence of SOCS3 regulation induces unregulated gp130 activation and atypical gene expression in the cardiomyocyte, which leads to contractile dysfunction and ventricular arrhythmias accompanied by abnormal myofilament Ca²⁺ sensitivity and SR Ca²⁺ overload. There are various factors that can inhibit SOCS3 protein expression such as alterations in transcription, promoter silencing through CpG methylation and microRNA expression that could potentially modulate the plasticity of gp130 signaling.