Direct toxicity of cigarette smoke extract on cardiac function mediated by mitochondrial dysfunction in Sprague-Dawley rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes.

Cigarette smoke has been recognized as a major risk factor for cardiovascular disease. However, its direct effects on rodent and human cardiomyocytes and its cellular mechanisms are not fully understood. In this study, we examined the direct effects of cigarette smoke extract (CSE) on contractile functions, intracellular Ca2+ dynamics, and mitochondrial function using cultured or freshly isolated rat ventricular myocytes and human induced pluripotent stem cell (iPS)-derived cardiomyocytes. In rat cardiomyocytes, CSE (≥0.1%) resulted in a time- and concentration-dependent cessation of spontaneous beating of cultured cardiomyocytes, eventually leading to cell death, which indicates direct toxicity. In addition, 1% CSE reduced contractile function of freshly isolated ventricular myocytes. Similar contractile dysfunction (declined spontaneous beating rate and contractility) was also observed in human iPS-derived cardiomyocytes. Regarding intracellular Ca2+ dynamics, 1% CSE increased the Ca2+ transient amplitude by greatly increasing systolic Ca2+ levels and slightly increasing diastolic Ca2+ levels. CSE also accelerated the decay of Ca2+ transients, and triggered spike-shaped Ca2+ transients in some cells. These results indicate that CSE causes abnormal Ca2+ dynamics in cardiomyocytes. Furthermore, CSE induced a cascade of mitochondrial dysfunctions, including increased mitochondrial reactive oxygen species, opening of mitochondrial permeability transition pore, reduction of mitochondrial membrane potential, and release of cytochrome c from mitochondria. These results suggest that CSE-induced contractile dysfunction and myocardial cell death is caused by abnormal Ca2+ dynamics and subsequent mitochondrial dysregulation, which would result in reduced bioenergetics and activation of cell death pathways.

Licorice metabolite 18ß-glycyrrhetinic acid activates G protein-gated inwardly rectifying K+ channels.

BACKGROUND AND PURPOSE: Licorice (liquorice) is a common food additive and is used in Chinese medicine. Excess licorice intake can induce atrial fibrillation. Patients with atrial fibrillation possess constitutively activated G protein-gated inwardly rectifying K+ (GIRK) channels. Whether licorice affects GIRK channel activity is unknown. We aimed to clarify the effects of licorice ingredients on GIRK current and the mechanism of action. EXPERIMENTAL APPROACH: A major component of licorice, glycyrrhizic acid (GA), and its metabolite, 18ß-glycyrrhetinic acid (18ß-GA), were tested. We performed electrophysiological recordings in Xenopus oocytes to examine the effects of GA and 18ß-GA on various GIRK subunits (Kir 3.1-Kir 3.4), mutagenesis analyses to identify the crucial residues for drug action and motion analysis in cultured rat atrial myocytes to clarify effects of 18ß-GA on atrial functions. KEY RESULTS: GA inhibited Kir 3.1-containing channels, while 18ß-GA activated all Kir 3.x subunits. A pore helix residue Phe137 in Kir 3.1 was critical for GA-mediated inhibition, and the corresponding Ser148 in Kir 3.2 was critical for 18ß-GA-mediated activation. 18ß-GA activated GIRK channel in a Gßγ -independent manner, whereas phosphatidylinositol 4,5-bisphosphate (PIP2 ) was essential for activation. Glu236 located at the cytoplasmic pore of Kir 3.2 appeared to be important to interactions with 18ß-GA. In rat atrial myocytes, 18ß-GA suppressed spontaneous beating via activation of GIRK channels. CONCLUSION AND IMPLICATIONS: GA acts as a novel GIRK inhibitor, and 18ß-GA acts as a novel GIRK activator. 18ß-GA alters atrial function via activation of GIRK channels. This study elucidates the pharmacological activity of licorice ingredients and provides information for drug design.

CL-705G: a novel chemical Kir6.2-specific KATP channel opener.

