I-band titin in cardiac muscle is a three-element molecular spring and is critical for maintaining thin filament structure.

In cardiac muscle, the giant protein titin exists in different length isoforms expressed in the molecule's I-band region. Both isoforms, termed N2-A and N2-B, comprise stretches of Ig-like modules separated by the PEVK domain. Central I-band titin also contains isoform-specific Ig-motifs and nonmodular sequences, notably a longer insertion in N2-B. We investigated the elastic behavior of the I-band isoforms by using single-myofibril mechanics, immunofluorescence microscopy, and immunoelectron microscopy of rabbit cardiac sarcomeres stained with sequence-assigned antibodies. Moreover, we overexpressed constructs from the N2-B region in chick cardiac cells to search for possible structural properties of this cardiac-specific segment. We found that cardiac titin contains three distinct elastic elements: poly-Ig regions, the PEVK domain, and the N2-B sequence insertion, which extends approximately 60 nm at high physiological stretch. Recruitment of all three elements allows cardiac titin to extend fully reversibly at physiological sarcomere lengths, without the need to unfold Ig domains. Overexpressing the entire N2-B region or its NH(2) terminus in cardiac myocytes greatly disrupted thin filament, but not thick filament structure. Our results strongly suggest that the NH(2)-terminal N2-B domains are necessary to stabilize thin filament integrity. N2-B-titin emerges as a unique region critical for both reversible extensibility and structural maintenance of cardiac myofibrils.

Assembly of thick, thin, and titin filaments in chick precardiac explants.

De novo cardiac myofibril assembly has been difficult to study due to the lack of available cell culture models that clearly and accurately reflect heart muscle development in vivo. However, within precardiac chick embryo explants, premyocardial cells differentiate and commence beating in a temporal pattern that corresponds closely with myocyte differentiation in the embryo. Immunofluorescence staining of explants followed by confocal microscopy revealed that distinct stages of cardiac myofibril assembly, ranging from the earliest detection of sarcomeric proteins to the late appearance of mature myofibrils, were consistently recognized in precardiac cultures. Assembly events involved in the early formation of sarcomeres were clearly visualized and accurately reflected observations described by others during chick heart muscle development. Specifically, the early colocalization of alpha-actinin and titin dots was observed near the cell periphery representing I-Z-I-like complex formation. Myosin-containing thick filaments assembled independently of actin-containing thin filaments and appeared centered within sarcomeres when titin was also linearly aligned at or near cell borders. An N-terminal epitope of titin was detected earlier than a C-terminal epitope; however, both epitopes were observed to alternate near the cell periphery concomitant with the earliest formation of myofibrils. Although vascular actin was detected within cells during early assembly stages, cardiac actin predominated as the major actin isoform in mature thin filaments. Well-aligned thin filaments were also observed in the absence of organized staining for tropomodulin at thin filament pointed ends, suggesting that tropomodulin is not required to define thin filament lengths. Based on these findings, we conclude that the use of the avian precardiac explant system accurately allows for direct investigation of the mechanisms regulating de novo cardiac myofibrillogenesis.