Nitric Oxide Modulates Ca2+ Leak and Arrhythmias via S-Nitrosylation of CaMKII.

BACKGROUND: Nitric oxide (NO) has been identified as a signaling molecule generated during ß-adrenergic receptor stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of CaMKIIδ (Ca2+/calmodulin kinase II delta) is emerging. NO donors are routinely used clinically for their cardioprotective effects on the heart, but it is unknown how NO donors modulate the proarrhythmic CaMKII to alter cardiac arrhythmia incidence. We test the role of S-nitrosylation of CaMKIIδ at the Cysteine-273 inhibitory site and cysteine-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during ß-adrenergic receptor stimulation. METHODS: We measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at cysteine-273 or cysteine-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 µM), sodium nitroprusside (200 µM), and ß-adrenergic agonist isoproterenol (100 nmol/L). RESULTS: Both WT and CaMKIIδ-KO cardiomyocytes responded to isoproterenol with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from isoproterenol-induced Ca2+ sparks and waves was mimicked by GSNO pretreatment in WT cardiomyocytes but lost in CaMKIIδ-C273S cardiomyocytes. When GSNO was applied after isoproterenol, this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pretreatment limited isoproterenol-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after isoproterenol sustained or exacerbated arrhythmic events. CONCLUSIONS: We conclude that prior S-nitrosylation of CaMKIIδ at cysteine-273 can limit subsequent ß-adrenergic receptor-induced arrhythmias, but that S-nitrosylation at cysteine-290 might worsen or sustain ß-adrenergic receptor-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

Whole-heart multiparametric optical imaging reveals sex-dependent heterogeneity in cAMP signaling and repolarization kinetics.

Cyclic adenosine 3',5'-monophosphate (cAMP) is a key second messenger in cardiomyocytes responsible for transducing autonomic signals into downstream electrophysiological responses. Previous studies have shown intracellular heterogeneity and compartmentalization of cAMP signaling. However, whether cAMP signaling occurs heterogeneously throughout the intact heart and how this drives sex-dependent functional responses are unknown. Here, we developed and validated a novel cardiac-specific fluorescence resonance energy transfer-based cAMP reporter mouse and a combined voltage-cAMP whole-heart imaging system. We showed that in male hearts, cAMP was uniformly activated in response to pharmacological ß-adrenergic stimulation. In contrast, female hearts showed that cAMP levels decayed faster in apical versus basal regions, which was associated with nonuniform action potential changes and notable changes in the direction of repolarization. Apical phosphodiesterase (PDE) activity was higher in female versus male hearts, and PDE inhibition prevented repolarization changes in female hearts. Thus, our imaging approach revealed sex-dependent regional breakdown of cAMP and associated electrophysiological differences.

AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and RYR.

BACKGROUND: The sarcoplasmic reticulum (SR) Ca2+-ATPase 2 (SERCA2) mediates Ca2+ reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca2+ release from SR and triggers contraction. Ca2+/CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. METHODS: A role for AKAP18δ (A-kinase anchoring protein 18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adenoassociated virus injection, structural modeling, surface plasmon resonance, and alpha screen technology. RESULTS: Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via 2 distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent Ca2+ threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca2+ reuptake by SERCA2 and Ca2+ release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca2+-frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR. CONCLUSIONS: AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge, this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP (A-kinase anchoring protein) reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.

CaMKII Serine 280 O-GlcNAcylation Links Diabetic Hyperglycemia to Proarrhythmia.

[Figure: see text].

CaMKII and PKA-dependent phosphorylation co-regulate nuclear localization of HDAC4 in adult cardiomyocytes.

