Changes in myofilament proteins, but not Ca²âº regulation, are associated with a high-fat diet-induced improvement in contractile function in heart failure.

Pathological conditions such as diabetes, insulin resistance, and obesity are characterized by elevated plasma and myocardial lipid levels and have been reported to exacerbate the progression of heart failure (HF). Alterations in cardiomyocyte Ca(2+) regulatory properties and myofilament proteins have also been implicated in contractile dysfunction in HF. However, our prior studies reported that high saturated fat (SAT) feeding improves in vivo myocardial contractile function, thereby exerting a cardioprotective effect in HF. Therefore, we hypothesized that SAT feeding improves contractile function by altering Ca(2+) regulatory properties and myofilament protein expression in HF. Male Wistar rats underwent coronary artery ligation (HF) or sham surgery (SH) and were fed normal chow (SHNC and HFNC groups) or a SAT diet (SHSAT and HFSAT groups) for 8 wk. Contractile properties were measured in vivo [echocardiography and left ventricular (LV) cannulation] and in isolated LV cardiomyocytes. In vivo measures of contractility (peak LV +dP/dt and -dP/dt) were depressed in the HFNC versus SHNC group but improved in the HFSAT group. Isolated cardiomyocytes from both HF groups were hypertrophied and had decreased percent cell shortening and a prolonged time to half-decay of the Ca(2+) transient versus the SH group; however, SAT feeding reduced in vivo myocyte hypertrophy in the HFSAT group only. The peak velocity of cell shortening was reduced in the HFNC group but not the HFSAT group and was positively correlated with in vivo contractile function (peak LV +dP/dt). The HFNC group demonstrated a myosin heavy chain (MHC) isoform switch from fast MHC-α to slow MHC-ß, which was prevented in the HFSAT group. Alterations in Ca(2+) transients, L-type Ca(2+) currents, and protein expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase and phosphorylated phospholamban could not account for the changes in the in vivo contractile properties. In conclusion, the cardioprotective effects associated with SAT feeding in HF may occur at the level of the isolated cardiomyocyte, specifically involving changes in myofilament function but not sarcoplasmic reticulum Ca(2+) regulatory properties.

Ascorbate attenuates atrial pacing-induced peroxynitrite formation and electrical remodeling and decreases the incidence of postoperative atrial fibrillation.

Atrial fibrillation (AF), the most common chronic arrhythmia, increases the risk of stroke and is an independent predictor of mortality. Available pharmacological treatments have limited efficacy. Once initiated, AF tends to self-perpetuate, owing in part to electrophysiological remodeling in the atria; however, the fundamental mechanisms underlying this process are still unclear. We have recently demonstrated that chronic human AF is associated with increased atrial oxidative stress and peroxynitrite formation; we have now tested the hypothesis that these events participate in both pacing-induced atrial electrophysiological remodeling and in the occurrence of AF following cardiac surgery. In chronically instrumented dogs, we found that rapid (400 min(-1)) atrial pacing was associated with attenuation of the atrial effective refractory period (ERP). Treatment with ascorbate, an antioxidant and peroxynitrite decomposition catalyst, did not directly modify the ERP, but attenuated the pacing-induced atrial ERP shortening following 24 to 48 hours of pacing. Biochemical studies revealed that pacing was associated with decreased tissue ascorbate levels and increased protein nitration (a biomarker of peroxynitrite formation). Oral ascorbate supplementation attenuated both of these changes. To evaluate the clinical significance of these observations, supplemental ascorbate was given to 43 patients before, and for 5 days following, cardiac bypass graft surgery. Patients receiving ascorbate had a 16.3% incidence of postoperative AF, compared with 34.9% in control subjects. In combination, these studies suggest that oxidative stress underlies early atrial electrophysiological remodeling and offer novel insight into the etiology and potential treatment of an enigmatic and difficult to control arrhythmia. The full text of this article is available at http://www.circresaha.org.

