Sarcoplasmic Reticulum Fractions Research Articles

Calcium Handling Remodeling Underlies Impaired Sympathetic Stress Response in Ventricular Myocardium from Cacna1c Haploinsufficient Rats.

CACNA1C encodes the pore-forming α1C subunit of the L-type Ca2+ channel, Cav1.2. Mutations and polymorphisms of the gene are associated with neuropsychiatric and cardiac disease. Haploinsufficient Cacna1c+/- rats represent a recently developed model with a behavioral phenotype, but its cardiac phenotype is unknown. Here, we unraveled the cardiac phenotype of Cacna1c+/- rats with a main focus on cellular Ca2+ handling mechanisms. Under basal conditions, isolated ventricular Cacna1c+/- myocytes exhibited unaltered L-type Ca2+ current, Ca2+ transients (CaTs), sarcoplasmic reticulum (SR) Ca2+ load, fractional release, and sarcomere shortenings. However, immunoblotting of left ventricular (LV) tissue revealed reduced expression of Cav1.2, increased expression of SERCA2a and NCX, and augmented phosphorylation of RyR2 (at S2808) in Cacna1c+/- rats. The β-adrenergic agonist isoprenaline increased amplitude and accelerated decay of CaTs and sarcomere shortenings in both Cacna1c+/- and WT myocytes. However, the isoprenaline effect on CaT amplitude and fractional shortening (but not CaT decay) was impaired in Cacna1c+/- myocytes exhibiting both reduced potency and efficacy. Moreover, sarcolemmal Ca2+ influx and fractional SR Ca2+ release after treatment with isoprenaline were smaller in Cacna1c+/- than in WT myocytes. In Langendorff-perfused hearts, the isoprenaline-induced increase in RyR2 phosphorylation at S2808 and S2814 was attenuated in Cacna1c+/- compared to WT hearts. Despite unaltered CaTs and sarcomere shortenings, Cacna1c+/- myocytes display remodeling of Ca2+ handling proteins under basal conditions. Mimicking sympathetic stress with isoprenaline unmasks an impaired ability to stimulate Ca2+ influx, SR Ca2+ release, and CaTs caused, in part, by reduced phosphorylation reserve of RyR2 in Cacna1c+/- cardiomyocytes.

Activation of Ca2+ transport in cardiac microsomes enriches functional sets of ER and SR proteins

The importance of sarcoplasmic reticulum (SR) Ca2+-handling in heart has led to detailed understanding of Ca2+-release and re-uptake protein complexes, while less is known about other endoplasmic reticulum (ER) functions in the heart. To more fully understand cardiac SR and ER functions, we analyzed cardiac microsomes based on their increased density through the actions of the SR Ca2+-ATPase (SERCA) and the ryanodine receptor that are highly active in cardiomyocytes. Crude cardiac microsomal vesicles loaded with Ca oxalate produced two higher density subfractions, MedSR and HighSR. Proteins from 20.0 μg of MV, MedSR, and HighSR protein were fractionated using SDS-PAGE, then trypsinized from 20 separate gel pieces, and analyzed by LC–MS/MS to determine protein content. From 62,000 individual peptide spectra obtained, we identified 1105 different proteins, of which 354 were enriched ≥ 2.0-fold in SR fractions compared to the crude membrane preparation. Previously studied SR proteins were all enriched, as were proteins associated with canonical ER functions. Contractile, mitochondrial, and sarcolemmal proteins were not enriched. Comparing the levels of SERCA-positive SR proteins in MedSR versus HighSR vesicles produced a range of SR subfraction enrichments signifying differing levels of Ca2+ leak co-localized in the same membrane patch. All known junctional SR proteins were more enriched in MedSR, while canonical ER proteins were more enriched in HighSR membrane. Proteins constituting other putative ER/SR subdomains also exhibited average Esub enrichment values (mean ± S.D.) that spanned the range of possible Esub values, suggesting that functional sets of proteins are localized to the same areas of the ER/SR membrane. We conclude that active Ca2+ loading of cardiac microsomes, reflecting the combined activities of Ca2+ uptake by SERCA, and Ca2+ leak by RyR, permits evaluation of multiple functional ER/SR subdomains. Sets of proteins from these subdomains exhibited similar enrichment patterns across membrane subfractions, reflecting the relative levels of SERCA and RyR present within individual patches of cardiac ER and SR.

