Clustering properties of the cardiac ryanodine receptor in health and heart failure.

The cardiac ryanodine receptor (RyR2) is an intracellular Ca2+ release channel vital for the function of the heart. Physiologically, RyR2 is triggered to release Ca2+ from the sarcoplasmic reticulum (SR) which enables cardiac contraction; however, spontaneous Ca2+ leak from RyR2 has been implicated in the pathophysiology of heart failure (HF). RyR2 channels have been well documented to assemble into clusters within the SR membrane, with the organisation of RyR2 clusters recently gaining interest as a mechanism by which the occurrence of pathological Ca2+ leak is regulated, including in HF. In this review, we explain the terminology relating to key nanoscale RyR2 clustering properties as both single clusters and functionally grouped Ca2+ release units, with a focus on the advancements in super-resolution imaging approaches which have enabled the detailed study of cluster organisation. Further, we discuss proposed mechanisms for modulating RyR2 channel organisation and the debate regarding the potential impact of cluster organisation on Ca2+ leak activity. Finally, recent experimental evidence investigating the nanoscale remodelling and functional alterations of RyR2 clusters in HF is discussed with consideration of the clinical implications.

Direct regulation of the cardiac ryanodine receptor (RyR2) by O-GlcNAcylation.

BACKGROUND: O-GlcNAcylation is the enzymatic addition of a sugar, O-linked ß-N-Acetylglucosamine, to the serine and threonine residues of proteins, and is abundant in diabetic conditions. We have previously shown that O-GlcNAcylation can trigger arrhythmias by indirectly increasing pathological Ca2+ leak through the cardiac ryanodine receptor (RyR2) via Ca2+/calmodulin-dependent kinase II (CaMKII). However, RyR2 is well known to be directly regulated by other forms of serine and threonine modification, therefore, this study aimed to determine whether RyR2 is directly modified by O-GlcNAcylation and if this also alters the function of RyR2 and Ca2+ leak. METHODS: O-GlcNAcylation of RyR2 in diabetic human and animal hearts was determined using western blotting. O-GlcNAcylation of RyR2 was pharmacologically controlled and the propensity for Ca2+ leak was determined using single cell imaging. The site of O-GlcNAcylation within RyR2 was determined using site-directed mutagenesis of RyR2. RESULTS: We found that RyR2 is modified by O-GlcNAcylation in human, animal and HEK293 cell models. Under hyperglycaemic conditions O-GlcNAcylation was associated with an increase in Ca2+ leak through RyR2 which persisted after CaMKII inhibition. Conversion of serine-2808 to alanine prevented an O-GlcNAcylation induced increase in Ca2+ leak. CONCLUSIONS: These data suggest that the function of RyR2 can be directly regulated by O-GlcNAcylation and requires the presence of serine-2808.

Depressed HCN4 function in the type 2 diabetic sinoatrial node.

Diabetic patients often have impaired heart rate (HR) control. HR is regulated both intrinsically within the sinoatrial node (SAN) and via neuronal input. Previously, we found lower ex vivo HR in type 2 diabetic rat hearts, suggesting impaired HR generation within the SAN. The major driver of pacemaking within the SAN is the activity of hyperpolarisation-activated cyclic nucleotide-gated 4 (HCN(4)) channels. This study aimed to investigate whether the lower intrinsic HR in the type 2 diabetic heart is due to changes in HCN4 function, protein expression and/ or distribution. The intrinsic HR response to HCN4 blockade was determined in isolated Langendorff-perfused hearts of Zucker type 2 Diabetic Fatty (ZDF) rats (DM) and their non-diabetic ZDF littermates (nDM). HCN4 protein expression and membrane localisation were determined using western blot and immunofluorescence, respectively. We found that the intrinsic HR was lower in DM compared to nDM hearts. The change in intrinsic HR in response to HCN4 blockade with ivabradine was diminished in DM hearts, which normalised the intrinsic HR between the groups. HCN4 protein expression was decreased in the SAN of DM compared to nDM controls with no change in the fraction of HCN4 localised to the membrane of SAN cardiomyocytes. The lower intrinsic HR in DM is likely due to decreased HCN4 expression and depressed HCN4 function. Our study provides a novel understanding into the intrinsic mechanisms underlying altered HR control in type 2 diabetes.

The function and regulation of calsequestrin-2: implications in calcium-mediated arrhythmias.

