Contents

๐Ÿ“’ Li 2014

Inhibiting Na+/K+ ATPase can impair mitochondrial energetics and induce abnormal Ca2+ cycling and automaticity in guinea pig cardiomyocytes1

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Introduction

  • Na+/K+-ATPase (NKA) inhibiting glycosides in the clinic was partially due to their well-known side effects such as cardiac arrhythmias, gastrointestinal symptoms, and central nervous system abnormalities

  • SR Ca2+ overload is not the only root cause of glycoside-induced cardiac arrhythmias

    • glycosides (e.g. ouabain) may impair mitochondrial energy metabolism and raise oxidative stress, especially at increased workload
    • Na+ accumulation caused mitochondrial Ca2+ deficiency, NADH imbalance, and increased reactive oxygen species (ROS) accumulation
    • ouabain-induced mitochondrial dysfunction can disturb Ca2+ cycling and elicit erratic action potentials
  • mitochondrial Ca2+ is regulated not only by mNCE but also by mitochondrial Ca2+ uniporters (MCU)

    • low affinity of MCU to Ca2+ (Km ~ 10-20 ฮผM, 10x the Ca2+ amplitude in CICR)
    • mitochondria are functionally and physically tethered to SR by a mitochondrial fusion protein => high Ca2+ microdomain, facilitating dynamic interorganellar coupling
  • impairment of mitochondrial function, such as dissipation of mitochondrial membrane potential or inhibition of ATP synthesis, promotes proarrhythmic Ca2+ alternans

  • both Ca2+ and AP alternans could be elicited when severe ATP depletion occurred as the result of high frequency pacing and concurrent NKA inhibition

Methods

Model Development

https://journals.plos.org/plosone/article/figure/image?size=large&id=info:doi/10.1371/journal.pone.0093928.g001

  • ECME-RIRR model (Zhou’s model)
  • refined mitochondrial model consisting of Na+, H+ and Pi + channels was incorporated (Wei’s model)
  • a detailed antioxidant subsystem (Kembro’s model)
  • For simplicity, the pathway of isocitrate dehydrogenase 2 (IDH2) and diffusion of reduced glutathione (GSH) in the Kembro model were not considered in the current work

Modeling Mitochondrial Ca2+ Uptake through MCU

  • hypothesized that MCU distribute non-uniformly along the mitochondrial membrane, with expression abundant in areas facing the SR microdomain and scarce on membrane away from the SR
  • kinetics formulas and parameters of Ca2+ uptake remained the same as those in the ECME-RIRR model. Only changed the Vmax and distribution of MCU, and normalize them to the original ECME-RIRR model

Model Simulation Protocol

  • The formulas of other processes, such as membrane potential, ion channels and metabolic reactions, and model parameters were the same as those in the ECME-RIRR model
  • Paced @ 0.25 Hz until the steady state was reached
  • The effect of cardiac glycosides was mimicked by inhibiting NKA activity by 50%, which caused a 2.4-fold increase in cytosolic Na+
  • examined the effect of NKA inhibition-induced mitochondrial dysfunction on Ca2+ transients and AP under low (i.e. the fraction of electron transport chain O2 โˆ’ production shunt โ€Š=โ€Š1%) and relatively high (i.e. shuntโ€Š=โ€Š2.5%) O2 โˆ’ production conditions
  • The simulation results were post-processed and plotted using Origin 8.6

Results

Effects of MCU Ca2+ Pools and NKA Inhibition on Intracellular Ca2+ Dynamics during the Transition of Increasing Workload

  • impacts of different subcellular mitochondrial Ca2+ uptake pools (i.e. cytosol vs. mitochondria-SR microdomain or p1:p2 ratio) on Ca2+ dynamic https://journals.plos.org/plosone/article/figure/image?size=large&id=10.1371/journal.pone.0093928.g002

  • NKA inhibition (by 50%) monotonically enhanced the accumulation of [Ca2+]i and amplified the influence of MCU Ca2+ uptake pool

  • The effect of blocking NKA on pacing-induced [Ca2+]m elevation was complex and dependent upon the p1:p2 ratio

  • NKA inhibition started to suppress the pacing-induced [Ca2+]m elevation with the pool shifting to the microdomain

  • when p1:p2โ€Š=โ€Š1โˆถ3, NKA inhibition caused about 15% reduction in [Ca2+]m accumulation, which was comparable to the experimental data. p1:p2 ratio of 1โˆถ3 was used in all subsequent simulations.

