Defects in ryanodine receptor calcium release in skeletal muscle from post-myocardial infarct rats.

Defective calcium (Ca2+) signaling and impaired contractile function have been observed in skeletal muscle secondary to impaired myocardial function. However, the molecular basis for these muscle defects have not been identified. In this study, we evaluated the alterations of the ryanodine-sensitive Ca2+ release channels (RyR1) by analyzing global and local Ca2+ signaling in a rat postmyocardial infarction (PMI) model of myocardial overload. Ca2+ transients, measured with multiphoton imaging in individual fibers within a whole extensor digitorum longus (EDL) muscle, exhibited significantly reduced amplitude and a prolonged time course in PMI. Spatio-temporal properties of spontaneous Ca2+ sparks in fibers isolated from PMI EDL muscles were also significantly altered. In addition, RyR1 from PMI skeletal muscles were PKA-hyperphosphorylated and depleted of the FK506 binding protein (FKBP12). These data show that PMI skeletal muscles exhibit altered local Ca2+ signaling, associated with hyperphosphorylation of RyR1. The observed changes in Ca2+ signaling may contribute to defective excitation-contraction coupling in muscle that can contribute to the reduced exercise capacity in PMI, out of proportion to the degree of cardiac dysfunction.

beta-adrenergic receptor blockers restore cardiac calcium release channel (ryanodine receptor) structure and function in heart failure.

BACKGROUND: beta-Adrenergic receptor blockade is one of the most effective treatments for heart failure, a leading cause of mortality worldwide. The use of beta-adrenergic receptor blockers in patients with heart failure is counterintuitive, however, because they are known to decrease contractility in normal hearts. The ryanodine receptor (RyR2) on cardiac sarcoplasmic reticulum is the key calcium release channel required for excitation-contraction coupling. In failing hearts, the stoichiometry and function of the RyR2 macromolecular complex is altered. Decreased levels of phosphatases (PP1 and PP2A) and hyperphosphorylation by protein kinase A result in dissociation of the regulatory protein FKBP12.6 and channels with increased open probability. METHODS AND RESULTS: Here, we show that systemic oral administration of a beta-adrenergic receptor blocker reverses protein kinase A hyperphosphorylation of RyR2, restores the stoichiometry of the RyR2 macromolecular complex, and normalizes single-channel function in a canine model of heart failure. CONCLUSIONS: These results may, in part, explain the improved cardiac function observed in heart failure patients treated with beta-adrenergic receptor blockers.

Dilated cardiomyopathy and sudden death resulting from constitutive activation of protein kinase a.

beta-Adrenergic receptor (betaAR) signaling, which elevates intracellular cAMP and enhances cardiac contractility, is severely impaired in the failing heart. Protein kinase A (PKA) is activated by cAMP, but the long-term physiological effect of PKA activation on cardiac function is unclear. To investigate the consequences of chronic cardiac PKA activation in the absence of upstream events associated with betaAR signaling, we generated transgenic mice that expressed the catalytic subunit of PKA in the heart. These mice developed dilated cardiomyopathy with reduced cardiac contractility, arrhythmias, and susceptibility to sudden death. As seen in human heart failure, these abnormalities correlated with PKA-mediated hyperphosphorylation of the cardiac ryanodine receptor/Ca(2+)-release channel, which enhances Ca(2+) release from the sarcoplasmic reticulum, and phospholamban, which regulates the sarcoplasmic reticulum Ca(2+)-ATPase. These findings demonstrate a specific role for PKA in the pathogenesis of heart failure, independent of more proximal events in betaAR signaling, and support the notion that PKA activity is involved in the adverse effects of chronic betaAR signaling.

Phosphorylation-dependent regulation of ryanodine receptors: a novel role for leucine/isoleucine zippers.

