Indo-1 Fluorescence Research Articles

Stretch-induced increase in cardiac contractility is independent of myocyte Ca2+ while block of stretch channels by streptomycin improves contractility after ischemic stunning.

Stretching the cardiac left ventricle (LV) enhances contractility but its effect on myoplasmic [Ca2+] is controversial. We measured LV pressure (LVP) and [Ca2+] as a function of intra-LV stretch in guinea pig intact hearts before and after 15 min global stunning ± perfusion with streptomycin (STM), a stretch-activated channel blocker. LV wall [Ca2+] was measured by indo-1 fluorescence and LVP by a saline-filled latex balloon inflated in 50 μL steps to stretch the LV. We implemented a mathematical model to interpret cross-bridge dynamics and myofilament Ca2+ responsiveness from the instantaneous relationship between [Ca2+] and LVP ± stretching. We found that: (1) stretch enhanced LVP but not [Ca2+] before and after stunning in either control (CON) and STM groups, (2) after stunning [Ca2+] increased in both groups although higher in STM versus CON (56% vs. 39%), (3) STM-enhanced LVP after stunning compared to CON (98% vs. 76% of prestunning values), and (4) stretch-induced effects on LVP were independent of [Ca2+] before or after stunning in both groups. Mathematical modeling suggested: (1) cooperativity in cross-bridge kinetics and myofilament Ca2+ handling is reduced after stunning in the unstretched heart, (2) stunning results in depressed myofilament Ca2+ sensitivity in the presence of attached cross-bridges regardless of stretch, and (3) the initial mechanism responsible for increased contractility during stretch may be enhanced formation of cross-bridges. Thus stretch-induced enhancement of contractility is not due to increased [Ca2+], whereas enhanced contractility after stunning in STM versus CON hearts results from improved Ca2+ handling and/or enhanced actinomyosin cross-bridge cycling.

NOS1 induces NADPH oxidases and impairs contraction kinetics in aged murine ventricular myocytes.

Nitric oxide (NO) modulates calcium transients and contraction of cardiomyocytes. However, it is largely unknown whether NO contributes also to alterations in the contractile function of cardiomyocytes during aging. Therefore, we analyzed the putative role of nitric oxide synthases and NO for the age-related alterations of cardiomyocyte contraction. We used C57BL/6 mice, nitric oxide synthase 1 (NOS1)-deficient mice (NOS1(-/-)) and mice with cardiomyocyte-specific NOS1-overexpression to analyze contractions, calcium transients (Indo-1 fluorescence), acto-myosin ATPase activity (malachite green assay), NADPH oxidase activity (lucigenin chemiluminescence) of isolated ventricular myocytes and cardiac gene expression (Western blots, qPCR). In C57BL/6 mice, cardiac expression of NOS1 was upregulated by aging. Since we found a negative regulation of NOS1 expression by cAMP in isolated cardiomyocytes, we suggest that reduced efficacy of β-adrenergic signaling that is evident in aged hearts promotes upregulation of NOS1. Shortening and relengthening of cardiomyocytes from aged C57BL/6 mice were decelerated, but were normalized by pharmacological inhibition of NOS1/NO. Cardiomyocytes from NOS1(-/-) mice displayed no age-related changes in contraction, calcium transients or acto-myosin ATPase activity. Aging increased cardiac expression of NADPH oxidase subunits NOX2 and NOX4 in C57BL/6 mice, but not in NOS1(-/-) mice. Similarly, cardiac expression of NOX2 and NOX4 was upregulated in a murine model with cardiomyocyte-specific overexpression of NOS1. We conclude that age-dependently upregulated NOS1, putatively via reduced efficacy of β-adrenergic signaling, induces NADPH oxidases. By increasing nitrosative and oxidative stress, both enzyme systems act synergistically to decelerate contraction of aged cardiomyocytes.

Subcellular distribution of glycogen and decreased tetanic calcium in fatigued single intact mouse muscle fibres.

Subcellular distribution of glycogen and decreased tetanic calcium in fatigued single intact mouse muscle fibres.

