MYL7 Human

What is the structure and function of MYL7 in human cardiac tissue?

MYL7, also known as Atrial Light Chain-2 (ALC-2) or Myosin regulatory light chain 2, atrial isoform (MLC2a), is a protein of 175 amino acids with a molecular weight of approximately 19.4 kDa . It is an EF hand protein that binds to the neck region of alpha myosin heavy chain . Structurally, MYL7 shares 59% homology with its ventricular counterpart (VLC-2), with significant differences at the N-termini and at regulatory phosphorylation sites, particularly Serine-15 and Serine/Asparagine-14 .

Functionally, MYL7 expression is restricted to cardiac muscle atria in healthy individuals . It plays a crucial role in cardiac development and contractility, functioning in the stabilization of thick filaments and regulation of heart muscle contraction . MYL7 has proven to be an important marker for cardiac muscle chamber distinction and differentiation during development . Studies have demonstrated that deletion of MYL7 can lead to abnormal atrial contraction, highlighting its essential role in proper cardiac function .

The expression of MYL7 correlates with alpha-myosin heavy chain expression in cardiac atria, suggesting coordinated regulation of these proteins in heart development and function . This restricted expression pattern makes MYL7 a valuable marker for studying cardiac chamber development and differentiation.

How is MYL7 expression regulated during cardiac development?

MYL7 displays a distinct developmental expression pattern that provides valuable insights into cardiac chamber specification and differentiation. During embryonic development, MYL7 expression is cardiac-specific throughout embryonic days 8-16 in mice . From embryonic day 12 onward, MYL7 expression becomes increasingly restricted to atria, showing very low levels in the aorta and becoming undetectable in ventricles, skeletal muscle, uterus, and liver .

Notably, this atrial-specific expression pattern occurs prior to cardiac septation, suggesting MYL7 may play a role in early chamber specification . MYL7 shows a pattern distinct from atrial essential light chain (ALC-1) during cardiogenesis, indicating differential regulation of these related proteins . The timing of this restricted expression suggests MYL7 may serve as an early marker of atrial fate determination.

Research using zebrafish models has identified myl7 as the ortholog to human MYL7, making this a valuable model organism for studying developmental regulation . Transgenic zebrafish strains using myl7 promoters to drive reporter gene expression have been developed to visualize cardiac development in real-time, demonstrating the utility of MYL7 regulatory elements in developmental studies .

Interestingly, in certain pathological conditions, the strict chamber-specific expression pattern of MYL7 can be altered, suggesting that the regulatory mechanisms controlling MYL7 expression can be modified in disease states .

What experimental models are commonly used to study MYL7 function?

Several experimental models have proven valuable for investigating MYL7 function across different research contexts:

Zebrafish models have emerged as particularly useful systems for studying MYL7. The zebrafish myl7 gene is orthologous to human MYL7, making it an excellent model for translational research . Researchers have developed sophisticated transgenic zebrafish lines such as Casper/myl7:RFP;annexin-5:YFP that combine cardiac-specific fluorescent reporters with transparent body phenotypes . These models enable real-time in vivo confocal microscopy to study cardiomyocyte morphology and function throughout development and in response to cardiac injury .

Mouse models with genetic modifications of MYL7 have been instrumental in understanding its functional significance. Knockout studies have demonstrated that deletion of MYL7 leads to abnormal atrial contraction, highlighting its essential role in cardiac function . Additionally, models like the VASN-deficient mice (VASN-/-) have revealed previously unknown relationships between MYL7 and other regulatory pathways, showing that VASN deficiency leads to cardiac hypertrophy by downregulating MYL7 production .

For molecular studies, recombinant protein expression systems using E. coli have been developed to produce human MYL7 protein for structural and biochemical analyses . These systems typically generate a single polypeptide chain containing 175 amino acids with a molecular mass of 22.0 kDa, often fused to a His-tag for purification purposes .

Transgenic models using MYL7 regulatory elements have also proven valuable. The Tg(myl7.L-cre)1141Tmhn mouse model uses the Xenopus laevis myosin light chain 2 regulatory cardiac slow promoter to drive expression of Cre recombinase specifically in cardiac tissue, enabling cardiac-specific gene manipulation .

