Manipulating L-type calcium channels in cardiomyocytes using split-intein protein transsplicing

Area of Science:

• Cardiovascular Biology
• Molecular Cardiology
• Gene Therapy

Background:

• Adult cardiomyocytes have limited viral vector packaging capacity (4-7 kb), hindering the study of large genes (>6 kb).
• Understanding structure-function relationships of large proteins, such as L-type calcium channels (LTCCs), is crucial for cardiac physiology.
• LTCCs play vital roles in cardiac excitation-contraction coupling, excitability, and gene expression.

Purpose of the Study:

• To overcome the technical challenge of expressing large genes, specifically the 6.6 kb α1C-subunit of LTCCs, in adult cardiomyocytes.
• To enable detailed investigation into the multidimensional signaling roles of LTCCs.

Main Methods:

• Utilized split-intein-mediated protein trans-splicing to reconstitute the full-length LTCC α1C-subunit from two separate halves.
• Engineered split-intein-tagged α1C fragments into adenovirus vectors for cardiomyocyte transduction.
• Reconstituted functional LTCCs within adult cardiomyocytes.

Main Results:

• Successfully expressed a full-length, functional LTCC α1C-subunit in adult cardiomyocytes, overcoming packaging limitations.
• Recombinant LTCCs demonstrated correct subcellular localization to dyads and functional activity, including triggering Ca(2+) transients.
• The reconstituted channels associated with caveolin-3 and supported normal β-adrenergic regulation of excitation-contraction coupling.

Conclusions:

• Split-intein protein trans-splicing provides an effective strategy to express large proteins, like LTCC α1C-subunits, in adult cardiomyocytes.
• This method significantly advances the ability to study large protein function in the heart.
• The developed approach removes a major technical barrier in cardiac molecular research.