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. 2025 Nov 13;83(9):719–732. doi: 10.1002/cm.70063

Four New Muscle Myosin II Binding Properties of Titin: Implications for Myofibrillogenesis

Prokash Chowrashi 1, Balraj Mittal 2, Yingli Fan 2, Zhen Ma 3, Jushuo Wang 2, Jean M Sanger 2, Joseph W Sanger 2,✉
PMCID: PMC13550713  PMID: 41230616

ABSTRACT

Before muscle specific proteins are expressed in precursor cells of cardiac muscle, only the nonmuscle myosin II isoform of myosin II is present in the cells. It provides contractile force for the cell divisions that occur, and it is present together with actin in fibers in the cytoplasm. When expression of muscle proteins occurs, both muscle and nonmuscle isoforms of myosin II co‐exist in the same cells. Nonmuscle myosin II isoforms are organized in minisarcomeric bands in premyofibrils with alternating bands of muscle‐specific alpha‐actinin. In the same cell, muscle myosin II is present in mature myofibrils that are unassociated with nonmuscle myosin II isoforms. There is an intermediate group of fibrils, i.e., nascent myofibrils, in which both isoforms of myosin II are present in the cardiomyocytes. Since muscle and nonmuscle myosin II form copolymers in solution, we asked whether any of the myofibril proteins that interact with muscle myosin could prevent nonmuscle and muscle myosin from copolymerizing, thus explaining the absence of nonmuscle myosin II in mature myofibrils. We examined the effects of two myosin‐binding proteins involved in cardiomyopathies: C‐Protein (myosin binding protein or MyBP‐C) and titin, on the filament forming properties of the two different types of myosin II. In filament forming conditions, neither C‐protein nor titin bind nonmuscle myosin II. Both C‐protein and titin, as expected, bind muscle myosin II. C‐Protein does not inhibit the copolymerization of the two different types of myosin IIs. Co‐polymerization of nonmuscle and muscle myosin IIs is prevented in the presence of either full length titin isolated from cardiac muscles or a bacterially expressed titin peptide containing just one myosin‐binding region. In the presence of titin, paracrystals of Light Meromyosin (LMM) change their normal 14 nm periodicities in pure LMM paracrystals to 42 nm in copolymerization of LMM and full‐length titin. Our experiments suggest four novel roles of titin in myofibrillogenesis: one: prevention of copolymerization of nonmuscle myosin II and muscle myosin II in mature myofibrils, and two: formation of thick filaments with linear cross‐bridges separated by 42 nm repeats in the absence of C‐Protein. Two additional properties of titin in myofibrillogenesis would be titin's capture and transport of copolymers of myosin filaments to premyofibrils to form nascent myofibrils, and the start of release of nonmuscle myosin II from copolymeric myosin II filaments resulting in mature myofibrils lacking nonmuscle myosins.

Keywords: muscle myosin II, myofibrillogenesis, myosin binding protein‐C, nascent myofibrils, nonmuscle myosin II, titin

1. Introduction

A three‐step model for myofibril assembly in embryonic chick cardiomyocytes was shown by Rhee et al. (1994) to begin assembly sequentially from premyofibrils to nascent myofibrils, and ultimately to mature myofibrils (Figure 1). This assembly model has been observed in a number of different types of cardiomyocytes: spreading adult cat and rat adult cardiomyocytes (LoRusso et al. 1997); living embryonic cardiomyocytes transfected with plasmids encoding GFP‐alpha‐actinin (Dabiri et al. 1997); the first cardiomyocytes formed from isolated quail precardiac mesoderm (Du et al. 2003); myofibrillogenesis in situ in embryonic hearts (Du et al. 2008); myofibril assembly in cultured mouse neonatal cardiomyocytes (White et al. 2018); and myofibrillogenesis in human inducible Pluripotent Stem Cell‐derived cardiomyocytes (Wang, Fan, Sanger, and Sanger 2022; Wang, Fan, Mittal, et al. 2022).

FIGURE 1.

FIGURE 1

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Three step model for myofibrillogenesis. Bipolarity of Actin filaments (black lines) is established in the premyofibril stage by the bipolar nonmuscle myosin II filaments (orange color). Muscle myosin II (black cross‐bridges) and titin (red color) are detected for the first time in the nascent myofibril. Some myosin II copolymer filaments (long arrow in the nascent myofibril) contain both nonmuscle myosin II proteins (orange cross‐bridges) along with muscle myosin II proteins (black cross‐bridges). In the mature myofibril, nonmuscle myosin II is not detected, and muscle myosin II is the only myosin II. The alpha‐actinin containing Z‐bodies (green solid circles) align and fuse to form linear Z‐Bands (green color). The mature myofibril exhibits two new late assembling proteins, i.e., the M‐line protein myomesin (brown color), and the muscle myosin II binding protein or C‐Protein (blue solid circles). Titin filaments (red color) become tightly bound along the length of the thick muscle myosin II filaments in the mature myofibrils. Another late assembling protein in the mature myofibril is the titin binding protein telethonin (not indicated in the diagram) that is in the Z‐Bands (green color).

