External Validation of a Clinical Score to Differentiate Takotsubo Syndrome From Non-ST-Elevation Myocardial Infarction in Women.

BACKGROUND AND AIMS: Clinical presentation of Takotsubo Syndrome (TS) mimics acute coronary syndrome (ACS). A score to differentiate TS from ACS would be helpful to facilitate appropriate investigation and management. We have previously developed a clinical score (NSTE-Takotsubo Score) to distinguish women with non-ST-segment elevation myocardial infarction (NSTEMI) from TS with non-ST-segment elevation (NSTE-TS). This study sought to assess the diagnostic validity of this score in an external validation cohort. METHODS: The external cohort consisted of women with NSTE-TS (n=110) and NSTEMI (n=113) from two major tertiary hospitals in New Zealand. The five variables in the arithmetic score (range -6 to +5) and their relative weights are: T-wave inversion (TWI) in ≥6 leads (3 points), recent stress (2 points), diabetes mellitus (DM) (-1 point), prior cardiovascular disease (CVD) (-2 points) and presence of ST depression (-3 points). Two clinicians blinded to the diagnoses calculated the score using clinical and electrocardiogram (ECG) data on day 1 post-admission. RESULTS: The NSTE-Takotsubo Score discriminated well between NSTE-TS and NSTEMI. The sensitivity and specificity of a score ≥1 to distinguish NSTE-TS from NSTEMI were 78% and 85%, respectively. The area under the receiver operator curve was 0.78 (95% CI 0.72 to 0.84). CONCLUSION: In an external validation cohort, the NSTE-Takotsubo Score was easy to apply and useful to identify women likely to have NSTE-TS on day 1 post-admission.

The carboxyl-terminal sequence of PUMA binds to both anti-apoptotic proteins and membranes.

Anti-apoptotic proteins such as BCL-XL promote cell survival by sequestering pro-apoptotic BCL-2 family members, an activity that frequently contributes to tumorigenesis. Thus, the development of small-molecule inhibitors for anti-apoptotic proteins, termed BH3-mimetics, is revolutionizing how we treat cancer. BH3 mimetics kill cells by displacing sequestered pro-apoptotic proteins to initiate tumor-cell death. Recent evidence has demonstrated that in live cells the BH3-only proteins PUMA and BIM resist displacement by BH3-mimetics, while others like tBID do not. Analysis of the molecular mechanism by which PUMA resists BH3-mimetic mediated displacement from full-length anti-apoptotic proteins (BCL-XL, BCL-2, BCL-W, and MCL-1) reveals that both the BH3-motif and a novel binding site within the carboxyl-terminal sequence (CTS) of PUMA contribute to binding. Together these sequences bind to anti-apoptotic proteins, which effectively 'double-bolt locks' the proteins to resist BH3-mimetic displacement. The pro-apoptotic protein BIM has also been shown to double-bolt lock to anti-apoptotic proteins however, the novel binding sequence in PUMA is unrelated to that in the CTS of BIM and functions independent of PUMA binding to membranes. Moreover, contrary to previous reports, we find that when exogenously expressed, the CTS of PUMA directs the protein primarily to the endoplasmic reticulum (ER) rather than mitochondria and that residues I175 and P180 within the CTS are required for both ER localization and BH3-mimetic resistance. Understanding how PUMA resists BH3-mimetic displacement will be useful in designing more efficacious small-molecule inhibitors of anti-apoptotic BCL-2 proteins.

Endoplasmic reticulum protein BIK binds to and inhibits mitochondria-localized antiapoptotic proteins.

The proapoptotic BCL-2 homology (BH3)-only endoplasmic reticulum (ER)-resident protein BCL-2 interacting killer (BIK) positively regulates mitochondrial outer membrane permeabilization, the point of no return in apoptosis. It is generally accepted that BIK functions at a distance from mitochondria by binding and sequestering antiapoptotic proteins at the ER, thereby promoting ER calcium release. Although BIK is predominantly localized to the ER, we detect by fluorescence lifetime imaging microscopy-FRET microscopy, BH3 region-dependent direct binding between BIK and mitochondria-localized chimeric mutants of the antiapoptotic proteins BCL-XL and BCL-2 in both baby mouse kidney (BMK) and MCF-7 cells. Direct binding was accompanied by cell type-specific differential relocalization in response to coexpression of either BIK or one of its target binding partners, BCL-XL, when coexpressed in cells. In BMK cells with genetic deletion of both BAX and BAK (BMK-double KO), our data suggest that a fraction of BIK protein moves toward mitochondria in response to the expression of a mitochondria-localized BCL-XL mutant. In contrast, in MCF-7 cells, our data suggest that BIK is localized at both ER and mitochondria-associated ER membranes and binds to the mitochondria-localized BCL-XL mutant via relocalization of BCL-XL to ER and mitochondria-associated ER membrane. Rather than functioning at a distance, our data suggest that BIK initiates mitochondrial outer membrane permeabilization via direct interactions with ER and mitochondria-localized antiapoptotic proteins, which occur via ER-mitochondria contact sites, and/or by relocalization of either BIK or antiapoptotic proteins in cells.