Background: KATP channels have diverse roles, including regulation of insulin secretion and blood flow, and protection against biological stress responses and are excellent therapeutic targets. Different subclasses of KATP channels exist in various tissue types due to the unique assemblies of specific pore-forming (Kir6.x) and accessory (SURx) subunits. The majority of pharmacological openers and blockers act by binding to SURx and are poorly selective against the various KATP channel subclasses. Methods and Results: We used 3D models of the Kir6.2/SUR homotetramers based on existing cryo-EM structures of channels in both the open and closed states to identify a potential agonist binding pocket in a functionally critical area of the channel. Computational docking screens of this pocket with the Chembridge Core chemical library of 492,000 drug-like compounds yielded 15 top-ranked "hits", which were tested for activity against KATP channels using patch clamping and thallium (Tl+) flux assays with a Kir6.2/SUR2A HEK-293 stable cell line. Several of the compounds increased Tl+ fluxes. One of them (CL-705G) opened Kir6.2/SUR2A channels with a similar potency as pinacidil (EC50 of 9 µM and 11 µM, respectively). Remarkably, compound CL-705G had no or minimal effects on other Kir channels, including Kir6.1/SUR2B, Kir2.1, or Kir3.1/Kir3.4 channels, or Na+ currents of TE671 medulloblastoma cells. CL-705G activated Kir6.2Δ36 in the presence of SUR2A, but not when expressed by itself. CL-705G activated Kir6.2/SUR2A channels even after PIP2 depletion. The compound has cardioprotective effects in a cellular model of pharmacological preconditioning. It also partially rescued activity of the gating-defective Kir6.2-R301C mutant that is associated with congenital hyperinsulinism. Conclusion: CL-705G is a new Kir6.2 opener with little cross-reactivity with other channels tested, including the structurally similar Kir6.1. This, to our knowledge, is the first Kir-specific channel opener.

Chemogenetic activation of central gastrin-releasing peptide-expressing neurons elicits itch-related scratching behavior in male and female mice.

Several lines of evidence have clarified that the key transmission pathways of itching sensation travel from the periphery to the central nervous system (CNS). Despite the functional significance of gastrin-releasing peptide (GRP) and its cognate receptor in the itch processing mechanism in the spinal dorsal horn (SDH), the roles of GRP-expressing (GRP+ ) neurons in different regions remain unclear. This study aimed to determine whether GRP+ neurons in the CNS directly modulated itch processing. To specifically activate spinal and supraspinal GRP neurons by the designer receptors exclusively activated by designer drugs (DREADDs) system, CAG-LSL-Gq-DREADD mice were crossed with GRP-Cre mice, resulting in the development of GRP-hM3Dq mice. Immunohistochemistry showed that hM3Dq was highly expressed in the SDH and brainstem closely related to sensory processing. The intraperitoneal, intrathecal, or intracerebroventricular administration of clozapine-N-oxide, an agonist of hM3Dq, strongly elicited dermatome-dependent itch-related scratching behavior, but did not change pain sensitivity. Importantly, GRP-Gq-DREADD-mediated scratching behavior in GRP-hM3Dq mice was not affected by the ablation of transient receptor potential vanilloid 1+ sensory C-fibers, and it was also observed to a similar degree under chronic itch conditions. Furthermore, there were no significant sex differences in the scratching behavior elicited by GRP-Gq-DREADD, suggesting that itch-dominant roles of central GRP+ neurons might be common in both sexes, at least under normal physiological conditions. These novel findings not only contribute to understanding the functional roles of central GRP+ neurons further, but also propose the development of future effective therapeutics for intractable itching.

Chemogenetic Regulation of CX3CR1-Expressing Microglia Using Gi-DREADD Exerts Sex-Dependent Anti-Allodynic Effects in Mouse Models of Neuropathic Pain.

