Sir2 is an NAD-dependent deacetylase that connects metabolism with longevity in yeast, flies, and worms. Mammals have seven Sir2 homologs (SIRT1-7). We show that SIRT4 is a mitochondrial enzyme that uses NAD to ADP-ribosylate and downregulate glutamate dehydrogenase (GDH) activity. GDH is known to promote the metabolism of glutamate and glutamine, generating ATP, which promotes insulin secretion. Loss of SIRT4 in insulinoma cells activates GDH, thereby upregulating amino acid-stimulated insulin secretion. A similar effect is observed in pancreatic beta cells from mice deficient in SIRT4 or on the dietary regimen of calorie restriction (CR). Furthermore, GDH from SIRT4-deficient or CR mice is insensitive to phosphodiesterase, an enzyme that cleaves ADP-ribose, suggesting the absence of ADP-ribosylation. These results indicate that SIRT4 functions in beta cell mitochondria to repress the activity of GDH by ADP-ribosylation, thereby downregulating insulin secretion in response to amino acids, effects that are alleviated during CR.

BACKGROUND Dilated cardiomyopathy and hypertrophic cardiomyopathy arise from mutations in many genes. TTN, the gene encoding the sarcomere protein titin, has been insufficiently analyzed for cardiomyopathy mutations because of its enormous size. METHODS We analyzed TTN in 312 subjects with dilated cardiomyopathy, 231 subjects with hyper-trophic cardiomyopathy, and 249 controls by using next-generation or dideoxy sequencing. We evaluated deleterious variants for cosegregation in families and assessed clinical characteristics. RESULTS We identified 72 unique mutations (25 nonsense, 23 frameshift, 23 splicing, and 1 large tandem insertion) that altered full-length titin. Among subjects studied by means of next-generation sequencing, the frequency of TTN mutations was significantly higher among subjects with dilated cardiomyopathy (54 of 203 [27%]) than among subjects with hypertrophic cardiomyopathy (3 of 231 [1%], P = 3×10−16) or controls (7 of 249 [3%], P = 9×10−14). TTN mutations cosegregated with dilated cardiomyopathy in families (combined lod score, 11.1) with high (>95%) observed penetrance after the age of 40 years. Mutations associated with dilated cardiomyopathy were overrepresented in the titin A-band but were absent from the Z-disk and M-band regions of titin (P≤0.01 for all comparisons). Overall, the rates of cardiac outcomes were similar in subjects with and those without TTN mutations, but adverse events occurred earlier in male mutation carriers than in female carriers (P = 4×10−5). CONCLUSIONS TTN truncating mutations are a common cause of dilated cardiomyopathy, occurring in approximately 25% of familial cases of idiopathic dilated cardiomyopathy and in 18% of sporadic cases. Incorporation of sequencing approaches that detect TTN truncations into genetic testing for dilated cardiomyopathy should substantially increase test sensitivity, thereby allowing earlier diagnosis and therapeutic intervention for many patients with dilated cardiomyopathy. Defining the functional effects of TTN truncating mutations should improve our understanding of the pathophysiology of dilated cardiomyopathy. (Funded by the Howard Hughes Medical Institute and others.)

Rationale Epigenetic marks are crucial for organogenesis, but their role in heart development is poorly understood. Polycomb Repressive Complex 2 (PRC2) trimethylates histone H3 at lysine 27, establishing H3K27me3 repressive epigenetic marks that promote tissue-specific differentiation by silencing ectopic gene programs. Objective We studied the function of PRC2 in murine heart development using a tissue-restricted conditional inactivation strategy. Methods and Results Inactivation of the PRC2 subunit Ezh2 by Nkx2-5Cre (Ezh2NK) caused lethal congenital heart malformations, namely compact myocardial hypoplasia, hypertrabeculation, and ventricular septal defect. Candidate and genome-wide RNA expression profiling and chromatin immunoprecipitation analyses of Ezh2NK heart identified genes directly repressed by EZH2. Among these were the potent cell cycle inhibitors Ink4a/b, whose upregulation was associated with decreased cardiomyocyte proliferation in Ezh2NK. EZH2-repressed genes were enriched for transcriptional regulators of non-cardiomyocyte expression programs, such as Pax6, Isl1, Six1. EZH2 was also required for proper spatiotemporal regulation of cardiac gene expression, as Hcn4, Mlc2a, and Bmp10 were inappropriately upregulated in ventricular RNA. PRC2 was also required later in heart development, as indicated by cardiomyocyte-restricted TNT-Cre inactivation of the PRC2 subunit Eed. However, Ezh2 inactivation by TNT-Cre did not cause an overt phenotype, likely due to functional redundancy with Ezh1. Thus early Ezh2 inactivation by Nk2-5Cre caused later disruption of cardiomyocyte gene expression and heart development. Conclusions Our study reveals a previously undescribed role of EZH2 in regulating heart formation and shows that perturbation of the epigenetic landscape early in cardiogenesis has sustained disruptive effects at later developmental stages.