Calcium Transient Amplitude Research Articles

Phosphodiesterase 5 inhibition improves contractile function and restores transverse tubule loss and catecholamine responsiveness in heart failure

Heart failure (HF) is characterized by poor survival, a loss of catecholamine reserve and cellular structural remodeling in the form of disorganization and loss of the transverse tubule network. Indeed, survival rates for HF are worse than many common cancers and have not improved over time. Tadalafil is a clinically relevant drug that blocks phosphodiesterase 5 with high specificity and is used to treat erectile dysfunction. Using a sheep model of advanced HF, we show that tadalafil treatment improves contractile function, reverses transverse tubule loss, restores calcium transient amplitude and the heart’s response to catecholamines. Accompanying these effects, tadalafil treatment normalized BNP mRNA and prevented development of subjective signs of HF. These effects were independent of changes in myocardial cGMP content and were associated with upregulation of both monomeric and dimerized forms of protein kinase G and of the cGMP hydrolyzing phosphodiesterases 2 and 3. We propose that the molecular switch for the loss of transverse tubules in HF and their restoration following tadalafil treatment involves the BAR domain protein Amphiphysin II (BIN1) and the restoration of catecholamine sensitivity is through reductions in G-protein receptor kinase 2, protein phosphatase 1 and protein phosphatase 2 A abundance following phosphodiesterase 5 inhibition.

Cardiac arrhythmias after renal I/R depend on IL-1β

AimsCardiac arrhythmias are one of the most important remote complications after kidney injury. Renal ischemia reperfusion (I/R) is a major cause of acute renal injury predisposing to several remote dysfunctions, including cardiac electrical disturbance. Since IL-1β production dependent on NLRP3 represents a link between tissue malfunctioning and cardiac arrhythmias, here we tested the hypothesis that longer ventricular repolarization and arrhythmias after renal I/R depend on this innate immunity sensor. Methods and resultsNlrp3−/− and Casp1−/− mice reacted to renal I/R with no increase in plasma IL-1β, different from WT (wild-type) I/R. A prolonged QJ interval and an increased susceptibility to ventricular arrhythmias were found after I/R compared to Sham controls in wild-type mice at 15 days post-perfusion, but not in Nlrp3−/− or CASP1−/− I/R, indicating that the absence of NLRP3 or CASP1 totally prevented longer QJ interval after renal I/R. In contrast with WT mice, we found no renal atrophy and no renal dysfunction in Nlrp3−/− and Casp1−/− mice after renal I/R. Depletion of macrophages in vivo after I/R and a day before IL-1β peak (at 7 days post-perfusion) totally prevented prolongation of QJ interval, suggesting that macrophages might participate as sensors of tissue injury. Moreover, treatment of I/R-WT mice with IL-1r antagonist (IL-1ra) from 8 to 15 days post perfusion did not interfere with renal function, but reversed QJ prolongation, prevented the increase in susceptibility to ventricular arrhythmias and rescued a close to normal duration and amplitude of calcium transient. ConclusionTaken together, these results corroborate the hypothesis that IL-1β is produced after sensing renal injury through NRLP3-CASP1, and IL-1β on its turn triggers longer ventricular repolarization and increase susceptibility to cardiac arrhythmias. Still, they offer a therapeutic approach to treat cardiac arrhythmias that arise after renal I/R.

Preconception Exposure to Fine Particulate Matter Leads to Cardiac Dysfunction in Adult Male Offspring.

