Formation and function of a highly specialised type of organelle in cardiac valve cells.

Within a cell, vesicles play a crucial role in the transport of membrane material and proteins to a given target membrane, and thus regulate a variety of cellular functions. Vesicular transport occurs by means of, among others, endocytosis, where cargoes are taken up by the cell and are processed further upon vesicular trafficking, i.e. transported back to the plasma membrane via recycling endosomes or the degraded by fusion of the vesicles with lysosomes. During evolution, a variety of vesicles with individual functions arose, with some of them building up highly specialised subcellular compartments. In this study, we have analysed the biosynthesis of a new vesicular compartment present in the valve cells of Drosophila melanogaster. We show that the compartment is formed by invaginations of the plasma membrane and grows via re-routing of the recycling endosomal pathway. This is achieved by inactivation of other membrane-consuming pathways and a plasma membrane-like molecular signature of the compartment in these highly specialised heart cells.

Activation of the nonneuronal cholinergic cardiac system by hypoxic preconditioning protects isolated adult cardiomyocytes from hypoxia/reoxygenation injury.

Activation of the vagus nerve mediates cardioprotection and attenuates myocardial ischemia/reperfusion (I/R) injury. In response to vagal activation, acetylcholine (ACh) is released from the intracardiac nervous system (ICNS) and activates intracellular cardioprotective signaling cascades. Recently, however, a nonneuronal cholinergic cardiac system (NNCCS) in cardiomyocytes has been described as an additional source of ACh. To investigate whether the NNCCS mediates cardioprotection in the absence of vagal and ICNS activation, we used a reductionist approach of isolated adult rat ventricular cardiomyocytes without neuronal cells, using hypoxic preconditioning (HPC) as a protective stimulus. Adult rat ventricular cardiomyocytes were isolated, the absence of neuronal cells was confirmed, and HPC was induced by 10/20 min hypoxia/reoxygenation (H/R) before subjection to 30/5 min H/R to simulate I/R injury. Cardiomyocyte viability was assessed by trypan blue staining at baseline and after HPC+H/R or H/R. Intra- and extracellular ACh was quantified using liquid chromatography-coupled mass spectrometry at baseline, after HPC, after hypoxia, and after reoxygenation, respectively. In a subset of experiments, muscarinic and nicotinic ACh receptor (m- and nAChR) antagonists were added during HPC or during H/R. Cardiomyocyte viability at baseline (69 ± 4%) was reduced by H/R (10 ± 3%). With HPC, cardiomyocyte viability was preserved after H/R (25 ± 6%). Intra- and extracellular ACh increased during hypoxia; HPC further increased both intra- and extracellular ACh (from 0.9 ± 0.7 to 1.5 ± 1.0 nmol/mg; from 0.7 ± 0.6 to 1.1 ± 0.7 nmol/mg, respectively). The addition of mAChR and nAChR antagonists during HPC had no impact on HPC's protection; however, protection was abrogated when antagonists were added during H/R (cardiomyocyte viability after H/R: 23 ± 5%; 13 ± 4%). In conclusion, activation of the NNCCS is involved in cardiomyocyte protection; HPC increases intra- and extracellular ACh during H/R, and m- and nAChRs are causally involved in HPC's cardiomyocyte protection during H/R. The interplay between upstream ICNS activation and NNCCS activation in myocardial cholinergic metabolism and cardioprotection needs to be investigated in future studies.NEW & NOTEWORTHY The intracardiac nervous system is considered to be involved in ischemic conditioning's cardioprotection through the release of acetylcholine (ACh). However, we demonstrate that hypoxic preconditioning (HPC) protects from hypoxia/reoxygenation injury and increases intra- and extracellular ACh during hypoxia in isolated adult ventricular rat cardiomyocytes. HPC's protection involves cardiomyocyte muscarinic and nicotinic ACh receptor activation. Thus, besides the intracardiac nervous system, a nonneuronal cholinergic cardiac system may also be causally involved in cardiomyocyte protection by ischemic conditioning.

Alary muscles and thoracic alary-related muscles are atypical striated muscles involved in maintaining the position of internal organs.

