Compensatory role of neuregulin-1 in diabetic cardiomyopathy.

Diabetes mellitus is a prevalent risk factor for congestive heart failure. Diabetic cardiomyopathy patients present with left ventricular (LV) diastolic dysfunction at an early stage, then systolic dysfunction as the disease progresses. The mechanism underlying the development of diabetic cardiomyopathy has not yet been fully understood. This study aimed to elucidate the mechanisms by which diastolic dysfunction precedes systolic dysfunction at the early stage of diabetic cardiomyopathy. We hypothesized that the downregulation of cardioprotective factors is involved in the pathogenesis of diabetic cardiomyopathy. LV diastolic dysfunction, but not systolic dysfunction, was observed in type-1 diabetes mellitus model mice 4 weeks after STZ administration (STZ-4W), mimicking the early stage of diabetic cardiomyopathy. Counter to expectations, neuregulin-1 (NRG1) was markedly upregulated in the vascular endothelial cell in the ventricles of STZ-4W mice. To clarify the functional significance of the upregulated NRG1, we blocked its receptor ErbB2 with trastuzumab (TRZ). In STZ-4W mice, TRZ significantly reduced the systolic function without affecting diastolic function and caused a more prominent reduction in Akt phosphorylation levels. These results indicate that the compensatory upregulated NRG1 contributes to maintaining the LV systolic function, which explains why diastolic dysfunction precedes systolic dysfunction at the early stage of diabetic cardiomyopathy.

Pathogenic Mechanism of Dry Eye-Induced Chronic Ocular Pain and a Mechanism-Based Therapeutic Approach.

Purpose: Dry eye-induced chronic ocular pain is also called ocular neuropathic pain. However, details of the pathogenic mechanism remain unknown. The purpose of this study was to elucidate the pathogenic mechanism of dry eye-induced chronic pain in the anterior eye area and develop a pathophysiology-based therapeutic strategy. Methods: We used a rat dry eye model with lacrimal gland excision (LGE) to elucidate the pathogenic mechanism of ocular neuropathic pain. Corneal epithelial damage, hypersensitivity, and hyperalgesia were evaluated on the LGE side and compared with the sham surgery side. We analyzed neuronal activity, microglial and astrocytic activity, α2δ-1 subunit expression, and inhibitory interneurons in the trigeminal nucleus. We also evaluated the therapeutic effects of ophthalmic treatment and chronic pregabalin administration on dry eye-induced ocular neuropathic pain. Results: Dry eye caused hypersensitivity and hyperalgesia on the LGE side. In the trigeminal nucleus of the LGE side, neuronal hyperactivation, transient activation of microglia, persistent activation of astrocytes, α2δ-1 subunit upregulation, and reduced numbers of inhibitory interneurons were observed. Ophthalmic treatment alone did not improve hyperalgesia. In contrast, continuous treatment with pregabalin effectively ameliorated hypersensitivity and hyperalgesia and normalized neural activity, α2δ-1 subunit upregulation, and astrocyte activation. Conclusions: These results suggest that dry eye-induced hypersensitivity and hyperalgesia are caused by central sensitization in the trigeminal nucleus with upregulation of the α2δ-1 subunit. Here, we showed that pregabalin is effective for treating dry eye-induced ocular neuropathic pain even after chronic pain has been established.

A Novel Non-Invasive Method for Estimating Elevated Pulmonary Vascular Resistance Based on Echocardiographic Assessment of Pulmonary Artery Wave Reflection.