Ryanodine receptors (RyRs), intracellular calcium release channels required for cardiac and skeletal muscle contraction, are macromolecular complexes that include kinases and phosphatases. Phosphorylation/dephosphorylation plays a key role in regulating the function of many ion channels, including RyRs. However, the mechanism by which kinases and phosphatases are targeted to ion channels is not well understood. We have identified a novel mechanism involved in the formation of ion channel macromolecular complexes: kinase and phosphatase targeting proteins binding to ion channels via leucine/isoleucine zipper (LZ) motifs. Activation of kinases and phosphatases bound to RyR2 via LZs regulates phosphorylation of the channel, and disruption of kinase binding via LZ motifs prevents phosphorylation of RyR2. Elucidation of this new role for LZs in ion channel macromolecular complexes now permits: (a) rapid mapping of kinase and phosphatase targeting protein binding sites on ion channels; (b) predicting which kinases and phosphatases are likely to regulate a given ion channel; (c) rapid identification of novel kinase and phosphatase targeting proteins; and (d) tools for dissecting the role of kinases and phosphatases as modulators of ion channel function.

FKBP12 binding modulates ryanodine receptor channel gating.

The ryanodine receptor (RyR1)/calcium release channel on the sarcoplasmic reticulum of skeletal muscle is comprised of four 565,000-dalton RyR1s, each of which binds one FK506 binding protein (FKBP12). RyR1 is required for excitation-contraction coupling in skeletal muscle. FKBP12, a cis-trans peptidyl-prolyl isomerase, is required for the normal gating of the RyR1 channel. In the absence of FKBP12, RyR1 channels exhibit increased gating frequency, suggesting that FKBP12 "stabilizes" the channel in the open and closed states. We now show that substitution of a Gly, Glu, or Ile for Val2461 in RyR1 prevents FKBP12 binding to RyR1, resulting in channels with increased gating frequency. In the case of the V2461I mutant RyR1, normal channel function can be restored by adding FKBP12.6, an isoform of FKBP12. These data identify Val2461 as a critical residue required for FKBP12 binding to RyR1 and demonstrate the functional role for FKBP12 in the RyR1 channel complex.

Chronic unloading by left ventricular assist device reverses contractile dysfunction and alters gene expression in end-stage heart failure.

BACKGROUND: Left ventricular (LV) assist devices (LVADs) can improve contractile strength and normalize characteristics of the Ca(2+) transient in myocytes isolated from failing human hearts. The purpose of the present study was to determine whether LVAD support also improves contractile strength at different frequencies of contraction (the force-frequency relationship [FFR]) of intact myocardium and alters the expression of genes encoding for proteins involved in Ca(2+) handling. METHODS AND RESULTS: The isometric FFRs of LV trabeculae isolated from 15 patients with end-stage heart failure were compared with those of 7 LVAD-supported patients and demonstrated improved contractile force at 1-Hz stimulation, with reversal of a negative FFR after LVAD implantation. In 20 failing hearts, Northern blot analysis for sarcoplasmic endoreticular Ca(2+)-ATPase subtype 2a (SERCA2a), the ryanodine receptor, and the sarcolemmal Na(+)-Ca(2+) exchanger was performed on LV tissue obtained before and after LVAD implantation. These paired data demonstrated an upregulation of all 3 genes after LVAD support. In tissue obtained from subsets of these patients, Western blot analysis was performed, and oxalate-supported Ca(2+) uptake by isolated sarcoplasmic reticular membranes was determined. Despite higher mRNA for all genes after LVAD support, only SERCA2a protein was increased. Functional significance of increased SERCA2a was confirmed by augmented Ca(2+) uptake by sarcoplasmic reticular membranes isolated from LVAD-supported hearts. CONCLUSIONS: LVAD support can improve contractile strength of intact myocardium and reverse the negative FFR associated with end-stage heart failure. The expression of genes encoding for proteins involved in Ca(2+) cycling is upregulated (reverse molecular remodeling), but only the protein content of SERCA2a is increased.

PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts.

