Safety and Feasibility of Skin-to-Skin Contact in the Delivery Room for High-Risk Cardiac Neonates.

Early skin-to-skin contact (SSC), beginning in the delivery room, provides myriad health benefits for mother and baby. Early SSC in the delivery room is the standard of care for healthy neonates following both vaginal and cesarean delivery. However, there is little published evidence on the safety of this practice in infants with congenital anomalies requiring immediate postnatal evaluation, including critical congenital heart disease (CCHD). Currently, the standard practice following delivery of infants with CCHD in many delivery centers has been immediate separation of mother and baby for neonatal stabilization and transfer to a different hospital unit or a different hospital altogether. However, most neonates with prenatally diagnosed congenital heart disease, even those with ductal-dependent lesions, are clinically stable in the immediate newborn period. Therefore, we sought to increase the percentage of newborns with prenatally diagnosed CCHD who are born in our regional level II-III delivery hospitals who receive mother-baby SSC in the delivery room. Using quality improvement methodology, through a series of Plan-Do-Study-Act cycles we successfully increased mother-baby skin-to-skin contact in the delivery room for eligible cardiac patients born across our city-wide delivery hospitals from a baseline 15% to greater than 50%.

S-nitrosylation and S-glutathionylation of Cys134 on troponin I have opposing competitive actions on Ca2+ sensitivity in rat fast-twitch muscle fibers.

Nitric oxide is generated in skeletal muscle with activity and decreases Ca2+ sensitivity of the contractile apparatus, putatively by S-nitrosylation of an unidentified protein. We investigated the mechanistic basis of this effect and its relationship to the oxidation-induced increase in Ca2+ sensitivity in mammalian fast-twitch (FT) fibers mediated by S-glutathionylation of Cys134 on fast troponin I (TnIf). Force-[Ca2+] characteristics of the contractile apparatus in mechanically skinned fibers were assessed by direct activation with heavily Ca2+-buffered solutions. Treatment with S-nitrosylating agents, S-nitrosoglutathione (GSNO) or S-nitroso-N-acetyl-penicillamine (SNAP), decreased pCa50 ( = -log10 [Ca2+] at half-maximal activation) by ~-0.07 pCa units in rat and human FT fibers without affecting maximum force, but had no effect on rat and human slow-twitch fibers or toad or chicken FT fibers, which all lack Cys134. The Ca2+ sensitivity decrease was 1) fully reversed with dithiothreitol or reduced glutathione, 2) at least partially reversed with ascorbate, indicative of involvement of S-nitrosylation, and 3) irreversibly blocked by low concentration of the alkylating agent, N-ethylmaleimide (NEM). The biotin-switch assay showed that both GSNO and SNAP treatments caused S-nitrosylation of TnIfS-glutathionylation pretreatment blocked the effects of S-nitrosylation on Ca2+ sensitivity, and vice-versa. S-nitrosylation pretreatment prevented NEM from irreversibly blocking S-glutathionylation of TnIf and its effects on Ca2+ sensitivity, and likewise S-glutathionylation pretreatment prevented NEM block of S-nitrosylation. Following substitution of TnIf into rat slow-twitch fibers, S-nitrosylation treatment caused decreased Ca2+ sensitivity. These findings demonstrate that S-nitrosylation and S-glutathionylation exert opposing effects on Ca2+ sensitivity in mammalian FT muscle fibers, mediated by competitive actions on Cys134 of TnIf.

S-glutathionylation of troponin I (fast) increases contractile apparatus Ca2+ sensitivity in fast-twitch muscle fibres of rats and humans.

