Quantitative model analysis of the resting membrane potential in insect skeletal muscle: Implications for low temperature tolerance.

Abiotic stressors, such as cold exposure, can depolarize insect cells substantially causing cold coma and cell death. During cold exposure, insect skeletal muscle depolarization occurs through a 2-stage process. Firstly, short-term cold exposure reduces the activity of electrogenic ion pumps, which depolarize insect muscle markedly. Secondly, during long-term cold exposure, extracellular ion homeostasis is disrupted causing further depolarization. Consequently, many cold hardy insects improve membrane potential stability during cold exposure through adaptations that secure maintenance of ion homeostasis during cold exposure. Less is known about the adaptations permitting cold hardy insects to maintain membrane potential stability during the initial phase of cold exposure, before ion balance is disrupted. To address this problem it is critical to understand the membrane components (channels and transporters) that determine the membrane potential and to examine this question the present study constructed a mathematical "charge difference" model of the insect muscle membrane potential. This model was parameterized with known literature values for ion permeabilities, ion concentrations and membrane capacitance and the model was then further developed by comparing model predictions against empirical measurements following pharmacological inhibitors of the Na+/K+ ATPase, Cl- channels and symporters. Subsequently, we compared simulated and recorded membrane potentials at 0 and 31 °C and at 10-50 mM extracellular [K+] to examine if the model could describe membrane potentials during the perturbations occurring during cold exposure. Our results confirm the importance of both Na+/K+ ATPase activity and ion-selective Na+, K+ and Cl- channels, but the model also highlights that additional electroneutral flux of Na+ and K+ is needed to describe how membrane potentials respond to temperature and [K+] in insect muscle. While considerable further work is still needed, we argue that this "charge difference" model can be used to generate testable hypotheses of how insects can preserve membrane polarization in the face of stressful cold exposure.

Cold acclimation increases depolarization resistance and tolerance in muscle fibers from a chill-susceptible insect, Locusta migratoria.

Cold exposure depolarizes cells in insects due to a reduced electrogenic ion transport and a gradual increase in extracellular K+ concentration ([K+]). Cold-induced depolarization is linked to cold injury in chill-susceptible insects, and the locust, Locusta migratoria, has been shown to improve cold tolerance following cold acclimation through depolarization resistance. Here we investigate how cold acclimation influences depolarization resistance and how this resistance relates to improved cold tolerance. To address this question, we investigated if cold acclimation affects the electrogenic transport capacity and/or the relative K+ permeability during cold exposure by measuring membrane potentials of warm- and cold-acclimated locusts in the presence and absence of ouabain (Na+-K+ pump blocker) or 4-aminopyridine (4-AP; voltage-gated K+ channel blocker). In addition, we compared the membrane lipid composition of muscle tissue from warm- and cold-acclimated locust and the abundance of a range transcripts related to ion transport and cell injury accumulation. We found that cold-acclimated locusts are depolarization resistant due to an elevated K+ permeability, facilitated by opening of 4-AP-sensitive K+ channels. In accordance, cold acclimation was associated with an increased abundance of Shaker transcripts (gene encoding 4-AP-sensitive voltage-gated K+ channels). Furthermore, we found that cold acclimation improved muscle cell viability following exposure to cold and hyperkalemia even when muscles were depolarized substantially. Thus cold acclimation confers resistance to depolarization by altering the relative ion permeability, but cold-acclimated locusts are also more tolerant to depolarization.

Cold acclimation modulates voltage gated Ca2+ channel currents and fiber excitability in skeletal muscles of Locusta migratoria.

