Robert W. Wiseman

Skeletal myoblast transplantation for repair of myocardial necrosis

Myocardial infarcts heal by scarring because myocardium cannot regenerate. To determine if skeletal myoblasts could establish new contractile tissue, hearts of adult inbred rats were injured by freeze-thaw, and 3-4.5 x 10(6) neonatal skeletal muscle cells were transplanted immediately thereafter. At 1 d the graft cells were proliferating and did not express myosin heavy chain (MHC). By 3 d, multinucleated myotubes were present which expressed both embryonic and fast fiber MHCs. At 2 wk, electron microscopy demonstrated possible satellite stem cells. By 7 wk the grafts began expressing beta-MHC, a hallmark of the slow fiber phenotype; coexpression of embryonic, fast, and beta-MHC continued through 3 mo. Transplanting myoblasts 1 wk after injury yielded comparable results, except that grafts expressed beta-MHC sooner (by 2 wk). Grafts never expressed cardiac-specific MHC-alpha. Wounds containing 2-wk-old myoblast grafts contracted when stimulated ex vivo, and high frequency stimulation induced tetanus. Furthermore, the grafts could perform a cardiac-like duty cycle, alternating tetanus and relaxation, for at least 6 min. Thus, skeletal myoblasts can establish new muscle tissue when grafted into injured hearts, and this muscle can contract when stimulated electrically. Because the grafts convert to fatigue-resistant, slow twitch fibers, this new muscle may be suited to a cardiac work load.

The signal transduction function for oxidative phosphorylation is at least second order in ADP

To maintain ATP constant in the cell, mitochondria must sense cellular ATP utilization and transduce this demand to F0-F1-ATPase. In spite of a considerable research effort over the past three decades, no combination of signal(s) and kinetic function has emerged with the power to explain ATP homeostasis in all mammalian cells. We studied this signal transduction problem in intact human muscle using 31P NMR spectroscopy. We find that the apparent kinetic order of the transduction function of the signal cytosolic ADP concentration ([ADP]) is at least second order and not first order as has been assumed. We show that amplified mitochondrial sensitivity to cytosolic [ADP] harmonizes with in vitro kinetics of [ADP] stimulation of respiration and explains ATP homeostasis also in mouse liver and canine heart. This result may well be generalizable to all mammalian cells.

High-performance liquid chromatographic assays for free and phosphorylated derivatives of the creatine analogues β-guanidopropionic acid and 1-carboxymethyl-2-iminoimidazolidine (cyclocreatine)

Creatine and phosphocreatine are substrates for creatine kinase which is a key enzyme involved in energy transfer within the cell. Analogues of creatine have been fed to animals to determine the role this enzyme plays in energy metabolism, but progress in interpretation has been hampered by the lack of quantitative techniques to determine tissue content of these compounds. We describe the separation and quantitation of substituted guanidino compounds and their phosphorylated forms by high-performance liquid chromatography. First, a cation-exchange column is used to assay free creatine and its unphosphorylated analogues, and then phosphocreatine and its phosphorylated analogues as well as adenylate content (AMP, ADP, ATP) are assayed on an anion-exchange column. These methods have proven successful in measuring the chemical contents of these compounds in neutralized perchloric acid extracts of mammalian skeletal muscles. The sensitivity of this method ranges from 50 to 200 pmol, which is adequate to provide information from tissue extracts of 5- to 10-mg samples.

Hypoxia suppresses runx2 independent of modeled microgravity

Bone loss is a consequence of skeletal unloading as seen in bed rest and space flight. Unloading decreases oxygenation and osteoblast differentiation/function in bone. Previously we demonstrated that simulation of unloading in vitro, by culturing differentiating mouse osteoblasts in a horizontal rotating wall vessel (RWV), results in suppressed expression of runx2, a master transcriptional regulator of osteoblast differentiation. However, the RWV is able to reproduce in a controlled fashion at least two aspects of disuse that are directly linked, model microgravity and hypoxia. Hypoxia in the RWV is indicated by reduced medium oxygen tension and increased expression of GAPDH and VEGF. To uncouple the role of model microgravity from hypoxia in suppressed runx2 expression, we cultured osteoblasts under modeled microgravity (oxygenated, horizontal RWV rotation), hypoxia (vertical RWV rotation), or both conditions (horizontal RWV rotation). The expression, DNA binding activity and promoter activity of runx2, was suppressed under hypoxic but not normoxic modeled microgravity RWV conditions. Consistent with a role for hypoxia in suppression of runx2, direct exposure to hypoxia alone is sufficient to suppress runx2 expression in osteoblasts grown in standard tissue culture plates. Taken together, our findings indicate that hypoxia associated with skeletal unloading could be major suppressor of runx2 expression leading to suppressed osteoblast differentiation and bone formation.