Myofibril assembly in different types of skeletal muscle cells was observed as well to follow the same three‐step model: cultured avian skeletal muscle cells (Sanger et al. 2002; Siebrands et al. 2004; Golson et al. 2004; Wang, Shaner, et al. 2005; Wang, Sanger, and Sanger 2005; Stout et al. 2008; Wang, Fan, Mittal, et al. 2022); skeletal muscle cells in zebrafish (Sanger et al. 2009); cultured mouse primary skeletal myotubes (White et al. 2018). Figure 1 illustrates the latest three‐step rendition of this premyofibril model. Premyofibrils are characterized by the presence of one type of myosin II, i.e., nonmuscle myosin II in the middle of the minisarcomeres, whose boundaries are marked by muscle alpha‐actinin in Z‐Bodies. The bipolar nonmuscle myosin II filaments have been suggested to produce the alignment of oppositely polarized actin filaments in the middle of the premyofibril's minisarcomeres (Rhee et al. 1994; Sanger et al. 2017). Huxley (1963) was the first to report that the barbed ends of muscle actin filaments were embedded in the Z‐Bands of myofibrils, allowing the bipolar arrangement of actin filaments in a muscle sarcomere to interdigitate and interact with the bipolar thick myosin II filaments. Sanger and Sanger (1980) reported a similar arrangement of barbed ends of nonmuscle actin filaments emanating from dense bodies of stress fibers, and cleavage furrows in nonmuscle cells. Putative nonmuscle myosin II short filaments were found between the dense bodies, and in between the oppositely oriented heavy meromyosin decorated actin filaments (Sanger and Sanger 1980). The dense bodies of the stress fibers were later reported to be the sites of alpha‐actinin molecules (Sanger et al. 1983). Rhee et al. (1994) reported that muscle myosin II and titin make their first appearances in the nascent myofibrils (Figure 1). The increased resolution using STED (Stimulated Emission Depletion) microscopy revealed that muscle myosin II is in an overlapping pattern over a core of periodic arrangement of nonmuscle myosin II bands (Wang, Fan, Mittal, et al. 2022) as first suggested by Rhee et al. (1994) using wide‐field fluorescent microscopy. Wang, Fan, et al. (2018) using FRET (Fluorescence Resonance Energy Transfer) techniques reported that some of the muscle myosin II and nonmuscle myosin II molecules are copolymerized in the nascent myofibrils (Figure 1). The mature myofibril exhibits only one type of myosin II, i.e., muscle myosin II in A‐Bands, and highly organized sarcomeric arrays of titin (Figure 1). Wang et al. (2020); Wang, Fan, Mittal, et al. (2022) reported that the Ubiquitin Proteasome System is essential in permitting the progression of nascent myofibrils to form mature myofibrils.

Two aspects of the premyofibril model have been left unresolved (Sanger et al. 2005). It is unclear what molecular mechanisms are responsible for the exclusion of nonmuscle myosin II from the mature myofibrils. It is known that purified nonmuscle myosin IIA derived from platelets, and skeletal muscle myosin II, can copolymerize in solution (Pollard 1975). Rhee et al. (1994) suggested based on the myosin binding protein‐C initial appearance in mature myofibrils, that this protein might also prevent the assembly of nonmuscle myosin II with muscle myosin II in mature myofibrils. Chowrashi and Pepe (1977) suggested that C‐Protein was the basis for the linear arrangement of the 42 nm periodicities of the muscle myosin II cross bridges along the thick filaments. This idea was based on the results that C‐Protein changed the 14 nm repeats of pure LMM paracrystals to 42 nm periodicity in copolymerization of LMM and C‐Protein. However, Harris et al. (2002) and Luther et al. (2008) demonstrated that MyBP‐C is not needed for the formation of mature myofibrils in mice bred to eliminate MyBP‐C.

Our biochemical experiments in this report demonstrate that (a) titin can prevent the copolymerization of nonmuscle myosin II and muscle myosin II, and (b) titin induced the change of the 14 nm repeat of pure LMM to 42 nm periodicities. Titin is essential for the formation and stability of mature myofibrils (Turnacioglu et al. 1997a; van der Ven et al. 2000; Ayoob et al. 2000; Miller et al. 2003; Kellermayer et al. 2019; Hinson and Campbell 2024). These findings suggest new roles of titin in the assembly of mature myofibrils, and support the three‐step model of myofibrillogenesis in determining how nonmuscle myosin II is excluded from mature myofibrils, and how mature myofibrils can form in the absence of MyBP‐C.

2. Results

2.1. Formation of Short Nonmuscle Myosin II B and Long Cardiac Muscle Myosin II Filaments

Nonmuscle myosin IIB was prepared from fresh calf brains as described in the Materials and Methods sections. Cardiac muscle myosin II was isolated from calf hearts using techniques described previously (Sanger 1971; Chowrashi and Pepe 1977). The myosins were in a buffered high salt solution (0.6 M KCl). Separate overnight dialysis of the nonmuscle and muscle myosin IIs against a low salt solution (0.15 KCl) led to the formation of short filaments of nonmuscle myosin II B (Figures 2a and 3a), and much longer filaments composed of muscle myosin II (Figures 2b and 3b).

FIGURE 2.

FIGURE 2

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Negatively stained filaments formed from (a) nonmuscle myosin II B, and (b) cardiac muscle myosin II are shown at the same magnification. Bar in (a) equals 0.5 μm.

FIGURE 3.

FIGURE 3

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Distribution of lengths of negatively stained filaments formed from (a) nonmuscle myosin IIB, and from (b) cardiac muscle myosin II.

2.2. Effects of Cardiac Muscle Myosin Binding Protein or C‐Protein on Nonmuscle and Muscle Myosin II Filaments

Figure 4a,b demonstrate the presence of C‐Protein only in the mature myofibrils in a human‐induced pluripotent stem cell (hiPSC) derived cardiomyocyte. The elimination of C‐Protein in an hiPSC derived cardiomyocyte (Figure 4a,b) shows the presence of mature myofibrils in fluorescently labeled phalloidin staining (Figure 4c) despite the absence of C‐Protein (Figure 4d). The cardiomyocyte lacking C‐Protein was stained with both phalloidin for F‐actin (Figure 5a) and with an anti‐muscle alpha‐actinin antibody (Figure 5b). The mature myofibrils are detected with both reagents in the center of the cardiomyocyte. The precursors to the mature myofibrils reveal minisarcomeric spacings near the periphery of the cell when stained with the alpha‐actinin antibody (Figure 5b).

FIGURE 4.

FIGURE 4

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hiPSC‐derived cardiomyocytes stained with anti‐human Muscle Myosin Binding Protein‐C (MyBP‐C) (b, d), and fluorescent phalloidin (a, c) to indicate the positions of F‐actin in these same cells. In the wild‐type cardiomyocyte (a, b), MyBP‐C is present only in the mature myofibrils (b). The arrows in (a) and (b) mark the outer borders of the same cell. The outer F‐actin staining in (a) indicates nonstriated fibrils where premyofibrils and nascent myofibrils would be located. The cardiomyocyte in (c, d) lacks MyBP‐C. The arrows in (d) show the edges of the same phalloidin stained cell in (c). Note that mature myofibrils are detected with phalloidin in the center of the cardiomyocyte in (c), and there is no staining of these mature myofibrils with the MyBP‐C antibody (d). Scale = 10 μm.

FIGURE 5.

FIGURE 5

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The cardiomyocyte in (a, b) lacks MyBP‐C. The cell has been stained with both (a) phalloidin for F‐actin and (b) an anti‐alpha‐actinin antibody. The mature myofibrils are detected with both reagents in the center of the cardiomyocyte. The precursors to the mature myofibrils reveal minisarcomeric spacings near the periphery of the cell when stained with the alpha‐actinin antibody (b). Scale = 10 μm.

Rhee et al. (1994) first reported the absence of C‐Protein in premyofibrils and nascent myofibrils in their three‐step model of mature myofibrils assembly (updated model in Figure 1). Figure 6 shows the length distribution of the short nonmuscle myosin II filaments is unchanged in the absence (Figure 6a) or presence of C‐protein (Figure 6b). The lack of C‐Protein affecting the lengths of nonmuscle filaments is not surprising since we did not see any staining of premyofibrils with antibodies against C‐Protein in cardiomyocytes (Rhee et al. 1994) or in skeletal muscle cells (Golson et al. 2004; White et al. 2014; Sanger et al. 2017). Figure 7a demonstrates the distribution of filaments formed by copolymerization of equal concentrations of nonmuscle myosin IIB and cardiac muscle myosin II. The length of copolymerized filaments lies between the filament formed with pure nonmuscle myosin II and muscle myosin II (Figure 3). Figure 7b indicates that the presence of C‐Protein during copolymerization of equal concentrations of nonmuscle myosin IIB and cardiac muscle myosin II leads to an increase in lengths of the copolymerized myosin II filaments.

FIGURE 6.

FIGURE 6

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Distribution of lengths of negatively stained filaments formed from (a) nonmuscle myosin II B in the absence of C‐Protein, and (b) in the presence of C‐Protein. There is no detection of changes in the lengths of nonmuscle myosin IIB filaments in the presence of C‐Protein. This is not unexpected since there is no C‐Protein in either premyofibrils or nascent myofibrils where nonmuscle myosin IIB is present.

FIGURE 7.

