• Calcium sparks in embryonic and postnatal mouse skeletal muscle

      Chun, Lois Grace; Schneider, Martin F. (2002)
      Brief elevations of myoplasmic Ca2+ (Ca2+ sparks) have been visualized in embryonic mammalian skeletal muscle but are rarely seen in adult skeletal muscle (Conklin et al., 1998; Gyorke and Gyorke, 1996; Shirokova et al. 1998). I have monitored the occurrence of Ca2+ sparks in dissociated mouse diaphragm and the hindlimb (EDL) muscle fibers as a function of development using a confocal microscope and the fluorescent dye, Fluo4-AM. Ca2+ sparks were identified from many series of XY images of fiber sections using a computer analysis program. The frequency of events decreased to significantly low levels within two weeks in the diaphragm fibers and within 1 week in EDL fibers. The spatial properties of events in XY images demonstrated a decreasing trend as development progressed, even though there were no significant differences in temporal parameters of events measured in line-scan mode. To demonstrate that the occurrence of Ca2+ sparks in embryonic skeletal muscle was not related to the isoform type of RyR, Western blot analysis was performed on diaphragm muscles at developmental ages corresponding to Ca2+ spark frequency data. The decline of Ca 2+ spark frequency did not correlate with RyR3 expression suggesting that the event frequency is not attributed to RyR isoform. To further elucidate the production of Ca2+ sparks in embryonic mammalian skeletal muscle, embryonic diaphragm fibers (E18) were bathed in Ringer's solution containing 0Ca2+ with 1 mM EGTA, 1.8mM Ca2+ (physiological Ca2+ concentration) or 8mM Ca2+. The Ca2+ event frequency increased in fibers bathed in solution containing high concentrations of external Ca2+ and decreased in fibers bathed in 0Ca2+ indicating that Ca2+ influx could modulate the frequency of Ca2+ events in embryonic mammalian skeletal muscle. To confirm this finding, Ca2+ channels were blocked using CoCl2 and specifically the L-type Ca 2+ channels were inhibited using the dihydropyridine antagonist, nifedipine. The frequency of Ca2+ events decreased significantly after treatment with both types of Ca2+ channel blockers, indicating that Ca2+ spark production in embryonic mouse skeletal muscle is influenced by Ca2+ influx from L-type Ca2+ channels.
    • Calcium sparks: Elementary events underlying excitation-contraction coupling in heart muscle

      Cheng, Heping (1995)
      Spontaneous local increases in the concentration of intracellular calcium ( (Ca{dollar}\sp{lcub}2+{rcub}\rbrack \rm\sb{lcub}i{rcub}{dollar}), called "calcium sparks", were detected in quiescent rat heart cells with a confocal laser scanning microscope and the fluorescent calcium indicator fluo-3. A calcium spark is associated with an elevation of (Ca{dollar}\sp{lcub}2+{rcub}\rbrack \rm\sb{lcub}i{rcub}{dollar} by {dollar}\sim{dollar}200 nM within a volume of {dollar}\sim{dollar}10 fl, and to decline with an half time of {dollar}\sim{dollar}20 msec. Estimates of calcium flux associated with the local increase in (Ca{dollar}\sp{lcub}2+{rcub}\rbrack \rm\sb{lcub}i{rcub}{dollar} suggest that calcium sparks arise from the spontaneous openings of single or a few sarcoplasmic reticulum (SR) calcium-release channels (also known as ryanodine receptors, RyRs) acting in concert, a finding supported by ryanodine modification of spark kinetics. Thus calcium sparks represent the functional elementary events of the SR release of calcium. By measuring the occurrence of calcium, the in vivo open probability of RyR/channels is shown to be around 0.0001 S{dollar}\sp{lcub}-1{rcub}{dollar} at resting (Ca{dollar}\sp{lcub}2+{rcub}\rbrack \rm\sb{lcub}i{rcub}{dollar}. It is generally agreed that during cardiac excitation-contraction (EC) coupling, calcium release from the SR is triggered by the sarcolemmal calcium current (I{dollar}\rm\sb{lcub}Ca{rcub}{dollar}) via the calcium-induced calcium release mechanism. However, it is unclear how a mechanism with intrinsic positive feedback can provide graded responses. This work reveals, for the first time, that at the microscopic level, EC coupling takes the form of I{dollar}\rm\sb{lcub}Ca{rcub}{dollar}-evoked calcium sparks. Direct visualization of evoked calcium sparks was possible when I{dollar}\rm\sb{lcub}Ca{rcub}{dollar} was reduced by calcium channel antagonists D600 or cadmium, or during small ramp depolarization under whole-cell voltage-clamp conditions. These evoked calcium sparks resemble spontaneous calcium sparks observed at rest, in amplitude and in spatio-temporal properties. The activation of calcium sparks is controlled by local I{dollar}\rm\sb{lcub}Ca{rcub}{dollar} in a stochastic manner. Once activated, calcium release from the SR during a calcium spark is essentially independent of the triggering calcium influx and does not activate neighboring SR release sites. These novel findings are used to develop a new mechanistic model for cardiac EC coupling in which the graded amplification of the triggering I{dollar}\rm\sb{lcub}Ca{rcub}{dollar} by the SR can be explained by altering the extent of spatial and temporal summation of the elementary release events.
    • Calcium(2+)-signaling in cardiac muscle: From development to heart failure