Dynamic perifusion to maintain and assess isolated pancreatic islets

Advances in human islet transplant techniques are hampered by the inability to assess the quality of isolated islets. A flow culture system was developed to perifuse isolated pancreatic islets or cultured beta-cell lines in order to continuously and noninvasively assess cell function and viability with high kinetic resolution. Continuous perifusion of large amounts of islet tissue as isolated from human pancreata enables the use of noninvasive measurement technologies not previously applied to islets. To compare dynamic perifusion of tissue at high density with conventional static cultures, we measured glucose-stimulated insulin secretion and O2 consumption of large amounts of INS-1 cells (45-65 x 10(6)) to confirm that perifused cells were adequately supplied with oxygen and nutrients and remained functionally responsive. Isolated human and monkey islets that were perifused for 18 h showed robust biphasic insulin secretion in response to a step increase in glucose, demonstrating the ability to maintain islets and the high kinetic resolution of the system. As an example of the systems ability to resolve multiple indicator dilution experiments, the retention of [3H]-glibenclamide was kinetically distinguished from that of an extracellular marker. In summary, the perifusion system is able to maintain healthy cells, assess insulin secretion and metabolite fluxes such as oxygen consumption and lactate production, and characterize the kinetics of the interaction between radiopharmaceuticals and islet cells. The ability to systematically assess the metabolic and functional viability of islets will facilitate the optimization of islet isolation procedures, islet transplantation studies, and islet storage methodologies.

Neither changes in phosphorus metabolite levels nor myosin isoforms can explain the weakness in aged mouse muscle.

1. The contractile force, phosphorus metabolite levels, intracellular pH and myosin isoforms were compared in isolated soleus and extensor digitorum longus (EDL) muscles from young (6 month old) and aged (28 month old) mice, at 23 degrees C. 2. The isometric force per unit cross‐sectional area was significantly lower by 21 +/‐ 5% in soleus muscles from aged mice compared to those from young mice (mean +/‐ S.E.M., n = 11 and 9 respectively). 3. The EDL muscle contained twice as much total creatine and phosphocreatine as the soleus, 1.7 times as much ATP, and 0.4 times the inorganic phosphate (Pi) per unit weight. The intracellular pH and free ADP levels were not significantly different between these muscle types. 4. There was no significant difference in resting metabolite levels between young and old EDL or soleus despite the difference in mechanical strength. 5. Examination of the expression of myosin isoforms by non‐denaturing gel electrophoresis has shown that the percentage of each isoform does not change with respect to age; thus, if there is an atrophic process occurring, it is not fibre type specific. 6. We have determined that neither the Pi levels nor the intracellular pH can explain the differences seen in muscle strength with age. There is also no correlation between muscle weakness and any of the other metabolites responsible for energy transduction (phosphocreatine, ATP or ADP).

Kinetic characterization of hexokinase isoenzymes from glioma cells: Implications for FDG imaging of human brain tumors

Quantitative imaging of glucose metabolism of human brain tumors with PET utilizes 2-[(18)F]-fluorodeoxy-D-glucose (FDG) and a conversion factor called the lumped constant (LC), which relates the metabolic rate of FDG to glucose. Since tumors have greater uptake of FDG than would be predicted by the metabolism of native glucose, the characteristic of tumors that governs the uptake of FDG must be part of the LC. The LC is chiefly determined by the phosphorylation ratio (PR), which is comprised of the kinetic parameters (Km and Vmax) of hexokinase (HK) for glucose as well as for FDG (LC proportional to (Km(glc) x Vmax(FDG))/(Km(FDG) x Vmax(glc)). The value of the LC has been estimated from imaging studies, but not validated in vitro from HK kinetic parameters. In this study we measured the kinetic constants of bovine and 36B-10 rat glioma HK I (predominant in normal brain) and 36B-10 glioma HK II (increased in brain tumors) for the hexose substrates glucose, 2-deoxy-D-glucose (2DG) and FDG. Our principal results show that the KmGlc < KmFDG << Km2DG and that PR2DG < PRFDG. The FDG LC calculated from our kinetic parameters for normal brain, possessing predominantly HK I, would be higher than the normal brain LC predicted from animal studies using 2DG or human PET studies using FDG or 2DG. These results also suggest that a shift from HK I to HK II, which has been observed to increase in brain tumors, would have little effect on the value of the tumor LC.

Phosphocreatine as an energy source for actin cytoskeletal rearrangements during myoblast fusion.

