Altered fusion dynamics underlie unique morphological changes in mitochondria during hypoxia-reoxygenation stress - PubMed (original) (raw)
Altered fusion dynamics underlie unique morphological changes in mitochondria during hypoxia-reoxygenation stress
X Liu et al. Cell Death Differ. 2011 Oct.
Abstract
Functional states of mitochondria are often reflected in characteristic mitochondrial morphology. One of the most fundamental stress conditions, hypoxia-reoxygenation has been known to cause impaired mitochondrial function accompanied by structural abnormalities, but the underlying mechanisms need further investigation. Here, we monitored bioenergetics and mitochondrial fusion-fission in real time to determine how changes in mitochondrial dynamics contribute to structural abnormalities during hypoxia-reoxygenation. Hypoxia-reoxygenation resulted in the appearance of shorter mitochondria and a decrease in fusion activity. This fusion inhibition was a result of impaired ATP synthesis rather than Opa1 cleavage. A striking feature that appeared during hypoxia in glucose-free and during reoxygenation in glucose-containing medium was the formation of donut-shaped (toroidal) mitochondria. Donut formation was triggered by opening of the permeability transition pore or K(+) channels, which in turn caused mitochondrial swelling and partial detachment from the cytoskeleton. This then favored anomalous fusion events (autofusion and fusion at several sites among 2-3 mitochondria) to produce the characteristic donuts. Donuts effectively tolerate matrix volume increases and give rise to offspring that can regain ΔΨ(m). Thus, the metabolic stress during hypoxia-reoxygenation alters mitochondrial morphology by inducing distinct patterns of mitochondrial dynamics, which includes processes that could aid mitochondrial adaptation and functional recovery.
Figures
Figure 1
Effect of hypoxia–reoxygenation on ΔΨm and mitochondrial morphology. (a) Images of two H9c2 cells transfected with mtYFP (green) and loaded with TMRE (red) acquired before (left) and during hypoxia (H) (60 min: left middle) and during reoxygenation (R) (30 min: right middle; 60 min: right). (b) Cumulative results for TMRE fluorescence in +G medium (upper left, _n_=35) and in −G medium (upper right, _n_=29) for mitochondrial morphology and distribution (middle left and right, +G medium: _n_=35, −G medium: _n_=56) and for the amount of donuts (lower left and right, +G medium: _n_=35, −G medium: _n_=29). *P<0.001
Figure 2
Effect of hypoxia–reoxygenation, FCCP and oligomycin on mitochondrial fusion and Opa1 cleavage. (a) Images showing the spread of photoactivated mtPAGFP in three subsets of mitochondria in naïve, hypoxia (H) in −G medium or FCCP-pretreated H9c2 cells. (b) In the graphs, the total number of mitochondrial fusion events per cell per 7 min is shown in each condition (_n_=4–6 cells, *_P_⩽0.01), whereas the immunoblots show the Opa1 levels. The higher molecular weight band is unaffected by H, reoxygenation (R), oligomycin, but gradually disappears during FCCP treatment
Figure 3
Role of PTP opening in donut formation. (a) Effect of mastoparan (Mas, 5 _μ_M) on ΔΨm, mitochondrial morphology and Opa1 cleavage. (b) Effect of CSA, Me-Val-CSA (mCSA) and FK506 on the FCCP-induced donut formation and Opa1 cleavage. (c) Effect of CSA on the hypoxia–reoxygenation (H–R) induced donut formation (*_P_⩽0.001)
Figure 4
Role of K+ channel activity in donut formation. (a) Images of mtDsRed-transfected H9c2 cells show that a high concentration valinomycin (Val) causes mitochondria swelling (middle) that is insensitive to CSA (right). (b) Effect of various doses of valinomycin on Opa1 cleavage (b) and on ΔΨm (c). Hollow circles show the H9c2 cells treated with valinomycin, whereas the filled symbols show the corresponding time control. (d) Images of mtYFP-expressing (green) and TMRE-loaded H9c2 cells show donut formation by a low concentration valinomycin (0.5 nM) that did not evoke ΔΨm loss, Opa1 cleavage and massive matrix swelling. (e) Lack of protection by CSA against valinomycin-induced donut formation. (f and g) Dependence of donut formation during H–R on K+ channel opening. The concentrations of drugs were as follows: diazoxide (KATP agonist, 100 _μ_M), NS1619 (KCa agonist, 30 _μ_M), 5HD (KATP inhibitor, 500 _μ_M), paxilline (Pax, KCa inhibitor, 10 _μ_M). H9c2 cells were pretreated with drugs for 20 min before hypoxia (H) (*_P_⩽0.004)
Figure 5
Distinct mechanisms of donut formation. (a–d) Time-lapse images show formation of individual donuts in H9c2 cells exposed to hypoxia (H) (45–60 min in −G medium) (a) or to FCCP (0–15 min) (b–d). (e) Matrix continuity is indicated by rapid spreading of photoactivated mtPAGFP throughout two donuts formed during FCCP treatment (30–40 min). In each image sequence, the first image shown is labeled as 0 s
Figure 6
Spatial relationship between microtubules and mitochondria during donut formation. (a and b) Time-lapse images of RBL cells expressing tubulin-GFP (green) and mtDsRed (red) treated with FCCP (5 _μ_M for 0–15 min). The first image shown is labeled as 0 s. (c) 3D reconstruction of a _z_-series
Figure 7
Donut structure and possible roles in mitochondrial adaptation to metabolic stress. (a) Images show mitochondrial morphology (mtYFP, green) and TMRE uptake (red) during FCCP 30 min treatment and washout (1 and 8 min) in H9c2 cells. (b) Time-lapse images of individual donuts during FCCP washout incubated without (upper row) or with rotenone and oligomycin (middle row). In each image sequence, the first image shown is labeled as 0 s. (c) Cumulative data on donut number during FCCP washout in the absence or presence of rotenone, oligomycin (added 25 min before washout) and dominant-negative Drp1 (_n_=6, *P<0.02). (d and e) Volume increase-induced reshaping of tubular- and donut-shaped model mitochondria. Drawing shows the diameter and length changes evoked by 35% volume increase (d). Calculated volume–diameter relationships at two different initial length values (_L_0: 3 and 6 _μ_m, e). (f and g) Images and bar charts show faster TMRE uptake by donuts descendents (_n_=95) than by linear mitochondria (_n_=44) during FCCP washout with rotenone (*P<0.001). (h) Summary of the cause–effect relationship between changes in mitochondrial composition, function and dynamics during hypoxia and reoxygenation (H–R)
References
- Di Lisa F, Canton M, Menabo R, Kaludercic N, Bernardi P. Mitochondria and cardioprotection. Heart Fail Rev. 2007;12:249–260. - PubMed
- Halestrap AP. Mitochondria and reperfusion injury of the heart – a holey death but not beyond salvation. J Bioenerg Biomembr. 2009;41:113–121. - PubMed
- Lemasters JJ. Modulation of mitochondrial membrane permeability in pathogenesis, autophagy and control of metabolism. J Gastroenterol Hepatol. 2007;22 (Suppl 1:S31–S37. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources