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

Inhibition of aggregate (A–C) and amyloid oligomer formation (D–E) by GGA treatment in HSPB5 R120G TG (R120G TG) mice at 30 weeks of age.

<p>(A) Representative pictures of the immunohistochemistry are shown. The aggregates containing the mutant HSPB5 R120G protein were observed in the R120G TG mice, and long-term GGA treatment reduced the aggregates. (B) Typical picture of the filter assay for the detection of the aggregates. (C) Quantitative analysis of the aggregates containing the mutant HSPB5 R120G protein (n = 4 mice). (D) Effect of GGA on expression level of the amyloid oligomer. The amyloid oligomer (green) was stained by the anti-oligomer antibody as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005351#s2" target="_blank">Materials and Methods</a> section. To distinguish the cardiomyocytes, cardiac troponin I was stained (red). (E) Quantitative analysis of the amyloid oligomer. Amyloid oligomer levels were measured by fluorescence intensity. GGA treatment reduced the HSPB5 R120G-induced amyloid oligomer formation. Values shown are the -fold increase relative to NTG mice, whose value was set to 1. (n = 4 mice) *** <i>p</i><0.001 vs. NTG mice; # <i>p</i><0.05, ### <i>p</i><0.001 vs. R120G TG mice.</p

Effect of long-term GGA treatment on development of cardiomyopathy in HSPB5 R120G TG (R120G TG) mice.

<p>(A) Representative pictures of the hearts in the R120G TG mice at 30 weeks of age. (B) The ratios of heart weight per body weight. Long-term GGA treatment inhibited hypertrophy in R120G TG mice (n = 6–8 mice). (C) Masson's trichrome stain in the R120G TG mouse heart with or without GGA treatment (2% GGA). (D) Fractional shortening. Cardiac functional measurement was carried out at 30 weeks of age (n = 10–15 mice). (E) Survival curves. GGA treatment effectively attenuated premature death in the R120G TG mice (n = 15–20 mice). (F) Heat shock factor (HSF1) in the hearts. Localization of HSF1 is shown in heart from R120G TG mice. ***<i>p</i><0.001 vs. NTG mice and # <i>p</i><0.05, ## <i>p</i><0.01, and ### <i>p</i><0.001 vs. R120G TG mice with 0% GGA (0% GGA in the R120G TG mice).</p

HSPB8 was overexpressed using tTA/inducible α-myosin heavy chain promoter system as described in Materials and Methods.

<p>(A) Representative Western blots using hearts from tTA/HSPB8 double transgenic (tTA/HSPB8 TG) mice, HSPB5 R120G transgenic (R120G TG) and tTA/HSPB8/HSPB5 R120G triple transgenic (Triple TG) mice. (B) Quantitative analysis of small HSP levels (n = 4 mice). Values are the -fold increase relative to those in non-transgenic (NTG) mice whose values are arbitrarily set to 1. (C) Representative pictures of R120G TG, tTA/HSPB8 TG and Triple TG hearts at 30 weeks of age. (D) The ratios of heart weight per body weight. Overexpression of HSPB8 inhibited cardiac enlargement in R120G TG mice (n = 6–8 mice). (E) Masson's trichrome stain. Overexpression of HSPB8 attenuated the interstitial fibrosis in R120G TG mice. (F) Fractional shortening assessed by the echocardiogram. Cardiac functional measurements were made at 30 weeks (n = 10–15 mice). (G) Survival curves. Overexpression of HSPB8 effectively attenuated premature death in R120G TG mice (n = 15–20 mice). *** <i>p</i><0.001 vs. NTG mice and # <i>p</i><0.05, and ### <i>p</i><0.0001 vs. the R120G TG.</p

Induction of small HSPs by GGA in neonatal rat cardiomyocytes.

<p>(A) Western blot analysis. GGA induced the expression levels of small HSPs in a dose-dependent manner. (B) Quantitative analysis of small HSP expression (n = 4). Values are the -fold increase relative to cardiomyocyte cultures treated with vehicle (0 nM of GGA) whose values are arbitrarily set to 1. * <i>p</i><0.05, ** <i>p</i><0.01 and *** <i>p</i><0.001 vs. 0 nM of GGA. (C) Representative pictures of immunohistochemical analyses in the HSPB5 R120G-infected cardiomyocytes. Cardiomyocytes were infected with the adenovirus vector containing the wild-type HSPB5 (WT) or mutant HSPB5 R120G (R120G). An amyloid oligomer (green) was detected by the anti-oligomer antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005351#s2" target="_blank">Materials and Methods</a>. Nuclei were stained with DAPI. (D) Quantitative analysis of the amyloid oligomer. Amyloid oligomer levels were measured by fluorescence intensity (n = 4). *** <i>p</i><0.001 vs. cardiomyocytes infected with WT, and ### <i>p</i><0.001 vs. cardiomyocytes infected with R120G. (E) Protective effects of GGA on the cellular viability of the HSPB5 R120G-infected cardiomyocytes. Cellular viability was determined by the MTT assay. Values are the -fold increase relative to untreated cardiomyocyte cultures whose values are arbitrarily set to 1 (n = 6). *** <i>p</i><0.001 vs. untreated cardiomyocytes (UN), ### <i>p</i><0.001 vs. cardiomyocytes infected with WT, and aa <i>p</i><0.01 vs. cardiomyocytes infected with R120G.</p

