Brian D. Fink

CaMKII determines mitochondrial stress responses in heart

Myocardial cell death is initiated by excessive mitochondrial Ca2+ entry causing Ca2+ overload, mitochondrial permeability transition pore (mPTP) opening and dissipation of the mitochondrial inner membrane potential (ΔΨm). However, the signalling pathways that control mitochondrial Ca2+ entry through the inner membrane mitochondrial Ca2+ uniporter (MCU) are not known. The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated in ischaemia reperfusion, myocardial infarction and neurohumoral injury, common causes of myocardial death and heart failure; these findings suggest that CaMKII could couple disease stress to mitochondrial injury. Here we show that CaMKII promotes mPTP opening and myocardial death by increasing MCU current (IMCU). Mitochondrial-targeted CaMKII inhibitory protein or cyclosporin A, an mPTP antagonist with clinical efficacy in ischaemia reperfusion injury, equivalently prevent mPTP opening, ΔΨm deterioration and diminish mitochondrial disruption and programmed cell death in response to ischaemia reperfusion injury. Mice with myocardial and mitochondrial-targeted CaMKII inhibition have reduced IMCU and are resistant to ischaemia reperfusion injury, myocardial infarction and neurohumoral injury, suggesting that pathological actions of CaMKII are substantially mediated by increasing IMCU. Our findings identify CaMKII activity as a central mechanism for mitochondrial Ca2+ entry in myocardial cell death, and indicate that mitochondrial-targeted CaMKII inhibition could prevent or reduce myocardial death and heart failure in response to common experimental forms of pathophysiological stress.

Effects of Adenoviral Overexpression of Uncoupling Protein-2 and -3 on Mitochondrial Respiration in Insulinoma Cells1

The brown adipose tissue uncoupling protein 1 (UCP1) catalyzes proton reentry without ATP synthesis, thereby dissipating energy as heat. In contrast, the function(s) of the recently described homologs, UCP2 and UCP3, are less clear. The aim of the present study was to determine whether overexpressed UCP subtypes affect mitochondrial respiration and substrate oxidation in cultured insulin-secreting INS-1 insulinoma cells. Adenoviral overexpression of UCP2 significantly decreased the ADP/O ratio by 31% and 39% in comparison to β-galactosidase (β-gal) or the mitochondrial protein manganese superoxide dismutase (MnSOD), respectively, and increased state 4 respiration in the presence of succinate and oligomycin by 52% and 59% in comparison toβ -gal or MnSOD, respectively. Adenoviral overexpression of UCP3 also decreased the ADP/O ratio by 18% (nonsignificant) and increased state 4 respiration by 24% (nonsignificant) in comparison to β-gal and significantly decreased the ADP/O ratio by 32% and increased state 4...

REACTIVE OXYGEN AND TARGETED ANTIOXIDANT ADMINISTRATION IN ENDOTHELIAL CELL MITOCHONDRIA

We used fluorescent probes and EPR to study the mechanism(s) underlying reactive oxygen species (ROS) production by endothelial cell mitochondria and the action of mitoquinol, a mitochondria-targeted antioxidant. ROS measured by fluorescence resulted from complex I superoxide released to the matrix and converted to H2O2. In contrast, EPR largely detected superoxide generated at complex III and effluxed outward. ROS fluorescence by mitochondria fueled by the complex II substrate, succinate, was substantial but markedly inhibited by rotenone. Superoxide, detected by EPR, in succinate-fueled mitochondria was not inhibited by rotenone and likely derived from semiquinone formation at complex III. Mitoquinol decreased H2O2 fluorescence by succinate-fueled mitochondria but had little effect on the EPR signal for superoxide. This was not associated with a detectable decrease in membrane potential. Mitoquinol markedly enhanced ROS fluorescence in mitochondria fueled by the complex I substrates, glutamate and malate. Inhibitor studies suggested that this occurred in complex I, at one or more Q binding pockets. The above effects of mitoquinol were determined in mitochondria isolated and subsequently exposed to the targeted antioxidant. However, similar effects were observed in mitochondria after antecedent exposure to mitoquinol/mitoquinone in culture, suggesting that the agent is retained after isolation of the organelles. In conclusion, ROS production in bovine aortic endothelial cell mitochondria results largely from reverse transport to complex I and through the Q cycle in complex III. Mitoquinol blocks ROS from reverse electron transport but increases superoxide production derived from forward transport. These effects likely occur at one or more Q binding sites in complex I.

