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Introduction: Developmental and epileptic encephalopathies (DEEs) are rare epilepsy conditions that collectively impact 1 in 2000 children. They are highly genetically heterogeneous, resulting in significant barriers to accurate and adequate information for caregivers. This can lead to increased distress and dissatisfaction with the healthcare system. To address this gap, we developed 'GenE Compass' to provide caregivers with the highest-quality possible, understandable and relevant information in response to specific questions about their child's DEE. Using a mixed-method design, we will now pilot GenE Compass to evaluate the acceptability to caregivers and clinicians, feasibility and impact to caregivers., Methods and Analysis: We will recruit 88 caregivers (estimated final sample of 50 at follow-up) who have a child under 18 years of age with a suspected or confirmed DEE diagnosis. Following consent and a baseline questionnaire (questionnaire 1 (Q1)), participants will be able to submit questions to GenE Compass over a 3-month period. After 3 months, participants will complete a follow-up questionnaire (Q2) and an optional telephone interview to answer the research questions. Primary outcomes are acceptability of GenE Compass and feasibility of delivering the intervention (eg, cost of the intervention, number of questions submitted and time taken to respond to questions). Secondary outcomes include the impact of GenE Compass on caregivers' quality of life, information searching behaviours, perceptions of their child's illness and activation., Ethics and Discussion: The study protocol (V.2, dated 16 September 2021) has been approved by the Sydney Children's Hospitals Network Human Research Ethics Committee (ETH11277). The results will be disseminated in peer-reviewed journals and at scientific conferences. A lay summary will be disseminated to all participants., Trial Registration Number: ACTRN12621001544864., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The sinoatrial node (SAN), the leading pacemaker region, generates electrical impulses that propagate throughout the heart. SAN dysfunction with bradyarrhythmia is well documented in heart failure (HF). However, the underlying mechanisms are not completely understood. Mitochondria are critical to cellular processes that determine the life or death of the cell. The release of Ca 2+ from the ryanodine receptors 2 (RyR2) on the sarcoplasmic reticulum (SR) at mitochondria-SR microdomains serves as the critical communication to match energy production to meet metabolic demands. Therefore, we tested the hypothesis that alterations in the mitochondria-SR connectomics contribute to SAN dysfunction in HF. We took advantage of a mouse model of chronic pressure overload-induced HF by transverse aortic constriction (TAC) and a SAN-specific CRISPR-Cas9-mediated knockdown of mitofusin-2 ( Mfn2 ), the mitochondria-SR tethering GTPase protein. TAC mice exhibited impaired cardiac function with HF, cardiac fibrosis, and profound SAN dysfunction. Ultrastructural imaging using electron microscope (EM) tomography revealed abnormal mitochondrial structure with increased mitochondria-SR distance. The expression of Mfn2 was significantly down-regulated and showed reduced colocalization with RyR2 in HF SAN cells. Indeed, SAN-specific Mfn2 knockdown led to alterations in the mitochondria-SR microdomains and SAN dysfunction. Finally, disruptions in the mitochondria-SR microdomains resulted in abnormal mitochondrial Ca 2+ handling, alterations in localized protein kinase A (PKA) activity, and impaired mitochondrial function in HF SAN cells. The current study provides insights into the role of mitochondria-SR microdomains in SAN automaticity and possible therapeutic targets for SAN dysfunction in HF patients.

The process of urine removal from the kidney occurs via the renal pelvis (RP). The RP demarcates the beginning of the upper urinary tract and is endowed with smooth muscle cells. Along the RP, organized contraction of smooth muscle cells generates the force required to move urine boluses toward the ureters and bladder. This process is mediated by specialized pacemaker cells that are highly expressed in the proximal RP that generate spontaneous rhythmic electrical activity to drive smooth muscle depolarization. The mechanisms by which peristaltic contractions propagate from the proximal to distal RP are not fully understood. In this study, we utilized a transgenic mouse that expresses the genetically encoded Ca 2+ indicator, GCaMP3, under a myosin heavy chain promotor to visualize spreading peristaltic contractions in high spatial detail. Using this approach, we discovered variable effects of L-type Ca 2+ channel antagonists on contraction parameters. Inhibition of T-type Ca 2+ channels reduced the frequency and propagation distance of contractions. Similarly, antagonizing Ca 2+ -activated Cl - channels or altering the transmembrane Cl - gradient decreased contractile frequency and significantly inhibited peristaltic propagation. These data suggest that voltage-gated Ca 2+ channels are important determinants of contraction initiation and maintain the fidelity of peristalsis as the spreading contraction moves further toward the ureter. Recruitment of Ca 2+ -activated Cl - channels, likely Anoctamin-1, and T-type Ca 2+ channels are required for efficiently conducting the depolarizing current throughout the length of the RP. These mechanisms are necessary for the efficient removal of urine from the kidney., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Physiological Society.)

