Calcium Handling In Cardiomyocytes Research Articles

Calcium Dobesilate (CaD) Attenuates High Glucose and High Lipid-Induced Impairment of Sarcoplasmic Reticulum Calcium Handling in Cardiomyocytes.

Calcium dobesilate (CaD) is used effectively in patients with diabetic microvascular disorder, retinopathy, and nephropathy. Here we sought to determine whether it has an effect on cardiomyocytes calcium mishandling that is characteristic of diabetic cardiomyopathy. Cardiomyocytes were sterile isolated and cultured from 1 to 3 days neonatal rats and treated with vehicle (Control), 25 mM glucose+300 μM Palmitic acid (HG+PA), 100 μM CaD (CaD), or HG+PA+CaD to test the effects on calcium signaling (Ca2+ sparks, transients, and SR loads) and reactive oxygen species (ROS) production by confocal imaging. Compared to Control, HG+PA treatment significantly reduced field stimulation-induced calcium transient amplitudes (2.22 ± 0.19 vs. 3.56 ± 0.21, p < 0.01) and the levels of caffeine-induced calcium transients (3.19 ± 0.14 vs. 3.72 ± 0.15, p < 0.01), however significantly increased spontaneous Ca2+ sparks firing levels in single cardiomyocytes (spontaneous frequency 2.65 ± 0.23 vs. 1.72 ± 0.12, p < 0.01) and ROS production (67.12 ± 4.4 vs. 47.65 ± 2.12, p < 0.05), which suggest that HG+PA treatment increases the Spontaneity Ca2+ spark frequency, and then induced partial reduction of SR Ca2+ content and subsequently weaken systolic Ca2+ transient in cardiomyocyte. Remarkably, these impairments in calcium signaling and ROS production were largely prevented by pre-treatment of the cells with CaD. Therefore, CaD may contribute to a good protective effect on patients with calcium mishandling and contractile dysfunction in cardiomyocytes associated with diabetic cardiomyopathy.

Cardiac Myosin Activation for the Treatment of Systolic Heart Failure.

Left ventricular systolic dysfunction is the hallmark pathology in heart failure with reduced ejection fraction. Increasing left ventricular contractility with beta-adrenergic receptor agonists, phosphodiesterase-3 inhibitors, or levosimendan has failed to improve clinical outcomes and, in some situations, increased the risk of sudden cardiac death. Beta-adrenergic receptor agonists and phosphodiesterase-3 inhibitors retain an important role in advanced heart failure. Thus, there remains an unmet need for safe and effective therapies to improve left ventricular systolic function. Two novel cardiac myotropes, omecamtiv mecarbil and danicamtiv, target cardiac myosin to increase left ventricular systolic performance. Neither omecamtiv mecarbil nor danicamtiv affects cardiomyocyte calcium handling, the proposed mechanism underlying the life-threatening arrhythmias associated with cardiac calcitropes and calcium sensitizers. Phase 2 clinical trials have demonstrated that these cardiac myosin activators prolong left ventricular systolic ejection time and promote left ventricular and atrial reverse remodeling. At higher plasma concentrations, these agents may be associated with myocardial ischemia and impaired diastolic function. An ongoing phase 3 clinical trial will estimate the clinical efficacy and safety of omecamtiv mecarbil. An additional study of these agents, which have minimal hemodynamic and renal effects, is warranted in patients with advanced heart failure refractory to guideline-directed neurohormonal blockers.

Altered calcium handling in cardiomyocytes from arginine-glycine amidinotransferase-knockout mice is rescued by creatine.

