Stiffness anisotropy coordinates supracellular contractility driving long-range myotube-ECM alignment.

The ability of cells to organize into tissues with proper structure and function requires the effective coordination of proliferation, migration, polarization, and differentiation across length scales. Skeletal muscle is innately anisotropic; however, few biomaterials can emulate mechanical anisotropy to determine its influence on tissue patterning without introducing confounding topography. Here, we demonstrate that substrate stiffness anisotropy coordinates contractility-driven collective cellular dynamics resulting in C2C12 myotube alignment over millimeter-scale distances. When cultured on mechanically anisotropic liquid crystalline polymer networks (LCNs) lacking topography, C2C12 myoblasts collectively polarize in the stiffest direction. Cellular coordination is amplified through reciprocal cell-ECM dynamics that emerge during fusion, driving global myotube-ECM ordering. Conversely, myotube alignment was restricted to small local domains with no directional preference on mechanically isotropic LCNs of the same chemical formulation. These findings provide valuable insights for designing biomaterials that mimic anisotropic microenvironments and underscore the importance of stiffness anisotropy in orchestrating tissue morphogenesis.

Facile Physicochemical Reprogramming of PEG-Dithiolane Microgels.

Granular biomaterials have found widespread applications in tissue engineering, in part because of their inherent porosity, tunable properties, injectability, and 3D printability. However, the assembly of granular hydrogels typically relies on spherical microparticles and more complex particle geometries have been limited in scope, often requiring templating of individual microgels by microfluidics or in-mold polymerization. Here, we use dithiolane-functionalized synthetic macromolecules to fabricate photopolymerized microgels via batch emulsion, and then harness the dynamic disulfide crosslinks to rearrange the network. Through unconfined compression between parallel plates in the presence of photoinitiated radicals, we transform the isotropic microgels are transformed into disks. Characterizing this process, we find that the areas of the microgel surface in contact with the compressive plates are flattened while the curvature of the uncompressed microgel boundaries increases. When cultured with C2C12 myoblasts, cells localize to regions of higher curvature on the disk-shaped microgel surfaces. This altered localization affects cell-driven construction of large supraparticle scaffold assemblies, with spherical particles assembling without specific junction structure while disk microgels assemble preferentially on their curved surfaces. These results represent a unique spatiotemporal process for rapid reprocessing of microgels into anisotropic shapes, providing new opportunities to study shape-driven mechanobiological cues during and after granular hydrogel assembly.

Stiffness anisotropy coordinates supracellular contractility driving long-range myotube-ECM alignment.

In skeletal muscle tissue, injury-related changes in stiffness activate muscle stem cells through mechanosensitive signaling pathways. Functional muscle tissue regeneration also requires the effective coordination of myoblast proliferation, migration, polarization, differentiation, and fusion across multiple length scales. Here, we demonstrate that substrate stiffness anisotropy coordinates contractility-driven collective cellular dynamics resulting in C2C12 myotube alignment over millimeter-scale distances. When cultured on mechanically anisotropic liquid crystalline polymer networks (LCNs) lacking topographic features that could confer contact guidance, C2C12 myoblasts collectively polarize in the stiffest direction of the substrate. Cellular coordination is amplified through reciprocal cell-ECM dynamics that emerge during fusion, driving global myotube-ECM ordering. Conversely, myotube alignment was restricted to small local domains with no directional preference on mechanically isotropic LCNs of same chemical formulation. These findings reveal a role for stiffness anisotropy in coordinating emergent collective cellular dynamics, with implications for understanding skeletal muscle tissue development and regeneration.

Photoinduced Dithiolane Crosslinking for Multiresponsive Dynamic Hydrogels.

While many hydrogels are elastic networks crosslinked by covalent bonds, viscoelastic hydrogels with adaptable crosslinks are increasingly being developed to better recapitulate time and position-dependent processes found in many tissues. In this work, 1,2-dithiolanes are presented as dynamic covalent photocrosslinkers of hydrogels, resulting in disulfide bonds throughout the hydrogel that respond to multiple stimuli. Using lipoic acid as a model dithiolane, disulfide crosslinks are formed under physiological conditions, enabling cell encapsulation via an initiator-free light-induced dithiolane ring-opening photopolymerization. The resulting hydrogels allow for multiple photoinduced dynamic responses including stress relaxation, stiffening, softening, and network functionalization using a single chemistry, which can be supplemented by permanent reaction with alkenes to further control network properties and connectivity using irreversible thioether crosslinks. Moreover, complementary photochemical approaches are used to achieve rapid and complete sample degradation via radical scission and post-gelation network stiffening when irradiated in the presence of reactive gel precursor. The results herein demonstrate the versatility of this material chemistry to study and direct 2D and 3D cell-material interactions. This work highlights dithiolane-based hydrogel photocrosslinking as a robust method for generating adaptable hydrogels with a range of biologically relevant mechanical and chemical properties that are varied on demand.

