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Rationale: Atrial fibrillation (AF) and heart failure often coexist, but their interaction is poorly understood. Clinical data indicate that the arrhythmic component of AF may contribute to left ventricular (LV) dysfunction. Objective: This study investigates the effects and molecular mechanisms of AF on the human LV. Methods and Results: Ventricular myocardium from patients with aortic stenosis and preserved LV function with sinus rhythm or rate-controlled AF was studied. LV myocardium from patients with sinus rhythm and patients with AF showed no differences in fibrosis. In functional studies, systolic Ca 2+ transient amplitude of LV cardiomyocytes was reduced in patients with AF, while diastolic Ca 2+ levels and Ca 2+ transient kinetics were not statistically different. These results were confirmed in LV cardiomyocytes from nonfailing donors with sinus rhythm or AF. Moreover, normofrequent AF was simulated in vitro using arrhythmic or rhythmic pacing (both at 60 bpm). After 24 hours of AF-simulation, human LV cardiomyocytes from nonfailing donors showed an impaired Ca 2+ transient amplitude. For a standardized investigation of AF-simulation, human iPSC-cardiomyocytes were tested. Seven days of AF-simulation caused reduced systolic Ca 2+ transient amplitude and sarcoplasmic reticulum Ca 2+ load likely because of an increased diastolic sarcoplasmic reticulum Ca 2+ leak. Moreover, cytosolic Na + concentration was elevated and action potential duration was prolonged after AF-simulation. We detected an increased late Na + current as a potential trigger for the detrimentally altered Ca 2+ /Na + -interplay. Mechanistically, reactive oxygen species were higher in the LV of patients with AF. CaMKII (Ca 2+ /calmodulin-dependent protein kinase IIδc) was found to be more oxidized at Met281/282 in the LV of patients with AF leading to an increased CaMKII activity and consequent increased RyR2 phosphorylation. CaMKII inhibition and ROS scavenging ameliorated impaired systolic Ca 2+ handling after AF-simulation. Conclusions: AF causes distinct functional and molecular remodeling of the human LV. This translational study provides the first mechanistic characterization and the potential negative impact of AF in the absence of tachycardia on the human ventricle.

Introduction: The effects of atrial fibrillation (AF) on left ventricular (LV) function are poorly understood. Clinical data indicate that the arrhythmic component of AF may contribute to LV dysfunction. This study investigated the effects of AF on the human LV. Methods and Results: LV myocardium from patients with preserved LV function (EF>50%) with sinus rhythm (SR, n=31) or rate-controlled AF (n=24) obtained during surgery for aortic stenosis was studied. Clinical data were mostly balanced without changes in LV function and remodeling. LV myocardium from SR and AF patients showed no changes in fibrosis. In functional studies, systolic Ca 2+ transient amplitude of human LV cardiomyocytes (CM) was reduced in AF patients, while diastolic Ca 2+ levels and Ca 2+ transient kinetics were unaltered. These results were confirmed in LV CM from non-failing donors with AF (n=4) vs. SR (n=8). In addition, normofrequent AF was simulated in vitro using arrhythmic or rhythmic electrical culture pacing (both at 60 bpm). After 24h of AF-simulation, human LV CM from non-failing donors (n=9) showed also an impaired Ca 2+ transient amplitude. For a standardized chronic AF-simulation, human iPSC-CM (n=22 differentiations/5 individuals) were tested. 7 days of AF-simulation caused reduced systolic Ca 2+ transient amplitude and sarcoplasmic reticulum Ca 2+ load, which could be explained by an increased diastolic Ca 2+ leak. Mechanistically, oxidative stress markers were higher in the LV of AF patients. Ca 2+ /calmodulin-dependent protein kinase IIδ (CaMKII) was found to be more oxidized at Met281/282 in the LV of AF patients leading to an increased CaMKII activity. Thus, RyR2 phosphorylation was elevated, which likely underlies the depression of LV Ca 2+ handling in AF. In translational contractility experiments of human ventricular trabecula from patients with heart failure (n=8), in vitro AF-simulation for 8h worsened systolic and diastolic contractile function. Conclusions: Here we could firstly show that AF impairs human LV function via increased oxidative stress and CaMKII activity in different patient populations and models. Thus, this translational study demonstrates the detrimental effects and mechanisms of AF in the absence of tachycardia on the human LV.

