Interaction between the sarcoplasmic reticulum and mitochondria in the control of contractile calcium in guinea-pig atria--does it exist?

The inhibitory action of caffeine on calcium (Ca2+) release from the sarcoplasmic reticulum (SR) and interference with mitochondrial (Ca2+) fluxes by a mitochondrial uncoupler protonfore CCCP were utilized to define a calcium pool responsible for potentiation of post-rest twitch tension in guinea-pig atria. The Ca2+ fluxes were assessed by means of 45Ca2+. Caffeine and CCCP when applied separately did not affect post-rat 45Ca2+ content. Yet, when they were applied together it was markedly reduced to the resting level. It is concluded that a possible source of contractile Ca2+ may be located in mitochondria and an eventual shift of Ca2+ between mitochondria and the SR seems to be a plausible assumption.     

The positive force–frequency relationship is maintained in absence of sarcoplasmic reticulum function in rabbit,but not in rat myocardium

Myocardial calcium handling differs between species, mainly in the relative contribution between the sources for activator calcium. To investigate the role of the myofilaments and intracellular calcium decline in governing the relaxation phase of cardiac muscle, and to elucidate additional determinants of relaxation other than the sarcoplasmic reticulum (SR) at various frequencies within the in vivo range, the present study was performed by altering the calcium handling in rat and rabbit. Trabeculae, iontophoretically loaded with bis-fura-2 to monitor cytoplasmic calcium levels, were subjected to ryanodine and cyclopiazonic acid to inhibit SR function. Simultaneous force and [Ca²⁺]_i measurements were obtained at 1–4 Hz in rabbit and at 4–8 Hz in rat before and after SR inhibition. Inhibition of the SR resulted in increased diastolic and peak calcium levels as well as decreased developed force in both species. Calcium transient amplitude decreased in rat, but increased in rabbit after SR inhibition. Time to peak tension, time from peak tension to 50% relaxation, time to peak calcium, and time from peak calcium to 50% calcium decline were all prolonged. Results suggest that L-type calcium channel current is responsible for increases in calcium with increasing frequency, and that the SR amplifies this effect in response to increased L-type current. The response of the myofilaments to alterations in calcium handling plays a critical role in the final determination of force, and may differ between species. These results imply the balance between force relaxation and calcium decline is significantly different in larger mammals, necessitating a critical re-evaluation of how myocardial relaxation is governed, specifically regarding frequency-dependent activation.     

Defective pro-IL-1β responses in macrophages from aged mice

Background

Cytokines regulated by the inflammasome pathway have been extensively implicated in various age-related immune pathologies. We set out to elucidate the contribution of the nod-like receptor protein 3 (NLRP3) inflammasome pathway to the previously described deficiencies in IL-1β production by macrophages from aged mice. We examined the production of pro-IL-1β and its conversion into IL-1β as two separate steps and compared these cytokine responses in bone marrow derived macrophages from young (6–8 weeks) and aged (18–24 months) C57BL/6 mice.

Findings

Relative to macrophages from young mice, macrophages from aged mice produced less pro-IL-1β after TLR4 stimulation with LPS. However upon activation of the NLRP3 inflammasome with ATP, macrophages from young and aged mice were able to efficiently convert and secrete intracellular pro-cytokines as functional cytokines.

Conclusions

Lower levels of IL-1β production are a result of slower and lower overall production of pro-IL-1β in macrophages from aged mice.

     

Lack of effects of calcium · calmodulin-dependent phosphorylation on Ca2+ release from cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum is phosphorylated by an endogenous calcium · calmodulin-dependent protein kinase and phosphorylation occurs mainly on a 27 kDa proteolipid, called phospholamban. To determine whether this phosphorylation has any effect on Ca²⁺ release, sarcoplasmic reticulum vesicles were phosphorylated by the calcium · calmodulin-dependent protein kinase, while non-phosphorylated vesicles were preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both non-phosphorylated and phosphorylated vesicles were centrifuged to remove calmodulin, and subsequently used for Ca²⁺ release studies. Calcium loading was carried out either by the active calcium pump or by incubation with high (5 mM) calcium for longer periods. Phosphorylation of sarcoplasmic reticulum by calcium · calmodulin-dependent protein kinase had no appreciable effect on the initial rates of Ca²⁺ released from cardiac sarcoplasmic reticulum vesicles loaded under passive conditions and on the apparent ⁴⁵Ca²⁺ − ⁴⁰Ca²⁺ exchange from cardiac sarcoplasmic reticulum vesicles loaded under active conditions. Thus, it appears that calcium · calmodulin-dependent protein kinase mediated phosphorylation of cardiac sarcoplasmic reticulum is not involved in the regulation of Ca²⁺ release and ⁴⁵Ca²⁺ − ⁴⁰Ca²⁺ exchange.     

