Calcium Sensitivity Research Articles

A dynamic calcium-force relationship model for sag behavior in fast skeletal muscle.

In vitro studies using isolated or skinned muscle fibers suggest that the sigmoidal relationship between the intracellular calcium concentration and force production may depend upon muscle type and activity. The goal of this study was to investigate whether and how the calcium-force relationship changes during force production under physiological conditions of muscle excitation and length in fast skeletal muscles. A computational framework was developed to identify the dynamic variation in the calcium-force relationship during force generation over a full physiological range of stimulation frequencies and muscle lengths in cat gastrocnemius muscles. In contrast to the situation in slow muscles such as the soleus, the calcium concentration for the half-maximal force needed to drift rightward to reproduce the progressive force decline, or sag behavior, observed during unfused isometric contractions at the intermediate length under low-frequency stimulation (i.e., 20 Hz). The slope at the calcium concentration for the half-maximal force was required to drift upward for force enhancement during unfused isometric contractions at the intermediate length under high-frequency stimulation (i.e., 40 Hz). The slope variation in the calcium-force relationship played a crucial role in shaping sag behavior across different muscle lengths. The muscle model with dynamic variations in the calcium-force relationship also accounted for the length-force and velocity-force properties measured under full excitation. These results imply that the calcium sensitivity and cooperativity of force-inducing crossbridge formation between actin and myosin filaments may be operationally altered in accordance with the mode of neural excitation and muscle movement in intact fast muscles.

Stretch-Induced Down-Regulation of HCN2 Suppresses Contractile Activity.

Although hyperpolarization-activated and cyclic nucleotide-gated 2 channels (HCN2) are expressed in multiple cell types in the gut, the role of HCN2 in intestinal motility is poorly understood. HCN2 is down-regulated in intestinal smooth muscle in a rodent model of ileus. Thus, the purpose of this study was to determine the effects of HCN inhibition on intestinal motility. HCN inhibition with ZD7288 or zatebradine significantly suppressed both spontaneous and agonist-induced contractile activity in the small intestine in a dose-dependent and tetrodotoxin-independent manner. HCN inhibition significantly suppressed intestinal tone but not contractile amplitude. The calcium sensitivity of contractile activity was significantly suppressed by HCN inhibition. Inflammatory mediators did not affect the suppression of intestinal contractile activity by HCN inhibition but increased stretch of the intestinal tissue partially attenuated the effects of HCN inhibition on agonist-induced intestinal contractile activity. HCN2 protein and mRNA levels in intestinal smooth muscle tissue were significantly down-regulated by increased mechanical stretch compared to unstretched tissue. Increased cyclical stretch down-regulated HCN2 protein and mRNA levels in primary human intestinal smooth muscle cells and macrophages. Overall, our results suggest that decreased HCN2 expression induced by mechanical signals, such as intestinal wall distension or edema development, may contribute to the development of ileus.

