TRPM2 Channels Protect against Cardiac Ischemia-Reperfusion Injury: ROLE OF MITOCHONDRIA*

Cardiac TRPM2 channels were activated by intracellular adenosine diphosphate-ribose and blocked by flufenamic acid. In adult cardiac myocytes the ratio of G_Ca to G_Na of TRPM2 channels was 0.56 ± 0.02. To explore the cellular mechanisms by which TRPM2 channels protect against cardiac ischemia/reperfusion (I/R) injury, we analyzed proteomes from WT and TRPM2 KO hearts subjected to I/R. The canonical pathways that exhibited the largest difference between WT-I/R and KO-I/R hearts were mitochondrial dysfunction and the tricarboxylic acid cycle. Complexes I, III, and IV were down-regulated, whereas complexes II and V were up-regulated in KO-I/R compared with WT-I/R hearts. Western blots confirmed reduced expression of the Complex I subunit and other mitochondria-associated proteins in KO-I/R hearts. Bioenergetic analyses revealed that KO myocytes had a lower mitochondrial membrane potential, mitochondrial Ca²⁺ uptake, ATP levels, and O₂ consumption but higher mitochondrial superoxide levels. Additionally, mitochondrial Ca²⁺ uniporter (MCU) currents were lower in KO myocytes, indicating reduced mitochondrial Ca²⁺ uptake was likely due to both lower ψ_m and MCU activity. Similar to isolated myocytes, O₂ consumption and ATP levels were also reduced in KO hearts. Under a simulated I/R model, aberrant mitochondrial bioenergetics was exacerbated in KO myocytes. Reactive oxygen species levels were also significantly higher in KO-I/R compared with WT-I/R heart slices, consistent with mitochondrial dysfunction in KO-I/R hearts. We conclude that TRPM2 channels protect the heart from I/R injury by ameliorating mitochondrial dysfunction and reducing reactive oxygen species levels.     

Modulation of Inositol 1,4,5-Trisphosphate Receptor Type 2 Channel Activity by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)-mediated Phosphorylation

InsP₃-mediated calcium release through the type 2 inositol 1,4,5-trisphosphate receptor (InsP₃R2) in cardiac myocytes results in the activation of associated CaMKII, thus enabling the kinase to act on downstream targets, such as histone deacetylases 4 and 5 (HDAC4 and HDAC5). The CaMKII activity also feedback modulates InsP₃R2 function by direct phosphorylation and results in a dramatic decrease in the receptor-channel open probability (P_o). We have identified S150 in the InsP₃R2 core suppressor domain (amino acids 1–225) as the specific residue that is phosphorylated by CaMKII. Site-directed mutagenesis reveals that S150 is the CaMKII phosphorylation site responsible for modulation of channel activity. Nonphosphorylatable (S150A) and phosphomimetic (S150E) mutations were studied in planar lipid bilayers. The InsP₃R2 S150A channel showed no decrease in activity when treated with CaMKII. Conversely, the phosphomimetic (S150E) channel displayed a very low P_o under normal recording conditions in the absence of CaMKII (2 μm InsP₃ and 250 nm [Ca²⁺]_FREE) and mimicked a WT channel that has been phosphorylated by CaMKII. Phopho-specific antibodies demonstrate that InsP₃R2 Ser-150 is phosphorylated in vivo by CaMKIIδ. The results of this study show that serine 150 of the InsP₃R2 is phosphorylated by CaMKII and results in a decrease in the channel open probability.     

Sodium-Calcium Exchange Is Essential for Effective Triggering of Calcium Release in Mouse Heart

