Lactation Is a Risk Factor of Postpartum Heart Failure in Mice with Cardiomyocyte-specific Apelin Receptor (APJ) Overexpression.

The G protein-coupled receptor APJ and its ligand apelin are highly expressed in cardiovascular tissues and are associated with the regulation of blood pressure and cardiac function. Although accumulating evidence suggests that APJ plays a crucial role in the heart, it remains unclear whether up-regulation of APJ affects cardiac function. Here we generated cardiomyocyte-specific APJ-overexpressing (APJ-TG) mice and investigated the cardiac phenotype in APJ-TG mice. Male and non-pregnant APJ-TG mice showed cardiac hypertrophy, contractile dysfunction, and elevation of B-type natriuretic peptide gene expression in the heart but not cardiac fibrosis and symptoms of heart failure, including breathing abnormality and pleural effusion. We further examined the influence of APJ overexpression in response to physiological stress induced by pregnancy and lactation in the heart. Interestingly, repeating pregnancy and lactation (pregnancy-lactation cycle) exacerbated cardiac hypertrophy and systolic dysfunction and induced cardiac fibrosis, lung congestion, pleural effusion, and abnormal breathing in APJ-TG mice. These data indicate that female APJ-TG mice develop postpartum cardiomyopathy. We showed that lactation, but not parturition, was critical for the onset of postpartum cardiomyopathy in APJ-TG mice. Furthermore, we found that lactating APJ-TG mice showed impaired myocardial angiogenesis and imbalance of pro- and antiangiogenic gene expression in the heart. These results demonstrate that overexpression of APJ in cardiomyocytes has adverse effects on cardiac function in male and non-pregnant mice and that lactation contributes to the development of postpartum cardiomyopathy in the heart with APJ overexpression.

Podocyte injury-driven lipid peroxidation accelerates the infiltration of glomerular foam cells in focal segmental glomerulosclerosis.

Intracapillary foam cell infiltration with podocyte alterations is a characteristic pathology of focal segmental glomerulosclerosis (FSGS). We investigated the possible role of podocyte injury in glomerular macrophage and foam cell infiltration in a podocyte-selective injury model (NEP25 mice) and hypercholesterolemic model [low-density lipoprotein receptor deficiency (LDLR(-/-)) mice] with doxorubicin-induced nephropathy. Acute podocyte selective injury alone failed to induce glomerular macrophages in the NEP25 mice. However, in the doxorubicin-treated hypercholesterolemic LDLR(-/-) mice, glomerular macrophages/foam cells significantly increased and were accompanied by lipid deposition and the formation and ingestion of oxidized phospholipids (oxPLs). Glomerular macrophages significantly correlated with the amount of glomerular oxPL. The NEP25/LDLR(-/-) mice exhibited severe hypercholesterolemia, glomerular lipid deposition, and renal dysfunction. Imaging mass spectrometry revealed that a major component of oxidized low-density lipoprotein, lysophosphatidylcholine 16:0 and 18:0, was present only in the glomeruli of NEP25/LDLR(-/-) mice. Lysophosphatidylcholine 16:0 stimulated mesangial cells and macrophages, and lysophosphatidylcholine 18:0 stimulated glomerular endothelial cells to express adhesion molecules and chemokines, promoting macrophage adhesion and migration in vitro. In human FSGS, glomerular macrophage-derived foam cells contained oxPLs accompanied by the expression of chemokines in the tuft. In conclusion, glomerular lipid modification represents a novel pathology by podocyte injury, promoting FSGS. Podocyte injury-driven lysophosphatidylcholine de novo accelerated glomerular macrophage-derived foam cell infiltration via lysophosphatidylcholine-mediated expression of adhesion molecules and chemokines in glomerular resident cells.

Possible involvement of downregulation of the apelin-APJ system in doxorubicin-induced cardiotoxicity.

Apelin peptide is an endogenous ligand of APJ (a putative receptor protein related to the angiotensin II type 1 receptor), which is a member of a G protein-coupled receptor superfamily with seven transmembrane domains. Recent findings have suggested that the apelin-APJ system plays a potential role in cardiac contraction and cardioprotection. In the present study, we show that the apelin-APJ system is disrupted in doxorubicin (Dox)-induced cardiotoxicity. We found downregulation of apelin and APJ mRNA expression in C57Bl/6J mouse hearts on days 1 and 5 after Dox administration (20 mg/kg ip). Plasma apelin levels and cardiac APJ protein expression were significantly decreased on day 5 after Dox injection. Cardiac apelin contents were reduced on day 1 but increased to basal levels on day 5 after Dox injection. We also examined the effects of APJ gene deletion on Dox-induced cardiotoxicity. Compared with wild-type mice, APJ knockout mice showed a significant depression in cardiac contractility on day 5 after Dox (15 mg/kg ip) treatment followed by a decrease in 14-day survival rates. Moreover, Dox-induced myocardial damage, cardiac protein carbonylation, and autophagic dysfunction were accelerated in APJ knockout mice. Rat cardiac H9c2 cells showed Dox-induced decreases in viability, which were prevented by APJ overexpression and the combination with apelin treatment. These results suggest that the suppression of APJ expression after Dox administration can exacerbate Dox-induced cardiotoxicity, which may be responsible for depressed protective function of the endogenous apelin-APJ system. Modulation of the apelin-APJ system may hold promise for the treatment of Dox-induced cardiotoxicity.

