Cardiac mitochondria in heart failure: Normal cardiolipin profile and increased threonine phosphorylation of complex IV

Biochimica et Biophysica Acta 1807 (2011) 1373–1382

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a b i o

Cardiac mitochondria in heart failure: Normal cardiolipin profile and increased threonine phosphorylation of complex IV Mariana Rosca a,b, Paul Minkler a,b, Charles L. Hoppel a,b,c,⁎ a b c

Center for Mitochondrial Diseases Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA Department of Medicine, Case Western Reserve University, Cleveland, OH, USA

a r t i c l e

i n f o

Article history: Received 18 October 2010 Received in revised form 3 February 2011 Accepted 5 February 2011 Available online 12 February 2011 Keywords: Mitochondria Supercomplex Oxidative phosphorylation Cardiolipin Protein threonine phosphorylation

a b s t r a c t Mitochondrial dysfunction is a major contributor in heart failure (HF). We investigated whether the decrease in respirasome organization reported by us previously in cardiac mitochondria in HF is due to changes in the phospholipids of the mitochondrial inner membrane or modifications of the subunits of the electron transport chain (ETC) complexes. The contents of the main phospholipid species, including cardiolipin, as well as the molecular species of cardiolipin were unchanged in cardiac mitochondria in HF. Oxidized cardiolipin molecular species were not observed. In heart mitochondria isolated from HF, complex IV not incorporated into respirasomes exhibits increased threonine phosphorylation. Since HF is associated with increased adrenergic drive to cardiomyocytes, this increased protein phosphorylation might be explained by the involvement of cAMP-activated protein kinase. Does the preservation of cAMP-induced phosphorylation changes of mitochondrial proteins or the addition of exogenous cAMP have similar effects on oxidative phosphorylation? The usage of phosphatase inhibitors revealed a specific decrease in complex I-supported respiration with glutamate. In saponin-permeabilized cardiac fibers, pre-incubation with cAMP decreases oxidative phosphorylation due to a defect localized at complex IV of the ETC inter alia. We propose that phosphorylation of specific complex IV subunits decreases oxidative phosphorylation either by limiting the incorporation of complex IV in supercomplexes or by decreasing supercomplex stability. © 2011 Published by Elsevier B.V.

1. Introduction The heart has a steady and uninterrupted requirement for energy in the form of ATP produced by mitochondria. A link between a decline in mitochondrial energy transduction and mechanical dysfunction in heart failure (HF) is suggested by studies performed in animal models [1,2] and in patients with genetic mitochondrial diseases [3] or acquired cardiomyopathies [4–6]. In moderately severe HF in dogs we found a 50% decrease in oxidative phosphorylation in both populations of heart mitochondria, subsarcolemmal (SSM) and interfibrillar (IFM) [7] without changes in either the content or activities of individual electron transport chain (ETC) complexes. The defect in oxidative phosphorylation is due to the decrease in the amount of the supercomplex that consists of complex I/complex III dimer/complex IV (I/III2/IV). This finding led to the conclusion that the mitochondrial defect lies in respirasomes, and suggests either impaired formation or decreased stability.

⁎ Corresponding author at: Case Western Reserve University, School of Medicine, Department of Pharmacology, 10900 Euclid Avenue, Cleveland, OH 44106–4981, USA. Tel.: + 1 216 368 3147; fax: + 1 216 368 5162. E-mail address: [email protected] (C.L. Hoppel). 0005-2728/$ – see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.bbabio.2011.02.003

The ETC complexes, whether unincorporated in respirasomes or organized in supercomplexes, are embedded in the phospholipid bilayer structure of the mitochondrial inner membrane. Cardiolipin (CL) is an anionic phospholipid present almost exclusively in the mitochondrial inner membrane of eukaryotic cells. Tetra-linoleoyl-CL [(C18:2)4-CL] is the predominant form of all CL species. The content of (C18:2)4-CL is maintained by a continuous remodeling process under the control of tafazzin (catalyzes the re-acylation reactions) and mitochondrial phospholipase A (catalyzes the de-acylation reactions). Cardiolipin provides structural and functional support to components of both the mitochondrial ETC and phosphorylation apparatus [8–18]. Cardiolipin binding sites for all ETC components have been described, and CL depletion results in reduced activities of complexes I, III [10] and IV along with the dissociation of subunits VIa and IVb from complex IV [19]. Mitochondrial dysfunction in the ischemic heart [20,21] and HF [22] is reported to be associated with CL peroxidation, loss of total CL content and decrease in (C18:2)4-CL. Recent studies suggest that CL plays a central role in the higher order organization of mitochondrial ETC in supercomplexes. It was reported that CL is essential for either formation [23] or stabilization [15] of respiratory supercomplexes. Yeast cells lacking Taz1, the orthologue of human taffazin enzyme essential for CL remodeling, revealed a decreased stability of the III2/IV2 supercomplex [24]. In