Impaired myofibrillar energetics and oxidative injury during human atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is associated with severe contractile dysfunction and structural and electrophysiological remodeling. Mechanisms responsible for impaired contractility are undefined, and current therapies do not address this dysfunction. We have found that myofibrillar creatine kinase (MM-CK), an important controller of myocyte contractility, is highly sensitive to oxidative injury, and we hypothesized that increased oxidative stress and energetic impairment during AF could contribute to contractile dysfunction. Methods and Results-- Right atrial appendages were obtained from AF patients undergoing the Maze procedure and from control patients who were in normal sinus rhythm and undergoing cardiac surgery. MM-CK activity was reduced in AF patients compared with controls (25.4+/-3.4 versus 18.2+/-3.8 micromol/mg of myofibrillar protein per minute; control versus AF; P<0.05). No reduction in total CK activity or myosin ATPase activity was detected. This selective reduction in MM-CK activity was associated with increased relative expression of the beta-myosin isoform (25+/-6 versus 63+/-5%beta, CTRL versus AF; P<0.05). Western blotting of AF myofibrillar isolates demonstrated no changes in protein composition but showed increased prevalence of protein oxidation as detected by Western blotting for 3-nitrotyrosine (peroxynitrite biomarker) and protein carbonyls (hydroxyl radical biomarker; P<0.05). Patterns of these oxidative markers were distinct, which suggests discrete chemical events and differential protein vulnerabilities in vivo. MM-CK inhibition was statistically correlated to extent of nitration (P<0.01) but not to carbonyl presence. CONCLUSIONS: The present results provide novel evidence of oxidative damage in human AF that altered myofibrillar energetics may contribute to atrial contractile dysfunction and that protein nitration may be an important participant in this condition.

C-reactive protein elevation in patients with atrial arrhythmias: inflammatory mechanisms and persistence of atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) may persist due to structural changes in the atria that are promoted by inflammation. C-reactive protein (CRP), a marker of systemic inflammation, predicts cardiovascular events and stroke, a common sequela of AF. We hypothesized that CRP is elevated in patients with atrial arrhythmias. METHODS AND RESULTS: Using a case-control study design, CRP in 131 patients with atrial arrhythmias was compared with CRP in 71 control patients. Among arrhythmia patients, 6 had frequent atrial ectopy or tachycardia, 86 had paroxysmal AF, 39 had persistent AF lasting >30 days, and 70 had lone arrhythmias. CRP was higher in arrhythmia than in control patients (median, 0.21 versus 0.096 mg/dL; P<0.001). Arrhythmia patients in AF within 24 hours before sampling had higher CRP than those in sinus rhythm (0.30 versus 0.15 mg/dL; P<0.001). CRP in controls was not different than in patients with atrial ectopy or tachycardia. Lone arrhythmia patients had a CRP of 0.21 mg/dL, which was not significantly lower than arrhythmia patients with structural heart disease (CRP, 0.23 mg/dL) but higher than controls (P=0.002). Persistent AF patients had a higher CRP (0.34 mg/dL) than paroxysmal AF patients (0.18 mg/dL; P=0.008); both groups had higher CRP levels than controls (P

Adenosine modulates methylmercuric chloride (MeHgCl)-induced D-aspartate release from neonatal rat primary astrocyte cultures.

The effects of adenosine, and selective adenosine receptor agonists and antagonists on methylmercury (MeHg)-induced aspartate release were studied in neonatal rat primary astrocyte cultures. Whereas basal levels of D-[3H]aspartate release were unchanged upon treatment with adenosine or the selective A1 receptor agonists, N6-cyclopentyladenosine (CPA), cyclohexyladenosine (CHA), and R-phenylisopropyladenosine (R-PIA), all partially reversed the MeHg-induced release of D-aspartate. Treatment of astrocytes with the xanthine derivative, theophylline, an adenosine antagonist, reversed the inhibitory effect of adenosine on MeHg-induced D-[3H]aspartate release. Since the effect of MeHg on D-[3H]aspartate release is known to be associated with sulfhydryl (-SH) groups which are controlled by intracellular glutathione concentrations [GSH]i, we also evaluated the effects of adenosine, the A1 agonists CPA and CHP, and the adenosine antagonist, theophylline, on astrocytic [GSH]i. Attenuation of the stimulatory effect of MeHg on D-[3H]aspartate release by adenosine and its agonists occurred in the presence of reduced astrocytic [GSH]i, suggesting that other mechanisms must be invoked for this protective effect. Whilst the mechanism of MeHg-induced D-[3H]aspartate release is not known, the data suggest a role for adenosine in its regulation.

