Adult Rat Ventricular Cardiomyocytes Research Articles

Role of Orai1 and Ca2+-stimulated adenylyl cyclase 8 in the adverse cardiac remodeling after myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agencia Estatal de Investigación. Background It is well established that abnormalities in the regulation of the intracellular concentration of Ca2+ ([Ca2+]i) occurs in diseased heart such as under acute myocardial infarction (AMI). Recent studies have demonstrated that proteins related to the Store Operated Calcium Entry (SOCE), such as Orai1/2/3 and TRPC channels, play a role in the adverse cardiac remodelling. Previously, we demonstrated that the expression of Orai1 increased in heart subjected to ischemia and reperfusion (I/R) syndrome. However, the signalling pathway implicated in Orai1 regulation has not been addressed. Objective The aim of this study was to examine the molecular mechanism and signalling pathways involved in the of Orai1 upregulation after I/R in cardiomyocytes. Materials and Methods The experiments were performed in adult (ARVM) and neonatal rat ventricular cardiomyocytes (NRVM), in a rat model of myocardial I/R and in ventricular biopsies of patients with ischemic heart failure. Ca2+ studies were realized using microfluorimetric technique in FURA-2AM loaded cardiomyocytes. Immunofluorescence was used to investigate CREB activation using a specific phospho-CREB antibody and Orai1 and AC8 protein expression were analysed by Western Blot. This study was performed in accordance with the recommendations of the Royal Decree 53/2013 in agreement to the Directive 2010/63/EU of the European Parliament and approved by the local Ethics Committee on human Research and the Animal Research Committee. Results We found that under I/R diastolic level of [Ca2+]i was significantly increased in cardiac myocytes isolated from I/R hearts, which was inhibited by SOCE blockers. Moreover, we found that Orai1 and Ca2+-sensitive adenylyl cyclase 8 (AC8) are overexpressed in the heart isolated from I/R rat model, as compared to sham. Similarly, we found significant increase in the expression of Orai1 and AC8 in ventricle biopsies of post-infarction patients with HF. We also observed that I/R stimulated cAMP production, involving Orai1 and AC8 activation in rats, indicating that Ca2+ entry through Orai1 stimulated AC8. Moreover, I/R phosphorylated cAMP response element-binding protein (CREB) through PKA activation, but not via EPAC2 or ERK1/2. Interestingly, we found that CREB facilitated I/R-induced Orai1 upregulation and related exacerbated SOCE by its activation of Orai1 promoter under I/R. Finally, the intramyocardial administration of AAV9 holding AC8 shRNA decreased the expression of AC8, Orai1 and CREB, which restored diastolic [Ca2+]i. Conclusions Our results show that the activation of Orai1/AC8/CREB axis plays a key role in the I/R-induced Orai1 upregulation, which participated in diastolic [Ca2+]i mishandling related to the adverse cardiac remodelling.

Activation of the non-neuronal 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 non-neuronal cholinergic cardiac system (NNCCS) in cardiomyocytes has been described as an additional source of ACh. To investigate whether the NNCCS mediates cardioprotection in absence of vagal and ICNS activation. For that, we used a reductionist approach of isolated adult rat ventricular cardiomyocytes in absence of neuronal cells with hypoxic preconditioning (HPC) as protective stimulus. Adult rat ventricular cardiomyocytes were isolated, absence of neuronal cells was confirmed, 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. 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/nicotinic ACh receptor (mAChR/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). 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%). Activation of the NNCCS is involved in cardiomyocyte protection: HPC increases intra- and extracellular ACh during H/R, and m-/nAChRs are causally involved in HPC´s cardiomyocyte protection during H/R. The interplay between upstream ICNS activation and the NNCCS activation to myocardial cholinergic metabolism and cardioprotection needs to be investigated in future studies.

Store-Operated Calcium Entry Increases Nuclear Calcium in Adult Rat Atrial and Ventricular Cardiomyocytes.

Store-operated calcium entry (SOCE) in cardiomyocytes may be involved in cardiac remodeling, but the underlying mechanisms remain elusive. We hypothesized that SOCE may increase nuclear calcium, which alters gene expression via calcium/calmodulin-dependent enzyme signaling, and elucidated the underlying cellular mechanisms. An experimental protocol was established in isolated adult rat cardiomyocytes to elicit SOCE by re-addition of calcium following complete depletion of sarcoplasmic reticulum (SR) calcium and to quantify SOCE in relation to the electrically stimulated calcium transient (CaT) measured in the same cell before SR depletion. Using confocal imaging, calcium changes were recorded simultaneously in the cytosol and in the nucleus of the cell. In ventricular myocytes, SOCE was observed in the cytosol and nucleus amounting to ≈15% and ≈25% of the respective CaT. There was a linear correlation between the SOCE-mediated calcium increase in the cytosol and nucleus. Inhibitors of TRPC or Orai channels reduced SOCE by ≈33-67%, whereas detubulation did not. In atrial myocytes, SOCE with similar characteristics was observed in the cytosol and nucleus. However, the SOCE amplitudes in atrial myocytes were ≈two-fold larger than in ventricular myocytes, and this was associated with ≈1.4- to 3.6-fold larger expression of putative SOCE proteins (TRPC1, 3, 6, and STIM1) in atrial tissue. The results indicated that SOCE in atrial and ventricular myocytes is able to cause robust calcium increases in the nucleus and that both TRPC and Orai channels may contribute to SOCE in adult cardiomyocytes.