Hematological effects of Levosimendan therapy in patients with heart failure

Abstract Funding Acknowledgements Type of funding sources: None. Introduction Levosimendan is an inotropic drug, acting as calcium sensitizer, used in the management of acute heart failure. Despite its hemodynamic benefits, Levosimendan is associated with several adverse reactions, including hematological effects, with a hemoglobin value decrease, causing anemia. Acute coronary syndromes (ACS) might be a precipitant of acute heart failure, in a substantial proportion of cases. These situations may require the use of inotropic drugs, such as Levosimendan. Antithrombotic therapy is the cornerstone therapy for management of acute coronary syndromes, and the decision of which strategy is determined by the balance of ischemic and hemorrhagic risks. Therefore, in the management of acute heart failure, associated with ACS, the use of Levosimendan might be associated with the increase of hemorrhagic risk, due to hemoglobin decrease, influencing the antithrombotic therapy strategy choice. Purpose This study aims to evaluate the effect of the use of Levosimendan on the hemoglobin value, comparing with Dobutamine, in patients hospitalized for ACS. Methods We analyzed a retrospective, multicenter, observational cohort of 317 patients, admitted for ACS, requiring inotropic therapy. The population was divided into two groups: a group that used Levosimendan and a group that used Dobutamine. The mean decrease in hemoglobin value, and the minimum hemoglobin value, during hospitalization, was compared between both groups. Results Levosimendan sample included 39 patients, of which 40,1% (n=15) were women, with a mean age of 83±12,1 year old, while Dobutamine sample had 278 elements, of which 38% (n=105) were women with a mean age of 82±12,1. There was no statistically significant difference between both samples on age and sex distribution. Levosimendan group had a mean minimum hemoglobin value of 10,7±2,2 g/dL and a mean decrease in hemoglobin value, during hospitalization, of 2,7±1,9 g/dL, while Dobutamine group had a mean minimum hemoglobin value of 10,7±2,2 g/dL and a decrease in hemoglobin value, during hospitalization, 2,1±1,9g/dL. There was no statically significant difference on mean minimum hemoglobin value on both samples (p>0,05), however, there was a statistically significant difference, with a higher decrease in mean hemoglobin value, during hospitalization in patients using Levosimendan, comparing with patients using Dobutamine (2,7±1,9g/dL vs 2,1±1,9g/dL, p=0,04). Conclusion Levosimendan is associated with a higher decrease in hemoglobin value. As heart failure might complicate ACS episode, and might require inotropic therapy, we should be aware that the choice of using Levosimendan in these situations may confer a greater risk of bleeding, associated with the higher decrease in hemoglobin value, in patients who need intense antithrombotic therapy.

Targeting Troponin C with Small Molecules Containing Diphenyl Moieties: Calcium Sensitivity Effects on Striated Muscles and Structure-Activity Relationship.

Despite large investments from academia and industry, heart failure, which results from a disruption of the contractile apparatus, remains a leading cause of death. Cardiac muscle contraction is a calcium-dependent mechanism, which is regulated by the troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium-binding subunit (cNTnC). There is an increasing need for the development of small molecules that increase calcium sensitivity without altering the systolic calcium concentration, thereby strengthening the cardiac function. Here, we examined the effect of our previously identified calcium-sensitizing small molecule, ChemBridge compound 7930079, in the context of several homologous muscle systems. The effect of this molecule on force generation in isolated cardiac trabeculae and slow skeletal muscle fibers was measured. Furthermore, we explored the use of Gaussian accelerated molecular dynamics in sampling highly predictive receptor conformations based on NMR-derived starting structures. Additionally, we took a rational computational approach for lead optimization based on lipophilic diphenyl moieties. This integrated structural-biochemical-physiological approach led to the identification of three novel low-affinity binders, which had similar binding affinities to the known positive inotrope trifluoperazine. The most potent identified calcium sensitizer was compound 16 with an apparent affinity of 117 ± 17 μM.

C42 OR 1896 EXERTS POSITIVE INOTROPIC EFFECTS IN ISOLATED HUMAN RIGHT ATRIAL MUSCLE

Abstract Levosimendan is approved for use as an intravenous agent to treat temporarily patients with severe systolic heart failure. It is thought, but hitherto never tested in isolated human cardiac tissue, that the main metabolite of levosimendan, called OR 1896, might cause the positive inotropic effect of levosimendan in patients. For comparison, we studied mouse atrial preparations. OR alone (up to 10 µM) failed to increase force of contraction in mouse atrial preparations while in the presence of the phosphodiesterase (PDE) IV–inhibitor rolipram (0.1 µM) profound positive inotropic effects and positive chronotropic effects of OR 1895 were recorded in mouse atrial preparations. In contrast to mouse atrium, we noticed that OR 1896 alone (0.01 µM to 10 µM cumulatively applied) exerted time– and concentration–dependent positive inotropic effects 1 µM increase by 72 ± 14.7%, p<0.05, n=7) and shortened the time of relaxation (by 7 .3 %, p<0.05, n=7) in isolated electrically paced human right atrial muscle preparations (obtained during cardiac surgery), that started at 0.1 µM and plateaued at 1 µM OR 1896. The positive inotropic effect of OR 1896 was antagonized by 10 µM propranolol. The positive inotropic effect of OR 1896 in mouse atrial preparations and human right atrial preparations was less effective than that of 1 µM isoprenaline (human atrium by 154 %). The positive inotropic effect of OR 1896 was diminished by pre–incubation of the samples with the PDE III inhibitor cilostamide (10 µM). EMD 57033 (10 µM), a calcium sensitizer, increased force of contraction further in the additional presence of 1 µM OR 1896. Our data suggest that OR 1896 is indeed a positive inotropic agent in the human heart and that OR 1896 acts mainly as a PDE III – inhibitor because the effect of OR 1896 was attenuated by a β–adrenoceptor antagonist and thus OR 1896 is unlikely to act as a calcium sensitizer in the human heart.

