Increase In Calcium Transient Amplitude Research Articles

The Role of NAADP-Mediated Endo-Lysosomal Calcium Release in the Cardiac Atria

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a highly potent calcium-mobilising second messenger which stimulates calcium release from endo-lysosomes via the opening of TPC2 channels. In cardiac ventricular myocytes, lysosomes form nano-junctions with the sarcoplasmic reticulum (SR) and mitochondria and exposure to NAADP (synthesised by CD38 upon beta-adrenergic stimulation), leads to an augmented calcium transient and SR calcium content, and contributes to the hypertrophy and arrhythmogenesis associated with chronic beta-adrenergic stress. We utilised transmission electron microscopy (TEM) and optical mapping in lapine and murine models to investigate endo-lysosomal calcium signalling in the cardiac atria. TEM of fixed, imbedded rabbit atrial myocytes or tissue (n=3 animals) confirmed that lysosomes form nano-junctions with both the SR (median distance 17nm, IQR 13-21nm, n=15 lysosomes) and mitochondria (median distance 13nm, IQR 10-57nm, n=16 lysosomes). These are close enough to have the potential for localised calcium signalling. In intact murine left atria, 10nM isoprenaline caused a 59.3±4.8% increase in calcium transient amplitude (n=6, fluorophore rhod-2). Abolishing endo-lysosomal calcium storage using 5µM Bafilomycin A1 (Baf) significantly reduced the calcium transient response to isoprenaline (to 35.3±2.7%, n=6, P<0.01). In CD38KO mice, left atrial responses were significantly reduced (35.9±2.6%, n=6) in comparison to wild-type controls (59.3±4.9%, n=6, P<0.01) and there was no longer an effect of Baf on isoprenaline response (35.9±2.6% and 33.6±4.0% in CD38KO control and Baf respectively, P>0.05, both n=6). Our data suggest that, endo-lysosomes can form nanojunctions for calcium signalling within cardiac atrial myocytes and that the endo-lysosomal NAADP pathway is functionally relevant during beta-adrenergic stimulation. The changes to and effects of this stereotyped architecture and pathway in atrial disease states are unknown and will be a vital avenue for future research.

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.

The TRPV4 Ion Channel Alters Intracellular Calcium Transients in Cardiomyocytes of Aged Mice

Cardiac excitation-contraction coupling (ECC) occurs via calcium-induced calcium release and is therefore critically dependent on precise regulation of intracellular calcium homeostasis. Cardiomyocytes of the aged heart exhibit alterations in calcium homeostasis which predisposes the heart to calcium overload, contractile dysfunction, and arrhythmia. In this investigation we tested the hypothesis that the transient receptor potential vanilloid member 4 (TRPV4) osmotically-sensitive cation channel is expressed in cardiomyocytes of the aged mouse heart and alters intracellular calcium transients. Western blot analysis of whole heart homogenates of Young (3-6 month) and Aged (24-26 month) C57BL/6 male mice revealed increased expression of TRPV4 protein in hearts of Aged. Enzymatically isolated left-ventricular cardiomyocytes were loaded with the calcium indicator dye fluo-4/AM and electrically stimulated at 0.5 Hz to induce intracellular calcium transients. Pharmacological activation of TRPV4 with the agonist RN1747 (100 nM) produced an increase in calcium transient amplitude in cardiomyocytes of Aged (F/F0:3.3±0.4 RN1747 versus 2.6±0.4 control, P<0.05), but not Young (F/F0:2.8±0.3 RN1747 versus 2.9±0.2 control) mice. Following hypo-osmotic stress (250 mOsm, 40 minutes), cardiomyocytes of Aged exhibited enhanced responsiveness to RN1747 (F/F0:4.3±0.6 RN1747 versus 3.5±0.4 control, P<0.05) which associated with altered transient decay kinetics and cellular calcium overload (7/8 cells); these effects were prevented by the TRPV4 antagonist HC067047 (1 uM; F/F0:2.9±0.3 RN1747+HC versus 3.0±0.2 HC, 0/5 cells). In conclusion, the TRPV4 ion channel is expressed in cardiomyocytes of the aged heart and alters calcium transients during ECC.

