Miller Barbara A, Wang JuFang, Song Jianliang, Zhang Xue-Qian, Hirschler-Laszkiewicz Iwona, Shanmughapriya Santhanam, Tomar Dhanendra, Rajan Sudasan, Feldman Arthur M, Madesh Muniswamy, Sheu Shey-Shing, Cheung Joseph Y
Department of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania.
Center for Translational Medicine, Department of Pharmacology, Lewis Katz School of Medicine of Temple University, Philadelphia, Pennsylvania.
J Cell Physiol. 2019 Sep;234(9):15048-15060. doi: 10.1002/jcp.28146. Epub 2019 Jan 13.
The mechanisms by which Trpm2 channels enhance mitochondrial bioenergetics and protect against oxidative stress-induced cardiac injury remain unclear. Here, the role of proline-rich tyrosine kinase 2 (Pyk2) in Trpm2 signaling is explored. Activation of Trpm2 in adult myocytes with H O resulted in 10- to 21-fold increases in Pyk2 phosphorylation in wild-type (WT) myocytes which was significantly lower (40%) in Trpm2 knockout (KO) myocytes. Pyk2 phosphorylation was inhibited (54%) by the Trpm2 blocker clotrimazole. Buffering Trpm2-mediated Ca increase with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) resulted in significantly reduced pPyk2 in WT but not in KO myocytes, indicating Ca influx through activated Trpm2 channels phosphorylated Pyk2. Part of phosphorylated Pyk2 translocated from cytosol to mitochondria which has been previously shown to augment mitochondrial Ca uptake and enhance adenosine triphosphate generation. Although Trpm2-mediated Ca influx phosphorylated Ca -calmodulin kinase II (CaMKII), the CaMKII inhibitor KN93 did not significantly affect Pyk2 phosphorylation in H O -treated WT myocytes. After ischemia/reperfusion (I/R), Pyk2 phosphorylation and its downstream prosurvival signaling molecules (pERK1/2 and pAkt) were significantly lower in KO-I/R when compared with WT-I/R hearts. After hypoxia/reoxygenation, mitochondrial membrane potential was lower and superoxide level was higher in KO myocytes, and were restored to WT values by the mitochondria-targeted superoxide scavenger MitoTempo. Our results suggested that Ca influx via tonically activated Trpm2 phosphorylated Pyk2, part of which translocated to mitochondria, resulting in better mitochondrial bioenergetics to maintain cardiac health. After I/R, Pyk2 activated prosurvival signaling molecules and prevented excessive increases in reactive oxygen species, thereby affording protection from I/R injury.
瞬时受体电位M2型（Trpm2）通道增强线粒体生物能量学并保护心脏免受氧化应激诱导损伤的机制仍不清楚。在此，我们探究了富含脯氨酸的酪氨酸激酶2（Pyk2）在Trpm2信号传导中的作用。用H₂O₂激活成年心肌细胞中的Trpm2，导致野生型（WT）心肌细胞中Pyk2磷酸化增加10至21倍，而在Trpm2基因敲除（KO）心肌细胞中显著降低（约40%）。Trpm2阻滞剂克霉唑抑制了Pyk2磷酸化（约54%）。用1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA）缓冲Trpm2介导的Ca²⁺增加，导致WT心肌细胞中磷酸化Pyk2（pPyk2）显著降低，但在KO心肌细胞中未降低，表明通过激活的Trpm2通道的Ca²⁺内流使Pyk2磷酸化。部分磷酸化的Pyk2从细胞质转移到线粒体，先前已证明这会增加线粒体Ca²⁺摄取并增强三磷酸腺苷生成。尽管Trpm2介导的Ca²⁺内流使Ca²⁺-钙调蛋白激酶II（CaMKII）磷酸化，但CaMKII抑制剂KN93对H₂O₂处理的WT心肌细胞中Pyk2磷酸化没有显著影响。缺血/再灌注（I/R）后，与WT-I/R心脏相比，KO-I/R心脏中Pyk2磷酸化及其下游促生存信号分子（磷酸化细胞外信号调节激酶1/2，pERK1/2和磷酸化蛋白激酶B，pAkt）显著降低。缺氧/复氧后，KO心肌细胞中的线粒体膜电位较低，超氧化物水平较高，而线粒体靶向超氧化物清除剂MitoTempo将其恢复到WT值。我们的结果表明，通过持续激活的Trpm2的Ca²⁺内流使Pyk2磷酸化，其中一部分转移到线粒体，导致更好的线粒体生物能量学以维持心脏健康。I/R后，Pyk2激活促生存信号分子并防止活性氧过度增加，从而提供对I/R损伤的保护。
