Robert S Balaban

• Ph.D.
• Scientist Emeritus at National Institutes of Health

Background Commercial low-field-strength MRI systems are generally not equipped with state-of-the-art MRI hardware, and are not suitable for demanding imaging techniques. An MRI system was developed that combines low field strength (0.55 T) with high-performance imaging technology. Purpose To evaluate applications of a high-performance low-field-st...

The design of the energy metabolism system in striated muscle remains a major area of investigation. Here, we review our current understanding and emerging hypotheses regarding the metabolic support of muscle contraction. Maintenance of ATP free energy, so called energy homeostasis, via mitochondrial oxidative phosphorylation is critical to sustain...

Mitochondrial adaptations are fundamental to differentiated function and energetic homeostasis in mammalian cells. But the mechanisms that underlie these relationships remain poorly understood. Here, we investigated organ-specific mitochondrial morphology, connectivity and protein composition in a model of extreme mammalian metabolism, the Least sh...

Intracellular energy distribution has attracted much interest and has been proposed to occur in skeletal muscle via metabolite-facilitated diffusion1, 2; however, genetic evidence suggests that facilitated diffusion is not critical for normal function3, 4, 5, 6, 7. We hypothesized that mitochondrial structure minimizes metabolite diffusion distance...

Vascular production of nitric oxide (NO) regulates vascular tone. However, highly permeable NO entering the cardiomyocyte would profoundly impact metabolism and signalling without scavenging mechanisms. The purpose of this study was to establish mechanisms of cardiac NO scavenging. Quantitative optical studies of normoxic working hearts demonstrate...

Mitochondrial Ca2+ overload is proposed to regulate cell death via opening of the mitochondrial permeability transition pore. It is hypothesized that inhibition of the mitochondrial Ca2+ uniporter (MCU) will prevent Ca2+ accumulation during ischemia/reperfusion and thereby reduce cell death. To address this, we evaluate mitochondrial Ca2+ in ex-viv...

The catalytic cycle of cytochrome c oxidase (COX) couples the reduction of oxygen to the translocation of protons across the inner mitochondrial membrane and involves several intermediate states of the heme a3-CuB binuclear center with distinct absorbance properties. The absorbance maximum close to 605 nm observed during respiration is commonly ass...

Retinal diseases exhibit extensive genetic heterogeneity and complex etiology with varying onset and severity. Mutations in over 200 genes can lead to photoreceptor dysfunction and/or cell death in retinal neurodegeneration. To deduce molecular pathways that initiate and/or drive cell death, we adopted a temporal multi-omics approach and examined m...

Paracoccus denitrificans is a model organism for the study of oxidative phosphorylation. We demonstrate a very high respiratory capacity compared to mitochondria when normalizing to cytochrome aa3 content even in the absence of alternative terminal oxidases. To gain insight into conserved mechanisms of energy homeostasis, we characterized the metab...

Mitochondrial adaptations are fundamental to differentiated function and energetic homeostasis in mammalian cells. But the mechanisms that underlie these relationships remain poorly understood. Here, we investigated organ‐specific mitochondrial morphology, connectivity and protein composition in a model of extreme mammalian metabolism, the least sh...

Optical methods for measuring intracellular ions including Ca2+ revolutionized our understanding of signal transduction. However, these methods are not extensively applied to intact organs due to issues including inner filter effects, motion, and available probes. Mitochondrial Ca2+ is postulated to regulate cell energetics and death pathways that...

Objectives: Contemporary lower-field magnetic resonance imaging (MRI) may offer advantages for lung imaging by virtue of the improved field homogeneity. The aim of this study was to evaluate the utility of lower-field MRI for combined morphologic imaging and regional lung function assessment. We evaluate low-field MRI in patients with lymphangiole...

Neurodegenerative diseases exhibit extensive genetic heterogeneity and complex etiology with varying onset and severity. To deduce the mechanism leading to retinal degeneration, we adopted a temporal multi-omics approach and examined molecular and cellular events before the onset of photoreceptor cell death in the widely-used Pde6b rd1/rd1 ( rd1 )...

The purpose of this study was to evaluate oxygen-enhanced pulmonary imaging at 0.55 T with 3D stack-of-spirals ultrashort-TE (UTE) acquisition. Oxygen-enhanced pulmonary MRI offers the measurement of regional lung ventilation and perfusion using inhaled oxygen as a contrast agent. Low-field MRI systems equipped with contemporary hardware can provid...

Large changes in energy metabolism are associated with minimal alterations in surface temperature of isolated mammalian cells.

Cardiac mitochondria uptake calcium through the mitochondrial calcium uniporter (MCU). To better understand the role of MCU and mitochondrial calcium in regulating heart physiology and pathophysiology, we developed a method to measure mitochondrial matrix calcium in beating, perfused hearts. Langendorff perfusing hearts are loaded with 4.5 uM rhod-...

