MYLPF Human

What is MYLPF and what is its role in human skeletal muscle?

MYLPF (myosin light chain, phosphorylatable, fast skeletal muscle) is a regulatory light chain component of the myosin motor protein complex predominantly expressed in fast-twitch skeletal muscle fibers. It plays a crucial role in muscle contraction by regulating myosin motor activity through its phosphorylation state. In human muscle fibers, MYLPF is specifically associated with fast-twitch (type IIx) fibers, which contain different mixtures of myosin heavy and light chains compared to slow-twitch fibers . The protein interacts directly with myosin heavy chains and contributes to the structural stability and functional properties of the myosin molecule, ultimately influencing muscle contractile force and speed.

How does MYLPF differ from other myosin light chains in human muscle?

MYLPF represents one of several myosin light chain isoforms expressed in human skeletal muscle, each with distinct tissue distribution and functional properties:

• MYLPF (fast skeletal muscle regulatory light chain) is predominantly expressed in fast-twitch muscle fibers

• MYL3 (slow skeletal muscle essential light chain) is predominantly found in slow-twitch muscle fibers

• MYL1 (fast skeletal muscle essential light chain) is predominantly expressed in fast-twitch muscle fibers

These light chains differ in their amino acid sequences, phosphorylation sites, and calcium-binding properties. Immunoblot analyses reveal that MYL1 and MYLPF show higher expression in muscles with greater proportions of fast-twitch fibers, while MYL3 expression correlates with slow-twitch fiber content . This differential expression pattern is consistent with the specialized contractile properties of different muscle fiber types.

What are the structural characteristics of the MYLPF protein?

The human MYLPF protein belongs to the EF-hand calcium-binding protein family. Key structural features include:

• Multiple EF-hand domains that form helix-loop-helix structures

• A phosphorylation site that regulates its activity

• Binding regions that mediate interactions with myosin heavy chains

Sequence alignment studies show that MYLPF shares structural similarities with other regulatory light chains including MLC2, MLC5, MLC7, MLC9, MLC10, MLC12A, and MLC12B, as well as essential light chains MLC1, MLC3, MLC4, and MLC6 . These homologous proteins have diverse tissue expression patterns and functions. Models of MYLPF suggest that specific residues (such as Ala33) directly interact with myosin, while others (like Cys157 and Gly163) affect this interaction indirectly, which explains the different inheritance patterns of disease-causing mutations .

What are the most effective methods for isolating and studying MYLPF in human muscle samples?

For isolation and characterization of MYLPF from human muscle samples, researchers employ a combination of techniques:

• Laser Capture Microdissection (LCM): This technique allows precise isolation of specific fiber types from muscle biopsies. As demonstrated in published protocols, LCM can be used to obtain approximately 300 transversely cut fibers of a single type (types I, IIa, or IIx), representing about 2.0 mm² of tissue . This approach enables fiber type-specific protein and gene expression analysis.

• Immunoblotting: Using specific antibodies against MYLPF (such as anti-MYL1 antibodies, e.g., SAB1409338 from Sigma-Aldrich), researchers can detect and quantify MYLPF protein levels in muscle homogenates or LCM-isolated fiber samples .

• Mass Spectrometry Proteomics: This technique provides comprehensive identification and quantification of myosin isoforms and their post-translational modifications in muscle samples.

• Next-Generation Sequencing: RNA-seq analysis of isolated muscle fibers can reveal the expression patterns of MYLPF and related genes across different fiber types and disease states.

These methods can be combined to correlate MYLPF expression with muscle fiber composition and functional properties.

How can researchers effectively model MYLPF dysfunction in laboratory settings?

Several experimental models have proven valuable for studying MYLPF function and dysfunction:

• Zebrafish Models: Zebrafish provide an excellent system for investigating muscle development and function. Researchers have successfully generated MYLPF-deficient zebrafish by knocking out the mylpfa gene (the zebrafish ortholog of human MYLPF) using CRISPR-Cas9 genome editing. These models can be created using established protocols with guide RNAs targeting specific exons (e.g., exon 2 or 3) . Transgenic zebrafish lines expressing fluorescent markers in muscle tissues (such as mylz2:GFP, myog:H2B-mRFP, and smyhc1:EGFP) facilitate visualization of muscle development and function .

• Mouse Models: Complete knockout of Mylpf in mice results in absence of skeletal muscle and perinatal lethality due to respiratory failure, indicating the essential role of this protein in muscle development . Conditional knockout or knock-in models with specific mutations can provide insights into the tissue-specific and temporal requirements of MYLPF.

