Postnatal Pdzrn3 deficiency causes acute muscle atrophy without alterations in endplate morphology.

PDZ domain-containing RING finger family protein 3 (PDZRN3) is expressed in various tissues, including the skeletal muscle. Although PDZRN3 plays a crucial role in the terminal differentiation of myoblasts and synaptic growth/maturation in myogenesis, the role of this molecule in postnatal muscles is completely unknown despite its lifelong expression in myofibers. In this study, we aimed to elucidate the function of PDZRN3 in mature myofibers using myofiber-specific conditional knockout mice. After tamoxifen injection, PDZRN3 deficiency was confirmed in both fast and slow myofibers of Myf6-CreERT2; Pdzrn3flox/flox (Pdzrn3mcKO) mice. Transcriptome analysis of the skeletal muscles of Pdzrn3mcKO mice identified differentially expressed genes, including muscle atrophy-related genes such as Smox, Amd1/2, and Mt1/2, suggesting that PDZRN3 is involved in the homeostatic maintenance of postnatal muscles. PDZRN3 deficiency caused muscle atrophy, predominantly in fast-twitch (type II) myofibers, and reduced muscle strength. While myofiber-specific PDZRN3 deficiency did not influence endplate morphology or expression of neuromuscular synaptic formation-related genes in postnatal muscles, indicating that the relationship between PDZRN3 and neuromuscular junctions might be limited during muscle development. Considering that the expression of Pdzrn3 in skeletal muscles was significantly lower in aged mice than in mature adult mice, we speculated that the PDZRN3-mediated muscle maintenance system might be associated with the pathophysiology of age-related muscle decline, such as sarcopenia.

Generation of Specific Aptamers.

Nucleic acid aptamers are therapeutic agents consisting of short single-strand DNA or RNA oligonucleotides, which have the ability to bind to target therapeutic molecules with high affinity and specificity and have been developed as potent drugs for the treatment of rheumatoid arthritis. Aptamers have unique and advantageous features over antibodies, such as superior affinity with nano- or pico-molar dissociation constants and ease of chemical synthesis, modification, and inactivation by designing antisense sequences. In this chapter, using a DNA-oligonucleotide pool, the technology of proteoliposome-systematic evolution of ligands by exponential enrichment (SELEX) is introduced. By using this technique, potential therapeutic agents with high affinity and specificity could be obtained.

Local immune checkpoint blockade therapy by an adenovirus encoding a novel PD-L1 inhibitory peptide inhibits the growth of colon carcinoma in immunocompetent mice.

A novel peptide that interferes with the PD-1/PD-L1 immune checkpoint pathway, termed PD-L1 inhibitory peptide 3 (PD-L1ip3), was computationally designed, experimentally validated for its specific binding to PD-L1, and evaluated for its antitumor effects in cell culture and in a mouse colon carcinoma syngeneic murine model. In several cell culture studies, direct treatment with PD-L1ip3, but not a similar peptide with a scrambled sequence, substantially increased death of CT26 colon carcinoma cells when co-cultured with murine CD8+ T cells primed by CT26 cell antigens. In a syngeneic mouse tumor model, the growth of CT26 tumor cells transduced with the PD-L1ip3 gene by an adenovirus vector was significantly slower than that of un-transduced CT26 cells in immunocompetent mice. This tumor growth attenuation was further enhanced by the coadministration of the peptide form of PD-L1ip3 (10 mg/kg/day). The current study suggests that this peptide can stimulate host antitumor immunity via blockade of the PD-1/PD-L1 pathway, thereby increasing CD8+ T cell-induced death of colon carcinoma cells. The tumor site-specific inhibition of PD-L1 by an adenovirus carrying the PD-L1ip3 gene, together with direct peptide treatment, may be used as a local immune checkpoint blockade therapy to inhibit colon carcinoma growth.

Calcium/calmodulin-dependent regulation of Rac GTPases and Akt in histamine-induced chemotaxis of mast cells.