Myoblast fusion is essential for muscle development, postnatal growth and muscle repair after injury. Recent studies have demonstrated roles for actin polymerization during myoblast fusion. Dynamic cytoskeletal assemblies directing cell–cell contact, membrane coalescence and ultimately fusion require substantial cellular energy demands. Various energy generating systems exist in cells but the partitioning of energy sources during myoblast fusion is unknown. Here, we demonstrate a novel role for phosphocreatine (PCr) as a spatiotemporal energy buffer during primary mouse myoblast fusion with nascent myotubes. Creatine treatment enhanced cell fusion in a creatine kinase (CK)‐dependent manner suggesting that ATP‐consuming reactions are replenished through the PCr/CK system. Furthermore, selective inhibition of actin polymerization prevented myonuclear addition following creatine treatment. As myotube formation is dependent on cytoskeletal reorganization, our findings suggest that PCr hydrolysis is coupled to actin dynamics during myoblast fusion. We conclude that myoblast fusion is a high‐energy process, and can be enhanced by PCr buffering of energy demands during actin cytoskeletal rearrangements in myoblast fusion. These findings implicate roles for PCr as a high‐energy phosphate buffer in the fusion of multiple cell types including sperm/oocyte, trophoblasts and macrophages. Furthermore, our results suggest the observed beneficial effects of oral creatine supplementation in humans may result in part from enhanced myoblast fusion.

The effect of metabolic fuel on force production and resting inorganic phosphate levels in mouse skeletal muscle.

1. The effect of different metabolic fuels (glucose, pyruvate and lactate) and no exogenous metabolic fuel on force production was studied in isolated mouse soleus and extensor digitorum longus (EDL) muscles. Force was measured, at 25 degrees C, during isometric tetanic contractions and during contractions with isovelocity stretching and shortening. In parallel experiments, measurements were made of the resting phosphorus metabolite levels using 31P NMR. 2. In soleus muscles, the isometric tetanic force was potentiated with pyruvate (20 mM) as metabolic fuel, compared with glucose (11 mM), by 17.8 +/‐ 3.6% (mean +/‐ S.E.M., n = 6). The force was the same with no exogenous metabolic fuel, with glucose, or with lactate as metabolic fuel. The force exerted during shortening was also potentiated by pyruvate and by the same proportion as isometric force. However, during rapid stretching there was no force enhancement with pyruvate. The changes in the force seen with pyruvate are qualitatively similar to those produced when inorganic phosphate (Pi) is lowered in skinned rabbit psoas muscle fibres. 3. We tested whether the Pi content decreased in the presence of pyruvate by measuring resting Pi using 31P NMR spectroscopy. We found that, in soleus muscles, resting Pi was present with glucose and absent with pyruvate as metabolic fuel, and the effect was reversible. 4. EDL muscles produced the same isometric force whether the metabolic fuel was glucose, pyruvate, lactate or if no exogenous metabolic fuel was supplied. EDL muscles already had Pi levels below detectability at rest in glucose. There were no changes in the 31P NMR spectrum with pyruvate as metabolic fuel. 5. It appears therefore that the force potentiation in soleus muscles with pyruvate is due to a lowering of Pi. EDL muscles, which have a very low resting Pi in glucose, therefore have very little potential for force enhancement by this mechanism.

Intracellular Buffering Capacity in Molluscan Muscle: Superfused Muscle versus Homogenates

In situ estimates of intracellular pH (pHi) in isolated bands of radula protractor muscles from the whelk Busycon canaliculatum were made using phosphorus nuclear magnetic resonance spectroscopy (31P-NMR). The 31P-NMR spectra contained distinct resonances for arginine phosphate and the α, β, and γ phosphates of ATP. In addition, initial spectra displayed a small inorganic phosphate (Pi) peak, which disappeared upon continued superfusion under aerobic conditions. Addition of 2 mM 2-deoxyglucose to the medium resulted in the generation of an additional resonance corresponding to 2-deoxyglucose-6-phosphate (2DGP). Using the chemical shift differences of both the Pi and 2DGP peaks relative to the internal reference arginine phosphate, the resting aerobic pH, was found to be 7.29 ± 0.07 SD (n = 3) and 7.32 ± 0.04 SD (n = 5), respectively. Intracellular buffering capacity (β) was estimated in situ using NH₄Cl prepulse and weak acid loading techniques. The β values were 30 ± 3.9 SD (n = 7) and 31 ± 3.8 SD (n = 7) μ mol·pH⁻¹·g intracellular water⁻¹, respectively. A third method, by titration of tissue homogenates, yielded a β value of 85.8 ± 17.5 SD (n = 8) μ mol·pH⁻¹·g intracellular water⁻¹. This large discrepancy can be partially explained by the rapid hydrolysis of arginine phosphate immediately after homogenization, generating free inorganic phosphate, an excellent buffer in the physiological pH range. The chemical nature of intracellular buffers in B. canaliculatum radula protractor muscle was examined through crude acid fractionation and subsequent amino acid analysis. The total histidine content in this muscle accounted for 72% of the in situ buffering capacity. Unlike some vertebrate systems where large amounts of histidine-containing dipeptides function as buffers, most of the histidine is associated with the protein fraction of the tissue.