TY - JOUR
T1 - Imaging ATP Consumption in Resting Skeletal Muscle
T2 - One Molecule at a Time
AU - Nelson, Shane R.
AU - Li, Amy
AU - Beck-Previs, Samantha
AU - Kennedy, Guy G.
AU - Warshaw, David M.
N1 - Funding Information:
Funding: National Institutes of Health Grants AR067279 , HL126909 , and HL150953 (to D.M.W.) and supported in part by a generous gift to D.M.W. from Arnold and Mariel Goran.
Funding Information:
The authors wish to acknowledge George Osol and Marilynn Cipolla (University of Vermont) for contribution of biological samples and insightful conversations early on with Michael Geeves and Neil Kad (University of Kent). Funding: National Institutes of Health Grants AR067279, HL126909, and HL150953 (to D.M.W.) and supported in part by a generous gift to D.M.W. from Arnold and Mariel Goran.
Publisher Copyright:
© 2020 Biophysical Society
PY - 2020/9/15
Y1 - 2020/9/15
N2 - Striated muscle contraction is the result of sarcomeres, the basic contractile unit, shortening because of hydrolysis of adenosine triphosphate (ATP) by myosin molecular motors. In noncontracting, “relaxed” muscle, myosin still hydrolyzes ATP slowly, contributing to the muscle's overall resting metabolic rate. Furthermore, when relaxed, myosin partition into two kinetically distinct subpopulations: a faster-hydrolyzing “relaxed” population, and a slower-hydrolyzing “super relaxed” (SRX) population. How these two myosin subpopulations are spatially arranged in the sarcomere is unclear, although it has been proposed that myosin-binding protein C (MyBP-C) may stabilize the SRX state. Because MyBP-C is found only in a distinct region of the sarcomere, i.e., the C-zone, are SRX myosin similarly colocalized in the C-zone? Here, we imaged the binding lifetime and location (38-nm resolution) of single, fluorescently labeled boron-dipyrromethene-labeled ATP molecules in relaxed skeletal muscle sarcomeres from rat soleus. The lifetime distribution of fluorescent ATP-binding events was well fitted as an admixture of two subpopulations with time constants of 26 ± 2 and 146 ± 16 s, with the longer-lived population being 28 ± 4% of the total. These values agree with reported kinetics from bulk studies of skeletal muscle for the relaxed and SRX subpopulations, respectively. Subsarcomeric localization of these events revealed that SRX-nucleotide-binding events are fivefold more frequent in the C-zone (where MyBP-C exists) than in flanking regions devoid of MyBP-C. Treatment with the small molecule myosin inhibitor, mavacamten, caused no change in SRX event frequency in the C-zone but increased their frequency fivefold outside the C-zone, indicating that all myosin are in a dynamic equilibrium between the relaxed and SRX states. With SRX myosin found predominantly in the C-zone, these data suggest that MyBP-C may stabilize and possibly regulate the SRX state.
AB - Striated muscle contraction is the result of sarcomeres, the basic contractile unit, shortening because of hydrolysis of adenosine triphosphate (ATP) by myosin molecular motors. In noncontracting, “relaxed” muscle, myosin still hydrolyzes ATP slowly, contributing to the muscle's overall resting metabolic rate. Furthermore, when relaxed, myosin partition into two kinetically distinct subpopulations: a faster-hydrolyzing “relaxed” population, and a slower-hydrolyzing “super relaxed” (SRX) population. How these two myosin subpopulations are spatially arranged in the sarcomere is unclear, although it has been proposed that myosin-binding protein C (MyBP-C) may stabilize the SRX state. Because MyBP-C is found only in a distinct region of the sarcomere, i.e., the C-zone, are SRX myosin similarly colocalized in the C-zone? Here, we imaged the binding lifetime and location (38-nm resolution) of single, fluorescently labeled boron-dipyrromethene-labeled ATP molecules in relaxed skeletal muscle sarcomeres from rat soleus. The lifetime distribution of fluorescent ATP-binding events was well fitted as an admixture of two subpopulations with time constants of 26 ± 2 and 146 ± 16 s, with the longer-lived population being 28 ± 4% of the total. These values agree with reported kinetics from bulk studies of skeletal muscle for the relaxed and SRX subpopulations, respectively. Subsarcomeric localization of these events revealed that SRX-nucleotide-binding events are fivefold more frequent in the C-zone (where MyBP-C exists) than in flanking regions devoid of MyBP-C. Treatment with the small molecule myosin inhibitor, mavacamten, caused no change in SRX event frequency in the C-zone but increased their frequency fivefold outside the C-zone, indicating that all myosin are in a dynamic equilibrium between the relaxed and SRX states. With SRX myosin found predominantly in the C-zone, these data suggest that MyBP-C may stabilize and possibly regulate the SRX state.
UR - http://www.scopus.com/inward/record.url?scp=85089824968&partnerID=8YFLogxK
U2 - 10.1016/j.bpj.2020.07.036
DO - 10.1016/j.bpj.2020.07.036
M3 - Article
C2 - 32857963
AN - SCOPUS:85089824968
SN - 0006-3495
VL - 119
SP - 1050
EP - 1055
JO - Biophysical Journal
JF - Biophysical Journal
IS - 6
ER -