A probable role of dihydropyridine receptors in repression of Ca2+ sparks demonstrated in cultured mammalian muscle

To activate skeletal muscle contraction, action potentials must be sensed by dihydropyridine receptors (DHPRs) in the T tubule, which signal the Ca²⁺ release channels or ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) to open. We demonstrate here an inhibitory effect of the T tubule on the production of sparks of Ca²⁺ release. Murine primary cultures were confocally imaged for Ca²⁺ detection and T tubule visualization. After 72 h of differentiation, T tubules extended from the periphery for less than one-third of the myotube radius. Spontaneous Ca²⁺ sparks were found away from the region of cells where tubules were found. Immunostaining showed RyR1 and RyR3 isoforms in all areas, implying inhibition of both isoforms by a T tubule component. To test for a role of DHPRs in this inhibition, we imaged myotubes from dysgenic mice (mdg) that lack DHPRs. These exhibited T tubule development similar to that of normal myotubes, but produced few sparks, even in regions where tubules were absent. To increase spark frequency, a high-Ca²⁺ saline with 1 mM caffeine was used. Wild-type cells in this saline plus 50 µM nifedipine retained the topographic suppression pattern of sparks, but dysgenic cells in high-Ca²⁺ saline did not. Shifted excitation and emission ratios of indo-1 in the cytosol or mag-indo-1 in the SR were used to image [Ca²⁺] in these compartments. Under the conditions of interest, wild-type and mdg cells had similar levels of free [Ca²⁺] in cytosol and SR. These data suggest that DHPRs play a critical role in reducing the rate of spontaneous opening of Ca²⁺ release channels and/or their susceptibility to Ca²⁺-induced activation, thereby suppressing the production of Ca²⁺ sparks. excitation-contraction coupling; sarcoplasmic reticulum; ryanodine receptors; Ca²⁺ imaging     

Further study on the role of HSP70 on Ca2+ homeostasis in rat ventricular myocytes subjected to simulated ischemia

We hypothesized that activation of heat shock protein 70 (HSP70) by preconditioning, which is known to confer delayed cardioprotection, attenuates the impaired handling of Ca²⁺ at multiple sites. To test the hypothesis, we determined how the ryanodine receptor (RyR), sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA), and Na⁺/Ca²⁺ exchanger (NCX) handled Ca²⁺ in rat ventricular myocytes preconditioned with a -opioid receptor agonist, U50488H (UP), followed by blockade of HSP70 with a selective antisense oligonucleotide and subsequently subjected to simulated ischemia. We determined the following: 1) the Ca²⁺ transients induced by electrical stimulation and caffeine, which provide the overall picture of Ca²⁺ homeostasis; 2) expression of RyR, SERCA, and NCX; and 3) Ca²⁺ fluxes via NCX by the use of ⁴⁵Ca²⁺ in the rat ventricular myocyte. We found that UP increased the activity of RyR, SERCA, and NCX and the expression of RyR and SERCA. These effects led to increases in the release of Ca²⁺ from the sarcoplasmic reticulum via RyR and in the removal of Ca²⁺ from the cytoplasm by reuptake of Ca²⁺ to the SR via SERCA and by extrusion of Ca²⁺ out of the cell via NCX. UP also reduced mitochondrial Ca²⁺ accumulation. All of the effects of UP were either abolished or significantly attenuated by blockade of HSP70 synthesis with a selective antisense oligonucleotide. The results are evidence that activation of HSP70 by preconditioning improves the ischemia-impaired Ca²⁺ homeostasis at multiple sites in the heart, which may be responsible, at least partly, for attenuated Ca²⁺ overload, improved recovery in contractile function, and cardioprotection. intracellular Ca²⁺, -opioid receptor; Na⁺/Ca²⁺ exchanger; ryanodine receptor; sarco(endo)plasmic reticulum Ca²⁺-ATPase     

Ca2+ Release in Muscle Fibers Expressing R4892W and G4896V Type 1 Ryanodine Receptor Disease Mutants

