Cardiomyocytes, cardiac endothelial cells and fibroblasts contribute to anthracycline-induced cardiac injury through RAS-homologous small GTPases RAC1 and CDC42.

The clinical use of the DNA damaging anticancer drug doxorubicin (DOX) is limited by irreversible cardiotoxicity, which depends on the cumulative dose. The RAS-homologous (RHO) small GTPase RAC1 contributes to DOX-induced DNA damage formation and cardiotoxicity. However, the pathophysiological relevance of other RHO GTPases than RAC1 and different cardiac cell types (i.e., cardiomyocytes, non-cardiomyocytes) for DOX-triggered cardiac damage is unclear. Employing diverse in vitro and in vivo models, we comparatively investigated the level of DOX-induced DNA damage in cardiomyocytes versus non-cardiomyocytes (endothelial cells and fibroblasts), in the presence or absence of selected RHO GTPase inhibitors. Non-cardiomyocytes exhibited the highest number of DOX-induced DNA double-strand breaks (DSB), which were efficiently repaired in vitro. By contrast, rather low levels of DSB were formed in cardiomyocytes, which however remained largely unrepaired. Moreover, DOX-induced apoptosis was detected only in non-cardiomyocytes but not in cardiomyocytes. Pharmacological inhibitors of RAC1 and CDC42 most efficiently attenuated DOX-induced DNA damage in all cell types examined in vitro. Consistently, immunohistochemical analyses revealed that the RAC1 inhibitor NSC23766 and the pan-RHO GTPase inhibitor lovastatin reduced the level of DOX-induced residual DNA damage in both cardiomyocytes and non-cardiomyocytes in vivo. Overall, we conclude that endothelial cells, fibroblasts and cardiomyocytes contribute to the pathophysiology of DOX-induced cardiotoxicity, with RAC1- and CDC42-regulated signaling pathways being especially relevant for DOX-stimulated DSB formation and DNA damage response (DDR) activation. Hence, we suggest dual targeting of RAC1/CDC42-dependent mechanisms in multiple cardiac cell types to mitigate DNA damage-dependent cardiac injury evoked by DOX-based anticancer therapy.

Molecular and cellular evidence for the impact of a hypertrophic cardiomyopathy-associated RAF1 variant on the structure and function of contractile machinery in bioartificial cardiac tissues.

Noonan syndrome (NS), the most common among RASopathies, is caused by germline variants in genes encoding components of the RAS-MAPK pathway. Distinct variants, including the recurrent Ser257Leu substitution in RAF1, are associated with severe hypertrophic cardiomyopathy (HCM). Here, we investigated the elusive mechanistic link between NS-associated RAF1S257L and HCM using three-dimensional cardiac bodies and bioartificial cardiac tissues generated from patient-derived induced pluripotent stem cells (iPSCs) harboring the pathogenic RAF1 c.770 C > T missense change. We characterize the molecular, structural, and functional consequences of aberrant RAF1-associated signaling on the cardiac models. Ultrastructural assessment of the sarcomere revealed a shortening of the I-bands along the Z disc area in both iPSC-derived RAF1S257L cardiomyocytes and myocardial tissue biopsies. The aforementioned changes correlated with the isoform shift of titin from a longer (N2BA) to a shorter isoform (N2B) that also affected the active force generation and contractile tensions. The genotype-phenotype correlation was confirmed using cardiomyocyte progeny of an isogenic gene-corrected RAF1S257L-iPSC line and was mainly reversed by MEK inhibition. Collectively, our findings uncovered a direct link between a RASopathy gene variant and the abnormal sarcomere structure resulting in a cardiac dysfunction that remarkably recapitulates the human disease.

Implementation of 2D Barcode Medication Labels and Smart Pumps in Pediatric Acute Care: Lessons Learned.