Evaluation of novel cyclic analogues of apelin.

Apelin regulates various cell signaling processes through interaction with its specific cell-surface receptor, APJ, which is a member of a seven transmembrane G protein-coupled receptor superfamily. To develop a novel apelin analogue, we synthesized cyclic analogues of minimal apelin fragment RPRLSHKGPMPF (apelin-12), and evaluated their bioactivities in a recombinant human APJ-expressed cell line. Three cyclic analogues were synthesized: cyclo apelin-12 (C1) in combination with amino-terminal to carboxy-terminal, cyclourea apelin-12 (C3) in combination with amino-terminal and amino acid side chain at positions 7, and cyclic apelin-12 (C4) in combination with amino acid side chain at positions 7 to carboxy-terminal. All cyclic analogues exhibited dose-dependent inhibitory effects against forskolin-induced cyclic adenosine monophosphate (cAMP) accumulation, and the maximal effects were almost abolished by pertussis toxin (PTx) treatment. Moreover, they could modulate the intracellular signaling pathways composed of Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) serine/threonine protein kinases in PTx-sensitive manner. This is the first approach to apply cyclization on apelin, and these results provide the basis for the development of drug-like apelin analogues.

Regulation of sympathetic and parasympathetic nerve activities by BIT/SHPS-1.

The hypothalamus plays a central role in the homeostatic regulation of internal physiological conditions such as body temperature and energy balance. We have previously shown that cold exposure enhances tyrosine phosphorylation of BIT/SHPS-1 (brain immunoglobulin-like molecule with tyrosine-based activation motifs/SHP substrate-1) in hypothalamic nuclei including the suprachiasmatic nucleus. In order to elucidate the function of BIT/SHPS-1 in the hypothalamus, we stimulated BIT/SHPS-1 in vivo by using the anti-BIT monoclonal antibody (mAb) 1D4, which reacts with the extracellular domain of BIT/SHPS-1 and induces its tyrosine phosphorylation. Administration of mAb 1D4 into the third cerebral ventricle enhanced the electrical activity of the renal sympathetic nerves, while it suppressed that of the gastric parasympathetic nerves. Similarly, blood pressure increased in response to the mAb 1D4 injection, and additionally, temperatures of the abdomen and brown adipose tissue increased. These results indicate that BIT/SHPS-1 is involved in the hypothalamic regulation of thermogenesis via the autonomic nervous system.

Ground-based assessment of JAXA mouse habitat cage unit by mouse phenotypic studies.

The Japan Aerospace Exploration Agency developed the mouse Habitat Cage Unit (HCU) for installation in the Cell Biology Experiment Facility (CBEF) onboard the Japanese Experimental Module ("Kibo") on the International Space Station. The CBEF provides "space-based controls" by generating artificial gravity in the HCU through a centrifuge, enabling a comparison of the biological consequences of microgravity and artificial gravity of 1 g on mice housed in space. Therefore, prior to the space experiment, a ground-based study to validate the habitability of the HCU is necessary to conduct space experiments using the HCU in the CBEF. Here, we investigated the ground-based effect of a 32-day housing period in the HCU breadboard model on male mice in comparison with the control cage mice. Morphology of skeletal muscle, the thymus, heart, and kidney, and the sperm function showed no critical abnormalities between the control mice and HCU mice. Slight but significant changes caused by the HCU itself were observed, including decreased body weight, increased weights of the thymus and gastrocnemius, reduced thickness of cortical bone of the femur, and several gene expressions from 11 tissues. Results suggest that the HCU provides acceptable conditions for mouse phenotypic analysis using CBEF in space, as long as its characteristic features are considered. Thus, the HCU is a feasible device for future space experiments.

PRMT8 as a phospholipase regulates Purkinje cell dendritic arborization and motor coordination.

