Background
Left ventricular dyssynchrony is an adverse consequence of ST-elevation myocardial infarction (STEMI) and bears an unfavorable prognosis. Mechanical dyssynchrony as measured by phase analysis from gated single photon emission computed tomography (GSPECT) correlates well with other imaging methods of assessing dyssynchrony but has not been studied in STEMI. We hypothesized that systolic dyssynchrony as measured by GSPECT would correlate with adverse remodeling after STEMI.
Methods
In 28 subjects suffering STEMI, GSPECT with technetium-99m sestamibi was performed immediately after presentation (day 5) and remotely (6 months). Parameters of left ventricular dyssynchrony (QRS width, histogram bandwidth (HBW) and phase standard deviation (PSD)) were measured from GSPECT using the Emory Cardiac Toolbox. Left ventricular volumes, ejection fraction (LVEF) and infarct size were also assessed.
Results
After successful primary percutaneous coronary intervention to the infarct-related artery, subjects had an LVEF of 46.4% ± 11% and a resting perfusion defect of 27.4% ± 16% at baseline. Baseline QRS width was normal (91.5 ± 17.5 ms). Subjects with STEMI had dyssynchrony compared with a cohort of 22 normal subjects (age 57.2 ± 10.6 years, <5% perfusion defect) by both HBW (100.3° ± 70.7° vs 26.5° ± 5.3°, P < .0001) and PSD (35.3° ± 16.9° vs 7.9° ± 2.1°, P < .0001). Baseline HBW correlated with resting perfusion defect size (r = 0.67, P < .001), end-systolic volume (r = 0.72, P < .001), end-diastolic volume (r = 0.63, P = .001), and inversely with LVEF (r =−0.74, P < .001). HBW and PSD improved over the follow-up period (−24.1 ± 35.9 degrees, P = .003 and −8.7° ± 14.6°, P = .006, respectively), and improvement in HBW correlated with reduction in LV end-systolic volumes (r = 0.43, P = .034). Baseline HBW and PSD, however, did not independently predict LVEF at 6 months follow-up.
Conclusions
After STEMI, subjects exhibit mechanical dyssynchrony as measured by GSPECT phase analysis without evidence of electrical dyssynchrony. Improvement in mechanical dyssynchrony correlates with beneficial ventricular remodeling. The full predictive value of this measure in post-infarct patients warrants further study.
In contrast to other cell types, vascular smooth muscle cells modify their phenotype in response to external signals. NADPH oxidase 4 (Nox4) is critical for maintenance of smooth muscle gene expression; however, the underlying mechanisms are incompletely characterized. Using smooth muscle α-actin (SMA) as a prototypical smooth muscle gene and transforming growth factor-β (TGF-β) as a differentiating agent, we examined Nox4-dependent signaling. TGF-β increases Nox4 expression and activity in human aortic smooth muscle cells (HASMC). Transfection of HASMC with siRNA against Nox4 (siNox4) abolishes TGF-β-induced SMA expression and stress fiber formation. siNox4 also significantly inhibits TGF-β-stimulated p38MAPK phosphorylation, as well as that of its substrate, mitogen-activated protein kinase-activated protein kinase-2 (MK-2). Moreover, the p38MAPK inhibitor SB-203580 nearly completely blocks the SMA increase induced by TGF-β. Inhibition of either p38MAPK or NADPH oxidase-derived reactive oxygen species impairs the TGF-β-induced phosphorylation of Ser103 on serum response factor (SRF) and reduces its transcriptional activity. Binding of SRF to myocardin-related transcription factor (MRTF) is also necessary, because downregulation of MRTF by siRNA abolishes TGF-β-induced SMA expression. Taken together, these data suggest that Nox4 regulates SMA expression via activation of a p38MAPK/SRF/MRTF pathway in response to TGF-β.