Cardiac arrhythmias are life-threatening events in which the heart develops an irregular rhythm. Mishandling of Ca2+ within the myocytes of the heart has been widely demonstrated to be an underlying mechanism of arrhythmogenesis. This includes altered function of the ryanodine receptor (RyR2)-the primary Ca2+ release channel located to the sarcoplasmic reticulum (SR). The spontaneous leak of SR Ca2+ via RyR2 is a well-established contributor in the development of arrhythmic contractions. This leak is associated with increased channel activity in response to changes in SR Ca2+ load. RyR2 activity can be regulated through several avenues, including interactions with numerous accessory proteins. One such protein is calsequestrin-2 (CSQ2), which is the primary Ca2+-buffering protein within the SR. The capacity of CSQ2 to buffer Ca2+ is tightly associated with the ability of the protein to polymerise in response to changing Ca2+ levels. CSQ2 can itself be regulated through phosphorylation and glycosylation modifications, which impact protein polymerisation and trafficking. Changes in CSQ2 modifications are implicated in cardiac pathologies, while mutations in CSQ2 have been identified in arrhythmic patients. Here, we review the role of CSQ2 in arrhythmogenesis including evidence for the indirect and direct regulation of RyR2 by CSQ2, and the consequences of a loss of functional CSQ2 in Ca2+ homeostasis and Ca2+-mediated arrhythmias. Supplementary Information: The online version contains supplementary material available at 10.1007/s12551-021-00914-6.

BESPOKE study protocol: a multicentre, prospective observational study to evaluate the impact of circulating tumour DNA guided therapy on patients with colorectal cancer.

INTRODUCTION: Colorectal cancer (CRC) is a highly prevalent disease, wherein, ~30%-40% of patients with CRC relapse postresection. In some patients with CRC, adjuvant chemotherapy can help delay recurrence or be curative. However, current biomarkers show limited clinical utility in determining if/when chemotherapy should be administered, to provide benefit. Circulating tumour DNA (ctDNA) can measure molecular residual disease (MRD) and relapse with high specificity and sensitivity. This study protocol investigates the clinical utility of ctDNA for optimal use of adjuvant chemotherapy in patients with surgically resected CRC and to detect early disease progression in the surveillance setting. METHODS AND ANALYSIS: This is a multicentre prospective, observational cohort study. A total of 2000 stage I-IV patients will be enrolled in up to 200 US sites, and patients will be followed for up to 2 years with serial ctDNA analysis, timed with the standard-of-care visits. The primary endpoints are to observe the impact of bespoke ctDNA testing on adjuvant treatment decisions and to measure CRC recurrence rates while asymptomatic and without imaging correlate. The secondary endpoints are MRD clearance rate (MRD+ to MRD-) during or after adjuvant chemotherapy, percentage of patients that undergo surgery for oligometastatic recurrence, survival of MRD-negative patients treated with adjuvant chemotherapy versus no adjuvant chemotherapy (active surveillance), overall survival, examine the number of stage I CRC that have recurrent disease detected postsurgery, and patient-reported outcomes. ETHICS AND DISSEMINATION: This study has received ethical approval from the Advarra Institutional Review Board (IRB) protocol: Natera-20-041-NCP/3766.01, BESPOKE Study of ctDNA Guided Therapy in Colorectal Cancer (BESPOKE CRC) (Pro00041473) on 10 June 2021. Data protection and privacy regulations will be strictly observed in the capturing, forwarding, processing and storing of patients' data. Publication of any study results will be approved by Natera in accordance with the site-specific contract. TRIAL REGISTRATION NUMBER: NCT04264702.

Human Atrial Fibrillation Is Not Associated With Remodeling of Ryanodine Receptor Clusters.

The release of Ca2+ by ryanodine receptor (RyR2) channels is critical for cardiac function. However, abnormal RyR2 activity has been linked to the development of arrhythmias, including increased spontaneous Ca2+ release in human atrial fibrillation (AF). Clustering properties of RyR2 have been suggested to alter the activity of the channel, with remodeling of RyR2 clusters identified in pre-clinical models of AF and heart failure. Whether such remodeling occurs in human cardiac disease remains unclear. This study aimed to investigate the nanoscale organization of RyR2 clusters in AF patients - the first known study to examine this potential remodeling in diseased human cardiomyocytes. Right atrial appendage from cardiac surgery patients with paroxysmal or persistent AF, or without AF (non-AF) were examined using super-resolution (dSTORM) imaging. Significant atrial dilation and cardiomyocyte hypertrophy was observed in persistent AF patients compared to non-AF, with these two parameters significantly correlated. Interestingly, the clustering properties of RyR2 were remarkably unaltered in the AF patients. No significant differences were identified in cluster size (mean ∼18 RyR2 channels), density or channel packing within clusters between patient groups. The spatial organization of clusters throughout the cardiomyocyte was also unchanged across the groups. RyR2 clustering properties did not significantly correlate with patient characteristics. In this first study to examine nanoscale RyR2 organization in human cardiac disease, these findings indicate that RyR2 cluster remodeling is not an underlying mechanism contributing to altered channel function and subsequent arrhythmogenesis in human AF.

