Structural modeling of protein-RNA complexes using crosslinking of segmentally isotope-labeled RNA and MS/MS.

Ribonucleoproteins (RNPs) are key regulators of cellular function. We established an efficient approach, crosslinking of segmentally isotope-labeled RNA and tandem mass spectrometry (CLIR-MS/MS), to localize protein-RNA interactions simultaneously at amino acid and nucleotide resolution. The approach was tested on polypyrimidine tract binding protein 1 and U1 small nuclear RNP. Our method provides distance restraints to support integrative atomic-scale structural modeling and to gain mechanistic insights into RNP-regulated processes.

Hearts deficient in both Mfn1 and Mfn2 are protected against acute myocardial infarction.

Mitochondria alter their shape by undergoing cycles of fusion and fission. Changes in mitochondrial morphology impact on the cellular response to stress, and their interactions with other organelles such as the sarcoplasmic reticulum (SR). Inhibiting mitochondrial fission can protect the heart against acute ischemia/reperfusion (I/R) injury. However, the role of the mitochondrial fusion proteins, Mfn1 and Mfn2, in the response of the adult heart to acute I/R injury is not clear, and is investigated in this study. To determine the effect of combined Mfn1/Mfn2 ablation on the susceptibility to acute myocardial I/R injury, cardiac-specific ablation of both Mfn1 and Mfn2 (DKO) was initiated in mice aged 4-6 weeks, leading to knockout of both these proteins in 8-10-week-old animals. This resulted in fragmented mitochondria (electron microscopy), decreased mitochondrial respiratory function (respirometry), and impaired myocardial contractile function (echocardiography). In DKO mice subjected to in vivo regional myocardial ischemia (30 min) followed by 24 h reperfusion, myocardial infarct size (IS, expressed as a % of the area-at-risk) was reduced by 46% compared with wild-type (WT) hearts. In addition, mitochondria from DKO animals had decreased MPTP opening susceptibility (assessed by Ca(2+)-induced mitochondrial swelling), compared with WT hearts. Mfn2 is a key mediator of mitochondrial/SR tethering, and accordingly, the loss of Mfn2 in DKO hearts reduced the number of interactions measured between these organelles (quantified by proximal ligation assay), attenuated mitochondrial calcium overload (Rhod2 confocal microscopy), and decreased reactive oxygen species production (DCF confocal microscopy) in response to acute I/R injury. No differences in isolated mitochondrial ROS emissions (Amplex Red) were detected in response to Ca(2+) and Antimycin A, further implicating disruption of mitochondria/SR tethering as the protective mechanism. In summary, despite apparent mitochondrial dysfunction, hearts deficient in both Mfn1 and Mfn2 are protected against acute myocardial infarction due to impaired mitochondria/SR tethering.

Mitochondrial G protein coupled receptor kinase 2 regulates proinflammatory responses in macrophages.

G-protein-coupled receptor kinase 2 (GRK2) levels are elevated in inflammation but its role is not clear yet. Here we show that GRK2 expression is dependent on NFκB transcriptional activity. In macrophages, LPS induces GRK2 accumulation in mitochondria increasing biogenesis. The overexpression of the carboxy-terminal domain of GRK2 (ßARK-ct), known to displace GRK2 from plasma membranes, induces earlier localization of GRK2 to mitochondria in response to LPS leading to increased mt-DNA transcription and reduced ROS production and cytokine expression. Our study shows the relevance of GRK2 subcellular localization in macrophage biology and its potential therapeutic properties in inflammation.

Knowledge-assisted visualization and segmentation of geologic features.

A 3D visualization environment contains domain information about geologic features in seismic data and lets users interactively steer segmentations on the basis of visual sources of information and knowledge. A user study demonstrates this approach's ability to transfer domain knowledge to non experts.

Reduced troponin I phosphorylation and increased Ca(2+)-dependent ATP-consumption in triton X-skinned fiber preparations from Galphaq overexpressor mice.

