Human atrial beta1L-adrenoceptor but not beta3-adrenoceptor activation increases force and Ca2+ current at physiological temperature

BACKGROUND AND PURPOSE It has been proposed that BRL37344, SR58611 and CGP12177 activate b3-adrenoceptors in human atrium to increase contractility and L-type Ca2+ current (ICa-L). b3-adrenoceptor agonists are potentially beneficial for the treatment of a variety of diseases but concomitant cardiostimulation would be potentially harmful. It has also been proposed that (-)-CGP12177 activates the low affinity binding site of the b1-adrenoceptor in human atrium. We therefore used BRL37344, SR58611 and (-)-CGP12177 with selective b-adrenoceptor subtype antagonists to clarify cardiostimulant b-adrenoceptor subtypes in human atrium. EXPERIMENTAL APPROACH Human right atrium was obtained from patients without heart failure undergoing coronary artery bypass or valve surgery. Cardiomyocytes were prepared to test BRL37344, SR58611 and CGP12177 effects on ICa-L. Contractile effects were determined on right atrial trabeculae. KEY RESULTS BRL37344 increased force which was antagonized by blockade of b1- and b2-adrenoceptors but not by blockade of b3-adrenoceptors with b3-adrenoceptor-selective L-748,337 (1 mM). The b3-adrenoceptor agonist SR58611 (1 nM–10 mM) did not affect atrial force. BRL37344 and SR58611 did not increase ICa-L at 37°C, but did at 24°C which was prevented by L-748,337. (-)-CGP12177 increased force and ICa-L at both 24°C and 37°C which was prevented by (-)-bupranolol (1–10 mM), but not L-748,337. CONCLUSIONS AND IMPLICATIONS We conclude that the inotropic responses to BRL37344 are mediated through b1- and b2-adrenoceptors. The inotropic and ICa-L responses to (-)-CGP12177 are mediated through the low affinity site b1L-adrenoceptor of the b1-adrenoceptor. b3-adrenoceptor-mediated increases in ICa-L are restricted to low temperatures. Human atrial b3-adrenoceptors do not change contractility and ICa-L at physiological temperature.

