Calcium Homeostasis In Cardiomyocytes Research Articles

ZEB2 regulates a transcriptional network of calcium-handling genes in the injured heart

Abstract Intracellular calcium (Ca2+) overload is known to play a critical role in the development of cardiac dysfunction. Despite the remarkable progress in managing the progression of the disease, the development of effective therapies for heart failure (HF) remains challenging. Therefore, it is of great importance to understand the molecular mechanisms that maintain calcium level and contractility in homeostatic conditions. Here we identified a transcription factor ZEB2 that regulates the expression of numerous contractile and calcium-related genes. Zinc finger E-box-binding homeobox2 (ZEB2) is a transcription factor that plays a role during early fetal development and epithelial-to-mesenchymal transition (EMT); however, its function in the heart remains to be determined. Recently, we found that ZEB2 is upregulated in murine cardiomyocytes shortly after an ischemic event, but returns to baseline levels as the disease progresses. Gain- and loss-of-function genetic mouse models revealed the necessity and sufficiency of ZEB2 to maintain proper cardiac function after ischemic injury. We show that cardiomyocyte-specific ZEB2 overexpression (Zeb2 cTG) protected from ischemia-induced diastolic dysfunction and attenuated the structural remodeling of the heart. Moreover, RNA-sequencing of Zeb2 cTG hearts post-injury implicated ZEB2 in the regulation of numerous calcium-handling and contractile-related genes when compared to wildtype mice. Mechanistically, ZEB2 overexpression increased the phosphorylation of phospholamban (PLN) at both serine-16 and threonine-17, implying enhanced activity of the sarcoplasmic reticulum Ca2+-ATPase (SERCA2A), thereby augmenting contractility. Improved cardiac function in ZEB2-overexpressing hearts correlated with higher expression of several sarcomeric proteins like myosin-binding protein C3 (MYBPC3), desmin (DES) and myosin regulatory light chain 2 (MYL2) further contributing to the observed protective phenotype. Furthermore, we observed a decrease in the activity of Ca2+-depended calcineurin/NFAT signaling, which is the main driver of pathological cardiac remodeling. Conversely to Zeb2 cTg mice, loss of ZEB2 from cardiomyocytes perturbed the expression of calcium- and contractile-related proteins and increased the activity of calcineurin/NFAT pathway, exacerbating cardiac dysfunction. Together, we show that ZEB2 is a central regulator of contractile and calcium-handling components, consequently mediating contractility in the mammalian heart. Further mechanistic understanding of the role of ZEB2 in the regulation of calcium homeostasis in cardiomyocytes is a critical step towards the development of improved therapies for various forms of heart failure. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): DR. E. Dekker from Dutch Heart Foundation

Klotho modulates electrical activity and calcium homeostasis in pulmonary vein cardiomyocytes via PI3K/Akt signalling.

Klotho, a potential antiageing protein has remarkable cardiovascular effects, which is lower in the patients with chronic kidney disease (CKD). Chronic kidney disease increases the risk of atrial fibrillation, majorly triggered by pulmonary vein (PV) arrhythmogenesis. This study investigated whether klotho protein can modulate PV electrical activity and the underlying potential mechanisms. A conventional microelectrode and whole-cell patch clamp were used to investigate the action potentials and ionic currents in isolated rabbit PV tissue preparations and single cardiomyocytes before and after klotho administration. Phosphoinositide 3-kinase (PI3K)/Akt signalling was studied using western blotting. Klotho significantly reduced PV spontaneous beating rates in PV tissue preparations at 1.0 and 3.0 ng/mL (but not at 0.1 and 0.3 ng/mL). In the presence of the Akt inhibitor (10 µM), klotho (1.0 and 3.0 ng/mL) did not change PV electrical activities. Klotho (1.0 ng/mL) significantly decreased the late sodium current (INa-Late) and L-type calcium current (ICa-L), similar to the Akt inhibitor (10 µM). Western blots demonstrated that klotho (1.0 ng/mL)-treated PV cardiomyocytes had less phosphorylation of Akt (Ser473) compared with klotho-untreated cardiomyocytes. Compared with control PVs, klotho at relatively lower concentrations (0.1 and 0.3 ng/mL) significantly reduced beating rates and decreased the amplitudes of delay afterdepolarizations in CKD PVs. Klotho modulated PV electrical activity by inhibiting PI3K/Akt signalling, which may provide a novel insight into CKD-induced arrhythmogenesis.