Overexpression of the Galphaq-protein has been shown to result in hypertrophic and dilated cardiomyopathy. This study investigated Ca(2+ )sensitivity of tension and myosin-ATPase activity in skinned fiber preparations of male and female wildtype (WT; n = 12) and transgenic mice with a cardiac specific overexpression of the Galphaq-protein (Galphaq-OE; n = 11). In addition, the phosphorylation status of troponin I was measured. Ca(2+) sensitivity of tension was increased in Galphaq-OE with a significant reduction in the half-maximum Ca(2+) concentration (EC(50)) compared to WT. Similarly, Ca(2+) sensitivity of myosin ATPase activity was increased in Galphaq-OE when comparing Galphaq-OE to WT. Maximum Ca(2+)-dependent tension and ATPase activity were both enhanced in Galphaq-OE compared to WT littermates. Phosphorylation of troponin I was significantly reduced in Galphaq-OE compared to WT. In the above experiments, no gender specific differences were observed in either Gaq-OE or in WT. We conclude that, in mice, increased expression of the Galphaq-protein induces alterations of myofibrillar function and energy consumption, which are also characteristics of human heart failure. This may result from a decreased phosphorylation of troponin I in Galphaq-OE.

Cardiac reanimation: targeting cardiomyocyte death by BNIP3 and NIX/BNIP3L.

Programmed cardiac myocyte death contributes to pathological ventricular remodeling and the progression of myocardial infarction or pressure overload hypertrophy to dilated cardiomyopathy. Recent work has identified importance of stress-mediated transcriptional induction of BNIP3 (BCL2 and 19-kDa interacting protein-3) and NIX/BNIP3L in cardiac remodeling. Here, the regulatory mechanisms for these two factors in the heart and their effects on programmed cardiomyocyte death are reviewed, with a focus on information derived from studies using mouse models of cardiac BNIP3 and NIX/BNIP3L overexpression and gene ablation.

The clinical and economic impact of competing management strategies for gastro-oesophageal reflux disease.

BACKGROUND: Gastro-oesophageal reflux disease (GERD) is a common disorder in the primary care setting. Traditional management strategies consist of sequentially intensive therapeutic trials followed by invasive diagnostic testing for nonresponders. A high dose proton pump inhibitor trial (the "proton pump inhibitor test") has been shown to be an accurate diagnostic alternative, and may be an efficient initial approach to patients with GERD symptoms. AIM: To examine the clinical, economic and policy implications of alternative management strategies for GERD. METHODS: Decision analysis was used to calculate the clinical and economic outcomes of competing management strategies. The traditional strategy incorporates sequential therapeutic trials with more intensive therapy ("step-up" approach) followed by sequential invasive diagnostic testing of nonresponders. The "proton pump inhibitor test" strategy includes an initial "proton pump inhibitor test" (7 days of omeprazole; 40 mg AM + 20 mg PM daily) followed by less intensive therapeutic trials in those testing positive ("step-down" approach) with sequential invasive diagnostic testing as needed. Cost estimates were based on Medicare reimbursement and average wholesale drug prices. Probability estimates were derived from a systematic review of the published medical literature. Model results are reported as the average and incremental cost-per-symptom free patient and cost-per-quality-adjusted life-years (QALYs) gained. RESULTS: The average cost per patient was 1045 US dollars for the traditional step-up management strategy, compared to 1172 US dollars for the "proton pump inhibitor test" and step-down strategy. The percentage of patients who were symptom-free at 1 year was 50% for the traditional management strategy compared to 75% for the "proton pump inhibitor test" strategy. The "proton pump inhibitor test" strategy results in QALY gains of 0.01-0.05 depending on the utility estimate employed. The incremental cost-effectiveness ratio for the "proton pump inhibitor test" strategy is 510 US dollars per additional symptomatic cure over 1 year, and between 2822-10,160 US dollars per QALY gained. The traditional management strategy resulted in a greater than 5-fold increase in the utilization of upper endoscopy, which was partially offset by a 47% reduction in the use of ambulatory 24-h oesophageal pH monitoring. The reduced effectiveness of the traditional management strategy may be attributed in part to a 118% increase in the use of "high-dose" H2RAs while reducing the use of standard dose proton pump inhibitors by only 42% and "high-dose" proton pump inhibitors by 57%. CONCLUSIONS: Based on the results of this analysis, strategies utilizing the initial PPI test followed by a "step-down" approach may result in improved symptom relief and quality of life over 1 year, and more appropriate utilization of invasive diagnostic testing at a small marginal increase in total costs. These findings warrant a prospective trial comparing these competing management strategies.

