Bioenergetic functions in subpopulations of heart mitochondria are preserved in a non-obese type 2 diabetes rat model (Goto-Kakizaki).

A distinct bioenergetic impairment of heart mitochondrial subpopulations in diabetic cardiomyopathy is associated with obesity; however, many type 2 diabetic (T2DM) patients with high-risk for cardiovascular disease are not obese. In the absence of obesity, it is unclear whether bioenergetic function in the subpopulations of mitochondria is affected in heart with T2DM. To address this issue, a rat model of non-obese T2DM was used to study heart mitochondrial energy metabolism, measuring bioenergetics and enzyme activities of the electron transport chain (ETC). Oxidative phosphorylation in the presence of substrates for ETC and ETC activities in both populations of heart mitochondria in T2DM rats were unchanged. Despite the preservation of mitochondrial function, aconitase activity in T2DM heart was reduced, suggesting oxidative stress in mitochondria. Our study indicate that metabolic function of heart mitochondria is unchanged in the face of oxidative stress and point to a critical role of obesity in T2DM cardiomyopathy.

Mitochondria as targets of drug toxicity: Lessons from the R118 phase I experience.

Low Wang et al. report the results of the phase I program for R118 in this issue.(2) R118 was designed as an activator of adenosine monophosphate activated protein kinase (AMPK) to treat claudication. The single ascending dose study in healthy subjects was characterized by an unacceptable number of serious adverse events and substantial risk to the participants. The probable mitochondrial mechanism underlying these adverse events suggests important lessons for future drug development.

Multiple mitochondrial alterations in a case of myopathy.

Mitochondrial alterations are the most common feature of human myopathies. A biopsy of quadriceps muscle from a 50-year-old woman exhibiting myopathic symptoms was examined by transmission electron microscopy. Biopsied fibers from quadriceps muscle displayed numerous subsarcolemmal mitochondria that contained crystalloids. Numbering 1-6 per organelle, these consisted of rows of punctuate densities measuring ∼0.34 nm; the parallel rows of these dots had a periodicity of ∼0.8 nm. The crystalloids were ensconced within cristae or in the outer compartment. Some mitochondria without crystalloids had circumferential cristae, leaving a membrane-free center that was filled with a farinaceous material. Other scattered fibrocyte defects included disruption of the contractile apparatus or its sporadic replacement by a finely punctuate material in some myofibers. Intramitochondrial crystalloids, although morphologically striking, do not impair organelle physiology to a significant degree, so the muscle weakness of the patient must originate elsewhere.

Mitochondria in the human heart.

The heart relies mainly on mitochondrial metabolism to provide the energy needed for pumping blood to oxygenate the organs of the body. The study of mitochondrial function in the human heart faces many obstacles and elucidation of the role of mitochondria in cardiac diseases has relied mainly on studies with animal models. Cardiac diseases are the leading cause of mortality worldwide. With the emergence of new therapies to treat and prevent heart disease, some aiming at metabolic modulation, a need for acquiring a better understanding of mitochondrial function in the human heart becomes apparent. Our review is aimed at specific evaluation of the human heart in terms of (1) methods to understand mitochondrial function, with particular emphasis on integrated function, (2) data on the role of mitochondrial dysfunction in cardiovascular disease, and (3) possible applications of this knowledge in the treatment of patients with cardiac disease.

A novel mutation in the mitochondrial tRNA(Ser(AGY)) gene associated with mitochondrial myopathy, encephalopathy, and complex I deficiency.