HomeJournal of the American Heart AssociationVol. 8, No. 8Preconception Exposure to Fine Particulate Matter Leads to Cardiac Dysfunction in Adult Male Offspring Open AccessCorrectionPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citations ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toOpen AccessCorrectionPDF/EPUBPreconception Exposure to Fine Particulate Matter Leads to Cardiac Dysfunction in Adult Male Offspring Originally published8 Apr 2019https://doi.org/10.1161/JAHA.118.002293Journal of the American Heart Association. 2019;8:e002293This article corrects the followingPreconception Exposure to Fine Particulate Matter Leads to Cardiac Dysfunction in Adult Male OffspringIn the article by Tanwar et al, “Preconception Exposure to Fine Particulate Matter Leads to Cardiac Dysfunction in Adult Male Offspring,” which published online December 5, 2018, and appeared in the December 18, 2018 issue of the journal (J Am Heart Assoc. 2018;7:e010797. DOI: 10.1161/JAHA.118.010797), several errors were found. On page 3, second column, last paragraph, fifth line, “*, †, ‡ represent" was corrected to “*, **, *** represent”. On page 5, second column, fourth line, “(Figure 3A, 3B, and 3D)” was corrected to “(Figure 3A, 3B, and 3C).” On page 6, Figure 3 was a duplicate image of Figure 1. The correct Figure 3 is presented below and corrected in the online version.Also, on page 6, first paragraph, fourth line, “(Figure 3C and 3E)” was corrected to “(Figure 3D and 3E).” On page 11, the caption for Figure 8, fifth line, “methyltransferases” was corrected to “methyltransferases‐3a.”Download figureDownload PowerPointFigure 3. In vitro cardiomyocyte functional and calcium signaling parameters obtained from 3‐month‐old mice that were born to preconception filtered air or particulate matter (<2.5 μm in diameter) exposed parents. Graphs depicted are representative of (A) %peak shortening (%PS), (B) negative velocity (‐dL/dT), (C) time‐to‐peak shortening (TPS90), (D) fluorescence decay rate (τ), (E) calcium transient amplitude (Δ340/380). Data were collected from 70 to 80 cardiomyocytes isolated from 9 to 10 mice per group taken from individual litters. Data are expressed as mean±SEM. %PS indicates %peak shortening; ‐dL/dT, negative velocity; PC FA indicates preconception filtered air; PC PM2.5, preconception particulate matter (<2.5 μm in diameter); TPS90, time‐to‐peak shortening. *P<0.05, **P<0.01 vs PC FA controls.The publisher regrets the errors.The online version of the article has been updated and is available at https://www.ahajournals.org/doi/10.1161/JAHA.118.010797Footnotes Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesPreconception Exposure to Fine Particulate Matter Leads to Cardiac Dysfunction in Adult Male OffspringVineeta Tanwar, et al. Journal of the American Heart Association. 2018;7 April 16, 2019Vol 8, Issue 8Article InformationMetrics Copyright © 2019 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley BlackwellThis is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.https://doi.org/10.1161/JAHA.118.002293PMID: 30957626 Originally publishedApril 8, 2019 PDF download

Particulate matter 2.5 induced arrhythmogenesis mediated by TRPC3 in human induced pluripotent stem cell-derived cardiomyocytes

Particulate matter (PM) is one of the most important environmental issues worldwide, which is associated with not only pulmonary but also cardiovascular diseases. However, the underlying biological mechanisms of PM related cardiovascular dysfunction remained poorly defined, especially mediated by the pathway of direct impact on vascular and heart. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide an ideal platform for studying PM-exposed cellular diseases model in vitro. Here, we investigated the direct effects of particulate matter 2.5 (PM2.5) on hiPSC-CMs and the potential mechanism at non-cytotoxic concentrations. Cell viability, contraction amplitude and spontaneous beating rate of iPSC-CMs after direct exposure to PM2.5 showed that the concentration of lower than 100µg/ml would not lead to cytotoxic effects. Calcium-mediated optical mapping illustrated that there was a concentration-dependent reduction in quantification of calcium transient amplitude and an increase in the incidence of early after depolarizations due to PM2.5 treatment. Furthermore, there were dramatic dosage-dependent shortening in action potential duration and decrease in L-type calcium peak current density. The Ingenuity Pathway Analysis of our transcriptive study indicated that PM2.5 exposure preferentially influenced the expression of genes involved in calcium signaling. Among them the up-regulation of TRPC3 potentially played an important role in the electrophysiological alteration of PM2.5 on hiPSC-CMs, which could be ameliorated by pretreatment with pyr3, the inhibitor of TRPC3. In conclusion, our results demonstrated that exposure to PM2.5 was capable of increasing propensity to cardiac arrhythmias which could be attenuated with TRPC3 inhibition.

Acute Genetic Ablation of Sodium-Calcium Exchange: Adaptations of Excitation-Contraction Coupling and Calcium Regulation

Previously described murine models of sodium-calcium exchange (NCX) ablation exhibit cardiac remodeling that complicates interpretation, likely a result of embryonic excision of the NCX gene and chronic adaptation over time. To investigate the effect of acute ablation of NCX, we created a cardiac specific NCX KO in adult mice using the tamoxifen-inducible cardiac specific (αMHC-MerCreMer) promoter. We injected tamoxifen (40mg/kg/day IP) for 5 consecutive days. One week post tamoxifen, NCX protein expression was reduced by 96% in ventricular tissue and NCX current was completely eliminated in 4/9 cells (85% average reduction). LV function by echocardiography was preserved and, unlike ventricular-specific NCX KO mice produced by the MLC2v promoter, we observed no fibrosis or hypertrophy. We observed a 12% increase of resting calcium and normal Ca transient amplitudes in isolated cardiomyocytes loaded with fura-2 AM (30 min, 2.5 μM), despite a 37% reduction in ICa (P < 0.001), a two-fold increase of SERCA2 (P<0.01) and a two-fold increase of PMCA (P<0.05). Reduced ICa and preserved Ca release are indicative of increased EC coupling gain, which we have observed in MLC2v NCX KO mice. However, changes in SERCA and PMCA are distinct from the MLC2v NCX KO at 8-12 weeks, where resting Ca was similar to wildtype and SERCA and PMCA protein expression were unchanged. 4 weeks after tamoxifen, NCX expression and current were virtually eliminated and ICa was reduced by 47%. SERCA and PMCA continued to increase but fibrosis and hypertrophy developed despite normalization of diastolic Ca. In conclusion, mice survive after acute ablation of NCX by reducing Ca influx, increasing EC coupling gain, and increasing alternative Ca efflux and SR uptake mechanisms. However, prolonged absence of NCX results in cardiac remodeling, possibly by activating Ca-dependent gene programming.