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humans, reduced (C18:2)4-CL, due to defective CL remodeling (Barth syndrome), causes dilated cardiomyopathy associated with destabilization of all supercomplexes containing complex IV, loss of complex I from the supermolecular assembly and decrease of the individual enzymatic activities of complexes I, III and IV [25]. There is an accumulating evidence that phosphorylation of mitochondrial proteins induced by cAMP/cAMP-activated protein kinase (PKA) signaling pathway alters both the assembly [26–29] and the activity of mitochondrial complexes [30]. In cardiomyocytes there are two distinct cAMP generators, the transmembrane adenylyl cyclase (AC) linked to β1-adrenergic receptors and activated by sympathetic stimulation, and the mitochondrial soluble AC activated by calcium and bicarbonate. Additional evidence shows that cardiac mitochondria have a complete cAMP-PKA signaling pathway, with PKA present on both mitochondrial membranes and matrix [31–39] and signalterminating mechanisms, such as phosphodiesterases [40] and phosphatases [41,42] present in the matrix. cAMP/PKA-induced phosphorylation events that change the function of proteins occur on components of all mitochondrial ETC complexes and phosphorylation apparatus (for review see [30]), suggesting that cAMP represents a major regulator of mitochondrial oxidative phosphorylation. Complex IV seems to be the main target of this regulation. The allosteric control of complex IV activity by intramitochondrial ADP/ATP ratio is dependent on cAMP-induced phosphorylation of complex IV specific subunits [43,44]. Phosphorylation of complex IV emerges as either beneficial or detrimental for complex IV activity. Phosphorylation of complex IV subunits induced by overexpression of mitochondrial soluble AC improves complex IV activity and mitochondrial respiration in cultured cells and animals with complex IV deficiency [45]. In contrast, PKA-dependent phosphorylation of complex IV in hypoxia and cardiac ischemia causes a decrease in complex IV activity [46,47]. The two distinct effects on mitochondrial function may be explained by the presence of two individual ACs and cAMP subcellular pools that can be regulated differently in response to either extra- or intracellular signals. The aim of the present study was to identify the mechanism responsible for the impaired supramolecular organization of the ETC in cardiac mitochondria in HF. We hypothesized that the decrease in the content of supercomplexes in HF is due either to 1) changes in the phospholipid composition of the mitochondrial inner membrane or to 2) postranslational modifications of the ETC complexes by phosphorylation. Our approach was first to investigate the changes in the main phospholipid species, including cardiolipin, and in the molecular species of cardiolipin as potential causes of the decreased supercomplex amount in HF. Second, we investigated postranslational modifications of mitochondrial proteins, specifically serine and threonine phosphorylation in HF, a state which is associated with an in vivo chronic stimulation of the β1-adrenergic receptors/transmembrane AC/cAMP/PKA signaling pathway in the heart. Third, to examine the effect of acute activation of the mitochondrial PKA we used rat cardiac fibers incubated with membrane permeable cAMP. Fourth, by preserving the phosphorylation status of mitochondrial proteins with protein phosphatase inhibitors we studied the effect of increased amount of phosphorylation changes on mitochondrial function. 2. Methods 2.1. Materials, reagents Unless specified, all reagents were purchased from Sigma-Aldrich and were of the highest purity grade. 2.2. Animal model The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health

(NIH publication no. 85–23, revised 1996). All experimental protocols were approved by the Case Western Reserve University Institutional Animal Care and Use Committee. The intracoronary microembolization-induced model as described [7,48,49] was used to induce HF in dogs. Both groups were described in our previous study [7]. Male Sprague–Dawley rats weighing 300–400 g were used to isolate cardiac fibers and cardiac mitochondria. 2.3. Isolation of cardiac mitochondria and fibers The dog heart SSM and IFM were isolated, studied and their respiratory properties reported [7]. At that time, freshly-isolated cardiac mitochondria were used for a comprehensive study of mitochondrial function in HF. A portion of each mitochondrial sample was divided into small aliquots and stored at − 60 °C until analyzed in the current study. Rat cardiac SSM and IFM were isolated following the protocol of Palmer et al. [50] except that trypsin (5 mg/g wet weight cardiac tissue) was used as a protease to isolate IFM. In two experiments, both the isolation and storage buffers were supplemented with protein phosphatase inhibitors (sodium orthovanadate 1 mM, sodium fluoride 10 mM, glycerophosphate 4 mM and sodium diphosphate 1 mM). Mitochondrial protein concentration was determined by the Lowry method using bovine serum albumin as a standard [51]. Individual heart muscle fibers were obtained in high-energy relaxing buffer (EGTA 10 mM, imidazole 20 mM, MES 50 mM, dithiothreitol 0.5 mM, MgCl2 3 mM, ATP 5.77 mM, phosphocreatine 15 mM, pH 7.1) as described [52]. 2.4. Measurement of respiratory properties of isolated cardiac mitochondria and permeabilized fibers Oxygen consumption was measured in freshly-isolated intact cardiac mitochondria with a Clark-type electrode as described [53,54] and expressed as nA oxygen per minute per milligram mitochondrial protein. High-resolution respirometry was used to measure oxygen consumption in permeabilized rat heart fibers. Individual heart muscle fibers were permeabilized with saponin (50 μg/ml for 30 min at 4 °C) and transferred into two chambers of an OROBOROS Oxygraph 2K containing air-saturated assay medium (EGTA 0.5 mM, MgCl2 3 mM, K lactobionate 60 mM, taurine 20 mM, K phosphate 10 mM, HEPES 20 mM, sucrose 110 mM, bovine serum albumin 1 g/l) at 37 °C. Fibers were incubated for 30 min with either assay buffer or assay buffer supplemented with 200 μM dibutyryl cAMP to activate protein kinase A (PKA) and 6.4 μM theophylline to inhibit phosphodiesterases and maintain an increased cAMP content. Oxygen flux in heart muscle fibers was assessed after sequential additions of substrates, ADP (2.5 mM), dinitrophenol (uncoupler) and inhibitors, and expressed as picomole O2 per seconds per milligram wet weight. The following substrates were used: glutamate (10 mM) + malate (2 mM), succinate (10 mM), and TMPD (5 mM) + ascorbate (2 mM). 2.5. Separation and quantitation of individual phospholipid species Total lipids from frozen–thawed heart mitochondria of control and HF dogs (1.5 mg mitochondrial proteins diluted to a final volume of 0.25 ml with 50 mM KCl) were extracted with chloroform/ methanol (3:1, by volume) [55]. Organic extracts were fractionated by silica gel column chromatography into main lipid classes, which were serially eluted using solvents of increasing polarity [56]. The phospholipid (PPL) fraction was further separated by normal phase high-performance liquid chromatography into different phospholipid species and quantified by organic phosphate assay, as described [57]. Briefly, the phospholipid fraction was collected in methanol. One portion was used to measure the total organic phosphate in the PPL