Despite growing evidence suggesting that spinal microglia play an important role in the molecular mechanism underlying experimental neuropathic pain (NP) in male rodents, evidence regarding the sex-dependent role of these microglia in NP is insufficient. In this study, we evaluated the effects of microglial regulation on NP using Gi-designer receptors exclusively activated by designer drugs (Gi-DREADD) driven by the microglia-specific Cx3cr1 promoter. For the Cre-dependent expression of human Gi-coupled M4 muscarinic receptors (hM4Di) in CX3C chemokine receptor 1-expressing (CX3CR1+) cells, R26-LSL-hM4Di-DREADD mice were crossed with CX3CR1-Cre mice. Mouse models of NP were generated by partial sciatic nerve ligation (PSL) and treatment with anti-cancer agent paclitaxel (PTX) or oxaliplatin (OXA), and mechanical allodynia was evaluated using the von Frey test. Immunohistochemistry revealed that hM4Di was specifically expressed on Iba1+ microglia, but not on astrocytes or neurons in the spinal dorsal horn of CX3CR1-hM4Di mice. PSL-induced mechanical allodynia was significantly attenuated by systemic (intraperitoneal, i.p.) administration of 10 mg/kg of clozapine N-oxide (CNO), a hM4Di-selective ligand, in male CX3CR1-hM4Di mice. The mechanical threshold in naive CX3CR1-hM4Di mice was not altered by i.p. administration of CNO. Consistently, local (intrathecal, i.t.) administration of CNO (20 nmol) significantly relieved PSL-induced mechanical allodynia in male CX3CR1-hM4Di mice. However, neither i.p. nor i.t. administration of CNO affected PSL-induced mechanical allodynia in female CX3CR1-hM4Di mice. Both i.p. and i.t. administration of CNO relieved PTX-induced mechanical allodynia in male CX3CR1-hM4Di mice, and a limited effect of i.p. CNO was observed in female CX3CR1-hM4Di mice. Unlike PTX-induced allodynia, OXA-induced mechanical allodynia was slightly improved, but not significantly relieved, by i.p. administration of CNO in both male and female CX3CR1-hM4Di mice. These results suggest that spinal microglia can be regulated by Gi-DREADD and support the notion that CX3CR1+ spinal microglia play sex-dependent roles in nerve injury-induced NP; however, their roles may vary among different models of NP.

Emerging Roles of Neuronal Ca2+ Sensor-1 in Cardiac and Neuronal Tissues: A Mini Review.

The EF-hand calcium (Ca2+)-binding protein, neuronal Ca2+ sensor-1 (NCS-1/frequenin), is predominantly expressed in neuronal tissues and plays a crucial role in neuronal functions, including synaptic transmission and plasticity. NCS-1 has diverse functional roles, as elucidated in the past 15 years, which include the regulation of phosphatidylinositol 4-kinase IIIß (PI-4K-ß) and several ion channels such as voltage-gated K+ and Ca2+ channels, the D2 dopamine receptors, and inositol 1,4,5-trisphosphate receptors (InsP3Rs). Functional analyses demonstrated that NCS-1 enhances exocytosis and neuronal survival after injury, as well as promotes learning and memory in mice. NCS-1 is also expressed in the heart including the Purkinje fibers (PFs) of the conduction system. NCS-1 interacts with KV4 K+ channels together with dipeptidyl peptidase-like protein-6 (DPP-6), and this macromolecule then composes the transient outward current in PFs and contributes to the repolarization of PF action potential, thus being responsible for idiopathic arrhythmia. Moreover, NCS-1 expression was reported to be significantly high at the immature stage and at hypertrophy in adults. That report demonstrated that NCS-1 positively regulates cardiac contraction in immature hearts by increasing intracellular Ca2+ signals through interaction with InsP3Rs. With the related signals, NCS-1 activates nuclear Ca2+ signals, which would be a mechanism underlying hormone-induced cardiac hypertrophy. Furthermore, NCS-1 contributes to stress tolerance in cardiomyocytes by activating mitochondrial detoxification pathways, with a key role in Ca2+-dependent pathways. In this review, we will discuss recent findings supporting the functional significance of NCS-1 in the brain and heart and will address possible underlying molecular mechanisms.

Possible Signaling Pathways Mediating Neuronal Calcium Sensor-1-Dependent Spatial Learning and Memory in Mice.