In cardiac myocytes, excitation-contraction coupling depends upon sarcoplasmic reticular Ca²⁺ release triggered by Ca²⁺ influx through L-type Ca²⁺ channels. Although Na⁺-Ca²⁺ exchange (NCX) is essential for Ca²⁺ extrusion, its participation in the trigger process of excitation-contraction coupling is controversial. To investigate the role of NCX in triggering, we examined Ca²⁺ sparks in ventricular cardiomyocytes isolated from wild-type (WT) and cardiac-specific NCX knockout (KO) mice. Myocytes from young NCX KO mice are known to exhibit normal resting cytosolic Ca²⁺ and normal Ca²⁺ transients despite reduced L-type Ca²⁺ current. We loaded myocytes with fluo-3 to image Ca²⁺ sparks using confocal microscopy in line-scan mode. The frequency of spontaneous Ca²⁺ sparks was reduced in KO myocytes compared with WT. However, spark amplitude and width were increased in KO mice. Permeabilizing the myocytes with saponin eliminated differences between spontaneous sparks in WT and KO mice. These results suggest that sarcolemmal processes are responsible for the reduced spark frequency and increased spark width and amplitude in KO mice. When myocytes were loaded with 1 mM fluo-3 and 3 mM EGTA via the patch pipette to buffer diadic cleft Ca²⁺, the number of sparks triggered by action potentials was reduced by 60% in KO cells compared to WT cells, despite similar SR Ca²⁺ content in both cell types. When EGTA was omitted from the pipette solution, the number of sparks triggered in KO and WT myocytes was similar. Although the number of sparks was restored in KO cells, Ca²⁺ release was asynchronous. These results suggest that high subsarcolemmal Ca²⁺ is required to ensure synchronous triggering with short spark latency in the absence of NCX. In WT mice, high subsarcolemmal Ca²⁺ is not required for synchronous triggering, because NCX is capable of priming the diadic cleft with sufficient Ca²⁺ for normal triggering, even when subsarcolemmal Ca²⁺ is lowered by EGTA. Thus, reducing subsarcolemmal Ca²⁺ with EGTA in NCX KO mice reveals the dependence of Ca²⁺ release on NCX.     

Regulation of the Tyrosine Kinase Pyk2 by Calcium Is through Production of Reactive Oxygen Species in Cytotoxic T Lymphocytes

Pyk2 was identified as a Ca²⁺-dependent kinase, however, the regulation of Pyk2 by Ca²⁺ in T cells remains controversial. We found that Ca²⁺ mobilization preferentially induced Pyk2 phosphorylation in cytotoxic T lymphocytes (CTL). Furthermore, Pyk2 phosphorylation in CTL was not absolutely Ca²⁺ dependent but relied on the strength of T cell receptor stimulation. Ionomycin-stimulated Pyk2 phosphorylation did not require calmodulin activity, because phosphorylation was not inhibited by the calmodulin inhibitor W7, and we detected no Ca²⁺-regulated association between Pyk2 and calmodulin. Ca²⁺-stimulated Pyk2 phosphorylation was dependent on Src-family kinase activity, even at the Pyk2 autophosphorylation site. We sought to identify a Ca²⁺-regulated pathway that could trigger Pyk2 phosphorylation in T cells and found that ionomycin stimulated the production of reactive oxygen species and an H₂O₂ scavenger inhibited ionomycin-induced Pyk2 phosphorylation. Additionally, H₂O₂ induced strong Erk activation and ionomycin-stimulated Pyk2 phosphorylation was Erk dependent. These data support the conclusion that Ca²⁺ mobilization induces the production of reactive oxygen species, which in turn activate the Erk pathway, leading to Src-family kinase-dependent Pyk2 phosphorylation. Our data demonstrate that Pyk2 is not a Ca²⁺-dependent kinase in T cells but instead, increased intracellular Ca²⁺ induces Pyk2 phosphorylation through production of reactive oxygen species. These findings are consistent with the possibility that Pyk2 acts as an early sensor of numerous extracellular signals that trigger a Ca²⁺ flux and/or reactive oxygen species to amplify tyrosine phosphorylation signaling events.     

CaMKII knockdown attenuates H2O2-induced phosphorylation of ERK1/2, PKB/Akt, and IGF-1R in vascular smooth muscle cells

We have shown earlier a requirement for Ca²⁺ and calmodulin (CaM) in the H₂O₂-induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), key mediators of growth-promoting, proliferative, and hypertrophic responses in vascular smooth muscle cells (VSMC). Because the effect of CaM is mediated through CaM-dependent protein kinase II (CaMKII), we have investigated here the potential role of CaMKII in H₂O₂-induced ERK1/2 and PKB phosphorylation by using pharmacological inhibitors of CaM and CaMKII, a CaMKII inhibitor peptide, and siRNA knockdown strategies for CaMKIIα. Calmidazolium and W-7, antagonists of CaM, as well as KN-93, a specific inhibitor of CaMKII, attenuated H₂O₂-induced responses of ERK1/2 and PKB phosphorylation in a dose-dependent fashion. Similar to H₂O₂, calmidazolium and KN-93 also exhibited an inhibitory effect on glucose/glucose oxidase-induced phosphorylation of ERK1/2 and PKB in these cells. Transfection of VSMC with CaMKII autoinhibitory peptide corresponding to the autoinhibitory domain (aa 281–309) of CaMKII and with siRNA of CaMKIIα attenuated the H₂O₂-induced phosphorylation of ERK1/2 and PKB. In addition, calmidazolium and KN-93 blocked H₂O₂-induced Pyk2 and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation. Moreover, treatment of VSMC with CaMKIIα siRNA abolished the H₂O₂-induced IGF-1R phosphorylation. H₂O₂ treatment also induced Thr²⁸⁶ phosphorylation of CaMKII, which was inhibited by both calmidazolium and KN-93. These results demonstrate that CaMKII plays a critical upstream role in mediating the effects of H₂O₂ on ERK1/2, PKB, and IGF-1R phosphorylation.     