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fraction and another portion was subjected to normal-phase HPLC performed on a Hypersil silica column (4.6 × 250 mm, 5 μm). Elution was performed with an initial mobile phase of hexane:2-propanol:25 mM potassium acetate (pH 7.0):ethanol:glacial acetic acid (367:490:62:100:0.6, by volume) followed by a gradient (increasing hexane and decreasing ethanol) to the mobile phase. The individual chromatographic peaks noted in Table 1 were identified by comparison of retention times of standards. Organic phosphate was measured by reading the absorbance in a plate reader at 815 nm. The recovery was calculated as a percentage of the sum of individual PPL species (total phosphate in the PPL, total PPL in Table 1) from the total organic phosphate (the starting material). 2.6. Separation and quantitation of cardiolipin molecular species Reversed-phase ion pair high performance liquid chromatographymass spectrometry was used to separate, identify and quantify the CL molecular species as described [58].

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the Box–Cox transformation was used with λ = 0.52 (Fig. 4) and 0.23 (Fig. 5) set per the R function box–cox in the R package MASS: λ = 0 corresponds to a log, λ = 0.5 to a square root, and λ = 1 to no transformation. Quantile–quantile and residual-versus-fitted-value plots were used to confirm the normality assumptions of the models. 3. Results The hemodynamic parameters of control dogs and dogs with HF, as well as the respiratory properties and the amount of free ETC complexes and of the I/III2/IV1 respirasome in cardiac mitochondria, were reported elsewhere [7]. In brief, repeated coronary microembolization in dogs caused a progressive and moderately severe worsening of the left ventricle (LV) performance manifested as a decrease in ejection fraction (from 34 ± 1 to 28 ± 1) and increases in the LV end-systolic volume (from 42 ± 1 to 47 ± 1 ml) and enddiastolic volume (from 64 ± 2 to 67 ± 2 ml) [7]. 3.1. Separation and quantitation of individual phospholipid species

2.7. Separation of mitochondrial supramolecular complexes by Blue Native PAGE electrophoresis and Western blot analysis BN-PAGE was performed following the general approach of Schagger and Wittig [59]. Three hundred micrograms of the stored mitochondrial proteins were extracted with digitonin (detergent: protein ratio of 6:1 g/g) for 30 min at 4 °C in solubilization buffer (imidazole 50 mM, NaCl 50 mM, 6-aminocaproic acid 2 mM, EDTA 1 mM, pH 7.0), combined with Coomassie blue G-250 dye (detergent/ dye ratio of 8:1 g/g), and separated on precast 3%–12% acrylamide– bisacrylamide gradient gels (Invitrogen NativePAGE minigels) at 75 μg proteins per well. Proteins were electroblotted onto PVDF membranes and probed with either phosphoserine or phosphothreonine antibodies (Santa Cruz Biotechnology, 1:5000 dilution). Appropriate HRP-linked secondary antibodies were used (BioRad, 1:10,000 dilution). Membranes were developed using Amersham ECL™ substrates. Densitometric evaluation of the gels was carried out using Image J from the National Institutes of Health and Multi Gauge V3.0 from Fujifilm Life Science. 2.8. Statistical analysis The results were analyzed using the student's 2 tailed-t test except the data in Figs. 4 and 5. Data are reported as mean ± SEM. To be considered significant, the p value had to be less than 0.05. Linear statistical models were fitted to the data in Figs. 4 and 5 to obtain p values for difference parameters. For variance stabilization Table 1 Content of phospholipids in heart subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria from control and HF dogs (nanomole phospholipid phosphate per milligram mitochondrial protein). Mean ± SEM. PE, phosphatidylethanolamine; PI, phosphatidylinositol; CL, cardiolipin; PC, phosphatidylcholine.

