scholarly journals A four-electrode method to study dynamics of ion activity and transport in skeletal muscle fibers

2019 ◽  
Vol 151 (9) ◽  
pp. 1146-1155 ◽  
Author(s):  
Judith A. Heiny ◽  
Stephen C. Cannon ◽  
Marino DiFranco
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Ionic Current ◽  
Transport Mechanisms ◽  
Electrical Stability ◽  
Ion Concentrations ◽  
Cell Functions ◽  
Skeletal Muscle Fibers ◽  
Intracellular Ion Concentrations ◽  
Ion Movements

Ion movements across biological membranes, driven by electrochemical gradients or active transport mechanisms, control essential cell functions. Membrane ion movements can manifest as electrogenic currents or electroneutral fluxes, and either process can alter the extracellular and/or intracellular concentration of the transported ions. Classic electrophysiological methods allow accurate measurement of membrane ion movements when the transport mechanism produces a net ionic current; however, they cannot directly measure electroneutral fluxes and do not detect any accompanying change in intracellular ion concentrations. Here, we developed a method for simultaneously measuring ion movements and the accompanying dynamic changes in intracellular ion concentrations in intact skeletal muscle fibers under voltage or current clamp in real time. The method combines a two-microelectrode voltage clamp with ion-selective and reference microelectrodes (four-electrode system). We validate the electrical stability of the system and the viability of the preparation for periods of ∼1 h. We demonstrate the power of this method with measurements of intracellular Cl−, H+, and Na+ to show (a) voltage-dependent redistribution of Cl− ions; (b) intracellular pH changes induced by changes in extracellular pCO2; and (c) electroneutral and electrogenic Na+ movements controlled by the Na,K-ATPase. The method is useful for studying a range of transport mechanisms in many cell types, particularly when the transmembrane ion movements are electrically silent and/or when the transport activity measurably changes the intracellular activity of a transported ion.

scholarly journals A Four Electrode Method to Study Dynamics of Ion Activity and Transport in Skeletal Muscle Fibers

2019 ◽  
Author(s):  
Judith A. Heiny ◽  
Stephen C. Cannon ◽  
Marino DiFranco
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Ionic Current ◽  
Cell Types ◽  
Ion Concentration ◽  
Transport Mechanisms ◽  
Electrical Stability ◽  
Cell Functions ◽  
Skeletal Muscle Fibers ◽  
Ion Movements

ABSTRACTIon movements across biological membranes, driven by electrochemical gradients or active transport mechanisms, control essential cell functions. Membrane ion movements can manifest as electrogenic currents or electroneutral fluxes, and either process can alter the extracellular and/or intracellular concentration of the transported ion(s). Classical electrophysiological methods allow accurate measurement of membrane ion movements when the transport mechanism produces a net ionic current; however, they cannot directly measure electroneutral fluxes and do not detect any accompanying change in intracellular ion concentrations.Here, we developed a method for simultaneously measuring ion movements and the accompanying dynamic changes in intracellular ion concentration(s) in intact skeletal muscle fibers under voltage– or current clamp in real time. The method combines a two-microelectrode voltage-clamp with ion-selective and reference microelectrodes (4 electrode system). We validate the electrical stability of the system and the viability of the preparation for periods of approximately 1 h. We demonstrate the power of this method with measurements of intracellular Cl-, H+, and Na+ to show: 1) voltage-dependent redistribution of Cl- ions; 2) intracellular pH changes induced by changes in extracellular pCO2; and 3) electroneutral and electrogenic Na+ movements controlled by the Na,K-ATPase. The method is useful for studying a range of transport mechanisms in many cell types, particularly when the transmembrane ion movements are electrically silent and/or when the transport activity measurably changes the intracellular activity of a transported ion.

scholarly journals Kinetic properties of intramembrane charge movement under depolarized conditions in frog skeletal muscle fibers.

