Calcium Mishandling Research Articles

MURC/CAVIN-4 facilitates store-operated calcium entry in neonatal cardiomyocytes

Intact store-operated calcium entry (SOCE) mechanisms ensure the maintenance of Ca2+ homeostasis in cardiomyocytes while their dysregulation promotes the development of cardiomyopathies. To better understand this calcium handling process in cardiomyocytes, we sought to identify unknown protein partners of stromal interaction molecule 1 (STIM1), a main regulatory protein of SOCE. We identified the muscle-related coiled-coil protein (MURC), also known as Cavin-4, as a candidate and showed that MURC interacts with STIM1 in cardiomyocytes. This interaction occurs via the HR1 and ERM domains of MURC and STIM1, respectively. Our results also demonstrated that the overexpression of MURC in neonatal rat ventricular myocytes (NRVM) is sufficient to potentiate SOCE and that its HR1 domain is required to mediate this effect. Interestingly, the R140W-MURC mutant, a missense variant of the HR1 domain associated with human dilated cardiomyopathy, exacerbates the SOCE increase in NRVM. Although the endogenous expression of STIM1 and Ca2+ channel Orai1 is not modulated under these conditions, we showed that MURC increases the interaction between these proteins under resting conditions. Our study provides novel evidence that MURC regulates SOCE by interacting with STIM1 in cardiomyocytes. In addition, we identified a first potential mechanism by which the R140W mutation of MURC may contribute to calcium mishandling and the development of cardiomyopathies.

Molecular basis of high glucose-mediated cardiac calcium mishandling

Epidemiological and preclinical studies have pointed out a correlation between hyperglycemia and increasing risk of heart failure and cardiac death. In cardiomyocytes, hyperglycemia has been shown to alter Ca2 + signalling via CaMKII. Yet, the underlying mechanisms are still unclear. To determine the molecular basis of high glucose-mediated Ca2 + mishandling. Ventricular cardiomyocytes were isolated from control and Epac2-KO mice. Parallel experiments were repeated in human cardiomyocytes differentiated from induced pluripotent stem cells (h-iPSC-CM). Ca2 + signaling was studied in cells loaded with a fluorescent Ca2 + dye and treated with high glucose (HG) ± ESI-05 (Epac2 inhibitor). We assessed ryanodine receptor (RyR2) phosphorylation state by western blot and measured single RyR2 channel activity incorporated into bilayers. HG-mediated SR Ca2 + leak and pro-arrhythmic events were prevented with Epac2 blocker (ESI-05) in normal mice cells and with Epac2 deletion (Epac2-KO) (Ca2 + sparks frequency N /100 μm/s: 0.23 ± 0.15 for HG + ESI-05, 0.09 ± 0.04 for Epac2-KO + HG vs. 0.16 ± 0.06 for normal glucose, P = N.S.). HG treatment increased RyR2 phosphorylation at the CaMKII site (S2814, ∼ 29% increase, P < 0.05) leading to a dramatic increase in RyR2 open probability (P0 = 0.76 ± 0.07 in HG vs. 0.22 ± 0.07 in NG, P < 0.01) fully prevented with ESI-05 (P0 = 0.15 ± 0.03, P = N.S.). Similarly, in h-iPSC-CM, chronic HG (1 week) increased the percentage of cells presenting pro-arrhythmic events (85% of cells). Electrically evoked [Ca2 + ]i transients were decreased in h-IPSC-CM (F/F0 = 2.10 ± 0.07 vs. 2.94 ± 0.17, P < 0.01) and associated to higher RyR-S2814 phosphorylation. Those effects were blunted by ESI-05. Our data suggests that HG alters Ca2 + signalling via Epac2–mediated pro-arrhythmic events due a CaMKII-dependent increase of RyR2 activity. Overtime, this newly described mechanism lowers systolic Ca2 + release as seen in diabetic-associated cardiomyopathy.