Caffeine attenuates contraction-induced diminutions of the intracellular calcium transient in mouse lumbrical muscle ex vivo

The amount of calcium released from the sarcoplasmic reticulum in skeletal muscle rapidly declines during repeated twitch contractions. In this study, we test the hypothesis that caffeine can mitigate these contraction-induced declines in calcium release. Lumbrical muscles were isolated from male C57BL/6 mice and loaded with the calcium-sensitive indicator, AM-furaptra. Muscles were then stimulated at 8 Hz for 2.0 s in the presence or absence of 0.5 mM caffeine, at either 30 °C or 37 °C. The amplitude and area of the furaptra-based intracellular calcium transients and force produced during twitch contractions were calculated. For each of these measures, the values for twitch 16 relative to twitch 1 were higher in the presence of caffeine than in the absence of caffeine at both temperatures. We conclude that caffeine can attenuate contraction-induced diminutions of calcium release during repeated twitch contractions, thereby contributing to the inotropic effects of caffeine.

Screening of novel HSP-inducing compounds to conserve cardiomyocyte function in experimental atrial fibrillation.

BackgroundThe heat shock protein (HSP) inducer, geranylgeranylacetone (GGA), was previously found to protect against atrial fibrillation (AF) remodeling in experimental model systems. Clinical application of GGA in AF is limited, due to low systemic concentrations owing to the hydrophobic character of GGA.ObjectivesTo identify novel HSP-inducing compounds, with improved physicochemical properties, that prevent contractile dysfunction in experimental model systems for AF.MethodsEighty-one GGA-derivatives were synthesized and explored for their HSP-inducing properties by assessment of HSP expression in HL-1 cardiomyocytes pretreated with or without a mild heat shock (HS), followed by incubation with 10 µM GGA or GGA-derivative. Subsequently, the most potent HSP-inducers were tested for preservation of calcium transient (CaT) amplitudes or heart wall contraction in pretreated tachypaced HL-1 cardiomyocytes (with or without HSPB1 siRNA) and Drosophilas, respectively. Finally, CaT recovery in tachypaced HL-1 cardiomyocytes posttreated with GGA or protective GGA-derivatives was determined.ResultsThirty GGA-derivatives significantly induced HSPA1A expression after HS, and seven showed exceeding HSPA1A expression compared to GGA. GGA and nine GGA-derivatives protected significantly from tachypacing (TP)-induced CaT loss, which was abrogated by HSPB1 suppression. GGA and four potent GGA-derivatives protected against heart wall dysfunction after TP compared to non-paced control Drosophilas. Of these compounds, GGA and three GGA-derivatives induced a significant restoration from CaT loss after TP of HL-1 cardiomyocytes.ConclusionWe identified novel GGA-derivatives with improved physicochemical properties compared to GGA. GGA-derivatives, particularly GGA*-59, boost HSP expression resulting in prevention and restoration from TP-induced remodeling, substantiating their role as novel therapeutics in clinical AF.

Empagliflozin directly improves diastolic function in human heart failure.