Abstract 12951: Electrical and Calcium-Dependent Mechanisms That Contribute to Increase the Risk for Atrial Fibrillation in Obesity

Obesity (OB) is an independent predictor for atrial fibrillation (AF), but mechanisms by which OB increases AF risk remain poorly characterized. We hypothesized that OB modulates AF risk through electrical remodeling and impaired Ca 2+ dynamics that can be reversed after weight loss (WL). Using transesophageal rapid pacing we characterized AF inducibility and burden in lean, OB, and OB mice that lost 20% of body weight after modified vertical sleeve gastrectomy. Local field fluorescence microscopy was used to simultaneously record action potentials (APs) (di-8-ANEPPS or glass microelectrodes) and Ca 2+ transients (CaT) (Rhod-2) in Langendorff heart preparations. We evaluated AP duration (APD) dependency of frequency from 4 to 14 Hz (1 Hz incremental steps). AP parameters analyzed included APD30, APD50, APD90 and spontaneous arrhythmogenic events. We assessed Ca 2+ dynamics by studying CaT and fractional sarcoplasmic reticulum Ca 2+ release in the absence and presence of ryanodine (Ry) and thapsigargin (Tg). Since CaT Ry+Tg represents sarcolemmal Ca 2+ influx, we calculated the gain of Ca 2+ induced Ca 2+ release (CICR gain ) as the ratio between CaT in the absence and presence of Ry+Tg. Experiments were done in the presence of blebbistatin at 37° C. Mean±SE, t-test. After burst pacing, both AF prevalence and burden were higher in OB mice vs lean (94 % vs 47 %, 227±19 vs 20±1.2 s, n=16 mice/group, p<0.0001). About 76 % of OB hearts showed afterdepolarizations and fibrillation vs no events in lean. APD50 and APD90 significantly decreased as stimulation frequency increased from 4 to 14 Hz in OB hearts vs lean (n=6 mice/group, p<0.05). Peak CaT from OB hearts were significantly decreased compared to peak CaT from lean (0.3±0.05 vs. 0.8±0.06, n=9, p<0.001). CICR gain in OB hearts was also significantly compromised compared to lean (5.2±1 vs 23.3±1.4, n=7 mice/group, p<0.0001). After WL, OB mice showed a decreased AF burden (21.7±8 s), increased APD, increased peak CaT (0.8±0.15) and increased CICR gain (20±2.3) back to lean levels (n=8, p<0.001). In conclusion, shortened APs, spontaneous afterdepolarizations and impaired CICR significantly contribute to the development of AF in OB. WL reversed the electrical phenotype and the impaired Ca 2+ dynamics and prevented AF.

An Investigation of the Altered Textural Property in Woody Breast Myopathy Using an Integrative Omics Approach.

Woody breast (WB) is a myopathy observed in broiler Pectoralis major (PM) characterized by its tough and rubbery texture with greater level of calcium content. The objective of this study was to investigate the functionality/integrity of WB sarcoplasmic reticulum (SR), which may contribute to the elevated calcium content observed in WB and other factors that may influence WB texture. Fourteen Ross line broiler PM [7 severe WB and 7 normal (N)] were selected, packaged, and frozen at −20°C at 8 h postmortem from a commercial processing plant. Samples were used to measure pH, sarcomere length, proteolysis, calpain activity, collagenase activity, collagen content, collagen crosslinks density, and connective tissue peak transitional temperature. Exudate was also collected from each sample to evaluate free calcium concentration. The SR fraction of the samples was separated and utilized for proteomic and lipidomic analysis. The WB PM had a higher pH, shorter sarcomeres, lower % of intact troponin-T, more autolyzed μ/m calpain, more activated collagenase, greater collagen content, greater mature collagen crosslinks density, and higher connective tissue peak transitional temperature than the N PM (p ≤ 0.05). Exudate from WB PM had higher levels of free calcium than those from N PM (p < 0.05). Proteomics data revealed an upregulation of calcium transport proteins and a downregulation of proteins responsible for calcium release (p < 0.05) in WB SR. Interestingly, there was an upregulation of phospholipase A2 (PLA2), and cholinesterase exhibited a 7.6-fold increase in WB SR (p < 0.01). Lipidomics data revealed WB SR had less relative % of phosphatidylcholine (PC) and more lysophosphatidylcholine (LPC; p < 0.05). The results indicated that upregulation of calcium transport proteins and downregulation of calcium-release proteins in WB SR may be the muscle’s attempt to regulate this proposed excessive signaling of calcium release due to multiple factors, such as upregulation of PLA2 resulting in PC hydrolysis and presence of cholinesterase inhibitors in the system prolonging action potential. In addition, the textural abnormality of WB may be the combined effects of shorter sarcomere length and more collagen with greater crosslink density being deposited in the broiler PM.

The Role of T-Tubule Organization in Regulation of Intracellular Ca2+ Handling by Muscarinic Receptor Stimulation in Ventricular Myocytes

