Functional differences in human aortic valve interstitial cells from patients with varying calcific aortic valve disease.

Calcific aortic valve disease (CAVD) is characterized by progressive stiffening of aortic valve (AV) tissues, inducing stenosis and insufficiency. Bicuspid aortic valve (BAV) is a common congenital defect in which the AV has two leaflets rather than three, with BAV patients developing CAVD decades years earlier than in the general population. Current treatment for CAVD remains surgical replacement with its continued durability problems, as there are no pharmaceutical therapies or other alternative treatments available. Before such therapeutic approaches can be developed, a deeper understanding of CAVD disease mechanisms is clearly required. It is known that AV interstitial cells (AVICs) maintain the AV extracellular matrix and are typically quiescent in the normal state, transitioning into an activated, myofibroblast-like state during periods of growth or disease. One proposed mechanism of CAVD is the subsequent transition of AVICs into an osteoblast-like phenotype. A sensitive indicator of AVIC phenotypic state is enhanced basal contractility (tonus), so that AVICs from diseased AV will exhibit a higher basal tonus level. The goals of the present study were thus to assess the hypothesis that different human CAVD states lead to different biophysical AVIC states. To accomplish this, we characterized AVIC basal tonus behaviors from diseased human AV tissues embedded in 3D hydrogels. Established methods were utilized to track AVIC-induced gel displacements and shape changes after the application of Cytochalasin D (an actin polymerization inhibitor) to depolymerize the AVIC stress fibers. Results indicated that human diseased AVICs from the non-calcified region of TAVs were significantly more activated than AVICs from the corresponding calcified region. In addition, AVICs from the raphe region of BAVs were more activated than from the non-raphe region. Interestingly, we observed significantly greater basal tonus levels in females compared to males. Furthermore, the overall AVIC shape changes after Cytochalasin suggested that AVICs from TAVs and BAVs develop different stress fiber architectures. These findings are the first evidence of sex-specific differences in basal tonus state in human AVICs in varying disease states. Future studies are underway to quantify stress fiber mechanical behaviors to further elucidate CAVD disease mechanisms.

The effects of strain history on aortic valve interstitial cell activation in a 3D hydrogel environment.

Aortic valves (AVs) undergo unique stretch histories that include high rates and magnitudes. While major differences in deformation patterns have been observed between normal and congenitally defective bicuspid aortic valves (BAVs), the relation to underlying mechanisms of rapid disease onset in BAV patients remains unknown. To evaluate how the variations in stretch history affect AV interstitial cell (AVIC) activation, high-throughput methods were developed to impart varied cyclical biaxial stretch histories into 3D poly(ethylene) glycol hydrogels seeded with AVICs for 48 h. Specifically, a physiologically mimicking stretch history was compared to two stretch histories with varied peak stretch and stretch rate. Post-conditioned AVICs were imaged for nuclear shape, alpha smooth muscle actin (αSMA) and vimentin (VMN) polymerization, and small mothers against decapentaplegic homologs 2 and 3 (SMAD 2/3) nuclear activity. The results indicated that bulk gel deformations were accurately transduced to the AVICs. Lower peak stretches lead to increased αSMA polymerization. In contrast, VMN polymerization was a function of stretch rate, with SMAD 2/3 nuclear localization and nuclear shape also trending toward stretch rate dependency. Lower than physiological levels of stretch rate led to higher SMAD 2/3 activity, higher VMN polymerization around the nucleus, and lower nuclear elongation. αSMA polymerization did not correlate with VMN polymerization, SMAD 2/3 activity, nor nuclear shape. These results suggest that a negative feedback loop may form between SMAD 2/3, VMN, and nuclear shape to maintain AVIC homeostatic nuclear deformations, which is dependent on stretch rate. These novel results suggest that AVIC mechanobiological responses are sensitive to stretch history and provide insight into the mechanisms of AV disease.

Carvedilol induces biased ß1 adrenergic receptor-nitric oxide synthase 3-cyclic guanylyl monophosphate signalling to promote cardiac contractility.

