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Maintenance of protein quality control is a critical function of the ubiquitin proteasome system (UPS). Evidence is rapidly mounting to link proteasome dysfunction with a multitude of cardiac diseases, including ischemia, reperfusion, atherosclerosis, hypertrophy, heart failure, and cardiomyopathies. Recent studies have demonstrated a remarkable level of complexity in the regulation of the UPS in the heart and suggest that our understanding of how UPS dysfunction might contribute to the pathophysiology of such a wide range of cardiac afflictions is still very limited. Whereas experimental systems, including animal models, are invaluable for exploring mechanisms and establishing pathogenicity of UPS dysfunction in cardiac disease, studies using human heart tissue provide a vital adjunct for establishing clinical relevance of experimental findings and promoting new hypotheses. Accordingly, this review will focus on UPS dysfunction in human dilated and hypertrophic cardiomyopathies and highlight areas rich for further study in this expanding field.

Background: This study examined the hypothesis that 26S proteasome dysfunction in human end-stage heart failure is associated with decreased docking of the 19S regulatory particle to the 20S proteasome. Previous studies have demonstrated that 26S proteasome activity is diminished in human end-stage heart failure associated with oxidation of the 19S regulatory particle Rpt5 subunit. Docking of the 19S regulatory particle to the 20S proteasome requires functional Rpt subunit ATPase activity and phosphorylation of the α-type subunits., Methods and Results: An enriched proteasome fraction was prepared from 7 human nonfailing and 10 failing heart explants. Native gel electrophoresis assessed docking of 19S to 20S proteasome revealing 3 proteasome populations (20S, 26S, and 30S proteasomes). In failing hearts, 30S proteasomes were significantly lower (P=0.048) by 37% suggesting diminished docking. Mass spectrometry-based phosphopeptide analysis demonstrated that the relative ratio of phosphorylated:non phosphorylated α7 subunit (serine250) of the 20S proteasome was significantly less (P=0.011) by almost 80% in failing hearts. Rpt ATPase activity was determined in the enriched fraction and after immunoprecipitation with an Rpt6 antibody. ATPase activity (ρmol PO4/μg protein per hour) of the total fraction was lowered from 291±97 to 194±27 and in the immunoprecipitated fraction from 42±12 to 3±2 (P=0.005) in failing hearts., Conclusions: These studies suggest that diminished 26S activity in failing human hearts may be related to impaired docking of the 19S to the 20S as a result of decreased Rpt subunit ATPase activity and α7 subunit phosphorylation.

Objectives: The aim of this study was to determine the efficacy of implantable cardioverter-defibrillators (ICDs) in children and adolescents with hypertrophic cardiomyopathy (HCM)., Background: HCM is the most common cause of sudden death in the young. The availability of ICDs over the past decade for HCM has demonstrated the potential for sudden death prevention, predominantly in adult patients., Methods: A multicenter international registry of ICDs implanted (1987 to 2011) in 224 unrelated children and adolescents with HCM judged at high risk for sudden death was assembled. Patients received ICDs for primary (n = 188) or secondary (n = 36) prevention after undergoing evaluation at 22 referral and nonreferral institutions in the United States, Canada, Europe, and Australia., Results: Defibrillators were activated appropriately to terminate ventricular tachycardia or ventricular fibrillation in 43 of 224 patients (19%) over a mean of 4.3 ± 3.3 years. ICD intervention rates were 4.5% per year overall, 14.0% per year for secondary prevention after cardiac arrest, and 3.1% per year for primary prevention on the basis of risk factors (5-year cumulative probability 17%). The mean time from implantation to first appropriate discharge was 2.9 ± 2.7 years (range to 8.6 years). The primary prevention discharge rate terminating ventricular tachycardia or ventricular fibrillation was the same in patients who underwent implantation for 1, 2, or ≥3 risk factors (12 of 88 [14%], 10 of 71 [14%], and 4 of 29 [14%], respectively, p = 1.00). Extreme left ventricular hypertrophy was the most common risk factor present (alone or in combination with other markers) in patients experiencing primary prevention interventions (17 of 26 [65%]). ICD-related complications, particularly inappropriate shocks and lead malfunction, occurred in 91 patients (41%) at 17 ± 5 years of age., Conclusions: In a high-risk pediatric HCM cohort, ICD interventions terminating life-threatening ventricular tachyarrhythmias were frequent. Extreme left ventricular hypertrophy was most frequently associated with appropriate interventions. The rate of device complications adds a measure of complexity to ICD decisions in this age group., (Copyright © 2013 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

The clinical expression of hypertrophic cardiomyopathy (HC) is undoubtedly influenced by modifying genetic and environmental factors. Lifestyle practices such as tobacco and alcohol use, poor nutritional intake, and physical inactivity are strongly associated with adverse cardiovascular outcomes and increased mortality in the general population. Before addressing the direct effect of such modifiable factors on the natural history of HC, it is critical to define their prevalence in this population. A voluntary survey, drawing questions in part from the 2007 to 2008 National Health and Nutrition Examination Survey (NHANES), was posted on the HC Association website and administered to patients with HC at the University of Michigan. Propensity score matching to NHANES participants was used. Dichotomous and continuous health behaviors were analyzed using logistic and linear regression, respectively, and adjusted for body mass index and propensity score quintile. Compared to the matched NHANES participants, the patients with HC reported significantly less alcohol and tobacco use but also less time engaged in physical activity at work and for leisure. Time spent participating in vigorous or moderate activity was a strong predictor of self-reported exercise capacity. The body mass index was greater in the HC cohort than in the NHANES cohort. Exercise restrictions negatively affected emotional well-being in most surveyed subjects. In conclusion, patients with HC are less active than the general United States population. The well-established relation of inactivity, obesity, and cardiovascular mortality might be exaggerated in patients with HC. More data are needed on exercise in those with HC to strike a balance between acute risks and the long-term health benefits of exercise., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Elevated protein kinase C βII (PKCβII) expression develops during heart failure and yet the role of this isoform in modulating contractile function remains controversial. The present study examines the impact of agonist-induced PKCβII activation on contractile function in adult cardiac myocytes. Diminished contractile function develops in response to low dose phenylephrine (PHE, 100 nM) in controls, while function is preserved in response to PHE in PKCβII-expressing myocytes. PHE also caused PKCβII translocation and a punctate distribution pattern in myocytes expressing this isoform. The preserved contractile function and translocation responses to PHE are blocked by the inhibitor, LY379196 (30 nM) in PKCβII-expressing myocytes. Further analysis showed downstream protein kinase D (PKD) phosphorylation and phosphatase activation are associated with the LY379196-sensitive contractile response. PHE also triggered a complex pattern of end-target phosphorylation in PKCβII-expressing myocytes. These patterns are consistent with bifurcated activation of downstream signaling activity by PKCβII.