Ryanodine receptor phosphorylation in human heart failure

Heart failure is a complex disorder, characterized by activation of the sympathetic nervous system, leading to dysregulated Ca2+ homeostasis in cardiac myocytes and tissue remodeling. In a variety of diseases, cardiac malfunction is associated with aberrant fluxes of Ca2+ across both the surface membrane and the internal Ca2+ store, the sarcoplasmic reticulum (SR). One prominent hypothesis residues is that in heart failure, the activity of the ryanodine receptor (RyR2) Ca2+ release channel in the SR is increased due to excess phosphorylation and that this contributes to excess SR Ca2+ leak in diastole, reduced SR Ca2+ load and decreased contractility (Huke & Bers, 2008). There is controversy over which serine residues in RyR2 are hyperphosphorylated in animal models of heart failure and whether this is via the CaMKII or the PKA-linked signaling pathway. S2808, S2814 and S2030 in RyR2 have been variously claimed to be hyperphosphorylated. Our aim was to examine the degree of phosphorylation of these residues in RyR2 from failing human hearts. The use of human tissue was approved by the Human Research Ethics Committee, The Prince Charles Hospital, EC28114. Left ventricular tissue samples were obtained from an explanted heart of a patient with endstage heart failure (Emery Dreifuss Muscular Dystrophy with cardiomyopathy) and non-failing tissue was from a patient with cystic fibrosis undergoing heart-lung transplantation with no history of heart disease. SR vesicles were prepared as described by Laver et al. (1995) and examined with SDS-Page and Western Blot. Transferred proteins were probed with antibodies to detect total protein phosphorylation, phosphorylation of RyR2 serine residues S2808, S2814, S2030 and for the key proteins calsequestrin, triadin, junctin and FKBP12.6. To avoid membrane stripping artifact, each membrane was exposed to one phosphorylation-specific antibody and signal densities quantified using Bio-Rad Quantity One software. We found no distinguishable difference between failing and healthy hearts in the protein expression levels of RyR2, triadin, junctin or calsequestrin. We found an expected upregulation of total RyR2 phosphorylation in the failing heart sample, compared to a matched amount of RyR2 (quantified using densiometry) in healthy heart. Probing with antibodies detecting only the phosphorylated form of the specific RyR2 residues showed that the increase in total RyR2 phosphorylation in the failing heart was due to hyperphosphorylation of S2808 and S2814. We found that S2030 phosphorylation levels were unchanged in human heart failure. Interestingly, we found that S2030 has a basal level of phosphorylation in the healthy human heart, different from the absence of basal phosphorylation recently reported in rodent heart (Huke & Bers, 2008). Finally, preliminary results indicate that less FKBP 12.6 is associated with RyR2 in the failing heart, possibly as a consequence of PKA activation. In conclusion, residues S2808 and S2814 are hyperphosphorylated in human heart failure, presumably due to upregulation of the CaMKII and/or PKA signaling pathway as a result of chronic activation of the sympathetic nervous system. Such changes in RyR2 phosphorylation are believed to contribute to the leaky RyR2 phenotype associated with heart failure, which increases the incidence of arrhythmia and contributes to the severely impaired contractile performance of the failing heart. Huke S & Bers DM. (2008). Ryanodine receptor phosphorylation at serine 2030, 2808 and 2814 in rat cardiomyocytes. Biochemical and Biophysical Research Communications 376, 80-85. Laver DR, Roden LD, Ahern GP, Eager KR, Junankar PR & Dulhunty AF. (1995). Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle. Journal of Membrane Biology 147, 7-22. Proceedings

Patient education to prevent falls among older hospital inpatients : a randomized controlled trial

Background Falls are a common adverse event during hospitalization of older adults, and few interventions have been shown to prevent then. Methods This study was a 3-group randomized trial to evaluate the efficacy of 2 forms of multimedia patient education compared with usual care for the prevention of in-hospital falls. Older hospital patients (n = 1206) admitted to a mixture of acute (orthopedic, respiratory, and medical) and subacute (geriatric and neurorehabilitation) hospital wards at 2 Australian hospitals were recruited between January 2008 and April 2009. The interventions were a multimedia patient education program based on the health-belief model combined with trained health professional follow-up (complete program), multi-media patient education materials alone (materials only), and usual care (control). Falls data were collected by blinded research assistants by reviewing hospital incident reports, hand searching medical records, and conducting weekly patient interviews. Results Rates of falls per 1000 patient-days did not differ significantly between groups (control, 9.27; materials only, 8.61; and complete program, 7.63). However, there was a significant interaction between the intervention and presence of cognitive impairment. Falls were less frequent among cognitively intact patients in the complete program group (4.01 per 1000 patient-days) than among cognitively intact patients in the materials-only group (8.18 per 1000 patient-days) (adjusted hazard ratio, 0.51; 95% confidence interval, 0.28-0.93]) and control group (8.72 per 1000 patient-days) (adjusted hazard ratio, 0.43; 95% confidence interval, 0.24-0.78). Conclusion Multimedia patient education with trained health professional follow-up reduced falls among patients with intact cognitive function admitted to a range of hospital wards.

