Comparing the effects of chemical Ca2+ dyes and R-GECO on contractility and Ca2+ transients in adult and human iPSC cardiomyocytes.

We compared commonly used BAPTA-derived chemical Ca2+ dyes (fura2, Fluo-4, and Rhod-2) with a newer genetically encoded indicator (R-GECO) in single cell models of the heart. We assessed their performance and effects on cardiomyocyte contractility, determining fluorescent signal-to-noise ratios and sarcomere shortening in primary ventricular myocytes from adult mouse and guinea pig, and in human iPSC-derived cardiomyocytes. Chemical Ca2+ dyes displayed dose-dependent contractile impairment in all cell types, and we observed a negative correlation between contraction and fluorescence signal-to-noise ratio, particularly for fura2 and Fluo-4. R-GECO had no effect on sarcomere shortening. BAPTA-based dyes, but not R-GECO, inhibited in vitro acto-myosin ATPase activity. The presence of fura2 accentuated or diminished changes in contractility and Ca2+ handling caused by small molecule modulators of contractility and intracellular ionic homeostasis (mavacamten, levosimendan, and flecainide), but this was not observed when using R-GECO in adult guinea pig left ventricular cardiomyocytes. Ca2+ handling studies are necessary for cardiotoxicity assessments of small molecules intended for clinical use. Caution should be exercised when interpreting small molecule studies assessing contractile effects and Ca2+ transients derived from BAPTA-like chemical Ca2+ dyes in cellular assays, a common platform for cardiac toxicology testing and mechanistic investigation of cardiac disease physiology and treatment.

The Future of LAAC-In 5, 10, and 20 Years.

Early experience with percutaneous LAA closure documented complication rates of ∼10%, with failure to implant devices in ∼10% of patients. These numbers are unrecognizable in contemporary practice due to the iterative changes made largely in the last 10 years. Here we look forward to ask what might change, and when, to bring percutaneous LAA closure out of the niche early adopter centers into routine use. We consider the opportunity to incorporate different technologies into LAAc devices in the context of managing patient with atrial fibrillation. Finally, we consider how to make the procedure safer and more effective.

Comparison of the BioFire Joint Infection Panel to 16S Ribosomal RNA Gene-Based Targeted Metagenomic Sequencing for Testing Synovial Fluid from Patients with Knee Arthroplasty Failure.

The diagnosis of periprosthetic joint infection (PJI) is challenging, often requiring multiple clinical specimens and diagnostic techniques, some with prolonged result turnaround times. Here, the diagnostic performance of the Investigational Use Only (IUO) BioFire Joint Infection (JI) Panel was compared to 16S rRNA gene-based targeted metagenomic sequencing (tMGS) applied to synovial fluid for PJI diagnosis. Sixty synovial fluid samples from knee arthroplasty failure archived at -80°C were tested. Infectious Diseases Society of America (IDSA) diagnostic criteria were used to classify PJI. For culture-positive PJI with pathogens targeted by the JI panel, JI panel sensitivity was 91% (21/23; 95% confidence interval [CI], 73 to 98%), and tMGS sensitivity was 96% (23/24; 95% CI, 80 to 99%) (P = 0.56). Overall sensitivities of the JI panel and tMGS for PJI diagnosis were 56% (24/43; 95% CI, 41 to 70%) and 93% (41/44; 95% CI, 82 to 98%), respectively (P < 0.001). JI panel and tMGS overall specificities were 100% (16/16; 95% CI, 81 to 100%) and 94% (15/16; 95% CI, 72 to 99%), respectively. While the clinical sensitivity of the JI panel was excellent for on-panel microorganisms, overall sensitivity for PJI diagnosis was low due to the absence of Staphylococcus epidermidis, a common causative pathogen of PJI, on the panel. A PJI diagnostic algorithm for the use of both molecular tests is proposed.

Transplanted organoids empower human preclinical assessment of drug candidate for the clinic.

