Dataset Information

Mitofusin 2-containing mitochondrial-reticular microdomains direct rapid cardiomyocyte bioenergetic responses via interorganelle Ca(2+) crosstalk.

ABSTRACT: RATIONALE:Mitochondrial Ca(2+) uptake is essential for the bioenergetic feedback response through stimulation of Krebs cycle dehydrogenases. Close association of mitochondria to the sarcoplasmic reticulum (SR) may explain efficient mitochondrial Ca(2+) uptake despite low Ca(2+) affinity of the mitochondrial Ca(2+) uniporter. However, the existence of such mitochondrial Ca(2+) microdomains and their functional role are presently unresolved. Mitofusin (Mfn) 1 and 2 mediate mitochondrial outer membrane fusion, whereas Mfn2 but not Mfn1 tethers endoplasmic reticulum to mitochondria in noncardiac cells. OBJECTIVE:To elucidate roles for Mfn1 and 2 in SR-mitochondrial tethering, Ca(2+) signaling, and bioenergetic regulation in cardiac myocytes. METHODS AND RESULTS:Fruit fly heart tubes deficient of the Drosophila Mfn ortholog MARF had increased contraction-associated and caffeine-sensitive Ca(2+) release, suggesting a role for Mfn in SR Ca(2+) handling. Whereas cardiac-specific Mfn1 ablation had no effects on murine heart function or Ca(2+) cycling, Mfn2 deficiency decreased cardiomyocyte SR-mitochondrial contact length by 30% and reduced the content of SR-associated proteins in mitochondria-associated membranes. This was associated with decreased mitochondrial Ca(2+) uptake (despite unchanged mitochondrial membrane potential) but increased steady-state and caffeine-induced SR Ca(2+) release. Accordingly, Ca(2+)-induced stimulation of Krebs cycle dehydrogenases during ?-adrenergic stimulation was hampered in Mfn2-KO but not Mfn1-KO myocytes, evidenced by oxidation of the redox states of NAD(P)H/NAD(P)(+) and FADH(2)/FAD. CONCLUSIONS:Physical tethering of SR and mitochondria via Mfn2 is essential for normal interorganelle Ca(2+) signaling in the myocardium, consistent with a requirement for SR-mitochondrial Ca(2+) signaling through microdomains in the cardiomyocyte bioenergetic feedback response to physiological stress.

SUBMITTER: Chen Y

PROVIDER: S-EPMC3444672 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Mitofusin 2-containing mitochondrial-reticular microdomains direct rapid cardiomyocyte bioenergetic responses via interorganelle Ca(2+) crosstalk.

Chen Yun Y Csordás György G Jowdy Casey C Schneider Timothy G TG Csordás Norbert N Wang Wei W Liu Yingqiu Y Kohlhaas Michael M Meiser Maxie M Bergem Stefanie S Nerbonne Jeanne M JM Dorn Gerald W GW Maack Christoph C

Circulation research 20120709 7

<h4>Rationale</h4>Mitochondrial Ca(2+) uptake is essential for the bioenergetic feedback response through stimulation of Krebs cycle dehydrogenases. Close association of mitochondria to the sarcoplasmic reticulum (SR) may explain efficient mitochondrial Ca(2+) uptake despite low Ca(2+) affinity of the mitochondrial Ca(2+) uniporter. However, the existence of such mitochondrial Ca(2+) microdomains and their functional role are presently unresolved. Mitofusin (Mfn) 1 and 2 mediate mitochondrial ou ...[more]

