Impaired myogenic differentiation and maturation in mice with PINCH1 being ablated in skeletal muscle progenitors
To investigate the specific role of PINCH in skeletal muscle development, we generated a mouse line in which PINCH1 was deleted in skeletal muscle progenitor cells using MyoD-Cre[30]. PINCH1 mutant mice (MyoD-Cre; PINCH1f/f, SMUT) were born at expected mendelian ratio and appeared normal at birth. But with postnatal development, SMUT mice exhibited mild growth retardation with shorter body length and lower body weight compared to control littermates (Fig. 1A, B). PINCH1 mutants appeared less active, otherwise fertile and lived a normal lifespan.
The skeletal muscles are composed of bundles of multinucleated myofibers, each of which is filled with myofibrils with its nuclei placed at the periphery underneath sarcolemma[31, 32]. To investigate if there were any morphological change in SMUT skeletal muscles, we dissected the quadriceps muscles from 2-month adult SMUT mice and control littermates and performed H&E (Hematoxylin-Eosin staining) histological analyses. We found that ablation of PINCH1 in skeletal muscle progenitors resulted in a significant myopathic change. In longitudinal (Fig. 1C, D) and cross (Fig. 1F, G) sections, myofibers of control samples were compact and the nuclei were spindle-like and evenly located immediately below the plasma membrane (Fig. 1C, D). However, SMUT myofibers were loosely packed and significantly hypoplastic and variable in size, and there was a significant decrease in average cross area of SMUT myofibers compared to controls (Fig. 1C-G). Nuclei of SMUT myofibers exhibited a more rounded morphology and centrally localized, a feature of myopathies (Fig. 1D, arrow). The number of nuclei and percentage of centrally localized nuclei in SMUT myofibers were significantly increased compared to the control (Fig. 1H, I). These results suggested that deletion of PINCH1 in skeletal muscle progenitors leads to impaired terminal differentiation and maturation of multinucleated myofibers.
Ablation of PINCH1 and PINCH2 resulted in reduced size of muscle fibers and impaired multinucleation
Two PINCH isoforms (PINCH1 and 2) are expressed in mammals with partial functional redundancy[23, 24]. Relative mild phenotype in PINCH1 mutants could be attributed in part to the compensatory role by PINCH2. To examine this possibility, we generated a muscle-specific PINCH1 and PINCH2 double knockout mouse line (MyoD-Cre;PINCH1f/f; PINCH2-/-, DMUT). A majority of DMUT mice would die within 48 hours after birth, and all remaining mice die within 7-10 days (Fig. 2A). Neonatal DMUT pups appeared weak, slim with an empty stomach, suggesting defects in sucking and motor function. We found a marked reduction in the size of muscles in the back and thighs (Fig. 2A, B, boxed) in DMUT pups compared to control littermates. The diaphragm muscles of DMUT pups appeared shorter, and in most cases were readily detached from body wall (Fig. 2C-E, arrow).
During myogenesis, mononucleated myoblasts fuse to form multinucleated muscle cells that further differentiate to become mature myofibers. We examined the H&E-stained longitudinal sections of thigh and diaphragm muscles from postnatal day 1 (P1) DMUT mice and littermate controls. The control muscles were composed of elongated, cylindrical, well-aligned and compact myofibers that were filled with striated myofibrils, whereas the mutant myofibers were markedly reduced in size and disconnected from each other (Fig. 2F-J). Many mutant muscle cells appeared spindle-shaped without a clear striated pattern (Fig. 2G). Although both control and DMUT myofibers were multinucleated, quantitative analysis revealed a significant reduction in the number of nuclei in PINCH mutant myofibers, suggesting impaired myoblast fusion (Fig. 2K). To examine whether decreased nuclear number in PINCH mutant myofibers could result from a change in nuclear division, we performed immunostaining with a mitosis-specific antibody to phosphohistone H3 (Ph3), and observed instead a significant increase in mitotic nuclei in P1 DMUT muscle fibers compared to that in controls (Fig. 2L). In addition, significantly increased apoptosis was also observed in DMUT muscle cells (Fig. 2M).
MyoD is expressed early during myogenesis in myogenic progenitors and undifferentiated myoblasts and required for muscle lineage commitment. To determine whether PINCHs play a role in early myogenesis, we traced skeletal muscle lineage (MyoD-Cre+) by β-Galactosidase (β-gal) staining of E13.5 DMUT and control mice (on Rosa-lacZ background). We found that the overall shape and size of somites and muscle primordial blocks of the head, body and limbs were comparable between PINCH mutant and control mice (Fig. 2N-Q), suggesting that PINCH is dispensable for early stage myogenesis, but it is required for subsequent differentiation and maturation of muscle cells.
