Modification of cardiac disease by transgenically altered histone deacetylase 6.

Histone deacetylase 6 (HDAC6) is known to deacetylate amino acid lysine in alpha-tubulin. However, the functional role of HDAC6 in the progression of cardiac disease remains uncertain. The functional role of HDAC6 in the hearts was examined using transgenic (TG) mice expressing either human wild-type HDAC6, deacetylase inactive HDAC6 (HDAC6H216A, H611A), and human HDAC6 replaced all serine or threonine residues with aspartic acid at N-terminal 1- 43 amino acids (HDAC6NT-allD) specifically in the hearts. Overexpression of wild-type HDAC6 significantly reduced acetylated tubulin levels, and overexpression of HDAC6H216A, H611A significantly increased it in the mouse hearts. Detectable acetylated tubulin disappeared in HDAC6NT-allD TG mouse hearts. Neither histological alteration nor alteration of cardiac function was observed in the HDAC6 TG mouse hearts. To analyze the role of HDAC6 and acetylated tubulin in disease conditions, we examined HDAC6 in isoprenaline-induced hypertrophy or pressure-overload hypertrophy (TAC). No obvious alteration in the heart weight/body weight ratio or gene expressions of hypertrophic markers between NTG and HDAC6NT-allD mice was observed following treatment with isoprenaline. In contrast, a marked reduction in the shortening fraction and dilated chamber dilatation was detected in the HDAC6NT-allD TG mouse hearts 2 weeks after TAC. A sustained low level of acetylated tubulin and acetylated cortactin was observed in the TAC HDAC6NT-allD TG mouse hearts. Cardiac HDAC6 activity that can regulate acetylated levels of tubulin and cortactin may be critical factors involved in cardiac disease such as pressure-overload hypertrophy.

Chronic HDAC6 Activation Induces Atrial Fibrillation Through Atrial Electrical and Structural Remodeling in Transgenic Mice.

Atrial fibrillation (AF) is a relatively common complication of hypertension. Chronic hypertension induces cardiac HDAC6 catalytic activity. However, whether HDAC6 activation contributes to hypertension-induced AF is still uncertain. We examined whether chronic cardiac HDAC6 activation-induced atrial remodeling, leading to AF induction.The HDAC6 constitutively active transgenic (TG) (HDAC6 active TG) mouse overexpressing the active HDAC6 protein, specifically in cardiomyocytes, was created to examine the effects of chronic HDAC6 activation on atrial electrical and structural remodeling and AF induction in HDAC6 active TG and non-transgenic (NTG) mice. Left atrial burst pacing (S1S1 = 30 msec) for 15-30 sec significantly increased the frequency of sustained AF in HDAC6 active-TG mice compared with NTG mice. Left steady-state atrial pacing (S1S1 = 80 msec) decreased the atrial conduction velocity in isolated HDAC6 active TG compared with NTG mouse atria. The atrial size was similar between HDAC6 active TG and NTG mice. In contrast, atrial interstitial fibrosis increased in HDAC6 active TG compared with that of NTG mouse atria. While protein expression levels of both CX40 and CX43 were similar between HDAC6 active TG and NTG mouse atria, a heterogeneous distribution of CX40 and CX43 occurred in HDAC6 active-TG mouse atria but not in NTG mouse atria. Gene expression of interleukin 6 increased in HDAC6 active TG compared with NTG mouse atria.Chronic cardiac HDAC6 activation induced atrial electrical and structural remodeling, and sustained AF. Hypertension-induced cardiac HDAC6 catalytic activity may play important roles in the development of AF.

Topical administration of a ROCK inhibitor prevents anterior subcapsular cataract induced by UV-B irradiation.