PE PI CL PC Total PPL Total organic phosphate Recovery (%) Heart IFM PE PI CL PC Total PPL Total organic phosphate Recovery (%)

Control

Heart failure

P values

90 ± 7 12 ± 2 42 ± 4 130 ± 8 274 ± 20 273 ± 30 102 ± 14 N=5 95 ± 5 15 ± 3 51 ± 4 151 ± 7 311 ± 15 366 ± 30 85 ± 6

110 ± 13 18 ± 2 49 ± 6 177 ± 22 353 ± 38 346 ± 16 106 ± 15 N=4 118 ± 8 18 ± 4 52 ± 7 189 ± 9 376 ± 21 369 ± 34 101 ± 4

0.29 0.08 0.50 0.16 0.19 0.08 0.96 0.09 0.42 0.89 0.03 0.08 0.96 0.08

The content of mitochondrial phospholipids (phosphatidylethanolamine, phosphatidylinositol, CL and phosphatidylcholine) is given in Table 1. The analytical accuracy of these determinations is highlighted by the recovery of phospholipids that averaged 85%– 100%. The amount of phosphatidylcholine is increased in HF whereas the amount of CL is not changed. Our data rule out the alteration in the total content of mitochondrial CL as a cause for the decrease in the supercomplex assembly in HF. 3.2. Separation and quantitation of cardiolipin molecular species Cardiolipin separated by normal phase HPLC was characterized by reverse-phase ion-pair HPLC coupled with a hybrid triple quadruple linear ion-trap MS. Molecular species of CL from cardiac SSM and IFM of control dogs and dogs with HF were separated and their molecular mass identified. The percentage of CL species was determined from total CL content. Fig. 1A and B show the total ion chromatograms of the CL molecular species eluting between 15 and 40 min. Unmodified tetra-linoleoyl-CL [(C18:2)4-CL] elutes at ~ 30 min, and was the predominant CL species in heart SSM and IFM isolated from both control dogs and dogs with HF (Fig. 1A and B, and Table 2). The proportional amount of (C18:2)4-CL was not changed in cardiac mitochondria isolated from HF compared with the control hearts. Oxidation products of CL, shown to elute earlier than the unmodified CL [60] due to their decreased hydrophobicity, are not observed in HF in either cardiac SSM or IFM. Other CL species identified were trilinoleoyl-mono-oleoyl-CL [(C18:2)3, (C18:1)], di-linoleoyl-di-oleoylCL [(C18:2)2(C18:1)], tri-linoleoyl-mono-palmitoleoyl-CL [(C18:2)3 (C16:1)] and tri-linoleoyl-mono-linolenoyl-CL [(C18:2)3(C18:3)1] (Fig. 1A and B); the amount of those species did not change in cardiac mitochondria isolated from HF compared to those isolated from control dogs. We conclude that alteration in the mitochondrial CL species and acyl composition is not the cause of the decrease in supercomplex content in moderately severe HF in dogs. 3.3. Separation of mitochondrial supramolecular complexes by Blue Native PAGE electrophoresis and Western blot analysis We compared the status of phosphorylation at serine (Fig. 2) and threonine (Fig. 3) residues of supercomplexes and complexes not incorporated within the main respirasome (I/III2/IV) in cardiac mitochondria from control and HF dogs. There is a large variability in the amount of both serine and threonine modifications especially on proteins assembled in supercomplexes in cardiac SSM isolated from HF dogs. This may be a reflection of either the variability of phosphorylation modifications on mitochondrial proteins from this

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Time (min) Fig. 1. Representative total ion chromatograms generated from cardiac SSM (A) and IFM (B) isolated from control dogs and dogs with HF.

experimental group or the loss of phosphorylation modifications due to mitochondrial phosphatases during the freezing-thawing procedure. The levels of phosphorylation at both serine and threonine residues I/III2/IV supercomplex were significantly decreased in heart IFM isolated from HF, with a similar trend in heart SSM. These results are in agreement with the decreased amount of the supercomplex in HF in both types of cardiac mitochondria.

As shown in Fig. 3, complex IV unincorporated in the supercomplex contained a significant increased content of threonine phosphorylation in both types of heart mitochondria isolated from HF compared to the control. Since there is a pronounced activation of the β-adrenergic stimulation in cardiomyocytes in HF, our data suggest that cyclic AMP may have a role in phosphorylation of complex IV subunits at threonine residues. We also detected threonine

M. Rosca et al. / Biochimica et Biophysica Acta 1807 (2011) 1373–1382 Table 2 Cardiolipin species in heart subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria from control and HF dogs. Results are expressed as percentage of total cardiolipin content. Mean ± SEM. The abbreviations are in section 3.2. Heart SSM

Control

Heart failure

N=6

N=5

(C18:2)4 (C18:2)3 (C18:1)1 (C18:2)2(C18:1)2 (C18:2)3 (C16:1)1 (C18:2)3 (C18:3)1 Heart IFM (C18:2)4 (C18:2)3 (C18:1)1 (C18:2)2 (C18:1)2 (C18:2)3 (C16:1)1 (C18:2)3 (C18:3)1