A myocardin-adjacent lncRNA balances SRF-dependent gene transcription in the heart.

Myocardin, a potent coactivator of serum response factor (SRF), competes with ternary complex factor (TCF) proteins for SRF binding to balance opposing mitogenic and myogenic gene programs in cardiac and smooth muscle. Here we identify a cardiac lncRNA transcribed adjacent to myocardin, named CARDINAL, which antagonizes SRF-dependent mitogenic gene transcription in the heart. CARDINAL-deficient mice show ectopic TCF/SRF-dependent mitogenic gene expression and decreased cardiac contractility in response to age and ischemic stress. CARDINAL forms a nuclear complex with SRF and inhibits TCF-mediated transactivation of the promitogenic gene c-fos, suggesting CARDINAL functions as an RNA cofactor for SRF in the heart.

Stac proteins associate with the critical domain for excitation-contraction coupling in the II-III loop of CaV1.1.

In skeletal muscle, residues 720-764/5 within the CaV1.1 II-III loop form a critical domain that plays an essential role in transmitting the excitation-contraction (EC) coupling Ca2+ release signal to the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum. However, the identities of proteins that interact with the loop and its critical domain and the mechanism by which the II-III loop regulates RyR1 gating remain unknown. Recent work has shown that EC coupling in skeletal muscle of fish and mice depends on the presence of Stac3, an adaptor protein that is highly expressed only in skeletal muscle. Here, by using colocalization as an indicator of molecular interactions, we show that Stac3, as well as Stac1 and Stac2 (predominantly neuronal Stac isoforms), interact with the II-III loop of CaV1.1. Further, we find that these Stac proteins promote the functional expression of CaV1.1 in tsA201 cells and support EC coupling in Stac3-null myotubes and that Stac3 is the most effective. Coexpression in tsA201 cells reveals that Stac3 interacts only with II-III loop constructs containing the majority of the CaV1.1 critical domain residues. By coexpressing Stac3 in dysgenic (CaV1.1-null) myotubes together with CaV1 constructs whose chimeric II-III loops had previously been tested for functionality, we reveal that the ability of Stac3 to interact with them parallels the ability of these constructs to mediate skeletal type EC coupling. Based on coexpression in tsA201 cells, the interaction of Stac3 with the II-III loop critical domain does not require the presence of the PKC C1 domain in Stac3, but it does require the first of the two SH3 domains. Collectively, our results indicate that activation of RyR1 Ca2+ release by CaV1.1 depends on Stac3 being bound to critical domain residues in the II-III loop.

Stac3 has a direct role in skeletal muscle-type excitation-contraction coupling that is disrupted by a myopathy-causing mutation.

In skeletal muscle, conformational coupling between CaV1.1 in the plasma membrane and type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR) is thought to underlie both excitation-contraction (EC) coupling Ca(2+) release from the SR and retrograde coupling by which RyR1 increases the magnitude of the Ca(2+) current via CaV1.1. Recent work has shown that EC coupling fails in muscle from mice and fish null for the protein Stac3 (SH3 and cysteine-rich domain 3) but did not establish the functional role of Stac3 in the CaV1.1-RyR1 interaction. We investigated this using both tsA201 cells and Stac3 KO myotubes. While confirming in tsA201 cells that Stac3 could support surface expression of CaV1.1 (coexpressed with its auxiliary ß1a and α2-δ1 subunits) and the generation of large Ca(2+) currents, we found that without Stac3 the auxiliary γ1 subunit also supported membrane expression of CaV1.1/ß1a/α2-δ1, but that this combination generated only tiny Ca(2+) currents. In Stac3 KO myotubes, there was reduced, but still substantial CaV1.1 in the plasma membrane. However, the CaV1.1 remaining in Stac3 KO myotubes did not generate appreciable Ca(2+) currents or EC coupling Ca(2+) release. Expression of WT Stac3 in Stac3 KO myotubes fully restored Ca(2+) currents and EC coupling Ca(2+) release, whereas expression of Stac3W280S (containing the Native American myopathy mutation) partially restored Ca(2+) currents but only marginally restored EC coupling. We conclude that membrane trafficking of CaV1.1 is facilitated by, but does not require, Stac3, and that Stac3 is directly involved in conformational coupling between CaV1.1 and RyR1.

