Feedback contributions to excitation-contraction coupling in native functioning striated muscle.

Skeletal and cardiac muscle excitation-contraction coupling commences with Nav1.4/Nav1.5-mediated, surface and transverse (T-) tubular, action potential generation. This initiates feedforward, allosteric or Ca2+-mediated, T-sarcoplasmic reticular (SR) junctional, voltage sensor-Cav1.1/Cav1.2 and ryanodine receptor-RyR1/RyR2 interaction. We review recent structural, physiological and translational studies on possible feedback actions of the resulting SR Ca2+ release on Nav1.4/Nav1.5 function in native muscle. Finite-element modelling predicted potentially regulatory T-SR junctional [Ca2+]TSR domains. Nav1.4/Nav1.5, III-IV linker and C-terminal domain structures included Ca2+ and/or calmodulin-binding sites whose mutations corresponded to specific clinical conditions. Loose-patch-clamped native murine skeletal muscle fibres and cardiomyocytes showed reduced Na+ currents (INa) following SR Ca2+ release induced by the Epac and direct RyR1/RyR2 activators, 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphate and caffeine, abrogated by the RyR inhibitor dantrolene. Conversely, dantrolene and the Ca2+-ATPase inhibitor cyclopiazonic acid increased INa. Experimental, catecholaminergic polymorphic ventricular tachycardic RyR2-P2328S and metabolically deficient Pgc1ß-/- cardiomyocytes also showed reduced INa accompanying [Ca2+]i abnormalities rescued by dantrolene- and flecainide-mediated RyR block. Finally, hydroxychloroquine challenge implicated action potential (AP) prolongation in slowing AP conduction through modifying Ca2+ transients. The corresponding tissue/organ preparations each showed pro-arrhythmic, slowed AP upstrokes and conduction velocities. We finally extend discussion of possible Ca2+-mediated effects to further, Ca2+, K+ and Cl-, channel types. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

The ß3-subunit modulates the effect of venom peptides ProTx-II and OD1 on NaV 1.7 gating.

The voltage-gated sodium channel NaV 1.7 is involved in various pain phenotypes and is physiologically regulated by the NaV -ß3-subunit. Venom toxins ProTx-II and OD1 modulate NaV 1.7 channel function and may be useful as therapeutic agents and/or research tools. Here, we use patch-clamp recordings to investigate how the ß3-subunit can influence and modulate the toxin-mediated effects on NaV 1.7 function, and we propose a putative binding mode of OD1 on NaV 1.7 to rationalise its activating effects. The inhibitor ProTx-II slowed the rate of NaV 1.7 activation, whilst the activator OD1 reduced the rate of fast inactivation and accelerated recovery from inactivation. The ß3-subunit partially abrogated these effects. OD1 induced a hyperpolarising shift in the V1/2 of steady-state activation, which was not observed in the presence of ß3. Consequently, OD1-treated NaV 1.7 exhibited an enhanced window current compared with OD1-treated NaV 1.7-ß3 complex. We identify candidate OD1 residues that are likely to prevent the upward movement of the DIV S4 helix and thus impede fast inactivation. The binding sites for each of the toxins and the predicted location of the ß3-subunit on the NaV 1.7 channel are distinct. Therefore, we infer that the ß3-subunit influences the interaction of toxins with NaV 1.7 via indirect allosteric mechanisms. The enhanced window current shown by OD1-treated NaV 1.7 compared with OD1-treated NaV 1.7-ß3 is discussed in the context of differing cellular expressions of NaV 1.7 and the ß3-subunit in dorsal root ganglion (DRG) neurons. We propose that ß3, as the native binding partner for NaV 1.7 in DRG neurons, should be included during screening of molecules against NaV 1.7 in relevant analgesic discovery campaigns.

Validating the Simplified Endoscopic Mucosal Assessment for Crohn's Disease: A Novel Method for Assessing Disease Activity.