BACKGROUND: Several non-invasive methods for pulmonary vascular resistance (PVR) measurement are proposed, but none are sufficiently accurate for use in clinical practice. This study proposes a new echocardiographic method of pulmonary artery wave reflection and investigates its efficacy in managing patients with pulmonary hypertension.Methods and Results:In total, 83 patients with left heart disease, pulmonary arterial hypertension, and chronic thromboembolic pulmonary hypertension (CTEPH), who underwent Doppler echocardiography and right heart catheterization, were included in the study. Pulmonary artery wave reflection was characterized by separating the pulmonary artery pressure waveform into forward and backward (Pb) waves, based on wave intensity. Pulmonary artery pressure waveforms were estimated from continuous Doppler tracings of tricuspid regurgitation velocity, and flow velocity was measured using pulsed Doppler of the right ventricular outflow tract. Pb-peak was compared with catheter hemodynamic indices, and with PVR by Abbas 2003, 2013 and Haddad in relation to increased catheter PVR. Catheter PVR and Pb were strongly correlated (r=0.77, P<0.001). The areas under the receiver operator characteristic curve for Pb-peak, PVR by Abbas 2003, 2013 and Haddad were 0.91, 0.72, 0.80, and 0.80, respectively, and were used to detect an increase in PVR (>3 Woods units). CONCLUSIONS: This study describes a novel, simple, and non-invasive echocardiography method to assess pulmonary wave reflected pressure to identify patients with pulmonary hypertension due to increased PVR.

Quantification of BIM mRNA in circulating tumor cells of osimertinib-treated patients with EGFR mutation-positive lung cancer.

BACKGROUND: The response rate for osimertinib is high among patients with untreated epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer (NSCLC). However, there exist no biomarkers to predict the efficacy of the same. This study investigated whether BIM-γ mRNA expression in circulating tumor cells (CTCs) predicts poor outcomes for osimertinib treatment in patients with EGFR mutation-positive NSCLC. METHODS: Patients with advanced EGFR-tyrosine kinase inhibitor-untreated NSCLC or post-operative recurrence with EGFR-sensitive mutations (exon 19 deletion or L858R mutation) were included. Informed consent was obtained from all participants. The candidate biomarker BIM-γ was measured in CTCs after blood collection (10 mL of whole blood) at baseline. CTCs were collected with the ClearCell FX system, and quantitative real-time PCR was performed. Relative expression of BIM-γ mRNA from CTCs, as normalized to the reference gene (GAPDH mRNA), was calculated using the KCL22 cell line for calibration. RESULTS: We enrolled 30 EGFR mutation-positive NSCLC patients treated with osimertinib during the period from April 2018 through December 2019. All the patients had an EGFR mutation at the primary site: exon 19 deletion in 15 cases and L858R in 15 cases. Median CTC count at baseline was 12 (range 3-127)/7.5 mL, and median BIM-γ mRNA expression was 0.073 (range 0-1.37). Furthermore, the response rate to osimertinib was worse in patients with high than in those with low BIM-γ mRNA expression (n = 15 each) (26.6% vs. 73.3%, respectively; p = 0.011). Progression-free survival did not significantly differ between groups (p = 0.13). CONCLUSIONS: BIM-γ mRNA overexpression in CTCs from EGFR mutation-positive NSCLC patients is a potential a biomarker for poor response to osimertinib. CLINICAL TRIAL REGISTRATION NUMBER: UMIN:00032055.

Interleukin-11-expressing fibroblasts have a unique gene signature correlated with poor prognosis of colorectal cancer.

Interleukin (IL)-11 is a member of the IL-6 family of cytokines and is involved in multiple cellular responses, including tumor development. However, the origin and functions of IL-11-producing (IL-11+) cells are not fully understood. To characterize IL-11+ cells in vivo, we generate Il11 reporter mice. IL-11+ cells appear in the colon in murine tumor and acute colitis models. Il11ra1 or Il11 deletion attenuates the development of colitis-associated colorectal cancer. IL-11+ cells express fibroblast markers and genes associated with cell proliferation and tissue repair. IL-11 induces the activation of colonic fibroblasts and epithelial cells through phosphorylation of STAT3. Human cancer database analysis reveals that the expression of genes enriched in IL-11+ fibroblasts is elevated in human colorectal cancer and correlated with reduced recurrence-free survival. IL-11+ fibroblasts activate both tumor cells and fibroblasts via secretion of IL-11, thereby constituting a feed-forward loop between tumor cells and fibroblasts in the tumor microenvironment.

[Roles of p38 MAPK signaling in the skeletal muscle formation, regeneration, and pathology].

