Robert S. Balaban

Mitochondria, Oxidants, and Aging

The free radical theory of aging postulates that the production of intracellular reactive oxygen species is the major determinant of life span. Numerous cell culture, invertebrate, and mammalian models exist that lend support to this half-century-old hypothesis. Here we review the evidence that both supports and conflicts with the free radical theory and examine the growing link between mitochondrial metabolism, oxidant formation, and the biology of aging.

The Mammalian Target of Rapamycin (mTOR) Pathway Regulates Mitochondrial Oxygen Consumption and Oxidative Capacity

Metabolic rate and the subsequent production of reactive oxygen species are thought to contribute to the rate of aging in a wide range of species. The target of rapamycin (TOR) is a well conserved serine/threonine kinase that regulates cell growth in response to nutrient status. Here we demonstrate that in mammalian cells the mammalian TOR (mTOR) pathway plays a significant role in determining both resting oxygen consumption and oxidative capacity. In particular, we demonstrate that the level of complex formation between mTOR and one of its known protein partners, raptor, correlated with overall mitochondrial activity. Disruption of this complex following treatment with the mTOR pharmacological inhibitor rapamycin lowered mitochondrial membrane potential, oxygen consumption, and ATP synthetic capacity. Subcellular fractionation revealed that mTOR as well as mTOR-raptor complexes can be purified in the mitochondrial fraction. Using two-dimensional difference gel electrophoresis, we further demonstrated that inhibiting mTOR with rapamycin resulted in a dramatic alteration in the mitochondrial phosphoproteome. RNA interference-mediated knockdown of TSC2, p70 S6 kinase (S6K1), raptor, or rictor demonstrates that mTOR regulates mitochondrial activity independently of its previously identified cellular targets. Finally we demonstrate that mTOR activity may play an important role in determining the relative balance between mitochondrial and non-mitochondrial sources of ATP generation. These results may provide insight into recent observations linking the TOR pathway to life span regulation of lower organisms.

Detecting Acute Coronary Syndrome in the Emergency Department With Cardiac Magnetic Resonance Imaging

Background—Managing chest pain in the emergency department remains a challenge with current diagnostic strategies. We hypothesized that cardiac MRI could accurately identify patients with possible or probable acute coronary syndrome. Methods and Results—The diagnostic performance of MRI was evaluated in a prospective study of 161 consecutive patients. Enrollment required 30 minutes of chest pain compatible with myocardial ischemia but an ECG not diagnostic of acute myocardial infarction. MRI was performed at rest within 12 hours of presentation and included perfusion, left ventricular function, and gadolinium-enhanced myocardial infarction detection. MRI was interpreted qualitatively but also analyzed quantitatively. The sensitivity and specificity, respectively, for detecting acute coronary syndrome were 84% and 85% by MRI, 80% and 61% by an abnormal ECG, 16% and 95% for strict ECG criteria for ischemia (ST depression or T-wave inversion), 40% and 97% for peak troponin-I, and 48% and 85% for a TIMI risk score ≥3. The MRI was more sensitive than strict ECG criteria for ischemia (P <0.001), peak troponin-I (P <0.001), and the TIMI risk score (P =0.004), and MRI was more specific than an abnormal ECG (P <0.001). Multivariate logistic regression analysis showed MRI was the strongest predictor of acute coronary syndrome and added diagnostic value over clinical parameters (P <0.001). Conclusions—Resting cardiac MRI exhibited diagnostic operating characteristics suitable for triage of patients with chest pain in the emergency department. Performed urgently to evaluate chest pain, MRI accurately detected a high fraction of patients with acute coronary syndrome, including patients with enzyme-negative unstable angina.

Determination of pH using water protons and chemical exchange dependent saturation transfer (CEST).

Solution pH was measured using water proton NMR via chemical exchange dependent saturation transfer (CEST) with selected chemical exchange sites. Several useful pH‐sensitive proton chemical exchange agents were found: 5,6‐dihydrouracil, 5‐hydroxytryptophan, and a combination of 5‐hydroxytryptophan and 2‐imidazolidinethione. A ratiometric approach was developed that permitted pH determinations that were independent of water T1 or exchange site concentration. Magn Reson Med 44:799–802, 2000. Published 2000 Wiley‐Liss, Inc.

Role of mitochondrial Ca2+ in the regulation of cellular energetics.