Rationale: Kv1.5 (KCNA5) is expressed in the heart, where it underlies the I(Kur) current that controls atrial repolarization, and in the pulmonary vasculature, where it regulates vessel contractility in response to changes in oxygen tension. Atrial fibrillation and hypoxic pulmonary hypertension are characterized by downregulation of Kv1.5 protein expression, as well as with oxidative stress. Formation of sulfenic acid on cysteine residues of proteins is an important, dynamic mechanism for protein regulation under oxidative stress. Kv1.5 is widely reported to be redox-sensitive, and the channel possesses 6 potentially redox-sensitive intracellular cysteines. We therefore hypothesized that sulfenic acid modification of the channel itself may regulate Kv1.5 in response to oxidative stress., Objective: To investigate how oxidative stress, via redox-sensitive modification of the channel with sulfenic acid, regulates trafficking and expression of Kv1.5., Methods and Results: Labeling studies with the sulfenic acid-specific probe DAz and horseradish peroxidase-streptavidin Western blotting demonstrated a global increase in sulfenic acid-modified proteins in human patients with atrial fibrillation, as well as sulfenic acid modification to Kv1.5 in the heart. Further studies showed that Kv1.5 is modified with sulfenic acid on a single COOH-terminal cysteine (C581), and the level of sulfenic acid increases in response to oxidant exposure. Using live-cell immunofluorescence and whole-cell voltage-clamping, we found that modification of this cysteine is necessary and sufficient to reduce channel surface expression, promote its internalization, and block channel recycling back to the cell surface. Moreover, Western blotting demonstrated that sulfenic acid modification is a trigger for channel degradation under prolonged oxidative stress., Conclusions: Sulfenic acid modification to proteins, which is elevated in diseased human heart, regulates Kv1.5 channel surface expression and stability under oxidative stress and diverts channel from a recycling pathway to degradation. This provides a molecular mechanism linking oxidative stress and downregulation of channel expression observed in cardiovascular diseases.

Significant up-regulation of the protein kinase Cβ(II) (PKCβ(II)) develops during heart failure and yet divergent functional outcomes are reported in animal models. The goal here is to investigate PKCβ(II) modulation of contractile function and gain insights into downstream targets in adult cardiac myocytes. Increased PKCβ(II) protein expression and phosphorylation developed after gene transfer into adult myocytes while expression remained undetectable in controls. The PKCβ(II) was distributed in a peri-nuclear pattern and this expression resulted in diminished rates and amplitude of shortening and re-lengthening compared to controls and myocytes expressing dominant negative PKCβ(II) (PKCβDN). Similar decreases were observed in the Ca(2+) transient and the Ca(2+) decay rate slowed in response to caffeine in PKCβ(II)-expressing myocytes. Parallel phosphorylation studies indicated PKCβ(II) targets phosphatase activity to reduce phospholamban (PLB) phosphorylation at residue Thr17 (pThr17-PLB). The PKCβ inhibitor, LY379196 (LY) restored pThr17-PLB to control levels. In contrast, myofilament protein phosphorylation was enhanced by PKCβ(II) expression, and individually, LY and the phosphatase inhibitor, calyculin A each failed to block this response. Further work showed PKCβ(II) increased Ca(2+)-activated, calmodulin-dependent kinase IIδ (CaMKIIδ) expression and enhanced both CaMKIIδ and protein kinase D (PKD) phosphorylation. Phosphorylation of both signaling targets also was resistant to acute inhibition by LY. These later results provide evidence PKCβ(II) modulates contractile function via intermediate downstream pathway(s) in cardiac myocytes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The myocardial extracellular matrix (ECM), an interwoven meshwork of proteins, glycoproteins, proteoglycans, and glycosaminoglycans that is dominated by polymeric fibrils of type I collagen, serves as the mechanical scaffold on which myocytes are arrayed for coordinated and synergistic force transduction. Following ischemic injury, cardiac ECM remodeling is initiated via localized proteolysis, the bulk of which has been assigned to matrix metalloproteinase (MMP) family members. Nevertheless, the key effector(s) of myocardial type I collagenolysis both in vitro and in vivo have remained unidentified. In this study, using cardiac explants from mice deficient in each of the major type I collagenolytic MMPs, including MMP-13, MMP-8, MMP-2, MMP-9, or MT1-MMP, we identify the membrane-anchored MMP, MT1-MMP, as the dominant collagenase that is operative within myocardial tissues in vitro. Extending these observations to an in vivo setting, mice heterozygous for an MT1-MMP-null allele display a distinct survival advantage and retain myocardial function relative to wild-type littermates in an experimental model of myocardial infarction, effects associated with preservation of the myocardial type I collagen network as a consequence of the decreased collagenolytic potential of cardiac fibroblasts. This study identifies MT1-MMP as a key MMP responsible for effecting postinfarction cardiac ECM remodeling and cardiac dysfunction., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Histone H3 lysine 4 (H3K4me) methyltransferases and their cofactors are essential for embryonic development and the establishment of gene expression patterns in a cell-specific and heritable manner. However, the importance of such epigenetic marks in maintaining gene expression in adults and in initiating human disease is unclear. Here, we addressed this question using a mouse model in which we could inducibly ablate PAX interacting (with transcription-activation domain) protein 1 (PTIP), a key component of the H3K4me complex, in cardiac cells. Reducing H3K4me3 marks in differentiated cardiomyocytes was sufficient to alter gene expression profiles. One gene regulated by H3K4me3 was Kv channel-interacting protein 2 (Kcnip2), which regulates a cardiac repolarization current that is downregulated in heart failure and functions in arrhythmogenesis. This regulation led to a decreased sodium current and action potential upstroke velocity and significantly prolonged action potential duration (APD). The prolonged APD augmented intracellular calcium and in vivo systolic heart function. Treatment with isoproterenol and caffeine in this mouse model resulted in the generation of premature ventricular beats, a harbinger of lethal ventricular arrhythmias. These results suggest that the maintenance of H3K4me3 marks is necessary for the stability of a transcriptional program in differentiated cells and point to an essential function for H3K4me3 epigenetic marks in cellular homeostasis.

