Contents

📒 Li 2015

Mitochondria-derived ROS bursts disturb Ca cycling and induce abnormal automaticity in guinea pig cardiomyocytes: a theoretical study1

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Introduction

  • tetrameric RyRs have 89 cysteine residues, of which ∼21 are susceptible to oxidation by free radicals, reversible activation of channel activity
  • H2O2 and superoxide (a.k.a. O2·−) inhibit SERCA activity by directly oxidizing its thiol groups
  • difficulty to induce controllable endogenous ROS production in the cell in the experimental setting
  • Mitochondria and SR are in close proximity and physically tethered via mitofusin proteins
    • rapid mitochondrial Ca2+ uptake that stimulates oxidative phosphorylation
    • interorganellar redox signaling by mtROS
    • ROS-induced ROS release (RIRR) are closely correlated to enhanced Ca2+ sparks in resting guinea pig cardiomyocytes
  • A model that simulates mdROS bursts rapidly induce cytosolic Ca2+ ([Ca2+]i) overload by stimulating RyRs and inhibiting SERCA.

Methods

Model Development

https://user-images.githubusercontent.com/40054455/86703455-7ae08680-c046-11ea-9295-98699fdab930.png

  • ECME-RIRR
  • local control Ca2+-induced Ca2+-release
  • mitochondria-SR microdomain (MSM)
  • mtROS modulations of RyRs and SERCA
  • assumed that all mitochondria in the cardiomyocyte depolarize/oscillate synchronously
  • the mitochondria were lumped as a giant mitochondrion and mitochondria-SR subspaces were modeled as a single compartment https://user-images.githubusercontent.com/40054455/86703465-7caa4a00-c046-11ea-9ac3-846cf6dbdbe1.png

mdO2·− diffusion

  • Fick’s second law

  • MSM was discretized as two subcompartments, with one (MSM_SR) adjacent to the peri-SR space and another (MSM_mito) adjacent to the perimitochondrial space https://user-images.githubusercontent.com/40054455/86703446-7916c300-c046-11ea-8ad6-db4051da5164.png

  • The values of newly added model parameters (e.g., DO2·− and X) were taken from the literature https://www.physiology.org/na101/home/literatum/publisher/physio/journals/content/ajpheart/2015/ajpheart.2015.308.issue-6/ajpheart.00493.2014/production/images/large/zh40051514130001.jpeg

Local Ca2+ control.

  • coupled 40-state LCC-RyR model developed by Gauthier et al. into the ECME-RIRR model
  • SR is separated into two compartments (i.e., NSR and JSR), several model equations and parameters were modified
  • the number of Ca2+ release unit (NCaRU) was set to 300,000
  • ∼37% decrease of SR Ca2+ content during a normal AP cycle, which is comparable to that reported in the previous studies (∼35%)

ROS and RyR Ca2+ release

  • ROS exponentially increase RyR open probability (PO_ryr), with the enhancement effect determined by both the ROS concentration and the state of the channel https://user-images.githubusercontent.com/40054455/86703454-7a47f000-c046-11ea-8f58-1c45efc038c9.png
  • Parameters obtained by the least-square curve fitting using experimental data from the literature

ROS and SERCA Ca2+ uptake

  • SERCA Ca2+ uptake (Jup) is exponentially inhibited by ROS

$$ J_{up}^{ROS} = J_{up} \cdot \left[ c_{SERCA} \cdot \exp \left(-k_{SERCA} \cdot [O_2^-]_{SR} \right) \right] $$

  • Parameters obtained by the least-square curve fitting using experimental data from the literature

Other aspects

  • APD90 was defined as the interval between the time of the maximum upstroke velocity of the AP, [dV/dt]max, and 90% repolarization.
  • The whole cell model was coded in C++ (Visual Studio).
  • The nonlinear ordinary differential equations were integrated numerically with CVODE
  • Mitochondrial depolarization by superoxide efflux was obtained by increasing shunt from 2% to 10%.

Results

Model Validation

  • Increasing shunt from 2 to 10% triggered sustained mitochondrial oscillations including membrane potential (ΔΨm) and mdO2·− production, as well as NADH oxidation and GSH depletion.
  • addition of new model components (e.g., mdO2·− modulation of RyRs and local Ca2+ control) did not influence the dynamics of the existing model subsystems

Simulating mdROS-mediated abnormal APs

https://www.physiology.org/na101/home/literatum/publisher/physio/journals/content/ajpheart/2015/ajpheart.2015.308.issue-6/ajpheart.00493.2014/production/images/large/zh40051514130002.jpeg

  • mdO2·− burst induced before AP firing led to an early afterdepolarization (EAD)
  • ∼2.5-fold APD90 prolongation
  • significant increase in Ca2+ transient (1.63-fold)
  • reduction in SR Ca2+ storage (∼69%
  • mdO2·− burst induced during phase 4 of the AP cycle elicited a delayed afterdepolarization (DADs) https://www.physiology.org/na101/home/literatum/publisher/physio/journals/content/ajpheart/2015/ajpheart.2015.308.issue-6/ajpheart.00493.2014/production/images/large/zh40051514130003.jpeg

Effect of mdO2·− on Ca2+ Handling Channels

  • mdO2·− burst was induced before AP firing => EAD
    • activated Jrel
    • inhibited Jup
    • [Ca2+]i elevation & [Ca2+]SR reduction
    • enhanced the Na+/Ca2+ exchanger current & Ca2+-sensitive nonspecific cationic current
    • reversal of AP repolarization reactivated the ICaL, triggered a second CICR
    • further [Ca2+]i elevation and [Ca2+]SR depletion
    • further INCX and InsCa activation => EAD
  • DAD
    • activated Jrel evoked an extra Ca2+ transient
    • SR Ca2+ depletion
    • both INaCa and InsCa were enhanced => DAD
    • ICaL was not reactivated

