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Abstract：
Duchenne muscular dystrophy (DMD) is a progressive muscle degenerative disorder, culminating in a complete loss of ambulation, hypertrophic cardiomyopathy and a fatal cardiorespiratory failure. Necroptosis is the form of necrosis that is dependent upon the receptor-interacting protein kinase (RIPK) 3; it is involved in several inflammatory and neurodegenerative conditions. We previously identified RIPK3 as a key player in the acute myonecrosis affecting the hindlimb muscles of the mdx dystrophic mouse model. Whether necroptosis also mediates respiratory and heart disorders in DMD is currently unknown.
Evidence of activation of the necroptotic axis was examined in dystrophic tissues from Golden retriever muscular dystrophy (GRMD) dogs and R-DMDdel52 rats. A functional assessment of the involvement of necroptosis in dystrophic animals was performed on mdx mice that were genetically depleted for RIPK3. Dystrophic mice aged from 12 to 18 months were analysed by histology and molecular biology to compare the phenotype of muscles from mdxRipk3+/+ and mdxRipk3-/- mice. Heart function was also examined by echocardiography in 40-week-old mice.
RIPK3 expression in sartorius and biceps femoris muscles from GRMD dogs positively correlated to myonecrosis levels (r = 0.81; P = 0.0076). RIPK3 was also found elevated in the diaphragm (P ≤ 0.05). In the slow-progressing heart phenotype of GRMD dogs, the phosphorylated form of RIPK1 at the Serine 161 site was dramatically increased in cardiomyocytes. A similar p-RIPK1 upregulation characterized the cardiomyocytes of the severe DMDdel52 rat model, associated with a marked overexpression of Ripk1 (P = 0.007) and Ripk3 (P = 0.008), indicating primed activation of the necroptotic pathway in the dystrophic heart. MdxRipk3-/- mice displayed decreased compensatory hypertrophy of the heart (P = 0.014), and echocardiography showed a 19% increase in the relative wall thickness (P < 0.05) and 29% reduction in the left ventricle mass (P = 0.0144). Besides, mdxRipk3-/- mice presented no evidence of a regenerative default or sarcopenia in skeletal muscles, moreover around 50% less affected by fibrosis (P < 0.05).
Our data highlight molecular and histological evidence that the necroptotic pathway is activated in degenerative tissues from dystrophic animal models, including the diaphragm and the heart. We also provide the genetic proof of concept that selective inhibition of necroptosis in dystrophic condition improves both histological features of muscles and cardiac function, suggesting that prevention of necroptosis is susceptible to providing multiorgan beneficial effects for DMD.
Evidence of activation of the necroptotic axis was examined in dystrophic tissues from Golden retriever muscular dystrophy (GRMD) dogs and R-DMDdel52 rats. A functional assessment of the involvement of necroptosis in dystrophic animals was performed on mdx mice that were genetically depleted for RIPK3. Dystrophic mice aged from 12 to 18 months were analysed by histology and molecular biology to compare the phenotype of muscles from mdxRipk3+/+ and mdxRipk3-/- mice. Heart function was also examined by echocardiography in 40-week-old mice.
RIPK3 expression in sartorius and biceps femoris muscles from GRMD dogs positively correlated to myonecrosis levels (r = 0.81; P = 0.0076). RIPK3 was also found elevated in the diaphragm (P ≤ 0.05). In the slow-progressing heart phenotype of GRMD dogs, the phosphorylated form of RIPK1 at the Serine 161 site was dramatically increased in cardiomyocytes. A similar p-RIPK1 upregulation characterized the cardiomyocytes of the severe DMDdel52 rat model, associated with a marked overexpression of Ripk1 (P = 0.007) and Ripk3 (P = 0.008), indicating primed activation of the necroptotic pathway in the dystrophic heart. MdxRipk3-/- mice displayed decreased compensatory hypertrophy of the heart (P = 0.014), and echocardiography showed a 19% increase in the relative wall thickness (P < 0.05) and 29% reduction in the left ventricle mass (P = 0.0144). Besides, mdxRipk3-/- mice presented no evidence of a regenerative default or sarcopenia in skeletal muscles, moreover around 50% less affected by fibrosis (P < 0.05).