A monounsaturated fatty acid (oleic acid) modulates electrical activity in atrial myocytes with calcium and sodium dysregulation

BackgroundObesity and metabolic syndrome are important risk factors for atrial fibrillation. High plasma concentrations of monounsaturated fatty acids, including oleic acid (OLA), are frequently noted in obese individuals and patients with metabolic syndrome. However, it is not clear whether monounsaturated fatty acids (MUFAs) can directly modulate the electrophysiological characteristics of atrial myocytes. MethodsWhole-cell patch clamp, indo-1 fluorescence, and Western blot analyses were used to record the action potentials (APs), ionic currents, and protein expressions of HL-1 myocytes incubated with and without (control) OLA (0.5mM) for 24h. ResultsCompared to control myocytes (n=14), OLA-treated myocytes (n=16) had shorter APD90 (65±6 vs. 85±6ms, p<0.05) and APD50 (24±6 vs. 38±4ms, p<0.05) with a higher incidence of delayed afterdepolarizations (35.7% vs. 7%, p<0.05), which were suppressed by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, a blocker of the calcium-activated chloride current). In addition, OLA-treated myocytes (n=19) exhibited larger calcium transients (0.54±0.06 vs. 0.38±0.05 R410/485, p<0.05), and sarcoplasmic reticular calcium contents (0.91±0.05 vs. 0.64±0.08 R410/485, p<0.05) than control myocytes (n=15). OLA-treated myocytes had larger late sodium currents, smaller sodium–calcium exchanger currents, and smaller sodium–potassium pump currents. Moreover OLA-treated myocytes had higher expressions of sarcoplasmic reticular Ca2+-ATPase and calmodulin kinase II, but lower expression of the sodium–potassium ATPase protein than control myocytes. ConclusionsMUFAs can regulate atrial electrophysiological characteristics with calcium and sodium dysregulation, which may contribute to atrial arrhythmogenesis.

Leptin modulates electrophysiological characteristics and isoproterenol-induced arrhythmogenesis in atrial myocytes

BackgroundObesity is an important risk factor for atrial fibrillation (AF). Leptin is an important adipokine. However, it is not clear whether leptin directly modulates the electrophysiological characteristics of atrial myocytes.ResultsWhole cell patch clamp and indo-1 fluorescence were used to record the action potentials (APs) and ionic currents in isolated rabbit left atrial (LA) myocytes incubated with and without (control) leptin (100 nM) for 1 h to investigate the role of leptin on atrial electrophysiology. Leptin-treated LA myocytes (n = 19) had longer 20% of AP duration (28 ± 3 vs. 21 ± 2 ms, p < 0.05), but similar 50% of AP duration (51 ± 4 vs. 50 ± 3 ms, p > 0.05), and 90% of AP duration (89 ± 5 vs. 94 ± 4 ms, p > 0.05), as compared to the control (n = 22). In the presence of isoproterenol (10 nM), leptin-treated LA myocytes (n = 21) showed a lower incidence (19% vs. 54.2%, p < 0.05) of delayed afterdepolarization (DAD) than the control (n = 24). Leptin-treated LA myocytes showed a larger sodium current, but a smaller ultra-rapid delayed rectifier potassium current, and sodium-calcium exchanger current than the control. Leptin-treated and control LA myocytes exhibited a similar late sodium current, inward rectifier potassium current, transient outward current and L-type calcium current. In addition, the leptin-treated LA myocytes (n = 38) exhibited a smaller intracellular Ca2+ transient (0.21 ± 0.01 vs. 0.26 ± 0.01 R410/485, p < 0.05) and sarcoplasmic reticulum Ca2+ content (0.35 ± 0.02 vs. 0.43 ± 0.03 R410/485, p < 0.05) than the control LA myocytes (n = 42).ConclusionsLeptin regulates the LA electrophysiological characteristics and attenuates isoproterenol-induced arrhythmogenesis.

Type 1 Inositol (1,4,5)-Trisphosphate Receptor Activates Ryanodine Receptor 1 to Mediate Calcium Spark Signaling in Adult Mammalian Skeletal Muscle

Functional coupling between inositol (1,4,5)-trisphosphate receptor (IP(3)R) and ryanodine receptor (RyR) represents a critical component of intracellular Ca(2+) signaling in many excitable cells; however, the role of this mechanism in skeletal muscle remains elusive. In skeletal muscle, RyR-mediated Ca(2+) sparks are suppressed in resting conditions, whereas application of transient osmotic stress can trigger activation of Ca(2+) sparks that are restricted to the periphery of the fiber. Here we show that onset of these spatially confined Ca(2+) sparks involves interaction between activation of IP(3)R and RyR near the sarcolemmal membrane. Pharmacological prevention of IP(3) production or inhibition of IP(3)R channel activity abolishes stress-induced Ca(2+) sparks in skeletal muscle. Although genetic ablation of the type 2 IP(3)R does not appear to affect Ca(2+) sparks in skeletal muscle, specific silencing of the type 1 IP(3)R leads to ablation of stress-induced Ca(2+) sparks. Our data indicate that membrane-delimited signaling involving cross-talk between IP(3)R1 and RyR1 contributes to Ca(2+) spark activation in skeletal muscle.

