Theodore F. Wiesner

Dynamic Regulation of Sarcoplasmic Reticulum Ca2+ Content and Release by Luminal Ca2+-Sensitive Leak in Rat Ventricular Myocytes

In cardiac muscle, excitation-contraction (E-C) coupling is determined by the ability of the sarcoplasmic reticulum (SR) to store and release Ca(2+). It has been hypothesized that the Ca(2+) sequestration and release mechanisms might be functionally linked to optimize the E-C coupling process. To explore the relationships between the loading status of the SR and functional state of the Ca(2+) release mechanism, we examined the effects of changes in SR Ca(2+) content on spontaneous Ca(2+) sparks in saponin-permeabilized and patch-clamped rat ventricular myocytes. SR Ca(2+) content was manipulated by pharmacologically altering the capacities of either Ca(2+) uptake or leak. Ca(2+) sparks were recorded using a confocal microscope and Fluo-3 and were quantified considering missed events. SR Ca(2+) content was assessed by application of caffeine. Exposure of permeabilized cells to anti-phospholamban antibodies elevated the SR Ca(2+) content and increased the frequency of sparks. Suppression of the SR Ca(2+) pump by thapsigargin lowered [Ca(2+)](SR) and reduced the frequency of sparks. The ryanodine receptor (RyR) blockers tetracaine and Mg(2+) transiently suppressed the frequency of sparks. Upon washout of the drugs, sparking activity transiently overshot control levels. Low doses of caffeine transiently potentiated sparking activity upon application and transiently depressed the sparks upon removal. In patch-clamped cardiac myocytes, exposure to caffeine produced only a transient increase in the probability of sparks induced by depolarization. We interpret these results in terms of a novel dynamic control scheme for SR Ca(2+) cycling. A central element of this scheme is a luminal Ca(2+) sensor that links the functional activity of RyRs to the loading state of the SR, allowing cells to auto-regulate the size and functional state of their SR Ca(2+) pool. These results are important for understanding the regulation of intracellular Ca(2+) release and contractility in cardiac muscle.

The role of luminal Ca2+ in the generation of Ca2+ waves in rat ventricular myocytes

1 We used confocal Ca2+ imaging and fluo‐3 to investigate the transition of localized Ca2+ releases induced by focal caffeine stimulation into propagating Ca2+ waves in isolated rat ventricular myocytes. 2 Self‐sustaining Ca2+ waves could be initiated when the cellular Ca2+ load was increased by elevating the extracellular [Ca2+] ([Ca2+]o) and they could also be initiated at normal Ca2+ loads when the sensitivity of the release sites to cytosolic Ca2+ was enhanced by low doses of caffeine. When we prevented the accumulation of extra Ca2+ in the luminal compartment of the sarcoplasmic reticulum (SR) with thapsigargin, focal caffeine pulses failed to trigger self‐sustaining Ca2+ waves on elevation of [Ca2+]o. Inhibition of SR Ca2+ uptake by thapsigargin in cells already preloaded with Ca2+ above normal levels did not prevent local Ca2+ elevations from triggering propagating waves. Moreover, wave velocity increased by 20 %. Tetracaine (0·75 mM) caused transient complete inhibition of both local and propagating Ca2+ signals, followed by full recovery of the responses due to increased SR Ca2+ accumulation. 3 Computer simulations using a numerical model with spatially distinct Ca2+ release sites suggested that increased amounts of releasable Ca2+ might not be sufficient to generate self‐sustaining Ca2+ waves under conditions of Ca2+ overload unless the threshold of release site Ca2+ activation was set at relatively low levels (< 1·5 μM). 4 We conclude that the potentiation of SR Ca2+ release channels by luminal Ca2+ is an important factor in Ca2+ wave generation. Wave propagation does not require the translocation of Ca2+ from the spreading wave front into the SR. Instead, it relies on luminal Ca2+ sensitizing Ca2+ release channels to cytosolic Ca2+.

Termination of Ca2+ release during Ca2+ sparks in rat ventricular myocytes

1 Confocal Ca2+ imaging was used to measure spontaneous release events (Ca2+ sparks) in fluo‐3‐loaded isolated rat ventricular myocytes. 2 The microscopic Ca2+ release flux underlying Ca2+ sparks was derived by adapting the methods used previously to describe macroscopic Ca2+ release from cell‐averaged Ca2+ transients. 3 The magnitude of the local release fluxes varied from 2 to 5 μM ms−1, depending on SR Ca2+ loading conditions. Following spontaneous activation, the release flux rapidly decayed (τ= 6–12 ms). The rate of termination of release flux was found to be directly related to the magnitude of the flux (r2= 0.88). 4 The rate of termination of local release flux was slowed in the presence of FK506, a compound that is known to reduce inactivation of SR Ca2+ channels in vitro. 5 These results suggest that termination of release flux during sparks is not due to a spontaneous stochastic decay process or local depletion of Ca2+ from the SR, but rather involves an active extinguishing mechanism such as Ca2+‐dependent inactivation or adaptation.

Inhibition of Ca(2+) sparks by ruthenium red in permeabilized rat ventricular myocytes.

