Statement from the Asia Summit: Current state of arrhythmia care in Asia.

On May 27, 2022, the Asia Pacific Heart Rhythm Society and the Heart Rhythm Society convened a meeting of leaders from different professional societies of healthcare providers committed to arrhythmia care from the Asia Pacific region. The overriding goals of the meeting were to discuss clinical and health policy issues that face each country for providing care for patients with electrophysiologic issues, share experiences and best practices, and discuss potential future solutions. Participants were asked to address a series of questions in preparation for the meeting. The format of the meeting was a series of individual country reports presented by the leaders from each of the professional societies followed by open discussion. The recorded presentations from the Asia Summit can be accessed at https://www.heartrhythm365.org/URL/asiasummit-22. Three major themes arose from the discussion. First, the major clinical problems faced by different countries vary. Although atrial fibrillation is common throughout the region, the most important issues also include more general issues such as hypertension, rheumatic heart disease, tobacco abuse, and management of potentially life-threatening problems such as sudden cardiac arrest or profound bradycardia. Second, there is significant variability in the access to advanced arrhythmia care throughout the region due to differences in workforce availability, resources, drug availability, and national health policies. Third, collaboration in the area already occurs between individual countries, but no systematic regional method for working together is present.

HLA-DPB1 genotype variants predict DP molecule cell surface expression and DP donor specific antibody binding capacity.

The contribution of alloresponses to mismatched HLA-DP in solid organ transplantation and hematopoietic stem cell transplantation (HCT) has been well documented. Exploring the regulatory mechanisms of DPB1 alleles has become an important question to be answered. In this study, our initial investigation focused on examining the correlation between the rs9277534G/A SNP and DPB1 mRNA expression. The result showed that there was a significant increase in DPB1 mRNA expression in B lymphoblastoid cell lines (BLCLs) with the rs9277534GG genotype compared to rs9277534AA genotype. In addition, B cells with the rs9277534GG exhibited significantly higher DP protein expression than those carrying the rs9277534AA genotype in primary B cells. Furthermore, we observed a significant upregulation of DP expression in B cells following treatment with Interleukin 13 (IL-13) compared to untreated B cells carrying rs9277534GG-linked DPB1 alleles. Fluorescence in situ hybridization (FISH) analysis of DPB1 in BLCL demonstrated significant differences in both the cytoplasmic (p=0.0003) and nuclear (p=0.0001) localization of DP mRNA expression comparing DPB1*04:01 (rs9277534AA) and DPB1*05:01 (rs9277534GG) homozygous cells. The study of the correlation between differential DPB1 expression and long non-coding RNAs (lncRNAs) showed that lnc-HLA-DPB1-13:1 is strongly associated with DP expression (r=0.85), suggesting the potential involvement of lncRNA in regulating DP expression. The correlation of DP donor specific antibody (DSA) with B cell flow crossmatch (B-FCXM) results showed a better linear correlation of DP DSA against GG and AG donor cells (R2 = 0.4243, p=0.0025 and R2 = 0.6172, p=0.0003, respectively), compared to DSA against AA donor cells (R2 = 0.0649, p=0.4244). This explained why strong DP DSA with a low expression DP leads to negative B-FCXM. In conclusion, this study provides evidence supporting the involvement of lncRNA in modulating HLA-DP expression, shedding lights on the intricate regulatory mechanisms of DP, particularly under inflammatory conditions in transplantation.

Novel two-tiered developmental screening programme for Singaporean toddlers: a quality improvement report.