Introduction: It has been nearly 20 yr since the first published astronaut mortality analysis. Using astronaut vital data and general population mortality rates, we calculate Standardized Mortality Ratios (SMR) for both total and specific causes of death among astronauts between January 1980 and June 2009 to look for changes in mortality patterns over time., Methods: Astronaut vital data were derived from the Johnson Space Center website and the Astronaut Fact Book. General population mortality rates were taken from the Human Mortality Database and the Centers for Disease Control. SMR were computed as the ratio of observed deaths to expected deaths using indirect standardization to several comparison populations., Results: All SMR declined from the 1980s to the 2000s, though astronauts are still at increased risk of accidental death (SMR = 574, 95% C.I. 335-919). Astronauts are at greatly reduced risk of death by cardiovascular disease (SMR = 27, 95% C.I. 9-63) and cancer (SMR = 47, 95% C.I. 19-97), and astronauts are now at decreased risk of all-cause mortality compared with the general population., Discussion: The SMR show that mortality from circulatory disease, cancer, and accidents have all declined from previous estimates, though astronauts are still at increased risk of accidental death. Improvements in circulatory disease mortality are likely due to intensive health screening and physical fitness within the Astronaut Corps. Similarly, physical fitness may be contributing to the reduction in cancer mortality. Fewer airplane crashes have contributed to the decreased risk of fatal accidents, which in turn is driving the reduction in all-cause mortality risk.

Aim: To determine whether intrathecal baclofen (ITB) changes mortality risk in persons with cerebral palsy (CP)., Method: Records were reviewed for all persons with CP who were managed with ITB for hypertonicity at a specialty hospital in Minnesota between May 1993 and August 2007. A comparison cohort was randomly selected from clients of the California Department of Developmental Services who were initially evaluated between 1987 and 1990 and were matched to those with ITB for age, sex, Gross Motor Function Classification System (GMFCS) level, presence or absence of epilepsy, and feeding-tube use. Survival probabilities were estimated using the Kaplan-Meier method, and differences were tested via log-rank., Results: Three hundred and fifty-nine persons with CP (202 males, 157 females) receiving ITB for hypertonicity (mean age 12y 8mo, SD 7y 9mo, range 3y 1mo to 39y 9mo) were matched to 349 persons without ITB pumps (195 males, 154 females; mean age 12y 7mo, SD 8y 4mo, range 2y 7mo to 40y). The proportion of patients at different GMFCS levels in the ITB and in the non-ITB cohorts, respectively, was as follows: level II 3% and 3%, level III 16% and 16%, level IV 38% and 37%, and level V 43% and 44%. Survival at 8 years of follow-up was 92% (SD 1.9%) in the ITB cohort and 82% (SD 2.4%) in the non-ITB cohort (p<0.001). After adjustment to account for recent trends in improved survival in CP, 8-year survival in the non-ITB cohort was 88%, which was not significantly different from the ITB cohort (p=0.073)., Interpretation: ITB therapy does not increase mortality in individuals with CP and may suggest an increase in life expectancy.

Context: A recent cluster of sudden cardiac deaths in marathon runners has attracted considerable media attention and evoked concern over the safety of long-distance running and competition. This review discusses the acute and potential long-term risks associated with marathon running and puts these into perspective with the many health benefits afforded by habitual vigorous exercise., Evidence Acquisition: Data sources included peer-reviewed publications from 1979 to January 2010 as identified via PubMed and popular media., Results: Marathon running is associated with a transient and low risk of sudden cardiac death. This risk appears to be even lower in women and is independent of marathon experience or the presence of previously reported symptoms. Most deaths are due to underlying coronary artery disease. The value of preparticipation screening is limited by its insensitivity and impracticality of widespread implementation. Appropriate preparation and deployment of trained medical personnel and availability of automatic external defibrillators are expected to have a major impact on survival from cardiac arrests during marathons. Cardiac biochemical and functional abnormalities are commonly observed transiently following completion of a marathon, although their clinical significance is unknown., Conclusions: Sudden cardiac deaths associated with marathon running are exceedingly rare events. Prevention should focus on recognition and investigation of prodromal symptoms, if present, and access to rapid defibrillation and trained medical personnel. The robust association of endurance running with improved quality of life and longevity underscores the importance of putting risks into perspective with other well-established health benefits of regular vigorous exercise.

Context: An athlete's health may be endangered if he or she continues to compete after diagnosis of certain cardiovascular conditions. The most worrisome risk is sudden cardiac death; the annual rate in US athletes is 1 in 50 000 to 200 000., Evidence Acquisition: Part 2 of this review highlights the current guidelines and controversies surrounding compatibility of participation with a variety of cardiac conditions in competitive and recreational athletics. Data sources were limited to peer-reviewed publications from 1984 to the April 2009., Results: The guidelines published by the American College of Cardiology and the European Society of Cardiology provide a framework for safe competitive and recreational sports participation in athletes with a broad spectrum of inherited and acquired cardiovascular disorders. These guidelines are necessarily conservative because it is not currently possible to individualize risk prediction. Few data are available in many areas, particularly in the noncompetitive arena or in older athletes., Conclusions: Published national guidelines are currently the foundation governing return-to-play decisions in athletes with cardiovascular conditions. Further studies are needed to refine risk stratification algorithms to allow athletes with cardiovascular conditions to reap the health benefits of regular exercise and sports participation without undue risk.