We recently identified a protective MHC class Ib-restricted CD8 T cell response to infection with mouse polyomavirus. These CD8 T cells recognize a peptide from aa 139–147 of the VP2 viral capsid protein bound to the nonpolymorphic H-2Q9 molecule, a member of the Qa-2 family of β2m-associated MHC-Ib molecules. Q9:VP2.139-specific CD8 T cells exhibit an unusual inflationary response characterized by a gradual expansion over 3 months followed by a stable maintenance phase. We previously demonstrated that Q9:VP2.139-specific CD8 T cells are dependent on Ag for expansion, but not for the long-term maintenance. Here, we tested the hypothesis that the expansion and maintenance components of the Q9:VP2.139-specific T cell response are differentially dependent on CD4 T cell help and CD28 costimulation. Depletion of CD4+ cells and CD28/CD40L blockade impaired expansion of Q9:VP2.139-specific CD8 T cells, and intrinsic CD28 signaling was sufficient for expansion. In contrast, CD4 T cell-insufficiency, but not CD28/CD40L blockade, resulted in a decline in frequency of Q9:VP2.139-specific CD8 T cells during the maintenance phase. These results indicate that the Q9:VP2.139-specific CD8 T cell response to mouse polyomavirus infection depends on CD4 T cell help and CD28 costimulation for inflationary expansion, but only on CD4 T cell help for maintenance.
The desmosomal cadherin desmoglein-2 (Dsg2) is a transmembrane cell adhesion protein that is widely expressed in epithelial and non-epithelial tissues, such as the intestine, epidermis, testis and heart. Dsg2 has been shown to regulate numerous cellular processes, including proliferation and apoptosis, and we have previously reported that intracellular fragments of Dsg2 promote apoptosis in colonic epithelial cells. While several studies have shown that both the extracellular and intracellular domains of Dsg2 can be targeted by proteases, identification of these putative Dsg2 fragments in colonic epithelial cells has not been performed. Here, we report that the mouse monoclonal antibody (mAb) AH12.2 binds to the first extracellular domain of Dsg2. Using this antibody along with previously described mAb against the extracellular (6D8) and intracellular (DG3.10) domains of Dsg2, we characterize the expression and identify the cleavage fragments of Dsg2 in colonic epithelial cells. This study provides a detailed description of the extracellular and intracellular Dsg2 cleavage fragments that are generated in the simple epithelium of the colon and will guide future studies examining the relationship of these fragments to cellular fate and disease states.
Nuclear factor kappa B (NF-κB) is a key signaling molecule in the elaboration of the inflammatory response. Data indicate that curcumin, a natural ingredient of the curry spice turmeric, acts as a NF-κB inhibitor and exhibits both anti-inflammatory and anti-cancer properties. Curcumin analogues with enhanced activity on the NF-κB and other inflammatory signaling pathways have been developed including the synthetic monoketone compound termed 3,5-Bis(2-fluorobenzylidene)-4-piperidone (EF24). 3,5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31) is a structurally-related curcumin analogue whose potency for NF-κB inhibition has yet to be determined. To examine the activity of EF31 compared to EF24 and curcumin, mouse RAW264.7 macrophages were treated with EF31, EF24, curcumin (1–100µM) or vehicle (DMSO 1%) for 1 hour. NF-κB pathway activity was assessed following treatment with lipopolysaccharide (LPS) (1µg/mL). EF31 (IC50 ~5µM) exhibited significantly more potent inhibition of LPS-induced NF-κB DNA binding compared to both EF24 (IC50~35µM) and curcumin (IC50 >50µM). In addition, EF31 exhibited significantly greater inhibition of NF-κB nuclear translocation as well as the induction of downstream inflammatory mediators including pro-inflammatory cytokine mRNA and protein (tumor necrosis factor-α, interleukin-1β, and interleukin-6). Regarding the mechanism of these effects on NF-κB activity, EF31 (IC50~1.92µM) exhibited significantly greater inhibition of IκB kinase β compared to EF24 (IC50~131µM). Finally, EF31 demonstrated potent toxicity in NF-κB-dependent cancer cell lines while having minimal and reversible toxicity in RAW264.7 macrophages. These data indicate that EF31 is a more potent inhibitor of NF-κB activity than either EF24 or curcumin while exhibiting both anti-inflammatory and anticancer activities. Thus, EF31 represents a promising curcumin analogue for further therapeutic development.