Severe muscle wasting and denervation in mice lacking the RNA-binding protein ZFP106.

Innervation of skeletal muscle by motor neurons occurs through the neuromuscular junction, a cholinergic synapse essential for normal muscle growth and function. Defects in nerve-muscle signaling cause a variety of neuromuscular disorders with features of ataxia, paralysis, skeletal muscle wasting, and degeneration. Here we show that the nuclear zinc finger protein ZFP106 is highly enriched in skeletal muscle and is required for postnatal maintenance of myofiber innervation by motor neurons. Genetic disruption of Zfp106 in mice results in progressive ataxia and hindlimb paralysis associated with motor neuron degeneration, severe muscle wasting, and premature death by 6 mo of age. We show that ZFP106 is an RNA-binding protein that associates with the core splicing factor RNA binding motif protein 39 (RBM39) and localizes to nuclear speckles adjacent to spliceosomes. Upon inhibition of pre-mRNA synthesis, ZFP106 translocates with other splicing factors to the nucleolus. Muscle and spinal cord of Zfp106 knockout mice displayed a gene expression signature of neuromuscular degeneration. Strikingly, altered splicing of the Nogo (Rtn4) gene locus in skeletal muscle of Zfp106 knockout mice resulted in ectopic expression of NOGO-A, the neurite outgrowth factor that inhibits nerve regeneration and destabilizes neuromuscular junctions. These findings reveal a central role for Zfp106 in the maintenance of nerve-muscle signaling, and highlight the involvement of aberrant RNA processing in neuromuscular disease pathogenesis.

A MED13-dependent skeletal muscle gene program controls systemic glucose homeostasis and hepatic metabolism.

The Mediator complex governs gene expression by linking upstream signaling pathways with the basal transcriptional machinery. However, how individual Mediator subunits may function in different tissues remains to be investigated. Through skeletal muscle-specific deletion of the Mediator subunit MED13 in mice, we discovered a gene regulatory mechanism by which skeletal muscle modulates the response of the liver to a high-fat diet. Skeletal muscle-specific deletion of MED13 in mice conferred resistance to hepatic steatosis by activating a metabolic gene program that enhances muscle glucose uptake and storage as glycogen. The consequent insulin-sensitizing effect within skeletal muscle lowered systemic glucose and insulin levels independently of weight gain and adiposity and prevented hepatic lipid accumulation. MED13 suppressed the expression of genes involved in glucose uptake and metabolism in skeletal muscle by inhibiting the nuclear receptor NURR1 and the MEF2 transcription factor. These findings reveal a fundamental molecular mechanism for the governance of glucose metabolism and the control of hepatic lipid accumulation by skeletal muscle. Intriguingly, MED13 exerts opposing metabolic actions in skeletal muscle and the heart, highlighting the customized, tissue-specific functions of the Mediator complex.

A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle.

Muscle contraction depends on release of Ca(2+) from the sarcoplasmic reticulum (SR) and reuptake by the Ca(2+)adenosine triphosphatase SERCA. We discovered a putative muscle-specific long noncoding RNA that encodes a peptide of 34 amino acids and that we named dwarf open reading frame (DWORF). DWORF localizes to the SR membrane, where it enhances SERCA activity by displacing the SERCA inhibitors, phospholamban, sarcolipin, and myoregulin. In mice, overexpression of DWORF in cardiomyocytes increases peak Ca(2+) transient amplitude and SR Ca(2+) load while reducing the time constant of cytosolic Ca(2+) decay during each cycle of contraction-relaxation. Conversely, slow skeletal muscle lacking DWORF exhibits delayed Ca(2+) clearance and relaxation and reduced SERCA activity. DWORF is the only endogenous peptide known to activate the SERCA pump by physical interaction and provides a means for enhancing muscle contractility.

A micropeptide encoded by a putative long noncoding RNA regulates muscle performance.