What are the common techniques for studying MYL7 expression patterns?

Researchers employ a variety of complementary techniques to characterize MYL7 expression patterns across tissues, developmental stages, and disease conditions:

Immunohistochemistry and immunofluorescence are widely used to visualize MYL7 protein localization in tissue sections. The Human Protein Atlas has documented selective cytoplasmic expression of MYL7 in heart muscle using these techniques . These approaches allow researchers to observe the chamber-specific expression pattern of MYL7, confirming its predominant localization in atrial tissue rather than ventricular myocardium .

Real-time quantitative PCR (RT-qPCR) provides a sensitive method for measuring MYL7 mRNA expression levels across different tissues and experimental conditions. This technique has been valuable in documenting the upregulation of MYL7 as a marker of cardiac hypertrophy in animal models . RT-qPCR also enables precise quantification of expression changes during development or in disease states.

RNA sequencing (RNA-seq) offers a comprehensive approach to analyze MYL7 expression within the broader transcriptional landscape. This technique has been instrumental in identifying MYL7 downregulation as a consequence of VASN deficiency in mouse models . RNA-seq provides the advantage of simultaneously measuring all expressed genes, allowing researchers to identify co-regulated genes and potential regulatory networks.

Transgenic reporter systems using MYL7 promoter elements to drive fluorescent protein expression enable dynamic visualization of expression patterns in living organisms. The Casper/myl7:RFP zebrafish line uses the myl7 promoter to drive red fluorescent protein expression specifically in cardiac tissue . This approach allows real-time tracking of MYL7-expressing cells during development and in response to experimental manipulations.

Western blotting provides a method for quantifying MYL7 protein levels and analyzing post-translational modifications. This technique has been used to confirm the downregulation of MYL7 production in cardiac hypertrophy models .

What methodologies are most effective for studying MYL7 phosphorylation states in cardiac tissue?

Studying MYL7 phosphorylation states in cardiac tissue requires specialized techniques that can detect specific post-translational modifications with high sensitivity and specificity. The most effective methodologies combine multiple complementary approaches:

Phospho-specific antibodies targeted to known phosphorylation sites (particularly Serine-15 and Serine/Asparagine-14) provide a direct method for detecting phosphorylated MYL7 in tissue samples . Western blotting with these antibodies can quantify relative phosphorylation levels, while immunohistochemistry can reveal the spatial distribution of phosphorylated MYL7 within cardiac chambers. When using this approach, researchers should validate antibody specificity using dephosphorylated controls and competing peptides.

Mass spectrometry-based phosphoproteomics offers the most comprehensive analysis of MYL7 phosphorylation states. This technique can identify all phosphorylation sites simultaneously, including previously unknown modifications. Sample preparation typically involves enrichment of phosphopeptides using titanium dioxide (TiO₂) or immobilized metal affinity chromatography (IMAC) before LC-MS/MS analysis. Targeted approaches like parallel reaction monitoring (PRM) or multiple reaction monitoring (MRM) can quantify specific phosphopeptides with high sensitivity.

Phos-tag SDS-PAGE provides a gel-based separation method that can resolve different phosphorylated forms of MYL7 based on their mobility shift. This technique is particularly useful for visualizing the distribution of differentially phosphorylated species without requiring phospho-specific antibodies. Combined with Western blotting using total MYL7 antibodies, this approach can reveal the proportion of MYL7 in different phosphorylation states.

In vitro kinase assays using recombinant MYL7 protein can identify which kinases are capable of phosphorylating MYL7 at specific sites . This approach typically uses purified recombinant MYL7, incubated with candidate kinases and ATP, followed by detection of phosphorylation using mass spectrometry or radioisotope labeling.

For functional studies, site-directed mutagenesis of phosphorylation sites (Ser to Ala for preventing phosphorylation, or Ser to Asp/Glu for phosphomimetic mutations) in transgenic animal models or cell culture systems can reveal the physiological significance of specific phosphorylation events in MYL7.

How can researchers resolve contradictory data regarding MYL7 expression in cardiac hypertrophy models?