Nuclear histone deacetylase 4 (HDAC4) represses MEF2-mediated transcription, implicated in the development of heart failure. CaMKII-dependent phosphorylation drives nucleus-to-cytoplasm HDAC4 shuttling, but protein kinase A (PKA) is also linked to HDAC4 translocation. However, the interplay of CaMKII and PKA in regulating adult cardiomyocyte HDAC4 translocation is unclear. Here we sought to determine the interplay of PKA- and CaMKII-dependent HDAC4 phosphorylation and translocation in adult mouse, rabbit and human ventricular myocytes. Confocal imaging and protein analyses revealed that inhibition of CaMKII-but not PKA, PKC or PKD-raised nucleo-to-cytoplasmic HDAC4 fluorescence ratio (FNuc/FCyto) by ~ 50%, indicating baseline CaMKII activity that limits HDAC4 nuclear localization. Further CaMKII activation (via increased extracellular [Ca2+], high pacing frequencies, angiotensin II or overexpression of CaM or CaMKIIδC) led to significant HDAC4 nuclear export. In contrast, PKA activation by isoproterenol or forskolin drove HDAC4 into the nucleus (raising FNuc/FCyto by > 60%). These PKA-mediated effects were abolished in cells pretreated with PKA inhibitors and in cells expressing mutant HDAC4 in S265/266A mutant. In physiological conditions where both kinases are active, PKA-dependent nuclear accumulation of HDAC4 was predominant in the very early response, while CaMKII-dependent HDAC4 export prevailed upon prolonged stimuli. This orchestrated co-regulation was shifted in failing cardiomyocytes, where CaMKII-dependent effects predominated over PKA-dependent response. Importantly, human cardiomyocytes showed similar CaMKII- and PKA-dependent HDAC4 shifts. Collectively, CaMKII limits nuclear localization of HDAC4, while PKA favors HDAC4 nuclear retention and S265/266 is essential for PKA-mediated regulation. These pathways thus compete in HDAC4 nuclear localization and transcriptional regulation in cardiac signaling.

Intracellular ß1-Adrenergic Receptors and Organic Cation Transporter 3 Mediate Phospholamban Phosphorylation to Enhance Cardiac Contractility.

RATIONALE: ß1ARs (ß1-adrenoceptors) exist at intracellular membranes and OCT3 (organic cation transporter 3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular ß1AR in cardiac contractility remains to be elucidated. OBJECTIVE: Test localization and function of intracellular ß1AR on cardiac contractility. METHODS AND RESULTS: Membrane fractionation, super-resolution imaging, proximity ligation, coimmunoprecipitation, and single-molecule pull-down demonstrated a pool of ß1ARs in mouse hearts that were associated with sarco/endoplasmic reticulum Ca2+-ATPase at the sarcoplasmic reticulum (SR). Local PKA (protein kinase A) activation was measured using a PKA biosensor targeted at either the plasma membrane (PM) or SR. Compared with wild-type, myocytes lacking OCT3 (OCT3-KO [OCT3 knockout]) responded identically to the membrane-permeant ßAR agonist isoproterenol in PKA activation at both PM and SR. The same was true at the PM for membrane-impermeant norepinephrine, but the SR response to norepinephrine was suppressed in OCT3-KO myocytes. This differential effect was recapitulated in phosphorylation of the SR-pump regulator phospholamban. Similarly, OCT3-KO selectively suppressed calcium transients and contraction responses to norepinephrine but not isoproterenol. Furthermore, sotalol, a membrane-impermeant ßAR-blocker, suppressed isoproterenol-induced PKA activation at the PM but permitted PKA activation at the SR, phospholamban phosphorylation, and contractility. Moreover, pretreatment with sotalol in OCT3-KO myocytes prevented norepinephrine-induced PKA activation at both PM and the SR and contractility. CONCLUSIONS: Functional ß1ARs exists at the SR and is critical for PKA-mediated phosphorylation of phospholamban and cardiac contractility upon catecholamine stimulation. Activation of these intracellular ß1ARs requires catecholamine transport via OCT3.

Hyperglycemia regulates cardiac K+ channels via O-GlcNAc-CaMKII and NOX2-ROS-PKC pathways.

