MYH6 Antibody

What is MYH6 and why is it important in cardiac research?

MYH6 encodes the alpha chain of cardiac myosin, a muscle motor protein with two regions that bind actin at amino acids 657-679 and 759-773 . It plays critical roles in:

• Embryonic and adult heart development

• Muscle contraction, particularly in atrial tissue

• Force development, which occurs much faster in atrial tissue with higher ATP consumption

In human hearts, while the beta chain isoform (MYH7) predominates in ventricular tissue, MYH6 is found in variable proportions in atrial tissue . Defects in MYH6 are associated with atrial septal defect, hypertrophic cardiac myopathy, and sick sinus syndrome .

What applications can MYH6 antibodies be used for?

MYH6 antibodies have been validated for multiple applications:

Most antibodies detect MYH6 in the cytoplasm of cardiomyocytes, with particularly strong staining in atrial cardiomyocytes .

How should I design experiments to accurately detect MYH6 in cardiac tissue samples?

For optimal MYH6 detection in cardiac tissues:

• Sample preparation:

  • For IHC/IF: Use paraformaldehyde fixation (4%) for 24-48 hours

  • For WB: Fresh/frozen tissue extraction in RIPA buffer with protease inhibitors

• Protocol optimization:

  • For IHC-P: Perform heat-mediated antigen retrieval with citrate buffer pH 6.0 or TE buffer pH 9.0 as recommended for specific antibodies

  • For IF: Use fluorescent secondary antibodies with appropriate excitation/emission wavelengths (e.g., NorthernLights™ 557 for MYH6 with DAPI counterstain)

• Controls:

  • Positive control: Human/mouse heart tissue (atrium preferred over ventricle)

  • Negative control: Human/mouse prostate or endometrium tissues

  • For specificity: Consider skeletal muscle which may show cross-reactivity

• Dilution optimization:

  • Perform titration series (e.g., 1:50, 1:100, 1:200, 1:500) for each new antibody lot

What are the recommended procedures for MYH6 detection in stem cell-derived cardiomyocytes?

For iPSC/ESC-derived cardiomyocytes:

• Cell culture conditions:

  • Fix cells at appropriate differentiation stage (typically days 15-30 post-differentiation)

  • Use 4% paraformaldehyde for 15-20 minutes at room temperature

• Immunostaining protocol:

  • Permeabilize with 0.1-0.5% Triton X-100

  • Block with 5% normal serum (match species of secondary antibody)

  • Incubate with MYH6 antibody overnight at 4°C (typical concentration: 10 μg/ml)

  • Use fluorescent secondary antibodies and DAPI nuclear counterstain

• Co-staining options:

  • Pair with additional cardiac markers (cTnT, cTnI, α-actinin) for comprehensive characterization

  • Consider cardiac transcription factors (NKX2.5, GATA4) for developmental studies

This approach has been successfully used for detecting MYH6 in BG01V human embryonic stem cells differentiated into cardiomyocytes and human iPSC-derived cardiomyocytes .

How does MYH6 expression differ across species and what implications does this have for antibody selection?

MYH6 expression shows important species differences:

These differences are critical because:

• In rodent models (mice/rats), MYH6 is the predominant ventricular isoform, unlike humans where MYH7 dominates ventricles

• For translational research, these differences make mouse models imperfect for studying human cardiac conditions

• When validating antibodies across species, different tissues may be optimal for detection

Researchers should select antibodies validated in their species of interest, with clone S46 (monoclonal) showing confirmed reactivity across chicken, human, mouse, quail, rat, and zebrafish .

Beyond cardiac tissue, where else might MYH6 be expressed and detected?

While MYH6 is primarily considered a cardiac-specific marker, research has revealed expression in:

• Embryonic tissues:

  • During development in cardiac progenitor cells

  • Detected in human embryonic stem cells during cardiac differentiation

• Specialized muscle fibers:

  • Slow tonic myosin heavy chain in human extraocular muscles

  • Intrafusal muscle fibers

• Pathological contexts:

  • Detected in some skeletal muscle samples under certain conditions

  • Expression has been observed in certain cancer tissues, with research showing potential tumor suppressor activity in prostate cancer

For non-cardiac applications, validation is essential as expression levels are typically much lower than in cardiac tissue. When studying MYH6 in non-cardiac contexts, higher antibody concentrations may be required, and specificity controls become particularly important.

What are common issues when working with MYH6 antibodies and how can they be resolved?

For Western blot applications specifically:

• Use reducing conditions with Western Blot Buffer Group 1

• For optimal transfer of this large protein (~224 kDa), consider wet transfer methods with extended transfer times

• When detecting from heart tissues, sample from atrial regions for stronger MYH6 signal in human samples

How can researchers validate MYH6 antibody specificity in their experiments?

Comprehensive validation should include:

• Positive controls:

  • Human/mouse heart tissues (preferably atrium for human samples)

  • Known MYH6-expressing cell lines or differentiated cardiomyocytes

• Negative controls:

  • Tissues known not to express MYH6 (e.g., prostate, endometrium)

  • Secondary antibody-only controls to assess background

• Advanced validation approaches:

  • CRISPR/Cas9 knockout or knockdown of MYH6 in appropriate cell lines

  • Peptide competition assays with the immunizing peptide

  • Correlation with mRNA expression data (RT-qPCR)

  • Comparison of staining patterns with multiple antibodies targeting different MYH6 epitopes

• Critical control for cross-reactivity:

  • Test antibody against recombinant MYH6 and MYH7 to assess specificity

  • Compare staining patterns in tissues with known differential expression of MYH6 vs. MYH7

How can MYH6 antibodies be used to study cardiac disease mechanisms?