Empagliflozin, a clinically used oral antidiabetic drug that inhibits the sodium-dependent glucose co-transporter 2, has recently been evaluated for its cardiovascular safety. Surprisingly, empagliflozin reduced mortality and hospitalization for heart failure (HF) compared to placebo. However, the underlying mechanisms remain unclear. Therefore, our study aims to investigate whether empagliflozin may cause direct pleiotropic effects on the myocardium. In order to assess possible direct myocardial effects of empagliflozin, we performed contractility experiments with in toto-isolated human systolic end-stage HF ventricular trabeculae. Empagliflozin significantly reduced diastolic tension, whereas systolic force was not changed. These results were confirmed in murine myocardium from diabetic and non-diabetic mice, suggesting independent effects from diabetic conditions. In human HF cardiomyocytes, empagliflozin did not influence calcium transient amplitude or diastolic calcium level. The mechanisms underlying the improved diastolic function were further elucidated by studying myocardial fibres from patients and rats with diastolic HF (HF with preserved ejection fraction, HFpEF). Empagliflozin beneficially reduced myofilament passive stiffness by enhancing phosphorylation levels of myofilament regulatory proteins. Intravenous injection of empagliflozin in anaesthetized HFpEF rats significantly improved diastolic function measured by echocardiography, while systolic contractility was unaffected. Empagliflozin causes direct pleiotropic effects on the myocardium by improving diastolic stiffness and hence diastolic function. These effects were independent of diabetic conditions. Since pharmacological therapy of diastolic dysfunction and HF is an unmet need, our results provide a rationale for new translational studies and might also contribute to the understanding of the EMPA-REG OUTCOME trial.

Converse role of class I and class IIa HDACs in the progression of atrial fibrillation

Atrial fibrillation (AF), the most common persistent clinical tachyarrhythmia, is associated with altered gene transcription which underlies cardiomyocyte dysfunction, AF susceptibility and progression. Recent research showed class I and class IIa histone deacetylases (HDACs) to regulate pathological and fetal gene expression, and thereby induce hypertrophy and cardiac contractile dysfunction. Whether class I and class IIa HDACs are involved in AF promotion is unknown. We aim to elucidate the role of class I and class IIa HDACs in tachypacing-induced contractile dysfunction in experimental model systems for AF and clinical AF. Methods and resultsClass I and IIa HDACs were overexpressed in HL-1 cardiomyocytes followed by calcium transient (CaT) measurements. Overexpression of class I HDACs, HDAC1 or HDAC3, significantly reduced CaT amplitude in control normal-paced (1 Hz) cardiomyocytes, which was further reduced by tachypacing (5 Hz) in HDAC3 overexpressing cardiomyocytes. HDAC3 inhibition by shRNA or by the specific inhibitor, RGFP966, prevented contractile dysfunction in both tachypaced HL-1 cardiomyocytes and Drosophila prepupae. Conversely, overexpression of class IIa HDACs (HDAC4, HDAC5, HDAC7 or HDAC9) did not affect CaT in controls, with HDAC5 and HDAC7 overexpression even protecting against tachypacing-induced CaT loss. Notably, the protective effect of HDAC5 and HDAC7 was abolished in cardiomyocytes overexpressing a dominant negative HDAC5 or HDAC7 mutant, bearing a mutation in the binding domain for myosin enhancer factor 2 (MEF2). Furthermore, tachypacing induced phosphorylation of HDAC5 and promoted its translocation from the nucleus to cytoplasm, leading to up-regulation of MEF2-related fetal gene expression (β-MHC, BNP). In accord, boosting nuclear localization of HDAC5 by MC1568 or Go6983 attenuated CaT loss in tachypaced HL-1 cardiomyocytes and preserved contractile function in Drosophila prepupae. Findings were expanded to clinical AF. Here, patients with AF showed a significant increase in expression levels and activity of HDAC3, phosphorylated HDAC5 and fetal genes (β-MHC, BNP) in atrial tissue compared to controls in sinus rhythm. ConclusionClass I and class IIa HDACs display converse roles in AF progression. Whereas overexpression of Class I HDAC3 induces cardiomyocyte dysfunction, class IIa HDAC5 overexpression reveals protective properties. Accordingly, HDAC3 inhibitors and HDAC5 nuclear boosters show protection from tachypacing-induced changes and therefore may represent interesting therapeutic options in clinical AF.

Effect of KN93 on delayed afterdepolarization and calcium ion in ventricular myocytes of rabbits with heart failure