Alary muscles (AMs) have been described as a component of the cardiac system in various arthropods. Lineage-related thoracic muscles (TARMs), linking the exoskeleton to specific gut regions, have recently been discovered in Drosophila Asymmetrical attachments of AMs and TARMs, to the exoskeleton on one side and internal organs on the other, suggested an architectural function in moving larvae. Here, we analysed the shape and sarcomeric organisation of AMs and TARMs, and imaged their atypical deformability in crawling larvae. We then selectively eliminated AMs and TARMs by targeted apoptosis. Elimination of AMs revealed that AMs are required for suspending the heart in proper intra-haemocelic position and for opening of the heart lumen, and that AMs constrain the curvature of the respiratory tracheal system during crawling; TARMs are required for proper positioning of visceral organs and efficient food transit. AM/TARM cardiac versus visceral attachment depends on Hox control, with visceral attachment being the ground state. TARMs and AMs are the first example of multinucleate striated muscles connecting the skeleton to the cardiac and visceral systems in bilaterians, with multiple physiological functions.

Optogenetic Manipulation of the Vagus Nerve.

The vagus nerve plays a pivotal role in communication between the brain and peripheral organs involved in the sensory detection and the autonomic control of visceral activity. While the lack of appropriate experimental techniques to manipulate the physiological activity of the vagus nerve has been a long-standing problem, recent advancements in optogenetic tools, including viral vectors and photostimulation devices, during the late 2010s have begun to overcome this technical hurdle. Furthermore, identifying promoters for expressing transgenes in a cell-type-specific subpopulation of vagal neurons enables the selective photoactivation of afferent/efferent vagal neurons and specific visceral organ-innervating vagal neurons. In this chapter, we describe recent optogenetic approaches to study vagus nerve physiology and describe how these approaches have provided novel findings on the roles of vagus nerve signals in the cardiac, respiratory, and gastrointestinal systems. Compared with studies of the central nervous system, there are still few insights into vagus nerve physiology. Further studies with optogenetic tools will be useful for understanding the fundamental characteristics of vagus nerve signals transferred throughout the body.

Simulation Study for Designing a Dedicated Cardiac TOF-PET System.

The development of dedicated positron emission tomography scanners is an active area of research, especially aiming at the improvement of lesion detection and in support of cancer treatment and management. Recently, dedicated Positron Emission Tomography (PET) systems with different configurations for specific organs have been developed for improving detection effectiveness. Open geometries are always subject to distortion and artifacts in the reconstructed images. Therefore, the aim of this work is to determine the optimal geometry for a novel cardiac PET system that will be developed by our team, and determine the time resolution needed to achieve reasonable image quality for the chosen geometry. The proposed geometries consist of 36 modules. These modules are arranged in two sets of two plates, each one with different configurations. We performed Monte Carlo simulations with different TOF resolutions, in order to test the image quality improvement in each case. Our results show, as expected, that increasing TOF resolution reduces distortion and artifact effects. We can conclude that a TOF resolution of the order of 200 ps is needed to reduce the artifacts, to acceptable levels, generated in the simulated cardiac-PET open geometries.

Comparative iTRAQ analysis of protein abundance in the human sinoatrial node and working cardiomyocytes.

Our objective was to assess the changes in protein abundance in the human sinoatrial node (SAN) compared with working cardiomyocytes to identify SAN-specific protein signatures. Four pairs of samples (the SAN and working cardiomyocytes) were obtained postmortem from four human donors with no evidence of cardiovascular disease. We performed protein identification and quantitation using two-dimensional chromatography-tandem mass spectrometry with isobaric peptide labeling (iTRAQ). We identified 451 different proteins expressed in both the SAN and working cardiomyocytes, 166 of which were differentially regulated (110 were upregulated in the SAN and 56 in the working cardiomyocytes). We identified sarcomere structural proteins in both tissues, although they were differently distributed among the tested samples. For example, myosin light chain 4, myosin regulatory light chain 2-atrial isoform, and tropomyosin alpha-3 chain levels were twofold higher in the SAN than in working cardiomyocytes, and myosin light chain 3 and myosin regulatory light chain 2-ventricular/cardiac muscle isoform levels were twofold higher in the ventricle tissue than in SAN. We identified many mitochondrial oxidative phosphorylation, ß-oxidation, and tricarboxylic acid cycle proteins that were predominantly associated with working cardiomyocytes tissue. We detected upregulation of the fatty acid omega activation pathway proteins in the SAN samples. Some proteins specific for smooth muscle tissue were highly upregulated in the SAN (e.g. transgelin), which indicates that the SAN tissue might act as the bridge between the working myocardium and the smooth muscle. Our results show possible implementation of proteomic strategies to identify in-depth functional differences between various heart sub-structures.