Expression of Glia Maturation Factor β mRNA and Protein in Rat Organs and Cells

Abstract: Rat glia maturation factor β (GMF‐β) cDNA was obtained by reverse transcription of rat brain mRNA followed by polymerase chain reaction amplification, using primers from the human sequence. The deduced amino acid sequence of rat GMF‐β differed from the human counterpart in only three places: His27 in place of Asn, Val51 in place of Ile, and Leu93 in place of Val. The high degree of evolutionary conservation suggests that GMF‐β plays an essential role in animal cell physiology. The expression of GMF‐β mRNA in the rat was studied by the northern blot technique, using a rat cRNA probe corresponding to the entire coding region. GMF‐β mRNA was predominantly expressed in the brain and spinal cord, although trace levels were found in other organs, including testis and ovary. In the brain GMF‐β mRNA was detectable at as early as embryonic day 10, and persisted through as late as postnatal month 14, with minor variations in between. On the other hand, GMF‐β protein exhibited more obvious developmental changes, with its level increasing slowly prenatally and plateauing at 1 week after birth. GMF‐β mRNA and protein were also observed in several cultured cells. Some cells of neural origin contained higher levels of GMF‐β protein compared with cells derived from other sources. Through demonstration of mRNA and confirmation by immunoblotting, we conclude that GMF‐β is synthesized by rat organs and that GMF‐β is predominantly a brain protein.

Superoxide and Respiratory Coupling in Mitochondria of Insulin-Deficient Diabetic Rats

Mitochondrial reactive oxygen species have been implicated in both diabetic complications and the progression of the underlying diabetic state. However, it is not clear whether mitochondria of diabetic origin are intrinsically altered to generate excess reactive oxygen species independent of the surrounding diabetic milieu. Mitochondria were isolated from gastrocnemius, heart, and liver of 2-wk and 2-month streptozotocin diabetic rats and controls. We rigidly quantified mitochondrial superoxide, respiration and ATP production, respiratory coupling, the expression of several proteins with antioxidant properties, and the redox state of glutathione. Both fluorescent assessment and electron paramagnetic spectroscopy revealed that superoxide production was unchanged or reduced in the 2-month diabetic mitochondria compared with controls. Kinetic analysis of the proton leak showed that diabetic heart and muscle mitochondria were actually more coupled compared with control despite an approximate 2- to 4-fold increase in uncoupling protein-3 content. Adenine nucleotide translocator type 1 expression was reduced by approximately 50% in diabetic muscle mitochondria. Catalase was significantly up-regulated in muscle and heart tissue and in heart mitochondria, whereas glutathione peroxidase expression was increased in liver mitochondria of diabetic rats. We conclude that gastrocnemius, heart, and liver mitochondria of streptozotocin diabetic rats are not irrevocably altered toward excess superoxide production either by complex I or complex III. Moreover, gastrocnemius and heart mitochondria demonstrate increased, not decreased, respiratory coupling. Mitochondria of insulin-deficient diabetic rats do show signs of adaptation to antecedent oxidative stress manifested as tissue-specific enzyme and uncoupling protein expression but remain remarkably robust with respect to superoxide production.

