Search

Although protection against necrosis has been observed in both hibernating (HIB) and ischemic preconditioned hearts in the second window of protection (SWOP), a comparison of the mitochondrial proteome between the two entities has not been previously performed. Anesthetized swine underwent instrumentation with a fixed constrictor around the LAD artery and were followed for 12 weeks (HIB; N=7). A second group of anesthetized swine underwent ischemic preconditioning by inflating a balloon within the LAD artery 10 times for 2 min, each separated by 2 min reperfusion and were sacrificed 24h later (SWOP; N=7). Myocardial blood flow and high-energy nucleotides were obtained in the LAD region and normalized to remote regions. Post-sacrifice, protein content as measured with iTRAQ was compared in isolated mitochondria from the LAD area of a Sham heart. Basal regional blood flow in the LAD region when normalized to the remote region was 0.86±0.04 in HIB and 1.02±0.02 in SWOP tissue (P<0.05). Despite reduced regional blood flows in HIB hearts, ATP content in the LAD region, when normalized to the remote region was similar in HIB versus SWOP (1.06±0.06 and 1.02±0.05 respectively; NS) as was the transmural phosphocreatine (PCr) to ATP ratio (2.1±0.2 and 2.2±0.2 respectively; NS). Using iTRAQ, 64 common proteins were identified in HIB and SWOP hearts. Compared with SWOP, the relative abundance of mitochondrial proteins involved with electron transport chain (ETC) were reduced in HIB including NADH dehydrogenase, Cytochrome c reductase and oxidase, ATP synthase, and nicotinamide nucleotide transhydrogenase. Within chronically HIB heart tissue with reduced blood flow, the relative abundance of mitochondrial ETC proteins is decreased when compared with SWOP tissue. These data support the concept that HIB heart tissue subjected to chronically reduced blood flow is associated with a down-regulation in the expression of key mitochondrial proteins involved in electron transport., (Published by Elsevier Ltd.)

Altered expression of mitochondrial electron transport proteins has been shown in early preconditioned myocardial tissue. We wished to determine whether these alterations persist in the Second Window of Protection (SWOP) and if so, whether a favorable energetic state is facilitated during subsequent ischemia. Fourteen pigs underwent a SWOP protocol with ten 2-minute balloon inflations in the LAD artery, each separated by 2 minutes reperfusion. Twenty-four hours later, mitochondria were isolated from SWOP and SHAM pig hearts and analyzed for uncoupling protein (UCP)-2 content by western blot analysis, proteomic changes by iTRAQ(®) and respiration by an oxygen electrode. In parallel in vivo studies, high-energy nucleotides were obtained by transmural biopsy from anesthetized SWOP and SHAM pigs at baseline and during sustained low-flow ischemia. Compared with SHAM mitochondria, ex vivo SWOP heart tissue demonstrated increased expression of UCP-2, Complex IV (cytochrome c oxidase) and Complex V (ATPase) proteins. In comparison with SHAM pigs during in vivo conditions, transmural energetics in SWOP hearts, as estimated by the free energy of ATP hydrolysis (ΔG(0)), were similar at baseline but had decreased by the end of low-flow ischemia (-57.0 ± 2.1 versus -51.1 ± 1.4 kJ/mol; P < 0.05). In conclusion, within isolated mitochondria from preconditioned SWOP hearts, UCP-2 is increased and in concert with enhanced Complex IV and V proteins, imparts a favorable energetic state during low-flow ischemia. These data support the notion that mitochondrial adaptations that may reduce oxidant damage do not reduce the overall efficiency of energetics during sustained oxygen deprivation.

Objective: Clinical studies indicate incomplete functional recovery of hibernating myocardium after coronary artery bypass grafting. We hypothesized that persistent contractile abnormalities after coronary artery bypass grafting are associated with decreased mitochondrial proteins involving electron transport chain that might limit maximal oxygen consumption., Methods: Seven pigs with hibernating myocardium underwent off-pump revascularization with left internal thoracic artery to mid left anterior descending artery. At 4 weeks, left internal thoracic artery anastomosis was patent by multidetector computed tomography. Regional function (transthoracic echocardiography) and blood flow (microspheres) were assessed at rest and during high-dose dobutamine (40 μg/[kg · min]). Expression of electron transport chain proteins was analyzed with isobaric tags for relative and absolute quantification., Results: After revascularization, multidetector computed tomography confirmed severe left anterior descending stenosis and patent left internal thoracic artery graft. Regional function and blood flow normalized at rest; however, function in left anterior descending distribution remained depressed relative to remote regions, and myocardial blood flow in that region did not increase normally when challenged with high-work state. Concomitant with reduced maximal blood flow response in left anterior descending region was more than 40% reduction in electron transport chain proteins essential to adenosine triphosphate production., Conclusions: Despite successful revascularization of hibernating myocardium, regional function and blood flow remained depressed during catecholamine stress. Electron transport chain proteins known to be downregulated during adaptive process within hibernating myocardium did not normalize after revascularization. These data demonstrate a potential bioenergetic cause of persistent dysfunction and heart failure within successfully revascularized hibernating myocardium., (Copyright © 2011 The American Association for Thoracic Surgery. All rights reserved.)