Abstract 17093: The 3D T-Tubule Network Significantly Remodels in Cardiomyocytes Subjected to Mechanical Load

Introduction: T-tubules (TT) are critical for excitation-contraction coupling in cardiomyocytes (CMs). Past work studying heart failure has spatially localized TT loss with inhibited Ca2+ release in the 2D space. This limited view does not consider tubules in other z-planes, necessitating a 3D understanding of the network. Hypothesis: We hypothesize that the ultrastructure of the 3D TT network significantly remodels in response to increase in culture substrate stiffness. Methods: Adult rat ventricular CMs were cultured ex vivo on substrate with either physiological stiffness (10 kPa) or pathological stiffness (50 kPa) for 2 days. Live CMs were stained for TTs (Di-8-ANEPPS) and Ca2+ (Fluo-4). 3D renderings of the TT network were reconstructed from z-stacks of 2D confocal images in the Arivis software. Within a set 3D region of interest, orientation (longitudinal, transverse, axial), volume, and surface area of tubules were measured. Ca2+ transients were taken from a line scan. Data from 18 CMs cultured at 10 kPa and 15 CMs cultured at 50 kPa were acquired. Results: CMs cultured on 50 kPa surfaces exhibited a decrease in the number of transverse elements (147 to 112) and an increase for longitudinal elements (3 to 16) compared to control CMs cultured on 10 kPa surfaces. For all 3 orientations, the volume of the average tubule increased by at least 20% following culture on 50 kPa surfaces. The total surface area of all axial and longitudinal tubules increased by at least 130% and decreased by 13% for all transverse tubules following culture on 50 kPa surfaces. Conclusions: Two days of culture on stiffened surfaces, designed to mimic the stiffness of diseased hearts, produces significant TT remodeling. Our work suggests that the remodeled TT network has less spatial capacity to release Ca2+ and worsened access to actin-myosin sites. In the future, we will associate the distance between each pixel on the line scan to the nearest TT with Ca2+ release parameters. This model system will permit further elucidation of how particular aspects of TT remodeling affect Ca2+ release and will facilitate testing of therapeutic interventions designed to prevent load-dependent defects in excitation-contraction coupling.

Monoamine Oxidase A Contributes to Serotonin-But Not Norepinephrine-Dependent Damage of Rat Ventricular Myocytes.

Serotonin effects on cardiac hypertrophy, senescence, and failure are dependent either on activation of specific receptors or serotonin uptake and serotonin degradation by monoamine oxidases (MAOs). Receptor-dependent effects are specific for serotonin, but MAO-dependent effects are nonspecific as MAOs also metabolize other substrates such as catecholamines. Our study evaluates the role of MAO-A in serotonin- and norepinephrine-dependent cell damage. Experiments were performed in vivo to study the regulation of MAOA and MAOB expression and in vitro on isolated cultured adult rat ventricular cardiomyocytes (cultured for 24 h) to study the function of MAO-A. MAOA but not MAOB expression increased in maladaptive hypertrophic stages. Serotonin and norepinephrine induced morphologic cell damage (loss of rod-shaped cell structure). However, MAO-A inhibition suppressed serotonin-dependent but not norepinephrine-dependent damages. Serotonin but not norepinephrine caused a reduction in cell shortening in nondamaged cells. Serotonin induced mitochondria-dependent oxidative stress. In vivo, MAOA was induced during aging and hypertension but the expression of the corresponding serotonin uptake receptor (SLC6A4) was reduced and enzymes that reduce either oxidative stress (CAT) or accumulation of 5-hydroxyindolacetaldehyde (ALDH2) were induced. In summary, the data show that MAO-A potentially affects cardiomyocytes' function but that serotonin is not necessarily the native substrate.

CRISPR/Cas9 Knock-Out in Primary Neonatal and Adult Cardiomyocytes Reveals Distinct cAMP Dynamics Regulation by Various PDE2A and PDE3A Isoforms.