1991 ◽  
Vol 98 (2) ◽  
pp. 365-378 ◽  
Author(s):  
G Szücs ◽  
Z Papp ◽  
L Csernoch ◽  
L Kovács
Keyword(s):  
Skeletal Muscle ◽  
Voltage Dependence ◽  
Slow Component ◽  
Muscle Fibers ◽  
Ionic Current ◽  
Kinetic Properties ◽  
Constant Field ◽  
Charge Movement ◽  
Skeletal Muscle Fibers ◽  
Intramembrane Charge Movement

Intramembrane charge movement was measured on skeletal muscle fibers of the frog in a single Vaseline-gap voltage clamp. Charge movements determined both under polarized conditions (holding potential, VH = -100 mV; Qmax = 30.4 +/- 4.7 nC/micro(F), V = -44.4 mV, k = 14.1 mV; charge 1) and in depolarized states (VH = 0 mV; Qmax = 50.0 +/- 6.7 nC/micro(F), V = -109.1 mV, k = 26.6 mV; charge 2) had properties as reported earlier. Linear capacitance (LC) of the polarized fibers was increased by 8.8 +/- 4.0% compared with that of the depolarized fibers. Using control pulses measured under depolarized conditions to calculate charge 1, a minor change in the voltage dependence (to V = -44.6 mV and k = 14.5 mV) and a small increase in the maximal charge (to Qmax = 31.4 +/- 5.5 nC/micro(F] were observed. While in most cases charge 1 transients seemed to decay with a single exponential time course, charge 2 currents showed a characteristic biexponential behavior at membrane potentials between -90 and -180 mV. The voltage dependence of the rate constant of the slower component was fitted with a simple constant field diffusion model (alpha m = 28.7 s-1, V = -124.0 mV, and k = 15.6 mV). The midpoint voltage (V) was similar to that obtained from the Q-V fit of charge 2, while the steepness factor (k) resembled that of charge 1. This slow component could also be isolated using a stepped OFF protocol; that is, by hyperpolarizing the membrane to -190 mV for 200 ms and then coming back to 0 mV in two steps. The faster component was identified as an ionic current insensitive to 20 mM Co2+ but blocked by large hyperpolarizing pulses. These findings are consistent with the model implying that charge 1 and the slower component of charge 2 interconvert when the holding potential is changed. They also explain the difference previously found when comparing the steepness factors of the voltage dependence of charge 1 and charge 2.

The “KIMWIPE” Effect in Ultra-Thin Cryosections

1985 ◽  
Vol 43 ◽  
pp. 458-459
Author(s):  
I. Taylor ◽  
P. Ingram ◽  
J.R. Sommer
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Muscle Fibers ◽  
Ice Crystals ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Thin Sections ◽  
Skeletal Muscle Fibers ◽  
Freeze Dried ◽  
Ice Crystal Formation

In studying quick-frozen single intact skeletal muscle fibers for structural and microchemical alterations that occur milliseconds, and fractions thereof, after electrical stimulation, we have developed a method to compare, directly, ice crystal formation in freeze-substituted thin sections adjacent to all, and beneath the last, freeze-dried cryosections. We have observed images in the cryosections that to our knowledge have not been published heretofore (Figs.1-4). The main features are that isolated, sometimes large regions of the sections appear hazy and have much less contrast than adjacent regions. Sometimes within the hazy regions there are smaller areas that appear crinkled and have much more contrast. We have also observed that while the hazy areas remain still, the regions of higher contrast visibly contract in the beam, often causing tears in the sections that are clearly not caused by ice crystals (Fig.3, arrows).

Effect of External Calcium and other Divalent Cations on K+ Contractures in Denervated Slow Skeletal Muscle Fibers of the Frog

2019 ◽  
Author(s):  
Leonardo Hernández
Keyword(s):  
Skeletal Muscle ◽  
Binding Capacity ◽  
Divalent Cations ◽  
Muscle Fibers ◽  
Free Calcium ◽  
Slow Skeletal Muscle ◽  
Skeletal Muscle Fibers ◽  
Free Calcium Concentration ◽  
Chronic Denervation ◽  
Denervated Muscles