The deposition of extracellular matrix (ECM)-which is mainly composed of type I collagen-in anterior subcapsular cataracts (ASCs) during epithelial-to-mesenchymal transition (EMT) of lens epithelial cells (LECs) decreases visual function. Transforming growth factor (TGF)-ß is a key factor in the induction of EMT in LECs. Although Rho kinase (ROCK) plays an important role in EMT induced by TGF-ß, it is unknown whether ROCK inhibition affects type I collagen expression in TGF-ß-stimulated LECs and ASC formation. This was investigated in the present study both in vitro using human lens epithelium (HLE)-B3 cells and in vivo using mice with ultraviolet radiation (UVR)-B-induced cataracts. We found that TGF-ß2 increased type I collagen mRNA expression in HLE-B3 cells; this was inhibited in a dose-dependent manner by treatment with the ROCK inhibitor Y-27632. UVR-B exposure caused ASC formation in mice. A histopathological examination revealed that LECs in the anterior subcapsular area were flattened and multi-layered, and had a spindle shape in cross section. Immunohistochemical analysis revealed the presence of α-smooth muscle actin and type I collagen around these flattened LECs; these opacities were reduced by topical instillation of Y-27632. These findings suggest that suppression of TGF-ß signaling in LECs by topical application of a ROCK inhibitor can prevent the formation of ASCs.

Bcl-2-associated athanogene 3 (BAG3) is an enhancer of small heat shock protein turnover via activation of autophagy in the heart.

Bcl-2-associated athanogene 3 (BAG3) is strongly expressed in both cardiac and skeletal muscle. A recent study showed that BAG3 may play a protective role in muscles. Little is known, however, regarding the detailed role of BAG3 in cardiac muscle. To better understand the functional role of cardiac BAG3 in the heart, we generated transgenic (TG) mice that overexpress BAG3. A decrease in fractional shortening, and the induction of cardiac atrial natriuretic peptide, were observed in BAG3 TG mice. Moreover, a marked reduction in the protein level of small HSPs was detected in BAG3 TG mouse hearts. We analyzed the cardiac small HSP levels when either the ubiquitin-proteasome system (UPS) or the autophagy system (AS) was inhibited in BAG3 TG mice. The protein turnovers of small HSPs by the AS were activated in BAG3 TG mouse hearts. Thus, BAG3 is critical for the protein turnover of small HSPs via activation of autophagy in the heart.

Circadian rhythms govern cardiac repolarization and arrhythmogenesis.

Sudden cardiac death exhibits diurnal variation in both acquired and hereditary forms of heart disease, but the molecular basis of this variation is unknown. A common mechanism that underlies susceptibility to ventricular arrhythmias is abnormalities in the duration (for example, short or long QT syndromes and heart failure) or pattern (for example, Brugada's syndrome) of myocardial repolarization. Here we provide molecular evidence that links circadian rhythms to vulnerability in ventricular arrhythmias in mice. Specifically, we show that cardiac ion-channel expression and QT-interval duration (an index of myocardial repolarization) exhibit endogenous circadian rhythmicity under the control of a clock-dependent oscillator, krüppel-like factor 15 (Klf15). Klf15 transcriptionally controls rhythmic expression of Kv channel-interacting protein 2 (KChIP2), a critical subunit required for generating the transient outward potassium current. Deficiency or excess of Klf15 causes loss of rhythmic QT variation, abnormal repolarization and enhanced susceptibility to ventricular arrhythmias. These findings identify circadian transcription of ion channels as a mechanism for cardiac arrhythmogenesis.

Chronic Pressure Overload Induces Cardiac Hypertrophy and Fibrosis via Increases in SGLT1 and IL-18 Gene Expression in Mice.