Cyclic nucleotide phosphodiesterases 2A (PDE2A) and PDE3A play an important role in the regulation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)-to-cAMP crosstalk. Each of these PDEs has up to three distinct isoforms. However, their specific contributions to cAMP dynamics are difficult to explore because it has been challenging to generate isoform-specific knock-out mice or cells using conventional methods. Here, we studied whether the CRISPR/Cas9 approach for precise genome editing can be used to knock out Pde2a and Pde3a genes and their distinct isoforms using adenoviral gene transfer in neonatal and adult rat cardiomyocytes. Cas9 and several specific gRNA constructs were cloned and introduced into adenoviral vectors. Primary adult and neonatal rat ventricular cardiomyocytes were transduced with different amounts of Cas9 adenovirus in combination with PDE2A or PDE3A gRNA constructs and cultured for up to 6 (adult) or 14 (neonatal) days to analyze PDE expression and live cell cAMP dynamics. A decline in mRNA expression for PDE2A (~80%) and PDE3A (~45%) was detected as soon as 3 days post transduction, with both PDEs being reduced at the protein level by >50-60% in neonatal cardiomyocytes (after 14 days) and >95% in adult cardiomyocytes (after 6 days). This correlated with the abrogated effects of selective PDE inhibitors in the live cell imaging experiments based on using cAMP biosensor measurements. Reverse transcription PCR analysis revealed that only the PDE2A2 isoform was expressed in neonatal myocytes, while adult cardiomyocytes expressed all three PDE2A isoforms (A1, A2, and A3) which contributed to the regulation of cAMP dynamics as detected by live cell imaging. In conclusion, CRISPR/Cas9 is an effective tool for the in vitro knock-out of PDEs and their specific isoforms in primary somatic cells. This novel approach suggests distinct regulation of live cell cAMP dynamics by various PDE2A and PDE3A isoforms in neonatal vs. adult cardiomyocytes.

Preclinical characterization of CK-4021586, a new class of cardiac myosin inhibitors for the treatment of hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common monogenetic heart disease, with an estimated carrier prevalence of ∼1 in 500. Hypercontractility of the cardiac sarcomere is a common driver of the pathological hypertrophic cardiac remodeling that is a hallmark of the disease. Clinical trials of the selective cardiac myosin inhibitors mavacamten and aficamten have demonstrated that inhibition of the biochemical activity of cardiac myosin can induce cardiac remodeling and improve both patient symptoms and exercise capacity in HCM with left ventricular outflow tract obstruction (oHCM). The diverse genetic underpinnings of HCM suggest that it is possible that additional classes of cardiac myosin inhibitors with distinct biochemical mechanisms-of-action may also have a salutary impact on disease expression. While both mavacamten and aficamten bind to the single-headed motor domain of myosin (subfragment-1) and inhibit its ATPase activity, CK-4021586 (CK-586) is a new class of cardiac myosin inhibitor that inhibits the ATPase activity of two-headed heavy meromyosin (HMM) but not single-headed subfragment-1. CK-586 is a partial inhibitor of cardiac myofibrillar ATPase activity (EC50 2.9 µM, maximal inhibition ∼50%) that requires the regulatory light chain. Notably, fractional shortening of electrically-paced adult rat ventricular cardiomyocytes was inhibited almost completely (>80% at 5 µM) without alterations in the calcium (Ca2+) transient. In normal Sprague Dawley rats, CK-586 reduced cardiac fractional shortening in a dose and concentration-dependent manner. In conclusion, CK-586 is a novel, small molecule, cardiac myosin inhibitor that reduces cardiac contractility in vitro and in vivo. CK-586 has a biochemical mechanism of action distinct from both mavacamten and aficamten, providing an additional tool to decrease the number of functionally-available myosin heads and treat the cardiac hypercontractility that forms the pathologic basis of HCM.

Uric Acid Deteriorates Load-Free Cell Shortening of Cultured Adult Rat Ventricular Cardiomyocytes via Stimulation of Arginine Turnover.

Hyperuricemia is a risk factor for heart disease. Cardiomyocytes produce uric acid via xanthine oxidase. The enzymatic reaction leads to oxidative stress in uric-acid-producing cells. However, extracellular uric acid is the largest scavenger of reactive oxygen species, specifically to nitrosative stress, which can directly affect cells. Here, the effect of plasma-relevant concentrations of uric acid on adult rat ventricular cardiomyocytes is analyzed. A concentration- and time-dependent reduction of load-free cell shortening is found. This is accompanied by an increased protein expression of ornithine decarboxylase, the rate-limiting enzyme of the polyamine metabolism, suggesting a higher arginine turnover. Subsequently, the effect of uric acid was attenuated if other arginine consumers, such as nitric oxide synthase, are blocked or arginine is added. In the presence of uric acid, calcium transients are increased in cardiomyocytes irrespective of the reduced cell shortening, indicating calcium desensitization. Supplementation of extracellular calcium or stimulation of intracellular calcium release by β-adrenergic receptor stimulation attenuates the uric-acid-dependent effect. The effects of uric acid are attenuated in the presence of a protein kinase C inhibitor, suggesting that the PKC-dependent phosphorylation of troponin triggers the desensitizing effect. In conclusion, high levels of uric acid stress cardiomyocytes by accelerating the arginine metabolism via the upregulation of ornithine decarboxylase.

