Edited by: Roberta A. Gottlieb, Cedars-Sinai Medical Center, USA
Reviewed by: Amadou K. S. Camara, Medical College of Wisconsin, USA; Aleksandr Stotland, Cedars-Sinai Medical Center, USA
*Correspondence: Bernard Geny
This article was submitted to Mitochondrial Research, a section of the journal Frontiers in Physiology
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The mammalian heart presents both structural and functional heterogeneities across the left ventricular (LV) wall. Blood flow, structure, metabolism, electrophysiological, and contractile properties differ between the LV sub-endocardium (Endo) and sub-epicardium (Epi), generally supporting increased mechanical stress, contractility, and susceptibility to ischemia in Endo (Van der Vusse et al.,
Mitochondria play a critical role in ischemia/reperfusion injury and are involved both as target and source of oxidative damage (Gustafsson and Gottlieb,
However, to date, the mechanisms involved in the transmural energetic gradient of the normal LV still remain to be determined. Key factors that deserve to be studied are reactive oxygen species (ROS) and nitric oxide (NO) productions. Indeed, high oxidative stress has been related to mitochondrial dysfunction both in skeletal and in cardiac muscles (Thaveau et al.,
Interestingly, NO also modulates mitochondrial respiratory chain (MRC) activity and mitochondrial permeability transition pore (mPTP) function which opening leads to the apoptotic cascade (Brown and Borutaite,
The objectives of this study were to determine mitochondrial respiratory chain complexes activities in the normal LV and to investigate whether NO and ROS productions are specifically increased in Endo, supporting their participation in LV transmural respiratory gradient.
This study conformed to the
Anesthesia was first induced by 4% isoflurane gas and then maintained by reducing the isoflurane concentration to 2%. Heparin was administered intraperitoneally (10,000 UI/L) to prevent any thrombi formation in the heart and coronary arteries.
A sternotomy was performed. The still-beating heart was rapidly harvested and rinsed in ice-cold 0.9% NaCl solution, and the heart and the LV were weighed. The LV-free wall was dissected, and Endo and Epi myocardial samples were extracted under binocular microscopy (Endo and Epi were the innermost and outermost layers of the LV-free wall respectively). Tissue samples were used immediately for the measurement of mitochondrial functions and H2O2 production.
The MRC activity was studied in saponin-skinned fibers to ensure that the global function was assessed in intact mitochondria, in their normal intracellular milieu, preserving essential interactions with other organelles (Kindo et al.,
The relative contribution of respiratory chain complexes I, III, and IV to the global mitochondrial respiratory rate was also determined using different mitochondrial substrates and inhibitors (Thaveau et al.,
Mitochondrial coupling is inferred from the acceptor control ratio (Vmax/V0) which indicates the degree of coupling between oxidation and phosphorylation. Vmax and V0 represent the maximal and the basal mitochondrial respiratory rates, respectively.
Small LV tissue samples (3.0−5.0 mg dry fiber weight) were minced and placed into separate wells of a 24-well plate with 2 mL KHB (99 mM NaCl, 4.69 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 25 mM NaHCO3, 1.03 mM KH2PO4, 5.6 mM D-(+)-glucose, and 20 mM Na-HEPES, pH 7.4) that contained 200 μM FeSO4 and 415 μM DETC, which were previously degassed with N2. The tissue pieces were then incubated at 37°C with 20 mmHg O2 for 1 h and then placed on ice.
LV tissue preparation was performed as previously described (Fink et al.,
To investigate the effect of NO on MRC activity, the maximal fiber respiration (Vmax) rate was also measured in the presence of increasing concentrations of the NO donor MAHMA NONOate (10, 40, 60, and 80 μM) with a saturating amount of ADP as a phosphate acceptor. MAHMA NONOate is spontaneously dissociated with a half-life of 3 min at 22−25°C and liberates 2 moles of NO per mole of parent compound.
The fibers were then harvested and dried for 15 min at 150°C. The respiration rates are expressed as μmol O2/min/g dry weight.
The H2O2 production in response to the sequential addition of substrates and inhibitors was assessed in permeabilized Endo and Epi fibers (15). H2O2 production was measured with Amplex Red reagent (Invitrogen); this reagent reacts in 1:1 stoichiometry with H2O2 in a reaction catalyzed by HRP (horseradish peroxidase; Fluka Biochemika) to yield the fluorescent compound resorufin and a molar equivalent O2. Resorufin has excitation/emission wavelengths of 563/587 nm and is highly stable once formed. Fluorescence was measured continuously [change in fluorescence (F)/s] with a Fluoromax 4 (Jobin Yvon) spectrofluorometer equipped with temperature control and magnetic stirring.