PMID: 22777004

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Project description:A finite element analysis modelled diffusional generation of steady-state Ca2+ microdomains within skeletal muscle transverse (T)-tubular-sarcoplasmic reticular (SR) junctions, sites of ryanodine receptor (RyR)-mediated SR Ca2+ release. It used established quantifications of sarcomere and T-SR anatomy (radial diameter [Formula: see text]; axial distance [Formula: see text]). Its boundary SR Ca2+ influx densities,[Formula: see text], reflected step impositions of influxes, [Formula: see text] deduced from previously measured Ca2+ signals following muscle fibre depolarization. Predicted steady-state T-SR junctional edge [Ca2+], [Ca2+]edge, matched reported corresponding experimental cytosolic [Ca2+] elevations given diffusional boundary efflux [Formula: see text] established cytosolic Ca2+ diffusion coefficients [Formula: see text] and exit length [Formula: see text]. Dependences of predicted [Ca2+]edge upon [Formula: see text] then matched those of experimental [Ca2+] upon Ca2+ release through their entire test voltage range. The resulting model consistently predicted elevated steady-state T-SR junctional ~ µM-[Ca2+] elevations radially declining from maxima at the T-SR junction centre along the entire axial T-SR distance. These [Ca2+] heterogeneities persisted through 104- and fivefold, variations in D and w around, and fivefold reductions in d below, control values, and through reported resting muscle cytosolic [Ca2+] values, whilst preserving the flux conservation ([Formula: see text] condition, [Formula: see text]. Skeletal muscle thus potentially forms physiologically significant ~ µM-[Ca2+] T-SR microdomains that could regulate cytosolic and membrane signalling molecules including calmodulin and RyR, These findings directly fulfil recent experimental predictions invoking such Ca2+ microdomains in observed regulatory effects upon Na+ channel function, in a mechanism potentially occurring in similar restricted intracellular spaces in other cell types.

Project description:Endothelial cells (EC) serve a paracrine function to enhance signaling in cardiomyocytes (CM), and conversely, CM secrete factors that impact EC function. Understanding how EC interact with CM may be critically important in the context of ischemia-reperfusion injury, where EC might promote CM survival. We used isoflurane as a pharmacological stimulus to enhance EC protection of CM against hypoxia and reoxygenation injury. Triggering of intracellular signal transduction pathways culminating in the enhanced production of nitric oxide (NO) appears to be a central component of pharmacologically induced cardioprotection. Although the endothelium is well recognized as a regulator for vascular tone, little attention has been given to its potential importance in mediating cardioprotection. In the current investigation, EC-CM in co-culture were used to test the hypothesis that EC contribute to isoflurane-enhanced protection of CM against hypoxia and reoxygenation injury and that this protection depends on hypoxia-inducible factor (HIF1?) and NO. CM were protected against cell injury [lactate dehydrogenase (LDH) release] to a greater extent in the presence vs. absence of isoflurane-stimulated EC (1.7 ± 0.2 vs. 4.58 ± 0.8 fold change LDH release), and this protection was NO-dependent. Isoflurane enhanced release of NO in EC (1103 ± 58 vs. 702 ± 92 pmol/mg protein) and EC-CM in co-culture sustained NO release during reoxygenation. In contrast, lentiviral mediated HIF1? knockdown in EC decreased basal and isoflurane stimulated NO release in an eNOS dependent manner (517 ± 32 vs. 493 ± 38 pmol/mg protein) and prevented the sustained increase in NO during reoxygenation when co-cultured. Opening of mitochondrial permeability transition pore (mPTP), an index of mitochondrial integrity, was delayed in the presence vs. absence of EC (141 ± 2 vs. 128 ± 2.5 arbitrary mPTP opening time). Isoflurane stimulated an increase in HIF1? in EC but not in CM under normal oxygen tension (3.5 ± 0.1 vs. 0.79 ± 0.15 fold change density) and this action was blocked by pretreatment with the Mitogen-activated Protein/Extracellular Signal-regulated Kinase inhibitor U0126. Expression and nuclear translocation of HIF1? were confirmed by Western blot and immunofluorescence. Taken together, these data support the concept that EC are stimulated by isoflurane to produce important cardioprotective factors that may contribute to protection of myocardium during ischemia and reperfusion injury.

