MYOZ1 Human

What is MYOZ1 and what are its primary functions in human muscle cells?

MYOZ1 (Myozenin-1), also known as calsarcin-2 or FATZ1, belongs to the myozenin family of proteins that function primarily at the Z-disc of striated muscle. It serves as an intracellular binding protein that links Z-disk proteins such as alpha-actinin, gamma-filamin, TCAP/telethonin, and LDB3/ZASP while localizing calcineurin signaling to the sarcomere. The 299-amino acid protein plays a crucial role in the modulation of calcineurin signaling and may participate in myofibrillogenesis .

MYOZ1 functions as a structural scaffold for sarcomere organization and has recently been identified as an interaction hub for multiple Z-line proteins. Structurally, MYOZ1 forms a 2:1 complex with α-actinin-2, which stabilizes MYOZ1 proteins at the Z-disc, creating a foundation for proper sarcomere assembly .

How does MYOZ1 expression vary across different muscle types?

MYOZ1 demonstrates tissue-specific expression patterns, with predominant expression in fast-twitch skeletal muscles. This distribution pattern has been consistently observed across humans, mice, and chickens. Expression analysis reveals:

• Highest expression in leg and breast muscles (fast-twitch fibers)

• Lower expression in liver and heart tissues

• Expression levels that vary by sex and age

• Increased expression in response to frequent exercise

The selective expression in fast-twitch muscle fibers makes MYOZ1 a valuable marker for muscle fiber type analysis. Notably, studies in chickens have found that MYOZ1 is not expressed in variants with predominantly fast-twitch fibers, suggesting species-specific regulation patterns that may influence muscle development .

What are the known protein interaction partners of MYOZ1?

MYOZ1 functions as part of a complex protein interaction network within the Z-disc of skeletal muscle. Based on STRING database analysis and experimental validation, the principal interaction partners include:

These interactions have been verified through co-immunoprecipitation studies and yeast two-hybrid screens, establishing MYOZ1 as a central player in Z-disc architecture .

How is MYOZ1 protein stability regulated at the post-translational level?

MYOZ1 undergoes precise post-translational regulation through the ubiquitin-proteasome system. The E3 ligase FBXL21 has been identified as a critical regulator of MYOZ1 protein stability through the following mechanisms:

• Direct binding: FBXL21 physically interacts with MYOZ1 as demonstrated by co-immunoprecipitation studies in both cell culture models and skeletal muscle tissue.

• Ubiquitin-mediated degradation: FBXL21 promotes MYOZ1 proteasomal degradation in a dose-dependent manner. Treatment with the proteasome inhibitor MG132 prevents FBXL21-mediated MYOZ1 degradation.

• Circadian regulation: MYOZ1 displays an anti-phasic circadian rhythm compared to FBXL21 in skeletal muscle, suggesting temporal regulation of protein levels throughout the day .

Experimental half-life measurements reveal that MYOZ1 has a baseline half-life of approximately 15.4 hours, which is dramatically reduced to 4.3 hours in the presence of FBXL21. This degradation is further accelerated to 2.2 hours when GSK-3β is co-expressed, demonstrating the importance of phosphorylation in this regulatory process .

What is the relationship between MYOZ1 and calcineurin/NFAT signaling?

MYOZ1 functions as a negative regulator of calcineurin/NFAT signaling in skeletal muscle. The relationship between these components involves:

• Localization control: MYOZ1 tethers calcineurin to the Z-disc, spatially constraining its activity.

• Signaling modulation: MYOZ1 deficiency leads to excessive CaN/NFAT activation, demonstrating its inhibitory role.

• Muscle fiber type regulation: NFAT activation induced by MYOZ1 deficiency can trigger a muscle fiber type switch toward a slow-twitch and oxidative phenotype, with increased expression of type I myofiber-associated genes like myoglobin and troponin I slow .

This regulatory mechanism represents a critical link between sarcomere structure and muscle fiber type determination, with implications for both development and disease states .

How does GSK-3β phosphorylation affect MYOZ1 function and stability?

GSK-3β (Glycogen Synthase Kinase-3β) plays a significant role in the post-translational regulation of MYOZ1 through phosphorylation. Key experimental findings demonstrate:

• Direct interaction: GSK-3β physically interacts with both MYOZ1 and FBXL21 as demonstrated by co-immunoprecipitation assays.

• Kinase activity: In vitro kinase assays confirm that GSK-3β can phosphorylate MYOZ1, affecting its stability and interaction properties.

• Degradation kinetics: Ectopic expression of GSK-3β accelerates FBXL21-mediated MYOZ1 degradation, reducing MYOZ1's half-life from 4.3 hours to 2.2 hours.

• Inhibition effects: Treatment with the GSK-3 inhibitor CHIR-99021 decelerates FBXL21-mediated MYOZ1 degradation, extending MYOZ1's half-life to approximately 30.4 hours .