Muscarinic receptors expressed in cardiac myocytes play critical role in mediating regulation of heart function by the parasympathetic nervous system. How structural organization of cardiac myocytes affects muscarinic receptor mediated regulation of Ca2+ handling is not well defined. Using confocal Ca2+ imaging and patch-clamp techniques we studied the relationships between t-tubule density and cholinergic regulation of intracellular Ca2+ handling in normal murine ventricular myocytes and in myocytes in which the t-tubule system was disrupted by formamide treatment. Muscarinic receptor agonist CCh (10 μM) significantly inhibited amplitude of Ca2+ transients evoked by electrical field-stimulation in the presence of 100 nM isoproterenol (Iso), a beta-adrenergic agonist, in formamide-treated but not in control ventricular myocytes. Following Iso stimulation, formamide-treated cells exhibited reduced density of the inward Ca2+ current (ICa), decreased sarcoplasmic reticulum (SR) Ca2+ content, and enhanced fractional SR Ca2+ release when compared to control myocytes. CCh did not change SR Ca2+ content in Iso-stimulated formamide-treated myocytes but significantly reduced fractional release. This effect was associated with reduced Ca2+ release at coupled SR Ca2+ release sites and decreased Ca2+ sensitivity of the uncoupled release sites. CCh inhibited peak ICa recorded in a presence of Iso by ∼20 % in both control and detubulated ventricular myocytes. Reducing ICa amplitude by up to 40% by changing voltage step levels from 0 to −25 mV did not affect Ca2+ transients in control myocytes in the presence of Iso. In contrast, in formamide-treated myocytes similar decrease in ICa significantly diminished Ca2+ transients. We conclude that downregulation of the t-tubule system, as often observed in failing hearts, augments the inhibitory effects of muscarinic receptor agonists on Ca2+ transients by reducing functional reserve of excitation-contraction coupling.

Oxidation of ryanodine receptor after ischemia-reperfusion increases propensity of Ca2+ waves during β-adrenergic receptor stimulation.

β-Adrenergic receptor (β-AR) activation produces the main positive inotropic response of the heart. During ischemia-reperfusion (I/R), however, β-AR activation can trigger life-threatening arrhythmias. Because I/R is frequently associated with oxidative stress, we investigated whether ryanodine receptor (RyR) oxidation contributes to proarrythmogenic Ca2+ waves during β-AR activation. Measurements of contractile and electrical activity from Langendorff-perfused rabbit hearts revealed that I/R produces tachyarrhythmias. Ventricular myocytes isolated from I/R hearts had an increased level of oxidized glutathione (i.e., oxidative stress) and a decreased level of free thiols in RyRs (i.e., RyR oxidation). Furthermore, myocytes from I/R hearts were characterized by increased sarcoplasmic reticulum (SR) Ca2+ leak and enhanced fractional SR Ca2+ release. In myocytes from nonischemic hearts, β-AR activation with isoproterenol (10 nM) produced only a positive inotropic effect, whereas in myocytes from ischemic hearts, isoproterenol at the same concentration triggered spontaneous Ca2+ waves. β-AR activation produced a similar effect on RyR phosphorylation in control and I/R myocytes. Treatment of myocytes from I/R hearts with the reducing agent mercaptopropionylglycine (100 μM) attenuated RyR oxidization and decreased Ca2+ wave frequency during β-AR activation. On the other hand, treatment of myocytes from nonischemic hearts with H2O2 (50 μM) increased SR Ca2+ leak and triggered Ca2+ waves during β-AR activation. Collectively, these results suggest that RyR oxidation after I/R plays a critical role in the transition from positive inotropic to arrhythmogenic effects during β-AR stimulation. Prevention of RyR oxidation can be a promising strategy to inhibit arrhythmias and preserve positive inotropic effect of β-AR activation during myocardial infarction. NEW & NOTEWORTHY Oxidative stress induced by ischemia plays a critical role in triggering arrhythmias during adrenergic stimulation. The combined increase in sarcoplasmic reticulum Ca2+ leak (because of ryanodine receptor oxidation) and sarcoplasmic reticulum Ca2+ load (because of adrenergic stimulation) can trigger proarrythmogenic Ca2+ waves. Restoring normal ryanodine receptor redox status can be a promising strategy to prevent arrhythmias and preserve positive inotropic effect of adrenergic stimulation during myocardial infarction.

Signaling Mechanisms of Drp1 Translocation to the Mitochondria-SR Associations in Adult Murine Cardiomyocytes

Mitochondrial dynamics, including fission, fusion, and movement, is a fundamental mechanism in regulating mitochondrial function. The high level of Drp1 in the heart seems to be at odds with numerous studies showing that mitochondrial dynamics in adult ventricular myocytes occurs rather infrequently. Electron micrographs show that in adult mammalian cardiac myocytes, the majority of inter-myofibrillar mitochondria appear to be fragmented and tethered to the sarcoplasmic reticulum (SR) near the dyads. We tested the hypothesis that Drp1 is strategically accumulated at the mitochondria-SR associations (MAM) and that during continuous excitation-contraction coupling processes (e.g. heartbeats), the localized high Ca2+ in the SR-mitochondria junction further increases translocation of cytosolic Drp1 to the MAM. Western blots analysis shows a Drp1 presence in the SR and crude mitochondria fractions. Furthermore, proteomic results show that Drp1 is mostly presented and enriched in the Percoll purified MAM but not in the pure mitochondrial fraction. To confirm this data, the distance between SR(RyR2) and Drp1 in adult cardiomyocytes was quantified in nanometer scale by using confocal microscope-Airy-scan detector. Over 80% of Drp1 is located within 200nm radius of RyR2. Similar results were observed in preparations by using immunogold-TEM. Drp1 distribution along the dyads was studied by the localization of Drp1 along the transversal side of the mitochondria(TOM20). A significantly increased translocation of Drp1 to the transversal sides was observed upon 15min-2Hz electric-field stimulation in cardiomyocytes superfused with isoproterenol+Ca2+(1mM) in comparison to cardiomyocytes incubated with quasi-Ca2+-free(2.5μM) buffer. Finally, isolated hearts form rat retrogradely perfused (Langendorff) with Krebs buffer supplemented with isoproterenol+Ca2+(1.8mM) showed a significantly elevated Drp1 accumulation at the MAM. Conclusion: According to these results, we conclude that Drp1 is preferentially positioned at the mitochondria-SR associations and its recruitment likely involves Ca2+ signaling and/or beta-adrenergic activity.