Background
Cytomegalovirus (CMV) is the most common opportunistic infection after solid-organ transplant. Valganciclovir prophylaxis significantly reduces disease, but limited data are available on its use in children. Recently, an increase in delayed-onset CMV disease has been noted with some arguing that longer prophylaxis may decrease late-onset disease.
Methods
Single-center, retrospective analysis of pediatric renal transplant patients receiving 24 weeks valganciclovir prophylaxis (15 mg/kg/day, maximum 900 mg/day) from January 2004 to December 2008, aiming to measure the incidence of CMV disease and toxicity of valganciclovir.
Results
We enrolled 111 patients, 60% males, 46% African Americans, and median age at transplant 14.5 years (range 1.4–20.4 years). Sixty-nine percent of donors and 44% of recipients were seropositive pretransplant. Median duration of valganciclovir use was 5.9 months (range 0.5–24 months). CMV viremia and disease occurred in 27% and 4.5%, respectively. All patients with disease presented after prophylaxis ended and all were D+/R−. Thymoglobulin use (P=0.04) and positive donor CMV status (P=0.02) were associated with a higher risk of CMV viremia. Twenty-four percent had hematologic toxicity directly associated with valganciclovir.
Conclusions
Valganciclovir use in children was effective as prophylaxis against CMV disease; no children at our institution developed disease while on therapy. Our regimen of 24 weeks of prophylaxis was associated with a lower rate of late-onset disease than previous reports with 12-week regimens. Further controlled studies should be considered to compare longer versus shorter periods of prophylaxis and dose reductions and their impact on prevention of late-onset disease, resistance, cost, and toxicity.
Living systems have three major types of cell signaling systems that are dependent upon high-energy chemicals, redox environment and transmembranal ion gating mechanisms. Development of integrated systems biology descriptions of cell signaling require conceptual models incorporating all three. Recent advances in redox biology show that thiol/disulfide redox systems are regulated under dynamic, non-equilibrium conditions, progressively oxidized with the life cycle of cells and distinct in terms of redox potentials among subcellular compartments. The present article uses these observations as a basis to distinguish “redox-sensing” mechanisms, which are more global biologic redox control mechanisms, from “redox signaling”, which involves conveyance of discrete activating or inactivating signals. Both redox sensing and redox signaling use sulfur switches, especially cysteine (Cys) residues in proteins which are sensitive to reversible oxidation, nitrosylation, glutathionylation, acylation, sulfhydration or metal binding. Unlike specific signaling mechanisms, the redox-sensing mechanisms provide means to globally affect the rates and activities of the high-energy, ion gating and redox-signaling systems by controlling sensitivity, distribution, macromolecular interactions and mobility of signaling proteins. Effects mediated through Cys residues not directly involved in signaling means redox-sensing control can be orthogonal to the signaling mechanisms. This provides a capability to integrate signals according to cell cycle and physiologic state without fundamentally altering the signaling mechanisms. Recent findings that thiol/disulfide pools in humans are oxidized with age, environmental exposures and disease risk suggest that redox-sensing thiols could provide a central mechanistic link in disease development and progression.
Background
The purpose of this study was to develop a multi-harmonic phase analysis method to measure diastolic dyssynchrony from conventional gated SPECT myocardial perfusion imaging(MPI) data and to compare it with systolic dyssynchrony in normal subjects and in patients with end-stage renal disease (ESRD) and normal left-ventricular ejection fraction (LVEF).
Methods
121 consecutive patients with ESRD and normal LVEF and 30 consecutive normal controls were enrolled. Diastolic dyssynchrony parameters were calculated using 3-harmonic phase analysis. Systolic dyssynchrony parameters were calculated using the established 1-harmonic phase analysis.