FIGURE 7

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Effect of C‐protein on the co‐polymerization of nonmuscle myosin II B and cardiac muscle myosin IIB. (a) Copolymerization of nonmuscle myosin II B and cardiac muscle myosin IIB in the absence of C‐Protein leads to a distribution of filament lengths intermediate between those observed for pure nonmuscle myosin IIB filaments and pure cardiac muscle myosin II filaments. (b) Copolymerization of nonmuscle myosin IIB and cardiac muscle myosin II in the presence of C‐Protein leads to a wider distribution of filament lengths. The presence of C‐Protein did not separate the filaments into two groups of short and long filaments.

2.3. Titin Prevents the Copolymerization of Nonmuscle Myosin IIB and Cardiac Muscle Myosin II

In the three‐step model of myofibrillogenesis, nonmuscle myosin is absent in the mature myofibrils, and titin is a late assembling protein for the mature myofibrils (Figure 1). Figure 8a demonstrates that when nonmuscle myosin IIB and muscle myosin are allowed to copolymerize in the presence of purified full length titin molecules, the copolymerization of the two myosins is blocked, and two groups of filaments (short and long filaments) are present. To determine if a shorter region of the myosin II binding region of titin could also block copolymerization, we assembled a 41 kD fragment of the myosin binding region of titin that encoded one myosin binding site of titin. The surprising result of this experiment shows that just one myosin binding region of titin is enough to prevent the copolymerization of the nonmuscle and muscle myosin II molecules, resulting in the presence of two groups of myosin filaments (short and long filaments) (Figure 8b).

FIGURE 8.

FIGURE 8

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Effect of full‐length and 41 kD truncated titin on the copolymerization of nonmuscle myosin II B and cardiac muscle myosin IIB. (a) Copolymerization of nonmuscle myosin IIB and cardiac muscle myosin II in the presence of purified full‐length titin. The presence of titin at a ratio of 1 titin to 12.5 muscle myosin II molecules leads to the formation of two distinct groups of filaments, a shorter class typical of nonmuscle myosin IIB filaments, and a longer cohort typical of filaments formed from muscle myosin II. (b) Copolymerization of nonmuscle myosin IIB and cardiac muscle myosin II in the presence of truncated 41 kD titin molecules that contain one muscle myosin II binding repeat. This short titin fragment also resulted in two groups of short and long myosin II filaments.

2.4. Full Length Titin Induces the 14 nm Repeat of Crystalized Light Meromyosin (LMM) to Change to 42 nm

It is well known that Light Meromyosin (LMM) is soluble at 0.6 M KCl concentrations, and that when dialyzed against a 0.15 M KCl solution, LMM forms paracrystals with 14 nm periodicities along its long axis (Chowrashi and Pepe 1977). Figure 9 demonstrates the results of the formation of Light Meromyosin paracrystals in the absence (Figure 9a) and presence of full‐length titin molecules (Figure 9b,c). Figure 9a illustrates 14 nm periodicities of paracrystals of cardiac muscle LMM formed in the absence of titin. Figure 9b,c illustrate the paracrystalline arrangements of LMM when combined LMM and full‐length titin at 0.6 M KCl are dialyzed against 0.15 M KCl; the paracrystals that form now have a periodicity of 42 nm (Figure 9b,c). Formation of LMM paracrystals in the presence of titin molecules at a ratio of 1 titin filament per 12 LMM molecules had both 14 nm repeats and an unexpected 42 nm repeat (Figure 9b). Increasing the amount of titin filaments to a ratio of one titin per 6 LMM leads to all the LMM paracrystals exhibiting 42 nm repeats (Figure 9c). The smaller titin 41 kD fragment of titin did not alter the 14 nm repeat of the LMM paracrystals when it was present in the formation of LMM paracrystals. The 41,000 Da titin fragment has a much smaller length and molecular weight than the full‐length titin, which is approximately one micron in length and has a molecular weight of 3,000,000 Da (Labeit and Kolmerer 1995).

FIGURE 9.

FIGURE 9

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Formation of Light Meromyosin (LMM) paracrystals in the (a) absence and (b, c) presence of full‐length titin molecules. Negative staining of LMM paracrystals formed in the absence of titin molecules revealed the expected 14 nm repeat. (b) Formation of LMM paracrystals in the presence of titin molecules at a ratio of 1 titin filament per 12 LMM molecules had both 14 nm repeats and an unexpected 42 nm repeat. (c) Increasing the amount of titin filaments to a ratio of one titin per 6 LMM led to LMM paracrystals with all 42 nm repeats. Scale bar = 50 nm.