Nogo-C causes post-MI arrhythmia through increasing calcium leakage from sarcoplasmic reticulum

Disturbance of calcium homeostasis in cardiomyocytes plays a pivotal role in the pathogenesis of ventricular arrhythmias after myocardial infarction(MI). Nogo-C is a sarcoplasmic reticulum (SR) protein ubiquitously expressed in tissues including heart. We previously reported that Nogo-C played a critical role in the pathogenesis of MI through inducing cardiomyocyte apoptosis. However, the role and mechanisms of Nogo-C in the regulation of calcium homeostasis and in the pathogenesis of arrhythmia post-MI remain unclear. In the present study, wildtype and Nogo-C knockout mice were subjected with MI operation followed by intraperitoneal injection of caffeine and isoproterenol to induce arrhythmia. We found that Nogo-C knockout mice displayed lower incidence of arrhythmia compared with the high incidence of arrhythmia of wildtype mice. Moreover, Nogo-C overexpression by adenovirus in cardiomyocytes caused decreased SR calcium content, reduced contractile ability, and increased diastolic cytoplasma calcium concentration, as compared with control cells. Oxalate-facilitated 45Ca uptake assay showed no difference of SR calcium reuptake ability between control and Nogo-C overexpressing cardiomyocytes, indicating that the decreased SR calcium content was not through disturbing SR calcium reuptake in Nogo-C overexpressing cells. In contrast, the rate of SR calcium leak, spontaneous calcium wave, and calcium spark markedly increased in Nogo-C overexpressing cardiomyocytes, suggesting that the increased SR calcium leak causes the decreased SR calcium content. Furthermore, We found that Nogo-C overexpression markedly increased, while Nogo-C knockdown decreased Sec61 protein, a newly found calcium leak channel, in cardiomyocytes. Nogo-C bound with Sec61 through its transmembrane domains loop-6 and loop-8, thus inhibited the ubiquitination of Sec61, leading to the increased calcium leak from SR. Together, our data show that Nogo-C is increased in MI hearts, and the increased Nogo-C inhibits the degradation of Sec61 by ubiquitination, leading to increased SR calcium leak, thus promoting arrhythmia after MI.

20th Annual Feigenbaum Lecture: Echocardiography for Precision Medicine—Digital Biopsy to Deconstruct Biology

Heart failure with preserved ejection fraction (HFpEF) is a complex, heterogeneous syndrome in need of improved classification given its high morbidity and mortality and few effective treatment options. HFpEF represents an ideal setting to examine the utility and feasibility of a precision medicine approach. This article (based on the 20th annual Feigenbaum Lecture, presented at the 2019 American Society of Echocardiography Scientific Sessions) describes the utility of echocardiography as a "digital biopsy" and how deep quantitative echocardiographic phenotyping, coupled with machine learning, can be used to identify novel HFpEF phenotypes. The cellular and ultrastructural basis of abnormal speckle-tracking echocardiography- (STE-) based measurements of cardiac mechanics can provide a window into cardiomyocyte calcium homeostasis. STE-based measurements of longitudinal strain can thus inform the extent of myocardial involvement in patients with HFpEF, which may help to determine responsiveness to cardiac-specific HF medications. However, classifying the complex, systemic, multiorgan nature of HFpEF appropriately likely requires more advanced methods. Using unsupervised machine learning, HFpEF can be classified into three distinct phenogroups with differing clinical and echocardiographic characteristics and outcomes: (1) natriuretic peptide deficiency syndrome; (2) extreme cardiometabolic syndrome; and (3) right ventricle-cardio-abdomino-renal syndrome. Each can be probed to determine their biological basis. The goal of improved classification of HFpEF is to match the right patient with the right treatment, with the hope of improving the track record of HFpEF clinical trials. This article emphasizes the central role of echocardiography in advancing precision medicine and illustrates the integration of basic, translational, clinical, and population research in echocardiography with the goal of better understanding the pathobiology of a complex cardiovascular syndrome.

Macrophage migration inhibitory factor increases atrial arrhythmogenesis through CD74 signaling

Macrophage migration inhibitory factor (MIF), a pleiotropic inflammatory cytokine, is highly expressed in patients with atrial fibrillation (AF). CD74 (major histocompatibility complex, class II invariant chain) is the main receptor for MIF. However, the role of the MIF/CD74 axis in atrial arrhythmogenesis is unclear. In this study, we investigated the effects of MIF/CD74 signaling on atrial electrophysiological characteristics and determined its underlying mechanisms. Confocal fluorescence microscopy, patch clamp, and western blot analysis were used to study calcium homeostasis, ionic currents, and calcium-related signaling in MIF-treated HL-1 atrial cardiomyocytes with or without anti-CD74 neutralized antibodies treatment. Furthermore, electrocardiographic telemetry recording and echocardiography were obtained from mice treated with MIF. Compared with controls, MIF-treated HL-1 myocytes had increased calcium transients, sarcoplasmic reticulum (SR) calcium content, Na+/Ca2+ exchanger (NCX) efflux rate, calcium leak, transient outward potassium current, and ultra-rapid delayed rectifier potassium current. Furthermore, MIF could induce expression of SR Ca2+ATPase, NCX, phosphorylation of ryanodine receptor 2 (RyR2), and activation of calcium/calmodulin kinase II (CaMKII) when compared with control cells. MIF-mediated electrical dysregulation and CaMKII-RyR2 signaling activation were attenuated through blocking of CD74. Moreover, MIF-injected mice had lesser left atrium fractional shortening, greater atrial fibrosis, and atrial ectopic beats than control (nonspecific immunoglobulin treated) or MIF combined with anti-CD74 neutralized antibody-treated mice. Consequently, our study on MIF/CD74 signaling has pointed out a new potential therapeutic intervention of AF patients with MIF elevation.

