Sean Lal

Antibody arrays: an embryonic but rapidly growing technology.

Protein arrays are now an attractive proposition as they can measure a diverse range of protein interactions not possible with traditional DNA arrays. Antibody arrays are a specific subset of this technology. Originally conceived as multi-analyte detectors, antibody arrays are now used in a wide variety of applications. For instance, the potential of this technology to diagnose human diseases, such as leukemia, breast cancer and, potentially, heart failure, has stimulated much interest. Furthermore, identification of new protein targets in particular disease states will prove to be an invaluable tool in drug discovery and development. Patient prognosis and treatment are also potential applications of the technology. Antibody arrays have proved to be dynamic in response to these broad range of possibilities. This review examines variations in antibody array design and discusses current and potential applications of this novel and interesting technology.

Genome-wide identification of expression quantitative trait loci (eQTLs) in human heart.

In recent years genome-wide association studies (GWAS) have uncovered numerous chromosomal loci associated with various electrocardiographic traits and cardiac arrhythmia predisposition. A considerable fraction of these loci lie within inter-genic regions. The underlying trait-associated variants likely reside in regulatory regions and exert their effect by modulating gene expression. Hence, the key to unraveling the molecular mechanisms underlying these cardiac traits is to interrogate variants for association with differential transcript abundance by expression quantitative trait locus (eQTL) analysis. In this study we conducted an eQTL analysis of human heart. For a total of 129 left ventricular samples that were collected from non-diseased human donor hearts, genome-wide transcript abundance and genotyping was determined using microarrays. Each of the 18,402 transcripts and 897,683 SNP genotypes that remained after pre-processing and stringent quality control were tested for eQTL effects. We identified 771 eQTLs, regulating 429 unique transcripts. Overlaying these eQTLs with cardiac GWAS loci identified novel candidates for studies aimed at elucidating the functional and transcriptional impact of these loci. Thus, this work provides for the first time a comprehensive eQTL map of human heart: a powerful and unique resource that enables systems genetics approaches for the study of cardiac traits.

Introduction of a new observation chart and education programme is associated with higher rates of vital-sign ascertainment in hospital wards

Introduction Local and national awareness of the need to improve the recognition and response to the clinical deterioration of hospital inpatients is high. The authors designed and implemented a programme to improve recognition of deteriorating patients in their hospital; a new observation chart for vital signs was one of the major elements. The aim of the study is to evaluate the impact of the new chart and associated education programme on the completeness of vital-sign recording in ward areas. Methods The setting is a university-affiliated teaching hospital in Sydney, Australia. Three study periods, each lasting 14 days (preintervention, 2 weeks postintervention, 3 months postintervention), were carried out in three wards. The new observation chart was supported by an education programme. The primary outcome measures were the ascertainment rates of individual vital signs as a proportion of total observation sets. Results Documentation of respiratory rate increased from 47.8% to 97.8% (p<0.001) and was sustained at 3 months postintervention (98.5%). Collection of a full set of vital signs also improved by a similar magnitude. Basic neurological observation for all patients was introduced in the new chart; the uptake of this was very good (93.1%). Ascertainment rates of blood pressure and oxygen saturation also increased by small but significant amounts from good baseline rates of 97% or higher. Conclusion The introduction of a new observation chart, and education regarding its use and importance, was associated with a major improvement in the recording of respiratory rate and other vital signs.

Ablation of cardiac myosin binding protein-C disrupts the super-relaxed state of myosin in murine cardiomyocytes

Cardiac myosin binding protein-C (cMyBP-C) is a structural and regulatory component of cardiac thick filaments. It is observed in electron micrographs as seven to nine transverse stripes in the central portion of each half of the A band. Its C-terminus binds tightly to the myosin rod and contributes to thick filament structure, while the N-terminus can bind both myosin S2 and actin, influencing their structure and function. Mutations in the MYBPC3 gene (encoding cMyBP-C) are commonly associated with hypertrophic cardiomyopathy (HCM). In cardiac cells there exists a population of myosin heads in the super-relaxed (SRX) state, which are bound to the thick filament core with a highly inhibited ATPase activity. This report examines the role cMyBP-C plays in regulating the population of the SRX state of cardiac myosin by using an assay that measures single ATP turnover of myosin. We report a significant decrease in the proportion of myosin heads in the SRX state in homozygous cMyBP-C knockout mice, however heterozygous cMyBP-C knockout mice do not significantly differ from the wild type. A smaller, non-significant decrease is observed when thoracic aortic constriction is used to induce cardiac hypertrophy in mutation negative mice. These results support the proposal that cMyBP-C stabilises the thick filament and that the loss of cMyBP-C results in an untethering of myosin heads. This results in an increased myosin ATP turnover, further consolidating the relationship between thick filament structure and the myosin ATPase.

Using antibody arrays to detect microparticles from acute coronary syndrome patients based on cluster of differentiation (CD) antigen expression.

