Sieck, Gary C.

Neuregulin (NRG) is a member of the epidermal growth factor family, with 3 types varying in their structure and cellular expression but not in their receptor‐binding domain. NRG‐2 and NRG‐3 are primarily membrane bound, while NRG‐1 can be secreted. NRG‐1 is present in phrenic motor neurons. We previously reported that exogenous NRG containing only the receptor‐binding domain, increases protein synthesis in rat diaphragm. This effect was significantly inhibited by LY294002 indicating a PI 3‐kinase dependent mechanism. We also reported that NRG increases Akt phosphorylation. Interestingly, Akt phosphorylation is positively correlated with skeletal muscle growth and inversely correlated with atrophy. In this study, we hypothesized that NRG reduces protein degradation in rat diaphragm. Diaphragms were harvested from 4‐week old rats and incubated in the presence or absence of exogenous NRG. Protein degradation was measured by quantifying muscle tyrosine release via a fluorimetric assay. We found that NRG treatment resulted in a 50% reduction in tyrosine release from diaphragm preparations, indicating a reduction in basal protein degradation rates. These results suggest that phrenic motoneuron‐derived NRG‐1 regulates muscle protein synthesis and degradation rates, thereby playing a role in matching muscle fiber and motoneuron properties within motor units.

Neuregulin (NRG) is part of the epidermal growth factor family of proteins. Previous studies have shown that NRG activates PI3K/Akt pathways, leading to regulation of protein synthesis via downstream regulation of translation initiators. The principal translation initiator involved in protein synthesis is probably eIF4E, and the principal transcription factor regulating protein degradation is FOXO. We hypothesized that NRG signaling involves activation of the PI3K/Akt pathway, thus leading to an increase in expression of eIF4E and a decrease in expression of FOXO. An increase in eIF4E expression increases protein synthesis, and a decrease in FOXO expression reduces protein degradation. In this study we used dexamethasone, a synthetic glucocorticoid, to induce atrophy and cause an imbalance between protein synthesis and degradation in L6 myotubes. As expected, we found that after 48 hours of treatment with 10–100nM dexamethasone, eIF4E expression decreased and FOXO expression increased in a dose‐dependent manner. We found that treatment with 200 ng/ml NRG increased eIF4E expression. When L6 cells were treated with both NRG and dexamethasone for 48 hours, NRG blunted the catabolic effects of dexamethasone, thus increasing eIF4E expression and decreasing FOXO expression. These results indicate that NRG promotes protein synthesis and prevents protein degradation. Supported by NIH grant AR51173.

The role of gap junctional intercellular communication during the inflammatory process is not well understood. In particular, the role of vascular connexins (gap junction proteins) in regulating blood flow in response to inflammatory agents such as lipopolysaccharide (LPS) has not been fully investigated. Our hypothesis is that agonist‐mediated vasoconstriction is dependent on the presence of connexin 40 (Cx40) within the microvessel wall. Wild‐type (WT) and Cx40 −/− animals were treated with LPS (15 mg/kg ip) or saline for 18 hours. Small mesenteric resistance arteries (190–220 μm) were isolated, mounted on glass cannulae, pressurized at 60 mmHg with no lumen flow and placed on the stage of an inverted microscope. External arteriolar diameter was measured and concentration‐response curves to phenylephrine (PE, 10 −9 −10 −4 M) and to extracellular Ca 2+ (0 to 2 mM) under depolarizing conditions (120 mM K + ) were conducted. The half‐maximal effect (EC 50 ) was determined for each dose response curve. LPS treatment resulted in hyporesponsiveness to PE in WT mice (EC 50 2.07±0.45 mM vs 0.56±0.15 mM in shams) but resulted in hyperresponsiveness in Cx40 −/‐ (EC 50 0.27±0.03 mM vs 3.15±1.50 mM in shams). However, LPS treatment resulted in hyporesponsiveness to Ca 2+ in both WT mice (EC 50 0.39±0.03 mM vs 0.23±0.01mM in shams) and Cx40 −/− (EC 50 0.75±0.24 mM vs 0.23±0.03 mM in shams). We conclude that agonist‐mediated but not Ca 2+ ‐mediated vasoconstriction is modulated by the presence of Cx40.