Live-Cell FRET Biosensors for High-Throughput Screening Targeting the SERCA2a/Plb Complex

We have engineered cell-based high-throughput screening (HTS) methods using time-resolved fluorescence energy transfer (TR-FRET) sensitive to binding and structural dynamics of the SERCA2a/PLB complex. The membrane protein complex between the sarcoplasmic reticulum Ca-ATPase 2a (SERCA2a) and its regulator phospholamban (PLB) is a validated therapeutic target for reversing contractile dysfunction caused by aberrant calcium handling in the heart. Unfortunately, efforts to discover compounds with specificity for this complex have yet to yield an efficacious drug. GFP-SERCA2a (donor) was co-expressed in the endoplasmic reticulum of HEK293 cells with and without RFP-PLB (acceptor), and FRET was measured in a unique fluorescence lifetime microplate reader (FLT-PR), which increases the throughput of high-precision FLT measurements by several orders of magnitude. A triplicate screen against a small-molecule library containing 1500 compounds (LOPAC = Library of Pharmaceutically Active Compounds) identified ten hit compounds that reproducibly changed FRET by >3 SD and alter SERCA's ATPase activity. One compound increases SERCA2a Ca affinity and activity at physiological calcium levels in cardiac SR, but not in skeletal SR, suggesting that the compound is acting specifically on the SERCA2a/PLB complex (Stroik et al., Nature Scientific Reports 8:12560). These properties characterize a drug that could reverse calcium mishandling, as required for treatment of heart disease. We will present recent developments in the development and applications of live-cell FRET biosensors. This work was supported by NIH grants (GM27906, HL129814, AR07612, and DA037622).

TFAM overexpression reduces pathological cardiac remodeling

Heart failure (HF) is a functional lack of myocardial performance due to a loss of molecular control over increases in calcium and ROS, resulting in proteolytic degradative advances and cardiac remodeling. Mitochondria are the molecular powerhouse of cells, shifting the sphere of cardiomyocyte stability and performance. Functional mitochondria rely on the molecular abilities of safety factors such as TFAM to maintain physiological parameters. Mitochondrial transcription factor A (TFAM) creates a mitochondrial nucleoid structure around mtDNA, protecting it from mutation, inhibiting NFAT (ROS activator/hypertrophic stimulator), and transcriptionally activates Serca2a to decrease calcium mishandling. Calpain1 and MMP9 are proteolytic degratory factors that play a major role in cardiomyocyte decline in HF. Current literature depicts major decreases in TFAM as HF progresses. We aim to assess TFAM function against Calpain1 and MMP9 proteolytic activity and its role in cardiac remodeling. To this date, no publication has surfaced describing the effects of aortic banding (AB) as a surgical HF model in TFAM-TG mice. HF models were created via AB in TFAM transgenic (TFAM-TG) and C57BLJ-6 (WT) mice. Eight weeks post AB, functional analysis revealed a successful banding procedure, resulting in cardiac hypertrophy as observed via echocardiography. Pulse wave and color doppler show increased aortic flow rates as well as turbulent flow at the banding site. Preliminary results of cardiac tissue immuno-histochemistry of HF-control mice show decreased TFAM and compensatory increases in Serca2a fluorescent expression, along with increased Calpain1 and MMP9 expression. Protein, RNA, and IHC analysis will further assess TFAM-TG results post-banding. Echocardiography shows more cardiac stability and functionality in HF-induced TFAM-TG mice than the control counterpart. These findings complement our published in vitro results. Overall, this suggests that TFAM has molecular therapeutic potential to reduce protease expression.

P5700Pro-resolving mediators prevent myocarditis-induced calcium mishandling and cardiac dysfunction: involvement of nrf2

P5700Pro-resolving mediators prevent myocarditis-induced calcium mishandling and cardiac dysfunction: involvement of nrf2

P929Soluble klotho, an antiaging factor, prevents cardiomyocyte calcium mishandling and arrhythmia in an experimental model of chronic kidney disease

P929Soluble klotho, an antiaging factor, prevents cardiomyocyte calcium mishandling and arrhythmia in an experimental model of chronic kidney disease

The effect of myotonic dystrophy on intracellular calcium handling and mitochondrial function.