The large and rapidly increasing number of potentially pathological mutants in the type 1 ryanodine receptor (RyR1) prompts the need to characterize their effects on voltage-activated sarcoplasmic reticulum (SR) Ca²⁺ release in skeletal muscle. Here we evaluated the function of the R4892W and G4896V RyR1 mutants, both associated with central core disease (CCD) in humans, in myotubes and in adult muscle fibers. For both mutants expressed in RyR1-null (dyspedic) myotubes, voltage-gated Ca²⁺ release was absent following homotypic expression and only partially restored following heterotypic expression with wild-type (WT) RyR1. In muscle fibers from adult WT mice, both mutants were expressed in restricted regions of the fibers with a pattern consistent with triadic localization. Voltage-clamp-activated confocal Ca²⁺ signals showed that fiber regions endowed with G4896V-RyR1s exhibited an ∼30% reduction in the peak rate of SR Ca²⁺ release, with no significant change in SR Ca²⁺ content. Immunostaining revealed no associated change in the expression of either α1S subunit (Cav1.1) of the dihydropyridine receptor (DHPR) or type 1 sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA1), indicating that the reduced Ca²⁺ release resulted from defective RyR1 function. Interestingly, in spite of robust localized junctional expression, the R4892W mutant did not affect SR Ca²⁺ release in adult muscle fibers, consistent with a low functional penetrance of this particular CCD-associated mutant.     

Vectorial Ca2+ release via ryanodine receptors contributes to Ca2+ extrusion from freshly isolated rabbit aortic endothelial cells

In this study, we identified ryanodine receptors (RyRs) as a component of a cytosolic Ca(2+) removal pathway in freshly isolated rabbit aortic endothelial cells. In an earlier article, we reported that the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and Na(+)/Ca(2+) exchanger (NCX) function in series to extrude cytosolic Ca(2+) to the extracellular space. Here we employed caffeine and ryanodine as modulators of RyR and showed that they act as the linkage between SERCA and NCX in removing Ca(2+) from the cytoplasm. Our data indicate that both 15 mM caffeine and 1 microM ryanodine facilitated Ca(2+) extrusion by activating RyRs while 100 microM ryanodine had the opposite effect by blocking RyRs. A further attempt to investigate RyR pharmacology revealed that in the absence of extracellular Ca(2+), ryanodine at 1 microM, but not 100 microM, stimulated Ca(2+) loss from the endoplasmic reticulum (ER). Blockade of RyR had no effect on the Ca(2+) removal rate when NCX had been previously blocked. In addition, the localization of RyR was determined using confocal microscopy of BODIPY TR-X fluorescent staining. Taken together, our findings suggest that in freshly isolated endothelial cells Ca(2+) is removed in part by transport through SERCA, RyR, and eventually NCX, and that RyR and NCX are in close functional proximity near the plasma membrane. After blockade of this component, Ca(2+) extrusion could be further inhibited by carboxyeosin, indicating a parallel contribution by the plasmalemmal Ca(2+)-ATPase (PMCA).     

Ca2+ influx through {alpha}1S DHPR may play a role in regulating Ca2+ release from RyR1 in skeletal muscle

Differentiated primary myotubes isolated from wild-type mice exhibit ryanodine-sensitive, spontaneous global Ca²⁺ oscillations as well as spontaneous depolarizations in the plasma membrane. Immunolabeling of these myotubes showed expression of both _1S dihydropyridine receptors (DHPRs) and ryanodine-sensitive Ca²⁺-release channel 1 (RyR1), the two key proteins in skeletal excitation-contraction (E-C) coupling. Spontaneous global Ca²⁺ oscillations could be inhibited by addition of 0.1 mM CdCl₂/0.5 mM LaCl₃ or 5 µM nifedipine to the extracellular bathing solution. After either treatment, Ca²⁺ oscillations could be restored upon extensive washing. Although exposure to DHPR antagonists completely blocked Ca²⁺ oscillations, normal orthograde signaling between DHPRs and RyRs, such as that elicited by 80 mM KCl depolarization, was still observed. In addition, we showed that spontaneous Ca²⁺ oscillations were never present in cultured mdg myotubes, which lack the expression of _1SDHPRs. These results suggest that under physiological conditions in conjunction with the mechanical coupling between the _1SDHPRs and RyR1, the initiation of Ca²⁺ oscillations in myotubes may be facilitated, in part, by the Ca²⁺ influx through the _1s-subunit of the DHPR. calcium-induced calcium release; dihydropyridine receptors; excitation-contraction coupling; ryanodine receptors; skeletal muscle     

Malondialdehyde and 4-hydroxynonenal adducts are not formed on cardiac ryanodine receptor (RyR2) and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) in diabetes