AIMS: ß-blockers are widely used in therapy for heart failure and hypertension. ß-blockers are also known to evoke additional diversified pharmacological and physiological effects in patients. We aim to characterize the underlying molecular signalling and effects on cardiac inotropy induced by ß-blockers in animal hearts. METHODS AND RESULTS: Wild-type mice fed high-fat diet (HFD) were treated with carvedilol, metoprolol, or vehicle and echocardiogram analysis was performed. Heart tissues were used for biochemical and histological analyses. Cardiomyocytes were isolated from normal and HFD mice and rats for analysis of adrenergic signalling, calcium handling, contraction, and western blot. Biosensors were used to measure ß-blocker-induced cyclic guanosine monophosphate (cGMP) signal and protein kinase A activity in myocytes. Acute stimulation of myocytes with carvedilol promotes ß1 adrenergic receptor (ß1AR)- and protein kinase G (PKG)-dependent inotropic cardiac contractility with minimal increases in calcium amplitude. Carvedilol acts as a biased ligand to promote ß1AR coupling to a Gi-PI3K-Akt-nitric oxide synthase 3 (NOS3) cascade and induces robust ß1AR-cGMP-PKG signal. Deletion of NOS3 selectively blocks carvedilol, but not isoproterenol-induced ß1AR-dependent cGMP signal and inotropic contractility. Moreover, therapy with carvedilol restores inotropic contractility and sensitizes cardiac adrenergic reserves in diabetic mice with minimal impact in calcium signal, as well as reduced cell apoptosis and hypertrophy in diabetic hearts. CONCLUSION: These observations present a novel ß1AR-NOS3 signalling pathway to promote cardiac inotropy in the heart, indicating that this signalling paradigm may be targeted in therapy of heart diseases with reduced ejection fraction.

Biology and Biomechanics of the Heart Valve Extracellular Matrix.

Heart valves are dynamic structures that, in the average human, open and close over 100,000 times per day, and 3 × 109 times per lifetime to maintain unidirectional blood flow. Efficient, coordinated movement of the valve structures during the cardiac cycle is mediated by the intricate and sophisticated network of extracellular matrix (ECM) components that provide the necessary biomechanical properties to meet these mechanical demands. Organized in layers that accommodate passive functional movements of the valve leaflets, heart valve ECM is synthesized during embryonic development, and remodeled and maintained by resident cells throughout life. The failure of ECM organization compromises biomechanical function, and may lead to obstruction or leaking, which if left untreated can lead to heart failure. At present, effective treatment for heart valve dysfunction is limited and frequently ends with surgical repair or replacement, which comes with insuperable complications for many high-risk patients including aged and pediatric populations. Therefore, there is a critical need to fully appreciate the pathobiology of biomechanical valve failure in order to develop better, alternative therapies. To date, the majority of studies have focused on delineating valve disease mechanisms at the cellular level, namely the interstitial and endothelial lineages. However, less focus has been on the ECM, shown previously in other systems, to be a promising mechanism-inspired therapeutic target. Here, we highlight and review the biology and biomechanical contributions of key components of the heart valve ECM. Furthermore, we discuss how human diseases, including connective tissue disorders lead to aberrations in the abundance, organization and quality of these matrix proteins, resulting in instability of the valve infrastructure and gross functional impairment.

Phosphodiesterase 5 Associates With ß2 Adrenergic Receptor to Modulate Cardiac Function in Type 2 Diabetic Hearts.

Background In murine heart failure models and in humans with diabetic-related heart hypertrophy, inhibition of phosphodiesterase 5 (PDE5) by sildenafil improves cardiac outcomes. However, the mechanism by which sildenafil improves cardiac function is unclear. We have observed a relationship between PDE5 and ß2 adrenergic receptor (ß2AR), which is characterized here as a novel mechanistic axis by which sildenafil improves symptoms of diabetic cardiomyopathy. Methods and Results Wild-type and ß2AR knockout mice fed a high fat diet (HFD) were treated with sildenafil, and echocardiogram analysis was performed. Cardiomyocytes were isolated for excitation-contraction (E-C) coupling, fluorescence resonant energy transfer, and proximity ligation assays; while heart tissues were implemented for biochemical and histological analyses. PDE5 selectively associates with ß2AR, but not ß1 adrenergic receptor, and inhibition of PDE5 with sildenafil restores the impaired response to adrenergic stimulation in HFD mice and isolated ventriculomyocytes. Sildenafil enhances ß adrenergic receptor (ßAR)-stimulated cGMP and cAMP signals in HFD myocytes. Consequently, inhibition of PDE5 leads to protein kinase G-, and to a lesser extent, calcium/calmodulin-dependent kinase II-dependent improvements in adrenergically stimulated E-C coupling. Deletion of ß2AR abolishes sildenafil's effect. Although the PDE5-ß2AR association is not altered in HFD, phosphodiesterase 3 displays an increased association with the ß2AR-PDE5 complex in HFD myocytes. Conclusions This study elucidates mechanisms by which the ß2AR-PDE5 axis can be targeted for treating diabetic cardiomyopathy. Inhibition of PDE5 enhances ß2AR stimulation of cGMP and cAMP signals, as well as protein kinase G-dependent E-C coupling in HFD myocytes.

Illuminating cell signaling with genetically encoded FRET biosensors in adult mouse cardiomyocytes.