BACKGROUND: In pediatric intensive care, prescription, administration, and interpretation of drug doses are weight dependent. The use of standardized concentrations simplifies the preparation of drugs and increases safety. For safe administration as well as easy interpretation of intravenous drug dosing regimens with standardized concentrations, the display of weight-related dose rates on the infusion device is of pivotal significance. OBJECTIVES: We report on challenges in the implementation of a new information technology-supported medication workflow. The workflow was introduced on eight beds in the pediatric heart surgery intensive care unit as well as in the pediatric anesthesia at the University of Bonn Medical Center. The proposed workflow utilizes medication labels generated from prescription data from the electronic health record. The generated labels include a two-dimensional barcode to transfer data to the infusion devices. METHODS: Clinical and technical processes were agilely developed. The reliability of the system under real-life conditions was monitored. User satisfaction and potential for improvement were assessed. In addition, a structured survey among the nursing staff was performed. The questionnaire addressed usability as well as the end-users' perception of the effects on patient safety. RESULTS: The workflow has been applied 44,111 times during the pilot phase. A total of 114 known failures in the technical infrastructure were observed. The survey showed good ratings for usability and safety (median "school grade" 2 or B for patient safety, intelligibility, patient identification, and handling). The medical management of the involved acute care facilities rated the process as clearly beneficial regarding patient safety, suggesting a rollout to all pediatric intensive care areas. CONCLUSION: A medical information technology-supported medication workflow can increase user satisfaction and patient safety as perceived by the clinical end-users in pediatric acute care. The successful implementation benefits from an interdisciplinary team, active investigation of possible associated risks, and technical redundancy.

Phospholamban pentamerization increases sensitivity and dynamic range of cardiac relaxation.

AIMS: A key event in the regulation of cardiac contraction and relaxation is the phosphorylation of phospholamban (PLN) that relieves the inhibition of the sarco/endoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a). PLN exists in an equilibrium between monomers and pentamers. While only monomers can inhibit SERCA2a by direct interaction, the functional role of pentamers is still unclear. This study investigates the functional consequences of PLN pentamerization. METHODS AND RESULTS: We generated transgenic mouse models expressing either a PLN mutant that cannot form pentamers (TgAFA-PLN) or wild-type PLN (TgPLN) in a PLN-deficient background. TgAFA-PLN hearts demonstrated three-fold stronger phosphorylation of monomeric PLN, accelerated Ca2+ cycling of cardiomyocytes, and enhanced contraction and relaxation of sarcomeres and whole hearts in vivo. All of these effects were observed under baseline conditions and abrogated upon inhibition of protein kinase A (PKA). Mechanistically, far western kinase assays revealed that PLN pentamers are phosphorylated by PKA directly and independent of any subunit exchange for free monomers. In vitro phosphorylation of synthetic PLN demonstrated that pentamers even provide a preferred PKA substrate and compete with monomers for the kinase, thereby reducing monomer phosphorylation and maximizing SERCA2a inhibition. However, ß-adrenergic stimulation induced strong PLN monomer phosphorylation in TgPLN hearts and sharp acceleration of cardiomyocyte Ca2+ cycling and haemodynamic values that now were indistinguishable from TgAFA-PLN and PLN-KO hearts. The pathophysiological relevance of PLN pentamerization was evaluated using transverse aortic constriction (TAC) to induce left ventricular pressure overload. Compared to TgPLN, TgAFA-PLN mice demonstrated reduced survival after TAC, impaired cardiac haemodynamics, failure to respond to adrenergic stimulation, higher heart weight, and increased myocardial fibrosis. CONCLUSIONS: The findings show that PLN pentamerization greatly impacts on SERCA2a activity as it mediates the full range of PLN effects from maximum inhibition to full release of SERCA2a function. This regulation is important for myocardial adaptation to sustained pressure overload.

Diabetes disturbs functional adaptation of the remote myocardium after ischemia/reperfusion.