Recently, we reported an elevated level of glucose-generated carbonyl adducts on cardiac ryanodine receptor (RyR2) and sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2) in hearts of streptozotocin(STZ)-induced diabetic rats. We also showed these adduct impaired RyR2 and SERCA2 activities, and altered evoked Ca²⁺ transients. What is less clear is if lipid-derived malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) also chemically react with and impair RyR2 and SERCA2 activities in diabetes? This study used western blot assays with adduct-specific antibodies and confocal microscopy to assess levels of MDA, 4-HNE, N ^ε-carboxy(methyl)lysine (CML), pentosidine, and pyrraline adducts on RyR2 and SERCA2 and evoked intracellular transient Ca²⁺ kinetics in myocytes from control, diabetic, and treated-diabetic rats. MDA and 4-HNE adducts were not detected on RyR2 and SERCA2 from either control or 8 weeks diabetic rats with altered evoked Ca²⁺ transients. However, CML, pentosidine, and pyrraline adducts were elevated three- to five-fold (p < 0.05). Treating diabetic rats with pyridoxamine (a scavenger of reactive carbonyl species, RCS) or aminoguanidine (a mixed reactive oxygen species-RCS scavenger) reduced CML, pentosidine, and pyrraline adducts on RyR2 and SERCA2 and blunted SR Ca²⁺ cycling changes. Treating diabetic rats with the superoxide dismutase mimetic tempol had no impact on MDA and 4-HNE adducts on RyR2 and SERCA2, and on SR Ca²⁺ cycling. From these data we conclude that lipid-derived MDA and 4-HNE adducts are not formed on RyR2 and SERCA2 in this model of diabetes, and are therefore unlikely to be directly contributing to the SR Ca²⁺ dysregulation.     

Skeletal muscle sarcoplasmic reticulum glycogen status influences Ca2+ uptake supported by endogenously synthesized ATP

The purpose of this investigation was to determine whether there is a link between sarcoplasmic reticulum (SR) glycogen status and SR Ca²⁺ handling. In this investigation, skeletal muscle SR was purified from female Sprague-Dawley rats (200–250 g). Glycogen was extracted from the SR purified from one hindlimb, whereas the SR purified from the contralateral limb served as control. Before removal of the tissue, the animals were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). Both -amylase treatment (AM) and removal of EDTA from the homogenization and storage buffers reduced the amount of glycogen associated with the SR (P < 0.05). AM treatment reduced the glycogen phosphorylase content of SR (P < 0.05). In contrast, creatine kinase (CK) and pyruvate kinase (PK) contents were increased after both glycogen extraction protocols (P < 0.05). Under exogenous ATP conditions, both AM and EDTA-free (EF) treatments resulted in an increase in Ca²⁺-stimulated ATPase activity when normalized to sarco(endo)plasmic reticulum calcium-ATPase (SERCA) content (P < 0.05). CK and PK-supported SR Ca²⁺ uptake was decreased (P < 0.05) in the AM group when normalized to SERCA and CK or SERCA and PK content, respectively. AM was more effective than the EF for extracting glycogen associated with purified SR. Glycogen extraction alters the yield of purified SR proteins and must be taken into account when investigating SR calcium handling. Removal of glycogen from purified SR causes a change in Ca²⁺-handling properties as measured by ATPase and uptake activities. glycogen extraction; fatigue; SERCA     

Role of plasma membrane Ca2+-ATPase in contraction-relaxation processes of the bladder: evidence from PMCA gene-ablated mice

We investigated the roles and relationships of plasma membrane Ca²⁺-ATPase (PMCA), sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA)2, and Na⁺/Ca²⁺ exchanger (NCX) in bladder smooth muscle contractility in Pmca-ablated mice: Pmca4-null mutant (Pmca4^–/–) and heterozygous Pmca1 and homozygous Pmca4 double gene-targeted (Pmca1^+/–Pmca4^–/–) mice. Gene manipulation did not alter the amounts of PMCA1, SERCA2, and NCX. To study the role of each Ca²⁺ transport system, contraction of circular ring preparations was elicited with KCl (80 mM) plus atropine, and then the muscle was relaxed with Ca²⁺-free physiological salt solution containing EGTA. We measured the contributions of Ca²⁺ clearance components by inhibiting SERCA2 (with 10 µM cyclopiazonic acid) and/or NCX (by replacing NaCl with N-methyl-D-glucamine/HCl plus 10 µM KB-R7943). Contraction half-time (time to 50% of maximum tension) was prolonged in the gene-targeted muscles but marginally shortened when SERCA2 or NCX was inhibited. The inhibition of NCX significantly inhibited this prolongation, suggesting that NCX activity might be augmented to compensate for PMCA4 function in the gene-targeted muscles under nonstimulated conditions. Inhibition of SERCA2 and NCX as well as gene targeting all prolonged the relaxation half-time. The contribution of PMCA to relaxation was calculated to be 25–30%, with that of SERCA2 being 20% and that of NCX being 70%. PMCA and SERCA2 appeared to function additively, but the function of NCX might overlap with those of other components. In summary, gene manipulation of PMCA indicates that PMCA, in addition to SERCA2 and NCX, plays a significant role in both excitation-contraction coupling and the Ca²⁺ extrusion-relaxation relationship, i.e., Ca²⁺ homeostasis, of bladder smooth muscle. ATP2B; sarco(endo)plasmic reticulum Ca²⁺-ATPase 2; Na⁺/Ca²⁺ exchanger; homeostasis     