NAADP AS A NOVEL DOWNSTREAM MEDIATOR OF CARDIAC BETA-ADRENERGIC PATHWAY

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent endogenous Ca2+ mobilizing second messenger that is known to release Ca2+ from the acidic endolysosomal vesicles. Our previous work showed that intracellular application of NAADP led to the increase in contractions of ventricular cardiomyocytes, accompanied by an increase in sarcoplasmic reticulum Ca2+ load. Previously, it has been reported that beta-adrenergic activation increases NAADP level in guinea pig heart, and we therefore hypothesized that the inotropic effect of beta-adrenergic activation is partly mediated by NAADP. Two-pore channel 2 (Tpc2) is known to mediate NAADP-regulated endolysosomal calcium release. We showed that intracellular application of NAADP on electrically stimulated ventricular myocytes increased calcium transient amplitude in wild-type but not Tpc2-KO mice. Strikingly, Tpc2-KO ventricular cardiomyocytes also showed a reduced isoprenaline-induced increase in contraction and calcium transient amplitudes compared to wild-type mice, establishing the contribution of NAADP to the beta-adrenergic pathway-regulated inotropic effect. To dissect the downstream mechanism, we showed that Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor, KN93 preincubation abolished the NAADP-induced increase in calcium transient amplitude. This is consistent with CaMKII acting as a downstream effector of beta-adrenergic activation that amplifies SR calcium uptake. We report here that the NAADP signaling cascade is a novel downstream pathway of beta-adrenergic activation in ventricular cardiomyocytes. This pathway requires functional Tpc2 channels and is partly if not entirely mediated through the activation of CaMKII by the NAADP-released Ca2+, which subsequently enhances SR Ca2+ uptake, contributing to the inotropic effect of cardiac beta-adrenergic activation.

CaMKII-Mediated Amplification is Essential to NAADP Signalling in Cardiac Myocytes

NAADP is a highly potent endogenous Ca2+-mobilising second-messenger forming part of the beta-adrenergic response in cardiac myocytes12. Our previous work suggests NAADP causes Ca2+-release from acidic endolysosomal stores, leading to additional uptake by the SR12. Questions have arisen regarding whether the magnitude NAADP-mediated responses can be accounted for by acidic-store-mediated Ca2+ release through TPC channels34. This study aimed to confirm that TPC2 channels are required for NAADP responses in cardiac myocytes and investigate the possibility of amplification in the pathway.Transgenic mice were utilised to investigate the role of TPCs. Rapid application of NAADP-AM to WT murine ventricular myocytes elicited a significant increase in calcium transient amplitude (16±5%, P 0.05).NAADP photorelease in guinea pig atrial or ventricular myocytes caused a significant increase in calcium transient amplitude (of 37±8% and 38±9% respectively, both P 0.05, both measures, both cell types). Similarly, no changes in Ca2+ transients were observed during photorelease in the presence of the NAADP receptor antagonist, Ned-19 (P>0.05, both measures, both cell types).These data support the hypothesis that NAADP-induced Ca2+ release requires TPC2, and suggest CaMKII is the major effector for its actions in cardiac myocytes.1. Collins et al. (2011) Cell Calcium 50: 449.2. Macgregor et al. (2007) J Biol Chem 282: 15302.3. Pitt et al. (2010) J Biol Chem 285: 35039.4. Wang et al. (2012) Cell 151: 372.

The tumor suppressor RAS-association domain family protein 1A (RASSF1A) regulates TNF-alpha signaling in cardiomyocytes

Purpose: Tumour necrosis factor-α (TNF-α) is a pro-inflammatory cytokine which plays key roles in the pathogenesis of heart failure. Cardiomyocytes express the TNF-α receptor (TNFR), however, the mechanism of TNF-α signal transmission in cardiomyocytes is not completely understood. Here we show a novel regulator of TNF-α signaling, the Ras-association domain family 1 isoform A (RASSF1A), which regulates cardiac contractility and intracellular calcium through interaction with TNFR1. Methods and results: We used RASSF1A knockout (KO) mice and wild type (WT) controls and stimulated them with TNF-α (10μg/kg i.v.). In WT mice acute treatment with low dose of TNF-α increased cardiac contractility as indicated by the change in end systolic elastance (Ees) (baseline Ees (mmHg/mL), 3.3±0.5; Ees after TNF-α stimulation, 5.7±0.8, P<0.05) which is consistent with previously published data (Circulation 2004; 109:406-411). However, KO mice showed a blunted contractile response following acute TNF-a treatment (baseline Ees: 3.05±0.4 vs Ees after TNF-α: 2.54±0.3). Consistently, isolated cardiomyocytes from WT mice showed 40% increase in calcium transient amplitude in response to TNF-α (10 ng/ml) stimulation (n=24 cells, p<0.05). However, KO cardiomyocytes showed no significant increase in calcium transient amplitude following the same stimulation (n=24 cells). We also found that RASSF1A formed a molecular complex with TNFR1 in cardiomyocytes and this interaction was essential in the recruitment of TRADD and TRAF2, the major downstream effectors of TNF-α signalling. By mapping the interaction domain we found that the C-terminal region of RASSF1A was responsible for the formation of TNF-α receptor complex. Mechanistically, RASSF1A is essential in regulating calcium transients and contractility in cardiomyocytes downstream of TNF-α signaling through regulation of cytoplasmic phospholipase A2 (cPLA2) and phosphorylation of calcium handling molecules. Furthermore, using an adenoviral-mediated shRNA construct we found that cardiomyocytes lacking RASSF1A exhibited reduced activation of NFκB, a downstream target of TNF-α. Conclusion: Our data indicates an essential role of RASSF1A in regulating TNF-α signaling in cardiomyocytes. Moreover, our present work contributes a novel effector pathway via RASSF1A which transmits the positive inotropic effect of TNF-α and should therefore be preserved or even stimulated in the treatment of heart failure. In addition, enhancement of RASSF1A function/expression might well benefit patients with heart failure.