MYH6 antibodies are valuable tools for investigating various cardiac pathologies:

• Cardiomyopathies:

  • Track alterations in MYH6/MYH7 ratio in hypertrophic and dilated cardiomyopathy

  • Study MYH6 mutations associated with atrial septal defects using patient-derived iPSC-CMs

• Developmental cardiac defects:

  • Analyze MYH6 expression during heart development in disease models

  • Evaluate the impact of congenital MYH6 mutations on sarcomere organization

• Autoimmune cardiac disease:

  • Investigate autoimmune myocarditis, where impaired thymic tolerance to α-myosin (MYH6) drives autoimmunity

  • Study T-cell responses to MYH6 in myocarditis patients

• Experimental approach example:
  For studying MYH7/MYH6 dual mutations in hypertrophic cardiomyopathy:

  • Generate patient-specific iPSCs and differentiate to cardiomyocytes

  • Use MYH6 antibodies to assess sarcomere organization and protein levels

  • Combine with functional assays to correlate structure and function

  • This approach revealed that dual mutations dysregulate extracellular matrix remodeling and disrupt cell-ECM adhesion

What are the considerations for using MYH6 antibodies in gene-edited cardiac models?

When using MYH6 antibodies in CRISPR/Cas9 or other gene-edited models:

• Epitope preservation:

  • Ensure the antibody's epitope is not affected by your genetic modification

  • For C-terminal tags, choose antibodies targeting N-terminal regions and vice versa

• Knock-in reporter systems:

  • When creating αMHC-EGFP knock-in models using CRISPR/Cas9 , validate that the tag doesn't interfere with antibody binding

  • Consider dual validation with antibodies against both MYH6 and the reporter tag

• Quantification strategies:

  • Use image analysis software for accurate quantification of IF/IHC signals

  • For WB, include recombinant protein standards for absolute quantification

• Validation in heterozygous models:

  • In heterozygous knockout/mutation models, antibodies can help quantify the remaining wild-type protein

  • This is especially important for dominant-negative mutations where protein levels may not correlate with phenotype severity

How does MYH6 expression relate to thymic tolerance and autoimmunity in cardiovascular research?

Advanced research has revealed critical insights about MYH6 and autoimmunity:

• Thymic expression patterns:

  • MYH6 is barely detectable or absent in human medullary thymic epithelial cells (mTECs) and cortical thymic epithelial cells (cTECs)

  • This limited thymic expression leads to impaired negative selection of MYH6-reactive T cells

• Experimental findings:

  • Humans have high frequencies of α-MyHC–specific T cells in peripheral blood

  • T cell responses to α-MyHC are markedly augmented in patients with myocarditis

  • Transgenic expression of α-MyHC in thymic epithelium conferred tolerance to cardiac myosin and prevented myocarditis in animal models

• Research implications:

  • The limited thymic expression of MYH6, not its cardiac abundance, explains why it becomes an autoimmune target

  • When studying autoimmune myocarditis, researchers should evaluate both MYH6 protein expression and T-cell reactivity

• Methodological considerations:

  • For autoimmunity studies, pair MYH6 antibodies with T-cell profiling techniques

  • Consider both cellular and humoral immune responses to MYH6

What role might MYH6 play in cancer research, and how can antibodies facilitate these investigations?

Recent research has uncovered potential roles for MYH6 in cancer biology:

• Tumor suppressor function:

  • MYH6 shows reduced expression in prostate cancer tissues

  • Lower MYH6 gene expression correlates with worse clinical outcomes in prostate cancer patients

  • In vitro and in vivo studies demonstrate that overexpressed MYH6 suppresses proliferation and migration of prostate cancer cells

• Mechanistic insights:

  • RNA-seq investigations identified KIT Proto-Oncogene as a downstream target of MYH6

  • MYH6 appears to suppress tumor progression by downregulating KIT

  • MYH6 is frequently mutated in chemotherapy-treated residual small cell lung cancer tumors, potentially contributing to chemoresistance

• Experimental approaches using antibodies:

  • Use MYH6 antibodies for IHC to assess expression in cancer tissue microarrays

  • Combine with Ki-67 staining to correlate MYH6 expression with proliferation

  • Employ in vitro models with forced MYH6 expression and analyze downstream signaling pathways

This emerging research area suggests MYH6 may have broader roles beyond cardiac function, opening new avenues for investigation in cancer biology.

How can researchers effectively use MYH6 antibodies in stem cell-derived cardiomyocyte maturation studies?

MYH6 antibodies are valuable tools for assessing cardiomyocyte maturation:

• Developmental expression patterns:

  • MYH6 expression changes during cardiomyocyte maturation

  • The MYH6/MYH7 ratio serves as a key indicator of maturation state

• Experimental design considerations:

  • Time course analysis: Sample cells at multiple time points (days 10, 20, 30, 60, 90) post-differentiation

  • Combine with other maturation markers: sarcomeric organization, T-tubule formation

  • Use flow cytometry with MYH6 antibodies to quantify positive cell populations at different stages

• Advanced applications:

  • Single-cell analysis: Correlate MYH6 expression with electrophysiological properties

  • 3D cardiac organoids: Assess spatial distribution of MYH6 throughout organoid development

  • Mechanical stimulation studies: Determine how mechanical forces affect MYH6/MYH7 switching

• Practical methodology:

  • For accurate quantification, use consistent exposure settings in fluorescence microscopy

  • Implement high-content imaging systems for large-scale, unbiased analysis

  • Consider using MYH6 reporter lines in parallel with antibody detection for live cell tracking