Our data highlight molecular and histological evidence that the necroptotic pathway is activated in degenerative tissues from dystrophic animal models, including the diaphragm and the heart. We also provide the genetic proof of concept that selective inhibition of necroptosis in dystrophic condition improves both histological features of muscles and cardiac function, suggesting that prevention of necroptosis is susceptible to providing multiorgan beneficial effects for DMD.
摘要：
背景：杜氏肌营养不良症（DMD）是一种进行性肌肉退行性疾病，最终完全失去了步行能力，肥厚型心肌病和致命的心肺功能衰竭。坏死性凋亡是依赖于受体相互作用蛋白激酶(RIPK)3的坏死形式;它与几种炎症和神经退行性疾病有关。我们先前确定RIPK3是影响mdx营养不良小鼠模型后肢肌肉的急性心肌坏死的关键参与者。目前尚不清楚坏死是否也介导DMD中的呼吸和心脏疾病。
方法：在金毛猎犬肌营养不良（GRMD）犬和R-DMDDel52大鼠的营养不良组织中检查了坏死轴激活的证据。对营养不良动物中坏死的参与进行了功能评估，这些小鼠在遗传上耗尽了RIPK3。通过组织学和分子生物学分析12至18月龄的营养不良小鼠,以比较mdxRipk3+/+和mdxRipk3-/-小鼠的肌肉表型。还通过超声心动图检查了40周龄小鼠的心脏功能。
结果：GRMD犬的缝纫肌和股二头肌中RIPK3的表达与心肌坏死水平呈正相关（r=0.81；P=0.0076）。RIPK3在隔膜中也发现升高（P≤0.05）。在GRMD犬缓慢进展的心脏表型中，RIPK1在丝氨酸161位点的磷酸化形式在心肌细胞中显著增加.一个类似的p-RIPK1上调表征重度DMDDel52大鼠模型的心肌细胞,与Ripk1（P=0.007）和Ripk3（P=0.008）的明显过表达有关，表明营养不良心脏中坏死途径的激活。MdxRipk3-/-小鼠心脏代偿性肥大减少(P=0.014),超声心动图显示相对壁厚增加19％（P<0.05），左心室质量减少29％（P=0.0144）。此外,mdxRipk3-/-小鼠在骨骼肌中没有出现再生缺陷或肌肉减少症的证据，此外，约50%的纤维化影响（P<0.05）。
结论：我们的数据突出了分子和组织学证据，表明在营养不良动物模型的变性组织中，坏死途径被激活，包括隔膜和心脏.我们还提供了遗传证据的概念，即在营养不良状态下选择性抑制坏死可以改善肌肉和心脏功能的组织学特征。提示预防坏死性凋亡易于为DMD提供多器官有益作用。
方法：在金毛猎犬肌营养不良（GRMD）犬和R-DMDDel52大鼠的营养不良组织中检查了坏死轴激活的证据。对营养不良动物中坏死的参与进行了功能评估，这些小鼠在遗传上耗尽了RIPK3。通过组织学和分子生物学分析12至18月龄的营养不良小鼠,以比较mdxRipk3+/+和mdxRipk3-/-小鼠的肌肉表型。还通过超声心动图检查了40周龄小鼠的心脏功能。
结果：GRMD犬的缝纫肌和股二头肌中RIPK3的表达与心肌坏死水平呈正相关（r=0.81；P=0.0076）。RIPK3在隔膜中也发现升高（P≤0.05）。在GRMD犬缓慢进展的心脏表型中，RIPK1在丝氨酸161位点的磷酸化形式在心肌细胞中显著增加.一个类似的p-RIPK1上调表征重度DMDDel52大鼠模型的心肌细胞,与Ripk1（P=0.007）和Ripk3（P=0.008）的明显过表达有关，表明营养不良心脏中坏死途径的激活。MdxRipk3-/-小鼠心脏代偿性肥大减少(P=0.014),超声心动图显示相对壁厚增加19％（P<0.05），左心室质量减少29％（P=0.0144）。此外,mdxRipk3-/-小鼠在骨骼肌中没有出现再生缺陷或肌肉减少症的证据，此外，约50%的纤维化影响（P<0.05）。
结论：我们的数据突出了分子和组织学证据，表明在营养不良动物模型的变性组织中，坏死途径被激活，包括隔膜和心脏.我们还提供了遗传证据的概念，即在营养不良状态下选择性抑制坏死可以改善肌肉和心脏功能的组织学特征。提示预防坏死性凋亡易于为DMD提供多器官有益作用。