BACKGROUND: To demonstrate treatment efficacy in Crohn's disease (CD), regulatory authorities require that trials include an endoscopic remission/response end point; however, standardized endoscopic assessment of disease activity, such as the Simple Endoscopic Score for Crohn's Disease (SES-CD), is not typically recorded by clinicians in practice or outside of clinical trials. The novel Simplified Endoscopic Mucosal Assessment for Crohn's Disease (SEMA-CD) was developed to be easy to use in routine clinical practice and as a trial end point. We conducted a study to assess and validate the reliability and feasibility of SEMA-CD as a measure of endoscopic disease activity. METHODS: Pre- and post-treatment ileocolonoscopy videos of pediatric (n = 36) and adult (n = 74) CD patients from 2 ustekinumab clinical trials were each scored with SEMA-CD by 2 to 3 professional central readers, blinded to clinical history and other video scorings; the correlation between SEMA-CD and SES-CD previously completed during the trials was assessed. Sensitivity to change, inter- and intrarater reliability, and comparative ease of scoring were also assessed. RESULTS: The SEMA-CD strongly correlated with SES-CD (Spearman ρ = 0.89; 95% confidence interval, 0.86-0.92). Pre- to post-treatment changes in SEMA-CD vs in SES-CD were strongly correlated, and the correlation remained strong between the scores when compared by study population (pediatric, adult), disease severity, and video quality. Intra- and inter-rater reliability were good, and SEMA-CD was rated easier than SES-CD to score 63.0% of the time, although slightly more difficult than SES-CD to score <1.0% of the time. CONCLUSIONS: The SEMA-CD is reliable, reproducible, sensitive to change, and easy to use in both pediatric and adult patients with CD.

Rapid Personalised Virtual Planning and On-Demand Surgery for Acute Spinal Trauma Using 3D-Printing, Biomodelling and Patient-Specific Implant Manufacture.

Three-dimensional printing is a rapidly growing field, with extensive application to orthopaedics and spinal surgery. Three-dimensional-printed (3DP) patient-specific implants (PSIs) offer multiple potential benefits over generic alternatives, with their use increasingly being described in the spinal literature. This report details a unique, emergency case of a traumatic spinal injury in a 31-year-old male, acquired rurally and treated with a 3DP PSI in a tertiary unit. With increasing design automation and process improvements, rapid, on-demand virtual surgical planning (VSP) and 3DP PSIs may present the future of orthopaedics and trauma care, enabling faster, safer, and more cost-effective patient-specific procedures.

Cutibacterium recovered from deep specimens at the time of revision shoulder arthroplasty samples has increased biofilm-forming capacity and hemolytic activity compared with Cutibacterium skin isolates from normal subjects.

BACKGROUND: Biofilm formation and hemolytic activity are factors that may correlate with the virulence of Cutibacterium. We sought to compare the prevalence of these potential markers of pathogenicity between Cutibacterium recovered from deep specimens obtained at the time of surgical revision for failed shoulder arthroplasty and Cutibacterium recovered from skin samples from normal subjects. METHODS: We compared 42 deep-tissue or explant isolates with 43 control Cutibacterium samples obtained from skin isolates from normal subjects. Subtyping information was available for all isolates. Biofilm-forming capacity was measured by inoculating a normalized amount of each isolate onto a 96-well plate. Planktonic bacteria were removed, the remaining adherent bacteria were stained with crystal violet, the crystal violet was re-solubilized in ethyl alcohol, and biofilm-forming capacity was quantitated by optical density (OD). Hemolytic activity was measured by plating a normalized amount of isolate onto agar plates. The area of the colony and the surrounding area of blood lysis were measured and reported as minimal, moderate, or severe hemolysis. RESULTS: Biofilm-forming capacity was significantly higher in the tissue and explant samples compared with the control skin samples (OD of 0.34 ± 0.30 for deep tissue vs. 0.20 ± 0.28 for skin, P = .002). Hemolytic activity was also significantly higher in the tissue and explant samples than in the control skin samples (P < .0001). Samples with hemolytic activity had significantly higher biofilm-forming capacity compared with samples without hemolytic activity (OD of 0.27 ± 0.29 vs. 0.12 ± 0.15, P = .015). No difference in biofilm-forming capacity or hemolytic activity was found between subtypes. CONCLUSIONS: Cutibacterium obtained from deep specimens at the time of revision shoulder arthroplasty has higher biofilm-forming capacity and hemolytic activity than Cutibacterium recovered from the skin of normal subjects. These data add support for the view that Cutibacterium harvested from deep tissues may have clinically significant virulence characteristics. The lack of correlation between these clinically relevant phenotypes and subtypes indicates that additional study is needed to identify genotypic markers that better correlate with biofilm and hemolytic activity.