Oxidation can decrease or increase the Ca2+ sensitivity of the contractile apparatus in rodent fast-twitch (type II) skeletal muscle fibres, but the reactions and molecular targets involved are unknown. This study examined whether increased Ca2+ sensitivity is due to S-glutathionylation of particular cysteine residues. Skinned muscle fibres were directly activated in heavily buffered Ca2+ solutions to assess contractile apparatus Ca2+ sensitivity. Rat type II fibres were subjected to S-glutathionylation by successive treatments with 2,2'-dithiodipyridine (DTDP) and glutathione (GSH), and displayed a maximal increase in pCa50 (−log10 [Ca2+] at half-maximal force) of ∼0.24 pCa units, with little or no effect on maximum force or Hill coefficient. Partial similar effect was produced by exposure to oxidized gluthathione (GSSG, 10 mM) for 10 min at pH 7.1, and near-maximal effect by GSSG treatment at pH 8.5. None of these treatments significantly altered Ca2+ sensitivity in rat type I fibres. Western blotting showed that both the DTDP­GSH and GSSG­pH 8.5 treatments caused marked S-glutathionylation of the fast troponin I isoform (TnI(f)) present in type II fibres, but not of troponin C (TnC) or myosin light chain 2. Both the increased Ca2+ sensitivity and glutathionylation of TnI(f) were blocked by N-ethylmaleimide (NEM). S-nitrosoglutathione (GSNO) also increased Ca2+ sensitivity, but only in conditions where it caused S-glutathionylation of TnI(f). In human type II fibres from vastus lateralis muscle, DTDP­GSH treatment also caused similar increased Ca2+ sensitivity and S-glutathionylation of TnI(f). When the slow isoform of TnI in type I fibres of rat was partially substituted (∼30%) with TnI(f), DTDP­GSH treatment caused a significant increase in Ca2+ sensitivity (∼0.08 pCa units). TnIf in type II fibres from toad and chicken muscle lack Cys133 present in mammalian TnIf, and such fibres showed no change in Ca2+ sensitivity with DTDP­GSH nor any S-glutathionylation of TnI(f) (latter examined only in toad). Following 40 min of cycling exercise in human subjects (at ∼60% peak oxygen consumption), TnI(f) in vastus lateralis muscle displayed a marked increase in S-glutathionylation (∼4-fold). These findings show that S-glutathionylation of TnI(f), most probably at Cys133, increases the Ca2+ sensitivity of the contractile apparatus, and that this occurs in exercising humans, with likely beneficial effects on performance.

Differential effects of peroxynitrite on contractile protein properties in fast- and slow-twitch skeletal muscle fibers of rat.

Oxidative modification of contractile proteins is thought to be a key factor in muscle weakness observed in many pathophysiological conditions. In particular, peroxynitrite (ONOO(-)), a potent short-lived oxidant, is a likely candidate responsible for this contractile dysfunction. In this study ONOO(-) or 3-morpholinosydnonimine (Sin-1, a ONOO(-) donor) was applied to rat skinned muscle fibers to characterize the effects on contractile properties. Both ONOO(-) and Sin-1 exposure markedly reduced maximum force in slow-twitch fibers but had much less effect in fast-twitch fibers. The rate of isometric force development was also reduced without change in the number of active cross bridges. Sin-1 exposure caused a disproportionately large decrease in Ca(2+) sensitivity, evidently due to coproduction of superoxide, as it was prevented by Tempol, a superoxide dismutase mimetic. The decline in maximum force with Sin-1 and ONOO(-) treatments could be partially reversed by DTT, provided it was applied before the fiber was activated. Reversal by DTT indicates that the decrease in maximum force was due at least in part to oxidation of cysteine residues. Ascorbate caused similar reversal, further suggesting that the cysteine residues had undergone S-nitrosylation. The reduction in Ca(2+) sensitivity, however, was not reversed by either DTT or ascorbate. Western blot analysis showed cross-linking of myosin heavy chain (MHC) I, appearing as larger protein complexes after ONOO(-) exposure. The findings suggest that ONOO(-) initially decreases maximum force primarily by oxidation of cysteine residues on the myosin heads, and that the accompanying decrease in Ca(2+) sensitivity is likely due to other, less reversible actions of hydroxyl or related radicals.

Modulation of contractile apparatus Ca2+ sensitivity and disruption of excitation-contraction coupling by S-nitrosoglutathione in rat muscle fibres.

S-Nitrosoglutathione (GSNO) is generated in muscle and may S-glutathionylate and/or S-nitrosylate various proteins involved in excitation­contraction (EC) coupling, such as Na+-K+-ATPases, voltage-sensors (VSs) and Ca2+ release channels (ryanodine receptors,RyRs), possibly changing their properties. Using mechanically skinned fibres from rat extensor digitorum longus muscle, we sought to identify which EC coupling processes are most susceptible to GSNO-modulated changes and whether these changes could be important in muscle function and fatigue. For comparison, we examined the effect of other oxidation, nitrosylation, or glutathionylation treatments (S-nitroso-N-acetyl-penicillamine (SNAP), hydrogen peroxide,2,2-dithiodipyridine and reduced glutathione) on twitch and tetanic force, action potential (AP) repriming, sarcoplasmic reticulum (SR) Ca2+ loading and leakage, and contractile apparatus properties. None of the treatments detectably altered AP repriming, indicating that t-system excitability was relatively insensitive to such oxidative modification. Importantly, the overall effect on twitch and tetanic force of a given treatment was determined primarily by its action on Ca2+ sensitivity of the contractile apparatus. For example, S-nitrosylation with the NO• donor,SNAP, caused matching decreases in the contractile Ca2+ sensitivity and twitch response, and GSNO applied ∼10 min after preparation had very similar effects. The only exception was when GSNO was applied immediately after preparation, which resulted in irreversible decreases in twitch and tetanic responses even though it concomitantly increased Ca2+ sensitivity by∼0.1 pCaunits, the latter evidently due to S-glutathionylation of the contractile apparatus. This decrease in AP-mediated force responses was due to impaired VS­RyR coupling and was accompanied by increased Ca2+ leakage through RyRs. Such oxidation-related impairment of coupling could be responsible for prolonged low frequency fatigue in certain circumstances.