FRET-based biosensor experiments in adult cardiomyocytes are a powerful way of dissecting the spatiotemporal dynamics of the complicated signaling networks that regulate cardiac health and disease. However, although much information has been gleaned from FRET studies on cardiomyocytes from larger species, experiments on adult cardiomyocytes from mice have been difficult at best. Thus the large variety of genetic mouse models cannot be easily used for this type of study. Here we develop cell culture conditions for adult mouse cardiomyocytes that permit robust expression of adenoviral FRET biosensors and reproducible FRET experimentation. We find that addition of 6.25 µM blebbistatin or 20 µM (S)-nitro-blebbistatin to a minimal essential medium containing 10 mM HEPES and 0.2% BSA maintains morphology of cardiomyocytes from physiological, pathological, and transgenic mouse models for up to 50 h after adenoviral infection. This provides a 10-15-h time window to perform reproducible FRET readings using a variety of CFP/YFP sensors between 30 and 50 h postinfection. The culture is applicable to cardiomyocytes isolated from transgenic mouse models as well as models with cardiac diseases. Therefore, this study helps scientists to disentangle complicated signaling networks important in health and disease of cardiomyocytes.

Induction of anaplastic lymphoma kinase (ALK) as a novel mechanism of EGFR inhibitor resistance in head and neck squamous cell carcinoma patient-derived models.

Head and neck squamous cell carcinoma (HNSCC) currently only has one FDA-approved cancer intrinsic targeted therapy, the epidermal growth factor receptor (EGFR) inhibitor cetuximab, to which only approximately 10% of tumors are sensitive. In order to extend therapy options, we subjected patient-derived HNSCC cells to small-molecule inhibitor and siRNA screens, first, to find effective combination therapies with an EGFR inhibitor, and second, to determine a potential mechanistic basis for repurposing the FDA approved agents for HNSCC. The combinations of EGFR inhibitor with anaplastic lymphoma kinase (ALK) inhibitors demonstrated synergy at the highest ratio in our cohort, 4/8 HNSCC patients' derived tumor cells, and this corresponded with an effectiveness of siRNA targeting ALK combined with the EGFR inhibitor gefitinib. Co-targeting EGFR and ALK decreased HNSCC cell number and colony formation ability and increased annexin V staining. Because ALK expression is low and ALK fusions are infrequent in HNSCC, we hypothesized that gefitinib treatment could induce ALK expression. We show that ALK expression was induced in HNSCC patient-derived cells both in 2D and 3D patient-derived cell culture models, and in patient-derived xenografts in mice. Four different ALK inhibitors, including two (ceritinib and brigatinib) FDA approved for lung cancer, were effective in combination with gefitinib. Together, we identified induction of ALK by EGFR inhibitor as a novel mechanism potentially relevant to resistance to EGFR inhibitor, a high ratio of response of HNSCC patient-derived tumor cells to a combination of ALK and EGFR inhibitors, and applicability of repurposing ALK inhibitors to HNSCC that lack ALK aberrations.

Cross-Talk Between Insulin Signaling and G Protein-Coupled Receptors.

Diabetes is a major risk factor for the development of heart failure. One of the hallmarks of diabetes is insulin resistance associated with hyperinsulinemia. The literature shows that insulin and adrenergic signaling is intimately linked to each other; however, whether and how insulin may modulate cardiac adrenergic signaling and cardiac function remains unknown. Notably, recent studies have revealed that insulin receptor and ß2 adrenergic receptor (ß2AR) forms a membrane complex in animal hearts, bringing together the direct contact between 2 receptor signaling systems, and forming an integrated and dynamic network. Moreover, insulin can drive cardiac adrenergic desensitization via protein kinase A and G protein-receptor kinases phosphorylation of the ß2AR, which compromises adrenergic regulation of cardiac contractile function. In this review, we will explore the current state of knowledge linking insulin and G protein-coupled receptor signaling, especially ß-adrenergic receptor signaling in the heart, with emphasis on molecular insights regarding its role in diabetic cardiomyopathy.

Use of aromatherapy to promote a therapeutic nurse environment.

BACKGROUND: Workplace stress can affect nurse satisfaction. Aroma therapy as a therapeutic use of essential oil can be beneficial in reducing stress. PURPOSE: Assess perceived stress pre-post introduction of Essential Oil Lavender among registered nurses, charge nurses, and patient care technicians in a trauma intensive care unit, surgical specialty care unit and an orthopedic trauma unit. METHODS: Pre-post intervention with a quasi-experimental design. After a pre-survey, Essential Oil Lavender was diffused 24h per day over 30days in a designated nursing area that all nurses were not required to enter on each unit. RESULTS: Dependent sample t-test for "how often do nurses feel stressed a work in a typical week" revealed pre-survey mean 2.97 (SD=0.99) which was significantly higher than post-survey mean 2.70 (SD=0.92) with significance, t(69)=2.36, p=0.021, suggesting a difference in how often staff felt stressed at work in a typical week, trending down from "feeling stressed half of time" to "once in a while". There were no statistically significant differences in pre-post survey scores for TICU, TOU, or SSC as separate units. RELEVANCE: Use of essential oils to decrease work-related stress among nursing staff may improve retention, workplace environment, and increase nurse satisfaction.