• Cell Culture Systems: C2C12 myoblasts and primary human myoblasts can be differentiated into myotubes in vitro, allowing for manipulation of MYLPF expression through siRNA knockdown, CRISPR editing, or overexpression of wild-type or mutant forms.

• Patient-Derived iPSCs: Induced pluripotent stem cells from patients with MYLPF mutations can be differentiated into skeletal muscle cells to study disease mechanisms in a human genetic background.

These models enable functional analyses including muscle contractility measurements, histological evaluation, and molecular profiling.

What techniques are most sensitive for detecting MYLPF mutations in clinical samples?

The detection of MYLPF mutations in clinical samples typically employs a combination of approaches:

• Exome Sequencing: This comprehensive approach has successfully identified pathogenic variants in MYLPF in multiple families with distal arthrogryposis . Exome sequencing provides coverage of all exons and can detect both homozygous and heterozygous variants.

• Targeted Gene Panel Sequencing: For conditions with clinical features suggestive of distal arthrogryposis or congenital myopathies, targeted panels including MYLPF and other related genes can provide more focused and cost-effective genetic testing.

• Sanger Sequencing: This remains valuable for validating variants identified through next-generation sequencing approaches and for cascade family testing once a pathogenic variant has been identified .

• High-Resolution Melting Analysis (HRMA): This technique can be used for rapid screening of specific variants, as demonstrated in identifying CRISPR-induced mutations in zebrafish models .

• Data Sharing Platforms: Utilizing resources like MatchMaker Exchange (MME), MyGene2, and GeneMatcher facilitates identification of additional cases with rare variants in the same gene, which is crucial for establishing genotype-phenotype correlations .

For comprehensive analysis, researchers should consider both dominant and recessive inheritance patterns when interpreting MYLPF variants, as both patterns have been observed in association with distal arthrogryposis .

What is the spectrum of human diseases associated with MYLPF mutations?

MYLPF mutations have been primarily associated with distal arthrogryposis (DA), a group of disorders characterized by congenital contractures predominantly affecting the distal limbs. The specific phenotypic features include:

• Congenital contractures: Affecting hands, wrists, elbows, knees, and feet

• Scoliosis: Present in many affected individuals

• Short stature: A common finding among patients

• Clubfeet: Frequently observed bilateral talipes equinovarus

• Segmental amyoplasia: Complete absence of skeletal muscle in affected limb segments in some cases

Additional clinical features observed in some patients include undescended testicles, shoulder contractures, adducted thumbs, flexed metacarpophalangeal joints, blepharophimosis, and fifth finger clinodactyly . The condition can present with both autosomal dominant and autosomal recessive inheritance patterns, depending on the specific mutation and its functional impact on the MYLPF protein.

How do different MYLPF mutations correlate with phenotypic severity and inheritance patterns?

Research has identified several specific mutations in MYLPF associated with distal arthrogryposis, with clear genotype-phenotype correlations:

• Recessive mutations:

  • c.470G>T (p.Cys157Phe)

  • c.469T>C (p.Cys157Arg)

  • c.487G>A (p.Gly163Ser)

• Dominant mutations:

  • c.98C>T (p.Ala33Val)

The inheritance pattern appears to correlate with the functional impact of the mutation. Protein modeling suggests that residues associated with dominant disease (e.g., Ala33) directly interact with myosin, while residues altered in recessive cases (e.g., Cys157, Gly163) only indirectly impair this interaction . This molecular distinction explains why heterozygous carriers of recessive mutations remain unaffected while carriers of dominant mutations display the disease phenotype.

The severity of the phenotype also varies, with some mutations leading to complete absence of skeletal muscle in affected limb regions (segmental amyoplasia), while others result in muscle weakness without complete tissue loss .

What are the functional consequences of MYLPF mutations at the molecular and cellular levels?

MYLPF mutations disrupt skeletal muscle development and function through several mechanisms:

• Impaired myosin motor activity: MYLPF mutations can reduce myosin ATPase activity and force generation, as demonstrated in zebrafish models where mylpfa knockout resulted in reduced trunk contractile force and complete pectoral fin paralysis .

• Muscle fiber degeneration: Histological analyses of zebrafish models show that MYLPF deficiency leads to progressive muscle fiber degeneration, particularly affecting appendicular (limb) muscles more severely than axial musculature .

• Developmental defects: Complete loss of MYLPF function in mouse models results in absence of skeletal muscle development, indicating its critical role in myogenesis . Partial loss of function allows initial development but leads to subsequent degeneration.