In preconditioned myocardium, activation of the mitogen-activated protein kinase (MAPK) p38 leads to increased glucose uptake via enhanced GLUT-4 translocation. Glucose uptake is also increased in chronic hibernating myocardium, but the role of p38 MAPK and GLUT-4 translocation has not been studied. Nine swine underwent instrumentation of the proximal left anterior descending coronary artery (LAD) with a small, external constrictor. At 3 mo after instrumentation, myocardial glucose uptake by PET imaging was higher in the LAD than in the remote region under basal, fasted conditions (0.08 +/- 0.02 vs. 0.04 +/- 0.01 micromol.min(-1).g(-1), P < 0.05). Compared with the remote region, the LAD region demonstrated increased membrane-bound GLUT-4 relative to total content (61 +/- 04 vs. 45 +/- 06%, P < 0.05), higher glycogen (28.37 +/- 4.41 vs. 19.26 +/- 1.87 mg/g wet wt, P < 0.05), and increased inducible nitric oxide synthase (NOS) activity (1.43 +/- 0.34 vs. 0.51 +/- 0.21 activity/mg protein, P < 0.05). p38 MAPK was 47 +/- 14% higher in the LAD than in the remote region (P < 0.05) and correlated well with the absolute degree of GLUT-4 membrane-bound translocation (r = 0.81, P < 0.01), relative increase in glycogen (r = 0.70, P < 0.05), and total NOS activity (r = 0.68, P < 0.05). In chronic hibernating myocardial tissue, p38 MAPK activation is increased under basal fasted conditions and correlates well with the increased degree of GLUT-4 translocation, glycogen accumulation, and NOS activity. As in preconditioned myocardium, activation of p38 MAPK may play an important role in the metabolic adaptations that characterize chronic hibernating myocardium.

Background: In patients with hibernating myocardium, regional uptake of the glucose analog 2-fluorine 18-fluoro-2-deoxy-d-glucose (FDG) is increased under resting conditions. It is unclear whether the degree of increased FDG uptake correlates with the degree of impaired blood flow response and whether chronic changes in the glucose transporters may play a role in the enhanced FDG uptake under fasted conditions., Methods and Results: Twelve swine were instrumented with a constrictor on the left anterior descending (LAD) artery. Serial echocardiography and positron emission tomography studies were done to assess temporal changes in myocardial function, blood flow, and FDG uptake. One week after surgery (early study), wall thickening, blood flow, and postdobutamine FDG uptake in LAD and remote territories were similar. By approximately 6 weeks (late study), baseline wall thickening in the LAD region was lower than in remote regions (20% +/- 7% and 36% +/- 6%, P <.05), as was dobutamine-stimulated blood flow (0.92 +/- 0.16 mL. min(-1). g(-1) and 1.17 +/- 0.20 mL. min(-1). g(-1) in LAD and remote regions, respectively; P <.05). After the dobutamine infusion, FDG uptake in the LAD region during fasted conditions was higher than in remote regions (0.128 +/- 0.053 micromol. min(-1). g(-1) and 0.098 +/- 0.044 micromol. min(-1). g(-1), respectively; P <.05), and the increase was proportional to the impairment in dobutamine blood flow (r(2) = 0.62, P <.001). After the animals were killed, the LAD region showed a higher content of GLUT4 by immunoblots and a greater degree of translocation as estimated by immunohistochemistry. In 5 additional hibernating pigs studied under resting fasted conditions, FDG uptake and GLUT4 translocation were also higher in the LAD region, in the absence of dobutamine stimulation., Conclusions: In hibernating myocardium, regional FDG uptake under fasting conditions is higher than in remote regions, both at rest and after an infusion of dobutamine. The degree of poststress FDG uptake is proportional to the impaired stress-induced blood flow. Total GLUT4 content as well as membrane-bound protein is higher in the hibernating tissue, and these changes may facilitate the observed increase in FDG uptake.