These findings establish a phosphorylation-dependent regulatory mechanism where GSK-3β primes MYOZ1 for FBXL21-mediated degradation, creating a signaling nexus that potentially integrates metabolic signals with muscle structural regulation.

What role does MYOZ1 play in myoblast differentiation?

MYOZ1 contributes significantly to myoblast differentiation through its role in sarcomere organization and signaling regulation. Experimental evidence from FBXL21 knockout studies in C2C12 cells reveals:

• MYOZ1 accumulation: FBXL21 knockout leads to a 2.5-fold increase in MYOZ1 levels in myoblasts (Day 0) and a 2.2-fold increase in myotubes (Day 6).

• Differentiation impairment: Elevated MYOZ1 levels correlate with reduced expression of myosin heavy chain (MyHC), a terminal differentiation marker for muscle cells.

• Reduced fusion index: FBXL21 knockout C2C12 cells exhibit a significantly lower fusion index compared to control cells during differentiation (Days 4 and 6) .

These findings suggest that precise regulation of MYOZ1 protein levels is essential for proper myoblast differentiation. Excessive MYOZ1 accumulation disrupts the stoichiometry of sarcomeric components, potentially affecting the assembly and function of the contractile apparatus during muscle development .

How do mutations in the MYOZ1 gene affect muscle phenotype?

Mutations in the MYOZ1 gene have been associated with altered muscle phenotypes across various species. Studies in chickens have identified:

• Sequence variations: Substitution mutations in the MYOZ1 gene, including T117C, G119C, G120C, C121T, and T140C, among others.

• Insertion mutations: G23 and A25 insertions in specific chicken breeds.

• Phenotypic associations: These mutations correlate with variations in leg and breast muscle weight, suggesting functional consequences for muscle development.

• Protein structure alterations: Similar to findings with other genes like GAPDH, substitution mutations in MYOZ1 can potentially alter amino acid sequences and three-dimensional protein structure .

The consequences of MYOZ1 deficiency include decreased performance due to reduced body weight and fast-twitch muscles, resulting from excessive CaN/NFAT activation. This activation induces a muscle fiber type switch toward a slow-twitch and oxidative phenotype, affecting the expression profile of muscle-specific genes .

What evidence links MYOZ1 to cardiac pathologies?

MYOZ1 has been implicated in cardiac pathologies through several lines of evidence:

• Gene association studies: MYOZ1 has been reported as a candidate gene for determining heart failure and hypertrophy.

• Expression changes: Altered MYOZ1 expression patterns have been observed in cardiac tissue under pathological conditions.

• Signaling dysregulation: Through its interaction with calcineurin/NFAT signaling, MYOZ1 dysfunction may contribute to pathological cardiac remodeling.

• Sarcomere integrity: As a Z-disc component, MYOZ1 alterations may compromise sarcomere structure in cardiac muscle .

The dual role of MYOZ1 in structural organization and signaling regulation positions it as a potential therapeutic target for cardiac hypertrophy and heart failure, though additional research is needed to fully elucidate its role in these conditions.

What techniques are most effective for studying MYOZ1 protein-protein interactions?

Several complementary techniques have proven effective for investigating MYOZ1 protein-protein interactions:

• Yeast Two-Hybrid (Y2H) screening: Successfully used to identify MYOZ1 interaction with FBXL21 and TCAP. The assay employs the Gal4 DNA-binding domain (BD) and the Gal4 activation domain (AD) fusion constructs .

• Co-immunoprecipitation (co-IP): Powerful for validating interactions in both cell culture and tissue samples. Studies have employed both ectopic expression systems (HEK293T cells) and endogenous protein analysis in gastrocnemius tissues from mice at different time points (ZT0 and ZT12) .

• GST pull-down assays: Useful for mapping specific binding domains within MYOZ1 and its interaction partners.

• Protein stability assays: Cycloheximide (CHX) chase assays have effectively demonstrated how MYOZ1 stability is regulated by interacting proteins like FBXL21, with half-life calculations providing quantitative measurements of these effects .

• Immunofluorescence co-localization: Valuable for confirming interactions within cellular contexts, particularly at the Z-disc of muscle cells.

These methodologies have collectively established MYOZ1 as a hub for protein interactions at the Z-disc, creating a foundation for understanding its role in muscle structure and function.

How can researchers effectively manipulate MYOZ1 expression in experimental models?

Several approaches have been successfully implemented to manipulate MYOZ1 expression in experimental models:

• CRISPR-Cas9 gene editing: Successfully used to generate FBXL21 knockout C2C12 cells, leading to MYOZ1 accumulation. This approach enables studying the consequences of elevated MYOZ1 levels .