Warm acclimation and oxygen depletion induce species-specific responses in salmonids.

Anthropogenic activities are greatly altering the habitats of animals, whereby fish are already encountering several stressors simultaneously. The purpose of the current study was to investigate the capacity of fish to respond to two different environmental stressors (high temperature and overnight hypoxia) separately and together. We found that acclimation to increased temperature (from 7.7±0.02°C to 14.9±0.05°C) and overnight hypoxia (daily changes from normoxia to 63-67% oxygen saturation), simulating climate change and eutrophication, had both antagonistic and synergistic effects on the capacity of fish to tolerate these stressors. The thermal tolerance of Arctic char (Salvelinus alpinus) and landlocked salmon (Salmo salar m. sebago) increased with warm acclimation by 1.3 and 2.2°C, respectively, but decreased when warm temperature was combined with overnight hypoxia (by 0.2 and 0.4°C, respectively). In contrast, the combination of the stressors more than doubled hypoxia tolerance in salmon and also increased hypoxia tolerance in char by 22%. Salmon had 1.2°C higher thermal tolerance than char, but char tolerated much lower oxygen levels than salmon at a given temperature. The changes in hypoxia tolerance were connected to the responses of the oxygen supply and delivery system. The relative ventricle mass was higher in cold- than in warm-acclimated salmon but the thickness of the compact layer of the ventricle increased with the combination of warm and hypoxia acclimation in both species. Char had also significantly larger hearts and thicker compact layers than salmon. The results illustrate that while fish can have protective responses when encountering a single environmental stressor, the combination of stressors can have unexpected species-specific effects that will influence their survival capacity.

In vitro rescue study of a malignant familial hypertrophic cardiomyopathy phenotype by pseudo-phosphorylation of myosin regulatory light chain.

Pseudo-phosphorylation of cardiac myosin regulatory light chain (RLC) has never been examined as a rescue method to alleviate a cardiomyopathy phenotype brought about by a disease causing mutation in the myosin RLC. This study focuses on the aspartic acid to valine substitution (D166V) in the myosin RLC shown to be associated with a malignant phenotype of familial hypertrophic cardiomyopathy (FHC). The mutation has also been demonstrated to cause severe functional abnormalities in transgenic mice expressing D166V in the heart. To explore this novel rescue strategy, pseudo-phosphorylation of D166V was used to determine whether the D166V-induced detrimental phenotype could be brought back to the level of wild-type (WT) RLC. The S15D substitution at the phosphorylation site of RLC was inserted into the recombinant WT and D166V mutant to mimic constitutively phosphorylated RLC proteins. Non-phosphorylatable (S15A) constructs were used as controls. A multi-faceted approach was taken to determine the effect of pseudo-phosphorylation on the ability of myosin to generate force and motion. Using mutant reconstituted porcine cardiac muscle preparations, we showed an S15D-induced rescue of both the enzymatic and binding properties of D166V-myosin to actin. A significant increase in force production capacity was noted in the in vitro motility assays for S15D-D166V vs. D166V reconstituted myosin. A similar pseudo-phosphorylation induced effect was observed on the D166V-elicited abnormal Ca(2+) sensitivity of force in porcine papillary muscle strips reconstituted with phosphomimic recombinant RLCs. Results from this study demonstrate a novel in vitro rescue strategy that could be utilized in vivo to ameliorate a malignant cardiomyopathic phenotype. We show for the first time that pseudo-RLC phosphorylation can reverse the majority of the mutation-induced phenotypes highlighting the importance of RLC phosphorylation in combating cardiac disease.

Airborne polystyrene nanoplastics exposure leads to heart failure via ECM-receptor interaction and PI3K/AKT/BCL-2 pathways.