Evidence of disagreement between patient-perceived change and conventional longitudinal evaluation of change in health-related quality of life among older adults

Objective: To identify agreement levels between conventional longitudinal evaluation of change (post–pre) and patient-perceived change (post–then test) in health-related quality of life. Design: A prospective cohort investigation with two assessment points (baseline and six-month follow-up) was implemented. Setting: Community rehabilitation setting. Subjects: Frail older adults accessing community-based rehabilitation services. Intervention: Nil as part of this investigation. Main measures: Conventional longitudinal change in health-related quality of life was considered the difference between standard EQ-5D assessments completed at baseline and follow-up. To evaluate patient-perceived change a ‘then test’ was also completed at the follow-up assessment. This required participants to report (from their current perspective) how they believe their health-related quality of life was at baseline (using the EQ-5D). Patient-perceived change was considered the difference between ‘then test’ and standard follow-up EQ-5D assessments. Results: The mean (SD) age of participants was 78.8 (7.3). Of the 70 participants 62 (89%) of data sets were complete and included in analysis. Agreement between conventional (post–pre) and patient-perceived (post–then test) change was low to moderate (EQ-5D utility intraclass correlation coefficient (ICC)¼0.41, EQ-5D visual analogue scale (VAS) ICC¼0.21). Neither approach inferred greater change than the other (utility P¼0.925, VAS P¼0.506). Mean (95% confidence interval (CI)) conventional change in EQ-5D utility and VAS were 0.140 (0.045,0.236) and 8.8 (3.3,14.3) respectively, while patient-perceived change was 0.147 (0.055,0.238) and 6.4 (1.7,11.1) respectively. Conclusions: Substantial disagreement exists between conventional longitudinal evaluation of change in health-related quality of life and patient-perceived change in health-related quality of life (as measured using a then test) within individuals.

Patient preference for falls prevention in hospitals revealed through willingness-to-pay, contingent valuation survey

Rationale, aims and objectives: Patient preference for interventions aimed at preventing in-hospital falls has not previously been investigated. This study aims to contrast the amount patients are willing to pay to prevent falls through six intervention approaches. ----- ----- Methods: This was a cross-sectional willingness-to-pay (WTP), contingent valuation survey conducted among hospital inpatients (n = 125) during their first week on a geriatric rehabilitation unit in Queensland, Australia. Contingent valuation scenarios were constructed for six falls prevention interventions: a falls consultation, an exercise programme, a face-to-face education programme, a booklet and video education programme, hip protectors and a targeted, multifactorial intervention programme. The benefit to participants in terms of reduction in risk of falls was held constant (30% risk reduction) within each scenario. ----- ----- Results: Participants valued the targeted, multifactorial intervention programme the highest [mean WTP (95% CI): $(AUD)268 ($240, $296)], followed by the falls consultation [$215 ($196, $234)], exercise [$174 ($156, $191)], face-to-face education [$164 ($146, $182)], hip protector [$74 ($62, $87)] and booklet and video education interventions [$68 ($57, $80)]. A ‘cost of provision’ bias was identified, which adversely affected the valuation of the booklet and video education intervention. ----- ----- Conclusion: There may be considerable indirect and intangible costs associated with interventions to prevent falls in hospitals that can substantially affect patient preferences. These costs could substantially influence the ability of these interventions to generate a net benefit in a cost–benefit analysis.

Apoptosis is induced in Chlamydia trachomatis infected HEp-2 cells by the addition of a combination innate immune activation compounds and the inhibitor wedelolactone

Problem: Innate immune activation of human cells, for some intracellular pathogens, is advantageous for vacuole morphology and pathogenic viability. It is unknown whether innate immune activation is advantageous to Chlamydia trachomatis viability. ----- ----- Method of study: Innate immune activation of HEp-2 cells during Chlamydia infection was conducted using lipopolysaccharide (LPS), polyI:C, and wedelolactone (innate immune inhibitor) to investigate the impact of these conditions on viability of Chlamydia. ----- ----- Results: The addition of LPS and polyI:C to stimulate activation of the two distinct innate immune pathways (nuclear factor kappa beta and interferon regulatory factor) had no impact on the viability of Chlamydia. However, when compounds targeting either pathway were added in combination with the specific innate immune inhibitor (wedelolactone) a major impact on Chlamydia viability was observed. This impact was found to be due to the induction of apoptosis of the HEp-2 cells under these conditions. ----- ----- Conclusion: This is the first time that induction of apoptosis has been reported in C. trachomatis-infected cells when treated with a combination of innate immune activators and wedelolactone.

