ACTN2 Antibody

What is alpha-actinin 2 (ACTN2) and why is it important in research?

Alpha-actinin 2 (ACTN2) is an actin-binding protein with multiple roles in different cell types. It belongs to the alpha-actinin family and is predominantly expressed in both skeletal and cardiac muscles where it functions to anchor myofibrillar actin thin filaments and titin to Z-discs . In non-muscle cells, ACTN2 is found along microfilament bundles and adherens-type junctions, where it mediates the binding of actin to the cell membrane. In contrast, skeletal, cardiac, and smooth muscle isoforms are localized to the Z-disc and analogous dense bodies, where they help anchor myofibrillar actin filaments .

ACTN2 is particularly important in research because mutations in this gene have been associated with multiple cardiac and skeletal muscle disorders, including hypertrophic cardiomyopathy (HCM) and "Multiple structured Core Disease" (MsCD) . Understanding ACTN2's normal function and pathological variants provides crucial insights into muscle physiology and disease mechanisms.

In properly prepared samples, ACTN2 should display a distinctive cross-striated pattern when visualized in cardiac and skeletal muscle tissues. This reflects its normal localization to the Z-discs of sarcomeres. Immunofluorescence analysis of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with an α-actinin 2 antibody typically shows this cross-striated pattern at 30 days in vitro, indicating proper formation of sarcomeres .

How can ACTN2 antibodies be used to investigate cardiomyopathy-associated mutations?

ACTN2 antibodies provide critical tools for investigating the structural and functional consequences of cardiomyopathy-associated mutations. In advanced research, these antibodies can be employed to:

• Detect protein aggregation: Immunofluorescence and live cell imaging with ACTN2 antibodies can reveal protein aggregation in cells expressing mutant ACTN2. Research has shown that ACTN2mut hiPSC-CMs present higher indices of protein aggregation compared to wild-type cells .

• Examine myofibrillar organization: ACTN2 antibodies can be used to quantify myofibrillar disarray in mutant cardiomyocytes. Studies have demonstrated that ACTN2 mutations can lead to significant disruption of sarcomere structure, which can be visualized and quantified using appropriate antibodies .

• Evaluate hypertrophic responses: Paired with other cellular markers, ACTN2 antibodies can help assess hypertrophic changes in cardiomyocytes. Research has shown that ACTN2mut hiPSC-CMs exhibit increased cell area and volume compared to wild-type cells .

• Track multinucleation: ACTN2 immunostaining combined with nuclear staining can reveal multinucleation, which has been observed at higher rates in cells with ACTN2 mutations .

For robust experimental design, researchers should include appropriate controls and quantitative methods to assess these parameters objectively.

What approaches can be used to differentiate between wild-type and mutant ACTN2 in heterozygous models?

Differentiating between wild-type and mutant ACTN2 in heterozygous models requires specialized approaches:

• Allele-specific expression analysis: RT-PCR fragments can be subcloned and sequenced to determine the relative expression of wild-type versus mutant alleles. Research has shown that in HCM patient samples, analysis of multiple clones revealed 48% and 60% mutant clones in engineered heart tissues and left ventricular septum, respectively, suggesting allelic balance and stable missense transcripts .

• RNA sequencing: This approach can confirm allelic balance in heterozygous models .

• Live-cell imaging with tagged constructs: Researchers have used AAV6-mediated delivery of HaloTag®-labeled wild-type or mutant ACTN2 to visualize their differential behavior. Studies have shown that exogenous mutant-ACTN2 in wild-type cells induced aggregation in approximately 83% of cells, while wild-type ACTN2 in mutant cells reduced aggregation to only 17% . This demonstrates that mutant ACTN2 causes aggregation in wild-type cells, while wild-type ACTN2 can partially rescue the aggregation phenotype in mutant cells.

• Quantitative immunofluorescence: By analyzing the pattern and intensity of ACTN2 staining, researchers can identify differences in localization and aggregation between wild-type and mutant proteins.

These approaches provide complementary information about the expression, localization, and function of wild-type versus mutant ACTN2 in heterozygous models.

How does ACTN2 interact with proteolytic systems in cardiomyopathy models?

