Muscle-specific stress fibers give rise to sarcomeres in cardiomyocytes

Abstract

The sarcomere is the contractile unit within cardiomyocytes driving heart muscle contraction. We sought to test the mechanisms regulating actin and myosin filament assembly during sarcomere formation. Therefore, we developed an assay using human cardiomyocytes to monitor sarcomere assembly. We report a population of muscle stress fibers, similar to actin arcs in non-muscle cells, which are essential sarcomere precursors. We show sarcomeric actin filaments arise directly from muscle stress fibers. This requires formins (e.g., FHOD3), non-muscle myosin IIA and non-muscle myosin IIB. Furthermore, we show short cardiac myosin II filaments grow to form ~1.5 μm long filaments that then ‘stitch’ together to form the stack of filaments at the core of the sarcomere (i.e., the A-band). A-band assembly is dependent on the proper organization of actin filaments and, as such, is also dependent on FHOD3 and myosin IIB. We use this experimental paradigm to present evidence for a unifying model of sarcomere assembly.

Single-nucleus RNA-seq identifies transcriptional heterogeneity in multinucleated skeletal myofibers

“…including Nrap, Fhod3, Enah, and Flnc, as well as the non-muscle myosins Myh9 and Myh10, which have been postulated as components of "pre-myofibrils" laid down as scaffolds before mature muscle-specific myosins are assembled ( Fig. 2d) [22][23][24][25][26][27] . Multiple transcription factors were enriched in transient myonuclei, including both known drivers of skeletal muscle differentiation (Ifrd1, Nfat5, Mef2a) [28][29][30] and numerous TFs with no previous associations in muscle (Ell, Creb5, Zfp697) (Extended Data Fig.…”

Defective sarcomere organization and reduced larval locomotion and fish survival in slow muscle heavy chain 1 (smyhc1) mutants

“…9 As one of most important skeletal myofibrillar proteins, myosin molecules form bipolar thick filaments which move along the thin filaments to produce contraction forces in sarcomeres, the basic contractile units in skeletal and cardiac muscle fibers. [17][18][19][20][21][22] The "Stitching Model" proposes that parts of a sarcomere, namely the I-Z-I complexes and bipolar myosin filaments are assembled independently and then brought together during sarcomere assembly. The M-lines cross-link and stabilize the central portion of myosin filaments, and the Z-lines anchor the thin filaments.…”

“…[13][14][15][16] Several models have been proposed to explain the process of sarcomere assembly. 22 Genetic analysis reveals that MyHC plays important roles in muscle development, growth and function. 18 The "Pre-Myofibril Model," also known as the "Templating Model," argues that stress fiber-like structures containing both nonmuscle and sarcomeric proteins serve as a template for the formation of sarcomeres.…”

Defective heart chamber growth and myofibrillogenesis after knockout ofadprhl1gene function by targeted disruption of the ancestral catalytic active site

“…The starting points for assembly are aggregations of α-actin and α-actinin 2 (termed Z-bodies) that associate with the cell membrane at integrin adhesion sites (proto-costameres) (Reviewed Sparrow and Schöck, 2009). For recruitment of the motor proteins, intermediate steps involve incorporation of non-muscle myosin type II into filaments before it is then replaced by muscle myosin II protein to establish the correct sarcomere spacing Fenix et al, 2018). There is a mutual dependency between proper formation of actin and myosin filaments in muscle, although recent experiments have observed part assembled components of each in the absence of the other structure (Fenix et al, 2018;Rui et al, 2010).…”

“…For recruitment of the motor proteins, intermediate steps involve incorporation of non-muscle myosin type II into filaments before it is then replaced by muscle myosin II protein to establish the correct sarcomere spacing Fenix et al, 2018). There is a mutual dependency between proper formation of actin and myosin filaments in muscle, although recent experiments have observed part assembled components of each in the absence of the other structure (Fenix et al, 2018;Rui et al, 2010). Chaperones and co-chaperones such as Unc45b that facilitate folding of the myofilament proteins have been identified (Reviewed Carlisle et al, 2017) and the number will likely increase, given the size and complexity of sarcomere architecture (Reviewed Gautel and Djinović-Carugo, 2016).…”