Increased gene expression levels of sodium-glucose cotransporter 1 (SGLT1) are associated with hypertrophic and ischemic cardiomyopathy. However, it remains unclear whether chronic pressure overload increases SGLT1 expression, which in turn induces hypertrophic cardiomyopathy. We hypothesized that pressure overload could increase SGLT1 gene expression, leading to the development of hypertrophic cardiomyopathy.To create pressure overload-induced cardiomyopathy, transverse aortic constriction (TAC) was performed in SGLT1-deficient (SGLT1-/-) and wild-type (WT) mice. Six weeks after surgery, all mice were investigated. We observed a reduction of left ventricular fractional shortening and left ventricular dilatation in TAC-operated WT but not in TAC-operated SGLT1-/- mice. SGLT1, interleukin 18, connective tissue growth factor, and collagen type 1 gene expression levels were increased in TAC-operated WT mouse hearts compared with that of sham-operated WT mouse hearts. Moreover, heart/body weight ratio and ventricular interstitial fibrosis were increased in TAC-operated WT mice compared with that of sham-operated WT mice. Interestingly, these factors did not increase in TAC-operated SGLT1-/- mice compared with that of sham-operated WT and SGLT1-/- mice. Phenylephrine, an adrenergic α1 receptor agonist, caused cardiomyocyte hypertrophy in neonatal WT mouse hearts to a significantly larger extent than in neonatal SGLT1-/- mouse hearts.In conclusion, the results indicate that chronic pressure overload increases SGLT1 and IL-18 gene expressions, leading to the development of hypertrophic cardiomyopathy. These results make SGLT1 a potential candidate for the therapeutic target for hypertension-induced cardiomyopathy.

Analysis of serotonin concentrations in human milk by high-performance liquid chromatography with fluorescence detection.

Serotonin (5-hydroxytryptamine, 5-HT) plays an important role in milk volume homeostasis in the mammary gland during lactation; 5-HT in milk may also affect infant development. However, there are few reports on 5-HT concentrations in human breast milk. To address this issue, we developed a simple method based on high-performance liquid chromatography with fluorescence detection (HPLC-FD) for measuring 5-HT concentrations in human breast milk. Breast milk samples were provided by four healthy Japanese women. Calibration curves for 5-HT in each sample were prepared with the standard addition method between 5 and 1000 ng/ml, and all had correlation coefficients >0.999. The recovery of 5-HT was 96.1%-101.0%, with a coefficient of variation of 3.39%-8.62%. The range of 5-HT concentrations estimated from the calibration curves was 11.1-51.1 ng/ml. Thus, the HPLC-FD method described here can effectively extract 5-HT from human breast milk with high reproducibility.

Serotonin suppresses ß-casein expression via PTP1B activation in human mammary epithelial cells.

Serotonin (5-hydroxytriptamine, 5-HT) has an important role in milk volume homeostasis within the mammary gland during lactation. We have previously shown that the expression of ß-casein, a differentiation marker in mammary epithelial cells, is suppressed via 5-HT-mediated inhibition of signal transduction and activator of transcription 5 (STAT5) phosphorylation in the human mammary epithelial MCF-12A cell line. In addition, the reduction of ß-casein in turn was associated with 5-HT7 receptor expression in the cells. The objective of this study was to determine the mechanisms underlying the 5-HT-mediated suppression of ß-casein and STAT5 phosphorylation. The ß-casein level and phosphorylated STAT5 (pSTAT5)/STAT5 ratio in the cells co-treated with 5-HT and a protein kinase A (PKA) inhibitor (KT5720) were significantly higher than those of cells treated with 5-HT alone. Exposure to 100 µM db-cAMP for 6 h significantly decreased the protein levels of ß-casein and pSTAT5 and the pSTAT5/STAT5 ratio, and significantly increased PTP1B protein levels. In the cells co-treated with 5-HT and an extracellular signal-regulated kinase1/2 (ERK) inhibitor (FR180294) or Akt inhibitor (124005), the ß-casein level and pSTAT5/STAT5 ratio were equal to those of cells treated with 5-HT alone. Treatment with 5-HT significantly induced PTP1B protein levels, whereas its increase was inhibited by KT5720. In addition, the PTP1B inhibitor sc-222227 increased the expression levels of ß-casein and the pSTAT5/STAT5 ratio. Our observations indicate that PTP1B directly regulates STAT5 phosphorylation and that its activation via the cAMP/PKA pathway downstream of the 5-HT7 receptor is involved in the suppression of ß-casein expression in MCF-12A cells.