Diabetes mellitus type 2 is associated with adverse clinical outcome after myocardial infarction. To better understand the underlying causes we here investigated sarcomere protein function and its calcium-dependent regulation in the non-ischemic remote myocardium (RM) of diabetic mice (db/db) after transient occlusion of the left anterior descending coronary artery. Before and 24 h after surgery db/db and non-diabetic db/+ underwent magnetic resonance imaging followed by histological and biochemical analyses of heart tissue. Intracellular calcium transients and sarcomere function were measured in isolated cardiomyocytes. Active and passive force generation was assessed in skinned fibers and papillary muscle preparations. Before ischemia and reperfusion (I/R), beat-to-beat calcium cycling was depressed in diabetic cardiomyocytes. Nevertheless, contractile function was preserved owing to increased myofilament calcium sensitivity and higher responsiveness of myocardial force production to ß-adrenergic stimulation in db/db compared to db/+. In addition, protein kinase C activity was elevated in db/db hearts leading to strong phosphorylation of the titin PEVK region and increased titin-based tension of myofilaments. I/R impaired the function of whole hearts and RM sarcomeres in db/db to a larger extent than in non-diabetic db/+, and we identified several reasons. First, the amplitude and the kinetics of cardiomyocyte calcium transients were further reduced in the RM of db/db. Underlying causes involved altered expression of calcium regulatory proteins. Diabetes and I/R additively reduced phospholamban S16-phosphorylation by 80% (P < 000.1) leading to strong inhibition of the calcium ATPase SERCA2a. Second, titin stiffening was only observed in the RM of db/+, but not in the RM of db/db. Finally, db/db myofilament calcium sensitivity and force generation upon ß-adrenergic stimulation were no longer enhanced over db/+ in the RM. The findings demonstrate that impaired cardiomyocyte calcium cycling of db/db hearts is compensated by increased myofilament calcium sensitivity and increased titin-based stiffness prior to I/R. In contrast, sarcomere function of the RM 24 h after I/R is poor because both these compensatory mechanisms fail and myocyte calcium handling is further depressed.

4-Methylumbelliferone Attenuates Macrophage Invasion and Myocardial Remodeling in Pressure-Overloaded Mouse Hearts.

[Figure: see text].

Distinct contribution of Rac1 expression in cardiomyocytes to anthracycline-induced cardiac injury.

Cardiotoxicity is the dose limiting adverse effect of anthracycline-based anticancer therapy. Inhibitor studies point to Rac1 as therapeutic target to prevent anthracycline-induced cardiotoxicity. Yet, supporting genetic evidence is still missing and the pathophysiological relevance of different cardiac cell types is unclear. Here, we employed a tamoxifen-inducible cardiomyocyte-specific rac1 knock-out mouse model (Rac1flox/flox/MHC-MerCreMer) to investigate the impact of Rac1 expression in cardiomyocytes on cardiac injury following doxorubicin treatment. Distinctive stress responses resulting from doxorubicin treatment were observed, including upregulation of systemic markers of inflammation (IL-6, IL-1α, MCP-1), cardiac damage (ANP, BNP), DNA damage (i.e. DNA double-strand breaks (DSB)), DNA damage response (DDR) and cell death. Measuring the acute doxorubicin response, the serum level of MCP-1 was elevated, cardiac mRNA expression of Hsp70 was reduced and cardiac DDR was specifically enhanced in Rac1 deficient mice. The frequency of apoptotic heart cells remained unaffected by Rac1. Employing a subactue model, the number of doxorubicin-induced DSB was significantly reduced if Rac1 is absent. Yet, the doxorubicin-triggered increase in serum ANP and BNP levels remained unaffected by Rac1. Overall, knock-out of rac1 in cardiomyocytes confers partial protection against doxorubicin-induced cardiac injury. Hence, the data provide first genetic evidence supporting the view that pharmacological targeting of Rac1 is useful to widen the therapeutic window of anthracycline-based anticancer therapy by alleviating acute/subacute cardiomyocyte damage. Furthermore, considering published data obtained from the use of pharmacological Rac1 inhibitors, the results of our study indicate that Rac1-regulated functions of cardiac cell types others than cardiomyocytes additionally influence the adverse outcomes of anthracycline treatment on the heart.

Is there an effect of ischemic conditioning on myocardial contractile function following acute myocardial ischemia/reperfusion injury?