Intracellular Ca2+ signaling regulates diverse functions of the nervous system. Many of these neuronal functions, including learning and memory, are regulated by neuronal calcium sensor-1 (NCS-1). However, the pathways by which NCS-1 regulates these functions remain poorly understood. Consistent with the findings of previous reports, we revealed that NCS-1 deficient (Ncs1-/-) mice exhibit impaired spatial learning and memory function in the Morris water maze test, although there was little change in their exercise activity, as determined via treadmill-analysis. Expression of brain-derived neurotrophic factor (BDNF; a key regulator of memory function) and dopamine was significantly reduced in the Ncs1-/- mouse brain, without changes in the levels of glial cell-line derived neurotrophic factor or nerve growth factor. Although there were no gross structural abnormalities in the hippocampi of Ncs1-/- mice, electron microscopy analysis revealed that the density of large dense core vesicles in CA1 presynaptic neurons, which release BDNF and dopamine, was decreased. Phosphorylation of Ca2+/calmodulin-dependent protein kinase II-α (CaMKII-α, which is known to trigger long-term potentiation and increase BDNF levels, was significantly reduced in the Ncs1-/- mouse brain. Furthermore, high voltage electric potential stimulation, which increases the levels of BDNF and promotes spatial learning, significantly increased the levels of NCS-1 concomitant with phosphorylated CaMKII-α in the hippocampus; suggesting a close relationship between NCS-1 and CaMKII-α. Our findings indicate that NCS-1 may regulate spatial learning and memory function at least in part through activation of CaMKII-α signaling, which may directly or indirectly increase BDNF production.

Neuronal Ca2+ sensor-1 contributes to stress tolerance in cardiomyocytes via activation of mitochondrial detoxification pathways.

Identification of the molecules involved in cell death/survival pathways is important for understanding the mechanisms of cell loss in cardiac disease, and thus is clinically relevant. Ca2+-dependent signals are often involved in these pathways. Here, we found that neuronal Ca2+-sensor-1 (NCS-1), a Ca2+-binding protein, has an important role in cardiac survival during stress. Cardiomyocytes derived from NCS-1-deficient (Ncs1-/-) mice were more susceptible to oxidative and metabolic stress than wild-type (WT) myocytes. Cellular ATP levels and mitochondrial respiration rates, as well as the levels of mitochondrial marker proteins, were lower in Ncs1-/- myocytes. Although oxidative stress elevated mitochondrial proton leak, which exerts a protective effect by inhibiting the production of reactive oxygen species in WT myocytes, this response was considerably diminished in Ncs1-/- cardiomyocytes, and this would be a major reason for cell death. Consistently, H2O2-induced loss of mitochondrial membrane potential, a critical early event in cell death, was accelerated in Ncs1-/- myocytes. Furthermore, NCS-1 was upregulated in hearts subjected to ischemia-reperfusion, and ischemia-reperfusion injury was more severe in Ncs1-/- hearts. Activation of stress-induced Ca2+-dependent survival pathways, such as Akt and PGC-1α (which promotes mitochondrial biogenesis and function), was diminished in Ncs1-/- hearts. Overall, these data demonstrate that NCS-1 contributes to stress tolerance in cardiomyocytes at least in part by activating certain Ca2+-dependent survival pathways that promote mitochondrial biosynthesis/function and detoxification pathways.

Calcineurin B homologous protein 3 negatively regulates cardiomyocyte hypertrophy via inhibition of glycogen synthase kinase 3 phosphorylation.

Cardiac hypertrophy is a leading cause of serious heart diseases. Although many signaling molecules are involved in hypertrophy, the functions of some proteins in this process are still unknown. Calcineurin B homologous protein 3 (CHP3)/tescalcin is an EF-hand Ca(2+)-binding protein that is abundantly expressed in the heart; however, the function of CHP3 is unclear. Here, we aimed to identify the cardiac functions of CHP3. CHP3 was expressed in hearts at a wide range of developmental stages and was specifically detected in neonatal rat ventricular myocytes (NRVMs) but not in cardiac fibroblasts in culture. Moreover, knockdown of CHP3 expression using adenoviral-based RNA interference in NRVMs resulted in enlargement of cardiomyocyte size, concomitant with increased expression of a pathological hypertrophy marker ANP. This same treatment elevated glycogen synthase kinase (GSK3α/ß) phosphorylation, which is known to inhibit GSK3 function. In contrast, CHP3 overexpression blocked the insulin-induced phosphorylation of GSK3α/ß without affecting the phosphorylation of Akt, which is an upstream kinase of GSK3α/ß, in HEK293 cells, and it inhibited both IGF-1-induced phosphorylation of GSK3ß and cardiomyocyte hypertrophy in NRVMs. Co-immunoprecipitation experiments revealed that GSK3ß interacted with CHP3. However, a Ca(2+)-binding-defective mutation of CHP3 (CHP3-D123A) also interacted with GSK3ß and had the same inhibitory effect on GSK3α/ß phosphorylation, suggesting that the action of CHP3 was independent of Ca(2+). These findings suggest that CHP3 functions as a novel negative regulator of cardiomyocyte hypertrophy via inhibition of GSK3α/ß phosphorylation and subsequent enzymatic activation of GSK3α/ß.