Neuropeptide Substance-P Modulates Electrical Characteristics of Rabbit Atrial Myocytes

Background: The cardiac nervous system plays an important role in the initiation and maintenance of atrial fibrillation (AF). Besides the classical neurotransmitters, the nerves innervating the atria also release a wide range of neuropeptides that may play a role in the pathophysiology of AF. Since the direct effect of these neuropeptides on atrial myocytes is largely unknown, we investigated the electrophysiological response of atrial myocytes to several neuropeptides, importantly substance-P (Sub-P).Methods and Results: Single left atrial myocytes from rabbit hearts were obtained by enzymatic dissociation and the effects of Sub-P, neuropeptide-Y (NPY), somatostatin-14 (SOM-14) and vasoactive intestinal peptide (VIP), were studied using patch-clamp and indo-1 fluorescence methodologies. NPY, SOM-14 and VIP (1 μM) did not affect atrial action potential (AP) characteristics. Sub-P however, reduced both resting membrane potential (RMP) and action potential amplitude, and increased action potential duration at 90% of repolarisation (APD90) by 40%. The effects on APD90 were dose-dependent and occurred from a concentration of 10 nM. Voltage-clamp analysis revealed that Sub-P significantly diminished L-type calcium current, inward rectifier K+ current (IK1) and an unknown steady-state outward current. Transient outward current, Ca2+- activated chloride current and Na+-Ca2+-exchanger current were unaffected, as was the intracellular calcium handling. The reduction in RMP and increase in APD90 are likely due to the decrease in IK1 and steady-state outward current, respectively. Our experiments indicate that the latter is probably carried by background-like potassium channels.Conclusion: Atrial myocyte electrophysiology is significantly modulated by the neuropeptide Sub-P, mainly characterized by a substantial AP prolongation due to inhibition of a background potassium current. Since lengthening of atrial repolarization is potentially anti-arrhythmic, we hypothesize that release of Sub-P may be beneficial to the prevention and/or termination of AF.

Hydralazine-induced promoter demethylation enhances sarcoplasmic reticulum Ca2+-ATPase and calcium homeostasis in cardiac myocytes

Sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) plays an essential role in Ca2+ homeostasis and cardiac functions. The promoter region of SERCA2a has a high content of CpG islands; thus, epigenetic modification by inhibiting methylation can enhance SERCA2a expression in cardiomyocytes. Hydralazine, a drug frequently used in heart failure, is a potential DNA methylation inhibitor. We evaluated whether hydralazine can modulate Ca2+ handling through an increase in SERCA2a expression via regulating methylation. We used indo-1 fluorescence, real-time RT-PCR, immunoblotting, and methylation-specific PCR to investigate intracellular Ca2+, the expressions of RNA and protein, and methylation of SERCA2a in HL-1 cardiomyocytes with and without (control) the administration of hydralazine (1, 10, and 30 μM) for 72 h. Hydralazine (10 and 30 μM) increased the intracellular Ca2+ transients and SR Ca2+ contents. Hydralazine (10 and 30 μM) decreased methylation in the SERCA2a promoter region and increased the RNA and protein expressions of SERCA2a. Additionally, hydralazine (10 and 30 μM) decreased the expression of DNA methyltransferase 1. Moreover, treatment with hydralazine in isoproterenol-induced heart failure rats decreased the promoter methylation of SERCA2a and increased SERCA2a RNA expression. In conclusion, hydralazine-induced promoter demethylation may improve cardiac function through increasing SERCA2a and modulating calcium homeostasis in cardiomyocytes.

Eicosapentaenoic acid reduces the pulmonary vein arrhythmias through nitric oxide