Deciphering Functions of Intracellular Formaldehyde: Linking Cancer and Aldehyde Metabolism.

Carnosine (beta-alanyl-L-histidine), which is present in millimolar concentrations in skeletal muscles, induces Ca2+ release from the heavy fraction of rabbit skeletal muscle sarcoplasmic reticulum by activation ruthenium red-sensitive Ca-release channels. The effect of carnosine is dose-dependent, which indicates the presence of saturable carnosine-binding sites in the Ca-release channel molecule. The half-maximal Ca2+ release is observed in the presence of 8.7 mM carnosine. At the same time, carnosine addition to the medium increases the affinity of sarcoplasmic reticulum Ca-channels for the Ca-release activators, caffeine and adenine nucleotides. It is concluded that carnosine is an endogenous regulator of skeletal muscle sarcoplasmic reticulum Ca-channels which modulates the affinity of these channels for different ligands.

CD38-cADPR-SERCA Signaling Axis Determines Skeletal Muscle Contractile Force in Response to β-Adrenergic Stimulation

Background/Aims: Cyclic ADP-ribose (cADPR) is a Ca²⁺ -mobilization messenger that acts on ryanodine-sensitive Ca²⁺ channels in the sarcoplasmic reticulum (SR) Ca²⁺ stores. Moreover, it has been proposed that cADPR serves an additional role in activating the sarcoendoplasmic reticulum Ca²⁺ -ATPase (SERCA) pump. The aim of this study was to determine the exact mechanism by which cADPR regulates SR Ca²⁺ stores in physiologically relevant systems. Methods: We analyzed Ca²⁺ signals as well as the production of Ca²⁺ mobilizing messengers in the skeletal muscle cells of mice subjected to intensive exercise or in the SR fractions from skeletal muscle cells after β-adrenergic receptor (β-AR) stimulation. Results: We show that cADPR enhances SERCA activity in skeletal muscle cells in response to β-AR agonists, increasing SR Ca²⁺ uptake. We demonstrate that cADPR is generated by CD38, a cADPR-synthesizing enzyme, increasing muscle Ca²⁺ signals and contractile force during exercise. CD38 is upregulated by the cAMP response element–binding protein (CREB) transcription factor upon β-AR stimuli and exercise. CD38 knockout (KO) mice show defects in their exercise and cADPR synthesis capabilities, lacking a β-AR agonist-induced muscle contraction when compared to wild-type mice. The skeletal muscle of CD38 KO mice exhibits delayed cytosolic Ca²⁺ clearance and reduced SERCA activity upon exercise. Conclusion: These findings provide insight into the physiological adaptive mechanism by which the CD38- cADPR-SERCA signaling axis plays an essential role in muscle contraction under exercise, and define cADPR as an endogenous activator of SERCA in enhancing the SR Ca²⁺ load.

Computational modeling of amylin-induced calcium dysregulation in rat ventricular cardiomyocytes

Hyperamylinemia is a condition that accompanies obesity and precedes type II diabetes, and it is characterized by above-normal blood levels of amylin, the pancreas-derived peptide. Human amylin oligomerizes easily and can deposit in the pancreas [1], brain [2], and heart [3], where they have been associated with calcium dysregulation. In the heart, accumulating evidence suggests that human amylin oligomers form moderately cation-selective [4,5] channels that embed in the cell sarcolemma (SL). The oligomers increase membrane conductance in a concentration-dependent manner [5], which is correlated with elevated cytosolic Ca2+. These findings motivate our core hypothesis that non-selective inward Ca2+ conduction afforded by human amylin oligomers increase cytosolic and sarcoplasmic reticulum (SR) Ca2+ load, which thereby magnifies intracellular Ca2+ transients. Questions remain however regarding the mechanism of amylin-induced Ca2+ dysregulation, including whether enhanced SL Ca2+ influx is sufficient to elevate cytosolic Ca2+ load [6], and if so, how might amplified Ca2+ transients perturb Ca2+-dependent cardiac pathways. To investigate these questions, we modified a computational model of cardiomyocytes Ca2+ signaling to reflect experimentally-measured changes in SL membrane permeation and decreased sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) function stemming from acute and transgenic human amylin peptide exposure. With this model, we confirmed the hypothesis that increasing SL permeation alone was sufficient to enhance Ca2+ transient amplitudes. Our model indicated that amplified cytosolic transients are driven by increased Ca2+ loading of the SR and that greater fractional release may contribute to the Ca2+-dependent activation of calmodulin, which could prime the activation of myocyte remodeling pathways. Importantly, elevated Ca2+ in the SR and dyadic space collectively drive greater fractional SR Ca2+ release for human amylin expressing rats (HIP) and acute amylin-exposed rats (+Amylin) mice, which contributes to the inotropic rise in cytosolic Ca2+ transients. These findings suggest that increased membrane permeation induced by oligomeratization of amylin peptide in cell sarcolemma contributes to Ca2+ dysregulation in pre-diabetes.

