Cardioprotective Effect of Nicorandil, a Mitochondrial ATP-Sensitive Potassium Channel Opener, Prolongs Survival in HSPB5 R120G Transgenic Mice

BACKGROUND: Transgenic (TG) mice with overexpression of an arg120gly (R120G) missense mutation in HSPB5 display desmin-related cardiomyopathy, which is characterized by formation of aggresomes. It is also known that progressive mitochondrial abnormalities and apoptotic cell death occur in the hearts of R120G TG mice. The role of mitochondrial dysfunction and apoptosis in disease progression, however, remains uncertain. METHODS AND RESULTS: Mitochondrial abnormalities and apoptotic cell death induced by overexpression of HSPB5 R120G were analyzed in neonatal rat cardiomyocytes. Overexpression of mutant HSPB5 led to development of aggresomes with a concomitant reduction in cell viability in the myocytes. Overexpression of mutant HSPB5 induced a reduction in the cytochrome c level in the mitochondrial fraction and a corresponding increase in the cytoplasmic fraction in the myocytes. Down-regulation of BCL2 and up-regulation of BAX were detected in the myocytes expressing the mutant HSPB5. Concomitant with mitochondrial abnormality, the activation of caspase-3 and increased apoptotic cell death was observed. Cell viability was dose-dependently recovered in myocytes overexpressing HSPB5 R120G by treatment with nicorandil a mitochondrial ATP-sensitive potassium channel opener. Nicorandil treatment also inhibited the increase in BAX, the decrease in BCL2, activation of caspase-3 and apoptotic cell death by mutant HSPB5. To confirm the results of the in-vitro study, we analyzed the effect of nicorandil in HSPB5 R120G TG mice. Nicorandil treatment appeared to reduce mitochondrial impairment and apoptotic cell death and prolonged survival in HSPB5 R120G TG mice. CONCLUSIONS: Nicorandil may prolong survival in HSPB5 R120G TG mice by protecting against mitochondrial impairments

Regulation of neurite growth by inorganic pyrophosphatase 1 via JNK dephosphorylation.

Neural cell differentiation during development is controlled by multiple signaling pathways, in which protein phosphorylation and dephosphorylation play an important role. In this study, we examined the role of pyrophosphatase1 (PPA1) in neuronal differentiation using the loss and gain of function analysis. Neuronal differentiation induced by external factors was studied using a mouse neuroblastoma cell line (N1E115). The neuronal like differentiation in N1E115 cells was determined by morphological analysis based on neurite growth length. In order to analyze the loss of the PPA1 function in N1E115, si-RNA specifically targeting PPA1 was generated. To study the effect of PPA1 overexpression, an adenoviral gene vector containing the PPA1 gene was utilized to infect N1E115 cells. To address the need for pyrophosphatase activity in PPA1, D117A PPA1, which has inactive pyrophosphatase, was overexpressed in N1E115 cells. We used valproic acid (VPA) as a neuronal differentiator to examine the effect of PPA1 in actively differentiated N1E115 cells. Si-PPA1 treatment reduced the PPA1 protein level and led to enhanced neurite growth in N1E115 cells. In contrast, PPA1 overexpression suppressed neurite growth in N1E115 cells treated with VPA, whereas this effect was abolished in D117A PPA1. PPA1 knockdown enhanced the JNK phosphorylation level, and PPA1 overexpression suppressed it in N1E115 cells. It seems that recombinant PPA1 can dephosphorylate JNK while no alteration of JNK phosphorylation level was seen after treatment with recombinant PPA1 D117A. Enhanced neurite growth by PPA1 knockdown was also observed in rat cortical neurons. Thus, PPA1 may play a role in neuronal differentiation via JNK dephosphorylation

Radiofluorinated probe for PET imaging of fatty acid binding protein 4 in cancer.

【Introduction】Cancer-associated adipocytes metabolically interact with adjacent cancer cells to promote tumor proliferation and metastasis. Fatty acid binding protein 4 (FABP4) participates in this interaction, and is gathering attention as a therapeutic and diagnostic target. Positron emission tomography (PET) is a useful diagnostic method that enables noninvasive in vivo quantitative imaging of biofunctional molecules with probes labeled with positron-emitting radioisotopes. Here a novel 18F labeled probe for PET FABP4 imaging developed through dedicated drug design from a radioiodinated probe we recently reported is evaluated in vitro and in vivo.【Methods】We designed the [18F]-labeled FTAP1 and FTAP3 probe, composed of a single or triple oxyethylene linker and a triazolopyrimidine scaffold derived from an FABP4 inhibitor. FABP4 binding affinities for chemically synthesized FTAP1 and FTAP3 were measured using FABP4 and 8-anilino-1-naphthalene sulfonic acid. Cell membrane permeability was measured using a commercially available plate assay system. After radiosynthesis, [18F]FTAP1 affinity and selectivity were evaluated using immobilized FABP3, FABP4, and FABP5. Cell uptake was investigated using differentiated adipocytes expressing FABP4 with inhibitor treatment. Following biodistribution studies in C6 glioblastoma-bearing mice, ex vivo autoradiography and immunohistochemistry were performed using thin sliced tumor sections. PET/CT imaging was then performed on C6 tumor bearing mice.【[Results】FTAP1 showed high FABP4 affinity (Ki = 68 ± 8.9 nM) and adequate cell permeability. [18F]FTAP1 with ≥ 98% radiochemical purity was shown to selectively bind to FABP4 (16.3- and 9.3-fold higher than for FABP3 and FABP5, respectively). [18F]FTAP1 was taken up by FABP4 expressing cells, and this uptake could be blocked by an inhibitor, indicating very low non-specific cell binding. [18F]FTAP1 showed high tumor accumulation, which demonstrates its potential use for in vivo tumor PET imaging, and the intratumoral radioactivity distribution corresponded to the FABP4 expression profile.【Conclusion】][18F]FTAP1 is a promising PET probe to target FABP4