The role of -SH groups in methylmercuric chloride-induced D-aspartate and rubidium release from rat primary astrocyte cultures.

Methylmercuric chloride (MeHgCl) was shown to increase D-aspartate and rubidium (Rb; a marker for potassium) release from preloaded astrocytes in a dose- and time-dependent fashion. Two sulfhydryl (-SH) protecting agents: a cell membrane non-penetrating compound, reduced glutathione (GSH), and the membrane permeable dithiothreitol (DTT), were found to inhibit the stimulatory action of MeHgCl on the efflux of radiolabeled D-aspartate as well as Rb. MeHgCl-induced D-aspartate and Rb release was completely inhibited by the addition of 1 mM DTT or GSH during the actual 5 min perfusion period with MeHgCl (10 microM). However, when added after MeHgCl treatment, this inhibition could not be fully sustained by GSH, while DTT fully inhibited the MeHgCl-induced release of D-aspartate. Neither DTT or GSH alone had any effect on the rate of astrocytic D-aspartate release. Accordingly, it is postulated that the stimulatory effect exerted by MeHgCl on astrocytic D-aspartate release is associated with vulnerable -SH groups located within, but not on the surface of the cell membrane. Omission of Na+ from the perfusion solution did not accelerate MeHgCl-induced D-aspartate release, suggesting that reversal of the D-aspartate carrier cannot be invoked to explain MeHgCl-induced D-aspartate release. Omission of Ca2+ from the perfusion solution increased the time-dependent MeHgCl-induced D-aspartate release.

The role of sulfhydryl groups in D-aspartate and rubidium release from neonatal rat primary astrocyte cultures.

We have recently demonstrated that both methylmercury (MeHg) and mercuric chloride (MC) induce D-aspartate release from neonatal rat primary astrocyte cultures maintained in isotonic conditions. In the present study, we compare several other sulfhydryl-(-SH) selective alkylating reagents [methyl methanethiosulfonate (MMTS), N-ethylmaleimide (NEM), and iodoacetamide (IA)] in isotonic, as well as hypotonic conditions to discern the functional importance of -SH groups in [3H]D-aspartate and 86rubidium (86Rb) release from astrocytes. Treatment of astrocytes (5 min) in isotonic buffer with the hydrophobic reagent NEM (10 microM) caused a marked increase in 86Rb release but had no effect on [3H]D-aspartate release. Neither IA-, nor MMTS-treatment (both at 10 microM) induced increase in [3H]D-aspartate or 86Rb release in isotonic buffer. In hypotonic condition (-50 mM Na+), astrocytes were most sensitive to MC exposure (5 microM), exhibiting an increase in both [3H]D-aspartate and 86Rb efflux. The hydrophobic compounds MMTS and NEM, and the hydrophilic -SH modifying reagent, IA, attenuated the hypotonic-induced efflux of [3H]D-aspartate, in the absence of an effect on 86Rb release. These observations are consistent with a critical role for -SH groups both in basal (i.e. isotonic) and hypotonic-induced release of D-aspartate and Rb from astrocytes. Lack of uniformity of these effects may be attributed to site-specificity, related to the physicochemical properties of these -SH alkylating reagents.

Intracellular glutathione (GSH) levels modulate mercuric chloride (MC)- and methylmercuric chloride (MeHgCl)-induced amino acid release from neonatal rat primary astrocytes cultures.