Can the SARS-CoV-2 Spike Protein Bind Integrins Independent of the RGD Sequence?

The RGD motif in the Severe Acute Syndrome Coronavirus 2 (SARS-CoV-2) spike protein has been predicted to bind RGD-recognizing integrins. Recent studies have shown that the spike protein does, indeed, interact with αVß3 and α5ß1 integrins, both of which bind to RGD-containing ligands. However, computational studies have suggested that binding between the spike RGD motif and integrins is not favourable, even when unfolding occurs after conformational changes induced by binding to the canonical host entry receptor, angiotensin-converting enzyme 2 (ACE2). Furthermore, non-RGD-binding integrins, such as αx, have been suggested to interact with the SARS-CoV-2 spike protein. Other viral pathogens, such as rotaviruses, have been recorded to bind integrins in an RGD-independent manner to initiate host cell entry. Thus, in order to consider the potential for the SARS-CoV-2 spike protein to bind integrins independent of the RGD sequence, we investigate several factors related to the involvement of integrins in SARS-CoV-2 infection. First, we review changes in integrin expression during SARS-CoV-2 infection to identify which integrins might be of interest. Then, all known non-RGD integrin-binding motifs are collected and mapped to the spike protein receptor-binding domain and analyzed for their 3D availability. Several integrin-binding motifs are shown to exhibit high sequence similarity with solvent accessible regions of the spike receptor-binding domain. Comparisons of these motifs with other betacoronavirus spike proteins, such as SARS-CoV and RaTG13, reveal that some have recently evolved while others are more conserved throughout phylogenetically similar betacoronaviruses. Interestingly, all of the potential integrin-binding motifs, including the RGD sequence, are conserved in one of the known pangolin coronavirus strains. Of note, the most recently recorded mutations in the spike protein receptor-binding domain were found outside of the putative integrin-binding sequences, although several mutations formed inside and close to one motif, in particular, may potentially enhance binding. These data suggest that the SARS-CoV-2 spike protein may interact with integrins independent of the RGD sequence and may help further explain how SARS-CoV-2 and other viruses can evolve to bind to integrins.

Deep learning neural network analysis of human blastocyst expansion from time-lapse image files.

RESEARCH QUESTION: Can artificial intelligence (AI) discriminate a blastocyst's cellular area from unedited time-lapse image files using semantic segmentation and a deep learning optimized U-Net architecture for use in selecting single blastocysts for transfer? DESIGN: This platform was retrospectively applied to time-lapse files from 101 sequentially transferred single blastocysts that were prospectively selected for transfer by their highest expansion ranking within cohorts using a 10 h expansion assay rather than standard grading. RESULTS: The AI platform provides expansion curves and raw data files to classify and compare blastocyst phenotypes within both cohorts and populations. Of 35 sequential unbiopsied single blastocyst transfers, 23 (65.7%) resulted in a live birth. Of 66 sequential single euploid blastocyst transfers, also selected for their most robust expansion, 49 (74.2%) resulted in live birth. The AI platform revealed that the averaged expansion rate was significantly (P = 0.007) greater in euploid blastocysts that resulted in live births compared with those resulting in failure to give a live birth. The platform further provides a framework to analyse fragmentation phenotypes that can test new hypotheses for developmental regulation during the preimplantation period. CONCLUSIONS: AI can be used to quantitatively describe blastocyst expansion from unedited time-lapse image files and can be used to quantitatively rank-order blastocysts for transfer. Early clinical results from such single blastocyst selection suggests that live birth rates without biopsy may be comparable to those found using single euploid blastocysts in younger, good responder patients.

Clinically compatible advances in blood-derived endothelial progenitor cell isolation and reprogramming for translational applications.