Early identification of developmental delays with timely intervention, especially before the age of 3 years, can improve child development. In Singapore, however, diagnosis and intervention for developmental delays occur at a median age of 44 months. As early detection and intervention depends on an effective developmental screening programme, we aimed to improve the detection of developmental delays before the age of 3 years in a primary care setting. We did this by implementing a novel two-tiered screening programme which uses three standardised screening tools (Parents' Evaluation of Developmental Status, PEDS-Developmental Milestones and Ages and Stages Questionnaire-3). We used quality improvement methods to integrate and optimise this two-tiered programme into the existing 9-month and 18-month screening schedule, with an additional screening at 30 months to replace the pre-existing 36-month screening of the National Child Health Surveillance Programme. A total of three Plan-Do-Study-Act cycles were performed to ensure programme feasibility and sustainability. They focused on adequately training the primary care nurses, targeting an 80% screening rate and aiming for 20 min screening tool administration time per child. We assessed the proportion of children referred to the child development units after positive screening for developmental concerns under the new programme, with a pre-post and with-without intervention comparison, and reviewed the screening rates and screening tool administration time. The proportion of 18-month old children referred for developmental concerns improved from 3.5%-7.1% over a 6-month period. For those who received further assessment by developmental specialists after the two-tiered screening, 100% received a definitive diagnosis of developmental delays, similar to the situation before programme introduction. Our quality improvement efforts facilitated successful integration of the two-tiered programme into the pre-existing screening schedule with minimal impact to the clinic workflow. While we highlight challenges in implementation that need to be addressed, our findings support a potential nationwide adoption of the two-tiered programme.

The Opsin 3/Teleost multiple tissue opsin system: mRNA localization in the retina and brain of medaka (Oryzias latipes).

The photoreceptor protein, opsin, is one of the major components for vision and photoreceptive function in animals. Although many opsins have been discovered from animal genomes, only a few non-image-forming functions mediated by opsins have been identified. Understanding the mRNA distribution of photoreceptor proteins is one crucial step in uncovering their photoreceptive function in animals. Here we focus on the medaka fish (Oryzias latipes) Opsin 3 (Opn3)/Teleost multiple opsin (Tmt) system, which constitutes a separate phylogenetic group, having putative blue light photoreceptors for non-image-forming functions. In medaka, there is one opn3 and five tmt-opsin orthologs. The expression pattern of the opn3/tmt-opsins in the retina and brain was investigated by in situ hybridization. mRNAs for opn3/tmt-opsins were distributed in the retinal ganglion cells as well as interneurons and specific brain nuclei. Specifically, hybridization signals were observed in the glutamate decarboxylase 1 (gad1)-expressing amacrine cells for opn3, tmt1a, tmt1b, and tmt2, in the caudal lobe of the cerebellum for tmt1b and tmt2, in the cranial nerve nuclei for opn3, tmt1a, tmt1b, tmt2, and in the rostral pars distalis (adenohypophysis) for opn3. These expression patterns suggest that blue light sensing in the fish retina and brain may be involved in the integration of visual inputs, vestibular function, somatosensation, motor outputs, and pituitary endocrine regulation. This article is protected by copyright. All rights reserved.

HLA Class I and Class II-Induced Intracellular Signaling and Molecular Associations in Primary Human Endothelial Cells.

The signaling capacity of HLA molecules in vascular cells has been well established. Intracellular signaling and association with the coreceptor integrin ß4 has been well-studied for HLA class I. However, little is known regarding HLA class II intracellular signaling in human endothelial cells. Investigation of HLA class II has been challenging due to the loss of HLA class II expression in cultured primary cells. Herein, we describe methods for inducing expression of endogenous alleles and loci of HLA class II molecules, as well as for studying intracellular signaling. This includes siRNA knockdown of proteins and coimmunoprecipitation of putative coreceptors for HLA in primary human aortic endothelial cells.

Prevalence of cardiovascular morbidities in Myanmar.