      Dilly, Keith Wayne; Lederer, W. Jonathan (2001)
      Some changes observed in heart failure include altered ultrastructure, defects in excitation contraction coupling (ECC) and serious changes in beta-adrenergic signaling. I have examined these topics with an emphasis on cellular Ca 2+ signaling. Using immunohistochemical techniques combined with fluorescence confocal laser scanning microscopy (FCLSM) I have examined the localization and distribution of structural components of ECC in isolated ventricular myocytes from neonatal rats, and rat models of hypertrophy (Dahl SS/jr) and heart failure (SHHF). Using whole cell patch clamp techniques to record membrane voltage or currents, and simultaneously measuring intracellular [Ca2+] i using fluorescent Ca2+ sensitive indicators and FCLSM I have examined cardiac function in MLP-/- and transgenic V12HRas murine models of heart failure. I also examined the effectiveness of therapeutic strategies at preventing ECC defects in these models, Even at the early stages of development examined, primitive striations of a number of ECC protein structures were observed. Striations of sarcolemmal membrane (T-tubules) appear to form from the outside inwards, whereas proteins localized more intracellularly are first seen more internally. Structural changes observed in hypertrophic and failing myocytes include altered distribution of the Na 2+/Ca2+ exchanger. Increased Na2+/Ca 2+ exchanger density at the external surface is seen in these myocytes. In Ras myocytes unchanged Ca2+ currents (ICa(L)), decreased [Ca2+]i transients, defective ECC and action potential (AP) alterations were seen. Crossing Ras and PLBKO mice prevented AP alterations. In MLP-/- unchanged ICa(L ) and Ca2+ spark characteristics but decreased [Ca 2+]i transients, contractile responses and defective ECC were seen. Cellular defects were prevented in MLP-/- mice expressing a cardiac-targeted transgene blocking the function of beta-adrenergic receptor kinase-1 (betaARKct). These data suggest both defective SR function and down-regulation/de-sensitization of beta-adrenergic receptors play a pivotal role pathogenesis of heart failure. As such, PLBKO and betaARKct may prove effective therapies for preventing and possibly rescuing the cellular defects seen in heart failure. The mechanism(s) responsible for targeting ECC proteins to specific intracellular localizations remains to be discovered. Further studies on these and other methods for preventing and reversing Ca 2+ signaling defects seen in heart failure may provide valuable therapeutic tools for human heart failure treatment.
    • Coupling mechanisms to activate store-operated and TRP channels

