Generation of a Matrix Gla (Mgp) floxed mouse, followed by conditional knockout, uncovers a new Mgp function in the eye.

The ability to ablate a gene in a given tissue by generating a conditional knockout (cKO) is crucial for determining its function in the targeted tissue. Such tissue-specific ablation is even more critical when the gene's conventional knockout (KO) is lethal, which precludes studying the consequences of its deletion in other tissues. Therefore, here we describe a successful strategy that generated a Matrix Gla floxed mouse (Mgp.floxed) by the CRISPR/Cas9 system, that subsequently allowed the generation of cKOs by local viral delivery of the Cre-recombinase enzyme. MGP is a well-established inhibitor of calcification gene, highly expressed in arteries' smooth muscle cells and chondrocytes. MGP is also one of the most abundant genes in the trabecular meshwork, the eye tissue responsible for maintenance of intraocular pressure (IOP) and development of Glaucoma. Our strategy entailed one-step injection of two gRNAs, Cas9 protein and a long-single-stranded-circular DNA donor vector (lsscDNA, 6.7 kb) containing two loxP sites in cis and 900-700 bp 5'/3' homology arms. Ocular intracameral injection of Mgp.floxed mice with a Cre-adenovirus, led to an Mgp.TMcKO mouse which developed elevated IOP. Our study discovered a new role for the Mgp gene as a keeper of physiological IOP in the eye.

BRG1 and BRM SWI/SNF ATPases redundantly maintain cardiomyocyte homeostasis by regulating cardiomyocyte mitophagy and mitochondrial dynamics in vivo.

There has been an increasing recognition that mitochondrial perturbations play a central role in human heart failure. Mitochondrial networks, whose function is to maintain the regulation of mitochondrial biogenesis, autophagy ('mitophagy') and mitochondrial fusion/fission, are new potential therapeutic targets. Yet our understanding of the molecular underpinning of these processes is just emerging. We recently identified a role of the SWI/SNF ATP-dependent chromatin remodeling complexes in the metabolic homeostasis of the adult cardiomyocyte using cardiomyocyte-specific and inducible deletion of the SWI/SNF ATPases BRG1 and BRM in adult mice (Brg1/Brm double mutant mice). To build upon these observations in early altered metabolism, the present study looks at the subsequent alterations in mitochondrial quality control mechanisms in the impaired adult cardiomyocyte. We identified that Brg1/Brm double-mutant mice exhibited increased mitochondrial biogenesis, increases in 'mitophagy', and alterations in mitochondrial fission and fusion that led to small, fragmented mitochondria. Mechanistically, increases in the autophagy and mitophagy-regulated proteins Beclin1 and Bnip3 were identified, paralleling changes seen in human heart failure. Evidence for perturbed cardiac mitochondrial dynamics included decreased mitochondria size, reduced numbers of mitochondria, and an altered expression of genes regulating fusion (Mfn1, Opa1) and fission (Drp1). We also identified cardiac protein amyloid accumulation (aggregated fibrils) during disease progression along with an increase in pre-amyloid oligomers and an upregulated unfolded protein response including increased GRP78, CHOP, and IRE-1 signaling. Together, these findings described a role for BRG1 and BRM in mitochondrial quality control, by regulating mitochondrial number, mitophagy, and mitochondrial dynamics not previously recognized in the adult cardiomyocyte. As critical to the pathogenesis of heart failure, epigenetic mechanisms like SWI/SNF chromatin remodeling seem more intimately linked to cardiac function and mitochondrial quality control mechanisms than previously realized.

Registered report: A coding-independent function of gene and pseudogene mRNAs regulates tumour biology.

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered report describes the proposed replication plan of key experiments from 'A coding-independent function of gene and pseudogene mRNAs regulates tumour biology' by Poliseno et al. (2010), published in Nature in 2010. The key experiments to be replicated are reported in Figures 1D, 2F-H, and 4A. In these experiments, Poliseno and colleagues report microRNAs miR-19b and miR-20a transcriptionally suppress both PTEN and PTENP1 in prostate cancer cells (Figure 1D; Poliseno et al., 2010). Decreased expression of PTEN and/or PTENP1 resulted in downregulated PTEN protein levels (Figure 2H), downregulation of both mRNAs (Figure 2G), and increased tumor cell proliferation (Figure 2F; Poliseno et al., 2010). Furthermore, overexpression of the PTEN 3' UTR enhanced PTENP1 mRNA abundance limiting tumor cell proliferation, providing additional evidence for the co-regulation of PTEN and PTENP1 (Figure 4A; Poliseno et al., 2010). The Reproducibility Project: Cancer Biology is collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published in eLife.