Clinically Acquired High Sensitivity Cardiac Troponin T is a Poor Predictor of Reduced Left Ventricular Ejection Fraction After ST Elevation Myocardial Infarction: A National Cohort Study-ANZACS-QI 65.

OBJECTIVE: Cardiac troponins (cTn) have been used historically to estimate infarct size in ST elevation myocardial infarction (STEMI). Within a resource constrained health care environment, cTn could therefore be used for prioritisation of patients for cardiac imaging, in particular echocardiography. We aimed to determine how useful routinely collected cTn would be in predicting significant left ventricular (LV) impairment. METHODS: All patients in the All New Zealand Acute Coronary Syndrome Quality Improvement (ANZACS-QI) registry with their first episode of STEMI between January 2013 and November 2018, who had high sensitivity troponin T measured, were included. We excluded patients with no left ventricular ejection fraction (LVEF) assessment, known LV dysfunction, or prior myocardial infarction. RESULTS: In total, 3,698 patients were included in the analysis. A higher mean hsTnT (admission and peak) was seen in patients with more severely impaired LV function but there was significant overlap in the range of hsTnT between the different LVEF categories. Cardiac troponins demonstrated poor discriminative ability to either predict or exclude significant LV impairment (LVEF <40%). At an optimal cutpoint of 3,405 ng/L, peak hsTnT had a sensitivity of 56.5% (95% confidence interval [CI] 42-62%), a specificity of 65.3% (95% CI 62-79%) and an area under the receiver operating curve of 0.62 (95% CI 0.60-0.64). CONCLUSION: This is the largest study comparing clinically measured troponin levels and LV function in patients presenting with STEMI. A definite, but weak, association was seen between peak troponin and the degree of LV dysfunction, with significant overlap in troponin levels between levels of myocardial dysfunction. Routinely acquired troponin is not suitable for clinical use as a method of prioritising patients for cardiac imaging.

Efficacy and specificity of inhibitors of BCL-2 family protein interactions assessed by affinity measurements in live cells.

Cytoplasmic and membrane-bound BCL-2 family proteins regulate apoptosis, a form of programmed cell death, via dozens of binary protein interactions confounding measurement of the effects of inhibitors in live cells. In cancer, apoptosis is frequently dysregulated, and cell survival depends on antiapoptotic proteins binding to and inhibiting proapoptotic BH3 proteins. The clinical success of BH3 mimetic inhibitors of antiapoptotic proteins has spawned major efforts by the pharmaceutical industry to develop molecules with different specificities and higher affinities. Here, quantitative fast fluorescence lifetime imaging microscopy enabled comparison of BH3 mimetic drugs in trials and preclinical development by measuring drug effects on binding affinities of interacting protein pairs in live cells. Both selectivity and efficacy were assessed for 15 inhibitors of four antiapoptotic proteins for each of six BH3 protein ligands. While many drugs target the designed interaction, most also have unexpected selectivity and poor efficacy in cells.

Type of Stressor and Medium-Term Outcomes After Takotsubo Syndrome: What Becomes of the Broken Hearted? (ANZACS-QI 59).

BACKGROUND: Takotsubo syndrome (TS) is often triggered by an acute physical or emotional stressor. We hypothesised that medium-term prognosis may be better for TS patients with an associated emotional stressor than for those with an acute physical illness. METHODS: We identified consecutive TS patients presenting in New Zealand (2006-2018). The clinical presentation and outcomes of TS patients according to types of stressor (physical, emotional or no stressor) were assessed. Post-discharge survival after TS was compared with age- and gender-matched patients after myocardial infarction (MI) and people in the community without known cardiovascular disease (CVD). RESULTS: Of 632 TS patients (95.9% women, mean age 65.0±11.1 years), 27.4% had an associated acute physical stressor, 46.4% an emotional stressor and 26.2% no evident stressor. In-hospital mortality was similar for each group (1.7%, 1.2%, 0.3% respectively, p=0.29). In a median 4.4 years post-discharge there were 54 deaths (53 non-cardiac). Compared with patients without known CVD, TS patients with physical stress and those with MI were less likely to survive (HR 4.46, 95%CI 3.10-6.42; HR 4.23, 95%CI 3.81-4.70 respectively) but survival for TS patients associated with emotional stress or no stressor was similar (HR 1.11, 95%CI 0.66-1.85; HR 1.08, 95%CI 0.54-2.18, respectively). Recurrence was similar among the three groups (p=0.14). CONCLUSION: Takotsubo syndrome associated with physical stressor has a post-discharge mortality risk as high as after MI. In contrast, prognosis for TS triggered by an emotional stressor is excellent, and similar to that of those without known CVD.