The promise of using induced pluripotent stem cells (iPSCs) for cellular therapies has been hampered by the lack of easily isolatable and well characterized source cells whose genomes have undergone minimal changes during their processing. Blood-derived late-outgrowth endothelial progenitor cells (EPCs) are used for disease modeling and have potential therapeutic uses including cell transplantation and the translation of induced pluripotent stem cell (iPSC) derivatives. However, the current isolation of EPCs has been inconsistent and requires at least 40-80 mL of blood, limiting their wider use. In addition, previous EPC reprogramming methods precluded the translation of EPC-derived iPSCs to the clinic. Here a series of clinically-compatible advances in the isolation and reprogramming of EPCs is presented, including a reduction of blood sampling volumes to 10 mL and use of highly efficient RNA-based reprogramming methods together with autologous human serum, resulting in clinically relevant iPSCs carrying minimal copy number variations (CNVs) compared to their parent line.

Adjacent-Level Ossification Development in Single-Level Standalone Anterior Cervical Discectomy and Fusion Versus Anterior Cervical Discectomy and Fusion With Plate.

STUDY DESIGN: Retrospective case control study. OBJECTIVES: Adjacent-level ossification development (ALOD) is a distinct form of adjacent segmental degeneration that has been recognized to occur after anterior cervical discectomy and fusion (ACDF). It is unclear whether ACDF with plate versus standalone has an effect on rates of ALOD. This retrospective case-control study aims to assess the rate of ALOD in a large series of patients undergoing ACDF with and without plate and factors causing ALOD. METHODS: Data was collected for patients undergoing ACDF from January 2009 to July 2016. Data collected was from multiple centers and included demographic data, surgical data, radiological imaging at time of surgery, and serial follow-up imaging. The radiology for ALOD was independently reviewed. Cohorts were divided into ACDF with plate (Group P = plate) and ACDF without plate (Groups S = standalone) and outcomes were compared. RESULTS: There were 260 patients with 138 (53%) in Group P and 122 (47%) in Group S. ALOD was observed in 15.3% of patients overall, 29% in group P and 2.8% in group S (P < .001). Following multivariate adjustment, statistically significant association was found between use of plate and ALOD (odds ratio = 12.8, 95% confidence interval = 3.52-45.45, P < .001). Plate-to-disc distance <5 mm was significantly associated with ALOD (odds ratio = 13.5, 95% confidence interval = 3.83-47.62, P < .001). CONCLUSION: The use of anterior plate with ACDF was associated with ALOD. Plate-to-disc distance <5 mm was significantly associated with ALOD even after adjustment for confounding factors. We conclude utilization of standalone cages or cages with plate with more than 5 mm distance from adjacent disc to minimize ALOD.

Factors affecting the rates of adherence to surveillance recommendations for incidental pancreatic cystic lesions in a large urban safety net hospital.

BACKGROUND: Pancreatic cystic lesions (PCLs) are a heterogenous group of lesions with varying degrees of malignant potential. PCLs are often incidentally detected on imaging. Management for patients without an immediate indication for resection or tissue sampling entails radiographic surveillance to assess for features concerning for malignant transformation. This study aims to determine the rates of adherence to surveillance recommendations for incidental PCLs, and identify factors associated with adherence or loss of follow-up. METHODS: We conducted a single-centre retrospective study of patients at a tertiary safety net hospital with incidentally discovered asymptomatic PCLs. Follow-up was defined as having undergone repeat imaging as recommended in the radiology report. Data were analysed using logistic regression. RESULTS: Within our cohort (n=172), 123 (71.5%) subjects completed follow-up imaging. Attending a gastroenterology appointment was most strongly associated with completing follow-up for PCLs and remained significant (p=0.001) in a multivariate logistic regression model. Subjects without a documented primary care provider were less likely to have follow-up (p=0.028). Larger cyst size was associated with completion of follow-up in univariate only (p=0.067). CONCLUSION: We found that follow-up of an incidentally discovered PCLs was completed in the majority of our subjects. Incomplete follow-up for PCLs occurred in up to one in three to four patients in our cohort. Access to primary care and utilisation of subspecialty gastroenterology care are associated with completion of follow-up for PCLs. If validated, our findings can guide potential interventions to improve follow-up rates for PCLs.

