Estimation of crossbridge-state during cardiomyocyte beating using second harmonic generation.

Area of Science:

• Cardiology
• Biophysics
• Cell Biology

Background:

• Understanding dynamic crossbridge formation in cardiomyocytes is vital for elucidating cardiomyopathy mechanisms and evaluating therapeutic interventions.
• Current methods may not fully capture the dynamic nature of actomyosin interactions in living cells.

Purpose of the Study:

• To establish a novel assay system for dynamically measuring second harmonic generation (SHG) anisotropy in pulsating cardiomyocytes.
• To correlate SHG anisotropy with myosin crossbridge status and assess actomyosin activity.

Main Methods:

• Developed an assay to dynamically measure SHG anisotropy from myosin filaments, reflecting crossbridge status in pulsating cardiomyocytes.
• Utilized inheritable mutations causing excessive myosin-actin interactions to validate the assay.
• Employed infrared two-photon excitation for intravital evaluation in a *Drosophila* disease model.

Main Results:

• Established a correlation between sarcomere length and SHG anisotropy, representing the crossbridge formation ratio during pulsation.
• Demonstrated that ultraviolet irradiation increases non-force-generating attached crossbridges during myocardial differentiation.
• Successfully evaluated myocardial dysfunction in a *Drosophila* model, showcasing the method's applicability.

Conclusions:

• The developed SHG anisotropy assay effectively evaluates actomyosin activity in cardiomyocytes, crucial for drug or genetic defect assessment.
• This method offers a valuable tool for risk assessment of future heart failure, complementing genomic inspection.