PURPOSE: To identify molecular defects in a girl with clinical features of MELAS (mitochondrial encephalomyopathy and lactic acidosis) and MERRF (ragged-red fibres) syndromes. METHODS: The enzyme complex activities of the mitochondrial respiratory chain were assayed. Temporal temperature gradient gel electrophoresis was used to scan the entire mitochondrial genome for unknown mitochondrial DNA (mtDNA) alterations, which were then identified by direct DNA sequencing. RESULTS: A novel heteroplasmic mtDNA mutation, G12207A, in the tRNA(Ser(AGY)) gene was identified in the patient who had a history of developmental delay, feeding difficulty, lesions within her basal ganglia, cerebral atrophy, proximal muscle weakness, increased blood lactate, liver dysfunction, and fatty infiltration of her muscle. Muscle biopsy revealed ragged red fibres and pleomorphic mitochondria. Study of skeletal muscle mitochondria revealed complex I deficiency associated with mitochondrial proliferation. Real time quantitative PCR analysis showed elevated mtDNA content, 2.5 times higher than normal. The tRNA(Ser(AGY)) mutation was found in heteroplasmic state (92%) in the patient's skeletal muscle. It was not present in her unaffected mother's blood or in 200 healthy controls. This mutation occurs at the first nucleotide of the 5' end of tRNA, which is involved in the formation of the stem region of the amino acid acceptor arm. Mutation at this position may affect processing of the precursor RNA, the stability and amino acid charging efficiency of the tRNA, and overall efficiency of protein translation. CONCLUSION: This case underscores the importance of comprehensive mutational analysis of the entire mitochondrial genome when a mtDNA defect is strongly suggested.

Relationship of primary mitochondrial respiratory chain dysfunction to fiber type abnormalities in skeletal muscle.

Variation in the size and relative proportion of type 1 and type 2 muscle fibers can occur in a number of conditions, including structural myopathies, neuropathies, and various syndromes. In most cases, the pathogenesis of such fiber type changes is unknown and the etiology is heterogeneous. Skeletal muscle mitochondrial respiratory chain analysis was performed in 10 children aged 3 weeks to 5 years with abnormalities in muscle fiber type, size, and proportion. Five children were classified as having definite, four as probable, and one as possible mitochondrial disease. Type 1 fiber predominance was the most common histological finding (six of 10). On light microscopy, four cases had subtle concomitants of a mitochondriopathy, including mildly increased glycogen, lipid, and/or succinate dehydrogenase staining, and one case had more prominent evidence of underlying mitochondrial disease with marked subsarcolemmal staining. Most cases (nine of 10) had abnormal mitochondrial morphology on electron microscopy. All were found to have mitochondrial electron transport chain (ETC) abnormalities and met diagnostic criteria for mitochondrial disease. We did not ascertain any patients who had isolated fiber type abnormalities and normal respiratory chain analysis during the period of study. We conclude that mitochondrial ETC disorders may represent an etiology of at least a subset of muscle fiber type abnormalities. To establish an etiologic diagnosis and to determine the frequency of such changes in mitochondrial disease, we suggest analysis of ETC function in individuals with fiber type changes in skeletal muscle, even in the absence of light histological features suggestive of mitochondrial disorders.

Successful long-term treatment of hepatic carnitine palmitoyltransferase I deficiency and a novel mutation.

Individuals with carnitine palmitoyltransferase I (CPT-I) deficiency cannot metabolize long-chain fatty acids and can develop life-threatening hypoglycaemia. We present a boy with CPT-I deficiency maintained on a very low-fat diet with nighttime uncooked cornstarch feedings for 5(1/2) years with good success. He has had normal growth and no episodes of hypoglycaemia or adverse side-effects. We found that he was homozygous for a previously undescribed mutation, T314I, in the CPT1A protein.

Autosomal dominant acute necrotizing encephalopathy.

OBJECTIVE: To define the clinical and biochemical abnormalities of an autosomal dominant form of acute encephalopathy. METHODS: The clinical details of 11 affected family members in comparison with 63 unaffected relatives were analyzed. RESULTS: Affected children become comatose after onset of a febrile illness. Outcomes include full recovery, permanent neurologic impairment, and death. Recurrences produce more severe impairments. Lesions of necrotizing encephalopathy of the thalamus and brainstem are present on autopsy and MRI. Oxidative phosphorylation of intact mitochondria from a muscle biopsy shows loose coupling. Unaffected family members, including obligate carriers, share no clinical characteristics, demonstrating incomplete penetrance. CONCLUSIONS: Characteristic pathology and MRI findings define this disorder of autosomal dominant acute encephalopathy. Leigh syndrome and sporadic acute necrotizing encephalopathy share similarities but are distinct.

Aging skeletal muscle mitochondria in the rat: decreased uncoupling protein-3 content.