Histamine induces chemotaxis of mast cells through the histamine H4 receptor. This involves the activation of small GTPases, Rac1 and Rac2, downstream of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). Activation of the H4 receptor also results in phospholipase C (PLC)-mediated calcium mobilization; however, it is unclear whether the PLC­calcium pathway interacts with the PI3K-Rac pathway. Here, we demonstrated that calcium mobilization regulates the PI3K-dependent activation of Rac GTPases through calmodulin. A PLC inhibitor (U73122) and an intracellular calcium chelator (BAPTA-AM) suppressed the histamine-induced activation of Rac, whereas the calcium ionophore ionomycin increased the active Rac GTPases, suggesting that intracellular calcium regulates the activation of Rac. The calmodulin antagonist (W-7) inhibited the histamine-induced activation of Rac and migration of mast cells, indicating that calmodulin mediates the effect of calcium. Inhibition of calcium/calmodulin signaling suppressed histamine-induced phosphorylation of Akt. The Akt inhibitor MK-2206 attenuated histamine-induced migration of mast cells. However, it did not suppress the activation of Rac GTPases. These results suggest that Rac GTPases and Akt play independent roles in the histamine-induced chemotaxis of mast cells. Our findings enable further elucidation of the molecular mechanism of histamine-induced chemotaxis of mast cells and help identify therapeutic targets for allergic and inflammatory conditions involving mast cell accumulation.

PDZRN3 protects against apoptosis in myoblasts by maintaining cyclin A2 expression.

PDZRN3 is a PDZ domain-containing RING-finger family protein that functions in various developmental processes. We previously showed that expression of PDZRN3 is induced together with that of MyoD during the early phase of skeletal muscle regeneration in vivo. We here show that PDZRN3 suppresses apoptosis and promotes proliferation in myoblasts in a manner dependent on cyclin A2. Depletion of PDZRN3 in mouse C2C12 myoblasts by RNA interference reduced the proportion of Ki-67-positive cells and the level of Akt phosphorylation, implicating PDZRN3 in regulation of both cell proliferation and apoptosis. Exposure of C2C12 cells as well as of C3H10T1/2 mesenchymal stem cells and NIH-3T3 fibroblasts to various inducers of apoptosis including serum deprivation resulted in a greater increase in the amount of cleaved caspase-3 in PDZRN3-depleted cells than in control cells. The abundance of cyclin A2 was reduced in PDZRN3-depleted C2C12 myoblasts, as was that of Mre11, which contributes to the repair of DNA damage. Overexpression of cyclin A2 restored the expression of Mre11 and Ki-67 as well as attenuated caspase-3 cleavage in PDZRN3-depleted cells deprived of serum. These results indicate that PDZRN3 suppresses apoptosis and promotes proliferation in myoblasts and other cell types by maintaining cyclin A2 expression.

PDZRN3 regulates differentiation of myoblasts into myotubes through transcriptional and posttranslational control of Id2.

PDZRN3 (also known as LNX3) is a member of the PDZ domain-containing RING finger protein family. We previously showed that PDZRN3 is essential for differentiation of myoblasts into myotubes and that depletion of PDZRN3 inhibits such differentiation downstream of the upregulation of myogenin, a basic helix-loop-helix (bHLH) transcription factor required for completion of the differentiation process. However, the mechanism by which PDZRN3 controls this process has remained unclear. Myogenin is rendered active during the late stage of myogenic differentiation by the downregulation of Id2, a negative regulator of bHLH transcription factors. We now show that depletion of PDZRN3 inhibits the differentiation of C2C12 cells by inducing the upregulation of Id2 and thereby delaying its downregulation. Knockdown of Id2 by RNA interference restores the differentiation of PDZRN3-depleted cells. Luciferase reporter assays revealed that a putative binding site for STAT5b in the Id2 gene promoter is required for the upregulation of Id2 expression by PDZRN3 depletion. In addition, the amount of phosphorylated Id2 was reduced and that of the nonphosphorylated protein concomitantly increased in PDZRN3-depleted cells, with the inhibitory effect of Id2 on bHLH transcription factors having previously been shown to be attenuated by phosphorylation of Id2 catalyzed by the complex of Cdk2 with cyclin A2 or E1. Indeed, the expression of cyclin A2, but not that of cyclin E1, was reduced in PDZRN3-depleted cells. Our results thus indicate that PDZRN3 plays a key role in the differentiation of myoblasts into myotubes by regulating Id2 at both transcriptional and posttranslational levels.

