Christopher Lotz

Phosphoproteome analysis reveals regulatory sites in major pathways of cardiac mitochondria

Mitochondrial functions are dynamically regulated in the heart. In particular, protein phosphorylation has been shown to be a key mechanism modulating mitochondrial function in diverse cardiovascular phenotypes. However, site-specific phosphorylation information remains scarce for this organ. Accordingly, we performed a comprehensive characterization of murine cardiac mitochondrial phosphoproteome in the context of mitochondrial functional pathways. A platform using the complementary fragmentation technologies of collision-induced dissociation (CID) and electron transfer dissociation (ETD) demonstrated successful identification of a total of 236 phosphorylation sites in the murine heart; 210 of these sites were novel. These 236 sites were mapped to 181 phosphoproteins and 203 phosphopeptides. Among those identified, 45 phosphorylation sites were captured only by CID, whereas 185 phosphorylation sites, including a novel modification on ubiquinol-cytochrome c reductase protein 1 (Ser-212), were identified only by ETD, underscoring the advantage of a combined CID and ETD approach. The biological significance of the cardiac mitochondrial phosphoproteome was evaluated. Our investigations illustrated key regulatory sites in murine cardiac mitochondrial pathways as targets of phosphorylation regulation, including components of the electron transport chain (ETC) complexes and enzymes involved in metabolic pathways (e.g. tricarboxylic acid cycle). Furthermore, calcium overload injured cardiac mitochondrial ETC function, whereas enhanced phosphorylation of ETC via application of phosphatase inhibitors restored calcium-attenuated ETC complex I and complex III activities, demonstrating positive regulation of ETC function by phosphorylation. Moreover, in silico analyses of the identified phosphopeptide motifs illuminated the molecular nature of participating kinases, which included several known mitochondrial kinases (e.g. pyruvate dehydrogenase kinase) as well as kinases whose mitochondrial location was not previously appreciated (e.g. Src). In conclusion, the phosphorylation events defined herein advance our understanding of cardiac mitochondrial biology, facilitating the integration of the still fragmentary knowledge about mitochondrial signaling networks, metabolic pathways, and intrinsic mechanisms of functional regulation in the heart.

Activation of mitochondrial large-conductance calcium-activated K+ channels via protein kinase A mediates desflurane-induced preconditioning.

BACKGROUND:ATP-regulated K+ channels are involved in anesthetic-induced preconditioning (APC). The role of other K+ channels in APC is unclear. We tested the hypothesis that APC is mediated by large-conductance calcium-activated K+ channels (KCa). METHODS:Pentobarbital-anesthetized male C57BL/6 mice were subjected to 45 min of coronary artery occlusion and 3 h reperfusion. Thirty minutes before coronary artery occlusion, 1.0 MAC desflurane was administered for 15 min alone or in combination with the large-conductance KCa channel activator NS1619 (1 &mgr;g/g i.p.), its respective vehicle dimethylsulfoxide (10 &mgr;L/g i.p.), the large-conductance KCa channel blocker iberiotoxin (0.05 &mgr;g/g i.p.), or the protein kinase A (PKA) inhibitor H-89 (0.5 &mgr;g/g intraventricular). Infarct size was determined with triphenyltetrazolium chloride and area at risk with Evans blue. Mitochondrial and sarcolemmal localization of large-conductance KCa channels in cardiac myocytes was investigated with immunocytochemical staining of isolated cardiac myocytes. RESULTS:Desflurane significantly reduced infarct size compared with control animals (7.4% ± 0.8% vs 51.3% ± 6.1%; P < 0.05). Activation of large-conductance KCa channels by NS1619 (7.5% ± 1.8%; P < 0.05) mimicked and blockade of large-conductance KCa channels by iberiotoxin (49.1% ± 7.5%) abrogated desflurane-induced preconditioning. PKA blockade by H-89 abolished desflurane-induced (45.1% ± 4.0%) but not NS1619-induced (9.0% ± 2.4%, P < 0.05) preconditioning. Immunocytochemical staining revealed that large-conductance KCa channels were localized in the mitochondria but not in the sarcolemma of cardiac myocytes. CONCLUSION:These data suggest that desflurane-induced APC is mediated in part by activation of mitochondrial large-conductance KCa channels, and that activation of these channels by desflurane is mediated by PKA.