Mitochondrial and sarcoplasmic reticulum abnormalities in cancer cachexia: Altered energetic efficiency?

Background

Cachexia is a wasting condition that manifests in several types of cancer, and the main characteristic is the profound loss of muscle mass.

Methods

The Yoshida AH-130 tumor model has been used and the samples have been analyzed using transmission electronic microscopy, real-time PCR and Western blot techniques.

Results

Using in vivo cancer cachectic model in rats, here we show that skeletal muscle loss is accompanied by fiber morphologic alterations such as mitochondrial disruption, dilatation of sarcoplasmic reticulum and apoptotic nuclei. Analyzing the expression of some factors related to proteolytic and thermogenic processes, we observed in tumor-bearing animals an increased expression of genes involved in proteolysis such as ubiquitin ligases Muscle Ring Finger 1 (MuRF-1) and Muscle Atrophy F-box protein (MAFBx). Moreover, an overexpression of both sarco/endoplasmic Ca^2 +-ATPase (SERCA1) and adenine nucleotide translocator (ANT1), both factors related to cellular energetic efficiency, was observed. Tumor burden also leads to a marked decreased in muscle ATP content.

Conclusions

In addition to muscle proteolysis, other ATP-related pathways may have a key role in muscle wasting, both directly by increasing energetic inefficiency, and indirectly, by affecting the sarcoplasmic reticulum–mitochondrial assembly that is essential for muscle function and homeostasis.

General significance

The present study reports profound morphological changes in cancer cachectic muscle, which are visualized mainly in alterations in sarcoplasmic reticulum and mitochondria. These alterations are linked to pathways that can account for energy inefficiency associated with cancer cachexia.     

Alternative splicing generates a CaM kinase IIβ isoform in myocardium that targets the sarcoplasmic reticulum through a putative αKAP and regulates GAPDH

In the postgenomic era the elucidation of the physiological function of genes has become the rate-limiting step in the quest to understand the development and function of living organisms. Double-stranded RNA (dsRNA) interferes with gene expression in various species, a phenomenon known as RNA interference (RNAi). We show here that RNAi is also effective in modifying gene expression in neural stem cell differentiation. The progenitor cells were obtained from E14 mouse embryonic forebrain and maintained using N-2 medium containing basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and B27.A gene (NM017084.1) was previously discovered and validated to express obviously differently between differentiated and undifferentiated neural stem cells in our laboratory. Here we report a long double-stranded RNA to knock out or knock down this gene. The results demonstrated that following RNAi inhibition of expression of the NM017084.1 gene, the differentiation of neural stem cells is accelerated. Thus the NM017084.1 gene may play a pivotal role in the process of differentiation of neural stem cells.     

GLP-1 promotes mitochondrial metabolism in vascular smooth muscle cells by enhancing endoplasmic reticulum–mitochondria coupling