Arginine vasopressin modulates electrical activity and calcium homeostasis in pulmonary vein cardiomyocytes

BackgroundAtrial fibrillation (AF) frequently coexists with congestive heart failure (HF) and arginine vasopressin (AVP) V1 receptor antagonists are used to treat hyponatremia in HF. However, the role of AVP in HF-induced AF still remains unclear. Pulmonary veins (PVs) are central in the genesis of AF. The purpose of this study was to determine if AVP is directly involved in the regulation of PV electrophysiological properties and calcium (Ca2+) homeostasis as well as the identification of the underlying mechanisms.MethodsPatch clamp, confocal microscopy with Fluo-3 fluorescence, and Western blot analyses were used to evaluate the electrophysiological characteristics, Ca2+ homeostasis, and Ca2+ regulatory proteins in isolated rabbit single PV cardiomyocytes incubated with and without AVP (1 μM), OPC 21268 (0.1 μM, AVP V1 antagonist), or OPC 41061 (10 nM, AVP V2 antagonist) for 4–6 h.ResultsAVP (0.1 and 1 μM)-treated PV cardiomyocytes had a faster beating rate (108 to 152%) than the control cells. AVP (1 μM) treated PV cardiomyocytes had higher late sodium (Na+) and Na+/Ca2+ exchanger (NCX) currents than control PV cardiomyocytes. AVP (1 μM) treated PV cardiomyocytes had smaller Ca2+i transients, and sarcoplasmic reticulum (SR) Ca2+ content as well as higher Ca2+ leak. However, combined AVP (1 μM) and OPC 21268 (0.1 μM) treated PV cardiomyocytes had a slower PV beating rate, larger Ca2+i transients and SR Ca2+ content, smaller late Na+ and NCX currents than AVP (1 μM)-treated PV cardiomyocytes. Western blot experiments showed that AVP (1 μM) treated PV cardiomyocytes had higher expression of NCX and p-CaMKII, and a higher ratio of p-CaMKII/CaMKII.ConclusionsAVP increases PV arrhythmogenesis with dysregulated Ca2+ homeostasis through vasopressin V1 signaling.

Maurocalcine effects on calcium homeostasis in adult rat cardiomyocytes and human iPS cells derived cardiomyocytes

Ryanodine receptors type 2 (RyR2) the intracellular channels that control excitation-contraction (EC) coupling in cardiomyocytes represent an important target for treatment of arrhythmias accompanying cardiac pathologies. Maurocalcine (MCa), a peptide isolated from scorpion venom, has been described as a high affinity activator of RyR in skeletal muscle. Phosphorylation of MCa has been shown to induce a complete remodelling of its effect on RyR, making it a negative allosteric modulator of RyR. The purpose of this study is to characterize the effects of MCa and different analogues on EC coupling in isolated adult ventricular cardiomyocytes and hiPS-derived cardiomyocytes (hiPS-CMs). Adult ventricular myocytes were isolated from 3–6 months-old Sprague Dawley rats with conventional collagenase type II and protease type XIV mix, and hiPS-CMs were obtained from urine cells of healthy volunteers. Changes of cytoplasmic calcium concentration were measured using Fura-2 and IonOptix software. hiPS-CMs contractions and extrafield potentials were recorded with CardioExcyte system (Nanion). We first confirmed binding between MCa and RyR2. Next, we showed that MCa (500nM) induces asynchronous calcium release and contractions of isolated adult rat cardiomyocytes under electrical stimulation at 1Hz. Similar results were observed on hiPS-CMs in the presence of MCa. Effects of native MCa and analogues on isoprenaline-mediated stimulation of RyR2 are under investigation. Our results demonstrate that MCa penetrates adult cardiomyocytes as well as human iPS derived cardiomyocytes and modulates RyR2 activity. MCa analogues exhibit different effects on RyR2, from positive to negative allosteric modulation. These results provide promising evidence that MCa could be used as an in cellulo modulator of RyR2 to control abnormal RyR activity in pathological situations.