  • we proposed that mitochondria take up Ca2+ ions mainly, if not exclusively, from mitochondrial-SR microdomain.

Effect of NKA Inhibition on Intracellular Ion Homeostasis and Mitochondrial Energetics

https://journals.plos.org/plosone/article/figure/image?size=large&id=10.1371/journal.pone.0093928.g003

  • Increasing pacing frequency from 0.25 Hz to 2 Hz caused a 1.6-fold elevation in [Na+]i and a 1.5-fold increase in mitochondrial Na+ concentration ([Na+]m)
  • The NADH concentration decreased transiently and then returned to the basal level
  • Increasing pacing rate alone caused a slight increase in ROS (Fig. 3F) and had minor effects on mitochondrial membrane potential (ฮ”ฮจm) (Fig. 3G), [ATP]i and [ATP]m
  • Inhibiting NKA led to further increases of [Na+]i and [Na+]m, and enhanced [Ca2+]i accumulation and [Ca2+]m attenuation; the NADH level decreased by 9% and did not return to the basal level
    • along with a large (โˆผ30- fold) increase of ROS (Fig. 3F) and small loss of ฮ”ฮจm

As heart failure often associates with increased ROS production, we simulated the effect of NKA inhibition on mitochondrial energetics under a relatively higher ROS production condition (e.g. shuntโ€Š=โ€Š2.5%) https://journals.plos.org/plosone/article/figure/image?size=large&id=10.1371/journal.pone.0093928.g004

  • simultaneously blocking NKA (by 50%) and increasing energy demand (from 0.25 Hz to 2 Hz) caused detriment on mitochondrial energetics under the higher ROS production condition
  • NKA inhibition triggered sustained mitochondrial oscillations, including NADH (Fig. 4E), ROS production (Fig. 4F) and ฮ”ฮจm.

The Effect of Modulating mNCE or MCU on NKA Inhibition-induced Mitochondrial Dysfunction

  • mitochondrial energetics largely relies on mitochondrial Ca2+ retention and NADH homeostasis

Inhibitin mNCE

  • concurrent inhibition of mNCE (e.g. by 60%) significantly ameliorated NKA inhibition-induced mitochondrial dysfunction, without abolishing the inotropic effect of NKA inhibition , consistent with previous experimental findings
  • too much mNCE blocking (e.g. by 90%) completely suppressed NKA inhibition-induced [Ca2+]i elevation

Increasing MCU activity

https://journals.plos.org/plosone/article/figure/image?size=large&id=10.1371/journal.pone.0093928.g005

  • effects on NKA inhibition-induced [Na+]i, [Na+]m, and [Ca2+]i alterations were not significant, ROS accumulation and NADH depletion not prevented.

Effect of NKA Inhibition on Ca2+ Cycling and AP under more Stressed Conditions

https://journals.plos.org/plosone/article/figure/image?size=large&id=10.1371/journal.pone.0093928.g006

  • pacing rate from 0.25 Hz to 4 Hz resulted in mitochondrial depolarization and oscillations and cyclic ATP depletions when the shunt was set to 2.5%.