Incretin GLP-1 has important metabolic effects on several tissues, mainly through the regulation of glucose uptake and usage. One mechanism for increasing cell metabolism is modulating endoplasmic reticulum (ER)–mitochondria communication, as it allows for a more efficient transfer of Ca²⁺ into the mitochondria, thereby increasing activity. Control of glucose metabolism is essential for proper vascular smooth muscle cell (VSMC) function. GLP-1 has been shown to produce varied metabolic actions, but whether it regulates glucose metabolism in VSMC remains unknown. In this report, we show that GLP-1 increases mitochondrial activity in the aortic cell line A7r5 by increasing ER–mitochondria coupling. GLP-1 increases intracellular glucose and diminishes glucose uptake without altering glycogen content. ATP, mitochondrial potential and oxygen consumption increase at 3 h of GLP-1 treatment, paralleled by increased Ca²⁺ transfer from the ER to the mitochondria. Furthermore, GLP-1 increases levels of Mitofusin-2 (Mfn2), an ER-mitochondria tethering protein, via a PKA-dependent mechanism. Accordingly, PKA inhibition and Mfn2 down-regulation prevented mitochondrial Ca²⁺ increases in GLP-1 treated cells. Inhibiting both Ca²⁺ release from the ER and Ca²⁺ entry into mitochondria as well as diminishing Mfn2 levels blunted the increase in mitochondrial activity in response to GLP-1. Altogether, these results strongly suggest that GLP-1 increases ER–mitochondria communication in VSMC, resulting in higher mitochondrial activity.     

Quantitative measurement of Ca²(+) in the sarcoplasmic reticulum lumen of mammalian skeletal muscle

Skeletal muscle stores Ca²⁺ in the sarcoplasmic reticulum (SR) and releases it to initiate contraction, but the concentration of luminal Ca²⁺ in the SR ([Ca²⁺]_SR) and the amount that is released by physiological or pharmacological stimulation has been difficult to measure. Here we present a novel, yet simple and direct, method that provides the first quantitative estimates of static content and dynamic changes in [Ca²⁺]_SR in mammalian skeletal muscle, to our knowledge. The method uses fluo-5N loaded into the SR of single, mammalian skeletal muscle cells (murine flexor digitorum brevis myofibers) and confocal imaging to detect and calibrate the signals. Using this method, we have determined that [Ca²⁺]_{SR, free} is 390 μM. 4-Chloro-m-cresol, an activator of the skeletal muscle ryanodine receptor, reduces [Ca²⁺]_{SR, free} to ∼8 μM, when values are corrected for background fluorescence from cytoplasmic pools of dye. Prolonged electrical stimulation (10 s) at 50 Hz releases 88% of the SR Ca²⁺ content, whereas stimulation at 1 Hz (10 s) releases only 20%. Our results lay the foundation for molecular modeling of the dynamics of luminal SR Ca²⁺ and for future studies of the role of SR Ca²⁺ in healthy and diseased mammalian muscle.     

Endoplasmic reticulum–plasma membrane contacts: Principals of phosphoinositide and calcium signaling

The endoplasmic reticulum (ER) forms an extensive network of membrane contact sites with intra-cellular organelles and the plasma membrane (PM). Interorganelle contacts have vital roles in membrane lipid and ion dynamics. In particular, ER–PM contacts are integral to numerous inter-cellular and intra-cellular signaling pathways including phosphoinositide lipid and calcium signaling, mechanotransduction, metabolic regulation, and cell stress responses. Accordingly, ER–PM contacts serve important signaling functions in excitable cells including neurons and muscle and endocrine cells. This review highlights recent advances in our understanding of the vital roles for ER–PM contacts in phosphoinositide and calcium signaling and how signaling pathways in turn regulate proteins that form and function at ER–PM contacts.     

Does intrinsic fluorescence reflect conformational changes in the Ca2+-ATPase of sarcoplasmic reticulum?

We have investigated the kinetics of the intrinsic fluorescence drop observed when ATP is added to purified sarcoplasmic reticulum ATPase in a potassium-free medium containing magnesium and calcium, at pH 6 and 20°C. Under these conditions, analysis of the fluorescence drop is complex. Several events contributed to the rate of the fluorescence drop initiated by turnover, including phosphorylation, conformational transition of the phosphorylated complex, and dephosphorylation. On the other hand, when 75% of total fluorescence was quenched by energy transfer to the membrane-bound ionophore A23187, the observed turnoverdependent drop in residual fluorescence mainly reflected the conformational transition of the phosphorylated ATPase. Combination of fast kinetics with the quenching of selected tryptophan residues is suggested to be a promising tool for the study of proteins containing many of these residues.     