O22-3 - Upregulation of AMP Deaminase in the Vicinity of the SR via Translational Regulation by miR-301b Contributes to Diabetic Cardiomyopathy

Background: We recently reported that ATP loss by upregulated activity of AMP deaminase (AMPD) contributes to afterload-induced diastolic dysfunction in OLETF, type 2 diabetic rats. Here we examined the mechanism underlying the upregulation of AMPD. Methods and Results: Western blot analysis revealed that protein level of 90-kDa full-length AMPD3 was significantly increased in the sarcoplasmic reticulum (SR) fraction (117%) as well as in whole cell lysates (171%) in OLETF compared to its level in LETO, non-diabetic control rats. Levels of AMPD3 mRNA determined by RT-PCR was comparable in OLETF and LETO. Inhibitory effect of MG-132, a proteasome inhibitor, on degradation of the 90-kDa AMPD3 ex vivo was larger in OLETF than in LETO, arguing against suppressed degradation of AMPD3 in OLETF. MicroRNA array analysis revealed downregulation (>50%) of 57 microRNAs in OLETF compared to those in LETO. Transfection of H9c2 cells with an inhibitor of miR-301b, one of the 57 microRNAs, significantly increased level of the 90-kDa AMPD3. Binding of miR-301b to the 3'UTR of AMPD3 mRNA was confirmed by a luciferase reporter assay. Conclusion: Up-regulated AMPD3 translation by reduced miR-301b mediates increased AMPD3 in the diabetic heart. Since ATP generated by SR-associated glycolytic enzymes contributes to Ca2+ uptake by SERCA2a and promote diastolic relaxation, AMPD3 in the vicinity of the SR is a therapeutic target for diabetic cardiomyopathy.

Effect of ovariectomy on intracellular Ca2+ regulation in guinea pig cardiomyocytes.

This study addressed the hypothesis that long-term deficiency of ovarian hormones after ovariectomy (OVx) alters cellular Ca2+-handling mechanisms in the heart, resulting in the formation of a proarrhythmic substrate. It also tested whether estrogen supplementation to OVx animals reverses any alterations to cardiac Ca2+ handling and rescues proarrhythmic behavior. OVx or sham operations were performed on female guinea pigs using appropriate anesthetic and analgesic regimes. Pellets containing 17β-estradiol (1 mg, 60-day release) were placed subcutaneously in selected OVx animals (OVx + E). Cardiac myocytes were enzymatically isolated, and electrophysiological measurements were conducted with a switch-clamp system. In fluo-4-loaded cells, Ca2+ transients were 20% larger, and fractional sarcoplasmic reticulum (SR) Ca2+ release was 7% greater in the OVx group compared with the sham group. Peak L-type Ca2+ current was 16% larger in OVx myocytes with channel inactivation shifting to more positive membrane potentials, creating a larger "window" current. SR Ca2+ stores were 22% greater in the OVx group, and these cells showed a higher frequency of Ca2+ sparks and waves and shorter wave-free intervals. OVx myocytes showed higher frequencies of early afterdepolarizations, and a greater percentage of these cells showed delayed afterdepolarizations after exposure to isoprenaline compared with sham myocytes. The altered Ca2+ regulation occurring in the OVx group was not observed in the OVx + E group. These findings suggest that long-term deprivation of ovarian hormones in guinea pigs lead to changes in myocyte Ca2+-handling mechanisms that are considered proarrhythmogenic. 17β-Estradiol replacement prevented these adverse effects.NEW & NOTEWORTHY Ovariectomized guinea pig cardiomyocytes have higher frequencies of Ca2+ waves, and isoprenaline-challenged cells display more early afterdepolarizations, delayed afterdepolarizations, and extra beats compared with sham myocytes. These alterations to Ca2+ regulation were not observed in myocytes from ovariectomized guinea pigs supplemented with 17β-estradiol, suggesting that ovarian hormone deficiency modifies cardiac Ca2+ regulation, potentially creating proarrhythmic substrates.

