The Huntington's disease-related cardiomyopathy prevents a hypertrophic response in the R6/2 mouse model

<div><p>Huntington's disease (HD) is neurodegenerative disorder for which the mutation results in an extra-long tract of glutamines that causes the huntingtin protein to aggregate. It is characterized by neurological symptoms and brain pathology that is associated with nuclear and cytoplasmic aggregates and with transcriptional deregulation. Despite the fact that HD has been recognized principally as a neurological disease, there are multiple epidemiological studies showing that HD patients exhibit a high rate of cardiovascular events leading to heart failure. To unravel the mechanistic basis of cardiac dysfunction in HD, we employed a wide range of molecular techniques using the well-established genetic R6/2 mouse model that develop a considerable degree of the cardiac atrophy at end stage disease. We found that chronic treatment with isoproterenol, a potent beta-adrenoreceptor agonist, did not change the overall gross morphology of the HD murine hearts. However, there was a partial response to the beta-adrenergenic stimulation by the further re-expression of foetal genes. In addition we have profiled the expression level of <i>Hdacs</i> in the R6/2 murine hearts and found that the isoproterenol stimulation of <i>Hdac</i> expression was partially blocked. For the first time we established the <i>Hdac</i> transcriptional profile under hypertrophic conditions and found 10 out of 18 <i>Hdacs</i> to be markedly deregulated. Therefore, we conclude that R6/2 murine hearts are not able to respond to the chronic isoproterenol treatment to the same degree as wild type hearts and some of the hypertrophic signals are likely attenuated in the symptomatic HD animals.</p></div

Gross cardiac morphology of the hearts treated with isoproterenol.

<p>Representative phalloidin staining (green) shows left ventricle myocyte hypertrophy in WT mice but not in 12 week old R6/2 mice. Nuclei (blue) were visualized with DAPI. Scale bar 30 µm.</p

Transcriptional deregulation of HD markers involved in heart failure.

<p>(A) <i>S100A4</i> (S100 calcium binding protein A4), Vgl-3 (vestigial related factor 3), <i>Vgl-4</i> (vestigial related factor 4) were up-regulated while <i>Mck</i> (muscle creatine kinase) and <i>Bdnf</i> (brain derived neurotophic factor) transcripts were significantly decreased in the heart of R6/2 mice. (B) Transcripts of Transcriptional Enhancer Family members (TEAD or TEFs) were significantly deregulated in isoproterenol treated mice. All Taqman qPCR values were normalized to the geometric mean of three housekeeping genes: <i>Actb</i>, <i>Cyc1</i> and <i>Gapdh</i>. Error bars are SEM (n = 6). Two-way ANOVA with Bonferroni <i>post-hoc</i> test: *<i>p</i><0.05, **<i>p</i><0.01; ***<i>p</i><0.001.</p

Partial re-activation of foetal gene markers in the hearts of R6/2 treated with isoproterenol.

<p><i>Anp</i> (atrial natriuretic peptide), <i>Bnp</i> (brain natriuretic protein) and members of the four and half LIM family <i>Fhl1</i> and <i>Fhl2 were</i> elevated in the heart of WT and R6/2 mice. All Taqman qPCR values were normalized to the geometric mean of three housekeeping genes: <i>Actb</i>, <i>Cyc1</i> and <i>Gapdh</i>. Error bars are SEM (n = 6). Two-way ANOVA with Bonferroni <i>post-hoc</i> test: *<i>p</i><0.05, **<i>p</i><0.01; ***<i>p</i><0.001.</p

HDAC4 Does Not Act as a Protein Deacetylase in the Postnatal Murine Brain In Vivo

Reversible protein acetylation provides a central mechanism for controlling gene expression and cellular signaling events. It is governed by the antagonistic commitment of two enzymes families: the histone acetyltransferases (HATs) and the histone deacetylases (HDACs). HDAC4, like its class IIa counterparts, is a potent transcriptional repressor through interactions with tissue specific transcription factors via its N-terminal domain. Whilst the lysine deacetylase activity of the class IIa HDACs is much less potent than that of the class I enzymes, HDAC4 has been reported to influence protein deacetylation through its interaction with HDAC3. To investigate the influence of HDAC4 on protein acetylation we employed the immunoaffinity-based AcetylScan proteomic method. We identified many proteins known to be modified by acetylation, but found that the absence of HDAC4 had no effect on the acetylation profile of the murine neonate brain. This is consistent with the biochemical data suggesting that HDAC4 may not function as a lysine deacetylase, but these in vivo data do not support the previous report showing that the enzymatic activity of HDAC3 might be modified by its interaction with HDAC4. To complement this work, we used Affymetrix arrays to investigate the effect of HDAC4 knock-out on the transcriptional profile of the postnatal murine brain. There was no effect on global transcription, consistent with the absence of a differential histone acetylation profile. Validation of the array data by Taq-man qPCR indicated that only protamine 1 and Igfbp6 mRNA levels were increased by more than one-fold and only Calml4 was decreased. The lack of a major effect on the transcriptional profile is consistent with the cytoplasmic location of HDAC4 in the P3 murine brain

Chronic administration of isoproterenol causes a significant transcriptional deregulation of many <i>Hdacs</i> and <i>Sirtuins</i>.