The influence of Ca2+ and other divalent cations on contractile responses of slow skeletal muscle fibers of the frog (Rana pipiens) under conditions of chronic denervation was investigated.Isometric tension was recorded from slow bundles of normal and denervated cruralis muscle in normal solution and in solutions with free calcium concentration solution or in solutions where other divalent cations (Sr2+, Ni2+, Co2+ or Mn2+) substituted for calcium. In the second week after nerve section, in Ca2+-free solutions, we observed that contractures (evoked from 40 to 80 mM-K+) of non-denervated muscles showed significantly higher tensions (p<0.05), than those from denervated bundles. Likewise, in solutions where calcium was substituted by all divalent cations tested, with exception of Mn2+, the denervated bundles displayed lower tension than non-denervated, also in the second week of denervation. In this case, the Ca2+ substitution by Sr2+ caused the higher decrease in tension, followed by Co2+ and Ni2+, which were different to non-denervated bundles, as the lowest tension was developed by Mn2+, followed by Co2+, and then Ni2+ and Sr2+. After the third week, we observed a recovery in tension. These results suggest that denervation altering the binding capacity to divalent cations of the voltage sensor.

scholarly journals Development of a human neuromuscular tissue-on-a-chip model on a 24-well-plate-format compartmentalized microfluidic device

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Kazuki Yamamoto ◽  
Nao Yamaoka ◽  
Yu Imaizumi ◽  
Takunori Nagashima ◽  
Taiki Furutani ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Microfluidic Device ◽  
Motor Neurons ◽  
Muscle Fibers ◽  
Three Dimensional ◽  
Tissue Model ◽  
Skeletal Muscle Fibers

A three-dimensional human neuromuscular tissue model that mimics the physically separated structures of motor neurons and skeletal muscle fibers is presented.

scholarly journals Glycogenin is Dispensable for Glycogen Synthesis in Human Muscle, and Glycogenin Deficiency Causes Polyglucosan Storage

2019 ◽  
Vol 105 (2) ◽  
pp. 557-566 ◽  
Author(s):  
Kittichate Visuttijai ◽  
Carola Hedberg-Oldfors ◽  
Christer Thomsen ◽  
Emma Glamuzina ◽  
Cornelia Kornblum ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Skeletal Muscle ◽  
Western Blot ◽  
Cardiac Muscle ◽  
Glycogen Synthesis ◽  
Muscle Fibers ◽  
Human Muscle ◽  
Missense Mutations ◽  
Skeletal Muscle Fibers ◽  
Glycogen Biosynthesis

Abstract Context Glycogenin is considered to be an essential primer for glycogen biosynthesis. Nevertheless, patients with glycogenin-1 deficiency due to biallelic GYG1 (NM_004130.3) mutations can store glycogen in muscle. Glycogenin-2 has been suggested as an alternative primer for glycogen synthesis in patients with glycogenin-1 deficiency. Objective The objective of this article is to investigate the importance of glycogenin-1 and glycogenin-2 for glycogen synthesis in skeletal and cardiac muscle. Design, Setting, and Patients Glycogenin-1 and glycogenin-2 expression was analyzed by Western blot, mass spectrometry, and immunohistochemistry in liver, heart, and skeletal muscle from controls and in skeletal and cardiac muscle from patients with glycogenin-1 deficiency. Results Glycogenin-1 and glycogenin-2 both were found to be expressed in the liver, but only glycogenin-1 was identified in heart and skeletal muscle from controls. In patients with truncating GYG1 mutations, neither glycogenin-1 nor glycogenin-2 was expressed in skeletal muscle. However, nonfunctional glycogenin-1 but not glycogenin-2 was identified in cardiac muscle from patients with cardiomyopathy due to GYG1 missense mutations. By immunohistochemistry, the mutated glycogenin-1 colocalized with the storage of glycogen and polyglucosan in cardiomyocytes. Conclusions Glycogen can be synthesized in the absence of glycogenin, and glycogenin-1 deficiency is not compensated for by upregulation of functional glycogenin-2. Absence of glycogenin-1 leads to the focal accumulation of glycogen and polyglucosan in skeletal muscle fibers. Expression of mutated glycogenin-1 in the heart is deleterious, and it leads to storage of abnormal glycogen and cardiomyopathy.

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