The Unified Myofibrillar Matrix for Force Generation in Muscle
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ARTICLE https://doi.org/10.1038/s41467-020-17579-6 OPEN The unified myofibrillar matrix for force generation in muscle ✉ T. Bradley Willingham1, Yuho Kim 1, Eric Lindberg1, Christopher K. E. Bleck 1 & Brian Glancy 1,2 Human movement occurs through contraction of the basic unit of the muscle cell, the sarcomere. Sarcomeres have long been considered to be arranged end-to-end in series along the length of the muscle into tube-like myofibrils with many individual, parallel myofibrils 1234567890():,; comprising the bulk of the muscle cell volume. Here, we demonstrate that striated muscle cells form a continuous myofibrillar matrix linked together by frequently branching sarco- meres. We find that all muscle cells contain highly connected myofibrillar networks though the frequency of sarcomere branching goes down from early to late postnatal development and is higher in slow-twitch than fast-twitch mature muscles. Moreover, we show that the myofibrillar matrix is united across the entire width of the muscle cell both at birth and in mature muscle. We propose that striated muscle force is generated by a singular, mesh-like myofibrillar network rather than many individual, parallel myofibrils. 1 National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. 2 National Institute of Arthritis and Musculoskeletal and ✉ Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA. email: [email protected] NATURE COMMUNICATIONS | (2020) 11:3722 | https://doi.org/10.1038/s41467-020-17579-6 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17579-6 he mechanisms underlying how muscle contraction gen- muscle cell structures for abnormalities. Mitochondria, sarco- erates the forces that propel human movement have been plasmic reticulum, and t-tubules were all normal in appearance T 1–3 of fundamental interest for centuries , and much of our with no evidence of swelling, suggesting that physiological insight into muscle contractile function comes from our under- osmotic and ionic conditions were maintained through sample standing of the highly patterned nature of muscle cell structure4,5. fixation (Supplementary Movie 1). Contractile structures were The long, cylindrical muscle cell comprises many repeating sar- continuous and tightly packed together, and the visible presence comeres, which are understood to be arranged end to end in of I-bands confirmed that the muscles were not hypercontracted. series to form tube-like myofibrils, which generate force long- Moreover, z-disks were properly aligned in parallel for sarco- itudinally down the length of the cell6,7. The majority of muscle meres both with and without branching (Supplementary Movie 5, cell volume is thus considered to be made up of many individual, upper), and instances of Vernier displacement of z-disk parallel myofibrils, and this textbook view has changed little since sheets23,24 (Supplementary Movie 5, lower) appeared to occur the 1800s2,8,9. Over 35 years ago, it was proposed that contractile at a far lower frequency and much larger scale than branching of force could also be transmitted laterally along the transverse axis individual sarcomeres, further indicating that the presence of the of the muscle10. Since that time, the role of perpendicular myofibrillar matrix is not due to muscle damage or pathology. cytoskeletal connections between individual myofibrils in lateral These data demonstrate that the contractile apparatus of healthy, force transmission has been extensively studied11–14. However, fast-twitch skeletal muscle cells comprises a highly connected, despite the clear importance of desmin and other cytoskeletal nonlinear myofibrillar matrix linked together by branching proteins to normal muscle function, the precise mechanisms of sarcomeres. lateral force transmission remain unknown15–18. Recent developments in 3D electron microscopy have led to fundamental changes in our understanding of how cellular Developmental, fiber-type regulation of sarcomere branching. architecture translates into cellular function19–21. Here we use To determine whether the myofibrillar matrix was limited to fast- high-resolution, 3D electron microscopy to demonstrate that twitch muscle fibers or ubiquitous to all muscle types, we next striated muscle cells form a unified, nonlinear myofibrillar matrix evaluated contractile structures from slow-twitch and cardiac connected across both the length and width of the cell by fre- muscle cells. Both slow-twitch muscle fibers, as characterized by quently branching sarcomeres. We observe myofibrillar networks their more elongated and tortuous sarcomere cross-sectional in all muscle cells, although the frequency of sarcomere branching shapes, high mitochondrial content, and