Comparison of isoflurane-, sevoflurane-, and desflurane-induced pre- and postconditioning against myocardial infarction in mice in vivo.

The murine in vivo model of acute myocardial infarction is increasingly used to investigate anesthetic-induced preconditioning (APC) and postconditioning (APOST). However, in mice the potency of different volatile anesthetics to reduce myocardial infarct size (IS) has never been investigated systematically nor in a head to head comparison with regard to ischemic preconditioning (IPC) and postconditioning (IPOST). Male C57BL/6 mice were subjected to 45 min of coronary artery occlusion (CAO) and 180 min of reperfusion. To induce APC, 1.0 MAC isoflurane (ISO), sevoflurane (SEVO) or desflurane (DES) was administered 30 min prior to CAO for 15 min. In an additional group, ISO was administered 45 min prior to CAO for 30 min. To induce APOST, 1.0 MAC ISO, SEVO or DES was administered for 18 min starting 3 min prior to the end of CAO. IPC was induced by 3 or 6 cycles of 5 min ischemia/reperfusion, 40 or 60 min prior to CAO, respectively. IPOST was induced by 3 cycles of 30 sec reperfusion/ischemia at the beginning of reperfusion. Area at risk (AAR) and IS were determined with Evans Blue and TTC staining, respectively. IS (IS/AAR) was 50 ± 4% (mean ± SEM) in the control group and was significantly (*P < 0.05) reduced by 3×5 IPC (26 ± 3%*), 6×5 IPC (26 ± 4%*), IPOST (20 ± 2%*), ISO APOST (19 ± 1%*), SEVO APOST (15 ± 1%*), DES APOST (14 ± 2%*) and SEVO APC (27 ± 6%*). ISO APC significantly reduced IS compared to control when administered 30 min (33 ± 4%*), but not when administered 15 min (48 ± 6%). DES APC significantly reduced IS compared to control and to SEVO APC (7 ± 1%*). Within the paradigm of preconditioning, the potency of volatile anesthetics to reduce myocardial infarct size in mice significantly increases from ISO over SEVO to DES, whereas within the paradigm of postconditioning the potency of these volatile anesthetics to reduce myocardial infarct size in mice is similar.

Desflurane-induced Postconditioning Is Mediated by β-Adrenergic SignalingRole of β1- and β2-Adrenergic Receptors, Protein Kinase A, and Calcium/Calmodulin-dependent Protein Kinase II

Background:Anesthetic preconditioning is mediated by β- adrenergic signaling. This study was designed to elucidate the role of β-adrenergic signaling in desflurane-induced postconditioning. Methods:Pentobarbital-anesthetized New Zealand White rabbits were subjected to 30 min of coronary artery occlusion followed by 3 h of reperfusion and were randomly assigned to receive vehicle (control), 1.0 minimum alveolar concentration of desflurane, esmolol (30 mg · kg−1 · h−1) for the initial 30 min of reperfusion or throughout reperfusion, the β2-adrenergic receptor blocker ICI 118,551 (0.2 mg/kg), the protein kinase A inhibitor H-89 (250 μg/kg), or the calcium/calmodulin-dependent protein kinase II inhibitor KN-93 (300 μg/kg) in the presence or absence of desflurane. Protein expression of protein kinase B, calcium/calmodulin-dependent protein kinase II, and phospholamban was measured by Western immunoblotting. Myocardial infarct size was assessed by triphenyltetrazolium staining. Results:Infarct size was 57 ± 5% in control. Desflurane postconditioning reduced infarct size to 36 ± 5%. Esmolol given during the initial 30 min of reperfusion had no effect on infarct size (54 ± 4%) but blocked desflurane-induced postconditioning (58 ± 5%), whereas esmolol administered throughout reperfusion reduced infarct size in the absence or presence of desflurane to 42 ± 6% and 41 ± 7%, respectively. ICI 118,551 and KN-93 did not affect infarct size (62 ± 4% and 62 ± 6%, respectively) but abolished desflurane-induced postconditioning (57 ± 5% and 64 ± 3%, respectively). H-89 decreased infarct size in the absence (36 ± 5%) or presence (33 ± 5%) of desflurane. Conclusions:Desflurane-induced postconditioning is mediated by β-adrenergic signaling. However, β-adrenergic signaling displays a differential role in cardioprotection during reperfusion.