The risk of sudden cardiac death (SCD) associated with vigorous athletic competition in individuals with hypertrophic cardiomyopathy (HCM) is well documented, and is the basis for recommended exclusion from competitive sports independent of other risk factors. Although SCD risk with recreational exercise is less well defined, published guidelines for participation in recreational sports, based on a consensus of expert opinion, offer a valuable framework for counseling patients. Exercise stress testing is an important diagnostic and prognostic tool in the evaluation of HCM patients, providing an objective measure of functional capacity, physiologic hemodynamic responses to stress, presence of ischemia, and provocable left ventricular outflow tract obstruction. The value of cardiopulmonary exercise testing in HCM is less well studied than in the heart failure population, but can be used to set a safe target for exercise intensity as part of an individualized exercise prescription. The long-term effects of exercise on HCM pathophysiology are largely theoretical at this stage. Potential harmful effects of fatiguing exercise include prolonged contractile dysfunction resulting from microvascular ischemia and energetic compromise. Conversely, several animal studies have shown that voluntary exercise prevents or reverses many pathologic features of HCM, including those related to apoptosis and energetics. Substantial evidence for health-promoting benefits of exercise in the general population, in addition to promising safety and efficacy data in patients with chronic heart failure, emphasizes the need to attain a reasonable balance between potential risks and benefits of aerobic fitness in individuals with HCM.

Context: Identification of potentially fatal cardiac conditions in otherwise healthy athletes presents a major challenge to the sports medicine community. The requirements for preparticipation screening vary among countries and even from state to state within the United States. The mandated use of an electrocardiogram as a screening implement has provoked international controversy., Evidence Acquisition: Part 1 of this review highlights the current guidelines and controversies surrounding cardiovascular screening, with a focus on the diagnostic challenges associated with identifying athletes with inheritable cardiomyopathies. Data sources were limited to peer-reviewed publications from 1984 to the present., Results: Preparticipation screening should include at least a history and a physical examination for all athletes, whereas use of an electrocardiogram is still controversial. Diagnosis of inherited cardiomyopathies presents unique challenges, particularly in hypertrophic cardiomyopathy, where many features can mimic those found in the "athlete's heart.", Conclusions: Recognizing cardiac conditions in athletes that can predispose them to sudden cardiac death or other adverse outcomes is of vital importance, as is the appropriate exclusion of these athletes from competition. Further studies are needed to determine the most efficient and cost-effective means of screening and to increase the sensitivity and specificity of diagnostic testing for inheritable cardiovascular diseases.

Aims: Contractile dysfunction associated with myocardial ischaemia is a significant cause of morbidity and mortality in the elderly. Strategies to protect the aged heart from ischaemia-mediated pump failure are needed. We hypothesized that troponin I-mediated augmentation of myofilament calcium sensitivity would protect cardiac function in aged mice., Methods and Results: To address this, we investigated transgenic (Tg) mice expressing a histidine-substituted form of adult cardiac troponin I (cTnI A164H), which increases myofilament calcium sensitivity in a pH-dependent manner. Serial echocardiography revealed that Tg hearts showed significantly improved systolic function at 4 months, which was sustained for 2 years based on ejection fraction and velocity of circumferential fibre shortening. Age-related diastolic dysfunction was also attenuated in Tg mice as assessed by Doppler measurements of the mitral valve inflow and lateral annulus Doppler tissue imaging. During acute hypoxia, cardiac contractility significantly improved in aged Tg mice made evident by increased stroke volume, end systolic pressure, and +dP/dt compared with non-transgenic mice., Conclusion: This study shows that increasing myofilament function by means of a pH-responsive histidine button engineered into cTnI results in enhanced baseline heart function in Tg mice over their lifetime, and during acute hypoxia improves survival in aged mice by maintaining cardiac contractility.

Relaxation abnormalities are prevalent in heart failure and contribute to clinical outcomes. Disruption of Ca2+ homeostasis in heart failure delays relaxation by prolonging the intracellular Ca2+ transient. We sought to speed cardiac relaxation in vivo by cardiac-directed transgene expression of parvalbumin (Parv), a cytosolic Ca2+ buffer normally expressed in fast-twitch skeletal muscle. A key feature of Parv's function resides in its Ca2+/Mg2+ binding affinities that account for delayed Ca2+ buffering in response to the intracellular Ca2+ transient. Cardiac Parv expression decreased sarcoplasmic reticulum Ca2+ content without otherwise altering intracellular Ca2+ homeostasis. At high physiological mouse heart rates in vivo, Parv modestly accelerated relaxation without affecting cardiac morphology or systolic function. Ex vivo pacing of the isolated heart revealed a marked heart rate dependence of Parv's delayed Ca2+ buffering effects on myocardial performance. As the pacing frequency was lowered (7 to 2.5 Hz), the relaxation rates increased in Parv hearts. However, as pacing rates approached the dynamic range in humans, Parv hearts demonstrated decreased contractility, consistent with Parv buffering systolic Ca2+. Mathematical modeling and in vitro studies provide the underlying mechanism responsible for the frequency-dependent fractional Ca2+ buffering action of Parv. Future studies directed toward refining the dose and frequency-response relationships of Parv in the heart or engineering novel Parv-based Ca2+ buffers with modified Mg2+ and Ca2+ affinities to limit systolic Ca2+ buffering may hold promise for the development of new therapies to remediate relaxation abnormalities in heart failure.

The United States has had 43 presidents. We examined whether they survive significantly longer or shorter than their contemporaries. We found that survival was better for presidents elected in the 1789-1841 and 1933-2001 periods (SMRs of 0.7 and 0.6, respectively), but worse for those elected in 1845-1929 (SMR = 2.9). We also found increased mortality during the years lived in office (SMR = 1.4), but no increase in mortality after leaving office (SMR = 1.0).

