Fibronectin increases the force production of mouse papillary muscles via α5β1 integrin

The extracellular matrix (ECM) protein-integrin-cytoskeleton axis plays a central role as a mechanotransducing protein assemblage in many cell types. However, how the process of mechanotransduction and the mechanically generated signals arising from this axis affect myofilament function in cardiac muscle are not completely understood. We hypothesize that ECM proteins can regulate cardiac function through integrin binding, and thereby alter the intracellular calcium concentration ([Ca²⁺]_i) and/or modulate myofilament activation processes. Force measurements made in mouse papillary muscle demonstrated that in the presence of the soluble form of the ECM protein, fibronectin (FN), active force was increased significantly by 40% at 1 Hz, 54% at 2 Hz, 35% at 5 Hz and 16% at 9 Hz stimulation frequencies. Furthermore, increased active force in the presence of FN was associated with 12-33% increase in [Ca²⁺]_i and 20-50% increase in active force per unit Ca²⁺. A function blocking antibody for α5 integrin prevented the effects of the FN on the changes in force and [Ca²⁺]_i, whereas a function blocking α3 integrin antibody did not reverse the effects of FN. The effects of FN were reversed by an L-type Ca²⁺ channel blocker, verapamil or PKA inhibitor. Freshly isolated cardiomyocytes exhibited a 39% increase in contraction force and a 36% increase in L-type Ca²⁺ current in the presence of FN. Fibers treated with FN showed a significant increase in the phosphorylation of phospholamban; however, the phosphorylation of troponin I was unchanged. These results demonstrate that FN acts via α5β1 integrin to increase force production in myocardium and that this effect is partly mediated by increases in [Ca²⁺]_i and Ca²⁺ sensitivity, PKA activation and phosphorylation of phospholamban.     

The cAMP binding protein Epac regulates cardiac myofilament function

In the heart, cAMP is a key regulator of excitation–contraction coupling and its biological effects are mainly associated with the activity of protein kinase A (PKA). The aim of this study was to investigate the contribution of the cAMP-binding protein Epac (Exchange protein directly activated by cAMP) in the regulation of the contractile properties of rat ventricular cardiac myocytes. We report that both PKA and Epac increased cardiac sarcomere contraction but through opposite mechanisms. Differently from PKA, selective Epac activation by the cAMP analog 8-(4-chlorophenylthio)-2′-O-methyl-cAMP (8-pCPT) reduced Ca²⁺ transient amplitude and increased cell shortening in intact cardiomyocytes and myofilament Ca²⁺ sensitivity in permeabilized cardiomyocytes. Moreover, ventricular myocytes, which were infected in vivo with a constitutively active form of Epac, showed enhanced myofilament Ca²⁺ sensitivity compared to control cells infected with green fluorescent protein (GFP) alone. At the molecular level, Epac increased phosphorylation of 2 key sarcomeric proteins, cardiac Troponin I (cTnI) and cardiac Myosin Binding Protein-C (cMyBP-C). The effects of Epac activation on myofilament Ca²⁺ sensitivity and on cTnI and cMyBP-C phosphorylation were independent of PKA and were blocked by protein kinase C (PKC) and Ca²⁺ calmodulin kinase II (CaMKII) inhibitors. Altogether these findings identify Epac as a new regulator of myofilament function.     

Protein kinase C α and ε phosphorylation of troponin and myosin binding protein C reduce Ca2+ sensitivity in human myocardium