The development of vertebrate neurons requires a change in membrane phosphatidylcholine (PC) metabolism. Although PC hydrolysis is essential for enhanced axonal outgrowth mediated by phospholipase D (PLD), less is known about the determinants of PC metabolism on dendritic arborization. We show that protein arginine methyltransferase 8 (PRMT8) acts as a phospholipase that directly hydrolyzes PC, generating choline and phosphatidic acid. We found that PRMT8 knockout mice (prmt8 (-/-)) displayed abnormal motor behaviors, including hindlimb clasping and hyperactivity. Moreover, prmt8 (-/-) mice and TALEN-induced zebrafish prmt8 mutants and morphants showed abnormal phenotypes, including the development of dendritic trees in Purkinje cells and altered cerebellar structure. Choline and acetylcholine levels were significantly decreased, whereas PC levels were increased, in the cerebellum of prmt8 (-/-) mice. Our findings suggest that PRMT8 acts both as an arginine methyltransferase and as a PC-hydrolyzing PLD that is essential for proper neurological functions.

Tyrosine phosphorylation of BIT on photic stimulation in the rat retina.

BIT is a transmembrane glycoprotein with three immunoglobulin-like domains in its extracellular region and tyrosine phosphorylation sites in its cytosolic region. We have previously shown that BIT was tyrosine phosphorylated in the hypothalamic suprachiasmatic nucleus in response to light exposure during the dark period, and suggested that it was involved in the light entrainment of the circadian clock. To further investigate the function of BIT in the nervous system, we examined the effect of photic stimulation on its tyrosine phosphorylation in the rat retina. It was found that the tyrosine phosphorylation level of BIT in the retina was higher in the light period than in the dark period. In addition, a light stimulation during the dark period resulted in a rapid phosphorylation of BIT and a subsequent association of BIT with SHP-2. The phosphorylation state was quickly reverted when the light was turned off. The light-dependent phosphorylation of BIT was also observed in isolated cultured retinas, and this was blocked by a specific Src-family inhibitor, PP-2. Immunohistochemical study showed that BIT was highly enriched in the inner and outer plexiform layers in the retina, where the immunoreactivity to anti-SHP-2 antibody was also detected. These results suggest that tyrosine phosphorylation of BIT is involved in neuronal transmission in the retina.

Stimulation of BIT induces a circadian phase shift of locomotor activity in rats.

Circadian rhythms of mammals are generated by a circadian oscillation of master pacemaker genes in the suprachiasmatic nucleus of the hypothalamus (SCN), and entrained by environmental factors such as 24-h light-dark cycles. We have previously shown that light exposure during the dark period enhanced tyrosine phosphorylation of brain immunoglobulin-like molecule with tyrosine-based activation motifs (BIT) in the rat SCN. To elucidate the functional roles of BIT in the circadian clock, we stimulated BIT using an anti-BIT monoclonal antibody (mAb) 1D4, which reacts with its extracellular region and induces phosphorylation of its intracellular tyrosine residues. Administration of mAb 1D4 into the third cerebral ventricle induced tyrosine phosphorylation of BIT in the SCN. Behavioral analyses showed that the SCN-injection of the antibody at CT15 induced a phase delay of the circadian rhythm of locomotor activity, and that at CT20 induced a phase advance. Pretreatment with MK801, a non-competitive antagonist of NMDARs, diminished the 1D4-induced phase shift at CT20, but not at CT15. These results suggest that BIT is involved in the entrainment of circadian rhythms through the function of NMDARs and non-NMDARs.

Effect of lactation on postpartum cardiac function of pregnancy-associated hypertensive mice.

Preeclampsia is a serious complication during pregnancy, and recent epidemiological studies indicate the association between preeclampsia and cardiac morbidity and mortality during the postpartum period. Although the risk of cardiovascular diseases in the postpartum period is affected by lactation, its role in maternal heart with a history of preeclampsia remains unclear. In this study, we investigated postpartum change in cardiac remodeling and function of pregnancy-associated hypertensive (PAH) mice with and without lactation. The systolic blood pressure was increased in PAH mice at day 19 of gestation (E19) and was reduced to normal levels in both lactating and nonlactating (NL) groups in the postpartum period. Histological analyses revealed that cardiac hypertrophy and macrophage infiltration in PAH mice at E19 were improved in both lactating and NL groups at 4 weeks postpartum (4W-PP), while marked fibrosis remained. Increased mRNA expression of profibrotic genes and proinflammatory cytokines in PAH mice at E19 was significantly reduced in both lactating and NL groups at 4W-PP. Echocardiographic analysis found no significant differences in fractional shortening between PAH mice and C57BL/6J mice at E19. On the other hand, at 4W-PP, NL PAH mice showed normal fractional shortening, but lactating PAH mice exhibited significant decreases in cardiac contractility compared with NL PAH mice. These results show that cardiac remodeling induced by hypertension during pregnancy are improved in the postpartum period except fibrosis, whereas lactation induces cardiac contractile dysfunction in mice with a history of pregnancy-associated hypertension.

