Recombinant Human Myosin-14 (MYH14), partial

What is MYH14 and what is its biological significance?

MYH14 encodes one of the heavy chains of class II nonmuscle myosins. These proteins are implicated in various motile processes, including organelle translocation, ion-channel gating, and cytoskeleton reorganization. MYH14 has been definitively linked to autosomal dominant hearing impairment (DFNA4), confirming the crucial role of the myosin superfamily in auditory functions . Recent research has also implicated MYH14 in protection against noise-induced hearing loss and in mitochondrial dynamics related to peripheral neuropathy .

How is MYH14 expression characterized in different tissues?

MYH14 expression can be effectively detected through multiple methodologies:

• RT-PCR: Studies have confirmed MYH14 expression in mouse cochlea by amplifying a 768 bp fragment from cochlear cDNA .

• Immunohistochemistry: MYH14 is primarily expressed in or near the reticular lamina, specifically in the apical junctional complexes (AJCs) of the cochlea .

• Western blot analysis: Using specific anti-NMHC IIC antibodies, MYH14 protein can be detected in tissue samples such as the cerebellum, where expression is particularly high .

What genetic disorders are associated with MYH14 mutations?

Mutations in MYH14 have been causally linked to:

• DFNA4-type hearing impairment, an autosomal dominant form of progressive hearing loss

• Peripheral neuropathy, particularly through mutations that disrupt mitochondrial fission

• Increased susceptibility to noise-induced hearing loss

The discovery of nonsense and missense mutations in the MYH14 gene in large pedigrees with hearing impairment has definitively established its role in auditory function .

What is the recommended approach for generating MYH14 knockout models?

The CRISPR/Cas9 genome-editing technology has been successfully employed to establish MYH14 knockout mouse lines. The detailed protocol includes:

• Design of sgRNA targeting a specific exon (e.g., exon 9 with target sequence 5′-CCTGAAGAAAGAGCGCAATA-3′)

• In vitro transcription of sgRNA using T7 as promoter with the MEGAshortscript kit

• Synthesis of hCas9 mRNA using the mMESSAGE mMACHINE T7 kit

• Purification of both sgRNA and Cas9 mRNA with the MEGAclear kit

• Microinjection into pronuclear stage mouse embryos

• Genotyping through sequencing analysis to identify successful mutants

• Breeding strategy to establish homozygous lines

The effectiveness of the knockout should be verified through Western blot and immunocytochemistry to confirm complete abolishment of MYH14 protein expression .

How can recombinant MYH14 be produced for functional studies?

Recombinant MYH14 motor domains can be produced through:

• Cloning of the motor domain region (such as short subfragment 1, sS1, corresponding to residues 1-808)

• Expression in differentiated muscle cells (e.g., C2C12 cell line)

• Purification of the recombinant protein for subsequent kinetic analysis

This approach has been successfully employed for related myosin motors and allows for extensive kinetic characterization of both wild-type and mutant forms .

What methodologies are most effective for identifying novel MYH14 mutations?

A comprehensive approach to identify novel MYH14 mutations includes:

• Linkage analysis: Establish linkage to the DFNA4 locus (chromosome 19)

• Haplotype construction: Using dinucleotide markers surrounding the MYH14 gene

• Direct sequencing: Analysis of all exons and exon-intron boundaries

• Segregation analysis: Verification that identified variants co-segregate with the hearing impairment phenotype

• Control screening: Confirming absence of variants in 200+ healthy individuals

• Next-generation sequencing: For comprehensive analysis of the entire candidate region

For specific known mutations, more targeted approaches like restriction fragment length polymorphism (RFLP) analysis may be applicable. For example, the R726S mutation creates an AlwNI restriction site that can be detected after PCR amplification .

What are the phenotypic characteristics of MYH14 knockout mice?

MYH14 knockout mice on the CBA/CaJ background demonstrate:

• Normal appearance and cochlear development initially

• High-frequency hearing loss developing by five months of age

• Moderate hair cell loss in the basal turn of the cochlea starting around the same time period

• Significantly increased vulnerability to high-intensity noise compared to wild-type controls

• More substantial outer hair cell loss following acoustic trauma

Interestingly, background strain significantly influences the phenotype. While CBA/CaJ-based knockouts show the above characteristics, previous studies using C57/B6 and 129/Sv strains reported no obvious differences between MYH14 knockout and wild-type mice .

How do specific mutations affect MYH14 function and disease phenotypes?

Different mutations in MYH14 result in distinct clinical presentations:

What mechanisms underlie MYH14's role in noise-induced hearing loss protection?