Myotonic dystrophy (DM) is a multisystemic genetic disorder that leads to skeletal muscle, cardiac and neurological impairment. DM is characterized by multiple CUG repeats along the DMPK gene, with the severity of the disease increasing with the number of repeats. DM patients exhibit extended muscle contractions due to misregulated ion channels. As a result, normal ion balance, notably calcium, is disrupted leading to cellular damage and eventual death. While research has been aimed at identifying how the CUG repeats regulate gene expression, little is known regarding the effects of DM on organelle function. This study works to understand how DM affects calcium handling between the mitochondria and smooth endoplasmic reticulum to cause extended muscle contractions. An in vitro model of DM will be created by transfecting C2C12 cells with a plasmid containing 950 CUG repeats. This will allow for testing of overall calcium distribution and organelle functions in the control versus diseased state. We will run a mixture of fluorescent microscopy experiments to evaluate calcium distribution and organelle structure, as well as biochemical and functional assays to assess the difference in organelle function between these two groups. I hypothesize that DM cells will exhibit a misregulation of calcium handling leading to calcium overload in the mitochondria, thereby decreasing mitochondrial function. Results should indicate calcium mishandling and inhibited ATP production as shown by the results of the various assays. Therefore, if results should hold to be true, DM will indeed induce calcium mishandling through misregulation of calcium handling protein channels and a decrease in ATP synthesis due to mitochondrial dysfunction. Currently, there are limited DM treatments available and the identification of a new possible cell target may lead to the development of new pharmacological agents to combat the disease.Support or Funding InformationThe project will be supported and funded by the project advisor, Dr. Katrina Yamazaki.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Discovery of SERCA2a/PLB Activators and Inhibitors by Structure-Based High-throughput Screening using Live Cell FRET Biosensors

We have developed a cell-based high-throughput screening (HTS) method using time-resolved fluorescence energy transfer (TR-FRET) sensitive to binding and structural dynamics of the SERCA2a/PLB complex. Overwhelming evidence establishes the membrane protein complex between the sarcoplasmic reticulum Ca-ATPase 2a (SERCA2a) and its regulator phospholamban (PLB) as a therapeutic target for reversing cardiac contractile dysfunction caused by aberrant calcium handling. Unfortunately, efforts to discover compounds with specificity for this complex have yet to yield an efficacious drug. GFP-SERCA2a (donor) was co-expressed in the endoplasmic reticulum of HEK293 cells with and without RFP-PLB (acceptor), and FRET was measured in a fluorescence lifetime microplate reader. We carried out a triplicate screen against a small chemical library (LOPAC) and identified 21 compounds that reproducibly changed FRET by >3 SD. Dose-response of FRET and ATPase activity readouts reveal that ten hits reproducibly alter SERCA2a/PLB structure and function. One compound increases SERCA2a Ca affinity and activity (EC50 = 23 µM) at physiological calcium levels in cardiac SR, but not in skeletal SR, suggesting that the compound is acting specifically on the SERCA2a/PLB complex. These properties characterize a drug that could reverse calcium mishandling. The excellent Z’ factor and correlation between structural and functional assays validate this method to discover SERCA2a-PLB complex effectors for large-scale HTS campaigns. This work was supported by NIH grants (GM27906, HL129814, AR07612, and DA037622).

PINK1-mediated phosphorylation of LETM1 regulates mitochondrial calcium transport and protects neurons against mitochondrial stress

Mutations in PTEN-induced kinase 1 (PINK1) result in a recessive familial form of Parkinson’s disease (PD). PINK1 loss is associated with mitochondrial Ca2+ mishandling, mitochondrial dysfunction, as well as increased neuronal vulnerability. Here we demonstrate that PINK1 directly interacts with and phosphorylates LETM1 at Thr192 in vitro. Phosphorylated LETM1 or the phospho-mimetic LETM1-T192E increase calcium release in artificial liposomes and facilitates calcium transport in intact mitochondria. Expression of LETM1-T192E but not LETM1-wild type (WT) rescues mitochondrial calcium mishandling in PINK1-deficient neurons. Expression of both LETM1-WT and LETM1-T192E protects neurons against MPP+–MPTP-induced neuronal death in PINK1 WT neurons, whereas only LETM1-T192E protects neurons under conditions of PINK1 loss. Our findings delineate a mechanism by which PINK1 regulates mitochondrial Ca2+ level through LETM1 and suggest a model by which PINK1 loss leads to deficient phosphorylation of LETM1 and impaired mitochondrial Ca2+ transport..