Effects of peroxynitrite on sarco/endoplasmic reticulum Ca2+ pump isoforms SERCA2b and SERCA3a

Sarco(endo)plasmic reticulum Ca²⁺ (SERCA) pumps are important for cell signaling. Three different genes, SERCA1, 2, and 3, encode these pumps. Most tissues, including vascular smooth muscle, express a splice variant of SERCA2 (SERCA2b), whereas SERCA3a is widely distributed in tissues such as vascular endothelium, tracheal epithelium, mast cells, and lymphoid cells. SERCA2b protein is readily inactivated by peroxynitrite that may be formed during cardiac ischemia reperfusion or during immune response after infection. Here, we compared the peroxynitrite sensitivity of SERCA2b and SERCA3a by using microsomes prepared from HEK-293T cells overexpressing the pumps. We incubated the microsomes with different concentrations of peroxynitrite and determined Ca²⁺ uptake, Ca²⁺-Mg²⁺-ATPase, Ca²⁺-dependent formation of acylphosphate intermediate, and protein mobility in Western blots. Ca²⁺ uptake, Ca²⁺-Mg²⁺-ATPase, and Ca²⁺-dependent formation of acylphosphate intermediate were inactivated for both SERCA2b and SERCA3a, but the latter was more resistant to the inactivation. Western blots showed that SERCA2b and SERCA3a proteins oligomerized after treatment with peroxynitrite, but each with a slightly different pattern. Compared with monomers, the oligomers may be less efficient in forming the acylphosphate intermediate and in conducting the remainder of the steps in the reaction cycle. We conclude that the resistance of SERCA3a to peroxynitrite may aid the cells expressing them in functioning during exposure to oxidative stress. free radicals; Ca²⁺-Mg²⁺-ATPase; ischemia; coronary artery; vascular smooth muscle; sarco(endo)plasmic reticulum Ca²⁺ pumps     

Mg2+ activates the ryanodine receptor type 2 (RyR2) at intermediate Ca2+ concentrations

To clarify whether activity of the ryanodine receptor type 2 (RyR2) is reduced in the sarcoplasmic reticulum (SR) of cardiac muscle, as is the case with the ryanodine receptor type 1 (RyR1), Ca²⁺-dependent [³H]ryanodine binding, a biochemical measure of Ca²⁺-induced Ca²⁺ release (CICR), was determined using SR vesicle fractions isolated from rabbit and rat cardiac muscles. In the absence of an adenine nucleotide or caffeine, the rat SR showed a complicated Ca²⁺ dependence, instead of the well-documented biphasic dependence of the rabbit SR. In the rat SR, [³H]ryanodine binding initially increased as [Ca²⁺] increased, with a plateau in the range of 10–100 µM Ca²⁺, and thereafter further increased to an apparent peak around 1 mM Ca²⁺, followed by a decrease. In the presence of these modulators, this complicated dependence prevailed, irrespective of the source. Addition of 0.3–1 mM Mg²⁺ unexpectedly increased the binding two- to threefold and enhanced the affinity for [³H]ryanodine at 10–100 µM Ca²⁺, resulting in the well-known biphasic dependence. In other words, the partial suppression of RyR2 is relieved by Mg²⁺. Ca²⁺ could be a substitute for Mg²⁺. Mg²⁺ also amplifies the responses of RyR2 to inhibitory and stimulatory modulators. This stimulating effect of Mg²⁺ on RyR2 is entirely new, and is referred to as the third effect, in addition to the well-known dual inhibitory effects. This effect is critical to describe the role of RyR2 in excitation-contraction coupling of cardiac muscle, in view of the intracellular Mg²⁺ concentration. [³H]ryanodine binding; CICR; stimulation by physiological Mg²⁺, excitation-contraction coupling in the heart     

Activators of protein kinase C decrease Ca2+ spark frequency in smooth muscle cells from cerebral arteries