Research has uncovered important interactions between ACTN2 and cellular proteolytic systems in cardiomyopathy models:

These findings indicate that therapeutic strategies targeting proteolytic systems might be beneficial in treating ACTN2-associated cardiomyopathies.

What are the optimal protocols for detecting ACTN2 in different tissue types?

The optimal protocols for detecting ACTN2 vary by tissue type and application:

For Western Blot:

• Use PVDF membrane probed with 0.2 μg/mL of anti-ACTN2 antibody

• Follow with HRP-conjugated secondary antibody

• Perform under reducing conditions using appropriate buffer groups

• ACTN2 should be detected at approximately 100-103 kDa

• Recommended dilutions range from 1:5000 to 1:100000, depending on the specific antibody

For Immunohistochemistry of paraffin-embedded tissues:

• Perform heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic or comparable solution

• Apply primary ACTN2 antibody at 10 μg/ml for 1 hour at room temperature

• Follow with HRP-conjugated secondary antibody or HRP Polymer Antibody

• Develop with DAB (brown) and counterstain with hematoxylin (blue)

• ACTN2 should show specific staining localized to the cytoplasm in muscle tissues

• Recommended dilutions typically range from 1:500 to 1:2000

For Immunofluorescence:

• For cell lines: Apply 3 μg/mL of ACTN2 antibody for 3 hours at room temperature

• Use fluorophore-conjugated secondary antibodies (e.g., NorthernLights™ 557-conjugated Anti-Rabbit IgG)

• Counterstain nuclei with DAPI

• Specific staining should be localized to cytoplasm and possibly nuclei

• Recommended dilutions typically range from 1:50 to 1:500 for IF/ICC and 1:200 to 1:800 for IF-P

Different tissue types may require optimization of these basic protocols to account for tissue-specific characteristics and antibody penetration issues.

What controls should be included when using ACTN2 antibodies in experimental designs?

Robust experimental designs using ACTN2 antibodies should include multiple controls:

• Positive tissue controls: Include known positive tissues such as human/mouse skeletal muscle, heart tissue, or C2C12 mouse myoblast cell line where ACTN2 is highly expressed .

• Negative controls: Include tissues known to have minimal or no ACTN2 expression, or use isotype-matched control antibodies to assess non-specific binding.

• Peptide competition assays: Pre-incubate the ACTN2 antibody with excess immunizing peptide to demonstrate binding specificity.

• Knockout/knockdown controls: When available, include ACTN2 knockout or knockdown samples to confirm antibody specificity.

• Cross-reactivity controls: For multi-species studies, include species-specific positive controls to confirm cross-reactivity as claimed by the manufacturer.

• Dilution series: Perform antibody titration experiments to determine optimal concentration for each application and tissue type.

• Secondary antibody only controls: Include samples treated only with secondary antibody to assess background signal.

• Wild-type vs. mutant comparisons: When studying mutations, include both wild-type and mutant samples to assess differential patterns of expression or localization .

• Loading controls: For Western blots, include appropriate loading controls (GAPDH, β-tubulin, etc.) to ensure equal protein loading across samples .

These controls collectively ensure the specificity, sensitivity, and reliability of results obtained using ACTN2 antibodies.

How can ACTN2 antibodies be optimized for use in human iPSC-derived cardiomyocytes?

Optimizing ACTN2 antibodies for use in human iPSC-derived cardiomyocytes (hiPSC-CMs) requires specific considerations:

• Timing of differentiation: ACTN2 antibodies typically show optimal cross-striated patterns in hiPSC-CMs after at least 30 days of differentiation, when sarcomeres are well-formed . Earlier timepoints may show incomplete sarcomere organization.

• Fixation methods: For immunofluorescence applications in hiPSC-CMs, immersion fixation with 4% paraformaldehyde for 15-20 minutes at room temperature typically provides good results.

• Permeabilization: Gentle permeabilization with 0.1-0.2% Triton X-100 for 10 minutes often yields optimal antibody penetration without disrupting sarcomeric structures.

• Blocking: Using 3-5% BSA or 5-10% normal serum (from the species in which the secondary antibody was raised) reduces background staining.