Arrhythmic Risk Stratification among Patients with Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a cardiac muscle disorder characterized by generally asymmetric abnormal hypertrophy of the left ventricle without abnormal loading conditions (such as hypertension or valvular heart disease) accounting for the left ventricular wall thickness or mass. The incidence of sudden cardiac death (SCD) in HCM patients is about 1% yearly in adults, but it is far higher in adolescence. HCM is the most frequent cause of death in athletes in the Unites States of America. HCM is an autosomal-dominant genetic cardiomyopathy, and mutations in the genes encoding sarcomeric proteins are identified in 30-60% of cases. The presence of this genetic mutation carries more than 2-fold increased risk for all outcomes, including ventricular arrhythmias. Genetic and myocardial substrate, including fibrosis and intraventricular dispersion of conduction, ventricular hypertrophy and microvascular ischemia, increased myofilament calcium sensitivity and abnormal calcium handling, all play a role as arrhythmogenic determinants. Cardiac imaging studies provide important information for risk stratification. Transthoracic echocardiography can be helpful to evaluate left ventricular (LV) wall thickness, LV outflow-tract gradient and left atrial size. Additionally, cardiac magnetic resonance can evaluate the prevalence of late gadolinium enhancement, which when higher than 15% of LV mass is a prognostic maker of SCD. Age, family history of SCD, syncope and non-sustained ventricular tachycardia at Holter ECG have also been validated as independent prognostic markers of SCD. Arrhythmic risk stratification in HCM requires careful evaluation of several clinical aspects. Symptoms combined with electrocardiogram, cardiac imaging tools and genetic counselling are the modern cornerstone for proper risk stratification.

Structural and Functional Properties of Kappa Tropomyosin.

In the myocardium, the TPM1 gene expresses two isoforms of tropomyosin (Tpm), alpha (αTpm; Tpm 1.1) and kappa (κTpm; Tpm 1.2). κTpm is the result of alternative splicing of the TPM1 gene. We studied the structural features of κTpm and its regulatory function in the atrial and ventricular myocardium using an in vitro motility assay. We tested the possibility of Tpm heterodimer formation from α- and κ-chains. Our result shows that the formation of ακTpm heterodimer is thermodynamically favorable, and in the myocardium, κTpm most likely exists as ακTpm heterodimer. Using circular dichroism, we compared the thermal unfolding of ααTpm, ακTpm, and κκTpm. κκTpm had the lowest stability, while the ακTpm was more stable than ααTpm. The differential scanning calorimetry results indicated that the thermal stability of the N-terminal part of κκTpm is much lower than that of ααTpm. The affinity of ααTpm and κκTpm to F-actin did not differ, and ακTpm interacted with F-actin significantly worse. The troponin T1 fragment enhanced the κκTpm and ακTpm affinity to F-actin. κκTpm differently affected the calcium regulation of the interaction of pig and rat ventricular myosin with the thin filament. With rat myosin, calcium sensitivity of thin filaments containing κκTpm was significantly lower than that with ααTpm and with pig myosin, and the sensitivity did not differ. Thin filaments containing κκTpm and ακTpm were better activated by pig atrial myosin than those containing ααTpm.