LocalCa²⁺ transients("Ca²⁺ sparks") caused bythe opening of one or the coordinated opening of a number of tightlyclustered ryanodine-sensitiveCa²⁺-release (RyR) channels in thesarcoplasmic reticulum (SR) activate nearbyCa²⁺-dependentK⁺(K_Ca) channels to cause anoutward current [referred to as a "spontaneous transientoutward current" (STOC)]. TheseK_Ca currents cause membranepotential hyperpolarization of arterial myocytes, which would lead tovasodilation through decreasingCa²⁺ entry throughvoltage-dependent Ca²⁺ channels.Therefore, modulation of Ca²⁺spark frequency should be a means to regulation ofK_Ca channel currents and hencemembrane potential. We examined the frequency modulation ofCa²⁺ sparks and STOCs byactivation of protein kinase C (PKC). The PKC activators, phorbol12-myristate 13-acetate (PMA; 10 nM) and 1,2-dioctanoyl-sn-glycerol (1 µM),decreased Ca²⁺ spark frequency by72% and 60%, respectively, and PMA reduced STOC frequency by 83%.PMA also decreased STOC amplitude by 22%, which could be explained byan observed reduction (29%) inK_Ca channel open probability inthe absence of Ca²⁺ sparks. Thereduction in STOC frequency occurred in the presence of an inorganicblocker (Cd²⁺) ofvoltage-dependent Ca²⁺ channels.The reduction in Ca²⁺ sparkfrequency did not result from SRCa²⁺ depletion, sincecaffeine-induced Ca²⁺ transientsdid not decrease in the presence of PMA. These results suggest thatactivators of PKC can modulate the frequency ofCa²⁺ sparks, through an effect onthe RyR channel, which would decrease STOC frequency (i.e.,K_Ca channel activity).

     

The Ca 2+ leak paradox and rogue ryanodine receptors: SR Ca 2+ efflux theory and practice

Ca²⁺ efflux from the sarcoplasmic reticulum (SR) is routed primarily through SR Ca²⁺ release channels (ryanodine receptors, RyRs). When clusters of RyRs are activated by trigger Ca²⁺ influx through L-type Ca²⁺ channels (dihydropyridine receptors, DHPR), Ca²⁺ sparks are observed. Close spatial coupling between DHPRs and RyR clusters and the relative insensitivity of RyRs to be triggered by Ca²⁺ together ensure the stability of this positive-feedback system of Ca²⁺ amplification. Despite evidence from single channel RyR gating experiments that phosphorylation of RyRs by protein kinase A (PKA) or calcium-calmodulin dependent protein kinase II (CAMK II) causes an increase in the sensitivity of the RyR to be triggered by [Ca²⁺]_i there is little clear evidence to date showing an increase in Ca²⁺ spark rate. Indeed, there is some evidence that the SR Ca²⁺ content may be decreased in hyperadrenergic disease states. The question is whether or not these observations are compatible with each other and with the development of arrhythmogenic extrasystoles that can occur under these conditions. Furthermore, the appearance of an increase in the SR Ca²⁺ “leak” under these conditions is perplexing. These and related complexities are analyzed and discussed in this report. Using simple mathematical modeling discussed in the context of recent experimental findings, a possible resolution to this paradox is proposed. The resolution depends upon two features of SR function that have not been confirmed directly but are broadly consistent with several lines of indirect evidence: (1) the existence of unclustered or “rogue” RyRs that may respond differently to local [Ca²⁺]_i in diastole and during the [Ca²⁺]_i transient; and (2) a decrease in cooperative or coupled gating between clustered RyRs in response to physiologic phosphorylation or hyper-phosphorylation of RyRs in disease states such as heart failure. Taken together, these two features may provide a framework that allows for an improved understanding of cardiac Ca²⁺ signaling.     

Differential Ca(2+) sensitivity of skeletal and cardiac muscle ryanodine receptors in the presence of calmodulin

Calmodulin (CaM) activates the skeletal muscle ryanodine receptorCa²⁺ release channel (RyR1) in the presence of nanomolarCa²⁺ concentrations. However, the role of CaM activation inthe mechanisms that control Ca²⁺ release from thesarcoplasmic reticulum (SR) in skeletal muscle and in the heart remainsunclear. In media that contained 100 nM Ca²⁺, the rate of⁴⁵Ca²⁺ release from porcine skeletal muscle SRvesicles was increased approximately threefold in the presence of CaM(1 µM). In contrast, cardiac SR vesicle⁴⁵Ca²⁺ release was unaffected by CaM,suggesting that CaM activated the skeletal RyR1 but not the cardiacRyR2 channel isoform. The activation of RyR1 by CaM was associated withan approximately sixfold increase in the Ca²⁺ sensitivityof [³H]ryanodine binding to skeletal muscle SR, whereasthe Ca²⁺ sensitivity of cardiac SR[³H]ryanodine binding was similar in the absence andpresence of CaM. Cross-linking experiments identified both RyR1 andRyR2 as predominant CaM binding proteins in skeletal and cardiac SR,respectively, and [³⁵S]CaM binding determinations furtherindicated comparable CaM binding to the two isoforms in the presence ofmicromolar Ca²⁺. In nanomolar Ca²⁺, however,the affinity and stoichiometry of RyR2 [³⁵S]CaM bindingwas reduced compared with that of RyR1. Together, our results indicatethat CaM activates RyR1 by increasing the Ca²⁺ sensitivityof the channel, and further suggest differences in CaM's functionalinteractions with the RyR1 and RyR2 isoforms that may potentiallycontribute to differences in the Ca²⁺ dependence of channelactivation in skeletal and cardiac muscle.