Previous studies indicated that the increase in protein kinase C (PKC)-mediated myofilament protein phosphorylation observed in failing myocardium might be detrimental for contractile function. This study was designed to reveal and compare the effects of PKCα- and PKCε-mediated phosphorylation on myofilament function in human myocardium. Isometric force was measured at different [Ca²⁺] in single permeabilized cardiomyocytes from failing human left ventricular tissue. Activated PKCα and PKCε equally reduced Ca²⁺ sensitivity in failing cardiomyocytes (ΔpCa₅₀ = 0.08 ± 0.01). Both PKC isoforms increased phosphorylation of troponin I- (cTnI) and myosin binding protein C (cMyBP-C) in failing cardiomyocytes. Subsequent incubation of failing cardiomyocytes with the catalytic subunit of protein kinase A (PKA) resulted in a further reduction in Ca²⁺ sensitivity, indicating that the effects of both PKC isoforms were not caused by cross-phosphorylation of PKA sites. Both isozymes showed no effects on maximal force and only PKCα resulted in a modest significant reduction in passive force. Effects of PKCα were only minor in donor cardiomyocytes, presumably because of already saturated cTnI and cMyBP-C phosphorylation levels. Donor tissue could therefore be used as a tool to reveal the functional effects of troponin T (cTnT) phosphorylation by PKCα. Massive dephosphorylation of cTnT with alkaline phosphatase increased Ca²⁺ sensitivity. Subsequently, PKCα treatment of donor cardiomyocytes reduced Ca²⁺ sensitivity (ΔpCa₅₀ = 0.08 ± 0.02) and solely increased phosphorylation of cTnT, but did not affect maximal and passive force. PKCα- and PKCε-mediated phosphorylation of cMyBP-C and cTnI as well as cTnT decrease myofilament Ca²⁺ sensitivity and may thereby reduce contractility and enhance relaxation of human myocardium.     

Tonic Regulation of Excitation–Contraction Coupling by Basal Protein Kinase C Activity in Isolated Cardiac Myocytes

A high-speed imaging technique was used to investigate the effects of inhibitors and activators of protein kinase C (PKC) on the [Ca²⁺]_i transients and contraction of fura-2 loaded rat ventricular cardiac myocytes. The amplitude of the [Ca²⁺]_i transient was reduced following treatment with 100 nm phorbol 12,13-dibutyrate (PDBu), whereas the PKC inhibitors staurosporine (0.5μm) and calphostin C (10μm) increased [Ca²⁺]_i transient amplitude, elevated basal [Ca²⁺]_i and slowed the decay of the [Ca²⁺]_i transient. These changes were paralleled by similar alterations in the rate and extent of cell shortening. The activity of nitrendipine-sensitive Ca²⁺ channels was monitored indirectly as the rate of Mn²⁺ quench of cytosolic fura-2 in electrically-paced cells. PDBu reduced Mn²⁺ influx by six-fold, whereas staurosporine and calphostin C increased the influx rate by eight-fold and seven-fold over basal quench, respectively. The caffeine releasable Ca²⁺ pool was reduced in the presence of PDBu and increased transiently in presence of staurosporine. The effects of PKC activation and inhibition on sarcoplasmic reticulum Ca²⁺ content may be secondary to alterations of sarcolemmal Ca²⁺ influx. However, the PKC inhibitors also decreased the rate of sarcoplasmic reticulum Ca²⁺ uptake in permeabilized myocytes, suggesting that a direct effect of PKC on the sarcoplasmic reticulum may contribute to the prolongation of the [Ca²⁺]_i transient under these conditions. The present work demonstrates that basal PKC activity has a potent depressant effect, mediated primarily through inhibition of sarcolemmal Ca²⁺ influx, which may play a key role in setting the basal tone of cardiac muscle.     

Involvement of protein kinase C and intracellular Ca in goldfish brain somatostatin-28 inhibitory action on growth hormone release in goldfish

Goldfish brain somatostatin-28 (gbSS-28) is present in brain and pituitary tissues of goldfish. We assessed whether gbSS-28 targets Ca²⁺ and/or protein kinase C (PKC)-dependent signaling cascades in inhibiting growth hormone (GH) release. gbSS-28 decreased basal GH release from primary cultures of dispersed goldfish pituitary cells and intracellular free calcium levels ([Ca²⁺]_i) in goldfish somatotropes. gbSS-28 partially reduced [Ca²⁺]_i and GH responses induced by two endogeneous gonadotropin-releasing hormones (GnRHs), salmon (s)GnRH and chicken (c)GnRH-II. Furthermore, gbSS-28 reduced GH increases and abolished [Ca²⁺]_i elevations elicited by two PKC activators, tetradecanoyl 4β-phorbol-13-acetate and dioctanyl glycerol. The PKC inhibitors Gö6976 and Bis II abolished [Ca²⁺]_i responses to PKC activators, but only attenuated GnRH-induced increases in [Ca²⁺]_i and did not alter basal [Ca²⁺]_i. In cells pretreated with Bis II, gbSS-28 further reduced basal [Ca²⁺]_i. Our results suggest that gbSS-28 inhibits GnRH-induced GH release in part by attenuating PKC-mediated GnRH [Ca²⁺]_i signals. gbSS-28 reduces basal GH release also via reduction in [Ca²⁺]_i but PKC is not involved in this regard.     

