The Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA.
The Section of Cardiology, The Department of Medicine, Baylor College of Medicine, Houston, TX, 77030, USA.
Cardiovasc Drugs Ther. 2018 Dec;32(6):553-558. doi: 10.1007/s10557-018-6837-3.
We assessed whether the SGLT-2 inhibitor dapagliflozin (Dapa) attenuates the upregulation of the cardiac Na/H exchanger (NHE-1) in vitro in mouse cardiofibroblasts stimulated with lipopolysaccharides (LPS) and whether this effect is dependent on adenosine monophosphate kinase (AMPK) activation.
Mouse cardiofibroblasts were exposed for 16 h to Dapa (0.4 μM), AMPK activator (A769662 (10 μM)), AMPK inhibitor (compound C (CC) (10 μM)), an SGLT-1 and SGLT-2 inhibitor (phlorizin (PZ) (100 μM)), Dapa+CC, or Dapa+PZ, and then stimulated with LPS (10 ng/ml) for 3 h. NHE-1 mRNA levels were assessed by rt-PCR and total AMPK, phosphorylated-AMPK (P-AMPK), NHE-1, and heat shock protein-70 (Hsp70) protein levels in the whole cell lysate by immunoblotting. In addition, NHE-1 protein levels attached to Hsp70 were assessed by immunoprecipitation.
Exposure to LPS significantly reduced P-AMPK levels in the cardiofibroblasts. A769662 and Dapa equally increased P-AMPK. The effect was blocked by CC. Phlorizin had no effect on P-AMPK. LPS exposure significantly increased NHE-1 mRNA levels. Both Dapa and A769662 equally attenuated this increase. The effect of Dapa was blocked with CC. Interestingly, none of the compounds significantly affected NHE-1 and Hsp70 protein levels in the whole cell lysate. However, LPS significantly increased the concentration of NHE-1 attached to Hsp70. Both Dapa and A69662 attenuated this association and CC blocked the effect of Dapa. Again, phlorizin had no effect and did not alter the effect of Dapa.
Dapa increases P-AMPK in cardiofibroblasts exposed to LPS. Dapa attenuated the increase in NHE-1 mRNA and the association between NHE-1 and Hsp70. This effect was dependent on AMPK.
我们评估 SGLT-2 抑制剂达格列净（Dapa）是否能减弱脂多糖（LPS）刺激的体外鼠心脏成纤维细胞中钠/氢交换蛋白（NHE-1）的上调，并评估这种作用是否依赖于单磷酸腺苷激活的蛋白激酶（AMPK）的激活。
用 0.4 μM 的达格列净、AMPK 激活剂（A769662，10 μM）、AMPK 抑制剂（化合物 C，CC，10 μM）、SGLT-1 和 SGLT-2 抑制剂（根皮苷，PZ，100 μM）、达格列净+CC 或达格列净+PZ 处理鼠心脏成纤维细胞 16 小时，然后用 LPS（10 ng/ml）刺激 3 小时。通过逆转录聚合酶链反应评估 NHE-1 mRNA 水平，通过免疫印迹法评估全细胞裂解物中总 AMPK、磷酸化 AMPK（P-AMPK）、NHE-1 和热休克蛋白-70（Hsp70）的蛋白水平。此外，通过免疫沉淀法评估附着在 Hsp70 上的 NHE-1 蛋白水平。
LPS 处理显著降低了心脏成纤维细胞中的 P-AMPK 水平。A769662 和达格列净同样增加了 P-AMPK。CC 阻断了这一作用。根皮苷对 P-AMPK 没有影响。LPS 暴露显著增加了 NHE-1 mRNA 水平。达格列净和 A769662 同样减弱了这种增加。CC 阻断了达格列净的作用。有趣的是，没有一种化合物显著影响全细胞裂解物中的 NHE-1 和 Hsp70 蛋白水平。然而，LPS 显著增加了与 Hsp70 结合的 NHE-1 浓度。达格列净和 A769662 减弱了这种结合，CC 阻断了达格列净的作用。根皮苷没有作用，也没有改变达格列净的作用。
达格列净增加了 LPS 刺激的心脏成纤维细胞中的 P-AMPK。达格列净减弱了 NHE-1 mRNA 的增加和 NHE-1 与 Hsp70 之间的关联。这种作用依赖于 AMPK。