Generation of Specific Aptamers.

Nucleic acid aptamers are therapeutic agents consisting of short single-strand DNA or RNA oligonucleotides, which have the ability to bind to target therapeutic molecules with high affinity and specificity, and have been developed as potent drugs for the treatment of rheumatoid arthritis. Aptamers have unique and advantageous features over antibodies, such as superior affinity with nano- or pico-molar dissociation constants, and ease of chemical synthesis, modification, and inactivation by designing antisense sequences. In this chapter, using a DNA-oligonucleotide pool, the technology of proteoliposome-systematic evolution of ligands by exponential enrichment (SELEX) is introduced. By using this technique, potential therapeutic agents with high affinity and specificity could be obtained.

Distinct Roles of Small GTPases Rac1 and Rac2 in Histamine H4 Receptor-Mediated Chemotaxis of Mast Cells.

Histamine induces chemotaxis of mast cells through the H4 receptor. However, little is known about the precise intracellular signaling pathway that mediates this process. In this study, we identified small GTPases Rac1 and Rac2 as intracellular binding partners of the H4 receptor and characterized their roles in H4 receptor signaling. We showed that histamine induced Rac GTPase activation via the H4 receptor. A Rac inhibitor NSC23766 attenuated chemotaxis of mast cells toward histamine, as well as histamine-induced calcium mobilization and extracellular signal-regulated kinase (ERK) activation. Histamine-induced migration of mast cells was also sensitive to PD98059, an inhibitor of the mitogen-activated protein kinase kinase, indicating that the Rac-ERK pathway was involved in chemotaxis through the H4 receptor. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) by LY294002 suppressed the histamine-induced chemotaxis and activation of Rac GTPases, suggesting that PI3K regulates chemotaxis upstream of Rac activation. Specific knockdown of Rac1 and Rac2 by short-hairpin RNA revealed that both Rac GTPases are necessary for histamine-induced migration. Downregulation of Rac1 and Rac2 led to attenuated response in calcium mobilization and ERK activation, respectively. These observations suggested that Rac1 and Rac2 have distinct and essential roles in intracellular signaling downstream of H4 receptor-PI3K in histamine-induced chemotaxis of mast cells.

Long-term Functioning of Allogeneic Islets in Subcutaneous Tissue Pretreated With a Novel Cyclic Peptide Without Immunosuppressive Medication.

BACKGROUND: There exists a need for a minimally invasive method of islet transplantation without immunosuppressive drugs for the treatment of type 1 diabetes. METHODS: In diabetic August Copenhagen Irish rats, an agarose rod containing the cyclic oligopeptide SEK-1005 (agarose-SEK rod) was implanted at 2 dorsal subcutaneous sites. Then these rods were removed, and 1500 islets of Langerhans isolated from Fischer 344 rats were transplanted into each of the pockets. RESULTS: Ten days after implantation of agarose-SEK rods, vascularized pockets were present. Nonfasting blood glucose levels confirmed long-term survival of the allogeneic islet grafts, without immunosuppressive therapy, in 8 of 10 recipients. Flow cytometry and gene expression analyses were performed to investigate the mechanisms underlying graft acceptance. Agarose-SEK rod implantation led to the formation of granulomatous tissue containing regulatory T cells that suppressed immune reactions against the allogeneic islet grafts. CONCLUSIONS: These results indicate that the use of an agarose-SEK rod to prevascularize a subcutaneous site may be a useful method for achieving successful allogeneic islet transplantation without immunosuppression.