Ca disorder caused by rapid electrical field stimulation can be modulated by CaMKIIδ expression in primary rat atrial myocytes

Abnormalities in intracellular Ca²⁺ handing are believed to contribute to arrhythmogenesis during atrial fibrillation (AF). Ca²⁺/calmodulin-dependent protein kinaseII δ (CaMKIIδ) overexpression was detected in atrial myocytes from patients and animal models with persistent AF. In the present study, we found that rapid electrical field stimulation applied to primary atrial myocytes altered the CaMKIIδ activity, not expression level, resulting in Ca²⁺ disorder. By lentivirus mediated delivery of CaMKIIδ gene or siRNA into atrial myocytes, cells with different CaMKIIδ expression were generated. Changes of CaMKIIδ expression altered the sarcoplasmic reticulum (SR) Ca²⁺ release and L-type Ca²⁺ channels current (I_Ca) in both steady and electrical stimulating state. These results revealed the important role of CaMKIIδ in Ca²⁺ disorder caused by electrical field stimulation. It also provided a potential method to improve Ca²⁺ disorder in AF by modulating CaMKIIδ expression level.     

Mitochondrial dysfunction in human immunodeficiency virus-1 transgenic mouse cardiac myocytes

The pathophysiology of human immunodeficiency virus (HIV)-associated cardiomyopathy remains uncertain. We used HIV-1 transgenic (Tg26) mice to explore mechanisms by which HIV-related proteins impacted on myocyte function. Compared to adult ventricular myocytes isolated from nontransgenic (wild type [WT]) littermates, Tg26 myocytes had similar mitochondrial membrane potential (ΔΨ _m) under normoxic conditions but lower Δ Ψ _m after hypoxia/reoxygenation (H/R). In addition, Δ Ψ _m in Tg26 myocytes failed to recover after Ca ²⁺ challenge. Functionally, mitochondrial Ca ²⁺ uptake was severely impaired in Tg26 myocytes. Basal and maximal oxygen consumption rates (OCR) were lower in normoxic Tg26 myocytes, and further reduced after H/R. Complex I subunit and ATP levels were lower in Tg26 hearts. Post-H/R, mitochondrial superoxide (O ₂ ^•–) levels were higher in Tg26 compared to WT myocytes. Overexpression of B-cell lymphoma 2-associated athanogene 3 (BAG3) reduced O ₂ ^•– levels in hypoxic WT and Tg26 myocytes back to normal. Under normoxic conditions, single myocyte contraction dynamics were similar between WT and Tg26 myocytes. Post-H/R and in the presence of isoproterenol, myocyte contraction amplitudes were lower in Tg26 myocytes. BAG3 overexpression restored Tg26 myocyte contraction amplitudes to those measured in WT myocytes post-H/R. Coimmunoprecipitation experiments demonstrated physical association of BAG3 and the HIV protein Tat. We conclude: (a) Under basal conditions, mitochondrial Ca ²⁺ uptake, OCR, and ATP levels were lower in Tg26 myocytes; (b) post-H/R, Δ Ψ _m was lower, mitochondrial O ₂ ^•– levels were higher, and contraction amplitudes were reduced in Tg26 myocytes; and (c) BAG3 overexpression decreased O ₂ ^•– levels and restored contraction amplitudes to normal in Tg26 myocytes post-H/R in the presence of isoproterenol.     

A Role for the Tyrosine Kinase Pyk2 in Depolarization-induced Contraction of Vascular Smooth Muscle