3. Discussion

Muscle myosin II and nonmuscle myosin IIB molecules can be induced to produce filaments intermediate in length (Figure 7) between the longer filaments formed from pure muscle myosin II molecules and the shorter filaments formed from pure nonmuscle myosin IIB molecules (Figure 3). Our published Fluorescence Resonance Energy Transfer (FRET) experiments of living muscle cells cotransfected with CeFP‐nonmuscle myosin IIB heavy chains and YFP‐muscle myosin II heavy chains, support the conclusions that premyofibrils contain only nonmuscle myosin II, and the mature myofibrils contain only muscle myosin II molecules. Muscle titin was not detected in premyofibrils (Rhee et al. 1994), suggesting that titin does not bind nonmuscle myosin II. The FRET experiments indicate that nascent myofibrils contain myosin filaments formed of both muscle and nonmuscle myosin II molecules (Wang, Fan, et al. 2018). Wang, Fan, et al. (2018) demonstrated using FRET techniques on living cultured skeletal muscle cells that muscle myosin II and nonmuscle myosin IIA (or IIB or IIC) were as close together in nascent myofibrils as the muscle myosin II molecules were in the thick filaments arranged in A‐Bands of mature myofibrils. Thus, the nonmuscle myosin II heavy chains (IIA, IIB, or IIC) and muscle myosin heavy chain IIs are copolymerized in the nascent myofibrils (Wang, Fan, et al. 2018). Titin makes its first appearance in nascent myofibrils but becomes highly organized in the A‐Bands of the mature myofibrils (Figure 1). Our biochemical experiments in this report demonstrated that entire titin molecules (3000 kD molecular weight) or even one much smaller muscle myosin II binding domain of titin (41 kD) can prevent copolymerization of nonmuscle myosin IIs and muscle myosin II (Figure 8a,b). There are six identical muscle myosin binding regions on titin (Bennett et al. 2020), and the 41 kD titin fragment would be expected to bind muscle myosin II, acting as an inhibitor for the copolymerization of the two different types of myosin IIs. Thus, this inhibition of copolymerization would allow the formation of short filaments of nonmuscle myosin II and longer filaments of muscle myosin II (Figure 8b). These inhibition results may also indicate that the copolymer binding site of muscle myosin IIs and nonmuscle myosin IIs is close to the muscle myosin binding site to titin.

C‐Protein binds to myosin filaments composed of muscle myosin II, and induces an increase in their lengths (Davis 1988). C‐Protein also induces an increase in lengths of filaments from copolymerized muscle myosin II and nonmuscle myosin IIB (Figure 7b). C‐Protein, however, does not bind to nonmuscle myosin IIB (Rhee et al. 1994), nor does it induce an increase in the filament lengths of nonmuscle myosin IIB (Figure 6b) or prevent copolymerization of nonmuscle myosin IIB and muscle myosin II (Figure 7b). Both full length titin molecules (Figure 8a) or one 41 kD myosin‐binding region of titin (Figure 8b) prevent the copolymerization of the two different myosin II molecules (nonmuscle II and muscle myosin II) in vitro. In the updated premyofibril model of myofibrillogenesis (Figure 1), titin molecules and muscle myosin filaments are recruited to premyofibrils to form nascent myofibrils (Rhee et al. 1994; Welchons et al. 2023). Rhee et al. (1994) demonstrated titin is first detected in nascent myofibrils, while C‐Protein is first detected in mature myofibrils (Figure 1). Our results support the hypothesis that titin may have at least four roles in myofibrillogenesis: (a) the delivery of myosin II filaments to the aligned Z‐bodies and actin filaments in the premyofibrils that form the nascent myofibrils, (b) separation and (c) release of the nonmuscle myosin IIs in the nascent myofibrils, and (d) prevention of the copolymerization of the two different types of myosin IIs in mature myofibrils (Figure 1). The loss of nonmuscle myosin IIB from nascent myofibrils to form the mature myofibrils should allow tighter binding of titin to muscle myosin II filaments. The release of nonmuscle myosin II from the copolymeric myosin filaments to the spreading edges of the muscle cells would allow nonmuscle myosin II essential role in forming premyofibrils, where titin and muscle myosin II are absent (Figure 1). Isaacs et al. (1989) estimated that the synthesis of full‐length titin takes 1 to 1.5 h, while the synthetic half‐live of the titin molecule is about 70 h. Lopes et al. (2011) demonstrated using FRAP on cultured mouse cardiomyocytes expressing eGFP‐titin, an unexpected movement of fluorescently labeled titin from one sarcomere to another. These results indicate that titin is not an inactive largest protein of three million Daltons molecular weight. Evidence for titin regulating the length of thick myosin filaments in sarcomeric A‐Bands have been presented by two different groups (Du et al. 2008; Tonino et al. 2017). Several cancer drugs inhibit the formation of mature myofibrils from nascent myofibrils, potentially due to their off‐target effects on titin and muscle myosin II (Wang, Fan, Mittal, et al. 2022; Wang et al. 2021, Wang et al. 2025).