Co-treatment with a C1B5 peptide of protein kinase Cγ and a low dose of gemcitabine strongly attenuated pancreatic cancer growth in mice through T cell activation.

Although gemcitabine is an effective chemotherapeutic for pancreatic cancer, severe side effects often accompany its use. Since we have discovered that locally administered C1B domain peptides effectively control tumor growth without any side effects, the efficacy of co-treatment with this peptide and a low dose of gemcitabine on the growth of pancreatic cancer was examined. Two- and three-dimensional cell culture studies clarified that a co-treatment with C1B5 peptide and gemcitabine significantly attenuated growth of PAN02 mouse and PANC-1 human pancreatic cancer cells in 2D and 3D cultures. Although treatment with the low dose of gemcitabine alone (76%) or the C1B5 peptide alone (39%) inhibited tumor growth moderately, a co-treatment with C1B5 peptide and a low dose of gemcitabine markedly inhibited the growth of PAN02 autografts in the mouse peritoneal cavity (94% inhibition) without any noticeable adverse effect. The number of peritoneal cavity-infiltrating neutrophils and granzyme B+ lymphocytes was significantly higher in the co-treatment group than in the control group. A significant increase of granzyme B mRNA expression was also detected in human T cells by the co-treatment. Taken together, the current study suggests that C1B5 peptide offers a remarkably effective combination treatment strategy to reduce side effects associated with gemcitabine, without losing its tumoricidal effect.

A pyridone derivative activates SERCA2a by attenuating the inhibitory effect of phospholamban.

The cardiac sarco/endoplasmic reticulum Ca2+-dependent ATPase 2a (SERCA2a) plays a central role in Ca2+ handling within cardiomyocytes and is negatively regulated by phospholamban (PLN), a sarcoplasmic reticulum (SR) membrane protein. The activation of SERCA2a, which has been reported to improve cardiac dysfunction in heart failure, is a potential therapeutic approach for heart failure. Therefore, we developed a novel small molecule, compound A and characterized it both in vitro and in vivo. Compound A activated the Ca2+-dependent ATPase activity of cardiac SR vesicles but not that of skeletal muscle SR vesicles that lack PLN. The surface plasmon resonance assay revealed a direct interaction between compound A and PLN, suggesting that the binding of compound A to PLN attenuates its inhibition of SERCA2a, resulting in SERCA2a activation. This was substantiated by inhibition of the compound A-mediated increase in Ca2+ levels within the SR of HL-1 cells by thapsigargin, a SERCA inhibitor. Compound A also increased the Ca2+ transients and contraction and relaxation of isolated adult rat cardiomyocytes. In isolated perfused rat hearts, the compound A enhanced systolic and diastolic functions. Further, an infusion of compound A (30mg/kg, i.v. bolus followed by 2mg/kg/min, i.v. infusion) significantly enhanced the diastolic function in anesthetized normal rats. These results indicate that compound A is a novel SERCA2a activator, which attenuates PLN inhibition and enhances the systolic and diastolic functions of the heart in vitro and in vivo. Therefore, compound A might be a novel therapeutic lead for heart failure.

Involvement of angiotensin II type 2 receptor (AT2R) signaling in human pancreatic ductal adenocarcinoma (PDAC): a novel AT2R agonist effectively attenuates growth of PDAC grafts in mice.