Enhanced myofilament responsiveness upon β-adrenergic stimulation in post-infarct remodeled myocardium

Previously we showed that left ventricular (LV) responsiveness to exercise-induced increases in noradrenaline was blunted in pigs with a recent myocardial infarction (MI) [van der Velden et al. Circ Res. 2004], consistent with perturbed β-adrenergic receptor (β-AR) signaling. Here we tested the hypothesis that abnormalities at the myofilament level underlie impaired LV responsiveness to catecholamines in MI. Myofilament function and protein composition were studied in remote LV biopsies taken at baseline and during dobutamine stimulation 3 weeks after MI or sham. Single permeabilized cardiomyocytes demonstrated reduced maximal force (F_max) and higher Ca²⁺-sensitivity in MI compared to sham. F_max did not change during dobutamine infusion in sham, but markedly increased in MI. Moreover, the dobutamine-induced decrease in Ca²⁺-sensitivity was significantly larger in MI than sham. Baseline phosphorylation assessed by phosphostaining of β-AR target proteins myosin binding protein C (cMyBP-C) and troponin I (cTnI) in MI and sham was the same. However, the dobutamine-induced increase in overall cTnI phosphorylation and cTnI phosphorylation at protein kinase A (PKA)-sites (Ser23/24) was less in MI compared to sham. In contrast, the dobutamine-induced phosphorylation of cMyBP-C at Ser282 was preserved in MI, and coincided with increased autophosphorylation (at Thr282) of the cytosolic Ca²⁺-dependent calmodulin kinase II (CaMKII-δC). In conclusion, in post-infarct remodeled myocardium myofilament responsiveness to dobutamine is significantly enhanced despite the lower increase in PKA-mediated phosphorylation of cTnI. The increased myofilament responsiveness in MI may depend on the preserved cMyBP-C phosphorylation possibly resulting from increased CaMKII-δC activity and may help to maintain proper diastolic performance during exercise.     

Differential effects of phosphorylation of regions of troponin I in modifying cooperative activation of cardiac thin filaments

Ischemia and heart failure are associated with protein kinase C (PKC) dependent phosphorylation of cardiac troponin I (cTnI). We investigated the effect of phosphorylation of cTnI PKC sites S43, S45 and T144 under normal (pH 7.0) and acidic (pH 6.5) conditions on tension in skinned fiber bundles from a mouse heart. To mimic the PKC phosphorylation, we exchanged troponin (cTn) in these fiber bundles with cTn complex containing either cTnI-(S43E/S45E) or cTnI-(T144E). We determined how pseudo-phosphorylation and acidic pH affect activation of thin filaments by strongly bound crossbridges by use of n-ethyl maleimide (NEM-S1) to mimic rigor. We hypothesized that PKC phosphorylation of cTnI amplifies the effect of ischemic/hypoxic conditions to depress myofilament force and Ca²⁺-responsiveness by reducing the ability of rigor crossbridge to activate force. Pseudo-phosphorylation of cTnI at S43/S45 exacerbated the effect of acidic pH to induce a rightward shift in the Ca²⁺-tension relation. Under acidic conditions, fibers regulated by cTnI-(S43E/S45E) demonstrated a significant reduction in the ability of NEM-S1 to recruit cycling crossbridges, when compared to controls regulated by cTnI. Similar effects of pseudo-phosphorylation of cTnI-(T144) occurred, but to a lesser extent that those of pseudo-phosphorylation of S43/S45. We conclude that under acidic conditions PKC phosphorylation of cTnI residues at S43/S45 and at T144 is likely to have differential, but significant effects in depressing the ability of both Ca²⁺ and rigor crossbridges to activate force generation. Although these effects of PKC dependent phosphorylation may be maladaptive in heart failure, they may also spare ATP consumption and be cardio-protective in ischemia.     