Characterization, Design, and Function of the Mitochondrial Proteome: From Organs to Organisms

Mitochondria are a common energy source for organs and organisms; their diverse functions are specialized according to the unique phenotypes of their hosting environment. Perturbation of mitochondrial homeostasis accompanies significant pathological phenotypes. However, the connections between mitochondrial proteome properties and function remain to be experimentally established on a systematic level. This uncertainty impedes the contextualization and translation of proteomic data to the molecular derivations of mitochondrial diseases. We present a collection of mitochondrial features and functions from four model systems, including two cardiac mitochondrial proteomes from distinct genomes (human and mouse), two unique organ mitochondrial proteomes from identical genetic codons (mouse heart and mouse liver), as well as a relevant metazoan out-group (drosophila). The data, composed of mitochondrial protein abundance and their biochemical activities, capture the core functionalities of these mitochondria. This investigation allowed us to redefine the core mitochondrial proteome from organs and organisms, as well as the relevant contributions from genetic information and hosting milieu. Our study has identified significant enrichment of disease-associated genes and their products. Furthermore, correlational analyses suggest that mitochondrial proteome design is primarily driven by cellular environment. Taken together, these results connect proteome feature with mitochondrial function, providing a prospective resource for mitochondrial pathophysiology and developing novel therapeutic targets in medicine.

Activation of peroxisome-proliferator-activated receptors α and γ mediates remote ischemic preconditioning against myocardial infarction in vivo

Remote ischemic preconditioning (remote IPC) elicits a protective cardiac phenotype against myocardial ischemic injury. The remote stimulus has been hypothesized to act on major signaling pathways; however, its molecular targets remain largely undefined. We hypothesized that remote IPC exerts its effects by activating the peroxisome-proliferator-activated receptors (PPARs) α and γ, which have been previously implicated in cardioprotective signaling. Male New Zealand white rabbits (n = 78) were subjected to a 30-min coronary artery occlusion followed by three hours of reperfusion. Three cycles of remote IPC consisting of 10-min renal ischemia/reperfusion were performed. The animals either received the PPARα-antagonist GW6471 or the PPARγ-antagonist GW9662 alone or combined with remote IPC. Infarct size was determined gravimetrically. Tissue levels of 15d-prostaglandin J2 (15d-PGJ2), as well as the PPAR DNA binding were measured using specific assays. Reverse transcriptase polymerase chain reaction was used to analyze changes in endothelial nitric oxide synthase or inducible nitric oxide synthase (iNOS) mRNA expression in relative quantity (RQ). Data are mean ± SD. As a result, remote IPC significantly reduced the myocardial infarct size (42.2 ± 4.9%* versus 61 ± 1.9%), accompanied by an increased PPAR DNA-binding (189.6 ± 19.8RLU* versus 44.4 ± 9RLU), increased iNOS expression (3.5 ± 1RQ* versus 1RQ), as well as 15d-PGJ2 levels (179.7 ± 7.9 pg/mL* versus 127.9 ± 7.6 pg/mL). The protective response elicited by remote IPC, as well as the accompanying molecular changes were abolished by inhibiting PPARα (56.8 ± 4.7%; 61.1 ± 14.2RLU; and 1.91 ± 0.96RQ, respectively) or PPARγ (57.4 ± 3.3%; 52.7 ± 16.9RLU; and 1.54 ± 0.25RQ, respectively). (*Significantly different from control P < 0.05). In conclusion, the obtained results indicate that both PPARα and PPARγ play an essential role in remote IPC against myocardial infarction, impinging on the transcriptional control of iNOS expression.