Another new role for titin (Sanger and Sanger 2001) is its ability to change the 14 nm repeat of pure paracrystals of Light Meromyosin to 42 nm when LMM forms paracrystals in the presence of titin molecules (Figure 9). The 42 nm repeat equals the 42 nm cross bridge repeats along the lateral sides of the thick filaments (Huxley and Brown 1967). Houmeida et al. (1995) demonstrated in EM studies of muscle myosin II, LMM and full‐length titin, that titin bound to near the tip or C‐terminus of muscle myosin and LMM. Chowrashi and Pepe (1977) reported that copolymerization of LMM and C‐Protein resulted in paracrystals with 44 nm repeats suggesting that C‐Protein played an important role in myosin II filament formation. Harris et al. (2002) reported the surprising results that mice bred to eliminate the presence of C‐Protein in their hearts formed beating hearts, and mature myofibrils. These mice, however, had a shorter life span of one‐year instead of the normal two‐year lifespan. The mice lacking C‐Protein also developed hypertrophic cardiomyopathy (Harris et al. 2002). Isolated human cardiomyocytes prepared from wild type hiPSC (Figure 4a,b) and from hiPSC lacking C‐Protein (Figures 4c,d and 5) (Ma et al. 2018) revealed that mature myofibrils formed in the absence of C‐Protein in tissue culture. The absence of C‐Protein did not interfere with the formation of premyofibrils and nascent myofibrils and their progression to form mature myofibrils (Figures 4c,d and 5a,b). Two proteins can affect the periodic formation of LMM paracrystals: C‐Protein (Chowrashi and Pepe 1977) and titin (this paper). Mature myofibrils can be formed and maintained in the absence of C‐protein (Harris et al. 2002), but not in the absence of full‐length titin in muscle cells (Turnacioglu et al. 1996, 1997a, 1997b; Ayoob et al. 2000). A line of myofibroblasts derived from Baby Hamster Kidney cells (BHK‐21/C13), cells can assemble myofibrils with muscle specific proteins including titin (Schaart et al. 1991). A subline of BHK‐21/C13, i.e., BHK‐21‐Bi, arose that lacked part of the N‐terminus of titin (Jäckel et al. 1997), and were unable to assemble myofibrils (van der Ven et al. 2000), demonstrating again the important roles of full‐length titin in the assembly and maintenance of Z‐Bands and A‐Bands in mature myofibrils. These two myosin‐binding proteins, C‐Protein and titin are also important because of their involvement in cardiomyopathies. Fifty percent of hypertrophic cardiomyopathy are due to mutations and truncation of myosin binding protein C (Wang, Geist, et al. 2018). The cardiomyopathies may be due to interference with myofibril assembly and maintenance by these mutated and truncated proteins.

Cardiac and skeletal muscle cells assemble their myofibrils in a three‐step process (Sanger et al. 2017). There is a third type of muscle, i.e., smooth muscle which gained its name for the lack of striations on a light microscope level. However, the contractile unit of smooth muscle included actin filaments, alpha‐actinin dense bodies and thick filaments composed of smooth muscle myosin. These smooth muscle cells also contain a type of titin and nonmuscle myosin II (Sellers 2000). Smooth muscle cells will need to keep nonmuscle myosin II and smooth muscle myosin II from copolymerization. Extending the three‐step model to smooth muscles would include similar steps proposed for the assembly of mature myofibrils in cardiac and skeletal muscle cells. Thus, the premyofibrils in smooth muscle would have actin filaments attached with their barbed ends embedded in the alpha‐actinin containing dense bodies. The nonmuscle myosin IIs would be found interacting with the F‐actin filaments extending from the dense bodies. Nascent myofibrils in smooth muscle would have titin filaments starting to be added to the dense bodies and copolymers of nonmuscle and smooth muscle myosins interacting with the F‐actin filaments. A titin‐like protein will make its appearance in nascent myofibrils, e.g., Smitin (Kim and Keller 3rd 2002). The mature myofibril in smooth muscle would have lost the nonmuscle myosin II leaving just thick filaments containing smooth muscle myosin II. The ultrastructure of a molluscan smooth muscle shows some of the details of a presumptive mature smooth muscle myofibril in contraction (50% of rest length), rest length and stretched conditions (150% of rest length) (Sanger and Hill 1973). This molluscan smooth muscle can contract to 25% of rest length, typical of vertebrate smooth muscles. This shortening property is due to the ability of the thick filaments to slide pass the dense bodies that are attached to thin filaments at their opposite ends (Sanger and Hill 1973). The cardiotoxic effects and skeletal muscles atrophies caused by cancer drugs (Wang, Fan, Mittal, et al. 2022, Wang et al. 2025) would thus be a result of these drugs acting on the assembly of contractile units in all three types of muscles, i.e., cardiac, skeletal, and smooth muscles.