Abstract 109: Regulation of TNF-α Signaling in Cardiomyocytes via a Novel Interaction Between TNF-α Receptor and RASSF1A

Tumour necrosis factor-α (TNF-α) is a pro-inflammatory cytokine that plays key roles in the pathogenesis of heart failure. Cardiomyocytes express the TNF-α receptor (TNFR), however, the mechanism of TNF-α signal transmission in cardiomyocytes is not completely understood. Recent studies showed that in cancer cells TNFR is regulated by Ras-association domain family 1 isoformA (RASSF1A). Therefore, we investigated whether RASSF1A modulates TNF-α signalling in cardiomyocytes. We used RASSF1A knockout (KO) mice and wild type (WT) controls and stimulated them with TNF-α (10µg/kg i.v.). In WT mice acute treatment with low dose of TNF-α increased cardiac contractility as indicated by the change in end systolic elastance (Ees) (baseline Ees (mmHg/μl), 3.3±0.5; Ees after TNF-α stimulation, 5.7±0.8, P&lt;0.05) which is consistent with previously published data (Circulation 2004; 109:406–411). However, KO mice showed a blunted contractile response following acute TNF-α treatment (baseline Ees: 3.05±0.4 vs Ees after TNF-α: 2.54±0.3). Consistently, isolated cardiomyocytes from WT mice showed 40% increase in calcium transient amplitude in response to TNF-α (10 ng/ml) stimulation (n=24 cells, p&lt;0.05). However, KO cardiomyocytes showed no significant increase in calcium transient amplitude following the same stimulation (n=24 cells). We also found that RASSF1A formed a molecular complex with TNF-α receptor in cardiomyocytes and this interaction was essential in the recruitment of TRADD and TRAF2, the major downstream effectors of TNF-α signalling. By mapping the interaction domain we found that the C-terminal region of RASSF1A was responsible for the formation of TNF-α receptor complex. Furthermore, using an adenoviral-mediated shRNA construct we found that cardiomyocytes lacking RASSF1A exhibited reduced activation of NFkB, a downstream target of TNF-α. Overall, our data indicate an essential role of RASSF1A in regulating TNF-α signalling in cardiomyocytes, with RASSF1A being key in the formation of TNF receptor complex and in the signal transmission to the downstream targets.

Role of autocrine/paracrine mechanisms in response to myocardial strain

Myocardial strain triggers an autocrine/paracrine mechanism known to participate in myocardial hypertrophy development. After the onset of stretch, there is a rapid augmentation in developed tension due to an increase in myofilament calcium sensitivity (the Frank Starling mechanism) followed by a gradual increase in tension over the next 10-15min. This second phase is called the slow force response (SFR) to stretch and is known to be the result of an increase in calcium transient amplitude. In the present review, we will discuss what is known thus far about the SFR, which is the in vitro equivalent of the Anrep effect and the mechanical counterpart of the autocrine/paracrine mechanism elicited by myocardial stretch. The chain of events triggered by myocardial stretch comprises: (1) release of angiotensin II, (2) release/formation of endothelin, (3) NADPH oxidase activation and transactivation of the EGFR, (4) mitochondrial reactive oxygen species production, (5) activation of redox-sensitive kinases, (6) NHE-1 hyperactivity, (7) increase in intracellular Na(+) concentration, and (8) increase in Ca(2+) transient amplitude through the Na(+)/Ca(2+) exchanger. The evidence for each step of the intracellular signaling pathway leading to the development of SFR and their relationship with the mechanisms proposed for cardiac hypertrophy development will be analyzed.

