MYL4 Human

What is MYL4 and what is its normal function in human cardiac tissue?

MYL4 encodes an essential myosin light chain that is specifically expressed in human atria after birth. The essential light chain forms part of the actomyosin cross-bridge, providing structural support for the α-helical neck region of the myosin heavy chain and modulating actin-activated ATPase activity. Research demonstrates that MYL4 is essential for atrial electrical, functional, and structural integrity .

Unlike its ventricular analog (encoded by MYL3), MYL4 shows strong atrial-selective expression in humans, which explains the atrial-selective phenotypes observed in dysfunction cases. Notably, under ventricular pathological conditions including heart failure or hypertrophy, MYL4 can be re-expressed in ventricles as part of the fetal-pattern remodeling .

What mutations in MYL4 have been identified and associated with human disease?

Several significant mutations have been identified:

The c.31G>A mutation changes a negatively charged residue (glutamic acid) to a positively charged one (lysine) at a position that is highly conserved across species, predicted to be pathogenic by multiple computational tools .

What experimental models are available for studying MYL4 function and dysfunction?

A variety of experimental models have been developed to study MYL4:

• Rat models created via CRISPR/Cas9 genome editing:

  • MYL4 p.E11K knock-in rats

  • MYL4 knockout (KO) rats

  • MYL4 deletion rats with 4-amino-acid deletion

• Zebrafish models:

  • Orthologous mutation (E17K in cMLC-1)

  • Myl4 knockout zebrafish

• Cell culture models:

  • In vitro adenoviral transfer of mutant genes to neonatal rat cardiomyocytes

  • Human pluripotent stem cell-derived cardiomyocytes displaying atrial characteristics (using MYH6:mCherry knock-in reporter line)

These models demonstrate progressive atrial electrophysiological, contractile, and fibrotic abnormalities similar to affected patients .

What are the clinical manifestations of MYL4 dysfunction in humans?

MYL4 dysfunction causes a progressive atrial-selective cardiomyopathy characterized by:

• Initial electrical and contractile abnormalities even with sinus rhythm at a young age

• Development of atrial arrhythmias (including atrial fibrillation)

• Progression to atrioventricular block

• Eventually total atrial standstill requiring pacemaker implantation

In early stages, ECG may appear normal with minimal symptoms, but transthoracic echocardiography (TTE) can detect reduced mechanical activity in the atria, making TTE potentially useful for early identification of affected individuals in disease-bearing kindreds .

How does MYL4 expression differ between cardiac tissues and throughout development?

MYL4 exhibits tissue-specific and developmental expression patterns:

• In humans and rats: Strong atrial-selective expression after birth

• In zebrafish: Expression in both atria and ventricles

• During cardiac pathology: Re-expression in ventricles during heart failure or hypertrophy as part of fetal gene program reactivation

• In disease models: MYL4 protein expression is reduced in MYL4 p.E11K knock-in rats, suggesting loss of protein stability

What methodological approaches are recommended for characterizing novel MYL4 variants?

For thorough characterization of novel MYL4 variants, a multi-level approach is recommended:

• Genetic and Bioinformatic Analysis:

  • Segregation analysis in familial cases (e.g., logarithm of odds scoring)

  • Computational prediction using multiple algorithms (SIFT, MutationAssessor, GERP++)

  • Conservation analysis across species

• In Vitro Functional Assessment:

  • Protein stability evaluation via Western blot

  • Myosin ATPase activity measurement (Km and Vmax)

  • Adenoviral gene transfer to cardiomyocytes to assess direct effects

• Animal Model Validation:

  • CRISPR/Cas9-mediated genome editing to generate knock-in models

  • Comprehensive phenotyping including:

    • ECG parameters (P-wave amplitude, PR interval)

    • Echocardiography (atrial dimensions, contractility)

    • Histological examination (fibrosis development)

    • Force generation in isolated atrial preparations

• Stem Cell Models:

  • Development of human pluripotent stem cell-derived atrial cardiomyocytes

  • Molecular and physiological characterization

What molecular mechanisms underlie MYL4-associated atrial cardiomyopathy?