4. Materials and Methods

4.1. Preparation of Nonmuslce Myosin IIB

The procedure of nonmuscle myosin IIB preparation is very similar to that of Doctor Robert Adelstein (Pato et al. 1996). Fresh calf brain was obtained from a slaughterhouse and immediately kept under ice. Meninges, blood vessels and fats were removed by rubbing and removing the tissue with Kimwipes. Only the cerebral cortex portion of the brain was used for the nonmuscle myosin IIB preparation. The tissues were cut into small pieces. About 345 g of tissue was transferred to a 2‐L beaker and 1 L of PBS solution was added for washing. After stirring for 1–2 min, the entire suspension was passed through several layers of cheese cloth with the process repeated three times. Half of the tissue was transferred to a blender and 700 mL of the homogenizing solution (30 mM MOPS, 2 mM MgCl2, 2 mM EGTA, 0.5 mM DTT, Leupeptin 2.5 mg/2 L, 0.5 mL of PI cocktail/L, pH 7.0) was added and homogenized for 30 s at medium speed. The other half was treated in the same way. The homogenized tissue was centrifuged for 15 min at 11,000 rpm. The pellet was collected and transferred to a two‐liter beaker. About 30 mL of extracting solution was added to each centrifuge bottle to transfer the pellet completely, and the rest of the solution was added. It was extracted for 30 min at 4°C. Stirring was accomplished with an overhead Teflon stirrer. After extraction, 200 mM ATP solution was added and stirred for another 20 min. The total extract was centrifuged for 1 h at 35,000 rpm. The collected supernatant was passed through two layers of large Kimwipes and the total volume was measured. Solid powdery (NH4)2SO4, 22.6 g per 100 mL of the above filtered myosin solution was added slowly with stirring at 4°C to make a 40% saturation, stirred for15 min and centrifuged. Supernatants were collected., and more ammonium sulfate was added to make it 60% saturation (12 g/100 mL). The 60% ammonium sulfate cloudy solution was centrifuged and this time the pellet was collected, discarding the supernatant. The pellet was dissolved in gel filtration column buffer (0.9 M NaCl, 2 mM EDTA, 0.2 mM EGTA, 25 mM TRIS, 0.2 mM DTT, 0.2% sodium azide, pH 7.5), clarified with centrifugation, and applied to a Sepharose CL6B column, and the myosin IIB fraction was collected. The protein was dialyzed against 25 mM Tris/HCl, 2 mM EDTA,0.1 mM EGTA and sodium azide. After dialysis the salt concentration should be 0.4 M KCl. The myosin was concentrated by Aquacide absorption of salt solution from the dialysis bag.

4.2. Full Length Titin Preparation

The method for the preparation of titin from chicken skeletal muscles was essentially the same as that of Soteriou et al. (1993) who used rabbit skeletal muscles. White breast muscle was removed from freshly killed chickens and cut into pieces and chopped in a meat grinder. The ground muscle was transferred into a blender with equal volumes of a wash buffer added (50 mM TRIS, 5 mM EGTA,1 mM NaHCO3, 5 mM,5 mM E‐64, pH 7.0) and homogenized for 30 s at low speed, then centrifuged at 9000 rpm for 8 min. The process was repeated for another three cycles. In the last two cycles, the blending was active for 5 s only. In the last cycle the blending was done without the E‐64. The total volume of the tissue was about 200 mL. It was transferred to a 2‐L beaker, and three volumes of extracting solution were added (0.9 M KCl, 2 mM EGTA,10 mM Imidazole, 2 mM MgCl2, 1 mM PMSF, 0.5 mM DTT, 5 μM E‐64, 10 μg/mL trypsin inhibitor, pH 7.0), and extracted for 6 min, and then centrifuged at 35,000 rpm for 1 h. The supernatant was collected, and the pellet was discarded. The volume of the solution was measured and diluted to 0.22 M KCl by slowly adding cold DI water. The solution looked turbid and was left undisturbed for 1 h before the solution was centrifuged for 15 min at 12,000 rpm. The supernatant was collected and further diluted to 0.05 M KCl and centrifuged. This time, the pellet was collected. The crude titin pellet was dissolved in 0.6 M KCl, 30 mM potassium phosphate, pH 7.0 buffer and clarified by centrifugation. A 200 mM ATP solution was added to make it 2 mM in ATP and it was centrifuged at 35,000 rpm for 2 h. The supernatant was collected and dialyzed against 0.6 M KCl,10 mM imidazole, 0.02% sodium azide, pH 7.0. The protein solution was concentrated, and the protein concentration was determined by the Bradford method (Bio‐Rad).

4.3. Preparation of the 41 kD Fragment of Titin

A 388 amino acid fragment from human titin amino acid 3685–4073 was expressed as a GST fusion protein in E. coli . The fragment comprised one IG‐like and three fibronectin repeats and is known to bind the LMM (Light Mero Myosin) region of myosin (Labeit et al. 1992). The cDNA for the titin fragment was generated by RT‐PCR from total human skeletal muscle RNA (StrateGene), using the Gibco‐BRL Superscript One‐step RT PCR kit for long templates. The RT PCR product was gel‐purified and directionally cloned into the pGex‐Precision 6p vector (Amersham‐Pharmacia Biotech, Piscataway, NJ). The sequences of the insert and junction regions were confirmed by automated DNA sequencing (Napcore, The Children Hospital of Philadelphia). The fusion protein was expressed in E. coli BL‐21 (DE3) pLysS after inducing with 1 mM IPTG for 4 h. The GST‐titin fragment was purified by affinity chromatography as described (Freeman et al. 2000). The protein was dialyzed in binding buffer (0.6 M KCl, 10 mM Imidazole and 0.02% sodium azide, pH 7.0) and kept on ice. The protein concentration was determined by the Bradford method (Bio‐Rad).