We have recently discovered the potential involvement of angiotensin II type 2 receptor (AT2R) signaling in pancreatic cancer using AT2R deficient mice. To examine the involvement of AT2R expression in human PDAC, expressions of AT2R as well as the major angiotensin II receptor (type 1 receptor, AT1R) in human PDAC and adjacent normal tissue was evaluated by immunohistochemistry and real time PCR using surgically dissected human PDAC specimens. In immunohistochemical analysis, relatively strong AT1R expression was detected consistently in both normal pancreas and PDAC areas, whereas moderate AT2R expression was detected in 78.5% of PDAC specimens and 100% of normal area of the pancreas. AT1R, but not AT2R, mRNA levels were significantly higher in the PDAC area than in the normal pancreas. AT2R mRNA levels showed a negative correlation trend with overall survival. In cell cultures, treatment with a novel AT2R agonist significantly attenuated both murine and human PDAC cell growth with negligible cytotoxicity in normal epithelial cells. In a mouse study, administrations of the AT2R agonist in tumor surrounding connective tissue markedly attenuated growth of only AT2R expressing PAN02 murine PDAC grafts in syngeneic mice. The AT2R agonist treatment induced apoptosis primarily in tumor cells but not in stromal cells. Taken together, our findings offer clinical and preclinical evidence for the involvement of AT2R signaling in PDAC development and pinpoint that the novel AT2R agonist could serve as an effective therapeutic for PDAC treatment.

Peptide therapies for ocular surface disturbances based on fibronectin-integrin interactions.

The condition of the corneal epithelium is a critical determinant of corneal transparency and clear vision. The corneal epithelium serves as a barrier to protect the eye from external insults, with its smooth surface being essential for its optical properties. Disorders of the corneal epithelium include superficial punctate keratopathy, corneal erosion, and persistent epithelial defects (PEDs). The prompt resolution of these disorders is important for minimization of further damage to the cornea. Currently available treatment modalities for corneal epithelial disorders are based on protection of the ocular surface in order to allow natural healing to proceed. PEDs remain among the most difficult corneal conditions to treat, however. On the basis of characterization of the pathobiology of PEDs at the cell and molecular biological levels, we have strived to develop new modes of treatment for these defects. These treatments rely on two key concepts: provision of a substrate, such as the adhesive glycoprotein fibronectin, for the attachment and migration of corneal epithelial cells, and activation of these cells by biological agents such as the combination of substance P and insulin-like growth factor-1 (IGF-1). Central to both approaches is the role of the fibronectin-integrin system in corneal epithelial wound healing. Determination of the minimum amino acid sequences required for the promotion of corneal epithelial wound closure by fibronectin (PHSRN) and by substance P (FGLM-amide) plus IGF-1 (SSSR) has led to the development of peptide eyedrops for the treatment of PEDs that are free of adverse effects of the parent molecules.

Signaling mechanism underlying the promotion of keratinocyte migration by angiotensin II.

Re-epithelialization begins early during skin wound healing and is regulated by various growth factors and cytokines. Angiotensin II promotes the migration of keratinocytes and thereby contributes to wound healing. We investigated the mechanism by which angiotensin II stimulates human keratinocyte migration. Angiotensin II-induced keratinocyte migration was inhibited by an angiotensin II type 1 receptor (AT1R) antagonist (candesartan) or an angiotensin II type 2 receptor (AT2R) antagonist (PD123319) as well as by depletion of AT1R or AT2R. A biased agonist for AT1R, [Sar(1),Ile(4),Ile(8)]angiotensin II, induced cell migration, whereas depletion of ß-arrestin2 inhibited angiotensin II-induced migration. Angiotensin II-induced migration was blocked by neutralizing antibodies to transforming growth factor-ß (TGF-ß) as well as by the TGF-ß receptor inhibitor SB431542. The amount of TGF-ß1 was increased in the culture medium of angiotensin II-treated cells, and this effect was inhibited by candesartan or PD123319. Both angiotensin II- and TGF-ß-induced cell migration were inhibited by neutralizing antibodies to the epidermal growth factor (EGF) receptor but not by those to EGF receptor ligands. Angiotensin II-induced phosphorylation of the EGF receptor, and this effect was inhibited by candesartan, PD123319, SB431542, or depletion of ß-arrestin2, but not by neutralizing antibodies to heparin-binding EGF-like growth factor. Our results indicate that ß-arrestin-dependent signaling downstream of AT1R as well as AT2R signaling are necessary for angiotensin II-induced keratinocyte migration, and that such signaling promotes generation of the active form of TGF-ß, consequent activation of the TGF-ß receptor, and transactivation of the EGF receptor by the TGF-ß receptor.