Although mitochondrial DNA (mtDNA) is prone to accumulate mutations and lacks conventional DNA repair mechanisms, deleterious mutations are exceedingly rare. How the transmission of detrimental mtDNA mutations is restricted through the maternal lineage is debated. Here, we demonstrate that mitochondrial fission, together with the lack of mtDNA repl...

This chapter introduces to those with little or no experience the basic principles of cardiovascular magnetic resonance (CMR). The chapter describes how CMR images are created, using high static magnetic fields and magnetic gradients. The concepts of spin and precession at the Larmor frequency are explained. Radiofrequency (RF) excitation and subse...

The isolated saline-perfused heart is utilized extensively to study cardiac physiology. Previous isolated heart studies demonstrated lower tissue oxygenation than in vivo based on myoglobin oxygenation and mitochondria redox state. These data, consistent with small anoxic regions, suggest that the homeostatic balance between work and oxygen deliver...

(Cell Reports 19, 487–496; April 18, 2017) In the originally published version of this article, Armel Femnou's name was misspelled as Armel Fenmou in the author list. This has now been corrected. The authors regret this error.

Mitochondria exert an immense amount of cytophysiological functions, but the structural basis of most of these processes is still poorly understood. Here we use cross-linking mass spectrometry to probe the organization of proteins in native mouse heart mitochondria. Our approach provides the largest survey of mitochondrial protein interactions repo...

The left ventricular (LV) working, crystalloid-perfused heart is used extensively to evaluate basic cardiac function, pathophysiology, and pharmacology. Crystalloid-perfused hearts may be limited by oxygen delivery, as adding oxygen carriers increases myoglobin oxygenation and improves myocardial function. However, whether decreased myoglobin oxyge...

Absorbance spectroscopy of intrinsic cardiac chromophores provides non-destructive assessment of cytosolic oxygenation and mitochondria redox state. Isolated perfused heart spectroscopy is usually conducted by collecting reflected light from the heart surface, which represents a combination of surface scattering events and light that traversed port...

en The ability to monitor micropipette injections with a high‐resolution fluorescent microscope has utility for a variety of applications. Herein, different approaches were tested for creating broad‐band fluorescently labelled glass micropipettes including: UV cured glass glues, baked glass enamel containing fluorescent dyes as well as nanodiamonds...

T cells undergo metabolic reprogramming with major changes in cellular energy metabolism during activation. In patients with mitochondrial disease, clinical data were marked by frequent infections and immunodeficiency, prompting us to explore the consequences of oxidative phosphorylation dysfunction in T cells. Since cytochrome c oxidase (COX) is a...

Graphical Abstract Highlights d Mitochondrial networks are functionally linked through intermitochondrial junctions d Regional mitochondrial subnetworks proactively limit spread of local dysfunction d Dysfunctional mitochondria are electrically separated from the network in seconds d Physical network separation involves mitochondrial retraction In...

Significance Low water permeability is critical to the pressurized blood conduit function of the artery. Arterial wall permeability is altered in diseases including diabetes and atherosclerosis, in acute shock, and with the aging process. Here we directly map the water permeability and the associated pressure profile across intact pressurized arter...

Mitochondrial network connectivity enables rapid communication and distribution of potential energy throughout the cell. However, this connectivity puts the energy conversion system at risk, because damaged elements could jeopardize the entire network. Here, we demonstrate the mechanisms for mitochondrial network protection in heart and skeletal mu...

Background: Cardiomyocytes are organized in microstructures termed sheetlets that reorientate during left ventricular thickening. Diffusion tensor cardiac magnetic resonance (DT-CMR) may enable noninvasive interrogation of in vivo cardiac microstructural dynamics. Dilated cardiomyopathy (DCM) is a condition of abnormal myocardium with unknown shee...

While genomics, epigenomics, transcriptomics, proteomics, and metabolomics provide valuable insights into the identities of molecules in cells, these methods do not give a global picture of protein 3D structures or protein–protein interactions, both of which are important for protein function and rapid regulation. Protein structures and protein–pro...

Cardiovascular disease is a major leading cause of morbidity and mortality in the United States and elsewhere. Alterations in mitochondrial function are increasingly being recognized as a contributing factor in myocardial infarction and in patients presenting with cardiomyopathy. Recent understanding of the complex interaction of the mitochondria i...

The Virtual Physiological Human (VPH) project aims to develop integrative, explanatory and predictive computational models (C-Models) as numerical investigational tools to study disease, identify and design effective therapies and provide an in silico platform for drug screening. Ultimately, these models rely on the analysis and integration of expe...