Abstract 6: Restoration of Impaired Diastolic Function in Hypertrophic Cardiomyopathy Induced Pluripotent Stem Cell-derived Cardiomyocytes by Re-balancing the Calcium Homeostasis

Background: Diastolic dysfunction is commonly seen in hypertrophic cardiomyopathy (HCM). However, the cellular mechanism is not fully understood, and no effective treatment so far has been developed. We hypothesize here that HCM patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) can recapitulate the cellular mechanism, and provide us a platform for mechanistic study and for drug screening of diastolic dysfunctions in HCM. Methods and Results: We generated beating iPSC-CMs from healthy individuals and HCM patients carrying familial mutations (MYH7 R663H (n=2 lines) and MYBPC3 R943ter (n=2 lines)). Sarcomere shortening measurement in patterned iPSC-CMs with live cell confocal imaging showed significantly prolonged diastolic phase and slower relaxation velocity in HCM iPSC-CMs compared to WT cells. To elucidate the cellular mechanism, Fura-2 AM ratiometric calcium imaging showed marked elevation of resting calcium level and increased abnormal calcium handlings in HCM iPSC-CMs, which were exaggerated by β-adrenergic activation with isoproterenol. By applying calcium transient and contractile force simultaneous recording, we defined a “risk index of diastolic dysfunction” (measured as transient-contraction gain factor), which was significantly increased in HCM iPSC-CMs. Thus, both elevated basal calcium level and increased calcium sensitivity of myofilament contribute to the abnormal diastolic function in HCM iPSC-CMs. Gene expression profiling of HCM and WT iPSC-CMs indicated that increased calcium channels may underlie the increased basal calcium concentration in HCM cells. Indeed, partially blocking the calcium influx by calcium blockers reset the basal calcium level, attenuated calcium mishandling, and restored the diastolic function in HCM iPSC-CMs. Moreover, re-balancing calcium homeostasis significantly improved long-term survival rate of HCM iPSC-CMs at both basal level and under β-adrenergic stress. Conclusion: The iPSC-CM models carrying patient-specific HCM mutations recapitulated diastolic dysfunction on single cell level. Future studies using these platform may reveal additional novel cellular mechanisms and therapeutic targets of diastolic dysfunction in HCM disease.

INHIBITING THE MITOCHONDRIAL CALCIUM UNIPORTER INCREASE SYSTOLIC CALCIUM AND INDUCES UNSOLICITED CALCIUM WAVES WITHOUT AFFECTING SARCOPLASMIC RETICULUM CALCIUM CONTENT

Background: Ventricular arrhythmias are prevalent in cardiac diseases, including heart failure and myocardial ischemia where calcium mishandling appears as an underlying mechanism. Recently, it has been suggested that mitochondria importantly contributes as cytosolic Ca2+ buffer in a beat-to-beat

Genotype-Dependent and -Independent Calcium Signaling Dysregulation in Human Hypertrophic Cardiomyopathy.