77.5 ± 1.2 13.4 ± 0.7 3.9 ± 0.1 3.7 ± 0.3 1.5 ± 0.2 N=4 79.5 ± 0.8 12.1 ± 0.3 3.7 ± 0.2 3.3 ± 0.3 1.4 ± 0.1

80.7 ± 3.0 13.3 ± 1.4 3.0 ± 0.9 2.1 ± 1.0 0.9 ± 0.1 N=6 80.3 ± 2.2 13.6 ± 1.0 2.9 ± 0.7 2.1 ± 0.7 1.0 ± 0.2

phosphorylation on complexes I and III, but the content did not differ between mitochondria from control hearts and heart with HF. Our procedure did not detect any threonine phosphorylation in complex V. In contrast, the level of phosphorylation at serine of subunits of complex IV unincorporated within the respirasome was not changed in cardiac mitochondria isolated from HF compared with the control (Fig. 2). Complex III unincorporated in supercomplexes has a large amount of serine phosphorylation in both SSM and IFM isolated from control heart and the amount is not changed by HF. 3.4. The effect of protein phosphorylation on mitochondrial function 3.4.1. The effect of protein phosphatase inhibitors (PPI) In order to preserve the steady-state level of phosphorylation by preventing the action of phosphatases in removing the phosphate group from mitochondrial proteins, we isolated the two populations of rat cardiac mitochondria with protein phosphatase inhibitors (PPI)

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present in the isolation and storage buffers. ADP-dependent oxidative phosphorylation rates (state 3 rates) of rat heart SSM and IFM isolated and stored in the presence of PPI were compared with state 3 respiratory rates of rat heart mitochondria without PPI. Mitochondria isolated and investigated in the presence of PPI showed a decrease in the respiratory state 3 rates with complex I substrate glutamate (p = 0.0003 and 0.0001 for SSM and IFM, respectively), while the oxidative rates with complex II (succinate), III (duroquinol) and IV (TMPD + ascorbate) substrates are unchanged (Fig. 4). Uncoupling does not restore respiration, showing that the defect is localized at the level of oxidation side of oxidative phosphorylation rather than in the ATP-synthesis machinery. Moreover, the preservation of the steadystate level of phosphorylation on mitochondrial proteins with PPI does not change the amount of respirasomes in both populations of rat heart mitochondria (data not shown). Therefore, the addition of PPI reveals a selective defect in glutamate-supported respiration at the level of either glutamate transporter, glutamate dehydrogenase, or complex I. 3.4.2. The effect of exogenously-added cyclic AMP on mitochondrial oxidative phosphorylation High-resolution respirometry was used to investigate the effect of cAMP on mitochondrial oxidative phosphorylation in saponinpermeabilized rat heart fibers (Fig. 5). The respiratory rate with complex I substrates glutamate + malate in the absence of ADP is not changed by cAMP. The ADP-dependent (state 3) respiratory rates of cAMP-incubated fibers in the presence of complex I substrates (electron transport through complexes I/III/IV) was 70% of the control cardiac fibers. Respiratory rates did not significantly increase upon the addition of cytochrome c, showing that the high mitochondrial outer membrane integrity of cardiac fibers is maintained in the presence of cAMP. The addition of succinate (electron input to complex II) resulted in an increase in oxygen consumption in the control (80%) with a much smaller increase in the cAMP-incubated fibers (27%). Therefore, the oxygen flux through complexes I + II/III/IV in the cAMP-incubated fibers was approximately 50% of the control.

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Fig. 3. (A) Western blot analysis of supercomplexes and individual complexes of heart SSM and IFM isolated by BN PAGE and probed with threonine phosphorylation antibodies. (B) Densitometric analysis. *p b 0.05 control vs HF. Mean ± SEM.

Uncoupling does not relieve this decrease in oxygen flux (668.9 vs 357.4 pmol/s/mg wet weight in control and cAMP-incubated fibers, respectively), showing that the defect induced by cAMP resides within a component of the oxidative side of oxidative phosphorylation and not in the phosphorylation apparatus. Oxygen flux through complexes II/III/IV in the cAMP-incubated fibers measured upon the addition of rotenone was 33% of the control (140.6 vs 421.2 pmol/s/mg wet weight in cAMP-incubated fibers and control, respectively). These data indicate a greater

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inhibition by cAMP of complex II-supported respiration than of complex I-supported respiration, in contrast with the major effect of PPI on complex I-driven respiration, with preservation of respiration initiated by the complex II substrate (Fig. 4). Subsequent inhibition of complex III with antimycin A blunted the oxygen flux in both samples. TMPD + ascorbate donates reducing equivalents to cytochrome c, which is then oxidized by cytochrome c oxidase with final tetravalent reduction of oxygen to water. Incubation with cAMP resulted in a decrease in TMPD + ascorbate supported respiration

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Fig. 4. State 3 and uncoupled respiratory rates of rat heart SSM (A) and IFM (B). State 3 was induced by 200 μM ADP when mitochondria oxidize complex I substrates (Glutamate), and 100 μM ADP when mitochondria oxidize complexes II (Succinate+rotenone), III (Duroquinol) and IV via cytochrome c (TMPD + ascorbate + rotenone). The uncoupled rates were measured with 200 μM dinitrophenol. Abbreviations: TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine; PPI, protein phosphatase inhibitors. The values are the mean of two independent experiments. *p b 0.05 control vs HF. Mean ± SEM.