Sex differences in distribution of cannabinoid receptors (CB1 and CB2), S100A6 and CacyBP/SIP in human ageing hearts

BackgroundWomen live about 4 years longer due to lower prevalence of cardiovascular complication with ageing. However, the mechanisms involved in the preservation of heart functionality in women have not been fully elucidated.The endocannabinoid system fulfils a significant role in the regulation of cardiovascular system functioning. Cannabinoids, acting through specific receptors (CB1 and CB2), influence on blood pressure, heart rate and myocardial contractility. The function of cardiac muscle cells is strictly dependent on calcium ions. Calcium homeostasis in cardiomyocytes is subjected to complex regulation via calcium-binding proteins. Among them, increasing attention has been paid to the recently discovered S100A6 and CacyBP/SIP.In order to better understand sex differences in the regulation of cardiomyocyte function during ageing, we undertook the present research aimed at immunohistochemical identification and comparative evaluation of cannabinoid receptors, S100A6 and CacyBP/SIP, in the myocardium of ageing men and women.MethodsThe study was conducted on the hearts of 12 men and 10 women (organ donors) without a history of cardiovascular disease. The subjects were divided into two age groups: subjects older than 50 years and subjects under 50 years old. Paraffin heart sections were processed by immunohistochemistry for detection of cannabinoids receptors, S100A6 and CacyBP/SIP. In the heart samples from each study, participant’s expression of genes coding for CB1, CB2, S100A6 and CacyBP/SIP using real-time PCR method was measured.ResultsCB1 and CB2 immunoreactivity in the cytoplasm of cardiomyocytes in the heart of subjects over 50 was weaker than in younger individuals. In the heart of younger men, CB1-immunoreactivity was weaker and CB2-immunoreaction was stronger compared to women. In the hearts of older men, the CB1-immunostaining was more intense and CB2-immunoreactivity was weaker than in women. Immunodetection of CB1 shoved the presence of receptor in the intercalated discs, but only in the hearts of individuals over the 50 years old. In the hearts of older individuals, stronger immunolabelling was observed for S100A6 and CacyBP/SIP. Male hearts had greater S100A6-immunoreactivity (both age groups) but less CacyBP/SIP immunostaining (individuals over 50 years) compared to the age-matched women. The expression of genes coding CB1, CB2, S100A6 and CacyBP/SIP in the human heart was sex and age-dependent. Observed changes between men and women as well as between subject under and over 50 years were consistent with immunohistochemically stated changes in peptide content.ConclusionTogether, the data presented here indicate a close interaction between ageing and sex on the distribution and levels of cannabinoid receptors (CB1, CB2), S100A6 and CacyBP/SIP in the human heart.

Deteriorated gene expression of selected calcium transporters in streptozotocin-induced diabetic hearts of Wistar rats

Abstract Aim: The aim is to identify the possible changes in the expression of genes, that regulate calcium homeostasis in cardiomyocytes in diabetes mellitus. Methods: Male Wistar rats were randomized into two experimental protocols: short-term 5-days streptozotocin-induced diabetes protocol with 20 weeks old animals at the end of the protocol (total N = 20) and long-term 4-weeks protocol with 18 weeks of age at the end of the protocol (total N = 38). 50 mg/kg of streptozotocin (STZ) was administered in both protocols by a single intraperitoneal injection in 0,1M citrate buffer (pH = 4.5). Control group (CON) received only vehiculum. Gene expressions in samples of left heart ventricle were measured by RT-qPCR method. Results: The expression of SERCA2a in short-term protocol was decreased. In long-term protocol, decreased SERCA2a, TRPC4 and TRPC6 mRNA levels were observed (*p < 0.05). SERCA2a and TRPC4 mRNA levels exhibited statistical monotonic correlation in STZ-treated group in long-term protocol. Conclusions: In diabetes mellitus, the calcium homeostasis in cardiomyocytes is altered and there could be a relation between alteration of internal sarcoplasmatic stores and store-operated calcium entry.

Abstract 21413: Molecular Basis of Maternal Obesity Induced Fetal Cardiac Contractile Dysfunction

One third of the women at reproductive age are overweight or obese in US. Epidemiological studies in humans and studies on animals have shown that obesity during pregnancy ( i.e. maternal obesity, MO) increases incidences of cardiovascular diseases in offspring. Uterus environment is essential for fetal cardiac development and MO as a stress condition in uterus could program cardiovascular disease in prenatal and postnatal offspring. However, the mechanisms of the origin and development of MO-induced cardiovascular disease later in life remain unclear. For this purpose, we use sheep (sharing many similarities with human pregnancy) as a model to study the impact of MO on fetal heart contractile function. Previously, we have demonstrated that MO impaired fetal sheep cardiomyocyte contractility by reducing the peak shortening and velocity of shortening/relengthening and prolonged the time to peak shortening/relengthening. We also found that MO disrupted the calcium homeostasis in fetal cardiomyocytes by increasing intracellular Ca 2+ level and inducing the calcium insensitivity in the cell. In current study, we found that MO increased the phosphorylation of protein kinase A (PKA) as well as the phosphorylation of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII), which further promoted phosphorylation level of ryanodine receptor type 2 (Ryr2) at Ser2808 and Ser2814 respectively. Expression level of FKBP12.6, a key protein interacting with Ryr2 for calcium releasing, was upregulated in MO fetal myocardium. On the other hand, MO reduced expression of myosin heavy chain in fetal myocardium. Moreover, the cardiac troponin T and troponin I expression level was upregulated in MO fetuses, whereas MO downregulated the expression of troponin C in fetal myocardium. Taken together, our findings suggest that increased CaMKII and PKA phosphorylation of Ryr2 plays a critical role in the development of MO-induced sarcoplasmic reticulum Ca 2+ leak in fetal cardiomyocytes. Furthermore, our data indicate that the change in expression levels of myosin heavy chain and troponin C, I and T is the cause of calcium insensitivity and contractile dysfunction in MO fetal cardiomyocytes. Our studies have provided a fundamental molecular basis of MO programmed heart disease.