Stimulus-dependent regulation of nuclear Ca2+ signaling in cardiomyocytes: a role of neuronal calcium sensor-1.

In cardiomyocytes, intracellular calcium (Ca2+) transients are elicited by electrical and receptor stimulations, leading to muscle contraction and gene expression, respectively. Although such elevations of Ca2+levels ([Ca2+]) also occur in the nucleus, the precise mechanism of nuclear [Ca2+] regulation during different kinds of stimuli, and its relationship with cytoplasmic [Ca2+] regulation are not fully understood. To address these issues, we used a new region-specific fluorescent protein-based Ca2+ indicator, GECO, together with the conventional probe Fluo-4 AM. We confirmed that nuclear Ca2+ transients were elicited by both electrical and receptor stimulations in neonatal mouse ventricular myocytes. Kinetic analysis revealed that electrical stimulation-elicited nuclear Ca2+ transients are slower than cytoplasmic Ca2+ transients, and chelating cytoplasmic Ca2+ abolished nuclear Ca2+ transients, suggesting that nuclear Ca2+ are mainly derived from the cytoplasm during electrical stimulation. On the other hand, receptor stimulation such as with insulin-like growth factor-1 (IGF-1) preferentially increased nuclear [Ca2+] compared to cytoplasmic [Ca2+]. Experiments using inhibitors revealed that electrical and receptor stimulation-elicited Ca2+ transients were mainly mediated by ryanodine receptors and inositol 1,4,5-trisphosphate receptors (IP3Rs), respectively, suggesting different mechanisms for the two signals. Furthermore, IGF-1-elicited nuclear Ca2+ transient amplitude was significantly lower in myocytes lacking neuronal Ca2+ sensor-1 (NCS-1), a Ca2+ binding protein implicated in IP3R-mediated pathway in the heart. Moreover, IGF-1 strengthened the interaction between NCS-1 and IP3R. These results suggest a novel mechanism for receptor stimulation-induced nuclear [Ca2+] regulation mediated by IP3R and NCS-1 that may further fine-tune cardiac Ca2+ signal regulation.

Deficiency of a lipid droplet protein, perilipin 5, suppresses myocardial lipid accumulation, thereby preventing type 1 diabetes-induced heart malfunction.

Lipid droplet (LD) is a ubiquitous organelle that stores triacylglycerol and other neutral lipids. Perilipin 5 (Plin5), a member of the perilipin protein family that is abundantly expressed in the heart, is essential to protect LDs from attack by lipases, including adipose triglyceride lipase. Plin5 controls heart metabolism and performance by maintaining LDs under physiological conditions. Aberrant lipid accumulation in the heart leads to organ malfunction, or cardiomyopathy. To elucidate the role of Plin5 in a metabolically disordered state and the mechanism of lipid-induced cardiomyopathy, we studied the effects of streptozotocin-induced type 1 diabetes in Plin5-knockout (KO) mice. In contrast to diabetic wild-type mice, diabetic Plin5-KO mice lacked detectable LDs in the heart and did not exhibit aberrant lipid accumulation, excessive reactive oxygen species (ROS) generation, or heart malfunction. Moreover, diabetic Plin5-KO mice exhibited lower heart levels of lipotoxic molecules, such as diacylglycerol and ceramide, than wild-type mice. Membrane translocation of protein kinase C and the assembly of NADPH oxidase 2 complex on the membrane were also suppressed. The results suggest that diabetic Plin5-KO mice are resistant to type 1 diabetes-induced heart malfunction due to the suppression of the diacylglycerol/ceramide-protein kinase C pathway and of excessive ROS generation by NADPH oxidase.

