Protective Effect of Geranylgeranylacetone via Enhancement of HSPB8 Induction in Desmin-Related Cardiomyopathy

An arg120gly (R120G) missense mutation in HSPB5 (alpha-beta-crystallin ), which belongs to the small heat shock protein (HSP) family, causes desmin-related cardiomyopathy (DRM), a muscle disease that is characterized by the formation of inclusion bodies, which can contain pre-amyloid oligomer intermediates (amyloid oligomer). While we have shown that small HSPs can directly interrupt amyloid oligomer formation, the in vivo protective effects of the small HSPs on the development of DRM is still uncertain.In order to extend the previous in vitro findings to in vivo, we used geranylgeranylacetone (GGA), a potent HSP inducer. Oral administration of GGA resulted not only in up-regulation of the expression level of HSPB8 and HSPB1 in the heart of HSPB5 R120G transgenic (R120G TG) mice, but also reduced amyloid oligomer levels and aggregates. Furthermore, R120G TG mice treated with GGA exhibited decreased heart size and less interstitial fibrosis, as well as improved cardiac function and survival compared to untreated R120G TG mice. To address possible mechanism(s) for these beneficial effects, cardiac-specific transgenic mice expressing HSPB8 were generated. Overexpression of HSPB8 led to a reduction in amyloid oligomer and aggregate formation, resulting in improved cardiac function and survival. Treatment with GGA as well as the overexpression of HSPB8 also inhibited cytochrome c release from mitochondria, activation of caspase-3 and TUNEL-positive cardiomyocyte death in the R120G TG mice.Expression of small HSPs such as HSPB8 and HSPB1 by GGA may be a new therapeutic strategy for patients with DRM

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