Chronic hyperglycemia and diabetes lead to impaired cardiac repolarization, K+ channel remodeling and increased arrhythmia risk. However, the exact signaling mechanism by which diabetic hyperglycemia regulates cardiac K+ channels remains elusive. Here, we show that acute hyperglycemia increases inward rectifier K+ current (IK1), but reduces the amplitude and inactivation recovery time of the transient outward K+ current (Ito) in mouse, rat, and rabbit myocytes. These changes were all critically dependent on intracellular O-GlcNAcylation. Additionally, IK1 amplitude and Ito recovery effects (but not Ito amplitude) were prevented by the Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitor autocamtide-2-related inhibitory peptide, CaMKIIδ-knockout, and O-GlcNAc-resistant CaMKIIδ-S280A knock-in. Ito reduction was prevented by inhibition of protein kinase C (PKC) and NADPH oxidase 2 (NOX2)-derived reactive oxygen species (ROS). In mouse models of chronic diabetes (streptozotocin, db/db, and high-fat diet), heart failure, and CaMKIIδ overexpression, both Ito and IK1 were reduced in line with the downregulated K+ channel expression. However, IK1 downregulation in diabetes was markedly attenuated in CaMKIIδ-S280A. We conclude that acute hyperglycemia enhances IK1 and Ito recovery via CaMKIIδ-S280 O-GlcNAcylation, but reduces Ito amplitude via a NOX2-ROS-PKC pathway. Moreover, chronic hyperglycemia during diabetes and CaMKII activation downregulate K+ channel expression and function, which may further increase arrhythmia susceptibility.

A 20/20 view of ANT function in mitochondrial biology and necrotic cell death.

The adenosine nucleotide translocase (ANT) family of proteins are inner mitochondrial membrane proteins involved in energy homeostasis and cell death. The primary function of ANT proteins is to exchange cytosolic ADP with matrix ATP, facilitating the export of newly synthesized ATP to the cell while providing new ADP substrate to the mitochondria. As such, the ANT proteins are central to maintaining energy homeostasis in all eukaryotic cells. Evidence also suggests that the ANTs constitute a pore-forming component of the mitochondrial permeability transition pore (MPTP), a structure that forms in the inner mitochondrial membrane that is thought to underlie regulated necrotic cell death. Additionally, emerging studies suggest that ANT proteins are also critical for mitochondrial uncoupling and for promoting mitophagy. Thus, the ANTs are multifunctional proteins that are poised to participate in several aspects of mitochondrial biology and the greater regulation of cell death, which will be discussed here.

Hyperglycemia Acutely Increases Cytosolic Reactive Oxygen Species via O-linked GlcNAcylation and CaMKII Activation in Mouse Ventricular Myocytes.

