Subcellular Remodeling in Filamin C Deficient Mouse Hearts Impairs Myocyte Tension Development during Progression of Dilated Cardiomyopathy

Dilated cardiomyopathy (DCM) is a life-threatening form of heart disease that is typically characterized by progressive thinning of the ventricular walls, chamber dilation, and systolic dysfunction. Multiple mutations in the gene encoding filamin C (FLNC), an actin-binding cytoskeletal protein in cardiomyocytes, have been found in patients with DCM. However, the mechanisms that lead to contractile impairment and DCM in patients with FLNC variants are poorly understood. To determine how FLNC regulates systolic force transmission and DCM remodeling, we used an inducible, cardiac-specific FLNC-knockout (icKO) model to produce a rapid onset of DCM in adult mice. Loss of FLNC reduced systolic force development in single cardiomyocytes and isolated papillary muscles but did not affect twitch kinetics or calcium transients. Electron and immunofluorescence microscopy showed significant defects in Z-disk alignment in icKO mice and altered myofilament lattice geometry. Moreover, a loss of FLNC induces a softening myocyte cortex and structural adaptations at the subcellular level that contribute to disrupted longitudinal force production during contraction. Spatially explicit computational models showed that these structural defects could be explained by a loss of inter-myofibril elastic coupling at the Z-disk. Our work identifies FLNC as a key regulator of the multiscale ultrastructure of cardiomyocytes and therefore plays an important role in maintaining systolic mechanotransmission pathways, the dysfunction of which may be key in driving progressive DCM.

PHD2 deletion in endothelial or arterial smooth muscle cells reveals vascular cell type-specific responses in pulmonary hypertension and fibrosis

Hypoxia plays an important regulatory role in the vasculature to adjust blood flow to meet metabolic requirements. At the level of gene transcription, the responses are mediated by hypoxia-inducible factor (HIF) the stability of which is controlled by the HIF prolyl 4-hydroxylase-2 (PHD2). In the lungs hypoxia results in vasoconstriction, however, the pathophysiological relevance of PHD2 in the major arterial cell types; endothelial cells (ECs) and arterial smooth muscle cells (aSMCs) in the adult vasculature is incompletely characterized. Here, we investigated PHD2-dependent vascular homeostasis utilizing inducible deletions of PHD2 either in ECs (Phd2∆ECi) or in aSMCs (Phd2∆aSMC). Cardiovascular function and lung pathologies were studied using echocardiography, Doppler ultrasonography, intraventricular pressure measurement, histological, ultrastructural, and transcriptional methods. Cell intrinsic responses were investigated in hypoxia and in conditions mimicking hypertension-induced hemodynamic stress. Phd2∆ECi resulted in progressive pulmonary disease characterized by a thickened respiratory basement membrane (BM), alveolar fibrosis, increased pulmonary artery pressure, and adaptive hypertrophy of the right ventricle (RV). A low oxygen environment resulted in alterations in cultured ECs similar to those in Phd2∆ECi mice, involving BM components and vascular tone regulators favoring the contraction of SMCs. In contrast, Phd2∆aSMC resulted in elevated RV pressure without alterations in vascular tone regulators. Mechanistically, PHD2 inhibition in aSMCs involved actin polymerization -related tension development via activated cofilin. The results also indicated that hemodynamic stress, rather than PHD2-dependent hypoxia response alone, potentiates structural remodeling of the extracellular matrix in the pulmonary microvasculature and respiratory failure.

X-ray Diffraction Analysis to Explore Molecular Traces of Eccentric Contraction on Rat Skeletal Muscle Parallelly Evaluated with Signal Protein Phosphorylation Levels