The ryanodine receptor (RyR)/calcium release channel on the sarcoplasmic reticulum (SR) is the major source of calcium (Ca2+) required for cardiac muscle excitation-contraction (EC) coupling. The channel is a tetramer comprised of four type 2 RyR polypeptides (RyR2) and four FK506 binding proteins (FKBP12.6). We show that protein kinase A (PKA) phosphorylation of RyR2 dissociates FKBP12.6 and regulates the channel open probability (Po). Using cosedimentation and coimmunoprecipitation we have defined a macromolecular complex comprised of RyR2, FKBP12.6, PKA, the protein phosphatases PP1 and PP2A, and an anchoring protein, mAKAP. In failing human hearts, RyR2 is PKA hyperphosphorylated, resulting in defective channel function due to increased sensitivity to Ca2+-induced activation.

Intracellular calcium dynamics during photolysis.

The objective of this investigation was to gain a deeper understanding of the intracellular events that precede photolysis of cells. A model system, consisting of malignant melanoma cells pretreated with the calcium sensitive fluorescent dye, Fluo-3, was used to examine the intracellular calcium dynamics in single-cell photolysis experiments. Exposure of the cells to 632 nm laser light in the presence of photosensitizer, tin chlorin e6, resulted in a rise in intracellular calcium. The increase in intracellular calcium was blocked using a variety of calcium channel blocking agents, including verapamil, nifedipine, and nickel. Treatment with the channel blockers was also effective in either decreasing or eliminating cell death despite the presence of lethal doses of photosensitizer and irradiation. These results show that intracellular calcium rises prior to plasma membrane lysis, and that this early rise in intracellular calcium is necessary for membrane rupture.

Targeted antisense modulation of inflammatory cytokine receptors.

Antisense technology is potentially a powerful means by which to selectively control gene expression. We have used antisense oligonucleotides to modulate the response of the hepatoma cell line, HepG2, to the inflammatory cytokine, IL-6, by inhibiting the expression of its multifunctional signal transducer, gp130. HepG2 cells respond to IL-6 by upregulating acute phase proteins, such as haptoglobin, by five- to tenfold. Gp130 is central to this response, as the upregulation of haptoglobin is almost completely blocked by the addition of high concentrations ( approximately 100 microg/ml) of a monoclonal antibody to gp 130. Antisense oligodeoxynucleotides complementary to the mRNA encoding gp 130 inhibited the upregulation of haptoglobin by IL-6-stimulated HepG2 cells by about 50%. However, a nonsense sequence also inhibited haptoglobin secretion by about 20%. To improve the specificity and efficiency of action, we targeted the antisense oligonucleotides to HepG2 cells using a conjugate of asialoglycoprotein-poly-L-lysine. The targeted antisense reduced the binding of IL-6 to HepG2 cells, virtually eliminating high affinity binding. In addition, it inhibited haptoglobin upregulation by over 70%. Furthermore, the dose of targeted antisense required for biological effect was reduced by about an order of magnitude as compared with unconjugated antisense. These results demonstrate the potential of antisense oligonucleotides as a means to control the acute phase response as well as the need for a greater understanding of the mechanism and dynamics of antisense molecules as they are developed toward therapeutic application.

Control of hypertrophic scar growth using selective photothermolysis.

BACKGROUND AND OBJECTIVE: Previous studies have shown a clinical improvement of hypertrophic scars (HS) after treatment with a pulsed dye laser. The objective of this study was to investigate the effects of variations in pulse wavelength and energy density on HS tissue using human HS implanted in athymic mice. STUDY DESIGN/MATERIALS AND METHODS: Small pieces (approximately 1 mm3) of HS tissue were implanted into athymic mice and allowed to grow for 5 days. The implant site was then exposed to a single 450 microseconds pulse, and implant growth and histology were monitored for an additional 12 days. Laser wavelength and energy density ranges tested were 585-600 nm and 2-10 J/cm2, respectively. RESULTS: Using a wavelength of 585 nm, laser treatment inhibited implant growth by 70% at 6 J/cm2 and 92% at 10 J/cm2, respectively. The inhibitory effect decreased as the laser wavelength was increased from 585 to 600 nm. A widespread destruction of the implant microvasculature with a minor effect on surrounding extracellular matrix at the highest light dose were observed. CONCLUSION: Pulsed laser treatment inhibits HS implant growth in nude mice. This effect is likely mediated by selective photo-thermolysis of the implant microvasculature.