Defects in myoblast fusion and cytoskeleton assembly in PINCH double mutant mice
To further investigate the role of PINCH in sarcomere genesis, we examined the ultrastructure of muscle fibers by transmission electron microscopy (TEM). Control myofibers of the limb (Fig. 3A) and diaphragm (Fig. 3C) exhibited a typical striated pattern and well-organized sarcomere structures, including Z- and M-lines and I and A bands, and well-packed actin and myosin myofilaments (Fig. 3A-D). However, PINCH mutant limb myofibers largely lost their striated appearance with disorganized and truncated myofibrils. Sarcomere structures were severely disrupted, and only residual Z-lines and loosely packed myofilaments were visible (Fig. 3B, arrow). Sarcomere structure of PINCH mutant diaphragm myofibers appeared to be preserved, however, the myofibrils were markedly thinner and mal-aligned (Fig. 3D). Consistent with this, α-actinin immunostaining of hindlimb muscles showed that, compared to the controls, the myofibrils of the mutant myofibers were markedly thinner and lost their striated pattern (Fig. 3E, F), suggesting PINCH is essential for assembly of myofibril in vivo.
We further analyzed myoblast fusion and cytoskeleton assembly in vitro. Myoblasts were isolated from hindlimb of control and DMUT mice at E18.5 and cultured in 10% Matrigel-coated dishes. After 3 days in culture, cells were fixed and co-immunostained with antibody to cadherins and a-actinin. We found that the majority of control myoblasts were fused, elongated and become multinucleated myotubes with clear striated appearance (Fig. 3J). However, a majority of DMUT myoblasts remained mono- or binucleated, and were markedly smaller and spindle-like with no clear striations (Fig. 3H), even with extended culture time, which, however, resulted in excessive cell death in mutant cultures. Quantification revealed a significant reduction in the number of nuclei in mutant myofibers (Fig. 3I). However, we frequently observed cells that were aggregated (Fig. 3H yellow arrow) or aligned and fused to preexisting primary myofibers (Fig. 3H red arrow), suggesting those might be the cells in the process of cell fusion. In addition, expression of cadherins, known to regulate cell-cell adhesion and myoblast fusion, were correctly localized at the sites of cell contact and fusion, despite its level of expression was reduced (Fig. 3J, K, arrow, boxed inlet 1 and 2), suggesting PINCH may play a direct role in regulating myoblast fusion.
Defects in expression of cytoskeleton proteins and proteins involved in myogenesis in DMUT skeletal muscles
Integrin signaling pathway regulates cytoskeleton organization and is required for skeletal myogenesis[33]. Therefore, we analyzed the expression of proteins of integrin pathway and cytoskeleton, and of those involved in late stage myogenesis. DMUT and control myoblasts from E18.5 embryos were cultured for 48-72 hours to avoid excessive cell death. Cells were fixed and immunostained with antibodies to desmin, myosin, talin and vinculin to reveal the structures of cytoskeleton. DMUT muscle cells were markedly smaller and remained a myoblast-shape compared to controls, suggesting defects in cell spreading and cytoskeleton remodeling. In control muscle cells, expression of desmin, myosin and talin were cytoskeleton associated, and vinculin was concentrated at focal adhesion sites. In DMUT muscle cells, however, expression of myosin and talin were downregulated and aggregated. Vinculin was expressed in a pattern similar with that in control cells, although its expression was notably downregulated. These results suggested impaired assembly and organization of cytoskeleton, but relatively normal adhesion of PINCH mutant cells. Consistent with this observation, western blot analysis of P1 mouse skeletal muscles revealed a significant decrease in the expression of a number of cytoskeleton-associated proteins, including β1-integrin, talin, cadherin, vinculin, myosin and Titin. Interestingly, expression of ILK, known to interact with PINCH in focal adhesion complex, was slightly but significantly upregulated in DMUT, which might compensate partially for the loss of PINCH in cell adhesion. In DMUT skeletal muscles, phosphorylation of AKT and ERK1/2, the downstream mediators of integrin-focal adhesion, were significantly reduced, and total AKT is also slightly decreased. MRF4 is the predominant myogenic factor at late stages of myogenesis and is required for myoblast fusion and cytoskeleton gene expression. Mutation of MYF4 is associated with centronuclear myopathy in humans. MRF4 expression was abolished in DMUT muscles that might contribute in part to observed defects in myoblast fusion and cytoskeleton organization.