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+AA TMPD+A Azide

Fig. 5. Oxygen flux in control and cAMP-incubated permeabilized rat heart fibers. Abbreviation: G+M, Glutamate + Malate; cyt c, cytochrome c; S, Succinate; DNP, Dinitrophenol; Rot, Rotenone; AA, Antimycin A; TMPD+A, N,N,N′,N′-tetramethyl-pphenylenediamine + Ascorbate. cAMP = dibutyryl cAMP 200 μM. The values are the mean of two independent experiments. *p b 0.05 control vs HF. Mean ± SEM.

(1111.8 vs 667.6 pmol/s/mg wet weight in control and cAMPincubated fibers, respectively). Azide blunted the oxygen flux in both samples. In conclusion, cAMP leads to a decrease in oxidative phosphorylation with complex I, II and IV substrates, suggesting defects in the ETC localized at least at the level of cytochrome c oxidase. 4. Discussion We recently reported that the 50% reduction in oxidative phosphorylation of cardiac mitochondria in moderately severe HF may be explained by ~30% and 50% decrease in the amount of the major mitochondrial I/III2/IV1 respirasome in cardiac SSM and IFM, respectively [7]. We concluded that the mitochondrial defect in HF either limits the incorporation of individual complexes in supercomplexes or enhances supercomplex degradation. In follow-up to our previous study, we asked if the decrease in the supercomplex content is due to changes in the phospholipids of the mitochondrial inner membrane or postranslational modifications of subunits of the ETC complexes. The primary findings of the present study are: 1) the amount of total phospholipids did not change in both cardiac SSM and IFM isolated from HF compared with the control; 2) from phospholipid species, phosphatidylcholine is increased in HF whereas the amount of cardiolipin does not change; 2) (C18:2)4-CL represents the most abundant CL species, making up more than 80% of the total CL in both cardiac SSM and IFM isolated from control dogs and dogs with HF; 3) there were no changes in the content of (C18:2)4-CL in both cardiac SSM and IFM; 4) the amount of minor CL species also did not change in HF in both populations of cardiac mitochondria; 5) no oxidized CL species were observed in HF; 6) complex IV not incorporated into the respirasome organization exhibits a significant increase in threonine phosphorylation in heart mitochondria isolated from HF compared with the control. These data indicate that the damage of the ETC assembly in this model of HF of moderate severity is not due to changes in the CL profile, and suggest that cAMPdependent phosphorylation at threonine residues of mitochondrial proteins may play a role. We next asked if either exogenously-added cAMP or preservation of the cAMP-induced phosphorylation of mitochondrial proteins have similar effects on mitochondrial oxidative phosphorylation. We found that both approaches reveal defects localized in the oxidative side of oxidative phosphorylation rather than in the ATP-synthesis machinery. In permeabilized cardiac fibers, cAMP causes a decrease in oxidative phosphorylation due to defect(s) localized at least at the level of cytochrome c oxidase. In contrast, the preservation of the steady-state level of phosphorylation on mitochondrial proteins with protein phosphatase inhibitors reveals defect (s) localized at either glutamate transporter, glutamate dehydrogenase, or complex I. ETC complexes are assembled into high-molecular weight supercomplexes [61,62] which represent the structural basis for oxidative phosphorylation. In the I1III2IV1 supercomplex (respirasome) that