Increased myocardial Rab GTPase expression: a consequence and cause of cardiomyopathy.

The Ras-like Rab GTPases regulate vesicle transport in endocytosis and exocytosis. We found that cardiac Rabs1, 4, and 6 are upregulated in a dilated cardiomyopathy model overexpressing beta(2)-adrenergic receptors. To determine if increased Rab GTPase expression can contribute to cardiomyopathy, we transgenically overexpressed in mouse hearts prototypical Rab1a, the small G protein that regulates vesicle transport from endoplasmic reticulum to and through Golgi. In multiple independent mouse lines, Rab1a overexpression caused cardiac hypertrophy that progressed in a time- and transgene dose-dependent manner to heart failure. Isolated cardiac myocytes were hypertrophied and exhibited contractile depression with impaired calcium reuptake. Ultrastructural analysis revealed enlarged Golgi stacks and increased transitional vesicles in ventricular myocytes, with increased secretory atrial natriuretic peptide granules and degenerative myelin figures in atrial myocytes; immunogold studies localized Rab1a to these abnormal vesicular structures. A survey of hypertrophy signaling molecules revealed increased protein kinase C (PKC) alpha and delta, and confocal microscopy showed abnormal subcellular distribution of PKCalpha in Rab1a transgenics. These results indicate that increased expression of Rab1 GTPase in myocardium distorts subcellular localization of proteins and is sufficient to cause cardiac hypertrophy and failure.

Opposing cardioprotective actions and parallel hypertrophic effects of delta PKC and epsilon PKC.

Conflicting roles for protein kinase C (PKC) isozymes in cardiac disease have been reported. Here, deltaPKC-selective activator and inhibitor peptides were designed rationally, based on molecular modeling and structural homology analyses. Together with previously identified activator and inhibitor peptides of epsilonPKC, deltaPKC peptides were used to identify cardiac functions of these isozymes. In isolated cardiomyocytes, perfused hearts, and transgenic mice, deltaPKC and epsilonPKC had opposing actions on protection from ischemia-induced damage. Specifically, activation of epsilonPKC caused cardioprotection whereas activation of deltaPKC increased damage induced by ischemia in vitro and in vivo. In contrast, deltaPKC and epsilonPKC caused identical nonpathological cardiac hypertrophy; activation of either isozyme caused nonpathological hypertrophy of the heart. These results demonstrate that two related PKC isozymes have both parallel and opposing effects in the heart, indicating the danger in the use of therapeutics with nonselective isozyme inhibitors and activators. Moreover, reduction in cardiac damage caused by ischemia by perfusion of selective regulator peptides of PKC through the coronary arteries constitutes a major step toward developing a therapeutic agent for acute cardiac ischemia.

Specific inhibition of the rat ligand-gated ion channel P2X3 function via methoxyethoxy-modified phosphorothioated antisense oligonucleotides.