Highly variable contractile performance correlates with myocyte content in trabeculae from failing human hearts.

Heart failure (HF) is defined by compromised contractile function and is associated with changes in excitation-contraction (EC) coupling and cardiomyocyte organisation. Tissue level changes often include fibrosis, while changes within cardiomyocytes often affect structures critical to EC coupling, including the ryanodine receptor (RyR), the associated protein junctophilin-2 (JPH2) and the transverse tubular system architecture. Using a novel approach, we aimed to directly correlate the influence of structural alterations with force development in ventricular trabeculae from failing human hearts. Trabeculae were excised from explanted human hearts in end-stage failure and immediately subjected to force measurements. Following functional experiments, each trabecula was fixed, sectioned and immuno-stained for structural investigations. Peak stress was highly variable between trabeculae from both within and between failing hearts and was strongly correlated with the cross-sectional area occupied by myocytes (MCSA), rather than total trabecula cross-sectional area. At the cellular level, myocytes exhibited extensive microtubule densification which was linked via JPH2 to time-to-peak stress. Trabeculae fractional MCSA variability was much higher than that in adjacent free wall samples. Together, these findings identify several structural parameters implicated in functional impairment in human HF and highlight the structural variability of ventricular trabeculae which should be considered when interpreting functional data.

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.

Junctophilin-2 in the nanoscale organisation and functional signalling of ryanodine receptor clusters in cardiomyocytes.

Signalling nanodomains requiring close contact between the plasma membrane and internal compartments, known as 'junctions', are fast communication hubs within excitable cells such as neurones and muscle. Here, we have examined two transgenic murine models probing the role of junctophilin-2, a membrane-tethering protein crucial for the formation and molecular organisation of sub-microscopic junctions in ventricular muscle cells of the heart. Quantitative single-molecule localisation microscopy showed that junctions in animals producing above-normal levels of junctophilin-2 were enlarged, allowing the re-organisation of the primary functional protein within it, the ryanodine receptor (RyR; in this paper, we use RyR to refer to the myocardial isoform RyR2). Although this change was associated with much enlarged RyR clusters that, due to their size, should be more excitable, functionally it caused a mild inhibition in the Ca2+ signalling output of the junctions (Ca2+ sparks). Analysis of the single-molecule densities of both RyR and junctophilin-2 revealed an ∼3-fold increase in the junctophilin-2 to RyR ratio. This molecular rearrangement is compatible with direct inhibition of RyR opening by junctophilin-2 to intrinsically stabilise the Ca2+ signalling properties of the junction and thus the contractile function of the cell.

Early transverse tubule development begins in utero in the sheep heart.

The ventricular cardiomyocytes of adult mammals contain invaginations of the plasma membrane known as transverse (t)-tubules. These regular structures are essential for the synchronisation of excitation-contraction (EC) coupling throughout the cell, which is a vital process for cardiac function. T-tubules form a close association with the sarcoplasmic reticulum (SR) to form junctions, where several key proteins involved in EC coupling are localised, including the SR calcium release channels-the ryanodine receptors (RyR). The lipophilic SR protein junctophilin-2 (JPH2) has been implicated in the development of both the junctions and t-tubules. Several studies have identified that t-tubules develop only postnatally in rodents, while historical electron microscopy data indicate that this is not the case in larger mammals, including humans. We have performed, to our knowledge, the first fluorescent, target-specific study to characterise t-tubule development in the large mammalian fetal heart, focussing on the sheep. T-tubules were present in fetal sheep hearts from 114 days gestation (with term being 145 days), with occurrence progressively increasing with gestational age, and further maturation after birth. This was accompanied by an increasing intracellular localisation of JPH2, which progressively increased its association with RyR within the cardiomyocytes as they undergo hypertrophy. These findings indicate that large mammalian hearts exhibit a significantly different temporal pattern of development compared to that of the rodent. Our findings have potential implications for human cardiac development, including the future investigation of congenital heart disease.

Use of a facilitated discussion model for antenatal care to improve communication.