Aberrant calcium signaling may contribute to arrhythmias and adverse remodeling in hypertrophic cardiomyopathy (HCM). Mutations in sarcomere genes may distinctly alter calcium handling pathways. We analyzed gene expression, protein levels, and functional assays for calcium regulatory pathways in human HCM surgical samples with (n=25) and without (n=10) sarcomere mutations compared with control hearts (n=8). Gene expression and protein levels for calsequestrin, L-type calcium channel, sodium-calcium exchanger, phospholamban, calcineurin, and calcium/calmodulin-dependent protein kinase type II (CaMKII) were similar in HCM samples compared with controls. CaMKII protein abundance was increased only in sarcomere-mutation HCM (P<0.001). The CaMKII target pT17-phospholamban was 5.5-fold increased only in sarcomere-mutation HCM (P=0.01), as was autophosphorylated CaMKII (P<0.01), suggestive of constitutive activation. Calcineurin (PPP3CB) mRNA was not increased, nor was RCAN1 mRNA level, indicating a lack of calcineurin activation. Furthermore, myocyte enhancer factor 2 and nuclear factor of activated T cell transcription factor activity was not increased in HCM, suggesting that calcineurin pathway activation is not an upstream cause of increased CAMKII protein abundance or activation. SERCA2A mRNA transcript levels were reduced in HCM regardless of genotype, as was sarcoplasmic endoplasmic reticular calcium ATPase 2/phospholamban protein ratio (45% reduced; P=0.03). 45Ca sarcoplasmic endoplasmic reticular calcium ATPaseuptake assay showed reduced uptake velocity in HCM regardless of genotype (P=0.01). The cardiac ryanodine receptor was not altered in transcript, protein, or phosphorylated (pS2808, pS2814) protein abundance, and [3H]ryanodine binding was not different in HCM, consistent with no major modification of the ryanodine receptor. Human HCM demonstrates calcium mishandling through both genotype-specific and common pathways. Posttranslational activation of the CaMKII pathway is specific to sarcomere mutation-positive HCM, whereas sarcoplasmic endoplasmic reticular calcium ATPase 2 abundance and sarcoplasmic reticulum Ca uptake are depressed in both sarcomere mutation-positive and -negative HCM.

Loss of myocardial retinoic acid receptor α induces diastolic dysfunction by promoting intracellular oxidative stress and calcium mishandling in adult mice

Retinoic acid receptor (RAR) has been implicated in pathological stimuli-induced cardiac remodeling. To determine whether the impairment of RARα signaling directly contributes to the development of heart dysfunction and the involved mechanisms, tamoxifen-induced myocardial specific RARα deletion (RARαKO) mice were utilized. Echocardiographic and cardiac catheterization studies showed significant diastolic dysfunction after 16wks of gene deletion. However, no significant differences were observed in left ventricular ejection fraction (LVEF), between RARαKO and wild type (WT) control mice. DHE staining showed increased intracellular reactive oxygen species (ROS) generation in the hearts of RARαKO mice. Significantly increased NOX2 (NADPH oxidase 2) and NOX4 levels and decreased SOD1 and SOD2 levels were observed in RARαKO mouse hearts, which were rescued by overexpression of RARα in cardiomyocytes. Decreased SERCA2a expression and phosphorylation of phospholamban (PLB), along with decreased phosphorylation of Akt and Ca2+/calmodulin-dependent protein kinase II δ (CaMKII δ) was observed in RARαKO mouse hearts. Ca2+ reuptake and cardiomyocyte relaxation were delayed by RARα deletion. Overexpression of RARα or inhibition of ROS generation or NOX activation prevented RARα deletion-induced decrease in SERCA2a expression/activation and delayed Ca2+ reuptake. Moreover, the gene and protein expression of RARα was significantly decreased in aged or metabolic stressed mouse hearts. RARα deletion accelerated the development of diastolic dysfunction in streptozotocin (STZ)-induced type 1 diabetic mice or in high fat diet fed mice. In conclusion, myocardial RARα deletion promoted diastolic dysfunction, with a relative preserved LVEF. Increased oxidative stress have an important role in the decreased expression/activation of SERCA2a and Ca2+ mishandling in RARαKO mice, which are major contributing factors in the development of diastolic dysfunction. These data suggest that impairment of cardiac RARα signaling may be a novel mechanism that is directly linked to pathological stimuli-induced diastolic dysfunction.

Mitochondrial pathways to cardiac recovery: TFAM.