Resolving contradictory data regarding MYL7 expression in cardiac hypertrophy models requires a systematic approach that addresses potential sources of variability across studies:

First, carefully evaluate the specific cardiac hypertrophy models used in conflicting studies. Different models (pressure overload, genetic, pharmacological) may induce distinct molecular mechanisms that differentially affect MYL7 expression . For example, VASN deficiency-induced cardiac hypertrophy in mice shows downregulation of MYL7, but this pattern may not be universal across all hypertrophy models . Design experiments that directly compare multiple hypertrophy models within the same study to identify model-specific versus conserved responses.

Consider temporal dynamics in your experimental design. MYL7 expression may change during different phases of hypertrophy development (acute versus chronic, compensated versus decompensated). Sequential sampling at multiple time points can reveal transient changes that might be missed in single-timepoint studies. The sudden death of VASN-/- mice occurring 21-28 days after birth suggests critical developmental windows where MYL7 dysregulation becomes lethal .

Employ multiple complementary techniques to measure MYL7 expression. Contradictions may arise when different studies rely on single techniques with distinct limitations. Combining mRNA quantification (RT-qPCR, RNA-seq) with protein analysis (Western blotting, immunohistochemistry, mass spectrometry) provides a more comprehensive assessment . Additionally, distinguish between total MYL7 levels and post-translational modifications, which may change independently.

Assess chamber-specific expression patterns, as MYL7 is normally expressed predominantly in atria but not ventricles in adults . Hypertrophy may affect chamber-specific expression differently, so separate analysis of atrial versus ventricular tissue is crucial. Some contradictions may result from studies examining different cardiac chambers without clear distinction.

Finally, consider species differences when comparing results across model organisms. While MYL7 function is conserved across vertebrates, regulatory mechanisms may vary between species . When evaluating studies using zebrafish myl7 or rodent Myl7 models, consider the degree of functional conservation with human MYL7.

What are the current techniques to identify novel protein interactions with MYL7 in human atrial tissue?

Identifying novel protein interactions with MYL7 in human atrial tissue requires specialized approaches that preserve physiologically relevant interactions while providing sufficient sensitivity to detect transient or weak binding partners:

Proximity labeling methods represent cutting-edge approaches for identifying protein interactions in native contexts. BioID or TurboID fused to MYL7 can biotinylate nearby proteins when expressed in cardiomyocytes, followed by streptavidin pulldown and mass spectrometry identification. This approach captures both stable and transient interactions within the cellular environment. Alternatively, APEX2 fusion proteins can generate biotin-phenol radicals upon H₂O₂ exposure, labeling proximal proteins for subsequent purification and analysis.

Co-immunoprecipitation (Co-IP) coupled with mass spectrometry remains a foundational technique for protein interaction studies. For MYL7, using antibodies against endogenous protein or epitope-tagged versions in transfected cardiomyocytes can pull down interaction partners. Crosslinking prior to lysis (chemical or photoactivatable) can stabilize transient interactions. The technique has successfully identified interactions between MYL7 and alpha myosin heavy chain .

Yeast two-hybrid (Y2H) screening using MYL7 as bait against human heart cDNA libraries can identify direct binary interactions. Consider screening with different MYL7 domains separately, as the EF-hand domains may have distinct interaction partners from other regions. Split-luciferase complementation assays provide an alternative approach for validating Y2H hits in mammalian cells.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map interaction interfaces by measuring changes in deuterium uptake when MYL7 binds to partners. This technique requires purified recombinant MYL7 protein but provides structural insights into interaction mechanisms .

Native mass spectrometry of intact complexes isolated from human atrial tissue provides information about the stoichiometry and composition of physiological MYL7-containing complexes. This approach preserves non-covalent interactions and can reveal complex assembly dynamics.

For validation, in situ proximity ligation assays (PLA) can visualize and quantify specific protein-protein interactions in tissue sections, allowing researchers to confirm interactions in their native cellular context and examine their spatial distribution within atrial tissue.

How do I design experiments to differentiate the specific roles of MYL7 versus other myosin light chains in heart contractility?

Designing experiments to differentiate the specific roles of MYL7 versus other myosin light chains (particularly VLC-2) in heart contractility requires sophisticated approaches that exploit their differential expression patterns and unique structural features:

Utilize chamber-specific gene manipulation techniques that target MYL7 selectively in atria without affecting ventricular myosin light chains. This can be achieved using atrial-specific promoters driving Cre recombinase expression in floxed MYL7 mouse models . The Tg(myl7.L-cre) system provides a potential tool for this approach . Complementary studies can use ventricular-specific promoters to manipulate VLC-2 independently.