Rapid 3D phenotypic analysis of neurons and organoids using data-driven cell segmentation-free machine learning.

Phenotypic profiling of large three-dimensional microscopy data sets has not been widely adopted due to the challenges posed by cell segmentation and feature selection. The computational demands of automated processing further limit analysis of hard-to-segment images such as of neurons and organoids. Here we describe a comprehensive shallow-learning framework for automated quantitative phenotyping of three-dimensional (3D) image data using unsupervised data-driven voxel-based feature learning, which enables computationally facile classification, clustering and advanced data visualization. We demonstrate the analysis potential on complex 3D images by investigating the phenotypic alterations of: neurons in response to apoptosis-inducing treatments and morphogenesis for oncogene-expressing human mammary gland acinar organoids. Our novel implementation of image analysis algorithms called Phindr3D allowed rapid implementation of data-driven voxel-based feature learning into 3D high content analysis (HCA) operations and constitutes a major practical advance as the computed assignments represent the biology while preserving the heterogeneity of the underlying data. Phindr3D is provided as Matlab code and as a stand-alone program (https://github.com/DWALab/Phindr3D).

Neuronal cell life, death, and axonal degeneration as regulated by the BCL-2 family proteins.

Axonal degeneration and neuronal cell death are fundamental processes in development and contribute to the pathology of neurological disease in adults. Both processes are regulated by BCL-2 family proteins which orchestrate the permeabilization of the mitochondrial outer membrane (MOM). MOM permeabilization (MOMP) results in the activation of pro-apoptotic molecules that commit neurons to either die or degenerate. With the success of small-molecule inhibitors targeting anti-apoptotic BCL-2 proteins for the treatment of lymphoma, we can now envision the use of inhibitors of apoptosis with exquisite selectivity for BCL-2 family protein regulation of neuronal apoptosis in the treatment of nervous system disease. Critical to this development is deciphering which subset of proteins is required for neuronal apoptosis and axon degeneration, and how these two different outcomes are separately regulated. Moreover, noncanonical BCL-2 family protein functions unrelated to the regulation of MOMP, including impacting necroptosis and other modes of cell death may reveal additional potential targets and/or confounders. This review highlights our current understanding of BCL-2 family mediated neuronal cell death and axon degeneration, while identifying future research questions to be resolved to enable regulating neuronal survival pharmacologically.

A comparison of the clinical features and outcomes of Takotsubo syndrome across five metropolitan hospitals in New Zealand.

AIM: Takotsubo syndrome (TS) mimics acute coronary syndrome but has a distinct pathophysiology. This study aimed to compare and contrast the clinical presentation, management and outcomes of patients with TS in five large New Zealand hospitals. METHODS: We identified 632 consecutive patients presenting to the five major tertiary hospitals in New Zealand (Middlemore Hospital, Auckland City Hospital, North Shore Hospital, Christchurch Hospital and Dunedin Hospital) between January 2006 and June 2018 and obtained clinical, laboratory, electrocardiography, echocardiography, coronary angiography and long-term follow-up data. RESULTS: Six hundred and thirty-two consecutive patients with TS (606 women, mean age 65.0+11.1 years) were included. An associated stressor was identified in two-thirds of patients, and emotional triggers were more frequent than physical triggers (62.9% and 37.1%, respectively). Overall, 12.7% of patient had depression and 11.7% anxiety but this was more common in patients from Christchurch Hospital (20.4% and 23.4%, respectively). The in-hospital mortality among the five hospitals ranges between 0 to 2.0%. The mean follow-up was 4.9+3.4 years (median 4.4 years). Fifty-four people died post-discharge, all but one from a non-cardiac cause. Forty patients had recurrent TS. Mortality post-discharge (p=0.63) and TS recurrence (p=0.38) did not differ significantly among the five hospitals. CONCLUSION: In this large New Zealand TS cohort, the clinical characteristics and presentation were similar among the five hospitals. A subset of patients had a complicated in-hospital course, but late deaths were almost all from non-cardiac causes and recurrence was infrequent. Mortality post-discharge and recurrence was similar between the hospitals.