BACKGROUND: Cardiovascular diseases (CVDs) are now in a rising trend in South East Asia including Myanmar due to increase in major cardiovascular risk factors in both urban and rural areas, such as smoking, obesity and diabetes mellitus. It is necessary to determine CVD morbidities in Myanmar for planning of prevention and control activities for CVDs. The cross-sectional household survey was conducted in 2012 with 600 people aged 40 years and above in four townships (Kyauk-Tan, Mawlamyaing, Pathein and Pyay) and used face-to-face interview with standard questionnaire [Rose Angina Questionnaire and Questionnaire by European Cardiovascular Indicators Surveillance Set (EUROCISS) Research Group] to determine the level of reported CVD morbidities in adult population. RESULTS: Age of the study population ranged from 40 to 99 years with the mean age of 56 years. Seventy-one percent of the study population was women. Nine percent of the study population have suffered from angina according to Rose Angina Questionnaire. Prevalence of possible heart attack, stroke and heart failure was 7.5, 1.5 and 2.8%. Prevalence of hypertension was 51%. CONCLUSION: The CVD morbidities are high. There is a need for strengthening prevention and control activities of CVDs.

A multiparameter live cell imaging approach to monitor cyclic AMP and protein kinase A dynamics in parallel.

Parallel detection of signaling activities allows us to correlate activity dynamics between signaling molecules. In this review, we detail a multiparameter live cell imaging method to monitor 3',5'-cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) activities in parallel.

G6PD testing in support of treatment and elimination of malaria: recommendations for evaluation of G6PD tests.

Malaria elimination will be possible only with serious attempts to address asymptomatic infection and chronic infection by both Plasmodium falciparum and Plasmodium vivax. Currently available drugs that can completely clear a human of P. vivax (known as "radical cure"), and that can reduce transmission of malaria parasites, are those in the 8-aminoquinoline drug family, such as primaquine. Unfortunately, people with glucose-6-phosphate dehydrogenase (G6PD) deficiency risk having severe adverse reactions if exposed to these drugs at certain doses. G6PD deficiency is the most common human enzyme defect, affecting approximately 400 million people worldwide.Scaling up radical cure regimens will require testing for G6PD deficiency, at two levels: 1) the individual level to ensure safe case management, and 2) the population level to understand the risk in the local population to guide Plasmodium vivax treatment policy. Several technical and operational knowledge gaps must be addressed to expand access to G6PD deficiency testing and to ensure that a patient's G6PD status is known before deciding to administer an 8-aminoquinoline-based drug.In this report from a stakeholder meeting held in Thailand on October 4 and 5, 2012, G6PD testing in support of radical cure is discussed in detail. The focus is on challenges to the development and evaluation of G6PD diagnostic tests, and on challenges related to the operational aspects of implementing G6PD testing in support of radical cure. The report also describes recommendations for evaluation of diagnostic tests for G6PD deficiency in support of radical cure.

Cyclophilin A is required for angiotensin II-induced p47phox translocation to caveolae in vascular smooth muscle cells.

OBJECTIVE: Angiotensin II (AngII) signal transduction in vascular smooth muscle cells (VSMC) is mediated by reactive oxygen species (ROS). Cyclophilin A (CyPA) is a ubiquitously expressed cytosolic protein that possesses peptidyl-prolyl cis-trans isomerase activity, scaffold function, and significantly enhances AngII-induced ROS production in VSMC. We hypothesized that CyPA regulates AngII-induced ROS generation by promoting translocation of NADPH oxidase cytosolic subunit p47phox to caveolae of the plasma membrane. APPROACH AND RESULTS: Overexpression of CyPA in CyPA-deficient VSMC (CyPA(-/-)VSMC) significantly increased AngII-stimulated ROS production. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors (VAS2870 or diphenylene iodonium) significantly attenuated AngII-induced ROS production in CyPA and p47phox-overexpressing CyPA(-/-)VSMC. Cell fractionation and sucrose gradient analyses showed that AngII-induced p47phox plasma membrane translocation, specifically to the caveolae, was reduced in CyPA(-/-)VSMC compared with wild-type-VSMC. Immunofluorescence studies demonstrated that AngII increased p47phox and CyPA colocalization and translocation to the plasma membrane. In addition, immunoprecipitation of CyPA followed by immunoblotting of p47phox and actin showed that AngII increased CyPA and p47phox interaction. AngII-induced p47phox and actin cell cytoskeleton association was attenuated in CyPA(-/-)VSMC. Mechanistically, inhibition of p47phox phosphorylation and phox homology domain deletion attenuated CyPA and p47phox interaction. Finally, cyclosporine A and CyPA-peptidyl-prolyl cis-trans isomerase mutant, R55A, inhibited AngII-stimulated CyPA and p47phox association in VSMC, suggesting that peptidyl-prolyl cis-trans isomerase activity was required for their interaction. CONCLUSIONS: These findings provide the mechanism by which CyPA is an important regulator for AngII-induced ROS generation in VSMC through interaction with p47phox and cell cytoskeleton, which enhances the translocation of p47phox to caveolae.