P2X3 is one receptor of a family of seven ligand-gated ion channels responding to purines. Increasing evidence indicates its involvement in neuronal signaling and in pain. However, there is currently no selective inhibitor known for this subtype. In order to obtain such a specific inhibitor, a variety of antisense oligonucleotides (ASO) against rat P2X3 was tested, and dose-dependent, sequence-specific downregulation of the rat P2X3 receptor (expressed in a Chinese hamster ovary cell line [CHO-K1]) on the mRNA, protein, and functional levels was observed. Using real-time quantitative PCR, a dose-dependent downregulation of P2X3 mRNA by ASO, as compared with untreated and mismatch controls, was demonstrated. Subsequently, downregulation by the two most potent ASO was confirmed at the protein level by Western blot. Sequence specificity was shown by titration of mismatches to the original selected oligonucleotide, and this correlated with progressive loss of P2X3 inhibition. The functional response of the P2X3 receptor was examined using whole-cell voltage clamping. Upon application of 10 microM of a nonspecific agonist, alpha,beta-methylene-ATP (alphabeta meATP), pretreatment with increasing amounts of the most active ASO 5037 correlated with a decrease in depolarization. The ability to specifically downregulate the P2X3 receptor by ASO treatment will allow investigation of the biologic role of this receptor in neuronal tissues and eventually in in vivo models of chronic pain.

Divergent transcriptional responses to independent genetic causes of cardiac hypertrophy.

To define molecular mechanisms of cardiac hypertrophy, genes whose expression was perturbed by any of four different transgenic mouse hypertrophy models [protein kinase C-epsilon activation peptide (PsiepsilonRACK), calsequestrin (CSQ), calcineurin (CN), and Galpha(q)] were compared by DNA microarray analyses using the approximately 8,800 genes present on the Incyte mouse GEM1. The total numbers of regulated genes (tens to hundreds) correlated with phenotypic severity of the model (Galpha(q) > CN > CSQ > PsiepsilonRACK), but demonstrated that no single gene was consistently upregulated. Of the three models exhibiting pathological hypertrophy, only atrial natriuretic peptide was consistently upregulated, suggesting that transcriptional alterations are highly specific to individual genetic causes of hypertrophy. However, hierarchical-tree and K-means clustering analyses revealed that subsets of the upregulated genes did exhibit coordinate regulatory patterns that were unique or overlapping across the different hypertrophy models. One striking set consisted of apoptotic genes uniquely regulated in the apoptosis-prone Galpha(q) model. Thus, rather than identifying a single common hypertrophic cardiomyopathy gene program, these data suggest that extensive groups of genes may be useful for the prediction of specific underlying genetic determinants and condition-specific therapeutic approaches.

Mouse model of desmin-related cardiomyopathy.

BACKGROUND: The consequence of upregulation of desmin in the heart is unknown. Mutations in desmin have been linked to desmin-related myopathy (DRM), which is characterized by abnormal intrasarcoplasmic accumulation of desmin, but direct causative evidence that a desmin mutation leads to aberrant intrasarcoplasmic desmin accumulation, aggregation, and cardiomyopathy is lacking. METHODS AND RESULTS: Multiple transgenic mouse lines that expressed either murine wild-type desmin or a 7-amino acid deletion (R173 through E179) desmin (D7-des) mutation linked to DRM were made. The distribution of desmin protein was unchanged, and no overt phenotype was detected in the wild-type desmin transgenic mice. In contrast, the D7-des mouse heart showed aberrant intrasarcoplasmic and electron-dense granular filamentous aggregates that were desmin-positive and characteristic of human DRM. The desmin filament network was significantly disrupted, and myofibril alignment was visibly compromised. Although systolic function at the whole-organ level was substantially conserved in the young adult animals, the ability of the heart to respond to beta-agonist stimulation, as measured in the intact animal, was significantly blunted. CONCLUSIONS: Upregulation of desmin protein at moderate levels is not detrimental. However, the D7-des mutation is dominant negative, and expression of the mutant protein leads to the appearance of aggregates that are characteristic of and diagnostic for human desmin-related cardiomyopathy.

Superinhibition of sarcoplasmic reticulum function by phospholamban induces cardiac contractile failure.

