Decline of cardiomyocyte contractile performance and bioenergetic function in socially stressed male rats

Chronic social stress has been epidemiologically linked to increased risk for cardiovascular disease, yet the underlying pathophysiological mechanisms are still largely elusive. Mitochondrial (dys)function represents a potential intersection point between social stress exposure and (mal)adaptive cardiac responses. In this study, we used a rodent model of social stress to study the extent to which alterations in the cellular mechanical properties of the heart were associated with changes in indexes of mitochondrial function. Male adult rats were exposed to repeated episodes of social defeat stress or left undisturbed (controls). ECG signals were recorded during and after social defeat stress. Twenty-four hours after the last social defeat, cardiomyocytes were isolated for analyses of mechanical properties and intracellular Ca(2+) dynamics, mitochondrial respiration, and ATP content. Results indicated that social defeat stress induced potent cardiac sympathetic activation that lasted well beyond stress exposure. Moreover, cardiomyocytes of stressed rats showed poor contractile performance (e.g., slower contraction and relaxation rates) and intracellular Ca(2+) derangement (e.g., slower Ca(2+) clearing), which were associated with indexes of reduced reserve respiratory capacity and decreased ATP production. In conclusion, this study suggests that repeated social stress provokes impaired cardiomyocyte contractile performance and signs of altered mitochondrial bioenergetics in the rat heart. Future studies are needed to clarify the causal link between cardiac and mitochondrial functional remodeling under conditions of chronic social stress

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

Phase-contrast microtomography: are the tracers necessary for stem cell tracking in infarcted hearts?

Recent literature has identified innovative approaches of cellular therapy to generate new myocardium involving transcoronary and intramyocardial injection of cardiac progenitor cells (CPCs). One of the limiting factors in the overall interpretation of these preclinical results is the lack of reliable methods for 3D imaging and quantification of the injected cells and for the assessment of their fate within the myocardium. Here, for the first time to the authors' knowledge, we support by demonstrative experiments the hypothesis that phase-contrast microtomography (PhC-microCT) could offer an efficient 3D imaging approach to track the injected cells within the myocardium, without the need of any cell tracer. This deduction has been validated by several observations: i) a strong phase-contrast signal was observed in infarcted hearts injected with unlabeled cells; ii) the PhC-microCT 3D reconstructions of hearts injected with only vehicle saline solution and rhodamine particles, i.e. without CPCs, did not show any contrast; (iii) in the 3D PhC-microCT reconstructions of non infarcted hearts, injected with unlabeled CPCs, the contrast signal of the cells was present but differently distributed; and iv) the contrast signal of injected cells diminished over time apparently following the same timing of cell engraftment and differentiation, as confirmed in literature by histology and fluorescence analysis. The chance to avoid cell tracers is of paramount interest in determining the fate of transplanted stem cells because the quantification of the signal will not be any more dependent on injected dose, concentration of the tracer, cell proliferation and tracer uptake kinetics

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

Cardiac electromechanical performance following stem cell based regenerative therapies in infarcted rat heart