Control of hypertrophic scar growth using antibody-targeted photolysis.

Hypertrophic scar is marked by excess collagen accumulation secondary to an increased vascularization response in the scar and an increase in fibroblast cell density. It is currently the most debilitating long-term complication of the surviving burn patient, and at present, there is no routinely effective form of therapy. In this study, we investigated the potential use of antibody-targeted photolysis (ATPL) in treating hypertrophic scars. An immunoconjugate consisting of a photosensitizer (Sn-chlorin e6) linked to a monoclonal antibody that binds to human myofibroblasts (PR2D3) was prepared, which in response to photoactivation produces singlet oxygen in close proximity to the target cell surface. The model used for these studies consisted of 1-mm 3 human hypertrophic scar tissue implants in athymic mice. These implants increase approximately 20-fold in volume over a period of 15 days. Four days after implantation immunoconjugate was injected directly into scar implants allowed to diffuse throughout for 24 hr before implants were illuminated with laser light at 630 nm (120 J/cm 2). ATPL treatment caused a significant reduction in total growth compared to the untreated controls (P < 0.05). No effect was observed when an irrelevant conjugate (anti-Pseudomonas aeruginosa) was used. Histological examination of the ATPL-treated implants 24 hr post-ATPL revealed the presence of a large number of lipid droplets indicative of massive cell damage and infiltration by mononuclear cells and neutrophils.

Bispecific antibody modification of nicotinic acetylcholine receptors for biosensing.

Recent results show that bispecific antibodies can be used to tailor the selectivity of nicotinic acetylcholine receptors for biosensing purposes. The nicotinic acetylcholine receptors reconstituted in bilayer lipid membranes are inactivated when two bispecific antibodies, attached to the same receptor, bind to a single antigen molecule. Experiments with patch clamp recording equipment reveal that antigen levels of 10(-8) M completely and irreversibly inactivate small numbers of nicotinic acetylcholine receptors. This approach may lead to the construction of biosensors capable of detecting individual antibody-antigen (Ab-Ag) binding events.

A highly stable and selective biosensor using modified nicotinic acetylcholine receptor (nAChR).

Methods for developing stable, sensitive and selective bilayer lipid membrane (BLM)-based biosensors are discussed. Stable BLMs were formed over micromachined polyimide apertures. Selective sensors were made by incorporating nicotinic acetylcholine receptors (nAChRs) modified with bispecific antibodies (BsAbs). When two BsAbs, attached to one nAChR, encounter antigen (Ag), channels are blocked. Sensitivity to single Ag molecules would be possible by monitoring closure of individual nAChRs.

Efficient optimization of ELISAs.

In this paper, we describe a technique for using the statistical method of fractional factorial design in the optimization of an ELISA. Fractional factorial design dramatically reduces the total number of experiments required in the optimization. In addition, this technique enables us to determine the parameters that give the maximum sensitivity range with the most accuracy for the ELISA studied.

Antibody-targeted photolysis of bacteria in vivo.

We have evaluated the efficacy of antibody-targeted photolysis to kill bacteria in vivo using specific antibacterial photosensitizer (PS) immunconjugates. After infecting the dorsal skin in mice with Pseudomonas aeruginosa, both specific and nonspecific tin (IV) chlorin e6-monoclonal antibody conjugates were injected at the infection site. After a 15 min incubation period, the site was exposed to 630 nm light with a power density of 100 mW/cm2 for 1600 seconds. Irradiation resulted in a greater then 75% decrease in the number of viable bacteria at sites treated with a specific conjugate, whereas normal bacterial growth was observed in animals that were untreated or treated with a nonspecific conjugate. Antibody-targeted photolysis may be a selective and versatile tool for treating a variety of infections.

The use of an enzyme single fiber reactor in the study of leukemic cell proliferation: in vitro experiments and computer simulation.