Sarcopenia and frailty in aging, or cancer cachexia shows an abnormal decrease in skeletal muscle mass and muscle strength. However, the underlying mechanisms are not clear, and the promising drug seeds have not been discovered. The formation of skeletal muscle occurs not only during embryonic development but also in adulthood, and the muscle can be regenerated even if it is damaged by exercise overload or physical injury. Although p38MAPK is ubiquitous among tissues and transmits signal of inflammation and environmental stress into the nucleus, it has been revealed that this kinase is deeply involved in maintaining skeletal muscle homeostasis. Knowledge of p38MAPK accumulated so far suggests that it not only functions as an on-off switch for gene expression, but also it balances cell proliferation and differentiation of progenitor cells to properly respond to muscle damage and repair muscle according to its surrounding environmental cues. In addition, its role in cell fusion to induce myotube formation has been recently revealed. On the other hand, it has been pointed out that in aging and chronic inflammation, excessive enhancement of the p38MAPK activity may disrupt skeletal muscle homeostasis and lead to muscle pathology. Interestingly, animal models have shown that pharmacological manipulation of p38MAPK activity can re-activate aged muscle satellite cells, suggesting the possibility of plastically manipulating skeletal muscle aging. Furthermore, it has become possible to track the dynamics of intracellular signaling of skeletal muscle cells or muscle progenitor cells in time and space by using advanced imaging techniques. In this review, we focus on the functional roles and regulatory mechanism of p38MAPK in skeletal muscle and its relation to the pathology in the context of dysregulation of skeletal muscle formation and regeneration.

Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono-phosphorylated Pbs2 MAP2K.

The MAP kinase (MAPK) Hog1 is the central regulator of osmoadaptation in yeast. When cells are exposed to high osmolarity, the functionally redundant Sho1 and Sln1 osmosensors, respectively, activate the Ste11-Pbs2-Hog1 MAPK cascade and the Ssk2/Ssk22-Pbs2-Hog1 MAPK cascade. In a canonical MAPK cascade, a MAPK kinase kinase (MAP3K) activates a MAPK kinase (MAP2K) by phosphorylating two conserved Ser/Thr residues in the activation loop. Here, we report that the MAP3K Ste11 phosphorylates only one activating phosphorylation site (Thr-518) in Pbs2, whereas the MAP3Ks Ssk2/Ssk22 can phosphorylate both Ser-514 and Thr-518 under optimal osmostress conditions. Mono-phosphorylated Pbs2 cannot phosphorylate Hog1 unless the reaction between Pbs2 and Hog1 is enhanced by osmostress. The lack of the osmotic enhancement of the Pbs2-Hog1 reaction suppresses Hog1 activation by basal MAP3K activities and prevents pheromone-to-Hog1 crosstalk in the absence of osmostress. We also report that the rapid-and-transient Hog1 activation kinetics at mildly high osmolarities and the slow and prolonged activation kinetics at severely high osmolarities are both caused by a common feedback mechanism.

Addendum: A FRET biosensor for necroptosis uncovers two different modes of the release of DAMPs.

The cDNA sequence of human SMART described in this Article was misreported, as described in the accompanying Addendum. This error does not affect the results or any conclusion of the Article.

A FRET biosensor for necroptosis uncovers two different modes of the release of DAMPs.

Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like (MLKL). While danger-associated molecular pattern (DAMP)s are involved in various pathological conditions and released from dead cells, the underlying mechanisms are not fully understood. Here we develop a fluorescence resonance energy transfer (FRET) biosensor, termed SMART (a sensor for MLKL activation by RIPK3 based on FRET). SMART is composed of a fragment of MLKL and monitors necroptosis, but not apoptosis or necrosis. Mechanistically, SMART monitors plasma membrane translocation of oligomerized MLKL, which is induced by RIPK3 or mutational activation. SMART in combination with imaging of the release of nuclear DAMPs and Live-Cell Imaging for Secretion activity (LCI-S) reveals two different modes of the release of High Mobility Group Box 1 from necroptotic cells. Thus, SMART and LCI-S uncover novel regulation of the release of DAMPs during necroptosis.

Immunolocalization of muscarinic M1 receptor in the rat medial prefrontal cortex.