Depolarization of the vascular smooth muscle cell membrane evokes a rapid (phasic) contractile response followed by a sustained (tonic) contraction. We showed previously that the sustained contraction involves genistein-sensitive tyrosine phosphorylation upstream of the RhoA/Rho-associated kinase (ROK) pathway leading to phosphorylation of MYPT1 (the myosin-targeting subunit of myosin light chain phosphatase (MLCP)) and myosin regulatory light chains (LC₂₀). In this study, we addressed the hypothesis that membrane depolarization elicits activation of the Ca²⁺-dependent tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2). Pyk2 was identified as the major tyrosine-phosphorylated protein in response to membrane depolarization. The tonic phase of K⁺-induced contraction was inhibited by the Pyk2 inhibitor sodium salicylate, which abolished the sustained elevation of LC₂₀ phosphorylation. Membrane depolarization induced autophosphorylation (activation) of Pyk2 with a time course that correlated with the sustained contractile response. The Pyk2/focal adhesion kinase (FAK) inhibitor PF-431396 inhibited both phasic and tonic components of the contractile response to K⁺, Pyk2 autophosphorylation, and LC₂₀ phosphorylation but had no effect on the calyculin A (MLCP inhibitor)-induced contraction. Ionomycin, in the presence of extracellular Ca²⁺, elicited a slow, sustained contraction and Pyk2 autophosphorylation, which were blocked by pre-treatment with PF-431396. Furthermore, the Ca²⁺ channel blocker nifedipine inhibited peak and sustained K⁺-induced force and Pyk2 autophosphorylation. Inhibition of Pyk2 abolished the K⁺-induced translocation of RhoA to the particulate fraction and the phosphorylation of MYPT1 at Thr-697 and Thr-855. We conclude that depolarization-induced entry of Ca²⁺ activates Pyk2 upstream of the RhoA/ROK pathway, leading to MYPT1 phosphorylation and MLCP inhibition. The resulting sustained elevation of LC₂₀ phosphorylation then accounts for the tonic contractile response to membrane depolarization.     

Redox Signal-mediated Enhancement of the Temperature Sensitivity of Transient Receptor Potential Melastatin 2 (TRPM2) Elevates Glucose-induced Insulin Secretion from Pancreatic Islets

Transient receptor potential melastatin 2 (TRPM2) is a thermosensitive Ca²⁺-permeable cation channel expressed by pancreatic β cells where channel function is constantly affected by body temperature. We focused on the physiological functions of redox signal-mediated TRPM2 activity at body temperature. H₂O₂, an important molecule in redox signaling, reduced the temperature threshold for TRPM2 activation in pancreatic β cells of WT mice but not in TRPM2KO cells. TRPM2-mediated [Ca²⁺]_i increases were likely caused by Ca²⁺ influx through the plasma membrane because the responses were abolished in the absence of extracellular Ca²⁺. In addition, TRPM2 activation downstream from the redox signal plus glucose stimulation enhanced glucose-induced insulin secretion. H₂O₂ application at 37 °C induced [Ca²⁺]_i increases not only in WT but also in TRPM2KO β cells. This was likely due to the effect of H₂O₂ on K_ATP channel activity. However, the N-acetylcysteine-sensitive fraction of insulin secretion by WT islets was increased by temperature elevation, and this temperature-dependent enhancement was diminished significantly in TRPM2KO islets. These data suggest that endogenous redox signals in pancreatic β cells elevate insulin secretion via TRPM2 sensitization and activity at body temperature. The results in this study could provide new therapeutic approaches for the regulation of diabetic conditions by focusing on the physiological function of TRPM2 and redox signals.     

Domain-dependent modulation of insulin-induced AS160 phosphorylation and glucose uptake by Ca2+/calmodulin-dependent protein kinase II in L6 myotubes

In skeletal muscle, the molecular mechanisms by which insulin stimulates glucose transport remains incompletely understood. Our study investigated the cellular dynamics of intracellular Ca²⁺ mobilisation and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) activation on insulin-induced skeletal muscle glucose transport. L6 myotubes were treated without or with insulin [100 nM] for 15 min and subsequently monitored for glucose uptake using isotope-labelled 2-deoxyglucose (I-2DOG), intracellular Ca²⁺ (Ca_i²⁺) release using Fluo-4AM and protein phosphorylation using Western blotting. Acute exposure of myotubes to insulin increased both Akt substrate-160 kDa (AS160) phosphorylation and I-2DOG uptake. Insulin concurrently increased Ca_i²⁺ and activated CaMKII. Exposing myotubes to either BAPTA/AM to sequester Ca_i²⁺ or KN-93 to inhibit CaMKII activity, decreased insulin-induced glucose uptake without affecting AS160 phosphorylation. On the other hand, blocking either calmodulin or the autoregulatory domain of CaMKII blocked the effect of insulin on both AS160 phosphorylation and glucose transport. Likewise, genetic knockdown of CaMKII in myotubes using siRNA completely abolished insulin-mediated glucose uptake. These results illustrate impairments in Ca_i²⁺ mobilisation and CaMKII activation are sufficient to negatively influence insulin-dependent glucose transport by L6 myotubes. Additionally, our results show for the first time that Ca_i²⁺ and domain-dependent CaMKII signalling differentially affect insulin-induced AS160 phosphorylation, and establish that Ca²⁺ and CaMKII are components of the insulin signalling pathway in L6 myotubes.     