Unlabelled: The goal of this study was to determine whether myocardial glucose uptake after repetitive ischemia differs in response to coronary occlusion-reperfusion versus supply-demand ischemia induced by dobutamine. Although glucose metabolism is increased after myocardial ischemia, the metabolic effect of supply-demand ischemia induced by dobutamine may increase glucose metabolism within remote myocardium. This would make it difficult to discriminate postischemic from remote myocardium with glucose tracers., Methods: Eighteen swine with a hydraulic occluder and flow probe on the circumflex artery underwent repetitive ischemia. In group 1 (n = 9), the circumflex artery was occluded, whereas in group 2 (n = 9), circumflex flow was decreased by 30% before dobutamine (40 micro g/kg/min intravenously). Each pig underwent 15 min of ischemia, twice per day for 5 d. Echocardiography and PET to determine myocardial glucose ((18)F-FDG) uptake were performed after final ischemia, and tissue was later analyzed for activation of Akt, p38 mitogen-activated protein, and adenosine monophosphate (AMP) kinase., Results: Wall thickening in the circumflex region was lower than in remote regions in both groups. (18)F-FDG uptake in the circumflex region was similar in groups 1 and 2 (0.22 +/- 0.03 and 0.23 +/- 0.04 micro mol/min/g, respectively; not statistically significant). In the remote region, (18)F-FDG uptake was lower than in the circumflex region in group 1 (0.14 +/- 0.03 micro mol/min/g; P < 0.05) but was similar to that in the circumflex region in group 2 (0.20 +/- 0.03 micro mol/min/g; not statistically significant). AMP kinase activity in the remote region was significantly lower than in the circumflex region in group 1 but was similar to that in the circumflex region in group 2., Conclusion: Unlike repetitive coronary artery occlusion-reperfusion, repetitive supply-demand ischemia with dobutamine alters glucose uptake within the remote myocardium, possibly as a result of AMP kinase activation. Clinically, these data suggest that (18)F-FDG studies have a limited role in discriminating postischemic from remote myocardium after dobutamine stress.

Repetitive myocardial ischemia increases glucose uptake, but the effect on glycogen is unclear. Thirteen swine instrumented with a hydraulic occluder on the circumflex (Cx) artery underwent 10-min occlusions twice per day for 4 days. After 24 h postfinal ischemia and in the fasted state, echocardiogram and positron emission tomography imaging for blood flow ([(13)N]-ammonia) and 2-[(18)F]fluoro-2-deoxy-D-glucose (FDG) uptake were obtained. Tissue was then collected for ATP, creatine phosphate (CP), glycogen, and glucose transporter-4 content, and hexokinase activity. After reperfusion, regional function and CP-to-ATP ratios in the Cx and remote regions were similar. Despite the absence of stunning, the Cx region demonstrated higher glycogen levels (33 +/- 11 vs. 24 +/- 11 micromol/g; P < 0.05), and this increase correlated well with the increase in FDG uptake (r(2) = 0.78; P < 0.01). Hexokinase activity was also increased relative to remote regions (0.62 +/- 0.29 vs. 0.37 +/- 0.19 IU/g; P < 0.05), with no difference in GLUT-4 content. In summary, 24 h after repetitive ischemia, glucose uptake and glycogen levels are increased at a time that functional and bioenergetic markers of stunning have recovered. The significant correlation between glycogen content and FDG accumulation in the postischemic region suggests that increased rates of glucose transport and/or phosphorylation are linked to increased glycogen levels in hearts subjected to repetitive bouts of ischemia.

Within hibernating myocardium, it is uncertain whether a normal energetic state is present at baseline and whether maintaining that energy state during a catecholamine challenge is dependent on ATP-dependent potassium channel opening. In this study, 16 swine underwent a thoracotomy with placement of an external constrictor on the left anterior descending coronary artery (LAD) (hibernation model). Seven additional swine underwent a sham operation. At 10 wk, the myocardial energetic state in the LAD region was assessed by 31P-NMR spectroscopy, and the ratio of phosphocreatine to ATP (PCr/ATP) was determined at baseline, during glibenclamide treatment (0.5 mg/kg bolus with 50 μg/min iv), and during addition of dobutamine (40 μg·kg-1·min-1 iv). At baseline, transmural blood flow in the LAD and remote region was 0.75 ± 0.11 and 0.88 ± 0.09 ml·min-1·g-1, respectively (P < 0.01), in hibernating hearts and 0.83 ± 0.12 and 0.88 ± 0.15 ml·min-1·g-1, respectively (not significant), in sham-operated hearts. Under basal conditions, PCr/ATP in the LAD region of hibernating and sham pigs was 2.15 ± 0.04 and 2.11 ± 0.05, respectively (not significant). In sham pigs, addition of dobutamine to glibenclamide increased the double product from 10.4 ± 0.8 to 23.9 ± 4.0 mmHg·beats·min-1 X 1,000 (P < 0.05) and decreased transmural PCr/ATP from 2.06 ± 0.06 to 1.69 ± 0.06 (P < 0.05). Dobutamine increased the double product in hibernating pigs in a similar fashion and, despite a 40% lower blood flow response, induced an equivalent decrease in PCr/ATP from 2.04 ± 0.04 to 1.73 ± 0.08 (P < 0.05). In conclusion, we found that, in chronic hibernating swine myocardium with reduced basal blood flow and perfusion reserve, the transmural energetic state, defined by PCr/ATP, is normal during addition of dobutamine, despite inhibition of ATP-dependent potassium channel opening with glibenclamide. These data suggest that important adaptations other than the ATP-dependent potassium channel opening allow hibernating myocardium to operate over a lower range of the oxygen supply-demand relationship to protect against myocardial ischemia. [ABSTRACT FROM AUTHOR]