Mercuric chloride (MC) and methylmercury (MeHg) were found to increase amino acid release from astrocytes. This suggests interaction with sulfhydryl (-SH) groups which are controlled by glutathione [GSH] levels. In the present study, we evaluated the effects of alterations in intracellular glutathione concentrations [GSH]i on the outcome of MC and MeHg treatment. [GSH]i were increased in a time-dependent fashion by incubating the astrocytes with 1 mM L-2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine precursor. OTC attenuated the release of [2,3-3H]D-aspartic acid from astrocytes exposed to MC- (5 microM) and MeHg-(10 microM). MeHg-induced [3H]D-taurine release was also reduced by pretreatment of astrocytes with OTC. Treatment with BSO (50 microM) decreased [GSH]i in astrocytes, and increased [2,3-3H]D-aspartate release from MC- and MeHg-treated astrocytes, and [3H]D-taurine release from MeHg-treated cells. Neither OTC nor BSO when added to cultures in the absence of MC or MeHg had an effect on amino acid release by astrocytes. The current study underscores both the sensitivity of astrocytes to mercurials in terms of amino acid release and the relationship of these effects of astrocytic [GSH]i.

Educational benefits and cost savings from an interactive multimedia "best practice".

BACKGROUND: Developing and deploying "clinical practice guidelines" and "best practices" is a major strategic goal of our organization. We developed and evaluated a novel way of educating our colleagues on the office management of shoulder pain, using an interactive multimedia computer program to allow rapid access to user-selected audiovisual educational materials through a simple point and click interface. METHODS: Pre- and post-testing of 30 practicing primary care providers was conducted to assess ability to evaluate and manage patients who present with shoulder pain, and a post-use survey was administered to assess clinician acceptance and confidence, ability to use as a just-in-time tool, and changes in utilization of medical resources. RESULTS: Pre- and post-testing demonstrated that clinicians used this tool to improve their scores in a wide variety of clinical areas including history, physical examination, diagnosis, and management of conditions causing painful shoulder above their scores using textbooks and other traditional educational materials alone. The post-use survey showed outstanding acceptance by clinicians and their reported increased confidence as well as the potential for their increased abilities to translate into significant cost savings through more focused use of radiographic studies, orthopedic consultations, and physical therapy treatments. CONCLUSIONS: It was concluded that "The Multimedia Reference for Office Management of Shoulder Pain" is an effective and valuable learning resource for practicing clinicians, and demonstrates the potential to use an interactive multimedia tool to augment clinical skills and promote cost effective management of patients with this common clinical problem.

Mechanosensitive gating of atrial ATP-sensitive potassium channels.

Cell-attached and inside-out excised-patch recording techniques were used to search for mechanosensitive ion channels in neonatal and adult rat atrial myocytes. A channel activated by negative pressure applied to the patch, with a single-channel conductance of 52 pS in symmetric potassium solutions, was frequently observed. This channel has been identified as the atrial ATP-sensitive potassium (KATP) channel on the basis of its potassium selectivity, as well as its inhibition by ATP or tolbutamide in the inside-out excised patch. Mechanosensitive modulation of the KATP channel has not previously been reported. In the presence of 1 mM ATP, 10-50 microM pinacidil (a specific KATP channel agonist) does not significantly increase basal KATP channel activity; however, these concentrations of pinacidil potentiated the mechanosensitive modulation of the KATP channel. A hypotonic swelling protocol (a mechanical stimulus) was used in an effort to determine whether mechanosensitive modulation of this channel can generate significant whole-cell currents. Under perforated-patch whole-cell recording conditions, superfusion of atrial myocytes with a 240 mosm/kg solution (control solution, 290 mosm/kg) stimulated whole-cell currents with a magnitude similar to those activated by 10 microM pinacidil. These results demonstrate that the gating of the atrial KATP channel is mechanosensitive and suggest that mechanosensitive modulation may be an additional and significant mechanism, modulating channel activity under both physiological and pathological conditions.

Molecular basis of electrical remodeling in atrial fibrillation.