      Venkatachalam, Kartik; Gill, Donald L. (2002)
      Despite the precision with which spatial and temporal details of Ca 2+ signals have been resolved, a fundamental aspect of the generation of Ca2+ signals, the activation of "store-operated channels" (SOCs), remains a molecular and mechanistic mystery. The TRP family of receptor-operated channels share several operational parameters with SOCs and the question of whether activation of SOCs and TRP channels is mediated by the inositol-1,4,5-trisphosphate (InsP3) receptor (InsP3R) is examined. The permeant InsP3R-antagonist, 2-aminoethoxydiphenyl borate (2-APB) was previously reported to block SOCs and TRPs in an InsP3R-dependent fashion. Electroretinogram recordings of the light-induced current in Drosophila revealed that the TRP channel-mediated light response is inhibited by 2-APB. This action of 2-APB is likely to be InsP3R-independent since InsP3Rs are dispensable for the light response. We used a triple InsP3R knockout variant line of DT40 chicken B cells to further assess the role of InsP3Rs in SOC and TRP activation. 45Ca2+ flux analysis revealed that only the wild-type DT40 cells retain normal InsP3R-mediated, 2-APB-sensitive, Ca 2+ release. In intact cells, all parameters of Ca2+ store-function and coupling to activate SOCs were identical in DT40 wt and DT40InsP3R-k/o cells. Moreover, in both cell lines SOC-activation is completely blocked by 2-APB with identical kinetics of inhibition. We transiently transfected TRPC3 channels into the DT40 cells, and found that (a) endogenous B-cell receptors (BCR) coupled to phospholipase C-gamma (PLC-gamma); (b) exogenously expressed muscarinic receptors coupled to phospholipase c-gamma (PLC-gamma), and (c) the diacylglycerol (DAG) analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG) activate the expressed TRPC3 channels in both DT40w/t and DT40InsP3R-k/o cells. BCR-induced TRPC3 activation was blocked by the PLC enzymic inhibitor U73122, and by wortmannin-induced PLC substrate depletion. However OAG-induced TRPC3 activation and store-operated channel activation remained unaffected under these conditions. We found that in the DT40 cells, 2-APB was a powerful InsP3R independent activator of store-operated Ca2+ entry between 1--10 muM. 2-APB activated authentic SOCs since the entry was selective for Ca2+ and highly sensitive to inhibition by La3+. With both w/t and InsP3R-k/o DT40 cells, the actions of 2-APB were restricted to SOCs in a store-coupled state. The results indicate that SOC and TRPC3 activation can occur independent of functional InsP 3Rs, and that in the DT40 cells TRPC3 channels are activated almost entirely by DAG following the stimulation of PLC-beta or -gamma. Furthermore, the biphasic effects of 2-APB reflect activation of authentic SOCs and 2-APB appears to modify SOCs by targeting the elusive coupling mechanism.
    • Local calcium release in muscle

      Santana Ferrer, Luis Fernando; Lederer, W. Jonathan (1996)
      Using a confocal microscope discrete foci of elevated intracellular Ca2+ concentration ([Ca2+]i), called "Ca2+ sparks", were observed in cardiac, skeletal and vascular smooth muscle cells. In all three muscle types Ca2+ sparks could be attributed to the opening of a single (or a few) ryanodine receptors (RyR). Simultaneous measurement of whole-cell Ca2+ currents (ICa) and Ca2+ sparks in rat ventricular myocytes showed that the probability of Ca2+ spark occurrence (Ps) depends on the square of iCa. Furthermore, it was found that at negative potentials the opening of a single L-type Ca2+ channel can provide enough Ca2+ to activate a single Ca2+ spark by the mechanism of calcium-induced calcium-release (CICR). In skeletal muscle fibers, however, Ca2+ sparks were activated by the voltage-sensor and by CICR. In marked contrast to striated muscle Ca2+ sparks were found to hyperpolarize and relax myogenic cerebral arteries because Ca2+ sparks occupied a small volume of the cell (0.8%), had at low frequency (about 1 Hz), and in occurred close to the sarcolemma were they could activate hyperpolarizing Ca2+-activated potassium (KCa) currents.
    • Modification of calcium pool function by fatty acids and their coenzyme A esters