The creatine kinase system facilitates energy transfer between mitochondria and the major ATPases in the heart. Creatine-deficient mice, which lack arginine-glycine amidinotransferase (AGAT) to synthesize creatine and homoarginine, exhibit reduced cardiac contractility. We studied how the absence of a functional CK system influences calcium handling in isolated cardiomyocytes from AGAT-knockouts and wild-type littermates as well as in AGAT-knockout mice receiving lifelong creatine supplementation via the food. Using a combination of whole cell patch clamp and fluorescence microscopy, we demonstrate that the L-type calcium channel (LTCC) current amplitude and voltage range of activation were significantly lower in AGAT-knockout compared with wild-type littermates. Additionally, the inactivation of LTCC and the calcium transient decay were significantly slower. According to our modeling results, these changes can be reproduced by reducing three parameters in knockout mice when compared with wild-type: LTCC conductance, the exchange constant of Ca2+ transfer between subspace and cytosol, and SERCA activity. Because tissue expression of LTCC and SERCA protein were not significantly different between genotypes, this suggests the involvement of posttranslational regulatory mechanisms or structural reorganization. The AGAT-knockout phenotype of calcium handling was fully reversed by dietary creatine supplementation throughout life. Our results indicate reduced calcium cycling in cardiomyocytes from AGAT-knockouts and suggest that the creatine kinase system is important for the development of calcium handling in the heart.NEW & NOTEWORTHY Creatine-deficient mice lacking arginine-glycine amidinotransferase exhibit compromised cardiac function. Here, we show that this is at least partially due to an overall slowing of calcium dynamics. Calcium influx into the cytosol via the L-type calcium current (LTCC) is diminished, and the rate of the sarcoendoplasmic reticulum calcium ATPase (SERCA) pumping calcium back into the sarcoplasmic reticulum is slower. The expression of LTCC and SERCA did not change, suggesting that the changes are regulatory.

Abstract 14803: Activin A Directly Impaired Human IPSC-cardiomyocyte Contractility and Electrophysiology

Activin A is a homo-dimeric TGF-β family member involved in embryonic development, tissue morphogenesis, and cellular differentiation. Levels of activin A correlate with NYHA functional classification and age dependent cardiac dysfunction; however, no data are available to demonstrate a direct role for activin A in regulating human cardiomyocyte dysfunction. The purpose of this study was to characterize the functional impact of chronic activin A on human cardiomyocyte electrophysiology, contractility, calcium handling, and gene expression. Methods: Human iPSC-cardiomyocytes (iPSC-CM) were supplied by Fujifilm Cellular Dynamics. Calcium transients were captured using the FLIPR Tetra imager and impedance/electrophysiology by the Nanion CardioExcyte96. Cells were plated (50k/well), allowed to reach a stable synchronous monolayer, and dosed with 1nM of activin A (A) at each media change alongside a media control (C) or in the presence of inhibitory anti-activin A antibodies. Gene expression was determined using Taqman. Results: Activin A reduced contractile impedance amplitude (C=9.82±0.30, A=6.82±0.21 Ohms, p&lt;0.01) and slowed relaxation velocity kinetics (C=48.78±2.65, A=35.07±1.07 Ohms/sec, p&lt;0.01). Elongated extracellular field potential durations were observed with activin A treatment compared to control (C=0.49±0.02, A=0.56±0.01 sec, p&lt;0.01). Impaired calcium handling peak amplitude (C=609±99, A= C=447±33 RFU, p&lt;0.05) was also observed alongside slower calcium flux falling times (C=0.52±0.05, A=0.70±0.04 sec, p&lt;0.01). An inhibitory activin-A antibody restored all parameters to control values. Chronic exposure of iPSC-CM to activin A reduced expression of calcium handling genes RYR2 (C=1.01±0.07, A=0.75±0.03, p&lt;0.01) and ATP2A2 (C=1.00±0.05, A=0.89±0.05, p&lt;0.05). Conclusions: Chronic activin A treatment reduced cardiomyocyte contractile amplitude, slowed contractile kinetics, impaired cardiomyocyte calcium handling, elongated the action potential, and reduced expression of genes regulating key calcium handling proteins. These data demonstrate that elevated levels of activin A can directly act on cardiomyocytes and therefore may contribute to cardiac dysfunction in heart failure and aged populations.