Comparison between levosimendan versus beta agonists in preservation of renal function in cardiac surgery patients with low cardiac output syndrome

ABSTRACT Background Post cardiac surgery acute kidney injury (AKI) secondary to postoperative low cardiac output syndrome (LCOS), is a serious complication. Positive inotropic agents are the main line of treatment for LCOS with different degrees of improvement of cardiac function & nephroprotective efficacy Levosimendan is a calcium sensitizer in cardiac muscles, which has a comparable positive inotropic and nephroprotective effects to that of classic beta2 agonists. Objective To evaluate the possible nephroprotective effect of levosimendan as compared to beta agonists in cardiac surgery patients with LCOS. Patients and Methods It is a prospective, randomized and comparative study conducted at Ain Shams University Hospitals over the period from December 2020 to May 2021. A total of 60 patients with post cardiac surgery low cardiac output syndrome were divided into two groups of 30 patients each. Group A (control) received beta-agonists (dobutamine or adrenaline) and Group B (study) received levosimendan. The incidence of AKI at the diagnosis of LCOS & its progression to renal failure in both groups were assessed. Results The incidence of AKI at diagnosis of LCOS postoperative was 30% (n = 9) in each group; 44% of them in the control group (n = 4) developed renal failure at discharge from ICU and none of the study group patients developed renal failure at discharge. At the time of discharge from ICU, the incidence of renal failure in beta-agonist group was 13.3%, while the incidence in the levosimendan group was 0% with statistically significant P value of 0.038. Conclusion In comparison to beta-agonists, Levosimendan may have a better nephroprotective effect that plays a role in decreasing the incidence of kidney failure in patients with post cardiac surgery LCOS. A larger randomized, controlled trials are recommended to prove such a beneficial nephroprotective effect and its exact mechanism.

Application of an artificial intelligence ensemble for detection of important secondary findings on lung ventilation and perfusion SPECT-CT

RationaleSingle-photon-emission-computerized-tomography/computed-tomography(SPECT/CT) is commonly used for pulmonary disease. Scant work has been done to determine ability of AI for secondary findings using low-dose-CT(LDCT) attenuation correction series of SPECT/CT. Methods120 patients with ventilation-perfusion-SPECT/CT from 9/1/21–5/1/22 were included in this retrospective study. AI-RAD companion(VA10A,Siemens-Healthineers, Erlangen, Germany), an ensemble of deep-convolutional-neural-networks was evaluated for the detection of pulmonary nodules, coronary artery calcium, aortic ectasia/aneurysm, and vertebral height loss. Accuracy, sensitivity, specificity was measured for the outcomes. Inter-rater reliability were measured. Inter-rater reliability was measured using the intraclass correlation coefficient (ICC) by comparing the number of nodules identified by the AI to radiologist. ResultsOverall per-nodule accuracy, sensitivity, and specificity for detection of lung nodules were 0.678(95%CI 0.615–0.732), 0.956(95%CI 0.900–0.985), and 0.456(95%CI 0.376–0.543), respectively, with an intraclass correlation coefficient (ICC) between AI and radiologist of 0.78(95%CI 0.71–0.83). Overall per-patient accuracy for AI detection of coronary artery calcium, aortic ectasia/aneurysm, and vertebral height loss was 0.939(95%CI 0.878–0.975), 0.974(95%CI 0.925–0.995), and 0.857(95%CI 0.781–0.915), respectively. Sensitivity for coronary artery calcium, aortic ectasia/aneurysm, and vertebral height loss was 0.898(95%CI 0.778–0.966), 1 (95%CI 0.958–1), and 1 (95%CI 0.961–1), respectively. Specificity for coronary artery calcium, aortic ectasia/aneurysm, and vertebral height loss was 0.969(95% CI 0.893–0.996), 0.897 (95% CI 0.726–0.978), and 0.346 (95% CI 0.172–0.557), respectively. ConclusionAI ensemble was accurate for coronary artery calcium and aortic ectasia/aneurysm, while sensitive for aortic ectasia/aneurysm, lung nodules and vertebral height loss on LDCT attenuation correction series of SPECT/CT.