Effect of Timing of mdROS Burst Induction on AP Morphology

https://www.physiology.org/na101/home/literatum/publisher/physio/journals/content/ajpheart/2015/ajpheart.2015.308.issue-6/ajpheart.00493.2014/production/images/large/zh40051514130004.jpeg

Effect of mdO2·−-Mediated RyR Activation or SERCA Inhibition Alone on Ca2+ Cycling and AP

https://www.physiology.org/na101/home/literatum/publisher/physio/journals/content/ajpheart/2015/ajpheart.2015.308.issue-6/ajpheart.00493.2014/production/images/large/zh40051514130005.jpeg

  • Jrel solely: substantial [Ca2+]SR reduction + [Ca2+]i elevation + single EAD
    • could not elicit multiple EADs
  • Jup solely: only cause APD prolongation

Effect of Shunt or Mitochondria-SR Distance on AP Morphology

https://www.physiology.org/na101/home/literatum/publisher/physio/journals/content/ajpheart/2015/ajpheart.2015.308.issue-6/ajpheart.00493.2014/production/images/large/zh40051514130006.jpeg

  • increasing shunt enhanced mdO2·− burst-induced Ca2+ elevation, earlier occurrence of EAD, shifting APD prolongation to EAD and single EAD to multiple EADs
  • Reducing X had the similar effect
  • Ca2+ transient became “nonphysiologically” large when O2·− production was very high (shunt=20%) or X is small (X = 25 nm)
  • mdO2·− caused dramatic SR Ca2+ depletions

Effect of Blocking Ca2+ Handling Channel on mdROS-Induced EADs

https://www.physiology.org/na101/home/literatum/publisher/physio/journals/content/ajpheart/2015/ajpheart.2015.308.issue-6/ajpheart.00493.2014/production/images/large/zh40051514130007.jpeg

  • Blocking InsCa did not significantly affect AP (Fig. 7Bi) and Ca2+ transient
  • blockade of INaCa inhibited Iti (Under normal mdO2·− production conditions, blocking INaCa had minimal effects on both AP and [Ca2+]i)
  • mdO2·−-induced EAD was suppressed but APD remained prolonged when ICaL was blocked. mdO2·−-induced RyR activation and SERCA inhibition, which resulted in a relatively small but prolonged [Ca2+]i elevation

DISCUSSION

  • Findings

    1. mdO2·−-induced AP abnormality involves alterations of multiple Ca2+ handling channels in a coordinated fashion, including RyRs, SERCA, InsCa, INaCa, and ICaL
    2. variance of mdO2·− burst-induced AP abnormality largely depends on the time interval between its induction and AP firing
    3. the mdO2·−-induced AP abnormality is also influenced by mdO2·− dosage and/or mitochondria-SR distance.
  • ROS-induced Ca2+ release (RICR): mdROS activates RyRs => erratic APs

  • mdO2·−-mediated SERCA inhibition had smaller effects. Sustained SERCA inhibition, such as that induced by continuous H2O2 perfusion, would have a much more prominent effect on Ca2+ handling.

  • mdO2·− burst can elicit various patterns of AP abnormality such as APD prolongation, single EAD, and multiple EADs and DAD. They may occur asynchronously in various cells, resulting in regional electrical heterogeneity that increases the propensity for arrhythmogenesis.

  • IcaL reactivation

    1. Amplitude of mdO2·− burst-induced SR Ca2+ release enhancing INaCa and InsCa, Iti can reverse AP repolarization
    2. The membrane potential needs to be in the range where ICaL can be activated
    3. When the situaion is right, it can be reactivated repeatedly, producing multiple EADs
  • Transient inward currents (Iti)

    • Trigger of EADs or DADs, eliciting Ca2+ overload-mediated arrhythmias
    • In human atrial myocytes: InsCa
    • In ventricular myocytes: ??
    • Simulation: blocking INaCa completely suppressed the mdROS burst-induced EAD

Model Limitations and Future Directions

  • Mitochondrial dysfunction inhibits cell contraction not in the model

    • enhanced ROS production could directly inhibit cardiomyocyte contraction
    • ATP depletion directly inhibits cell contraction (via KATP, myofibril). But 3% in this model.
  • The diffusion of mdROS in MSM cannot be validated by experimental study

  • Does not consider the effects of mitochondrial-derived H2O2 or other oxidizing species

  • H2O2 is more stable and can diffuse further, thus affecting ion channels not only on the proximal SR membrane but also on the sarcolemmal membrane

  • ROS can affect INa either directly (38) or indirectly by activating CaMKII

  • CaMKII activation can also enhance SERCA activity via phospholamban phosphorylation

  • Other redox-sensitive ion channels include K+ channels (Kir and Kv) and the Na+/Ca2+ exchanger (NCX)

  • The above would not impact our main conclusions

  • assumed that all mitochondria in the cardiomyocyte depolarize and release ROS simultaneously. In a real cardiomyocyte, the rate of ROS increase might be slower due to the heterogeneity of mitochondrial energetic state and network ultrastructure.

  • the present model did not incorporate mPTP


  1. Li Q, Su D, O’Rourke B, Pogwizd SM, Zhou L. Mitochondria-derived ROS bursts disturb Ca 2 + cycling and induce abnormal automaticity in guinea pig cardiomyocytes: a theoretical study. Am. J. Physiol. Heart Circ. Physiol. 2015;308(6):H623-36. doi:10.1152/ajpheart.00493.2014. PMC4360058 ↩︎