Microparticles circulate in plasma and have recently emerged as potential inflammatory markers in cardiovascular disease. They are fragments of cell membranes that express cluster of differentiation (CD) antigens and are present at elevated levels in patients with acute coronary syndrome. We have developed a novel method for the rapid detection of microparticles in plasma using a fluorescence-based antibody array system. Isolated microparticles are captured on anti-CD antibody spots immobilized on a nitrocellulose membrane. These CD antibodies are directed against extracellular epitopes, whereas the intracellular exposed surface of the microparticles is labeled with a fluorescent anti-annexin antibody. The array is then scanned and quantified. A pilot study was undertaken to compare microparticle CD antigen expression in acute coronary syndrome and healthy subjects. Ten CD antigens (44, 45, 54, 62E, 79, 102, 117, 130, 138, and 154) had significantly increased expression in the disease group relative to the healthy controls. These results were then verified using flow cytometry and scanning electron microscopy. Although we have focused our analysis on changes in microparticle CD antigen expression, this technique is amenable to analyzing other surface markers. Microparticles can be derived from a wide variety of cell types, so selection of the primary antibody can be tailored to the cell origin that is to be investigated.

MYBPC3 mutations are associated with a reduced super-relaxed state in patients with hypertrophic cardiomyopathy

The “super-relaxed state” (SRX) of myosin represents a ‘reserve’ of motors in the heart. Myosin heads in the SRX are bound to the thick filament and have a very low ATPase rate. Changes in the SRX are likely to modulate cardiac contractility. We previously demonstrated that the SRX is significantly reduced in mouse cardiomyocytes lacking cardiac myosin binding protein–C (cMyBP-C). Here, we report the effect of mutations in the cMyBP-C gene (MYBPC3) using samples from human patients with hypertrophic cardiomyopathy (HCM). Left ventricular (LV) samples from 11 HCM patients were obtained following myectomy surgery to relieve LV outflow tract obstruction. HCM samples were genotyped as either MYBPC3 mutation positive (MYBPC3mut) or negative (HCMsmn) and were compared to eight non-failing donor hearts. Compared to donors, only MYBPC3mut samples display a significantly diminished SRX, characterised by a decrease in both the number of myosin heads in the SRX and the lifetime of ATP turnover. These changes were not observed in HCMsmn samples. There was a positive correlation (p < 0.01) between the expression of cMyBP-C and the proportion of myosin heads in the SRX state, suggesting cMyBP-C modulates and maintains the SRX. Phosphorylation of the myosin regulatory light chain in MYBPC3mut samples was significantly decreased compared to the other groups, suggesting a potential mechanism to compensate for the diminished SRX. We conclude that by altering both contractility and sarcomeric energy requirements, a reduced SRX may be an important disease mechanism in patients with MYBPC3 mutations.

Increased collagen within the transverse tubules in human heart failure.

Aims In heart failure transverse-tubule (t-tubule) remodelling disrupts calcium release, and contraction. T-tubules in human failing hearts exhibit increased labelling by wheat germ agglutinin (WGA), a lectin that binds to the dystrophin-associated glycoprotein complex. We hypothesized changes in this complex may explain the increased WGA labelling and contribute to t-tubule remodelling in the failing human heart. In this study we sought to identify the molecules responsible for this increased WGA labelling. Methods and results Confocal and super-resolution fluorescence microscopy and proteomic analyses were used to quantify left ventricle samples from healthy donors and patients with idiopathic dilated cardiomyopathy (IDCM). Confocal microscopy demonstrated both WGA and dystrophin were located at t-tubules. Super-resolution microscopy revealed that WGA labelling of t-tubules is largely located within the lumen while dystrophin was restricted to near the sarcolemma. Western blots probed with WGA reveal a 5.7-fold increase in a 140 kDa band in IDCM. Mass spectrometry identified this band as type VI collagen (Col-VI) comprised of α1(VI), α2(VI), and α3(VI) chains. Pertinently, mutations in Col-VI cause muscular dystrophy. Western blotting identified a 2.4-fold increased expression and 3.2-fold increased WGA binding of Col-VI in IDCM. Confocal images showed that Col-VI is located in the t-tubules and that their diameter increased in the IDCM samples. Super-resolution imaging revealed Col-VI was restricted to the t-tubule lumen where increases were associated with displacement in the sarcolemma as identified from dystrophin labelling. Samples were also labelled for type I, III, and IV collagen. Both confocal and super-resolution imaging identified that these collagens were also present within t-tubule lumen. Conclusion Increased expression and labelling of collagen in IDCM samples indicates fibrosis may contribute to t-tubule remodelling in human heart failure.