Effect of troponin I Ser23/24 phosphorylation on Ca-sensitivity in human myocardium depends on the phosphorylation background

Protein kinase A (PKA)-mediated phosphorylation of Ser23/24 of cardiac troponin I (cTnI) causes a reduction in Ca²⁺-sensitivity of force development. This study aimed to determine whether the PKA-induced modulation of the Ca²⁺-sensitivity is solely due to cTnI phosphorylation or depends on the phosphorylation status of other sarcomeric proteins. Endogenous troponin (cTn) complex in donor cardiomyocytes was partially exchanged (up to 66 ± 1%) with recombinant unphosphorylated human cTn and in failing cells similar exchange was achieved using PKA-(bis)phosphorylated cTn complex. Cardiomyocytes immersed in exchange solution without complex added served as controls. Partial exchange of unphosphorylated cTn complex in donor tissue significantly increased Ca²⁺-sensitivity (pCa₅₀) to 5.50 ± 0.02 relative to the donor control value (pCa₅₀ = 5.43 ± 0.04). Exchange in failing tissue with PKA-phosphorylated cTn complex did not change Ca²⁺-sensitivity relative to the failing control (pCa₅₀ = 5.60 ± 0.02). Subsequent treatment of the cardiomyocytes with the catalytic subunit of PKA significantly decreased Ca²⁺-sensitivity in donor and failing tissue. Analysis of phosphorylated cTnI species revealed the same distribution of un-, mono- and bis-phosphorylated cTnI in donor control and in failing tissue exchanged with PKA-phosphorylated cTn complex. Phosphorylation of myosin-binding protein-C in failing tissue was significantly lower compared to donor tissue. These differences in Ca²⁺-sensitivity in donor and failing cells, despite similar distribution of cTnI species, could be abolished by subsequent PKA-treatment and indicate that other targets of PKA are involved the reduction of Ca²⁺-sensitivity. Our findings suggest that the sarcomeric phosphorylation background, which is altered in cardiac disease, influences the impact of cTnI Ser23/24 phosphorylation by PKA on Ca²⁺-sensitivity.     

Spontaneous and agonist-induced calcium oscillations in single human nonfunctioning adenoma cells

The effects of gonadotropin-releasing hormone (GnRH) and GnRH-associated peptide (GAP) on cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in 20 human nonfunctioning pituitary adenomas. We divided these tumors into three classes according to their response pattern to hypothalamic peptides. In type I adenomas (8 out of 20 adenomas), GnRH and GAP mobilized intracellular calcium ions stored in a thapsigargin (TG)-sensitive store. For the same concentration of agonist, two distinct patterns of GnRH-GAP-induced Ca²⁺ mobilization were observed (1) sinusoidal oscillations, and (2) monophasic transient. The latter is followed by a protein kinase C (PKC)-dependent increase in calcium influx through L-type channels. In type II adenomas (7 out of 20 adenomas), GnRH and GAP only stimulate calcium influx through dihydropyridine-sensitive Ca²⁺ channels by a PKC-dependent mechanism. TG (1 μM) did not affect [Ca²⁺]_i in these cells, suggesting that they do not possess TG-sensitive Ca²⁺ pools. All the effects of GnRH and GAP were blocked by an inhibitor of phospholipase C (PLC), suggesting that they were owing to the activation of the phosphoinositide turnover. Type I and type II adenoma cells showed spontaneous Ca²⁺ oscillations that were blocked by dihydropyridines and inhibition of PKC activity. GnRH and GAP had no effect on the [Ca²⁺]_i of type III adenoma cells that were also characterized by a low resting [Ca²⁺]_i and by the absence of spontaneous Ca²⁺ fluctuations. K⁺-induced depolarization provoked a reduced Ca²⁺ influx, whereas TG had no effect on the [Ca²⁺]_i of type III adenoma cells. The variety of [Ca²⁺]_i response patterns makes these cells a good cell model for studying calcium homeostasis in pituitary cells.     

