Recombinant Human Trans-2,3-enoyl-CoA reductase-like (TECRL)

What is TECRL and what is its role in cardiac pathophysiology?

TECRL (Trans-2,3-enoyl-CoA reductase-like) is an endoplasmic reticulum protein primarily expressed in cardiac and skeletal muscle tissue. The gene encodes the trans-2,3-enoyl-CoA reductase-like protein, which has emerged as a significant factor in cardiac electrophysiology . Biallelic pathogenic variants in TECRL have been associated with life-threatening inherited arrhythmias that present with overlapping features of both Long QT Syndrome (LQTS) and Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) .

Mechanistically, TECRL deficiency affects calcium handling in cardiomyocytes, characterized by:

• Smaller calcium transient amplitudes

• Elevated diastolic calcium concentration

• Prolonged action potential duration

• Increased susceptibility to delayed afterdepolarizations (DADs) upon adrenergic stimulation

These calcium handling abnormalities provide the pathophysiological basis for the observed clinical phenotypes, explaining both the QT prolongation (LQTS characteristic) and adrenergic-induced arrhythmias (CPVT characteristic) .

What experimental approaches are recommended for studying TECRL mutations?

When investigating TECRL mutations, a multi-faceted experimental approach is recommended:

Genetic Screening Methods

Whole-exome sequencing (WES) has proven effective for identifying TECRL mutations in patients with inherited arrhythmias where conventional genetic testing for common LQTS and CPVT genes is negative . For targeted analysis, PCR with primers designed to target specific TECRL exons can be used to identify splice variants .

Cellular Models

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) serve as an ideal platform for functional studies of TECRL mutations:

• Generate hiPSCs from affected individuals, heterozygous carriers, and healthy controls

• Differentiate hiPSCs into cardiomyocytes

• Validate cardiac phenotype using immunostaining (e.g., ACTN2)

Gene Manipulation Approaches

Alternative approaches include:

• Lentiviral-mediated knockdown using shRNA (achieving approximately 70% reduction in TECRL mRNA levels)

• CRISPR/Cas9-mediated knockout in cellular systems

• AAV9-mediated gene silencing for in vivo studies (particularly valuable for skeletal muscle research)

Functional Assays

Key assays include:

• Calcium imaging to measure calcium transient amplitude, diastolic calcium levels, and calcium handling kinetics

• Patch-clamp electrophysiology to assess action potential morphology and duration

• Western blotting to evaluate effects on calcium handling proteins (RYR2, CASQ2, SERCA2a, PLB, NCX, Cav1.2)

How do TECRL mutations manifest clinically and what are the genotype-phenotype correlations?

TECRL mutations demonstrate variable clinical presentations with notable genotype-phenotype patterns:

Clinical Spectrum

Patients with biallelic TECRL mutations typically present with:

• Stress-induced ventricular arrhythmias

• Cardiac arrest at young age (median age at onset: 8 years, range: 1-22 years)

• Features overlapping both LQTS and CPVT:

  • Normal QT interval at baseline with adrenergic-induced QT prolongation

  • Stress-induced atrial and ventricular arrhythmias

Specific Mutations and Associated Phenotypes

Two predominant mutations have been characterized:

• p.Arg196Gln mutation: Identified in French Canadian patients, results in a missense mutation predicted to be "probably damaging" by PolyPhen-2 and deleterious by SIFT

• c.331+1G>A mutation: Identified in a Sudanese family, causes complete skipping of exon 3 due to disruption of the splice donor site

Inheritance Pattern

TECRL mutations follow an autosomal recessive inheritance pattern:

• Homozygous carriers display the disease phenotype

• Heterozygous carriers (n=37 in published studies) remain clinically asymptomatic

• No cardiac disease or sudden death has been observed in heterozygous genotype carriers

Treatment Response

Important clinical observations indicate differential response to beta-blockers:

• Patients on metoprolol, bisoprolol, or atenolol showed failure of therapy

• Nadolol or propranolol appears to be superior as beta-blockers for these patients

What molecular mechanisms underlie TECRL deficiency-induced calcium handling abnormalities?

The molecular mechanisms by which TECRL deficiency affects calcium handling involve multiple interconnected pathways:

Changes in Calcium Handling Proteins

TECRL deficiency significantly alters the expression of key calcium handling proteins:

• 52% reduction in RYR2 (ryanodine receptor) protein levels

• 85% reduction in CASQ2 (calsequestrin) protein levels

• No significant changes in SERCA2a, PLB, NCX, and Cav1.2 levels

Similar findings were observed in shRNA-mediated TECRL knockdown experiments, confirming these changes are directly related to TECRL deficiency .