• Antibody concentration: For immunofluorescence in hiPSC-CMs, ACTN2 antibodies have been successfully used at concentrations of 3 μg/mL , though this may require optimization for specific antibody clones.

• Co-staining markers: Combining ACTN2 antibody with other sarcomeric markers (e.g., cardiac troponin T) provides comprehensive assessment of sarcomere organization. This approach has been used to analyze ACTN2 localization alongside other cardiac structural proteins .

• Live-cell imaging adaptations: For live-cell applications, fusion proteins like ACTN2-HaloTag® have been successfully used with appropriate fluorescent ligands (e.g., TMR-ligand) in conjunction with nuclear stains like Hoechst .

• Culture format considerations: ACTN2 detection protocols may need to be adjusted depending on whether hiPSC-CMs are cultured in 2D monolayers or 3D formats like engineered heart tissues (EHTs) .

By carefully optimizing these parameters, researchers can achieve reliable and reproducible results when using ACTN2 antibodies in hiPSC-CM research.

How can researchers troubleshoot weak or non-specific ACTN2 antibody signals?

When encountering weak or non-specific signals with ACTN2 antibodies, researchers can implement the following troubleshooting strategies:

• For weak signals:

  • Increase antibody concentration incrementally (staying within recommended ranges)

  • Extend primary antibody incubation time (overnight at 4°C can improve signal)

  • Optimize antigen retrieval methods (for IHC, try both citrate buffer pH 6.0 and TE buffer pH 9.0)

  • Use signal amplification systems (e.g., HRP polymer systems or tyramide signal amplification)

  • Ensure samples are properly fixed but not over-fixed, which can mask epitopes

  • Check antibody storage conditions and expiration date

• For non-specific signals:

  • Increase blocking time and concentration (5-10% normal serum or BSA)

  • Include 0.1-0.3% Triton X-100 in blocking and antibody diluent solutions

  • Reduce primary and secondary antibody concentrations

  • Include additional blocking agents (0.1-0.5% non-fat dry milk or 1-5% normal serum)

  • Perform additional washing steps with gentle agitation

  • Use more dilute antibody solutions but with longer incubation times

  • Pre-absorb the antibody with non-specific proteins

• For high background:

  • Use fresher blocking reagents

  • Ensure complete removal of OCT compound or paraffin

  • Quench endogenous peroxidase activity (for IHC applications)

  • Include 0.1-0.3% Tween-20 in wash buffers

  • Filter all solutions used in the protocol

Each antibody may require specific optimization, so systematic testing of these variables is recommended for optimal results.

What are the common pitfalls when comparing wild-type and mutant ACTN2 expression or localization?

When comparing wild-type and mutant ACTN2 expression or localization, researchers should be aware of several common pitfalls:

• Heterogeneous differentiation: hiPSC-CMs can show varied differentiation efficiency, leading to differences in sarcomere maturity that might be misinterpreted as mutation effects. Ensure comparable differentiation efficiency (>90% cardiac troponin T-positive cells) between wild-type and mutant lines .

• Maturation stage differences: ACTN2 localization changes during cardiomyocyte maturation. Compare wild-type and mutant cells at the same maturation stage, typically after at least 30 days of differentiation for well-formed sarcomeres .

• Z-disc versus aggregate misinterpretation: Distinguishing between normal Z-disc localization and small aggregates can be challenging. Use quantitative measures of sarcomere organization and proper controls to differentiate these patterns .

• Allelic expression imbalance: In heterozygous models, unequal expression of wild-type and mutant alleles may occur. Verify allelic balance through subcloning and sequencing of RT-PCR fragments or RNA sequencing .

• Antibody clone specificity: Some antibodies may have different affinities for wild-type versus mutant ACTN2. When possible, use multiple antibody clones targeting different epitopes.

• 3D versus 2D culture effects: ACTN2 expression and localization may differ between 2D cultured cardiomyocytes and 3D models like engineered heart tissues. Results from one system may not translate directly to the other .

• Overinterpretation of colocalization: When studying ACTN2 interactions with other proteins, be cautious about concluding direct interactions based solely on colocalization data.

• Neglecting time-dependent changes: Some mutation effects on ACTN2 may develop over time. Include multiple time points in experimental designs to capture temporal dynamics.