Calcium-sensitizing inotropic agents in the treatment of heart failure: A critical view

Summary Interventions that augment the contractile state of the heart are associated with, or caused by, alterations in Ca²⁺ exchange in heart muscles. New inotropic agents have been developed that increase the sensitivity of the myofilaments to Ca²⁺. To examine the effect of calcium-sensitizing agents on force development, we measured systolic and diastolic intracellular Ca²⁺ concentration ([Ca²⁺]_i) and constructed [Ca²⁺]_i-force relationships in normal (n=6) and myopathic human hearts (n=10). Using the bioluminescent calcium indicator aequorin, we found that the diastolic [Ca²⁺]_i was 225±52 nM in normal muscles, whereas in myopathic muscles diastolic [Ca²⁺]_i was significantly higher at 361±68 nM. Calcium-sensitizing agents that shift the [Ca²⁺]_i-force relationship toward lower [Ca²⁺]_i increase the diastolic force of myopathic hearts significantly more than in normal human hearts. This leads us to the conclusion that inotropic agents that increase the sensitivity of the myofilaments to Ca²⁺ further impair relaxation in myopathic hearts, resulting in a reduced contractile reserve and diminished actice force production.     

Sarcoplasmic Reticulum Function in Murine Ventricular Myocytes Overexpressing SR CaATPase

To examine the effects of the overexpression of sarcoplasmic reticulum (SR) CaATPase on function of the SR and Ca²⁺homeostasis, we measured [Ca²⁺]_itransients (fluo-3), and L-type Ca²⁺currents (I_Ca,L), Na/Ca exchanger currents (I_Na/Ca), and SR Ca²⁺content with voltage clamp in ventricular myocytes isolated from wild type (WT) mice and transgenic (SRTG) mice. The amplitude of [Ca²⁺]_itransients was insignificantly increased in SRTG myocytes, while the diastolic [Ca²⁺]_itended to be lower. The initial and terminal declines of [Ca²⁺]_itransients were significantly accelerated in SRTG myocytes, implying a functional upregulation of the SR CaATPase. We examined the functional contribution of only the SR CaATPase to the initial and the terminal phase of the decline of [Ca²⁺]_i, by abruptly inhibiting Na/Ca exchange with a rapid switcher device. The rate of [Ca²⁺] decline mediated by the SR CaATPase was increased by 40% in SRTG compared with WT myocytes. The function of the L-type Ca²⁺channel was unchanged in SRTG myocytes, while INa/Ca density was slightly (10%) decreased. Measured SR Ca²⁺content was significantly increased by 29% in SRTG myocytes. Thus, overexpression of SR CaATPase markedly accelerates the decline of [Ca²⁺]_itransients, and induces an increase in SR Ca²⁺content, with some downregulation of the Na/Ca exchanger.     

Migration of primary cultured rabbit gastric epithelial cells requires intact protein kinase C and Ca2+/calmodulin activity

Superficial gastric mucosal injury is rapidly repaired by epithelial cell migration. This study aims to characterize the intracellular signal transduction pathways underlying the repair process. Primary monolayer cultures of rabbit gastric epithelial cells were wounded. The measured spontaneous cell migration speed at the edge of the wound was 457 ± 89 m/24 hr. Epidermal growth factor stimulated and genistein (receptor tyrosine protein kinase inhibitor) inhibited cell migration significantly. Down-regulation of protein Kinase C (PKC) with long-term phorbol 12-myristate 13-acsetate or inhibition with calphostin-C significantly inhibited cell migration. Blocking of Ca²⁺ channels with verapamil and endogenous Ca²⁺ release with TMB-8 or inhibition of the Ca²⁺/calmodulin complex with calmidazolium likewise significantly inhibited migration speed and also abolished the rise of [Ca²⁺]_i, which was measured in migrating cells. Modulation of the cAMP-PKA pathway or prostaglandin synthesis had no influence on cell migration. Gastric epithelial cell migration implies activation of receptor tyrosine kinase. It is associated with increased [Ca²⁺]_i and requires an intact Ca²⁺/calmodulin complex. Intact PKC activity also is needed.     