Dyspnea in an HIV Patient: A Not so Typical Presentation of Lung Adenocarcinoma.

Dyspnea in a HIV patient often warrants an extensive workup. The most common etiology of this presentation is likely due to an infectious etiology. However, with the introduction of antiretroviral treatment, non-AIDS-defining illness including malignancies are increasingly being reported. We report the case of a 46-year-old African American female, nonsmoker who presented with dyspnea and found to have pericardial effusion. In patients with HIV presenting with dyspnea, pericardial effusion should be considered among the differential diagnosis, more so in patients in whom infectious etiologies have been ruled out. Further workup, including imaging and biopsy, revealed that our patient had metastatic lung adenocarcinoma. The introduction of antiretroviral treatment has significantly reduced mortality for those with AIDS from AIDS-defining illness and malignancies. However, the incidence of non-AIDS-defining malignancies like lung adenocarcinoma (most common non-AIDS-defining malignancy) is being increasingly reported. Lung adenocarcinoma often presents at a younger age in patients with HIV than the general population. Smoking rates are higher in patients with HIV and may be a contributing factor to the early onset of lung cancer; however, other factors such as long-term medications and immunomodulation in HIV may also play a role. Prognosis is also worse for HIV-positive patients having lung cancer compared with those who are HIV negative, even at a similar stage of cancer.

4PBA Restores Signaling of a Cysteine-substituted Mutant BMPR2 Receptor Found in Patients with Pulmonary Arterial Hypertension.

Mutations in the gene encoding BMPR2 (bone morphogenetic protein type 2 receptor) are the major cause of heritable pulmonary arterial hypertension (PAH). Point mutations in the BMPR2 ligand-binding domain involving cysteine residues (such as C118W) are causative of PAH and predicted to cause protein misfolding. Using heterologous overexpression systems, we showed previously that these mutations lead to retention of BMPR2 in the endoplasmic reticulum but are partially rescued by chemical chaperones. Here, we sought to determine whether the chemical chaperone 4-phenylbutyrate (4PBA) restores BMPR2 signaling in primary cells and in a knockin mouse harboring a C118W mutation. First, we confirmed dysfunctional BMP signaling in dermal fibroblasts isolated from a family with PAH segregating the BMPR2 C118W mutation. After BMP4 treatment, the induction of downstream signaling targets (Smad1/5, ID1 [inhibitor of DNA binding 1], and ID2) was significantly reduced in C118W mutant cells. Treatment with 4PBA significantly rescued Smad1/5, ID1, and ID2 expression. Pulmonary artery smooth muscle cells isolated from the lungs of heterozygous mice harboring the Bmpr2 C118W mutation exhibited significantly increased proliferation. In the presence of 4PBA, hyperproliferation was dramatically reduced. Furthermore, in vivo, 4PBA treatment of Bmpr2 C118W mice partially rescued Bmpr2 expression, restored downstream signaling, and improved vascular remodeling. These findings demonstrate in primary cells and in a knockin mouse that the repurposed small-molecule chemical chaperone 4PBA might be a promising precision medicine approach to treat PAH in patients with specific subtypes of BMPR2 mutation involving cysteine substitutions in the ligand-binding domain.

Supramolecular clustering of the cardiac sodium channel Nav1.5 in HEK293F cells, with and without the auxiliary ß3-subunit.