The goal of the present study was to discern the cellular mechanism(s) that contributes to the age-associated decrease in skeletal muscle aerobic capacity. Skeletal muscle mitochondrial content, a parameter of oxidative capacity, was significantly lower (25 and 20% calculated on the basis of citrate synthase and succinate dehydrogenase activities, respectively) in 24-mo-old Fischer 344 rats compared with 6-mo-old adult rats. Mitochondria isolated from skeletal muscle of both age groups had identical state 3 (ADP-stimulated) and ADP-stimulated maximal respiratory rates and phosphorylation potential (ADP-to-O ratios) with both nonlipid and lipid substrates. In contrast, mitochondria from 24-mo-old rats displayed significantly lower state 4 (ADP-limited) respiratory rates and, consequently, higher respiratory control ratios. Consistent with the tighter coupling, there was a 68% reduction in uncoupling protein-3 (UCP-3) abundance in mitochondria from elderly compared with adult rats. Congruent with the respiratory studies, there was no age-associated decrease in carnitine palmitoyltransferase I and carnitine palmitoyltransferase II activities in isolated skeletal muscle mitochondria. However, there was a small, significant decrease in tissue total carnitine content. It is concluded that the in vivo observed decrease in skeletal muscle aerobic capacity with advanced age is a consequence of the decreased mitochondrial density. On the basis of the dramatic reduction of UCP-3 content associated with decreased state 4 respiration of skeletal muscle mitochondria from elderly rats, we propose that an increased free radical production might contribute to the metabolic compromise in aging.

Mitochondrial dysfunction in cardiac disease: ischemia--reperfusion, aging, and heart failure.

Mitochondria contribute to cardiac dysfunction and myocyte injury via a loss of metabolic capacity and by the production and release of toxic products. This article discusses aspects of mitochondrial structure and metabolism that are pertinent to the role of mitochondria in cardiac disease. Generalized mechanisms of mitochondrial-derived myocyte injury are also discussed, as are the strengths and weaknesses of experimental models used to study the contribution of mitochondria to cardiac injury. Finally, the involvement of mitochondria in the pathogenesis of specific cardiac disease states (ischemia, reperfusion, aging, ischemic preconditioning, and cardiomyopathy) is addressed.

Myocardial ischemia selectively depletes cardiolipin in rabbit heart subsarcolemmal mitochondria.

Mitochondria contribute to myocyte injury during ischemia. After 30 and 45 min of ischemia in the isolated perfused rabbit heart, subsarcolemmal mitochondria (SSM), located beneath the plasma membrane, sustain a decrease in oxidative phosphorylation through cytochrome oxidase. In contrast, oxidation through cytochrome oxidase in interfibrillar mitochondria (IFM), located between the myofibrils, remains unaffected. Cytochrome oxidase activity in the intact membrane requires an inner mitochondrial membrane lipid environment enriched in cardiolipin. During ischemia, the content of cardiolipin decreased only in SSM, whereas the content of other phospholipids was preserved. Ischemia did not alter the composition of the cardiolipin that remained in SSM. Cardiolipin content was preserved in IFM during ischemia. Thus cardiolipin is a relatively early target of ischemic mitochondrial damage, leading to loss of oxidative phosphorylation through cytochrome oxidase in SSM.

Ischemic injury to mitochondrial electron transport in the aging heart: damage to the iron-sulfur protein subunit of electron transport complex III.

The aging heart sustains greater injury during ischemia and reperfusion compared to adult hearts. Aging decreases oxidative function in interfibrillar mitochondria (IFM) that reside among the myofibers, while subsarcolemmal mitochondria (SSM), located beneath the plasma membrane, remain unaltered. Aging decreases complex III activity selectively in IFM via alteration of the cytochrome c binding site. With 25 min of global ischemia, complex III activity decreases in SSM and further decreases in IFM in the aging heart. Ischemia leads to a marked decrease in the electron paramagnetic resonance signal of the iron-sulfur protein (ISP) in both SSM and IFM, despite a preserved content of ISP peptide. Thus, ischemia results in a functional decrease in the iron-sulfur center in ISP without subunit peptide loss. In the aging heart, at the onset of reperfusion, IFM contain two tandem defects in the path of electron flow through complex III, providing a likely mechanism for enhanced oxidant production and reperfusion damage.

Phase I clinical and pharmacokinetic study of rebeccamycin analog NSC 655649 given daily for five consecutive days.