Best practice BioBanking of human heart tissue

This review provides a guide to researchers who wish to establish a biobank. It also gives practical advice to investigators seeking access to samples of healthy or diseased human hearts. We begin with a brief history of the Sydney Heart Bank (SHB) from when it began in 1989, including the pivotal role played by the late Victor Chang. We discuss our standard operating procedures for tissue collection which include cryopreservation and the quality assurance needed to maintain the long-term molecular and cellular integrity of the samples. The SHB now contains about 16,000 heart samples derived from over 450 patients who underwent isotopic heart transplant procedures and from over 100 healthy organ donors. These enable us to provide samples from a wide range of categories of heart failure. So far, we have delivered heart samples to more than 50 laboratories over two decades, and we answer their most frequently asked questions. Other SHB services include the development of tissue microarrays (TMA). These enable end users to perform preliminary examinations of the expression and localisation of target molecules in diseased or aging donor hearts, all in a single section of the TMA. Finally, the processes involved in managing tissue requests from external users and logistics considerations for the shipment of human tissue are discussed in detail.

Abnormal contractility in human heart myofibrils from patients with dilated cardiomyopathy due to mutations in TTN and contractile protein genes

Dilated cardiomyopathy (DCM) is an important cause of heart failure. Single gene mutations in at least 50 genes have been proposed to account for 25–50% of DCM cases and up to 25% of inherited DCM has been attributed to truncating mutations in the sarcomeric structural protein titin (TTNtv). Whilst the primary molecular mechanism of some DCM-associated mutations in the contractile apparatus has been studied in vitro and in transgenic mice, the contractile defect in human heart muscle has not been studied. In this study we isolated cardiac myofibrils from 3 TTNtv mutants, and 3 with contractile protein mutations (TNNI3 K36Q, TNNC1 G159D and MYH7 E1426K) and measured their contractility and passive stiffness in comparison with donor heart muscle as a control. We found that the three contractile protein mutations but not the TTNtv mutations had faster relaxation kinetics. Passive stiffness was reduced about 38% in all the DCM mutant samples. However, there was no change in maximum force or the titin N2BA/N2B isoform ratio and there was no titin haploinsufficiency. The decrease in myofibril passive stiffness was a common feature in all hearts with DCM-associated mutations and may be causative of DCM.

Limitations in Translating Animal Studies to Humans in Cardiovascular Disease.

The last decade alone saw an unprecedented rise (75 %) in the number of animal-centred laboratory research projects funded by the National Institute of Health (NIH) [1]. And according to the National Association for Biomedical Research, 95 % of all non-human laboratory experiments aimed at modelling human diseases are in mice and rats [2]. Some reasons behind this increase in rodent use, as opposed to larger animals, are due to ease of access, lower cost of care and maintenance, and the revised legislation for rodents. One such example was the Farm Bill signed in 2002 that saw laboratory rats, mice and birds explicitly excluded from the Animal Welfare Act. There is no doubt that rodent models in cardiovascular disease have produced key findings that have led to new therapies in humans. For example, the landmark studies by Marc and Janice Pfeffer on the effects of ACE inhibitors in rats with myocardial infarction [3] that were later reproduced in the human trial SAVE [4]. More recently, the effects of novel angiotensin receptor inhibitors were demonstrated in the same animal model [5]. However, we must remember that the aim of animal models in say, heart failure, is to simplify an extremely complex syndrome, often complicated by multi-factorial aetiologies, into manageable research questions. This generally results in a trade-off between convenience and physiological applicability. These animal models are precisely defined in the context of a uniform genotype and in a uniform environment but stand in stark contrast to humans who have a comparatively varied genetic composition, highly variable diet and exposure to an array of environmental stresses. An editorial published in NatureMedicine in April 2013 [6] discussed the dangers of assuming direct correlation between the pathophysiology of mouse and human diseases. Notably, the genetic alterations between these species in inflammatory conditions were essentially equivalent to random chance (50 %). Another example is the shortcomings of mice as models for hyperlipidaemia in human heart transplantation. A recent editorial in the American Journal of Transplantation [7] found that Blong-term allograft tolerance is relatively easy to achieve in mice but remains an elusive goal in humans^. Therefore, we should be cautious in interpreting and drawing conclusions based on data obtained from animals alone because of factors such as genetic and environmental divergence. Concerns regarding the translatability of animal models is further emphasised by well-known structural and functional differences between human and rodent hearts. For example, a mouse heart beats at around 600 bpm compared to 70 bpm in humans. This likely reflects significant differences in calcium handling and ion currents. In addition, rodents also express different protein isoforms, the most notable being fast alphamyosin in their ventricles compared to the slower beta variant of human cardiac myosin [8]. Such differences can complicate the interpretation of results from animal models and their applicability to drug testing and human clinical trials. A recent meta-analysis evaluated 11 functional parameters of the heart comparing rodents with humans [9]. They reported a worrying trend where the correlation of mouse and human functional data varied from as low as 0.1:1 up to 16:1 in both the left and right ventricles. The results were only marginally better (0.15:1 through 7.8:1) when the data from rat strains were compared to humans. Only 1 of the 11 measured parameters (systolic pressure) was within an acceptable range for comparison. Of particular concern is the difference in relaxation and contraction kinetics—both of which are common measures of cardiac dysfunction in humans. Editor-in-Chief Jennifer L. Hall oversaw the review of this article