Changes to management of a non-pandemic illness during the COVID-19 pandemic: case study of invasive management of acute coronary syndrome.

The coronavirus 2019 (COVID-19) pandemic requires significant changes to standard operating procedures for non-COVID-19 related illnesses. Balancing the benefit from standard evidence-based treatments with the risks posed by COVID-19 to patients, healthcare workers and to the population at large is difficult due to incomplete and rapidly changing information. In this article, we use management of acute coronary syndromes as a case study to show how these competing risks and benefits can be resolved, albeit incompletely. While the risks due to COVID-19 in patients with acute coronary syndromes is unclear, the benefits of standard management are well established in this condition. As an aid to decision making, we recommend systematic estimation of the risks and benefits for management of any condition where there is likely to be an increase in non-COVID-19 related mortality and morbidity due to changes in routine care.

The carboxyl-terminal sequence of bim enables bax activation and killing of unprimed cells.

The Bcl-2 family BH3 protein Bim promotes apoptosis at mitochondria by activating the pore-forming proteins Bax and Bak and by inhibiting the anti-apoptotic proteins Bcl-XL, Bcl-2 and Mcl-1. Bim binds to these proteins via its BH3 domain and to the mitochondrial membrane by a carboxyl-terminal sequence (CTS). In cells killed by Bim, the expression of a Bim mutant in which the CTS was deleted (BimL-dCTS) triggered apoptosis that correlated with inhibition of anti-apoptotic proteins being sufficient to permeabilize mitochondria isolated from the same cells. Detailed analysis of the molecular mechanism demonstrated that BimL-dCTS inhibited Bcl-XL but did not activate Bax. Examination of additional point mutants unexpectedly revealed that the CTS of Bim directly interacts with Bax, is required for physiological concentrations of Bim to activate Bax and that different residues in the CTS enable Bax activation and binding to membranes.

Measuring Small-molecule Inhibition of Protein Interactions in Live Cells Using FLIM-FRET.

This protocol was designed to quantitatively measure small-molecule displacement of proteins in live mammalian cells using fluorescence lifetime imaging microscopy-Förster resonance energy transfer (FLIM-FRET). Tumour cell survival is often dependent on anti-apoptotic proteins, which bind to and inhibit pro-apoptotic proteins, thus preventing apoptosis. Small-molecule inhibitors that selectively target these proteins (termed BH3-mimetics) are therefore a promising avenue for the treatment of several cancers. Previous techniques used to study the efficacy of these drugs often use truncated versions of both pro- and anti-apoptotic proteins, as they are membrane bound and hydrophobic in nature. As a result, the true efficacy of these drugs to displace full-length pro-apoptotic proteins in their native environment within a cell is poorly understood. This protocol describes FLIM-FRET methods to directly measure the displacement (or lack of displacement) of full-length Bcl-2 family proteins in live mammalian cells.

Performance of Endocarditis-Specific Risk Scores in Surgery for Infective Endocarditis.

BACKGROUND: Risk models can play an important part in the decision-making of surgery for infective endocarditis, but they remain underutilized. Several endocarditis-specific risk models have been recently published with the aim to improve on existing general cardiac surgery scores such as EuroSCORE. We compared their prognostic utility of mortality and morbidities for infective endocarditis surgery. METHODS: The additive Society of Thoracic Surgeon's (STS) Endocarditis score, Costa score, De Feo-Cotrufo score, and Pulsuse score were calculated for consecutive patients undergoing cardiac surgery for active infective endocarditis during 2005 to 2011 at Auckland City Hospital and their discriminative value for adverse outcomes assessed. RESULTS: Mean scores for 146 endocarditis surgery patients with operative mortality 6.8% (10) were additive STS score: 32.2 ± 13.5, Costa score: 12.0 ± 6.8, De Feo-Cotrufo score: 14.6 ± 9.2, and Pulsuse score 2.2 ± 1.3. Areas under curve and 95% confidence intervals for operative mortality were 0.699 (0.534-0.865), 0.596 (0.426-0.765), 0.744 (0.590-0.899), and 0.673 (0.510-0.836), respectively. All four scores could also discriminate mortality during follow-up and composite morbidity, with the De Feo-Cotrufo score having the best overall performance. CONCLUSION: Endocarditis-specific risk models had moderate discrimination of operative mortality and most postoperative complications, and the De Feo-Cotrufo score is the preferred score to advise clinical decisions in this setting.