PURPOSE: Rebeccamycin analog (NSC 655649) is active against a variety of both solid and nonsolid tumor cell lines. We performed a phase I trial to determine the maximum-tolerated dose (MTD) of rebeccamycin analog when given on a daily x 5 schedule repeated every 3 weeks, characterize the toxicity profile using this schedule, observe patients for antitumor response, and determine the pharmacokinetics of the agent and pharmacodynamic interactions. PATIENTS AND METHODS: Thirty assessable patients received a total of 153 cycles according to the following dose escalation schema: 60, 80, 106, 141, and 188 mg/m(2)/d x 5 days. RESULTS: Grade 2 phlebitis occurred in all patients before the use of central venous access, placed at dose level 4 and higher. Dose-limiting toxicity (DLT), grade 4 neutropenia, occurred at 188 mg/m(2)/d x 5 days in both previously treated and chemotherapy-naive patients. Pharmacokinetic analysis revealed a three-compartmental model of drug elimination and a long terminal half-life (154 +/- 55 hours). The percentage drop in absolute neutrophil count correlates with the area under the curve infinity. The presence of a second peak during the elimination phase as well as a high concentration of NSC 655649 in biliary fluid compared with the corresponding plasma measurement (one patient) is suggestive of enterohepatic circulation. Two partial responses, two minor responses, and six prolonged (> 6 months) cases of stable disease were observed. Of these, three patients with gallbladder cancer and one patient with cholangiocarcinoma experienced either a minor response or a significant period of freedom from progression. CONCLUSION: The recommended phase II dose for NSC 665649 on a daily x 5 every 3 weeks schedule is 141 and 165 mg/m(2)/d for patients with prior and no prior therapy, respectively, with DLT being neutropenia. During this phase I trial, encouraging antitumor activity was been observed.

Mitochondrial expression and function of GAS-1 in Caenorhabditis elegans.

A mutation in the gene gas-1 alters sensitivity to volatile anesthetics, fecundity, and life span in the nematode Caenorhabditis elegans. gas-1 encodes a close homologue of the 49-kDa iron protein subunit of Complex I of the mitochondrial electron transport chain from bovine heart. gas-1 is widely expressed in the nematode neuromuscular system and in a subcellular pattern consistent with that of a mitochondrial protein. Pharmacological studies indicate that gas-1 functions partially via presynaptic effects. In addition, a mutation in the gas-1 gene profoundly decreases Complex I-dependent metabolism in mitochondria as measured by rates of both oxidative phosphorylation and electron transport. An increase in Complex II-dependent metabolism also is seen in mitochondria from gas-1 animals. There is no apparent alteration in physical structure in mitochondria from gas-1 nematodes compared with those from wild type. These data indicate that gas-1 is the major 49-kDa protein of complex I and that the GAS-1 protein is critical to mitochondrial function in C. elegans. They also reveal the importance of mitochondrial function in determining not only aging and life span, but also anesthetic sensitivity, in this model organism.

Phase II and pharmacokinetic/pharmacodynamic trial of sequential topoisomerase I and II inhibition with topotecan and etoposide in advanced non-small-cell lung cancer.

PURPOSE: In vitro and in vivo preclinical models have demonstrated synergistic activity when topoisomerase I and II inhibitors are administered sequentially. Topoisomerase I inhibitors increase topoisomerase II levels and increase cell kill induced by topoisomerase II poisons. We evaluated this hypothesis in a cohort of patients with advanced non-small-cell lung cancer (NSCLC). METHODS: A group of 19 patients with advanced NSCLC (70% adenocarcinoma) received topotecan at a dose of 0.85 mg/m2 per day as a continuous 72-h infusion from days 1 to 3. Etoposide was administered orally at a dose of 100 mg twice daily for 3 days on days 7-9 (schedule and dose derived from prior phase I trials). Total and lactone topotecan concentrations were measured at the end of the 72-h infusion. Blood samples were obtained immediately after each 72-h topotecan infusion in order to measure the mutational frequency at the hypoxanthine phosphoribosyl transferase (HPRT) locus in peripheral lymphocytes. RESULTS: A total of 55 cycles were administered. Toxicity was mainly hematologic with grade 4 neutropenia occurring in 7% of courses. Only one partial response and two stable diseases were observed. The 1-year survival rate was 33%. There was a statistically significant difference between steady-state lactone concentrations between cycle 1 and cycle 2 with decreasing concentrations with cycle 2 (P = 0.02). This was explained by a statistically significant increase in the clearance of topotecan lactone during cycle 2 (P = 0.03). Total but not lactone concentrations correlated with nadir WBC, ANC and platelet levels. Steady-state plasma lactone levels correlated with the mutational frequency at the HPRT locus (P = 0.06). In the one patient with a partial response a sixfold increase in HPRT mutational frequency was observed, which was not seen in patients with progressive disease. CONCLUSION: The combination of topotecan and etoposide in this schedule of administration has minimal activity in adenocarcinoma of the lung. This lack of activity may be due to the delay in administration of etoposide after the topotecan as studies have shown that the compensatory increase in topoisomerase II levels after treatment with topoisomerase I inhibitors is shortlived (<24 h). The HPRT mutational frequency results suggest that the lack of clinical response may be associated with failure to achieve sufficient cytotoxic dose as indicated by a lack of increase in mutational frequency in those patients with progressive disease. HPRT mutational frequency may correlate with plasma steady-state topotecan lactone levels. Future studies should be directed toward earlier administration of topoisomerase II inhibitors after topoisomerase I inhibition.