Neuregulin-1β1 rapidly modulates nitric oxide synthesis and calcium handling in rat cardiomyocytes

The ErbB-neuregulin-1β1 (Nrg1β1) pathway is required for cardiac development and exerts chronic effects on the postnatal adult heart. Long-term application of Nrg1β1 results in hypertrophy and protection against oxidative stress and cytotoxic agents. We performed experiments with acute Nrg1β1 treatment to find evidence for a further protective role due to rapid modulation of adult cardiomyocyte function. In confocal fluorimetric measurements, Nrg1β1 induced a calcium-independent increase in nitric oxide (NO) production in isolated adult rat ventricular myocytes (ARVCMs) that was blocked by the phosphoinositide-3-kinase (PI3K) inhibitor Wortmannin. Western blot analysis showed enhancement of endothelial nitric oxide synthase phosphorylation in Nrg1β1-treated ARVCMs, which was attenuated by Wortmannin. Nrg1β1 induced a significant increase in calcium transient amplitude (indo-1 ratiometric measurement) and accelerated the recovery of cytosolic calcium in the sarcoplasmic reticulum without affecting whole-cell L-type calcium current. Wortmannin or the protein kinaseG inhibiting peptide (DT-2) abolished the increase in calcium transient amplitude and the acceleration of calcium recovery induced by Nrg1β1 treatment. Immunofluorescence analysis revealed that Nrg1β1 treatment increased phospholamban phosphorylation, and the effect was blocked by PI3K and protein kinaseG inhibition. Caffeine-releasable sarcoplasmic reticulum calcium content was also higher during Nrg1β1 administration. Rapid activation of PI3K, endothelial nitric oxide synthase and protein kinaseG and a consequent improvement in diastolic calcium can be added to established Nrg1 protective roles.

Mitochondrial membrane fluidity, potential, and calcium transients in the myocardium from acute diabetic ratsThis paper is one of a selection of papers published in this Special Issue, entitled The Cellular and Molecular Basis of Cardiovascular Dysfunction, Dhalla 70th Birthday Tribute.

In this study, we report for the first time concurrent measurements of membrane potential and dynamics and respiratory chain activities in rat heart mitochondria, as well as calcium transients in the hearts of rats in an early phase of streptozotocin diabetes, not yet accompanied with diabetes-induced complications. Quantitative relationships among these variables were assessed. The mitochondria from diabetic rats exhibited decreased fluorescence anisotropy values of diphenylhexatriene. This indicates that hydrophobic core of the membranes was more fluid compared with controls (p<0.05). We discuss the changes in fluidity as having been associated with augmented energy transduction through the diabetic membranes. Reduced ratio of JC-1 fluorescence (aggregates to monomers) in the mitochondria from diabetic hearts reflected descendent transmembrane potential. A significant negative association between membrane fluidity and potential in the diabetic group was found (p<0.05; r=0.67). Further, we observed an increase in calcium transient amplitude (CTA) in the diabetic cardiomyocytes (p=0.048). We conclude that some of the calcium-induced regulatory events that dictate fuel selection and capacity for ATP production in diabetic heart occur at the membrane level. Our findings offer new insight into acute diabetes-induced changes in cardiac mitochondria.

β1-Receptors increase cAMP and induce abnormal Cai cycling in the German shepherd sudden death model

We studied the role of beta-adrenergic receptor subtype signaling to cAMP and calcium in the genesis of catecholamine-dependent arrhythmias in German shepherd dogs that develop lethal arrhythmias at ~5 mo of age. There were three major findings in this study: 1) isoproterenol induces similar increases in cAMP in afflicted and control dogs exclusively through beta(1)-receptors (not beta(2)), 2) cells from afflicted dogs display prolonged relaxation kinetics at long cycle lengths and large frequent spontaneous calcium oscillations (and aftercontractions) with little increase in calcium transient amplitude in response to beta(1)-receptor agonists, and 3) beta(2)-receptor agonists induce a similar marked increases in calcium transient and twitch amplitude, with only rare spontaneous calcium oscillations in afflicted and control cells. These results indicate that catecholamines provide inotropic support to canine cardiomyocytes through distinct beta(1)- and beta(2)-receptor pathways with differing requirements for cAMP. The propensity to develop arrhythmias is not induced by beta(2)-receptors (or a rise in calcium alone), but rather occurs in the context of beta(1)-receptor activation of the cAMP-dependent pathway.