RATIONALE: Diabetes mellitus is a complex, multisystem disease, affecting large populations worldwide. Chronic CaMKII (Ca2+/calmodulin-dependent kinase II) activation may occur in diabetes mellitus and be arrhythmogenic. Diabetic hyperglycemia was shown to activate CaMKII by (1) O-linked attachment of N-acetylglucosamine (O-GlcNAc) at S280 leading to arrhythmia and (2) a reactive oxygen species (ROS)-mediated oxidation of CaMKII that can increase postinfarction mortality. OBJECTIVE: To test whether high extracellular glucose (Hi-Glu) promotes ventricular myocyte ROS generation and the role played by CaMKII. METHODS AND RESULTS: We tested how extracellular Hi-Glu influences ROS production in adult ventricular myocytes, using DCF (2',7'-dichlorodihydrofluorescein diacetate) and genetically targeted Grx-roGFP2 redox sensors. Hi-Glu (30 mmol/L) significantly increased the rate of ROS generation-an effect prevented in myocytes pretreated with CaMKII inhibitor KN-93 or from either global or cardiac-specific CaMKIIδ KO (knockout) mice. CaMKII KO or inhibition also prevented Hi-Glu-induced sarcoplasmic reticulum Ca2+ release events (Ca2+ sparks). Thus, CaMKII activation is required for Hi-Glu-induced ROS generation and sarcoplasmic reticulum Ca2+ leak in cardiomyocytes. To test the involvement of O-GlcNAc-CaMKII pathway, we inhibited GlcNAcylation removal by Thiamet G (ThmG), which mimicked the Hi-Glu-induced ROS production. Conversely, inhibition of GlcNAcylation (OSMI-1 [(αR)-α-[[(1,2-dihydro-2-oxo-6-quinolinyl)sulfonyl]amino]-N-(2-furanylmethyl)-2-methoxy-N-(2-thienylmethyl)-benzeneacetamide]) prevented ROS induction in response to either Hi-Glu or ThmG. Moreover, in a CRSPR-based knock-in mouse in which the functional GlcNAcylation site on CaMKIIδ was ablated (S280A), neither Hi-Glu nor ThmG induced myocyte ROS generation. So CaMKIIδ-S280 is required for the Hi-Glu-induced (and GlcNAc dependent) ROS production. To identify the ROS source(s), we used different inhibitors of NOX (NADPH oxidase) 2 (Gp91ds-tat peptide), NOX4 (GKT137831), mitochondrial ROS (MitoTempo), and NOS (NO synthase) pathway inhibitors (L-NAME, L-NIO, and L-NPA). Only NOX2 inhibition or KO prevented Hi-Glu/ThmG-induced ROS generation. CONCLUSIONS: Diabetic hyperglycemia induces acute cardiac myocyte ROS production by NOX2 that requires O-GlcNAcylation of CaMKIIδ at S280. This novel ROS induction may exacerbate pathological consequences of diabetic hyperglycemia.

Balance Between Rapid Delayed Rectifier K+ Current and Late Na+ Current on Ventricular Repolarization: An Effective Antiarrhythmic Target?

BACKGROUND: Rapid delayed rectifier K+ current (IKr) and late Na+ current (INaL) significantly shape the cardiac action potential (AP). Changes in their magnitudes can cause either long or short QT syndromes associated with malignant ventricular arrhythmias and sudden cardiac death. METHODS: Physiological self AP-clamp was used to measure INaL and IKr during the AP in rabbit and porcine ventricular cardiomyocytes to test our hypothesis that the balance between IKr and INaL affects repolarization stability in health and disease conditions. RESULTS: We found comparable amount of net charge carried by IKr and INaL during the physiological AP, suggesting that outward K+ current via IKr and inward Na+ current via INaL are in balance during physiological repolarization. Remarkably, IKr and INaL integrals in each control myocyte were highly correlated in both healthy rabbit and pig myocytes, despite high overall cell-to-cell variability. This close correlation was lost in heart failure myocytes from both species. Pretreatment with E-4031 to block IKr (mimicking long QT syndrome 2) or with sea anemone toxin II to impair Na+ channel inactivation (mimicking long QT syndrome 3) prolonged AP duration (APD); however, using GS-967 to inhibit INaL sufficiently restored APD to control in both cases. Importantly, INaL inhibition significantly reduced the beat-to-beat and short-term variabilities of APD. Moreover, INaL inhibition also restored APD and repolarization stability in heart failure. Conversely, pretreatment with GS-967 shortened APD (mimicking short QT syndrome), and E-4031 reverted APD shortening. Furthermore, the amplitude of AP alternans occurring at high pacing frequency was decreased by INaL inhibition, increased by IKr inhibition, and restored by combined INaL and IKr inhibitions. CONCLUSIONS: Our data demonstrate that IKr and INaL are counterbalancing currents during the physiological ventricular AP and their integrals covary in individual myocytes. Targeting these ionic currents to normalize their balance may have significant therapeutic potential in heart diseases with repolarization abnormalities. Visual Overview: A visual overview is available for this article.