Mitochondrial dysfunction underlines a multitude of pathologies; however, studies are scarce that rescue the mitochondria for cellular resuscitation. Exploration into the protective role of mitochondrial transcription factor A (TFAM) and its mitochondrial functions respective to cardiomyocyte death are in need of further investigation. TFAM is a gene regulator that acts to mitigate calcium mishandling and ROS production by wrapping around mitochondrial DNA (mtDNA) complexes. TFAM's regulatory functions over serca2a, NFAT, and Lon protease contribute to cardiomyocyte stability. Calcium- and ROS-dependent proteases, calpains, and matrix metalloproteinases (MMPs) are abundantly found upregulated in the failing heart. TFAM's regulatory role over ROS production and calcium mishandling leads to further investigation into the cardioprotective role of exogenous TFAM. In an effort to restabilize physiological and contractile activity of cardiomyocytes in HF models, we propose that TFAM-packed exosomes (TFAM-PE) will act therapeutically by mitigating mitochondrial dysfunction. Notably, this is the first mention of exosomal delivery of transcription factors in the literature. Here we elucidate the role of TFAM in mitochondrial rescue and focus on its therapeutic potential.

Rheostatic Regulation of the SERCA/Phospholamban Membrane Protein Complex Using Non-Coding RNA and Single-Stranded DNA oligonucleotides.

The membrane protein complex between sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and phospholamban (PLN) is a prime therapeutic target for reversing cardiac contractile dysfunctions caused by calcium mishandling. So far, however, efforts to develop drugs specific for this protein complex have failed. Here, we show that non-coding RNAs and single-stranded DNAs (ssDNAs) interact with and regulate the function of the SERCA/PLN complex in a tunable manner. Both in HEK cells expressing the SERCA/PLN complex, as well as in cardiac sarcoplasmic reticulum preparations, these short oligonucleotides bind and reverse PLN’s inhibitory effects on SERCA, increasing the ATPase’s apparent Ca2+ affinity. Solid-state NMR experiments revealed that ssDNA interacts with PLN specifically, shifting the conformational equilibrium of the SERCA/PLN complex from an inhibitory to a non-inhibitory state. Importantly, we achieved rheostatic control of SERCA function by modulating the length of ssDNAs. Since restoration of Ca2+ flux to physiological levels represents a viable therapeutic avenue for cardiomyopathies, our results suggest that oligonucleotide-based drugs could be used to fine-tune SERCA function to counterbalance the extent of the pathological insults.

Abstract 361: Loss of Cardiac Retinoic Acid Receptor Alpha Promotes Diastolic Heart Failure With Preserved Ejection Fraction

Objectives: We have previously demonstrated that the expression/activation of retinoic acid receptor (RAR) is inhibited in diabetic hearts, and that activation of RARα prevents diabetes-induced diastolic heart failure, suggesting that impairment of RARα signaling may be a critical mechanism in heart failure. Methods and Results: Cardiac RARα gene deletion (KO) was achieved by tamoxifen injection at 6-weeks old RARαfl/fl α-MHC-MerCreMer mice, RARαfl/fl mice were used as control (WT). Heart function was monitored by echocardiograph for 64 wks. Mice were sacrificed at 20 or 64 wks post gene deletion, respectively. A significant decrease in E/A ratio and TDI E’ and increase in IVRT (isovolumic relaxation time) and DT (deceleration time) suggested diastolic dysfunction after 16 wks of gene deletion in KO mice, which was confirmed by cardiac catheterization (decreased dP/dtmin and increased tau). Concentric hypertrophy developed in KO mice after 56 wks of gene deletion, as confirmed by increased thickness of left ventricular wall and interventricular septum and elevated heart weight/tibia length ratio. However, no significant difference was observed in LVEF (LV ejection fraction), FS (fraction shortening) and dP/dtmax between KO and WT mice. Significantly increased gene expression of NOX2 (NADPH oxidase 2) and NOX4, decreased SOD1 and SOD2 levels and increased intracellular reactive oxygen species (ROS) were observed in KO mouse hearts, along with a significantly decreased protein expression of SERCA2a and CaMKIIδ, decreased phosphorylation of PLB, Akt and CaMKIIδ. Overexpression of RARα in cardiomyocytes rescued RARα deletion-induced changes in SERCA2a, PLB, Akt and CaMKIIδ. Deletion of RARα in cardiomyocytes impaired intracellular calcium reuptake into SR and cardiomyocyte relaxation. Inhibition of ROS by N-acetyl cysteine abolished RARα deletion-induced calcium mishandling and cardiomyocyte relaxation. Conclusion: Cardiac specific deletion of RARα induces diastolic heart failure with preserved ejection fraction (HFpEF), by promoting intracellular ROS and disrupting SERCA2a-mediated calcium reuptake and cardiomyocyte relaxation. Our study suggests that deficiency in RARα signaling is a novel mechanism leading to HFpEF.

