Integrity of the network sarcoplasmic reticulum in skeletal muscle requires small ankyrin 1
Maegen A. AckermannAndrew P. ZimanJohn StrongYing‐Hua ZhangApril K. HartfordChristopher W. WardWilliam R. RandallAikaterini Kontrogianni‐KonstantopoulosRobert J. Bloch
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Abstract:
Small ankyrin 1 (sAnk1; Ank1.5) is a ~20 kDa protein of striated muscle that concentrates in the network compartment of the sarcoplasmic reticulum (nSR). We used siRNA targeted to sAnk1 to assess its role in organizing the sarcoplasmic reticulum (SR) of skeletal myofibers in vitro. siRNA reduced sAnk1 mRNA and protein levels and disrupted the organization of the remaining sAnk1. Sarcomeric proteins were unchanged, but two other proteins of the nSR, SERCA and sarcolipin, decreased significantly in amount and segregated into distinct structures containing sarcolipin and sAnk1, and SERCA, respectively. Exogenous sAnk1 restored SERCA to its normal distribution. Ryanodine receptors and calsequestrin in the junctional SR, and L-type Ca2+ channels in the transverse tubules were not reduced, although their striated organization was mildly altered. Consistent with the loss of SERCA, uptake and release of Ca2+ were significantly inhibited. Our results show that sAnk1 stabilizes the nSR and that its absence causes the nSR to fragment into distinct membrane compartments.Keywords:
SERCA
Calsequestrin
Ankyrin
Cardiac muscle
Sarcoplasm
The cardiac ryanodine receptor (RyR2) is the sarcoplasmic reticulum (SR) Ca2+ release channel which is responsible for generation of the cytosolic Ca2+ transient required for activation of cardiac contraction. RyR2 functional activity is governed by changes in [Ca2+] on both the cytosolic and luminal phase of the RyR2 channel. Activation of RyR2 by cytosolic Ca2+ results in Ca2+-induced Ca2+ release (CICR) from the SR. The decline in luminal [Ca2+] following release contributes to termination of CICR and Ca2+ signalling refractoriness through the process of luminal Ca2+-dependent deactivation of RyR2s. The control of RyR2s by luminal Ca2+ involves coordinated interaction of the channel with several SR proteins, including the Ca2+-binding protein calsequestrin (CASQ2), and the integral proteins triadin 1 (TRD) and junctin (JCN). CASQ2 in addition to serving as a Ca2+ storage site and a luminal Ca2+ buffer modulates RyR2 function more directly as a putative luminal Ca2+ sensor. TRD and JCN, stimulatory by themselves, mediate the interactions between CASQ2 and RyR2. Acquired and genetic defects in proteins of this junctional Ca2+ signalling complex lead to disease states such as cardiac arrhythmia and heart failure by impairing luminal Ca2+ regulation of RyR2.
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瞄准：比较调整蛋白质在的 Ca (2+) 的表示上面胃肠(官方补给) 弛缓不能病人和健康志愿者并且到的道阐明他们在弛缓不能的角色。方法：肌质的蜂窝胃 Ca (2+) ATPase (SERCA ) isoforms 2a 和 2b， phospholamban (PLB ) ， calsequestrin (CSQ ) ，和 calreticulin (CRT ) 被在老鼠，兔子，和人的食管和心的量的西方的弄污估计。而且，在从有弛缓不能和健康志愿者的病人的更低的食道的括约肌和食管的活体检视的这些蛋白质的表示侧面被分析。结果：SERCA 2a 蛋白质表示与食管相比在人的心(心脏的室) 是高得多的。然而， SERCA 2b 在食管主要被表示。当尽管高度在心表示了， PLB 在我们在上面的官方补给的织物的察觉限制下面时，最高的 CRT 表示在人的食管被注意。在更低的食道的括约肌和远侧的食道的身体比作健康控制， CSQ 和 CRT 表示显著地与弛缓不能在病人被减少(P < 0.05 ) 。结论：在人的食管的 PLB 可能比在心具有为 SERCA 的规定的更小的重要性。Ca (2+) 存储蛋白质(CSQ 和 CRT ) 的更低的表示可能在弛缓不能贡献增加的更低的食道的括约肌压力，可能由增加免费细胞内部的 Ca (2+) 。