This study aimed to determine the probability that a child with cerebral palsy (CP) will lose or gain ambulatory ability through adolescence and young adulthood. We analyzed retrospectively data from 1987 to 2002 on Californians with CP initially aged 10 years (SD 0.9y; n=7550 [4304 males, 3246 females]) and 25 years (SD 0.8y; n=5721 [3261 males, 2460 females]) who had varying levels of ambulatory ability (initial Gross Motor Function Classification System Levels I-IV). We used the Aalen-Johansen estimator to estimate probabilities of transition to other levels of ambulatory ability in the future. Those who walked and climbed stairs without difficulty at age 10 had only a 23% chance of decline (to requiring a handrail to manage stairs, or worse) 15 years later. Those who ambulated with some difficulty but did not use a wheelchair had a significant chance (33%) of improvement (to being able to walk unsteadily alone at least 3m or better) and only a small chance (11%) of becoming non-ambulatory. Those who used a wheelchair were more likely to lose ambulatory ability (34%) or die (6%). Those who walked and climbed stairs well at age 25 were likely to maintain that ability 15 years later (76%), while those needing support to climb stairs were more likely to lose ability. Improvement in ambulation after age 25 was unlikely. Children and young adults with CP are likely to maintain their ambulatory ability during their next 15 years. Some who ambulate with difficulty at age 10 may improve through adolescence, but those who use a wheelchair are more likely to decline. By age 25 improvement in ambulation is unlikely and decline more likely. Most, however, will not change over the next 15 years.

The cardiac myofilaments are composed of highly ordered arrays of proteins that coordinate cardiac contraction and relaxation in response to the rhythmic waves of [Ca(2+)] during the cardiac cycle. Several cardiac disease states are associated with altered myofilament protein interactions that contribute to cardiac dysfunction. During acute myocardial ischemia, the sensitivity of the myofilaments to activating Ca(2+) is drastically reduced, largely due to the effects of intracellular acidosis on the contractile machinery. Myofilament Ca(2+) sensitivity remains compromised in post-ischemic or "stunned" myocardium even after complete restoration of blood flow and intracellular pH, likely because of covalent modifications of or proteolytic injury to contractile proteins. In contrast, myofilament Ca(2+) sensitivity can be increased in chronic heart failure, owing in part to decreased phosphorylation of troponin I, the inhibitory subunit of the troponin regulatory complex. We highlight, in this paper, the central role of the myofilaments in the pathophysiology of each of these distinct disease entities, with a particular focus on the molecular switch protein troponin I. We also discuss the beneficial effects of a genetically engineered cardiac troponin I, with a histidine button substitution at C-terminal residue 164, for a variety of pathophysiologic conditions, including hypoxia, ischemia, ischemia-reperfusion and chronic heart failure.

This study examined growth of children and adolescents with cerebral palsy (CP) who received services from the California Department of Developmental Services from 1987 to 2002. In all, 141 961 measurements of height and weight were taken from 24920 patients with CP (14103 males, 10817 females). Centiles of weight and height were determined by age, sex, and five levels of functional ability ranging from fully ambulatory to unable to walk, crawl, or feed self, and fed via gastrostomy tube. Resulting charts of height and weight centiles were compared with Centers for Disease Control and Prevention weight and height charts for the general population of the US. Centiles of height and weight of patients with CP were close to those of the general population for the highest functioning groups with CP, but lagged substantially for other groups. Presence of a feeding tube was associated with greater height and weight in the lowest functioning groups, with centiles for weight being 2 to 5 kg higher for those with gastrostomy tubes. The charts may assist in early identification of nutritional or metabolic difficulties beyond what might be expected for patients with similar functional disabilities.

The myofilament protein troponin I (TnI) has a key isoform-dependent role in the development of contractile failure during acidosis and ischemia. Here we show that cardiac performance in vitro and in vivo is enhanced when a single histidine residue present in the fetal cardiac TnI isoform is substituted into the adult cardiac TnI isoform at codon 164. The most marked effects are observed under the acute challenges of acidosis, hypoxia, ischemia and ischemia-reperfusion, in chronic heart failure in transgenic mice and in myocytes from failing human hearts. In the isolated heart, histidine-modified TnI improves systolic and diastolic function and mitigates reperfusion-associated ventricular arrhythmias. Cardiac performance is markedly enhanced in transgenic hearts during reperfusion despite a high-energy phosphate content similar to that in nontransgenic hearts, providing evidence for greater energetic economy. This pH-sensitive 'histidine button' engineered in TnI produces a titratable molecular switch that 'senses' changes in the intracellular milieu of the cardiac myocyte and responds by preferentially augmenting acute and long-term function under pathophysiological conditions. Myofilament-based inotropy may represent a therapeutic avenue to improve myocardial performance in the ischemic and failing heart.

Background/objective: Identify factors related to long-term survival, and quantify their effect on mortality and life expectancy., Setting: Model spinal cord injury systems of care across the United States., Study Design: Survival analysis of persons with traumatic spinal cord injury who are ventilator dependent at discharge from inpatient rehabilitation and who survive at least 1 year after injury., Methods: Logistic regression analysis on a data set of 1,986 person-years occurring among 319 individuals injured from 1973 through 2003., Results: The key factors related to long-term survival were age, time since injury, neurologic level, and degree of completeness of injury. The life expectancies were modestly lower than previous estimates. Pneumonia and other respiratory conditions remain the leading cause of death but account for only 31% of deaths of known causes., Conclusions: Whereas previous research has suggested a dramatic improvement in survival over the last few decades in this population, this is only the case during the critical first few years after injury. There was no evidence for such a trend in the subsequent period.

Dystrophin deficiency causes Duchenne muscular dystrophy (DMD) in humans, an inherited and progressive disease of striated muscle deterioration that frequently involves pronounced cardiomyopathy. Heart failure is the second leading cause of fatalities in DMD. Progress towards defining the molecular basis of disease in DMD has mostly come from studies on skeletal muscle, with comparatively little attention directed to cardiac muscle. The pathophysiological mechanisms involved in cardiac myocytes may differ significantly from skeletal myofibres; this is underscored by the presence of significant cardiac disease in patients with truncated or reduced levels of dystrophin but without skeletal muscle disease. Here we show that intact, isolated dystrophin-deficient cardiac myocytes have reduced compliance and increased susceptibility to stretch-mediated calcium overload, leading to cell contracture and death, and that application of the membrane sealant poloxamer 188 corrects these defects in vitro. In vivo administration of poloxamer 188 to dystrophic mice instantly improved ventricular geometry and blocked the development of acute cardiac failure during a dobutamine-mediated stress protocol. Once issues relating to optimal dosing and long-term effects of poloxamer 188 in humans have been resolved, chemical-based membrane sealants could represent a new therapeutic approach for preventing or reversing the progression of cardiomyopathy and heart failure in muscular dystrophy.