Ischemic conditioning induces cardioprotection; the final infarct size following a myocardial ischemic event is reduced. However, whether ischemic conditioning has long-term beneficial effects on myocardial contractile function following such an ischemic event needs further elucidation. To date, ex vivo studies have shown that ischemic conditioning improves the contractile recovery of isolated ventricular papillary muscle or atrial trabeculae following simulated ischemia. However, in vivo animal studies and studies in patients undergoing elective cardiac surgery show conflicting results. At the subcellular level, it is known that ischemic conditioning improved energy metabolism, preserved mitochondrial respiration, ATP production, and Ca2+ homeostasis in isolated mitochondria from the myocardium. Ischemic conditioning also presents with post-translational modifications of proteins in the contractile machinery of the myocardium. The beneficial effects on myocardial contractile function need further elucidation. This article is part of a Special Issue entitled: The power of metabolism: Linking energy supply and demand to contractile function edited by Torsten Doenst, Michael Schwarzer and Christine Des Rosiers.

Influence of Personalized Exercise Recommendations During Rehabilitation on the Sustainability of Objectively Measured Physical Activity Levels, Fatigue, and Fatigue-Related Biomarkers in Patients With Breast Cancer.

PURPOSE: Only one-third of patients with breast cancer reach the recommended activity level of 15 to 25 MET h/wk. The aim of this study was to determine the influence of personalized exercise recommendations during rehabilitation on patients' physical activity level, fatigue, and self-perceived cognitive function as well as on side effect-associated biomarkers. METHODS: Total metabolic rate, physical activity level, mean MET and steps, fatigue, self-perceived cognitive functioning , and biomarkers (C-reactive protein [CRP], interleukin 6, macrophage migration inhibiting factor [MIF], tumor necrosis factor [TNF]-α, brain-derived neurotrophic factor [BDNF], insulin-like growth factor 1 [IGF1]) were assessed in 60 patients with breast cancer in the aftercare phase before ( t0) and 8 months after ( t1) the intervention. The rehabilitation program consisted of an initial 3-week period and a 1-week stay after 4 months. RESULTS: Paired t-test indicated a statistically significant increase in all activity outcomes from t0 to t1. Patients' mean activity level significantly increased from 14.89 to 17.88 MET h/wk. Fatigue and self-perceived cognitive functioning significantly improved from t0 to t1. CRP levels significantly decreased, and BDNF as well as IGF1 levels significantly increased over time. Correlation analysis revealed statistically significant negative associations between fatigue, physical activity, and markers of inflammation (TNF-α and MIF). Furthermore, significant positive correlations between subjective cognitive functioning and all dimensions of fatigue were observed. CONCLUSIONS: The results support the importance of personalized exercise recommendations to increase physical activity levels in patients with breast cancer. Furthermore, the results highlighti an association between physical activity, fatigue, and inflammation.

Sustainable impact of an individualized exercise program on physical activity level and fatigue syndrome on breast cancer patients in two German rehabilitation centers.

PURPOSE: Although physical activity has been demonstrated to increase cancer survival in epidemiological studies, breast cancer patients tend toward inactivity after treatment. METHODS: Breast cancer patients were quasi-randomly allocated to two different groups, intervention (IG) and control (CG) groups. The intervention group (n = 111) received an individual 3-week exercise program with two additional 1-week inpatient stays after 4 and 8 months. At the end of the rehabilitation, a home-based exercise program was designed. The control group (n = 83) received a 3-week rehabilitation program and did not obtain any follow-up care. Patients from both groups were measured using questionnaires on physical activity, fatigue, and quality of life (QoL) at five time points, 4 months (t1), 8 months (t2), 12 months (t3), 18 months (t4), and 24 months (t5) after the beginning of the rehabilitation. RESULTS: After 2 years, the level of physical activity (total metabolic rate) increased significantly from 2733.16 ± 2547.95 (t0) to 4169.71 ± 3492.27 (t5) metabolic equivalent (MET)-min/week in the intervention group, but just slightly changed from 2858.38 ± 2393.79 (t0) to 2875.74 ± 2590.15 (t5) MET-min/week in the control group (means ± standard deviation). Furthermore, the internal group comparison showed significant differences after 2 years as well. These results came along with a significantly reduced fatigue syndrome and an increased health-related quality of life. CONCLUSIONS: The data indicate that an individual, according to their preferences, and physical-resource-adapted exercise program has a more sustainable impact on the physical activity level in breast cancer patients than the usual care. It is suggested that the rehabilitation program should be personalized for all breast cancer patients.