Differential Role of Pim-1 Kinase in Anesthetic-induced and Ischemic Preconditioning against Myocardial Infarction

Background:Ischemic preconditioning (IPC) and anesthetic-induced preconditioning against myocardial infarction are mediated via protein kinase B. Pim-1 kinase acts downstream of protein kinase B and was recently shown to regulate cardiomyocyte survival. The authors tested the hypothesis that IPC and anesthetic-induced preconditioning are mediated by Pim-1 kinase. Methods:Pentobarbital-anesthetized male C57Black/6 mice were subjected to 45 min of coronary artery occlusion and 3 h of reperfusion. Animals received no intervention, Pim-1 kinase inhibitor II (10 &mgr;g/g intraperitoneally), its vehicle dimethyl sulfoxide (10 &mgr;l/g intraperitoneally), or 1.0 minimum alveolar concentration desflurane alone or in combination with Pim-1 kinase inhibitor II (10 &mgr;g/g intraperitoneally). IPC was induced by three cycles of 5 min ischemia–reperfusion each, and animals received IPC either alone or in combination with Pim-1 kinase inhibitor II (10 &mgr;g/g intraperitoneally). Infarct size was determined with triphenyltetrazolium chloride, and area at risk was determined with Evans blue (Sigma-Aldrich, Taufkirchen, Germany). Protein expression of Pim-1 kinase, Bad, phospho-BadSer112, and cytosolic content of cytochrome c were measured using Western immunoblotting. Results:Infarct size in the control group was 47 ± 2%. Pim-1 kinase inhibitor II (44 ± 2%) had no effect on infarct size. Desflurane (17 ± 3%) and IPC (19 ± 2%) significantly reduced infarct size compared with control (both P < 0.05 vs. control). Blockade of Pim-1 kinase completely abrogated desflurane-induced preconditioning (43 ± 3%), whereas IPC (35 ± 3%) was blocked partially. Desflurane tended to reduce cytosolic content of cytochrome c, which was abrogated by Pim-1 kinase inhibitor II. Conclusion:These data suggest that Pim-1 kinase mediates at least in part desflurane-induced preconditioning and IPC against myocardial infarction in mice.

Differential Role of Calcium/Calmodulin-dependent Protein Kinase II in Desflurane-induced Preconditioning and Cardioprotection by Metoprolol : Metoprolol Blocks Desflurane-induced Preconditioning

Background:Anesthetic preconditioning is mediated by &bgr;- adrenergic signaling. This study tested the hypotheses that desflurane-induced preconditioning is dose-dependently blocked by metoprolol and mediated by calcium/calmodulin-dependent protein kinase II (CaMK II). Methods:Pentobarbital-anesthetized New Zealand White rabbits were instrumented for measurement of systemic hemodynamics and subjected to 30 min of coronary artery occlusion followed by 3 h of reperfusion. Rabbits were assigned to receive vehicle (control), 0.2, 1.0, 1.75, or 2.5 mg/kg metoprolol for 30 min, or the CaMK II inhibitor KN-93 in the absence or presence of 1.0 minimum alveolar concentration desflurane. Protein expression of CaMK II, phospholamban, and phospho-phospholamban was measured by Western blotting. Myocardial infarct size and area at risk were measured with triphenyltetrazolium staining and patent blue, respectively. Results:Baseline hemodynamics were not different among groups. Infarct size was 60 ± 3% in control and significantly (* P < 0.05) decreased to 33 ± 2%* by desflurane. The CaMK II inhibitor KN-93 did not affect infarct size (55 ± 4%) but blocked desflurane-induced preconditioning (57 ± 3%). Metoprolol at 0.2 and 1.0 mg/kg had no effect on infarct size (55 ± 3% and 53 ±3%), whereas metoprolol at 1.75 and 2.5 mg/kg reduced infarct size to 48 ± 4%* and 39 ± 5%*, respectively. Desflurane-induced preconditioning was attenuated by metoprolol at 0.2 mg/kg, leading to an infarct size of 46 ± 5%*, and was completely abolished by metoprolol at 1.0, 1.75, and 2.5 mg/kg, resulting in infarct sizes of 51 ± 3%, 52 ± 3%, and 55 ± 3%, respectively. Conclusions:Desflurane-induced preconditioning is dose-dependently blocked by metoprolol and mediated by CaMK II.