Arf6 guanine nucleotide exchange factor cytohesin-2 binds to CCDC120 and is transported along neurites to mediate neurite growth.

The mechanism of neurite growth is complicated, involving continuous cytoskeletal rearrangement and vesicular trafficking. Cytohesin-2 is a guanine nucleotide exchange factor for Arf6, an Arf family molecular switch protein, controlling cell morphological changes such as neuritogenesis. Here, we show that cytohesin-2 binds to a protein with a previously unknown function, CCDC120, which contains three coiled-coil domains, and is transported along neurites in differentiating N1E-115 cells. Transfection of the small interfering RNA (siRNA) specific for CCDC120 into cells inhibits neurite growth and Arf6 activation. When neurites start to extend, vesicles containing CCDC120 and cytohesin-2 are transported in an anterograde manner rather than a retrograde one. As neurites continue extension, anterograde vesicle transport decreases. CCDC120 knockdown inhibits cytohesin-2 localization into vesicles containing CCDC120 and diffuses cytohesin-2 in cytoplasmic regions, illustrating that CCDC120 determines cytohesin-2 localization in growing neurites. Reintroduction of the wild type CCDC120 construct into cells transfected with CCDC120 siRNA reverses blunted neurite growth and Arf6 activity, whereas the cytohesin-2-binding CC1 region-deficient CCDC120 construct does not. Thus, cytohesin-2 is transported along neurites by vesicles containing CCDC120, and it mediates neurite growth. These results suggest a mechanism by which guanine nucleotide exchange factor for Arf6 is transported to mediate neurite growth.

Serotonin regulates ß-casein expression via 5-HT7 receptors in human mammary epithelial MCF-12A cells.

We previously reported that serotonin (5-hydroxytryptamine; 5-HT) suppresses ß-casein expression, a differentiation marker in mammary epithelial cells, via inhibition of the signal transducer and activator of transcription 5 (STAT5) phosphorylation in the human mammary epithelial cell line, MCF-12A. In this study, we investigated the expression pattern of the different 5-HT receptor subtypes in MCF-12A cells, and identified the receptors involved in 5-HT-mediated suppression of ß-casein protein expression. ß-Casein mRNA expression was inhibited by 30 µM 5-HT in a time-dependent manner. Treatment with 30 µM 5-HT for 72 h decreased ß-casein protein levels and STAT5 phosphorylation (pSTAT5). The cells expressed four 5-HT receptors subtypes (5-HTR1D, 2B, 3A, and 7) at the mRNA and protein level, and their expression was elevated by prolactin (PRL) treatment. Additionally, the mRNA levels of 5-HTR1D and 5-HTR7 were significantly higher than the other 5-HT receptors in the cells. Tryptophan hydroxylase 1 mRNA was detectable in the cells in the absence of PRL, and PRL treatment significantly increased its expression. ß-Casein and pSTAT5/STAT5 levels in the cells co-treated with 5-HT and a selective 5-HTR1D inhibitor, BRL15572, were equal to those observed in cells treated with 5-HT alone. However, in the cells co-treated with 5-HT and a selective 5-HTR7 inhibitor, SB269970, ß-casein and pSTAT5/STAT5 levels increased in a SB269970 concentration-dependent manner. In conclusion, we showed that 5-HT regulates ß-casein expression via 5-HTR7 in MCF-12A human mammary epithelial cells.

Phenotype of cardiomyopathy in cardiac-specific heat shock protein B8 K141N transgenic mouse.