AimsOmega-3 polyunsaturated fatty acids can modulate cardiac electrophysiology and reduce the genesis of atrial fibrillation. This study investigates the potential mechanisms through which eicosapentaenoic acid (EPA) reduces pulmonary vein (PV) arrhythmogenesis. Main methodsConventional microelectrodes were used to record the action potentials (APs), before and after the EPA (0.1μM and 1.0μM) administration with and without the presence of a nitric oxide (NO) synthase inhibitor (L-NAME, 100μM) in isolated rabbit PV tissue preparations. Furthermore, indo-1 fluorimetric ratio technique was used to evaluate intracellular calcium in isolated single PV cardiomyocytes with or without incubation of EPA (1.0μM, 30min). Key findingsEPA concentration-dependently reduced the PV spontaneous beating rate (P<0.05). EPA (1.0μM) also reduced the amplitude of delayed afterdepolarizations (P<0.05). EPA hyperpolarized the maximal diastolic potential (MDP), shortened AP duration, increased AP amplitude (APA), and reduced diastolic tension and contractility. However, EPA in the presence of L-NAME or omega-9 fatty acids (oleic acid, 1.0μM) did not have any effect on PV spontaneous activity, AP morphology, or contractile force. A linear regression shows that the decrease in PV spontaneous beating rates induced by EPA correlated well with the changes of MDP, APA, diastolic tension, and contractile force of PVs. In addition, intracellular Ca2+ transient and sarcoplasmic reticulum Ca2+ content were significantly more decreased in the EPA-treated cardiomyocytes than in control PV cardiomyocytes as observed by indo-1 fluorescence. SignificanceEPA reduces PV arrhythmogenesis through the mechanoelectrical feedback generated by NO production.

Multiparameter flow cytometric kinetics of phagocyte stimulus responses

THE recently published Technical Note by Vines et al., ‘‘A Flow-Cytometric Method for Continuous Measurement of Intracellular Ca Concentration,’’ Cytometry Part A 77A:1091 1097, 2010, demonstrated that such measurements are possible in an Accuri C6 flow cytometer. Their contribution demonstrates that rapid measurements can be achieved when stimuli are added to cells (in an open tube) which then flow via a pump mechanism, yielding observations within 5 s, judging from their figures, albeit without thermostating or stirring the sample. Intracellular kinetic measurements of cytoplasmic Ca (and other events) using other flow cytometers (FACS 440, MoFlo, Aria) have been previously reported by a number of laboratories, including ours and extensively reviewed (1 9). Since our own first report in 1986 (10), we have published numerous flow cytometric measurements of cytoplasmic Ca fluxes ([Ca]in) in various blood cells, using a modified FACS 440 (BD) or MoFlo (Cytomation), injecting the compounds into a stirred thermostated flowing cell suspension. These flow cytometers, and the LSRII and FACSAria to which we are now adapting the sample handling system we described, use N2 pressure to move the cell stream, which permits the sample to be stoppered and reduces risk from potential biohazards. Thermostating and stirring assure physiologic conditions and rapid mixing. Injecting into pressure-driven, flowing cells a very short (<10 cm) distance from the nozzle, permits continuous observation of cellular responses within 4 s after reagent addition without using high flow rates. Using the technique above, we also have correlated cytoplasmic changes in Ca and pH, and consequent reactive oxygen and lytic entities, with specific receptor occupancy and subpopulation responses by and with the neutrophils’ receptor cross-linking requirements (11). Similar studies performed with monocytes (12) permitted differentiation between cells responding and not responding withD[Ca] by the presence or absence of a specific receptor (CD14) on their surface (Fig. 1). Our flow cytometric methods have now been applied to studies of the early kinetics of cytoplasmic and phagosomal changes as cells respond to stimulating organisms (e.g., changes in phagosomal pH, generation of reactive oxygen species (ROS), presence of lytic enzymes) by labeling the phagocytized entity so that it acts as an indicator of conditions in that compartment (11). Recently, we showed that neutrophil (PMN) stimulation by Staphylococcus epidermidis (13) requires opsonization of the organism. A transient spike in D[Ca]in is maximal within 18 s and originates from intra PMN stores. Using pHrodo (LifeSciences), we further showed that the phagosome enclosing S. epidermidis becomes increasingly alkaline, in contrast to the acidification we observed when a fungus, Cryptococcus neoformans, was phagocytized. By using flow cytometry and our adaptation for rapid kinetics (described above) in conjunction with multiple labels on the same organism, we were able to correlate the initial D[Ca]in signal not only with the phagosomal pH (pHp) but also with the ROS, generated in the phagosome and the receptor occupancy on the same PMN. Quenching the fluorescence of bound but not yet phagosome-enclosed S. epidermidis permitted us to focus on the conditions within it and to begin investigation of the role of extracellular Ca. This method gains greater potential relevance when it is used to investigate the very early, coordinated signaling events that occur upon binding and phagocytosis of organisms like mycobacteria that are able to alter the phagocyte response to evade killing and establish latent infection. Binding-induced fluxes resulting in rapid changes in local ion concentrations and their relationships to activation of membrane-localized

Activated human platelet products induce proarrhythmic effects in ventricular myocytes