Pancreatic beta-cells from obese-hyperglycemic mice are characterized by excessive firing of cytoplasmic Ca2+ transients

Pancreatic β-cells from obese-hyperglycemic (ob/ob) mice are widely used for studying the mechanisms of insulin release, including its regulation by the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). In this study, we compared changes of [Ca²⁺]_i in single β-cells isolated from ob/ob mice with those from lean mice using dual-wavelength microfluorometry and the indicator fura-2. There were no differences in the frequency, amplitude, and half-width of the slow oscillations induced by glucose. Most β-cells from the obese mice responded to 10 mM caffeine with transformation of the oscillations into sustained elevation of [Ca²⁺]_i, a process counteracted by ryanodine. The β-cells from the obese mice were characterized by ample generation of [Ca²⁺]_i transients, which increased in number in the presence of glucagon. The transients became less frequent when leptin was added at a concentration as low as 1 nM. It is suggested that the excessive firing of [Ca²⁺]_i transients in the ob/ob mice is owing to the absence of leptin and is mediated by activation of the phospholipase C signaling pathway.     

Diabetes-induced activation of protein kinase C inhibits store-operated Ca<Superscript>2+</Superscript> uptake in rat retinal microvascular smooth muscle

Aims/Hypothesis To assess the effects of diabetes-induced activation of protein kinase C (PKC) on voltage-dependent and voltage-independent Ca²⁺ influx pathways in retinal microvascular smooth muscle cells.Methods Cytosolic Ca²⁺ was estimated in freshly isolated rat retinal arterioles from streptozotocin-induced diabetic and non-diabetic rats using fura-2 microfluorimetry. Voltage-dependent Ca²⁺ influx was tested by measuring rises in [Ca²⁺]_i with KCl (100 mmol/l) and store-operated Ca²⁺ influx was assessed by depleting [Ca²⁺]_i stores with Ca²⁺ free medium containing 5 µmol/l cyclopiazonic acid over 10 min and subsequently measuring the rate of rise in Ca²⁺ on adding 2 mmol/l or 10 mmol/l Ca²⁺solution.Results Ca²⁺ entry through voltage-dependent L-type Ca²⁺ channels was unaffected by diabetes. In contrast, store-operated Ca²⁺ influx was attenuated. In microvessels from non-diabetic rats 20 mmol/l D-mannitol had no effect on store-operated Ca²⁺ influx. Diabetic rats injected daily with insulin had store-operated Ca²⁺ influx rates similar to non-diabetic control rats. The reduced Ca²⁺ entry in diabetic microvessels was reversed by 2-h exposure to 100 nmol/l staurosporine, a non-specific PKC antagonist and was mimicked in microvessels from non-diabetic rats by 10-min exposure to the PKC activator phorbol myristate acetate (100 nmol/l). The specific PKC antagonist LY379196 (100 nmol/l) also reversed the poor Ca²⁺ influx although its action was less efficacious than staurosporine.Conclusion/interpretation These results show that store-operated Ca²⁺ influx is inhibited in retinal arterioles from rats having sustained increased blood glucose and that PKC seems to play a role in mediating this effect.Abbreviations DAG Diacylglycerol - PKC protein kinase C - [Ca²⁺]_i intracellular calcium concentration - STZ streptozotocin - SPP staurosporine - SR sarcoplasmic reticulum - MVSM microvascular smooth muscle - CPA cyclopiazonic acid - PMA phorbol myristate acetate - VDCC voltage-dependent Ca²⁺ channels     

Experimental diabetes enhances Ca2+ mobilization and glutamate exocytosis in cerebral synaptosomes from mice