Pharmacodynamic (PD) studies are an essential component of preclinical drug discovery. Current approaches for PD studies, including the analysis of novel kidney disease targeting therapeutic agents, are limited to animal models with unclear translatability to the human condition. To address this challenge, we developed a novel approach for PD studies using transplanted, perfused human kidney organoids. We performed pharmacokinetic (PK) studies with GFB-887, an investigational new drug now in phase 2 trials. Orally dosed GFB-887 to athymic rats that had undergone organoid transplantation resulted in measurable drug exposure in transplanted organoids. We established the efficacy of orally dosed GFB-887 in PD studies, where quantitative analysis showed significant protection of kidney filter cells in human organoids and endogenous rat host kidneys. This widely applicable approach demonstrates feasibility of using transplanted human organoids in preclinical PD studies with an investigational new drug, empowering organoids to revolutionize drug discovery.

High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening.

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. Genetically encoded calcium indicators (GECIs) with multiple emission windows can be combined (e.g., for simultaneous observation of distinct subcellular events) or used in extended applications with other light-dependent actuators in excitable cells (e.g., combining genetically encoded optogenetic control with spectrally compatible calcium indicators). Such approaches have been used in primary or stem cell-derived neurons, cardiomyocytes, and pancreatic beta-cells. However, it has been challenging to increase the throughput, or duration of observation, of such approaches due to limitations of the instruments, analysis software, indicator performance, and gene delivery efficiency. Here, a high-performance green GECI, mNeonGreen-GECO (mNG-GECO), and red-shifted GECI, K-GECO, is combined with optogenetic control to achieve all-optical control and visualization of cellular activity in a high-throughput imaging format using a High-Content Imaging System. Applications demonstrating cardiotoxicity testing and phenotypic drug screening with healthy and patient-derived iPSC-CMs are shown. In addition, multi-parametric assessments using combinations of spectral and calcium affinity indicator variants (NIR-GECO, LAR-GECO, and mtGCEPIA or Orai1-G-GECO) are restricted to different cellular compartments are also demonstrated in the iPSC-CM model.

The Future of LAAC-In 5, 10, and 20 Years.

Early experience with percutaneous LAA closure documented complication rates of ∼10%, with failure to implant devices in ∼10% of patients. These numbers are unrecognizable in contemporary practice due to the iterative changes made largely in the last 10 years. Here we look forward to ask what might change, and when, to bring percutaneous LAA closure out of the niche early adopter centers into routine use. We consider the opportunity to incorporate different technologies into LAAc devices in the context of managing patient with atrial fibrillation. Finally, we consider how to make the procedure safer and more effective.

Targeted Metagenomic Sequencing-based Approach Applied to 2146 Tissue and Body Fluid Samples in Routine Clinical Practice.

BACKGROUND: The yield of next-generation sequencing (NGS) added to a Sanger sequencing-based 16S ribosomal RNA (rRNA) gene polymerase chain reaction (PCR) assay was evaluated in clinical practice for diagnosis of bacterial infection. METHODS: PCR targeting the V1 to V3 regions of the 16S rRNA gene was performed, with amplified DNA submitted to Sanger sequencing and/or NGS (Illumina MiSeq) or reported as negative, depending on the cycle threshold value. A total of 2146 normally sterile tissues or body fluids were tested between August 2020 and March 2021. Clinical sensitivity was assessed in 579 patients from whom clinical data were available. RESULTS: Compared with Sanger sequencing alone (400 positive tests), positivity increased by 87% by adding NGS (347 added positive tests). Clinical sensitivity of the assay that incorporated NGS was 53%, which was higher than culture (42%, P < .001), with an impact on clinical decision-making in 14% of infected cases. Clinical sensitivity in the subgroup that received antibiotics at sampling was 41% for culture and 63% for the sequencing assay (P < .001). CONCLUSIONS: Adding NGS to Sanger sequencing of the PCR-amplified 16S rRNA gene substantially improved test positivity. In the patient population studied, the assay was more sensitive than culture, especially in patients who had received antibiotic therapy.

