Identifying Features of Cardiac Disease Phenotypes Based on Mechanical Function in a Catecholaminergic Polymorphic Ventricular Tachycardia Model.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is characterized by an arrhythmogenic mechanism involving disruption of calcium handling. This genetic disease can lead to sudden death in children and young adults during physical or emotional stress. Prior CPVT studies have focused on calcium handling, but mechanical functionality has rarely been investigated in vitro. In this research we combine stem cell-derived cardiomyocytes from a CPVT patient (RyR2-H2464D mutation) and a healthy familial control with an engineered culture platform to evaluate mechanical function of cardiomyocytes. Substrates with Young's modulus ranging from 10 to 50 kPa were used in conjunction with microcontact printing of ECM proteins into defined patterns for subsequent attachment. Digital Image Correlation (DIC) was used to evaluate collections of contracting cells. The amplitude of contractile strain was utilized as a quantitative indicator of functionality and disease severity. We found statistically significant differences: the maximum contractile strain was consistently higher in patient samples compared to control samples on all substrate stiffnesses. Additionally, the patient cell line had a statistically significantly slower intrinsic contraction rate than the control, which agrees with prior literature. Differences in mechanical strain have not been previously reported, and hypercontractility is not a known characteristic of CPVT. However, functional changes can occur as the disease progresses, thus this observation may not represent behavior observed in adolescent and adult patients. These results add to the limited studies of mechanical function of CPVT CMs reported in literature and identify functional differences that should be further explored.

A structured community engagement strategy to support uptake of TB active case-finding.

BACKGROUND: In Lima, Peru, a mobile TB screening program ("TB Móvil") was implemented in high TB prevalence districts to increase TB screening. Community engagement activities to promote TB Móvil were simultaneously conducted. OBJECTIVE: To describe a structured, theory-driven community engagement strategy to support the uptake of TB Móvil. METHODS: We adapted Popular Opinion Leader (POL), an evidence-based social networking intervention previously used in Peru to promote HIV testing, for TB Móvil. Community health workers, women who run soup kitchens, and motorcycle taxi drivers served as "popular opinion leaders" who disseminated information about TB Móvil in everyday conversations, aided by a multi-media campaign. Performance indicators of POL included the number/characteristics of persons screened; number of multimedia elements; and proportion of persons with abnormal radiographs hearing about TB Móvil before attending. RESULTS: Between February 2019 and January 2020, 63,899 people attended the TB Móvil program at 210 sites; 60.1% were female. The multimedia campaign included 36 videos, 16 audio vignettes, flyers, posters, community murals and "jingles." Among attendees receiving an abnormal chest X-ray suggestive of TB, 48% (6,935/14,563) reported hearing about TB Móvil before attending. CONCLUSIONS: POL promotes the uptake of TB Móvil and should be considered as a strategy for increasing TB screening uptake.

CONTEXTE: À Lima, Pérou, un programme mobile de dépistage de la TB (« TB Móvil ¼) a été mis en place dans les quartiers à forte prévalence de TB afin d'accroître le dépistage de la maladie. Des activités de mobilisation communautaire visant à promouvoir TB Móvil ont été menées en parallèle. L'objectif de ce rapport est de décrire une stratégie structurée de mobilisation communautaire, fondée sur des principes théoriques, afin de soutenir le recours au programme TB Móvil. MÉTHODES: Nous avons adapté à TB Móvil l'intervention factuelle de réseautage social appelée « Popular Opinion Leader (POL; leader d'opinion) ¼, précédemment utilisée au Pérou pour promouvoir le dépistage du VIH. Les agents de santé communautaires, les femmes responsables de la soupe populaire et les chauffeurs de mototaxis étaient des leaders d'opinion. Ils communiquaient des informations sur TB Móvil lors de leurs conversations quotidiennes, qui étaient étayées par une campagne multimédia. Les indicateurs de performance des POL comprenaient le nombre/les caractéristiques des personnes dépistées, le nombre d'éléments multimédias et le pourcentage de personnes avec cliché radiographique anormal qui avaient entendu parler de TB Móvil avant de se faire dépister. RÉSULTATS: Entre février 2019 et janvier 2020, 63 899 personnes ont pris part au programme TB Móvil dans 210 sites ; 60,1% étaient des femmes. La campagne multimédia reposait sur 36 vidéos, 16 vignettes audio, des prospectus, des posters, des peintures murales dans la communauté et des « jingles ¼. Parmi les personnes dont la radiographie pulmonaire était anormale et évocatrice de TB, 48% (6 935/14 563) ont rapporté avoir entendu parler de TB Móvil avant de venir consulter. CONCLUSIONS: L'intervention POL, qui semblait renforcer le recours au programme TB Móvil, peut donc servir d'une stratégie de promotion du dépistage de la TB.