Impaired oxidative metabolism and calcium mishandling underlie cardiac dysfunction in a rat model of post-acute isoproterenol-induced cardiomyopathy.

Stress-induced cardiomyopathy, triggered by acute catecholamine discharge, is a syndrome characterized by transient, apical ballooning linked to acute heart failure and ventricular arrhythmias. Rats receiving an acute isoproterenol (ISO) overdose (OV) suffer cardiac apex ischemia-reperfusion damage and arrhythmia, and then undergo cardiac remodeling and dysfunction. Nevertheless, the subcellular mechanisms underlying cardiac dysfunction after acute damage subsides are not thoroughly understood. To address this question, Wistar rats received a single ISO injection (67 mg/kg). We found in vivo moderate systolic and diastolic dysfunction at 2 wk post-ISO-OV; however, systolic dysfunction recovered after 4 wk, while diastolic dysfunction worsened. At 2 wk post-ISO-OV, cardiac function was assessed ex vivo, while mitochondrial oxidative metabolism and stress were assessed in vitro, and Ca(2+) handling in ventricular myocytes. These were complemented with sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), phospholamban (PLB), and RyR2 expression studies. Ex vivo, basal mechanical performance index (MPI) and oxygen consumption rate (MVO2) were unchanged. Nevertheless, upon increase of metabolic demand, by β-adrenergic stimulation (1-100 nM ISO), the MPI versus MVO2 relation decreased and shifted to the right, suggesting MPI and mitochondrial energy production uncoupling. Mitochondria showed decreased oxidative metabolism, membrane fragility, and enhanced oxidative stress. Myocytes presented systolic and diastolic Ca(2+) mishandling, and blunted response to ISO (100 nM), and all these without apparent changes in SERCA, PLB, or RyR2 expression. We suggest that post-ISO-OV mitochondrial dysfunction may underlie decreased cardiac contractility, mainly by depletion of ATP needed for myofilaments and Ca(2+) transport by SERCA, while exacerbated oxidative stress may enhance diastolic RyR2 activity.

Reciprocal disruption of neuronal signaling and Aβ production mediated by extrasynaptic NMDA receptors: a downward spiral

It is becoming increasingly clear that aberrant neuronal activity can be the cause and the result of amyloid beta production. Synaptic activation facilitates non-amyloidogenic processing of amyloid precursor protein (APP) and cell survival, primarily through synaptic NMDA receptors (NMDARs) and perhaps specifically those containing GluN2A-subunits. In contrast, extrasynaptic and GluN2B-containing NMDARs promote beta-secretase cleavage of APP into amyloid-beta (Aβ). The opposing nature of these NMDAR populations is reflected in their control over cell survival and death pathways. Subtle changes in glutamate homeostasis may shift the balance between these pathways and could play a role in Alzheimer's disease (AD). Indeed, Aβ production, regional loss of brain connectivity and neurodegeneration correlate with neuronal activity in AD patients. From another perspective, Aβ oligomers (Aβo) alter neuronal signaling through several mechanisms involving NMDARs and intracellular calcium mishandling. While Aβo affect multiple receptors, GluN2B-NMDARs have emerged as primary mediators of altered synaptic plasticity and neurotoxicity. Memantine and its successor, NitroMemantine, are efficient at blocking or reversing the deleterious actions of Aβo largely due to their selectivity for extrasynaptic NMDARs. Recently, Aβo were shown to trigger astrocytic release of glutamate to the extrasynaptic space where it activates NMDARs to promote further Aβ production and synaptic depression. Combined with the reciprocal regulation between neuronal activity and Aβ production, extrasynaptic glutamate release adds to a maladaptive model and ultimately results in synaptotoxicity and neurodegeneration of AD. Extrasynaptic NMDAR antagonists remain as a promising therapeutic avenue by interfering with this cascade.