Abstract 14245: Fibroblast Growth Factor 23 and Long-term Cardiac Function: The Multi- Ethnic Study of Atherosclerosis

Background: While fibroblast growth factor 23 (FGF23) is associated with incident heart failure (HF) and atrial fibrillation (AF), the mechanisms driving these associations are unclear. FGF23 elevation leads to cardiomyocyte calcium handling abnormalities, suggesting that FGF23 may directly reduce myocardial function. Methods: In the Multi-Ethnic Study of Atherosclerosis, a cohort free of cardiovascular disease at recruitment, we evaluated the associations of serum FGF23 (2000-2002) with measures of left ventricular (LV) and left atrial (LA) mechanical function on cardiac magnetic resonance (CMR) at 10-year follow up (2010-2012). Results: Of 2,276 participants with baseline FGF23 and CMR at 10-year follow up, participants with higher FGF23 levels were more likely white race, taking anti-hypertensive medications, and had lower baseline glomerular filtration rate (GFR). After covariate adjustment, baseline FGF23 levels were independently associated with worse LV global circumferential strain, worse LV mid-wall circumferential strain, and lower LA total emptying fraction in later life ( Table ). The association of FGF23 and LV global circumferential strain was consistent across the spectrum of GFR ( Figure ). While higher FGF23 was associated with higher LV mass (β coefficient per SD higher: 1.14, 95% CI: 0.16, 2.12, P= 0.02), it was not associated with the presence of macroscopic myocardial scar (OR per SD higher: 1.12, 95% CI: 0.86-1.45, P= 0.42). Conclusions: Baseline FGF23 is independently associated with lower LV and LA systolic function in later life. These findings provide mechanistic insight driving the associations of FGF23 with development of both HF and AF.

Epitranscriptomics in the Heart: a Focus on m6A.

Post-transcriptional modifications are key regulators of gene expression that allow the cell to respond to environmental stimuli. The most abundant internal mRNA modification is N6-methyladenosine (m6A), which has been shown to be involved in the regulation of RNA splicing, localization, translation, and decay. It has also been implicated in a wide range of diseases, and here, we review recent evidence of m6A's involvement in cardiac pathologies and processes. Studies have primarily relied on gain and loss of function models for the enzymes responsible for adding and removing the m6A modification. Results have revealed a multifaceted role for m6A in the heart's response to myocardial infarction, pressure overload, and ischemia/reperfusion injuries. Genome-wide analyses of mRNAs that are differentially methylated during cardiac stress have highlighted the importance of m6A in regulating the translation of specific categories of transcripts implicated in pathways such as calcium handling, cell growth, autophagy, and adrenergic signaling in cardiomyocytes. Regulation of gene expression by m6A is critical for cardiomyocyte homeostasis and stress responses, suggesting a key role for this modification in cardiac pathophysiology.

The phospholamban p.(Arg14del) pathogenic variant leads to cardiomyopathy with heart failure and is unresponsive to standard heart failure therapy

Phospholamban (PLN) plays a role in cardiomyocyte calcium handling as primary inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). The p.(Arg14del) pathogenic variant in the PLN gene results in a high risk of developing dilated or arrhythmogenic cardiomyopathy with heart failure. There is no established treatment other than standard heart failure therapy or heart transplantation. In this study, we generated a novel mouse model with the PLN-R14del pathogenic variant, performed detailed phenotyping, and tested the efficacy of established heart failure therapies eplerenone or metoprolol. Heterozygous PLN-R14del mice demonstrated increased susceptibility to ex vivo induced arrhythmias, and cardiomyopathy at 18 months of age, which was not accelerated by isoproterenol infusion. Homozygous PLN-R14del mice exhibited an accelerated phenotype including cardiac dilatation, contractile dysfunction, decreased ECG potentials, high susceptibility to ex vivo induced arrhythmias, myocardial fibrosis, PLN protein aggregation, and early mortality. Neither eplerenone nor metoprolol administration improved cardiac function or survival. In conclusion, our novel PLN-R14del mouse model exhibits most features of human disease. Administration of standard heart failure therapy did not rescue the phenotype, underscoring the need for better understanding of the pathophysiology of PLN-R14del-associated cardiomyopathy. This model provides a great opportunity to study the pathophysiology, and to screen for potential therapeutic treatments.