Diabetic microcirculatory disturbances and pathologic erythropoiesis are provoked by deposition of amyloid-forming amylin in red blood cells and capillaries.

In the setting of type-2 diabetes, there are declines of structural stability and functionality of blood capillaries and red blood cells (RBCs), increasing the risk for microcirculatory disturbances. Correcting hyperglycemia is not entirely effective at reestablishing normal cellular metabolism and function. Therefore, identification of pathological changes occurring before the development of overt hyperglycemia may lead to novel therapeutic targets for reducing the risk of microvascular dysfunction. Here we determine whether RBC-capillary interactions are altered by prediabetic hypersecretion of amylin, an amyloid forming hormone co-synthesized with insulin, and is reversed by endothelial cell-secreted epoxyeicosatrienoic acids. In patients, we found amylin deposition in RBCs in association with type-2 diabetes, heart failure, cancer and stroke. Amylin-coated RBCs have altered shape and reduced functional (non-glycated) hemoglobin. Amylin-coated RBCs administered intravenously in control rats upregulated erythropoietin and renal arginase expression and activity. We also found that diabetic rats expressing amyloid-forming human amylin in the pancreas (the HIP rat model) have increased tissue levels of hypoxia-inducible transcription factors, compared to diabetic rats that express non-amyloid forming rat amylin (the UCD rat model). Upregulation of erythropoietin correlated with lower hematocrit in the HIP model indicating pathologic erythropoiesis. In the HIP model, pharmacological upregulation of endogenous epoxyeicosatrienoic acids protected the renal microvasculature against amylin deposition and also reduced renal accumulation of HIFs. Thus, prediabetes induces dysregulation of amylin homeostasis and promotes amylin deposition in RBCs and the microvasculature altering RBC-capillary interaction leading to activation of hypoxia signaling pathways and pathologic erythropoiesis. Hence, dysregulation of amylin homeostasis could be a therapeutic target for ameliorating diabetic vascular complications.

Disease-associated mutations in Niemann-Pick type C1 alter ER calcium signaling and neuronal plasticity.

Niemann-Pick type C1 (NPC1) protein is essential for the transport of externally derived cholesterol from lysosomes to other organelles. Deficiency of NPC1 underlies the progressive NPC1 neurodegenerative disorder. Currently, there are no curative therapies for this fatal disease. Given the Ca2+ hypothesis of neurodegeneration, which posits that altered Ca2+ dynamics contribute to neuropathology, we tested if disease mutations in NPC1 alter Ca2+ signaling and neuronal plasticity. We determine that NPC1 inhibition or disease mutations potentiate store-operated Ca2+ entry (SOCE) due to a presenilin 1 (PSEN1)-dependent reduction in ER Ca2+ levels alongside elevated expression of the molecular SOCE components ORAI1 and STIM1. Associated with this dysfunctional Ca2+ signaling is destabilization of neuronal dendritic spines. Knockdown of PSEN1 or inhibition of the SREBP pathway restores Ca2+ homeostasis, corrects differential protein expression, reduces cholesterol accumulation, and rescues spine density. These findings highlight lysosomes as a crucial signaling platform responsible for tuning ER Ca2+ signaling, SOCE, and synaptic architecture in health and disease.

Whole-Cell cAMP and PKA Activity are Epiphenomena, Nanodomain Signaling Matters.

Novel targeted fluorescent biosensors provide key insights into very local nanodomains of cAMP and PKA activity, and how they respond differently to ß-adrenergic activation in cardiac myocytes. This unique spatiotemporal detail in living cells is not available with biochemical measurements of total cellular cAMP and PKA, and provides unique physiological insights.

Dual role of inorganic polyphosphate in cardiac myocytes: The importance of polyP chain length for energy metabolism and mPTP activation.