A K141N missense mutation in heat shock protein (HSP) B8, which belongs to the small HSP family, causes distal hereditary motor neuropathy, which is characterized by the formation of inclusion bodies in cells. Although the HSPB8 gene causes hereditary motor neuropathy, obvious expression of HSPB8 is also observed in other tissues, such as the heart. The effects of a single mutation in HSPB8 upon the heart were analyzed using rat neonatal cardiomyocytes. Expression of HSPB8 K141N by adenoviral infection resulted in increased HSPB8-positive aggregates around nuclei, whereas no aggregates were observed in myocytes expressing wild-type HSPB8. HSPB8-positive aggresomes contained amyloid oligomer intermediates that were detected by a specific anti-oligomer antibody (A11). Expression of HSPB8 K141N induced slight cellular toxicity. Recombinant HSPB8 K141N protein showed reactivity against the anti-oligomer antibody, and reactivity of the mutant HSPB8 protein was much higher than that of wild-type HSPB8 protein. To extend our in vitro study, cardiac-specific HSPB8 K141N transgenic (TG) mice were generated. Echocardiography revealed that the HSPB8 K141N TG mice exhibited mild hypertrophy and apical fibrosis as well as slightly reduced cardiac function, although no phenotype was detected in wild-type HSPB8 TG mice. A single point mutation of HSPB8, such as K141N, can cause cardiac disease.

Serotonin suppresses ß-casein expression via inhibition of the signal transducer and activator of transcription 5 (STAT5) protein phosphorylation in human mammary epithelial cells MCF-12A.

Serotonin (5-hydroxytryptamine; 5-HT) has an important physiological role in controlling lactation, namely, milk volume homeostasis, within mammary glands. The objectives of this study were to evaluate whether exogenous 5-HT can suppress ß-casein expression, a differentiation marker, produced in human mammary epithelial cells, and to determine whether 5-HT can attenuate ß-casein signaling via the prolactin (PRL) receptor (PRLr) and Janus kinase 2/signal transducer and activator of transcription 5 (STAT5) pathway. PRL treatment increased the mRNA level of ß-casein in the MCF-12A human mammary epithelial cell line, and the highest level occurred at days 7 and 14 of culture. In contrast, PRLr expression was not affected significantly by PRL treatment. PRL treatment in MCF-12A cells increased levels of ß-casein and phosphorylated STAT5 (pSTAT5) proteins in a concentration-dependent manner, with a slight increase of STAT5 protein. ß-Casein expression was inhibited by 0.1 mM 5-HT in a time-dependent manner. Additionally, treatment with 0.1 mM 5-HT for 72 h decreased protein levels of ß-casein and pSTAT5, with a slight decrease in STAT5 levels. These results suggest that exogenous 5-HT can inhibit STAT5 phosphorylation, resulting in a decrease in ß-Casein expression. In conclusion, we showed that exogenous 5-HT decreased ß-casein expression in MCF-12A human mammary epithelial cells, and that 5-HT was responsible for inhibiting phosphorylation of STAT5, resulting in a decline in lactational function.

Nicorandil improves electrical remodelling, leading to the prevention of electrically induced ventricular tachyarrhythmia in a mouse model of desmin-related cardiomyopathy.

1. Transgenic (TG) mice overexpressing an arg120gly missense mutation in heat shock protein B5 (HSPB5; i.e. R120G TG mice) exhibit desmin-related cardiomyopathy. Recently, the cardioprotective effect of nicorandil has been shown to prolong the survival of R120G TG mice. However, whether the TG mice exhibit ventricular arrhythmias and whether nicorandil can inhibit these arrhythmias remain unknown. In the present study we examined the effects of chronic nicorandil administration on ventricular electrical remodelling and arrhythmias in R120G TG mice. 2. Mice were administered nicorandil (15 mg/kg per day) or vehicle (water) orally from 5 to 30 weeks of age. Electrocardiograms (ECG) and optical action potentials were recorded from R120G TG mouse hearts. In addition, the expression of ventricular connexin 43 and the cardiac Na(+) channel Nav1.5 was examined in TG mice. 3. All ECG parameters tested were prolonged in R120G TG compared with non-transgenic (NTG) mice. Nicorandil improved the prolonged P, PQ and QRS intervals in R120G TG mice. Interestingly, impulse conduction slowing and increases in the expression of total and phosphorylated connexin 43 and Nav1.5 were observed in ventricles from R120G TG compared with NTG mice. Nicorandil improved ventricular impulse conduction slowing and normalized the increased protein expression levels of total and phosphorylated connexin 43, but not of Nav1.5, in R120G TG mouse hearts. Electrical rapid pacing at the ventricle induced ventricular tachyarrhythmias (VT) in six of eight R120G TG mouse hearts, but not in any of the eight nicorandil-treated R120G TG mouse hearts (P < 0.05). 4. These findings demonstrate that nicorandil inhibits cardiac electrical remodelling and that the prevention of VT by nicorandil is associated with normalization of connexin 43 expression in this model.