Rewarming patients following profound hypothermia may result in acute heart failure and high mortality (50‐80%); however underlying pathophysiological mechanisms are largely unknown. We characterized myocardial dysfunction induced by hypothermia/rewarming (H/R) by measuring intracellular Ca 2+ concentration ([Ca 2+ ] i ) and twitch force in intact electrically stimulated left ventricular (LV) rat papillary muscles loaded with Fura‐2AM. In skinned papillary muscle fibers, we assessed maximal Ca 2+ ‐activated force (F max ) and Ca 2+ sensitivity of force. Following H/R, peak twitch force decreased by 30‐40%, but [Ca 2+ ] i was not significantly altered. In skinned preparations, maximal F max was decreased by ~30%, while the pCa required for 50% of F max (pCa 50 ) was reduced by ~0.14. Interestingly, in rewarmed papillary muscle, both total cTnI phosphorylation and protein kinase A (PKA)‐mediated cTnI phosphorylation at Ser23/24 were significantly increased compared to controls. We conclude that after H/R myocardial contractility is significantly reduced as evidenced by reduced twitch force and F max . Reduced myocardial contractility is attributed to decreased Ca 2+ sensitivity of force that likely results from increased cTnI phosphorylation.

Myocardial performance is depressed in both sepsis and endotoxin shock. Altered gap junctional coupling of ventricular myocytes may play an important role in myocardial dysfunction during sepsis. Since phosphorylation is known to regulate turnover of gap junction protein, we hypothesized that the phosphorylation status of connexin 43 (Cx43) is altered in vivo following exposure to lipopolysacccharide (LPS). Saline or LPS (25 mg/kg; E. coli 055:B5) was injected intraperitoneally into c57bl/6 mice. At 18 h, hearts were excised and snap frozen on dry ice. Tissue was homogenized in Triton X‐100 lysate buffer, centrifuged and prepared for Western blot. Cx43 was immunoprecipitated from the supernatant and tyrosine phosphorylation of Cx43, detected by immunoblot. Phosphorylated forms of Cx43 are seen as three major bands on Western blots. After LPS treatment, there is a decrease in the intensity of the fastest migrating band (Cx43‐P0) and an increase in the two slower migrating forms, (Cx43‐P1 and Cx43‐P2). This corresponds to a decrease in tyrosine phosphorylation of Cx43. LPS results in decrease in myocardial Cx43 tyrosine phosphorylation possibly mediating LPS‐induced myocardial dysfunction.

Neuregulin (NRG) is a nerve derived trophic factor that regulates acetylcholine receptor expression during neuromuscular junction development. NRG also has important trophic effects in the adult central nervous system. We hypothesized that NRG is a trophic factor for phrenic motoneurons. In adult male Sprague‐Dawley rats, we examined NRG‐1 immunoreactivity in the vicinity of phrenic motoneurons. Retrogradely‐labeled phrenic motoneurons were imaged and reconstructed in 3D. NRG‐1 boutons were found mainly around the soma and primary dendrites. The co‐localization of NRG boutons and synaptic markers was determined using multicolor confocal microscopy and a custom‐developed application (Metamorph; Universal Imaging). We found that 94 ± 3% of NRG‐containing boutons resided on synapses, as determined by co‐localization with synaptophysin. In addition, 6 ± 3% of synapses on phrenic motoneurons (within 1 micrometer of soma and primary dendrite surface) expressed NRG. Overall, 13 ± 5 NRG boutons were present at the soma vs. 6 ± 3 at each primary dendrite of a motoneuron.Thus, 57% of NRG‐containing boutons were surrounding the motoneuron soma. In summary, NRG‐1 synaptic boutons are present at phrenic motoneurons in the adult, although its role is presently unknown. Supported by NIH grant AR51173.

Retrograde transport of fluorescent dyes permits imaging of nearly the entire pool of motoneurons when administered in the target muscle or axon shaft, but the extent of dendritic labeling is usually limited. We used an intracellular injection of Lucifer Yellow CH (LY) into retrogradely labeled phrenic motoneurons (PMNs) to enhance dendritic structure. PMNs were specifically labeled by Alexa Fluor 488‐conjugated cholera toxin fragment B (CTB) injected into the pleural space of 18‐days old rats. After a 72‐hr survival period, animals were transcardially perfused with 4% paraformaldehyde and 4% sucrose in 0.1 M phosphate buffer. Cervical spinal cords were sectioned in coronal plane. Individual CTB‐labeled PMNs were intracellularly injected with LY under direct visual control and subsequently imaged with an Olympus FluoView 200 laser‐scanning confocal system. Image processing and analyses were performed using Metamorph 7.4 software. PMNs were extensively labeled and reconstructed in 3D. The range of visible primary, secondary and tertiary processes was 3 ‐ 7, 2 ‐ 4 and 0 ‐ 2 per PMN, respectively. Average length of a single dendritic tree was 218.0±20.1 μm and surface area was 1295±199 μm 2 . The described technique provides 3D quantitative morphometry of a large sample of retrogradely‐labeled PMNs in great detail. Supported by NIH grants HL37680 and AR51173.