Atrial fibrillation (AF) is the most common cardiac arrhythmia, and is often associated with other cardiovascular disorders and diseases. AF can lead to thromboembolism, reduced left ventricular function and stroke, and, importantly, it is independently associated with increased mortality. AF is a progressive disease; numerous lines of evidence suggest that disease progression results from cumulative electrophysiological and structural remodeling of the atria. There is considerable interest in delineating the molecular mechanisms involved in the remodeling that occurs in the atria of patients with AF. Cellular electrophysiological studies have revealed marked reductions in the densities of the L-type voltage-gated Ca2+ current, I(Ca,L), the transient outward K+ current, I(TO), and the ultrarapid delayed rectifier K+ current, I(Kur), in atrial myocytes from patients in chronic AF. Similar (but not identical) changes in currents are evident in myocytes isolated from a canine model of AF and, in this case, the changes in currents are correlated with reduced expression of the underlying channel forming subunits. In both human and canine AF, the reduction in I(Ca,L) appears to be sufficient to explain the observed decreases in action potential duration and effective refractory period that are characteristic features of the remodeled atria. In addition, expression of the sarcoplasmic reticulum Ca2+ ATPase is reduced, suggesting that calcium cycling is affected in AF. These recent studies suggest that calcium overload and perturbations in calcium handling play prominent roles in AF-induced atrial remodeling. Although considerable progress has been made, further studies focused on defining the detailed structural, cellular and molecular changes that accompany the different stages of AF in humans, as well as in animal models of AF, are clearly warranted. It is anticipated that molecular insights gleaned from these studies will facilitate the development of improved therapeutic approaches to treat AF and to prevent the progression of the arrhythmia.

Phenylephrine suppresses outward K+ currents in rat atrial myocytes.

The modulation of whole cell K+ currents by the alpha 1-adrenergic agonist, phenylephrine, was studied in isolated rat atrial myocytes by use of perforated-patch whole cell recording techniques. The out ward K+ current in these myocytes consists of two inactivating components (iK,f and iK,s), which differ in the kinetics of inactivation and recovery from inactivation, and a noninactivating component, (iK,ss). Superfusion of these myocytes with 10 microM phenylephrine caused a rapid suppression of iK,ss, with little effect on the other current components. This effect of phenylephrine could be mimicked by exogenous application of 1,2-dioctanoyl-sn-glycerol (20 microM), a membrane-permeant diacylglycerol analogue; however, it was clearly distinct from the effect of 5 nM alpha-dendrotoxin, which selectively suppressed the slowly inactivating current component, iK,s, while having no effect on iK,ss. At a dose of 50 microM, phenylephrine also suppressed iK,s. There was no significant effect of phenylephrine (10 or 50 microM) or alpha-dendrotoxin (5 nM) on the rapidly inactivating current component, iK,f. The kinetic and pharmacological differences between these current components suggest that they represent the activity of distinct K+ channels.

Astrocytes as potential modulators of mercuric chloride neurotoxicity.

1. MC has been shown to inhibit the uptake of L-glutamate and increase D-aspartate release from preloaded astrocytes in a dose-dependent fashion. 2. Two sulfhydryl (SH-)-protecting agents; reduced glutathione (GSH), a cell membrane-nonpenetrating compound, and the membrane permeable dithiothreitol (DTT), have been shown consistently to reverse the above effects. MC-induced D-aspartate release is completely inhibited by the addition of 1 mM DTT or GSH during the actual 5-min perfusion period with MC (5 microM); when added after MC treatment, DTT fully inhibits the MC-induced D-aspartate release, while GSH does not. 3. Neither DTT nor GSH, in the absence of MC, have any effect on the rate of astrocytic D-aspartate release. Other studies demonstrate that although MC treatment (5 microM) does not induce astrocytic swelling, its addition to astrocytes swollen by exposure to hypotonic medium leads to their failure to volume regulate. 4. Omission of calcium from the medium greatly potentiates the effect of MC on astrocytic D-aspartate release, an effect which can be reversed by cotreatment of astrocytes with the dihydropyridine Ca(2+)-channel antagonist nimodipine (10 microM), indicating that one possible route of MC entry into the cells is through voltage-gated L-type channels.