     

Adenylyl cyclase expression and modulation of cAMP in rat taste cells

The present study determined Ca²⁺ handling in the hearts of rats subjected to chronic hypoxia (CH). Spectrofluorometry was used to measure intracellular Ca²⁺ concentration ([Ca²⁺]_i) and its responses to electrical stimulation, caffeine, and isoproterenol in myocytes from the right ventricle of rats breathing 10% oxygen for 1, 3, 7, 14, 21, 28, and 56 days and age-matched controls. The protein expression of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) and its ryanodine receptor (RyR) were measured. The uptake of ⁴⁵Ca²⁺ by SERCA, release by RyR, and extrusion by Na⁺/Ca²⁺ exchange (NCX) were determined. It was found that Ca²⁺ homeostasis and Ca²⁺ responses to -adrenoceptor stimulation reached a new equilibrium after 4 wk of CH. Ca²⁺ content in the sarcoplasmic reticulum (SR) was reduced, but cytosolic Ca²⁺ remained unchanged after CH. Expression of SERCA and its Ca²⁺ uptake, Ca²⁺ release via RyR, and NCX activity were suppressed by CH. The results indicate impaired Ca²⁺ handling, which may be responsible for the attenuated Ca²⁺ responses to -adrenoceptor stimulation in CH. intracellular calcium ion concentration; calcium-adenosinetriphosphatase; ryanodine receptor; sodium/calcium exchange; sarcoplasmic reticulum; -adrenoceptor; chronic hypoxia     

New N-aryl-N-alkyl-thiophene-2-carboxamide compound enhances intracellular Ca2+ dynamics by increasing SERCA2a Ca2+ pumping

The type 2a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) plays a central role in the intracellular Ca²⁺ homeostasis of cardiac myocytes, pumping Ca²⁺ from the cytoplasm into the sarcoplasmic reticulum (SR) lumen to maintain relaxation (diastole) and prepare for contraction (systole). Diminished SERCA2a function has been reported in several pathological conditions, including heart failure. Therefore, development of new drugs that improve SERCA2a Ca²⁺ transport is of great clinical significance. In this study, we characterized the effect of a recently identified N-aryl-N-alkyl-thiophene-2-carboxamide (or compound 1) on SERCA2a Ca²⁺-ATPase and Ca²⁺ transport activities in cardiac SR vesicles, and on Ca²⁺ regulation in a HEK293 cell expression system and in mouse ventricular myocytes. We found that compound 1 enhances SERCA2a Ca²⁺-ATPase and Ca²⁺ transport in SR vesicles. Fluorescence lifetime measurements of fluorescence resonance energy transfer between SERCA2a and phospholamban indicated that compound 1 interacts with the SERCA-phospholamban complex. Measurement of endoplasmic reticulum Ca²⁺ dynamics in HEK293 cells expressing human SERCA2a showed that compound 1 increases endoplasmic reticulum Ca²⁺ load by enhancing SERCA2a-mediated Ca²⁺ transport. Analysis of cytosolic Ca²⁺ dynamics in mouse ventricular myocytes revealed that compound 1 increases the action potential-induced Ca²⁺ transients and SR Ca²⁺ load, with negligible effects on L-type Ca²⁺ channels and Na⁺/Ca²⁺ exchanger. However, during adrenergic receptor activation, compound 1 did not further increase Ca²⁺ transients and SR Ca²⁺ load, but it decreased the propensity toward Ca²⁺ waves. Suggestive of concurrent desirable effects of compound 1 on RyR2, [³H]-ryanodine binding to cardiac SR vesicles shows a small decrease in nM Ca²⁺ and a small increase in μM Ca²⁺. Accordingly, compound 1 slightly decreased Ca²⁺ sparks in permeabilized myocytes. Thus, this novel compound shows promising characteristics to improve intracellular Ca²⁺ dynamics in cardiomyocytes that exhibit reduced SERCA2a Ca²⁺ uptake, as found in failing hearts.     