The medial prefrontal cortex (mPFC) has been considered to participate in many higher cognitive functions, such as memory formation and spatial navigation. These cognitive functions are modulated by cholinergic afferents via muscarinic acetylcholine receptors. Previous pharmacological studies have strongly suggested that the M1 receptor (M1R) is the most important subtype among muscarinic receptors to perform these cognitive functions. Actually, M1R is abundant in mPFC. However, the proportion of somata containing M1R among cortical cellular types, and the precise intracellular localization of M1R remain unclear. In this study, to clarify the precise immunolocalization of M1R in rat mPFC, we examined three major cellular types, pyramidal neurons, inhibitory neurons, and astrocytes. M1R immunopositivity signals were found in the majority of the somata of both pyramidal neurons and inhibitory neurons. In pyramidal neurons, strong M1R immunopositivity signals were usually found throughout their somata and dendrites including spines. On the other hand, the signal strength of M1R immunopositivity in the somata of inhibitory neurons significantly varied. Some neurons showed strong signals. Whereas about 40% of GAD67-immunopositive neurons and 30% of parvalbumin-immunopositive neurons (PV neurons) showed only weak signals. In PV neurons, M1R immunopositivity signals were preferentially distributed in somata. Furthermore, we found that many astrocytes showed substantial M1R immunopositivity signals. These signals were also mainly distributed in their somata. Thus, the distribution pattern of M1R markedly differs between cellular types. This difference might underlie the cholinergic modulation of higher cognitive functions subserved by mPFC.

Epac1-dependent phospholamban phosphorylation mediates the cardiac response to stresses.

PKA phosphorylates multiple molecules involved in calcium (Ca2+) handling in cardiac myocytes and is considered to be the predominant regulator of ß-adrenergic receptor-mediated enhancement of cardiac contractility; however, recent identification of exchange protein activated by cAMP (EPAC), which is independently activated by cAMP, has challenged this paradigm. Mice lacking Epac1 (Epac1 KO) exhibited decreased cardiac contractility with reduced phospholamban (PLN) phosphorylation at serine-16, the major PKA-mediated phosphorylation site. In Epac1 KO mice, intracellular Ca2+ storage and the magnitude of Ca2+ movement were decreased; however, PKA expression remained unchanged, and activation of PKA with isoproterenol improved cardiac contractility. In contrast, direct activation of EPAC in cardiomyocytes led to increased PLN phosphorylation at serine-16, which was dependent on PLC and PKCε. Importantly, Epac1 deletion protected the heart from various stresses, while Epac2 deletion was not protective. Compared with WT mice, aortic banding induced a similar degree of cardiac hypertrophy in Epac1 KO; however, lack of Epac1 prevented subsequent cardiac dysfunction as a result of decreased cardiac myocyte apoptosis and fibrosis. Similarly, Epac1 KO animals showed resistance to isoproterenol- and aging-induced cardiomyopathy and attenuation of arrhythmogenic activity. These data support Epac1 as an important regulator of PKA-independent PLN phosphorylation and indicate that Epac1 regulates cardiac responsiveness to various stresses.

A subset of thalamocortical projections to the retrosplenial cortex possesses two vesicular glutamate transporter isoforms, VGluT1 and VGluT2, in axon terminals and somata.

Vesicular glutamate transporter isoforms, VGluT1-VGluT3, accumulate glutamate into synaptic vesicles and are considered to be important molecules in glutamatergic transmission. Among them, VGluT2 mRNA is expressed predominantly throughout the dorsal thalamus, whereas VGluT1 mRNA is expressed in a few thalamic nuclei. In the thalamic nuclei that project to the retrosplenial cortex (RSC), VGluT1 mRNA is expressed strongly in the anterodorsal thalamic nucleus (AD), is expressed moderately in the anteroventral and laterodorsal thalamic nuclei, and is not expressed in the anteromedial thalamic nucleus. Thus, it has been strongly suggested that a subset of thalamocortical projections to RSC possesses both VGluT1 and VGluT2. In this study, double-labeled neuronal somata showing both VGluT1 and VGluT2 immunolabelings were found exclusively in the ventral region of AD (vAD). Many double-labeled axon terminals were also found in two major targets of vAD, the rostral part of the reticular thalamic nucleus and layers Ia and III-IV of the retrosplenial granular b cortex (RSGb). Some were also found in layer Ia of the retrosplenial granular a cortex (RSGa). These axon terminals contain significant amounts of both VGluTs. Because the subset of thalamocortical projections to RSC has a unique molecular basis in the glutamatergic transmission system, it might play an important role in the higher cognitive functions processed in the RSC. Furthermore, double-labeled axon terminals of a different type were distributed in RSGb and RSGa. Because they are small and the immunoreactivity of VGluT2 is significantly weaker than that of VGluT1, they seemed to be a subset of corticocortical terminals.