BACKGROUND: Achieving health literacy is a critical step to improving health outcomes and the health of a nation. However, there is a lack of research on health literacy in low-resource countries, where maternal health outcomes are at their worst. OBJECTIVES: To examine the usefulness and feasibility of providing focused antenatal care (FANC) in a group setting using picture cards to improve patient-provider communication, patient engagement, and improve health literacy. DESIGN: An exploratory, mixed methods design was employed to gather pilot data using the Health Literacy Skills Framework. SETTINGS: A busy urban district hospital in the Ashanti Region of Ghana was used to gather data during 2014. PARTICIPANTS: A facility-driven convenience sample of midwives (n=6) aged 18 years or older, who could speak English or Twi, and had provided antenatal care at the participating hospital during the previous year prior to the start of the study participated in the study. METHODS: Data were collected using pre-test and post-test surveys, completed three months after the group FANC was implemented. A semi-structured focus group was conducted with four of the participating midwives and the registered nurse providing support and supervision for the study (n=5) at the time of the post-test. Data were analyzed concurrently to gain a broad understanding of patient communication, engagement, and group FANC. RESULTS: There were no significant differences in the mean communication (t(df=3)=0.541, p=0.626) and engagement (t(df=3)=-0.775, p=0.495) scores between the pre- and post-test. However, the focus group revealed the following themes: (a) improved communication through the use of picture cards; (b) enhanced information sharing and peer support through the facilitated group process and; and (c) an improved understanding of patient concerns. CONCLUSIONS: The improved communication noted through the use of picture cards and the enhanced information sharing and peer support elicited through the group FANC undoubtedly provided patients with additional tools to invoke self-determination, and carry out the behaviors they thought were most important to improve pregnancy outcomes.

A case series study on the effect of Ebola on facility-based deliveries in rural Liberia.

BACKGROUND: As communities' fears of Ebola virus disease (EVD) in West Africa exacerbate and their trust in healthcare providers diminishes, EVD has the potential to reverse the recent progress made in promoting facility-based delivery. Using retrospective data from a study focused on maternal and newborn health, this analysis examined the influence of EVD on the use of facility-based maternity care in Bong Country, Liberia, which shares a boarder with Sierra Leone - near the epicenter of the outbreak. METHODS: Using a case series design, retrospective data from logbooks were collected at 12 study sites in one county. These data were then analyzed to determine women's use of facility-based maternity care between January 2012 and October 2014. The primary outcome was the number of facility-based deliveries over time. The first suspected case of EVD in Bong County was reported on June 30, 2014. Heat maps were generated and the number of deliveries was normalized to the average number of deliveries during the full 12 months before the EVD outbreak (March 2013 - February 2014). RESULTS: Prior to the EVD outbreak, facility-based deliveries steadily increased in Bong County reaching an all-time high of over 500 per month at study sites in the first half of 2014 - indicating Liberia was making inroads in normalizing institutional maternal healthcare. However, as reports of EVD escalated, facility-based deliveries decreased to a low of 113 in August 2014. CONCLUSION: Ebola virus disease has negatively impacted the use of facility-based maternity services, placing childbearing women at increased risk for morbidity and death.

Outcomes of anatomic shoulder arthroplasty in primary osteoarthritis in type B glenoids.

BACKGROUND: Primary glenohumeral osteoarthritis with posterior wear of the glenoid and posterior subluxation of the humerus (Walch type B) presents a challenge to the treating surgeon. Our hypothesis was that glenoids with biconcavity (B2) would be associated with worse outcomes (functional scores and complications) than B1 glenoids. MATERIALS AND METHODS: We retrospectively analyzed prospectively collected data on 112 anatomic total shoulder arthroplasties (104 patients) with B glenoids. Preoperative computed tomography identified 64 B1 glenoids and 48 B2 glenoids (50 and 37 available for follow-up). RESULTS: A significant difference between B1 and B2 glenoids was noted in average retroversion (11° vs. 16°; P < .001) and average posterior humeral subluxation (65% vs. 75%; P < .001). No significant difference was seen in mean age (69.5 vs. 69.2 years) or body mass index (28.5 vs. 27.4) at time of surgery. At average follow-up of 60 months (range, 23-120 months), glenoid component radiolucencies (51.6%, B1; 47.9%, B2), range of motion, preoperative and postoperative scores of the shortened Disabilities of the Arm, Shoulder, and Hand questionnaire, and patient satisfaction were not significantly different between the 2 groups. Four revisions (4.6%) were documented for acute postoperative infection (2.3%), subscapularis failure (1.1%), and glenoid loosening (1.1%). CONCLUSIONS: Although biconcave glenoids commonly have more severe retroversion and posterior subluxation of the humerus, we were unable to find a clinical or radiographic difference in outcome of patients with B1 or B2 glenoids treated with anatomic total shoulder arthroplasty at intermediate-term follow-up. Continued clinical and radiographic follow-up of these cohorts will be necessary to assess any future divergence in outcome.