αB-Crystallin R120G variant causes cardiac arrhythmias and alterations in the expression of Ca(2+) -handling proteins and endoplasmic reticulum stress in mice.

Mutations of αB-crystallin (CryαB), a small heat shock protein abundantly expressed in cardiac and skeletal muscles, are known to cause desmin-related myopathies. The CryαB R120G allele has been linked to a familial desminopathy and, in transgenic mice, causes a sudden death at about 28 weeks of age. To investigate the mechanisms of the sudden cardiac arrest of CryαB R120G transgenic mice, we prepared protein samples from left ventricular tissues of two different age groups (10 and 28 weeks) and examined Ca(2+) -handling proteins. Expression of sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) 2, phospholamban, ryanodine receptor 2 and calsequestrin 2 was significantly decreased in 28- versus 10-week-old CryαB R120G transgenic mice. In addition, low heart rate variability, including heart rate, total power and low frequency, was observed and continuous electrocardiogram monitoring revealed cardiac arrhythmias, such as ventricular tachycardia, atrioventricular block and atrial flutter, in 28-week-old CryαB R120G transgenic mice. In contrast, expression of endoplasmic reticulum (ER) degradation enhancing α-mannosidase-like protein, inositol requirement 1 and X-box binding protein 1 were increased significantly in 28- versus 10-week-old CryαBR120G transgenic mice, suggesting that the CryαBR120G transgenic mice exhibit increased ER stress compared with wild-type mice. Together, the data suggest that the CryαB R120G dominant variant induces ER stress and impairs Ca(2+) regulation, leading to ageing-related cardiac dysfunction, arrhythmias and decreased autonomic tone with shortened lifespan.

Inhibition of Connective Tissue Growth Factor Expression in Adult Retinal Pigment Epithelial-19 Cells by Blocking Yes-Associated Protein/Transcriptional Coactivator with PDZ-Binding Motif Activity.

Purpose: To investigate the effect of yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) on connective tissue growth factor (CTGF) expression in adult retinal pigment epithelial (ARPE)-19 cells. We also studied the inhibitory effect of K-975, a new pan-transcriptional enhanced associate domain (TEAD) inhibitor, and luteolin, a plant-derived flavonoid on CTGF expression. Methods: ARPE-19 cells were transfected with either YAP or TAZ overexpression plasmid or treated with transforming growth factor (TGF)-ß2. The cells were cultured either with or without K-975 or luteolin. The expression of YAP, TAZ, and CTGF was examined using real-time PCR. Results: ARPE-19 cells overexpressing YAP or TAZ exhibited significantly increased CTGF expression. This increase was attenuated by K-975 or luteolin alone. TGF-ß2 treatment significantly raised the expression of not just YAP and TAZ, but also CTGF in ARPE-19 cells. TGF-ß2 treatment-enhanced CTGF expression was considerably lowered by the addition of K-975 or luteolin. Conclusions: Overexpression of YAP or TAZ and treatment with TGF-ß2 led to an increase in the expression of CTGF in ARPE-19 cells. These increases were attenuated by treatment with K-975 and luteolin. These findings suggest that YAP and TAZ may be related to the expression of CTGF in ARPE-19 cells and that K-975 and luteolin can be explored as potential therapeutic agents for preventing CTGF production in vitreoretinal fibrosis.