Effects of Circulating HMGB-1 and Histones on Cardiomyocytes-Hemadsorption of These DAMPs as Therapeutic Strategy after Multiple Trauma.

Background and purpose: The aim of the study was to determine the effects of post-traumatically released High Mobility Group Box-1 protein (HMGB1) and extracellular histones on cardiomyocytes (CM). We also evaluated a therapeutic option to capture circulating histones after trauma, using a hemadsorption filter to treat CM dysfunction. Experimental Approach: We evaluated cell viability, calcium handling and mitochondrial respiration of human cardiomyocytes in the presence of HMGB-1 and extracellular histones. In a translational approach, a hemadsorption filter was applied to either directly eliminate extracellular histones or to remove them from blood samples obtained from multiple injured patients. Key results: Incubation of human CM with HMGB-1 or histones is associated with changes in calcium handling, a reduction of cell viability and a substantial reduction of the mitochondrial respiratory capacity. Filtrating plasma from injured patients with a hemadsorption filter reduces histone concentration ex vivo and in vitro, depending on dosage. Conclusion and implications: Danger associated molecular patterns such as HMGB-1 and extracellular histones impair human CM in vitro. A hemadsorption filter could be a therapeutic option to reduce high concentrations of histones.

Amorphous SiO2 nanoparticles promote cardiac dysfunction via the opening of the mitochondrial permeability transition pore in rat heart and human cardiomyocytes

BackgroundSilica nanoparticles (nanoSiO2) are promising systems that can deliver biologically active compounds to tissues such as the heart in a controllable manner. However, cardiac toxicity induced by nanoSiO2 has been recently related to abnormal calcium handling and energetic failure in cardiomyocytes. Moreover, the precise mechanisms underlying this energetic debacle remain unclear. In order to elucidate these mechanisms, this article explores the ex vivo heart function and mitochondria after exposure to nanoSiO2.ResultsThe cumulative administration of nanoSiO2 reduced the mechanical performance index of the rat heart with a half-maximal inhibitory concentration (IC50) of 93 μg/mL, affecting the relaxation rate. In isolated mitochondria nanoSiO2 was found to be internalized, inhibiting oxidative phosphorylation and significantly reducing the mitochondrial membrane potential (ΔΨm). The mitochondrial permeability transition pore (mPTP) was also induced with an increasing dose of nanoSiO2 and partially recovered with, a potent blocker of the mPTP, Cyclosporine A (CsA). The activity of aconitase and thiol oxidation, in the adenine nucleotide translocase, were found to be reduced due to nanoSiO2 exposure, suggesting that nanoSiO2 induces the mPTP via thiol modification and ROS generation. In cardiac cells exposed to nanoSiO2, enhanced viability and reduction of H2O2 were observed after application of a specific mitochondrial antioxidant, MitoTEMPO. Concomitantly, CsA treatment in adult rat cardiac cells reduced the nanoSiO2-triggered cell death and recovered ATP production (from 32.4 to 65.4%). Additionally, we performed evaluation of the mitochondrial effect of nanoSiO2 in human cardiomyocytes. We observed a 40% inhibition of maximal oxygen consumption rate in mitochondria at 500 μg/mL. Under this condition we identified a remarkable diminution in the spare respiratory capacity. This data indicates that a reduction in the amount of extra ATP that can be produced by mitochondria during a sudden increase in energy demand. In human cardiomyocytes, increased LDH release and necrosis were found at increased doses of nanoSiO2, reaching 85 and 48%, respectively. Such deleterious effects were partially prevented by the application of CsA. Therefore, exposure to nanoSiO2 affects cardiac function via mitochondrial dysfunction through the opening of the mPTP.ConclusionThe aforementioned effects can be partially avoided reducing ROS or retarding the opening of the mPTP. These novel strategies which resulted in cardioprotection could be considered as potential therapies to decrease the side effects of nanoSiO2 exposure.