Parallel tracking of cAMP and PKA signaling dynamics in living cells with FRET-based fluorescent biosensors.

Proper regulation of cellular functions relies upon a network of intricately interwoven signaling cascades in which multiple components must be tightly coordinated both spatially and temporally. To better understand how this network operates within the cellular environment, it is important to define the parameters of various signaling activities and to reveal the characteristic activity structure of the signaling cascades. This task calls for molecular tools capable of parallelly tracking multiple activities in cellular time and space with high sensitivity and specificity. Here, we present new biosensors developed based on two conveniently co-imageable FRET pairs consisting of CFP-RFP and YFP-RFP, specifically Cerulean-mCherry and mVenus-mCherry, for parallel monitoring of PKA activity and cAMP dynamics in living cells. These biosensors provide orthogonal readouts in co-imaging experiments and display a comparable dynamic range to their cyan-yellow counterparts. Characterization of signaling responses induced by a panel of pathway activators using this co-imaging approach reveals distinct activity and kinetic patterns of cAMP and PKA dynamics arising from differential signal activation and processing. This technique is therefore useful for parallel monitoring of multiple signaling dynamics in single living cells and represents a promising approach towards a more precise characterization of the activity structure of the dynamic cellular signaling network.

Cyclophilin A promotes cardiac hypertrophy in apolipoprotein E-deficient mice.

OBJECTIVE: Cyclophilin A (CyPA, encoded by Ppia) is a proinflammatory protein secreted in response to oxidative stress in mice and humans. We recently demonstrated that CyPA increased angiotensin II (Ang II)-induced reactive oxygen species (ROS) production in the aortas of apolipoprotein E (Apoe)-/- mice. In this study, we sought to evaluate the role of CyPA in Ang II-induced cardiac hypertrophy. METHODS AND RESULTS: Cardiac hypertrophy was not significantly different between Ppia+/+ and Ppia-/- mice infused with Ang II (1000 ng/min per kg for 4 weeks). Therefore, we investigated the effect of CyPA under conditions of high ROS and inflammation using the Apoe-/- mice. In contrast to Apoe-/- mice, Apoe-/-Ppia-/- mice exhibited significantly less Ang II-induced cardiac hypertrophy. Bone marrow cell transplantation showed that CyPA in cells intrinsic to the heart plays an important role in the cardiac hypertrophic response. Ang II-induced ROS production, cardiac fibroblast proliferation, and cardiac fibroblast migration were markedly decreased in Apoe-/-Ppia-/- cardiac fibroblasts. Furthermore, CyPA directly induced the hypertrophy of cultured neonatal cardiac myocytes. CONCLUSIONS: CyPA is required for Ang II-mediated cardiac hypertrophy by directly potentiating ROS production, stimulating the proliferation and migration of cardiac fibroblasts, and promoting cardiac myocyte hypertrophy.

Signaling diversity of PKA achieved via a Ca2+-cAMP-PKA oscillatory circuit.