Na+/H+ exchanger 1 is regulated via its lipid-interacting domain, which functions as a molecular switch: a pharmacological approach using indolocarbazole compounds.

The plasma membrane Na(+)/H(+) exchanger 1 (NHE1) is rapidly activated in response to various stimuli. The membrane-proximal cytoplasmic region (∼60 residues), termed the lipid-interacting domain (LID), is an important regulatory domain of NHE1. Here, we used a pharmacological approach to further characterize the role of LID in the regulation of NHE1. Pharmacological analysis using staurosporine-like indolocarbazole and bisindolylmaleimide compounds suggested that the phorbol ester- and receptor agonist-induced activation of NHE1 occurs through a protein kinase C-independent mechanism. In particular, only indolocarbazole compounds that inhibited NHE1 activation were able to interact with the LID, suggesting that the inhibition of NHE1 activation is achieved through the direct action of these compounds on the LID. Furthermore, in addition to phorbol esters and a receptor agonist, okadaic acid and hyperosmotic stress, which are known to activate NHE1 through unknown mechanisms, were found to promote membrane association of the LID concomitant with NHE1 activation; these effects were inhibited by staurosporine, as well as by a mutation in the LID. Binding experiments using the fluorescent ATP analog trinitrophenyl ATP revealed that ATP and the NHE1 activator phosphatidylinositol 4,5-bisphosphate bind competitively to the LID. These findings suggest that modulation of NHE1 activity by various activators and inhibitors occurs through the direct binding of these molecules to the LID, which alters the association of the LID with the plasma membrane.

Regulation of the cardiac Na⁺/H⁺ exchanger in health and disease.

The Na(+) gradient produced across the cardiac sarcolemma by the ATP-dependent Na(+)-pump is a constant source of energy for Na(+)-dependent transporters. The plasma membrane Na(+)/H(+) exchanger (NHE) is one such secondary active transporter, regulating intracellular pH, Na(+) concentration, and cell volume. NHE1, the major isoform found in the heart, is activated in response to a variety of stimuli such as hormones and mechanical stress. This important characteristic of NHE1 is intimately linked to heart diseases, including maladaptive cardiac hypertrophy and subsequent heart failure, as well as acute ischemic-reperfusion injury. NHE1 activation results in elevation of pH and intracellular Na(+) concentration, which potentially enhance downstream signaling cascades in the myocardium. Therefore, in addition to determining the mechanism underlying regulation of NHE1 activity, it is important to understand how the ionic signal produced by NHE1 is transmitted to the downstream targets. Extensive studies have identified many accessory factors that interact with NHE1. Here, we have summarized the recent progress on understanding the molecular mechanism underlying NHE1 regulation and have shown a possible signaling pathway leading to cardiac remodeling, which is initiated from NHE1. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".

Evidence that Na+/H+ exchanger 1 is an ATP-binding protein.

Na(+)/H(+) exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP-binding protein. We designed a baculovirus vector carrying both epitope-tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1-CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8-azido-ATP-biotin. UV irradiation promoted the incorporation of 8-azido-ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µM in the presence of Mg(2+). The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mM, close to the ATP concentration giving the half-maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8-azido-ATP-binding site is within the C-terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1-derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542-Pro598), which is critical for ATP-dependent regulation of NHE1. These findings suggest that NHE1 is an ATP-binding transporter. Thus, ATP may serve as a direct activator of NHE1.

Role of neuronal calcium sensor-1 in the cardiovascular system.

Calcium (Ca(2+)) is an important intracellular messenger, regulating myocyte contraction via excitation-contraction (EC) coupling and gene transcription underlying hypertrophy in the heart. Although the mechanisms of EC coupling in the immature heart are believed to be different from those in the adult heart because of the structural immaturity of the sarcoplasmic reticulum in the young heart, the details of these mechanisms are not completely understood. Neuronal Ca(2+) sensor-1 (NCS-1) is an EF-hand Ca(2+)-binding protein that is highly expressed in young hearts; however, little is known about its cardiac functions. In this review, we summarize our recent findings indicating that NCS-1 acts as a novel regulator enhancing Ca(2+) signals in the heart and hence promoting contraction in the immature heart and hypertrophy in the adult heart. Possible signal transduction pathways are also discussed.