Environmental contamination has been recognized as a significant threat to human well-being, and recent findings of microplastic presence in human cardiac tissues have raised concerns. However, research on the effects of airborne nanoplastics (NPs) on cardiac physiology remains limited. We utilized a comprehensive body exposure apparatus to simulate the impact of airborne polystyrene NPs pollution, focusing on understanding how airborne NPs affect cardiac morphology and function. Following two weeks of NPs exposure, mice exhibited a 23.89 ± 8.30 % reduction in heart mass, a 20.05 ± 2.97 % decrease in heart rate as detected, and a myocardial electrical conduction block. Echocardiography showed significant changes in cardiac contractility, with increases in cardiac ejection fraction and stroke volume of 13.00 ± 3.00 % and 43.00 ± 17.00 %, respectively. In addition, histologic assessments revealed signs of ventricular hypertrophy, ventricular myocardial hypertrophy, and myocardial necrotic fibrosis. Of particular interest, our mechanistic investigations highlighted the harmful effects of NPs on cardiac structure and function, mediated through extracellular matrix (ECM) receptor interactions and the PI3K/AKT/BCL-2 signaling pathway. The insights gained provide a foundation for understanding the risks posed by airborne NPs to human cardiac health, emphasizing the need for increased vigilance and implementation of mitigation strategies in environmental management.

Myocardial infarction detection using ITD, DWT and deterministic learning based on ECG signals.

Nowadays, cardiovascular diseases (CVD) is one of the prime causes of human mortality, which has received tremendous and elaborative research interests regarding the prevention issue. Myocardial ischemia is a kind of CVD which will lead to myocardial infarction (MI). The diagnostic criterion of MI is supplemented with clinical judgement and several electrocardiographic (ECG) or vectorcardiographic (VCG) programs. However the visual inspection of ECG or VCG signals by cardiologists is tedious, laborious and subjective. To overcome such disadvantages, numerous MI detection techniques including signal processing and artificial intelligence tools have been developed. In this study, we propose a novel technique for automatic detection of MI based on disparity of cardiac system dynamics and synthesis of the standard 12-lead and Frank XYZ leads. First, 12-lead ECG signals are synthesized with Frank XYZ leads to build a hybrid 4-dimensional cardiac vector, which is decomposed into a series of proper rotation components (PRCs) by using the intrinsic time-scale decomposition (ITD) method. The novel cardiac vector may fully reflect the pathological alterations provoked by MI and may be correlated to the disparity of cardiac system dynamics between healthy and MI subjects. ITD is employed to measure the variability of cardiac vector and the first PRCs are extracted as predominant PRCs which contain most of the cardiac vector's energy. Second, four levels discrete wavelet transform with third-order Daubechies (db3) wavelet function is employed to decompose the predominant PRCs into different frequency bands, which combines with three-dimensional phase space reconstruction to derive features. The properties associated with the cardiac system dynamics are preserved. Since the frequency components above 40 Hz are lack of use in ECG analysis, in order to reduce the feature dimension, the advisable sub-band (D4) is selected for feature acquisition. Third, neural networks are then used to model, identify and classify cardiac system dynamics between normal (healthy) and MI cardiac vector signals. The difference of cardiac system dynamics between healthy control and MI cardiac vector is computed and used for the detection of MI based on a bank of estimators. Finally, experiments are carried out on the PhysioNet PTB database to assess the effectiveness of the proposed method, in which conventional 12-lead and Frank XYZ leads ECG signal fragments from 148 patients with MI and 52 healthy controls were extracted. By using the tenfold cross-validation style, the achieved average classification accuracy is reported to be 98.20%. Results verify the effectiveness of the proposed method which can serve as a potential candidate for the automatic detection of MI in the clinical application.

Sub-lethal Camphor Exposure Triggers Oxidative Stress, Cardiotoxicity, and Cardiac Physiology Alterations in Zebrafish Embryos.

