Dietary nitrate increases tetanic [Ca2+]i and contractile force in mouse fast-twitch muscle

Abstract

Dietary inorganic nitrate has profound effects on health and physiological responses to exercise. Here, we examined if nitrate, in doses readily achievable via a normal diet, could improve Ca(2+) handling and contractile function using fast- and slow-twitch skeletal muscles from C57bl/6 male mice given 1 mm sodium nitrate in water for 7 days. Age matched controls were provided water without added nitrate. In fast-twitch muscle fibres dissected from nitrate treated mice, myoplasmic free [Ca(2+)] was significantly greater than in Control fibres at stimulation frequencies from 20 to 150 Hz, which resulted in a major increase in contractile force at ≤ 50 Hz. At 100 Hz stimulation, the rate of force development was ∼35% faster in the nitrate group. These changes in nitrate treated mice were accompanied by increased expression of the Ca(2+) handling proteins calsequestrin 1 and the dihydropyridine receptor. No changes in force or calsequestrin 1 and dihydropyridine receptor expression were measured in slow-twitch muscles. In conclusion, these results show a striking effect of nitrate supplementation on intracellular Ca(2+) handling in fast-twitch muscle resulting in increased force production. A new mechanism is revealed by which nitrate can exert effects on muscle function with applications to performance and a potential therapeutic role in conditions with muscle weakness.

Figure 1. Nitrate feeding increases contractile force in fast-twitch EDL muscles

Mean force data (±SEM) for EDL (A) and soleus (B) vs. stimulation frequency. Note the break in the x-axis in panel A. Muscles from Control (filled symbols, n = 7) and Nitrate mice (open symbols; n = 7 for EDL, n = 6 for soleus). *P < 0.05.

Figure 2. Nitrate feeding increases expression of SR Ca²⁺ handling proteins

Representative Western blots (EDL: panel A; soleus: panel B). C, Control; N, Nitrate. Mean data (±SEM; EDL: panel C; soleus: panel D) for CASQ1, DHPR, and RyR. EDL for Control (n = 3–7) and Nitrate mice (n = 3–7). Soleus for Control (n = 6) and Nitrate mice (n = 4). Control is filled bar mean set to 100%; Nitrate is open bar. *P < 0.05.

Figure 3. Tetanic [Ca²⁺]_i and force are increased in FDB fibres of nitrate fed mice

[Ca²⁺]_i (A) and force traces (B) from representative Control and Nitrate fibres during 30 Hz stimulation. Mean data (±SEM) of tetanic [Ca²⁺]_i (C) and force (D) vs. stimulation frequency. Note that in C, two axes are used for tetanic [Ca²⁺]_i in order to show the marked differences between the groups both at low and high frequencies. Fibres from Control (filled symbols, n = 7) and Nitrate mice (open symbols, n = 5). *P < 0.05.

Figure 4. The effect of nitrate feeding on force is mediated via increased [Ca²⁺]_i

A, mean force–Ca²⁺ curves constructed from 15–150 Hz contractions given at 1 min intervals. Mean (±SEM) data for 30 and 100 Hz are included to illustrate how the force response to an increase in [Ca²⁺]_i depends on the location on the force–Ca²⁺ curve. B, mean force records illustrating the faster rate of development of force in 100 Hz tetani. C, mean data (±SEM) for time to 50% max force (t_1/2) in these tetani. Control (n = 7, filled symbols and bar, continuous line) and Nitrate (n = 5, open symbols and bar, dashed line). *P < 0.05.

Figure 5. Sarcoplasmic reticulum Ca²⁺ uptake is not altered in muscles of Nitrate mice

A, individual 120 Hz tetanic vs. resting [Ca²⁺]_i data points for Control (filled circles) and Nitrate (open circles) fibres. B, representative Western blots and mean data (±SEM) of SERCA1 protein expression in EDL muscles from Control (C, n = 7, filled bar mean set to 100%) and Nitrate (N, n = 6, open bar). C, average records of [Ca²⁺]_i tails after 30 and 100 Hz stimulation. Control (n = 7, continuous line) and Nitrate mice (n = 5, dashed line).