Cold exposure is known to induce stressful imbalances in chill susceptible insects, including loss of hemolymph water, hyperkalemia and cell depolarization. Cold induced depolarization induces uncontrolled Ca2+ influx and accumulation of injury through necrosis/apoptosis. Conversely cold induced Ca2+ influx has been shown to induce rapid cold hardening and therefore also play a role to reduce cold injury. Cold acclimation is known to reduce cold injury in insects and due to the involvement of depolarization and Ca2+ in the pathophysiology of hypothermia, we hypothesized that cold acclimation modulates voltage gated Ca2+ channels and fiber excitability. Using intracellular electrodes or force transducers, we measured the Ca2+ currents, fiber excitability and muscle contractility in warm (31 °C) and cold (11 °C) acclimated locusts. Experiments were performed under conditions ranging from mild conditions where the membrane potential is well regulated to stressful conditions, where the membrane potential is very depolarized and the tissue is at risk of accumulating injury. These experiments found that cold acclimation modulates Ca2+ currents and fiber excitability in a manner that depends on the cold exposure. Thus, under mild conditions, Ca2+ currents and fiber excitability was increased whilst muscle contractility was unaffected by cold acclimation. Conversely, fiber excitability and muscle contractility was decreased under stressful conditions. Further work is required to fully understand the adaptive effects of these modulations. However, we propose a model which reconciles the dualistic role of the Ca2+ ion in cold exposure and cold acclimation. Thus, increased Ca2+ currents at mild temperatures could help to enhance cold sensing capacity whereas reduced fiber excitability under stressful conditions could help to reduce catastrophic Ca2+ influx during periods of severe cold exposure.

Cold exposure causes cell death by depolarization-mediated Ca2+ overload in a chill-susceptible insect.

Cold tolerance of insects is arguably among the most important traits defining their geographical distribution. Even so, very little is known regarding the causes of cold injury in this species-rich group. In many insects it has been observed that cold injury coincides with a cellular depolarization caused by hypothermia and hyperkalemia that develop during chronic cold exposure. However, prior studies have been unable to determine if cold injury is caused by direct effects of hypothermia, by toxic effects of hyperkalemia, or by the depolarization that is associated with these perturbations. Here we use a fluorescent DNA-staining method to estimate cell viability of muscle and hindgut tissue from Locusta migratoria and show that the cellular injury is independent of the direct effects of hypothermia or toxic effects of hyperkalemia. Instead, we show that chill injury develops due to the associated cellular depolarization. We further hypothesized that the depolarization-induced injury was caused by opening of voltage-sensitive Ca2+ channels, causing a Ca2+ overload that triggers apoptotic/necrotic pathways. In accordance with this hypothesis, we show that hyperkalemic depolarization causes a marked increase in intracellular Ca2+ levels. Furthermore, using pharmacological manipulation of intra- and extracellular Ca2+ concentrations as well as Ca2+ channel conductance, we demonstrate that injury is prevented if transmembrane Ca2+ flux is prevented by removing extracellular Ca2+ or blocking Ca2+ influx. Together these findings demonstrate a causal relationship between cold-induced hyperkalemia, depolarization, and the development of chill injury through Ca2+-mediated necrosis/apoptosis.

Skeletal muscle dysfunction in the db/db mouse model of type 2 diabetes.

INTRODUCTION: In this study we examined the mechanisms of motor dysfunction in type 2 diabetes. METHODS: Contractile force was measured in isolated nerve-muscle preparations of db/db mice using various protocols for electrical stimulation. Sarcoplasmic reticulum Ca(2+) adenosine triphosphatase protein (SERCA) was quantified by comparing Ca(2+) -dependent and non-specific phosphorylation. RESULTS: Compared with controls, the muscle-nerve preparations of db/db mice displayed muscle atrophy, reduced axonal excitability, and force deficit when stimulated via the nerve. Muscle relaxation after contraction was slowed, and SERCA content was reduced. In contrast, the sensitivity of the neuromuscular junction to tubocurarine and muscle fiber excitability were not affected. CONCLUSIONS: The force deficit in db/db muscles was caused by atrophy and failure of neuromuscular signal transmission related to motor nerve axonal dysfunction. The slowed relaxation rate generally observed in diabetic muscles can, to a large extent, be explained by decreased SERCA pump content. Muscle Nerve 54: 460-468, 2016.