To determine whether selective impairment of cardiac sarcoplasmic reticulum (SR) Ca(2+) transport may drive the progressive functional deterioration leading to heart failure, transgenic mice, overexpressing a phospholamban Val(49) --> Gly mutant (2-fold), which is a superinhibitor of SR Ca(2+)-ATPase affinity for Ca(2+), were generated, and their cardiac phenotype was examined longitudinally. At 3 months of age, the increased EC(50) level of SR Ca(2+) uptake for Ca(2+) (0.67 +/- 0.09 microm) resulted in significantly higher depression of cardiomyocyte rates of shortening (57%), relengthening (31%), and prolongation of the Ca(2+) signal decay time (165%) than overexpression (2-fold) of wild type phospholamban (68%, 64%, and 125%, respectively), compared with controls (100%). Echocardiography also revealed significantly depressed function and impaired beta-adrenergic responses in mutant hearts. The depressed contractile parameters were associated with left ventricular remodeling, recapitulation of fetal gene expression, and hypertrophy, which progressed to dilated cardiomyopathy with interstitial tissue fibrosis and death by 6 months in males. Females also had ventricular hypertrophy at 3 months but exhibited normal systolic function up to 12 months of age. These results suggest a causal relationship between defective SR Ca(2+) cycling and cardiac remodeling leading to heart failure, with a gender-dependent influence on the time course of these alterations.

Gender and race differences in the predictors of daily health practices among older adults.

Preventive health behaviors are crucial for older adults' well-being. This study examined the factors that influence the practice of positive daily health behaviors over time in a sample of older adults (N = 1266) and investigated whether explanatory factors differ by health behavior, gender or race. Physical activity, weight maintenance, smoking, alcohol consumption and sleep patterns were examined as dependent variables. Independent variables included demographic characteristics, baseline health behavior, health status variables, psychological factors and social network characteristics. Results indicate that age and health status are important predictors of preventive health behaviors. However, the factors that predict preventive health behaviors vary by behavior, gender and race. The independent variables included in this study were most successful in explaining cigarette smoking and weight maintenance, and least successful in explaining amount of sleep. In addition, results suggest that social network variables are particularly influential for women's health behaviors, while health status is more influential among men. Greater education predicts better health behaviors among whites, while formal social integration seems particularly important for the health behaviors of older black women. These results indicate that examining older adults' health behaviors by race and gender leads to a fuller understanding of these behaviors.

Cytoplasmic signaling pathways that regulate cardiac hypertrophy.

This review discusses the rapidly progressing field of cardiomyocyte signal transduction and the regulation of the hypertrophic response. When stimulated by a wide array of neurohumoral factors or when faced with an increase in ventricular-wall tension, individual cardiomyocytes undergo hypertrophic growth as an adaptive response. However, sustained cardiac hypertrophy is a leading predictor of future heart failure. A growing number of intracellular signaling pathways have been characterized as important transducers of the hypertrophic response, including specific G protein isoforms, low-molecular-weight GTPases (Ras, RhoA, and Rac), mitogen-activated protein kinase cascades, protein kinase C, calcineurin, gp130-signal transducer and activator of transcription, insulin-like growth factor I receptor pathway, fibroblast growth factor and transforming growth factor beta receptor pathways, and many others. Each of these signaling pathways has been implicated as a hypertrophic transducer, which collectively suggests an emerging paradigm whereby multiple pathways operate in concert to orchestrate a hypertrophic response

Interactions between phospholamban and beta-adrenergic drive may lead to cardiomyopathy and early mortality.