Molecular aggregates of partially fluorinated quaternary ammonium salt gemini surfactants.

The size and shape of novel partially fluorinated gemini surfactant 1,2-bis[dimethyl-(3-perfluoroalkyl-2-hydroxypropyl)ammonium]ethane bromide (CnFC3-2-C3CnF, where n=4, 6, and 8) were investigated in aqueous solution by means of light scattering and transmission electron microscopy (TEM). The sizes of these molecular aggregates changed with increasing carbon number of the alkyl chain and concentration. For example, the apparent hydrodynamic radius by dynamic light scattering was 18 nm at a concentration of cmcx5 for n=4, 115 nm at the cmcx15 for n=6, and 62 nm at the cmcx30 for n=8, at 298.2 K. The shapes of CnFC3-2-C3CnF aggregates drastically changed with the alkyl chain length; the aggregates were mainly in the form of large or irregular small aggregates (n=4), string-like aggregates (n=6), and vesicles (n=8). The bromide-ion activity was measured using a bromide-ion-selective electrode to determine the degree of counterion binding to the aggregates. The degree of counterion binding to aggregate was very small compared with that in the typical hydrogenated gemini surfactants. These results indicated that the small curvature of large aggregates was not influenced by an electrostatic repulsion between the cationic head groups in the case of the bulky molecular volume of fluorinated gemini surfactants.

Axonal guidance protein FEZ1 associates with tubulin and kinesin motor protein to transport mitochondria in neurites of NGF-stimulated PC12 cells.

Fasciculation and elongation protein zeta-1 (FEZ1) promotes efficiently the neurite elongation of rat phaeochromocytoma PC12 cells. We here characterized FEZ1 in PC12 cells. Nerve growth factor (NGF) stimulation induces significant expression of endogenous FEZ1 in PC12 cells. Upon NGF stimulation FEZ1 localizes in both cytoplasm and neuritis, co-localizing with mitochondria. Silencing of FEZ1 by RNA interference efficiently reduces NGF-induced neurite elongation and the anterograde motility of mitochondria in PC12 cells. Immunoprecipitation and pulldown assay shows that FEZ1 interacts with kinesin superfamily protein 5 (KIF5) and tubulin. Thus, our results suggest that the FEZ1/kinesin complex functions for the transport of mitochondria along microtubules toward the extending neurites in differentiating PC12 cells.

Cold exposure induces tyrosine phosphorylation of BIT through NMDA receptors in the rat hypothalamus.

The hypothalamus has a central role in maintaining homeostases of physiological conditions including body temperature and energy balance. To examine molecular responses to cold exposure in the hypothalamus, we examined changes in protein tyrosine phosphorylation in the suprachiasmatic nucleus of the hypothalamus after acute cold exposure in rats. It was found that brain immunoglobulin-like molecule with tyrosine-based inhibitory motifs (BIT, also called SHPS-1, SIRPalpha or p84), a transmembrane glycoprotein with two ITIM motifs, showed enhanced tyrosine phosphorylation after cold exposure. Its tyrosine phosphorylation induced by cold exposure was also found in other hypothalamic nuclei including the paraventricular nucleus, lateral hypothalamic area, ventromedial hypothalamus, and arcuate nucleus. This phosphorylation was blocked by AP-5, an NMDA receptor antagonist, indicating that it was mediated by NMDA receptors. These results suggest that BIT is involved in the mechanism of neuronal responses to cold exposure in the hypothalamus.

Identification of a tissue-non-specific homologue of axonal fasciculation and elongation protein zeta-1.

Fasciculation and elongation protein zeta-1 (FEZ1) is a mammalian orthologue of the Caenorhabditis elegans UNC-76 protein involved in the axonal outgrowth and fasciculation and promotes neurite extension of PC12 cells through interaction with protein kinase C zeta (PKCzeta). The gene coding for FEZ2, a homologue of FEZ1, has also been reported in rat and human. In this study, we compared mRNA expression of FEZ1 and FEZ2 in adult rat tissues and mouse embryos by Northern blot and in situ hybridization analyses. In contrast to FEZ1 whose mRNA is expressed almost exclusively in rat brain and temporarily around the neurogenesis stage of mouse embryos, the message for FEZ2 is detected weakly in most tissues and abundantly throughout the mouse embryonic stages. Similar to FEZ1, FEZ2 interacted with PKCzeta and induced neurite extension of PC12 cells when coexpressed with a constitutively active mutant of PKCzeta. These results suggest that FEZ2 plays an important role in the morphological changes of various cells by associating with PKCzeta in a tissue-non-specific manner.