We have previously demonstrated that inorganic polyphosphate (polyP) is a potent activator of the mitochondrial permeability transition pore (mPTP) in cardiac myocytes. PolyP depletion protected against Ca2+-induced mPTP opening, however it did not prevent and even exacerbated cell death during ischemia-reperfusion (I/R). The central goal of this study was to investigate potential molecular mechanisms underlying these dichotomous effects of polyP on mitochondrial function. We utilized a Langendorff-perfused heart model of I/R to monitor changes in polyP size and chain length at baseline, 20 min no-flow ischemia, and 15 min reperfusion. Freshly isolated cardiac myocytes and mitochondria from C57BL/6J (WT) and cyclophilin D knock-out (CypD KO) mice were used to measure polyP uptake, mPTP activity, mitochondrial membrane potential, respiration and ATP generation. We found that I/R induced a significant decrease in polyP chain length. We, therefore, tested, the ability of synthetic polyPs with different chain length to accumulate in mitochondria and induce mPTP. Both short and long chain polyPs accumulated in mitochondria in oligomycin-sensitive manner implicating potential involvement of mitochondrial ATP synthase in polyP transport. Notably, only short-chain polyP activated mPTP in WT myocytes, and this effect was prevented by mPTP inhibitor cyclosprorin A and absent in CypD KO myocytes. To the contrary, long-chain polyP suppressed mPTP activation, and enhanced ADP-linked respiration and ATP production. Our data indicate that 1) effect of polyP on cardiac function strongly depends on polymer chain length; and 2) short-chain polyPs (as increased in ischemia-reperfusion) induce mPTP and mitochondrial uncoupling, while long-chain polyPs contribute to energy generation and cell metabolism.

ß-adrenergic regulation of late Na+ current during cardiac action potential is mediated by both PKA and CaMKII.

Late Na+ current (INaL) significantly contributes to shaping cardiac action potentials (APs) and increased INaL is associated with cardiac arrhythmias. ß-adrenergic receptor (ßAR) stimulation and its downstream signaling via protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathways are known to regulate INaL. However, it remains unclear how each of these pathways regulates INaL during the AP under physiological conditions. Here we performed AP-clamp experiments in rabbit ventricular myocytes to delineate the impact of each signaling pathway on INaL at different AP phases to understand the arrhythmogenic potential. During the physiological AP (2 Hz, 37 °C) we found that INaL had a basal level current independent of PKA, but partially dependent on CaMKII. ßAR activation (10 nM isoproterenol, ISO) further enhanced INaL via both PKA and CaMKII pathways. However, PKA predominantly increased INaL early during the AP plateau, whereas CaMKII mainly increased INaL later in the plateau and during rapid repolarization. We also tested the role of key signaling pathways through exchange protein activated by cAMP (Epac), nitric oxide synthase (NOS) and reactive oxygen species (ROS). Direct Epac stimulation enhanced INaL similar to the ßAR-induced CaMKII effect, while NOS inhibition prevented the ßAR-induced CaMKII-dependent INaL enhancement. ROS generated by NADPH oxidase 2 (NOX2) also contributed to the ISO-induced INaL activation early in the AP. Taken together, our data reveal differential modulations of INaL by PKA and CaMKII signaling pathways at different AP phases. This nuanced and comprehensive view on the changes in INaL during AP deepens our understanding of the important role of INaL in reshaping the cardiac AP and arrhythmogenic potential under elevated sympathetic stimulation, which is relevant for designing therapeutic treatment of arrhythmias under pathological conditions.

GRAM domain proteins specialize functionally distinct ER-PM contact sites in human cells.