Ryanoids and imperatoxin affect the modulation of cardiac ryanodine receptors by dihydropyridine receptor Peptide A

Ca²⁺-entry via L-type Ca²⁺ channels (DHPR) is known to trigger ryanodine receptor (RyR)-mediated Ca²⁺-release from sarcoplasmic reticulum (SR). The mechanism that terminates SR Ca²⁺ release is still unknown. Previous reports showed evidence of Ca²⁺-entry independent inhibition of Ca²⁺ sparks by DHPR in cardiomyocytes. A peptide from the DHPR loop II-III (PepA) was reported to modulate isolated RyRs. We found that PepA induced voltage-dependent “flicker block” and transition to substates of fully-activated cardiac RyRs in planar bilayers. Substates had less voltage-dependence than block and did not represent occupancy of a ryanoid site. However, ryanoids stabilized PepA-induced events while PepA increased RyR2 affinity for ryanodol, which suggests cooperative interactions. Ryanodol stabilized Imperatoxin A (IpTx_A) binding but when IpTx_A bound first, it prevented ryanodol binding. Moreover, IpTx_A and PepA excluded each other from their sites. This suggests that IpTx_A generates a vestibular gate (either sterically or allosterically) that prevents access to the peptides and ryanodol binding sites. Inactivating gate moieties (“ball peptides”) from K⁺ and Na⁺ channels (ShakerB and KIFMK, respectively) induced well resolved slow block and substates, which were sensitive to ryanoids and IpTx_A and allowed, by comparison, better understanding of PepA action. The RyR2 appears to interact with PepA or ball peptides through a two-step mechanism, reminiscent of the inactivation of voltage-gated channels, which includes binding to outer (substates) and inner (block) vestibular regions in the channel conduction pathway. Our results open the possibility that “ball peptide-like” moieties in RyR2-interacting proteins could modulate SR Ca²⁺ release in cells.     

Reconciling depressed Ca2+ sparks occurrence with enhanced RyR2 activity in failing mice cardiomyocytes

Abnormalities in cardiomyocyte Ca²⁺ handling contribute to impaired contractile function in heart failure (HF). Experiments on single ryanodine receptors (RyRs) incorporated into lipid bilayers have indicated that RyRs from failing hearts are more active than those from healthy hearts. Here, we analyzed spontaneous Ca²⁺ sparks (brief, localized increased in [Ca²⁺]_i) to evaluate RyR cluster activity in situ in a mouse post-myocardial infarction (PMI) model of HF. The cardiac ejection fraction of PMI mice was reduced to ∼30% of that of sham-operated (sham) mice, and their cardiomyocytes were hypertrophied. The [Ca²⁺]_i transient amplitude and sarcoplasmic reticulum (SR) Ca²⁺ load were decreased in intact PMI cardiomyocytes compared with those from sham mice, and spontaneous Ca²⁺ sparks were less frequent, whereas the fractional release and the frequency of Ca²⁺ waves were both increased, suggesting higher RyR activity. In permeabilized cardiomyocytes, in which the internal solution can be controlled, Ca²⁺ sparks were more frequent in PMI cells (under conditions of similar SR Ca²⁺ load), confirming the enhanced RyR activity. However, in intact cells from PMI mice, the Ca²⁺ sparks frequency normalized by the SR Ca²⁺ load in that cell were reduced compared with those in sham mice, indicating that the cytosolic environment in intact cells contributes to the decrease in Ca²⁺ spark frequency. Indeed, using an internal “failing solution” with less ATP (as found in HF), we observed a dramatic decrease in Ca²⁺ spark frequency in permeabilized PMI and sham myocytes. In conclusion, our data show that, even if isolated RyR channels show more activity in HF, concomitant alterations in intracellular media composition and SR Ca²⁺ load may mask these effects at the Ca²⁺ spark level in intact cells. Nonetheless, in this scenario, the probability of arrhythmogenic Ca²⁺ waves is enhanced, and they play a potential role in the increase in arrhythmia events in HF patients.     