Inhibiting Insulin-Mediated ß2-Adrenergic Receptor Activation Prevents Diabetes-Associated Cardiac Dysfunction.

BACKGROUND: Type 2 diabetes mellitus (DM) and obesity independently increase the risk of heart failure by incompletely understood mechanisms. We propose that hyperinsulinemia might promote adverse consequences in the hearts of subjects with type-2 DM and obesity. METHODS: High-fat diet feeding was used to induce obesity and DM in wild-type mice or mice lacking ß2-adrenergic receptor (ß2AR) or ß-arrestin2. Wild-type mice fed with high-fat diet were treated with a ß-blocker carvedilol or a GRK2 (G-protein-coupled receptor kinase 2) inhibitor. We examined signaling and cardiac contractile function. RESULTS: High-fat diet feeding selectively increases the expression of phosphodiesterase 4D (PDE4D) in mouse hearts, in concert with reduced protein kinase A phosphorylation of phospholamban, which contributes to systolic and diastolic dysfunction. The expression of PDE4D is also elevated in human hearts with DM. The induction of PDE4D expression is mediated by an insulin receptor, insulin receptor substrate, and GRK2 and ß-arrestin2-dependent transactivation of a ß2AR-extracellular regulated protein kinase signaling cascade. Thus, pharmacological inhibition of ß2AR or GRK2, or genetic deletion of ß2AR or ß-arrestin2, all significantly attenuate insulin-induced phosphorylation of extracellular regulated protein kinase and PDE4D induction to prevent DM-related contractile dysfunction. CONCLUSIONS: These studies elucidate a novel mechanism by which hyperinsulinemia contributes to heart failure by increasing PDE4D expression and identify ß2AR or GRK2 as plausible therapeutic targets for preventing or treating heart failure in subjects with type 2 DM.

Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure.

RATIONALE: In heart failure, myofilament proteins display abnormal phosphorylation, which contributes to contractile dysfunction. The mechanisms underlying the dysregulation of protein phosphorylation on myofilaments is not clear. OBJECTIVE: This study aims to understand the mechanisms underlying altered phosphorylation of myofilament proteins in heart failure. METHODS AND RESULTS: We generate a novel genetically encoded protein kinase A (PKA) biosensor anchored onto the myofilaments in rabbit cardiac myocytes to examine PKA activity at the myofilaments in responses to adrenergic stimulation. We show that PKA activity is shifted from the sarcolemma to the myofilaments in hypertrophic failing rabbit myocytes. In particular, the increased PKA activity on the myofilaments is because of an enhanced ß2 adrenergic receptor signal selectively directed to the myofilaments together with a reduced phosphodiesterase activity associated with the myofibrils. Mechanistically, the enhanced PKA activity on the myofilaments is associated with downregulation of caveolin-3 in the hypertrophic failing rabbit myocytes. Reintroduction of caveolin-3 in the failing myocytes is able to normalize the distribution of ß2 adrenergic receptor signal by preventing PKA signal access to the myofilaments and to restore contractile response to adrenergic stimulation. CONCLUSIONS: In hypertrophic rabbit myocytes, selectively enhanced ß2 adrenergic receptor signaling toward the myofilaments contributes to elevated PKA activity and PKA phosphorylation of myofilament proteins. Reintroduction of caveolin-3 is able to confine ß2 adrenergic receptor signaling and restore myocyte contractility in response to ß adrenergic stimulation.

Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS).

Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis.

A molecular case report: functional assay of tyrosine kinase inhibitors in cells from a patient's primary renal cell carcinoma.

Current therapies for Renal Cell Carcinoma favor vascular endothelial growth factor receptor (VEGF-R) tyrosine kinase (TK) inhibitors (TKIs). In theory, these are most applicable in tumors that have lost VHL-with subsequent stabilization of HIF and upregulation of VEGF. A subset of patients harbor primary-refractory disease, as in this case, where there was no evidence for loss of VHL or chromosome 3p. We evaluated molecular targeted agents in viable tumor cells cultured from a patient's clear cell renal cell carcinoma (RCC). Of 66 agents, only dasatinib, an inhibitor of Src tyrosine kinase, strongly reduced viability of the patient's cultured kidney tumor cells. Immunostaining of the original primary tumor revealed strong positivity for VHL and Src protein expression. Functional evaluation of a patient's tumor cells appears feasible in the setting of RCC.