Amblyopia due a dentigerous cyst. Case report and literature review.

PURPOSE: The goal of this article is to present an infrequent clinical case and to review the available literatura, with an emphasis on ophthalmological symptoms. METHODS: We present the case of a 4-year-old girl with a large dentigerous cyst on the maxillary bone, who had long-standing unilateral epiphora associated with progressive ocular dystopia, facial asymmetry and ipsilateral amblyopia. A multidisciplinary approach was taken by the maxillofacial surgery, ophthalmology and optometry teams. This included systemic antibiotic administration, surgical cyst drainage and amblyopia treatment. The literature review was carried out in the MEDLINE database through the free electronic access to PubMed in March 2020. RESULTS: At the 6-month follow-up, the patient was asymptomatic. The most common symptoms of dentigerous cysts are epiphora 36.8%, ocular dystopia 31.2%, diplopia 21.1%, proptosis, nasolacrimal duct obstruction and blurred vision at 10.5%. Amblyopia has not been reported. CONCLUSIONS: Dentigerous cysts are benign odontogenic cysts, which can be found in the jaw and less frequently on the maxillary bone. They are usually asymptomatic, and the occurrence of ophthalmic complications is very infrequent. Multidisciplinary management is essential to avoiding long-term morbidity of maxillary dentigerous cysts and should include an ophthalmologist.

Lacrimal Puncta Agenesis in Kabuki Syndrome.

Kabuki syndrome (KS) is a rare congenital disorder characterized by multiple systemic anomalies and facial characteristics. Here, the authors present the first case, to the best of the authors' knowledge, of bilateral lacrimal puncta agenesis in a patient with KS.#8232;The proband patient was a 29-year-old woman diagnosed with this syndrome, brought to our office due to recurrent conjunctivitis where agenesia of lacrimal puncta was observed. Therapeutic options were exposed but, as the concomitant medication (topiramate) produced ocular dryness, conservative treatment was decided. Diagnosis of KS is challenging because it is a complex syndrome with many associated findings. The authors recommend taking into account the agenesis of lacrimal points in the differential diagnosis of KS if it is associated with other phenotypic alterations as well as including lacrimal examination in patients with KS diagnosis. The authors emphasize the importance of individualizing treatment since drugs used for the systematic management of these patients can influence tear symptoms.

Scorpion venom components that affect ion-channels function.

The number and types of venom components that affect ion-channel function are reviewed. These are the most important venom components responsible for human intoxication, deserving medical attention, often requiring the use of specific anti-venoms. Special emphasis is given to peptides that recognize Na(+)-, K(+)- and Ca(++)-channels of excitable cells. Knowledge generated by direct isolation of peptides from venom and components deduced from cloned genes, whose amino acid sequences are deposited into databanks are nowadays in the order of 1.5 thousands, out of an estimate biodiversity closed to 300,000. Here the diversity of components is briefly reviewed with mention to specific references. Structural characteristic are discussed with examples taken from published work. The principal mechanisms of action of the three different types of peptides are also reviewed. Na(+)-channel specific venom components usually are modifier of the open and closing kinetic mechanisms of the ion-channels, whereas peptides affecting K(+)-channels are normally pore blocking agents. The Ryanodine Ca(++)-channel specific peptides are known for causing sub-conducting stages of the channels conductance and some were shown to be able to internalize penetrating inside the muscle cells.