thicker z-disks com- is regulated by muscle type and developmental stage. Addition- pared to fast-twitch fibers25,26 (Supplementary Movie 1), and ally, we found that sarcomere branching can occur either through cardiac myocytes (Fig. 2a) also comprise frequently branching splitting of a single sarcomere into two or through a transfer of sarcomeres, indicating the presence of a myofibrillar matrix in all myofilaments between two adjacent sarcomeres, and that the mature striated muscle types. To evaluate whether myofibrillar frequency of each branching mechanism also varies by muscle matrix structures were the predominant contractile structure type. The contiguous nature of the myofibrillar matrix across within muscle cells, we assessed how frequently branching both the length and width of the cell provides a direct pathway for myofibrillar structures occurred relative to the number of non- both lateral and longitudinal active force transmission throughout branching, individual myofibrils. By beginning with each parallel the muscle cell and argues against the presence of many indivi- sarcomere at one end of the dataset (see Supplementary Fig. 1) dual myofibrils running the entire length of the muscle cell. and tracking its structure along the length of the muscle within the field of view (Supplementary Movie 1, left), we determined whether any of the serial sarcomeres branched or if a single tube- Results like myofibril was apparent. Nearly all myofibrillar structures Branching sarcomeres lead to a connected myofibrillar matrix. (96.1 ± 1.4%, 99.4 ± 0.6%, and 94.4 ± 2.5% of fast-twitch, slow- To evaluate muscle contractile structure, we used focused ion twitch, and cardiac myofibrils, mean ± SE, n = 4, 3, and 5 muscle beam-scanning electron microscopy (FIB-SEM)19,20,22 to image volumes and 468, 299, and 344 myofibrils, respectively) contained mouse muscle volumes with 10 nm resolution in 3D (Supple- at least one branching sarcomere within the field of view, indi- mentary Movie 1). Initially, we attempted to visualize individual cating that the myofibrillar matrix was the primary contractile myofibrils within the intact fast-twitch muscle cell by choosing a structure in each adult muscle type (Fig. 2b). To further assess the sarcomere from the muscle cross-section (Supplementary Fig. 1) degree of connectivity of the myofibrillar matrix, we measured and tracing its shape and that of each subsequent sarcomere how frequently sarcomere branching occurred for each muscle connected in series through the z-disks along the length of the type. For every ten sarcomeres in series, 1.6 ± 0.2 sarcomeres muscle. However, we found that sarcomeres often branched at an contained branches in fast-twitch muscle (n = 4 muscle volumes, angle and resulted in a highly connected myofibrillar matrix 193 myofibrils, 3477 sarcomeres), 2.2 ± 0.2 sarcomere branches rather than the textbook linear, tube-like myofibril structure occurred in cardiac muscle (n = 5 muscle volumes, 167 myofi- (Fig. 1, Supplementary Movie 1). As a result, we modified our brils, 2114 sarcomeres), and 4.3 ± 0.4 sarcomere branches tracing routine to separate each myofibrillar segment within the occurred in slow-twitch muscle (n = 3 muscle volumes, 110 myofibrillar matrix at the branching points (Fig. 1, Supplemen- myofibrils, 1396 sarcomeres), significantly higher than either tary Movie 2). There was no apparent relationship between sar- cardiac or fast-twitch muscle (Fig. 2c). These data indicate that comere branch points and specific sarcomeric regions such as the while the myofibrillar matrix is ubiquitous across muscle types, I-band (actin but no myosin present), A-band (actin and myosin the degree of connectivity is regulated according to muscle fiber overlap), or H-zone (myosin but no actin present) as sarcomere type. branch points could be seen in all three regions as well as just To further demonstrate that a unified myofibrillar matrix is the before and after the z-disks (Fig. 1). Additionally, sarcomere normal structure of mature skeletal muscle cells, we evaluated branches did not appear to alter the normal spatial relationships myofibrillar structures in FIB-SEM datasets of the vastus lateralis between the sarcomere and mitochondria (Supplementary of three adult humans (<40 years of age) published as part of the Movie 3) or the sarcotubular systems (Supplementary Movie 4). Baltimore Longitudinal Study of Aging27. In human muscle, To exclude the possibility that sarcomere branches were caused sarcomere branching occurred in 100% of the myofibrils we by muscle damage or tissue fixation artifacts, we closely examined evaluated (n = 60 myofibrils, three muscle volumes). Moreover, 2 NATURE COMMUNICATIONS | (2020) 11:3722 | https://doi.org/10.1038/s41467-020-17579-6 | www.nature.com/naturecommunications