BACKGROUND: Relieving the inhibition of sarcoplasmic reticular function by phospholamban is a major target of beta-adrenergic stimulation. Chronic beta-adrenergic receptor activity has been suggested to be detrimental, on the basis of transgenic overexpression of the receptor or its signaling effectors. However, it is not known whether physiological levels of sympathetic tone, in the absence of preexisting heart failure, are similarly detrimental. METHODS AND RESULTS: Transgenic mice overexpressing phospholamban at 4-fold normal levels were generated, and at 3 months, they exhibited mildly depressed ventricular contractility without heart failure. As expected, transgenic cardiomyocyte mechanics and calcium kinetics were depressed, but isoproterenol reversed the inhibitory effects of phospholamban on these parameters. In vivo cardiac function was substantially depressed by propranolol administration, suggesting enhanced sympathetic tone. Indeed, plasma norepinephrine levels and the phosphorylation status of phospholamban were elevated, reflecting increased adrenergic drive in transgenic hearts. On aging, the chronic enhancement of adrenergic tone was associated with a desensitization of adenylyl cyclase (which intensified the inhibitory effects of phospholamban), the development of overt heart failure, and a premature mortality. CONCLUSIONS: The unique interaction between phospholamban and increased adrenergic drive, elucidated herein, provides the first evidence that compensatory increases in catecholamine stimulation can, even in the absence of preexisting heart failure, be a primary causative factor in the development of cardiomyopathy and early mortality.

RGS4 reduces contractile dysfunction and hypertrophic gene induction in Galpha q overexpressing mice.

The intrinsic GTPase activity of Galpha q is low, and RGS proteins which activate GTPase are expressed in the heart; however, their functional relevance in vivo is unknown. Transgenic mice with cardiac-specific overexpression of Galpha q in myocardium exhibit cardiac hypertrophy, enhanced PKC xi membrane translocation, embryonic gene expression, and depressed cardiac contractility. We recently reported that transgenic mice with cardiac-specific expression of RGS4, a Galpha q and Galpha i GTPase activator, exhibit decreased left ventricular hypertrophy and ANF induction in response to pressure overload. To test the hypothesis that RGS4 can act as a Galpha q-specific GTPase activating protein (GAP) in the in vivo heart, dual transgenic Galpha q-40xRGS4 mice were generated to determine if RGS4 co-expression would ameliorate the Galpha q-40 phenotype. At age 4 weeks, percent fractional shortening was normalized in dual transgenic mice as was left ventricular internal dimension and posterior and septal wall thicknesses. PKC xi membrane translocation and ANF and alpha -skeletal actin mRNA levels were also normalized. Compound transgenic mice eventually developed depressed cardiac contractility that was evident by 9 weeks of age. These studies establish for the first time a role for RGS4 as a GAP for Galpha q in the in vivo heart, and demonstrate that its regulated expression can have pathophysiologic consequences.

Rescue of contractile parameters and myocyte hypertrophy in calsequestrin overexpressing myocardium by phospholamban ablation.

Cardiac-specific overexpression of murine cardiac calsequestrin results in depressed cardiac contractile parameters, low Ca(2+)-induced Ca(2+) release from sarcoplasmic reticulum (SR) and cardiac hypertrophy in transgenic mice. To test the hypothesis that inhibition of phospholamban activity may rescue some of these phenotypic alterations, the calsequestrin overexpressing mice were cross-bred with phospholamban-knockout mice. Phospholamban ablation in calsequestrin overexpressing mice led to reversal of the depressed cardiac contractile parameters in Langendorff-perfused hearts or in vivo. This was associated with increases of SR Ca(2+) storage, assessed by caffeine-induced Na(+)-Ca(2+) exchanger currents. The inactivation time of the L-type Ca(2+) current (I(Ca)), which has an inverse correlation with Ca(2+)-induced SR Ca(2+) release, and the relation between the peak current density and half-inactivation time were also normalized, indicating a restoration in the ability of I(Ca) to trigger SR Ca(2+) release. The prolonged action potentials in calsequestrin overexpressing cardiomyocytes also reversed to normal upon phospholamban ablation. Furthermore, ablation of phospholamban restored the expression levels of atrial natriuretic factor and alpha-skeletal actin mRNA as well as ventricular myocyte size. These results indicate that attenuation of phospholamban function may prevent or overcome functional and remodeling defects in hypertrophied hearts.