Isoproterenol-induced hypertrophy of neonatal cardiac myocytes and H9c2 cell is dependent on TRPC3-regulated CaV1.2 expression

Ca_V1.2 and transient receptor potential canonical channel 3 (TRPC3) are two proteins known to have important roles in pathological cardiac hypertrophy; however, such roles still remain unclear. A better understanding of these roles is important for furthering the clinical understanding of heart failure. We previously reported that Trpc3-knockout (KO) mice are resistant to pathologic hypertrophy and that their Ca_V1.2 protein expression is reduced. In this study, we aimed to examine the relationship between these two proteins and characterize their role in neonatal cardiomyocytes. We measured Ca_V1.2 expression in the hearts of wild-type (WT) and Trpc3^−/− mice, and examined the effects of Trpc3 knockdown and overexpression in the rat cell line H9c2. We also compared the hypertrophic responses of neonatal cardiomyocytes cultured from Trpc3^−/− mice to a representative hypertrophy-causing drug, isoproterenol (ISO), and measured the activity of nuclear factor of activated T cells 3 (NFAT3) in neonatal cardiomyocytes (NCMCs). We inhibited the L-type current with nifedipine, and measured the intracellular calcium concentration using Fura-2 with 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced Ba²⁺ influx. When using the Trpc3-mediated Ca²⁺ influx, both intracellular calcium concentration and calcium influx were reduced in Trpc3-KO myocytes. Not only was the expression of Ca_V1.2 greatly reduced in Trpc3-KO cardiac lysate, but the size of the Ca_V1.2 currents in NCMCs was also greatly reduced. When NCMCs were treated with Trpc3 siRNA, it was confirmed that the expression of Ca_V1.2 and the intracellular nuclear transfer activity of NFAT decreased. In H9c2 cells, the ISO activated- and verapamil inhibited- Ca²⁺ influxes were dramatically attenuated by Trpc3 siRNA treatment. In addition, it was confirmed that both the expression of Ca_V1.2 and the size of H9c2 cells were regulated according to the expression and activation level of TRPC3. We found that after stimulation with ISO, cell hypertrophy occurred in WT myocytes, while the increase in size of Trpc3-KO myocytes was greatly reduced. These results suggest that not only the cell hypertrophy process in neonatal cardiac myocytes and H9c2 cells were regulated according to the expression level of Ca_V1.2, but also that the expression level of Ca_V1.2 was regulated by TRPC3 through the activation of NFAT.     

Activation of Ca2+/Calmodulin-Dependent Protein Kinase II by Extracellular Calcium in Cultured Hippocampal Pyramidal Neurons

Abstract: The ability of various stimuli to convert Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) into a Ca²⁺-independent (autonomous) form was examined in cultured embryonic rat hippocampal pyramidal neurons. The most effective stimulation by far was observed when cells were equilibrated in buffer containing low extracellular [Ca²⁺] ([Ca²⁺]_o) (～50 nM) and then shifted to normal [Ca²⁺]_o (～1.26 mM) by addition of CaCl₂ (referred to as “Ca²⁺ stimulation”). Virtually complete (>90%) conversion of the kinase to the autonomous form occurred within 30–50 s, with a return to baseline within 5 min. By contrast, depolarization of cells with high [K⁺] or treatment with glutamate or a Ca²⁺ ionophore caused insignificant increases (<10%) in levels of the autonomous form. The Ca²⁺-stimulated increase in CaMKII autonomy coincided with a two- to threefold increase in kinase subunit phosphorylation. In the first 40 s of Ca²⁺ stimulation, ³²P incorporation into the immunoprecipitated subunits of CaMKII occurred exclusively on threonine residues, including Thr²⁸⁶Thr²⁸⁷ of the α/β subunits. Longer incubation of cells resulted in a decline of phosphothreonine content, whereas levels of phosphoserine-containing peptides showed a significant increase. The activation of CaMKII by Ca²⁺ stimulation was accompanied by only a small rise in intracellular [Ca²⁺]. Inhibitor studies showed that Na⁺-dependent action potentials and Ca²⁺ influx through glutamate receptors or voltage-sensitive Ca²⁺ channels did not contribute to the activation. Moreover, CaMKII was not activated by extracellular addition of other cations, including Mn²⁺, Mg²⁺, Co²⁺, or Gd³⁺. Although the mechanism of Ca²⁺ stimulation is presently unclear, it may involve either activation of extracellular calcium receptors or capacitative calcium entry. The dramatic rise in CaMKII autonomy and the Ca²⁺ selectivity of the response suggest a direct and specific relationship between [Ca²⁺]_o and the state of activation of the kinase in intact neurons.     