Sympathetic inhibition, leptin, and uncoupling protein subtype expression in normal fasting rats

To further investigate neural effects on leptin and uncoupling proteins (UCPs), we studied in vivo perturbations intended to block adrenergic input to peripheral tissues. We examined plasma leptin, leptin mRNA, and adipose and muscle UCP subtype mRNA in rats treated with alpha-methyl-p-tyrosine methyl ester (AMPT-ME), which inhibits catecholamine synthesis and 6-hydroxydopamine (6HDA), which is toxic to catecholinergic nerve terminals but, unlike AMPT-ME, does not enter the central nervous system. Intraperitoneal AMPT-ME, 250 mg/kg, was administered at 1800 and 0700 the following day, and rats were killed at 1200-1400. All rats were fasted with free access to water during this time. Intraperitoneal AMPT-ME increased plasma leptin by 15-fold, increased interscapular brown adipose tissue (IBAT) and epididymal fat leptin mRNA by 2- to 2.5-fold, and also increased plasma insulin and glucose concentrations. Intraperitoneal AMPT-ME decreased IBAT UCP-3 mRNA to 40% of control, while it increased epididymal adipose UCP-3 mRNA approximately twofold. Intravenous AMPT-ME, 250 mg/kg, administered to conscious rats for 5 h decreased lumbar sympathetic nerve activity, increased plasma leptin (5.89 +/- 1.43 compared with 2.75 +/- 0.31 ng/ml in vehicle-treated rats, n = 7, P < 0.05), and decreased cardiac rate with no sustained change in blood pressure. Intraperitoneal 6HDA, 100 mg/kg, as a single dose at 1800, increased plasma leptin approximately twofold after 18-20 h, increased IBAT (but not epididymal fat) leptin mRNA by two- to threefold, and decreased IBAT UCP-3 mRNA to 30-40% of control. Neither AMPT-ME nor 6HDA significantly altered mRNA encoding gastrocnemius muscle UCP-3, IBAT UCP-1, or IBAT and epididymal UCP-2. In summary, AMPT-ME and 6HDA increased plasma leptin and upregulated leptin mRNA expression. AMPT-ME also resulted in complex tissue and subtype-specific modulation of adipose UCP mRNA. These data are consistent with interaction between leptin and sympathetic nerve activity (SNA) in regulation of fat cell energy utilization. However, the in vivo modulation of leptin and UCPs appears complex and, beyond a causal effect of SNA per se, may depend on concurrent changes in plasma insulin, glucose, and circulatory hemodynamics.To further investigate neural effects on leptin and uncoupling proteins (UCPs), we studied in vivo perturbations intended to block adrenergic input to peripheral tissues. We examined plasma leptin, leptin mRNA, and adipose and muscle UCP subtype mRNA in rats treated with α-methyl- p-tyrosine methyl ester (AMPT-ME), which inhibits catecholamine synthesis and 6-hydroxydopamine (6HDA), which is toxic to catecholinergic nerve terminals but, unlike AMPT-ME, does not enter the central nervous system. Intraperitoneal AMPT-ME, 250 mg/kg, was administered at 1800 and 0700 the following day, and rats were killed at 1200-1400. All rats were fasted with free access to water during this time. Intraperitoneal AMPT-ME increased plasma leptin by 15-fold, increased interscapular brown adipose tissue (IBAT) and epididymal fat leptin mRNA by 2- to 2.5-fold, and also increased plasma insulin and glucose concentrations. Intraperitoneal AMPT-ME decreased IBAT UCP-3 mRNA to 40% of control, while it increased epididymal adipose UCP-3 mRNA approximately twofold. Intravenous AMPT-ME, 250 mg/kg, administered to conscious rats for 5 h decreased lumbar sympathetic nerve activity, increased plasma leptin (5.89 ± 1.43 compared with 2.75 ± 0.31 ng/ml in vehicle-treated rats, n = 7, P < 0.05), and decreased cardiac rate with no sustained change in blood pressure. Intraperitoneal 6HDA, 100 mg/kg, as a single dose at 1800, increased plasma leptin approximately twofold after 18-20 h, increased IBAT (but not epididymal fat) leptin mRNA by two- to threefold, and decreased IBAT UCP-3 mRNA to 30-40% of control. Neither AMPT-ME nor 6HDA significantly altered mRNA encoding gastrocnemius muscle UCP-3, IBAT UCP-1, or IBAT and epididymal UCP-2. In summary, AMPT-ME and 6HDA increased plasma leptin and upregulated leptin mRNA expression. AMPT-ME also resulted in complex tissue and subtype-specific modulation of adipose UCP mRNA. These data are consistent with interaction between leptin and sympathetic nerve activity (SNA) in regulation of fat cell energy utilization. However, the in vivo modulation of leptin and UCPs appears complex and, beyond a causal effect of SNA per se, may depend on concurrent changes in plasma insulin, glucose, and circulatory hemodynamics.