Functional micropeptides can be concealed within RNAs that appear to be noncoding. We discovered a conserved micropeptide, which we named myoregulin (MLN), encoded by a skeletal muscle-specific RNA annotated as a putative long noncoding RNA. MLN shares structural and functional similarity with phospholamban (PLN) and sarcolipin (SLN), which inhibit SERCA, the membrane pump that controls muscle relaxation by regulating Ca(2+) uptake into the sarcoplasmic reticulum (SR). MLN interacts directly with SERCA and impedes Ca(2+) uptake into the SR. In contrast to PLN and SLN, which are expressed in cardiac and slow skeletal muscle in mice, MLN is robustly expressed in all skeletal muscle. Genetic deletion of MLN in mice enhances Ca(2+) handling in skeletal muscle and improves exercise performance. These findings identify MLN as an important regulator of skeletal muscle physiology and highlight the possibility that additional micropeptides are encoded in the many RNAs currently annotated as noncoding.

KLHL40 deficiency destabilizes thin filament proteins and promotes nemaline myopathy.

Nemaline myopathy (NM) is a congenital myopathy that can result in lethal muscle dysfunction and is thought to be a disease of the sarcomere thin filament. Recently, several proteins of unknown function have been implicated in NM, but the mechanistic basis of their contribution to disease remains unresolved. Here, we demonstrated that loss of a muscle-specific protein, kelch-like family member 40 (KLHL40), results in a nemaline-like myopathy in mice that closely phenocopies muscle abnormalities observed in KLHL40-deficient patients. We determined that KLHL40 localizes to the sarcomere I band and A band and binds to nebulin (NEB), a protein frequently implicated in NM, as well as a putative thin filament protein, leiomodin 3 (LMOD3). KLHL40 belongs to the BTB-BACK-kelch (BBK) family of proteins, some of which have been shown to promote degradation of their substrates. In contrast, we found that KLHL40 promotes stability of NEB and LMOD3 and blocks LMOD3 ubiquitination. Accordingly, NEB and LMOD3 were reduced in skeletal muscle of both Klhl40-/- mice and KLHL40-deficient patients. Loss of sarcomere thin filament proteins is a frequent cause of NM; therefore, our data that KLHL40 stabilizes NEB and LMOD3 provide a potential basis for the development of NM in KLHL40-deficient patients.

Requirement of MEF2A, C, and D for skeletal muscle regeneration.

Regeneration of adult skeletal muscle following injury occurs through the activation of satellite cells, an injury-sensitive muscle stem cell population that proliferates, differentiates, and fuses with injured myofibers. Members of the myocyte enhancer factor 2 (MEF2) family of transcription factors play essential roles in muscle differentiation during embryogenesis, but their potential contributions to adult muscle regeneration have not been systematically explored. To investigate the potential involvement of MEF2 factors in muscle regeneration, we conditionally deleted the Mef2a, c, and d genes, singly and in combination, within satellite cells in mice, using tamoxifen-inducible Cre recombinase under control of the satellite cell-specific Pax7 promoter. We show that deletion of individual Mef2 genes has no effect on muscle regeneration in response to cardiotoxin injury. However, combined deletion of the Mef2a, c, and d genes results in a blockade to regeneration. Satellite cell-derived myoblasts lacking MEF2A, C, and D proliferate normally in culture, but cannot differentiate. The absence of MEF2A, C, and D in satellite cells is associated with aberrant expression of a broad collection of known and unique protein-coding and long noncoding RNA genes. These findings reveal essential and redundant roles of MEF2A, C, and D in satellite cell differentiation and identify a MEF2-dependent transcriptome associated with skeletal muscle regeneration.

Skeletal muscle-specific T-tubule protein STAC3 mediates voltage-induced Ca2+ release and contractility.

Excitation-contraction (EC) coupling comprises events in muscle that convert electrical signals to Ca(2+) transients, which then trigger contraction of the sarcomere. Defects in these processes cause a spectrum of muscle diseases. We report that STAC3, a skeletal muscle-specific protein that localizes to T tubules, is essential for coupling membrane depolarization to Ca(2+) release from the sarcoplasmic reticulum (SR). Consequently, homozygous deletion of src homology 3 and cysteine rich domain 3 (Stac3) in mice results in complete paralysis and perinatal lethality with a range of musculoskeletal defects that reflect a blockade of EC coupling. Muscle contractility and Ca(2+) release from the SR of cultured myotubes from Stac3 mutant mice could be restored by application of 4-chloro-m-cresol, a ryanodine receptor agonist, indicating that the sarcomeres, SR Ca(2+) store, and ryanodine receptors are functional in Stac3 mutant skeletal muscle. These findings reveal a previously uncharacterized, but required, component of the EC coupling machinery of skeletal muscle and introduce a candidate for consideration in myopathic disorders.