Par4 is a coactivator for a splice isoform-specific transcriptional activation domain in WT1

The Wilms’ tumor suppressor protein WT1 is a transcriptional regulator involved in differentiation and the regulation of cell growth. WT1 is subject to alternative splicing, one isoform including a 17–amino acid region that is specific to mammals. The function of this 17–amino acid insertion is not clear, however. Here, we describe a transcriptional activation domain in WT1 that is specific to the WT1 splice isoform that contains the 17–amino acid insertion. We show that the function of this domain in transcriptional activation is dependent on a specific interaction with the prostate apoptosis response factor par4. A mutation in WT1 found in Wilms’ tumor disturbs the interaction with par4 and disrupts the function of the activation domain. Analysis of WT1 derivatives in cells treated to induce par4 expression showed a strong correlation between the transcription function of the WT1 17–amino acid insertion and the ability of WT1 to regulate cell survival and proliferation. Our results provide a molecular mechanism by which alternative splicing of WT1 can regulate cell growth in development and disease.

Patient-specific computational biomechanics for simulating adolescent scoliosis surgery : predicted vs clinical correction for a preliminary series of six patients

Scoliosis is a spinal deformity that requires surgical correction in progressive cases. In order to optimize surgical outcomes, patient-specific finite element models are being developed by our group. In this paper, a single rod anterior correction procedure is simulated for a group of six scoliosis patients. For each patient, personalised model geometry was derived from low-dose CT scans, and clinically measured intra-operative corrective forces were applied. However, tissue material properties were not patient-specific, being derived from existing literature. Clinically, the patient group had a mean initial Cobb angle of 47.3 degrees, which was corrected to 17.5 degrees after surgery. The mean simulated post-operative Cobb angle for the group was 18.1 degrees. Although this represents good agreement between clinical and simulated corrections, the discrepancy between clinical and simulated Cobb angle for individual patients varied between -10.3 and +8.6 degrees, with only three of the six patients matching the clinical result to within accepted Cobb measurement error of +-5 degrees. The results of this study suggest that spinal tissue material properties play an important role in governing the correction obtained during surgery, and that patient-specific modelling approaches must address the question of how to prescribe patient-specific soft tissue properties for spine surgery simulation.

Active Notch1 confers a transformed phenotype to primary human melanocytes

The importance of mitogen-activated protein kinase signaling in melanoma is underscored by the prevalence of activating mutations in N-Ras and B-Raf, yet clinical development of inhibitors of this pathway has been largely ineffective, suggesting that alternative oncogenes may also promote melanoma. Notch is an interesting candidate that has only been correlated with melanoma development and progression; a thorough assessment of tumor-initiating effects of activated Notch on human melanocytes would clarify the mounting correlative evidence and perhaps identify a novel target for an otherwise untreatable disease. Analysis of a substantial panel of cell lines and patient lesions showed that Notch activity is significantly higher in melanomas than their nontransformed counterparts. The use of a constitutively active, truncated Notch transgene construct (N(IC)) was exploited to determine if Notch activation is a "driving" event in melanocytic transformation or instead a "passenger" event associated with melanoma progression. N(IC)-infected melanocytes displayed increased proliferative capacity and biological features more reminiscent of melanoma, such as dysregulated cell adhesion and migration. Gene expression analyses supported these observations and aided in the identification of MCAM, an adhesion molecule associated with acquisition of the malignant phenotype, as a direct target of Notch transactivation. N(IC)-positive melanocytes grew at clonal density, proliferated in limiting media conditions, and also exhibited anchorage-independent growth, suggesting that Notch alone is a transforming oncogene in human melanocytes, a phenomenon not previously described for any melanoma oncogene. This new information yields valuable insight into the basic epidemiology of melanoma and launches a realm of possibilities for drug intervention in this deadly disease.