Nanospan, an alternatively spliced isoform of sarcospan, localizes to the sarcoplasmic reticulum in skeletal muscle and is absent in limb girdle muscular dystrophy 2F

BackgroundSarcospan (SSPN) is a transmembrane protein that interacts with the sarcoglycans (SGs) to form a tight subcomplex within the dystrophin-glycoprotein complex that spans the sarcolemma and interacts with laminin in the extracellular matrix. Overexpression of SSPN ameliorates Duchenne muscular dystrophy in murine models.MethodsStandard cloning approaches were used to identify nanospan, and nanospan-specific polyclonal antibodies were generated and validated. Biochemical isolation of skeletal muscle membranes and two-photon laser scanning microscopy were used to analyze nanospan localization in muscle from multiple murine models. Duchenne muscular dystrophy biopsies were analyzed by immunoblot analysis of protein lysates as well as indirect immunofluorescence analysis of muscle cryosections.ResultsNanospan is an alternatively spliced isoform of sarcospan. While SSPN has four transmembrane domains and is a core component of the sarcolemmal dystrophin-glycoprotein complex, nanospan is a type II transmembrane protein that does not associate with the dystrophin-glycoprotein complex. We demonstrate that nanospan is enriched in the sarcoplasmic reticulum (SR) fractions and is not present in the T-tubules. SR fractions contain membranes from three distinct structural regions: a region flanking the T-tubules (triadic SR), a SR region across the Z-line (ZSR), and a longitudinal SR region across the M-line (LSR). Analysis of isolated murine muscles reveals that nanospan is mostly associated with the ZSR and triadic SR, and only minimally with the LSR. Furthermore, nanospan is absent from the SR of δ-SG-null (Sgcd−/−) skeletal muscle, a murine model for limb girdle muscular dystrophy 2F. Analysis of skeletal muscle biopsies from Duchenne muscular dystrophy patients reveals that nanospan is preferentially expressed in type I (slow) fibers in both control and Duchenne samples. Furthermore, nanospan is significantly reduced in Duchenne biopsies.ConclusionsAlternative splicing of proteins from the SG-SSPN complex produces δ-SG3, microspan, and nanospan that localize to the ZSR and the triadic SR, where they may play a role in regulating resting calcium levels as supported by previous studies (Estrada et al., Biochem Biophys Res Commun 340:865–71, 2006). Thus, alternative splicing of SSPN mRNA generates three protein isoforms (SSPN, microspan, and nanospan) that differ in the number of transmembrane domains affecting subcellular membrane association into distinct protein complexes.

Reciprocal Polarization of the Mitochondrial Ca2+ Uniporter and NA+-CA2+ Exchanger in Cardiac Muscle

Control of myocardial energetics by Ca2+ signal propagation to the mitochondrial matrix includes local Ca2+ delivery from sarcoplasmic reticulum (SR) ryanodine receptors (RyR2) to the inner mitochondrial membrane (IMM) through the Ca2+ uniporter (mtCU). Ca2+ is extruded by the Na+-Ca2+ exchanger (NCLX), predominantly active in excitable tissues such as heart, and/or the H+-Ca2+ exchanger preferentially active in non-excitable cells. We have recently reported that the mtCU is strategically positioned in cardiac muscle at the mitochondria-junctional SR association in order to support more efficiently the local Ca2+ signals. In those studies we established a comparative abundance profile for a range of mitochondrial proteins between the mitochondrial and SR fractions of mouse and rat heart homogenates. While mtCU constituents were equally distributed (MCU, MICU1) or enriched in the SR fraction (EMRE), the NCLX was strongly (multiple fold) enriched in the mitochondrial fraction. We thus hypothesized, that NCLX was excluded from the mtCU hot spots at the dyad junctions and that it would be localized at areas where mitochondria associate with the network SR. Indeed, percoll purification of the crude mitochondrial fraction that decreased the abundance of MCU and EMRE lead to an enrichment of NCLX. Mitochondrial 45Ca2+ uptake assays using isolated mitochondria or SR fraction with vs. without NCLX inhibition (pharmacological or Na+ removal) revealed a robust Na+-dependent efflux working against the uptake in the mitochondrial but not the SR fraction. Direct measurement of the fractional Ca2+ release from the two membrane fractions also pointed toward much more efficient NCLX-mediated Ca2+ extrusion in the mitochondrial fraction than in the SR. Thus, mitochondrial Ca2+ uptake and Na+ dependent extrusion seem to be reciprocally polarized to optimize both the efficacy of receiving local Ca2+ signals at the dyadic interface and of feeding back the uptake Ca2+ to the SERCA pumps of the network SR.

A unified theory of calcium alternans in ventricular myocytes.

Intracellular calcium (Ca2+) alternans is a dynamical phenomenon in ventricular myocytes, which is linked to the genesis of lethal arrhythmias. Iterated map models of intracellular Ca2+ cycling dynamics in ventricular myocytes under periodic pacing have been developed to study the mechanisms of Ca2+ alternans. Two mechanisms of Ca2+ alternans have been demonstrated in these models: one relies mainly on fractional sarcoplasmic reticulum Ca2+ release and uptake, and the other on refractoriness and other properties of Ca2+ sparks. Each of the two mechanisms can partially explain the experimental observations, but both have their inconsistencies with the experimental results. Here we developed an iterated map model that is composed of two coupled iterated maps, which unifies the two mechanisms into a single cohesive mathematical framework. The unified theory can consistently explain the seemingly contradictory experimental observations and shows that the two mechanisms work synergistically to promote Ca2+ alternans. Predictions of the theory were examined in a physiologically-detailed spatial Ca2+ cycling model of ventricular myocytes.