Inter-organ communication in the regulation of lipid metabolism: focusing on the network between the liver, intestine, and heart.

Recent studies have shown that lipid metabolism is regulated through the orchestration of multiple organs. Gut microbiota influences the metabolism of the liver through the production of fatty acids and phosphatidylcholine as well as the modulation of bile acid profile. Microbiota also affects the cardiovascular system through the production of metabolites from nutrients. MicroRNAs (miRNAs) are non-coding RNAs comprised of around 22 nucleotides in length. MiRNAs are released into blood flow from organs and interfere with the gene expression of target organs. MiRNAs are involved in the regulation of metabolic homeostasis including lipoprotein production and cardiovascular functions. Fatty acids are also circulating and distributed to each organ by fatty acid transporting proteins. Fatty acids can act as a ligand of G protein-coupled receptors, such as GPR41 and GPR43, and nuclear receptor PPARα, which bear crucial roles in the regulation of energy expenditure. Therefore the inter-organ communication plays important roles in the systematic regulation of lipid metabolism. Studies on the inter-organ network system will contribute to the development of diagnostic and therapeutic strategies for metabolic diseases. This review discusses how lipid metabolism is regulated by the inter-organ communication, focusing on the network axis between the liver, intestine, and heart.

Cardiohemodynamic and electrophysiological effects of anti-influenza drug oseltamivir in vivo and in vitro.

Electropharmacological effects of oseltamivir were studied in comparison with pilsicainide using halothane-anesthetized dogs (n = 4) and isolated left atrium of guinea pigs (n = 5). Oseltamivir (0.3, 3 and 30 mg/kg, i.v.) or pilsicainide (1 and 3 mg/kg, i.v.) was additionally administered to the dogs. The low dose of oseltamivir provided clinically relevant plasma concentrations with C max of 4 µM. The low and middle doses of oseltamivir increased cardiac output, whereas the middle dose increased blood pressure and delayed intra-atrial conduction and ventricular repolarization. The high dose of oseltamivir exerted negative chronotropic, inotropic and hypotensive effects, while it delayed intra-atrial, atrioventricular nodal and intra-ventricular conduction and ventricular repolarization. Use-dependent delay of ventricular repolarization was observed after oseltamivir, whereas reverse use-dependent prolongation was induced by pilsicainide. Moreover, oseltamivir more selectively suppressed intra-atrial conduction than intra-ventricular conduction, which was less selective for pilsicainide. Action potential assay using isolated atrium indicated that oseltamivir (10 µM) decreased V max more than pilsicainide (10 µM) and that oseltamivir (10-100 µM) prolonged action potential duration, which was not induced by pilsicainide (1-10 µM). Thus, oseltamivir in clinically relevant to its 10 times higher doses is relatively safe, whereas 10-100 times higher doses possess unique electrophysiological profile.

Disruption of Stard10 gene alters the PPARα-mediated bile acid homeostasis.