The cardiac cycle starts when an action potential is produced by pacemaking cells in the sinoatrial node. This cycle is repeated approximately 100 000 times in humans and 1 million times in mice per day, imposing a monumental metabolic demand on the heart, requiring efficient blood supply via the coronary vasculature to maintain cardiac function. Although the ventricular coronary circulation has been extensively studied, the relationship between vascularization and cellular pacemaking modalities in the sinoatrial node is poorly understood. Here, we tested the hypothesis that the organization of the sinoatrial node microvasculature varies regionally, reflecting local myocyte firing properties. We show that vessel densities are higher in the superior versus inferior sinoatrial node. Accordingly, sinoatrial node myocytes are closer to vessels in the superior versus inferior regions. Superior and inferior sinoatrial node myocytes produce stochastic subthreshold voltage fluctuations and action potentials. However, the intrinsic action potential firing rate of sinoatrial node myocytes is higher in the superior versus inferior node. Our data support a model in which the microvascular densities vary regionally within the sinoatrial node to match the electrical and Ca 2+ dynamics of nearby myocytes, effectively determining the dominant pacemaking site within the node. In this model, the high vascular density in the superior sinoatrial node places myocytes with metabolically demanding, high-frequency action potentials near vessels. The lower vascularization and electrical activity of inferior sinoatrial node myocytes could limit these cells to function to support sinoatrial node periodicity with sporadic voltage fluctuations via a stochastic resonance mechanism., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Physiological Society.)

Key Points: Platelet-derived growth factor receptor-α (PDGFRα) is a novel biomarker along with smooth myosin heavy chain for the pacemaker cells (previously termed 'atypical' smooth muscle cells) in the murine and cynomolgus monkey pelvis-kidney junction. PDGFRα + cells present in adventitial and urothelial layers of murine renal pelvis do not express smooth muscle myosin heavy chain (smMHC) but are in close apposition to nerve fibres. Most c-Kit + cells in the renal pelvis are mast cells. Mast cells (CD117 + /CD45 + ) are more abundant in the proximal renal pelvis and pelvis-kidney junction regions whereas c-Kit + interstitial cells (CD117 + /CD45 - ) are found predominantly in the distal renal pelvis and ureteropelvic junction. PDGFRα + cells are distinct from c-Kit + interstitial cells. A subset of PDGFRα + cells express the Ca 2+ -activated Cl - channel, anoctamin-1, across the entire renal pelvis. Spontaneous Ca 2+ transients were observed in c-Kit + interstitial cells, smMHC + PDGFRα cells and smMHC - PDGFRα cells using mice expressing genetically encoded Ca 2+ sensors., Abstract: Rhythmic contractions of the renal pelvis transport urine from the kidneys into the ureter. Specialized pacemaker cells, termed atypical smooth muscle cells (ASMCs), are thought to drive the peristaltic contractions of typical smooth muscle cells (TSMCs) in the renal pelvis. Interstitial cells (ICs) in close proximity to ASMCs and TSMCs have been described, but the role of these cells is poorly understood. The presence and distributions of platelet-derived growth factor receptor-α + (PDGFRα + ) ICs in the pelvis-kidney junction (PKJ) and distal renal pelvis were evaluated. We found PDGFRα + ICs in the adventitial layers of the pelvis, the muscle layer of the PKJ and the adventitia of the distal pelvis. PDGFRα + ICs were distinct from c-Kit + ICs in the renal pelvis. c-Kit + ICs are a minor population of ICs in murine renal pelvis. The majority of c-Kit + cells were mast cells. PDGFRα + cells in the PKJ co-expressed smooth muscle myosin heavy chain (smMHC) and several other smooth muscle gene transcripts, indicating these cells are ASMCs, and PDGFRα is a novel biomarker for ASMCs. PDGFRα + cells also express Ano1, which encodes a Ca 2+ -activated Cl - conductance that serves as a primary pacemaker conductance in ICs of the GI tract. Spontaneous Ca 2+ transients were observed in c-Kit + ICs, smMHC + PDGFRα cells and smMHC - PDGFRα cells using genetically encoded Ca 2+ sensors. A reporter strain of mice with enhanced green fluorescent protein driven by the endogenous promotor for Pdgfra was shown to be a powerful new tool for isolating and characterizing the phenotype and functions of these cells in the renal pelvis., (© 2020 The Authors. The Journal of Physiology © 2020 The Physiological Society.)