4.4. Light Meromyosin Preparation

Light Meromyosin was purified using the techniques described by Chowrashi and Pepe (1977). Light meromyosin‐22 (LMM 22, where 22 denotes the time of digestion) was prepared by digesting fresh column pure chicken skeletal myosin II with trypsin. Myosin at a concentration of 6–7 mg/mL was dialyzed against a buffer of 0.5 M KCl, 30 mM potassium phosphate buffer, pH 6.2 overnight. For each 150 mg of myosin, 1 mL of 0.05% trypsin and 1 mL of 0.1% soybean‐trypsin inhibitor were used to stop the reaction. The digestion was done at room temperature. The digested myosin was dialyzed against 0.007 M potassium phosphate buffer pH 6.5 overnight. At this step, LMM and undigested myosin were precipitated. The mixture was centrifuged. The supernatant (HMM) was separated. The pellet was dissolved in 0.53 M potassium phosphate buffer, pH 6.5. Three times the volume of ethanol was added and stirred for 3 h at room temperature. The undigested myosin would be denatured and LMM would be precipitated. The precipitated protein in alcohol was centrifuged. The pellet was allowed to dissolve in 0.53 M potassium phosphate buffer, pH 7.1, and then dialyzed in 0.6 M KCl, 30 mM imidazole, pH 7.0 buffer. After dialysis it was centrifuged; the supernatant (LMM) was separated, and the pellet was discarded. The protein concentration of LMM was determined by measuring the OD of the solutions at 280 nm using 0.35 as the molar extinction coefficient of LMM at 1 mg/mL.

4.5. Study of the Interaction of LMM‐22 With Full‐Length and 41 kD of Titin

To study the interaction of light meromyosin with full‐length and 41 KD titin, light meromyosin paracrystals were made by dialyzing LMM alone and a mixture of light meromyosin and titin (full‐length and 41 KD fragments) together. LMM:Titin = 1:4 μM. Two different buffers were used in these experiments: 0.15 M KCl, 30 mM imidazole, pH 7.0 for dissolving the LMM and 0.05 M KCl, 0.05 M potassium phosphate buffer, pH 7.0 for paracrystal formation. A drop of LMM paracrystals was placed on a 400 mesh carbon‐coated copper grid, washed with buffer and stained with 1% uranyl acetate, dried with a piece of filter paper for negative stain. The formed paracrystals were observed under a JEOL electron microscope.

4.6. Cell Culture, Immunostaining, and Imaging

The hiPSC‐cardiomyocytes (WT and MyBP‐C3 −/−) were grown on MatTek dishes coated with gelatin (catalog number S006100 from Thermo Fisher Scientific) as previously described (Ma et al. 2018). The MyBP‐C3 −/− heart cells were generated as reported by Ma et al. (2018). Cells were fixed with 3% paraformaldehyde, permeabilized and stained with anti‐sarcomeric α‐actinin antibody (Sigma Aldrich, St. Louis, MO), or MyBP‐C3 Antibody (E‐7, Santa Cruz Biotechnology). Cells were also stained with Alexa Fluor 633‐labeled phalloidin (Invitrogen, Carlsbad, CA) to localize F‐actin. All images were acquired using a Leica SP8 confocal (Wang, Fan, Mittal, et al. 2022; Wang et al. 2021).

Author Contributions

All the authors contributed to the experiments and composition of the manuscript.

Ethics Statement

This article does not contain any studies on living animals performed by any of the authors. Animal by‐products, i.e., nine‐day‐old fertilized embryonic quail eggs, were used to isolate myoblasts from embryonic breast muscle, and cardiomyocytes were isolated from the heart of nine‐day‐old chick embryos as described in the Materials and Methods section. This article does not contain any studies involving human participants performed by any of the authors.

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgments

This contribution is dedicated to the memory of the late Professor Frank A. Pepe (1931–2023) who was a pioneer in the role of muscle myosin binding protein C in muscle structures.

Chowrashi, P. , Mittal B., Fan Y., et al. 2026. “Four New Muscle Myosin II Binding Properties of Titin: Implications for Myofibrillogenesis.” Cytoskeleton 83, no. 9: 719–732. 10.1002/cm.70063.

Funding: This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS RO1 AR‐57063) awarded to J.M.S. and J.W.S. and J.W.S. was also supported by a grant from the Hendricks Fund SUNY Upstate Medical University.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Articles from Cytoskeleton (Hoboken, N.j.) are provided here courtesy of Wiley

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