A 3-week multimodal intervention involving high-intensity interval training in female cancer survivors: a randomized controlled trial.

To compare the effects of a 3-week multimodal rehabilitation involving supervised high-intensity interval training (HIIT) on female breast cancer survivors with respect to key variables of aerobic fitness, body composition, energy expenditure, cancer-related fatigue, and quality of life to those of a standard multimodal rehabilitation program. A randomized controlled trial design was administered. Twenty-eight women, who had been treated for cancer were randomly assigned to either a group performing exercise of low-to-moderate intensity (LMIE; n = 14) or a group performing high-intensity interval training (HIIT; n = 14) as part of a 3-week multimodal rehabilitation program. No adverse events related to the exercise were reported. Work economy improved following both HIIT and LMIE, with improved peak oxygen uptake following LMIE. HIIT reduced mean total body fat mass with no change in body mass, muscle or fat-free mass (best P < 0.06). LMIE increased muscle and total fat-free body mass. Total energy expenditure (P = 0.45) did not change between the groups, whereas both improved quality of life to a similar high extent and lessened cancer-related fatigue. This randomized controlled study demonstrates that HIIT can be performed by female cancer survivors without adverse health effects. Here, HIIT and LMIE both improved work economy, quality of life and cancer-related fatigue, body composition or energy expenditure. Since the outcomes were similar, but HIIT takes less time, this may be a time-efficient strategy for improving certain aspects of the health of female cancer survivors.

Phospholamban pentamers attenuate PKA-dependent phosphorylation of monomers.

Phospholamban (PLN) is a key regulator of cardiac contraction and relaxation through its inhibition of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a). The inhibitory effect is attenuated upon protein kinase A (PKA) dependent phosphorylation of PLN. PLN exists in an equilibrium of pentamers and monomers. While monomers inhibit SERCA2a by direct interaction, the function of the pentamers is still unclear. Here, we tested the hypothesis that the PLN pentamer exhibits an important regulatory role by modifying PKA-dependent phosphorylation of inhibitory monomeric PLN subunits. Using Western blot analyses and antibodies specific for PKA-dependent phosphorylation of PLN, pentamers showed stronger signals than monomers both in transfected HEK293 cells and in cardiomyocytes. Upon activation of PKA, phosphorylation of protomers in the PLN pentamers increased faster and at lower levels of stimulation than PLN monomers, suggesting pentamers as the preferred PKA target. The comparison of phosphorylation patterns at different pentamer/monomer ratios revealed that pentamers delay phosphorylation of PLN monomers. A mechanistic explanation was provided by co-immunoprecipitation that suggested high affinity of PKA for PLN pentamers. Both monomers and pentamers were pulled down with SERCA2a indicating co-localization. Unlike pentamers, phosphorylated PLN monomers fully dissociated from the Ca(2+)-ATPase upon stimulation of PKA. These findings suggest a model where PLN pentamers reduce phosphorylation of monomers at baseline and delay monomer phosphorylation upon PKA stimulation leading to increased interaction of PLN monomers with SERCA2a.

Interleukin-6-dependent phenotypic modulation of cardiac fibroblasts after acute myocardial infarction.

