Pathogenesis associated with a restrictive cardiomyopathy mutant in cardiac troponin T is due to reduced protein stability and greatly increased myofilament Ca2+ sensitivity.
Pathogenesis associated with a restrictive cardiomyopathy mutant in cardiac troponin T is due to reduced protein stability and greatly increased myofilament Ca2+ sensitivity.
复制标题
与心肌肌钙蛋白 T 的限制性心肌病突变相关的发病机制是由于蛋白质稳定性降低和肌丝 Ca2+ 敏感性大大增加。
DOI：
10.1016/j.bbagen.2014.09.029
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Pinto,JoseRenato
中科院分区：
文献类型：
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作者：
Parvatiyar,MichelleS;Pinto,JoseRenato
背景扩张型和肥厚型心肌病肌钙蛋白突变可以减弱蛋白激酶A(PKA)磷酸化心肌肌钙蛋白I(CTnI)的作用，降低肌丝对钙的敏感性；然而，这种效应从未在限制性心肌病(RCM)突变中得到测试。本研究探讨心肌肌钙蛋白T突变体(cTnT-ΔE96)是否干扰心肌肌钙蛋白A的趋化调节，以及肌钙蛋白T的不稳定性是否有助于极大地增强猪心肌纤维对钙的敏感性。方法采用猪心肌肌钙蛋白T突变体(cTnT-cTnT-E96)对猪去皮心肌纤维进行收缩作用力和光谱分析。结果与WT或cTnT-−E96共同孵育后，猪去皮纤维的钙敏感性分别下降了0.26和0.25pCa单位。为了进一步评估cTnT-ΔE96是否单独干扰cTnI磷酸化效应的传递，用PKA伪磷酸化cTnI(cTnI-SS/DD.cTnC)重建皮肤纤维。CTnT-WT置换的纤维，与cTnI-SS/DD.cTnC重组后，与对照cTnI-WT.cTnC(pCa50=65.75)相比，力的钙敏感性降低(pCa50=55.61)，与对照(pCa50=66.14)相比，同样影响cTnT-ΔE96(pCa50=66.03)。由cTnT-ΔE96引起的cTnCIAANSCa2+亲和力变化的荧光研究表明，更高的复杂性(细丝)更好地概括了皮肤纤维对钙的敏感性变化。圆二色谱显示cTnT-αE96的Δ螺旋度降低，热折叠提前。结论cTnT-ΔE96虽然不能有效降低肌丝对钙离子的敏感性，但不干扰Δ的磷酸化作用；2)cTnT-ΔE96需要肌动蛋白来增加cTnC的钙亲和力；3)缺失E96降低了cTnT的稳定性，可能破坏了关键的细丝相互作用。
BackgroundDilated and hypertrophic cardiomyopathy mutations in troponin can blunt effects of protein kinase A (PKA) phosphorylation of cardiac troponin I (cTnI), decreasing myofilament Ca2+-sensitivity; however this effect has never been tested for restrictive cardiomyopathy (RCM) mutants. This study explores whether an RCM cardiac troponin T mutant (cTnT-ΔE96) interferes with convergent PKA regulation and if TnT instability contributes to greatly enhanced Ca2+-sensitivity in skinned fibers.MethodsForce of contraction in skinned cardiac porcine fiber and spectroscopic studies were performed.ResultsA decrease of − 0.26 and − 0.25pCa units in Ca2+-sensitivity of contraction after PKA incubation was observed for skinned fibers incorporated with WT or cTnT-ΔE96, respectively. To further assess whether cTnT-ΔE96 interferes solely with transmission of cTnI phosphorylation effects, skinned fibers were reconstituted with PKA pseudo-phosphorylated cTnI (cTnI-SS/DD.cTnC). Fibers displaced with cTnT-WT, reconstituted with cTnI-SS/DD.cTnC decreased Ca2+-sensitivity of force (pCa50= 5.61) compared to control cTnI-WT.cTnC (pCa50= 5.75), similarly affecting cTnT-ΔE96 (pCa50= 6.03) compared to control \cTnI-WT.cTnC (pCa50= 6.14). Fluorescence studies measuring cTnCIAANSCa2+-affinity changes due to cTnT-ΔE96 indicated that higher complexity (thin filament) better recapitulates skinned fiber Ca2+sensitive changes. Circular dichroism revealed reduced α-helicity and earlier thermal unfolding for cTnT-ΔE96 compared to WT.ConclusionsAlthough ineffective in decreasing myofilament Ca2+-sensitivity to normal levels, cTnT-ΔE96 does not interfere with PKA cTnI phosphorylation mediated effects; 2) cTnT-ΔE96 requires actin to increase cTnC Ca2+-affinity; and 3) deletion of E96 reduces cTnT stability, likely disrupting crucial thin filament interactions.General significanceThe pathological effect of cTnT-ΔE96 is largely manifested by dramatic myofilament Ca2+-sensitization which still persists even after PKA phosphorylation mediated Ca2+-desensitization.