Sarcoplasmic reticulum and excitation-contraction coupling at 20 and 10 °C in rainbow trout myocardium

Summary Isometric force and series membrane potential were recorded in isolated ventricular strips from rainbow trout at 20 and 10 °C. Preparations were electrically stimulated to contract at either 0.5 or 0.2 Hz. Single extrastimulations elicited a twitch force which diminished when the preceding diastole was shortened below the regular value. The stimulation following this extra stimulation evoked no potentiation of force. Apart from a marginal effect on the post extrasystolic force at 20 °C, ryanodine did not affect either of these responses or the steady-state force at 0.5 Hz. At 0.2 Hz the steady-state force was somewhat depressed by ryanodine at 20 but not at 10 °C. In contrast, extrastimulations preceded by diastoles of up to 1 h more than doubled extrasystolic force at 20 °C. This effect was removed by ryanodine. Both the potentiations and the effect of ryanodine were strongly reduced at 10 °C. Apparently, temperature acts on the release of Ca²⁺ from the sarcoplasmic reticulum, since Ca²⁺ seems to be taken up at both temperatures. Hence, at both 20 and 10 °C, the contractures evoked in a solution inhibiting sarcolemmal Ca²⁺ transfer and releasing Ca²⁺ from the sarcoplasmic reticulum were diminished by pretreatment with 15 mM caffeine. Action potential duration at 20 °C was less than half of that at 10 °C. At both temperatures it tended to be prolonged by periods of prolonged rest. No effect of ryanodine on action potential configuration was detected. The results suggest that trout myocardial sarcoplasmic reticulum, although powerful at unphysiologically low stimulation rates, does not partake in the beat-to-beat regulation of force at heart rates encountered in vivo.Abbreviations ESF extrasystolic force - SR sarcoplasmic reticulum - v _F maximal rate of force development - v _R maximal rate of relaxation - TPF time to peak force - TR _0.5 time for half relaxation - TTF duration of force development     

Malate–aspartate shuttle promotes l-lactate oxidation in mitochondria

Metabolism in cancer cells is rewired to generate sufficient energy equivalents and anabolic precursors to support high proliferative activity. Within the context of these competing drives aerobic glycolysis is inefficient for the cancer cellular energy economy. Therefore, many cancer types, including colon cancer, reprogram mitochondria-dependent processes to fulfill their elevated energy demands. Elevated glycolysis underlying the Warburg effect is an established signature of cancer metabolism. However, there are a growing number of studies that show that mitochondria remain highly oxidative under glycolytic conditions. We hypothesized that activities of glycolysis and oxidative phosphorylation are coordinated to maintain redox compartmentalization. We investigated the role of mitochondria-associated malate–aspartate and lactate shuttles in colon cancer cells as potential regulators that couple aerobic glycolysis and oxidative phosphorylation. We demonstrated that the malate–aspartate shuttle exerts control over NAD⁺/NADH homeostasis to maintain activity of mitochondrial lactate dehydrogenase and to enable aerobic oxidation of glycolytic l -lactate in mitochondria. The elevated glycolysis in cancer cells is proposed to be one of the mechanisms acquired to accelerate oxidative phosphorylation.     

Interpretation of relevance of sodium–calcium exchange in action potential of diabetic rat heart by mathematical model

Sarcolemmal Na⁺–Ca²⁺ exchange plays a central role in ion transport of the myocardium and the current carried with it contributes to the late phase of the action potential (AP) besides the contribution of outward K⁺-currents. In this study, the mathematical model for AP of the diabetic rat ventricular myocytes [34] was modified and used for the diabetic rat papillary muscle. We used our experimentally measured values of two K⁺-currents; transient outward current, I_to and steady-state outward current, I_ss, as well as L-type Ca²⁺-current, I_CaL, then compared with the simulated values. We have demonstrated that the prolongation in the AP of the papillary muscle of the diabetic rats are not due to the alteration of I_CaL but mainly due to the inhibition of the K⁺-currents and also the Na⁺–Ca²⁺ exchanger current, I_Na–Ca. In combination with our experimental data on sodium-selenite-treated diabetic rats, our simulation results provide new information concerning plausible ionic mechanisms, and second a possible positive effect of selenium treatment on the altered I_Na–Ca for the observed changes in the AP duration of streptozotocin-induced diabetic rat heart. (Mol Cell Biochem 269: 121–129, 2005)     

Localization of E1–E2 conformational transitions of sarcoplasmic reticulum Ca-ATPase by tryptic cleavage and hydrophobic labeling