Protein Kinase CK2 Modulates the Calcium Sensitivity of Type 3 Small-conductance Calcium-activated Potassium Channels in Colonic Platelet-derived Growth Factor Receptor Alpha-positive Cells From Streptozotocin-induced Diabetic Mice

The gastrointestinal symptom of diabetes mellitus, chronic constipation, seriously affects patients' life. Whereas, the mechanism of chronic constipation is still ambiguous, resulting in a lack of effective therapies for this symptom. As a part of the smooth muscle cells, interstitial cells of Cajal, and platelet-derived growth factor receptor alpha-positive (PDGFRα+) cells syncytium (SIP syncytium), PDGFRα+ cells play an important role in regulating colonic motility. According to our previous study, in PDGFRα+ cells in colons of diabetic mice, the function of the P2Y1 purinergic receptor/type 3 small-conductance calcium-activated potassium (SK3) channel signaling pathway is strengthened, which may lead to colonic dysmotility. The purpose of this study is to investigate the changes in SK3 channel properties of PDGFRα+ cells in diabetic mice. Whole-cell patch clamp, Western blotting, superoxide dismutase activity measurement, and malondialdehyde measurement were main methods in the present study. The present study revealed that when dialysed with low calcium ion (Ca2+) solution, the SK3 current density was significantly decreased in PDGFRα+ cells from diabetic mice. However, the SK3 current density in PDGFRα+ cells was enhanced from diabetic mice when dialysed with high Ca2+ solution. Moreover, hydrogen peroxide-treatment mimicked this phenomenon in SK3 transgenic HEK293 cells. The subunit of SK3 channels, protein kinase CK2, was up-regulated in colonic muscle layers and hydrogen peroxide-treated HEK293 cells. Additionally, protein phosphatase 2A, the subunit of SK3 channels, was not changed in streptozotocin-treated mouse colons or hydrogen peroxide-treated HEK293 cells. The diabetic oxidative stress-induced upregulation of CK2 contributed to modulating SK3 channel sensitivity to Ca2+ in colonic PDGFRα+ cells, which may result in colonic dysmotility in diabetic mice.

Modulating insulin secretion and inflammation against sodium arsenite toxicity by levosimendan as a novel pancreatic islets’ protector

Levosimendan (LEVO) is a calcium sensitizer with established inotropic and vasodilator impacts associated with ATP-sensitive potassium channels (KATP). Although LEVO’s role in modulating oxidative stress of cardiac cells has been studied, in the current study, LEVO was used, for the first time, to investigate its preventive and therapeutic roles in pancreatic islets through viability, functionality, and inflammation pathways as well as oxidative stress against the toxicity induced by sodium arsenite (NaAsO2). Following the optimization studies to select the proper concentrations of LEVO and NaAsO2, isolated pancreatic islets were exposed to different combinations of NaAsO2 and LEVO. MTT assay, glucose-dependent insulin secretion test, investigation of oxidative stress and inflammation biomarkers in addition to qualification of the expressions of P16, P38, and NF-κB genes were performed to assess different underlying mechanisms to investigate the protective role of LEVO against NaAsO2-induced toxicity. This study demonstrated that NaAsO2-treated pancreatic islets’ exposure to LEVO improved their viability and functionality while modulating the generation of oxidative stress and inflammatory biomarkers. Therefore LEVO caused significant recoveries in the characteristics of the islets. The main conclusion is that LEVO showed protective impacts in pancreatic islets against exposure to NaAsO2. However, therapeutic uses of LEVO in the field of diabetes need further investigations. HIGHLIGHTS Sodium arsenite is responsible for activating the oxidative stress and inflammation pathways in pancreatic islet cells and consequently decreasing their viability and function. Treatment of pancreatic islets with sodium arsenite results in the loss of glucose-dependent insulin secretion (GDIS) which can be alleviated by levosimendan. Levosimendan modulates the decreased level of secreted insulin by preventing formation of reactive oxygen species (ROS) and inactivating the inflammatory biomarkers. Levosimendan improves the viability and function of sodium arsenite-treated pancreatic islets by modulating insulin secretion and glucose control. Levosimendan acts as a protective agent against the toxicity induced by sodium arsenite in pancreatic islets through modulating the viability, oxidative stress, inflammation, and function pathways.