Redox and Activation of Protein Kinase A Dysregulates Calcium Homeostasis in Pulmonary Vein Cardiomyocytes of Chronic Kidney Disease.

BackgroundChronic kidney disease (CKD) increases the occurrence of atrial fibrillation and pulmonary vein (PV) arrhythmogenesis. Calcium dysregulation and reactive oxygen species (ROS) enhance PV arrhythmogenic activity. The purposes of this study were to investigate whether CKD modulates PV electrical activity through dysregulation of calcium homeostasis and ROS.Methods and ResultsBiochemical and electrocardiographic studies were conducted in rabbits with and without CKD (induced by 150 mg/kg per day neomycin sulfate and 500 mg/kg per day cefazolin). Confocal microscopy with fluorescence and a whole‐cell patch clamp were applied to study calcium homeostasis and electrical activities in control and CKD isolated single PV cardiomyocytes with or without treatment with H89 (1 μmol/L, a protein kinase A inhibitor) and MPG (N‐[2‐mercaptopropionyl]glycine; 100 μmol/L, a ROS scavenger). The ROS in mitochondria and cytosol were evaluated via intracellular dye fluorescence and lipid peroxidation. CKD rabbits had excessive atrial premature captures over those of control rabbits. Compared with the control, CKD PV cardiomyocytes had a faster beating rate and larger calcium transient amplitudes, sarcoplasmic reticulum calcium contents, sodium/calcium exchanger currents, and late sodium currents but smaller L‐type calcium current densities. CKD PV cardiomyocytes had a higher frequency and longer duration of calcium sparks and more ROS in the mitochondria and cytosol than did controls. Moreover, H89 suppressed all calcium sparks in CKD PV cardiomyocytes, and H89‐ and MPG‐treated CKD PV cardiomyocytes had similar calcium transients compared with control PV cardiomyocytes.Conclusions CKD increases PV arrhythmogenesis with enhanced calcium‐handling abnormalities through activation of protein kinase A and ROS.

TRPV4 Enhances Cardiomyocyte Calcium Transients and Cardiac Contractility Following Hypoosmotic Stress and Ischemia-Reperfusion

The Transient Receptor Potential (TRP) ion channel family plays an important role in cardiomyocyte calcium homeostasis, particularly in diseased states. Here, we test the hypothesis that the osmotically-activated TRP Vanilloid 4 (TRPV4) channel enhances cardiomyocyte calcium transients in the aged heart following hypoosmotic stress. Expression of TRPV4 was low in cardiomyocytes of Young (3-6 month) mice, but significantly increased in cardiomyocytes of Aged (24-26 month) mice. In cardiomyocytes of Aged, hypoosmotic stress (250 mOsm) induced an increase in calcium transient amplitude (F/F0:3.6±0.1 hypoosmotic versus 2.9±0.2 isosmotic, P<0.05). This effect was prevented by the TRPV4 inhibitor HC067047 (1 μM, F/F0:2.9±0.1 hypoosmotic) and was absent in cardiomyocytes of Young mice (F/F0:3.2±0.1 hypoosmotic versus 2.9±0.2 isosmotic). However, cardiomyocytes of Young mice with cardiac-specific TRPV4 Overexpression exhibited enhanced calcium transient amplitude following hypoosmotic stress (F/F0:3.5±0.2 hypoosmotic versus 2.4±0.1 isosmotic, P<0.05) that was prevented by HC067047 (F/F0:2.6±0.1 hypoosmotic). Ischemia-reperfusion (I-R) injury is a pathological scenario associated with pronounced osmotic stress on cardiomyocytes. We therefore monitored left-ventricular pressure development in Langendorff-perfused hearts of Young, Aged, Young TRPV4 Overexpressor, and Aged TRPV4 knock-out mice subjected to 45 minute global ischemia followed by reperfusion. Prior to ischemia, all hearts exhibited similar contractility (dP/dtMax range: 1934±206 to 2226±62 mmHg/s). Following I-R, hearts of Aged mice exhibited enhanced contractile performance (dP/dtMax: 2770±180 mmHg/s) versus hearts of Aged TRPV4 knock-out mice (dP/dtMax: 1400±300 mmHg/s, P<0.05). Similarly, hearts of Young TRPV4 Overexpressor mice exhibited enhanced contractile performance (dP/dtMax: 2750±180 mmHg/s) versus hearts of Young mice (dP/dtMax: 1650±260 mmHg/s, P<0.05). In conclusion, TRPV4 enhances calcium transients following hypoosmotic stress and contributes to hypercontractility following I-R. Our findings have potential clinical implications in the treatment of elderly populations at increased risk of myocardial infarction and reperfusion injury.