Background: Front-of-neck airway rescue in a cannot intubate, cannot oxygenate (CICO) scenario with impalpable anatomy is particularly challenging. Several techniques have been described based on a midline vertical neck incision with subsequent finger dissection, followed by either a cannula or scalpel puncture of the now palpated airway. We explored whether the speed of rescue oxygenation differs between these techniques., Methods: In a high-fidelity simulation of a CICO scenario in anaesthetised Merino sheep with impalpable front-of-neck anatomy, 35 consecutive eligible participants undergoing airway training performed scalpel-finger-cannula and scalpel-finger-bougie in a random order. The primary outcome was time from airway palpation to first oxygen delivery. Data, were analysed with Cox proportional hazards., Results: Scalpel-finger-cannula was associated with shorter time to first oxygen delivery on univariate (hazard ratio [HR]=11.37; 95% confidence interval [CI], 5.14-25.13; P<0.001) and multivariate (HR=8.87; 95% CI, 4.31-18.18; P<0.001) analyses. In the multivariable model, consultant grade was also associated with quicker first oxygen delivery compared with registrar grade (HR=3.28; 95% CI, 1.36-7.95; P=0.008). With scalpel-finger-cannula, successful oxygen delivery within 3 min of CICO declaration and ≤2 attempts was more frequent; 97% vs 63%, P<0.001. In analyses of successful cases only, scalpel-finger-cannula resulted in earlier improvement in arterial oxygen saturations (-25 s; 95% CI, -35 to -15; P<0.001), but a longer time to first capnography reading (+89 s; 95% CI, 69 to 110; P<0.001). No major complications occurred in either arm., Conclusions: The scalpel-finger-cannula technique was associated with superior oxygen delivery performance during a simulated CICO scenario in sheep with impalpable front-of-neck anatomy., (Copyright © 2020 British Journal of Anaesthesia. All rights reserved.)

Competing Interests: The authors declare no competing interest.

Excitation-contraction (EC) coupling is the coordinated process by which an action potential triggers cardiac myocyte contraction. EC coupling is initiated in dyads where the junctional sarcoplasmic reticulum (jSR) is in tight proximity to the sarcolemma of cardiac myocytes. Existing models of EC coupling critically depend on dyad stability to ensure the fidelity and strength of EC coupling, where even small variations in ryanodine receptor channel and voltage-gated calcium channel-α 1.2 subunit separation dramatically alter EC coupling. However, dyadic motility has never been studied. Here, we developed a novel strategy to track specific jSR units in dissociated adult ventricular myocytes using photoactivatable fluorescent proteins. We found that the jSR is not static. Instead, we observed dynamic formation and dissolution of multiple dyadic junctions regulated by the microtubule-associated molecular motors kinesin-1 and dynein. Our data support a model where reproducibility of EC coupling results from the activation of a temporally averaged number of SR Ca 2+ release units forming and dissolving SR-sarcolemmal junctions. These findings challenge the long-held view that the jSR is an immobile structure and provide insights into the mechanisms underlying its motility.