Research has revealed several interconnected pathological mechanisms:

• Sarcomeric Dysfunction:

  • Disruption of Z-disc structure

  • Reduced myosin ATPase activity

  • Altered transition from weakly (non-force-generating) to strongly attached (force-generating) states

• Pathological Signaling Activation:

  • Enhanced proapoptotic signaling

  • Increased profibrotic pathway activation

  • Disruption of macromolecular signaling complexes located at the Z-disc

• Cellular Homeostasis Disruption:

  • Cardiomyocyte apoptosis (evidenced by increased TUNEL staining)

  • Progressive atrial fibrosis

  • Reduced protein stability of mutant MYL4

• Altered Molecular Pathways:

  • Increased retinoic acid signaling in both human embryonic stem cells and zebrafish mutant models

  • Abnormal expression and localization of cytoskeletal proteins

Evidence suggests that apoptosis and atrial fibrosis occur very early in the disease process (as early as 2 days of age in rat models), preceding functional manifestations like P-wave amplitude reduction or atrial enlargement .

How can researchers reconcile differences between knockout and knock-in MYL4 models?

Important observations regarding model differences include:

• Phenotypic Variations:

  • MYL4 knockout rats exhibit earlier and more severe phenotypes than knock-in models

  • Spontaneous atrial arrhythmias are more readily detected in knockout rats

  • Both models ultimately develop similar atrial cardiomyopathy features

• Mechanistic Interpretations:

  • MYL4 p.E11K knock-in similarly reduces MYL4 protein expression as knockout, suggesting loss of protein stability

  • The similar phenotypes between knockout and knock-in models suggest a predominantly loss-of-function mechanism

  • Severity differences may reflect complete loss versus partial function

• Experimental Considerations:

  • Recording methodology limitations (standard ECG vs. continuous telemetry) may account for some discrepancies in arrhythmia detection

  • Genetic background may influence phenotype expression, particularly arrhythmogenesis

  • Developmental timing of phenotype manifestation offers insights into disease progression mechanisms

What are the optimal methods for assessing atrial contractile function in MYL4 dysfunction models?

A comprehensive approach to evaluating atrial function should include:

• In Vivo Assessment:

  • Echocardiography for atrial dimensions and contractility

  • ECG monitoring for P-wave characteristics and arrhythmia detection

  • Age-dependent longitudinal studies to capture disease progression

• Ex Vivo Functional Studies:

  • Force measurements in isolated atrial preparations

  • Assessment of contractile force generation

  • Electrophysiological studies of atrial conduction

• Molecular/Cellular Analysis:

  • Myosin ATPase activity measurement

  • Sarcomere integrity evaluation

  • Determination of atrial-specific mechanical properties

• Data Interpretation Considerations:

  • Correlation between structural changes (fibrosis) and functional parameters

  • Assessment of both left and right atrial function

  • Recognition of potential secondary effects from bradycardia or arrhythmias on ventricular function

How does early molecular remodeling contribute to disease progression in MYL4-related cardiomyopathy?

Research demonstrates that molecular alterations precede functional changes:

• Temporal Sequence of Pathological Events:

  • Evidence of apoptosis and atrial fibrosis as early as 2 days of age

  • These changes occur well before substantial loss of P-wave amplitude or increase in left atrial dimension

  • This suggests that molecular remodeling drives, rather than results from, functional changes

• Primary vs. Secondary Mechanisms:

  • Proapoptotic and profibrotic signaling appears to be a direct result of the mutation

  • In vitro gene transfer of the mutation reproduces signaling changes, indicating these are primary effects

  • Z-disc disruption affects macromolecular complexes involved in intracellular signaling

• Progression Patterns:

  • Initial molecular changes

  • Followed by cellular/structural alterations

  • Eventually leading to electrical and contractile dysfunction

  • Ultimately resulting in complete atrial standstill

What therapeutic approaches might be considered for MYL4-associated atrial cardiomyopathy?

Based on current understanding of pathophysiology, several therapeutic strategies could be explored:

• Targeting Cellular Processes:

  • Anti-apoptotic interventions to prevent early cardiomyocyte death

  • Anti-fibrotic agents to mitigate progressive atrial fibrosis

  • Modulators of retinoic acid signaling

• Gene-Based Approaches:

  • Gene therapy to supplement functional MYL4 in loss-of-function cases

  • Gene editing technologies to correct specific mutations

  • Selective silencing of toxic mutant alleles

• Pharmacological Interventions:

  • Drugs targeting disrupted signaling pathways

  • Agents that improve atrial contractile function

  • Compounds that stabilize sarcomere structure

• Clinical Management Considerations:

  • Early intervention before irreversible fibrosis develops

  • TTE as a screening tool for early detection in at-risk individuals

  • Pacemaker implantation for advanced cases with bradyarrhythmias