Calcium is an important signaling molecule involved in the regulation of many cellular functions. The large free energy in the Ca(2+) ion membrane gradients makes Ca(2+) signaling inherently sensitive to the available cellular free energy, primarily in the form of ATP. In addition, Ca(2+) regulates many cellular ATP-consuming reactions such as muscle contraction, exocytosis, biosynthesis, and neuronal signaling. Thus, Ca(2+) becomes a logical candidate as a signaling molecule for modulating ATP hydrolysis and synthesis during changes in numerous forms of cellular work. Mitochondria are the primary source of aerobic energy production in mammalian cells and also maintain a large Ca(2+) gradient across their inner membrane, providing a signaling potential for this molecule. The demonstrated link between cytosolic and mitochondrial Ca(2+) concentrations, identification of transport mechanisms, and the proximity of mitochondria to Ca(2+) release sites further supports the notion that Ca(2+) can be an important signaling molecule in the energy metabolism interplay of the cytosol with the mitochondria. Here we review sites within the mitochondria where Ca(2+) plays a role in the regulation of ATP generation and potentially contributes to the orchestration of cellular metabolic homeostasis. Early work on isolated enzymes pointed to several matrix dehydrogenases that are stimulated by Ca(2+), which were confirmed in the intact mitochondrion as well as cellular and in vivo systems. However, studies in these intact systems suggested a more expansive influence of Ca(2+) on mitochondrial energy conversion. Numerous noninvasive approaches monitoring NADH, mitochondrial membrane potential, oxygen consumption, and workloads suggest significant effects of Ca(2+) on other elements of NADH generation as well as downstream elements of oxidative phosphorylation, including the F(1)F(O)-ATPase and the cytochrome chain. These other potential elements of Ca(2+) modification of mitochondrial energy conversion will be the focus of this review. Though most specific molecular mechanisms have yet to be elucidated, it is clear that Ca(2+) provides a balanced activation of mitochondrial energy metabolism that exceeds the alteration of dehydrogenases alone.

Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes.

Nicotinamide adenine dinucleotide (NADH) plays a critical role in oxidative phosphorylation as the primary source of reducing equivalents to the respiratory chain. Using a modified fluorescence microscope, we have obtained spectra and images of the blue autofluorescence from single rat cardiac myocytes. The optical setup permitted rapid acquisition of fluorescence emission spectra (390-595 nm) or intensified digital video images of individual myocytes. The spectra showed a broad fluorescence centered at 447 +/- 0.2 nm, consistent with mitochondrial NADH. Addition of cyanide resulted in a 100 +/- 10% increase in fluorescence, while the uncoupler FCCP resulted in a 82 +/- 4% decrease. These two transitions were consistent with mitochondrial NADH and implied that the myocytes were 44 +/- 6% reduced under the resting control conditions. Intracellular fluorescent structures were observed that correlated with the distribution of a mitochondrial selective fluorescent probe (DASPMI), the mitochondrial distribution seen in published electron micrographs, and a metabolic digital subtraction image of the cyanide fluorescence transition. These data are consistent with the notion that the blue autofluorescence of rat cardiac myocytes originates from mitochondrial NADH.

Calcium and cyclic adenosine monophosphate as second messengers for vasopressin in the rat inner medullary collecting duct.

UNLABELLED Vasopressin increases both the urea permeability and osmotic water permeability in the terminal part of the renal inner medullary collecting duct (terminal IMCD). To identify the second messengers that mediate these responses, we measured urea permeability, osmotic water permeability, intracellular calcium concentration, and cyclic AMP accumulation in isolated terminal IMCDs. After addition of vasopressin, a transient rise in intracellular calcium occurred that was coincident with increases in cyclic AMP accumulation and urea permeability. Half-maximal increases in urea permeability and osmotic water permeability occurred with 0.01 nM vasopressin. The threshold concentration for a measurable increase in cyclic AMP accumulation was approximately 0.01 nM, while measurable increases in intracellular calcium required much higher vasopressin concentrations (greater than 0.1 nM). Exogenous cyclic AMP (1 mM 8-Br-cAMP) mimicked the effect of vasopressin on urea permeability but did not produce a measurable change in intracellular calcium concentration. CONCLUSIONS (a) Cyclic AMP is the second messenger that mediates the urea permeability response to vasopressin in the rat terminal IMCD. (b) Vasopressin increases the intracellular calcium concentration in the rat terminal IMCD, but the physiological role of this response is not yet known.