Treatment with levosimendan in an experimental model of early ventilator-induced diaphragmatic dysfunction.

Mechanical ventilation (MV) is a life-saving approach in critically ill patients. However, it may affect the diaphragmatic structure and function, beyond the lungs. Levosimendan is a calcium sensitizer widely used in clinics to improve cardiac contractility in acute heart failure patients. In vitro studies have demonstrated that levosimendan increased force-generating capacity of the diaphragm in chronic obstructive pulmonary disease patients. Thus the aim of this study was to evaluate the effects of levosimendan administration in an animal model of ventilator-induced diaphragmatic dysfunction (VIDD) on muscle contraction and diaphragm muscle cell viability. Sprague-Dawley rats underwent prolonged MV (5 hours). VIDD+Levo group received a starting bolus of levosimendan immediately after intratracheal intubation and then an intravenous infusion of levosimendan throughout the study. Diaphragms were collected for ex vivo contractility measurement (with electric stimulation), histological analysis and Western blot analysis. Healthy rats were used as the control. Levosimendan treatment maintained an adequate mean arterial pressure during the entire experimental protocol, preserved levels of autophagy-related proteins (LC3BI and LC3BII) and the muscular cell diameter demonstrated by histological analysis. Levosimendan did not affect the diaphragmatic contraction or the levels of proteins involved in the protein degradation (atrogin). Our data suggest that levosimendan preserves muscular cell structure (cross-sectional area) and muscle autophagy after 5 hours of MV in a rat model of VIDD. However, levosimendan did not improve diaphragm contractile efficiency.

Thermoluminescence investigations and kinetic analysis of highly sensitive LiCaAlF6: Eu,Y phosphor for dosimetric applications

The paper presents detailed thermoluminescence characterization and kinetic analysis of highly sensitive and dosimetric grade lithium calcium aluminum fluoride doped with europium and co-doped with yttrium (LiCaAlF6:Eu,Y). It is synthesized by melt processing technique. The thermoluminescence (TL) and optically stimulated luminescence (OSL) correlation was systematically studied. The study reveals that the OSL signal is attributed to the observed TL peaks in this material. Thus, same defects are responsible for TL and OSL in LiCaAlF6:Eu,Y. Further characterization involved measurement of TL emission spectrum, OSL emission spectrum, steady state photoluminescence (PL) spectra, phosphorescence, sensitivity, fading, dose response etc. The TL sensitivity of this phosphor was observed to be ∼5 times and ∼3 times to that of CaSO4:Dy and commercially available Al2O3:C respectively. PL, TL and OSL emission spectra suggest that Eu2+ ion acts as a luminescence center in this material. Minimum measureable dose for the phosphor in TL mode was found to be 0.3 μGy with negligible fading post 10 days of irradiation. The fading of TL signal in this material is similar to the one observed for OSL signal. The thermal stability and hygroscopic nature of LiCaAlF6:Eu, Y were studied using thermogravimetric and differential scanning calorimetry techniques. Owing to the attributes like high sensitivity, wide dynamic dose range it stands as a viable and excellent phosphor material for various radiological and dosimetric applications.

Discovery of novel cardiac troponin activators using fluorescence polarization-based high throughput screening assays

The large unmet demand for new heart failure therapeutics is widely acknowledged. Over the last decades the contractile myofilaments themselves have emerged as an attractive target for the development of new therapeutics for both systolic and diastolic heart failure. However, the clinical use of myofilament-directed drugs has been limited, and further progress has been hampered by incomplete understanding of myofilament function on the molecular level and screening technologies for small molecules that accurately reproduce this function in vitro. In this study we have designed, validated and characterized new high throughput screening platforms for small molecule effectors targeting the interactions between the troponin C and troponin I subunits of the cardiac troponin complex. Fluorescence polarization-based assays were used to screen commercially available compound libraries, and hits were validated using secondary screens and orthogonal assays. Hit compound-troponin interactions were characterized using isothermal titration calorimetry and NMR spectroscopy. We identified NS5806 as novel calcium sensitizer that stabilizes active troponin. In good agreement, NS5806 greatly increased the calcium sensitivity and maximal isometric force of demembranated human donor myocardium. Our results suggest that sarcomeric protein-directed screening platforms are suitable for the development of compounds that modulate cardiac myofilament function.

