Suet Nee Chen

Myozenin 2 Is a Novel Gene for Human Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a genetic disorder caused by mutations in sarcomeric proteins (excluding phenocopy). The causal genes in approximately one-third of the cases remain unknown. We identified a family comprised of 6 clinically affected members. The phenotype was characterized by early onset of symptoms, pronounced cardiac hypertrophy, and cardiac arrhythmias. We excluded MYH7, MYBPC3, TNNT2, and ACTC1 as the causal gene either by direct sequencing or by haplotype analysis. To map the putative candidate sarcomeric gene, we perforbold locus-specific haplotyping to detect cosegregation of the locus haplotype with the phenotype, followed by mutation screening. We genotyped 5 short-tandem-repeat markers that spanned a 4.4-centimorgan region on 4q26-q27 locus and encompassed myozenin 2 (MYOZ2), a Z-disk protein. The maximum logarithm of odds score was 2.03 (P=0.005). All affected members shared a common haplotype, implicating MYOZ2 as the causal gene. To detect the causal mutation, we sequenced all exons and exon–intron boundaries of MYOZ2 in 10 family members and identified a T→C missense mutation corresponding to S48P substitution, which cosegregated with inheritance of HCM (N=6). It was absent in 4 clinically normal family members and in 658 additional normal individuals. To determine frequency of the MYOZ2 mutations in HCM, we sequenced MYOZ2 in 516 HCM probands and detected another missense mutation (I246M). It was absent in 2 normal family members and 517 controls. Both mutations affect highly conserved amino acids. We conclude MYOZ2 is a novel causal gene for human HCM.

Prevention of Cardiac Hypertrophy by Atorvastatin in a Transgenic Rabbit Model of Human Hypertrophic Cardiomyopathy

Cardiac hypertrophy, a major determinant of morbidity and mortality in hypertrophic cardiomyopathy (HCM), is considered a secondary phenotype and potentially preventable. To test this hypothesis, we screened 30 5- to 6-month-old &bgr;-myosin heavy chain Q403 transgenic rabbits by echocardiography and selected 26 without cardiac hypertrophy. We randomized the transgenic rabbits to treatment with atorvastatin (2.5 mg/Kg/d), known to block hypertrophic signaling or a placebo. We included 15 nontransgenic rabbits as controls. Cardiac phenotype was analyzed serially before, 6 and 12 months after randomization. Serum total cholesterol levels were reduced by 49% with atorvastatin administration. Left-ventricular mass, wall thickness; myocyte size, myocardial levels of molecular markers of hypertrophy, lipid peroxides, and oxidized mitochondrial DNA; and the number of terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive myocytes were increased significantly in the placebo but not in the atorvastatin group. Myocardium catalase mRNA levels were decreased by 5-fold in the placebo but were normal in the atorvastatin group. Catalase protein level and activity were not significantly changed. Levels of membrane-bound Ras and phospho-p44/42 mitogen-activated-protein kinase (MAPK) were increased in the placebo group (≈2.5 fold) but were reduced in the atorvastatin group. Levels of GTP- and membrane-bound RhoA and Rac1, phospho-p38, and phospho-c-Jun NH2-terminal kinases were unchanged. Thus, atorvastatin prevented development of cardiac hypertrophy; determined at organ, cellular, and molecular levels, partly through reducing active Ras and p44/42 MAPK. The results indicate potential beneficial effects of atorvastatin in prevention of cardiac hypertrophy, a major determinant of morbidity in all forms of cardiovascular diseases, and beckon clinical studies in humans with HCM.

The Hippo Pathway Is Activated and Is a Causal Mechanism for Adipogenesis in Arrhythmogenic Cardiomyopathy

Rationale: Mutations in the intercalated disc proteins, such as plakophilin 2 (PKP2), cause arrhythmogenic cardiomyopathy (AC). AC is characterized by the replacement of cardiac myocytes by fibro-adipocytes, cardiac dysfunction, arrhythmias, and sudden death. Objective: To delineate the molecular pathogenesis of AC. Methods and Results: Localization and levels of selected intercalated disc proteins, including signaling molecules, were markedly reduced in human hearts with AC. Altered protein constituents of intercalated discs were associated with activation of the upstream Hippo molecules in the human hearts, in Nkx2.5-Cre:DspW/F and Myh6:Jup mouse models of AC, and in the PKP2 knockdown HL-1 myocytes (HL-1PKP2:shRNA). Level of active protein kinase C-&agr; isoform, which requires PKP2 for activity, was reduced. In contrast, neurofibromin 2 (or Merlin), a molecule upstream of the Hippo pathway and that is inactivated by protein kinase C-&agr; isoform, was activated. Consequently, the downstream Hippo molecules mammalian STE20-like protein kinases 1/2 (MST1/2), large tumor suppressor kinases 1/2 (LATS1/2), and Yes-associated protein (YAP) (the latter is the effector of the pathway) were phosphorylated. Coimmunoprecipitation detected binding of phosphorylated YAP, phosphorylated &bgr;-catenin, and junction protein plakoglobin (the latter translocated from the junction). RNA sequencing, transcript quantitative polymerase chain reaction, and reporter assays showed suppressed activity of SV40 transcriptional enhancer factor domain (TEAD) and transcription factor 7-like 2 (TCF7L2), which are transcription factors of the Hippo and the canonical Wnt signaling, respectively. In contrast, adipogenesis was enhanced. Simultaneous knockdown of Lats1/2, molecules upstream to YAP, rescued inactivation of YAP and &bgr;-catenin and adipogenesis in the HL-1PKP2:shRNA myocytes. Conclusions: Molecular remodeling of the intercalated discs leads to pathogenic activation of the Hippo pathway, suppression of the canonical Wnt signaling, and enhanced adipogenesis in AC. The findings offer novel mechanisms for the pathogenesis of AC.