Metabolic network control of oxidative phosphorylation: Multiple roles of inorganic phosphate

Phosphate (Pi) is a putative cytosolic signaling molecule in the regulation of oxidative phosphorylation. Here, by using a multiparameter monitoring system, we show that Pi controls oxidative phosphorylation in a balanced fashion, modulating both the generation of useful potential energy and the formation of ATP by F1F0-ATPase in heart and skeletal muscle mitochondria. In these studies the effect of Pi was determined on the mitochondria [NADH], NADH generating capacity, matrix pH, membrane potential, oxygen consumption, and cytochrome reduction level. Pi enhanced NADH generation and was obligatory for electron flow under uncoupled conditions. Pi oxidized cytochrome b (cyto-b) and reduced cytochrome c (cyto-c), potentially improving the coupling between the NADH free energy and the proton motive force. The apparent limitation in reducing equivalent flow between cyto-b and cyto-c in the absence of Pi was confirmed in the intact heart by using optical spectroscopic techniques under conditions with low cytosolic [Pi]. These results demonstrate that Pi signaling results in the balanced modulation of oxidative phosphorylation, by influencing both ΔGH+ generation and ATP production, which may contribute to the energy metabolism homeostasis observed in intact systems.

The role of Ca2+ signaling in the coordination of mitochondrial ATP production with cardiac work

The heart is capable of balancing the rate of mitochondrial ATP production with utilization continuously over a wide range of activity. This results in a constant phosphorylation potential despite a large change in metabolite turnover. The molecular mechanisms responsible for generating this energy homeostasis are poorly understood. The best candidate for a cytosolic signaling molecule reflecting ATP hydrolysis is Ca(2+). Since Ca(2+) initiates and powers muscle contraction as well as serves as the primary substrate for SERCA, Ca(2+) is an ideal feed-forward signal for priming ATP production. With the sarcoplasmic reticulum to cytosolic Ca(2+) gradient near equilibrium with the free energy of ATP, cytosolic Ca(2+) release is exquisitely sensitive to the cellular energy state providing a feedback signal. Thus, Ca(2+) can serve as a feed-forward and feedback regulator of ATP production. Consistent with this notion is the correlation of cytosolic and mitochondrial Ca(2+) with work in numerous preparations as well as the localization of mitochondria near Ca(2+) release sites. How cytosolic Ca(2+) signaling might regulate oxidative phosphorylation is a focus of this review. The relevant Ca(2+) sensitive sites include several dehydrogenases and substrate transporters together with a post-translational modification of F1-FO-ATPase and cytochrome oxidase. Thus, Ca(2+) apparently activates both the generation of the mitochondrial membrane potential as well as utilization to produce ATP. This balanced activation extends the energy homeostasis observed in the cytosol into the mitochondria matrix in the never resting heart.

p53 Improves Aerobic Exercise Capacity and Augments Skeletal Muscle Mitochondrial DNA Content

Rationale: Exercise capacity is a physiological characteristic associated with protection from both cardiovascular and all-cause mortality. p53 regulates mitochondrial function and its deletion markedly diminishes exercise capacity, but the underlying genetic mechanism orchestrating this is unclear. Understanding the biology of how p53 improves exercise capacity may provide useful insights for improving both cardiovascular as well as general health. Objective: The purpose of this study was to understand the genetic mechanism by which p53 regulates aerobic exercise capacity. Methods and Results: Using a variety of physiological, metabolic, and molecular techniques, we further characterized maximum exercise capacity and the effects of training, measured various nonmitochondrial and mitochondrial determinants of exercise capacity, and examined putative regulators of mitochondrial biogenesis. As p53 did not affect baseline cardiac function or inotropic reserve, we focused on the involvement of skeletal muscle and now report a wider role for p53 in modulating skeletal muscle mitochondrial function. p53 interacts with Mitochondrial Transcription Factor A (TFAM), a nuclear-encoded gene important for mitochondrial DNA (mtDNA) transcription and maintenance, and regulates mtDNA content. The increased mtDNA in p53+/+ compared to p53−/− mice was more marked in aerobic versus glycolytic skeletal muscle groups with no significant changes in cardiac tissue. These in vivo observations were further supported by in vitro studies showing overexpression of p53 in mouse myoblasts increases both TFAM and mtDNA levels whereas depletion of TFAM by shRNA decreases mtDNA content. Conclusions: Our current findings indicate that p53 promotes aerobic metabolism and exercise capacity by using different mitochondrial genes and mechanisms in a tissue-specific manner.