Effect of nitric oxide on single skeletal muscle fibres from the mouse

Abstract

1. Single skeletal muscle fibres from a mouse foot muscle were used to investigate the effects of nitric oxide on contractile function. 2. We measured force production and myoplasmic free Ca2+ concentration ([Ca2+]i) in single fibres exposed to the nitric oxide donors S-nitroso-N-acetylcysteine (SNAC) and nitroprusside. 3. The nitric oxide donors reduced myofibrillar Ca2+ sensitivity, whereas [Ca2+]i transients were increased during submaximal tetani. Force was largely unchanged. SNAC did not change maximum shortening velocity, the rate of force redevelopment, or force production at saturating [Ca2+]i. 4. The guanylyl cyclase inhibitor LY83583 increased tetanic [Ca2+]i but had no effect on Ca2+ sensitivity. LY83583 did not prevent the decrease in myofibrillar Ca2+ sensitivity in response to SNAC. The oxidizer sodium nitrite increased tetanic [Ca2+]i and decreased myofibrillar Ca2+ sensitivity. 5. We conclude that under our experimental conditions nitric oxide impairs Ca2+ activation of the actin filaments which results in decreased myofibrillar Ca2+ sensitivity.

Figure 1. Effect of SNAC on submaximal [Ca²⁺]_i and force

Original records from 40 Hz tetani produced in one fibre under control conditions, after 4 min exposure to 0.25 mM SNAC, and after wash-out. The upper part of the figure shows [Ca²⁺]_i records obtained with indo-1 and the lower part shows force. The bars show the mean [Ca²⁺]_i over the period where measurements were done. The short exposure to SNAC increased tetanic [Ca²⁺]_i from 0.69 to 0.77 μM. In this fibre the increase in [Ca²⁺]_i was accompanied by a small (3 %) decrease of force (measured during the same period as [Ca²⁺]_i). Both [Ca²⁺]_i and force had returned to their control values 1 min after wash-out of SNAC.

Figure 2. Nitric oxide donors increase tetanic [Ca²⁺]_i but not force

Relative changes of tetanic [Ca²⁺]_i (A) and force (B) as a result of exposure to the drugs indicated below the columns. Results are presented as means ±s.e.m. from fixed-frequency comparisons within each fibre; n= 12 for SNAC, 9 for NP, 7 for NO₂⁻, 8 for LY83583, and 9 for SNAC + LY83583. * Significant difference from control (P < 0.05). Tetanic [Ca²⁺]_i was elevated with all the treatments: SNAC, 109 ± 3 %; NP, 107 ± 2 %; NO₂⁻, 110 ± 3 %; LY83583, 107 ± 2 %; LY83583 + SNAC, 106 ± 1 %. LY83583 also increased force (108 ± 3 %).

Figure 3. SNAC decreases the SR Ca²⁺ pumping rate

A, averaged records from twelve fibres showing the recovery of [Ca²⁺]_i at the end of tetanic stimulation under control conditions and during exposure to SNAC. SNAC caused an upward and parallel shift of the [Ca²⁺]_i tail. B, analysis of the SR function according to eqn (1) performed on double exponential fits of the [Ca²⁺]_i tails (A, dashed lines). Control, • and dashed line; SNAC, ○ and continuous line. SNAC had no significant effect on the SR Ca²⁺ leak (L), whereas the rate of SR Ca²⁺ pumping (reflected by A in eqn (1)) was reduced to about 67 % of the control value.

Figure 4. Effect on force production at saturating [Ca²⁺]_i

Original records of [Ca²⁺]_i (upper traces) and force (lower traces) from 100 Hz tetani produced in the presence of 10 mM caffeine. [Ca²⁺]_i was high in both contractions and the forces were almost identical. Force during co-incubation with caffeine was 422 ± 18 kPa for control, and 420 ± 21 kPa for SNAC (n= 5).

Figure 5. Typical change of force-[Ca²⁺]_i relationship with SNAC

A, analysis of the force-[Ca²⁺]_i relationship in one fibre in control (•), with 0.25 mM SNAC (○), and after wash-out (▴). Curves were drawn by fitting data points in control (dashed line) and with SNAC (continuous line) to eqn (2). All the data points obtained during the exposure to SNAC lie to the right of the control points, and Ca₅₀ (eqn (2)) increased from 0.68 to 0.81 μM. This rightward shift due to SNAC was reversible with wash-out (▴). B, force-[Ca²⁺]_i relationship in one fibre in control (• and dashed line), and during co-incubation with 5 μM LY83483 and 0.5 mM SNAC (○ and continuous line). As with SNAC alone, co-incubation with LY83583 and SNAC shifted all the data points to the right of the control relationship, and Ca₅₀ increased from 0.44 to 0.57 μM.

Figure 6. Lack of effect on shortening velocity

Slack tests performed in control and with 0.25 mM SNAC. A, original force records from 120 μm releases; note that the time until force redevelopment starts is very similar with (dashed line) and without SNAC (continuous line). B, plot of the amplitude of shortening vs. the time to take up the slack. There was no noticeable difference between the times measured in control (• and dashed line) and with SNAC (○ and continuous line) at any of the four release steps studied.