Project description:Intracellular calcium (Ca(2+)) plays a significant role in many cell signaling pathways, some of which are localized to spatially restricted microdomains. Ca(2+) binding proteins (Ca(2+) buffers) play an important role in regulating Ca(2+) concentration ([Ca(2+)]). Buffers typically slow [Ca(2+)] temporal dynamics and increase the effective volume of Ca(2+) domains. Because fluctuations in [Ca(2+)] decrease in proportion to the square-root of a domain's physical volume, one might conjecture that buffers decrease [Ca(2+)] fluctuations and, consequently, mitigate the significance of small domain volume concerning Ca(2+) signaling. We test this hypothesis through mathematical and computational analysis of idealized buffer-containing domains and their stochastic dynamics during free Ca(2+) influx with passive exchange of both Ca(2+) and buffer with bulk concentrations. We derive Langevin equations for the fluctuating dynamics of Ca(2+) and buffer and use these stochastic differential equations to determine the magnitude of [Ca(2+)] fluctuations for different buffer parameters (e.g., dissociation constant and concentration). In marked contrast to expectations based on a naive application of the principle of effective volume as employed in deterministic models of Ca(2+) signaling, we find that mobile and rapid buffers typically increase the magnitude of domain [Ca(2+)] fluctuations during periods of Ca(2+) influx, whereas stationary (immobile) Ca(2+) buffers do not. Also contrary to expectations, we find that in the absence of Ca(2+) influx, buffers influence the temporal characteristics, but not the magnitude, of [Ca(2+)] fluctuations. We derive an analytical formula describing the influence of rapid Ca(2+) buffers on [Ca(2+)] fluctuations and, importantly, identify the stochastic analog of (deterministic) effective domain volume. Our results demonstrate that Ca(2+) buffers alter the dynamics of [Ca(2+)] fluctuations in a nonintuitive manner. The finding that Ca(2+) buffers do not suppress intrinsic domain [Ca(2+)] fluctuations raises the intriguing question of whether or not [Ca(2+)] fluctuations are a physiologically significant aspect of local Ca(2+) signaling.

Project description:BackgroundMitochondrial dynamics play a critical role in mitochondrial function. The mitofusin 2 (MFN2) gene encodes a mitochondrial membrane protein that participates in mitochondrial fusion to maintain and operate the mitochondrial network. Moreover, MFN2 is essential for mitophagy. In Ang II-induced cardiac remodeling, the combined effects of MFN2-mediated mitochondrial fusion and mitophagy are unclear. This study was designed to explore a novel strategy for preventing cardiomyocyte injury via modulation of mitochondrial dynamics.MethodsWe studied the function of MFN2 in mitochondrial fusion and mitophagy in Ang II-stimulated cardiomyocyte injury. Cardiomyocyte injury experiments, including reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), and apoptosis rate of cardiomyocytes were performed. The mitochondrial morphology in cardiomyocytes was examined via transmission electron microscopy (TEM) and confocal microscopy. Autophagic levels in response to Ang II were examined by immunoblotting of autophagy-related proteins. Moreover, PINK1/MFN2/Parkin pathway-related proteins were examined.ResultsWith stimulation by Ang II, MFN2 expression was progressively reduced. MFN2 deficiency impaired mitochondrial quality, resulting in exacerbated mitochondrial damage induced by Ang II. The Ang II-induced increases in ROS production and apoptosis rate were alleviated by MFN2 overexpression. Moreover, MFN2 alleviated the Ang II-induced reduction in MMP. MFN2 promoted mitochondrial fusion, and MFN2 promoted Parkin translocation and phosphorylation, leading to mitochondrial autophagy. The effects of MFN2 overexpression were reversed by autophagy inhibitors.ConclusionMitofusin 2 promotes Parkin translocation and phosphorylation, leading to mitophagy to clear damaged mitochondria. However, the beneficial effects of MFN2 were reversed by autophagy inhibitors. Additionally, MFN2 participates in mitochondrial fusion to maintain mitochondrial quality. Thus, MFN2 participated in mitophagy and mitochondrial fusion against Ang II-induced cardiomyocyte injury.

Dataset Information

Mitofusin 2-containing mitochondrial-reticular microdomains direct rapid cardiomyocyte bioenergetic responses via interorganelle Ca(2+) crosstalk.

Publications

Mitofusin 2-containing mitochondrial-reticular microdomains direct rapid cardiomyocyte bioenergetic responses via interorganelle Ca(2+) crosstalk.

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