Discovery and Optimization of Highly Selective Inhibitors of CDK5.

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent monogenic human disease, but to date, only one therapy (tolvaptan) is approved to treat kidney cysts in ADPKD patients. Cyclin-dependent kinase 5 (CDK5), an atypical member of the cyclin-dependent kinase family, has been implicated as a target for treating ADPKD. However, no compounds have been disclosed to date that selectively inhibit CDK5 while sparing the broader CDK family members. Herein, we report the discovery of CDK5 inhibitors, including GFB-12811, that are highly selective over the other tested kinases. In cellular assays, our compounds demonstrate CDK5 target engagement while avoiding anti-proliferative effects associated with inhibiting other CDKs. In addition, we show that the compounds in this series exhibit promising in vivo PK profiles, enabling their use as tool compounds for interrogating the role of CDK5 in ADPKD and other diseases.

Left Atrial Appendage Occlusion: Current Advances and Remaining Challenges.

The field of left atrial appendage occlusion is rapidly evolving. However, several issues remain including the limited randomized efficacy data, peri-device leak, device-related thrombus, and the ongoing refinement of procedural techniques. In this article, we provide a contemporary overview of left atrial appendage occlusion focusing on 4 key remaining challenges: efficacy data, peri-device leak, device-related thrombus, and procedural optimization.

Lockdown, slow down: impact of the COVID-19 pandemic on physical activity-an observational study.

AIMS: In response to the COVID-19 pandemic, the UK was placed under strict lockdown measures on 23 March 2020. The aim of this study was to quantify the effects on physical activity (PA) levels using data from the prospective Triage-HF Plus Evaluation study. METHODS: This study represents a cohort of adult patients with implanted cardiac devices capable of measuring activity by embedded accelerometery via a remote monitoring platform. Activity data were available for the 4 weeks pre-implementation and post implementation of 'stay at home' lockdown measures in the form of 'minutes active per day' (min/day). RESULTS: Data were analysed for 311 patients (77.2% men, mean age 68.8, frailty 55.9%. 92.2% established heart failure (HF) diagnosis, of these 51.2% New York Heart Association II), with comorbidities representative of a real-world cohort.Post-lockdown, a significant reduction in median PA equating to 20.8 active min/day was seen. The reduction was uniform with a slightly more pronounced drop in PA for women, but no statistically significant difference with respect to age, body mass index, frailty or device type. Activity dropped in the immediate 2-week period post-lockdown, but steadily returned thereafter. Median activity week 4 weeks post-lockdown remained significantly lower than 4 weeks pre-lockdown (p≤0.001). CONCLUSIONS: In a population of predominantly HF patients with cardiac devices, activity reduced by approximately 20 min active per day in the immediate aftermath of strict COVID-19 lockdown measures. TRIAL REGISTRATION NUMBER: NCT04177199.

An assessment of annual procedure volumes and therapy adoption of transcatheter closure of patent foramen ovale in four European countries.

INTRODUCTION: Patent foramen ovale closure reduces recurrence of cryptogenic ischaemic stroke compared to anti-platelet therapy. Our goal was to determine procedure volumes and closure utilisation as a proportion of candidates in four large European countries. PATIENTS AND METHODS: National statistics were obtained for Germany, England, France, and Italy for the last available five years (2014-2018). Eligibility was aligned to the enrolment criteria of pivotal trials and current consensus documents. Stroke and transient ischaemic attack incidences were obtained from epidemiological registries and claims data. The eligible candidate pool for analysis included current year candidates plus untreated patients from the prior two years. Absolute strokes avoided assumed the hazard ratio for ischaemic stroke recurrence from a recent meta-analysis. RESULTS: In 2018, closure incidence rates were 5.64, 0.53, 2.94 and 5.26 per 100,000 in Germany, England, France and Italy, respectively. This reflects five-year increases of 128% in Germany, 462% in France and 36% in Italy (p < 0.05 for all), and a decline of 37% in England. The proportions of treated patients versus candidates for the combined stroke and transient ischaemic attack pool were 55%, 30%, 80%, and 6%, respectively. DISCUSSION: Patent foramen ovale closure volumes increased after the 2017 announcement of positive trial results but still differ substantially across large European countries. If all closure candidates in 2018 with prior ischaemic stroke were treated, the resulting absolute reduction of recurrent ischaemic strokes, compared to anti-platelet therapy alone, would be between 782 and 2295 across the four countries over five years. CONCLUSION: Many eligible patients at risk for a recurrent cryptogenic event might remain untreated due to regional practice variations.