By addressing these potential pitfalls, researchers can ensure more robust and reproducible comparisons between wild-type and mutant ACTN2.

How can researchers quantitatively assess ACTN2 aggregation and myofibrillar disarray?

Quantitative assessment of ACTN2 aggregation and myofibrillar disarray requires rigorous methodological approaches:

Research has employed these methods to demonstrate that ACTN2mut hiPSC-CMs show significantly higher indices of myofibrillar disarray and more ACTN2 aggregates compared to ACTN2wt hiPSC-CMs . These quantitative approaches provide objective metrics for assessing the structural consequences of ACTN2 mutations.

How can ACTN2 antibodies contribute to understanding the relationship between cardiomyopathy and proteostasis mechanisms?

ACTN2 antibodies have become instrumental in elucidating the relationship between cardiomyopathy and proteostasis mechanisms:

• Identifying protein quality control activation: Research using ACTN2 antibodies has demonstrated that ACTN2 mutations activate both the ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) . This suggests that cardiomyocytes attempt to clear mutant or misfolded ACTN2 proteins through these proteolytic systems.

• Characterizing protein aggregation dynamics: ACTN2 antibodies enable visualization and quantification of protein aggregates in cardiomyocytes. Studies have shown that ACTN2mut presents higher protein aggregation compared to ACTN2wt, highlighting a potential proteotoxic mechanism in cardiomyopathy pathogenesis .

• Monitoring autophagic flux: By combining ACTN2 antibodies with markers of autophagy (like LC3-II), researchers can assess whether autophagic mechanisms are attempting to clear ACTN2 aggregates. Research has shown increased autophagic flux in ACTN2mut hiPSC-CMs, suggesting activation of this pathway in response to proteotoxic stress .

• Mapping UPS activation patterns: ACTN2 antibodies, used in conjunction with ubiquitin antibodies, help map the spatial relationship between ACTN2 aggregates and UPS activation sites in cardiomyocytes.

• Assessing therapeutic interventions: ACTN2 antibodies can be used to evaluate the efficacy of proteostasis-targeting therapeutics by monitoring changes in ACTN2 aggregation and localization following treatment.

These applications collectively support the emerging concept that proteopathy (protein misfolding and aggregation) is a central pathological feature in ACTN2-associated cardiomyopathies , opening new avenues for therapeutic intervention targeting proteostasis mechanisms.

What insights have ACTN2 antibodies provided into the molecular mechanisms of hypertrophic cardiomyopathy?

ACTN2 antibodies have facilitated several key insights into the molecular mechanisms of hypertrophic cardiomyopathy (HCM):

• Structural basis of sarcomere dysfunction: Immunofluorescence studies using ACTN2 antibodies have revealed that ACTN2 mutations can cause myofibrillar disarray and impaired sarcomere organization . This disorganization likely contributes to contractile dysfunction in HCM.

• Hypertrophic response characterization: ACTN2 antibodies have helped demonstrate that mutations in this protein lead to increased cardiomyocyte size (hypertrophy), a hallmark of HCM. Studies have shown that ACTN2mut hiPSC-CMs exhibit greater cell area and volume compared to wild-type cells .

• Multinucleation patterns: Research using ACTN2 antibodies has identified increased multinucleation in cardiomyocytes with ACTN2 mutations, suggesting alterations in cell division processes that may contribute to HCM pathogenesis .

• Mechanistic link to proteotoxicity: ACTN2 antibodies have helped establish that certain ACTN2 mutations lead to protein aggregation and activation of proteolytic systems, suggesting that proteotoxicity may be an important mechanism in HCM development .

• Force generation defects: In engineered heart tissues, ACTN2 mutations have been shown to reduce levels of sarcomere-associated proteins and impair force generation . ACTN2 antibodies have been crucial in establishing these connections between structural and functional defects.

• Allelic balance effects: Studies using ACTN2 antibodies have demonstrated that in heterozygous conditions (like those typically found in HCM patients), both wild-type and mutant ACTN2 are expressed in balanced proportions, suggesting that mutant ACTN2 exerts dominant negative effects rather than causing haploinsufficiency .