nNOS splice variants differentially regulate myofilament function but are dispensable for intracellular calcium and force transients in cardiac papillary muscles

Cardiac muscle expresses three neuronal nitric oxide synthase (nNOS) splice variants: nNOS alpha, nNOS mu and nNOS beta. The functions of these nNOS splice variants in cardiac muscle, particularly myofilament-associated nNOS beta are unclear. To decipher cardiac nNOS splice variant function we investigated myofilament function and intracellular calcium and force transients in demembranated and intact papillary muscles from two lines of nNOS knockout mice. The first line (KN1) lacks nNOS alpha and nNOS mu. The second line (KN2) lacks active nNOS alpha, nNOS mu and nNOS beta. Demembranated KN1 papillary muscles exhibited reduced myofilament ATPase activity (-35%) and specific force (-10%) relative to controls. Demembranated KN2 muscles exhibited a smaller decrease in myofilament ATPase activity (-21 %), but a greater reduction in specific force (-26%) relative to controls. Myofilament calcium sensitivity in demembranated KN1 and KN2 papillary muscles was similar to controls. Thus, papillary muscle-expressed nNOS splice variants are necessary for control levels of myofilament ATPase activity and force generation, but dispensable for myofilament calcium sensitivity. The greater reduction in myofilament ATPase relative to specific force in KN1, but not KN2 muscle, reduced the energy cost of muscle contraction, suggesting that nNOS beta increased the energetic efficiency of contraction in the absence of nNOS mu and nNOS alpha. Analyses of intact KN1 and KN2 papillary muscles showed that both intracellular calcium transients and their evoked force transients were similar to controls at stimulation frequencies between 1 and 3 Hz. Therefore, nNOS was dispensable for baseline excitation-contraction coupling. In summary, these data suggest that nNOS splice variants differentially regulate myofilament function, but not baseline calcium handling in papillary muscles. More importantly, they suggest that nNOS beta is a novel modulator of myofilament function, and ultimately the energetic efficiency of cardiac papillary muscle contraction.