Conditional neuronal nitric oxide synthase overexpression impairs myocardial contractility

Area of Science:

• Cardiovascular Physiology
• Molecular Cardiology
• Enzyme Function in the Heart

Background:

• The precise role of neuronal nitric oxide synthase (nNOS) in regulating cardiac function is not fully understood.
• Previous studies using nNOS knockout mice or pharmacological inhibition yielded conflicting results, potentially overlooking the importance of enzyme localization.

Purpose of the Study:

• To investigate the impact of close proximity between nNOS and effector molecules, such as L-type Ca(2+) channels, on myocardial contractility.
• To generate and characterize a novel transgenic mouse model for conditional, cardiac-specific overexpression of nNOS.

Main Methods:

• Generation of a conditional transgenic mouse model for cardiac-specific nNOS overexpression.
• Western blot analysis to confirm increased nNOS protein levels.
• Measurement of total NOS activity using radiolabeled l-arginine.
• In vivo cardiac function assessment (e.g., +dp/dt(max), ejection fraction).
• Coimmunoprecipitation assays to identify protein interactions.
• Electrophysiological studies on isolated cardiac myocytes to measure L-type Ca(2+) current (I(Ca,L)) and intracellular Ca(2+) transients.

Main Results:

• Transgenic mice exhibited a 6-fold increase in cardiac nNOS protein expression and a 30% increase in total NOS activity.
• nNOS overexpression led to significantly reduced myocardial contractility, including decreased +dp/dt(max) and ejection fraction.
• Coimmunoprecipitation revealed interactions between nNOS, SR Ca(2+) ATPase, and L-type Ca(2+) channels.
• Isolated cardiac myocytes from overexpressing mice showed decreased I(Ca,L) density, impaired intracellular Ca(2+) transients, and reduced fractional shortening.

Conclusions:

• Conditional, cardiac-specific overexpression of nNOS in a transgenic model demonstrably reduces myocardial contractility.
• nNOS appears to suppress L-type Ca(2+) channel function, leading to diminished Ca(2+) transients and a negative inotropic effect on the heart.