Nitric oxide synthase in cardiac sarcoplasmic reticulum

Area of Science:

• Cardiovascular Physiology
• Molecular Biology
• Cellular Signaling

Background:

• Nitric oxide (NO) is a critical signaling molecule influencing cardiac function and metabolism.
• The precise localization and function of NO-producing enzymes within cardiac cells remain areas of active investigation.

Purpose of the Study:

• To investigate the presence and function of nitric oxide synthase (NOS) within cardiac sarcoplasmic reticulum (SR) membranes.
• To determine the impact of endogenously produced NO on SR calcium handling in cardiac myocytes.

Main Methods:

• Biochemical assays measuring L-citrulline formation from L-arginine in isolated cardiac SR vesicles.
• Electron paramagnetic resonance (EPR) spin-trapping to detect endogenous NO production.
• Immunoelectron microscopy and confocal microscopy to localize NOS isoforms in cardiac SR.
• Western blotting to analyze NOS protein size and identify potential novel isoforms.
• Measurement of 45Ca uptake by cardiac SR vesicles and the effect of NOS inhibition.

Main Results:

• Neuronal-type NOS (nNOS) was identified on cardiac SR vesicles, distinct from endothelial (eNOS) and inducible (iNOS) isoforms.
• Endogenous NO production by SR-associated nNOS was detected and shown to inhibit SR Ca2+ uptake.
• Cardiac SR nNOS appears to be a distinct isoform, larger than brain nNOS and absent in nNOS knockout mice.
• Selective nNOS inhibition prevented the NO-mediated inhibition of SR Ca2+ transport.

Conclusions:

• A cardiac-specific nNOS isoform is localized to the SR of cardiac myocytes.
• This SR-associated nNOS produces NO that directly modulates SR Ca2+ uptake, suggesting a role in regulating cardiac contractility.
• The findings reveal a novel mechanism for NO-mediated regulation of calcium handling in the heart.