Dilated cardiomyopathy: a disease of the myocardium.

Cardiomyopathies are defined as cardiac diseases of the myocardium with associated cardiac dysfunction. They are cardiac diseases in which heart muscle disease and/or measurable deterioration of cardiac muscle function occurs due to various causes, such as genetic and sporadic mutations of muscle proteins, as well as external factors such as hypertension, ischemia, and inflammation. In 1995, the WHO/International Society and Federation of Cardiology (ISFC) classified primary cardiomyopathy caused by intrinsic factors into five groups according to the dominant pathophysiology: dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), restricted cardiomyopathy (RCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), and unclassified cardiomyopathy. Among these cardiomyopathies, DCM is the most prevalent and the most common reason for cardiac transplantation in adults and children. Many recent findings indicate that genetic and sporadic mutations of a number of muscle proteins, such as myofibrillar, structural, and Ca(2+) regulating proteins, can cause DCM. In such cases, certain mutations often induce DCM with cardiac arrhythmia that is recognized as a potential trigger of sudden cardiac death. Thus, effective prognostic determination and appropriate cardiac care depend on accurate molecular and genetic diagnoses.

Changes in small heat shock proteins HSPB1, HSPB5 and HSPB8 in mitochondria of the failing heart following myocardial infarction in rats.

The mechanisms underlying mitochondrial impairment in the failing heart are not yet clearly defined. In the present study, we examined the involvement of changes in small heat shock proteins (HSPs) such as HSPB1, HSPB5 and HSPB8 in mitochondrial dysfunction of the failing heart. Hemodynamic parameters of rats with myocardial infarction at the 2nd and 8th weeks (2W- and 8W-) after coronary artery ligation (CAL) were measured. The 8W-CAL rats, but not the 2W-CAL ones, showed the signs of the chronic heart failure concomitant with a reduced mitochondrial oxygen consumption rate. In the mitochondrial fraction prepared from the heart of the 2W-CAL animals, the contents of small HSPs and phosphorylated small HSPs were increased, suggesting that these increases contributed to the preservation of the mitochondrial energy-producing ability. In the failing heart, HSPB1 and HSPB8 contents and phosphorylated small HSP contents in the mitochondrial fraction were decreased, suggesting that a reduction in mitochondrial translocation of these small HSPs led to impaired mitochondrial energy-producing ability. To further define the submitochondrial locations of these small HSPs, we performed mitochondrial subfractionation. The contents of small HSPs in the 2W-CAL rats were increased in the mitochondrial inner-membrane fraction, whereas those of the 8W-CAL rats were reversed to those of the control animals. These findings suggest that small HSPs, at least in part, play an important role in the development of the impaired mitochondrial energy-producing ability that leads to heart failure after a myocardial infarction.

The atypical Guanine-nucleotide exchange factor, dock7, negatively regulates schwann cell differentiation and myelination.

In development of the peripheral nervous system, Schwann cells proliferate, migrate, and ultimately differentiate to form myelin sheath. In all of the myelination stages, Schwann cells continuously undergo morphological changes; however, little is known about their underlying molecular mechanisms. We previously cloned the dock7 gene encoding the atypical Rho family guanine-nucleotide exchange factor (GEF) and reported the positive role of Dock7, the target Rho GTPases Rac/Cdc42, and the downstream c-Jun N-terminal kinase in Schwann cell migration (Yamauchi et al., 2008). We investigated the role of Dock7 in Schwann cell differentiation and myelination. Knockdown of Dock7 by the specific small interfering (si)RNA in primary Schwann cells promotes dibutyryl cAMP-induced morphological differentiation, indicating the negative role of Dock7 in Schwann cell differentiation. It also results in a shorter duration of activation of Rac/Cdc42 and JNK, which is the negative regulator of myelination, and the earlier activation of Rho and Rho-kinase, which is the positive regulator of myelination. To obtain the in vivo evidence, we generated Dock7 short hairpin (sh)RNA transgenic mice. They exhibited a decreased expression of Dock7 in the sciatic nerves and enhanced myelin thickness, consistent with in vitro observation. The effects of the in vivo knockdown on the signals to Rho GTPases are similar to those of the in vitro knockdown. Collectively, the signaling through Dock7 negatively regulates Schwann cell differentiation and the onset of myelination, demonstrating the unexpected role of Dock7 in the interplay between Schwann cell migration and myelination.