• Fiber type-specific effects: Since MYLPF is predominantly expressed in fast-twitch fibers, mutations primarily affect these fiber types, potentially explaining the pattern of muscle involvement in patients .

The varying severity of these effects likely depends on the specific mutation and its impact on MYLPF structure, stability, and interaction with myosin heavy chains.

How does phosphorylation regulate MYLPF function in different physiological contexts?

As a phosphorylatable light chain, MYLPF activity is dynamically regulated through post-translational modifications:

• Phosphorylation mechanisms: MYLPF is phosphorylated by myosin light chain kinase (MLCK) and dephosphorylated by myosin light chain phosphatase (MLCP). These enzymes respond to calcium signaling and other regulatory inputs.

• Physiological significance: Phosphorylation of MYLPF increases myosin ATPase activity and enhances the rate and force of muscle contraction in fast-twitch fibers. This regulation is particularly important during rapid, high-force contractions.

• Fiber type-specific regulation: The phosphorylation state of MYLPF differs between resting and actively contracting muscles, and likely varies across fiber types with different contractile properties. Research methods to study this include phospho-specific antibodies, phosphoproteomics, and functional assays of contractile properties.

• Disease implications: Mutations affecting phosphorylation sites or interactions with regulatory kinases could alter the normal dynamic regulation of MYLPF, potentially contributing to muscle dysfunction even in the absence of structural abnormalities.

Future research should examine whether therapeutic approaches targeting MYLPF phosphorylation could ameliorate muscle weakness in patients with partial MYLPF deficiency or dysfunction.

What is the evolutionary conservation of MYLPF across species and what can this tell us about its function?

MYLPF shows significant evolutionary conservation, reflecting its fundamental role in muscle function:

• Cross-species comparison: MYLPF orthologs have been identified across vertebrates, including humans, mice, zebrafish, and other model organisms. Even invertebrates possess related myosin light chains, such as Mlc2 in Saccharomyces cerevisiae .

• Functional domains: The highest conservation is observed in domains critical for calcium binding and myosin heavy chain interaction. The EF-hand domains that form helix-loop-helix structures are particularly conserved.

• Zebrafish paralogs: Zebrafish possess two MYLPF paralogs (mylpfa and mylpfb), with mylpfa being more prominently expressed in fast-twitch muscles . This gene duplication may provide insights into subfunctionalization of MYLPF roles.

• Experimental implications: The conservation between human MYLPF and zebrafish mylpfa allows for meaningful disease modeling in zebrafish, as demonstrated by the recapitulation of limb-predominant muscle weakness in mylpfa knockout fish .

Comparative genomic approaches, combined with functional studies across model organisms, can reveal evolutionarily constrained regions of MYLPF that are likely critical for function and potentially intolerant to variation.

How does MYLPF expression and function change during muscle development, regeneration, and aging?

MYLPF expression and function undergo dynamic changes throughout the lifespan of muscle tissue:

• Developmental regulation: During embryonic development, MYLPF expression increases as muscle fiber type specification occurs. In zebrafish, mylpfa expression is detectable in developing fast-twitch muscles by 1 day post-fertilization (dpf) .

• Fiber type transitions: Changes in MYLPF expression may contribute to fiber type transitions that occur during development and in response to altered activity patterns or disease states. This can be studied using immunohistochemistry with fiber type-specific markers combined with MYLPF detection.

• Regenerative capacity: The role of MYLPF in muscle regeneration remains to be fully elucidated. Research questions include whether MYLPF expression in satellite cells differs from mature fibers, and how its regulation affects the success of muscle repair processes.

• Age-related changes: Studies of muscle biopsies from individuals across different age ranges (22-87 years) suggest potential age-related changes in myosin light chain composition . This may contribute to the altered contractile properties and fiber type distribution observed in aging muscle.

• Exercise adaptation: Exercise training can induce fiber type transitions and alter myosin composition. The role of MYLPF in these adaptations represents an important area for future investigation, particularly in the context of therapeutic exercise for muscle disorders.

What are the protein-protein interaction networks involving MYLPF in muscle cells?

MYLPF functions within a complex network of protein interactions in muscle cells:

• Myosin heavy chain interactions: MYLPF directly binds to the neck region of myosin heavy chains, particularly those expressed in fast-twitch fibers (MYH1, MYH2, and MYH4) . The specific binding interfaces and how mutations disrupt these interactions can be studied using co-immunoprecipitation, yeast two-hybrid assays, and structural biology approaches.