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contains coenzyme Q and cytochrome c, the electron carriers are closely apposed [63], supporting the concept of channeled electron transfer from NADH to reduce oxygen [64], decreasing electron leakage and superoxide production [65]. The individual complexes are fixed in this cohesive respiratory unit by either protein-lipid and/or protein-protein interactions. Although the concept suggested by Skulachev [66] that cardiolipin is a major factor for supercomplex stability was confirmed in yeast [23], further studies showed that CL is not essential for the formation of supercomplexes in the inner membrane, but stabilizes the ETC supercomplexes and individual complexes in yeast [15] and humans [25]. Cardiolipin facilitates specific protein-protein interactions by providing ionic bridges at the hydrophobic interfaces of the subunits of individual complexes, and by adjusting irregular protein surfaces into flexible interfaces, thus allowing the interaction between less tightly-associated proteins [23]. We report a decrease in the amount of the I1III2IV1 respirasome which is not associated with changes in either the content of total CL or CL molecular species profile in HF of moderate severity. Tetralinoleoyl-CL is the predominant CL in both populations of cardiac mitochondria isolated from HF, suggesting that the tafazzindependent remodeling step of CL is not altered in HF. These data differ from those reported for other models of HF by Sparagna et al. who found a decrease in the amount of [(C18:2)4]CL in animals with accelerated hypertensive HF [67] and humans with severe HF [22], associated with a decline in complex IV activity with unchanged complex amount. The authors, however, did not investigate the partition of complex IV, as part of the I1III2IV1 respirasome or unincorporated within the supercomplex. Our data generated in a less severe stage of HF in dogs indicate that the decrease in functional respirasomes is independent of both the mitochondrial CL content and CL molecular species profile and point to specific protein/protein interactions between subunits of individual complexes as a cause for the decrease in supercomplex content. We further asked if the primary event causing the defect in the ETC assembly is represented by modifications of protein subunits of ETC complexes. Phosphorylation seems to be a frequent postranslational modification in mitochondrial proteome. More than 60 mitochondrial proteins are phosphoproteins [68] and at least half are phosphorylated at serine and threonine residues. The increased adrenergic drive in HF implies a pronounced activation of the β-adrenergic receptor-stimulatory GTP-binding protein-adenylyl cyclase-cAMP signaling pathway in cardiomyocytes. Several observations provide the basis for a specific mitochondrial cAMP-protein kinase A (PKA) signaling pathway: 1) there are high concentrations of cAMP in mitochondria [69] that further increase within 2–3 min upon activation of β-adrenergic receptors by isoproterenol [70]; 2) the soluble mitochondrial adenyl cyclase regulates oxidative phosphorylation [40]; and 3) PKA is present in mitochondria [34–39]. As a first step in defining the role of cAMP-dependent phosphorylation in the decrease of the respirasome organization in HF, we compared the level of phosphorylation at serine and threonine residues of the main respirasome and of individual complexes in cardiac mitochondria isolated from control dogs and dogs with HF. We found that complex IV unincorporated in the supercomplex has a significant increase in the content of threonine phosphorylation in both types of heart mitochondria isolated from HF compared with the control. We also detected threonine phosphorylation on complexes III and I, but the content did not differ between mitochondria from control hearts and hearts with HF. We did not detect threonine phosphorylation in complex V, showing that complex V is not a major target for this substrate-specific phosphorylation. Phosphorylation controls complex IV activity and stability. Cyclic AMP-dependent phosphorylation at Tyr-304 on helix VIII of COX subunit I [71] causes a strong allosteric inhibition in the presence of ATP with no activity at physiologic levels of cytochrome c, when mitochondria were isolated in the presence of the phosphodiesterase

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inhibitor theophylline, but not in its absence. Ischemia/reperfusion causes cAMP-induced phosphorylation of COX subunits I (Ser115/ Ser116), IV (Thr52) and Vb (Ser40) [46] and increases reactive oxygen species production [47] by rabbit heart mitochondria isolated without protein phosphatase inhibitors. These serine phosphorylation sites in subunits IV and Vb apparently are not related to the regulation of COX activity, and it was suggested that they are involved in the binding affinity of COX with other proteins and COX incorporation in supercomplexes [72]. The increased threonine phosphorylation of the unincorporated complex IV in mitochondria isolated from HF in the absence of both phosphodiesterase and phosphatase inhibitors does not change the individual activity of complex IV which was reported in our previous study [7]. The data suggest that these threonine phosphorylation modifications involve small regulatory subunits of complex IV rather than catalytic subunits. Unfortunately, the exhaustion of mitochondrial supply limited the depth of the present study in identifying the specific subunits of complex IV that are phosphorylated. In our experiments, both electrophoretic separation of supercomplexes and Western blot analysis were performed with − 60 °C frozen/thawed mitochondria. Therefore, the post-translational modifications we detected in cardiac mitochondria in both Control and HF are stable ex vivo upon storage and freezing/thawing procedures and represent the steady-state level of phosphorylation events. The level of phosphorylation on mitochondrial proteins is under the control of protein kinases including PKA and the activity of mitochondrial phosphatases. We asked if the increased mitochondrial content of cAMP or preservation of the cAMP-induced phosphorylation of mitochondrial proteins have similar effects on mitochondrial oxidative phosphorylation. The content of cAMP within the mitochondria is balanced by its potential import from the cytosol and mitochondrial synthesis on one hand, and its degradation via mitochondrial phosphodiesterases on the other. Cyclic AMP released from the membrane-permeable dibutyryl cAMP by cellular esterases was used to increase the content of cAMP in mitochondria from saponinpermeabilized cardiac fibers in the presence of a phosphodiesterase inhibitor. Exogenously-added cAMP leads to a 50% decrease in oxidative phosphorylation through complex I + II/III/IV in rat cardiac fibers, suggesting defect(s) in the ETC localized at least at the level of complex IV. The analysis of the effect of cAMP on oxidative phosphorylation of mitochondria in situ in permeabilized rat heart fibers yields similar results as the investigation of oxidative phosphorylation in isolated mitochondria in HF [7]. When the pathway was dissected, we found that respiration through complexes II/III/IV is decreased to a greater extent than the respiration through complexes I/III/IV, suggesting an additional defect of the succinate transporter or complex II. In our experiments, dog mitochondria were isolated and stored in the presence of the Ca2+ chelator EGTA that disables the Ca2+dependent phosphatases in the absence of broad-spectrum protein phosphatase inhibitors. We therefore checked if we uncovered only an incomplete picture of phosphorylation events on mitochondrial proteins in both control heart mitochondria and in mitochondria isolated from dogs with HF due to the removal of the phosphate group from proteins by mitochondrial phosphatases during the mitochondrial isolation and storage. Interestingly, the addition of phosphatase inhibitors reveals a selective decrease in glutamate-supported respiration due to defect(s) located at the level of the substrate transport and processing (glutamate transporter and dehydrogenase) or complex I, with preservation of respiration driven by complex II, III and IV substrates. Both approaches used to investigate the effect of protein phosphorylation on mitochondrial function led to the conclusion that the ATP-synthesis machinery (ATP synthase, adenine nucleotide translocase, and phosphate transporter) is not a target for functional changes induced by cAMP. This conclusion is congruent with the