Repetitive use of levosimendan in severe functional tricuspid regurgitation.

Functional tricuspid regurgitation (FTR) has a very poor outcome and few patients are referred for surgery due to the important peri-operative mortality which averages 15%.1 Edge-to-edge percutaneous repair has been proposed as an alternative to cardiac surgery and preliminary results showed improvement in patient’s functional status and outcome. However, the feasibility is limited in patients with advanced FTR disease, i.e. in case of a large gap of leaflet coaptation.2 Levosimendan is a calcium sensitizer agent which enhances cardiac contractility and has a strong vasodilatation effect. Levosimendan seems particularly efficient in improving right ventricular (RV) function. The benefit of the repetitive use of levosimendan in chronic heart failure (HF) has been demonstrated,3 but no study has been conducted in patients with severe FTR. In this preliminary clinical study, we reported the clinical and echocardiography impacts of the repetitive use of levosimendan in HF patients with severe FTR. We prospectively included 33 consecutive patients (77 ± 7 years, 48.5% of women) admitted for HF with severe FTR [73% graded as massive or torrential, mean effective regurgitation orifice area (EROA) = 100 ± 56 m²]. All patients signed informed consent. Most of the patients were in NYHA Class III and IV (91%) with a median dose of furosemide at 250 mg daily (95; 500). The majority were in chronic atrial fibrillation (92%), 51.5% had a history of cardiac surgery, and 42.4% had a pacemaker or defibrillator lead. Surgery was denied by the Heart Team because of the operative risk (mean Triscore = 25 ± 17%) and the percutaneous approach was first excluded because of inappropriate anatomy or imaging. Levosimendan was delivered without bolus by intravenous perfusion (0.2 γ/kg/min over 24 h) and repeated at least one time after 2 or 3 weeks. The severity of TR was graded according to EROA using the PISA (proximal isovelocity surface area) method3 by transthoracic echocardiography. Tricuspid annulus diameter, tricuspid leaflet tenting and coaptation, and RV and right atrial (RA) dimensions were quantified from a four-chamber view and inferior vena cava (IVC) diameter was assessed in the subcostal view. Right ventricular function was assessed using tricuspid annular plane systolic excursion (TAPSE).

Clinical and functional characterization of a long survivor congenital titinopathy patient with a novel metatranscript-only titin variant

Congenital titinopathies are an emerging group of a potentially severe form of congenital myopathies caused by biallelic mutations in titin, encoding the largest existing human protein involved in the formation and stability of sarcomeres. In this study we describe a patient with a congenital myopathy characterized by multiple contractures, a rigid spine, non progressive muscular weakness, and a novel homozygous TTN pathogenic variant in a metatranscript-only exon: the c.36400A > T, p.Lys12134*. Muscle biopsies showed increased internalized nuclei, variability in fiber size, mild fibrosis, type 1 fiber predominance, and a slight increase in the number of satellite cells. RNA studies revealed the retention of intron 170 and 171 in the open reading frame, and immunoflourescence and western blot studies, a normal titin content. Single fiber functional studies showed a slight decrease in absolute maximal force and a cross-sectional area with no decreases in tension, suggesting that weakness is not sarcomere-based but due to hypotrophy. Passive properties of single fibers were not affected, but the observed increased calcium sensitivity of force generation might contribute to the contractural phenotype and rigid spine of the patient. Our findings provide evidence for a pathogenic, causative role of a metatranscript-only titin variant in a long survivor congenital titinopathy patient with distal arthrogryposis and rigid spine.