Introduction: Studies of hibernating myocardium suggest that functional recovery following CABG may be incomplete. Application of stem cells at the time of CABG may have promise for improving function, possibly by enhanced expression of mitochondrial antioxidant proteins. Hypothesis: The application of MSCs on a vicryl-based patch that is sutured onto the epicardium at the time of CABG enhances the expression of mitochondrial antioxidant proteins. Methods: Anesthetized swine underwent thoracotomy with placement of a constrictor around the LAD. At 12 weeks, pigs underwent off-pump revascularization with a LIMA graft and a vicryl patch seeded with (N=4) or without (N=4) MSCs (4 million cells, CD90+, CD105+ & CD45-) was sutured to the surface. At 4 weeks, regional function with MRI and expression of mitochondria proteins by TMT proteomics were determined. Results: At 4 weeks following CABG, swine that received the stem cell patch showed improvement in regional wall thickening in response to inotropic stimulation, indicating improved recovery of function as compared to revascularization alone (63±22% versus 47±2%; P<0,05). As shown in the Figure, expression of PGC1α as well as mitochondrial antioxidant proteins from the chronically ischemic territories were higher in animals with cell-based patch compared to the pigs that received CABG without a cell-based therapy. Conclusions: In summary, placement of an epicardial patch loaded with MSCs at the time of CABG improves contractile reserve within the revascularized territory, potentially by enhanced expression of mitochondrial antioxidant proteins, potentially by a PGC1α mechanism. These data support the utility of a cell-based approach for hibernating myocardial regions at the time of CABG, potentially by enhanced antioxidant pathways. [ABSTRACT FROM AUTHOR]

Introduction: Coenzyme Q10 (CoQ10) has been shown to reduce oxidant stress in ischemic heart tissue, though it is unclear whether it alters the expression of anti-oxidant proteins within mitochondria from the ischemic territory. Hypothesis: Using a swine model of chronic myocardial ischemia, we tested whether daily administration of CoQ10for 4-weeks would enhance the expression of key anti-oxidant proteins by TMT proteomics in mitochondria from the chronically ischemic regions. Methods: Twelve pigs underwent placement of a constrictor around the LAD artery and had reduced regional function at 12 weeks. Pigs were given daily dietary supplements of either CoQ10 (10 mg/kg/day) or placebo and studied with 2-D ECHO 4 weeks later. Animals were sacrificed and mitochondria were isolated from the LAD regions, for analysis of PGC1 alpha by western gels and mitochondrial antioxidant proteins by TMT Proteomics. Results: At 12 weeks following instrumentation, regional wall thickening in the LAD region of the Placebo and CoQ10 regions was lower than remote regions (34±7% versus 62±7%; P<0.01), consistent with the presence of chronic myocardial ischemia. Following 4-weeks of treatment, repeat estimates of regional wall thickening in the LAD and Remote regions were 41±5% versus 72±7% (P<0.01), suggesting minimal change in regional function. Post-sacrifice, mitochondria were isolated from the ischemic regions and analyzed for PGC1 alpha and anti-oxidant protein expression. As shown in the Figure, the CoQ10 treated-pigs demonstrated higher levels of PGC1alpha and enhanced anti-oxidant proteins, as measured by TMT proteomics compared with Placebo treated pigs (Significant in yellow). Conclusions: In summary, in this model of chronic myocardial ischemia, supplementation with CoQ10 at 4 weeks improves the expression of anti-oxidant proteins in the mitochondria, potentially by increased PGC1 alpha signaling. [ABSTRACT FROM AUTHOR]