Sarcoplasmic Reticulum Calcium Content

TECRL-deficient cardiomyocytes demonstrate:

• Lower SR calcium content (measured via caffeine-evoked transients)

• Smaller calcium transient amplitude

• Slower calcium transient upstroke

• Elevated diastolic calcium concentration

These changes create a substrate for delayed afterdepolarizations (DADs) when exposed to adrenergic stimulation, explaining the adrenergic-induced arrhythmias observed clinically .

Mitochondrial Function

Recent research has identified that TECRL deficiency also impacts mitochondrial function:

• Impaired mitochondrial respiration

• Reduced ATP production

• Increased fatty acid synthase (FAS) activity

• Elevated reactive oxygen species (ROS) production

• Decreased expression of MFN2, p-AKT (Ser473), and NRF2

This suggests that TECRL regulates mitochondrial function primarily through the PI3K/AKT signaling pathway and mitochondrial fusion protein MFN2 .

What is the role of TECRL in skeletal muscle and regenerative myogenesis?

Recent discoveries have expanded our understanding of TECRL's function beyond cardiac tissue:

Expression Patterns in Skeletal Muscle

TECRL is expressed in skeletal muscle in addition to cardiac tissue. Its expression increases in response to muscle injury, suggesting a role in the regenerative response .

Impact on Satellite Cell Function

Satellite cell-specific deletion of TECRL enhances muscle repair through:

• Increased expression of EGR2 (Early Growth Response 2)

• Activation of the ERK1/2 signaling pathway

• Subsequent promotion of PAX7 expression, a key regulator of satellite cell function

Epigenetic Regulation

TECRL deletion leads to upregulation of the histone acetyltransferase general control nonderepressible 5 (GCN5), which:

• Enhances transcription of EGR2 through increased acetylation

• Creates a permissive chromatin environment for satellite cell activation and proliferation

Therapeutic Potential

AAV9-mediated TECRL silencing has been demonstrated to improve muscle repair in mice, representing a potential therapeutic approach for enhancing skeletal muscle regeneration .

How can human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) be optimized for TECRL functional studies?

hiPSC-CMs have proven invaluable for studying TECRL function and disease pathophysiology:

Generation Protocol

For TECRL studies, the following approach has been validated:

• Obtain peripheral blood samples or skin biopsies from patients with TECRL mutations, heterozygous carriers, and healthy family members

• Reprogram somatic cells to hiPSCs using established methods

• Differentiate hiPSCs into cardiomyocytes using directed differentiation protocols

• Characterize cardiomyocyte phenotype using cardiac-specific markers (e.g., ACTN2, TNNT2)

Functional Assessment

Comprehensive functional assessment should include:

• Calcium imaging using fluorescent calcium indicators to measure transient amplitude, diastolic levels, and kinetics

• Electrophysiological recordings to assess action potential morphology and duration

• Response to adrenergic stimulation (e.g., noradrenaline) to recapitulate the stress-induced phenotype

• Pharmacological interventions to assess drug responses

Addressing Limitations

It is important to acknowledge limitations of hiPSC-CMs:

• The apparent immature phenotype (e.g., action potential duration <200ms) compared to adult ventricular cardiomyocytes

• This limitation does not preclude their use in disease modeling, as demonstrated by successful recapitulation of key disease features

• Consider maturation protocols (electrical stimulation, 3D culture) to enhance physiological relevance

Data Interpretation

When interpreting results from hiPSC-CM studies:

• Compare data from multiple lines: homozygous mutant, heterozygous carrier, and control lines

• Consider patient-specific factors that might influence phenotype

• Validate findings using alternative approaches (e.g., gene silencing in wild-type cells)

What are the recommended experimental designs for studying TECRL in the context of skeletal muscle regeneration?

For researchers focusing on TECRL's role in skeletal muscle, specific experimental designs have proven effective:

In Vivo Injury Models

Validated skeletal muscle injury models include:

• Cardiotoxin (CTX) injection into the tibialis anterior or gastrocnemius muscle

• Muscle laceration or crush injury

• Exercise-induced muscle damage protocols

Genetic Manipulation Approaches

Several approaches have been successful:

• Satellite cell-specific TECRL knockout using Pax7-CreER;TECRLflox/flox mice

• AAV9-mediated TECRL silencing for targeted in vivo modulation

• CRISPR/Cas9-based approaches for in vitro studies

Analytical Methods

Key analytical methods include:

• Histological analysis of muscle regeneration (H&E staining, embryonic myosin heavy chain)

• Flow cytometry for satellite cell isolation and quantification

• Colony formation assays to assess satellite cell proliferative capacity

• Single myofiber isolation and culture to assess satellite cell activation, proliferation, and differentiation

• RNA-seq analysis to identify transcriptional changes

• Dual luciferase reporter assays to assess transcriptional activity

Signaling Pathway Analysis

Focus on the following key pathways:

• ERK1/2 signaling: Assess phosphorylation status via Western blotting

• EGR2 expression and activity: RNA and protein levels, ChIP assays

• PAX7 regulation: Expression analysis, reporter assays

• GCN5-mediated histone acetylation: ChIP assays for H3K9ac and H3K14ac marks

How does TECRL function differ between cardiac and skeletal muscle tissues?