Introduction: Tenascin-C (TN-C) upregulation is linked to worse clinical outcome in diabetic patients. However, the role of TN-C in the development of diabetic cardiomyopathy is still unknown. Hypothesis: Whether TN-C plays a maladaptive role in diabetic cardiomyopathy. Methods: AJ (n=14) and TNC-KO (n=16) adult male mice were injected with streptozotocin (50mg/kg) to induce diabetes. Cardiac function was measured by echocardiography at 16-18 weeks follow-up. Additionally, cardiomyocyte passive stiffness , vascular endothelial function (aorta), and cardiac fibrosis were assessed. In addition, the hemodynamic effect of human recombinant TN-C (hrTN-C; 80 ng/ml) on the isolated working rat heart (n=3) was evaluated. Isolated primary mouse cardiomyocytes (IPMC) were used to assess TN-C expression. Human ventricular cardiac fibroblasts (HCF) were cultured and treated with 1) TGF-β (20ng/ml); 2) rhTN-C (10μg/ml) and TLR4 inhibitor (TAK-242, 50 mM) in combination with TN-C and mRNA expression of α-SMA, TN-C, Col-1, Col-3 and ACE1 were assessed by RT-qPCR. Finally, HUVEC were treated either with rhTN-C (10μg/ml) or combination with TLR-4 inhibitor and analysed of the expression of NADPH oxidase 1 and 4 (NOX1, NOX4). Results: TN-C deficiency was accompanied by preserved LV ejection fraction and endothelium function in aorta (p Conclusions: TN-C promotes a hostile environment that facilitates fibrosis and oxidative stress leading to cardiomyocyte and endothelial cell dysfunction. Thus, TN-C may play a crucial role in cardiovascular dysfunction in diabetes as well as a potential target for therapy.

INTRODUCTION AND OBJECTIVE:Kidney cancer is placed third in urologic cancers in Canada, right behind prostate and bladder cancer. Many new therapeutic options are being developed in the metastatic ...

Background: Direct oral anticoagulants (DOACs) are surpassing warfarin as the anticoagulant of choice for stroke prevention in nonvalvular atrial fibrillation. DOAC outcomes in elective periprocedural settings have not been well elucidated and remain a source of concern for clinicians. The aim of this meta-analysis was to evaluate the periprocedural safety and efficacy of DOACs versus warfarin in patients with nonvalvular atrial fibrillation. Methods: We reviewed the literature for data from phase III randomized controlled trials comparing DOACs with warfarin in the periprocedural period among patients with nonvalvular atrial fibrillation. Substudies from 4 trials (RE-LY [Randomized Evaluation of Long-Term Anticoagulation Therapy], ROCKET AF [Rivaroxaban Once Daily Oral Direct Factor Xa Inhibitor Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation], ARISTOTLE [Apixaban for the Prevention of Stroke in Subjects With Atrial Fibrillation], and ENGAGE-AF [Effective Anticoagulation With Factor xA Next Generation in Atrial Fibrillation]) were included in the meta-analysis. DOACs as a group and warfarin were compared in terms of the 30-day pooled risk for stroke/systemic embolism, major bleeding, and death, according to whether the study drug was interrupted or not periprocedurally. The overall relative risk (RR) was estimated with a random-effects model. The I 2 test was used to assess heterogeneity in RR among the studies. Results: In the uninterrupted anticoagulant strategy, there were no differences in the rates of stroke/systemic embolism (pooled risk, 0.6% [29 events/4519 procedures] versus 1.1% [31/2971]; RR, 0.70; 95% confidence interval [CI], 0.41–1.18) and death (1.4% versus 1.8%; RR, 0.77; 95% CI, 0.53–1.12) between DOACs and warfarin and significantly fewer major bleeding events (2.0% versus 3.3%; RR, 0.62; 95% CI, 0.47–0.82) with DOACs compared to warfarin. Under an interrupted strategy, there was no significant difference between DOACs versus warfarin for stroke/systemic embolism (0.4% [41/9260] versus 0.5% [31/7168]; RR, 0.95; 95% CI, 0.59–1.55), major bleeding (2.1% versus 2.0%; RR, 1.05; 95% CI, 0.85–1.30), and death (0.7% versus 0.6%; RR, 1.24; 95% CI, 0.76–2.04). The studies were homogeneous ( I 2 =0.0%) for all calculated pooled associations except for the RR of death in the interrupted strategy ( I 2 =26.3%). Conclusions: The short-term safety and efficacy of DOACs and warfarin are not different in patients with nonvalvular atrial fibrillation periprocedurally. Under an uninterrupted anticoagulation strategy, DOACs are associated with a 38% lower risk of major bleeding compared with warfarin.