X-ray fluorescence imaging reveals subcellular biometal disturbances in a childhood neurodegenerative disorder.

Biometals such as zinc, iron, copper and calcium play key roles in diverse physiological processes in the brain, but can be toxic in excess. A hallmark of neurodegeneration is a failure of homeostatic mechanisms controlling the concentration and distribution of these elements, resulting in overload, deficiency or mislocalization. A major roadblock to understanding the impact of altered biometal homeostasis in neurodegenerative disease is the lack of rapid, specific and sensitive techniques capable of providing quantitative subcellular information on biometal homeostasis in situ. Recent advances in X-ray fluorescence detectors have provided an opportunity to rapidly measure biometal content at subcellular resolution in cell populations using X-ray Fluorescence Microscopy (XFM). We applied this approach to investigate subcellular biometal homeostasis in a cerebellar cell line isolated from a natural mouse model of a childhood neurodegenerative disorder, the CLN6 form of neuronal ceroid lipofuscinosis, commonly known as Batten disease. Despite no global changes to whole cell concentrations of zinc or calcium, XFM revealed significant subcellular mislocalization of these important biological second messengers in cerebellar Cln6nclf (CbCln6nclf ) cells. XFM revealed that nuclear-to-cytoplasmic trafficking of zinc was severely perturbed in diseased cells and the subcellular distribution of calcium was drastically altered in CbCln6nclf cells. Subtle differences in the zinc K-edge X-ray Absorption Near Edge Structure (XANES) spectra of control and CbCln6nclf cells suggested that impaired zinc homeostasis may be associated with an altered ligand set in CbCln6nclf cells. Importantly, a zinc-complex, ZnII(atsm), restored the nuclear-to-cytoplasmic zinc ratios in CbCln6nclf cells via nuclear zinc delivery, and restored the relationship between subcellular zinc and calcium levels to that observed in healthy control cells. ZnII(atsm) treatment also resulted in a reduction in the number of calcium-rich puncta observed in CbCln6nclf cells. This study highlights the complementarities of bulk and single cell analysis of metal content for understanding disease states. We demonstrate the utility and broad applicability of XFM for subcellular analysis of perturbed biometal metabolism and mechanism of action studies for novel therapeutics to target neurodegeneration.

Probing Cardiac Membrane Proteins with Fluorescence Resonance Energy Transfer

We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to study the structural basis of regulation of sarcoplasmic reticulum Ca2+ ATPase (SERCA) by a single-pass transmembrane protein, phospholamban (PLB). The most prominent feature of heart failure is calcium mishandling, which is largely due to impaired activity of sarcoplasmic reticulum calcium ATPase (SERCA), which actively transports Ca2+ from cytosol into the SR to relax the muscle. Unphosphorylated phospholamban inhibits SERCA by decreasing its apparent Ca2+ affinity. This inhibition can be relieved by either micromolar Ca2+ or by phosphorylation of PLB. The structural basis for relief of inhibition remains controversial. Cross-linking studies support the canonical dissociation model. However, recent EPR, NMR and FRET studies support the subunit model, in which subtle structural rearrangements are required to relieve the inhibition. The goal of this study is to elucidate the structural rearrangements during relief of inhibition. Previous EPR and NMR studies found that the cytoplasmic domain of PLB exists in a dynamic equilibrium between T and R states. We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to probe the complex directly by measuring the distances between SERCA (C674) and a series of positions on PLB in co-reconstituted vesicles. We found that the distances between SERCA and the cytoplasmic domain of PLB are best fitted with two Gaussian distance distributions, suggesting that the cytoplasmic domain of bound PLB exists in two states. Results will be reported on the effects of perturbations in this system which are known to relieve inhibition, including PLB phosphorylation, PLB mutation, and calcium binding to SERCA. We conclude that the function of the SERCA-PLB complex depends primarily on the structural equilibrium between two principal structural states of the complex.