Camphor is a terpene ketone with aromatic and volatile properties in nature derived from the bark of Cinnamomum camphora or synthesized from turpentine. Camphor exhibits various biological properties such as anti-microbial, anti-viral, anti-coccidial, and anti-cancer. It is also used as a form of topical medication for skin irritation, joint pain, and as a relief for itching from insect bites. However, even though the high dose of camphor has been documented to be toxic/lethal in humans in different studies, camphor's developmental toxicity has not yet been explored, and its extensive mechanism of action is still unclear. In the present study, we aimed to assess the toxic effects of camphor in zebrafish embryos in the initial developmental stages. The obtained results demonstrated that a sub-lethal dose of camphor caused a decrease in hatching rate, body length, and substantial elevation in malformation rate on zebrafish embryos. On further observation, in the following time frame, curved body and pericardial edema of zebrafish were also observed. Furthermore, exposure to a sub-lethal dose of camphor was also able to trigger cardiotoxicity in zebrafish larvae. Later, on subsequent biochemical analysis, it was found that the antioxidant capacity inhibition and oxidative stress elevation that occurred after camphor exposure might be associated with the inhibition of total superoxide dismutase (SOD) activity and an increase in reactive oxygen species (ROS) and malondialdehyde (MDA) concentration. In addition, compared to the control group, several apoptotic cells in treated zebrafish were also found to be elevated. Finally, after further investigation on marker gene expressions, we conclude that the developmental toxicity of camphor exposure might be associated with apoptosis elevation and oxidative stress. Taken together, the current study provides a better understanding of the developmental toxicity of camphor on zebrafish, a promising alternative animal model to assess the developmental toxicity of chemical compounds.

Experimental data of labeling the heart and cardiac cultures with a retrograde tracer in vitro and in vivo.

Retrograde dyes are often used in basic research to investigate neuronal innervations of an organ. This article describes the experimental data on the application of retrograde dyes on the mouse heart in vivo and on the cardiac or neuronal cultures in vitro. By providing this information, cardiac or inneinnervations can be evaluated in vivo. Therefore, unknown cellular and molecular mechanisms and systemic interactions in the body can be investigated. In particular, we provided practical tips to lower mortality risks following the cardiac surgery and evaluated the staining capacity and fluorescent characteristics of the Di-8-ANEPPQ dye in the cardiac tissue and cell cultures. First, primary cultures of mouse nodose ganglia (NG) neurons and mouse neonatal cardiomyocytes were stained with Di-8-ANEPPQ. The Di-8-ANEPPQ signal from live cultures were visualized using spinning disk confocal microscopy to verify the lipophilic and fluorescent labeling capacity of Di-8-ANEPPQ. Next, the excitation and emission data of Di-8-ANEPPQ were collected between 415 nm and 690 nm using power spectrum module of confocal microscopy. This spectrum analysis could be useful for the researchers who plan to use Di-8-ANEPPQ in combination with other fluorescent dyes to eliminate any florescent overlap. In order to label the heart tissue with tracer dyes Di-8-ANEPPQ or DiI in vivo, the heart was exposed without damaging lungs or other tissues following anesthetization, then the retrograde dye was applied as a paste for DiI or injected to the apex of the heart for Di-8-ANEPPQ and the operation area was sutured. The surgical procedure required intubation to control the respiratory reflex without the need to perform a tracheotomy and yielded high viability. Following labeling the heart in vivo, the heart was dissected, and images of injection area were captured using confocal microscopy. All fluorescent images of Di-8-ANEPPQ labeled cells were analyzed by using the Fiji software. Overall, these data provide applicable data to other investigators to trace the sensory neurons innervating not only the heart but also other organs using Di-8-ANEPPQ. These data support the original research article titled "Evaluation of bilateral cardiac afferent distribution at the spinal and vagal ganglia by retrograde labeling" that was accepted for publication in Brain Research Journal [1].

Classification of myocardial infarction based on hybrid feature extraction and artificial intelligence tools by adopting tunable-Q wavelet transform (TQWT), variational mode decomposition (VMD) and neural networks.