Summary Tryptic peptides of Ca-ATPase in E_t and E₂ conformational states (Andersen, J. P., Jørgensen, P. L.,J. Membrane Biol. 88:187–198 (1985)) have been isolated by size exclusion high performance liquid chromatography in sodium dodecyl sulfate. This permitted unambiguous localization of a conformational sensitive tryptic split at Arg 198 by N-terminal amino acid sequence analysis. Other splits at Arg 505 and at Arg 819-Lys 825 were insensitive to E₁–E₂ transitions. Tryptic cleavage of Ca-ATPase after phosphorylation by inorganic phosphate showed that this enzyme form has a conformation similar to that of the vanadate-bound E₂ state, both in membranous and in soluble monomeric Ca-ATPase.Hydrophobic labeling of Ca-ATPase in sarcoplasmic reticulum vesicles with the photoactivable reagent trifluoromethyl-[¹²⁵I]iodophenyl-diazirine indicated that E₂ and E₂V states are more exposed to the membrane phase than E₁ and E₁P (Ca²⁺-occluded) states. The preferetial hydrophobic labeling in E₂ forms was found to be localized in the A₁ tryptic fragment.     

How does ryanodine modify ion handling in the sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel?

Under appropriate conditions, the interaction of the plant alkaloid ryanodine with a single cardiac sarcoplasmic reticulum Ca(2+)-release channel results in a profound modification of both channel gating and conduction. On modification, the channel undergoes a dramatic increase in open probability and a change in single-channel conductance. In this paper we aim to provide a mechanistic framework for the interpretation of the altered conductance seen after ryanodine binding to the channel protein. To do this we have characterized single-channel conductance with representative members of three classes of permeant cation; group 1a monovalent cations, alkaline earth divalent cations, and organic monovalent cations. We have quantified the change in single-channel conductance induced by ryanodine and have expressed this as a fraction of conductance in the absence of ryanodine. Fractional conductance seen in symmetrical 210 mM solutions is not fixed but varies with the nature of the permeant cation. The group 1a monovalent cations (K+, Na+, Cs+, Li+) have values of fractional conductance in a narrow range (0.60- 0.66). With divalent cations fractional conductance is considerably lower (Ba2+, 0.22 and Sr2+, 0.28), whereas values of fractional conductance vary considerably with the organic monovalent cations (ammonia 0.66, ethylamine 0.76, propanolamine 0.65, diethanolamine 0.92, diethylamine 1.2). To establish the mechanisms governing these differences, we have monitored the affinity of the conduction pathway for, and the relative permeability of, representative cations in the ryanodine-modified channel. These parameters have been compared with those obtained in previous studies from this laboratory using the channel in the absence of ryanodine and have been modeled by modifying our existing single-ion, four-barrier three-well rate theory model of conduction in the unmodified channel. Our findings indicate that the high affinity, essentially irreversible, interaction of ryanodine with the cardiac sarcoplasmic reticulum Ca(2+)-release channel produces a conformational alteration of the protein which results in modified ion handling. We suggest that, on modification, the affinity of the channel for the group 1a monovalent cations is increased while the relative permeability of this class of cations remains essentially unaltered. The affinity of the conduction pathway for the alkaline earth divalent cations is also increased, however the relative permeability of this class of cations is reduced compared to the unmodified channel. The influence of modification on the handling by the channel of the organic monovalent cations is determined by both the size and the nature of the cation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Novel triterpenoid AECHL-1 induces apoptosis in breast cancer cells by perturbing the mitochondria–endoplasmic reticulum interactions and targeting diverse apoptotic pathways