DAMP-mediated cardiac dysfunction: cardiomyocytes as actors and target of innate immune response

Abstract Objective Severe trauma often led to a systemic inflammatory response, accompanied by release of danger associated molecular patterns such as the high mobility group box-1 protein (HMGB-1) and extracellular histones. The aim of this study was to clarify the influence of HMGB-1 and extracellular histones on human cardiomyocytes, in context of posttraumatic cardiac dysfunction. Material and Methods Cell viability, mitochondrial respiration and calcium handling of human cardiomyocytes in presence of extracellular histones or HMGB-1 were evaluated. A hemadsorption filter was tested to eliminate extracellular histones in vitro and from blood samples of multiple trauma patients (mean injury severity score &amp;gt;16). Results Incubation of human cardiomyocytes with HMGB-1 or histones were associated with impaired calcium handling, mitochondrial respiration and reduction of cell viability. Furthermore, human cardiomyocytes release further cardio-depressive cytokines in the presence of extracellular histones and HMGB-1 such as IL-1b and TNF. Hemadsorption of human plasma after multiple trauma showed a significant reduction of histone levels. Furthermore, in vitro analysis of hemofiltration capacity detected a dose dependant reduction of extracellular histones. Conclusion Despite their positive intention as part of the immune reaction, HMGB-1 and extracellular histones are associated with detrimental effects on cardiomyocyte viability, calcium handling and mitochondrial function. Moreover, presence of DAMPs resulted in additional release of cardiodepressive cytokines. Therefore, in the context of tissue trauma cardiomyocytes are target and actor at the same time.

Cardiomyocyte calcium handling in health and disease: Insights from in vitro and in silico studies

Calcium (Ca2+) plays a central role in cardiomyocyte excitation-contraction coupling. To ensure an optimal electrical impulse propagation and cardiac contraction, Ca2+ levels are regulated by a variety of Ca2+-handling proteins. In turn, Ca2+ modulates numerous electrophysiological processes. Accordingly, Ca2+-handling abnormalities can promote cardiac arrhythmias via various mechanisms, including the promotion of afterdepolarizations, ion-channel modulation and structural remodeling. In the last 30 years, significant improvements have been made in the computational modeling of cardiomyocyte Ca2+ handling under physiological and pathological conditions. However, numerous questions involving the Ca2+-dependent regulation of different macromolecular complexes, cross-talk between Ca2+-dependent regulatory pathways operating over a wide range of time scales, and bidirectional interactions between electrophysiology and mechanics remain to be addressed by in vitro and in silico studies. A better understanding of disease-specific Ca2+-dependent proarrhythmic mechanisms may facilitate the development of improved therapeutic strategies. In this review, we describe the fundamental mechanisms of cardiomyocyte Ca2+ handling in health and disease, and provide an overview of currently available computational models for cardiomyocyte Ca2+ handling. Finally, we discuss important uncertainties and open questions about cardiomyocyte Ca2+ handling and highlight how synergy between in vitro and in silico studies may help to answer several of these issues.

088 Calcium Handling in Cardiomyocytes: does Ventricular Hypertrophy Impair the Response to Inotropic Stimulation?

088 Calcium Handling in Cardiomyocytes: does Ventricular Hypertrophy Impair the Response to Inotropic Stimulation?

Chronic vagal nerve stimulation has no effect on tachycardia-induced heart failure progression or excitation-contraction coupling.