Understanding the tissue-specific functions of TECRL is crucial for targeted therapeutic approaches:

Shared Characteristics

In both cardiac and skeletal muscle, TECRL:

• Localizes to the endoplasmic reticulum

• Influences calcium handling and homeostasis

• Impacts mitochondrial function and energy metabolism

• Shows increased expression in response to tissue stress/injury

Cardiac-Specific Functions

In cardiac tissue, TECRL primarily:

• Regulates calcium handling proteins critical for excitation-contraction coupling (RYR2, CASQ2)

• Influences action potential duration and repolarization

• Affects susceptibility to adrenergic-induced arrhythmias

• Impacts mitochondrial function through PI3K/AKT signaling

Skeletal Muscle-Specific Functions

In skeletal muscle, TECRL:

• Regulates satellite cell proliferation and differentiation

• Modulates the ERK1/2/EGR2/PAX7 signaling axis

• Influences epigenetic regulation through histone acetyltransferase activity

• Affects regenerative capacity following injury

Experimental Design Considerations

When designing experiments to compare cardiac and skeletal muscle functions:

• Use tissue-specific conditional knockout models to avoid developmental confounders

• Employ parallel analytical approaches across tissues

• Consider temporal dynamics of TECRL expression and function during development and in response to stress

• Evaluate shared vs. tissue-specific binding partners and signaling pathways

What is the current state of knowledge regarding TECRL's protein structure and enzymatic activity?

Despite its importance in disease, several aspects of TECRL's biochemical properties remain to be fully characterized:

Protein Structure

TECRL is a member of the short-chain dehydrogenase/reductase (SDR) protein family:

• The name "trans-2,3-enoyl-CoA reductase-like" suggests structural similarity to TECR, which is involved in fatty acid elongation

• The p.Arg196Gln mutation occurs at a highly conserved site with a high Genomic Evolutionary Rate Profiling (GERP) score (5.11)

• The c.331+1G>A mutation results in exon 3 skipping, producing a protein with a 45-bp deletion

Enzymatic Function

The precise enzymatic activity of TECRL remains incompletely characterized:

• Based on homology, TECRL is predicted to be involved in fatty acid metabolism

• Its role in calcium handling suggests additional functions beyond canonical enzymatic activity

• The connection to mitochondrial function indicates a potential role in energy metabolism

Protein-Protein Interactions

Limited data exists on TECRL's interactome:

• TECRL deficiency affects RYR2 and CASQ2 expression, suggesting potential direct or indirect interactions

• The influence on PI3K/AKT signaling in mitochondrial function suggests connections to this pathway

• In skeletal muscle, TECRL appears to influence ERK1/2 signaling and subsequent EGR2/PAX7 expression

Research Gaps

Key areas requiring further investigation:

• Crystal structure determination

• Substrate specificity characterization

• Identification of direct binding partners

• Elucidation of post-translational modifications and their functional significance

What are the recommended therapeutic strategies for targeting TECRL-related disorders?

Current evidence suggests several potential therapeutic approaches for TECRL-related disorders:

Pharmacological Management of Cardiac Arrhythmias

For inherited arrhythmias associated with TECRL mutations:

• Beta-blockers represent the first-line therapy, with nadolol and propranolol showing superior efficacy compared to metoprolol, bisoprolol, or atenolol

• Flecainide showed some efficacy in reducing delayed afterdepolarizations in TECRL-deficient cardiomyocytes, though its QT-prolonging effect may potentially offset benefits

• Implantable cardioverter-defibrillators (ICDs) should be considered for high-risk patients, particularly those who have experienced cardiac arrest

Gene Therapy Approaches

Emerging evidence suggests potential for gene therapy:

• AAV9-mediated TECRL silencing improved muscle repair in mouse models, suggesting therapeutic potential for skeletal muscle disorders

• For cardiac applications, gene therapy could potentially restore TECRL expression in recessive loss-of-function mutations

• For skeletal muscle enhancement, targeted TECRL inhibition might improve regenerative capacity

Small Molecule Development

Rational approaches for small molecule development could include:

• Compounds that stabilize RYR2 and CASQ2 expression in TECRL-deficient cardiomyocytes

• Modulators of calcium handling that reduce diastolic calcium levels

• Activators of the ERK1/2/EGR2/PAX7 axis to enhance skeletal muscle regeneration

Personalized Medicine Considerations

Important factors for individualized approaches:

• Specific TECRL mutation and its functional consequences

• Tissue-specific manifestations (cardiac vs. skeletal muscle)

• Age of onset and disease severity

• Response to conventional therapies like beta-blockers