Strategic Positioning and Biased Activity of the Mitochondrial Calcium Uniporter in Cardiac Muscle

Control of myocardial energetics by Ca2+ signal propagation to the mitochondrial matrix includes local Ca2+ delivery from sarcoplasmic reticulum (SR) ryanodine receptors (RyR2) to the inner mitochondrial membrane (IMM) Ca2+ uniporter (mtCU). mtCU activity in cardiac mitochondria is relatively low, whereas the IMM surface is large, due to extensive cristae folding. Hence, stochastically distributed mtCU may not suffice to support local Ca2+ transfer. We hypothesized that mtCU concentrated at mitochondria-SR associations would promote the effective Ca2+ transfer. mtCU distribution was determined by tracking MCU and EMRE, the proteins essential for channel formation. Both proteins were enriched in the IMM-outer mitochondrial membrane (OMM) contact point submitochondrial fraction and, as super-resolution microscopy revealed, located more to the mitochondrial periphery (inner boundary membrane) than inside the cristae, indicating high accessibility to cytosol-derived Ca2+ inputs. Furthermore, MCU immunofluorescence distribution was biased toward the mitochondria-SR interface (RyR2), and this bias was promoted by Ca2+ signaling activity in intact cardiomyocytes. The SR fraction of heart homogenate contains mitochondria with extensive SR associations, and these mitochondria are highly enriched in EMRE. Size exclusion chromatography suggested for EMRE- and MCU-containing complexes a wide size range and also revealed MCU-containing complexes devoid of EMRE (thus disabled) in the mitochondrial but not the SR fraction. Functional measurements suggested more effective mtCU-mediated Ca2+ uptake activity by the mitochondria of the SR than of the mitochondrial fraction. Thus, mtCU "hot spots" can be formed at the cardiac muscle mitochondria-SR associations via localization and assembly bias, serving local Ca2+ signaling and the excitation-energetics coupling.

L-Type Calcium and NCX Currents during Ischemia and Reperfusion in Intact Mouse Hearts

Cardiac ischemia is a pathological condition in which the blood supply to the myocardial tissue is interrupted producing an impairment of the mechanical and electrical function of the heart. Although there is reduction of Ca2+ transients during ischemia, the underlying mechanism of this Ca2+ mishandling is not fully understood. Currently, no reports exist in which Ca2+ currents and SR Ca2+ release are measured simultaneously in situ during global ischemia. Recently, we found that global ischemia induces large increases in diastolic Ca2+ leading to an increase in sarcoplasmic reticulum (SR) Ca2+ content. Contrary to what we expected there was a smaller fractional SR Ca2+ depletion during an action potential (AP). However, Ca2+ sparks measured in intact hearts during ischemia are kinetically undistinguishable from preischemic recordings. Our idea is that depressed Ca2+ transients during ischemia may result from a reduction of L-type Ca2+ currents and/or coupling between plasma membrane Ca2+ influx and SR Ca2+ release. Recently, we found using loose patch photolysis that Ca2+ dependent currents during an AP showed a fast early component driven by an L-type Ca2+influx and a slower late component mediated by a Na+-Ca2+ exchanger (NCX) forward current. To understand why Ca2+ transient decreases, we performed experiments to address if ischemia induces changes in Ca2+ dependent currents. Our results indicate that ischemia produced a decrease of the early Ca2+ current and the late NCX current. However, the reduction of the NCX current occurs before the L-type current attenuation. Although both Ca2+ mediated currents recover during reperfusion after ischemia, L-type Ca2+ currents recuperate faster. We conclude that the impairment of NCX during ischemia is an early event in both the contractility reduction and diastolic Ca2+ increase and that this impairment is maintained during postischemic diastolic dysfunction.

Dyscholesterolemia Protects Against Ischemia-Induced Ventricular Arrhythmias

Hypercholesterolemia protects against ventricular fibrillation in patients with myocardial infarction. We hypothesize that hypercholesterolemia protects against ischemia-induced reentrant arrhythmias because of altered ion channel function. ECGs were measured in low-density lipoprotein receptor knockout (LDLr(-/-)), apolipoprotein A1 knockout (ApoA1(-/-)), and wild-type (WT) mice. Action potentials, calcium handling, and ion currents were recorded in ventricular myocytes. Gene expression was determined by quantitative polymerase chain reaction and Western blot. In isolated perfused hearts, regional ischemia was induced and arrhythmia inducibility was tested. Serum low-density lipoprotein (LDL) cholesterol was higher in LDLr(-/-) mice than in WT mice (2.6 versus 0.4 mmol/L), and high-density lipoprotein cholesterol was significantly lower in ApoA1(-/-) mice than in WT mice (0.3 versus 1.8 mmol/L). LDLr(-/-) and ApoA1(-/-) myocytes contained more cholesterol than WT (34.4±2.8 and 36.5±2.4 versus 25.5±0.4 μmol/g protein). The major potassium currents were not different in LDLr(-/-) and ApoA1(-/-) compared with WT mice. The L-type calcium current (I(Ca)), however, was larger in LDLr(-/-) and ApoA1(-/-) than in WT (12.1±0.7 and 12.8±0.8 versus 9.4±1.1 pA/pF). Calcium transient amplitude and fractional sarcoplasmic reticulum calcium release were larger and action potential and QTc duration longer in LDLr(-/-) and ApoA1(-/-) than in WT mice (action potential duration at 90% of repolarization: 102±4 and 106±3 versus 84±3.1 ms; QTc: 50.9±1.3 and 52.8±0.8 versus 43.5±1.2 ms). During ischemia, ventricular tachycardia/ventricular fibrillation inducibility was larger in WT than in LDLr(-/-) and ApoA1(-/-) hearts. Expression of sodium channel and Ca-handling genes were not significantly different between groups. Dyscholesterolemia is associated with action potential prolongation because of increased I(Ca) and reduces occurrence of reentrant arrhythmias during ischemia.