Supplemental Digital Content is available in the text., OBJECTIVES: Pediatric Index of Mortality 3 is a validated tool including 11 variables for the assessment of mortality risk in PICU patients. With the recent advances in explainable machine learning algorithms, we aimed to assess feasibility of application of these machine learning models to simplify the Pediatric Index of Mortality 3 scoring system in order to decrease time and labor required for data collection and entry for Pediatric Index of Mortality 3. DESIGN: Single-center, retrospective cohort study. Data from the Virtual Pediatric Systems for patients admitted to Cleveland Clinic Children`s PICU between January 2008 and December 2019 was obtained. Light Gradient Boosting Machine Regressor (a gradient boosting decision tree algorithm) was used for building the machine learning models. Variable importance was analyzed by SHapley Additive exPlanations. All of the 11 Pediatric Index of Mortality 3 variables were used as input variables in the machine learning models to predict Pediatric Index of Mortality 3 risk of mortality as the outcome variable. Mean absolute error, root mean squared error, and R-squared were calculated for each of the 11 machine learning models as model performance parameters. SETTING: Quaternary children’s hospital. PATIENTS: PICU patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Five-thousand sixty-eight patients were analyzed. The machine learning models were able to maintain similar predictive error until the number of input variables decreased to four. The machine learning model with five input variables (mechanical ventilation in the first hour of PICU admission, very-high-risk diagnosis, surgical recovery from a noncardiac procedure, low-risk diagnosis, and base excess) produced lowest mean root mean squared error of 1.49 (95% CI, 1.05–1.93) and highest R-squared of 0.73 (95% CI, 0.6–0.86) with mean absolute error of 0.43 (95% CI, 0.35–0.5) among all the 11 machine learning models. CONCLUSIONS: Explainable machine learning methods were feasible in simplifying the Pediatric Index of Mortality 3 scoring system with similar risk of mortality predictions compared to the original Pediatric Index of Mortality 3 model tested in a single-center dataset.

INTRODUCTION AND OBJECTIVE:The development of immunotherapies (IOs) for the treatment of bladder cancer in first and second-line increased the economic burden of this disease. We aimed to use an ec...

The giant protein titin forms a unique filament network in cardiomyocytes, which engages in both mechanical and signaling functions of the heart. TTN , which encodes titin, is also a major human disease gene. In this review, we cover the roles of cardiac titin in normal and failing hearts, with a special emphasis on the contribution of titin to diastolic stiffness. We provide an update on disease-associated titin mutations in cardiac and skeletal muscles and summarize what is known about the impact of protein–protein interactions on titin properties and functions. We discuss the importance of titin-isoform shifts and titin phosphorylation, as well as titin modifications related to oxidative stress, in adjusting the diastolic stiffness of the healthy and the failing heart. Along the way we distinguish among titin alterations in systolic and in diastolic heart failure and ponder the evidence for titin stiffness as a potential target for pharmacological intervention in heart disease.