The Effects of the Levosimendan Metabolites OR-1855 and OR-1896 on Endothelial Pro-Inflammatory Responses

The calcium sensitizer levosimendan is used for the treatment of acute decompensated heart failure. A small portion (4-7%) of levosimendan is metabolized to the pharmacologically active metabolite OR-1896 via the inactive intermediate OR-1855. In addition, levosimendan has been shown to exert positive effects on the endothelium in vitro antagonizing vascular dysfunction and inflammation. However, the function of the levosimendan metabolites within this context is still unknown. In this study, we thus investigated the impact of the metabolites OR-1896 and OR-1855 on endothelial inflammatory processes in vitro. We observed a reduction of IL-1β-dependent endothelial adhesion molecule ICAM-1 and VCAM-1 as well as interleukin (IL) -6 expression upon levosimendan treatment but not after treatment with OR-1855 or OR-1896, as assessed by western blotting, flow cytometry, and qRT-PCR. Instead, the metabolites impaired IL-1β-induced ROS formation via inactivation of the MAPK p38, ERK1/2, and JNK. Our results suggest that the levosimendan metabolites OR-1896 and OR-1855 have certain anti-inflammatory properties, partly other than levosimendan. Importantly, they additionally show that the intermediate metabolite OR-1855 does, in fact, have pharmacological effects in the endothelium. This is interesting, as the metabolites are responsible for the long-term therapeutic effects of levosimendan, and heart failure is associated with vascular dysfunction and inflammation.

Fast and sensitive GCaMP calcium indicators for imaging neural populations

Calcium imaging with protein-based indicators1,2 is widely used to follow neural activity in intact nervous systems, but current protein sensors report neural activity at timescales much slower than electrical signalling and are limited by trade-offs between sensitivity and kinetics. Here we used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators3–8. The resulting ‘jGCaMP8’ sensors, based on the calcium-binding protein calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (half-rise times of 2 ms) and the highest sensitivity for neural activity reported for a protein-based calcium sensor. jGCaMP8 sensors will allow tracking of large populations of neurons on timescales relevant to neural computation.

Conversion of a Cardiac Muscle Modulator from an Inhibitor to an Activator.

The binding of calcium to cardiac troponin C (cTnC) enhances the binding of troponin I (cTnI) switch region to the regulatory domain of cTnC (cNTnC) and triggers muscle contraction. Several molecules alter the response of the sarcomere by targeting this interface; virtually all have an aromatic core that binds to the hydrophobic pocket of cNTnC and an aliphatic tail that interacts with the switch region of cTnI. W7 has been extensively studied, and the positively charged tail has been shown to be important for its inhibitory action. Herein we investigate the importance of the aromatic core of W7 by synthesizing compounds that have the core region of calcium activator dfbp-o with various lengths of the same tail (D-series). These compounds all bind more tightly to cNTnC-cTnI chimera (cChimera) than the analogous W-series compounds and show increased calcium sensitivity of force generation and ATPase activity, demonstrating that the cardiovascular system is tightly balanced.

Mechanoelectric effects in healthy cardiac function and under Left Bundle Branch Block pathology

Mechanoelectric feedback (MEF) in the heart operates through several mechanisms which serve to regulate cardiac function. Stretch activated channels (SACs) in the myocyte membrane open in response to cell lengthening, while tension generation depends on stretch, shortening velocity, and calcium concentration. How all of these mechanisms interact and their effect on cardiac output is still not fully understood. We sought to gauge the acute importance of the different MEF mechanisms on heart function.An electromechanical computer model of a dog heart was constructed, using a biventricular geometry of 500K tetrahedral elements. To describe cellular behavior, we used a detailed ionic model to which a SAC model and an active tension model, dependent on stretch and shortening velocity and with calcium sensitivity, were added. Ventricular inflow and outflow were connected to the CircAdapt model of cardiovascular circulation. Pressure–volume loops and activation times were used for model validation.Simulations showed that SACs did not affect acute mechanical response, although if their trigger level was decreased sufficiently, they could cause premature excitations. The stretch dependence of tension had a modest effect in reducing the maximum stretch, and stroke volume, while shortening velocity had a much bigger effect on both. MEF served to reduce the heterogeneity in stretch while increasing tension heterogeneity. In the context of left bundle branch block, a decreased SAC trigger level could restore cardiac output by reducing the maximal stretch when compared to cardiac resynchronization therapy. MEF is an important aspect of cardiac function and could potentially mitigate activation problems.