Objective To observe the effect of KN93, a CaMKⅡ inhibitor, on delayed afterdepolarization (DAD) and calcium ion in ventricular myocytes of rabbits with heart failure, and to investigate the effect of CaMKⅡ signaling pathway on trigged arrhythmia after heart failure. Methods Thirty male New Zealand White rabbits were randomized(random number) into the sham operated group (sham group), heart failure group (HF group) and heart failure with KN93 group (HF+KN93 group) (n=10 each group). The rabbit heart failure model was established by abdominal aortic constriction combined with aortic valve regurgitation. The ventricular myocytes were isolated by double enzyme digestion. The action potential and the transient inward current (Iti) were recorded by the whole-cell patch-clamp. The intracellular calcium transient was measured by the ion concentration measurement system. The main calcium transporter protein was detected by Western blotting. Data were analyzed by pCLAMP10.2. Statistical analysis was performed using SPSS 17.0. Comparisons among groups were conducted using ANOVA, and SNK-q multiple comparison procedure was utilized for post-hoc analysis. Results (1) After induction of heart failure, DAD and increment of trigger activity (TA) were observed in rabbit ventricular myocytes. Treatment of KN93 with 1.0 μmol/L reduced the events of DAD and TA. (2) After induction of heart failure, Iti densities were increased from -0.12±0.02 pA/pF to -0.95±0.06 pA/pF at the polarization potential of -50 mV (n=10, P<0.01). The current densities were reduced to -0.44±0.04 pA/pF after application of 1.0 μmol/L of KN93 (n=10, P<0.01). (3) KN93 led to decrement of intracellular calcium ion concentration and calcium transient amplitude, and acceleration of the decay process of calcium transient. (4) KN93 upregulated the expression of pPLN and SERCA2a, increased the uptake of intracellular calcium ion, downregulated the expression of NCX, decreased the Iti, and reduced the occurrence of DAD and TA. Conclusions KN93 can reduce the intracellular calcium ion concentration of the heart failure animal model, and the occurrence of the DAD and TA. CaMKⅡ may be a new therapeutic target for arrhythmias in the heart failure. Key words: KN93; Heart failure; Delayed afterdepolarization; Transient inward current; Calcium ion

PySight: plug and play photon counting for fast continuous volumetric intravital microscopy

Imaging increasingly large neuronal populations at high rates pushed multi-photon microscopy into the photon-deprived regime. We present PySight, an add-on hardware and software solution tailored for photon-deprived imaging conditions. PySight more than triples the measured median amplitude of neuronal calcium transients in awake mice and facilitates single-trial intravital voltage imaging in fruit flies. Its unique data streaming architecture allowed us to image a fruit fly’s brain olfactory response over 234 μm×600 μm×330 μm at 73 volumes per second, while retaining over 200 times lower data rates than those of a conventional data acquisition system with comparable voxel sizes (1.2 μm×1.2 μm×2.2 μm). PySight requires no electronics expertise or custom synchronization boards, and its open-source software is extensible to any imaging method based on single-pixel (bucket) detectors. PySight offers an optimal data acquisition scheme for ever increasing imaging volumes of turbid living tissue.

Electrical stimulation increases hypertrophy and metabolic flux in tissue-engineered human skeletal muscle

In vitro models of contractile human skeletal muscle hold promise for use in disease modeling and drug development, but exhibit immature properties compared to native adult muscle. To address this limitation, 3D tissue-engineered human muscles (myobundles) were electrically stimulated using intermittent stimulation regimes at 1 Hz and 10 Hz. Dystrophin in myotubes exhibited mature membrane localization suggesting a relatively advanced starting developmental maturation. One-week stimulation significantly increased myobundle size, sarcomeric protein abundance, calcium transient amplitude (∼2-fold), and tetanic force (∼3-fold) resulting in the highest specific force generation (19.3mN/mm2) reported for engineered human muscles to date. Compared to 1 Hz electrical stimulation, the 10 Hz stimulation protocol resulted in greater myotube hypertrophy and upregulated mTORC1 and ERK1/2 activity. Electrically stimulated myobundles also showed a decrease in fatigue resistance compared to control myobundles without changes in glycolytic or mitochondrial protein levels. Greater glucose consumption and decreased abundance of acetylcarnitine in stimulated myobundles indicated increased glycolytic and fatty acid metabolic flux. Moreover, electrical stimulation of myobundles resulted in a metabolic shift towards longer-chain fatty acid oxidation as evident from increased abundances of medium- and long-chain acylcarnitines. Taken together, our study provides an advanced in vitro model of human skeletal muscle with improved structure, function, maturation, and metabolic flux.

ATP Reduces the Entry of Calcium Ions into the Nerve Ending by Blocking L-type Calcium Channels

At neuromuscular junctions, ATP inhibits both the evoked and spontaneous acetylcholine release and inward calcium current operating via presynaptic P2Y receptors. It was shown in the experiments with the frog neuromuscular synapse using specific calcium-sensitive dye Oregon Green Bapta 1 that exogenous ATP reduces the amplitude of calcium transient, which reflects the changes in the entry of calcium ions in response to the nerve pulse. The depressing effect of ATP on the transient was prevented by suramin, the blocker of P2 receptors. Nitrendipine, a specific blocker of L-type calcium channels, per se decreased the calcium transient amplitude and significantly attenuated the effect of ATP on the calcium signal. Contrariwise, the preliminary application of ATP to the neuromuscular junction completely eliminated the depressing effect of nitrendipine on the calcium response. The obtained data suggest that an essential component in the inhibitory action of ATP on the calcium transient amplitude is provided by reduction of the entry of calcium ions into a frog nerve ending via L-type voltage-gated calcium channels.