High myocardial diastolic stiffness has usually been attributed to excessive myocardial collagen deposition. Over the last decennium, stiff cardiomyocytes were also identified as important contributors to high myocardial diastolic stiffness, especially in heart failure (HF) with preserved ejection fraction (HFPEF).1–3 Cardiomyocyte stiffness relates to elasticity of the giant cytoskeletal protein titin, which spans the sarcomere from the Z disk to the M line and functions as a bidirectional spring responsible for early diastolic recoil and late diastolic distensibility of cardiomyocytes.4 In HFPEF patients and in HFPEF animal models,5 the observed increase in cardiomyocyte stiffness was always accompanied by increased deposition of collagen; therefore, it remained unclear whether impaired elasticity of titin could be solely responsible for high myocardial diastolic stiffness and HFPEF. In this issue of Circulation , however, Chung et al6 provide compelling evidence for titin being the sole perpetrator in the diastolic left ventricular (LV) dysfunction of an HFPEF mouse model. They generated mice with a deletion of nine immunoglobulin (Ig)-like domains from the proximal tandem Ig segment of the titin spring region (IG KO). This deletion extended the remaining titin spring segments and increased overall titin stiffness. Despite unaltered myocardial collagen content or composition, the IG KO mice developed HFPEF, evident from a reduced exercise tolerance, an enlarged left atrium, and a steeper LV end-diastolic pressure-volume relationship. The elegant study by Chung et al therefore clearly establishes myocardial titin to be able to sufficiently compromise diastolic LV function to induce HFPEF. Article see p 19 Titin, with a molecular mass of up to 3800 kDa, spans half-sarcomeres from the Z disk to the M band and contains a molecular spring segment, the I-band region, that supports early diastolic recoil and late diastolic resistance to stretch (Figure 1A). The I-band region has …

Rationale: Myocardial diastolic stiffness and cardiomyocyte passive force (F passive ) depend in part on titin isoform composition and phosphorylation. Ca 2+ /calmodulin-dependent protein kinase-II (CaMKII) phosphorylates ion channels, Ca 2+ -handling proteins, and chromatin-modifying enzymes in the heart, but has not been known to target titin. Objective: To elucidate whether CaMKII phosphorylates titin and modulates F passive in normal and failing myocardium. Methods and Results: Titin phosphorylation was assessed in CaMKIIδ/γ double-knockout (DKO) mouse, transgenic CaMKIIδC-overexpressing mouse, and human hearts, by Pro-Q-Diamond/Sypro-Ruby staining, autoradiography, and immunoblotting using phosphoserine-specific titin-antibodies. CaMKII-dependent site-specific titin phosphorylation was quantified in vivo by mass spectrometry using s table isotope labeling by amino acids in cell culture mouse heart mixed with wild-type (WT) or DKO heart. F passive of single permeabilized cardiomyocytes was recorded before and after CaMKII-administration. All-titin phosphorylation was reduced by >50% in DKO but increased by up to ≈100% in transgenic versus WT hearts. Conserved CaMKII-dependent phosphosites were identified within the PEVK-domain of titin by quantitative mass spectrometry and confirmed in recombinant human PEVK-fragments. CaMKII also phosphorylated the cardiac titin N2B-unique sequence. Phosphorylation at specific PEVK/titin N2B-unique sequence sites was decreased in DKO and amplified in transgenic versus WT hearts. F passive was elevated in DKO and reduced in transgenic compared with WT cardiomyocytes. CaMKII-administration lowered F passive of WT and DKO cardiomyocytes, an effect blunted by titin antibody pretreatment. Human end-stage failing hearts revealed higher CaMKII expression/activity and phosphorylation at PEVK/titin N2B-unique sequence sites than nonfailing donor hearts. Conclusions: CaMKII phosphorylates the titin springs at conserved serines/threonines, thereby lowering F passive . Deranged CaMKII-dependent titin phosphorylation occurs in heart failure and contributes to altered diastolic stress.

Background— In vitro studies suggest that phosphorylation of titin reduces myocyte/myofiber stiffness. Titin can be phosphorylated by cGMP-activated protein kinase. Intracellular cGMP production is stimulated by B-type natriuretic peptide (BNP) and degraded by phosphodiesterases, including phosphodiesterase-5A. We hypothesized that a phosphodiesterase-5A inhibitor (sildenafil) alone or in combination with BNP would increase left ventricular diastolic distensibility by phosphorylating titin. Methods and Results— Eight elderly dogs with experimental hypertension and 4 young normal dogs underwent measurement of the end-diastolic pressure-volume relationship during caval occlusion at baseline, after sildenafil, and BNP infusion. To assess diastolic distensibility independently of load/extrinsic forces, the end-diastolic volume at a common end-diastolic pressure on the sequential end-diastolic pressure-volume relationships was measured (left ventricular capacitance). In a separate group of dogs (n=7 old hypertensive and 7 young normal), serial full-thickness left ventricular biopsies were harvested from the beating heart during identical infusions to measure myofilament protein phosphorylation. Plasma cGMP increased with sildenafil and further with BNP (7.31±2.37 to 26.9±10.3 to 70.3±8.1 pmol/mL; P P Conclusion— Short-term cGMP-enhancing treatment with sildenafil and BNP improves left ventricular diastolic distensibility in vivo, in part by phosphorylating titin.