In vivo left ventricular functional capacity is compromised in cMyBP-C null mice.

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament-associated protein that binds tightly to myosin and has a potential role for modulating myocardial contraction. We tested the hypothesis that cMyBP-C 1) contributes to the enhanced in vivo contractile state following beta-adrenergic stimulation and 2) is necessary for myocardial adaptation to chronic increases in afterload. In vivo pressure-volume relations demonstrated that left ventricular (LV) systolic and diastolic function were compromised under basal conditions in cMyBP-C(-/-) compared with WT mice. Moreover, whereas beta-adrenergic treatment significantly improved ejection fraction, peak elastance, and the time to peak elastance in WT mice, these functional indexes remained unchanged in cMyBP-C(-/-) mice. Morphological and functional changes were measured through echocardiography in anesthetized mice following 5 wk of aortic banding. Adaptation to pressure overload was diminished in cMyBP-C(-/-) mice as characterized by a lack of an increase in posterior wall thickness, increased LV diameter, deterioration of fractional shortening, and prolonged isovolumic relaxation time. These results suggest that the absence of cMyBP-C significantly diminishes in vivo LV function and markedly attenuates the increase in LV contractility following beta-adrenergic stimulation or adaptation to pressure overload.

Imperatoxin A (IpTx(a)) from Pandinus imperator stimulates [(3)H]ryanodine binding to RyR3 channels.

The effect of imperatoxin A (IpTx(a)) on the ryanodine receptor type 3 (RyR3) was studied. IpTx(a) stimulates [(3)H]ryanodine binding to RyR3-containing microsomes, but this effect requires toxin concentrations higher than those required to stimulate RyR1 channels. The effect of IpTx(a) on RyR3 channels was observed at calcium concentrations in the range 0.1 microM to 10 mM. By contrast, RyR2 channels were not significantly affected by IpTx(a) in the same calcium ranges. Single channel current measurements indicated that IpTx(a) induced subconductance state in RyR3 channels that was similar to those observed with RyR1 and RyR2 channels. These results indicate that IpTx(a) is capable of inducing similar subconductance states in all three RyR isoforms, while stimulation of [(3)H]ryanodine binding by this toxin results in isoform-specific responses, with RyR1 being the most sensitive channel, RyR3 displaying an intermediate response and RyR2 the least responsive ones.

Response mechanism of a neutral carrier Hg(II) polymeric membrane ion-selective electrode. SEM and EDAX study.

Scanning electron microscopy (SEM) and energy dispersive atomic X-ray spectrometry (EDAX) were used to study the response mechanism of a previously reported new Hg membrane ion-selective electrode (ISE) based on 1,3-diphenylthiourea. These techniques allowed the study of the membrane surface characteristics, such as the morphological homogeneity and chemical composition. A 'twice Nernstian' response at pH > or = 7 was explained by the detection of the Hg(OH)+ cation. A normal Nernstian response was found at acidic pH values. Using these techniques, both coordination compounds, [Ligand-Hg-OH] at pH 7 and [Ligand-Hg-Ligand] at pH 4.5, were confirmed on the electrode membrane surface activated with Hg(NO3)2 solution at both pH values. These methods provide results which are independent of the potential measurement data and in agreement with them. A successful response model has explained both independent and unbiased sets of results. These conclusions confirm the proposed response mechanisms for this new Hg membrane sensor.

cADP-ribose activates reconstituted ryanodine receptors from coronary arterial smooth muscle.