Effects of CaMKII-Mediated Phosphorylation of Ryanodine Receptor Type 2 on Islet Calcium Handling,Insulin Secretion,and Glucose Tolerance

Altered insulin secretion contributes to the pathogenesis of type 2 diabetes. This alteration is correlated with altered intracellular Ca²⁺-handling in pancreatic β cells. Insulin secretion is triggered by elevation in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) of β cells. This elevation in [Ca²⁺]_cyt leads to activation of Ca²⁺/calmodulin-dependent protein kinase II (CAMKII), which, in turn, controls multiple aspects of insulin secretion. CaMKII is known to phosphorylate ryanodine receptor 2 (RyR2), an intracellular Ca²⁺-release channel implicated in Ca²⁺-dependent steps of insulin secretion. Our data show that RyR2 is CaMKII phosphorylated in a pancreatic β-cell line in a glucose-sensitive manner. However, it is not clear whether any change in CaMKII-mediated phosphorylation underlies abnormal RyR2 function in β cells and whether such a change contributes to alterations in insulin secretion. Therefore, knock-in mice with a mutation in RyR2 that mimics its constitutive CaMKII phosphorylation, RyR2-S2814D, were studied. This mutation led to a gain-of-function defect in RyR2 indicated by increased basal RyR2-mediated Ca²⁺ leak in islets of these mice. This chronic in vivo defect in RyR2 resulted in basal hyperinsulinemia. In addition, S2814D mice also developed glucose intolerance, impaired glucose-stimulated insulin secretion and lowered [Ca²⁺]_cyt transients, which are hallmarks of pre-diabetes. The glucose-sensitive Ca²⁺ pool in islets from S2814D mice was also reduced. These observations were supported by immunohistochemical analyses of islets in diabetic human and mouse pancreata that revealed significantly enhanced CaMKII phosphorylation of RyR2 in type 2 diabetes. Together, these studies implicate that the chronic gain-of-function defect in RyR2 due to CaMKII hyperphosphorylation is a novel mechanism that contributes to pathogenesis of type 2 diabetes.     

Mechanism of calsequestrin regulation of single cardiac ryanodine receptor in normal and pathological conditions

Release of Ca²⁺ from the sarcoplasmic reticulum (SR) drives contractile function of cardiac myocytes. Luminal Ca²⁺ regulation of SR Ca²⁺ release is fundamental not only in physiology but also in physiopathology because abnormal luminal Ca²⁺ regulation is known to lead to arrhythmias, catecholaminergic polymorphic ventricular tachycardia (CPVT), and/or sudden cardiac arrest, as inferred from animal model studies. Luminal Ca²⁺ regulates ryanodine receptor (RyR)2-mediated SR Ca²⁺ release through mechanisms localized inside the SR; one of these involves luminal Ca²⁺ interacting with calsequestrin (CASQ), triadin, and/or junctin to regulate RyR2 function.CASQ2-RyR2 regulation was examined at the single RyR2 channel level. Single RyR2s were incorporated into planar lipid bilayers by the fusion of native SR vesicles isolated from either wild-type (WT), CASQ2 knockout (KO), or R33Q-CASQ2 knock-in (KI) mice. KO and KI mice have CPVT-like phenotypes. We show that CASQ2(WT) action on RyR2 function (either activation or inhibition) was strongly influenced by the presence of cytosolic MgATP. Function of the reconstituted CASQ2(WT)–RyR2 complex was unaffected by changes in luminal free [Ca²⁺] (from 0.1 to 1 mM). The inhibition exerted by CASQ2(WT) association with the RyR2 determined a reduction in cytosolic Ca²⁺ activation sensitivity. RyR2s from KO mice were significantly more sensitive to cytosolic Ca²⁺ activation and had significantly longer mean open times than RyR2s from WT mice. Sensitivity of RyR2s from KI mice was in between that of RyR2 channels from KO and WT mice. Enhanced cytosolic RyR2 Ca²⁺ sensitivity and longer RyR2 open times likely explain the CPVT-like phenotype of both KO and KI mice.     