Background— Aortic stenosis (AS) and diabetes mellitus (DM) are frequent comorbidities in aging populations. In heart failure, DM worsens diastolic left ventricular (LV) dysfunction, thereby adversely affecting symptoms and prognosis. Effects of DM on diastolic LV function were therefore assessed in aortic stenosis, and underlying myocardial mechanisms were identified. Methods and Results— Patients referred for aortic valve replacement were subdivided into patients with AS and no DM (AS; n=46) and patients with AS and DM (AS-DM; n=16). Preoperative Doppler echocardiography and hemodynamics were implemented with perioperative LV biopsies. Histomorphometry and immunohistochemistry quantified myocardial collagen volume fraction and myocardial advanced glycation end product deposition. Isolated cardiomyocytes were stretched to 2.2-μm sarcomere length to measure resting tension (F passive ). Expression and phosphorylation of titin isoforms were analyzed with gel electrophoresis with ProQ Diamond and SYPRO Ruby stains. Reduced LV end-diastolic distensibility in AS-DM was evident from higher LV end-diastolic pressure (21±1 mm Hg for AS versus 28±4 mm Hg for AS-DM; P =0.04) at comparable LV end-diastolic volume index and attributed to higher myocardial collagen volume fraction (AS, 12.9±1.1% versus AS-DM, 18.2±2.6%; P 2 versus AS-DM, 31.4±6.1 score per 1 mm 2 ; P =0.03), and higher F passive (AS, 3.5±1.7 kN/m 2 versus AS-DM, 5.1±0.7 kN/m 2 ; P =0.04). Significant hypophosphorylation of the stiff N2B titin isoform in AS-DM explained the higher F passive and normalization of F passive after in vitro treatment with protein kinase A. Conclusions— Worse diastolic LV dysfunction in AS-DM predisposes to heart failure and results from more myocardial fibrosis, more intramyocardial vascular advanced glycation end product deposition, and higher cardiomyocyte F passive , which was related to hypophosphorylation of the N2B titin isoform.

Oseltamivir, an orally bioavailable neuraminidase inhibitor, is licensed in the United States for treatment and chemoprophylaxis of influenza in children ≥1 year of age.1 Another neuraminidase inhibitor, zanamivir, also is licensed for use in children ≥7 years of age for treatment and ≥5 years of age for chemoprophylaxis.1 It is delivered via an inhaler device that is difficult for younger children to use. In a prospective, randomized, blinded, placebo-controlled study in influenza-infected children 1 to 12 years of age, a 5-day treatment course of oseltamivir was associated with a median reduction in overall clinical illness of 36 hours and a reduction in fever of 25 hours.2 Additionally, the incidence of acute otitis media was reduced by 44% compared with placebo recipients. A significant decrease in viral shedding was also noted in treated children, with few treated children still shedding virus on day 4 of therapy. The most common adverse drug effect was vomiting (14% of oseltamivir-treated subjects vs. 8% of placebo recipients).2 The safety of oseltamivir therapy in infants younger than 12 months of age is a potential concern. The oseltamivir package insert states that use of oseltamivir in this age group is not indicated,3 based on preclinical findings in juvenile rats.4 A single 1000 mg/kg dose of oseltamivir (about 250 times the recommended dose in children) in 7-day-old rats resulted in excess deaths in association with unusually high exposure to both the active metabolite of oseltamivir and the prodrug. Concentrations of the prodrug in juveniles were approximately 1500 times greater than those seen in adult animals, suggesting that these adverse outcomes may be related to an immature blood-brain barrier or to immaturity of conversion of prodrug to active metabolite in juvenile animals. While the applicability of these rat studies to human infants is questionable due to significant differences in oseltamivir metabolism between the 2 species and the extremely high doses employed in the animal trials, caution is warranted until appropriate pediatric studies can be conducted. Reassuringly, a retrospective study of infants younger than 1 year of age in Japan did not find an association between use of oseltamivir and either mortality or encephalopathy.5 Despite the wording of the package insert, off-label use of oseltamivir in infants less than 12 months of age occurs in the United States, particularly in infants who are seriously ill or considered to be at high risk of complications from influenza infection or in those who present with severe disease. To assess potential neurologic adverse effects of oseltamivir in infants, a retrospective chart review was performed in North America by the National Institute of Allergy and Infectious Diseases (NIAID) Collaborative Antiviral Study Group (CASG) of infants less than 12 months of age who received oseltamivir compared with either of the adamantanes (amantadine or rimantadine).