Interleukin-6 (IL-6) is a multifunctional cytokine that orchestrates the immune response to a wide variety of pathophysiologic challenges but also contributes to tissue homeostasis. Furthermore, IL-6 is elevated in patients with acute myocardial infarction. Hyaluronan (HA) is an extracellular carbohydrate that has been implicated in wound healing and accumulates after acute myocardial infarction (AMI). Aim of this study was to investigate the involvement of IL-6 in the regulation of the HA-matrix in the early phase of infarct healing. In the present study, we show by the use of a blocking anti-IL-6 antibody, that endogenous IL-6 rapidly but transiently increased HA-synthase (HAS) 1 and 2 expression resulting in the formation of a HA-rich matrix acutely after AMI in mice. In vitro, IL-6 induced HAS1 and 2 via STAT3 phosphorylation in cardiac fibroblasts (CF) and supported a myofibroblastic phenotype in a HA-dependent manner. Furthermore, CCL5 and MCP1 expression were dependent on IL-6, HA-synthesis and the HA-receptor CD44 as shown in cultured CF derived from CD44 knockout mice. In vivo after AMI, blocking IL-6 decreased HA-matrix formation in the peri-infarct region and alpha-smooth muscle actin-positive myofibroblasts. Blocking IL-6 also reduced neutrophil infiltration in infarcted left ventricles. Moreover, treatment with the blocking IL-6 antibody reduced cardiac ejection fraction and increased infarct size 3 weeks after AMI. These findings support a functionally important role for IL-6 in CF by transiently inducing a HA-rich matrix that in turn promotes a myofibroblastic phenotype and inflammatory responses, and ultimately establishes a cardioprotective program after AMI.

ß-Myosin heavy chain variant Val606Met causes very mild hypertrophic cardiomyopathy in mice, but exacerbates HCM phenotypes in mice carrying other HCM mutations.

RATIONALE: Approximately 40% of hypertrophic cardiomyopathy (HCM) is caused by heterozygous missense mutations in ß-cardiac myosin heavy chain (ß-MHC). Associating disease phenotype with mutation is confounded by extensive background genetic and lifestyle/environmental differences between subjects even from the same family. OBJECTIVE: To characterize disease caused by ß-cardiac myosin heavy chain Val606Met substitution (VM) that has been identified in several HCM families with wide variation of clinical outcomes, in mice. METHODS AND RESULTS: Unlike 2 mouse lines bearing the malignant myosin mutations Arg453Cys (RC/+) or Arg719Trp (RW/+), VM/+ mice with an identical inbred genetic background lacked hallmarks of HCM such as left ventricular hypertrophy, disarray of myofibers, and interstitial fibrosis. Even homozygous VM/VM mice were indistinguishable from wild-type animals, whereas RC/RC- and RW/RW-mutant mice died within 9 days after birth. However, hypertrophic effects of the VM mutation were observed both in mice treated with cyclosporine, a known stimulator of the HCM response, and compound VM/RC heterozygous mice, which developed a severe HCM phenotype. In contrast to all heterozygous mutants, both systolic and diastolic function of VM/RC hearts was severely impaired already before the onset of cardiac remodeling. CONCLUSIONS: The VM mutation per se causes mild HCM-related phenotypes; however, in combination with other HCM activators it exacerbates the HCM phenotype. Double-mutant mice are suitable for assessing the severity of benign mutations.

Elevated rates of force development and MgATP binding in F764L and S532P myosin mutations causing dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a disease characterized by dilation of the ventricular chambers and reduced contractile function. We examined the contractile performance of chemically-skinned ventricular strips from two heterozygous murine models of DCM-causing missense mutations of myosin, F764L/+ and S532P/+, in an α-myosin heavy chain (MyHC) background. In Ca(2+)-activated skinned myocardial strips, the maximum developed tension in F764L/+ was only ~50% that of litter-mate controls (+/+). The F764L/+ also exhibited significantly reduced rigor stiffness, loaded shortening velocity and power output. Corresponding indices for S532P/+ strips were not different from controls. Manipulation of MgATP concentration in conjunction with measures of viscoelasticity, which provides estimates of myosin detachment rate 2πc, allowed us to probe the molecular basis of changes in crossbridge kinetics that occur with the myosin mutations. By examining the response of detachment rate to varying MgATP we found the rate of MgADP release was unaffected by the myosin mutations. However, MgATP binding rate was higher in the DCM groups compared to controls (422±109mM(-1)·s(-1) in F764L/+, 483±74mM(-1)·s(-1) in S532P/+ and 303±18mM(-1)·s(-1) in +/+). In addition, the rate constant of force development, 2πb, was significantly higher in DCM groups compared to controls (at 5mM MgATP: 36.9±4.9s(-1) in F764L/+, 32.9±4.5s(-1) in S532P/+ and 18.2±1.7s(-1) in +/+). These results suggest that elevated rates of force development and MgATP binding are features of cardiac myofilament function that underlie the development of DCM.

