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,1
,
,
Departments of Medicine (Divisions of
* Cardiology and
Clinical Pharmacology) and
Pharmacology, Case Western Reserve University and Medical Service,
Louis Stokes Veterans Affairs Medical Center, Cleveland, Ohio, USA
1Correspondence: Cardiology Section, Medical Service 111(W), Louis Stokes VA Medical Center, 10701 East Blvd., Cleveland, OH 44106, USA. E-mail: EXL9{at}cwru.edu
SPECIFIC AIMS
Aging impairs mitochondrial oxidative metabolism in interfibrillar mitochondria (IFM) located between the myofibrils, with decreases in the rate of oxidative phosphorylation and decreased activity of electron transport complexes III and IV. The first aim of this study was to ask whether aging-induced defects in mitochondrial metabolism can be restored. The aged heart sustains greater damage during ischemia and reperfusion. The second aim of the study was to address the question, if the aging defects in mitochondrial respiration could be attenuated, would the aged heart now function as the adult heart and sustain less damage following ischemia and reperfusion?
PRINCIPAL FINDINGS
1. Acetylcarnitine treatment restores oxidative phosphorylation and complex III activity in IFM from the aged heart
Aging impairs cardiac mitochondrial metabolism, with decreases in the rate of oxidative phosphorylation and increased production of reactive oxygen species (ROS). Cardiac mitochondria exist in two functionally distinct populations. Subsarcolemmal mitochondria (SSM) lie beneath the plasma membrane. Interfibrillar mitochondria (IFM) are located between the myofibrils. Aging decreases oxidative phosphorylation and increases oxyradical production only in IFM, whereas SSM are unaffected. Aging decreases the activity of electron transport complexes III and IV in IFM. Treatment of aged rats with acetylcarnitine increased the maximal rate of oxidative phosphorylation in IFM to rates observed in the adult heart (glutamate respiration: 6 month Fischer 344 adult: 287±19 nAO/min/mg protein, n=8, ±SE; 24 month Fischer 344 aged 221±10, n=13, P<0.05 vs. adult; 24 month acetylcarnitine-treated Fischer 344 aged 307±15 n=6, p=NS vs. adult, P<0.05 vs. untreated aged). Respiration in acetylcarnitine-treated aging hearts remained well coupled with unaltered state 4 respiration, respiratory control ratios, and ADP:O ratios.
Aging decreased the activity of complex III in IFM. Acetylcarnitine treatment of aged rats restored the activity of complex III in IFM to that present in the adult heart (adult: 4860±290 mU/min/mg protein, n=8; 24 month aged 4040±480, n=11; 24 month acetylcarnitine-treated aged 6250±870 n=5, p=NS vs. adult, P<0.05 vs. untreated aged). Acetylcarnitine increased the content of cytochrome b in IFM (untreated aged: 0.24±0.01 nmol/mg protein, n=9 vs. acetylcarnitine-treated aged 0.29±0.01, n=5, P<0.05) and in SSM (untreated aged: 0.20±0.02 nmol/mg protein, n=9 vs. acetylcarnitine-treated aged 0.32±0.01, n=5, P<0.05).
2. Restoration of aging defects in IFM decreases ischemia-reperfusion injury in the aged heart
Adult and aged hearts underwent 25 min of global ischemia, followed by 30 min of reperfusion. Acetylcarnitine treatment did not alter ventricular function measured before ischemia. Diastolic pressure at the end of ischemia was unaltered by acetylcarnitine in adult and aged hearts. In the aged heart, acetylcarnitine enhanced the recovery of developed pressure (Fig. 1
) and positive and negative dp/dt. In the adult heart, contractile recovery during reperfusion was unchanged by acetylcarnitine (Fig. 1)
. In acetylcarnitine-treated aged hearts, recovery was similar to the recovery of treated or untreated adult hearts. Acetylcarnitine markedly reduced the release of lactate dehydrogenase (LDH) during reperfusion, a marker of myocyte necrosis, in the aged heart (Fig. 2
). In contrast, acetylcarnitine treatment did not alter LDH release during reperfusion in the adult heart (Fig. 2)
. Thus, acetylcarnitine treatment of the aged heart before ischemia improved functional recovery and decreased myocyte cell death following ischemia and reperfusion.
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CONCLUSIONS AND SIGNIFICANCE
Myocardial injury is increased in the aged heart following ischemia and reperfusion. In the aged heart, IFM exhibit decreased protein yield and reduced rates of oxidative phosphorylation, complex III activity, and respiration through complex IV. Acetylcarnitine treatment restored oxidative phosphorylation in IFM from the aged heart whereas protein content remained decreased. Respiration remained tightly coupled after treatment with acetylcarnitine.