After the baseline in the presence of F (reactants only) was established, the reaction was initiated by addition of a permeabilized fiber bundle to 600 μl of buffer Z with glutamate (5 mM) and malate (2.5 mM) as substrates for complex I and succinate (5 mM) as a substrate for complex II. The addition of ADP (2 mM) to the reaction buffer led to a reduction in H2O2 release, as expected when the electron flow through the respiratory chain is stimulated. Finally, addition of the complex I inhibitor amytal (2 mM) and the complex III inhibitor antimycin (8 μM) led to interruption of the normal electron flow and induced an increase in the H2O2 release. At the conclusion of each experiment, fibers were harvested and dried for 15 min at 150°C. H2O2 production was expressed as pmol/min/mg dry weight.
To evaluate
We evaluated the global mitochondrial content in Endo and Epi layers by measuring the activity of citrate synthase (Kindo et al.,
Permeabilized fibers without myosin were prepared as described previously (Halestrap,
Briefly, we measured the mitochondrial Ca2+ uptake after the addition of a single Ca2+ pulse (20 nM). After the pulse was applied, we monitored the decrease in the extramitochondrial calcium concentration with the fluorescent probe Calcium Green-5N (Invitrogen). The progressive uptake of Ca2+ by the mitochondria was monitored until mitochondrial Ca2+ release caused by mPTP opening was observed. The mitochondrial calcium retention capacity (CRC), which is a reliable index of mPTP sensitivity, was calculated as the total amount of Ca2+ taken up by mitochondria before Ca2+ release. The CRC values are expressed as mg dry fiber weight (Pottecher et al.,
The values are expressed as the mean ± standard error of mean (SEM). Differences in the group means were statistically analyzed by the Mann Whitney test or a one-way analysis of variance (ANOVA) with the Newman-Keuls post-test analysis. A
No difference through the LV wall was found regarding the global mitochondrial content by measuring the activity of citrate synthase (Figure
Similarly, the acceptor control ratio (Vmax/V0) indicating the degree of coupling between oxidation and phosphorylation, was similar in the two myocardial layers (Figure
However, maximal mitochondrial respiratory rates (Vmax, complexes I, III, and IV) were significantly reduced in the Endo compared with the Epi (Figure
Likewise, there was a consistent decrease in complex II, III, and IV activity in the presence of succinate substrate (Vsucci) in the Endo (Figure
No difference between the Endo and Epi respiratory activity was observed with TMPD-ascorbate substrates (complex IV) (Table
V0 | 5.80 ± 0.74 | 5.15 ± 0.48 | 0.738 |
Vmax | 31.38 ± 2.20 | 26.07 ± 1.54 | |
Vsucci | 22.63 ± 1.39 | 18.39 ± 0.94 | |
Vtmpd | 36.50 ± 2.77 | 30.70 ± 1.45 | 0.260 |
Thus, normal LV is characterized by transmural differences in mitochondrial respiratory chain complexes I, II, and III activities.
NO production was assessed using EPR (
With increasing concentrations of MAHMA NONOate, which is a NO donor, the maximal respiratory rates were significantly decreased in the Endo and the Epi (Figure
Mitochondrial H2O2 production was significantly increased in the Endo compared with the Epi (
Moreover, superoxide anion production, which was measured with DHE fluorescence intensity, was significantly increased in the Endo (Figures
The sensitivity of mPTP opening was assessed fluorometrically using Calcium Green (13 rats). There was no statistical difference between the mitochondrial Ca2+ retention capacity for the Endo and the Epi (Figure
The main results of this study are to confirm a lower oxidative capacity of the sub-endocardium as compared to the sub-epicardium across the normal left ventricle, together with an increased nitric oxide concentration in the endocardium. Reactive oxygen species production was also enhanced in LV endocardium. Mitochondrial content and coupling are similar in both LV layers.
Despite accumulating evidences regarding myocardial heterogeneity of the normal heart, upon blood flow, structure, metabolism, electrophysiological, and contractile properties, there are few data investigating potential energetic gradient across the LV wall.