Design domain-swap experiments to identify functional differences between MYL7 and VLC-2. Since these proteins share 59% homology but differ at their N-termini and phosphorylation sites , creating chimeric proteins that exchange these regions can pinpoint which domains confer atrial-specific functions. These constructs can be expressed in cardiomyocyte cultures or as transgenes in animal models to assess functional consequences.

Perform site-directed mutagenesis of the phosphorylation sites unique to MYL7 (Serine-15 and Serine/Asparagine-14) compared to VLC-2 . Creating phospho-null (S→A) and phosphomimetic (S→D/E) mutations can reveal how these regulatory sites contribute to the specific contractile properties of atrial versus ventricular myocardium.

Employ high-resolution imaging techniques to visualize contractile dynamics. Optical trapping combined with single-molecule fluorescence can measure the mechanical properties of individual myosin molecules containing either MYL7 or other myosin light chains. For tissue-level analysis, light sheet microscopy of transgenic zebrafish with fluorescently labeled myl7-expressing cells enables real-time visualization of contractile behavior .

Use isolated cardiomyocyte contractility assays with selective knockdown or overexpression of MYL7 versus other light chains. Measuring parameters such as contractile force, calcium sensitivity, and relaxation kinetics can reveal functional differences. Cell-specific CRISPR/Cas9 editing can achieve precise genetic manipulation for these experiments.

For translational relevance, analyze human cardiac tissue samples from patients with cardiomyopathies or heart failure to correlate changes in the ratio of MYL7 to other light chains with specific contractile abnormalities . Single-cell RNA sequencing of these samples can reveal cell-specific expression patterns and potential compensatory mechanisms.

What are the best approaches to study MYL7 mutations in the context of cardiomyopathies?

Studying MYL7 mutations in the context of cardiomyopathies requires an integrated approach that spans from molecular mechanisms to physiological consequences:

Begin with comprehensive genetic screening of cardiomyopathy patients to identify novel MYL7 variants. While MYL7 has been associated with various cardiomyopathies (hypertrophic, dilated, ischemic) , the complete spectrum of disease-causing mutations remains incompletely characterized. Next-generation sequencing panels that include MYL7 alongside other cardiomyopathy-associated genes can identify potential pathogenic variants. For population studies, genome-wide association studies (GWAS) can identify MYL7 variants associated with cardiac phenotypes.

Employ in silico structural analysis to predict the impact of identified mutations. Since MYL7 is an EF hand protein that binds to the neck region of alpha myosin heavy chain , computational modeling can predict how specific mutations might disrupt this interaction or affect calcium binding. Molecular dynamics simulations can provide insights into altered protein flexibility or stability resulting from mutations.

Create cellular models using patient-derived induced pluripotent stem cells (iPSCs) differentiated into cardiomyocytes. This approach maintains the patient's genetic background while allowing detailed functional studies. CRISPR/Cas9 gene editing can be used to correct mutations in patient cells or introduce them into control cells to confirm causality. These cardiomyocytes can be analyzed for contractile abnormalities, calcium handling, and electrophysiological properties.

Develop animal models carrying MYL7 mutations identified in cardiomyopathy patients. Transgenic mice or zebrafish expressing mutant human MYL7 can recapitulate disease phenotypes . The transparent Casper/myl7:RFP zebrafish model allows real-time visualization of cardiac morphology and function, making it particularly valuable for studying the developmental consequences of MYL7 mutations .

Perform detailed biochemical characterization of mutant MYL7 proteins. Recombinant expression systems can produce wild-type and mutant MYL7 for comparative analysis . Key properties to assess include protein stability, phosphorylation status, calcium binding affinity, and interaction with myosin heavy chain. Circular dichroism spectroscopy can detect structural changes, while isothermal titration calorimetry can measure binding affinities.

For clinical correlation, use cardiac imaging (echocardiography, cardiac MRI) to characterize the specific phenotypes associated with different MYL7 mutations in patients. This can establish genotype-phenotype correlations and inform prognosis and treatment decisions.