Many protein kinases are key nodal signaling molecules that regulate a wide range of cellular functions. These functions may require complex spatiotemporal regulation of kinase activities. Here, we show that protein kinase A (PKA), Ca(2+) and cyclic AMP (cAMP) oscillate in sync in insulin-secreting MIN6 beta cells, forming a highly integrated oscillatory circuit. We found that PKA activity was essential for this oscillatory circuit and was capable of not only initiating the signaling oscillations but also modulating their frequency, thereby diversifying the spatiotemporal control of downstream signaling. Our findings suggest that exquisite temporal control of kinase activity, mediated via signaling circuits resulting from cross-regulation of signaling pathways, can encode diverse inputs into temporal parameters such as oscillation frequency, which in turn contribute to proper regulation of complex cellular functions in a context-dependent manner.

Status of radiation protection in various interventional cardiology procedures in the Asia Pacific region.

OBJECTIVE: Increasing use of interventional procedures in cardiology with unknown levels of radiation protection in many countries of Asia-Pacific region necessitates the need for status assessment. The study was part of an International Atomic Energy Agency (IAEA) project for achieving improved radiation protection in interventional cardiology (IC) in developing countries. DESIGN: The survey covers 18 cardiac catheterisation laboratories in seven countries (Bangladesh, India, Malaysia, Myanmar, Singapore, Thailand and Vietnam). An important step was the creation of the 'Asian network of Cardiologists in Radiation Protection' and a newsletter. Data were collected on: radiation protection tools, number of IC laboratories, and annual number of various IC paediatric and adult procedures in the hospital and in the country. Patient radiation dose data were collected in terms of Kerma Area Product (KAP) and cumulative dose (CD). RESULTS: It is encouraging that protection devices for staff are largely used in the routine practice. Only 39% of the angiographic machines were equipped with a KAP meter. Operators' initial lack of awareness on radiation-protection optimisation improved significantly after participation in IAEA radiation-protection training. Only two out of five countries reporting patient percutaneous coronary intervention radiation-dose data were fully within the international guidance levels. Data from 51 patients who underwent multiple therapeutic procedures (median 2-3) indicated a total KAP reaching 995 Gy.cm(2) (range 10.1-995) and CD 15.1 Gy (range 0.4-15.1), stressing the importance of dose monitoring and optimisation. CONCLUSIONS: There is a need for interventional cardiology societies to play an active role in training actions and implementation of radiation protection.

Cyclophilin A is an inflammatory mediator that promotes atherosclerosis in apolipoprotein E-deficient mice.

Cyclophilin A (CyPA; encoded by Ppia) is a ubiquitously expressed protein secreted in response to inflammatory stimuli. CyPA stimulates vascular smooth muscle cell migration and proliferation, endothelial cell adhesion molecule expression, and inflammatory cell chemotaxis. Given these activities, we hypothesized that CyPA would promote atherosclerosis. Apolipoprotein E-deficient (Apoe(-/-)) mice fed a high-cholesterol diet for 16 wk developed more severe atherosclerosis compared with Apoe(-/-)Ppia(-/-) mice. Moreover, CyPA deficiency was associated with decreased low-density lipoprotein uptake, VCAM-1 (vascular cell adhesion molecule 1) expression, apoptosis, and increased eNOS (endothelial nitric oxide synthase) expression. To understand the vascular role of CyPA in atherosclerosis development, bone marrow (BM) cell transplantation was performed. Atherosclerosis was greater in Apoe(-/-) mice compared with Apoe(-/-)Ppia(-/-) mice after reconstitution with CyPA(+/+) BM cells, indicating that vascular-derived CyPA plays a crucial role in the progression of atherosclerosis. These data define a role for CyPA in atherosclerosis and suggest CyPA as a target for cardiovascular therapies.

Visualization of JNK activity dynamics with a genetically encoded fluorescent biosensor.

