Myofibrillar creatine kinase and cardiac contraction

This article is a review on the organization and function of myofibrillar creatine kinase in striated muscle. The first part describes myofibrillar creatine kinase as an integral structural part of the complex organization of myofibrils in striated muscle. The second part considers the intrinsic biochemical and mechanical properties of myofibrils and the functional coupling between myofibrillar CK and myosin ATPase. Skinned fiber studies have been developed to evidence this functional coupling and the consequences for cardiac contraction. The data show that creatine kinase in myofibrils is effective enough to sustain normal tension and relaxation, normal Ca sensitivity and kinetic characteristics. Moreover, the results suggest that myofibrillar creatine kinase is essential in maintaining adequate ATP/ADP ratio in the vicinity of myosin ATPase active site to prevent dysfunctioning of this enzyme. Implications for the physiology and physiopathology of cardiac muscle are discussed.     

Titin (Visco-) Elasticity in Skeletal Muscle Myofibrils

Titin is the third most abundant protein in sarcomeres and fulfills a number of mechanical and signaling functions. Specifically, titin is responsible for most of the passive forces in sarcomeres and the passive visco-elastic behaviour of myofibrils and muscles. It has been suggested, based on mechanical testing of isolated titin molecules, that titin is an essentially elastic spring if Ig domain un/refolding is prevented either by working at short titin lengths, prior to any unfolding of Ig domains, or at long sarcomere (and titin) lengths when Ig domain un/refolding is effectively prevented. However, these properties of titin, and by extension of muscles, have not been tested with titin in its natural structural environment within a sarcomere. The purpose of this study was to gain insight into the Ig domain un/refolding kinetics and test the idea that titin could behave essentially elastically at any sarcomere length by preventing Ig domain un/refolding during passive stretch-shortening cycles. Although not completely successful, we demonstrate here that titin’s visco-elastic properties appear to depend on the Ig domain un/refolding kinetics and that indeed, titin (and thus myofibrils) can become virtually elastic when Ig domain un/refolding is prevented.     

心肌特异表达的肌小节相关激酶基因p93的克隆与鉴定

心肌收缩受到由蛋白质因子构成的信号转导通路的调控 ,但确切机制尚未完全明了。从人心脏cDNA文库中克隆到心肌特异表达的可能参与信号转导调控的新基因 ,命名为p93基因。该基因定位于 1p31.1,属于MAP KKKs家族的相近亚家族。Northern印迹及含 76种组织的点杂交显示了p93仅在心肌组织中表达 ;免疫组化表明它主要定位于成人与胎儿的心肌细胞核 ,胞液次之 ;体外激酶活性实验证明野生型p93是一个可以进行自我磷酸化的功能性激酶分子 ;以该基因C端为诱饵质粒的酵母双杂交筛选表明 ,p93主要与心肌肌钙蛋白I(cTnI)等与收缩有关的肌小节蛋白发生相互作用 ,并以免疫共沉淀实验得到了验证。推测p93可能通过激酶信号转导通路的方式参与对肌小节收缩蛋白的调节。     

The interaction of tropomodulin with tropomyosin stabilizes thin filaments in cardiac myocytes

Actin (thin) filament length regulation and stability are essential for striated muscle function. To determine the role of the actin filament pointed end capping protein, tropomodulin1 (Tmod1), with tropomyosin, we generated monoclonal antibodies (mAb17 and mAb8) against Tmod1 that specifically disrupted its interaction with tropomyosin in vitro. Microinjection of mAb17 or mAb8 into chick cardiac myocytes caused a dramatic loss of the thin filaments, as revealed by immunofluorescence deconvolution microscopy. Real-time imaging of live myocytes expressing green fluorescent protein-alpha-tropomyosin and microinjected with mAb17 revealed that the thin filaments depolymerized from their pointed ends. In a thin filament reconstitution assay, stabilization of the filaments before the addition of mAb17 prevented the loss of thin filaments. These studies indicate that the interaction of Tmod1 with tropomyosin is critical for thin filament stability. These data, together with previous studies, indicate that Tmod1 is a multifunctional protein: its actin filament capping activity prevents thin filament elongation, whereas its interaction with tropomyosin prevents thin filament depolymerization.     

Titin mutations as the molecular basis for dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a heterogeneous cardiac disease characterized by ventricular dilatation and systolic dysfunction. Recent genetic studies have revealed that mutations in genes for cardiac sarcomere components lead to DCM. The cardiac sarcomere consists of thick and thin filaments and a giant protein, titin. Because one of the loci of familial DCM was mapped to the region of the titin gene, we searched for titin mutations in the patients and identified four possible disease-associated mutations. Two mutations, Val54Met and Ala743Val, were found in the Z-line region of titin and decreased binding affinities of titin to Z-line proteins T-cap/telethonin and alpha-actinin, respectively, in yeast two-hybrid assays. The other two mutations were found in the cardiac-specific N2-B region of titin and one of them was a nonsense mutation, Glu4053ter, presumably encoding for a truncated nonfunctional molecule. These observations suggest that titin mutations may cause DCM in a subset of the patients.     

The mechanisms of the residual force enhancement after stretch of skeletal muscle: non-uniformity in half-sarcomeres and stiffness of titin

When activated skeletal muscles are stretched, the force increases significantly. After the stretch, the force decreases and reaches a steady-state level that is higher than the force produced at the corresponding length during purely isometric contractions. This phenomenon, referred to as residual force enhancement, has been observed for more than 50 years, but the mechanism remains elusive, generating considerable debate in the literature. This paper reviews studies performed with single muscle fibres, myofibrils and sarcomeres to investigate the mechanisms of the stretch-induced force enhancement. First, the paper summarizes the characteristics of force enhancement and early hypotheses associated with non-uniformity of sarcomere length. Then, it reviews new evidence suggesting that force enhancement can also be associated with sarcomeric structures. Finally, this paper proposes that force enhancement is caused by: (i) half-sarcomere non-uniformities that will affect the levels of passive forces and overlap between myosin and actin filaments, and (ii) a Ca(2+)-induced stiffness of titin molecules. These mechanisms are compatible with most observations in the literature, and can be tested directly with emerging technologies in the near future.     

Automatic step-detection algorithm for analysis of sarcomere dynamics

Motile systems exhibit a stepwise nature, seen most prominently in muscle contraction. A novel algorithm has been developed that detects steps automatically in sarcomere-length change data and computes their size. The method is based on a nonlinear filter and a step detection protocol that detects local slope values in comparison to a threshold. The algorithm was first evaluated using artificial data with various degrees of Gaussian noise. Steps were faithfully detected even with significant noise. With actual data, the algorithm detected 2.7 nm steps and integer multiples thereof. The results confirm a quantal 2.7 nm step-size reported earlier. As stepwise phenomena become increasingly evident, automatic step-detection algorithms become increasingly useful since the limiting factor is almost always noise. The algorithm presented here offers a versatile and accurate tool that should be useful not only within muscle contraction and motility fields, but in fields which quantal phenomena play a role.