Regulation of Sarcoplasmic Reticulum Ca2+ ATPase 2 (SERCA2) Activity by Phosphodiesterase 3A (PDE3A) in Human Myocardium: PHOSPHORYLATION-DEPENDENT INTERACTION OF PDE3A1 WITH SERCA2

Cyclic nucleotide phosphodiesterase 3A (PDE3) regulates cAMP-mediated signaling in the heart, and PDE3 inhibitors augment contractility in patients with heart failure. Studies in mice showed that PDE3A, not PDE3B, is the subfamily responsible for these inotropic effects and that murine PDE3A1 associates with sarcoplasmic reticulum Ca(2+) ATPase 2 (SERCA2), phospholamban (PLB), and AKAP18 in a multiprotein signalosome in human sarcoplasmic reticulum (SR). Immunohistochemical staining demonstrated that PDE3A co-localizes in Z-bands of human cardiac myocytes with desmin, SERCA2, PLB, and AKAP18. In human SR fractions, cAMP increased PLB phosphorylation and SERCA2 activity; this was potentiated by PDE3 inhibition but not by PDE4 inhibition. During gel filtration chromatography of solubilized SR membranes, PDE3 activity was recovered in distinct high molecular weight (HMW) and low molecular weight (LMW) peaks. HMW peaks contained PDE3A1 and PDE3A2, whereas LMW peaks contained PDE3A1, PDE3A2, and PDE3A3. Western blotting showed that endogenous HMW PDE3A1 was the principal PKA-phosphorylated isoform. Phosphorylation of endogenous PDE3A by rPKAc increased cAMP-hydrolytic activity, correlated with shift of PDE3A from LMW to HMW peaks, and increased co-immunoprecipitation of SERCA2, cav3, PKA regulatory subunit (PKARII), PP2A, and AKAP18 with PDE3A. In experiments with recombinant proteins, phosphorylation of recombinant human PDE3A isoforms by recombinant PKA catalytic subunit increased co-immunoprecipitation with rSERCA2 and rat rAKAP18 (recombinant AKAP18). Deletion of the recombinant human PDE3A1/PDE3A2 N terminus blocked interactions with recombinant SERCA2. Serine-to-alanine substitutions identified Ser-292/Ser-293, a site unique to human PDE3A1, as the principal site regulating its interaction with SERCA2. These results indicate that phosphorylation of human PDE3A1 at a PKA site in its unique N-terminal extension promotes its incorporation into SERCA2/AKAP18 signalosomes, where it regulates a discrete cAMP pool that controls contractility by modulating phosphorylation-dependent protein-protein interactions, PLB phosphorylation, and SERCA2 activity.

CaMKII Does It Again

Although the recent identification of the mitochondrial Ca2+ uniporter (MCU) has resolved a long-standing mystery as to how Ca2+ freely enters the mitochondria, it has also evoked additional questions such as its mode of regulation and the identity of other associated factors. In an article recently published in Nature , Joiner et al1 provide data demonstrating that in the heart, matrix-localized Ca2+/calmodulin-dependent protein kinase II (CaMKII) can upregulate MCU activity in a manner requiring phosphorylation of the channel N terminus. They showed that inhibition of CaMKII-dependent MCU activity protected the heart from ischemic injury by presumably reducing Ca2+ influx and desensitizing the mitochondrial permeability transition pore (MPTP) to opening. Although these results demonstrate convincingly that CaMKII plays an important role in MCU regulation and subsequent response to cardiac injury, several questions remain unanswered. The ability of mitochondria to take up and sequester Ca2+ plays an important role in the buffering of cytosolic Ca2+, regulation of ATP production via the citric acid cycle, and regulation of apoptotic and necrotic cell death pathways.2 Although mitochondrial Ca2+ uptake was first described in the 1960s3 and the electrophysiological properties of the MCU were reported in 2004,4 it was not until 2011 that 2 articles were published revealing the genetic identity of the MCU.5,6 This pioneering work has initiated a search for additional members of the MCU complex (such as MICU17 and the recently discovered MCUR1),8 as well as an attempt to understand how MCU-mediated Ca2+ influx participates in the regulation of whole-cell Ca2+ signaling and whether well-described pathways that regulate other Ca2+ handling processes can similarly modulate MCU-dependent Ca2+ uptake. In the featured article, Joiner et al1 show that CaMKII serves …