The present study was designed to test the hypothesis that cADP-ribose (cADPR) increases Ca(2+) release through activation of ryanodine receptors (RYR) on the sarcoplasmic reticulum (SR) in coronary arterial smooth muscle cells (CASMCs). We reconstituted RYR from the SR of CASMCs into planar lipid bilayers and examined the effect of cADPR on the activity of these Ca(2+) release channels. In a symmetrical cesium methanesulfonate configuration, a 245 pS Cs(+) current was recorded. This current was characterized by the formation of a subconductance and increase in the open probability (NP(o)) of the channels in the presence of ryanodine (0.01-1 microM) and imperatoxin A (100 nM). A high concentration of ryanodine (50 microM) and ruthenium red (40-80 microM) substantially inhibited the activity of RYR/Ca(2+) release channels. Caffeine (0.5-5 mM) markedly increased the NP(o) of these Ca(2+) release channels of the SR, but D-myo-inositol 1,4,5-trisphospate and heparin were without effect. Cyclic ADPR significantly increased the NP(o) of these Ca(2+) release channels of SR in a concentration-dependent manner. Addition of cADPR (0.01 microM) into the cis bath solution produced a 2.9-fold increase in the NP(o) of these RYR/Ca(2+) release channels. An eightfold increase in the NP(o) of the RYR/Ca(2+) release channels (0.0056 +/- 0.001 vs. 0.048 +/- 0.017) was observed at a concentration of cADPR of 1 microM. The effect of cADPR was completely abolished by ryanodine (50 microM). In the presence of cADPR, Ca(2+)-induced activation of these channels was markedly enhanced. These results provide evidence that cADPR activates RYR/Ca(2+) release channels on the SR of CASMCs. It is concluded that cADPR stimulates Ca(2+) release through the activation of RYRs on the SR of these smooth mucle cells.

Characterization of a calcium-regulation domain of the skeletal-muscle ryanodine receptor.

A negatively charged region of the N-terminal portion of the skeletal ryanodine receptor (RyR), located between residues 1872-1923, is involved in Ca (2+)-dependent regulation of the Ca(2+)-release channel. This region is divergent between the skeletal (RyR1) and cardiac (RyR2) isoforms of the channel, and is known as D3. Ca(2+) exerts important regulatory functions on the RyR, being involved in both activation and inactivation functions of the channel, i.e. the effects occurring at micromolar and millimolar Ca(2+) concentrations respectively. To characterize the role of D3 in the Ca(2+)-dependent regulation of the Ca(2+)-release channel, we studied the functional consequences of deleting the D3 region from RyR1 (DeltaD3-RyR1) using a heterologous expression system, [(3)H]ryanodine binding assays and single-channel recordings in lipid bilayers. Deletion of the D3 region selectively affected Ca(2+)-dependent regulation of RyR1, but did not alter [(3)H]ryanodine binding or the effect of other modulators on the RyR. Compared with full-length RyR1 (wt-RyR1), the Ca(2+)-dependence curve of DeltaD3-RyR1 is broader, reflecting increased sensitivity to Ca(2+) activation and decreased sensitivity to Ca(2+) inactivation. In addition, DeltaD3-RyR1 was more resistant to inhibition by Mg(2+). Comparison of the effect of caffeine on wt-RyR1 and DeltaD3-RyR1 suggested that D3 is an important region of RyR that participates in Ca(2+)-dependent activation and inactivation of the Ca(2+)-release channel.

Effects of imperatoxin A on local sarcoplasmic reticulum Ca(2+) release in frog skeletal muscle.