Age-dependent effect of insulin in the regulation of intracellular calcium in ventricular cardiomyocytes.

In this study, we wanted to verify whether the effect of insulin on calcium homeostasis depends on the heart's development stage. Using a quantitative 3D confocal microscopy, we tested the effect of a high insulin concentration (100 µU) in freshly cultured ventricular cardiomyocytes from newborn and adult rats. Our results showed that the cytosolic basal level of calcium was higher in newborn cardiomyocytes with no change in the nuclear basal calcium level compared with the adult cardiomyocytes; in addition, insulin induced a slow increase of cytosolic and nuclear calcium in newborn ventricular cardiomyocytes, followed by two phases. However, the first phase of slow cytosolic and nuclear calcium increase was absent in adult rat ventricular cardiomyocytes. Furthermore, the time to the onset of increase of cytosolic and nuclear calcium was longer in newborn cardiomyocytes compared with adults. Moreover, the time to peak of the calcium transient was shorter in newborns than in adult cardiomyocytes. These results demonstrate that insulin differently regulates calcium homeostasis in newborns than in adult cardiomyocytes. Thus, newborn rat cardiomyocytes, commonly used in research as a model for adult cardiomyocytes, should be used with caution when dealing with insulin in normal and disease conditions.

Novel specific cardiac homing peptides to target failing cardiomyocytes

Heart failure (HF) is one of the most prevalent cardiovascular diseases worldwide with high mortality. Although β-blockers are now the cornerstone of HF treatment, they are ineffective in patients’ subpopulations and may cause several side effects. Thus, the development of novel drug delivery systems directly targeting the myocardium in a tissue-specific manner is a promising approach. Two ligands have been characterized to transduce cardiomyocytes (CMs) in vitro as well as cardiac tissue in vivo: the cardiac targeting peptide (CTP) and the primary cardio myocyte (PCM) peptide. However, very little is known about their uptake mechanisms, targeting efficacies, and intracellular distribution. Characterization and optimization of the CTP and PCM peptides through a structure-activity relationship study. Over 100 variants of CTP and PCM peptides have been engineered and screened. The targeting capacity of peptides was evaluated using flow cytometry on neonatal rat ventricular CMs (NRVC) cultured with 6-carboxyfluorescein conjugated peptides. Other cells lines, including human lung adenocarcinoma (A549) and human liver cancer (HepG2) cells, were used to assess the cardiospecificity of the new peptides. Peptide uptake was also monitored by confocal microscopy imaging in NRVC and adult rat ventricular CMs (ARVC), as well as in the ex vivo model. We have generated specific cardiac homing peptides-1 and 2 (i.e., SCHoP-1 and SCHoP-2) with improved properties and increased ability to target and transduce CMs specifically, compared to the parent CTP and PCM peptides. Flow cytometry assays support that SCHoP-1 and SCHoP-2 significantly increase cardiac targeting in living NRVC by 4-fold over the CTP and PCM peptides. No cellular toxicity was observed. SCHoP-1 and SCHoP-2 target preferentially CMs, over A549 and HepG2 cells. We confirmed that they internalize in NRVC and in ARVC localized in the cytosol. CMs internalization is also increased in ex vivo hearts. SCHoP-1 and SCHoP-2 open the way to explore their potential as a targeting moiety to address a therapeutic agent specifically to failing CMs for the treatment of HF.