Relationship Between Diabetic Variables and Outcomes After Coronary Artery Bypass Grafting in Diabetic Patients.

BACKGROUND: Nearly half of the patients undergoing coronary artery bypass grafting (CABG) have diabetes. There is mixed data as to whether preoperative (haemoglobin A1c{HbA1c}) and/or perioperative diabetes control is associated with mortality and morbidity after CABG. We reviewed the characteristics and outcomes of diabetic patients undergoing CABG with a focus on HbA1c, perioperative glucose levels and diabetic treatment regimens. METHODS: Diabetic patients undergoing CABG during July 2010 to June 2012 were studied (n=306). The last preoperative HbA1c levels, and perioperative glucose levels (mean and coefficient of variation {CV}) were retrospectively recorded, as well as the pre-existing and perioperative diabetes treatment regimens for analyses. RESULTS: Mean HbA1c was 7.7+/-1.6%, and 11.1% (34), 56.2% (172), and 32.7% (100) of patients were managed preoperatively with diet only, oral diabetic medications and insulin respectively. For operative mortality which occurred in 2.0%, C-statistics (95% confidence interval) was only significant for HbA1c, 0.855 (0.757-0.975), and glucose CV on the day of surgery, 0.722 (0.567-0.877). HbA1c also detected postoperative renal failure, C-statistic 0.617 (0.504-0.730), but not other complications or mortality during follow-up. In multivariate analysis, HbA1c was the only diabetes-related independent predictor of operative mortality, hazards ratio 4.13 (1.04-16.4), and none of the diabetes-related variables predicted mortality during follow-up or other postoperative complications. CONCLUSION: Preoperative HbA1c was the only diabetic variable to independently predict operative mortality after CABG, suggesting medium-term preoperative diabetes control is more important and prognostic of operative outcomes than perioperative diabetes control.

The not so innocent heart murmur: a 5-year experience.

BACKGROUND: Auckland City Hospital (ACH) established a Heart Murmur Clinic (HMC) with the aim of providing prompt assessment of patients with asymptomatic systolic murmurs. This may lead to early intervention and improved outcomes if significant structural heart disease is detected and reassurance if no significant findings are found. Similar clinics for children have proven beneficial; the benefit of a HMC in an adult population has been difficult to determine. AIM: To review the clinical demographics and echocardiographic information of patients presenting to our HMC, to assess what proportion of significance structural heart disease had and determine the common structural abnormalities in this population. METHODS: This is a retrospective review of patients aged ≥15 years presenting to our HMC between March 2010 and December 2015 with an asymptomatic systolic murmur. Patients with previous cardiac surgery or known congenital or valvular heart disease were excluded. RESULTS: A total of 1221 patients was reviewed over the 5-year period; 980 underwent echocardiography. Significant cardiac disease was detected in 156 patients, with 23 patients requiring surgical intervention over the 5-year period. Significant aortic stenosis (n = 43) and mitral regurgitation (n = 48) were the most common pathologies. Patients > 65 years were more likely to have structural heart disease (16% vs 11%, P < 0.05). CONCLUSION: Establishing a HMC has allowed the screening of a large number of patients who would otherwise have low priority for assessment. We have identified a large proportion with significant structural disease, which has allowed for early surgical intervention when appropriate and may potentially result in improved patient outcomes.

Adherence to the current guidelines for bradycardic pacing in the octogenarian and nonagenarian populations.

AIM: A significant proportion of single-chamber ventricular pacemakers are implanted in octogenarian and nonagenarian patients. We aimed to assess whether the current pacing guideline is adhered for these populations. METHODS: We retrospectively identified patients ≥80 years of age, who received their first pacemaker from July 2010 to June 2013. RESULTS: A total of 356 patients were identified. Mean age was 86.1 years and 82.6 years for single and dual-chamber pacemakers respectively (p<0.05). Total procedure-related complications occurred in 9.5% and were comparable between both groups (p=0.08). At the time of implantation, 185 patients who received single-chamber pacemaker were in sinus rhythm (52%). They were older (86.2±4.3 vs 82.6±2.9, p<0.05), more likely to have ischaemic and valvular heart disease (68 vs 27, p= 0.02 and 22 vs 13, p=0.01, respectively), and cognitive impairment (34 vs 0, p= 0.001). They were also more likely to be discharged to a residential care facility (17 vs 1, p<0.01). CONCLUSION: The utility of dual-chamber pacemaker in this age group remains below expectation and did not comply with current cardiac pacing guidelines. The presence of older age, multiple co-morbidities, cognitive impairment and residential care on discharge likely influenced the type of device implanted.