Glycosylated Chromogranin A in Heart Failure: Implications for Processing and Cardiomyocyte Calcium Homeostasis.

Chromogranin A (CgA) levels have previously been found to predict mortality in heart failure (HF), but currently no information is available regarding CgA processing in HF and whether the CgA fragment catestatin (CST) may directly influence cardiomyocyte function. CgA processing was characterized in postinfarction HF mice and in patients with acute HF, and the functional role of CST was explored in experimental models. Myocardial biopsies from HF, but not sham-operated mice, demonstrated high molecular weight CgA bands. Deglycosylation treatment attenuated high molecular weight bands, induced a mobility shift, and increased shorter CgA fragments. Adjusting for established risk indices and biomarkers, circulating CgA levels were found to be associated with mortality in patients with acute HF, but not in patients with acute exacerbation of chronic obstructive pulmonary disease. Low CgA-to-CST conversion was also associated with increased mortality in acute HF, thus, supporting functional relevance of impaired CgA processing in cardiovascular disease. CST was identified as a direct inhibitor of CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) activity, and CST reduced CaMKIIδ-dependent phosphorylation of phospholamban and the ryanodine receptor 2. In line with CaMKIIδ inhibition, CST reduced Ca2+ spark and wave frequency, reduced Ca2+ spark dimensions, increased sarcoplasmic reticulum Ca2+ content, and augmented the magnitude and kinetics of cardiomyocyte Ca2+ transients and contractions. CgA-to-CST conversion in HF is impaired because of hyperglycosylation, which is associated with clinical outcomes in acute HF. The mechanism for increased mortality may be dysregulated cardiomyocyte Ca2+ handling because of reduced CaMKIIδ inhibition.

Restoring calcium homeostasis in diabetic cardiomyocytes: an investigation through mathematical modelling

Calcium homeostasis is a key factor in the regulation of cardiac excitation-contraction coupling. Calcium dynamics in cardiomyocytes is governed by ATP which depends on insulin dependent glucose concentration, via the glucose transporter type 4 (GLUT4) transporter. It would therefore be interesting to see how calcium dynamics changes in a cardiomyocyte under diabetic conditions. We proposed and analysed a four dimensional ordinary differential equation (ODE) model to capture the interdependency of calcium dynamics on glucose uptake and ATP generation. More specifically, we looked for the role of GLUT4, energy metabolism, L-type channels, RyR2 channels, SERCA2a pumps and leakage rate in the normal functioning of cardiomyocytes. To understand the system dynamics, we first obtained the stability and Hopf-bifurcation criteria of steady state and then through parameter perturbation we captured the role of different parameters in maintaining normal calcium oscillation (frequency 40 to 180 beats per minute and amplitude ≥0.4 μM) and hence normal cardiac function. We observed that any divergence in the GLUT4 activity (especially a decrease in the glucose uptake rate) might cause abnormal calcium oscillation, leading to cardiac dysfunction (CD). Our study finally hypothesizes that a regulated sarcoplasmic reticulum (SR) calcium flux could be a possible therapeutic strategy to maintain normal calcium dynamics in diabetic heart and to prevent possible CD.

Disruption of calcium homeostasis by cardiac-specific over-expression of PPAR-γ in mice: A role in ventricular arrhythmia

