Kenneth A. Schenkman

An unexpected role for brain-type sodium channels in coupling of cell surface depolarization to contraction in the heart

Voltage-gated sodium channels composed of pore-forming α and auxiliary β subunits are responsible for the rising phase of the action potential in cardiac muscle, but the functional roles of distinct sodium channel subtypes have not been clearly defined. Immunocytochemical studies show that the principal cardiac pore-forming α subunit isoform Nav1.5 is preferentially localized in intercalated disks, whereas the brain α subunit isoforms Nav1.1, Nav1.3, and Nav1.6 are localized in the transverse tubules. Sodium currents due to the highly tetrodotoxin (TTX)-sensitive brain isoforms in the transverse tubules are small and are detectable only after activation with β scorpion toxin. Nevertheless, they play an important role in coupling depolarization of the cell surface membrane to contraction, because low TTX concentrations reduce left ventricular function. Our results suggest that the principal cardiac isoform in the intercalated disks is primarily responsible for action potential conduction between cells and reveal an unexpected role for brain sodium channel isoforms in the transverse tubules in coupling electrical excitation to contraction in cardiac muscle.

Mice with Mitochondrial Complex I Deficiency Develop a Fatal Encephalomyopathy

To study effects of mitochondrial complex I (CI, NADH:ubiquinone oxidoreductase) deficiency, we inactivated the Ndufs4 gene, which encodes an 18 kDa subunit of the 45-protein CI complex. Although small, Ndufs4 knockout (KO) mice appeared healthy until approximately 5 weeks of age, when ataxic signs began, progressing to death at approximately 7 weeks. KO mice manifested encephalomyopathy including a retarded growth rate, lethargy, loss of motor skill, blindness, and elevated serum lactate. CI activity in submitochondrial particles from KO mice was undetectable by spectrophotometric assays. However, CI-driven oxygen consumption by intact tissue was about half that of controls. Native gel electrophoresis revealed reduced levels of intact CI. These data suggest that CI fails to assemble properly or is unstable without NDUFS4. KO muscle has normal morphology but low NADH dehydrogenase activity and subsarcolemmal aggregates of mitochondria. Nonetheless, total oxygen consumption and muscle ATP and phosphocreatine concentrations measured in vivo were within normal parameters.

Propofol impairment of mitochondrial respiration in isolated perfused guinea pig hearts determined by reflectance spectroscopy

Objective: To simultaneously determine the effect of propofol on myocardial oxygenation, mitochondrial function, and whole organ function in an isolated heart model, using optical reflectance spectroscopy. Design: Controlled laboratory investigation. Setting: Research laboratory. Subjects: Twenty adult guinea pigs. Interventions: Isolated hearts were perfused alternately with a modified oxygenated Krebs‐Henseleit buffer and with buffer containing varied concentrations of propofol. Ninety seconds of ischemia were produced during perfusion with each solution studied. Measurements and Main Results: Myoglobin oxygen saturation, cytochrome c and cytochrome a/a3 redox state, and ventricular pressure were continuously measured from isolated guinea pig hearts during a 2‐hr period. Myoglobin oxygen saturation increased and both cytochromes became more oxidized in the presence of propofol. During ischemia, myoglobin desaturation and cytochrome reduction were delayed and less complete in the presence of propofol. The mean ischemic time to 50% myoglobin desaturation was, on average, 14.3 secs with buffer perfusion, and increased to 24.5, 27.9, and 41.8 secs, with 50, 100, and 200 μM propofol perfusion, respectively. Ventricular function decreased linearly with increasing propofol concentration. From baseline buffer perfusion, maximal dP/dt per cardiac cycle decreased on average by 30.4%, 40.9%, and 69.4%, with 50, 100, and 200 μM propofol perfusion, respectively. Conclusions: Propofol impairs either oxygen utilization or inhibits electron flow along the mitochondrial electron transport chain in the guinea pig cardiomyocyte. Propofol also significantly decreases ventricular performance in the isolated perfused heart. These effects are linearly correlated with propofol concentration in the range studied.

Reduced mitochondrial coupling in vivo alters cellular energetics in aged mouse skeletal muscle

The mitochondrial theory of ageing proposes that the accumulation of oxidative damage to mitochondria leads to mitochondrial dysfunction and tissue degeneration with age. However, no consensus has emerged regarding the effects of ageing on mitochondrial function, particularly for mitochondrial coupling (P/O). One of the main barriers to a better understanding of the effects of ageing on coupling has been the lack of in vivo approaches to measure P/O. We use optical and magnetic resonance spectroscopy to independently quantify mitochondrial ATP synthesis and O2 uptake to determine in vivo P/O. Resting ATP demand (equal to ATP synthesis) was lower in the skeletal muscle of 30‐month‐old C57Bl/6 mice compared to 7‐month‐old controls (21.9 ± 1.5 versus 13.6 ± 1.7 nmol ATP (g tissue)−1 s−1, P= 0.01). In contrast, there was no difference in the resting rates of O2 uptake between the groups (5.4 ± 0.6 versus 8.4 ± 1.6 nmol O2 (g tissue)−1 s−1). These results indicate a nearly 50% reduction in the mitochondrial P/O in the aged animals (2.05 ± 0.07 versus 1.05 ± 0.36, P= 0.02). The higher resting ADP (30.8 ± 6.8 versus 58.0 ± 9.5 μmol g−1, P= 0.05) and decreased energy charge (ATP/ADP) (274 ± 70 versus 84 ± 16, P= 0.03) in the aged mice is consistent with an impairment of oxidative ATP synthesis. Despite the reduced P/O, uncoupling protein 3 protein levels were not different in the muscles of the two groups. These results demonstrate reduced mitochondrial coupling in aged skeletal muscle that alters cellular metabolism and energetics.