Autonomic dysregulation plays a key role in the development and progression of heart failure (HF). Vagal nerve stimulation (VNS) may be a promising therapeutic approach. However, the outcomes from clinical trials evaluating VNS in HF have been mixed, and the mechanisms underlying this treatment remain poorly understood. Intermittent high‐frequency VNS (pulse width 300 µs, 30 Hz stimulation, 30 s on, and 300 s off) was used in healthy sheep and sheep in which established HF had been induced by 4 weeks rapid ventricular pacing to assess (a) the effects of VNS on intrinsic cardiac vagal tone, (b) whether VNS delays the progression of established HF, and (c) whether high‐frequency VNS affects the regulation of cardiomyocyte calcium handling in health and disease. VNS had no effect on resting heart rate or intrinsic vagal tone in the healthy heart. Although fewer VNS‐treated animals showed subjective signs of heart failure at 6 weeks, overall VNS did not slow the progression of clinical or echocardiographic signs of HF. Chronic VNS did not affect left ventricular cardiomyocyte calcium handling in healthy sheep. Rapid ventricular pacing decreased the L‐type calcium current and calcium transient amplitude, but chronic VNS did not rescue dysfunctional calcium handling. Overall, high‐frequency VNS did not prevent progression of established HF or influence cellular excitation–contraction coupling. However, a different model of HF or selection of different stimulation parameters may have yielded different results. These results highlight the need for greater insight into VNS dosing and parameter selection and a deeper understanding of its physiological effects.

Chronic aerobic exercise associated to low-dose L-NAME improves contractility without changing calcium handling in rat cardiomyocytes.

Nitric oxide (NO) inhibition by high-dose NG-nitro-L-arginine methyl ester (L-NAME) is associated with several detrimental effects on the cardiovascular system. However, low-dose L-NAME increases NO synthesis, which in turn induces physiological cardiovascular benefits, probably by activating a protective negative feedback mechanism. Aerobic exercise, likewise, improves several cardiovascular functions in healthy hearts, but its effects are not known when chronically associated with low-dose L-NAME. Thus, we tested whether the association between low-dose L-NAME administration and chronic aerobic exercise promotes beneficial effects to the cardiovascular system, evaluating the cardiac remodeling process. Male Wistar rats were randomly assigned to control (C), L-NAME (L), chronic aerobic exercise (Ex), and chronic aerobic exercise associated to L-NAME (ExL). Aerobic training was performed with progressive intensity for 12 weeks; L-NAME (1.5 mg·kg-1·day-1) was administered by orogastric gavage. Low-dose L-NAME alone did not change systolic blood pressure (SBP), but ExL significantly increased SBP at week 8 with normalization after 12 weeks. Furthermore, ExL promoted the elevation of left ventricle (LV) end-diastolic pressure without the presence of cardiac hypertrophy and fibrosis. Time to 50% shortening and relaxation were reduced in ExL, suggesting a cardiomyocyte contractile improvement. In addition, the time to 50% Ca2+ peak was increased without alterations in Ca2+ amplitude and time to 50% Ca2+ decay. In conclusion, the association of chronic aerobic exercise and low-dose L-NAME prevented cardiac pathological remodeling and induced cardiomyocyte contractile function improvement; however, it did not alter myocyte affinity and sensitivity to intracellular Ca2+ handling.

Assessing SSRIs’ effects on fetal cardiomyocytes utilizing placenta-fetus model

Selective serotonin reuptake inhibitors (SSRIs) have been shown to hinder cardiomyocyte signaling, raising concerns about their safety during pregnancy. Approaches to assess SSRI-induced effects on fetal cardiovascular cells following passage of drugs through the placental barrier in vitro have only recently become available. Herein, we report that the SSRIs, fluoxetine and sertraline, lead to slowed cardiomyocyte calcium oscillations and induce increased secretion of troponin T and creatine kinase-MB with reduced secretion of NT-proBNP, three key cardiac injury biomarkers. We show the cardiomyocyte calcium handling effects are further amplified following indirect exposure through a placental barrier model. These studies are the first to investigate the effects of placental barrier co-culture with cardiomyocytes in vitro and to show cardiotoxicity of SSRIs following passage through the placental barrier. Statement of SignificanceUse of selective serotonin reuptake inhibitors (SSRIs), a class of antidepressants, during pregnancy continues to rise despite multiple studies showing potential for detrimental effects on the developing fetus. SSRIs are particularly thought to slow cardiovascular electrical activity, such as ion signaling, yet few, if any, methods exist to rigorously study these drug-induced effects on human pregnancy and the developing fetus. Within this study, we utilized a placenta-fetus model to evaluate these drug-induced effects on cardiomyocytes, looking the drugs’ effects on calcium handling and secretion of multiple cardiac injury biomarkers. Together, with existing literature, this study provides a platform for assessing pharmacologic effects of drugs on cells mimicking the fetus and the role the placenta plays in this process.