Sudden cardiac death remains one of the most prevalent modes of death and is mainly caused by ventricular fibrillation (VF) in the setting of acute ischemia resulting from coronary thrombi. Animal experiments have shown that platelet activation may increase susceptibility of ischemic myocardium to VF, but the mechanism is unknown. In the present study, we evaluated the effects of activated blood platelet products (ABPPs) on electrophysiological properties and intracellular Ca 2+ (Ca 2+ i ) homeostasis. Platelets were collected from healthy volunteers. After activation, their secreted ABPPs were added to superfusion solutions. Rabbit ventricular myocytes were freshly isolated, and membrane potentials and Ca 2+ i were recorded using patch-clamp methodology and indo-1 fluorescence measurements, respectively. ABPPs prolonged action potential duration and induced early and delayed afterdepolarizations. ABPPs increased L-type Ca 2+ current ( I Ca,L) density, but left densities of sodium current, inward rectifier K + current, transient outward K + current, and rapid component of the delayed rectifier K + current unchanged. ABPPs did not affect kinetics or (in)activation properties of membrane currents. ABPPs increased systolic Ca 2+ i , Ca 2+ i transient amplitude, and sarcoplasmic reticulum Ca 2+ content. ABPPs did not affect the Na +− Ca 2+ exchange current ( I NCX) in Ca 2+-buffered conditions. Products secreted from activated human platelets induce changes in I Ca,L and Ca 2+ i , which result in action potential prolongation and the occurrence of early and delayed afterdepolarizations in rabbit myocytes. These changes may trigger and support reentrant arrhythmias in ischemia models of coronary thrombosis.

Extracellular Matrix of Collagen Modulates Intracellular Calcium Handling and Electrophysiological Characteristics of HL-1 Cardiomyocytes With Activation of Angiotensin II Type 1 Receptor

Background Myocardial fibrosis plays a critical role in heart failure, resulting in cardiac structural and electrical remodeling which can induce atrial arrhythmias. Collagen is the major element of fibrosis. However, it is not clear whether collagen can directly regulate the calcium homeostasis and the electrophysiologic characteristics of cardiomyocytes. The aim of this study was to determine the effects of collagen on calcium homeostasis and the electrical properties of atrial cardiomyocytes. Methods and Results HL-1 cardiomyocytes were cultured with and without collagen type I (1 or 10 μg/mL) or losartan (10 μmol/L). Whole-cell clamp, indo-1 fluorescence, and Western blotting were used to evaluate the action potential (AP) and ionic currents, intracellular calcium homeostasis, and calcium regulatory proteins. Compared with the control samples, there was no significant difference in collagen (1 μg/mL)–treated HL-1 cardiomyocytes. However, collagen (10 μg/mL)–treated HL-1 cardiomyocytes exhibited larger intracellular calcium ([Ca 2+] i) transients by 113% and a larger sarcoplasmic reticulum calcium content by 86%. Collagen (10 μg/mL)–treated HL-1 cardiomyocytes had higher expression of sarcoplasmic reticulum ATPase (SERCA2a) and Thr17-phosphorylated phospholamban but similar protein expressions of the Na +/Ca 2+ exchanger and ryanodine receptor. Collagen (10 μg/mL)–treated HL-1 cardiomyocytes (n = 11) had larger AP amplitude (104 ± 5 vs 83 ± 7 mV; P < .05), and shorter 90% of AP duration (25 ± 2 vs 33 ± 2 ms, P < .05) than control cells (n = 11). Moreover, collagen (10 μg/mL)–treated HL-1 cells had larger I to and I Ksus values than control cells. The administration of losartan (10 μmol/L) attenuated collagen-induced changes in [Ca 2+] i transients, [Ca 2+] i stores, AP morphology, ionic currents, SERCA2a, and Thr17-phosphorylated phospholamban expressions. Conclusions This study demonstrates that collagen can directly modulate the calcium dynamics and electrical activities of atrial cardiomyocytes, which are associated with the renin-angiotensin system. These findings suggest a critical role of collagen in electrical remodeling during fibrosis.