Particularly, there are only two reports using saponin-skinned fibers that ensure global mitochondrial functionnal assessment in intact mitochondria (i.e., not isolated and potentially fragilized mitochondria). MacDonald et al. observed no LV transmural respirational difference in healthy rats (MacDonald et al.,
In the present study, subendocardial oxidative capacity was significantly lower than subepicardial capacity when using glutamate/malate or succinate, supporting that complexes I, II, and III activities of the mitochondrial respiratory chain were reduced. This result gave further
NO is a free radical with a very short live in tissue. Under physiological conditions, NO detection and quantification in biological tissue is challenging (Hogg,
EPR spin-trapping showed that NO level was significantly higher in the LV Endo compared with the LV Epi. Our data highlight for the first time, to our knowledge, that a transmural NO gradient exists through the LV at the basal state. Accordingly, Brahmajothi et al. have shown heterogeneous expression gradients of NOS in the LV wall with NOS1 predominately localized in LV Endo and NOS3 in LV Epi (Brahmajothi and Campbell,
The physiological significance of such transmural NO gradient is still poorly known but increased NO concentration in the subendocardium may participate in the higher contractility and blood flow of the Endo compared with the Epi, previously observed in mammalian hearts (Brahmajothi and Campbell,
Further, some new informations might be inferred from our results. Indeed, NO increase in the Endo might have been linked with the reduced subendocardial mitochondrial respiratory chain complex activities. Thus, despite a similar global mitochondrial content through the LV wall, the NO gradient observed in this study was associated with reduced complexes I, II, and III activities in the Endo compared with the Epi. To further analyze a potential relationship between increased NO and decreased mitochondrial respiratory chain function, we determined the effect of increasing dose of NO donor on the maximal oxidative capacity of both Endo and Epi LV layers. Vmax activities decreased when the NO donor was added. This is consistent with previous data demonstrating that NO can inhibit mitochondrial respiration by reversible direct inhibition of cytochrome c oxidase and by reactive nitrate species production leading to respiratory complexes S-nitrosylation (Brown and Borutaite,
ROS include free radicals like superoxide anion, hydroxyl and the highly reactive compound hydrogen peroxide (H2O2). H2O2, resulting from superoxide dismutase action on superoxide anion, can give rise to hydroxyls radicals and may impair mitochondrial respiration rates (Veal et al.,
In the present study, H2O2 overproduction and increased DHE staining were observed in Endo, supporting an increased oxidative stress in the sub-endocardium as compared to the sub-epicardium. Such increased oxidative stress, specifically located in the Endo, might have been involved in sub-endocardium reduced mitochondrial function. However, alternatively, mitochondrial dysfunction is also a source of ROS production and mitochondrial reduced respiration might have been the origin of H2O2 overproduction and increased DHE staining (reflecting ROS production including superoxide anion, Pottecher et al.,
Interestingly, however a reduced Endo mitochondrial function was observed despite similar relative H2O2production in both Endo and Epi layers (Kindo et al.,
To investigate whether reduced sub-endocardial mitochondrial respiratory chain complex activities and/or whether increased sub-endocardial NO production might be considered as deleterious, we determined for the first time the mitochondrial permeability transition pore sensitivity in both Endo and Epi layers. Although mitochondrial membrane permeabilization can occur independent of mPTP formation, mPTP is a major large ion channel in the inner mitochondrial membrane that induces apoptosis upon opening (Halestrap,
In summary, our data give an overview of left ventricular wall physiology at the basal state. A transmural NO gradient, present in normal left ventricle might potentially participate in the endocardium higher contractility and blood flow. However, increased subendocardial NO concentration is associated with a decrease in the mitochondrial respiratory chain complex activity and with an increase in mitochondrial ROS production.
Such associations should probably not be viewed as impairments since they are observed in normal hearts and since the sensitivity of mPTP opening, involved in apoptosis, is similar in left ventricular wall layers.
Further studies will be useful to demonstrate if a defect in this transmural heterogeneity in NO expression modulates mitochondrial function and participate in the pathophysiology of cardiovascular diseases. Indeed, we previously observed that the level of the transmural gradient in mitochondrial respiration might be used as potential biomarker for transition from uncomplicated to complicated LV hypertrophy.
Conception or design of the work: MK, SG, JB, TH, AC, FP, BG. Acquisition, analysis: MK, SG, JB, TH, AC, JZ. Interpretation of data for the work: MK, SG, JM, JZ, FP, BG. Drafting or revising the work: MK, AC, JM, BG. Final approval: MK, SG, JB, TH, AC, JM, JZ, FP, BG. Agreement to be accountable of all aspects of the work: MK, SG, JB, TH, AC, JM, JZ, FP, BG.
This work was supported by a grant from the OCOVAS (‘Association des opérés du coeur et des vaisseaux à Strasbourg’) and the RITAC (‘Recherche et Innovations Technologiques dans les Affections Cardiovasculaires’).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AS and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.
We thank Professor Bernard Eisenmann for his generous support and encouragement during this study. We also thank Fabienne Goupilleau and Isabelle Bentz for their expert technical assistance.