Bioenergetic Effects of Mitochondrial-Targeted Coenzyme Q Analogs in Endothelial Cells

Mitochondrial-targeted analogs of coenzyme Q (CoQ) are under development to reduce oxidative damage induced by a variety of disease states. However, there is a need to understand the bioenergetic effects of these agents and whether or not these effects are related to redox properties, including their known pro-oxidant effects. We examined the bioenergetic effects of two mitochondrial-targeted CoQ analogs in their quinol forms, mitoquinol (MitoQ) and plastoquinonyl-decyl-triphenylphosphonium (SkQ1), in bovine aortic endothelial cells. We used an extracellular oxygen and proton flux analyzer to assess mitochondrial action at the intact-cell level. Both agents, in dose-dependent fashion, reduced the oxygen consumption rate (OCR) directed at ATP turnover (OCRATP) (IC50 values of 189 ± 13 nM for MitoQ and 181 ± 7 for SKQ1; difference not significant) while not affecting or mildly increasing basal oxygen consumption. Both compounds increased extracellular acidification in the basal state consistent with enhanced glycolysis. Both compounds enhanced mitochondrial superoxide production assessed by using mitochondrial-targeted dihydroethidium, and both increased H2O2 production from mitochondria of cells treated before isolation of the organelles. The manganese superoxide dismutase mimetic manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin did not alter or actually enhanced the actions of the targeted CoQ analogs to reduce OCRATP. In contrast, N-acetylcysteine mitigated this effect of MitoQ and SkQ1. In summary, our data demonstrate the important bioenergetic effects of targeted CoQ analogs. Moreover, these effects are mediated, at least in part, through superoxide production but depend on conversion to H2O2. These bioenergetic and redox actions need to be considered as these compounds are developed for therapeutic purposes.

Mitochondrial Function in Diabetes: Novel Methodology and New Insight

Interpreting mitochondrial function as affected by comparative physiologic conditions is confounding because individual functional parameters are interdependent. Here, we studied muscle mitochondrial function in insulin-deficient diabetes using a novel, highly sensitive, and specific method to quantify ATP production simultaneously with reactive oxygen species (ROS) at clamped levels of inner mitochondrial membrane potential (ΔΨ), enabling more detailed study. We used a 2-deoxyglucose (2DOG) energy clamp to set ΔΨ at fixed levels and to quantify ATP production as 2DOG conversion to 2DOG-phosphate measured by one-dimensional 1H and two-dimensional 1H/13C heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy. These techniques proved far more sensitive than conventional 31P nuclear magnetic resonance and allowed high-throughput study of small mitochondrial isolates. Over conditions ranging from state 4 to state 3 respiration, ATP production was lower and ROS per unit of ATP generated was greater in mitochondria isolated from diabetic muscle. Moreover, ROS began to increase at a lower threshold for inner membrane potential in diabetic mitochondria. Further, ATP production in diabetic mitochondria is limited not only by respiration but also by limited capacity to use ΔΨ for ATP synthesis. In summary, we describe novel methodology for measuring ATP and provide new mechanistic insight into the dysregulation of ATP production and ROS in mitochondria of insulin-deficient rodents.

