Source data for Kasik et al. Causal relevance of the JAK/STAT pathway for cardioprotection via cold acclimation

ABSTRACT
Cardioprotective effect of cold acclimation (5 weeks; 9°C; CA) and the same effect persisting 2 weeks after recovery from CA (CAR) are induced by different mechanisms. We showed that salvage β2-adrenoceptor/Gi/Akt pathway is only involved in the mechanism of cardioprotection after CAR. Since the mechanism of CA-elicited cardioprotection is not known, we examined the role of JAK2/STAT3 pathway. Male Wistar rats exposed to CA and controls (24°C) were treated with the JAK2 inhibitor AG490 affecting downstream STAT3 signaling in the heart (5 mg/kg/day) for three days prior to the end of experiment. AG490 administration abolished CA-elicited reduction of infarct-size and significant improvement of MPT pore opening. IL-6, as the main STAT3 upstream effector, was upregulated by CA, and AG490 reversed its level. CA had no effect on IL-1β and TNF-α, but was upregulated by AG490 in controls, and downregulated in CA-AG490 group. CA also reduced pro-apoptotic p38-MAPK, which was abolished by AG490 administration. Spatial expression analyses revealed CA-elicited translocation of total-STAT3 from mitochondria to sarcolemma compartment which was eliminated by JAK2/STAT3 inhibition. However, CA-induced loss of pSTAT3Y705 from sarcolemma compartment, and loss of pSTAT3S727 from nucleus. These results identify non-genomic, mitochondria-associated STAT3 activity as a confirmed mechanism of CA-elicited cardioprotection.

摘要
冷驯化（cold acclimation，CA，时长5周，温度9℃）所产生的心脏保护作用，以及冷驯化恢复（CAR）2周后仍持续存在的同款心脏保护作用，二者的诱导机制各不相同。本研究证实，补救性β2肾上腺素能受体/Gi蛋白/Akt信号通路仅参与CAR介导的心脏保护作用机制。鉴于CA诱导的心脏保护作用机制尚未明确，本研究针对JAK2/STAT3信号通路的作用展开了探究。将暴露于CA环境的雄性Wistar大鼠与对照组（饲养于24℃环境）均给予JAK2抑制剂AG490（每日5 mg/kg，通过影响心脏内下游STAT3信号通路发挥作用），于实验结束前连续给药3天。给予AG490可抵消CA诱导的梗死面积缩小以及线粒体通透性转换孔（MPT pore）开放状态的显著改善。作为STAT3的主要上游效应因子，白细胞介素6（IL-6）的表达在CA处理后被上调，而AG490可逆转这一表达变化。冷驯化对IL-1β与肿瘤坏死因子α（TNF-α）的表达无影响，但AG490可在对照组中上调二者的表达，而在CA-AG490联合处理组中则使其表达下调。CA还可降低促凋亡p38丝裂原活化蛋白激酶（p38-MAPK）的水平，这一效应可被AG490给药所抵消。空间表达分析结果显示，CA可诱导总STAT3蛋白从线粒体向肌膜区域发生转位，这一转位过程可被JAK2/STAT3通路抑制所消除。但CA可导致肌膜区域的酪氨酸705位点磷酸化STAT3（pSTAT3Y705）水平下降，同时使细胞核内的丝氨酸727位点磷酸化STAT3（pSTAT3S727）水平降低。上述研究结果证实，非基因组依赖、与线粒体相关的STAT3活性是CA诱导心脏保护作用的明确机制。