Chang Audrey N, Harada Keita, Ackerman Michael J, Potter James D
Department of Medicine, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA.
J Biol Chem. 2005 Oct 7;280(40):34343-9. doi: 10.1074/jbc.M505014200. Epub 2005 Jul 25.
To study the functional consequences of various cardiomyopathic mutations in human cardiac alpha-tropomyosin (Tm), a method of depletion/reconstitution of native Tm and troponin (Tn) complex (Tm-Tn) in cardiac myofibril preparations has been developed. The endogenous Tm-Tn complex was selectively removed from myofibrils and replaced with recombinant wild-type or mutant proteins. Successful depletion and reconstitution steps were verified by SDS-gel electrophoresis and by the loss and regain of Ca2+-dependent regulation of ATPase activity. Five Tm mutations were chosen for this study: the hypertrophic cardiomyopathy (HCM) mutations E62Q, E180G, and L185R and the dilated cardiomyopathy (DCM) mutations E40K and E54K. Through the use of this new depletion/reconstitution method, the functional consequences of these mutations were determined utilizing myofibrillar ATPase measurements. The results of our studies showed that 1) depletion of >80% of Tm-Tn from myofibrils resulted in a complete loss of the Ca2+-regulated ATPase activity and a significant loss in the maximal ATPase activity, 2) reconstitution of exogenous wild-type Tm-Tn resulted in complete regain in the calcium regulation and in the maximal ATPase activity, and 3) all HCM-associated Tm mutations increased the Ca2+ sensitivity of ATPase activity and all had decreased abilities to inhibit ATPase activity. In contrast, the DCM-associated mutations both decreased the Ca2+ sensitivity of ATPase activity and had no effect on the inhibition of ATPase activity. These findings have demonstrated that the mutations which cause HCM and DCM disrupt discrete mechanisms, which may culminate in the distinct cardiomyopathic phenotypes.
为了研究人类心脏α-原肌球蛋白（Tm）中各种心肌病突变的功能后果，已经开发了一种在心肌肌原纤维制剂中耗尽/重建天然Tm和肌钙蛋白（Tn）复合物（Tm-Tn）的方法。内源性Tm-Tn复合物从肌原纤维中被选择性去除，并用重组野生型或突变蛋白替代。通过SDS-凝胶电泳以及ATP酶活性的Ca2+依赖性调节的丧失和恢复来验证成功的耗尽和重建步骤。本研究选择了五个Tm突变：肥厚型心肌病（HCM）突变E62Q、E180G和L185R以及扩张型心肌病（DCM）突变E40K和E54K。通过使用这种新的耗尽/重建方法，利用肌原纤维ATP酶测量来确定这些突变的功能后果。我们的研究结果表明：1）从肌原纤维中耗尽>80%的Tm-Tn导致Ca2+调节的ATP酶活性完全丧失以及最大ATP酶活性显著丧失；2）外源性野生型Tm-Tn的重建导致钙调节和最大ATP酶活性完全恢复；3）所有与HCM相关的Tm突变均增加了ATP酶活性对Ca2+的敏感性，并且所有突变抑制ATP酶活性的能力均降低。相比之下，与DCM相关的突变既降低了ATP酶活性对Ca2+的敏感性，又对ATP酶活性的抑制没有影响。这些发现表明，导致HCM和DCM的突变破坏了不同的机制，这可能最终导致不同的心肌病表型。