Sustainable Resumption of Cardiac Catheterization Laboratory Procedures, and the Importance of Testing, During Endemic COVID-19.

PURPOSE: As second and third waves of the COVID-19 pandemic challenge healthcare in North America and Europe once again, we analyze the impact of the first wave on routine elective cardiovascular care, and the differential COVID risk emerging within our patient groups and staff. PERSPECTIVE: We describe the need to sustainably resume, and temporarily expand, routine elective cardiac services in the face of resurgent COVID-19. Some, but not all, cardiac patient groups are particularly vulnerable to adverse outcomes following COVID-19 infection. We explore mitigation measures at the institutional level to increase resilience within cardiac services to enable them to operate deep into subsequent waves of COVID infection which place unprecedented demands on intensive care infrastructure. As measures to eradicate the virus appear to have failed in many countries, and vaccine roll-out will take many months we take the view that the threat imposed by endemic COVID-19 alters the way elective procedural care should be offered to cardiovascular patients. CONCLUSION: Our patients are at definite risk from their cardiovascular disease, and a return to suspension of proven prognostic interventional treatments on an elective basis - the default for the first wave - must be avoided at all costs.

#SoMe for #IC: Optimal use of social media in interventional cardiology.

Social media allows interventional cardiologists to disseminate and discuss research and clinical cases in real-time, to demonstrate and learn innovative techniques, to build professional networks, and to reach out to patients and the general public. Social media provides a democratic platform for all participants to influence the conversation and demonstrate their expertise. This review addresses the use of social media for these purposes in interventional cardiology, as well as respect for patient privacy, how to get started on social media, the creation of high-impact social media content, and the role of traditional journals in the age of social media. In the future, we hope that interventional cardiology fellowship programs will incorporate social media training into their curricula. In addition, professional societies may adapt to the rapid dissemination of data on social media by developing processes to update guidelines more rapidly and more frequently.

Influence of Contextual Factors, Technical Performance, and Movement Demands on the Subjective Task Load Associated With Professional Rugby League Match-Play.

PURPOSE: To identify the association between several contextual match factors, technical performance, and external movement demands on the subjective task load of elite rugby league players. METHODS: Individual subjective task load, quantified using the National Aeronautics and Space Administration Task Load Index (NASA-TLX), was collected from 29 professional rugby league players from one club competing in the European Super League throughout the 2017 season. The sample consisted of 26 matches (441 individual data points). Linear mixed modeling revealed that various combinations of contextual factors, technical performance, and movement demands were associated with subjective task load. RESULTS: Greater number of tackles (effect size correlation ± 90% confidence intervals; η2 = .18 ± .11), errors (η2 = .15 ± .08), decelerations (η2 = .12 ± .08), increased sprint distance (η2 = .13 ± .08), losing matches (η2 = .36 ± .08), and increased perception of effort (η2 = .27 ± .08) led to most likely-very likely increases in subjective total task load. The independent variables included in the final model for subjective mental demand (match outcome, time played, and number of accelerations) were unclear, excluding a likely small correlation with technical errors (η2 = .10 ± .08). CONCLUSIONS: These data provide a greater understanding of the subjective task load and their association with several contextual factors, technical performance, and external movement demands during rugby league competition. Practitioners could use this detailed quantification of internal loads to inform recovery sessions and current training practices.