• Plasmid-based overexpression: Transfection of expression constructs (e.g., Flag-MYOZ1) in cell culture models like HEK293T cells for protein interaction and stability studies.

• RNA interference (RNAi): siRNA or shRNA targeting MYOZ1 for transient or stable knockdown, respectively.

• Knockout mouse models: Genetic ablation of MYOZ1 to study whole-organism phenotypes related to muscle development and function.

• Site-directed mutagenesis: Creation of specific MYOZ1 mutations to investigate the functional consequences of genetic variations identified in population studies.

When selecting a manipulation approach, researchers should consider the temporal requirements of their experiment, as protein stability assays may benefit from transient transfection methods, while developmental studies often require stable genetic modifications .

What analytical methods are recommended for quantifying MYOZ1 expression in muscle tissues?

Several analytical methods can be employed to quantify MYOZ1 expression in muscle tissues with varying degrees of resolution and information:

• Quantitative immunofluorescence: Provides spatial information about MYOZ1 distribution while enabling quantification of expression levels. This method has been effectively used to demonstrate a 2.5-fold increase in MYOZ1 levels in FBXL21 knockout C2C12 cells .

• Western blotting: Effective for quantifying total MYOZ1 protein levels in tissue lysates, particularly when combined with cycloheximide chase assays for stability assessment. Quantification can be performed using unnormalized optical density (OD) values .

• RT-qPCR: Measures MYOZ1 mRNA expression levels, allowing comparison across different tissues, developmental stages, or experimental conditions.

• RNA sequencing: Provides comprehensive transcriptomic data, contextualizing MYOZ1 expression within the broader gene expression landscape.

• Mass spectrometry: Enables absolute quantification of MYOZ1 protein levels and can detect post-translational modifications.

For optimal results, researchers should employ multiple complementary techniques to validate findings across different levels of biological organization, from mRNA to protein expression and localization .

How does the circadian regulation of MYOZ1 impact muscle function?

The discovery of anti-phasic circadian rhythms between MYOZ1 and its regulator FBXL21 opens new research questions about temporal control of muscle function:

• Expression patterns: MYOZ1 displays circadian oscillation in skeletal muscle that is anti-phasic to FBXL21, suggesting temporal regulation of Z-disc composition .

• Protein stability cycles: FBXL21-mediated degradation of MYOZ1 follows a circadian pattern, potentially linking day-night cycles to muscle structure and function.

• Functional implications: The circadian regulation of sarcomere proteins may facilitate adaptation to daily activity patterns and metabolic cycles.

• Experimental approaches: Researchers can investigate these connections using time-course sampling of muscle tissues at different zeitgeber times (ZT), combined with protein stability assays and functional measurements of muscle performance .

This emerging area connects chronobiology with muscle physiology, suggesting that Z-disc composition and function may be temporally regulated to match activity patterns and metabolic demands throughout the day-night cycle.

What is the potential of MYOZ1 as a biomarker for muscle-related conditions?

MYOZ1 shows promise as a biomarker for several muscle-related conditions:

• Muscle fiber type determination: As MYOZ1 is predominantly expressed in fast-twitch fibers, it serves as a potential marker for fiber type composition.

• Meat quality assessment: In animal husbandry, MYOZ1 gene expression and single-nucleotide polymorphisms have been associated with meat quality traits in chickens and pigs.

• Exercise adaptation: Increased MYOZ1 expression has been observed in response to frequent exercise, potentially reflecting muscle fiber type transitions.

• Cardiac pathology: As a candidate gene for heart failure and hypertrophy, MYOZ1 expression patterns might indicate cardiac stress or pathological remodeling .

Researchers can leverage various analytical approaches, including genetic screening for MYOZ1 mutations, expression profiling, and protein quantification, to evaluate MYOZ1's utility as a biomarker in both research and clinical settings .

How does MYOZ1 contribute to the stoichiometric balance of the Z-disc complex?

Recent structural studies have revealed that MYOZ1 serves as a critical component in maintaining the stoichiometric balance of the Z-disc complex:

• Interaction hub: MYOZ1 contains multiple binding sites for other sarcomere proteins, creating an architectural foundation for Z-line assembly.

• Complex formation: MYOZ1 forms a specific 2:1 complex with α-actinin-2, which stabilizes MYOZ1 at the Z-disc.

• Binding domains: The C-terminal region of MYOZ1 mediates binding to α-actinin-2/-3, myotilin, and filamin-C.

• Dysregulation consequences: Altered MYOZ1 levels due to FBXL21 deficiency disrupt the stoichiometry of sarcomeric components, potentially contributing to compromised Z-line architecture .

Understanding the precise stoichiometric relationships between MYOZ1 and its binding partners will be crucial for developing interventions that target muscle disorders characterized by Z-disc disruption. Future research should focus on determining the optimal ratios of these components for proper sarcomere function and muscle development .