      Rys-Sikora, Krystyna Ewa; Gill, Donald L. (1997)
      Intracellular Ca2+ pools are essential elements in the generation of Ca2+ signals within cells, however, their nature and identity have remained elusive. Ca2+ pools are complex and dynamic entities and are modified by G protein-induced membrane fusion events, allowing GTP-activated transfer of Ca2+ between discrete Ca2+ pools. Studies examined the modification of GTP-activated Ca2+ translocation process by fatty acyl-CoA esters and fatty acids since G protein action can be modified by fatty acylation. Using permeabilized DDT1MF-2 smooth muscle cells, palmitoyl-CoA (IC50 = 0.5 muM) was observed to completely block 45Ca2+ release activated by GTP, while having no effect on InsP3-induced Ca2+ release. Fatty acyl chain length was important, only C-13 to C-16 fatty acyl-CoA esters fully inhibited the action of GTP. CoA(10 muM) also blocked GTP-activated Ca2+ release, although the free sulfhydryl group and ATP requirements indicated that CoA must be fatty acylated to be effective. The nonhydrolyzable myristoyl-CoA analog, S-(2-oxopentadecyl)-CoA, blocked the GTP effect identically to myristoyl- and palmitoyl-CoA. Thus, fatty acyl transfer is not required indicating that the blockade is due to a direct allosteric modification of a component of the GTP-activated process. Palmitoyl-CoA not only inhibited but completely reversed GTP-activated Ca2+ release. In the presence of oxalate, GTP-activated Ca2+ transfer causes a substantial increase in Ca2+ accumulation; palmitoyl-CoA also completely reversed this effect. These results provide strong evidence that GTP-activated Ca2+ translocation does not reflect a full fusion event, but the formation of a reversible prefusion pore. The actions of fatty acids were very different from their acyl-CoA esters; 10-100 muM palmitate (C16:0) had a major stimulatory effect on GTP-mediated Ca2+accumulation. The biphasic nature of the palmitate effect was characteristically similar to the effect of oxalate; however, the EC50 for palmitate was 20 muM (approx. 100-fold lower that of oxalate). This activation was highly specific for chain length and degree of saturation. Only pentadecanoic acid (C15:0) duplicated this effect, unsaturated fatty acids were completely ineffective. Both palmitate- and oxalate-activated Ca2+ accumulation in the presence of GTP were inhibited by the anion transport inhibitor 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS). Hence, both Ca2+-complexing agents may enter anion-permeable Ca2+ subpools through similar anion channels. To further examine the nature of these Ca2+ complexes, a comparison of the releasability of Ca2+ using InsP3 and the Ca2+ ionophore, A23187 was undertaken. In the presence of oxalate, GTP-mediated accumulation of Ca2+ was only slowly releasable by InsP3 or A23187. Whereas, Ca2+ accumulated in the presence of palmitate and GTP was completely releasable by A23187, only a small fraction of the accumulated Ca2+was released by InsP3. These data suggest important differences between the state and possibly, the location of oxalate- and palmitate-Ca2+ complexes within Ca2+ pools. Thus, the formation of Ca2+-fatty acid complexes and, in turn, the activation of Ca2+ accumulation may reflect a major physiological role for fatty acids in stabilizing Ca2+ within the lumen of Ca2+ pools.
    • Novel Functions of Protein Kinase D in Cardiac Excitation-Contraction Coupling

      Goodall, Mariah; Rogers, Terry Birkby (2010)
      While the function of protein kinase D (PKD) in cardiac cells has remained enigmatic, recent work has shown that PKD phosphorylates the nuclear regulators HDAC5/7 and CREB implicating this kinase in the development of dysfunction seen in heart failure. Here we significantly extend our understanding of PKD signaling in heart using a molecular genetic approach to examine PKD through adenoviral vector expression of wild type (wt), constitutively active (ca) or dominant negative (dn) PKD in cultured adult rat ventricular myocytes. Confocal immunofluorescent images of these cells reveal a predominant distribution of all PKD forms in a non-nuclear, Z-line localized, striated-reticular pattern suggesting the importance of PKD in Ca<super>2+</super> signaling in heart. Thus, an initial hypothesis was that PKD plays unappreciated roles in regulation of excitation-contraction coupling in heart. Consistent with an established role of PKD in targeting cardiac troponin I (cTnI), caPKD expression led to a marked decrease in contractile myofilament Ca<super>2+</super> sensitivity with an unexpected electrical stimulus-dependence to this response. This desensitization was accompanied by stimulus-dependent increases in phosphorylation of cTnI and of regulatory site, Ser916, on PKD. The functional importance of this phospho-Ser916 event is demonstrated in experiments with a phosphorylation-defective mutant, caPKD-S916A, which is functionally inactive and blocks stimulus-dependent increases in cTnI phosphorylation. dnPKD expression resulted in sensitization of the myofilaments to Ca<super>2+</super> and blocked stimulus-dependent increases in cTnI phosphorylation. Furthermore, steady-state Ca<super>2+</super> transients were markedly increased in dnPKD cells and are explained, in part, by a marked increase in sarcoplasmic reticulum (SR) Ca<super>2+</super> load. In addition, changes in the cardiac Ca<super>2+</super> current (I<sub>Ca</sub>) and behavior of the phosphatase inhibitor, calyculin A (CalyA), support a role for PKD as a dynamic regulatory kinase of the L-type Ca<super>2+</super> channel (LTCC). Together, these data suggest a complex collection of novel functions carried out by PKD to dynamically regulate several components of the excitation-contraction coupling cascade in cardiomyocytes to allow for precise fine-tuning of cardiac cell function. Given that PKD activity is elevated in failing hearts, it will be important to determine the role of increased signaling at these newly appreciated cellular locales in the development of heart disease.
    • Regulation of calcium entry mechanisms by intracellular calcium pools