Voltage-gated sodium channels comprise an ion-selective α-subunit and one or more associated ß-subunits. The ß3-subunit (encoded by the SCN3B gene) is an important physiological regulator of the heart-specific sodium channel, Nav1.5. We have previously shown that when expressed alone in HEK293F cells, the full-length ß3-subunit forms trimers in the plasma membrane. We extend this result with biochemical assays and use the proximity ligation assay (PLA) to identify oligomeric ß3-subunits, not just at the plasma membrane, but throughout the secretory pathway. We then investigate the corresponding clustering properties of the α-subunit and the effects upon these of the ß3-subunits. The oligomeric status of the Nav1.5 α-subunit in vivo, with or without the ß3-subunit, has not been previously investigated. Using super-resolution fluorescence imaging, we show that under conditions typically used in electrophysiological studies, the Nav1.5 α-subunit assembles on the plasma membrane of HEK293F cells into spatially localized clusters rather than individual and randomly dispersed molecules. Quantitative analysis indicates that the ß3-subunit is not required for this clustering but ß3 does significantly change the distribution of cluster sizes and nearest-neighbor distances between Nav1.5 α-subunits. However, when assayed by PLA, the ß3-subunit increases the number of PLA-positive signals generated by anti-(Nav1.5 α-subunit) antibodies, mainly at the plasma membrane. Since PLA can be sensitive to the orientation of proteins within a cluster, we suggest that the ß3-subunit introduces a significant change in the relative alignment of individual Nav1.5 α-subunits, but the clustering itself depends on other factors. We also show that these structural and higher-order changes induced by the ß3-subunit do not alter the degree of electrophysiological gating cooperativity between Nav1.5 α-subunits. Our data provide new insights into the role of the ß3-subunit and the supramolecular organization of sodium channels, in an important model cell system that is widely used to study Nav channel behavior.

Principles of Optogenetic Methods and Their Application to Cardiac Experimental Systems.

Optogenetic techniques permit studies of excitable tissue through genetically expressed light-gated microbial channels or pumps permitting transmembrane ion movement. Light activation of these proteins modulates cellular excitability with millisecond precision. This review summarizes optogenetic approaches, using examples from neurobiological applications, and then explores their application in cardiac electrophysiology. We review the available opsins, including depolarizing and hyperpolarizing variants, as well as modulators of G-protein coupled intracellular signaling. We discuss the biophysical properties that determine the ability of microbial opsins to evoke reliable, precise stimulation or silencing of electrophysiological activity. We also review spectrally shifted variants offering possibilities for enhanced depth of tissue penetration, combinatorial stimulation for targeting different cell subpopulations, or all-optical read-in and read-out studies. Expression of the chosen optogenetic tool in the cardiac cell of interest then requires, at the single-cell level, introduction of opsin-encoding genes by viral transduction, or coupling "spark cells" to primary cardiomyocytes or a stem-cell derived counterpart. At the system-level, this requires construction of transgenic mice expressing ChR2 in their cardiomyocytes, or in vivo injection (myocardial or systemic) of adenoviral expression systems. Light delivery, by laser or LED, with widespread or multipoint illumination, although relatively straightforward in vitro may be technically challenged by cardiac motion and light-scattering in biological tissue. Physiological read outs from cardiac optogenetic stimulation include single cell patch clamp recordings, multi-unit microarray recordings from cell monolayers or slices, and electrical recordings from isolated Langendorff perfused hearts. Optical readouts of specific cellular events, including ion transients, voltage changes or activity in biochemical signaling cascades, using small detecting molecules or genetically encoded sensors now offer powerful opportunities for all-optical control and monitoring of cellular activity. Use of optogenetics has expanded in cardiac physiology, mainly using optically controlled depolarizing ion channels to control heart rate and for optogenetic defibrillation. ChR2-expressing cardiomyocytes show normal baseline and active excitable membrane and Ca2+ signaling properties and are sensitive even to ~1 ms light pulses. They have been employed in studies of the intrinsic cardiac adrenergic system and of cardiac arrhythmic properties.

Early blastocyst expansion in euploid and aneuploid human embryos: evidence for a non-invasive and quantitative marker for embryo selection.

RESEARCH QUESTION: How can the kinetics of human blastocyst expansion be used to evaluate an embryo's ploidy identified using preimplantation genetic testing for aneuploidy (PGT-A)? DESIGN: This was a retrospective observational study of 188 autologous blastocysts from 34 sequential treatment cycles using PGT-A and blastocyst biopsy. Using time-lapse imaging, blastocyst expansion was evaluated using a quantitative standardized expansion assay (qSEA). Trophectoderm cell division was examined in selected, unbiopsied embryos (n = 7) to evaluate the contribution of mitosis to the expansion rate. RESULTS: The averaged euploid blastocyst expansion rate was significantly (52.8%) faster than in aneuploid blastocysts (P = 0.0041). Scatterplots, representing 'expansion maps', revealed that both populations showed a similarly overlapping distribution of blastocyst formation times at 80-140 h from fertilization. Euploidy and aneuploidy were better distinguished in regions of higher and lower expansion, respectively, in expansion maps. Based upon the expansion slopes, rank-ordering of individual embryos within cohorts resulted in more than 90% euploid embryos in the first two ranks in patients less than 35 years of age. Additional detailed time-lapse image analysis provided evidence that rapid expansion was associated with robust, integrative cellular mitosis in trophectoderm cells. CONCLUSIONS: The kinetics of human blastocyst expansion are related to an embryo's ploidy. These preliminary observations describe a new quantitative, non-invasive approach to embryo assessment that may be useful to identify single blastocysts for transfer, particularly in younger patient groups. However, this approach may also be useful for euploid embryo selection after PGT-A. The results support the hypothesis that aneuploidy universally impairs general cellular processes, including cell division, in differentiated cells.