Endoplasmic reticulum (ER) membrane contact sites (MCSs) are crucial regulatory hubs in cells, playing roles in signaling, organelle dynamics, and ion and lipid homeostasis. Previous work demonstrated that the highly conserved yeast Ltc/Lam sterol transporters localize and function at ER MCSs. Our analysis of the human family members, GRAMD1a and GRAMD2a, demonstrates that they are ER-PM MCS proteins, which mark separate regions of the plasma membrane (PM) and perform distinct functions in vivo. GRAMD2a, but not GRAMD1a, co-localizes with the E-Syt2/3 tethers at ER-PM contacts in a PIP lipid-dependent manner and pre-marks the subset of PI(4,5)P2-enriched ER-PM MCSs utilized for STIM1 recruitment. Data from an analysis of cells lacking GRAMD2a suggest that it is an organizer of ER-PM MCSs with pleiotropic functions including calcium homeostasis. Thus, our data demonstrate the existence of multiple ER-PM domains in human cells that are functionally specialized by GRAM-domain containing proteins.

Amylin and diabetic cardiomyopathy - amylin-induced sarcolemmal Ca2+ leak is independent of diabetic remodeling of myocardium.

Amylin is a pancreatic ß-cell hormone co-secreted with insulin, plays a role in normal glucose homeostasis, and forms amyloid in the pancreatic islets of individuals with type-2 diabetes. Aggregated amylin is also found in blood and extra-pancreatic tissues, including myocardium. Myocardial amylin accumulation is associated with myocyte Ca2+ dysregulation in diabetic rats expressing human amylin. Whether deposition of amylin in the heart is a consequence of or a contributor to diabetic cardiomyopathy remains unknown. We used amylin knockout (AKO) mice intravenously infused with either human amylin (i.e, the aggregated form) or non-amyloidogenic (i.e., monomeric) rodent amylin to test the hypothesis that aggregated amylin accumulates in the heart in the absence of diabetes. AKO mice infused with human amylin, but not rodent amylin, showed amylin deposits in the myocardium. Cardiac amylin level was larger in males compared to females. Sarcolemmal Ca2+ leak and Ca2+ transients were increased in myocytes isolated from males infused with human amylin while no significant changes occurred in either females injected with human amylin or in rat amylin-infused mice. In isolated cardiac myocytes, the amylin receptor antagonist AC-187 did not effectively block the interaction of amylin with the sarcolemma. In conclusion, circulating aggregated amylin accumulates preferentially in male vs. female hearts and its effects on myocyte Ca2+ cycling do not require diabetic remodeling of the myocardium. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.

Subcellular localization of Na/K-ATPase isoforms in ventricular myocytes.

The sodium/potassium ATPase (NKA) is essential for establishing the normal intracellular [Na+] and [K+] and transmembrane gradients that are essential for many cellular functions, including cardiac electrophysiology and contractility. Different NKA isoforms exhibit differential expression levels, cellular localization, and function in different tissues and species. Prior work has indicated that the NKA-α1 isoform is quantitatively predominant in cardiac myocytes, but that the α2 isoform is preferentially concentrated in the transverse tubules (TT), possibly at junctions with the sarcoplasmic reticulum (SR) where α2 may preferentially modulate cardiac contractility. Here we measured subcellular localization of NKA-α1 and α2 using super-resolution microscopy (STED and STORM) and isoform-selective antibodies in mouse ventricular myocytes. We confirm the preferential localization of NKA-α2 in TT vs. surface sarcolemma, but also show that α2 is relatively excluded from longitudinal TT elements. In contrast NKA-α1 is relatively uniformly expressed in all three sarcolemmal regions. We also tested the hypothesis that NKA-α2 (vs. α1) is preferentially concentrated at SR junctional sites near ryanodine receptors (RyR2). The results refute this hypothesis, in that NKA-α1 and α2 were equally close to RyR2 at the TT, with no preferential NKA isoform localization near RyR2. We conclude that in contrast to relatively uniform NKA-α1 distribution, NKA-α2 is preferentially concentrated in the truly transverse (and not longitudinal) TT elements. However, NKA-α2 does not preferentially cluster at RyR2 junctions, so the TT NKA-α2 concentration may suffice for preferential effects of NKA-α2 inhibition on cardiac contractility.