Strigolactone signaling in root development and phosphate starvation.

Strigolactones (SLs), have recently been recognized as phytohormone involve in orchestrating shoot and root architecture. In, roots SLs positively regulate root hair length and density, suppress lateral root formation and promote primary root meristem cell number. The biosynthesis and exudation of SLs increases under low phosphate level to regulate root responses. This hormonal response suggests an adaptation strategy of plant to optimize growth and development under nutrient limitations. However, little is known on signal-transduction pathways associated with SL activities. In this review, we outline the current knowledge on SL biology by describing their role in the regulation of root development. Also, we discuss the recent findings on the non-cell autonomous signaling of SLs, that involve PIN polarization, vesicle trafficking, changes in actin architecture and dynamic in response to phosphate starvation.

Arabidopsis response to low-phosphate conditions includes active changes in actin filaments and PIN2 polarization and is dependent on strigolactone signalling.

Strigolactones (SLs) are plant hormones that regulate the plant response to phosphate (Pi) growth conditions. At least part of SL-signalling execution in roots involves MAX2-dependent effects on PIN2 polar localization in the plasma membrane (PM) and actin bundling and dynamics. We examined PIN2 expression, PIN2 PM localization, endosome trafficking, and actin bundling under low-Pi conditions: a MAX2-dependent reduction in PIN2 trafficking and polarization in the PM, reduced endosome trafficking, and increased actin-filament bundling were detected in root cells. The intracellular protein trafficking that is related to PIN proteins but unassociated with AUX1 PM localization was selectively inhibited. Exogenous supplementation of the synthetic SL GR24 to a SL-deficient mutant (max4) led to depletion of PIN2 from the PM under low-Pi conditions. Accordingly, roots of mutants in MAX2, MAX4, PIN2, TIR3, and ACTIN2 showed a reduced low-Pi response compared with the wild type, which could be restored by auxin (for all mutants) or GR24 (for all mutants except max2-1). Changes in PIN2 polarity, actin bundling, and vesicle trafficking may be involved in the response to low Pi in roots, dependent on SL/MAX2 signalling.

Strigolactone analog GR24 triggers changes in PIN2 polarity, vesicle trafficking and actin filament architecture.

Strigolactones (SLs) are plant hormones that regulate shoot and root development in a MAX2-dependent manner. The mechanism underlying SLs' effects on roots is unclear. We used root hair elongation to measure root response to SLs. We examined the effects of GR24 (a synthetic, biologically active SL analog) on localization of the auxin efflux transporter PIN2, endosomal trafficking, and F-actin architecture and dynamics in the plasma membrane (PM) of epidermal cells of the primary root elongation zone in wildtype (WT) Arabidopsis and the SL-insensitive mutant max2. We also recorded the response to GR24 of trafficking (tir3), actin (der1) and PIN2 (eir1) mutants. GR24 increased polar localization of PIN2 in the PM of epidermal cells and accumulation of PIN2-containing brefeldin A (BFA) bodies, increased ARA7-labeled endosomal trafficking, reduced F-actin bundling and enhanced actin dynamics, all in a MAX2-dependent manner. Most of the der1 and tir3 mutant lines also displayed reduced sensitivity to GR24 with respect to root hair elongation. We suggest that SLs increase PIN2 polar localization, PIN2 endocytosis, endosomal trafficking, actin debundling and actin dynamics in a MAX2-dependent fashion. This enhancement might underlie the WT root's response to SLs, and suggests noncell autonomous activity of SLs in roots.

Diuretics prevent thiazolidinedione-induced cardiac hypertrophy without compromising insulin-sensitizing effects in mice.

