Early incorporation of obscurin into nascent sarcomeres: implication for myofibril assembly during cardiac myogenesis

Abstract

Obscurin is a recently identified giant multidomain muscle protein whose functions remain poorly understood. The goal of this study was to investigate the process of assembly of obscurin into nascent sarcomeres during the transition from non-striated myofibril precursors to striated structure of differentiating myofibrils in cell cultures of neonatal rat cardiac myocytes. Double immunofluorescent labeling and high resolution confocal microscopy demonstrated intense incorporation of obscurin in the areas of transition from non-striated to striated regions on the tips of developing myofibrils and at the sites of lateral fusion of nascent sarcomere bundles. We found that obscurin rapidly and precisely accumulated in the middle of the A-band regions of the terminal newly assembled half-sarcomeres in the zones of transition from the continuous, non-striated pattern of sarcomeric α-actinin distribution to cross-striated structure of laterally expanding nascent Z-discs. The striated pattern of obscurin typically ended at these points. This occurred before the assembly of morphologically differentiated terminal Z-discs of the assembling sarcomeres on the tips of growing myofibrils. The presence of obscurin in the areas of the terminal Z-discs of each new sarcomere was detected at the same time or shortly after complete assembly of sarcomeric structure. Many non-striated fibers with very low concentration of obscurin were already immunopositive for sarcomeric actin and myosin. This suggests that obscurin may serve for organization and alignment of myofilaments into the striated pattern. The comparison of obscurin and titin localization in these areas showed that obscurin assembly into the A-bands occurred soon after or concomitantly with incorporation of titin. Electron microscopy of growing myofibrils demonstrated intense formation and integration of myosin filaments into the "open" half-assembled sarcomeres in the areas of the terminal Z-I structures and at the lateral surfaces of newly formed, terminally located nascent sarcomeres. This process progressed before the assembly of the second-formed, terminal Z-discs of new sarcomeres and before the development of ultrastructurally detectable mature M-lines that define the completion of myofibril assembly, which supports the data of immunocytochemical study. Abundant non-aligned sarcomeres in immature myofibrils located on the growing tips were spatially separated and underwent the transition to the registered, aligned pattern. The sarcoplasmic reticulum, © Springer-Verlag 2008 aborisov@umich.edu. This paper is dedicated to the memory of Professor Pavel P. Rumyantsev (1927Rumyantsev ( -1988, a pioneer in studies of cardiac muscle differentiation, who is a lasting inspiration to all who worked with him. the organelle known to interact with obscurin, assembled around each new sarcomere. These results suggest that obscurin is directly involved in the proper positioning and alignment of myofilaments within nascent sarcomeres and in the establishment of the registered ...

New aspects of obscurin in human striated muscles

“…In line with that suggestion is that, obscurin has been found indispensable for the organisation of myosin into A-bands during cycles of contraction and relaxation in cultured cardiomyocytes . A detailed study of obscurins involvement in the proper positioning and alignment of myoWlaments within nascent sarcomeres in rat cardiomyocytes was recently published by Borisov et al (2008). Obscurin was also suggested to be involved in the establishment of the registered pattern of newly assembled myoWbrils and the sarcoplasmic reticulum at advanced stages of myoWbrillogenesis (Borisov et al 2008).…”

“…Even though several studies on obscurin have been published in the last 5 years (Arimura et al 2007;Bagnato et al 2003;Borisov et al 2003Borisov et al , 2004Borisov et al , 2006Borisov et al , 2007Borisov et al , 2008Bowman et al 2007;Fukuzawa et al 2005;Geisler et al 2007;Kontrogianni-Konstantopoulos and Bloch 2006b;Kontrogianni-Konstantopoulos et al 2003, 2006aRaeker et al 2006), the complexity of obscurin biology is still obscure. Our study, which to our knowledge is the Wrst on human skeletal muscle and myocardium, gives novel information on the localisation and function of obscurin in developing and mature muscles.…”

“…It has a proposed function in sarcomere assembly Kontrogianni-Konstantopoulos et al 2004). Recent in vitro results have suggested that obscurin is indispensable for the assembly and organisation of myosin into regular A-bands during cardiac myoWbrillogenesis (Borisov et al , 2008Kontrogianni-Konstantopoulos et al 2004).…”

Obscurin Interacts with a Novel Isoform of MyBP-C Slow at the Periphery of the Sarcomeric M-Band and Regulates Thick Filament Assembly

“…Obscurin-MLCK may be expressed independently as smaller, alternatively spliced products that contain one (single MCLK; sMLCK) or both (tandem MLCK; tMLCK) kinase motifs or as part of the larger, ϳ720-kDa form of obscurin-A, referred to as obscurin-B or giant MLCK (gMLCK; ϳ860 kDa), in which the two MLCK motifs are arranged in tandem at the COOH terminus of the protein, replacing the nonmodular COOH terminus of obscurin-A (Russell et al, 2002;Fukuzawa et al, 2005;Borisov et al, 2008).…”

Laser-guided cell micropatterning system

“…For example, heart tissue is composed of rodlike cardiomyocytes aligned in specific directions with polarized intercellular binding. 1 This directionality is vital to correct mechanical activity, 2 electrical signal conduction, 3,4 and wound repair. 5 In the nervous system, the organization of neurons and their connections are intrinsic to sensing, information processing, and control functions.…”