Leptin stimulation of COXIV is impaired in obese skeletal muscle myotubes.

OBJECTIVE: To investigate the effects of leptin on the mRNA abundance of key genes involved in fatty acid oxidation and mitochondrial biogenesis in cultured skeletal muscle myotubes derived from lean and obese individuals. RESEARCH METHODS AND PROCEDURES: Rectus abdominus muscle biopsies were obtained from surgical patients to establish primary skeletal muscle cell cultures. Two distinct primary cell culture groups were established (Lean and Obese) n = 7 in each group. Differentiated cultures were then exposed to leptin (2.5 µg/ml) for 6 h. mRNA expression was subsequently measured by real-time PCR analysis. RESULTS: Basal mRNA expression of ßHAD, COXIII, COXIV, PGC-1α and SOCS3 in the cultured human skeletal muscle myotubes were similar, however, PDK4 mRNA was elevated (P < 0.05) in the myotubes derived from obese individuals. The addition of leptin resulted in a 2.5-fold increase in COXIV mRNA expression in the myotubes derived from Lean individuals only (P < 0.05). There was also a tendency for leptin to increase COXIII, ßHAD and PDK4 mRNA expression in this same group. Leptin had no impact on the gene expression of all measured transcripts in myotubes derived from obese individuals. CONCLUSION: Short-term exposure of human skeletal muscle myotubes to leptin stimulated the expression of the mitochondrial enzyme COXIV in myotubes derived from lean individuals, an effect that was abrogated in myotubes derived from obese individuals. These data demonstrate a novel capacity for leptin to increase mitochondrial biogenesis and thus, a possible increased capacity for lipid oxidation and the persistence of a defect in leptin signalling in human myotubes cultured from obese individuals.

Intense exercise up-regulates Na+,K+-ATPase isoform mRNA, but not protein expression in human skeletal muscle.

Characterization of expression of, and consequently also the acute exercise effects on, Na(+),K(+)-ATPase isoforms in human skeletal muscle remains incomplete and was therefore investigated. Fifteen healthy subjects (eight males, seven females) performed fatiguing, knee extensor exercise at approximately 40% of their maximal work output per contraction. A vastus lateralis muscle biopsy was taken at rest, fatigue and 3 and 24 h postexercise, and analysed for Na(+),K(+)-ATPase alpha(1), alpha(2), alpha(3), beta(1), beta(2) and beta(3) mRNA and crude homogenate protein expression, using Real-Time RT-PCR and immunoblotting, respectively. Each individual expressed gene transcripts and protein bands for each Na(+),K(+)-ATPase isoform. Each isoform was also expressed in a primary human skeletal muscle cell culture. Intense exercise (352 +/- 69 s; mean +/-s.e.m.) immediately increased alpha(3) and beta(2) mRNA by 2.4- and 1.7-fold, respectively (P < 0.05), whilst alpha(1) and alpha(2) mRNA were increased by 2.5- and 3.5-fold at 24 h and 3 h postexercise, respectively (P < 0.05). No significant change occurred for beta(1) and beta(3) mRNA, reflecting variable time-dependent responses. When the average postexercise value was contrasted to rest, mRNA increased for alpha(1), alpha(2), alpha(3), beta(1), beta(2) and beta(3) isoforms, by 1.4-, 2.2-, 1.4-, 1.1-, 1.0- and 1.0-fold, respectively (P < 0.05). However, exercise did not alter the protein abundance of the alpha(1)-alpha(3) and beta(1)-beta(3) isoforms. Thus, human skeletal muscle expresses each of the Na(+),K(+)-ATPase alpha(1), alpha(2), alpha(3), beta(1), beta(2) and beta(3) isoforms, evidenced at both transcription and protein levels. Whilst brief exercise increased Na(+),K(+)-ATPase isoform mRNA expression, there was no effect on isoform protein expression, suggesting that the exercise challenge was insufficient for muscle Na(+),K(+)-ATPase up-regulation.

The interaction between the endothelial cell protein C receptor and protein C is dictated by the gamma-carboxyglutamic acid domain of protein C.