The Absence of Active Creatine Kinase System Influences Cardiac Calcium Handling

Creatine kinase (CK) system is known as a phosphoryl buffering and transfer system between mitochondria and ATPases. Functional coupling between CK and adenine nucleotide translocase, ATPases, and ATP-sensing potassium channel, has been demonstrated suggesting a major role of the CK system in the heart. To study CK system role, we used two lines of creatine-deficient mice, which lack either guanidinoacetate methyltransferase (GAMT) or arginine-glycine amidinotransferase (AGAT) on creatine synthesis pathway. Intriguingly, there have been no significant adaptations to the absence of active CK system in GAMT and AGAT mice cardiomyocytes from bioenergetics perspective: mitochondrial arrangement, respiration kinetics, activities of alternative pathway enzymes, intracellular compartmentation are the same in wild-type (WT) and knock-out (KO) littermates. Using whole-cell patch clamp, we studied how the absence of CK system influences calcium handling in cardiomyocytes of AGAT KO and WT littermates. Voltage dependence of L-type calcium channel (LTCC) current was significantly different in KO and WT animals with the significant differences between sexes within groups. LTCC amplitude was influenced by the absence of active CK system only in female mice in control conditions but was found to be the same in KO and WT male mice and mice of both sexes after adrenergic stimulation. The absence of active CK system had a major impact on calcium uptake, with the KO mice requiring more time for reducing calcium concentration to the resting level at the lower uptake rates. The effects of the active CK system absence on LTCC and calcium uptake, presumingly mainly by SR calcium ATPase, were fully reversed by creatine feeding to the KO animals. Our results suggest that there is a major role of the CK system in providing energy to excitation-contraction machinery in the heart to maintain normal calcium homeostasis.

Abstract 261: Activation of the Thromboxane/Prostanoid Receptor Contributes to Elevated End-Diastolic Calcium in Cardiomyocytes and Cardiac Fibrosis Following Right Ventricular Pressure Overload

Like its systemic counterpart, pulmonary arterial hypertension (PAH) results in remodeling and fibrosis of the right ventricle as it attempts to adapt to the increased pressure overload. This eventually leads to contractile dysfunction, and RV failure is the primary cause of death in PAH patients. The G protein-coupled thromboxane/prostanoid (TP) receptor is expressed in vascular smooth muscle and immune cells, and is upregulated in cardiomyocytes following PAH. Activation of the cardiac TP receptor increases cardiomyocyte intracellular calcium and can lead to arrhythmias. We previously reported that oral treatment with the TP receptor antagonist ifetroban prevents RV fibrosis in a mouse pressure overload model of PAH. Here, we investigate the effects of TP receptor activation on calcium handling in RV cardiomyocytes and explore treatment of established RV remodeling with ifetroban, compared with prevention. Fixed pressure overload of the RV via pulmonary arterial banding (PAB) caused an increase in contractility and resting (end-diastolic) intracellular calcium in individual cardiomyocytes after 3 weeks; this occurred in conjunction with RV dilation, fibrosis, and stiffness. Surprisingly, total calcium content of the sarcoplasmic reticulum was increased following PAB. Antagonism with ifetroban decreased formation of fibrosis in a time-dependent manner. However, treatment with antagonist following establishment of RV fibrosis still prevented the cardiomyocyte increase in end-diastolic calcium. This suggests a multi-factorial contribution of the TP receptor in the RV response to PAH. Further studies continue to analyze changes in calcium-dependent signaling, as well as the contribution of the cardiomyocyte TP receptor to both fibrosis and calcium handling.

Correction: Acute exhaustive aerobic exercise training impair cardiomyocyte function and calcium handling in Sprague-Dawley rats

[This corrects the article DOI: 10.1371/journal.pone.0173449.].