AimsAdverse cardiovascular effects induced by peroxisome proliferator activator receptor-γ (PPAR-γ) activation were observed in clinical setting. But the underlying mechanism is unclear. Now, transgenic mice with cardiac specific peroxisome proliferator activator receptor-γ overexpression (TG-PPAR-γ) were used to explore the possible mechanisms. Materials and methodsCardiac tissues from TG-PPAR-γ mice, a PPAR-γ over-expressing human cardiomyocyte line AC16 cell, and PPAR-γ agonist-treated primary cardiomyocytes were used to evaluate the expression of cardiac calcium regulatory proteins as sarcoplasmic reticulum Ca2+ ATPase, Na+/Ca2+ exchanger 1, ryanodine receptor 2 and phospholamban. Intracellular Ca2+ levels were also examined by flow cytometry and confocal microscopy with Fluo-4/AM in these cells. Key findingsIn this study, frequent ventricular premature contraction and polymorphic ventricular tachycardia were observed in TG-PPAR-γ but not in wild-type mice. Besides, we found the calcium regulatory proteins expression were higher in the TG-PPAR-γ mice, PPAR-γ overexpressing human cardiomyocyte line AC16 cell and PPAR-γ agonist-treated primary cardiomyocytes than the control group respectively. In addition, an increase of intracellular calcium levels and CaMKII δ expression in PPAR-γ overexpression and PPAR-γ activation group. Moreover, Inhibition of CaMKII δ could improve the intracellular calcium levels and reduce the occurrence of ventricular arrhythmia. SignificancePPAR-γ over-expression perturbs the intracellular calcium homeostasis in cardiomyocytes which contribute to the ventricular arrhythmias and cardiac sudden death in TG-PPAR-γ mice.

Fibroblast growth factor 23 dysregulates late sodium current and calcium homeostasis with enhanced arrhythmogenesis in pulmonary vein cardiomyocytes

Fibroblast growth factor 23 (FGF23), elevated in chronic renal failure, increases atrial arrhythmogenesis and dysregulates calcium homeostasis. Late sodium currents (INa-Late) critically induces ectopic activity of pulmoanry vein (the most important atrial fibrillation trigger). This study was to investigate whether FGF23 activates the INa-Late leading to calcium dysregulation and increases PV arrhythmogenesis. Patch clamp, western blot, and confocal microscopy were used to evaluate the electrical activities, calcium homeostasis, and mitochondrial reactive oxygen species (ROS) in PV cardiomyocytes with or without FGF23 (0.1 or 1 ng/mL) incubation for 4~6 h. Compared to the control, FGF23 (1 ng/mL, but not 0.1 ng/mL)-treated PV cardiomyocytes had a faster beating rate. FGF23 (1 ng/mL)-treated PV cardiomyocytes had larger INa-Late, calcium transients, and mitochondrial ROS than controls. However, ranolazine (an inhibitor of INa-Late) attenuated FGF23 (1 ng/mL)-increased beating rates, calcium transients and mitochondrial ROS. FGF23 (1 ng/mL)-treated PV cardiomyocytes exhibited larger phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII). Chelerythrine chloride (an inhibitor of protein kinase C) decreased INa-Late in FGF23 (1 ng/mL)-treated PV cardiomyocytes. However, KN93 (a selective CaMKII blocker) decreased INa-Late in control and FGF23 (1 ng/mL)-treated PV cardiomyocytes to a similar extent. In conclusion, FGF23 increased PV arrhythmogenesis through sodium and calcium dysregulation by acting protein kinase C signaling.

0346 : Characterization of the calcium deregulation in cardiomyocytes from mdx mice, the main rodent model of the Duchenne muscular dystrophy

The Duchenne muscular dystrophy (DMD) is caused by an absence or mutation of the protein dystrophin, which leads to smooth, skeletal and cardiac muscles degeneration. Intracellular calcium deregulation, notably abnormally high cytosolic calcium concentration, is thought to contribute to muscle necrosis and to cardiomyopathy development. Despite its importance in cell necrosis, the mechanisms involved in this calcium deregulation are not fully understood. In our study, we have investigated several parameters of the calcium homeostasis in cardiomyocytes from mdx mice, the main rodent model of the DMD. All the parameters have been measured by using confocal microscopy calcium imaging experiments in cardiomyocytes loaded with the calcium indicator Fluo-3AM. Our results show an increase of the occurrence of spontaneous calcium events such as a higher frequency of calcium sparks and waves in the mdx cardiomyocytes compared with wild type mice. We also found that the calcium sensitivity of the ryanodine receptors (RyRs) is increased in cardiomyocytes from mdx mice. Finally, the sarcoplasmic reticulum (SR) calcium content has been evaluated by caffeine-induced calcium release. We found reduced calcium content of the SR in the mdx mice, which could be explained, at least in part, by an excessive spontaneous calcium activity due to the enhanced RyRs sensitivity. Our data suggest that targeting this SR calcium “leak” with pharmacological tools could be beneficial for the DMD patients. The author hereby declares no conflict of interest

Glucagon-like peptide-1 regulates calcium homeostasis and electrophysiological activities of HL-1 cardiomyocytes