Peroxisome‐proliferator‐activated receptor γ mediates the second window of anaesthetic‐induced preconditioning

The second window of anaesthetic‐induced preconditioning (APC) is afforded by the interplay of multiple signalling pathways, whereas a similar protective response is mediated by peroxisome‐proliferator‐activated receptor γ (PPARγ) agonists. However, a possible role of this nuclear receptor during APC has not been studied to date. We investigated the hypothesis that the second window of APC is mediated by the activation of PPARγ. New Zealand White rabbits (n= 48) were subjected to 30 min of coronary artery occlusion followed by 3 h of reperfusion. The animals received desflurane (1.0 minimal alveolar concentration), the PPARγ antagonist GW9662, as well as the combined application of both, respectively, 24 h prior to coronary artery occlusion. Infarct size was determined gravimetrically; tissue levels of 15‐deoxy‐Δ12,14‐prostaglandin J2 (15d‐PGJ2) and nitrite/nitrate (NOx), as well as PPAR DNA binding were measured using specific assays. Data are presented as means ±s.e.m. Desflurane led to a reduced myocardial infarct size (41.7 ± 2.5 versus 61.8 ± 2.8%, P < 0.05), accompanied by significantly increased PPAR DNA binding (289.9 ± 33 versus 102.9 ± 18 relative light units, P < 0.05), as well as elevated tissue levels of 15d‐PGJ2 (224.4 ± 10.2 versus 116.9 ± 14.2 pg ml−1, P < 0.05) and NOx (14.9 ± 0.7 versus 5.4 ± 0.7 μm, P < 0.05). Pharmacological inhibition of PPARγ abolished these protective effects, resetting the infarct size (56.5 ± 2.9%), as well as PPAR DNA‐binding activity (91.2 ± 31 relative light units) and NOx tissue levels (5.9 ± 0.9 μm) back to control levels. Desflurane governs a second window of APC by increasing the production of 15d‐PGJ2, subsequently activating PPARγ, resulting in a diminished myocardial infarct size by increasing the downstream availability of NO.

Isoflurane protects the myocardium against ischemic injury via the preservation of mitochondrial respiration and its supramolecular organization.

BACKGROUND:Isoflurane has been demonstrated to limit myocardial ischemic injury. This effect is hypothesized to be mediated in part via effects on mitochondria. We investigated the hypothesis that isoflurane maintains mitochondrial respiratory chain functionality, in turn limiting mitochondrial damage and mitochondrial membrane disintegration during myocardial ischemic injury. METHODS:Mice (9–12 weeks of age) received isoflurane (1.0 minimum alveolar concentration) 36 hours before a 30-minute coronary artery occlusion that was followed by 24 hours of reperfusion. Cardiac mitochondria were isolated at a time point corresponding to 4 hours of reperfusion. 2,3,5-Triphenyltetrazoliumchloride staining was used to determine myocardial infarct size. Mitochondrial respiratory chain functionality was investigated using blue native polyacrylamide gel electrophoresis, as well as specific biochemical assays. Mitochondrial lipid peroxidation was quantified via the formation of malondialdehyde; mitochondrial membrane integrity was assessed by Ca2+-induced swelling. Protein identification was achieved via liquid chromatography mass spectrometry/mass spectrometry. RESULTS:Thirty-one mice were studied. Mice receiving isoflurane displayed a reduced myocardial infarct size (P = 0.0011 versus ischemia/reperfusion [I/R]), accompanied by a preserved activity of respiratory complex III (P = 0.0008 versus I/R). Isoflurane stabilized mitochondrial supercomplexes consisting of oligomers from complex III/IV (P = 0.0086 versus I/R). Alleviation of mitochondrial damage after isoflurane treatment was further demonstrated as decreased malondialdehyde formation (P = 0.0019 versus I/R) as well as a diminished susceptibility to Ca2+-induced swelling (P = 0.0010 versus I/R). CONCLUSIONS:Our findings support the hypothesis that isoflurane protects the heart from ischemic injury by maintaining the in vivo functionality of the mitochondrial respiratory chain. These effects may result in part from the preservation of mitochondrial supramolecular organization and minimized oxidative damage, circumventing the loss of mitochondrial membrane integrity.