Agmatine modulates calcium handling in cardiomyocytes of hibernating ground squirrels through calcium-sensing receptor signaling

True hibernators are remarkable group of mammals whose hearts are resistant to such stressors as deep hypothermia, ischemia, arrhythmia. Capability of cardiac cells from hibernating species to effectively rule Ca2+ homeostasis during torpor is poorly studied. Better understanding of these mechanisms could allow to introduce new strategies for improvement the cardiac performance and may be useful for cardiovascular medicine. Here for the first time we have shown that the regulation of Ca2+ handling and thereby cardiomyocyte contractility by endogenous neurotransmitter agmatine occurs through the modulation of calcium-sensing receptor (CaSR). In isolated cardiocytes of hibernating ground squirrels generating stationary Ca2+ transients in the absence of actual myocellular excitation, low doses of this polyamine (up to 500 μM) induce the Gβγ-dependent activation of PI3-kinase with subsequent stimulation of Akt-kinase and nitric oxide (NO) production by endothelial NO-synthase (eNOS). NO production abolishes Ca2+ oscillations in virtue of the enhancement of Ca2+ reuptake by sarco(endo)plasmic Ca2+ ATPase (SERCA). Simultaneously, the activation of phospholipase A2 (PLA2) and arachidonic-acid dependent Ca2+ entry occur providing replenishment of Ca2+ store. High concentrations of agmatine (> 2 mM) induce other CaSR-mediated pathways involving phospholipase C (PLC) pathway, the formation of inositoltriphosphate (IP3) and diacylglicerol (DAG) followed by induction of their targets: IP3 receptors and protein kinase C isoforms (PKC), respectively. Furthermore, it is also responsible for the stimulation of PLA2 and elevation of intracellular calcium caused by arachidonic acid-regulated Ca2+-permeable (ARC) channels. Additionally, there is a potent store-operated Ca2+ entry (SOC) in cardiomyocyte. Negative (NPS 2143) and positive (R 568) allosteric modulators of CaSR recapitulate effects of low and high agmatine doses on Ca2+ handling and NO synthesis. These facts and the alteration of agmatine influence in response to an increase of extracellular Ca2+, which is the direct agonist of CaSR, may confirm the participation of CaSR in regulation of Ca2+ handling and excitability of cardiomyocytes by agmatine.

Inhibition of calcium/calmodulin-dependent kinase II restores contraction and relaxation in isolated cardiac muscle from type 2 diabetic rats

BackgroundCalcium/calmodulin-dependent kinase II-delta (CaMKIIδ) activity is enhanced during hyperglycemia and has been shown to alter intracellular calcium handling in cardiomyocytes, ultimately leading to reduced cardiac performance. However, the effects of CaMKIIδ on cardiac contractility during type 2 diabetes are undefined.MethodsWe examined the expression and activation of CaMKIIδ in right atrial appendages from non-diabetic and type 2 diabetic patients (n = 7 patients per group) with preserved ejection fraction, and also in right ventricular tissue from Zucker Diabetic Fatty rats (ZDF) (n = 5–10 animals per group) during early diabetic cardiac dysfunction, using immunoblot. We also measured whole heart function of ZDF and control rats using echocardiography. Then we measured contraction and relaxation parameters of isolated trabeculae from ZDF to control rats in the presence and absence of CaMKII inhibitors.ResultsCaMKIIδ phosphorylation (at Thr287) was increased in both the diabetic human and animal tissue, indicating increased CaMKIIδ activation in the type 2 diabetic heart. Basal cardiac contractility and relaxation were impaired in the cardiac muscles from the diabetic rats, and CaMKII inhibition with KN93 partially restored contractility and relaxation. Autocamtide-2-related-inhibitor peptide (AIP), another CaMKII inhibitor that acts via a different mechanism than KN93, fully restored cardiac contractility and relaxation.ConclusionsOur results indicate that CaMKIIδ plays a key role in modulating performance of the diabetic heart, and moreover, suggest a potential therapeutic role for CaMKII inhibitors in improving myocardial function during type 2 diabetes.