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

Figures

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  Fig 1. Exon structure of cardiac muscle expressed nNOS splice variants and their expression profile in the nNOS null mouse models used in this study. Coding exons of each splice variant are gray numbered boxes. Exon 2 encodes the PDZ (PSD95/Dlg1/ZO1) protein-protein interaction domain. Exon 6 encodes the heme binding domain essential for nitric oxide synthesis. Exon sequences that form unique 5’ untranslated sequences are white. Asterisks mark translation initiation sites. nNOS splice variant expression in control and murine KN1 and KN2 nNOS knockout models are shown on the right. A tick mark indicates expression. A cross mark indicates absence of expression and/or activity. This study employs two nNOS knockout models: first knockout of nNOS (KN1, exon 2 deletion) and second knockout of nNOS (KN2, exon 6 deletion). KN1 and KN2 mice have distinct isoform expression profiles. KN1 mice lack nNOSα and nNOSμ, but still express nNOSβ. KN2 mice do not express any active nNOS splice variants.

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  Fig 2. nNOS inhibition decreases maximum myofilament ATPase activity and specific force output in demembranated papillary muscle fibers. (A) Maximal myofilament acto-myosin ATPase activities in demembranated papillary muscles from WT, KN1 and KN2 mice. Maximum ATPase activities were significantly decreased in KN1 and KN2 papillary muscles relative to WT controls. ATPase activities in KN2 papillary muscles were intermediate between KN1 and wild type. (B) Demembranated papillary muscles from KN1 and KN2 showed a significant decrease in specific force output relative to WT controls. n = 10–12 mice per group (2–4 fibers per mouse). One factor ANOVA, * p < 0.05, ** p < 0.01, **** p < 0.001.

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  Fig 3. nNOS inhibition has no impact on the Ca2+ sensitivity of myofilament ATPase activity or force output in papillary muscle fibers. (A) Dependence of myofilament ATPase activity on Ca2+ concentrations in demembranated papillary muscles from WT, KN1 and KN2 mice. Normalized maximum ATPase activities were plotted against Ca2+ (pCa = -log10[Ca2+]). KN1, KN2 and WT control papillary muscles exhibited similar Ca2+ concentrations required for the activation of ATPase activity. (B) Papillary muscles from WT, KN1 and KN2 mice showed similar Ca2+ concentrations required for half maximal activation of ATPase activity (pCa50). (C) Dependence of demembranated papillary muscle force output on Ca2+ in WT, KN1 and KN2 mice. nNOS inhibition had no impact on the force-Ca2+ relationship which was similar between KN1, KN2 and wild type controls. (D) Papillary muscles from WT, KN1 and KN2 mice showed similar Ca2+ concentrations required for half maximal force output (pCa50). n = 10–12 mice per group (2–4 fibers per mouse).

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  Fig 4. KN1 mice exhibit a reduced energy cost of muscle contraction. The energetic cost (maximal ATPase activity divided by force output) of muscle contraction was calculated at different muscle fractional activation states. Muscle activation is normalized muscle force output over the range of Ca2+ concentrations in Fig 3. Demembranated papillary muscles from KN1 mice exhibited a lower energy cost of contraction than wild type controls. KN2 papillary muscles exhibited a similar energy cost of contraction to wild type controls. n = 10–12 mice (2–4 fibers per mouse). Two factor ANOVA, ***** p < 0.001 WT versus KN1, #### p < 0.001 KN1 versus KN2.

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  Fig 5. nNOS is dispensable for intracellular Ca2+ transients in intact cardiac papillary muscles. The impact of nNOS splice variant deficiency on Ca2+ transients was determined in intact papillary muscles loaded with Fura-2 Ca2+ indicator paced at different stimulation frequencies. Simultaneous measurements of intracellular Ca2+ (here) and force (Fig 6) were recorded when force reached a steady state following changes in stimulus frequencies. For each stimulation frequency, intracellular Ca2+ transients were averaged and plotted as a function of time. Ca2+ transients in intact papillary muscles from KN1 and KN2 mice were indistinguishable from wild type controls, and from each other, at all stimulation frequencies. The top right panel shows a high magnification image of intracellular Ca2+ concentrations between 250–300 ms (gray box in top left panel-1 Hz stimulation) in intact papillary muscles from wild type, KN1 and KN2 mice to highlight extensive data overlap. n = 4–6 mice per group.

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  Fig 6. nNOS inhibition has no impact on force responses to Ca2+ transients in intact papillary muscles. The impact of nNOS splice variant deficiency on the normalized force (fraction of maximum) response to Ca2+ transients (shown in Fig 5) was determined in intact papillary muscles at different stimulation frequencies. Normalized force output from intact papillary muscles from KN1 and KN2 mice was indistinguishable from wild type controls, and from each other, at stimulation frequencies between 1 and 3 Hz. The bottom right panel shows a high magnification image of normalized force output between 250–300 ms (gray box in bottom left panel-3 Hz stimulation) in intact papillary muscles from wild type, KN1 and KN2 mice to highlight extensive data overlap. n = 4–6 mice per group.