We have investigated the effects of imperatoxin A (IpTx(a)) on local calcium release events in permeabilized frog skeletal muscle fibers, using laser scanning confocal microscopy in linescan mode. IpTx(a) induced the appearance of Ca(2+) release events from the sarcoplasmic reticulum that are approximately 2 s and have a smaller amplitude (31 +/- 2%) than the "Ca(2+) sparks" normally seen in the absence of toxin. The frequency of occurrence of long-duration imperatoxin-induced Ca(2+) release events increased in proportion to IpTx(a) concentrations ranging from 10 nM to 50 nM. The mean duration of imperatoxin-induced events in muscle fibers was independent of toxin concentration and agreed closely with the channel open time in experiments on isolated frog ryanodine receptors (RyRs) reconstituted in planar lipid bilayer, where IpTx(a) induced opening of single Ca(2+) release channels to prolonged subconductance states. These results suggest involvement of a single molecule of IpTx(a) in the activation of a single Ca(2+) release channel to produce a long-duration event. Assuming the ratio of full conductance to subconductance to be the same in the fibers as in bilayer, the amplitude of a spark relative to the long event indicates involvement of at most four RyR Ca(2+) release channels in the production of short-duration Ca(2+) sparks.

Three-dimensional location of the imperatoxin A binding site on the ryanodine receptor.

Cryo-electron microscopy and three-dimensional, single-particle image analysis have been used to reveal the specific binding site of imperatoxin A (IpTx(a)) on the architecture of the calcium release channel/ryanodine receptor from skeletal muscle (RyR1). IpTx(a) is a peptide toxin that binds with high affinity to RyR1 and affects its functioning. The toxin was derivatized with biotin to enhance its detection with streptavidin. IpTx(a) binds to the cytoplasmic moiety of RyR1 between the clamp and handle domains, 11 nm away from the transmembrane pore. The proposed mimicry by IpTx(a) of the dihydropyridine receptor (DHPR) II-III loop, thought to be a main physiological excitation-contraction trigger, suggests that the IpTx(a) binding location is a potential excitation-contraction signal transduction site.

Conversion of an inactive cardiac dihydropyridine receptor II-III loop segment into forms that activate skeletal ryanodine receptors.

A 25 amino acid segment (Glu666-Pro691) of the II-III loop of the alpha1 subunit of the skeletal dihydropyridine receptor, but not the corresponding cardiac segment (Asp788-Pro814), activates skeletal ryanodine receptors. To identify the structural domains responsible for activation of skeletal ryanodine receptors, we systematically replaced amino acids of the cardiac II-III loop with their skeletal counterparts. A cluster of five basic residues of the skeletal II-III loop (681RKRRK685) was indispensable for activation of skeletal ryanodine receptors. In the cardiac segment, a negatively charged residue (Glu804) appears to diminish the electrostatic potential created by this basic cluster. In addition, Glu800 in the group of negatively charged residues 798EEEEE802 of the cardiac II-III loop may serve to prevent the binding of the activation domain.

Activation of ryanodine receptors by imperatoxin A and a peptide segment of the II-III loop of the dihydropyridine receptor.

Excitation-contraction coupling in skeletal muscle is believed to be triggered by direct protein-protein interactions between the sarcolemmal dihydropyridine-sensitive Ca2+ channel and the Ca2+ release channel/ryanodine receptor (RyR) of sarcoplasmic reticulum. A 138-amino acid cytoplasmic loop between repeats II and III of the alpha1 subunit of the skeletal dihydropyridine receptor (the II-III loop) interacts with a region of the RyR to elicit Ca2+ release. In addition, small segments (10-20 amino acid residues) of the II-III loop retain the capacity to activate Ca2+ release. Imperatoxin A, a 33-amino acid peptide from the scorpion Pandinus imperator, binds directly to the RyR and displays structural and functional homology with an activating segment of the II-III loop (Glu666-Leu690). Mutations in a structural motif composed of a cluster of basic amino acids followed by Ser or Thr dramatically reduce or completely abolish the capacity of the peptides to activate RyRs. Thus, the Imperatoxin A-RyR interaction mimics critical molecular characteristics of the II-III loop-RyR interaction and may be a useful tool to elucidate the molecular mechanism that couples membrane depolarization to sarcoplasmic reticulum Ca2+ release in vivo.