Within the mitochondrial reticulum of skeletal muscle, the I-Band segments (IBS) traverse the cell and form a contiguous matrix with the mitochondrial segments at the periphery (PS) of the cell. A tight electrical coupling via the matrix between the PS and IBS has been demonstrated. In addition, oxidative phosphorylation complexes that generate the...

Purpose: Cell death in neurodegeneration occurs at the convergence of diverse metabolic pathways. In the retina, a common underlying mechanism involves mitochondrial dysfunction since photoreceptor homeostasis and survival are highly susceptible to altered aerobic energy metabolism. We sought to develop an assay to directly measure oxygen consumpt...

This paper investigates a postprocessing approach to correct spatial distortion in two-photon fluorescence microscopy images for vascular network reconstruction. It is aimed at in vivo imaging of large field-of-view, deep-tissue studies of vascular structures. Based on simple geometric modelling of the object-of-interest, a distortion function is d...

Purpose: To provide a rapid method to reduce the radiofrequency (RF) E-field coupling and consequent heating in long conductors in an interventional MRI (iMRI) setup. Methods: A driving function for device heating (W) was defined as the integration of the E-field along the direction of the wire and calculated through a quasistatic approximation....

Various human diseases are associated with mitochondrial DNA (mtDNA) mutations, but heteroplasmy-the coexistence of mutant and wild-type mtDNA-complicates their study. We previously isolated a temperature lethal mtDNA mutation in Drosophila, mt:CoI(T300I) affecting the cytochrome c oxidase subunit I (CoI) locus. In the current study, we found that...

Using the intrinsic optical properties of collagen and elastin, two-photon microscopy was applied to evaluate the three-dimensional (3D) macromolecular structural development of the mouse thoracic aorta from birth to 60 days old. Baseline development was established in the Scavenger Receptor Class B Type I-Deficient, Hypomorphic Apolipoprotein ER61...

Cardiac oxidative ATP generation is finely tuned to match several-fold increases in energy demand. Calcium has been proposed to play a role in the activation of ATP production via PKA phosphorylation in response to intramitochondrial cAMP generation. We evaluated the effect of cAMP, its membrane permeable analogs (dibutyryl-cAMP, 8-bromo-cAMP), and...

In conventional multi-probe fluorescence microscopy, narrow bandwidth filters on detectors are used to avoid bleed-through artefacts between probes. The limited bandwidth reduces the signal-to-noise ratio of the detection, often severely compromising one or more channels. Herein, we describe a process of using independent component analysis to disc...

The structure and function of the renal artery ostium flow diverter on the caudal side of the renal branch point were previously reported; in this study, we further evaluate the diverter's possible functions. The protrusion of this structure into the abdominal aorta suggests that the diverter may preferentially direct blood flow to the renal arteri...

Objective Subendothelial LDL retention by intimal matrix proteoglycans is an initial step in atherosclerosis and calcific aortic valve disease. Herein, we identify decorin and biglycan as the proteoglycans that preferentially retain LDL in intimal matrix at disease-prone sites in normal valve and vessel wall. Methods The porcine aortic valve and r...

In this review, we focus on the impact of tissue motion on attempting to conduct subcellular resolution optical microscopy, in vivo. Our position is that tissue motion is one of the major barriers in conducting these studies along with light induced damage, optical probe loading as well as absorbing and scattering effects on the excitation point sp...

We describe a compact, non-contact design for a total emission detection (c-TED) system for intra-vital multiphoton imaging. To conform to a standard upright two-photon microscope design, this system uses a parabolic mirror surrounding a standard microscope objective in concert with an optical path that does not interfere with normal microscope ope...

Mitochondrial calcium has been postulated to regulate a wide range of processes from bioenergetics to cell death. Here, we characterize a mouse model that lacks expression of the recently discovered mitochondrial calcium uniporter (MCU). Mitochondria derived from MCU(-/-) mice have no apparent capacity to rapidly uptake calcium. Whereas basal metab...

Objective To provide insight into mitochondrial function in vivo, we evaluated the 3D spatial relationship between capillaries, mitochondria, and muscle fibers in live mice. Methods3D volumes of in vivo murine Tibialis anterior muscles were imaged by multi-photon microscopy (MPM). Muscle fiber type, mitochondrial distribution, number of capillaries...

The tendency of atherosclerotic plaques to develop at arterial branch points is likely due to both the hemodynamics and macromolecular environment associated with these branch points. Arterial branches experience flow separation, which results in regions of low shear stress[1–3], and contributes to longer residence times that may allow for depositi...

Recent evidence suggests that the activity of mitochondrial oxidative phosphorylation Complexes (MOPC) is modulated at multiple sites. Herein, a method of optically monitoring electron distribution within and between MOPC is described using a center-mounted sample in an integrating sphere (to minimize scattering effects) with a rapid-scanning spect...