This study investigated mortality and causes of death between 1988 and 1999 in 14781 persons (6702 female) with Down syndrome in California, comparing age, sex, ethnicity, and other factors. Mean age at the start of follow-up was 14 years 8 months (SD 14y 10mo). During the study period 600 persons died. The standardized mortality ratio (SMR) for the population was 5.5. Blacks were at greater risk than whites, Hispanics, or Asians (relative risk = 1.5). Mortality declined during the period, especially for children with congenital heart defects. Leukemia (SMR = 17), respiratory illnesses (SMR = 27), congenital anomalies (SMR = 72), and circulatory diseases (SMR = 5.3) accounted for most of the excess mortality. With the exception of leukemia, cancer mortality was not different from that of the general population.

Leukocytes and leukocyte-derived microparticles contain low levels of tissue factor (TF) and incorporate into forming thrombi. Although this circulating pool of TF has been proposed to play a key role in thrombosis, its functional significance relative to that of vascular wall TF is poorly defined. We tested the hypothesis that leukocyte-derived TF contributes to thrombus formation in vivo. Compared to wild-type mice, mice with severe TF deficiency (ie, TF(-/-), hTF-Tg+, or "low-TF") demonstrated markedly impaired thrombus formation after carotid artery injury or inferior vena cava ligation. A bone marrow transplantation strategy was used to modulate levels of leukocyte-derived TF. Transplantation of low-TF marrow into wild-type mice did not suppress arterial or venous thrombus formation. Similarly, transplantation of wild-type marrow into low-TF mice did not accelerate thrombosis. In vitro analyses revealed that TF activity in the blood was very low and was markedly exceeded by that present in the vessel wall. Therefore, our results suggest that thrombus formation in the arterial and venous macrovasculature is driven primarily by TF derived from the blood vessel wall as opposed to leukocytes.

Background: Nitrergic and prostanoid pathways have both been implicated in inflammatory processes., Objective: To investigate their respective contributions in a rat model of chronic arthritis., Methods: Male Wistar rats (n = 4-6/group) received either an intra-articular injection of 2% carrageenan/4% kaolin (C/K) or intra- and periarticular injections of Freund's complete adjuvant (FCA; 10 mg/ml M tuberculosis). Joint diameter, urinary nitric oxide metabolites (NO(x)), and prostaglandin E(2) (PGE(2)) levels were measured as indices of the inflammatory process. A prophylactic and therapeutic (day 5) dose ranging study of an inducible nitric oxide synthase inhibitor, L-N-(1-iminoethyl)-lysine (L-NIL), and a cyclo-oxygenase-2 (COX-2) inhibitor, SC-236, was performed with the drugs given subcutaneously. Submaximal doses were identified and used for combination studies. Appropriate vehicle controls were included., Results: L-NIL and SC-236 dose dependently inhibited C/K induced acute joint swelling, the magnitude being greatest when they were given in combination. Both prophylactic and therapeutic administration of SC-236 in the FCA induced model of chronic arthritis produced a dose dependent reduction in all the measures assessed. However, although L-NIL demonstrated similar dose dependent inhibition of urinary NO(x) and PGE(2) levels, joint swelling was significantly exacerbated in this model. Co-administration of the inhibitors nullified the benefits of SC-236., Conclusion: Whereas COX-2 derived prostaglandins are proinflammatory in both acute and chronic joint inflammation, NO seems to have divergent roles, being anti-inflammatory in chronic and proinflammatory in acute joint inflammation.

Objectives: To determine independent predictors of ambulation among children with cerebral palsy and to develop a simple tool that estimates the probability that a child will walk., Methods: In a retrospective study of all children with cerebral palsy who were not yet walking at 2 to 3(1/2) years of age, while receiving services from the California Department of Developmental Services during the years 1987-1999, we analyzed medical and functional data obtained annually by Department of Developmental Services physicians and social workers. Using logistic regression analyses, we determined independent predictors of a child's ability to walk well alone at least 20 feet, without assistive devices, by age 6. We then estimated the probabilities of walking at various levels of ability over time, using multistate survival analysis., Results: Of 5366 study subjects, 2295 (43%) were evaluated at age 6; 12.8% could walk independently and 18.4% walked with support. Independent predictors of successful ambulation included early motor milestones such as sitting (odds ratio: 12.5; 95% confidence interval: 5.8-27.2) and pulling to a stand (odds ratio: 28.5; 95% confidence interval: 13.4-60.4) when compared with lack of rolling at age 2, cerebral palsy type other than spastic quadriparesis (odds ratio: 2.2; 95% confidence interval: 1.5-3.1), and preserved visual function (odds ratio: 2.4; 95% confidence interval: 1.1-5.4). Our ambulation charts depict the probability of remaining nonambulatory, transitioning to 1 of 3 possible ambulatory states, or expiring at all subsequent ages through age 14., Conclusion: The ambulation charts provide a simple straightforward way to estimate the probability that a child with cerebral palsy who is nonambulatory at 2 to 3 12 years of age will eventually walk with or without support.

Due to exciting advances in molecular biology, the laboratory mouse has become an important and frequently used model for studying thrombosis. This article reviews several experimental approaches that have been used to study arterial, venous, and microvascular thrombosis in mice. The advantages and limitations of different models are examined. Related topics of mouse anesthesia, phlebotomy, and in vitro hemostasis testing are also reviewed.