Results
The systolic and diastolic dyssynchrony parameters were correlated, but significantly different in both control and ESRD groups, indicating they were physiologically related but measured different LV mechanisms. The systolic and diastolic dyssynchrony parameters were each significantly different between the control and the ESRD groups. Significant systolic and diastolic dyssynchrony were found in 47% and 65% of the entire ESRD group.
Conclusion
Multi-harmonic phase analysis has been developed to assess diastolic dyssynchrony, which measured a new LV mechanism of regional function from gated SPECT MPI and showed a significantly higher prevalence rate than systolic dyssynchrony in patients with ESRD and normal LVEF.
by
Omar Saeed;
Fumiyuki Otsuka;
Rohini Polavarapu;
Vinit Karmali;
Daiana Weiss;
Talina Davis;
Bradley Sverre Rostad;
Kimberly Pachura;
Lila Adams;
John Elliott;
W Robert Taylor;
Jagat Narula;
Frank Kolodgie;
Renu Virmani;
Charles C. Hong;
Aloke V Finn
Objectives
We recently reported that lowering of macrophage free intracellular iron increases expression of cholesterol efflux transporters ABCA1 and ABCG1 by reducing generation of reactive oxygen species. In this study, we explore whether reducing macrophage intracellular iron levels via pharmacologic suppression of hepcidin can increase macrophage-specific expression of cholesterol efflux transporters and reduce atherosclerosis.
Methods and Results
To suppress hepcidin, increase expression of the iron exporter ferroportin (FPN), and reduce macrophage intracellular iron, we used a small molecule inhibitor of BMP signaling, LDN 193189 (LDN). LDN (10 mg/kg i.p. bid) was administered to mice and its effects on atherosclerosis, intracellular iron, oxidative stress, lipid efflux, and foam cell formation were measured in plaques and peritoneal macrophages. Long-term LDN administration to Apo E (-/-) mice increased ABCA1 immunoreactivity within intraplaque macrophages by 3.7-fold (n=8; p=0.03), reduced oil-red-o positive lipid area by 50% (n=8; p=0.02) and decreased total plaque area by 43% (n=8; p=0.001). LDN suppressed liver hepcidin transcription and increased macrophage FPN, lowering intracellular iron and hydrogen peroxide production. LDN treatment increased macrophage ABCA1 and ABCG1 expression, significantly raised cholesterol efflux to ApoA-1 and decreased foam cell formation. All preceding LDN-induced effects on cholesterol efflux were reversed by exogenous hepcidin administration, suggesting that modulation of intracellular iron levels within macrophages as the mechanism by which LDN triggers these effects.
Conclusion
These data suggest that pharmacologic manipulation of iron homeostasis may be a promising target to increase macrophage reverse cholesterol transport and limit atherosclerosis.
Aims
Reduction of transient outward current (Ito) and excessive activation of Ca2+/Calmodulin-dependent kinase II (CaMKII) are general features of ventricular myocytes in heart failure. We hypothesize that alterations of Ito directly regulate CaMKII activation in cardiomyocytes.
Methods and results
A dynamic coupling of Ito channel subunit Kv4.3 and inactive CaMKII was discovered in cardiomyocytes with the membrane predominant distribution by co-immunoprecipitation and fluorescence resonance energy transfer techniques. CaMKII dissociation from Kv4.3–CaMKII units caused a significant increase in CaMKII autophosphorylation and L-type calcium current (ICa) facilitation. ICa facilitation was blunted by the compartmental Ca2+ chelator BAPTA but unaffected by bulk Ca2+ chelator EGTA, implicating membrane-localized CaMKII. Kv4.3 overexpression reduced basal CaMKII autophosphorylation in myocytes and eliminated Ca2+-induced CaMKII activation. Kv4.3 blocks CaMKII activation by binding to the calmodulin binding sites, whereas Kv4.3 uncoupling releases these sites and leads to a substantial CaMKII activation.
Conclusion
Our results uncovered an important mechanism that regulates CaMKII activation in the heart and implicate Ito channel alteration in pathological CaMKII activation.