Scopo: Gli effetti elettrofisiologici conseguenti all’utilizzo di terapie rigenerative basate su cellule staminali in campo cardiologico non sono ancora stati sufficientemente chiariti. Abbiamo voluto affrontare questo problema utilizzando un modello di ratto con infarto miocardico cronico sottoposto a diversi approcci rigenerativi: (i) iniezioni intramiocardiche di Fattore di crescita Insulino-simile (Insulin-like Growth Factor-1: IGF-1) e Fattore di crescita Epatico (Hepatocyte Growth Factor: HGF) al fine di attivare il pool di cellule progenitrici residenti; (ii) iniezioni locali di cellule progenitrici cardiache (CPCs), EGFP-positive (Enhanced-Green Fluorescent Protein) e marcate con QDots. Queste ultime sono state addizionate o meno con HGF+IGF-1 (GFs). Metodi: Ratti maschi Wistar con infarto miocardico cronico di 4 settimane sono stati sottoposti ad iniezioni di: (i) HGF+IGF-1 (gruppo MI-GF), (ii) CPCs (gruppo MI-cells) e (iii) CPCs addizionate con fattori di crescita (gruppo MI-cells+GF) e contemporaneamente strumentati con una pompa osmotica sottocutanea per il rilascio continuo di BrdC per la determinazione dei processi rigenerativi. Prima e due settimane dopo il trattamento si determinava la funzionalità elettrica e meccanica del cuore. In particolare, venivano eseguite delle registrazioni elettrocardiografiche telemetriche durante attivazione neurovegetativa (stress sociale), ottenuta sottoponendo l’animale ad interazione con un conspecifico aggressivo, per la valutazione degli eventi aritmici ventricolari (EAV). Sui tracciati elettrocardiografici sono stati determinati anche l’intervallo R-R e gli indici indiretti dell’input neurovegetativo al cuore SDRR e rMSSD. Prima del sacrificio, i ratti sono stati sottoposti ad esame ecocardiografico e/o a misure emodinamiche invasive. Infine, il cuore è stato arrestato in diastole, perfuso con formalina ed utilizzato per uno studio morfometrico ed immunoistochimico. Risultati: L’infarto miocardico ha determinato un peggioramento della funzionalità meccanica associato ad una aumentata incidenza e complessità degli EAV da stress rispetto agli animali sham-operated. Mentre l’iniezione di HGF+IGF-1 ha determinato una riduzione degli EAV da stress di circa due volte nel 68% degli animali del gruppo MI-GF, il trattamento con CPCs+GFs, ha portato ad una riduzione di circa tre volte nella totalità degli animali del gruppo MI-cells+GF. Il trattamento con CPCs si è dimostrato completamente inefficace da punto di vista antiaritmico. La frequenza cardiaca e gli indici SDRR e rMSSD non hanno mostrato differenze rilevanti tra i vari gruppi sperimentali. Il gruppo MI-GF ha mostrato un parziale recupero della funzionalità meccanica associato ad un rimodellamento ventricolare positivo. Un completo recupero della funzionalità meccanica ed una netta riduzione delle alterazioni strutturali del ventricolo sinistro conseguenti all’infarto si ottenevano invece mediante trattamento con CPCs o con CPCs+GFs. L’analisi immunoistochimica ha mostrato la formazione nell’area peri-infartuale e nell’area infartuata, sia di miociti elettricamente e meccanicamente connessi con il miocardio risparmiato dall’infarto sia di nuove strutture vascolari, per tutti i trattamenti analizzati. Tuttavia, l’analisi quantitativa ha mostrato che i processi rigenerativi erano più pronunciati nel gruppo MI-cells+GF. Conclusioni: I nostri risultati evidenziano che, tra le varie terapie rigenerative utilizzate in questo studio, l’iniezione di CPCs+GF oltre a costituire l’approccio più efficiente per il recupero delle proprietà elettrofisiologiche del cuore infartuato, è in grado di determinare anche più marcato recupero della funzionalità meccanica cardiaca. Questo suggerisce come il cambiamento del microambiente sia in grado di favorire l’engraftment e potenziare la capacità rigenerativa propria delle CPCs in termini sia di migrazione che differenziazione ed integrazione nel tessuto ospite.Aim: The electrophysiological effects of stem cells based therapies in heart repair have not been systematically analyzed. We addressed this issue in a rat model of chronic myocardial infarction (MI). Regenerative treatments consisted of: (i) intra-myocardial injection of Insulin-like Growth Factor-1 (IGF-1) and Hepatocyte Growth Factor (HGF) in order to activate resident cardiac progenitor cells; (ii) local injection of Enhanced-Green Fluorescent Protein (EGFP)-positive cardiac progenitors cells (CPCs) labeled with Quantum Dots. CPCs were used either alone or associated with HGF and IGF-1. Methods: Male Wistar rats with 4-week old MI were studied. Animals were subjected to injections of: (i) HGF+IGF1 (MI-GF group), (ii) CPCs (MI-cells group) and (iii) CPCs supplemented with HGF+IGF-1 (MI-cells+GF). To detect regenerative processes, a continuous infusion of BrdC via an osmotic pump was also started. Ventricular arrhythmias (VAEs) occurring during stress-induced autonomic stimulation (social stress: resident-intruder test) were telemetrically recorded in conscious and freely moving animals, prior and two weeks after treatment. On the ECG tracings, the mean heart rate and heart rate based indices of the autonomic input to the heart (SDRR: standard deviation of the mean R-R interval: rMSSD: square root of the mean squared differences of successive R-R intervals) were also determined. Before sacrifice, invasive echocardiographic and/or hemodynamic measurements were performed. The heart was then perfusion-fixed for morphometric and immunohistochemical studies. Results: Myocardial infarction worsened cardiac mechanical performance and increased the proneness to stress-induced VAEs as compared to sham operated rats. Cytokine injection significantly reduced VAEs by two-fold in 68% of MI-GF rats while the injection of CPCs supplemented with growth factors (GFs) resulted in a higher reduction of VAEs (three-fold), involving 100% of MI-cells+GF rats. Conversely, the treatment with CPCs alone was ineffective. No definite differences among groups were found in heart rate, SDRR and rMSSD values. In MI-GF group a partial recovery of cardiac hemodynamic parameters in association with a positive remodeling of the left ventricle was observed. Treatment with CPCs or CPCs+GFs determined a complete recovery of myocardial mechanical function and further reduced the alterations in left ventricular structure. Formation of electro-mechanically competent myocytes and vascular structures occurred in the infarcted and peri-infarcted areas in all rats submitted to regenerative approaches, as revealed by immunohistochemical studies. However, regenerative processes were more pronounced in MI-cells+GF group. Conclusions: Our results indicate that, among the different regenerative therapies employed in the study, the injection of CPCs+GFs constitutes the most efficient approach for the recovery of electrophysiological properties of the infarcted heart. This treatment also determined a significant improvement of mechanical function suggesting that cytokines by changing the unfavorable microenvironment, favored the engraftment and differentiation of the injected CPCs promoting their natural role in heart repair