How the expression of green fluorescent protein and human cardiac actin in the heart influences cardiac function and aerobic performance in zebrafish Danio rerio

The present study examined how the expression of enhanced green fluorescent protein (eGFP) and human cardiac actin (ACTC) in zebrafish Danio rerio influences embryonic heart rate (R_H) and the swim performance and metabolic rate of adult fish. Experiments with the adults involved determining the critical swimming speed (U_crit, the highest speed sustainable and measure of aerobic capacity) while measuring oxygen consumption. Two different transgenic D. rerio lines were examined: one expressed eGFP in the heart (tg(cmlc:egfp)), while the second expressed ACTC in the heart and eGFP throughout the body (tg(cmlc:actc,ba:egfp)). It was found that R_H was significantly lower in the tg(cmlc:actc,ba:egfp) embryos 4 days post‐fertilization compared to wild‐type (WT) and tg(cmlc:egfp). The swim experiments demonstrated that there was no significant difference in U_crit between the transgenic lines and the wild‐type fish, but metabolic rate and cost of transport (oxygen used to travel a set distance) was nearly two‐fold higher in the tg(cmlc:actc,ba:egfp) fish compared to WT at their respective U_crit. These results suggest that the expression of ACTC in the D. rerio heart and the expression of eGFP throughout the animal, alters cardiac function in the embryo and reduces the aerobic efficiency of the animal at high levels of activity.     

Enterococcus faecalis immunoregulates osteoclastogenesis of macrophages

Persistent apical periodontitis (PAP) is characterized by refractory inflammation and progressive bone destruction. Enterococcus faecalis infection is considered an important etiological factor for the development of PAP, although the exact mechanisms remain unknown. This study aimed at investigating the role of E. faecalis in cell proliferation, inflammatory reactions and osteoclast differentiation of macrophages using an in vitro infection model of osteoclast precursor RAW264.7 cells. A cell viability assay of cultured RAW264.7 cells exposed to live E. faecalis at a multiplicity of infection of 100 for 2 h, indicated that the infection exhibited no cytotoxic effect. Transmission electron microscopy images revealed no apoptotic changes but a rise of metabolic activity and phagocytic features in the infected RAW264.7 cells. Confocal laser scanning microscopic and flow cytometric analysis indicated that the phagocytosis of RAW264.7 cells was activated by E. faecalis infection. Furthermore, quantitative real-time PCR assays demonstrated that the expression of inflammatory cytokines was remarkably elevated in infected RAW264.7 cells. Differentiation of infected RAW264.7 cells into osteoclasts was remarkably attenuated, and expression of osteoclast marker genes as well as fusogenic genes significantly dropped. In summary, E. faecalis appears to attenuate osteoclastic differentiation of RAW264.7 precursor cells, rather stimulates them to function as macrophages.     

Fluid-structure interaction simulation of artificial textile reinforced aortic heart valve: Validation with an in-vitro test

Prosthetic heart valves deployed in the left heart (aortic and mitral) are subjected to harsh hemodynamical conditions. Most of the tissue engineered heart valves have been developed for the low pressure pulmonary position because of the difficulties in fabricating a mechanically strong valve, able to withstand the systemic circulation. This necessitates the use of reinforcing scaffolds, resulting in a tissue-engineered textile reinforced tubular aortic heart valve. Therefore, to better design these implants, material behaviour of the composite, valve kinematics and its hemodynamical response need to be evaluated. Experimental assessment can be immensely time consuming and expensive, paving way for numerical studies. In this work, the material properties obtained using the previously proposed multi-scale numerical method for textile composites was evaluated for its accuracy. An in silico immersed boundary (IB) fluid structure interaction (FSI) simulation emulating the in vitro experiment was set-up to evaluate and compare the geometric orifice area and flow rate for one beat cycle. Results from the in silico FSI simulation were found to be in good coherence with the in vitro test during the systolic phase, while mean deviation of approximately 9% was observed during the diastolic phase of a beat cycle. Merits and demerits of the in silico IB-FSI method for the presented case study has been discussed with the advantages outweighing the drawbacks, indicating the potential towards an effective use of this framework in the development and analysis of heart valves.     

