TY - JOUR
T1 - Dietary nitrate increases submaximal SERCA activity and ADP transfer to mitochondria in slow-twitch muscle of female mice
AU - Petrick, Heather L
AU - Brownell, Stuart
AU - Vachon, Bayley
AU - Brunetta, Henver S
AU - Handy, Rachel M
AU - van Loon, Luc J C
AU - Murrant, Coral L
AU - Holloway, Graham P
N1 - Funding Information:
To develop this approach, mitochondrial respiration (JO2) was monitored in permeabilized fibers from red gastrocnemius muscle (Fig. 3B). Respiration was supported by pyruvate + malate, followed by the addition of ATP (Fig. 3B). We then titrated sequentially increasing concentrations of CaCl2 in the presence of ATP to stimulate SERCA-mediated ATP hydrolysis and create ADP to support mitochondrial respiration. This revealed that maximal SERCA-supported respiration was ~25% of oxidative capacity in the presence of saturating ADP (Fig. 3, B and C). The addition of cyclopiazonic acid (CPA), a SERCA-specific inhibitor, fully attenuated mitochondrial respiration to that of pyruvate + malate (Fig. 3B), suggesting SERCA-derived ADP is capable of supporting mitochondrial respiration in our experimental design. Importantly, Ca2+ did not increase mitochondrial respiration following the prior addition of CPA (Fig. 3D), and titrations of Ca2+ after 500 µM ADP (~ADP Km) did not increase submaximal ADP-supported respiration or attenuate maximal respiratory capacity (Fig. 3E). Combined, this methodology enables the examination of a high-energy phosphate cycling microdomain between ATP hydrolysis from SERCA and mitochondrial ADP provision.
Publisher Copyright:
© 2022 the American Physiological Society.
PY - 2022/8/1
Y1 - 2022/8/1
N2 - Rapid oscillations in cytosolic calcium (Ca2+) coordinate muscle contraction, relaxation, and physical movement. Intriguingly, dietary nitrate decreases ATP cost of contraction, increases force production, and increases cytosolic Ca2+, which would seemingly necessitate a greater demand for sarcoplasmic reticulum Ca2+ ATPase (SERCA) to sequester Ca2+ within the sarcoplasmic reticulum (SR) during relaxation. As SERCA is highly regulated, we aimed to determine the effect of 7-day nitrate supplementation (1 mM via drinking water) on SERCA enzymatic properties and the functional interaction between SERCA and mitochondrial oxidative phosphorylation. In soleus, we report that dietary nitrate increased force production across all stimulation frequencies tested, and throughout a 25 min fatigue protocol. Mice supplemented with nitrate also displayed an ∼25% increase in submaximal SERCA activity and SERCA efficiency (P = 0.053) in the soleus. To examine a possible link between ATP consumption and production, we established a methodology coupling SERCA and mitochondria in permeabilized muscle fibers. The premise of this experiment is that the addition of Ca2+ in the presence of ATP generates ADP from SERCA to support mitochondrial respiration. Similar to submaximal SERCA activity, mitochondrial respiration supported by SERCA-derived ADP was increased by ∼20% following nitrate in red gastrocnemius. This effect was fully attenuated by the SERCA inhibitor cyclopiazonic acid and was not attributed to differences in mitochondrial oxidative capacity, ADP sensitivity, protein content, or reactive oxygen species emission. Overall, these findings suggest that improvements in submaximal SERCA kinetics may contribute to the effects of nitrate on force production during fatigue.NEW & NOTEWORTHY We show that nitrate supplementation increased force production during fatigue and increased submaximal SERCA activity. This was also evident regarding the high-energy phosphate transfer from SERCA to mitochondria, as nitrate increased mitochondrial respiration supported by SERCA-derived ADP. Surprisingly, these observations were only apparent in muscle primarily expressing type I (soleus) but not type II fibers (EDL). These findings suggest that alterations in SERCA properties are a possible mechanism in which nitrate increases force during fatiguing contractions.
AB - Rapid oscillations in cytosolic calcium (Ca2+) coordinate muscle contraction, relaxation, and physical movement. Intriguingly, dietary nitrate decreases ATP cost of contraction, increases force production, and increases cytosolic Ca2+, which would seemingly necessitate a greater demand for sarcoplasmic reticulum Ca2+ ATPase (SERCA) to sequester Ca2+ within the sarcoplasmic reticulum (SR) during relaxation. As SERCA is highly regulated, we aimed to determine the effect of 7-day nitrate supplementation (1 mM via drinking water) on SERCA enzymatic properties and the functional interaction between SERCA and mitochondrial oxidative phosphorylation. In soleus, we report that dietary nitrate increased force production across all stimulation frequencies tested, and throughout a 25 min fatigue protocol. Mice supplemented with nitrate also displayed an ∼25% increase in submaximal SERCA activity and SERCA efficiency (P = 0.053) in the soleus. To examine a possible link between ATP consumption and production, we established a methodology coupling SERCA and mitochondria in permeabilized muscle fibers. The premise of this experiment is that the addition of Ca2+ in the presence of ATP generates ADP from SERCA to support mitochondrial respiration. Similar to submaximal SERCA activity, mitochondrial respiration supported by SERCA-derived ADP was increased by ∼20% following nitrate in red gastrocnemius. This effect was fully attenuated by the SERCA inhibitor cyclopiazonic acid and was not attributed to differences in mitochondrial oxidative capacity, ADP sensitivity, protein content, or reactive oxygen species emission. Overall, these findings suggest that improvements in submaximal SERCA kinetics may contribute to the effects of nitrate on force production during fatigue.NEW & NOTEWORTHY We show that nitrate supplementation increased force production during fatigue and increased submaximal SERCA activity. This was also evident regarding the high-energy phosphate transfer from SERCA to mitochondria, as nitrate increased mitochondrial respiration supported by SERCA-derived ADP. Surprisingly, these observations were only apparent in muscle primarily expressing type I (soleus) but not type II fibers (EDL). These findings suggest that alterations in SERCA properties are a possible mechanism in which nitrate increases force during fatiguing contractions.
KW - Adenosine Diphosphate/metabolism
KW - Adenosine Triphosphate/metabolism
KW - Animals
KW - Calcium/metabolism
KW - Fatigue/metabolism
KW - Female
KW - Mice
KW - Mitochondria/metabolism
KW - Muscle Contraction/physiology
KW - Muscle Fibers, Slow-Twitch/metabolism
KW - Muscle, Skeletal/metabolism
KW - Nitrates/metabolism
KW - Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism
U2 - 10.1152/ajpendo.00371.2021
DO - 10.1152/ajpendo.00371.2021
M3 - Article
C2 - 35732003
SN - 0193-1849
VL - 323
SP - E171-E184
JO - American Journal of Physiology : Endocrinology and Metabolism
JF - American Journal of Physiology : Endocrinology and Metabolism
IS - 2
ER -