Decreased NADH dehydrogenase and ubiquinol-cytochrome c oxidoreductase in peripheral arterial disease.

Peripheral arterial disease (PAD) is associated with muscle metabolic changes that may contribute to the disability in these patients. However, the biochemical defects in PAD have not been identified. The present study was undertaken to test the hypothesis that PAD is associated with specific defects in skeletal muscle electron transport chain activity. Seventeen patients with PAD and nine age-matched controls underwent gastrocnemius muscle biopsies. There were no differences in the mitochondrial content per gram of skeletal muscle as assessed by citrate synthase activity between the PAD patients and the control subjects. Skeletal muscle NADH dehydrogenase activity was decreased by 27% compared with controls when expressed per unit of citrate synthase activity. Expression of enzyme activities normalized to cytochrome c-oxygen oxidoreductase activity confirmed a 26% decrease in NADH dehydrogenase activity and also demonstrated a 38% decrease in ubiquinol-cytochrome c oxidoreductase activity. Thus PAD is associated with specific changes in muscle mitochondrial electron transport chain activities characterized by relative decreases in NADH dehydrogenase and ubiquinol-cytochrome c oxidoreductase activities, which may contribute to the metabolic abnormalities and decreased exercise performance in these patients.

Aging decreases electron transport complex III activity in heart interfibrillar mitochondria by alteration of the cytochrome c binding site.

Aging alters cardiac physiology and structure and enhances damage during ischemia and reperfusion. Aging selectively decreases the rate of oxidative phosphorylation in the interfibrillar population of cardiac mitochondria (IFM) located among the myofibers, whereas subsarcolemmal mitochondria (SSM) located beneath the plasma membrane remain unaffected. Aging decreased the rate of oxidative phosphorylation using durohydroquinone, an electron donor to complex III, in IFM only. Complex III activity was decreased in IFM, but not SSM. Aging did not alter the content of catalytic centers of complex III (cytochromes b and c(1)and iron-sulfur protein). Complex III activity measured at physiologic ionic strength in IFM from aging hearts was decreased by 49% compared to IFM from adults, whereas activity measured at low ionic strength was unchanged, localizing the aging defect to the cytochrome c binding site of complex III. Subunits VIII and X of the cytochrome c binding site were present in complex III with the aging defect, indicating that loss of subunits did not occur. Study of aging damage to complex III will help clarify the contribution of altered electron transport in IFM to increased oxidant production during aging, formation of the aging cardiac phenotype, and the relationship of aging defects to increased damage following ischemia.

Separation and quantitation of phospholipids and lysophospholipids by high-performance liquid chromatography.

We describe a comprehensive approach to the separation, quantitation, and characterization of phospholipids and lysophospholipids present in complex biological samples. The central feature is a normal-phase HPLC separation of individual phospholipid and lysophospholipid classes. In this single chromatographic step, phospholipids and lysophospholipids are separated and recovered for quantitation by organic phosphate assay and characterization by acyl-group composition. Recovery of phospholipids and lysophospholipids from HPLC averages 80-90%. Isolated phospholipid and lysophospholipid fractions are available for separation of individual molecular species by second-dimension reverse-phase HPLC and characterization of individual molecular species by mass spectrometry.