Disruption of neonatal cardiomyocyte physiology following exposure to bisphenol-a

Bisphenol chemicals are commonly used in the manufacturing of polycarbonate plastics, polyvinyl chloride plastics, resins, and thermal printing applications. Humans are inadvertently exposed to bisphenols through contact with consumer products and/or medical devices. Recent reports have shown a link between bisphenol-a (BPA) exposure and adverse cardiovascular outcomes; although these studies have been limited to adult subjects and models. Since cardiac physiology differs significantly between the developing and adult heart, we aimed to assess the impact of BPA exposure on cardiac function, using a neonatal cardiomyocyte model. Neonatal rat ventricular myocytes were monitored to assess cell viability, spontaneous beating rate, beat rate variability, and calcium-handling parameters in the presence of control or bisphenol-supplemented media. A range of doses were tested to mimic environmental exposure (10−9–10−8M), maximum clinical exposure (10−5M), and supraphysiological exposure levels (10−4M). Acute BPA exposure altered cardiomyocyte functionality, resulting in a slowed spontaneous beating rate and increased beat rate variability. BPA exposure also impaired intracellular calcium handling, resulting in diminished calcium transient amplitudes, prolonged calcium transient upstroke and duration time. Alterations in calcium handling also increased the propensity for alternans and skipped beats. Notably, the effect of BPA-treatment on calcium handling was partially reversible. Our data suggest that acute BPA exposure could precipitate secondary adverse effects on contractile performance and/or electrical alternans, both of which are dependent on intracellular calcium homeostasis.

Environmental chemicals can disrupt neonatal cardiomyocyte physiology

BackgroundThe ubiquitous nature of plastics has raised concerns pertaining to continuous exposure to plastic polymers and human health risks. Of particular concern is the use of endocrine‐disrupting chemicals in plastic production, including bisphenol A (BPA). Widespread and continuous exposure to BPA can occur through dietary intake, inhalation, dermal and intravenous exposure via consumer products and medical devices. Recent studies have shown a link between BPA exposure and adverse cardiovascular outcomes. This is particularly concerning for vulnerable populations who are at risk of high BPA exposure due to medical treatment, including pediatric patients. Despite significant differences in the cardiac physiology of the developing and adult heart, our current understanding of BPA‐induced cardiac dysfunction has been limited to adult models.ObjectiveCardiac physiology differs significantly between the pediatric and adult heart. We aimed to determine the extent to which BPA exposure contributes to cardiovascular dysfunction, using a neonatal cardiomyocyte model.MethodsNeonatal rat ventricular myocytes (NRVM) were monitored to assess cell viability, spontaneous beating rate, beat rate variability, and calcium‐handling in the presence of control or bisphenol‐supplemented media. A range of environmental and clinically‐relevant exposure levels were tested [10−9 – 10−5M].ResultsAcute BPA exposure (15 – 20 min) altered cardiomyocyte functionality, resulting in a slowed spontaneous beating rate and increased beat rate variability beginning at low concentrations (10−8M BPA). BPA exposure also impaired intracellular calcium handling, resulting in diminished calcium transient amplitudes and prolonged calcium transient duration time, which replicated our previously published studies in isolated‐whole hearts. BPA‐induced changes in calcium handling increased the propensity for calcium transient alternans, beginning at low BPA concentrations (10−9M). The observed alterations in calcium kinetics following BPA exposure were not the result of cytotoxicity or altered cell viability.ConclusionOur studies show that acute BPA chemical exposure can alter neonatal cardiomyocyte physiology. Additional studies are necessary to determine the effects of bisphenol exposure on pediatric heart electrophysiology and mechanical function, as well as the impact of long‐term treatment that more closely mimics clinical exposure.Support or Funding InformationThis publication was supported by the National Institutes of Health (R00ES023477, UL1TR000075), Children's Research Institute and Children's National Heart Institute.

Haplo-insufficiency of Bcl2-associated athanogene 3 in mice results in progressive left ventricular dysfunction, β-adrenergic insensitivity, and increased apoptosis.

Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid protein that is found predominantly in the heart, skeletal muscle, and many cancers. Deletions and truncations in BAG3 that result in haplo-insufficiency have been associated with the development of dilated cardiomyopathy. To study the cellular and molecular events attributable to BAG3 haplo-insufficiency we generated a mouse in which one allele of BAG3 was flanked by loxP recombination sites (BAG3fl/+ ). Mice were crossed with α-MHC-Cre mice in order to generate mice with cardiac-specific haplo-insufficiency (cBAG3+/-) and underwent bi-weekly echocardiography to assess their cardiac phenotype. By 10 weeks of age, cBAG3+/- mice demonstrated increased heart size and diminished left ventricular ejection fraction when compared with non-transgenic littermates (Cre-/- BAG3fl/+ ). Contractility in adult myocytes isolated from cBAG3+/- mice were similar to those isolated from control mice at baseline, but showed a significantly decreased response to adrenergic stimulation. Intracellular calcium ([Ca2+ ]i ) transient amplitudes in myocytes isolated from cBAG3+/- mice were also similar to myocytes isolated from control mice at baseline but were significantly lower than myocytes from control mice in their response to isoproterenol. BAG3 haplo-insufficiency was also associated with decreased autophagy flux and increased apoptosis. Taken together, these results suggest that mice in which BAG3 has been deleted from a single allele provide a model that mirrors the biology seen in patients with heart failure and BAG3 haplo-insufficiency.

The Anti-Cancer Multikinase Inhibitor Sorafenib Impairs Cardiac Contractility by Reducing Phospholamban Phosphorylation and Sarcoplasmic Calcium Transients

Tyrosine-kinase inhibitors (TKIs) have revolutionized cancer therapy in recent years. Although more targeted than conventional chemotherapy, TKIs exhibit substantial cardiotoxicity, often manifesting as hypertension or heart failure. Here, we assessed myocyte intrinsic cardiotoxic effects of the TKI sorafenib and investigated underlying alterations of myocyte calcium homeostasis. We found that sorafenib reversibly decreased developed force in auxotonically contracting human myocardia (3 µM: −25 ± 4%, 10 µM: −29 ± 7%, 30 µM: −43 ± 12%, p < 0.01), reduced peak cytosolic calcium concentrations in isolated cardiomyocytes (10 µM: 52 ± 8.1% of baseline, p < 0.001), and slowed cytosolic calcium removal kinetics (RT50, RT10, Tau, p < 0.05). Beta-adrenergic stimulation induced augmentation of calcium transient (CaT) amplitude was attenuated in sorafenib-treated cells (2.7 ± 0.3-fold vs. 3.6 ± 0.2-fold in controls, p < 0.001). Sarcoplasmic reticulum (SR) calcium content was reduced to 67 ± 4% (p < 0.01), and SR calcium re-uptake slowed (p < 0.05). Sorafenib significantly reduced serine 16 phosphorylation of phospholamban (PLN, p < 0.05), while PLN threonine 17 and CaMKII (T286) phosphorylation were not altered. Our data demonstrate that sorafenib acutely impairs cardiac contractility by reducing S16 PLN phosphorylation, leading to reduced SR calcium content, CaT amplitude, and slowed cytosolic calcium removal. These results indicate myocyte intrinsic cardiotoxicity irrespective of effects on the vasculature and chronic cardiac remodeling.

Exosomal microRNA-21-5p Mediates Mesenchymal Stem Cell Paracrine Effects on Human Cardiac Tissue Contractility.

The promising clinical benefits of delivering human mesenchymal stem cells (hMSCs) for treating heart disease warrant a better understanding of underlying mechanisms of action. hMSC exosomes increase myocardial contractility; however, the exosomal cargo responsible for these effects remains unresolved. This study aims to identify lead cardioactive hMSC exosomal microRNAs to provide a mechanistic basis for optimizing future stem cell-based cardiotherapies. Integrating systems biology and human engineered cardiac tissue (hECT) technologies, partial least squares regression analysis of exosomal microRNA profiling data predicted microRNA-21-5p (miR-21-5p) levels positively correlate with contractile force and calcium handling gene expression responses in hECTs treated with conditioned media from multiple cell types. Furthermore, miR-21-5p levels were significantly elevated in hECTs treated with the exosome-enriched fraction of the hMSC secretome (hMSC-exo) versus untreated controls. This motivated experimentally testing the human-specific role of miR-21-5p in hMSC-exo-mediated increases of cardiac tissue contractility. Treating hECTs with miR-21-5p alone was sufficient to recapitulate effects observed with hMSC-exo on hECT developed force and expression of associated calcium handling genes (eg, SERCA2a and L-type calcium channel). Conversely, knockdown of miR-21-5p in hMSCs significantly diminished exosomal procontractile and associated calcium handling gene expression effects on hECTs. Western blots supported miR-21-5p effects on calcium handling gene expression at the protein level, corresponding to significantly increased calcium transient amplitude and decreased decay time constant in comparison to miR-scramble control. Mechanistically, cotreating with miR-21-5p and LY294002, a PI3K inhibitor, suppressed these effects. Finally, mathematical simulations predicted the translational capacity for miR-21-5p treatment to restore calcium handling in mature ischemic adult human cardiomyocytes. miR-21-5p plays a key role in hMSC-exo-mediated effects on cardiac contractility and calcium handling, likely via PI3K signaling. These findings may open new avenues of research to harness the role of miR-21-5p in optimizing future stem cell-based cardiotherapies.

