Elevated miR-34a expression and altered transcriptional profile are associated with adverse electromechanical remodeling in the heart of male rats exposed to social stress.

This study investigated epigenetic risk factors that may contribute to stress-related cardiac disease in a rodent model. Experiment 1 was designed to evaluate the expression of microRNA-34a (miR-34a), a known modulator of both stress responses and cardiac pathophysiology, in the heart of male adult rats exposed to a single or repeated episodes of social defeat stress. Moreover, RNA sequencing was conducted to identify transcriptomic profile changes in the heart of repeatedly stressed rats. Experiment 2 was designed to assess cardiac electromechanical changes induced by repeated social defeat stress that may predispose rats to cardiac dysfunction. Results indicated a larger cardiac miR-34a expression after repeated social defeat stress compared to a control condition. This molecular modification was associated with increased vulnerability to pharmacologically induced arrhythmias and signs of systolic left ventricular dysfunction. Gene expression analysis identified clusters of differentially expressed genes in the heart of repeatedly stressed rats that are mainly associated with morphological and functional properties of the mitochondria and may be directly regulated by miR-34a. These results suggest the presence of an association between miR-34a overexpression and signs of adverse electromechanical remodeling in the heart of rats exposed to repeated social defeat stress, and point to compromised mitochondria efficiency as a potential mediator of this link. This rat model may provide a useful tool for investigating the causal relationship between miR-34a expression, mitochondrial (dys)function, and cardiac alterations under stressful conditions, which could have important implications in the context of stress-related cardiac disease

Trimethylamine-N-Oxide (TMAO)-Induced Impairment of Cardiomyocyte Function and the Protective Role of Urolithin B-Glucuronide

One of the most recently proposed candidates as a potential trigger for cardiovascular diseases is trimethylamine-N-oxide (TMAO). Possible direct effects of TMAO on myocardial tissue, independent of vascular damage, have been only partially explored so far. In the present study, we assessed the detrimental direct effects of TMAO on cardiomyocyte contractility and intracellular calcium dynamics, and the ability of urolithin B-glucuronide (Uro B-gluc) in counteracting TMAO-induced cell damage. Cell mechanics and calcium transients were measured, and ultrastructural analysis was performed in ventricular cardiomyocytes isolated from the heart of normal adult rats. Cells were either untreated, exposed to TMAO, or to TMAO and Uro B-gluc. TMAO exposure worsened cardiomyocyte mechanics and intracellular calcium handling, as documented by the decrease in the fraction of shortening (FS) and the maximal rate of shortening and re-lengthening, associated with reduced efficiency in the intracellular calcium removal. Ultrastructurally, TMAO-treated cardiomyocytes also exhibited glycogen accumulation, a higher number of mitochondria and lipofuscin-like pigment deposition, suggesting an altered cellular energetic metabolism and a higher rate of protein oxidative damage, respectively. Uro B-gluc led to a complete recovery of cellular contractility and calcium dynamics, and morphologically to a reduced glycogen accumulation. We demonstrated for the first time a direct negative role of TMAO on cardiomyocyte functional properties and the ability of Uro B-gluc in counteracting these detrimental effects

Long-Term Oral Administration of Theaphenon-E Improves Cardiomyocyte Mechanics and Calcium Dynamics by Affecting Phospholamban Phosphorylation and ATP Production