Resolution of Established Cardiac Hypertrophy and Fibrosis and Prevention of Systolic Dysfunction in a Transgenic Rabbit Model of Human Cardiomyopathy Through Thiol-Sensitive Mechanisms

Background— Cardiac hypertrophy, the clinical hallmark of hypertrophic cardiomyopathy (HCM), is a major determinant of morbidity and mortality not only in HCM but also in a number of cardiovascular diseases. There is no effective therapy for HCM and generally for cardiac hypertrophy. Myocardial oxidative stress and thiol-sensitive signaling molecules are implicated in pathogenesis of hypertrophy and fibrosis. We posit that treatment with N-acetylcysteine, a precursor of glutathione, the largest intracellular thiol pool against oxidative stress, could reverse cardiac hypertrophy and fibrosis in HCM. Methods and Results— We treated 2-year-old β-myosin heavy-chain Q403 transgenic rabbits with established cardiac hypertrophy and preserved systolic function with N-acetylcysteine or a placebo for 12 months (n=10 per group). Transgenic rabbits in the placebo group had cardiac hypertrophy, fibrosis, systolic dysfunction, increased oxidized to total glutathione ratio, higher levels of activated thiol-sensitive active protein kinase G, dephosphorylated nuclear factor of activated T cells (NFATc1) and phospho-p38, and reduced levels of glutathiolated cardiac α-actin. Treatment with N-acetylcysteine restored oxidized to total glutathione ratio, normalized levels of glutathiolated cardiac α-actin, reversed cardiac and myocyte hypertrophy and interstitial fibrosis, reduced the propensity for ventricular arrhythmias, prevented cardiac dysfunction, restored myocardial levels of active protein kinase G, and dephosphorylated NFATc1 and phospho-p38. Conclusions— Treatment with N-acetylcysteine, a safe prodrug against oxidation, reversed established cardiac phenotype in a transgenic rabbit model of human HCM. Because there is no effective pharmacological therapy for HCM and given that hypertrophy, fibrosis, and cardiac dysfunction are common and major predictors of clinical outcomes, the findings could have implications in various cardiovascular disorders.

Human Molecular Genetic and Functional Studies Identify TRIM63, Encoding Muscle RING Finger Protein 1, as a Novel Gene for Human Hypertrophic Cardiomyopathy

Rationale: A delicate balance between protein synthesis and degradation maintains cardiac size and function. TRIM63 encoding Muscle RING Finger 1 (MuRF1) maintains muscle protein homeostasis by tagging the sarcomere proteins with ubiquitin for subsequent degradation by the ubiquitin-proteasome system (UPS). Objective: To determine the pathogenic role of TRIM63 in human hypertrophic cardiomyopathy (HCM). Methods and Results: Sequencing of TRIM63 gene in 302 HCM probands (250 white individuals) and 339 control subjects (262 white individuals) led to identification of 2 missense (p.A48V and p.I130M) and a deletion (p.Q247*) variants exclusively in the HCM probands. These 3 variants were absent in 751 additional control subjects screened by TaqMan assays. Likewise, rare variants were enriched in the white HCM population (11/250, 4.4% versus 3/262, 1.1%, respectively, P=0.024). Expression of the mutant TRIM63 was associated with mislocalization of TRIM63 to sarcomere Z disks, impaired auto-ubiquitination, reduced ubiquitination and UPS-mediated degradation of myosin heavy chain 6, cardiac myosin binding protein C, calcineurin (PPP3CB), and p-MTOR in adult cardiac myocytes. Induced expression of the mutant TRIM63 in the mouse heart was associated with cardiac hypertrophy, activation of the MTOR-S6K and calcineurin pathways, and expression of the hypertrophic markers, which were normalized on turning off expression of the mutant protein. Conclusions: TRIM63 mutations, identified in patients with HCM, impart loss-of-function effects on E3 ligase activity and are probably causal mutations in HCM. The findings implicate impaired protein degradation in the pathogenesis of HCM.