MarkoLAB: A simulator to study ionic channel's stochastic behavior.

Mathematical models of the cardiac cell have started to include markovian representations of the ionic channels instead of the traditional Hodgkin & Huxley formulations. There are many reasons for this: Markov models are not restricted to the idea of independent gates defining the channel, they allow more complex description with specific transitions between open, closed or inactivated states, and more importantly those states can be closely related to the underlying channel structure and conformational changes. METHODS: We used the LabVIEW® and MATLAB® programs to implement the simulator MarkoLAB that allow a dynamical 3D representation of the markovian model of the channel. The Monte Carlo simulation was used to implement the stochastic transitions among states. The user can specify the voltage protocol by setting the holding potential, the step-to voltage and the duration of the stimuli. RESULTS: The most studied feature of a channel is the current flowing through it. This happens when the channel stays in the open state, but most of the time, as revealed by the low open probability values, the channel remains on the inactive or closed states. By focusing only when the channel enters or leaves the open state we are missing most of its activity. MarkoLAB proved to be quite useful to visualize the whole behavior of the channel and not only when the channel produces a current. Such dynamic representation provides more complete information about channel kinetics and will be a powerful tool to demonstrate the effect of gene mutations or drugs on the channel function. CONCLUSIONS: MarkoLAB provides an original way of visualizing the stochastic behavior of a channel. It clarifies concepts, such as recovery from inactivation, calcium- versus voltage-dependent inactivation, and tail currents. It is not restricted to ionic channels only but it can be extended to other transporters, such as exchangers and pumps. This program is intended as a didactical tool to illustrate the dynamical behavior of a channel. It has been implemented in two platforms MATLAB® and LabVIEW® to enhance the target users of this new didactical tool. The computational cost of implementing a stochastic simulation is within the range of a personal computer performance; making MarkoLAB suitable to be run during a lecture or presentation.

ß-Adrenergic induced SR Ca2+ leak is mediated by an Epac-NOS pathway.

Cardiac ß-adrenergic receptors (ß-AR) and Ca2+-Calmodulin dependent protein kinase (CaMKII) regulate both physiological and pathophysiological Ca2+ signaling. Elevated diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) contributes to contractile dysfunction in heart failure and to arrhythmogenesis. ß-AR activation is known to increase SR Ca2+ leak via CaMKII-dependent phosphorylation of the ryanodine receptor. Two independent and reportedly parallel pathways have been implicated in this ß-AR-CaMKII cascade, one involving exchange protein directly activated by cAMP (Epac2) and another involving nitric oxide synthase 1 (NOS1). Here we tested whether Epac and NOS function in a single series pathway to increase ß-AR induced and CaMKII-dependent SR Ca2+ leak. Leak was measured as both Ca2+ spark frequency and tetracaine-induced shifts in SR Ca2+, in mouse and rabbit ventricular myocytes. Direct Epac activation by 8-CPT (8-(4-chlorophenylthio)-2'-O-methyl-cAMP) mimicked ß-AR-induced SR Ca2+ leak, and both were blocked by NOS inhibition. The same was true for myocyte CaMKII activation (assessed via a FRET-based reporter) and ryanodine receptor phosphorylation. Inhibitor and phosphorylation studies also implicated phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) downstream of Epac and above NOS activation in this pathway. We conclude that these two independently characterized parallel pathways function mainly via a single series arrangement (ß-AR-cAMP-Epac-PI3K-Akt-NOS1-CaMKII) to mediate increased SR Ca2+ leak. Thus, for ß-AR activation the cAMP-PKA branch effects inotropy and lusitropy (by effects on Ca2+ current and SR Ca2+-ATPase), this cAMP-Epac-NOS pathway increases pathological diastolic SR Ca2+leak. This pathway distinction may allow novel SR Ca2+ leak therapeutic targeting in treatment of arrhythmias in heart failure that spare the inotropic and lusitropic effects of the PKA branch.