Kininogen deficiency protects from ischemic neurodegeneration in mice by reducing thrombosis, blood-brain barrier damage, and inflammation.

Thrombosis and inflammation are hallmarks of ischemic stroke still unamenable to therapeutic interventions. High-molecular-weight kininogen (KNG) is a central constituent of the contact-kinin system which represents an interface between thrombotic and inflammatory circuits and is critically involved in stroke development. Kng(-/-) mice are protected from thrombosis after artificial vessel wall injury and lack the proinflammatory mediator bradykinin. We investigated the consequences of KNG deficiency in models of ischemic stroke. Kng(-/-) mice of either sex subjected to transient middle cerebral artery occlusion developed dramatically smaller brain infarctions and less severe neurologic deficits without an increase in infarct-associated hemorrhage. This protective effect was preserved at later stages of infarction as well as in elderly mice. Targeting KNG reduced thrombus formation in ischemic vessels and improved cerebral blood flow, and reconstitution of KNG-deficient mice with human KNG or bradykinin restored clot deposition and infarct susceptibility. Moreover, mice deficient in KNG showed less severe blood-brain barrier damage and edema formation, and the local inflammatory response was reduced compared with controls. Because KNG appears to be instrumental in pathologic thrombus formation and inflammation but dispensable for hemostasis, KNG inhibition may offer a selective and safe strategy for combating stroke and other thromboembolic diseases.

Critical role for stromal interaction molecule 1 in cardiac hypertrophy.

BACKGROUND: Cardiomyocytes use Ca2+ not only in excitation-contraction coupling but also as a signaling molecule promoting, for example, cardiac hypertrophy. It is largely unclear how Ca2+ triggers signaling in cardiomyocytes in the presence of the rapid and large Ca2+ fluctuations that occur during excitation-contraction coupling. A potential route is store-operated Ca2+ entry, a drug-inducible mechanism for Ca2+ signaling that requires stromal interaction molecule 1 (STIM1). Store-operated Ca2+ entry can also be induced in cardiomyocytes, which prompted us to study STIM1-dependent Ca2+ entry with respect to cardiac hypertrophy in vitro and in vivo. METHODS AND RESULTS: Consistent with earlier reports, we found drug-inducible store-operated Ca2+ entry in neonatal rat cardiomyocytes, which was dependent on STIM1. Although this STIM1-dependent, drug-inducible store-operated Ca2+ entry was only marginal in adult cardiomyocytes isolated from control hearts, it increased significantly in cardiomyocytes isolated from adult rats that had developed compensated cardiac hypertrophy after abdominal aortic banding. Moreover, we detected an inwardly rectifying current in hypertrophic cardiomyocytes that occurs under native conditions (i.e., in the absence of drug-induced store depletion) and is dependent on STIM1. By manipulating its expression, we found STIM1 to be both sufficient and necessary for cardiomyocyte hypertrophy in vitro and in the adult heart in vivo. Stim1 silencing by adeno-associated viruses of serotype 9-mediated gene transfer protected rats from pressure overload-induced cardiac hypertrophy. CONCLUSION: By controlling a previously unrecognized sarcolemmal current, STIM1 promotes cardiac hypertrophy.

Cardiac hypertrophy: targeting Raf/MEK/ERK1/2-signaling.