BackgroundThe modus operandi for an anti-cancer drug must allow for an efficient discrimination system between tumorigenic and non-tumorigenic cells. Targeting ER stress and mitochondrial function in cancer cells appears to be a suitable option, as these processes are dysregulated in tumor cells. AECHL-1, a novel triterpenoid, exhibits potent anticancer activity against an array of cancer cell lines however, its mechanism of action remains elusive.MethodsMolecular targets of AECHL-1 were investigated using breast adenocarcinoma cells MCF-7, MDA-MB-231 and mammary epithelial cell line MCF 10A in vitro and xenograft tumors in SCID mice in vivo. Western blotting, flow cytometry, and immunohistochemical studies were employed to delineate the molecular pathways.ResultsAECHL-1 caused a transient elevation of ER stress proteins along with a prolonged phosphorylation of eIF2α in breast cancer cells. This was accompanied by a simultaneous release of calcium from ER stores and subsequent mitochondrial accumulation. These effects could be reversed by using ER stress inhibitors. AECHL-1 brings about mitochondria mediated, caspase independent cell death via AIF in MCF-7 cells; MDA-MB-231 succumbed to caspase dependent extrinsic pathway. Xenograft studies closely echoed our in vitro results. AECHL-1 did not alter cellular and molecular parameters in MCF 10A.ConclusionThese findings reveal that, AECHL-1 targets the Achilles Heel of cancer cell, namely dysfunctional ER and mitochondria while being non toxic to normal parenchyma and can thus be further explored as a potential chemotherapeutic intervention.General significanceAggravation of ER stress by AECHL-1 uncovers a novel pathway for selective elimination of cancer cells.     

Calmodulin·(Ca2+)4 is the active calmodulin-calcium species activating the calcium-, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum in the regulation of the calcium pump

Calcium-, calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum increases the rate of calcium transport. The complex dependence of calmodulin-dependent phosphoester formation on free calcium and total calmodulin concentrations can be satisfactorily explained by assuming that CaM · (Ca²⁺)₄ is the sole calmodulin-calcium species which activates the calcium-, calmodulin-dependent, membrane-bound protein kinase. The apparent dissociation constant of the E · CaM · (Ca²⁺)₄ complex determined from the calcium dependence of calmodulin-dependent phosphoester formation over a 100-fold range of total calmodulin concentrations (0.01–1 μ M) was 0.9 nM; the respective apparent dissoclation constant at 0.8 mM free calcium, 1 mM free magnesium with low calmodulin concentrations (0.1–50 nM) was 2.60 nM. These results are in good agreement with the apparent dissociation constant of 2.54 nM of high affinity calmodulin binding determined by ¹²⁵I-labelled calmodulin binding to sarcoplasmic reticulum fractions at 1 mM free calcium, 1 mM free magnesium and total calmodulin concentration ranging from 0.1 to 150 nM, i.e. conditions where approximately 98% of the total calmodulin is present as CaM · (Ca²⁺)₄. The apparent dissociation constant of the calcium-free calmodulin-enzyme complex (E · CaM) is at least 100-fold greater than the apparent dissociation constant of the E · CaM · (Ca²⁺)₄ complex, as judged from non-saturation ¹²⁵I-labelled calmodulin binding at total calmodulin concentrations of up to 150 nM, in the absence of calcium.     

Defective human Ether-à-go-go-related gene trafficking linked to an endoplasmic reticulum retention signal in the C terminus

Mutations in the human Ether-à-go-go-Related gene (HERG), encoding the protein underlying the cardiac K(+) current, I(Kr), cause chromosome 7-linked long QT syndrome (LQT2). In this study, we show that deletion of the C-terminal 147 amino acids (HERG(Delta147)) abolished I(Kr), whereas a larger, 159-amino acid deletion (HERG(Delta159)) identified in an LQT2 kindred did generate I(Kr), albeit with reduced amplitude compared with the wild type. The 12 amino acids present in HERG(Delta147) and absent in HERG(Delta159) include a potential endoplasmic reticulum (ER) retention signal, RGR, which when mutated to LGL (HERG(Delta147-LGL)) restored I(Kr). Streptavidin selection of biotin-labeled surface proteins showed good expression of wild-type and HERG(Delta159) at the cell surface and low expression of HERG(Delta147-LGL) and HERG(Delta147). Additionally, a 100-amino acid peptide spanning the RGR triplet can rescue the defect in HERG(Delta147) when co-expressed as an ER-targeted minigene. Failure of HERG trafficking is known to cause LQT2, and this identified a molecular mechanism underlying this defect. Further, our data indicate that a key function of the C-terminal 104 amino acids is to mask the RGR ER retention signal, which becomes exposed when mutations truncate the HERG C terminus.