While the exact protective mechanism remains under investigation, experimental evidence clearly demonstrates that Myh14⁻/⁻ mice are more vulnerable to high-intensity noise exposure than wild-type controls . The increased outer hair cell loss observed after acoustic trauma suggests MYH14 may play a role in:

• Structural integrity of the cochlear sensory epithelium

• Mechanical resilience of hair cells to acoustic overstimulation

• Stress response pathways activated during noise exposure

This protective function adds another dimension to MYH14's importance beyond its role in hereditary hearing impairment .

How do mutations in MYH14 affect the kinetics of the myosin motor domain?

Motor domain mutations can significantly alter the cross-bridge cycle kinetics of myosin proteins. Key parameters that should be analyzed include:

• ATP binding and hydrolysis rates

• ADP release rates

• Actin-activated ATPase activity

• Motility characteristics

• Force generation capabilities

Research on related myosin motors has shown that mutations can affect these parameters to varying degrees, with changes of ≥2-fold in at least one parameter for each mutation studied . A comprehensive kinetic analysis is essential to understand how specific mutations impact motor function and contribute to disease phenotypes.

What are the challenges in correlating genotype with phenotype in MYH14-related disorders?

Several factors complicate genotype-phenotype correlations:

• Genetic background effects: The same mutation can produce different phenotypes in different genetic backgrounds, as demonstrated by the different outcomes of MYH14 knockout in different mouse strains .

• Variable expressivity: Within families carrying the same mutation, there can be significant variation in age of onset, progression rate, and severity of hearing loss.

• Environmental interactions: Factors such as noise exposure may interact with genetic mutations to accelerate or modify disease progression.

• Molecular complexity: The intricate biochemical and structural properties of myosin motors make it difficult to predict how specific mutations will affect function.

How might MYH14's role in mitochondrial dynamics connect to its function in hearing loss?

The R941L mutation in MYH14 has been implicated in disrupting mitochondrial fission , suggesting a potential link between mitochondrial dynamics and auditory function. This connection could involve:

• Energy production for high-metabolic-demand hair cells

• Calcium buffering in auditory neurons

• Apoptotic pathways activated during acoustic trauma

• Maintenance of specialized cytoskeletal structures in the inner ear

Research integrating these aspects could reveal how MYH14's diverse cellular functions contribute to its role in hearing maintenance and protection against noise-induced damage.

What are the key considerations for screening MYH14 in clinical settings?

Effective genetic screening approaches should account for:

• Mutational hotspots: Initial focus on exons where pathogenic mutations have been previously identified

• Inheritance patterns: Primarily autosomal dominant for hearing loss phenotypes

• Phenotypic indicators: Progressive sensorineural hearing loss, often with age of onset in the first or second decade

• Comprehensive analysis: Examination of the entire coding region and exon-intron boundaries

• Exclusion of non-pathogenic variants: Differentiation from common SNPs that do not segregate with disease phenotypes

How can researchers distinguish pathogenic variants from non-pathogenic polymorphisms?

Distinguishing pathogenic variants requires:

• Segregation analysis: Confirmation that the variant co-segregates with disease in affected families

• Population screening: Verification that the variant is absent in large numbers of healthy controls

• Conservation analysis: Assessment of whether the affected amino acid is conserved across species

• Functional studies: Experimental evaluation of the variant's impact on protein function

• In silico prediction: Use of computational tools to predict pathogenicity

Several known SNPs in MYH14 have been identified that do not cause disease, including synonymous substitutions at positions 657G > A (rs4801822) in exon 5 and 2127A > G (rs1651553) in exon 17, as well as 18 intronic variants .

What are the most promising approaches for therapeutic targeting of MYH14-related disorders?

Potential therapeutic strategies include:

• Gene therapy: Delivery of functional MYH14 to affected tissues

• Small molecule modulators: Compounds that might enhance residual MYH14 function

• Pathway-based interventions: Targeting downstream effectors or compensatory mechanisms

• Protective approaches: Methods to reduce susceptibility to noise-induced damage

• Mitochondrial-targeted therapies: For mutations affecting mitochondrial dynamics

How might multi-omics approaches advance understanding of MYH14 function?

Integration of multiple omics technologies could provide deeper insights:

• Transcriptomics: Identifying genes co-regulated with MYH14 or responsive to MYH14 dysfunction

• Proteomics: Mapping MYH14 interaction networks and post-translational modifications

• Metabolomics: Assessing metabolic consequences of MYH14 mutations

• Single-cell analyses: Characterizing cell-specific responses to MYH14 deficiency

• Systems biology: Modeling the broader impact of MYH14 dysfunction on cellular networks