      Ufret-Vincenty, Carmen Angeles; Gill, Donald L. (1998)
      Release of Ca2+ from intracellular pools is the main trigger for activation of Ca2+ entry during the generation of Ca2+ signals in non-excitable cells. The aim of these studies is to investigate the relationship between Ca2+ pool emptying and activation of Ca2+ influx. Using the smooth muscle cell line, DDT1MF-2, changes in cytosolic Ca2+ concentration were monitored by loading cells with the fluorescent Ca2+ indicator, fura-2. The studies reveal that Ca2+ pool depletion by treatment with the SERCA pump inhibitors, thapsigargin and 2,5-Di-tert-butylhydroquinone (DBHQ), stimulates the activation of a novel and distinct Ca2+ entry pathway sensitive to caffeine. Refilling of Ca2+ pools occurs concomitantly with the disappearance of the caffeine-sensitive Ca2+ entry pathway. Stimulation of the caffeine-sensitive Ca2+ entry pathway occurs independently of the means used to deplete intracellular Ca2+ pools (pump inhibition or ionophore-induced depletion). Therefore, activation of the caffeine-sensitive Ca2+ entry pathway is directly controlled by Ca2+ pool depletion. Ion permeability studies indicate that the Ca2+ entry pathway sensitive to caffeine is less selective for Ca2+ than store-operated Ca2+ entry, the major Ca2+ influx pathway activated by pool depletion in a wide variety of cells. Nitric oxide-induced thiol modification (nitrosylation) has recently been shown to have major modulatory effects on the activity of several Ca2+ channels. Therefore, it was important to investigate the role of thiol nitrosylation in controlling Ca2+ entry and its activation by pool depletion. Studies reveal that nitric oxide donors activate a substantial entry of Ca2+ through a direct pathway which is independent of guanylate cyclase activation, a well-studied target for nitric oxide. Cell permeant alkylating agents activate an entry of Ca2+ remarkably similar to nitric oxide-induced Ca2+ entry, indicating that activation of Ca2+ entry relies on modification of one or more thiol residues in the channel or a closely associated protein. Most significantly, Ca2+ pool emptying strongly stimulates the Ca2+ entry activated by both nitric oxide donors and alkylating agents, revealing a direct link between thiol nitrosylation and activation of a store-sensitive Ca2+ entry pathway. These studies provide further evidence for the strong involvement of Ca2+ pool content in controlling the activity of major Ca2+ entry mechanisms.
    • The Role of LRP1 in Inflammation and Vasculopathies

      Au, Dianaly; Strickland, Dudley K.; Catania, Selen M.; 0000-0002-8797-833X
      The prevalence of overweight and obesity and a growing aging population has resulted in higher incidences of type 2 diabetes (T2D) and cardiovascular disease (CVD). Although risk factors for T2D and CVD have been known for decades, the molecular mechanisms involved in the pathophysiology of these multifaceted diseases and their interrelationship remain unclear. The LDL receptor-related protein 1 (LRP1) is a large endocytic and signaling receptor that is abundantly expressed in several tissues and possesses diverse biological functions, including chylomicron remnant clearance, involvement in insulin signaling and glucose homeostasis, modulation of the inflammatory response, atheroprotection, and maintenance of vascular integrity. We hypothesized that LRP1 may serve as a molecular link between metabolic processes and CVD development and employed tissue-specific LRP1 knockout mouse models to identify potential molecular mechanisms. Studies performed on macrophage-specific LRP1-deficient mice generated on an LDL receptor knockout background (LDLR-/-; macLRP1-/-) and challenged with a Western diet revealed that LRP1 expression in macrophages promotes hepatic inflammation and the development of glucose intolerance and insulin resistance by modulating Wnt signaling. Interestingly, studies performed on smooth muscle-specific LRP1-deficient (smLRP1-/-) mice identified a novel and critical role for LRP1 in modulating vascular smooth muscle cell (VSMC) contraction by regulating calcium signaling events. These results uncovered a potential mechanism by which LRP1 protects against aneurysm development. Studies performed on VSMCs isolated from an aneurysm patient, who also contains two heterozygous missense mutations within the LRP1 gene, showed dysregulation of the TGF-β and p53 signaling pathways. These results provide further biological evidence supporting the association of LRP1 with aortic aneurysms. The role of LRP1 in vascular remodeling was also investigated by inducing remodeling in smLRP1-/- mice using the carotid artery ligation model. Our results suggest that LRP1 protects against excessive vascular remodeling by modulating angiotensin II-mediated signaling. Taken together, this work reveals the complex roles of LRP1 in various tissues and provides evidence supporting LRP1 as a critical molecule that integrates metabolic processes with inflammation and vascular disease.