Na(+)/H(+) exchanger 1 directly binds to calcineurin A and activates downstream NFAT signaling, leading to cardiomyocyte hypertrophy.

The calcineurin A (CaNA) subunit was identified as a novel binding partner of plasma membrane Na(+)/H(+) exchanger 1 (NHE1). CaN is a Ca(2+)-dependent phosphatase involved in many cellular functions, including cardiac hypertrophy. Direct binding of CaN to the (715)PVITID(720) sequence of NHE1, which resembles the consensus CaN-binding motif (PXIXIT), was observed. Overexpression of NHE1 promoted serum-induced CaN/nuclear factor of activated T cells (NFAT) signaling in fibroblasts, as indicated by enhancement of NFAT promoter activity and nuclear translocation, which was attenuated by NHE1 inhibitor. In neonatal rat cardiomyocytes, NHE1 stimulated hypertrophic gene expression and the NFAT pathway, which were inhibited by a CaN inhibitor, FK506. Importantly, CaN activity was strongly enhanced with increasing pH, so NHE1 may promote CaN/NFAT signaling via increased intracellular pH. Indeed, Na(+)/H(+) exchange activity was required for NHE1-dependent NFAT signaling. Moreover, interaction of CaN with NHE1 and clustering of NHE1 to lipid rafts were also required for this response. Based on these results, we propose that NHE1 activity may generate a localized membrane microdomain with higher pH, thereby sensitizing CaN to activation and promoting NFAT signaling. In cardiomyocytes, such signaling can be a pathway of NHE1-dependent hypertrophy.

Perilipin 5, a lipid droplet-binding protein, protects heart from oxidative burden by sequestering fatty acid from excessive oxidation.

Lipid droplets (LDs) are ubiquitous organelles storing neutral lipids, including triacylglycerol (TAG) and cholesterol ester. The properties of LDs vary greatly among tissues, and LD-binding proteins, the perilipin family in particular, play critical roles in determining such diversity. Overaccumulation of TAG in LDs of non-adipose tissues may cause lipotoxicity, leading to diseases such as diabetes and cardiomyopathy. However, the physiological significance of non-adipose LDs in a normal state is poorly understood. To address this issue, we generated and characterized mice deficient in perilipin 5 (Plin5), a member of the perilipin family particularly abundant in the heart. The mutant mice lacked detectable LDs, containing significantly less TAG in the heart. Particulate structures containing another LD-binding protein, Plin2, but negative for lipid staining, remained in mutant mice hearts. LDs were recovered by perfusing the heart with an inhibitor of lipase. Cultured cardiomyocytes from Plin5-null mice more actively oxidized fatty acid than those of wild-type mice. Production of reactive oxygen species was increased in the mutant mice hearts, leading to a greater decline in heart function with age. This was, however, reduced by the administration of N-acetylcysteine, a precursor of an antioxidant, glutathione. Thus, we conclude that Plin5 is essential for maintaining LDs at detectable sizes in the heart, by antagonizing lipase(s). LDs in turn prevent excess reactive oxygen species production by sequestering fatty acid from oxidation and hence suppress oxidative burden to the heart.

Neuronal calcium sensor-1 promotes immature heart function and hypertrophy by enhancing Ca2+ signals.