A cell-penetrating phospholamban-specific RNA aptamer enhances Ca2+ transients and contractile function in cardiomyocytes.

The sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a)-phospholamban (PLN) system of sarcoplasmic reticulum plays a pivotal role in regulation of intracellular Ca(2+) cycling in ventricular cardiomyocytes. Given that Ca(2+) cycling is impaired in heart failure, proteins that contribute to this process are potential targets for the treatment of this condition. We have now isolated PLN-specific aptamers with a phosphorothioate-modified backbone from a library of RNA molecules containing a randomized 40-nucleotide sequence by application of the systematic evolution of ligands by exponential enrichment (SELEX) protocol with a fusion protein containing the cytoplasmic region of human PLN. One of these aptamers was shortened to a 30-nucleotide oligomer (RNA-Apt30) without loss of function. RNA-Apt30 showed a high affinity for the cytoplasmic region of PLN (Kd=11 nM), but it did not bind to the phosphorylated form of PLN or to a phosphomimetic mutant. It also increased SERCA2a activity in isolated cardiac SR vesicles with an EC50 of 18 nM by relieving PLN-mediated inhibition. Conjugation of RNA-Apt30 to a cell-penetrating peptide allowed its delivery into adult rat cardiomyocytes, in which it enhanced both Ca(2+) transients and contractile function. These effects of the aptamer were also apparent in the presence of the ß-adrenergic receptor antagonist propranolol. This cell-penetrating PLN aptamer may thus provide a basis for the development of new therapeutic agents for heart failure without the need for gene transfer or a change in endogenous protein expression.

Regulation of adipocyte differentiation of 3T3-L1 cells by PDZRN3.

PDZRN3, a member of the PDZRN (or LNX) family of proteins, is essential for the differentiation of mesenchymal stem cells into myotubes, but it plays an inhibitory role in the differentiation of these cells into osteoblasts. Given that mesenchymal stem cells also differentiate into adipocytes, we examined the possible role of PDZRN3 in adipogenesis in mouse 3T3-L1 preadipocytes. The expression of PDZRN3 decreased at both the mRNA and protein levels during adipogenic differentiation. RNAi-mediated depletion of PDZRN3 enhanced the differentiation of 3T3-L1 cells into adipocytes as assessed on the basis of lipid accumulation. The upregulation of aP2 and CCAAT/enhancer-binding protein (C/EBP)-ß during adipocyte differentiation was also enhanced in the PDZRN3-depleted cells, as was the induction of peroxisome proliferator-activated receptor-γ (PPARγ), an upstream regulator of aP2 and C/EBPα, at both the mRNA and protein levels. Among transcription factors that control the expression of PPARγ, we found that STAT5b, but not STAT5a, was upregulated in PDZRN3-depleted cells at both mRNA and protein levels. Tyrosine phosphorylation of STAT5b, but not that of STAT5a, was also enhanced at an early stage of differentiation by PDZRN3 depletion. In addition, the expression of C/EBPß during the induction of differentiation was enhanced at the mRNA and protein levels in PDZRN3-depleted cells. Our results thus suggest that PDZRN3 negatively regulates adipogenesis in 3T3-L1 cells through downregulation of STAT5b and C/EBPß and consequent suppression of PPARγ expression.

Ischemia-reperfusion induces myocardial infarction through mitochondrial Ca²âº overload.