Unilateral denervation (DNV) of rat diaphragm muscle (DIAm) increases protein synthesis and degradation, with net protein breakdown by 14 days after DNV. We hypothesized that neural influences such as the nerve‐derived trophic factor neuregulin (NRG‐1) are essential in maintaining DIAm protein balance. NRG activates receptor tyrosine kinases of the ErbB family, with the ErbB2‐ErbB3 co‐receptor showing highest affinity for NRG‐1 and strong phosphoinositol‐3‐kinase (PI3K) activation. Both protein synthesis (via Akt phosphorylation) and degradation (via the transcription factor FoxO3A) are regulated by PI3K activity. We used Western blot analyses to assess NRG‐1, ErbB2, ErbB3 and FoxO3A protein expression at 1, 3, 7, and 14 days after DNV. By 1 day after DNV, NRG‐1 expression decreased, whereas ErbB2 and ErbB3 expression increased. Consistent with reduced NRG/ErbB signaling, association of the PI3K regulatory subunit p85 with ErbB3 decreased at 14 days after DNV. FoxO3A expression increased at 7 and 14 days after DNV, in agreement with delayed net protein breakdown. Importantly, DIAm inactivity induced by spinal hemisection had no effect on ErbB2, ErbB3, or FoxO3A expression. Taken together, these results indicate that DNV‐induced skeletal muscle adaptations result from removal of a trophic influence such as NRG‐1 rather than inactivity itself. Supported by NIH grant AR51173.

Intracellular Ca 2+ ([Ca 2+ ] i ) levels are increased in inflamed airway smooth muscle (ASM). Sarcoplasmic reticulum (SR) Ca 2+ release and reuptake are key components of ASM [Ca 2+ ] i regulation. Ca 2+ reuptake occurs via the ATPase SERCA. We hypothesized that inflammation disrupts SERCA function, contributing to increased [Ca 2+ ] i . We examined SERCA expression and regulation in human ASM exposed to pro‐inflammatory cytokines TNFα and IL‐13. Western blot analysis showed significantly decreased SERCA expression following overnight cytokine exposure. In other tissues, SERCA is regulated by the inhibitory protein phospholamban (PLB). We found that human ASM does not express PLB protein (although mRNA is detectable). Cytokine exposure did not induce PLB expression, raising the issue of how SERCA is regulated. We then found that direct SERCA phosphorylation (potentially via CaMKII) occurs in human ASM. In fura‐2 loaded human ASM cells, we examined [Ca 2+ ] i responses to ACh or bradykinin (zero extracellular Ca 2+ ) and found significant reduction in rates of fall of [Ca 2+ ] i transients in cells exposed to TNF‐α or IL‐13. We further found that CaMKII antagonist KN‐93 significantly slowed the fall of [Ca 2+ ] i transients in both controls and cytokine‐exposed human ASM cells. These data suggest that a) SERCA in human ASM is regulated by mechanisms such as CaMKII; and b) airway inflammation decreases SR refilling by blunting SERCA expression and function. Supported by NIH grants HL74309, the Flight Attendants Medical Research Institute (FAMRI), and the Department of Anesthesiology, Mayo Clinic.

We studied the effects of TNF on cell‐to‐cell communication in HeLa cells expressing vascular connexin 37, 40 or 43. HeLa cells were treated with TNF (20 ng/ml) for up to 2 h. In dye‐transfer experiments, carboxyfluroscein (HeLaCx40 and HeLaCx43) or Alexa Fluor‐480 (HeLaCx37) was injected into one cell for 10 s and cell transfer allowed to proceed for 10 m and the number of labeled cells counted. Cell lysates were prepared in Triton X‐100 lysate buffer and detergent‐soluble fractions collected. Cx37, 40 and 43 were detected by Western blot. After 1 hour, TNF treatment resulted in near total loss of dye‐coupling in HeLaCx37 and HeLaCx43 (p<0.02, n=14‐16) and remained constant up to 2 hours. Dye coupling in HeLaCx40 cells remained unchanged after 1 hour and decreased after 2 hours (p<0.05, n=10). Western blots indicated that TNF treatment did not affect detergent solubility of Cx40 and 43. However, TNF caused a significant increase in detergent solubility of Cx37. These results suggest that inflammatory mediators affect connexins differently, and that the loss of Cx37 function may be due to the loss of detergent resistance.