absence of serine and threonine phosphorylation in complex V in cardiac mitochondria isolated from both control and HF dogs. Both approaches instead point to changes in the oxidation side of oxidative phosphorylation located at different sites. However, since the oxidation of complex I substrates glutamate + malate depend upon the integrity of complex IV, we cannot exclude additional defects in either substrate processing or complex I upon the addition of exogenous cAMP on cardiac fibers. Specific changes in glutamate transporter and dehydrogenase may be induced by irreversible phosphorylation events due to the artificial inhibition of mitochondrial phosphatases. In addition, orthovanadate, used by us in the protein phosphatase inhibitor cocktail, also was reported to inhibit mitochondrial glutamate dehydrogenase [73] and succinyl CoA synthase [74], suggesting that phosphatase inhibitors lead to defects in glutamate-supported mitochondrial respiration that may be independent of protein phosphorylation. Alternatively, the unchanged complex IV-supported respiration upon preservation of protein phosphorylation events induced by a base-level of mitochondrial cAMP suggests that the defect in complex IV may be revealed only by elevated mitochondrial cAMP content. Based on our observations, we propose that in HF cAMP, either generated by the β-adrenergic receptor-linked adenylyl cyclase in cardiomyocytes or synthesized by soluble adenylyl cyclase in mitochondria, activates the mitochondrial PKA which phosphorylates threonine residues on subunits of complex IV. These postranslational modifications either impair the incorporation of complex IV into supercomplexes or cause the release of complex IV from the supercomplex, reducing the amount of functional respirasomes and decreasing the oxidative phosphorylation of heart mitochondria. Acknowledgments From our previous study, we thank Dr. Hani N. Sabbah for providing the dogs with heart failure, and Drs. William Stanley and Margaret Chandler for advice and technical assistance with regard to dog surgery. We thank Edwin Vazquez for isolating cardiac mitochondria and Lori Hezel for performing the phospholipid analysis. We appreciate the assistance of Dr. Tomas Radivoyevitch for statistical analysis. We acknowledge Dr. Bernard Tandler and the Hoppel lab Writing with style group for editorial assistance. Funding for this work was provided by the National Heart, Lung and Blood Institute, Program Project Grant PO1 (HL074237). References [1] A. Sanbe, K. Tanonaka, R. Kobayasi, S. Takeo, Effects of long-term therapy with ACE inhibitors, captopril, enalapril and trandolapril, on myocardial energy metabolism in rats with heart failure following myocardial infarction, J. Mol. Cell. Cardiol. 27 (1995) 2209–2222. [2] V.G. Sharov, A. Goussev, M. Lesch, S. Goldstein, H.N. Sabbah, Abnormal mitochondrial function in myocardium of dogs with chronic heart failure, J. Mol. Cell. Cardiol. 30 (1998) 1757–1762. [3] S. DiMauro, E.A. Schon, Mitochondrial DNA mutations in human disease, Am. J. Med. Genet. 106 (2001) 18–26. [4] A. Dorner, S. Giessen, R. Gaub, H. Grosse Siestrup, P.L. Schwimmbeck, R. Hetzer, W. Poller, H.P. Schultheiss, An isoform shift in the cardiac adenine nucleotide translocase expression alters the kinetic properties of the carrier in dilated cardiomyopathy, Eur. J. Heart Fail. 8 (2006) 81–89. [5] D. Jarreta, J. Orus, A. Barrientos, O. Miro, E. Roig, M. Heras, C.T. Moraes, F. Cardellach, J. Casademont, Mitochondrial function in heart muscle from patients with idiopathic dilated cardiomyopathy, Cardiovasc. Res. 45 (2000) 860–865. [6] V.G. Sharov, A.V. Todor, N. Silverman, S. Goldstein, H.N. Sabbah, Abnormal mitochondrial respiration in failed human myocardium, J. Mol. Cell. Cardiol. 32 (2000) 2361–2367. [7] M.G. Rosca, E.J. Vazquez, J. Kerner, W. Parland, M.P. Chandler, W. Stanley, H.N. Sabbah, C.L. Hoppel, Cardiac mitochondria in heart failure: decrease in respirasomes and oxidative phosphorylation, Cardiovasc. Res. 80 (2008) 30–39. [8] K. Beyer, B. Nuscher, Specific cardiolipin binding interferes with labeling of sulfhydryl residues in the adenosine diphosphate/adenosine triphosphate carrier protein from beef heart mitochondria, Biochemistry 35 (1996) 15784–15790. [9] C.E. Ellis, E.J. Murphy, D.C. Mitchell, M.Y. Golovko, F. Scaglia, G.C. Barcelo-Coblijn, R.L. Nussbaum, Mitochondrial lipid abnormality and electron transport chain

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