Key Points: Electrical pacemaking in gastrointestinal muscles is generated by specialized interstitial cells of Cajal that produce the patterns of contractions required for peristalsis and segmentation in the gut. The calcium-activated chloride conductance anoctamin-1 (Ano1) has been shown to be responsible for the generation of pacemaker activity in GI muscles, but this conclusion is established from studies of juvenile animals in which effects of reduced Ano1 on gastric emptying and motor patterns could not be evaluated. Knocking down Ano1 expression using Cre/LoxP technology caused dramatic changes in in gastric motor activity, with disrupted slow waves, abnormal phasic contractions and delayed gastric emptying; modest changes were noted in the small intestine. Comparison of the effects of Ano1 antagonists on muscles from juvenile and adult small intestinal muscles suggests that conductances in addition to Ano1 may develop with age and contribute to pacemaker activity., Abstract: Interstitial cells of Cajal (ICC) generate slow waves and transduce neurotransmitter signals in the gastrointestinal (GI) tract, facilitating normal motility patterns. ICC express a Ca 2+ -activated Cl - conductance (CaCC), and constitutive knockout of the channel protein anoctamin-1 leads to loss of slow waves in gastric and intestinal muscles. These knockout experiments were performed on juvenile mice. However, additional experiments demonstrated significant differences in the sensitivity of gastric and intestinal muscles to antagonists of anoctamin-1 channels. Furthermore, the significance of anoctamin-1 and the electrical and mechanical behaviours facilitated by this conductance have not been evaluated on the motor behaviours of adult animals. Cre/loxP technology was used to generate cell-specific knockdowns of anoctamin-1 in ICC (Kit CreERT2/+ ;Ano1 tm2jrr/+ ) in GI muscles. The recombination efficiency of Kit CreERT was evaluated with an eGFP reporter, molecular techniques and immunohistochemistry. Electrical and contractile experiments were used to examine the consequences of anoctamin-1 knockdown on pacemaker activity, mechanical responses, gastric motility patterns, gastric emptying and GI transit. Reduced anoctamin-1 caused loss of gastric, but not intestinal slow waves. Irregular spike complexes developed in gastric muscles, leading to uncoordinated antral contractions, delayed gastric emptying and increased total GI transit time. Slow waves in intestinal muscles of juvenile mice were more sensitive to anoctamin-1 antagonists than slow waves in adult muscles. The low susceptibility to anoctamin-1 knockdown and weak efficacy of anoctamin-1 antagonists in inhibiting slow waves in adult small intestinal muscles suggest that a conductance in addition to anoctamin-1 may develop in small intestinal ICC with ageing and contribute to pacemaker activity., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

Key Points: Activation of focal adhesion kinase (FAK) by integrin signalling facilitates smooth muscle contraction by transmitting the force generated by myofilament activation to the extracellular matrix and throughout the smooth muscle tissue. Here we report that electrical field stimulation (EFS) of cholinergic motor neurons activates FAK in gastric fundus smooth muscles, and that FAK activation by EFS is atropine-sensitive but nicardipine-insensitive. PDBu and calyculin A contracted gastric fundus muscles Ca 2+ -independently and also activated FAK. Inhibition of FAK activation inhibits the contractile responses evoked by EFS, and inhibits CPI-17 phosphorylation at T38. This study indicates that mechanical force or tension is sufficient to activate FAK, and that FAK appears to be involved in the activation of the protein kinase C-CPI-17 Ca 2+ sensitization pathway in gastric fundus smooth muscles. These results reveal a novel role for FAK in gastric fundus smooth muscle contraction by facilitating CPI-17 phosphorylation., Abstract: Smooth muscle contraction involves regulating myosin light chain phosphorylation and dephosphorylation by myosin light chain kinase and myosin light chain phosphatase. C-kinase potentiated protein phosphatase-1 inhibitor of 17 kDa (CPI-17) and myosin phosphatase targeting subunit of myosin light-chain phosphatase (MYPT1) are crucial for regulating gastrointestinal smooth muscle contraction by inhibiting myosin light chain phosphatase. Integrin signalling involves the dynamic recruitment of several proteins, including focal adhesion kinase (FAK), to focal adhesions. FAK tyrosine kinase activation is involved in cell adhesion to the extracellular matrix via integrin signalling. FAK participates in linking the force generated by myofilament activation to the extracellular matrix and throughout the smooth muscle tissue. Here, we show that cholinergic stimulation activates FAK in gastric fundus smooth muscles. Electrical field stimulation in the presence of N ω -nitro-l-arginine methyl ester and MRS2500 contracted gastric fundus smooth muscle strips and increased FAK Y397 phosphorylation (pY397). Atropine blocked the contractions and prevented the increase in pY397. The FAK inhibitor PF-431396 inhibited the contractions and the increase in pY397. PF-431396 also inhibited the electrical field stimulation-induced increase in CPI-17 T38 phosphorylation, and reduced MYPT1 T696 and T853, and myosin light chain S19 phosphorylation. Ca 2+ influx was unaffected by PF-431396. Nicardipine inhibited the contractions but had no effect on the increase in pY397. Phorbol 12,13-dibutyrate or calyculin A contracted gastric fundus smooth muscle strips Ca 2+ independently and increased pY397. Our findings suggest that FAK is activated by mechanical forces during contraction and reveal a novel role of FAK in the regulation of CPI-17 phosphorylation., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