Long noncoding RNA FTX regulates cardiomyocyte apoptosis by targeting miR-29b-1-5p and Bcl2l2

Cardiomyocyte apoptosis correlates with the pathogenesis of heart disease. Long noncoding RNA (LncRNA) emerges as a class of noncoding RNAs that regulate gene expression and participate in various cellular processes. However, the role of lncRNAs in cardiomyocyte apoptosis remains to be elucidated. In our study, we found that lncRNA FTX is significantly down-regulated upon ischemia/reperfusion injury and hydrogen peroxide treatment. Enhanced expression of FTX inhibits cardiomyocyte apoptosis induced by hydrogen peroxide. miR-29b-1-5p was found to interact with FTX and regulate the expression of Bcl2l2. Inhibition of miR-29b-1-5p attenuated cardiomyocyte apoptosis upon hydrogen peroxide treatment. We then found that FTX functions as endogenous sponge for miR-29b-1-5p and regulates the activity of miR-29b-1-5p. The results demonstrate that FTX regulates cardiomyocyte apoptosis through modulating the expression of Bcl2l2 which is mediated by miR-29b-1-5p. Our findings reveal a novel regulatory model which is composed of FTX, miR-29b-1-5p and Bcl2l2. Manipulating of their levels may become a new approach to tackling cardiomyocyte apoptosis related heart diseases.     

Monoamine oxidase-dependent histamine catabolism accounts for post-ischemic cardiac redox imbalance and injury

Monoamine oxidase (MAO), a mitochondrial enzyme that oxidizes biogenic amines generating hydrogen peroxide, is a major source of oxidative stress in cardiac injury. However, the molecular mechanisms underlying its overactivation in pathological conditions are still poorly characterized.Here, we investigated whether the enhanced MAO-dependent hydrogen peroxide production can be due to increased substrate availability using a metabolomic profiling method. We identified N¹-methylhistamine -the main catabolite of histamine- as an important substrate fueling MAO in Langendorff mouse hearts, directly perfused with a buffer containing hydrogen peroxide or subjected to ischemia/reperfusion protocol. Indeed, when these hearts were pretreated with the MAO inhibitor pargyline we observed N¹-methylhistamine accumulation along with reduced oxidative stress. Next, we showed that synaptic terminals are the major source of N¹-methylhistamine. Indeed, in vivo sympathectomy caused a decrease of N¹-methylhistamine levels, which was associated with a marked protection in post-ischemic reperfused hearts. As far as the mechanism is concerned, we demonstrate that exogenous histamine is transported into isolated cardiomyocytes and triggers a rise in the levels of reactive oxygen species (ROS). Once again, pargyline pretreatment induced intracellular accumulation of N¹-methylhistamine along with decrease in ROS levels. These findings uncover a receptor-independent mechanism for histamine in cardiomyocytes.In summary, our study reveals a novel and important pathophysiological causative link between MAO activation and histamine availability during pathophysiological conditions such as oxidative stress/cardiac injury.     

Human embryonic stem cell-derived cardiomyocytes as an in vitro model to study cardiac insulin resistance

Patients with type 2 diabetes (T2D) and/or insulin resistance (IR) have an increased risk for the development of heart failure (HF). Evidence indicates that this increased risk is linked to an altered cardiac substrate preference of the insulin resistant heart, which shifts from a balanced utilization of glucose and long-chain fatty acids (FAs) towards an almost complete reliance on FAs as main fuel source. This shift leads to a loss of endosomal proton pump activity and increased cardiac fat accumulation, which eventually triggers cardiac dysfunction. In this review, we describe the advantages and disadvantages of currently used in vitro models to study the underlying mechanism of IR-induced HF and provide insight into a human in vitro model: human embryonic stem cell-derived cardiomyocytes (hESC-CMs). Using functional metabolic assays we demonstrate that, similar to rodent studies, hESC-CMs subjected to 16 h of high palmitate (HP) treatment develop the main features of IR, i.e., decreased insulin-stimulated glucose and FA uptake, as well as loss of endosomal acidification and insulin signaling. Taken together, these data propose that HP-treated hESC-CMs are a promising in vitro model of lipid overload-induced IR for further research into the underlying mechanism of cardiac IR and for identifying new pharmacological agents and therapeutic strategies. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.     

Ryanodine receptor dysfunction in human disorders

Regulation of intracellular calcium (Ca²⁺) is critical in all cell types. The ryanodine receptor (RyR), an intracellular Ca²⁺ release channel located on the sarco/endoplasmic reticulum (SR/ER), releases Ca²⁺ from intracellular stores to activate critical functions including muscle contraction and neurotransmitter release. Dysfunctional RyR-mediated Ca²⁺ handling has been implicated in the pathogenesis of inherited and non-inherited conditions including heart failure, cardiac arrhythmias, skeletal myopathies, diabetes, and neurodegenerative diseases. Here we have reviewed the evidence linking human disorders to RyR dysfunction and describe novel approaches to RyR-targeted therapeutics.