Cholinergic action of spleen‐derived cardioprotective factors

IntroductionThere is an ambivalent relationship between the spleen and the heart in myocardial infarction (Circ Res 124; 2019; 26‐8). Sympathetic activation of the spleen initially increases myocardial inflammatory infiltration but resolves it later by initiating a myocardial healing response. Vagal activation of the spleen induces the release of cardioprotective factors: Perfusion of an isolated rat spleen with the vagomimetic carbachol resulted in the release of factors which then reduced infarct size (IS) in an isolated perfused rat heart.AimTo characterize cardioprotection of spleen‐derived factors, with a focus on their cholinergic action.MethodsRat spleens and hearts were isolated and buffer‐perfused at constant pressure. Isolated spleens were either challenged by infusion of saline (as control) or the vagomimetic carbachol (500 pmol/L), and splenic effluate (SEff; 100 mL) was sampled after saline infusion (SEffsaline) or carbachol infusion (SEffcarbachol), respectively. Cell‐free SEff was prepared by centrifugation and filtration. SEffsaline or SEffcarbachol was infused into isolated hearts which were then subjected to 30 min global ischemia and 120 min reperfusion (I/R). In subsets of experiments, the hearts were pretreated with the nicotinic receptor antagonist hexamethonium (50 µmol/L) or the muscarinic receptor antagonist atropine (100 pmol/L). IS was demarcated by triphenyltetrazolium chloride staining and calculated as percent of ventricular mass. To study the relevance of spleen‐derived protection on the cardiomyocyte level, adult rat ventricular cardiomyocytes were prepared. Cells were incubated for 30 minutes with SEffsaline or SEffcarbachol, respectively, and then subjected to 30 min hypoxia/ 5 min reoxygenation (H/R) or exposed to SEffsaline or SEffcarbachol for 35 minutes as time control (TC). Since nicotinic and muscarinic receptors are also expressed on cardiomyocytes, all experiments were repeated after pretreatment with hexamethonium (1 µmol/L) or atropine (100 nmol/L). Cardiomyocyte viability was quantified before (baseline) and after H/R or TC, as the percent fraction of rod‐shaped, unstained (5% trypan blue) cells over all cells. The estimated final concentration of carbachol in the SEffcarbachol of ≤10 pmol/L and the SEffsaline did neither reduce IS in isolated perfused rat hearts nor improve cardiomyocyte viability in isolated rat cardiomyocytes.ResultsWith SEffsaline, IS was 34±7%. With SEffcarbachol, IS was reduced to 19±8%. Hexamethonium or atropine had no impact on IS per se in hearts with SEffsaline (33±8%; 34±6%), but attenuated the cardioprotection by SEffcarbachol (27±10%; 26±10%; Figure A). The viability of cardiomyocytes subjected to H/R was better preserved with SEffcarbachol than with SEffsaline or saline alone. Again, this protection was attenuated by hexamethonium or atropine (Figure B, insert).ConclusionThe cardioprotection by spleen‐derived factors, which were released in response to the vagomimetic carbachol, is mediated through nicotinic and muscarinic receptors within the heart. Apparently, this protection does not need to involve neuronal structures within the myocardium – nicotinic and muscarinic receptors on cardiomyocytes per se mediate protection.

Inhibition of the NLRP3/IL-1β axis protects against sepsis-induced cardiomyopathy.

BackgroundSeptic cardiomyopathy worsens the prognosis of critically ill patients. Clinical data suggest that interleukin‐1β (IL‐1β), activated by the NLRP3 inflammasome, compromises cardiac function. Whether or not deleting Nlrp3 would prevent cardiac atrophy and improve diastolic cardiac function in sepsis was unclear. Here, we investigated the role of NLRP3/IL‐1β in sepsis‐induced cardiomyopathy and cardiac atrophy.Methods Male Nlrp3 knockout (KO) and wild‐type (WT) mice were exposed to polymicrobial sepsis by caecal ligation and puncture (CLP) surgery (KO, n = 27; WT, n = 33) to induce septic cardiomyopathy. Sham‐treated mice served as controls (KO, n = 11; WT, n = 16). Heart weights and morphology, echocardiography and analyses of gene and protein expression were used to evaluate septic cardiomyopathy and cardiac atrophy. IL‐1β effects on primary and immortalized cardiomyocytes were investigated by morphological and molecular analyses. IonOptix and real‐time deformability cytometry (RT‐DC) analysis were used to investigate functional and mechanical effects of IL‐1β on cardiomyocytes.ResultsHeart morphology and echocardiography revealed preserved systolic (stroke volume: WT sham vs. WT CLP: 33.1 ± 7.2 μL vs. 24.6 ± 8.7 μL, P < 0.05; KO sham vs. KO CLP: 28.3 ± 8.1 μL vs. 29.9 ± 9.9 μL, n.s.; P < 0.05 vs. WT CLP) and diastolic (peak E wave velocity: WT sham vs. WT CLP: 750 ± 132 vs. 522 ± 200 mm/s, P < 0.001; KO sham vs. KO CLP: 709 ± 152 vs. 639 ± 165 mm/s, n.s.; P < 0.05 vs. WT CLP) cardiac function and attenuated cardiac (heart weight–tibia length ratio: WT CLP vs. WT sham: −26.6%, P < 0.05; KO CLP vs. KO sham: −3.3%, n.s.; P < 0.05 vs. WT CLP) and cardiomyocyte atrophy in KO mice during sepsis. IonOptix measurements showed that IL‐1β decreased contractility (cell shortening: IL‐1β: −15.4 ± 2.3%, P < 0.001 vs. vehicle, IL‐1RA: −6.1 ± 3.3%, P < 0.05 vs. IL‐1β) and relaxation of adult rat ventricular cardiomyocytes (time‐to‐50% relengthening: IL‐1β: 2071 ± 225 ms, P < 0.001 vs. vehicle, IL‐1RA: 564 ± 247 ms, P < 0.001 vs. IL‐1β), which was attenuated by an IL‐1 receptor antagonist (IL‐1RA). RT‐DC analysis indicated that IL‐1β reduced cardiomyocyte size (P < 0.001) and deformation (P < 0.05). RNA sequencing showed that genes involved in NF‐κB signalling, autophagy and lysosomal protein degradation were enriched in hearts of septic WT but not in septic KO mice. Western blotting and qPCR disclosed that IL‐1β activated NF‐κB and its target genes, caused atrophy and decreased myosin protein in myocytes, which was accompanied by an increased autophagy gene expression. These effects were attenuated by IL‐1RA.ConclusionsIL‐1β causes atrophy, impairs contractility and relaxation and decreases deformation of cardiomyocytes. Because NLRP3/IL‐1β pathway inhibition attenuates cardiac atrophy and cardiomyopathy in sepsis, it could be useful to prevent septic cardiomyopathy.