Sodium current inhibition following stimulation of exchange protein directly activated by cyclic-3',5'-adenosine monophosphate (Epac) in murine skeletal muscle.

We investigated effects of pharmacological triggering of exchange protein directly activated by cyclic-3',5'-adenosine monophosphate (Epac) on Nav1.4 currents from intact murine (C67BL6) skeletal muscle fibres for the first time. This employed a loose patch clamp technique which examined ionic currents in response to superimposed 10-ms V1 steps to varying degrees of depolarisation, followed by V2 steps to a fixed, +100 mV depolarisation relative to resting membrane potential following 40 mV hyperpolarising prepulses of 50 ms duration. The activation and inactivation properties of the resulting Na+ membrane current densities revealed reduced maximum currents and steepnesses in their voltage dependences after addition of the Epac activator 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphate (1 µM) to the bathing Krebs-Henseleit solutions. Contrastingly, voltages at half-maximal current and timecourses of currents obtained in response to the V1 depolarising steps were unchanged. These effects were abolished by further addition of the RyR-inhibitor dantrolene (10 µM). In contrast, challenge by dantrolene alone left both currents and their parameters intact. These effects of Epac activation in inhibiting skeletal muscle, Nav1.4, currents, complement similar effects previously reported in the homologous Nav1.5 in murine cardiomyocytes. They are discussed in terms of a hypothesis implicating Epac actions in increasing RyR-mediated SR Ca2+ release resulting in a Ca2+-mediated inhibition of Nav1.4. The latter effect may form the basis for Ca2+-dependent Na+ channel dysregulation in SCN4A channelopathies associated with cold- and K+-aggravated myotonias.

Gene and Protein Expression Profile of Selected Molecular Targets Mediating Electrophysiological Function in Pgc-1α Deficient Murine Atria.

Increases in the prevalence of obesity, insulin resistance, and metabolic syndrome has led to the increase of atrial fibrillation (AF) cases in the developed world. These AF risk factors are associated with mitochondrial dysfunction, previously modelled using peroxisome proliferator activated receptor-γ (PPARγ) coactivator-1 (Pgc-1)-deficient murine cardiac models. We explored gene and protein expression profiles of selected molecular targets related to electrophysiological function in murine Pgc-1α-/- atria. qPCR analysis surveyed genes related to Na⁺-K⁺-ATPase, K⁺ conductance, hyperpolarisation-activated cyclic nucleotide-gated (Hcn), Na⁺ channels, Ca2+ channels, and indicators for adrenergic and cholinergic receptor modulation. Western blot analysis for molecular targets specific to conduction velocity (Nav1.5 channel and gap junctions) was performed. Transcription profiles revealed downregulation of molecules related to Na⁺-K⁺-ATPase transport, Hcn-dependent pacemaker function, Na⁺ channel-dependent action potential activation and propagation, Ca2+ current generation, calsequestrin-2 dependent Ca2+ homeostasis, and adrenergic α1D dependent protection from hypertrophic change. Nav1.5 channel protein expression but not gap junction expression was reduced in Pgc-1α-/- atria compared to WT. Nav1.5 reduction reflects corresponding reduction in its gene expression profile. These changes, as well as the underlying Pgc-1α-/- alteration, suggest potential pharmacological targets directed towards either upstream PGC-1 signalling mechanisms or downstream ion channel changes.