Hypertrophic cardiomyopathy mutations A63V and E180G in alpha-tropomyosin (alpha-Tm) have been shown to cause slow cardiac muscle relaxation. In this study, we used two complementary genetic strategies, gene transfer in isolated rat myocytes and transgenesis in mice, to ascertain whether parvalbumin (Parv), a myoplasmic calcium buffer, could correct the diastolic dysfunction caused by these mutations. Sarcomere shortening measurements in rat cardiac myocytes expressing the alpha-Tm A63V mutant revealed a slower time to 50% relengthening (T50R: 44.2+/-1.4 ms in A63V, 36.8+/-1.0 ms in controls; n=96 to 108; P<0.001) when compared with controls. Dual gene transfer of alpha-Tm A63V and Parv caused a marked decrease in T50R (29.8+/-1.0 ms). However, this increase in relaxation rate was accompanied with a decrease in shortening amplitude (114.6+/-4.4 nm in A63+Parv, 137.8+/-5.3 nm in controls). Using an asynchronous gene transfer strategy, Parv expression was reduced (from approximately 0.12 to approximately 0.016 mmol/L), slow relaxation redressed, and shortening amplitude maintained (T50R=33.9+/-1.6 ms, sarcomere shortening amplitude=132.2+/-7.0 nm in A63V+PVdelayed; n=56). Transgenic mice expressing the E180G alpha-Tm mutation and mice expressing Parv in the heart were crossed. In isolated adult myocytes, the alpha-Tm mutation alone (E180G+/PV-) had slower sarcomere relengthening kinetics than the controls (T90R: 199+/-7 ms in E180G+/PV-, 130+/-4 ms in E180G-/PV-; n=71 to 72), but when coexpressed with Parv, cellular relaxation was faster (T90R: 36+/-4 ms in E180G+/PV+). Collectively, these findings show that slow relaxation caused by alpha-Tm mutants can be corrected by modifying calcium handling with Parv.

Blood alcohol measurements determined by headspace gas chromatography have been challenged on the grounds that the presence of the preservative sodium fluoride in blood samples artificially increases headspace alcohol concentrations due to a salting out effect. Blood samples containing varying amounts of ethanol and sodium fluoride were tested using semi-automated headspace gas chromatography with n-propyl alcohol as the internal standard to assess the validity of this challenge. We find, in fact, that under these test conditions the measured alcohol levels are systematically depressed as the amount of sodium fluoride in the blood sample increases. The challenge thus has no basis.

Objectives: Nitric oxide and prostaglandins are both implicated in the pathogenesis of inflammatory conditions such as rheumatoid arthritis (RA). The hypothesis that simultaneous inhibition of nitric oxide synthase (NOS) and cyclooxygenase (COX) was more effective than inhibition of either enzyme alone was tested., Methods: J774 macrophages were pre-incubated with L-NAME and/or indomethacin, prior to activation with LPS (10 micrograms/ml)., Results: LPS significantly increased NO2-; PGE2 and TNF-alpha levels by 24 h. Quantitative real-time PCR demonstrated a dose-dependent reduction in the expression of COX-2 in the presence of increasing doses of L-NAME. NO2- and PGE2 production were inhibited in a dose-dependent manner by either indomethacin or L-NAME. Combined administration of L-NAME and indomethacin produced a significantly greater inhibition of NO2- and PGE2 than either inhibitor alone., Conclusion: The data supports the therapeutic potential of combined inhibition of the prostanoid and nitrergic systems as an anti-inflammatory treatment strategy and supports the progression of this work into models of arthritis.

The intra-articular injection of high molecular weight hyaluronic acid (HA) has been reported to be an effective treatment for pain of osteoarthritis of the knee. However, the mechanism by which HA exerts its effect is unknown. To explore HA's influence on the growth of U937 human macrophages, cells were incubated for 168 h with three concentrations, 1, 0.1 and 0.01 mg/mL, of Hyalgan, a high molecular weight HA preparation. At 24-h increments, the cells were examined for proliferation, cell cycle distribution as well as the number of apoptotic and dead cells. Exposing macrophages to 1 mg/mL Hyalgan significantly reduced the rate of cellular proliferation and altered the cell cycle distribution to yield decreased proportions of G0/G1 cells but increased S and G2/M cells. Concomitantly, a 10-fold increase in apoptotic cells and a 12-fold increase in dead cells were observed. The population doubling time (PDT) for cells treated with 1.0 mg/mL Hyalgan increased from 23.6 to 52.9 h. By contrast, the two lower Hyalgan concentrations significantly promoted macrophage proliferation in a dose-dependent manner. They also increased the proportion of G2/M cells, but had no effect on the number of apoptotic or dead cells. The PDTs of 21.5 and 22.2 h were less than the control time of 23.6 h. These results demonstrate that Hyalgan concentrations have a differential effect on macrophage growth dynamics and suggest an anti-inflammatory effect at high HA concentrations.

Background: Iron overload has been implicated in the pathogenesis of ischemic cardiovascular events. However, the effects of iron excess on vascular function and the thrombotic response to vascular injury are not well understood., Methods and Results: We examined the effects of chronic iron dextran administration (15 mg over 6 weeks) on thrombosis, systemic and vascular oxidative stress, and endothelium-dependent vascular reactivity in mice. Thrombus generation after photochemical carotid artery injury was accelerated in iron-loaded mice (mean time to occlusive thrombosis, 20.4+/-8.5 minutes; n=10) compared with control mice (54.5+/-35.5 minutes, n=10, P=0.009). Iron loading had no effect on plasma clotting, vessel wall tissue factor activity, or ADP-induced platelet aggregation. Acute administration of dl-cysteine, a reactive oxygen species scavenger, completely abrogated the effects of iron loading on thrombus formation, suggesting that iron accelerated thrombosis through a pro-oxidant mechanism. Iron loading enhanced both systemic and vascular reactive oxygen species production. Endothelium-dependent vasorelaxation was impaired in iron-loaded mice, indicating reduced NO bioavailability., Conclusions: Moderate iron loading markedly accelerates thrombus formation after arterial injury, increases vascular oxidative stress, and impairs vasoreactivity. Iron-induced vascular dysfunction may contribute to the increased incidence of ischemic cardiovascular events that have been associated with chronic iron overload.