Over the past two decades, basic research has revealed a complex network of regulatory mechanisms that control the ERK1/2-signaling cascade. ERK1/2 mediate cardiac hypertrophy, a major risk factor for the development of arrhythmias, heart failure and sudden death, but also beneficial effects, e.g. protection of the heart from cell death and ischemic injury. Selective targeting of these ambiguous ERK functions could provide a powerful tool in the treatment of cardiac disease. This short review will discuss new mechanistic insights into ERK1/2-dependent development of cardiac hypertrophy and the prospect to translate this knowledge into future therapeutic strategies.

Beta-trace protein--a marker of kidney function in children: "Original research communication-clinical investigation".

OBJECTIVES: To determine the pediatric reference interval for serum beta-trace protein (beta-TP) and to compare beta-TP with established LMW markers of GFR, i.e., cystatin C (CysC) and beta(2)-microglobulin (beta(2)-M). DESIGN AND METHODS: All three LMW markers were measured immunonephelometrically. In 106 children above the age of 2 years without evidence of kidney disease, non-parametric reference intervals were calculated. The relative rise of the GFR marker concentrations above the upper reference was studied in 107 samples from 96 patients covering the entire GFR range. RESULTS: Above 2 years, the reference range of beta-TP was constant at 0.43-1.04 mg/L. With decreasing Schwartz-GFR, there was a comparable rise in beta-TP and beta(2)-M, while CysC rose less in the group with GFR below 30 mL/min/1.73 m(2) (278+/-49% [CysC] versus 336+/-65% [beta-TP] and 342+/-76% [beta(2)-M]; p=0.043 and 0.027, respectively). CONCLUSIONS: These data confirm the potential of ss-TP as an endogenous GFR marker in children.

Comparison of the structures of triacylglycerols from native and transgenic medium-chain fatty acid-enriched rape seed oil by liquid chromatography--atmospheric pressure chemical ionization ion-trap mass spectrometry (LC-APCI-ITMS).

The sn position of fatty acids in seed oil lipids affects physiological function in pharmaceutical and dietary applications. In this study the composition of acyl-chain substituents in the sn positions of glycerol backbones in triacylglycerols (TAG) have been compared. TAG from native and transgenic medium-chain fatty acid-enriched rape seed oil were analyzed by reversed-phase high performance liquid chromatography coupled with online atmospheric-pressure chemical ionization ion-trap mass spectrometry. The transformation of summer rape with thioesterase and 3-ketoacyl-[ACP]-synthase genes of Cuphea lanceolata led to increased expression of 1.5% (w/w) caprylic acid (8:0), 6.7% (w/w) capric acid (10:0), 0.9% (w/w) lauric acid (12:0), and 0.2% (w/w) myristic acid (14:0). In contrast, linoleic (18:2n6) and alpha-linolenic acid (18:3n3) levels decreased compared with the original seed oil. The TAG sn position distribution of fatty acids was also modified. The original oil included eleven unique TAG species whereas the transgenic oil contained sixty. Twenty species were common to both oils. The transgenic oil included trioctadecenoyl-glycerol (18:1/18:1/18:1) and trioctadecatrienoyl-glycerol (18:3/18:3/18:3) whereas the native oil included only the latter. The transgenic TAG were dominated by combinations of caprylic, capric, lauric, myrisitic, palmitic (16:0), stearic (18:0), oleic (18:1n9), linoleic, arachidic (20:0), behenic (22:0), and lignoceric acids (24:0), which accounted for 52% of the total fat. In the original TAG palmitic, stearic, oleic, and linoleic acids accounted for 50% of the total fat. Medium-chain triacylglycerols with capric and lauric acids combined with stearic, oleic, linoleic, alpha-linolenic, arachidic, and gondoic acids (20:1n9) accounted for 25% of the transgenic oil. The medium-chain fatty acids were mainly integrated into the sn-1/3 position combined with the essential linoleic and alpha-linolenic acids at the sn-2 position. Eight species contained caprylic, capric, and lauric acids in the sn-2 position. The appearance of new TAG in the transgenic oil illustrates the extensive effect of genetic modification on fat metabolism by transformed plants and offers interesting possibilities for improved enteral applications.