Serum response factor (SRF) transcriptionally regulates expression of contractile genes in smooth muscle cells (SMC). Lack or decrease of SRF is directly linked to a phenotypic change of SMC, leading to hypomotility of smooth muscle in the gastrointestinal (GI) tract. However, the molecular mechanism behind SRF-induced hypomotility in GI smooth muscle is largely unknown. We describe here how SRF plays a functional role in the regulation of the SMC contractility via myotonic dystrophy protein kinase (DMPK) and L-type calcium channel CACNA1C. GI SMC expressed Dmpk and Cacna1c genes into multiple alternative transcriptional isoforms. Deficiency of SRF in SMC of Srf knockout (KO) mice led to reduction of SRF-dependent DMPK, which down-regulated the expression of CACNA1C. Reduction of CACNA1C in KO SMC not only decreased intracellular Ca2+ spikes but also disrupted their coupling between cells resulting in decreased contractility. The role of SRF in the regulation of SMC phenotype and function provides new insight into how SMC lose their contractility leading to hypomotility in pathophysiological conditions within the GI tract., Competing Interests: The authors have declared that no competing interests exist.

Objectives: Obtaining a diagnosis of an Autism Spectrum Disorder (ASD) for a child is a pivotal point in developing the treatment plan for the child but can also be regarded as highly stressful by parents. The current study examined the impact of different aspects of the diagnosis process on the self-reported mental health of mothers of children undergoing a diagnosis for ASD in a cross-sectional cohort design., Methods: One-hundred-fifty-eight mothers of consequently diagnosed children with ASD participated. The severity of the children's ASD and their intellectual functioning was assessed within twelve months of the diagnosis, and the mothers completed a psychometric assessment battery including the Hospital Anxiety and Depression Scale, General Health Questionnaire, and Questionnaire on Resources and Stress., Results: The actual time from first reporting a problem to obtaining a diagnosis, and the speed of the diagnostic process from first to last appointment, were both negatively related to patenting stress. In contrast, mothers' perceptions of the speed and helpfulness of the process were negatively related to levels of anxiety and depression. The number of professionals involved in the process and the perceived coherence of the diagnosis were also negatively related to aspects of mothers' functioning., Conclusions: Care is needed to help mothers through the diagnostic process with regard to their own functioning. Providing information and help sources throughout the process, while keeping the number of professionals involved to a minimum, may improve the parent perception of the process and reduce the negative impacts of the diagnosis on the family as a whole., Competing Interests: The authors declare no conflict of interest.

The mechanisms underlying slow-transit constipation (STC) are unclear. In 50% of patients with STC, some form of outlet obstruction has been reported; also an elongated colon has been linked to patients with STC. Our aims were 1) to develop a murine model of STC induced by partial outlet obstruction and 2) to determine whether this leads to colonic elongation and, consequently, activation of the inhibitory "occult reflex," which may contribute to STC in humans. Using a purse-string suture, we physically reduced the maximal anal sphincter opening in C57BL/6 mice. After 4 days, the mice were euthanized (acutely obstructed), the suture was removed (relieved), or the suture was removed and replaced repeatedly (chronically obstructed, over 24-31 days). In partially obstructed mice, we observed increased cyclooxygenase (COX)-2 levels in muscularis and mucosa, an elongated impacted large bowel, slowed transit, nonpropagating colonic migrating motor complexes (CMMCs), a lack of mucosal reflexes, a depolarized circular muscle with slow-wave activity due to a lack of spontaneous inhibitory junction potentials, muscle hypertrophy, and CMMCs in mucosa-free preparations. Elongation of the empty obstructed colon produced a pronounced occult reflex. Removal of the obstruction or addition of a COX-2 antagonist (in vitro and in vivo) restored membrane potential, spontaneous inhibitory junction potentials, CMMC propagation, and mucosal reflexes. We conclude that partial outlet obstruction increases COX-2 leading to a hyperexcitable colon. This hyperexcitability is largely due to suppression of only descending inhibitory nerve pathways by prostaglandins. The upregulation of motility is suppressed by the occult reflex activated by colonic elongation.