  • Concurrent induction of NKA inhibition by 50% prompted mitochondrial depolarization (Fig. 6A red curve) and accelerated ATP consumption

  • depletion of ATP significantly impaired SERCA Ca2+ uptake (Fig. 6C) and caused imbalance of SR Ca2+ cycling, increased amplitude of the larger Ca2+ transients within the large-small Ca2+ alternations

  • Concurrent inhibition of mNCE (by 90%) retarded mitochondrial oscillations and mitigated cytosolic ATP depletion, augmented SR Ca2+ uptake, abolished Ca2+ and AP alternans

https://journals.plos.org/plosone/article/figure/image?size=large&id=10.1371/journal.pone.0093928.g007

Discussion

  • main findings
    1. NKA inhibition simultaneously raises [Ca2+]i and blunts [Ca2+]m retention, causing ATP depletion and ROS accumulation that disturb SR Ca2+ handling and elicit abnormal APs
    2. NKA inhibition-induce adverse effects on mitochondrial energetics and Ca2+ cycling can be ameliorated by blocking mNCE, but not by enhancing MCU
    3. under more severe conditions, NKA inhibition can cause proarrhythmic Ca2+ and AP alternans
    4. mitochondrial uniporter takes up Ca2+ mainly from the mitochondria-SR microdomain instead of the cytoplasm.

https://journals.plos.org/plosone/article/figure/image?size=large&id=10.1371/journal.pone.0093928.g008

  1. NKA inhibition blunting [Ca2+]m accumulation, which reduces NADH production and therefore ROS removal
  2. NKA inhibition increasing [Ca2+]i and ATP hydrolysis
  • ouabain on mitochondrial ATP synthesis? contradictory results might be due to differences in experimental conditions, animal species or tissue variations

  • In this model, NKA inhibition differentially influence ATP production, depending on the severity of stress:

    1. concurrent NKA inhibition and moderate increase of workload stimulates oxidative phosphorylation and ATP production
    2. under more severe conditions such as higher frequency pacing, NKA inhibition caused dramatic ROS accumulation, mitochondrial depolarization, and tremendous ATP depletion.

Glycosides-induced Ca2+ Alternans: A New Mitochondria-originated Arrhythmic Substrate

  • NKA inhibition caused a significant ROS accumulation during the transition of moderate workload increment.
  • ROS may lead to abnormal Ca2+ handling and erratic APs
  • under more stressed conditions such as higher frequency pacing, NKA inhibition causes Ca2+ and AP alternans by reducing ATP, genesis of Ca2+ alternans was attributed to NKA inhibition-induced mitochondrial dysfunction
  • blocking mNCE ameliorated NKA inhibition-induced mitochondrial dysfunction, mitigating the decline of SR Ca2+ and suppressing the genesis of alternans

Can Modulating Mitochondrial Ca2+ Handling Alleviate Glycoside-induced Arrhythmogenesis?

  • CGP-17157, a mNCE inhibitor, mitigated ouabain-induced adverse effects on mitochondrial function and lowered the occurrence of cardiac arrhythmias in guinea pigs
  • mNCE inhibition retained [Ca2+]m accumulation and attenuated cytosolic Ca2+ accumulation
  • the majority of Ca2+ taken up by MCU is from the microdomain instead of cytoplasm. MCU are concentrated at mitochondria-SR contacting sites.

Study Limitations

  • subcellular Ca2+ compartmentation for MCU better represent Ca dynamics
  • The current model lacks an isoproterenol signaling pathway, not the same as Liu et al.
  • Ca2+ wave propagation plays a critical role in intracellular Ca2+ regulation and genesis of Ca2+ alternans (limited spatial distributions in the model)
  • The current model did not incorporate the direct link between mitochondrial ROS and redox sensitive SR Ca2+ proteins (in 2015, yes!)
  • the equations of complexes of the electron transport chain are lumped. We want Laura’s detailed ETC model.
  • The present model did not consider the effect of energy depletion on ATP-sensitive potassium channels (KATP). KATP would exacerbate the deleterious effect of NKA inhibition-induced mitochondrial dysfunction on cardiac action potentials

References


  1. Li Q, Pogwizd SM, Prabhu SD, Zhou L. Inhibiting Na+/K+ ATPase can impair mitochondrial energetics and induce abnormal Ca2+ cycling and automaticity in guinea pig cardiomyocytes. PLoS ONE 2014;9(4):e93928. doi:10.1371/journal.pone.0093928. PMC3983106 ↩︎