<p>(A) <i>Hdac1</i>, <i>Hdac2</i>, <i>Hdac4</i>, <i>Hdac6</i>, <i>Hdac7</i> and <i>Hdac8</i> transcript levels were increased while <i>Hdac3</i> mRNA was significantly reduced in the heart of WT mice treated with Isoproterenol. Only <i>Hdac4</i> and <i>Hdac6</i> were significantly increased in the hearts of R6/2 mice. (B) <i>Sirt3</i> and <i>Sirt5</i> transcript levels were significantly increased in the heart of WT mice treated with Isoproterenol. <i>Sirt 1</i> and <i>Sirt 2</i> were decreased in the hearts of WT mice, but only <i>Sirt2</i> was decreased in the hearts of R6/2 mice. All Taqman qPCR values were normalized to the geometric mean of three housekeeping genes: <i>Actb</i>, <i>Cyc1</i> and <i>Gapdh</i>. Error bars are SEM (n = 6). Two-way ANOVA with Bonferroni <i>post-hoc</i> test: *<i>p</i><0.05, **<i>p</i><0.01; ***<i>p</i><0.001.</p

Moderate fibrosis level based on collagen VI deposits is not attenuated in the hearts of R6/2 mice.

<p>(A) Representative confocal pictograms of whole heart sections from 12 week old WT and R6/2 mice. (B) Quantification of the collagen VI staining area. Fibrosis was detected with the anti-collagen VI antibody (green) and nuclei (blue) were visualised with DAPI. Scale bar 30 µm. Values are mean ± SEM (<i>n</i> = 3). Student's <i>t</i> test: *<i>p</i><0.05, ***<i>p</i><0.001.</p

Morphometric analysis of the isoproterenol treated mice.

<p>(A) Body weight at 10 weeks of age prior to implantation of the alzet pumps. (B) Body weight (C) tibia length (D) heart weight (E) heart weight to tibia length index (F) heart rate were measured at 12 weeks of age. The gender-combined analysis of body weight did not detect a decrease in the R6/2 group, which may be because of a gender imbalance between the R6/2 and WT groups. All values are mean ± SEM (<i>n</i> = 8 WTveh, <i>n</i> = 9 WTiso, <i>n</i> = 14 R6/2veh, <i>n</i> = 14 R6/2iso). One-way ANOVA with Bonferroni <i>post-hoc</i> test: *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Myostatin inhibition prevents skeletal muscle pathophysiology in Huntington's disease mice

Huntington’s disease (HD) is an inherited neurodegenerative disorder of which skeletal muscle atrophy is a common feature, and multiple lines of evidence support a muscle-based pathophysiology in HD mouse models. Inhibition of myostatin signaling increases muscle mass, and therapeutic approaches based on this are in clinical development. We have used a soluble ActRIIB decoy receptor (ACVR2B/Fc) to test the effects of myostatin/activin A inhibition in the R6/2 mouse model of HD. Weekly administration from 5 to 11 weeks of age prevented body weight loss, skeletal muscle atrophy, muscle weakness, contractile abnormalities, the loss of functional motor units in EDL muscles and delayed end-stage disease. Inhibition of myostatin/activin A signaling activated transcriptional profiles to increase muscle mass in wild type and R6/2 mice but did little to modulate the extensive Huntington’s disease-associated transcriptional dysregulation, consistent with treatment having little impact on HTT aggregation levels. Modalities that inhibit myostatin signaling are currently in clinical trials for a variety of indications, the outcomes of which will present the opportunity to assess the potential benefits of targeting this pathway in HD patients

Validation of the genes predicted to be downregulated in <i>Hdac4</i> knock-out P3 brains.

<p>(A) <i>Nrxn1</i> (neurexin1), <i>Gmbf</i> (glia maturation factor beta), Cdh7 (cadherin 7), <i>Bmpr2</i> (bone morphogenic protein receptor, type II), Stk25 (serine/threonine kinase 25), <i>Atrx</i> (alpha thalassemia/mental retardation syndrome X-linked homolog) and <i>Eif4g1</i> (eukaryotic translation initiation factor 4, gamma 1) were modestly upregulated in <i>Hdac4</i>KO P3 brains. <i>Calml4</i> (calmodulin-like 4) was the only gene that was downregulated (B) The expression level of Hif1α (hypoxia inducible factor 1 alpha subunit), <i>Crebbp</i> (CREB-binding protein), Syt1 (synaptotagemin 1) <i>Garln1</i> (GTPase activating RANGAP domain like 1 protein), <i>Rapgef6</i> (Rap guanine nucleotide exchange factor GEF6), Rock1 (Rho-associated coiled-coil containing protein kinase 1), <i>Nedd4</i> (NEDD4 binding protein), <i>Jmjd4</i> (jumonji containing protein 4), <i>Casc4</i> (cancer susceptibility candidate 4), <i>Pnmal</i> (PNMA-like 2), <i>Rlf</i> (rearranged L-myc fusion sequence) and Scd2 (stearoyl-Coenzyme A desaturase 2) did not change between <i>Hdac4</i>KO and WT P3 brains. All mRNA expression levels were assessed by Taqman qPCR and presented as a relative expression ratio to the geometric mean of three housekeeping genes <i>Atp5b</i>, <i>Canx</i>, <i>Rpl13α</i>. Error bars are S.E.M (n>7). *<i>p</i><0.05, **<i>p</i><0.01.</p