Continuous measurement of oxygen consumption by pancreatic islets.

The rate of oxygen consumption is an important measure of mitochondrial function in all aerobic cells. In pancreatic beta cells, it is linked to the transduction mechanism that mediates glucose-stimulated insulin secretion. However, measurement of oxygen consumption over long periods of time is technically difficult owing to the error resulting from baseline drift and the challenge of measuring small changes in oxygen tension. We have adapted an ultrastable oxygen sensor based on the detection of the decay of the phosphorescent emission from an oxygen-sensitive dye to a previously developed islet flow culture system. The drift of the sensor is approximately 0.3%/24 h, allowing for the continuous measurement of oxygen consumption by 300 islets (or about 6 x 10(5) cells) for hours or days. Rat islets placed in the perifusion chamber for 24 h were well maintained as reflected by membrane integrity, insulin secretion, and oxygen consumption. Both acute changes in oxygen consumption as induced by glucose and chronic changes as induced by sequential pulses of azide were resolved. The features of the flow culture system--aseptic conditions, fine temporal control of the composition of the media, and the collection of outflow fractions for measurement of insulin, and other products--facilitate a systematic approach to assessing metabolic and functional viability in responses to a variety of stimuli. Applications to the measurement of effects of hypoxia on insulin secretion, membrane integrity, and the redox state of cytochromes are demonstrated. The system has particular application to the field of human islet transplantation, where assessment and the study of islet viability have been hampered by a lack of experimental methods.

Cerebral hyperemia and impaired cerebral autoregulation associated with diabetic ketoacidosis in critically ill children

Objective:Cerebral edema associated with diabetic ketoacidosis is an uncommon but severe complication of insulin-dependent diabetes mellitus with unclear pathophysiology. We sought to determine whether cerebral edema in patients with diabetic ketoacidosis was related to changes in cerebral blood flow, autoregulation, regional cerebral saturation, or S100B. Design:Prospective case series. Setting:Pediatric intensive care unit of a tertiary children’s hospital. Patients:Six patients with diabetic ketoacidosis and altered mental status, requiring computed tomographic scan of the head. Interventions:Study evaluations included: 1) transcranial Doppler evaluations to determine middle cerebral artery flow velocities and cerebral autoregulation, defined by the autoregulatory index, at 6 and 36 hrs; 2) continuous monitoring of regional cerebral oxygenation on the left lateral forehead using near-infrared spectroscopy for the first 24 hrs of admission; 3) serial measurement of S100B as a marker of central nervous system injury; and 4) follow-up head computed tomographic scan. Results:Serial computed tomographic scans showed that four of six patients had changes in brain volume without overt cerebral edema. Initial scans showed narrowing of the third and lateral ventricles when compared with follow-up. There was no difference in middle cerebral artery flow velocities between admission and recovery at 36 hrs, despite Paco2 increasing during treatment. Cerebral flow was normal to increased, despite hypocapnia. Cerebral autoregulation was impaired in five of six patients at 6 hrs and normalized by 36 hrs. Mean regional cerebral oxygenation was measured in five of six patients and decreased linearly with time. Two patients showed maximal regional cerebral oxygenation before returning to baseline. There were no periods of low regional cerebral oxygenation in any patient at any time. No elevation in S100B was found. Conclusions:We found normal to increased cerebral blood flow, elevated regional cerebral oxygenation, impaired autoregulation, and changes in brain volume in clinically ill pediatric patients with diabetic ketoacidosis. We found no evidence of cerebral ischemia. These findings suggest that the pathophysiology of cerebral edema in diabetic ketoacidosis may involve a transient loss of cerebral autoregulation, allowing a paradoxic increase in cerebral blood flow and the development of vasogenic cerebral edema.