The signaling pathway mediated by JNK transduces different types of signals, such as stress stimuli and cytokines, into functional responses that mediate apoptosis, as well as proliferation, differentiation, and inflammation. To better characterize the dynamic information flow and signal processing of this pathway in the cellular context, a genetically encoded, fluorescent protein-based biosensor was engineered to detect endogenous JNK activity. This biosensor, named JNKAR1 (for JNK activity reporter), specifically detects stress- (ribotoxic and osmotic) and cytokine- (TNF-alpha) induced JNK activity in living cells with a 15 to 30% increase in the yellow-to-cyan emission ratio because of a phosphorylation-dependent increase in FRET between two fluorescent proteins. JNK activity was detected not only in the cytoplasm, but also in the nucleus, mitochondria, and plasma membrane with similar kinetics after induction of ribotoxic stress by anisomycin, suggesting relatively rapid signal propagation to the nuclear, mitochondrial, and plasma membrane compartments. Furthermore, quantitative single-cell analysis revealed that anisomycin-induced JNK activity exhibited ultrasensitivity, sustainability, and bimodality, features that are consistent with behaviors of bistable systems. The development of JNKAR1, therefore, laid a foundation for evaluating the signaling properties and behaviors of the JNK cascade in single living cells.

Angiotensin II-induced osteopontin expression in vascular smooth muscle cells involves Gq/11, Ras, ERK, Src and Ets-1.

Recent studies suggest that osteopontin (OPN) plays a critical role in the progression of atherosclerotic plaques and that angiotensin II (Ang II) is a potent upregulator of OPN expression. The goal of the present study was to characterize the signaling mechanisms whereby Ang II increases OPN expression in vascular smooth muscle cells (VSMC). YM-254890, a specific inhibitor of G(q/11), potently suppressed Ang II-induced OPN expression and ERK1/2 activation. Among dominant-negative (DN) mutants of small G proteins, only DN-Ras suppressed Ang II-induced OPN promoter activity. DN-MEK1 markedly inhibited Ang II-induced OPN promoter activity, while neither DN-JNK nor DN-p38 MAP kinase had any effect. DN-Src and DN-Fyn suppressed Ang II-induced OPN promoter activity. YM-254890 inhibited Ang II-induced Src and Ras activation, and PP2, a selective inhibitor for the Src kinase family, inhibited Ras activation, suggesting that the G(q/11)-Src-Ras axis is the upstream signaling cascade for Ang II-induced OPN expression. Finally, small interfering RNA against Ets-1 suppressed Ang II-induced OPN expression. In conclusion, these data suggest that Ang II-induced OPN expression in VSMC is mediated by signaling cascades involving G(q/11) the Ras-ERK axis, and the Src kinase family, and by the transcription factor, Ets-1. These signaling molecules may represent therapeutic targets for the prevention of pathological vascular remodeling.

Role of osteoprotegerin in arterial calcification: development of new animal model.

OBJECTIVES: Enhanced osteoclastogenesis, increased bone resorption, and osteoporosis have been reported in osteoprotegerin-deficient (OPG (-/-)) mice. OPG (-/-) mice available in Japan usually do not show vascular calcification. We have found that arterial calcification can be quickly induced by a simple procedure in OPG (-/-) mice. METHODS AND RESULTS: Male OPG (-/-), OPG (+/-), and OPG (+/+) mice were fed a high phosphate diet from 6 to 10 weeks after birth, and then 1alpha,25-dihydroxyvitamin D3 (calcitriol) was injected for 3 days. We found that severe calcification developed in the media of the aorta in OPG (-/-) mice. Under electron microscopy, calcium deposits were observed in the cytoplasm and extracellular matrix of vascular smooth muscle cells (VSMCs). Neither apoptosis of VSMCs nor infiltration of macrophages was observed. Alkaline phosphatase (ALP) activity of aortic tissue correlated with the calcified lesion area. Mouse aorta and bone extracts revealed an identical pattern by ALP electrophoresis. CONCLUSIONS: Our results demonstrated that OPG had anticalcification activity in the aorta, probably through the downregulation of ALP activity. Because the time course of arterial calcification after the injection of calcitriol is accurate and reproducible, this mouse model will be useful for further investigation of vascular calcification.