This paper describes the use of an immobilized enzyme reactor in the study of the in vitro effects of lysine deprivation on leukemic blood. L-lysine alpha-oxidase is immobilized in a single hollow fiber reactor to remove lysine from the blood of sheep infected by BLV. The treatment relies on the higher sensitivity of leukemic cells to nutrient depletion than that of normal cells. A population balance model is used to describe the changes in the leukocyte proliferative capacity after treatment. Additionally, preliminary data from in vitro tests with human blood demonstrate the potential of L-lysine alpha-oxidase and the enzymatic reactor in treating leukemia.

The effect of lysine deprivation on leukemic blood.

Studies have shown that specific amino acids are required for optimal growth of leukemic versus normal cells, and it is believed that the depletion of selected amino acids can abrogate tumor growth. We have developed a technique for studying the effect of amino acid deprivation on leukemic cell proliferation. The technique is based on the controlled enzymatic removal of the amino acid from leukemic blood and the subsequent measurement of cell proliferative capacity. The specific system being studied is the removal of lysine from blood using immobilized L-lysineα-oxidase.A reactor has been designed that consists of L-lysineα-oxidase and catalase co-immobilized within the void space of the porous region of asymmetric hollow fiber (ultrafiltration) membranes. Blood from leukemic sheep is currently being treatedin vitro with this reactor. By varying treatment time, the amount of enzyme immobilized, and the blood flow rate, the amount of lysine removed from the blood can be varied and controlled. Preliminary data indicate that 80% depletion of lysine from leukemic blood is enough to cause a significant (25%) decrease in total white cell count as well as a decrease in the proliferative capacity of the leukemic cells.

Evaluation of intrinsic immobilized kinetics in hollow fiber reactor systems.

Immobilized cell and enzyme hollow fiber reactors have been developed for a variety of biochemical and biomedical applications. Reported mathematical models for predicting substrate conversion in these reactors have been limited in accuracy because of the use of free-solution kinetic parameters. This paper describes a method for determining the intrinsic kinetics of enzymes immobilized in hollow fiber reactor systems using a mathematical model for diffusion and reaction in porous media and an optimization procedure to fit intrinsic kinetic parameters to experimental data. Two enzymes, a thermophilic beta-galactosidase that exhibits product inhibition and L-lysine alpha-oxidase, were used in the analysis. The intrinsic kinetic parameters show that immobilization enhanced the activity of the beta-galactosidase while decreasing the activity of L-lysine alpha-oxidase. Both immobilized enzymes had higher Km values than did the soluble enzyme, indicating less affinity for the substrate. These results are used to illustrate the significant improvement in the ability to predict substrate conversion in hollow fiber reactors.

The use of a single-fiber reactor for the enzymatic removal of amino acids from solutions.

In this article we describe the use of bench-scale single-fiber dialyzers for the development and testing of an immobilized enzyme reactor for the treatment of leukemia. The treatment is based on the enzymatic removal of specific amino acids from the blood of leukemia patients. L-Lysine alpha-oxidase and catalase were coimmobilized within the void space of the porous region of asymmetric hollow-fiber membranes for the removal of L-lysine from simulated human plasma solutions. Hollow-fiber reactor performance was evaluated using a small single-fiber dialyzer (SFD) consisting of a single fiber encased in a protective glass shell. This small reactor affords ease of use, requires small amounts of chemicals and biochemicals, and gives useful reactor performance data. Single-fiber dialyzers were constructed using polyamide fibers with a molecular weight cutoff of 10,000 (PA10 fibers); these fibers demonstrated the best compatibility with and retention of the enzymes. The SFD performance in removing L-lysine from solution was evaluated under both steady and pulsatile flow operation. Pulsatile flow was tested for two reasons: (1) to enhance the radial mass transfer of lysine within the SFD and (2) to simulate the pulsatile flow of blood in dialysis treatment. The use of pulsatile flow increased lysine conversion by 15% over the steady-flow case. Approximately 40% of the lysine was removed from simulated plasma by the SFD in a 4-h experiment using pulsatile flow in the recycle mode.