In vivo expression of G‐protein β1γ2 dimer in adult mouse skeletal muscle alters L‐type calcium current and excitation–contraction coupling

A number of G-protein-coupled receptors are expressed in skeletal muscle but their roles in muscle physiology and downstream effector systems remain poorly investigated. Here we explored the functional importance of the G-protein betagamma (Gbetagamma) signalling pathway on voltage-controlled Ca(2+) homeostasis in single isolated adult skeletal muscle fibres. A GFP-tagged Gbeta(1)gamma(2) dimer was expressed in vivo in mice muscle fibres. The GFP fluorescence pattern was consistent with a Gbeta(1)gamma(2) dimer localization in the transverse-tubule membrane. Membrane current and indo-1 fluorescence measurements performed under voltage-clamp conditions reveal a drastic reduction of both L-type Ca(2+) current density and of peak amplitude of the voltage-activated Ca(2+) transient in Gbeta(1)gamma(2)-expressing fibres. These effects were not observed upon expression of Gbeta(2)gamma(2), Gbeta(3)gamma(2) or Gbeta(4)gamma(2). Our data suggest that the G-protein beta(1)gamma(2) dimer may play an important regulatory role in skeletal muscle excitation-contraction coupling.

Differential Regulation of Calcium-Activated Potassium Channels by Dynamic Intracellular Calcium Signals

Calcium (Ca(2+))-activated K(+) (K(Ca)) channels regulate membrane excitability and are activated by an increase in cytosolic Ca(2+) concentration ([Ca(2+)](i)), leading to membrane hyperpolarization. Most patch clamp experiments that measure K(Ca) currents use steady-state [Ca(2+)] buffered within the patch pipette. However, when cells are stimulated physiologically, [Ca(2+)](i) changes dynamically, for example during [Ca(2+)](i) oscillations. Therefore, the aim of the present study was to examine the effect of dynamic changes in [Ca(2+)](i) on small (SK3), intermediate (hIK1), and large conductance (BK) channels. HEK293 cells stably expressing each K(Ca) subtype in isolation were used to simultaneously measure agonist-evoked [Ca(2+)](i) signals, using indo-1 fluorescence, and current/voltage, using perforated patch clamp. Agonist-evoked [Ca(2+)](i) oscillations induced a corresponding K(Ca) current that faithfully followed the [Ca(2+)](i) in 13-50% of cells, suggesting a good synchronization. However, [Ca(2+)](i) and K(Ca) current was much less synchronized in 50-76% of cells that exhibited Ca(2+)-independent current events (55% of SK3-, 50% of hIK1-, and 53% of BK-expressing cells) and current-independent [Ca(2+)](i) events (18% SK3- and 33% of BK-expressing cells). Moreover, in BK-expressing cells, where [Ca(2+)](i) and K(Ca) current was least synchronized, 36% of total [Ca(2+)](i) spikes occurred without activating a corresponding K(Ca) current spike, suggesting that BK(Ca) channels were either inhibited or had become desensitized. This desynchronization between dynamic [Ca(2+)](i) and K(Ca) current suggests that this relationship is more complex than could be predicted from steady-state [Ca(2+)](i) and K(Ca) current. These phenomena may be important for encoding stimulus-response coupling in various cell types.

NADPH oxidase-derived superoxide impairs calcium transients and contraction in aged murine ventricular myocytes

Since aging increases oxidative stress, we analyzed the contribution of reactive oxygen species (ROS) to the contractile dysfunction of aged ventricular myocytes and investigated whether short-term interference with ROS formation could normalize contractile performance. Isolated ventricular myocytes from young (2–4 months) and aged (24–26 months) male mice (C57BL/6) were used. We analyzed sarcomere shortening and calcium transients (Indo-1 fluorescence) of voltage clamped ventricular myocytes and myofilament ATPase activity (malachite green assay). Expression of calcium handling proteins (Western blots) and NADPH oxidase subunits (real-time PCR) was quantified, as well as NADPH oxidase activity (lucigenin chemiluminescence). We found that aged myocytes showed decelerated shortening/relengthening without changes in fractional shortening. Calcium transient decay was similarly decelerated, but the amplitude of calcium transients was increased with aging. Calcium sensitivity of myofilaments of aged myocytes was reduced. These age-dependent changes occurred without altered calcium handling protein expression but were reversed by the superoxide scavenger tiron. Aged myocytes showed increased NADPH oxidase expression and activity. Pharmacological inhibition of NADPH oxidase (diphenylene iodonium; apocynin) normalized age-dependent deceleration of shortening/relengthening. In summary, we show that increased superoxide formation by upregulated NADPH oxidase contributes significantly to age-dependent alterations in calcium handling and contractility of murine ventricular myocytes.