A phase II and pharmacokinetic study of weekly 72-h topotecan infusion in patients with platinum-resistant and paclitaxel-resistant ovarian carcinoma.

BACKGROUND: As suggested by preclinical trials, prolonged administration of topotecan, a reversible inhibitor of topoisomerase-I, may have a therapeutic advantage. Following a phase I trial of weekly 72-h topotecan infusion, we performed a phase II trial utilizing this schedule in ovarian carcinoma. METHODS: Eligibility included platinum-/paclitaxel-resistant ovarian carcinoma, measurable disease, and adequate hematologic, renal, and hepatic function. A dose of 2.0 mg/m(2) of topotecan was administered as a 72-h infusion weekly via an ambulatory pump. Plasma topotecan concentrations were determined prior to and at the completion of each weekly course. RESULTS: Twenty-four patients were entered and 23 patients were evaluable for toxicity and response. Two hundred eighteen weekly courses of therapy were administered (median 7 weeks, range 4-46 weeks). Toxicity was mild with grade 3 leukopenia, neutropenia, and anemia occurring in 13, 13, and 17% of patients, respectively. Two of 23 patients (9.1%) (CI 1-28%) had partial responses of 2 and 3 months' duration and 6 had stable disease. Steady state plasma topotecan lactone concentrations were a median of 1.2 ng/ml (range 0.4-8.00 ng/ml) following the first week of infusion. Steady state topotecan lactone concentrations after the first week of infusion were highest in 2 patients with partial responses. Mean steady state plasma topotecan lactone concentrations after the first week of infusion were 4.6, 2.0, and 1.3 ng/ml for partial response, stable disease, and progressive disease, respectively. An analysis of variance of steady state plasma topotecan concentrations after the first week of infusion over all administered cycles demonstrated a significant difference in steady state plasma topotecan lactone concentrations between patients with partial response and stable disease and between partial response and no response (significant at the 0.05 level after adjustment for multiple comparisons). Controlling for cycle number, steady state topotecan lactone concentrations are significantly greater for patients with responding or stable disease than those with progressive disease (P = 0.0003) and have a lower bound of > or = 1.9 ng/ml (95% confidence level). CONCLUSION: Steady state topotecan lactone concentrations are associated with responding or stable disease in platinum- and paclitaxel-resistant ovarian cancer. Steady state topotecan concentrations could potentially be utilized to modify tumor exposure and response.

Decreased carnitine biosynthesis in rats with secondary biliary cirrhosis.

Carnitine biosynthesis was investigated in rats with secondary biliary cirrhosis induced by bile duct ligation (BDL) for 4 weeks (n = 5) and in pair-fed, sham-operated control rats (n = 4). Control rats were pair-fed to BDL rats, and all rats were fed an artificial diet with negligible contents of carnitine, butyrobetaine, or trimethyllysine. Biosynthesis of carnitine and its precursors was determined by measuring their excretion in urine and accumulation in the body of the animals. Four weeks after BDL, total carnitine content was increased by 33% in livers from BDL rats when compared with control rats, but was unchanged in skeletal muscle and whole carcass. The plasma total carnitine concentration averaged 29.0 +/- 4.1 vs. 46.4 +/- 7.3 micromol/L in BDL rats and control rats, respectively. Urinary total carnitine excretion was reduced by 56% in BDL rats as compared with control rats. Carnitine biosynthesis was significantly decreased in BDL rats (0.45 +/- 0.19 vs. 0.93 +/- 0.08 micromol/100 g body weight/d in BDL and control rats, respectively). The tissue content of free and protein-linked trimethyllysine, a carnitine precursor, and trimethyllysine plasma concentrations were not different between BDL and control rats. However, urinary trimethyllysine excretion was increased 5-fold in BDL rats and approximated glomerular filtration. In contrast, urinary excretion of butyrobetaine, the direct carnitine precursor, was decreased by 40% in BDL rats as compared with control rats. Trimethyllysine biosynthesis was not different, but butyrobetaine biosynthesis was decreased by 51% in BDL as compared with control rats. In conclusion, carnitine biosynthesis is decreased in BDL rats as a result of a defect in the conversion of trimethyllysine to butyrobetaine.