Texting From the Bush: Data Collection Using SMS Text Messaging in Areas of Low Network Coverage From Low-Literacy Providers.

Mobile health technology, specifically Short Message Service (SMS), provides a low-cost medium to transmit data in real time. SMS has been used for data collection by highly literate and educated health care workers in low-resource countries; however, no previous studies have evaluated implementation of an SMS intervention by low-literacy providers. The Liberian Ministry of Health and Social Welfare identified a lack of accurate data on the number of pregnancies from rural areas. To capture these data from 11 rural communities in Liberia, 66 low-literate traditional midwives and 15 high-literate certified midwives were trained to report data via SMS. Data were reported via a 9-digit code sent from Java-based mobile phones. Study aims included determining the following components of SMS transmission: success rate, accuracy, predictors of successful transmission, and acceptance. Success rate of SMS transmission was significantly higher for certified midwives than for traditional midwives. The error rate was significantly higher for traditional midwives than for certified midwives. Years of education was the only predictor of successful SMS transmission. Traditional midwives and certified midwives accepted the intervention, although certified midwives found it easier to use. Certified midwives performed significantly better than did traditional midwives. SMS texting interventions should be targeted to health care workers with higher rates of literacy.

History, policy and nursing practice implications of the plan b(®) emergency contraceptive.

Numerous policy changes have expanded access to emergency contraception, such as Plan B®, in recent years. Plan B® is a progesterone-based medication that prevents pregnancy from occurring up to 120 hours after unprotected intercourse by preventing ovulation and tubal transport. Increased access to Plan B® allows women to make independent decisions regarding reproductive health. Nurses play an important role in providing education as well as comprehensive, compassionate and holistic care.

IMPORTANT BUT INCOMPLETE: PLAN B AS AN AVENUE FOR POST-ASSAULT CARE.

Many survivors of rape do not seek post-assault care. The recent change in status of emergency contraception (EC), such as Plan B, to an over-the-counter (OTC) product may be further changing post-assault care-seeking. This descriptive study will quantify OTC EC use in the post-assault period and elicit survivors' desires for care. Data were collected from women purchasing OTC EC at university pharmacies (n=55) and students in an undergraduate university class (n=165). Quantitative results indicate annual prevalence rates of post-assault OTC EC use as 5.4%-7.3%. Qualitative analyses indicate OTC EC is an important but incomplete form of post-assault care. Future work should focus on intervention development to provide all OTC EC users with information about post-assault resources to prevent long-term sequelae.

Revealing T-Tubules in Striated Muscle with New Optical Super-Resolution Microscopy Techniquess.

The t-tubular system plays a central role in the synchronisation of calcium signalling and excitation-contraction coupling in most striated muscle cells. Light microscopy has been used for imaging t-tubules for well over 100 years and together with electron microscopy (EM), has revealed the three-dimensional complexities of the t-system topology within cardiomyocytes and skeletal muscle fibres from a range of species. The emerging super-resolution single molecule localisation microscopy (SMLM) techniques are offering a near 10-fold improvement over the resolution of conventional fluorescence light microscopy methods, with the ability to spectrally resolve nanometre scale distributions of multiple molecular targets. In conjunction with the next generation of electron microscopy, SMLM has allowed the visualisation and quantification of intricate t-tubule morphologies within large areas of muscle cells at an unprecedented level of detail. In this paper, we review recent advancements in the t-tubule structural biology with the utility of various microscopy techniques. We outline the technical considerations in adapting SMLM to study t-tubules and its potential to further our understanding of the molecular processes that underlie the sub-micron scale structural alterations observed in a range of muscle pathologies.

Accuracy of a hand-held surgical navigation system for tibial resection in total knee arthroplasty.

BACKGROUND: Accuracy of total knee arthroplasty (TKA) implant placement and overall limb are important goals of TKA technique. METHODS: The accuracy and ease of use of an accelerometer-based hand-held navigation system for tibial resection during TKA was examined in 90 patients. Preoperative goals for sagittal alignment, navigation system assembly time, resection time, and tourniquet time were evaluated. Coronal and sagittal alignment was measured postoperatively. RESULTS: The average coronal tibial component alignment was 0.43° valgus; 6.7% of patients had tibial coronal alignment outside of ±3° varus/valgus. The difference between the intraoperative goal and radiographically measured posterior tibial slope was 0.5°. The average time to completion of the tibial cut was 4.6 minutes. CONCLUSION: The accelerometer-based hand-held navigation system was accurate for tibial coronal and sagittal alignment during TKA, with no additional surgical time compared with conventional instrumentation.