Background/Aims: Dietary polyphenols from green tea have been shown to possess cardioprotective activities in different experimental models of heart diseases and age-related ventricular dysfunction. The present study was aimed at evaluating whether long term in vivo administration of green tea extracts (GTE), can exert positive effects on the normal heart, with focus on the underlying mechanisms. Methods: The study population consisted of 20 male adult Wistar rats. Ten animals were given 40 mL/day tap water solution of GTE (concentration 0.3%) for 4 weeks (GTE group). The same volume of water was administered to the 10 remaining control rats (CTRL). Then, in vivo and ex vivo measurements of cardiac function were performed in the same animal, at the organ (hemodynamics) and cellular (cardiomyocyte mechanical properties and intracellular calcium dynamics) levels. On cardiomyocytes and myocardial tissue samples collected from the same in vivo studied animals, we evaluated: (1) the intracellular content of ATP, (2) the endogenous mitochondrial respiration, (3) the expression levels of the Sarcoplasmic Reticulum Ca2+-dependent ATPase 2a (SERCA2), the Phospholamban (PLB) and the phosphorylated form of PLB, the L-type Ca2+ channel, the Na+-Ca2+ exchanger, and the ryanodine receptor 2. Results: GTE cardiomyocytes exhibited a hyperdynamic contractility compared with CTRL (the rate of shortening and re-lengthening, the fraction of shortening, the amplitude of calcium transient, and the rate of cytosolic calcium removal were significantly increased). A faster isovolumic relaxation was also observed at the organ level. Consistent with functional data, we measured a significant increase in the intracellular ATP content supported by enhanced endogenous mitochondrial respiration in GTE cardiomyocytes, as well as higher values of the ratios phosphorylated-PLB/PLB and SERCA2/PLB. Conclusions: Long-term in vivo administration of GTE improves cell mechanical properties and intracellular calcium dynamics in normal cardiomyocytes, by increasing energy availability and removing the inhibitory effect of PLB on SERCA2

Effects of standardized green tea extract and its main component, EGCG, on mitochondrial function and contractile performance of healthy rat cardiomyocytes

We recently showed that the long-term in vivo administration of green tea catechin extract (GTE) resulted in hyperdynamic cardiomyocyte contractility. The present study investigates the mechanisms underlying GTE action in comparison to its major component, epigallocatechin-3-gallate (EGCG), given at the equivalent amount that would be in the entirety of GTE. Twenty-six male Wistar rats were given 40 mL/day of a tap water solution with either standardized GTE or pure EGCG for 4 weeks. Cardiomyocytes were then isolated for the study. Cellular bioenergetics was found to be significantly improved in both GTE- and EGCG-fed rats compared to that in controls as shown by measuring the maximal mitochondrial respiration rate and the cellular ATP level. Notably, the improvement of mitochondrial function was associated with increased levels of oxidative phosphorylation complexes, whereas the cellular mitochondrial mass was unchanged. However, only the GTE supplement improved cardiomyocyte mechanics and intracellular calcium dynamics, by lowering the expression of total phospholamban (PLB), which led to an increase of both the phosphorylated-PLB/PLB and the sarco-endoplasmic reticulum calcium ATPase/PLB ratios. Our findings suggest that GTE might be a valuable adjuvant tool for counteracting the occurrence and/or the progression of cardiomyopathies in which mitochondrial dysfunction and alteration of intracellular calcium dynamics constitute early pathogenic factors

HDAC Inhibition Improves the Sarcoendoplasmic Reticulum Ca2+-ATPase Activity in Cardiac Myocytes

SERCA2a is the Ca2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 \ub5M, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency

Titanium dioxide nanoparticles promote arrhythmias via a direct interaction with rat cardiac tissue

BackgroundIn light of recent developments in nanotechnologies, interest is growing to better comprehend the interaction of nanoparticles with body tissues, in particular within the cardiovascular system. Attention has recently focused on the link between environmental pollution and cardiovascular diseases. Nanoparticles <50 nm in size are known to pass the alveolar¿pulmonary barrier, enter into bloodstream and induce inflammation, but the direct pathogenic mechanisms still need to be evaluated. We thus focused our attention on titanium dioxide (TiO2) nanoparticles, the most diffuse nanomaterial in polluted environments and one generally considered inert for the human body.MethodsWe conducted functional studies on isolated adult rat cardiomyocytes exposed acutely in vitro to TiO2 and on healthy rats administered a single dose of 2 mg/Kg TiO2 NPs via the trachea. Transmission electron microscopy was used to verify the actual presence of TiO2 nanoparticles within cardiac tissue, toxicological assays were used to assess lipid peroxidation and DNA tissue damage, and an in silico method was used to model the effect on action potential.ResultsVentricular myocytes exposed in vitro to TiO2 had significantly reduced action potential duration, impairment of sarcomere shortening and decreased stability of resting membrane potential. In vivo, a single intra-tracheal administration of saline solution containing TiO2 nanoparticles increased cardiac conduction velocity and tissue excitability, resulting in an enhanced propensity for inducible arrhythmias. Computational modeling of ventricular action potential indicated that a membrane leakage could account for the nanoparticle-induced effects measured on real cardiomyocytes.ConclusionsAcute exposure to TiO2 nanoparticles acutely alters cardiac excitability and increases the likelihood of arrhythmic events