Mitochondrial Targeted Coenzyme Q, Superoxide, and Fuel Selectivity in Endothelial Cells

Background Previously, we reported that the “antioxidant” compound “mitoQ” (mitochondrial-targeted ubiquinol/ubiquinone) actually increased superoxide production by bovine aortic endothelial (BAE) cell mitochondria incubated with complex I but not complex II substrates. Methods and Results To further define the site of action of the targeted coenzyme Q compound, we extended these studies to include different substrate and inhibitor conditions. In addition, we assessed the effects of mitoquinone on mitochondrial respiration, measured respiration and mitochondrial membrane potential in intact cells, and tested the intriguing hypothesis that mitoquinone might impart fuel selectivity in intact BAE cells. In mitochondria respiring on differing concentrations of complex I substrates, mitoquinone and rotenone had interactive effects on ROS consistent with redox cycling at multiple sites within complex I. Mitoquinone increased respiration in isolated mitochondria respiring on complex I but not complex II substrates. Mitoquinone also increased oxygen consumption by intact BAE cells. Moreover, when added to intact cells at 50 to 1000 nM, mitoquinone increased glucose oxidation and reduced fat oxidation, at doses that did not alter membrane potential or induce cell toxicity. Although high dose mitoquinone reduced mitochondrial membrane potential, the positively charged mitochondrial-targeted cation, decyltriphenylphosphonium (mitoquinone without the coenzyme Q moiety), decreased membrane potential more than mitoquinone, but did not alter fuel selectivity. Therefore, non-specific effects of the positive charge were not responsible and the quinone moiety is required for altered nutrient selectivity. Conclusions In summary, the interactive effects of mitoquinone and rotenone are consistent with redox cycling at more than one site within complex I. In addition, mitoquinone has substrate dependent effects on mitochondrial respiration, increases repiration by intact cells, and alters fuel selectivity favoring glucose over fatty acid oxidation at the intact cell level.

Mitochondrial superoxide and coenzyme Q in insulin-deficient rats: increased electron leak

Mitochondrial superoxide is important in the pathogeneses of diabetes and its complications. However, there is uncertainty regarding the intrinsic propensity of mitochondria to generate this radical. Studies to date suggest that superoxide production by mitochondria of insulin-sensitive target tissues of insulin-deficient rodents is reduced or unchanged. Moreover, little is known of the role of the Coenzyme Q (CoQ), whose semiquinone form reacts with molecular oxygen to generate superoxide. We measured reactive oxygen species (ROS) production, respiratory parameters, and CoQ content in mitochondria from gastrocnemius muscle of control and streptozotocin (STZ)-diabetic rats. CoQ content did not differ between mitochondria isolated from vehicle- or STZ-treated animals. CoQ also was unaffected by weight loss in the absence of diabetes (induced by caloric restriction). Under state 4 or state 3 conditions, both respiration and ROS release were reduced in diabetic mitochondria fueled with succinate, glutamate plus malate, or with all three substrates (continuous TCA cycle). However, H(2)O(2) and directly measured superoxide production were substantially increased in gastrocnemius mitochondria of diabetic rats when expressed per unit oxygen consumed. On the basis of substrate and inhibitor effects, the mechanism involved multiple electron transport sites. More limited results using heart mitochondria were similar. ROS per unit respiration was greater in muscle mitochondria from diabetic compared with control rats during state 3, as well as state 4, while the reduction in ROS per unit respiration on transition to state 3 was less for diabetic mitochondria. In summary, ROS production is, in fact, increased in mitochondria from insulin-deficient muscle when considered relative to electron transport. This is evident on multiple energy substrates and in different respiratory states. CoQ is not reduced in diabetic mitochondria or with weight loss due to food restriction. The implications of these findings are discussed.