Role of pyrophosphatase activity on PPA1-induced inhibition of neuronal differentiation in N1E115 cells.

<p>(A) Recombinant wild-type PPA1 (his-PPA1) and PPA1 Asp117Ala (his-PPA1 D117A) proteins. Western blot using anti-PPA1 antibody showed that the his-PPA1 and his-PPA1 D117A protein amount is similar. (B) Recombinant protein pyrophosphatase activity. Pyrophosphatase activity was seen in his-PPA1 while no activity was detected in his-PPA1 D117A (n = 5). (C) Effect of either green fluorescence protein (AD-GFP), wild-type PPA1 (AD-PPA1), or PPA1 D117A (AD-PPA1 D117A) overexpression using adenoviral vector, on PPA1 protein levels in N1E115 cells treated with 1 mM valproic acid (VPA). (D) Typical morphological changes in N1E115 cells treated with VPA, using AD-GFP overexpression as a control (VPA+GFP), AD-PPA1 (VPA+AD-PPA1) or D117A (VPA+AD-PPA1 D117A). (E) Quantitative analysis of the neurite growth ratio is shown. Neurite growth is determined by morphological analysis as described in the Experimental procedure (n = 100). *p<0.05 vs. his-PPA1, #p<0.05 vs. N1E115 cells treated with VPA+GFP, a<0.05 vs. VPA+Ad-PPA1.</p

Effect of Nico (81 mg/l of drinking water) on cardiac disease at a relatively early stage in the HSPB5 R120G (R120G) TG mice.

<p>(A) Protocol was shown. Nico was administered via drinking water from 12 weeks for a total of 4 weeks in the R120G TG mice. (B) Typical pictures of Western blot analysis. (C) Quantitative analysis of HSPB5. Values are the x-fold increase relative to values in non-transgenic (NTG) mouse hearts whose values are arbitrarily set to 1. (D) Representative pictures of the immunohistochemistry are shown. (E) Typical picture of the filter assay for the detection of the aggregates containing HSPB5 proteins. (F) Quantitative analysis of the aggregates containing mutant R120G protein. (G) Fractional shortening assessed by the echocardiogram. Cardiac functional measurements were made at 16 weeks (n = 8 mice). (H) Typical pictures of Western blot analysis for ANF. (I) Quantitative analysis of ANF in the hearts in the R120G TG mice. (J) Masson's trichrome staining. (K) Cytochrome c levels of mitochondrial fraction isolated from the hearts from the R120G TG mice with or without Nico treatment. Typical pictures of Western blot analysis. Quantitative analysis of cytochrome c. GAPDH was used as a loading control in the cytosolic fraction and the voltage-dependent anion channel (VDAC) was used in the mitochondrial fraction. (L) Quantitative analysis of cytochrome c in mitochondrial and cytosolic fractions isolated from the hearts in the R120G TG mice. *p<0.05, ***p<0.001, vs. the NTG, #p<0.05 vs. the R120G TG mice (n = 6–8).</p

Cytochrome c levels of mitochondrial fraction isolated from the cardiomyocytes expressing HSPB5 R120G (R120G) with or without Nico treatment.

<p>(A) Typical pictures of Western blot analysis. Quantitative analysis of cytochrome c (B) and HSPB5 (C) in mitochondrial and cytosolic fractions isolated from the cardiomyocytes. GAPDH was used as a loading control in the cytosolic fraction and the voltage-dependent anion channel (VDAC) was used in mitochondrial fraction. *p<0.05, ** p<0.01, ***p<0.001 vs. the cardiomyocytes infected with LacZ (LacZ), ##p<0.01, ###p<0.001 vs. the cardiomyocytes infected with wild-type HSPB5 (HSPB5), ap<0.05 vs. cardiomyocytes infected with R120G treated with PBS. (n = 6).</p

Western blot analysis of apoptosis-related proteins, BAX and BCL2, and apoptotic cell death in HSPB5 R120G (R120G) TG mice with or without Nico treatment.

<p>(A) Typical pictures of Western blot analysis. (B and C) Quantitative analysis of BAX (B) and BCL2 (C) in mitochondrial fraction isolated from the TG mouse hearts. * p<0.05, **p<0.01 vs. non-transgenic (NTG) mice; # p<0.05 vs. R120G TG mice (n = 4).</p

Heart weight and blood pressure.

<p>Values are mean±SEM; TG: transgenic, NTG: non-transgenic; n = 6–8 nico, nicorandil.</p><p>***p<0.001 vs. NTG)mice.</p