When the aged heart was exposed to ischemia and reperfusion after restoration of oxidative phosphorylation, there was less myocardial damage and improved contractile recovery. In contrast, acetylcarnitine treatment had no effect on the extent of myocardial damage or contractile recovery in the adult heart. Thus, acetylcarnitine did not alter mechanisms of ischemic damage common to both the adult and aged hearts. These observations provide strong support for the contribution of aging-related defects in mitochondrial oxidative metabolism to the enhanced damage following ischemia and reperfusion.
Aging decreased the maximal activity of complex III in IFM despite a preserved content of subunit peptides, including cytochrome b. The aging defect in complex III is located within the quinol oxidation domain of cytochrome b. Acetylcarnitine restored complex III activity in IFM from aged hearts, accompanied by an increase in cytochrome b content. The increase in cytochrome b content following relief of the complex III enzyme defect is in line with cytochrome b as the locus of the aging defect. Increased cytochrome b content observed in both SSM and IFM after treatment is consistent with an increase in mitochondrial transcription and protein synthesis, perhaps leading to an increased content of complex III.
Aging decreased the rate of oxidative phosphorylation stimulated by TMPD-ascorbate, an electron donor to cytochrome oxidase via cytochrome c in intact IFM. Acetylcarnitine treatment improved respiration through cytochrome oxidase in both SSM and IFM, consistent with previous results from other investigators.
In the aged heart, ischemia-induced defects in mitochondrial electron transport are superimposed on aging defects. Aging increases the production of ROS from complex III in IFM. Ischemia damages complexes III and IV in the adult and aged heart. Mitochondria produce ROS during ischemia and also during reperfusion. Complex III in IFM is a prime source for the enhanced production of ROS during ischemia or reperfusion. Relative blockage of electron flow through complex IV augments oxyradical production from upstream redox centers. Thus, restoration of oxidative phosphorylation through complexes III and IV in interfibrillar mitochondria by acetylcarnitine treatment should decrease oxidative damage during ischemia and reperfusion in the aged heart.
Acetylcarnitine may improve aging defects in electron transport by two potential mechanisms. First, acetylcarnitine may increase the content of cardiolipin, a phospholipid present in mitochondria that is required for optimal activity of complex IV. However, aging does not decrease the content of cardiolipin in either SSM or IFM in the aged Fischer 344 rat heart, making increased cardiolipin synthesis a less likely mechanism of protection. Second, treatment with acetylcarnitine has been shown to increase transcription of mitochondrial DNA in the aged heart in a dose- and time-dependent manner. The optimal response to acetylcarnitine occurred at 3 h and 300 mg/kg in the previous studies, the treatment regimen used in the current study. In the previous work, the content of mtRNA normalized in the aged heart after treatment secondary to increased RNA synthesis, which was accompanied by increased mitochondrial protein synthesis and an increased content of mitochondrial-encoded electron transport subunits after acetylcarnitine treatment. Based on the increases seen in the contents of cytochrome aa3 and b in the present study, it appears that stimulation of mitochondrial protein synthesis is a potential mechanism of acetylcarnitine-mediated improvement in respiration in the aged heart. Future studies are required to explore whether acetylcarnitine increases cardiolipin content or, what is more likely, the synthesis of new electron transport complex III. If accumulation of aging induced oxidative damage to cytochrome b decreases complex III activity during aging, then a newly synthesized complex III would likely exhibit a nonaged phenotype.
In the current study, systemic administration of acetylcarnitine to the rat in vivo 3 h before ischemia protected the isolated heart subsequently exposed to in vitro ischemia and reperfusion. No additional drug was administered to the isolated heart. Thus, pretreatment of aging defects in the animal rendered the aged heart relatively resistant to ischemic injury without additional treatment of the heart in vitro. Previous studies found that acetylcarnitine administered in vitro directly to the isolated adult heart had a minimal protective effect. These studies also found that in vivo acetylcarnitine treatment did not increase the activity of electron transport chain enzymes in the adult heart, further supporting the selective aging-related effect observed in the current study.
Despite successful and sustained reperfusion of the infarct zone, the mortality of acute myocardial infarction in elderly patients remains elevated 6-fold compared to middle-aged patients. Although pharmacologic treatment at the onset of reperfusion can reduce myocardial injury, the option to utilize chronic pretreatment of high risk elders in a preemptive fashion before ischemia represents a novel cardioprotective strategy for the aged heart. The ability to modulate aging-induced defects in mitochondrial metabolism in order to reduce damage from ischemia and reperfusion is an attractive potential strategy to protect myocardium in the high-risk elderly patient.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4535fje
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