Application of plasma polymerized pyrrole nanoparticles to prevent or reduce de-differentiation of adult rat ventricular cardiomyocytes

Cardiovascular diseases are the leading cause of death in the world, cell therapies have been shown to recover cardiac function in animal models. Biomaterials used as scaffolds can solve some of the problems that cell therapies currently have, plasma polymerized pyrrole (PPPy) is a biomaterial that has been shown to promote cell adhesion and survival. The present research aimed to study PPPy nanoparticles (PPPyN) interaction with adult rat ventricular cardiomyocytes (ARVC), to explore whether PPPyN could be employed as a nanoscaffold and develop cardiac microtissues. PPPyN with a mean diameter of 330 nm were obtained, the infrared spectrum showed that some pyrrole rings are fragmented and that some fragments of the ring can be dehydrogenated during plasma synthesis, it also showed the presence of amino groups in the structure of PPPyN. PPPyN had a significant impact on the ARVC´s shape, delaying dedifferentiation, necrosis, and apoptosis processes, moreover, the cardiomyocytes formed cell aggregates up to 1.12 mm2 with some aligned cardiomyocytes and generated fibers on its surface similar to cardiac extracellular matrix. PPPyN served as a scaffold for adult ARVC. Our results indicate that PPPyN-scaffold is a biomaterial that could have potential application in cardiac cell therapy (CCT).

Divergent estimates of the ratio between Na+-Ca2+ current densities in t-tubular and surface membranes of rat ventricular cardiomyocytes.

The ratio between Na+-Ca2+ exchange current densities in t-tubular and surface membranes of rat ventricular cardiomyocytes (JNaCa-ratio) estimated from electrophysiological data published to date yields strikingly different values between 1.7 and nearly 40. Possible reasons for such divergence were analysed by Monte Carlo simulations assuming both normal and log-normal distribution of the measured data. The confidence intervals CI95 of the mean JNaCa-ratios computed from the reported data showed an overlap of values between 1 and 3, and between 0.3 and 4.3 in the case of normal and log-normal distribution, respectively. Further analyses revealed that the published high values likely result from a large scatter of data due to transmural differences in JNaCa, dispersion of cell membrane capacitances and variability in incomplete detubulation. Taking into account the asymmetric distribution of the measured data, the reduction of mean current densities after detubulation and the substantially smaller CI95 of lower values of the mean JNaCa-ratio, the values between 1.6 and 3.2 may be considered as the most accurate estimates. This implies that 40 to 60% of Na+-Ca2+ exchanger is located at the t-tubular membrane of adult rat ventricular cardiomyocytes.

Empagliflozin restores chronic kidney disease–induced impairment of endothelial regulation of cardiomyocyte relaxation and contraction

Chronic kidney disease (CKD) promotes development of cardiac abnormalities and is highly prevalent in patients with heart failure, particularly in those with preserved ejection fraction. CKD is associated with endothelial dysfunction, however, whether CKD can induce impairment of endothelium-to-cardiomyocyte crosstalk leading to impairment of cardiomyocyte function is not known. The sodium-glucose co-transporter 2 inhibitor, empagliflozin, reduced cardiovascular events in diabetic patients with or without CKD, suggesting its potential as a new treatment for heart failure with preserved ejection fraction. We hypothesized that uremic serum from patients with CKD would impair endothelial control of cardiomyocyte relaxation and contraction, and that empagliflozin would protect against this effect. Using a co-culture system of human cardiac microvascular endothelial cells with adult rat ventricular cardiomyocytes to measure cardiomyocyte relaxation and contraction, we showed that serum from patients with CKD impaired endothelial enhancement of cardiomyocyte function which was rescued by empagliflozin. Exposure to uremic serum reduced human cardiac microvascular endothelial cell nitric oxide bioavailability, and increased mitochondrial reactive oxygen species and 3-nitrotyrosine levels, indicating nitric oxide scavenging by reactive oxygen species. Empagliflozin attenuated uremic serum-induced generation of endothelial mitochondrial reactive oxygen species, leading to restoration of nitric oxide production and endothelium-mediated enhancement of nitric oxide levels in cardiomyocytes, an effect largely independent of sodium-hydrogen exchanger-1. Thus, empagliflozin restores the beneficial effect of cardiac microvascular endothelial cells on cardiomyocyte function by reducing mitochondrial oxidative damage, leading to reduced reactive oxygen species accumulation and increased endothelial nitric oxide bioavailability.