Role of protein kinase C in mediating effects of hydrogen peroxide in guinea-pig ventricular myocytes

The present study examined the effects of hydrogen peroxide (H 2O 2) on intracellular calcium transients and unloaded cell shortening in the presence of the protein kinase C (PKC) inhibitors 1-(5-isoquinolinesulfonyl-2-methylpiperazine (H7) or chelerythrine chloride (CHC) or the PKC activator phorbol 12-myristate 13-acetate (PMA). Calcium transient amplitudes and cell shortening were measured simultaneously in single, enzymatically dissociated ventricular myocytes loaded with fura2-AM. Exposure of myocytes to H 2O 2, 25 μ m or 75 μ m, for 15 min caused a time- and concentration-dependent increase in calcium transient amplitude, cell shortening and the diastolic 340 380 fluorescence ratio. Significant increases in calcium transient amplitude were observed from 7 to 15 min of superfusion with 25 μ m H 2O 2 and the transient amplitude remained elevated throughout the 10 min washout period. In the presence of 75 μ m H 2O 2, transient amplitude was elevated following 2 min and remained elevated for the remainder of the experiment. Significant increases in cell shortening were also observed from 7 to 15 min in the presence of either 25 or 75 μ m H 2O 2. This effect was reversed upon washout of the lower concentration of H 2O 2 but persisted during the initial 5 min of washout at the higher concentration. The diastolic 340 380 fluorescence ratio was unaltered in the presence of 25 μ m of H 2O 2, however this parameter was significantly increased from 7 to 15 min following exposure to 75 μ m H 2O 2 and remained elevated throughout the washout period. The H 2O 2-induced increases in calcium transient amplitude and cell shortening were significantly attenuated in myocytes which were pretreated with either H7 or CHC. In addition. H7 pretreatment abolished the increase in the diastolic ratio following exposure to the higher concentration of the oxidant. Addition of 10 −12 M PMA in the presence of 25 μ m H 2O 2 did not result in any further increase in either the calcium transient amplitude or in cell shortening, however PMA plus H 2O 2 did cause a significant increase in the diastolic ratio, an effect which had not been observed in the presence of this concentration of H 2O 2 alone. Pretreatment of cells with H7 prior to exposure to the combination of H 2O 2 and PMA significantly attenuated all changes seen in the absence of inhibitor. The results of this study suggest that the increase in the calcium transient amplitude and cell shortening following exposure of isolated myocytes to low micromolar concentrations of H 2O 2 are mediated through direct activation of PKC.

Modulation of sodium-hydrogen exchange activity in cardiac myocytes during acidosis and realkalinisation: effects on calcium, pHi, and cell shortening

The aim was to examine the effects of the Na+/H+ exchange inhibitor methylisobutylamiloride (MIA) as well as protein kinase C, a putative regulator of Na+/H+ exchange, on intracellular calcium, intracellular pH, and unloaded cell shortening in isolated guinea pig cardiac myocytes subjected to lactic acid induced acidosis followed by realkalinisation. Calcium transient amplitude and cell shortening were measured simultaneously in single isolated myocytes loaded with fura2-AM. Intracellular pH was measured in cells loaded with BCECF-AM. Exposure of cells to 5 min of lactate (20 mM) acidosis (pH 6.8) caused an increase in calcium transient amplitude and a decrease in cell shortening and intracellular pH. During realkalinisation (pH 7.3), the calcium transient gradually decreased while intracellular pH became more alkaline than pre-acidosis values. The cells underwent transient hypercontractility as evidenced by a marked increase in systolic cell shortening and a decrease in diastolic cell length. Inhibition of sodium/hydrogen exchange with MIA (1 microM) caused a significant attenuation of the increase in calcium transient amplitude during acidosis and further depressed cell shortening as well as intracellular pH. In addition, MIA significantly attenuated hypercontractility and abolished cell contracture upon realkalinisation. In contrast, phorbol 12-myristate 13-acetate (10(-12) M) exerted no effects on the response to acidosis; however, this treatment exacerbated cell hypercontractility and reduced functional recovery upon realkalinisation. Inhibition of Na+/H+ exchange activity during acidosis/realkalinisation enhances recovery of cell function.

Okadaic acid, a protein phosphatase inhibitor, increases the calcium transients in isolated ferret ventricular muscle.

Okadaic acid is a protein phosphatase inhibitor which has been found to produce a marked positive inotropic effect in isolated cardiac muscle. Using aequorin-injected ferret papillary muscles, we demonstrate that the increase in tension seen with okadaic acid is accompanied by a simultaneous increase in the amplitude of the calcium transients. By comparison with the effects of changing the extracellular calcium concentration, it is shown that the increase in calcium transient amplitude can account for the inotropic effect of okadaic acid.