TUNEL-positive cardiomyocyte death in HSPB5 R120G TG (R120G TG) mice.

<p>(A) Cytochrome c was released from mitochondria (Mito) into the cytosolic fraction (Cyto) in R120G TG mice while GGA treatment (GGA 2%) and overexpression of HSPB8 inhibited its release. (B) Immunostaining for activated caspase-3 in sections derived from hearts at 30 weeks of age. Cardiomyocytes were identified by phalloidin staining for actin (green). Activated caspase-3 (arrowheads) is apparent in the HSPB5 R120G (R120G TG) section. GGA treatment (GGA 2%) and overexpression of HSPB8 (tTA/HSPB8/HSPB5 R120G triple TG; Triple TG) reduced caspase-3 activation. (C) TUNEL assays in hearts from HSPB5 R120G TG mice at 30 weeks of age. Cardiomyocytes were identified by phalloidin staining for actin (green). Positive signals were apparent only in the HSPB5 R120G-derived sections and are indicated by an arrow. GGA treatment (GGA 2%) and overexpression of HSPB8 (Triple TG) decreased the TUNEL-positive signals. (D) Quantification of TUNEL signals. Between 5×10⁵ nuclei were counted for each sample and the percentage of TUNEL-positive cells were determined (n = 4 mice).</p

In vivo enhancement of small HSP induction by GGA.

<p>(A) Representative pictures of Western blot analysis of the R120G TG mouse heart. (B) Quantitative analysis of the small HSP expression levels (n = 4 mice). Values are the -fold increase relative to those in NTG mice whose values are arbitrarily set to 1. *** <i>p</i><0.001 vs. NTG. Values of wild-type HSPB5 TG mice (WT TG) are also shown as a control. (C) Diagram of the protocols. NTG and HSPB5 R120G TG mice at 4 weeks of age were administered GGA. Western blotting, morphological analysis, immunohistochemistry and echocardiography were performed on the mice at 30 weeks of age (arrow). (D) Representative pictures of Western blot analysis of hearts from R120G TG mice treated with GGA. GGA was administrated in R120G TG mice from 4 weeks until 30 weeks of age. (E) Quantitative analysis of the small HSP expression levels (n = 4–5 mice). Values are the -fold increase relative to those in NTG mice whose values are arbitrarily set to 1. * <i>p</i><0.05, ** <i>p</i><0.01 and *** <i>p</i><0.001 vs. 0% GGA.</p

Echocardiographic parameters.

<p>Values are mean±SEM. N = 10–15 in each group.</p><p>LVIDd indicates left ventricular internal end-diastolic dimension; LVIDs, left ventricular internal end-systolic dimension; LVESV, left ventricular end-systolic volume; LVEDV, left ventricular end-systolic volume.</p>*<p>p<0.05 vs. NTG.</p>#<p>p<0.05 vs. R120G TG.</p

Inhibition of aggregate formation (A–C) and amyloid oligomer formation (D–E) by overexpression of HSPB8 in HSPB5 R120G TG mice (R120G TG) at 30 weeks of age.

<p>(A) Representative pictures of the immunohistochemistry are shown. The aggregates containing the mutant HSPB5 R120G protein are observed in R120G TG mice, and the overexpression of HSPB8 reduced the aggregates in samples from HSPB8 TG mice (tTA/HSPB8/HSPB5 R120G triple TG; Triple TG). (B) Typical picture of the filter assay for the detection of the aggregates. (C) Quantitative analysis of the aggregates containing mutant HSPB5 R120G protein (n = 4 mice). (D) Effect of HSPB8 overexpression on amyloid oligomer formation (green). (E) Amyloid oligomer levels were measured by fluorescence intensity. The overexpression of HSPB8 reduced the HSPB5 R120G-induced amyloid oligomer formation (Triple TG). Values shown are the -fold increase relative to NTG mice, whose value was set to 1 (n = 4 mice). To distinguish the cardiomyocytes, cardiac troponin I was stained (red)(A and D). *** <i>p</i><0.001 vs. NTG mice; ### <i>p</i><0.001 vs. R120G TG mice.</p