Both mitochondria and the sarcoplasmic reticulum (SR) are essential for myocardial homeostasis and control of cardiac function. Uptake of Ca(2+) from the cytosol into SR is mediated by the Ca(2+)-dependent ATPase SERCA2a, which is reversibly inhibited by phospholamban (PLN). We previously showed that removal of PLN inhibition of SERCA2a with an antibody to (anti-) PLN reduces cytosolic Ca(2+) overload, thereby attenuating the spread of contraction bands and fodrin proteolysis, during reperfusion after cardiac ischemia. We have now examined the effects of anti-PLN injection into the heart on the development of myocardial infarction (MI) after ischemia-reperfusion in rats. Whereas anti-PLN injection attenuated cytosolic Ca(2+) overload, it did not affect MI size 6h after the onset of reperfusion and actually increased it at 30 min. The antibody also increased the release of apoptosis-inducing factor (AIF) from mitochondria into the cytosol, indicative of enhanced opening of the mitochondrial permeability transition pore (mPTP). Administration of an mPTP blocker at the time of reperfusion or of a blocker of the mitochondrial Ca(2+) uniporter significantly suppressed the release of AIF and the development of MI. These results indicate that the enhancement of SR Ca(2+) loading by anti-PLN injection facilitated Ca(2+) uniporter-dependent mitochondrial Ca(2+) uptake and thereby induced mPTP opening and MI development during early reperfusion. The enhancement of SR Ca(2+) loading thus aggravates MI in a manner independent of cytosolic Ca(2+) overload. Given that cytosolic Ca(2+) overload induces contraction bands, our findings are inconsistent with a causal relation between contraction bands and MI.

Heart failure-inducible gene therapy targeting protein phosphatase 1 prevents progressive left ventricular remodeling.

BACKGROUND: The targeting of Ca(2+) cycling has emerged as a potential therapy for the treatment of severe heart failure. These approaches include gene therapy directed at overexpressing sarcoplasmic reticulum (SR) Ca(2+) ATPase, or ablation of phospholamban (PLN) and associated protein phosphatase 1 (PP1) protein complexes. We previously reported that PP1ß, one of the PP1 catalytic subunits, predominantly suppresses Ca(2+) uptake in the SR among the three PP1 isoforms, thereby contributing to Ca(2+) downregulation in failing hearts. In the present study, we investigated whether heart-failure-inducible PP1ß-inhibition by adeno-associated viral-9 (AAV9) vector mediated gene therapy is beneficial for preventing disease progression in genetic cardiomyopathic mice. METHODS: We created an adeno-associated virus 9 (AAV9) vector encoding PP1ß short-hairpin RNA (shRNA) or negative control (NC) shRNA. A heart failure inducible gene expression system was employed using the B-type natriuretic protein (BNP) promoter conjugated to emerald-green fluorescence protein (EmGFP) and the shRNA sequence. AAV9 vectors (AAV9-BNP-EmGFP-PP1ßshRNA and AAV9-BNP-EmGFP-NCshRNA) were injected into the tail vein (2×10(11) GC/mouse) of muscle LIM protein deficient mice (MLPKO), followed by serial analysis of echocardiography, hemodynamic measurement, biochemical and histological analysis at 3 months. RESULTS: In the MLPKO mice, BNP promoter activity was shown to be increased by detecting both EmGFP expression and the induced reduction of PP1ß by 25% in the myocardium. Inducible PP1ßshRNA delivery preferentially ameliorated left ventricular diastolic function and mitigated adverse ventricular remodeling. PLN phosphorylation was significantly augmented in the AAV9-BNP-EmGFP-PP1ßshRNA injected hearts compared with the AAV9-BNP-EmGFP-NCshRNA group. Furthermore, BNP production was reduced, and cardiac interstitial fibrosis was abrogated at 3 months. CONCLUSION: Heart failure-inducible molecular targeting of PP1ß has potential as a novel therapeutic strategy for heart failure.

Concise synthesis of 2-benzazepine derivatives and their biological activity.

2-Benzazepines, which are potentially good candidates for new drug therapies to treat skin wounds, were readily prepared from substituted cinnamylamide via an intramolecular Friedel-Crafts reaction. With few steps and effective reactions, the procedure enables a rapid derivatization of 2-benzazepines. Moreover, optically active 4-substituted-2-benzazepines were prepared from chiral α-substituted cinnamylamides, which were readily prepared by asymmetric α-alkylation of chiral cinnamyl oxazolidinone amides. We have easily prepared a library of more than 20 derivatives and examined the biological activity of the compounds.