The present study was conducted to investigate the effects of the diabetic condition on the Ca²⁺ mobilization and glutamate release in cerebral nerve terminals (synaptosomes). Diabetes was induced in male mice by intraperitoneal injection of streptozotocin. Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and glutamate release in synaptosomes were determined using fura-2 and enzyme-linked fluorometric assay, respectively. Diabetes significantly enhanced the ability of the depolarizing agents K⁺ and 4-aminopyridine (4-AP) to increase [Ca²⁺]_i. In addition, diabetes significantly enhanced K⁺- and 4-AP-evoked Ca²⁺-dependent glutamate release. The pretreatment of synaptosomes with a combination of ω-agatoxin IVA (a P-type Ca²⁺ channel blocker) and ω-conotoxin GVIA (an N-type Ca²⁺ channel blocker) inhibited K⁺- or 4-AP-induced increases in [Ca²⁺]_i and Ca²⁺-dependent glutamate release in synaptosomes from the control and diabetic mice to a similar extent, respectively. These results indicate that diabetes enhances a K⁺- or 4-AP-evoked Ca²⁺-dependent glutamate release by increasing [Ca²⁺]_i via stimulation of Ca²⁺ entry through both P- and N-type Ca²⁺ channels.     

Intracellular Ca- and PKC-dependent upregulation of T-type Ca channels in LPC-stimulated cardiomyocytes

Lysophosphatidylcholine (LPC) accumulation in intracellular and/or interstitial space in cardiomyocytes may underlie as a mechanism for tachycardia and various arrhythmias during cardiac ischemia, which is usually accompanied by elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). The present study was therefore designed to investigate possible mechanisms responsible for [Ca²⁺]_i elevation by LPC focusing on T-type Ca²⁺ channel current (I_Ca.T). LPC as well as phorbol 12-myristate 13-acetate (PMA) significantly accelerated the beating rates of neonatal rat cardiomyocytes. Augmentation of I_Ca.T by LPC was dependent on the intracellular Ca²⁺ concentration: an increase of I_Ca.T was significantly larger in high [Ca²⁺]_i condition (pCa = 7) than those in low [Ca²⁺]_i condition (pCa = 11). In heterologous expression system by use of human cardiac Ca_V3.1 and Ca_V3.2 channels expressed in HEK293 cells, LPC augmented Ca_V3.2 channel current (I_Cav3.2) in a concentration-dependent manner but not Ca_V3.1 channel current (I_Cav3.1). Augmentation of I_Cav3.2 by LPC was highly [Ca²⁺]_i dependent: I_Cav3.2 was unchanged when pCa was 11 but was markedly increased when [Ca²⁺]_i was higher than 10⁻¹⁰ M (pCa ≤ 10) by LPC application (10-50 μM). A specific inhibitor of protein kinase Cα (Ro-32-0432) attenuated the increase of I_Cav3.2 by LPC. LPC stimulates I_Ca.T in a [Ca²⁺]_i-dependent manner via PKCα activation, which may play a role in triggering arrhythmias in pathophysiological conditions of the heart.     

Intracellular calcium activates TRPM2 and its alternative spliced isoforms

Melastatin-related transient receptor potential channel 2 (TRPM2) is a Ca²⁺-permeable, nonselective cation channel that is involved in oxidative stress-induced cell death and inflammation processes. Although TRPM2 can be activated by ADP-ribose (ADPR) in vitro, it was unknown how TRPM2 is gated in vivo. Moreover, several alternative spliced isoforms of TRPM2 identified recently are insensitive to ADPR, and their gating mechanisms remain unclear. Here, we report that intracellular Ca²⁺ ([Ca²⁺]_i) can activate TRPM2 as well as its spliced isoforms. We demonstrate that TRPM2 mutants with disrupted ADPR-binding sites can be activated readily by [Ca²⁺]_i, indicating that [Ca²⁺]_i gating of TRPM2 is independent of ADPR. The mechanism by which [Ca²⁺]_i activates TRPM2 is via a calmodulin (CaM)-binding domain in the N terminus of TRPM2. Whereas Ca²⁺-mediated TRPM2 activation is independent of ADPR and ADPR-binding sites, both [Ca²⁺]_i and the CaM-binding motif are required for ADPR-mediated TRPM2 gating. Importantly, we demonstrate that intracellular Ca²⁺ release activates both recombinant and endogenous TRPM2 in intact cells. Moreover, receptor activation-induced Ca²⁺ release is capable of activating TRPM2. These results indicate that [Ca²⁺]_i is a key activator of TRPM2 and the only known activator of the spliced isoforms of TRPM2. Our findings suggest that [Ca²⁺]_i-mediated activation of TRPM2 and its alternative spliced isoforms may represent a major gating mechanism in vivo, therefore conferring important physiological and pathological functions of TRPM2 and its spliced isoforms in response to elevation of [Ca²⁺]_i.     