Cardioprotection by GSK-3 inhibition: role of enhanced glycogen synthesis and attenuation of calcium overload

Glycogen synthase kinase-3 (GSK-3) is a multi-functional kinase that regulates signalling pathways affecting glycogen metabolism, protein synthesis, mitosis, and apoptosis. GSK-3 inhibition limits cardiac ischaemia-reperfusion (IR) injury, but mechanisms are not clearly defined. This study tested the hypothesis that acute GSK-3 inhibition stimulates glycogen synthesis, repartitions glucose away from glycolysis, reduces proton (H+) production from glucose metabolism, and attenuates intracellular Ca2+ (Ca2+(i)) overload. In isolated perfused working rat hearts subjected to global ischaemia and reperfusion, the selective GSK-3 inhibitor, SB-216763 (SB, 3 micromol/L), when added either prior to ischaemia or at the onset of reperfusion, improved recovery of left-ventricular (LV) work. SB increased glycogen synthesis during reperfusion while glycolysis and H+ production were reduced. Rates of glucose and palmitate oxidation were improved by SB. Measurement of Ca2+(i) concentration by rapid acquisition indo-1 fluorescence imaging showed that SB, when added either prior to ischaemia or at the onset of reperfusion, reduced diastolic Ca2+(i) overload during reperfusion. In aerobic hearts depleted of glycogen by substrate-free perfusion to a level similar to that measured at the onset of reperfusion, SB accelerated glycogen synthesis and reduced glycolysis and H+ production independent of changes in LV work. Our study indicates that reduction in H+ production by GSK-3 inhibition is an early and upstream event that lessens Ca2+(i) overload during ischaemia and early reperfusion independent of LV work which enhances the recovery of post-ischaemic LV function and that may ultimately contribute to previously observed reductions in cell death and infarction.

Arachidonic acid stimulates extracellular Ca2+ entry in rat pancreatic β cells via activation of the noncapacitative arachidonate-regulated Ca2+ (ARC) channels

Arachidonic acid (AA) is generated in the pancreatic islets during glucose stimulation. We investigated whether AA activated extracellular Ca2+ entry in rat pancreatic β cells via a pathway that was independent of the activation of voltage-gated Ca2+ channels. The AA triggered [Ca2+]i rise did not involve activation of GPR40 receptors or AA metabolism. When cells were voltage clamped at −70mV, the AA-mediated intracellular Ca2+ release was accompanied by extracellular Ca2+ entry. AA accelerated the rate of Mn2+ quench of indo-1 fluorescence (near the Ca2+-independent wavelength of indo-1), reflecting the activation of a Ca2+-permeable pathway. The AA-mediated acceleration of Mn2+ quench was inhibited by La3+ but not by 2-APB (a blocker of capacitative Ca2+ entry), suggesting the involvement of arachidonate-regulated Ca2+ (ARC) channels. Consistent with this, intracellular application of the charged membrane-impermeant analog of AA, arachidonyl-coenzyme A (ACoA) triggered extracellular Ca2+ entry, as well as the activation of a La3+-sensitive small inward current (1.7pA/pF) at −70mV. Our results indicate that the activation of ARC channels by intracellular AA triggers extracellular Ca2+ entry. This action may contribute to the effects of AA on Ca2+ signals and insulin secretion in rat β cells.

Effects of muscarinic receptor stimulation on Ca2+ transient, cAMP production and pacemaker frequency of rabbit sinoatrial node cells

We investigated the contribution of the intracellular calcium (Cai2+) transient to acetylcholine (ACh)-mediated reduction of pacemaker frequency and cAMP content in rabbit sinoatrial nodal (SAN) cells. Action potentials (whole cell perforated patch clamp) and Cai2+ transients (Indo-1 fluorescence) were recorded from single isolated rabbit SAN cells, whereas intracellular cAMP content was measured in SAN cell suspensions using a cAMP assay (LANCE®). Our data show that the Cai2+ transient, like the hyperpolarization-activated “funny current” (I f) and the ACh-sensitive potassium current (I K,ACh), is an important determinant of ACh-mediated pacemaker slowing. When I f and I K,ACh were both inhibited, by cesium (2 mM) and tertiapin (100 nM), respectively, 1 μM ACh was still able to reduce pacemaker frequency by 72%. In these I f and I K,ACh-inhibited SAN cells, good correlations were found between the ACh-mediated change in interbeat interval and the ACh-mediated change in Cai2+ transient decay (r 2 = 0.98) and slow diastolic Cai2+ rise (r 2 = 0.73). Inhibition of the Cai2+ transient by ryanodine (3 μM) or BAPTA-AM (5 μM) facilitated ACh-mediated pacemaker slowing. Furthermore, ACh depressed the Cai2+ transient and reduced the sarcoplasmic reticulum (SR) Ca2+ content, all in a concentration-dependent fashion. At 1 μM ACh, the spontaneous activity and Cai2+ transient were abolished, but completely recovered when cAMP production was stimulated by forskolin (10 μM) and I K,ACh was inhibited by tertiapin (100 nM). Also, inhibition of the Cai2+ transient by ryanodine (3 μM) or BAPTA-AM (25 μM) exaggerated the ACh-mediated inhibition of cAMP content, indicating that Cai2+ affects cAMP production in SAN cells. In conclusion, muscarinic receptor stimulation inhibits the Cai2+ transient via a cAMP-dependent signaling pathway. Inhibition of the Cai2+ transient contributes to pacemaker slowing and inhibits Cai2+-stimulated cAMP production. Thus, we provide functional evidence for the contribution of the Cai2+ transient to ACh-induced inhibition of pacemaker activity and cAMP content in rabbit SAN cells.