We have compared the effects of the sarcoplasmic reticulum (SR) Ca(2+) release inhibitor, ruthenium red (RR), on single ryanodine receptor (RyR) channels in lipid bilayers, and on Ca(2+) sparks in permeabilized rat ventricular myocytes. Ruthenium red at 5 microM inhibited the open probability (P(o)) of RyRs approximately 20-50-fold, without significantly affecting the conductance or mean open time of the channel. At the same concentration, RR inhibited the frequency of Ca(2+) sparks in permeabilized myocytes by approximately 10-fold, and reduced the amplitude of large amplitude events (with most probable localization on the line scan) by approximately 3-fold. According to our theoretical simulations, performed with a numerical model of Ca(2+) spark formation, this reduction in Ca(2+) spark amplitude corresponds to an approximately 4-fold decrease in Ca(2+) release flux underlying Ca(2+) sparks. Ruthenium red (5 microM) increased the SR Ca(2+) content by approximately 2-fold (from 151 to 312 micromol/l cytosol). Considering the degree of inhibition of local Ca(2+) release events, the increase in SR Ca(2+) load by RR, and the lack of effects of RR on single RyR open time and conductance, we have estimated that Ca(2+) sparks under normal conditions are generated by openings of at least 10 single RyRs.

Dynamic optimization of hybridoma growth in a fed-batch bioreactor

This study addressed the problem of maximizing cell mass and monoclonal antibody production from a fed-batch hybridoma cell culture. We hypothesized that inaccuracies in the process model limited the mathematical optimization. On the basis of shaker flask data, we established a simple phenomenological model with cell mass and lactate production as the controlled variables. We then formulated an optimal control algorithm, which calculated the process-model mismatch at each sampling time, updated the model parameters, and re-optimized the substrate concentrations dynamically throughout the time course of the batch. Manipulated variables were feed rates of glucose and glutamine. Dynamic parameter adjustment was done using a fuzzy logic technique, while a heuristic random optimizer (HRO) optimized the feed rates. The parameters selected for updating were specific growth rate and the yield coefficient of lactate from glucose. These were chosen by a sensitivity analysis. The cell mass produced using dynamic optimization was compared to the cell mass produced for an unoptimized case, and for a one-time optimization at the beginning of the batch. Substantial improvements in reactor productivity resulted from dynamic re-optimization and parameter adjustment. We demonstrated first that a single offline optimization of substrate concentration at the start of the batch significantly increased the yield of cell mass by 27% over an unoptimized fermentation. Periodic optimization online increased yield of cell mass per batch by 44% over the single offline optimization. Concomitantly, the yield of monoclonal antibody increased by 31% over the off-line optimization case. For batch and fed-batch processes, this appears to be a suitable arrangement to account for inaccuracies in process models. This suggests that implementation of advanced yet inexpensive techniques can improve performance of fed-batch reactors employed in hybridoma cell culture.

Underlying mechanisms of symmetric calcium wave propagation in rat ventricular myocytes.

Calcium waves in heart cells are mediated by diffusion-coupled calcium-induced calcium release. The waves propagate in circular fashion. This is counterintuitive in view of the accepted ultrastructure of the cardiac myocyte. The density of calcium release sites in the transverse direction is four times higher than in the longitudinal direction. Simulations with release sites localized along Z-lines and isotropic diffusion yielded highly elliptical, nonphysiological waves. We hypothesized that subcellular organelles counteracted the higher release site density along the Z-lines by acting as transverse diffusion barriers and sites of active calcium uptake. We quantified the reduction of transverse diffusion by microinjecting cells with the nonreactive dye fluorescein. The ratio of the radial diffusion coefficient to the longitudinal coefficient was 0.39. Inhibition of mitochondrial uptake by rotenone accelerated the wave in the transverse direction. Simulations with release sites clustered at the Z-lines and a transverse diffusion coefficient 50% of the longitudinal coefficient generated waves of ellipticity 2/1 (major axis along the Z-line). Introducing additional release sites between the Z-lines at a density 20% of that on the Z-lines produced circular waves. The experiments and simulations support the presence of transverse diffusion barriers, additional uptake sites, and possibly intermediate release sites as well.

Dynamic reoptimization of a fed-batch fermentor.

Traditionally, fed-batch biochemical process optimization and control use complicated models and off-line optimizers with no on-line model adaptation and re-optimization. This work demonstrates the applicability, effectiveness, and economic potential, of a simple phenomenological model for modeling, and a novel optimizer for on-line re-optimization and control of an aerobic fed-batch fermentor.

Nitrification-denitrification biological treatment of a high-nitrogen waste stream for water-reuse applications.

This research was conducted to evaluate the use of biological nitrification-denitrification systems as pre-processors for recycling wastewater to potable water in support of space exploration. A packed-bed bioreactor and membrane-aerated nitrification reactor were operated in series with a 10:1 recycle ratio over varying loading rates. The dissolved organic carbon (DOC) removal exceeded 80% for all loading rates (theta = 1 to 6.8 days), while total nitrogen removal generally increased with decreasing retention time, with a maximum removal of 55%. The degree of nitrification generally declined with decreasing retention time from a high of 80% to a low of 60%. Maximum DOC and total nitrogen volumetric removal rates exceeded 1000 and 800 g/m3 x d, respectively, and maximum nitrification volumetric conversion rates exceeded 300 g/m3 x d. At low hydraulic loading rates, the system was stoichiometrically limited, while kinetic limitations dominated at high hydraulic loading rates. Incomplete nitrification occurred at high loading rates, likely as a result of the high pH and large concentrations of ammonia.

Biological production of ethanol from CO 2 produced by a fossil-fueled power plant

Carbon dioxide, as the end product of fossil fuel combustion, is responsible for the global warming. To decrease emissions of CO2, a new method that directly converts CO2 into ethanol is proposed here, and hydrogen is demonstrated to be the most suitable electron donor. The bacteria, Clostridium ljungdahlii and Moorella sp. HUC22-1, are anaerobic microorganisms which may be used to facilitate the conversion. An ethanol production facility located adjacent a fossil fuel electric power plant was simulated in SuperPro Designer. For C. ljungdahli, the production cost of ethanol was estimated at

Technology in engineering education – essential for sustainable approaches to global technical challenges

0.83/gal, which compares quite favorably to a current ethanol selling price of