Oxidized Low-Density Lipoprotein Associates with Ventricular Stress in Young Adults and Triggers Intracellular Ca2+ Alterations in Adult Ventricular Cardiomyocytes.

Oxidized low-density lipoprotein (oxLDL) is associated with cardiac damage and causes injury to multiple cell types. We aimed to investigate the role of oxLDL in ventricular stress. We first examined the association between circulating oxLDL and N-terminal pro-brain natriuretic peptide (NT-proBNP), a marker of myocardial stress, in young subjects (30–50 years) with or without stable coronary artery disease (SCAD). oxLDL and NT-proBNP were significantly higher in subjects at high cardiovascular risk (CVR) than in subjects at low CVR and were associated independently of traditional CVR factors and C-reactive protein. Furthermore, the levels of oxLDL and NT-proBNP were significantly lower in subjects with SCAD than in peers at high CVR. To determine the intracellular mechanisms involved in the cardiac effects of oxLDL, we analyzed the in vitro effect of oxLDL on intracellular Ca2+ handling in adult rat ventricular cardiomyocytes using confocal microscopy. Acute challenge of adult ventricular cardiomyocytes to oxLDL reduced systolic Ca2+ transients and sarcoplasmic reticulum Ca2+ load. Moreover, diastolic spontaneous Ca2+ leak increased significantly after acute exposure to oxLDL. Thus, we demonstrate that oxLDL associates with NT-proBNP in young subjects, and can directly induce Ca2+ mishandling in adult ventricular cardiomyoyctes, predisposing cardiomyocytes to cardiac dysfunction and arrhythmogenicity.

Ellagic acid alleviates clozapine‑induced oxidative stress and mitochondrial dysfunction in cardiomyocytes

Clozapine (CLZ) as an antipsychotic agent is very effective in treating of psychosis disorders and resistant schizophrenia, but the risk of severe cardiac toxicity effects restricts its clinical use. There are several interrelated hypotheses to explain clozapine-induced cardiotoxicity which all of them may be related to oxidative stress. Therefore, the current study investigated the harmful effects of clozapine on cardiomyocytes and assessed the cytoprotective effect of ellagic acid (EA). Freshly isolated adult rat ventricular cardiomyocytes were incubated for 4 h at 37 °C with 00.05% ethanol as control, CLZ (50 µM), CLZ (50 µM) + a series of EA concentrations (10, 20 and 50 µM) and EA (50 µM). To evaluate the protective effect of EA, the markers of cell viability, reactive oxygen species (ROS) formation, mitochondria membrane potential (ΔΨm) collapse, lysosomal membrane integrity, malondialdehyde (MDA) and oxidized/reduced glutathione (GSH/GSSG) content were checked by biochemical and flowcytometry techniques. Our results demonstrated that EA (10, 20 and 50 µM) effectively inhibited CLZ-induced cytotoxicity which is associated with ROS overproduction and amelioration of mitochondrial and lysosomal damages. In addition, EA (10, 20 and 50 µM) in the presence of CLZ reduced the production of MDA as a specific marker lipid peroxidation and GSSG. Collectively, these findings suggested that EA protects cardiomyocytes from oxidative injury through inhibiting ROS formation, mitochondria dysfunction, and lysosomal damages, which suggest a potential therapeutic strategy of EA for CLZ-induced oxidative stress and cardiotoxicity.

Autocrine effects of PCSK9 on cardiomyocytes

Proprotein convertase subtilisin kexin type 9 (PCSK9) is in the focus of cardiovascular research due to its role in hepatic low density lipoprotein (LDL) clearance. However, extrahepatic expression of PCSK9 such as in cardiomyocytes and its regulation by oxidized LDL (oxLDL) put notion on extrahepatic effects of PCSK9 as well. This study was aimed to reveal the role of PCSK9 in oxLDL-dependent regulation of cardiomyocyte function. Adult rat and mouse ventricular cardiomyocytes and isolated perfused hearts were used. OxLDL was applied to increase PCSK9 expression in cardiomyocytes. Cell function was analyzed by load-free cell shortening as well as left ventricular developed pressure of isolated hearts. OxLDL decreased shortening in wild-type-derived mouse cardiomyocytes but not in those isolated from PCSK9 knockout mice. Overexpression of human PCSK9 in rat cardiomyocytes reduced shortening in the absence of oxLDL. Addition of recombinant PCSK9 mimicked these effects. In cardiomyocytes, oxLDL induced PCSK9 release into the supernatant. Inhibition of PCSK9 by Pep 2–8 or alirocumab attenuated the oxLDL-induced loss of cardiomyocyte shortening. Cardiomyocytes express surfeit locus protein 4 (SURF-4), a protein required for PCSK9 secretion in human embryonic kidney cells (HEK 293 T), and silencing of SURF-4 reduced the oxLDL effects on cardiomyocytes. In isolated perfused rat hearts PCSK9 inhibition by alirocumab improved the function. In addition, left ventricular function of isolated hearts from PCSK9 knockout mice was increased under basal conditions as well as at 10 min and 120 min of reperfusion following 45 min of ischemia. Collectively, the data show that cardiomyocytes express and release PCSK9 that acts in an autocrine way on cardiomyocytes and impairs their function.