The endothelial cell protein C receptor (EPCR) binds to both protein C and activated protein C (APC) with similar affinity. Removal of the Gla domain of protein C results in the loss of most of the binding affinity. This observation is compatible with at least two models: 1) the Gla domain of protein C interacts with phospholipid on cell surfaces to stabilize interaction with EPCR or 2) the Gla domain of protein C makes specific protein-protein interactions with EPCR. The latter model predicts that chimeric proteins containing the protein C Gla domain should interact with EPCR. To test this, we constructed a prothrombin chimera in which the Gla domain and aromatic stack of prothrombin were replaced with the corresponding region of protein C. The 125I-labeled chimera (Kd = 176 nM) and 125I-APC (Kd = 65 nM) both bound specifically to 293 cells stably transfected with EPCR, but both bound poorly to sham-transfected cells. The chimera also blocked APC binding to EPCR-transfected cells in a dose-dependent fashion (Ki approximately 139 nM) similarly to protein C (Ki approximately 75 nM). Chimera binding to EPCR-transfected cells was blocked by soluble EPCR, demonstrating direct protein-protein interaction between the chimera and EPCR. Consistent with this conclusion, the isolated Gla domain of protein C blocked APC binding to EPCR-transfected cells (IC50 = 2 microM). No inhibition was observed with the isolated prothrombin Gla domain. A protein C chimera with the prothrombin Gla domain and aromatic stack failed to bind to EPCR detectably. These data suggest that the Gla domain of protein C is responsible for much of the binding energy and specificity of the protein C-EPCR interaction.

The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex.

Protein C activation on the surface of the endothelium is critical to the negative regulation of blood coagulation. We now demonstrate that monoclonal antibodies that block protein C binding to the endothelial cell protein C receptor (EPCR) reduce protein C activation rates by the thrombin-thrombomodulin complex on endothelium, but that antibodies that bind to EPCR without blocking protein C binding have no effect. The kinetic result of blocking the EPCR-protein C interaction is an increased apparent Km for the activation without altering the affinity of thrombin for thrombomodulin. Activation rates of the protein C derivative lacking the gamma-carboxyglutamic acid domain, which is required for binding to EPCR, are not altered by the anti-EPCR antibodies. These data indicate that the protein C activation complex involves protein C, thrombin, thrombomodulin, and EPCR. These observations open new questions about the control of coagulation reactions on vascular endothelium.

The endothelial cell protein C receptor. Inhibition of activated protein C anticoagulant function without modulation of reaction with proteinase inhibitors.

A soluble form of the endothelial cell protein C receptor (EPCR) was analyzed for the ability to modulate the functional properties of protein C and activated protein C (APC). In a plasma clotting system initiated with factor Xa, EPCR blocked the anticoagulant activity of APC in a dose-dependent fashion. EPCR had no influence on clotting in the absence of APC. Consistent with the plasma results, EPCR slowed the proteolytic inactivation of factor Va by slowing both of the key proteolytic cleavages in the heavy chain of factor Va. EPCR did not prevent protein C activation by the soluble thrombin-thrombomodulin complex, did not alter the inactivation of APC by alpha1-antitrypsin or protein C inhibitor, and did not influence the kinetics of peptide paranitroanilide substrate cleavage significantly. We conclude that EPCR binds to an exosite on APC that selectively modulates the enzyme specificity in a manner reminiscent of the influence of thrombomodulin on thrombin.

Intranasal absorption of physostigmine and arecoline.

Physostigmine, an acetyl cholinesterase inhibitor, and arecoline, a muscarinic agonist, have been shown to improve Alzheimer presenile dementia in some patients when administered parenterally. Both of these compounds are ineffective orally due to first-pass metabolism. The nasal route was examined as an alternative route of administration for both drugs. Nasal bioavailabilities and dispositions for both drugs were determined in rats. Physostigmine nasal bioavailability was 100% as compared with iv bioavailability, and that of arecoline was 85% when compared with bioavailability following im administration.

Monitoring compliance through analysis of drug and metabolite levels.

Cold analytical methodology is usually available for drug and metabolite monitoring during clinical trials, since a procedure is required for the bioavailability, pharmacokinetic, and dose proportionality studies that must be conducted by the sponsor. Such methods can and have been applied to monitoring patient compliance. Examples from several classes of drugs with different pharmacokinetic profiles illustrate the type of data that can be obtained, along with their applicability and inherent limitation in assessing compliance. The effects of concomitant medication, drug half-life, volume of distribution, and sampling time on observed levels are also discussed. Several other approaches involving trace metals, microtaggants, and an electronic monitor are also presented.