FK506-binding proteins 12 and 12.6 (FKBPs) as regulators of cardiac Ryanodine Receptors: Insights from new functional and structural knowledge

Ryanodine Receptors (RyRs) are intracellular Ca²⁺ channels that mediate Ca²⁺ flux from the sarco(endo)plasmic reticulum in many cell types. The interaction of RyRs with FK506-binding proteins (FKBPs) has been proposed as an important regulatory mechanism, where the loss of this interaction leads to channel dysfunction. In the heart, phosphorylation of RyR has been suggested to disrupt the RyR-FKBP interaction promoting altered Ca²⁺ signaling, heart failure and arrhythmias. However, the functional result of FKBP interaction with RyR and how this interaction is regulated remains highly controversial. Recently, high resolution structures of RyR have provided novel aspects to the ongoing debate. This review will discuss the most recent functional data in light of these new structures.     

Voltage dependence of Ca2+ sparks in intact cerebral arteries

Ca²⁺ sparks have beenpreviously described in isolated smooth muscle cells. Here we presentthe first measurements of local Ca²⁺ transients("Ca²⁺ sparks") in an intactsmooth muscle preparation. Ca²⁺sparks appear to result from the opening of ryanodine-sensitive Ca²⁺ release (RyR) channels in thesarcoplasmic reticulum (SR). Intracellular Ca²⁺ concentration([Ca²⁺]_i)was measured in intact cerebral arteries (40-150 µm in diameter) from rats, using the fluorescentCa²⁺ indicator fluo 3 and a laserscanning confocal microscope. Membrane potential depolarization byelevation of external K⁺ from 6 to30 mM increased Ca²⁺ sparkfrequency (4.3-fold) and amplitude (~2-fold) as well as globalarterial wall[Ca²⁺]_i(~1.7-fold). The half time of decay (~50 ms) was not affected bymembrane potential depolarization. Ryanodine (10 µM), which inhibitsRyR channels and Ca²⁺ sparks inisolated cells, and thapsigargin (100 nM), which indirectly inhibitsRyR channels by blocking the SRCa²⁺-ATPase, completely inhibitedCa²⁺ sparks in intact cerebralarteries. Diltiazem, an inhibitor of voltage-dependentCa²⁺ channels, lowered global[Ca²⁺]_iand Ca²⁺ spark frequency andamplitude in intact cerebral arteries in a concentration-dependentmanner. The frequency of Ca²⁺sparks (<1s¹ · cell¹),even under conditions of steady depolarization, was too low tocontribute significant amounts ofCa²⁺ to globalCa²⁺ in intact arteries. Theseresults provide direct evidence that Ca²⁺ sparks exist in quiescentsmooth muscle cells in intact arteries and that changes of membranepotential that would simulate physiological changes modulate bothCa²⁺ spark frequency and amplitudein arterial smooth muscle.

     

Conformational coupling of DHPR and RyR1 in skeletal myotubes is influenced by long-range allosterism: evidence for a negative regulatory module

Four ryanodine receptor type 1 and 2 chimeras (R4, R9, R10, and R16) and their respective wild-type ryanodine receptors (type 1 and 2; wtRyR1 and wtRyR2) were expressed in dyspedic 1B5 to identify possible negative regulatory modules of the Ca²⁺ release channel that are under the influence of the dihydropyridine receptor (DHPR). Responses of intact 1B5 myotubes expressing each construct to caffeine in the absence or presence of either La³⁺ and Cd²⁺ or the organic DHPR blocker nifedipine were determined by imaging single 1B5 myotubes loaded with fluo 4. The presence of La³⁺ and Cd²⁺ or nifedipine in the external medium at concentrations known to block Ca²⁺ entry through the DHPRs significantly decreased the caffeine EC₅₀ of wtRyR1 (2.80 ± 0.12 to 0.83 ± 0.09 mM; P < 0.05). On the other hand, DHPR blockade did not significantly alter the caffeine EC₅₀ values of wtRyR2, chimeras R10 and R16, whereas the caffeine EC₅₀ values of chimeras R4 and R9 were significantly increased (1.27 ± 0.05 to 2.60 ± 0.16 mM, and 1.15 ± 0.03 to 2.11 ± 0.32 mM, respectively; P < 0.05). Despite the fact that all the chimeras form fully functional Ca²⁺ release channels in situ, sarcoplasmic reticulum (SR) containing R4, R10, and R16 did not possess high-affinity binding of [³H]ryanodine regardless of Ca²⁺ concentration. These results suggest the presence of an interaction between RyR1 and the DHPR, which is not present in RyR2, that contributes negative control of SR Ca²⁺ release induced by direct agonists such as caffeine. Although we were unable to define the negative module using RyR1-RyR2 chimeras, they further demonstrated that the RyR is very sensitive to long-range allosterism. ryanodine receptor type 1; dihydropyridine receptor; excitation-contraction coupling; negative module