Titin is the main determinant of myocardial passive tension and its distensibility is increased via phosphorylation by protein kinase G (PKG), activated by nitric oxide (NO) and natriuretic peptides (NP) upon acute overload. We hypothesized whether myocardial stretch led to decreased stiffness, optimizing diastolic filling along with the usual increase in contractility. Intact rat Langendorff hearts, strips dissected from the LV or right atrium of cardiac surgery patients and rabbit papillary muscles were acutely stretched for 15min. Passive tension (PT) was measured in skinned cardiomyocytes extracted from the LV of control and stretched rat hearts for sarcomere lengths (SL) 1.8-2.3μm before and after incubation with PKG. Rabbit muscles were incubated with a PKG inhibitor or, simultaneously, a NO synthase inhibitor, a NO scavenger, a NP receptor A antagonist. All-total titin phosphorylation was stained with Pro-Q Diamond and indexed to total-protein signals using SYPRO Ruby. Values are given as mean±SEM and statistical significance was set to p After acute stretch there was a progressive decrease in passive tension/diastolic pressure over 15min: 27±8% and 27±6% in human atrium and ventricular muscles, respectively, and 43±2% in rabbit papillary muscles and isolated hearts. This decrease in myocardial stiffness was significantly blunted by PKG inhibition (40%) and NO/NP pathway inhibition (29%). PT of cardiomyocytes was significantly lower (≈60%) in the previously stretched group for all SL. A similar effect, only significant in the control group, was observed after incubation with PKG. Titin phosphorylation increased markedly 15 minutes after acute myocardial stretch in human (atrial: 11±1% vs 41±8%; LV: 27±8% vs 71±21%) and rabbit (13±2% vs 23±3%) myocardium. The progressive decrease in myocardial stiffness after acute hemodynamic overload is preserved at the myofilamental level and seems to depend on PKG activity, representing a potential therapeutic target in patients with pathologically rigid myocardium. Moreover, blocking PKG activation seems to attenuate this adaptive diastolic response. Therefore, titin phosphorylation by this kinase is probably involved in this new myocardial response to stretch in both animals and humans.

To compare the bacteriologic and clinical efficacy of amoxicillin/clavulanate and azithromycin in patients with acute otitis media (AOM), particularly the ability to eradicate the predominant AOM pathogens from middle ear fluid as assessed by mandatory second tympanocentesis.In this single blind study 238 infants and children with AOM were randomized to receive amoxicillin/clavulanate (45/6.4 mg/kg/day in two divided doses for 10 days) or azithromycin (10 mg/kg on Day 1, then 5 mg/kg daily on Days 2 through 5). Tympanocentesis was performed before the first dose and repeated on Day 4, 5 or 6. Clinical response was assessed at end of therapy between Days 12 and 14 and at follow-up between Days 22 and 28.Amoxicillin/clavulanate was significantly more likely to eradicate all bacterial pathogens [83% (54 of 65) vs. 49% (35 of 71), P = 0.001] and Haemophilus influenzae [87% (26 of 30) vs. 39% (13 of 33), P = 0.0001] from middle ear fluid than was azithromycin. Amoxicillin/clavulanate was also more likely to eradicate Streptococcus pneumoniae, but the difference was not statistically significant [90% (18 of 20) vs. 68% (13 of 19) [corrected], P = 0.095]. On Days 12 to 14, signs and symptoms were more likely to resolve completely or improve in all culture-positive patients [86% (60 of 70) vs. 70% (51 of 73), P = 0.023] and in those with H. influenzae infections [91% (30 of 33) vs. 65% (22 of 34), P = 0.010] who received amoxicillin/clavulanate compared with those who received azithromycin. Otherwise there were no significant differences between groups in clinical outcomes on Days 12 to 14 or at follow-up.Our findings indicate that amoxicillin/clavulanate has superior bacteriologic and clinical efficacy compared with azithromycin in children with AOM.