STARD10, a member of the steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) protein family, is highly expressed in the liver and has been shown to transfer phosphatidylcholine. Therefore it has been assumed that STARD10 may function in the secretion of phospholipids into the bile. To help elucidate the physiological role of STARD10, we produced Stard10 knockout mice (Stard10(-/-)) and studied their phenotype. Neither liver content nor biliary secretion of phosphatidylcholine was altered in Stard10(-/-) mice. Unexpectedly, the biliary secretion of bile acids from the liver and the level of taurine-conjugated bile acids in the bile were significantly higher in Stard10(-/-) mice than wild type (WT) mice. In contrast, the levels of the secondary bile acids were lower in the liver of Stard10(-/-) mice, suggesting that the enterohepatic cycling is impaired. STARD10 was also expressed in the gallbladder and small intestine where the expression level of apical sodium dependent bile acid transporter (ASBT) turned out to be markedly lower in Stard10(-/-) mice than in WT mice when measured under fed condition. Consistent with the above results, the fecal excretion of bile acids was significantly increased in Stard10(-/-) mice. Interestingly, PPARα-dependent genes responsible for the regulation of bile acid metabolism were down-regulated in the liver of Stard10(-/-) mice. The loss of STARD10 impaired the PPARα activity and the expression of a PPARα-target gene such as Cyp8b1 in mouse hepatoma cells. These results indicate that STARD10 is involved in regulating bile acid metabolism through the modulation of PPARα-mediated mechanism.

Comparison of the effects of levofloxacin on QT/QTc interval assessed in both healthy Japanese and Caucasian subjects (pages.

AIMS: There is no consensus as to what extent the results of thorough QT interval/corrected QT interval (QT/QTc) studies need to be bridged. METHODS: The results of two studies using levofloxacin in Japanese and Caucasian subjects were compared in a post hoc analysis to investigate the similarity of dose­effect responses. RESULTS: Concentration­response analysis based on the change of QT interval corrected using Fridericia's formula (QTcF) from time-matched placebo was planned and performed in the combined data sets. At the geometric maximum mean concentration for the two doses in the Caucasian study, a predicted effect on QTcF comparable to the effects observed was found. For the Japanese study, the predicted effect was lower, but the difference was not statistically significant. CONCLUSIONS: No statistically significant differences in QTc-prolonging effect between Japanese and Caucasian subjects were observed following levofloxacin dosing. However, a trend suggests that Caucasian subjects may be more sensitive. Age and sex did not have an impact.

Spinal mechanism underlying the antiallodynic effect of gabapentin studied in the mouse spinal nerve ligation model.

We studied the antiallodynic effect of gabapentin (GBP) in the mouse model of neuropathic pain, aiming at clarifying the underlying mechanism. The L5 spinal nerve ligation induced tactile allodynia, an increase of CD11b expression, and an increase in the protein expression level of the voltage-dependent Ca(2+) channel α(2)/δ-1 subunit in the spinal dorsal horn on the injured side. The chronic intrathecal administration of GBP (100 µg/body per day) as well as ω-conotoxin MVIIA, an N-type Ca(2+)-channel blocker, completely suppressed allodynia, but did not attenuate the CD11b expression. The antiallodynic effect of GBP lasted for several days after the termination of the drug, while that of ω-conotoxin MVIIA disappeared immediately after the termination. GBP suppressed the elevation of the protein level of the α(2)/δ-1 subunit in the spinal dorsal horn, although it did not affect its mRNA level in the L5 DRG. These results suggest that GBP inhibits the development of allodynia by suppressing the up-regulation of N-type Ca(2+) channels, through normalization of the protein level of the α(2)/δ-1 subunit at the primary afferent nerve terminal via the inhibition of its anterograde transport. In addition, we propose that the nerve injury enhances the expression level of α(2)/δ-1 in the downstream of the activation of microglia.

Effects of S(+)-efonidipine on the rabbit sinus node action potential and calcium channel subunits Ca(V)1.2, Ca(V)1.3 and Ca(V)3.1.

The effect of S(+)-efonidipine on sinus node action potential and calcium channel α-subunits was examined. The slope of the phase 4 depolarization of isolated rabbit sinus node tissue was significantly reduced by S(+)-efonidipine (1 µM), slightly reduced by nifedipine (1 µM), but was not affected by R(-)-efonidipine. S(+)-efonidipine (1 µM), inhibited the expressed Ca(V)1.2, Ca(V)1.3 and Ca(V)3.1 channel currents by 75.7%, 75.3% and 94.0%, nifedipine 84.0%, 43.2% and 14.9%, and R(-)-efonidipine 30.0%, 19.6% and 92.8%, respectively. Thus, the prolongation of the phase 4 depolarization of the rabbit sinus node by S(+)-efonidipine may be explained by blockade of the Ca(V)1.3 channel current.