Calcium is believed to regulate mitochondrial oxidative phosphorylation, thereby contributing to the maintenance of cellular energy homeostasis. Skeletal muscle, with an energy conversion dynamic range of up to 100-fold, is an extreme case for evaluating the cellular balance of ATP production and consumption. This study examined the role of Ca(2+)...

There is growing evidence that alterations in metabolism may contribute to tumorigenesis. Here, we report on members of families with the Li-Fraumeni syndrome who carry germline mutations in TP53, the gene encoding the tumor-suppressor protein p53. As compared with family members who are not carriers and with healthy volunteers, family members with...

Allometry refers to biological scaling relationships (1). With regard to metabolic rate, the earliest allometric studies demonstrated a complex inverse relationship between body size and resting metabolic rate (2⇓–4). Subsequent studies have focused, without much success, on describing the cellular mechanisms of this relationship. The energy requir...

Rationale: Creatine is thought to be involved in the spatial and temporal buffering of ATP in energetic organs such as heart and skeletal muscle. Creatine depletion affects force generation during maximal stimulation, while reduced levels of myocardial creatine are a hallmark of the failing heart, leading to the widely held view that creatine is i...

Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light-scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the abil...

It has become appreciated over the last several years that protein phosphorylation within the cardiac mitochondrial matrix and respiratory complexes is extensive. Given the importance of oxidative phosphorylation and the balance of energy metabolism in the heart, the potential regulatory effect of these classical signaling events on mitochondrial f...

When conducting optical imaging experiments, in vivo, the signal to noise ratio and effective spatial and temporal resolution is fundamentally limited by physiological motion of the tissue. A three-dimensional (3D) motion tracking scheme, using a multiphoton excitation microscope with a resonant galvanometer, (512 × 512 pixels at 33 frames s(-1)) i...

The heart is nearly unique in the body in that it has a constant workload well beyond the normal maintenance of cellular integrity. In addition, the heart of a large animal such as man also has the capacity to increase its workload by nearly 10-fold for considerable amounts of time. A high steady

The purpose of this study was to evaluate the in vivo , 3D spatial relationship between capillaries (CAP), mitochondria (MITO), and three fiber types within murine Tibialis anterior (TA) muscle. Two photon excitation microscopy was utilized for in vivo imaging of MITO (NADH autofluorescence), vasculature, and interstitial space using injected dyes....

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 musc...

The concentration of mitochondrial oxidative phosphorylation complexes (MOPCs) is tuned to the maximum energy conversion requirements of a given tissue; however, whether the activity of MOPCs is altered in response to acute changes in energy conversion demand is unclear. We hypothesized that MOPCs activity is modulated by tissue metabolic stress to...

Despite identical cardiac outputs, the right (RV) and left ventricle (LV) have very different embryological origins and resting workload. These differences suggest that the ventricles have different protein programming with regard to energy metabolism and contractile elements. The objective of this study was to determine the relative RV and LV prot...

The measurement of unidirectional rates of reaction, in vivo, using magnetization transfer techniques is a unique capability of nuclear magnetic resonance spectroscopy (NMRS). However, the complicated network of cytosolic reactions as well as the potential existence of small exchanging metabolic pools or enzyme-substrate complexes with poorly defin...

SummaryA benefit of multiphoton fluorescence microscopy is the inherent optical sectioning that occurs during excitation at the diffraction-limited spot. The scanned collection of fluorescence emission is incoherent; that is, no real image needs to be formed on the detector plane. The nearly isotropic emission of fluorescence excited at the focal s...

Red and white muscles are faced with very different energetic demands. However, it is unclear whether relative mitochondrial protein expression is different between muscle types. Mitochondria from red and white porcine skeletal muscle were isolated with a Percoll gradient. Differences in protein composition were determined using blue native (BN)-PA...

Mitochondrial protein phosphorylation is a well-recognized metabolic control mechanism, with the classical example of pyruvate dehydrogenase (PDH) regulation by specific kinases and phosphatases of bacterial origin. However, despite the growing number of reported mitochondrial phosphoproteins, the identity of the protein kinases mediating these pho...

Micrometer-scale three-dimensional data from fluorescence microscopes offer unique insight into cellular morphology and function by resolving subcellular locations of fluorescent dyes and proteins. To increase field-of-view size while using a high-resolution multiphoton microscope, we have created an automated system of rapidly acquiring overlappin...

The Division of Lung Diseases of the National Heart, Lung, and Blood Institute, with the Office of Rare Diseases Research, held a workshop to identify priority areas and strategic goals to enhance and accelerate research that will result in improved understanding of the lung vasculature, translational research needs, and ultimately the care of pati...