Intracellular calcium ion response to glucose in beta-cells of calbindin-D28k nullmutant mice and in betaHC13 cells overexpressing calbindin-D28k

This article describes studies on the glucose-induced responses of intracellular Ca²⁺ concentration ([Ca²⁺]_i), insulin release, and redistribution of calbindin-D_28k, a calcium-binding regulatory protein, in β-cells of pancreatic islets of calbindin-D_28k knockout (KO) and wild-type mice (C57BL6) as well as in βHC-13 control cells and βHC-13 CaBP40 cells (β-cell line overexpressing calbindin-D_28k). Upon increasing the glucose concentration from 2.8 to 30 mM, islets of KO mice showed a significantly greater increase in [Ca²⁺]_i (mean increase in [Ca²⁺]_i, i.e., Δ[Ca²⁺], was 296 nM) compared with wild-type mice (Δ[Ca²⁺]_i=97 nM). βHC-13 CaBP40 cells showed little change in [Ca²⁺]_i upon elevation of glucose from 5.5 to 32.7 mM, whereas βHC-13 control cells exhibited significant increases in [Ca²⁺]_i (Δ[Ca²⁺]_i=510 nM). Similarly, upon addition of 30 mM glucose, the rate of insulin release increased from 25.2 (basal rate) to 145.2 pg/mL/min in βHC-13 control cells, whereas in βHC-13 CaBP40 cells the rate of insulin release was only 27.5 pg/mL/min in high glucose. Thus, levels of calbindin-D_28k in β-cells affect both [Ca²⁺]_i and insulin secretion in response to glucose. The three-dimensional reconstruct of confocal immunofluorescent images showed that glucose caused redistribution of calbindin-D_28k resulting in co-localization in the region of L-type voltage-dependent calcium channels (VDCC). This colocalization may be an important regulatory function concerning Ca²⁺ influx via L-type VDCC and exocytosis of insulin granules.     

Serotonin interferes with Ca2+ and PKC signaling to reduce gonadotropin-releasing hormone-stimulated GH secretion in goldfish pituitary cells

In goldfish, two endogenous gonadotropin-releasing hormones (GnRH), salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), are thought to stimulate growth hormone (GH) release via protein kinase C (PKC) and subsequent increases in intracellular Ca²⁺ levels ([Ca²⁺]_i). In contrast, the signaling mechanism for serotonin (5-HT) inhibition of GH secretion is still unknown. In this study, whether 5-HT inhibits GH release by actions at sites along the PKC and Ca²⁺ signal transduction pathways leading to hormone release were examined in primary cultures of goldfish pituitary cells. Under static incubation and column perifusion conditions, 5-HT reduced basal, as well as sGnRH- and cGnRH-II-stimulated, GH secretion. 5-HT also suppressed GH responses to two PKC activators but had no effect on the GH-releasing action of the Ca²⁺ ionophore ionomycin. Ca²⁺-imaging studies with identified somatotropes revealed that 5-HT did not alter basal [Ca²⁺]_i but attenuated the magnitude of the [Ca²⁺]_i responses to the two GnRHs. Prior treatment with 5-HT and cGnRH-II reduced the magnitude of the [Ca²⁺]_i responses induced by depolarizing levels of K⁺. Similar inhibition, however, was not observed with prior treatment of 5-HT and sGnRH. These results suggest that 5-HT, by direct actions at the somatotrope level, interferes with PKC and Ca²⁺ signaling pathways to reduce the GH-releasing effect of GnRH. 5-HT action may occur at the level of PKC activation or its downstream signaling events prior to the subsequent rise in [Ca²⁺]_i.. The differential Ca²⁺ responses by depolarizing doses of K⁺ is consistent with our previous findings that sGnRH and cGnRH-II are coupled to overlapping and yet distinct Ca²⁺-dependent mechanisms.