NEAR-INFRARED SPECTROSCOPIC MEASUREMENT OF MYOGLOBIN OXYGEN SATURATION IN THE PRESENCE OF HEMOGLOBIN USING PARTIAL LEAST-SQUARES ANALYSIS

Myoglobin is an important intracellular protein found in cardiac and skeletal muscle. It is involved in the intracellular transport of oxygen from the cell membrane to the mitochondria where oxidative phosphorylation takes place. The optical absorbance characteristics of myoglobin are similar to those of hemoglobin in the near-infrared spectral region. Distinguishing spectral information of myoglobin from hemoglobin should allow for determination of intracellular oxygen availability in muscle. Partial least-squares analysis is used in this report to determine the oxygen saturation of myoglobin, in the presence of hemoglobin, in vitro. Studies were performed with the use of both transmission and reflectance spectroscopic techniques. Transmission spectra of myoglobin solutions were determined with varying degrees of oxygen saturation achieved by deoxygenating the solution using E. coli. Calibration spectral data sets were developed with the use of varying concentrations of hemoglobin interference, and with varying degrees of myoglobin oxygen saturation. Reflectance spectra were obtained from myoglobin and hemoglobin solutions containing a scattering agent to mimic muscle tissue conditions. Predicted myoglobin saturation values were within 2% of the known saturation values from the use of this analysis. Partial least-squares analysis allows for accurate prediction of myoglobin oxygen saturation in the presence of hemoglobin from either transmission of reflectance near-infrared spectra.

Optical spectroscopic method for in vivo measurement of cardiac myoglobin oxygen saturation

A fiber-optic-based spectrophotometer was developed to acquire optical reflectance spectra from a living dog heart. A bullseye concentric optical probe with a 3 mm source-to-detector fiber separation was designed to obtain a 1.5 mm average tissue depth of light penetration. Spectra were analyzed in the near-infrared region from 660 to 840 nm. Myoglobin oxygen saturation was determined by partial least-squares analysis using a calibration spectral data set developed in vitro. Comparison of in vivo and in vitro spectra by Mahalanobis distance and residual ratio tests demonstrated good similarity, justifying use of partial least-squares analysis. Coronary perfusion with an oxygenated blood substitute, Fluosol®, was used to demonstrate that hemoglobin had little effect on the analysis. An increase in myoglobin saturation of 5% was noted when the animals were changed from inspired room air to 100% oxygen. Occlusion of the coronary artery resulted in prompt decrease in myoglobin saturation, and release of the occlusion was followed by rapid increase in saturation to a value above baseline. These experiments demonstrate that it is feasible to use partial least-squares analysis of near-infrared reflectance spectra to determine myoglobin saturation in the blood-perfused, beating heart.

Wavelength Shift Analysis: A Simple Method to Determine the Contribution of Hemoglobin and Myoglobin to In Vivo Optical Spectra

The ability to quantify the contributions of hemoglobin (Hb) and myoglobin (Mb) to in vivo optical spectra has many applications for clinical and research use such as noninvasive measurement of local tissue O2 uptake rates and regional blood content. Recent work has demonstrated an approach to independently measure oxygen saturations of Hb and Mb in optical spectra collected in vivo. However, the utility of this approach is limited without information on tissue concentrations of these species. Here we describe a strategy to quantify the contributions of Hb and Mb to in vivo optical spectra. We have found that the peak position of the deoxy-heme peak around 760 nm in the optical spectra of the deoxygenated tissue is a linear function of the relative contributions of Hb and Mb to the optical spectra. Therefore, analysis of this peak position, hereafter referred to as wavelength shift analysis, reveals the relative concentration of Hb to Mb in solutions and intact tissue. Biochemical analysis of muscle homogenates confirmed that the wavelength shift of the combined Hb/Mb peak in in vivo spectra reflects the ratio of concentrations (Hb/Mb) in muscle. The importance of quantifying the Hb contribution is illustrated by our data demonstrating that Hb accounts for approximately 80% of the optical signal in mouse skeletal muscle but only approximately 20% in human skeletal muscle. This advance will facilitate comparison of the metabolic properties between individual muscles and provides a fully noninvasive approach to measuring local respiration that can be adapted for clinical use.

Mitochondrial function in vivo: Spectroscopy provides window on cellular energetics

Mitochondria integrate the key metabolic fluxes in the cell. This role places this organelle at the center of cellular energetics and, hence, mitochondrial dysfunction underlies a growing number of human disorders and age-related degenerative diseases. Here we present novel analytical and technical methods for evaluating mitochondrial metabolism and (dys)function in human muscle in vivo. Three innovations involving advances in optical spectroscopy (OS) and magnetic resonance spectroscopy (MRS) permit quantifying key compounds in energy metabolism to yield mitochondrial oxidation and phosphorylation fluxes. The first of these uses analytical methods applied to optical spectra to measure hemoglobin (Hb) and myoglobin (Mb) oxygenation states and relative contents ([Hb]/[Mb]) to determine mitochondrial respiration (O2 uptake) in vivo. The second uses MRS methods to quantify key high-energy compounds (creatine phosphate, PCr, and adenosine triphosphate, ATP) to determine mitochondrial phosphorylation (ATP flux) in vivo. The third involves a functional test that combines these spectroscopic approaches to determine mitochondrial energy coupling (ATP/O2), phosphorylation capacity (ATP(max)) and oxidative capacity (O2max) of muscle. These new developments in optical and MR tools allow us to determine the function and capacity of mitochondria noninvasively in order to identify specific defects in vivo that are associated with disease in human and animal muscle. The clinical implication of this unique diagnostic probe is the insight into the nature and extent of dysfunction in metabolic and degenerative disorders, as well as the ability to follow the impact of interventions designed to reverse these disorders.