The role of different isoforms of cyclo-oxygenase (COX) in mediating the acute (0-6 h) and late (24 h) phases of inflammation was investigated in the rat knee joint following intra-articular injection of carrageenan. The hyperaemic response was assessed transcutaneously using laser Doppler imaging (LDI). Samples were taken at corresponding time points for detection of synovial COX-1, COX-2 and inducible nitric oxide synthase (iNOS) mRNA, and measurement of urinary prostaglandin (PG) and nitric oxide metabolites (NO(x)). A non-selective COX inhibitor (indomethacin, 15 mg kg(-1) I.P.), a selective COX-2 inhibitor (SC-236, 16.8 mg kg(-1) I.P.) or vehicle were administered 1 h prior to carrageenan in the acute phase study. LDI scans were taken hourly for 4 h post-induction. Inflammatory hyperaemia in the vehicle group was attenuated in the indomethacin- (P < 0.001, two-way ANOVA) and SC-236-treated groups (P < 0.0001), with no difference between these treatments. At 24 h, I.V. infusion of indomethacin (0.1 mg min(-1)), increased vascular resistance (24 +/- 7.1 %; P < 0.05) compared to vehicle infusion, whereas SC-236 (0.11 mg min(-1)) did not. Resistance changes to indomethacin also differed from SC-236 (P < 0.05). Knee joint diameter progressively increased over 24 h (P < 0.0001, one-way ANOVA). Urinary PG levels increased by 6 h (P < 0.05), but returned to baseline by 24 h. COX-1 mRNA was detectable at all time points; COX-2 mRNA only at 3 h. Urinary NO(x) levels increased progressively over 24 h (P < 0.05), paralleled by induction of iNOS in the 3 and 24 h samples. Prostaglandin production via COX-2 appears to mediate the development of acute inflammatory hyperaemia, but nitrergic mechanisms may supervene subsequently. COX-1 but not COX-2 contributes to the maintenance of basal blood flow in the hyperaemic joint at 24 h.

Apical ATP, ATP, UTP and UDP evoked transient increases in short circuit current (I(SC), a direct measure of transepithelial ion transport) in confluent Caco-2 cells grown on permeable supports. These responses were mediated by a population of at least three pharmacologically distinct receptors. Experiments using cells grown on glass coverslips showed that ATP and UTP consistently increased intracellular free calcium ([Ca(2+)](i)) whilst sensitivity to UDP was variable. Cross desensitization experiments suggested that the responses to UTP and ATP were mediated by a common receptor population. Messenger RNA transcripts corresponding to the P2Y(2), P2Y(4) and P2Y(6) receptors genes were detected in cells grown on Transwell membranes by the reverse transcriptase - polymerase chain reaction. Identical results were obtained for cells grown on glass. Experiments in which I(SC) and [Ca(2+)](i) were monitored simultaneously in cells on Transwell membranes, confirmed that apical ATP and UTP increased both parameters and showed that the UDP-evoked increase in I(SC) was accompanied by a [Ca(2+)](i)-signal. Ionomycin consistently increased [Ca(2+)](i) in such polarized cells but caused no discernible change in I(SC). However, subsequent application of apical ATP or UTP evoked a small rise in I(SC) but no rise in [Ca(2+)](i). UDP evoked no such response. As well as evoking increases in [Ca(2+)](i), the ATP/UTP-sensitive receptors present in Caco-2 cells thus allow direct control over ion channels in the apical membrane. The UDP-sensitive receptors, however, appear to simply evoke a rise in [Ca(2+)](i).

We determined estimates of survival in children, 3-15 years of age, in the vegetative state (VS) (n = 564), immobile minimally conscious state (MCS) (n = 705), and mobile MCS (n = 3,806). Data were extracted from the annual Client Development Evaluation Reports of the California Department of Developmental Services between 1988 and 1997 using the operational definitions for these three states on the basis of 15 descriptive behavioral categories. Patients were also categorized according to the following four etiologies: acquired (traumatic and nontraumatic) brain injury; perinatal/genetic; degenerative; and unknown/undetermined. The percentage of patients surviving 8 years was 63%, 65%, and 81%, for the VS, immobile MCS, and mobile MCS, respectively. Children in the VS and MCSs with acquired brain injury had lower mortality rates and those with degenerative diseases the highest mortality rates. We observed little difference in survival between patients in the VS and immobile MCS, suggesting that the presence of consciousness is not a critical variable in determining life expectancy. Furthermore, survival was much greater for patients in the mobile MCS than for those in the immobile MCS, suggesting that mobility is more important in predicting survival than the level of consciousness.

This study was undertaken to determine the prognostic significance of hypotension induced during preoperative dobutamine stress echocardiography (DSE) before vascular and noncardiac thoracic surgery. Wall motion abnormality during DSE predicts perioperative risk. Although hypotension during DSE has not been shown to correlate with the presence or severity of coronary artery disease, its significance in perioperative risk assessment is unknown. We retrospectively studied 300 patients who had DSE within 6 months of noncardiac surgery. Perioperative events including death, myocardial infarction, ischemia, and arrhythmias were recorded. Odds ratios with 95% confidence intervals were used to examine the association between clinical and echocardiographic variables and perioperative events. A hypotensive response during DSE was seen in 85 patients (28%). Forty-eight patients (16%) had 54 perioperative complications including 4 cardiac-related deaths, 10 myocardial infarctions, 12 myocardial ischemic events, and 28 arrhythmias. Hypotension during DSE was predictive of the combined end point of perioperative cardiac mortality, myocardial infarction, and ischemia (odds ratio 4.04, 95% confidence interval 1.72 to 9.51). In a multivariate logistic regression model, hypotension during DSE remained a significant predictor (odds ratio 4.10, p<0.01). DSE-related hypotension was predictive of perioperative cardiac events and therefore may have a role in risk stratification before vascular or noncardiac thoracic surgery.

Thromboembolic disorders are a hallmark of the antiphospholipid antibody syndrome. We describe a patient with IgM antiphospholipid antibodies associated with pulmonary emboli and in situ thrombosis within an otherwise normal right atrium. Echocardiography, particularly the transesophageal study, proved invaluable in providing a diagnosis and guiding our patient's evaluation and treatment.