Pathogenesis of Hypertrophic Cardiomyopathy Caused by Myozenin 2 Mutations Is Independent of Calcineurin Activity

AIMS The role of calcineurin protein phosphatase 2B (PP2B) in the pathogenesis of human hypertrophic cardiomyopathy (HCM) remains unsettled. We determined potential involvement of calcineurin in the pathogenesis of HCM caused by mutations in myozenin 2 (MYOZ2), an inhibitor of calcineurin. METHODS AND RESULTS We generated multiple lines of transgenic mice expressing either Flag-tagged wild-type (WT) (MYOZ2(WT)) or mutant MYOZ2(S48P) and MYOZ2(I246M), identified in families with HCM, in the heart. To mimic the human genotype, we generated bigenic mice expressing WT and mutant MYOZ2 in the background of hemizygous endogenous MYOZ2 (Myoz2(+/-)). Transgene proteins constituted 15-48% of the total MYOZ2 protein in the heart. Mutant MYOZ2 mice showed molecular, cellular, and gross cardiac hypertrophy, preserved systolic function, and interstitial fibrosis. Immunofluorescence staining showed co-localization of WT and mutant MYOZ2 proteins with α-actinin at the Z disks. Electron microscopy showed disrupted and mal-aligned Z disks in the mutant mice. Cardiac calcineurin activity, determined by quantifying Rcan1.4 mRNA and protein levels, luciferase activity in triple transgenic Myoz2(+/-):NFATc-Luc:MYOZ2(I246M) and Myoz2(+/-):NFATc-Luc:MYOZ2(WT) mice, and NFATc transcriptional activity assay, was unchanged in the mutant transgenic mice. However, levels of phospho-ERK1/2 and JNK54/46 were altered in the transgenic mice. Likewise, lentiviral-mediated expression of the MYOZ2(I246M) did not affect RCAN1.4 and calcineurin (PPP3CB) protein levels. CONCLUSIONS Thus, the cardiac phenotype in HCM caused by MYOZ2 mutations might be independent of calcineurin activity in the heart. Z disk abnormalities might provide the stimulus for the induction of cardiac hypertrophy caused by MYOZ2 mutations.

Cardiac Fibro-Adipocyte Progenitors Express Desmosome Proteins and Preferentially Differentiate to Adipocytes Upon Deletion of the Desmoplakin Gene

RATIONALE Mutations in desmosome proteins cause arrhythmogenic cardiomyopathy (AC), a disease characterized by excess myocardial fibroadipocytes. Cellular origin(s) of fibroadipocytes in AC is unknown. OBJECTIVE To identify the cellular origin of adipocytes in AC. METHODS AND RESULTS Human and mouse cardiac cells were depleted from myocytes and flow sorted to isolate cells expressing platelet-derived growth factor receptor-α and exclude those expressing other lineage and fibroblast markers (CD32, CD11B, CD45, Lys76, Ly(-6c) and Ly(6c), thymocyte differentiation antigen 1, and discoidin domain receptor 2). The PDGFRA(pos):Lin(neg):THY1(neg):DDR2(neg) cells were bipotential as the majority expressed collagen 1 α-1, a fibroblast marker, and a subset CCAAT/enhancer-binding protein α, a major adipogenic transcription factor, and therefore, they were referred to as fibroadipocyte progenitors (FAPs). FAPs expressed desmosome proteins, including desmoplakin, predominantly in the adipogenic but not fibrogenic subsets. Conditional heterozygous deletion of Dsp in mice using Pdgfra-Cre deleter led to increased fibroadipogenesis in the heart and mild cardiac dysfunction. Genetic fate mapping tagged 41.4±4.1% of the cardiac adipocytes in the Pdgfra-Cre:Eyfp:Dsp(W/F) mice, indicating an origin from FAPs. FAPs isolated from the Pdgfra-Cre:Eyfp:Dsp(W/F) mouse hearts showed enhanced differentiation to adipocytes. Mechanistically, deletion of Dsp was associated with suppressed canonical Wnt signaling and enhanced adipogenesis. In contrast, activation of the canonical Wnt signaling rescued adipogenesis in a dose-dependent manner. CONCLUSIONS A subset of cardiac FAPs, identified by the PDGFRA(pos):Lin(neg):THY1(neg):DDR2(neg) signature, expresses desmosome proteins and differentiates to adipocytes in AC through a Wnt-dependent mechanism. The findings expand the cellular spectrum of AC, commonly recognized as a disease of cardiac myocytes, to include nonmyocyte cells in the heart.

Passion and Determination Are the Genesis of Research

> In life, materials will decay over time, what is left in you is Passion. I might as well take a chance and do what I love . > > —Suet Nee Chen When I was 6 years old, it became clear that my blurry vision was not a “little problem.” My parents sent me to an ophthalmologist who made the diagnosis of severe myopia (>−6 diopters [D]). Since then, my glasses have progressively thickened. By age of 13, my myopia had worsened to −10 D. The ophthalmologist advised my father that I should not stress my eyes and must avoid intense physical activities. Unfortunately, this included avoiding Olympic handball, which was my passion at the time. The concern was that a severe hit to my eyes could cause retinal detachment. When I heard the recommendation, my eyes welled up with tears. Either I had to stop playing the sport that I loved or risk going blind because of trauma. My father did what any loving parent would do. He said: “My child, please be happy in life and be whoever you want to be. You have my full support.” Despite the risk of losing my eyesight, my father …