We performed X-ray diffraction analyses on rat plantaris muscle to determine if there are strain-specific structural changes at the molecular level after eccentric contraction (ECC). ECC was elicited in situ by supramaximal electrical stimulation through the tibial nerve. One hour after a series of ECC sessions, the structural changes that remained in the sarcomere were evaluated using X-ray diffraction. Proteins involved in cell signaling pathways in the muscle were also examined. ECC elicited by 100, 75, and 50 Hz stimulation respectively developed peak tension of 1.34, 1.12 and 0.79 times the isometric maximal tetanus tension. The series of ECC sessions phosphorylated the forkhead box O proteins (FoxO) in a tension-time integral-dependent manner, as well as phosphorylated the mitogen-activated protein kinases (MAPK) and a protein in the mammalian target of rapamycin (mTOR) pathway in a maximal tension dependent manner. Compared to isometric contractions, ECC was more efficient in phosphorylating the signaling proteins. X-ray diffraction revealed that the myofilament lattice was preserved even after intense ECC stimulation at 100 Hz. Additionally, ECC < 75 Hz preserved the molecular alignment of myoproteins along the myofilaments, while 75-Hz stimulation induced a slight but significant decrease in the intensity of meridional troponin reflection at 1/38 nm−1, and of myosin reflection at 1/14.4 nm−1. These two reflections demonstrated no appreciable decrease with triple repetitions of the standard series of ECC sessions at 50 Hz, suggesting that the intensity decrease depended on the instantaneous maximal tension development rather than the total load of contraction, and was more likely linked with the phosphorylation of MAPK and mTOR signaling proteins.

Adaptation of the Calcium-dependent Tension Development in Ventricular Cardiomyocytes

Today a variety of models describe the physiological behavior of the heart on a cellular level. The intracellular calcium concentration plays an important role, since it is the main driver for the active contraction of the heart. Due to different implementations of the calcium dynamics, simulating cardiac electromechanics can lead to severely different behaviors of the active tension when coupling the same tension model with different electrophysiological models. To handle these variations, we present an optimization tool that adapts the parameters of the most recent, human based tension model. The goal is to generate a physiologically valid tension development when coupled to an electrophysiological cellular model independent of the specifics of that model's calcium transient. In this work, we focus on a ventricular cell model. In order to identify the calcium-sensitive parameters, a sensitivity analysis of the tension model was carried out. In a further step, the cell model was adapted to reproduce the sarcomere length-dependent behavior of troponin C. With a maximum relative deviation of 20.3% per defined characteristic of the tension development, satisfactory results could be obtained for isometric twitch tension. Considering the length-dependent troponin handling, physiological behavior could be reproduced. In conclusion, we propose an algorithm to adapt the tension development model to any calcium transient input to achieve a physiologically valid active contraction on a cellular level. As a proof of concept, the algorithm is successfully applied to one of the most recent human ventricular cell models. This is an important step towards fully coupled electromechanical heart models, which are a valuable tool in personalized health care.

Comparison of Shear Wave Elastography and Dynamometer Test in Muscle Tissue Characterization for Potential Medical and Sport Application

Skeletal muscle status and its dynamic follow up are of particular importance in the management of several diseases where weight and muscle mass loss and, consequently, immobilization occurs, as in cancer and its treatment, as well as in neurodegenerative disorders. But immobilization is not the direct result of body and muscle mass loss, but rather the loss of the maximal tension capabilities of the skeletal muscle. Therefore, the development of a non-invasive and real-time method which can measure muscle tension capabilities in immobile patients is highly anticipated. Our aim was to introduce and evaluate a special ultrasound measurement technique to estimate a maximal muscle tension characteristic which can be used in medicine and also in sports diagnostics. Therefore, we determined the relationship between the results of shear wave elastography measurements and the dynamometric data of individuals. The measurements were concluded on the m. vastus lateralis. Twelve healthy elite athletes took part in our preliminary proof of principle study—five endurance (S) and seven strength (F) athletes showing unambiguously different muscle composition features, nine healthy subjects (H) without prior sports background, and four cancer patients in treatment for a stage 3 brain tumor (T). Results showed a high correlation between the maximal dynamometric isometric torque (Mmax) and mean elasticity value (E) for the non-athletes [(H + T), (r = 0.795)] and for the athletes [(S + F), (r = 0.79)]. For the athletes (S + F), the rate of tension development at contraction (RTDk) and E correlation was also determined (r = 0.84, p &lt; 0.05). Our measurements showed significantly greater E values for the strength athletes with fast muscle fiber dominance than endurance athletes with slow muscle fiber dominance (p &lt; 0.05). Our findings suggest that shear wave ultrasound elastography is a promising method for estimating maximal muscle tension and, also, the human skeletal muscle fiber ratio. These results warrant further investigations with a larger number of individuals, both in medicine and in sports science.