Cardiovascular diseases (CVD) is the leading cause of human mortality and morbidity around the world, in which myocardial infarction (MI) is a silent condition that irreversibly damages the heart muscles. Currently, electrocardiogram (ECG) is widely used by the clinicians to diagnose MI patients due to its inexpensiveness and non-invasive nature. Pathological alterations provoked by MI cause slow conduction by increasing axial resistance on coupling between cells. This issue may cause abnormal patterns in the dynamics of the tip of the cardiac vector in the ECG signals. However, manual interpretation of the pathological alternations induced by MI is a time-consuming, tedious and subjective task. To overcome such disadvantages, computer-aided diagnosis techniques including signal processing and artificial intelligence tools have been developed. In this study we propose a novel technique for automatic detection of MI based on hybrid feature extraction and artificial intelligence tools. Tunable quality factor (Q-factor) wavelet transform (TQWT), variational mode decomposition (VMD) and phase space reconstruction (PSR) are utilized to extract representative features to form cardiac vectors with synthesis of the standard 12-lead and Frank XYZ leads. They are combined with neural networks to model, identify and detect abnormal patterns in the dynamics of cardiac system caused by MI. First, 12-lead ECG signals are reduced to 3-dimensional VCG signals, which are synthesized with Frank XYZ leads to build a hybrid 4-dimensional cardiac vector. Second, this vector is decomposed into a set of frequency subbands with a number of decomposition levels by using the TQWT method. Third, VMD is employed to decompose the subband of the 4-dimensional cardiac vector into different intrinsic modes, in which the first intrinsic mode contains the majority of the cardiac vector's energy and is considered to be the predominant intrinsic mode. It is selected to construct the reference variable for analysis. Fourth, phase space of the reference variable is reconstructed, in which the properties associated with the nonlinear cardiac system dynamics are preserved. Three-dimensional (3D) PSR together with Euclidean distance (ED) has been utilized to derive features, which demonstrate significant difference in cardiac system dynamics between normal (healthy) and MI cardiac vector signals. Fifth, cardiac system dynamics can be modeled and identified using neural networks, which employ the ED of 3D PSR of the reference variable as the input features. The difference of cardiac system dynamics between healthy control and MI cardiac vector is computed and used for the detection of MI based on a bank of estimators. Finally, data sets, which include conventional 12-lead and Frank XYZ leads ECG signal fragments from 148 patients with MI and 52 healthy controls from PTB diagnostic ECG database, are used for evaluation. By using the 10-fold cross-validation style, the achieved average classification accuracy is reported to be 97.98%. Currently, ST segment evaluation is one of the major and traditional ways for the MI detection. However, there exist weak or even undetectable ST segments in many ECG signals. Since the proposed method does not rely on the information of ST waves, it can serve as a complementary MI detection algorithm in the intensive care unit (ICU) of hospitals to assist the clinicians in confirming their diagnosis. Overall, our results verify that the proposed features may satisfactorily reflect cardiac system dynamics, and are complementary to the existing ECG features for automatic cardiac function analysis.

QTc Prolongation in Patients with Dementia and Mild Cognitive Impairment: Neuropsychological and Brain Imaging Correlations.

The QTc interval is the electrocardiographic manifestation of ventricular depolarization and repolarization. This marker is often prolonged in acute and chronic neurological conditions. The cause of the cerebrogenic QT prolongation remains unclear. The aim of the study was to analyze the relation between QTc interval and the degree of cognitive impairment and structural brain imaging changes in patients with dementia and mild cognitive impairment (MCI). To this aim, 269 patients were screened, of whom 61 met one or more exclusion criteria. The remaining 208 patients (56 control subjects, 44 patients with MCI, and 108 with dementia) were recruited. Eighty-five patients using drugs causing prolongation of QT interval were further excluded. The QT interval was measured manually in all 12 leads by a single blinded observer, assuming the longest QT value adjusted for heart rate by using the Bazett's formula. All patients underwent a structural brain imaging and the following measures were obtained: the bicaudate ratio and the periventricular hyperintensity and deep white matter hyperintensity using the modified Fazekas scale. Prolonged QTc interval was prevalent in 1) patients with dementia, especially in those with moderate-severe degree; 2) subjects with impairment of praxis and attention, low functional status, and behavioral symptoms; 3) patients with global and temporal atrophy and with higher scores on the Fazekas or leukoaraiosis scales. Degenerative and vascular processes might play a main role in QTc interval prolongation because of the damage to brain areas involved in the control of the autonomic cardiac nervous system.