We present a case report of a term neonate with congenital human immunodeficiency virus (HIV) infection born with nonimmune hydrops fetalis who developed hepatitis shortly after birth. Maternal HIV infection was diagnosed after delivery. An extensive evaluation for known causes of nonimmune hydrops, both infectious and noninfectious, was negative. After beginning highly active antiretroviral therapy, hepatitis resolved and the HIV viral load became undetectable. We believe this is the first report of nonimmune hydrops fetalis and hepatitis in an infant with congenital HIV infection.

Introduction: Small heat-shock proteins (sHSPs) generally participate in cellular protein quality-control mechanisms. They are abundant in cardiomyocytes where they bind under diverse stress conditions preferentially to myofilament proteins. The functional role of this association is unclear. Hypothesis: HSP27 and αB-crystallin bind to human cardiac titin spring elements and exert a protective effect on cardiomyocyte passive stiffness under stress conditions. Methods: Binding of sHSPs to recombinant human titin constructs was characterized by GST-pulldown assay and confirmed by immunofluorescence staining of human donor and failing (DCM) cardiomyocytes. Sedimentation-velocity centrifugation, photometric and chromatographic methods were used to test for titin aggregation and protection from it by sHSPs. Passive force was measured in isolated human cardiomyocytes or single myofibrils, in search for a possible protective effect of sHSPs on mechanical function. Results: HSP27 interacted with distinct domains of the human titin-spring region in vitro and in cardiomyocytes, and independent of the presence of actin filaments in the sarcomeres. The binding sites on the elastic titin segment resemble those for αB-crystallin and include proximal Ig-domains, the N2-B and N2-A regions, but not the PEVK-domain. In-vitro assays revealed a monomeric organization of these titin-spring elements; however, unfolded N2-A domain (mainly composed of Ig-domains) aggregated in pH-6.6 buffer but not in normal-pH buffer, whereas αB-crystallin protected from this effect. The intrinsically disordered N2-Bus titin domain did not aggregate. Single skinned human cardiomyocytes showed greatly increased passive stiffness when pre-stretched under acidic stress (pH 6.6), but αB-crystallin or HSP27 corrected the stiffening. In failing patient heart tissue, both HSP27 and αB-crystallin frequently associated with the elastic I-band region, in contrast to a cytosolic and Z-disk location of these sHSPs in donor heart. Conclusions: In cardiomyocytes sHSPs bind to mechanically active titin domains under stress conditions such as intracellular acidosis ( e.g. , ischemia), protecting the titin springs from aggregation and helping reverse diastolic stiffening.

To the Editor: AMP kinase (AMPK) activation represents a unique molecular event in cardioprotection against ischemia/reperfusion injury, potentially linking myocardial metabolism to a number of cardioprotective signaling pathways such as nitric oxide and reactive oxygen species signaling. In a recently published article in Circulation , Lamberts et al1 demonstrated that AMPK activation contributed to the signaling cascade in the powerful cardioprotection conferred by sevoflurane-induced preconditioning. This finding is intriguing because AMPK activation has the potential to overcome the loss of anesthetic-induced preconditioning during diabetes mellitus.2 The published report provides important information and useful insights, particularly regarding the triangle of AMPK signaling, reactive oxygen species generation, …