Cytohesin-2/ARNO, through its interaction with focal adhesion adaptor protein paxillin, regulates preadipocyte migration via the downstream activation of Arf6.

The formation of primitive adipose tissue is the initial process in adipose tissue development followed by the migration of preadipocytes into adipocyte clusters. Comparatively little is known about the molecular mechanism controlling preadipocyte migration. Here, we show that cytohesin-2, the guanine-nucleotide exchange factor for the Arf family GTP-binding proteins, regulates migration of mouse preadipocyte 3T3-L1 cells through Arf6. SecinH3, a specific inhibitor of the cytohesin family, markedly inhibits migration of 3T3-L1 cells. 3T3-L1 cells express cytohesin-2 and cytohesin-3, and knockdown of cytohesin-2 with its small interfering RNA effectively decreases cell migration. Cytohesin-2 preferentially acts upstream of Arf6 in this signaling pathway. Furthermore, we find that the focal adhesion protein paxillin forms a complex with cytohesin-2. Paxillin colocalizes with cytohesin-2 at the leading edges of migrating cells. This interaction is mediated by the LIM2 domain of paxillin and the isolated polybasic region of cytohesin-2. Importantly, migration is inhibited by expression of the constructs containing these regions. These results suggest that cytohesin-2, through a previously unexplored complex formation with paxillin, regulates preadipocyte migration and that paxillin plays a previously unknown role as a scaffold protein of Arf guanine-nucleotide exchange factor.

Molecular mechanisms of α-crystallinopathy and its therapeutic strategy.

α-B-Crystallin (CryAB, gene map locus: 11q22.3-q23.1) is a member of the small heat shock protein (HSP) family, a group of proteins that prevent protein aggregation upon exposure of a cell to heat and/or restore the biological activity of cell substrates. The missense mutation and the deletion mutation of CryAB can cause various forms of muscular disorder, including restrictive, hypertrophic, and dilated cardiomyopathies, heart failure, and skeletal muscle weakness. Collectively, these diseases constitute a rare autosomal-dominant inherited disorder called α-crystallinopathy (crystallinopathy), also known as desmin-related cardiomyopathy. The disease is a misfolded protein-related disease characterized by the formation of insoluble protein aggregates consisting of the CryAB protein in the patient's cardiomyocytes and skeletal myocytes. The details of crystallinopathy are unclear at the present time; what has been discovered concerning the disease mechanisms underlying crystallinopathy has been through experiments with genetically modified mice such as the CryAB knockout mouse and various mutant CryAB transgenic (TG) mice. Crystallinopathy can be recapitulated in TG mice by expressing the mutant CryAB Arg120Gly (R120G) protein, a causal mutation of crystallinopathy, specifically in the cardiomyocytes. CryAB R120G causes perinuclear formation of aggresomes containing preamyloid oligomer intermediates, which are wellknown as a primary toxic species in neurodegenerative disease. This suggests that crystallinopathy caused by the CryAB mutation could be considered one of the aggresomal and amyloid-related diseases. Moreover, recent findings have indicated that enhancement of HSP induction and inhibition of apoptotic cell death by mitochondrial protection may be a new therapeutic strategy for patients with crystallinopathy.