TRPV4 Increases Cardiomyocyte Calcium Transients and Contributes to Cardiac Damage Following Ischemia-Reperfusion in Hearts of Aged Mice

Transient Receptor Potential Vanilloid 4 (TRPV4) is an osmotically-activated cation channel, and expression is increased in cardiomyocytes of Aged mice. The goal of this investigation was to determine the role of TRPV4 in calcium handling following hypoosmotic stress and in cardiac function following ischemia-reperfusion (I-R). Hypoosmotic stress (250 mOsm pretreatment) induced an increase in action potential-induced calcium transient amplitude (fluo-4) in cardiomyocytes isolated from Aged (24-26 month) mice. This effect was prevented by TRPV4 inhibition with HC067047 (1μM), and was absent in cardiomyocytes from Young (3-6 month) mice. The elevation in calcium transients in Aged associated with an increase in ryanodine receptor-mediated calcium sparks (3.4±0.7 sparks/s/100μm hypoosmotic versus 1.1±0.3 control, P<0.05). Cardiac contractile function was examined in Langendorff-perfused hearts prior to and following global I-R (45 minutes ischemia, two hours reperfusion), and hearts of Aged responded to I-R with an initial increase in contractile function during reperfusion (ΔdP/dtMax: gain of 674±226 mmHg/s from baseline). The enhanced contractility during reperfusion of Aged was prevented by TRPV4 inhibition with HC067047 (ΔdP/dtMax: loss of 290±102 mmHg/s from baseline) and was absent in hearts of Young (ΔdP/dtMax: loss of 621±400 mmHg/s from baseline). Following two hours of reperfusion, metabolically active tissue was assessed using triphenyltetrazolium chloride. In hearts of Aged, TRPV4 inhibition with HC067047 decreased the percentage of damaged tissue compared to untreated conditions (7±3% HC067047 versus 21±5% untreated, P<0.05), revealing a potential pathological role of TRPV4 in cardiomyocyte viability. Taken together, our data suggest that while TRPV4 may increase calcium transients and contractile function, it also contributes to cardiomyocyte damage following I-R.

Rapid Electrical Stimulation Increased Cardiac Apoptosis Through Disturbance of Calcium Homeostasis and Mitochondrial Dysfunction in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Background/Aims: Heart failure induced by tachycardia, the most common arrhythmia, is frequently observed in clinical practice. This study was designed to investigate the underlying mechanisms. Methods: Rapid electrical stimulation (RES) at a frequency of 3 Hz was applied on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for 7 days, with 8 h/day and 24 h/day set to represent short-term and long-term tachycardia, respectively. Age-matched hiPSC-CMs without electrical stimulation or with slow electrical stimulation (1 Hz) were set as no electrical stimulation (NES) control or low-frequency electrical stimulation (LES) control. Following stimulation, JC-1 staining flow cytometry analysis was performed to examine mitochondrial conditions. Apoptosis in hiPSC-CMs was evaluated using Hoechst staining and Annexin V/propidium iodide (AV/PI) staining flow cytometry analysis. Calcium transients and L-type calcium currents were recorded to evaluate calcium homeostasis. Western blotting and qPCR were performed to evaluate the protein and mRNA expression levels of apoptosis-related genes and calcium homeostasis-regulated genes. Results: Compared to the controls, hiPSC-CMs following RES presented mitochondrial dysfunction and an increased apoptotic percentage. Amplitudes of calcium transients and L-type calcium currents were significantly decreased in hiPSC-CMs with RES. Molecular analysis demonstrated upregulated expression of Caspase3 and increased Bax/Bcl-2 ratio. Genes related to calcium re-sequence were downregulated, while phosphorylated Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) was significantly upregulated following RES. There was no significant difference between the NES control and LES control groups in these aspects. Inhibition of CaMKII with 1 µM KN93 partly reversed these adverse effects of RES. Conclusion: RES on hiPSC-CMs disturbed calcium homeostasis, which led to mitochondrial stress, promoted cell apoptosis and caused electrophysiological remodeling in a time-dependent manner. CaMKII played a central role in the damages induced by RES, pharmacological inhibition of CaMKII activity partly reversed the adverse effects of RES on both structural and electrophysiological properties of cells.