The myocardium possesses intrinsic protective mechanisms against ischemia and reperfusion injury (I/R). 5′AMP-activated protein kinase (AMPK) is known as regulator of cellular energy status and is reduced during diabetes mellitus. Recently, AMPK is also implicated in ischemic preconditioning leading to cardioprotection against I/R. We hypothesize that AMPK is involved in anesthetic-induced cardioprotection and that this AMPK activation is evoked by production of reactive oxygen species (ROS). Isolated Langendorff-perfused rat hearts were subjected to 35 minutes of global ischemia followed by 120 minutes of reperfusion. Hearts were divided into 2 groups: a Control group and a Sevo group receiving three times 5-minute episodes of 2.5 vol% sevoflurane before I/R. AMPK phosphorylation was determined with Western Blot analysis. Cardioprotection was assessed as recovery of left ventricular pressure after I/R and as infarct size using triphenyltetrazolium chloride staining. In the Control group, I/R resulted in a twofold increase in phosphorylation levels of AMPK (Control 1.0 ± 0.1 vs. Control-I/R: 2.3 ± 0.1 a.u., n = 4, p < 0.05). Sevoflurane preconditioning did not immediately, prior to ischemia, affect the AMPK phosphorylation (Sevo 0.9 ± 0.2 vs. Control 1.0 ± 0.2, n = 6), but doubled the increase in AMPK phosphorylation to control after ischemia (Sevo-I: 2.0 ± 0.5 (vs. Control-I), n = 6, p < 0.05), as well as after I/R (Sevo-I/R: 2.1 ± 0.3 vs. (Control-I/R), n = 6, p < 0.05). The AMPK-inhibitor compound C (1 and 10 μM) reduced the increase in AMPK phosphorylation and abolished the cardioprotection derived from functional recovery and infarct size. In addition, the ROS-scavenger n-(2-mercaptopropionyl)-glycine (MPG, 1mM) also reduced the sevoflurane-mediated increase in AMPK phosphorylation and completely prevented cardioprotection. These results demonstrate for the first time a direct link between AMPK activation and the production of ROS in cardioprotection. We conclude that anesthetic-induced AMPK activation protects the heart against I/R and relies on production of ROS, which might be especially important in the context of impaired cardioprotection in the diabetic myocardium.

Concentric left ventricular (LV) remodeling and cardiomyocyte (CM) hypertrophy characterize heart failure with normal ejection fraction (HFNEF) whereas eccentric LV remodeling and low myofilamentary density characterize heart failure with reduced ejection fraction (HFREF). In rodents, low myocardial protein kinase G (PKG) activity has been implicated in the development of pathological CM hypertrophy induced by LV pressure-overload. The present study therefore compared LV remodeling, CM hypertrophy and myocardial PKG activity in patients (pts) with HFNEF (n=36) and HFREF (n=43). All pts had been admitted to hospital for worsening heart failure (NYHA 3–4) and were free of coronary artery disease. HFNEF pts had a LVEF > 50% and a LV end-diastolic pressure > 16 mmHg. HFREF pts had a LVEF ). As evident from (P)-VASP/VASP ratio, PKG activity was lower in HFNEF than in HFREF (Table ). Conclusion: HFNEF patients have lower myocardial PKG activity than HFREF patients. This low myocardial PKG activity could contribute to CM hypertrophy and concentric LV remodeling observed in HFNEF. Raising myocardial PKG activity by phosphodiesterase 5A inhibition could be useful to limit CM hypertrophy and concentric LV remodeling in HFNEF.

Raised diastolic left ventricular (LV) stiffness importantly contributes to heart failure (HF) in diabetes mellitus (DM) and results from myocardial deposition of advanced glycation endproducts (AGEs), interstitial fibrosis, cardiomyocyte hypertrophy and elevated diastolic resting tension (F passive ) of cardiomyocytes. The relative importance of these mechanisms for diastolic LV stiffness differs between HF with reduced LV ejection fraction (EF) (HFrEF) and HF with normal LV EF (HFnEF) as evident from more fibrosis in HFrEF and more hypertrophy and higher F passive in HFnEF. The present study therefore compared myocardial effects of β-blocker therapy in DM patients suffering of HFrEF (n=11) or of HFnEF (n=15). All patients had DM type 2 and were free of significant coronary artery disease. LV endomyocardial biopsies were used to assess AGEs deposition by carboxymethyllysine immunohistochemistry and collagen volume fraction (CVF) or cardiomyocyte diameter (MyD) by histomorphometry. Cardiomyocytes were also isolated from the biopsies, attached to a force transducer and stretched to 2.2 μm to measure F passive . AGEs deposition was expressed relative to an age and sex matched control group without DM. In both HFrEF and HFnEF groups, β-blocker therapy reduced DM-related myocardial AGEs deposition. In both HFrEF and HFnEF groups, β-blocker therapy also reduced MyD. In HFnEF, β-blocker therapy lowered CVF but raised F passive of cardiomyocytes. Conclusion: Myocardial effects of β-blocker therapy were uniformly beneficial for diastolic LV stiffness in DM patients suffering of HFrEF but not in DM patients suffering of HFnEF. In DM, HF phenotype could therefore affect outcome of β-blocker therapy.