Hydrolytic activity of mitochondrial F1Fo ATP synthase as a target for myocardial ischemia: discovery and in vitro evaluation of novel inhibitors

Abstract Background/Introduction F1Fo ATP synthase is the mitochondrial complex responsible for ATP production. During myocardial ischemia, ATP synthase reverses its activity to hydrolyse ATP leading to cellular energetic deficit, contracture and lethal cardiomyocyte injury. Therefore, inhibition of ATP hydrolase is of major significance and the discovery of selective hydrolase inhibitors could be of particular interest in terms of cardiac response to ischemia. Purpose We performed an in vitro screening in order to identify and evaluate novel specific inhibitors of the hydrolytic activity of ATP synthase. Methods Initially, we identified inhibitors of the hydrolytic activity of ATP synthase using virtual screening methods. Docking-scoring calculations were performed targeting the three major inhibition sites of a holistic model of ATP synthase of mus musculus that was modelled for the first time. In silico ligand-based virtual screening was carried out using known binders such as BMS199264. The workflow was implemented on our in-house library of 2000 compounds, Pharmalab, plus 266,151 compounds of the National Cancer Institute (NCI) database. The best candidates were evaluated in vitro on isolated murine heart mitochondria to verify their inhibitory effect on ATP synthase hydrolytic activity. Moreover, we confirmed their action at a cellular level. H9C2 cells were treated with the three best candidates in the presence of rotenone and their ability to maintain membrane potential was monitored. The experiment was repeated in absence of rotenone in order to detect toxic or non-specific effects (n=5 independent experiments). Finally, the compounds were tested using an assay in which primary adult rat ventricular cardiomyocytes (ARVCs) were challenged with the mitochondrial uncoupler CCCP to induce reverse ATPase activity, and time until contracture was determined. For the in vitro experiments, oligomycin, a non-selective ATPase inhibitor and BTB06584 a selective hydrolase inhibitor were used as positive controls. Results Different steps of filtering through the virtual screening provided 38 molecules from Pharmalab and 15 candidates form NCI database. From the 53 candidates in total, five compounds displayed in vitro inhibitory activity at 200μM on isolated mitochondria and their IC50 values were determined. Among them, three synthetic derivatives possessing a central pyrazolopyridine core displayed the best IC50 values (81.7±1.3 μM, 99.8±1.2 μM, 144.8±1.3μM) and were evaluated on the H9C2 cells. They exhibited significant inhibitory activity at 50μM (p&amp;lt;0.01 compared to vehicle) while the BTB06584 inhibitor was inactive at the same concentration and two of them were selective. Finally, the final novel inhibitors significantly extended (p&amp;lt;0.001) the time until shortening of the ARVCs. Conclusion We have discovered two novel agents that act as selective inhibitors of ATP hydrolase and can be tested against myocardial ischemia. Funding Acknowledgement Type of funding source: None

Fibroblast growth factor 23 decreases PDE4 expression in heart increasing the risk of cardiac arrhythmia; Klotho opposes these effects.

The concentration of fibroblast growth factor 23 (FGF23) rises progressively in renal failure (RF). High FGF23 concentrations have been consistently associated with adverse cardiovascular outcomes or death, in chronic kidney disease (CKD), heart failure or liver cirrhosis. We identified the mechanisms whereby high concentrations of FGF23 can increase the risk of death of cardiovascular origin. We studied the effects of FGF23 and Klotho in adult rat ventricular cardiomyocytes (ARVMs) and on the heart of mice with CKD. We show that FGF23 increases the frequency of spontaneous calcium waves (SCWs), a marker of cardiomyocyte arrhythmogenicity, in ARVMs. FGF23 increased sarcoplasmic reticulum Ca2+ leakage, basal phosphorylation of Ca2+-cycling proteins including phospholamban and ryanodine receptor type 2. These effects are secondary to a decrease in phosphodiesterase 4B (PDE4B) in ARVMs and in heart of mice with RF. Soluble Klotho, a circulating form of the FGF23 receptor, prevents FGF23 effects on ARVMs by increasing PDE3A and PDE3B expression. Our results suggest that the combination of high FGF23 and low sKlotho concentrations decreases PDE activity in ARVMs, which favors the occurrence of ventricular arrhythmias and may participate in the high death rate observed in patients with CKD.