FFAR1 activation attenuates histamine-induced myosin light chain phosphorylation and cortical tension development in human airway smooth muscle cells

Background Activation of free fatty acid receptors (FFAR1 and FFAR4) which are G protein-coupled receptors (GPCRs) with established (patho)physiological roles in a variety of obesity-related disorders, induce human airway smooth muscle (HASM) cell proliferation and shortening. We reported amplified agonist-induced cell shortening in HASM cells obtained from obese lung donors. We hypothesized that FFAR1 modulate excitation–contraction (EC) coupling in HASM cells and play a role in obesity-associated airway hyperresponsiveness. Methods In HASM cells pre-treated (30 min) with FFAR1 agonists TAK875 and GW9508, we measured histamine-induced Ca2+ mobilization, myosin light chain (MLC) phosphorylation, and cortical tension development with magnetic twisting cytometry (MTC). Phosphorylation of MLC phosphatase and Akt also were determined in the presence of the FFAR1 agonists or vehicle. In addition, the effects of TAK875 on MLC phosphorylation were measured in HASM cells desensitized to β2AR agonists by overnight salmeterol treatment. The inhibitory effect of TAK875 on MLC phosphorylation was compared between HASM cells from age and sex-matched non-obese and obese human lung donors. The mean measurements were compared using One-Way ANOVA with Dunnett’s test for multiple group comparisons or Student’s t-test two-group comparison. For cortical tension measurements by magnetic twisted cytometry, mixed effect model using SAS V.9.2 was applied. Means were considered significant when p ≤ 0.05. Results Unexpectedly, we found that TAK875, a synthetic FFAR1 agonist, attenuated histamine-induced MLC phosphorylation and cortical tension development in HASM cells. These physiological outcomes were unassociated with changes in histamine-evoked Ca2+ flux, protein kinase B (AKT) activation, or MLC phosphatase inhibition. Of note, TAK875-mediated inhibition of MLC phosphorylation was maintained in β2AR-desensitized HASM cells and across obese and non-obese donor-derived HASM cells. Conclusions Taken together, our findings identified the FFAR1 agonist TAK875 as a novel bronchoprotective agent that warrants further investigation to treat difficult-to-control asthma and/or airway hyperreactivity in obesity.

PECULIARITIES OF THE SANDPLAY THERAPY APPLICATION IN DEALING WITH AGGRESSION IN TEENAGE YEARS

The actuality of the research is connected with the necessity of studying psychological peculiarities of aggression manifestation in teenage years, which is not studied sufficiently enough and requires a more thorough research, namely, the development of the program aimed at decreasing the level of the aggression manifestation with the use of methods of the sandplay therapy. The article presents the theoretical analysis of the contemporary methods in psychology for investigating aggression in the teenage years. Furthermore, the types of aggression, factors contributing to the occurrence the teenage aggression have been defined. The study of aggression is one of the main courses of the research, diagnostic and treatment, and prophylactic work. Nowadays psychologists implement plenty of various techniques in the work with the aggressive behavior in teenagers, but the most effective, as we see it, is the sandplay therapy. Through the use of methods of the sandplay therapy teenagers can master the skills of self-control and self-regulation, foster the positive personality traits, ease the psychological tension, relieve of the unpleasant experiences and the negative life situation due to its projecting on playing with the sand and symbolic reliving. The results of the empiric research, which targets the diagnostics of the level of the severity of aggression, determination of the integral forms of the communicative aggression and the tendency to the suicidal risk, are reviewed. The correction program has been tested on the experimental group of teenagers, who had the high indices of aggression levels, low self-esteem, the high level of anxiety, difficulties in communication, and detected signs of self-aggression. The courses of work of the psychological training aimed at coping with aggression in teenagers are: directing the decrease of the level of aggression; boosting the level of self-esteem; easing emotional tension; development of initiative and activity; development of the social skills. The peculiarities of development and approbation of the correction program aimed on decreasing the manifestation of aggression in the teenage years have been revealed. The conducted approbation of the training has demonstrated that in teenagers of the experimental group there has been a boost of self-esteem, decrease of the level of anxiety, fears, also their negativism and indirect aggression have been lowered, and the emotional state has improved.