RATIONALE: Neuronal calcium sensor-1 (NCS-1) regulates various neuronal functions. Although it is expressed in the heart, very little is known about its cardiac functions. OBJECTIVE: This study aimed to identify the physiological and pathological roles of NCS-1 in the heart. METHODS AND RESULTS: We characterized the cardiac functions of knockout mice (Ncs1(-/-)) and identified NCS-1 as a novel regulator of cardiac Ca(2+) signaling, specifically in immature and hypertrophic hearts. NCS-1 was highly expressed in young hearts, and its deletion decreased survival and contractile function in young mice. Intracellular Ca(2+) levels and sarcoplasmic reticulum Ca(2+) content were significantly lower in Ncs1(-/-) myocytes than in wild-type cells. This was due to reduced Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity in Ncs1(-/-) myocytes, which led to reduced sarcoplasmic reticulum Ca(2+) uptake and release. NCS-1 physically and functionally interacted with inositol 1,4,5-trisphosphate receptors (IP(3)Rs) in the heart. In addition, IP(3)R stimulation resulted in phosphorylation of CaMKII-δ, which was enhanced by NCS-1 overexpression. These results suggest that a functional link exists between NCS-1, IP(3)R function, and CaMKII activation that may affect global Ca(2+) signals in the immature heart. Furthermore, NCS-1 was upregulated in hypertrophic hearts, and hormone-induced hypertrophy was largely prevented in Ncs1(-/-) hearts. Inhibitors of IP(3)Rs, CaMKII, and calcineurin all prevented NCS-1-induced hypertrophy, which suggests the involvement of these pathways. CONCLUSIONS: NCS-1 is an important regulator of immature heart function and hypertrophy, and it functions in part by promoting IP(3)R function, followed by CaMKII-dependent signal activation.

Novel phorbol ester-binding motif mediates hormonal activation of Na+/H+ exchanger.

Protein kinase C (PKC) is considered crucial for hormonal Na(+)/H(+) exchanger (NHE1) activation because phorbol esters (PEs) strongly activate NHE1. However, here we report that rather than PKC, direct binding of PEs/diacylglycerol to the NHE1 lipid-interacting domain (LID) and the subsequent tighter association of LID with the plasma membrane mainly underlies NHE1 activation. We show that (i) PEs directly interact with the LID of NHE1 in vitro, (ii) like PKC, green fluorescent protein (GFP)-labeled LID translocates to the plasma membrane in response to PEs and receptor agonists, (iii) LID mutations markedly inhibit these interactions and PE/receptor agonist-induced NHE1 activation, and (iv) PKC inhibitors ineffectively block NHE1 activation, except staurosporin, which itself inhibits NHE1 via LID. Thus, we propose a PKC-independent mechanism of NHE1 regulation via a PE-binding motif previously unrecognized.

Activation of Na+/H+ exchanger 1 is sufficient to generate Ca2+ signals that induce cardiac hypertrophy and heart failure.

Activation of the sarcolemmal Na(+)/H(+) exchanger (NHE)1 is increasingly documented as a process involved in cardiac hypertrophy and heart failure. However, whether NHE1 activation alone is sufficient to induce such remodeling remains unknown. We generated transgenic mice that overexpress a human NHE1 with high activity in hearts. The hearts of these mice developed cardiac hypertrophy, contractile dysfunction, and heart failure. In isolated transgenic myocytes, intracellular pH was elevated in Hepes buffer but not in physiological bicarbonate buffer, yet intracellular Na(+) concentrations were higher under both conditions. In addition, both diastolic and systolic Ca(2+) levels were increased as a consequence of Na(+)-induced Ca(2+) overload; this was accompanied by enhanced sarcoplasmic reticulum Ca(2+) loading via Ca(2+)/calmodulin-dependent protein kinase (CaMK)II-dependent phosphorylation of phospholamban. Negative force-frequency dependence was observed with preservation of high Ca(2+), suggesting a decrease in myofibril Ca(2+) sensitivity. Furthermore, the Ca(2+)-dependent prohypertrophic molecules calcineurin and CaMKII were highly activated in transgenic hearts. These effects observed in vivo and in vitro were largely prevented by the NHE1 inhibitor cariporide. Interestingly, overexpression of NHE1 in neonatal rat ventricular myocytes induced cariporide-sensitive nuclear translocation of NFAT (nuclear factor of activated T cells) and nuclear export of histone deacetylase 4, suggesting that increased Na(+)/H(+) exchange activity can alter hypertrophy-associated gene expression. However, in transgenic myocytes, contrary to exclusive translocation of histone deacetylase 4, NFAT only partially translocated to nucleus, possibly because of marked activation of p38, a negative regulator of NFAT signaling. We conclude that activation of NHE1 is sufficient to initiate cardiac hypertrophy and heart failure mainly through activation of CaMKII-histone deacetylase pathway.