Glucagon like-peptide-1 (GLP-1) is an incretin hormone with antidiabetic effects through stimulating insulin secretion, β cell neogenesis, satiety sensation, and inhibiting glucagon secretion. Administration of GLP-1 provides cardioprotective effects through attenuating cardiac inflammation and insulin resistance. GLP-1 also modulates the heart rate and systolic pressure, which suggests that GLP-1 may have cardiac electrical effects. Therefore, the purposes of this study were to evaluate whether GLP-1 has direct cardiac effects and identify the underlying mechanisms. Patch clamp, confocal microscopy with Fluo-3 fluorescence, and Western blot analyses were used to evaluate the electrophysiological characteristics, calcium homeostasis, and calcium regulatory proteins in HL-1 atrial myocytes with and without GLP-1 (1 and 10nM) incubation for 24h. GLP-1 (1 and 10nM) and control cells had similar action potential durations. However, GLP-1 at 10nM significantly increased calcium transients and sarcoplasmic reticular Ca2+ contents. Compared to the control, GLP-1 (10nM)—treated cells significantly decreased phosphorylation of the ryanodine receptor at S2814 and total phospholamban, but there were similar protein levels of sarcoplasmic reticular Ca2+-ATPase and the sodium–calcium exchanger. Moreover, exendin (9–39) amide (a GLP-1 receptor antagonist, 10nM) attenuated GLP-1-mediated effects on total SR content and phosphorylated ryanodine receptor S2814. This study demonstrates GLP-1 may regulate HL-1 cell arrhythmogenesis through modulating calcium handling proteins.

Calmodulin kinase II inhibitor regulates calcium homeostasis changes caused by acute β-adrenergic receptor agonist stimulation in mouse ventricular myocytes.

Ca(2+)/calmodulin-dependent kinase II (CaMKII) is an important regulatory molecule under chronic β-adrenergic receptor agonist stimulation but cardiac diseases also occur when β-adrenergic elevated acutely in the circulation, of which the most harmful is lethal arrhythmia. The purpose of this study was to explore the effects of acute isoproterenol (ISO) stimulation on intracellular calcium handling and evaluate whether CaMKII inhibitor may change the effects caused by isoproterenol. Mouse ventricular myocytes were acutely isolated by enzymatic method and divided into four groups: control group, ISO group, KN-93 group, ISO + KN-93 group. The whole-cell patch clamp was used to study the effect of ISO and KN-93 on L-type calcium current (IL-Ca) in isolated ventricular myocytes. The technology of laser scanning confocal microscopy was used to record cardiomyocyte calcium transients after ISO and KN-93 were used. ISO significantly increased current density of IL-Ca (p < 0.01) and decreased the half activation voltage (p < 0.01), half inactivation voltage (p < 0.01), and the recovery time constant (p < 0.01). In the presence of CaMKII inhibitor, KN-93 decreased the increased current density of IL-Ca (p < 0.05), increased the reduced half activation voltage caused by ISO (p < 0.01), and prolonged the shortened recovery time constant caused by ISO (p < 0.01). In addition, KN-93 alone can change the activation, inactivation, and recovery kinetics of L-type calcium channels. Moreover, ISO significantly increased the Ca(2+) transient amplitude during both stimulation frequencies (0.5 Hz: p < 0.01, 1 Hz: p < 0.01) and was easy to induce calcium disorders; in the presence of KN-93, these changes were weakened (0.5 Hz: p < 0.05, 1 Hz: p < 0.05). Therefore, changes of the calcium homeostasis in cardiomyocytes caused by ISO can be adjusted by KN-93, thus KN-93 plays a vital role in regulating calcium homeostasis changes caused by ISO.

Orphan nuclear receptor Nur77 affects cardiomyocyte calcium homeostasis and adverse cardiac remodelling.

Distinct stressors may induce heart failure. As compensation, β-adrenergic stimulation enhances myocardial contractility by elevating cardiomyocyte intracellular Ca2+ ([Ca2+]i). However, chronic β-adrenergic stimulation promotes adverse cardiac remodelling. Cardiac expression of nuclear receptor Nur77 is enhanced by β-adrenergic stimulation, but its role in cardiac remodelling is still unclear. We show high and rapid Nur77 upregulation in cardiomyocytes stimulated with β-adrenergic agonist isoproterenol. Nur77 knockdown in culture resulted in hypertrophic cardiomyocytes. Ventricular cardiomyocytes from Nur77-deficient (Nur77-KO) mice exhibited elevated diastolic and systolic [Ca2+]i and prolonged action potentials compared to wild type (WT). In vivo, these differences resulted in larger cardiomyocytes, increased expression of hypertrophic genes, and more cardiac fibrosis in Nur77-KO mice upon chronic isoproterenol stimulation. In line with the observed elevated [Ca2+]i, Ca2+-activated phosphatase calcineurin was more active in Nur77-KO mice compared to WT. In contrast, after cardiac pressure overload by aortic constriction, Nur77-KO mice exhibited attenuated remodelling compared to WT. Concluding, Nur77-deficiency results in significantly altered cardiac Ca2+ homeostasis and distinct remodelling outcome depending on the type of insult. Detailed knowledge on the role of Nur77 in maintaining cardiomyocyte Ca2+ homeostasis and the dual role Nur77 plays in cardiac remodelling will aid in developing personalized therapies against heart failure.