• Regulatory protein interactions: MYLPF interacts with regulatory enzymes including MLCK and MLCP that control its phosphorylation state. These interactions may be tissue-specific and dynamically regulated during muscle contraction cycles.

• Sarcomeric organization: Beyond its role in myosin motor function, MYLPF may contribute to sarcomere assembly and stability through interactions with other structural proteins. Proteomics approaches combined with proximity labeling techniques can identify the full interactome.

• Signaling pathways: MYLPF may participate in signaling pathways that regulate muscle growth, differentiation, and adaptation to mechanical stress. Phosphoproteomic analyses can reveal how MYLPF phosphorylation states change in response to various stimuli.

Understanding these interaction networks can provide insights into the molecular pathogenesis of MYLPF-related disorders and identify potential therapeutic targets.

What therapeutic strategies could potentially address MYLPF-related muscle disorders?

Several therapeutic approaches show potential for addressing MYLPF-related muscle disorders:

• Gene therapy: Delivery of functional MYLPF using adeno-associated viral vectors (AAVs) could potentially restore protein expression in recessive disorders. The highly specific expression pattern of MYLPF in fast-twitch fibers would require careful promoter selection for targeted expression.

• RNA-based therapies: For dominant negative mutations, antisense oligonucleotides or RNA interference approaches could selectively suppress the mutant allele while preserving wild-type function.

• Small molecule modulators: Compounds that enhance residual MYLPF activity or augment the function of related myosin light chains might compensate for partial MYLPF deficiency.

• Cell-based therapies: Transplantation of myogenic stem cells expressing functional MYLPF could potentially regenerate affected muscles, particularly in cases of segmental amyoplasia.

• Physical therapy interventions: For patients with partial MYLPF function, specialized exercise protocols might help preserve muscle mass and function, though this would need to be carefully tailored to avoid further muscle damage.

Preclinical evaluation of these approaches in zebrafish and mouse models would be essential before clinical translation.

What are the key unresolved questions about MYLPF that would advance our understanding of muscle biology and disease?

Despite significant progress, several important questions about MYLPF remain unanswered:

• Tissue specificity: Why do MYLPF mutations predominantly affect distal limb muscles despite the protein's broader expression in fast-twitch muscles throughout the body? Understanding this selective vulnerability could provide insights into the pathogenesis of distal arthrogryposis.

• Compensatory mechanisms: Do other myosin light chains compensate for MYLPF deficiency in certain muscle groups but not others? Comparative transcriptomic and proteomic analyses across affected and unaffected muscles could address this question.

• Temporal requirements: Is MYLPF primarily required during developmental myogenesis, for maintenance of mature muscles, or both? Conditional knockout models with temporal control of gene inactivation would help resolve this question.

• Modifier genes: What genetic modifiers influence the severity of MYLPF-related phenotypes? Genome-wide association studies in patient cohorts with variable disease severity might identify such modifiers.

• Non-muscle roles: Does MYLPF have functions outside of skeletal muscle that remain undiscovered? Comprehensive expression profiling across tissues and developmental stages could reveal unexpected sites of expression.

Addressing these questions would not only advance our understanding of MYLPF biology but could also provide insights into fundamental aspects of muscle development, maintenance, and disease.

How might emerging technologies advance MYLPF research and clinical applications?

Cutting-edge technologies offer new opportunities to study MYLPF and develop targeted therapies:

• Single-cell technologies: Single-cell RNA sequencing and proteomics can reveal cell-type-specific expression patterns and responses to MYLPF dysfunction at unprecedented resolution.

• Advanced imaging: Super-resolution microscopy and live-cell imaging techniques can visualize MYLPF localization and dynamics during muscle contraction and development.

• Organoid models: Skeletal muscle organoids derived from patient iPSCs can provide three-dimensional models of MYLPF-related muscle disorders for mechanistic studies and drug screening.

• CRISPR-based approaches: Beyond gene editing for disease modeling, CRISPR activation (CRISPRa) or interference (CRISPRi) systems could be used to modulate MYLPF expression in therapeutic contexts.

• Systems biology: Integration of multi-omics data (genomics, transcriptomics, proteomics, metabolomics) with clinical information can provide a comprehensive understanding of how MYLPF variations impact muscle function at multiple biological levels.

• Artificial intelligence: Machine learning approaches could help predict the pathogenicity of novel MYLPF variants and identify patterns in patient data that inform prognosis and treatment selection.

By leveraging these technologies, researchers can accelerate discovery in MYLPF biology and develop more effective, personalized approaches to treating MYLPF-related disorders.