ADP/ATP Antiport and ADP Phosphorylation Increase Mitochondrial Free Ca2+

Introduction: Matrix free [Ca2+] (m[Ca2+]) is believed to be a key regulator of mitochondrial function. The effect of differential buffering of calcium by ADP, ATP and Pi on m[Ca2+] levels has not been examined. We tested how m[Ca2+] is increased by ADP/ATP transport and phosphorylation, and if increased m[Ca2+] alters the bioenergetic state. Materials and Methods: Guinea pig heart mitochondria were isolated by differential centrifugation. Respiration and m[Ca2+], using indo-1 fluorescence, and corrected for NADH autofluorescence, were measured. After energizing mitochondria with 0.5 mM pyruvic acid, 0, 10, 25 μM CaCl2 (16, 88, 130 nM [Ca2+]) was added to the suspension before adding 250 μM ADP, in the presence or absence of ADP/ATP carrier blocker carboxyatractyloside (CATR) or F1F0ATPase blocker oligomycin (OMN). Results: m[Ca2+] increased proportionately with addition of CaCl2. ADP caused an additional increase to 100±6% in m[Ca2+] after 25 μM CaCl2. This was due to lesser binding of ADP vs. ATP to Ca2+. The rise in m[Ca2+] after ADP was reversed after all ADP was converted to ATP. With OMN the increase after ADP was lower (18±6%), but remained elevated as ADP was not phosphorylated to ATP. CATR completely blocked the ADP -induced increases in m[Ca2+] because matrix ADP transport was blocked. State 2 and 4 respiration, but not state 3, increased 14% and 18% with 25 μM CaCl2. NADH decreased with ADP alone, but NADH was not altered by adding CaCl2. Discussion: These results show that ADP transport into mitochondria and ADP conversion to ATP have significant effects on m[Ca2+]. Acutely changing buffer [CaCl2] has limited effects on redox state, although m[Ca2+] is believed to stimulate several dehydrogenases. However the k0.5 (1 μM) for this effect is only reached by adding ADP after 25 μM CaCl2.

Adult progenitor cell transplantation influences contractile performance and calcium handling of recipient cardiomyocytes

Adult progenitor cell transplantation has been proposed for the treatment of heart failure, but the mechanisms effecting functional improvements remain unknown. The aim of this study was to test the hypothesis that, in failing hearts treated with cell transplantation, the mechanical properties and excitation-contraction coupling of recipient cardiomyocytes are altered. Adult rats underwent coronary artery ligation, leading to myocardial infarction and chronic heart failure. After 3 wk, they received intramyocardial injections of either 10(7) green fluorescence protein (GFP)-positive bone marrow mononuclear cells or 5 x 10(6) GFP-positive skeletal myoblasts. Four weeks after injection, both cell types increased ejection fraction and reduced cardiomyocyte size. The contractility of isolated GFP-negative cardiomyocytes was monitored by sarcomere shortening assessment, Ca(2+) handling by indo-1 and fluo-4 fluorescence, and electrophysiology by patch-clamping techniques. Injection of either bone marrow cells or skeletal myoblasts normalized the impaired contractile performance and the prolonged time to peak of the Ca(2+) transient observed in failing cardiomyocytes. The smaller and slower L-type Ca(2+) current observed in heart failure normalized after skeletal myoblast, but not bone marrow cell, transplantation. Measurement of Ca(2+) sparks suggested a normalization of sarcoplasmic reticulum Ca(2+) leak after skeletal myoblast transplantation. The increased Ca(2+) wave frequency observed in failing myocytes was reduced by either bone marrow cells or skeletal myoblasts. In conclusion, the morphology, contractile performance, and excitation-contraction coupling of individual recipient cardiomyocytes are altered in failing hearts treated with adult progenitor cell transplantation.