Metabolic adaptation to the chronic loss of Ca2+ signaling induced by KO of IP3 receptors or the mitochondrial Ca2+ uniporter

Calcium signaling is essential for regulating many biological processes. Endoplasmic reticulum inositol trisphosphate receptors (IP₃Rs) and the mitochondrial Ca²⁺ uniporter (MCU) are key proteins that regulate intracellular Ca²⁺ concentration. Mitochondrial Ca²⁺ accumulation activates Ca²⁺-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle that maintain the biosynthetic and bioenergetic needs of both normal and cancer cells. However, the interplay between calcium signaling and metabolism is not well understood. In this study, we used human cancer cell lines (HEK293 and HeLa) with stable KOs of all three IP₃R isoforms (triple KO [TKO]) or MCU to examine metabolic and bioenergetic responses to the chronic loss of cytosolic and/or mitochondrial Ca²⁺ signaling. Our results show that TKO cells (exhibiting total loss of Ca²⁺ signaling) are viable, displaying a lower proliferation and oxygen consumption rate, with no significant changes in ATP levels, even when made to rely solely on the TCA cycle for energy production. MCU KO cells also maintained normal ATP levels but showed increased proliferation, oxygen consumption, and metabolism of both glucose and glutamine. However, MCU KO cells were unable to maintain ATP levels and died when relying solely on the TCA cycle for energy. We conclude that constitutive Ca²⁺ signaling is dispensable for the bioenergetic needs of both IP₃R TKO and MCU KO human cancer cells, likely because of adequate basal glycolytic and TCA cycle flux. However, in MCU KO cells, the higher energy expenditure associated with increased proliferation and oxygen consumption makes these cells more prone to bioenergetic failure under conditions of metabolic stress.     

Investigation of carvedilol-evoked Ca2+ movement and death in human oral cancer cells

The effect of carvedilol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in OC2 human oral cancer cells is unknown. This study examined if carvedilol altered basal [Ca²⁺]_i levels in suspended OC2 cells by using fura-2 as a Ca²⁺-sensitive fluorescent probe. Carvedilol at concentrations between 10 and 40 µM increased [Ca²⁺]_i in a concentration-dependent fashion. The Ca²⁺ signal was decreased by 50% by removing extracellular Ca²⁺. Carvedilol-induced Ca²⁺ entry was not affected by the store-operated Ca²⁺ channel blockers nifedipine, econazole, and SK&F96365, but was enhanced by activation or inhibition of protein kinase C. In Ca²⁺-free medium, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin did not change carvedilol-induced [Ca²⁺]_i rise; conversely, incubation with carvedilol did not reduce thapsigargin-induced Ca²⁺ release. Pretreatment with the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) inhibited carvedilol-induced [Ca²⁺]_i release. Inhibition of phospholipase C with U73122 did not alter carvedilol-induced [Ca²⁺]_i rise. Carvedilol at 5–50 µM induced cell death in a concentration-dependent manner. The death was not reversed when cytosolic Ca²⁺ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA/AM). Annexin V/propidium iodide staining assay suggests that apoptosis played a role in the death. Collectively, in OC2 cells, carvedilol induced [Ca²⁺]_i rise by causing phospholipase C-independent Ca²⁺ release from mitochondria and non-endoplasmic reticulum stores, and Ca²⁺ influx via protein kinase C-regulated channels. Carvedilol (up to 50 μM) induced cell death in a Ca²⁺-independent manner that involved apoptosis.     

Ca2+-independent Activation of Ca2+/Calmodulin-dependent Protein Kinase II Bound to the C-terminal Domain of CaV2.1 Calcium Channels

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) forms a major component of the postsynaptic density where its functions in synaptic plasticity are well established, but its presynaptic actions are poorly defined. Here we show that CaMKII binds directly to the C-terminal domain of Ca_V2.1 channels. Binding is enhanced by autophosphorylation, and the kinase-channel signaling complex persists after dephosphorylation and removal of the Ca²⁺/CaM stimulus. Autophosphorylated CaMKII can bind the Ca_V2.1 channel and synapsin-1 simultaneously. CaMKII binding to Ca_V2.1 channels induces Ca²⁺-independent activity of the kinase, which phosphorylates the enzyme itself as well as the neuronal substrate synapsin-1. Facilitation and inactivation of Ca_V2.1 channels by binding of Ca²⁺/CaM mediates short term synaptic plasticity in transfected superior cervical ganglion neurons, and these regulatory effects are prevented by a competing peptide and the endogenous brain inhibitor CaMKIIN, which blocks binding of CaMKII to Ca_V2.1 channels. These results define the functional properties of a signaling complex of CaMKII and Ca_V2.1 channels in which both binding partners are persistently activated by their association, and they further suggest that this complex is important in presynaptic terminals in regulating protein phosphorylation and short term synaptic plasticity.     