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Calsequestrin (CASQ2) is the primary Ca2+ buffering protein in the cardiac SR lumen and maintains a fine balance between free vs. bound calcium ion concentration. CASQ2, along with Triadin (TRD1) and Junctin forms a lumenal regulatory complex and controls Ryanodine receptor (RyR2) activity including channel opening, closing and refractoriness. Recent studies have clearly demonstrated that mutations in CASQ2 can result in altered RYR2 function/Ca2+ leak and triggered arrhythmias, especially CPVT (Catecholamine induced Polymorphic Ventricular Tachycardia). In this context we have been studying the importance of CASQ2 using a CASQ2 KO mouse model. These mice were able to maintain cardiac function and Ca2+ handling under resting conditions but were quite susceptible to triggered arrhythmias by catecholamines. We hypothesized that CASQ2 function is more important during increased SR loading, therefore we increased SR Ca2+ load by breeding PLB KO with CASQ2 KO mice. This resulted in an increased SR Ca2+ load but also increased RyR2 mediated Ca2+ leak and susceptibility to triggered arrhythmias. Interestingly the double KO mice developed severe, cardiac hypertrophy and die prematurely due to heart failure between 6 and 8 months. These data provide novel information to suggest that CASQ2 plays an important role in SR Ca2+ buffering function (Free vs. Bound Calcium) and stabilization of the Ryanodine receptor (open probability), thereby maintaining a fine balance between SR load and RyR2 mediated Ca2+ release. These studies also provide compelling evidence that chronic Ca2+ leak due to deregulated RyR2 acts as a causative mechanism to develop heart failure.
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Background Catecholaminergic polymorphic ventricular tachycardia ( CPVT ) is a familial arrhythmogenic syndrome characterized by sudden death. There are several genetic forms of CPVT associated with mutations in genes encoding the cardiac ryanodine receptor (RyR2) and its auxiliary proteins including calsequestrin ( CASQ 2) and calmodulin (CaM). It has been suggested that impairment of the ability of RyR2 to stay closed (ie, refractory) during diastole may be a common mechanism for these diseases. Here, we explore the possibility of engineering CaM variants that normalize abbreviated RyR2 refractoriness for subsequent viral‐mediated delivery to alleviate arrhythmias in non–CaM‐related CPVT . Methods and Results To that end, we have designed a CaM protein ( GSH ‐M37Q; dubbed as therapeutic CaM or T‐CaM) that exhibited a slowed N‐terminal Ca dissociation rate and prolonged RyR2 refractoriness in permeabilized myocytes derived from CPVT mice carrying the CASQ 2 mutation R33Q. This T‐CaM was introduced to the heart of R33Q mice through recombinant adeno‐associated viral vector serotype 9. Eight weeks postinfection, we performed confocal microscopy to assess Ca handling and recorded surface ECGs to assess susceptibility to arrhythmias in vivo. During catecholamine stimulation with isoproterenol, T‐CaM reduced isoproterenol‐promoted diastolic Ca waves in isolated CPVT cardiomyocytes. Importantly, T‐CaM exposure abolished ventricular tachycardia in CPVT mice challenged with catecholamines. Conclusions Our results suggest that gene transfer of T‐CaM by adeno‐associated viral vector serotype 9 improves myocyte Ca handling and alleviates arrhythmias in a calsequestrin‐associated CPVT model, thus supporting the potential of a CaM‐based antiarrhythmic approach as a therapeutic avenue for genetically distinct forms of CPVT .
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ABSTRACT Cardiac muscle contraction requires sarcoplasmic reticulum (SR) Ca2+ release mediated by the quaternary complex comprising the ryanodine receptor 2 (RyR2), calsequestrin 2 (CSQ2), junctin (encoded by ASPH) and triadin. Here, we demonstrate that a direct interaction exists between RyR2 and CSQ2. Topologically, CSQ2 binding occurs at the first luminal loop of RyR2. Co-expression of RyR2 and CSQ2 in a human cell line devoid of the other quaternary complex proteins results in altered Ca2+-release dynamics compared to cells expressing RyR2 only. These findings provide a new perspective for understanding the SR luminal Ca2+ sensor and its involvement in cardiac physiology and disease.
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Calcium in biology
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