Simultaneous measurements of electrical coupling and action potential transfer in pairs of ventricular cardiomyocytes

Spread and modulation of electrical activity in cardiac tissue requires intercellular transfer of current via gap junctions, specialised regions of densely packed ionic channels. Electrotonic interaction is determined not merely by intercellular electrical resistance (Rj) but rather by the interplay of Rj and sarcolemmal passive and active electrical properties (Zaniboni et al., Spitzer et al.). In this work we combined a well established protocol to measure Rj in cell pairs (Weingart e Maurer) with a stimulation protocol which allowed to simultaneously study parameters relative to action potential transfer during sequential stimulation. Current clamp experiments, performed on cardiomyocyte pairs held in double-patch configuration, allowed to simultaneously monitor, at a relatively high frequency (1 Hz), membrane resistance (Rm), resting potential (Vm), maximum depolarization rate (dv/dtmax) and time to peak of dv/dtmax in both cells as well as Rj. Spontaneous electrical uncoupling was observed in guinea pig cell pairs with little or no effect on action potential transfer. Pharmacological uncoupling with 40 μM β-glycyrrhetinic acid reached, in one case, a much higher level of Rj and dramatically increased time delay for action potential appearance. When only Rj was measured over a short time interval after approximately two minutes from cell-attachments, values of Rj ∼ 40 MΩ in rat cell pairs (n=20) and Rj ∼ 15 MΩ in guinea pig cell pairs (n=24) were obtained. The possibility of monitoring simultaneously active and intercellular/cellular passive electrical properties makes this protocol particularly suitable to study dynamic changes in Rj during action potential transfer

Effects of chronic psychosocial stress on cardiac autonomic responsiveness and myocardial structure in mice.

Repeated single exposures to social stressors induce robust shifts of cardiac sympathovagal balance toward sympathetic dominance both during and after each agonistic interaction. However, little evidence is available regarding possible persistent pathophysiological changes due to chronic social challenge. In this study, male CD-1 mice (n = 14) were implanted with a radiotelemetry system for electrocardiographic recordings. We assessed the effects of chronic psychosocial stress (15-day sensory contact with a dominant animal and daily 5-min defeat episodes) on 1) sympathovagal responsiveness to each defeat episode, as measured via time-domain indexes of heart rate variability (R-R interval, standard deviation of R-R interval, and root mean square of successive R-R interval differences), 2) circadian rhythmicity of heart rate across the chronic challenge (night phase, day phase, and rhythm amplitude values), and 3) amount of myocardial structural damage (volume fraction, density, and extension of fibrosis). This study indicated that there was habituation of acute cardiac autonomic responsiveness, i.e., the shift of sympathovagal balance toward sympathetic dominance was significantly reduced across repeated defeat episodes. Moreover, animals exhibited significant changes in heart rate rhythmicity, i.e., increments in day and night values and reductions in the rhythm amplitude, but these were limited to the first 5 days of chronic psychosocial stress. The volume fraction of fibrosis was sixfold larger than in control animals, because of the appearance of many microscopic scarrings. In summary, although mice appeared to adapt to chronic psychosocial stress in terms of acute cardiovascular responsiveness and heart rate rhythmicity, structural alterations occurred at the myocardial level