Gastrodin Inhibits Glutamate-Induced Apoptosis of PC12 Cells via Inhibition of CaMKII/ASK-1/p38 MAPK/p53 Signaling Cascade

Previous research demonstrated that glutamate induces neuronal injury partially by increasing intracellular Ca²⁺ concentrations ([Ca²⁺]_i), and inducing oxidative stress, leading to a neurodegenerative disorder. However, the mechanism of glutamate-induced injury remains elusive. Gastrodin, a major active component of the traditional herbal agent Gastrodia elata (GE) Blume, has been recognized as a potential neuroprotective drug. In the current study, a classical injury model based on glutamate-induced cell death of rat pheochromocytoma (PC12) cells was used to investigate the neuroprotective effect of gastrodin, and its potential mechanisms involved. In this paper, the presence of gastrodin inhibits glutamate-induced oxidative stress as measured by the formation of reactive oxygen species (ROS), the level of malondialdehyde (MDA), mitochondrial membrane potential (MMP), and superoxide dismutase (SOD); gastrodin also prevents glutamate-induced [Ca²⁺]_i influx, blocks the activation of the calmodulin-dependent kinase II (CaMKII) and the apoptosis signaling-regulating kinase-1 (ASK-1), inhibits phosphorylation of p38 mitogen-activated kinase (MAPK). Additionally, gastrodin blocked the expression of p53 phosphorylation, caspase-3 and cytochrome C, reduced bax/bcl-2 ratio induced by glutamate in PC12 cells. All these findings indicate that gastrodin protects PC12 cells from the apoptosis induced by glutamate through a new mechanism of the CaMKII/ASK-1/p38 MAPK/p53-signaling pathway.     

Channel Activity of Cardiac Ryanodine Receptors (RyR2) Determines Potency and Efficacy of Flecainide and R-Propafenone against Arrhythmogenic Calcium Waves in Ventricular Cardiomyocytes

Flecainide blocks ryanodine receptor type 2 (RyR2) channels in the open state, suppresses arrhythmogenic Ca²⁺ waves and prevents catecholaminergic polymorphic ventricular tachycardia (CPVT) in mice and humans. We hypothesized that differences in RyR2 activity induced by CPVT mutations determines the potency of open-state RyR2 blockers like flecainide (FLEC) and R-propafenone (RPROP) against Ca²⁺ waves in cardiomyocytes. Using confocal microscopy, we studied Ca²⁺ sparks and waves in isolated saponin-permeabilized ventricular myocytes from two CPVT mouse models (Casq2^-/-, RyR2-R4496C^+/-), wild-type (c57bl/6, WT) mice, and WT rabbits (New Zealand white rabbits). Consistent with increased RyR2 activity, Ca²⁺ spark and wave frequencies were significantly higher in CPVT compared to WT mouse myocytes. We next obtained concentration-response curves of Ca²⁺ wave inhibition for FLEC, RPROP (another open-state RyR2 blocker), and tetracaine (TET) (a state-independent RyR2 blocker). Both FLEC and RPROP inhibited Ca²⁺ waves with significantly higher potency (lower IC₅₀) and efficacy in CPVT compared to WT. In contrast, TET had similar potency in all groups studied. Increasing RyR2 activity of permeabilized WT myocytes by exposure to caffeine (150 µM) increased the potency of FLEC and RPROP but not of TET. RPROP and FLEC were also significantly more potent in rabbit ventricular myocytes that intrinsically exhibit higher Ca²⁺ spark rates than WT mouse ventricular myocytes. In conclusion, RyR2 activity determines the potency of open-state blockers FLEC and RPROP for suppressing arrhythmogenic Ca²⁺ waves in cardiomyocytes, a mechanism likely relevant to antiarrhythmic drug efficacy in CPVT.     

In?Vitro Reconstitution of a CaMKII Memory Switch by an NMDA Receptor-Derived Peptide

Ca²⁺/Calmodulin-dependent protein kinase II (CaMKII) has been shown to play a major role in establishing memories through complex molecular interactions including phosphorylation of multiple synaptic targets. However, it is still controversial whether CaMKII itself serves as a molecular memory because of a lack of direct evidence. Here, we show that a single holoenzyme of CaMKII per se serves as an erasable molecular memory switch. We reconstituted Ca²⁺/Calmodulin-dependent CaMKII autophosphorylation in the presence of protein phosphatase 1 in vitro, and found that CaMKII phosphorylation shows a switch-like response with history dependence (hysteresis) only in the presence of an N-methyl-D-aspartate receptor-derived peptide. This hysteresis is Ca²⁺ and protein phosphatase 1 concentration-dependent, indicating that the CaMKII memory switch is not simply caused by an N-methyl-D-aspartate receptor-derived peptide lock of CaMKII in an active conformation. Mutation of a phosphorylation site of the peptide shifted the Ca²⁺ range of hysteresis. These functions may be crucial for induction and maintenance of long-term synaptic plasticity at hippocampal synapses.