Much concern has arisen regarding critical adverse effects of thiazolidinediones (TZDs), including rosiglitazone and pioglitazone, on cardiac tissue. Although TZD-induced cardiac hypertrophy (CH) has been attributed to an increase in plasma volume or a change in cardiac nutrient preference, causative roles have not been established. To test the hypothesis that volume expansion directly mediates rosiglitazone-induced CH, mice were fed a high-fat diet with rosiglitazone, and cardiac and metabolic consequences were examined. Rosiglitazone treatment induced volume expansion and CH in wild-type and PPARγ heterozygous knockout (Pparg(+/-)) mice, but not in mice defective for ligand binding (Pparg(P465L/+)). Cotreatment with the diuretic furosemide in wild-type mice attenuated rosiglitazone-induced CH, hypertrophic gene reprogramming, cardiomyocyte apoptosis, hypertrophy-related signal activation, and left ventricular dysfunction. Similar changes were observed in mice treated with pioglitazone. The diuretics spironolactone and trichlormethiazide, but not amiloride, attenuated rosiglitazone effects on volume expansion and CH. Interestingly, expression of glucose and lipid metabolism genes in the heart was altered by rosiglitazone, but these changes were not attenuated by furosemide cotreatment. Importantly, rosiglitazone-mediated whole-body metabolic improvements were not affected by furosemide cotreatment. We conclude that releasing plasma volume reduces adverse effects of TZD-induced volume expansion and cardiac events without compromising TZD actions in metabolic switch in the heart and whole-body insulin sensitivity.

Distribution of histone3 lysine 4 trimethylation at T3-responsive loci in the heart during reversible changes in gene expression.

Expression in the adult heart of a number of cardiac genes, including the two genes comprising the cardiac myosin heavy chain locus (Myh), is controlled by thyroid hormone (T3) levels, but there is minimal information concerning the epigenetic status of the genes when their expressions change. We fed mice normal chow or a propyl thio uracil (PTU, an inhibitor of T3 production) diet for 6 weeks, or the PTU diet for 6 weeks followed by normal chow for a further 2 weeks. Heart ventricles from these groups were then used for ChIP-seq analyses with an antibody to H3K4me3, a well-documented epigenetic marker of gene activation. The resulting data show that, at the Myh7 locus, H3K4me3 modifications are induced primarily at 5' transcribed region in parallel with increased expression of beta myosin heavy chain (MHC). At the Myh6 locus, decreases in H3K4me3 modifications occurred at the promoter and 5' transcribed region. Extensive H3K4me3 modifications also occurred at the intergenic region between the two Myh genes, which extended into the 3' transcribed region of Myh7. The PTU-induced changes in H3K4me3 levels are, for the most part, reversible but are not invariably complete. We found full restoration of Myh6 gene expression upon PTU withdrawal; however, the H3K4me3 pattern was only partially restored at Myh6, suggesting that full reexpression of Myh6 does not require that the H3K4me3 modifications return fully to the untreated conditions. Together, our data show that the H3K4me3 modification is an epigenetic marker closely associated with changes in Myh gene expression.

MicroRNA-mediated in vitro and in vivo direct reprogramming of cardiac fibroblasts to cardiomyocytes.

RATIONALE: Repopulation of the injured heart with new, functional cardiomyocytes remains a daunting challenge for cardiac regenerative medicine. An ideal therapeutic approach would involve an effective method at achieving direct conversion of injured areas to functional tissue in situ. OBJECTIVE: The aim of this study was to develop a strategy that identified and evaluated the potential of specific micro (mi)RNAs capable of inducing reprogramming of cardiac fibroblasts directly to cardiomyocytes in vitro and in vivo. METHODS AND RESULTS: Using a combinatorial strategy, we identified a combination of miRNAs 1, 133, 208, and 499 capable of inducing direct cellular reprogramming of fibroblasts to cardiomyocyte-like cells in vitro. Detailed studies of the reprogrammed cells demonstrated that a single transient transfection of the miRNAs can direct a switch in cell fate as documented by expression of mature cardiomyocyte markers, sarcomeric organization, and exhibition of spontaneous calcium flux characteristic of a cardiomyocyte-like phenotype. Interestingly, we also found that miRNA-mediated reprogramming was enhanced 10-fold on JAK inhibitor I treatment. Importantly, administration of miRNAs into ischemic mouse myocardium resulted in evidence of direct conversion of cardiac fibroblasts to cardiomyocytes in situ. Genetic tracing analysis using Fsp1Cre-traced fibroblasts from both cardiac and noncardiac cell sources strongly suggests that induced cells are most likely of fibroblastic origin. CONCLUSIONS: The findings from this study provide proof-of-concept that miRNAs have the capability of directly converting fibroblasts to a cardiomyocyte-like phenotype in vitro. Also of significance is that this is the first report of direct cardiac reprogramming in vivo. Our approach may have broad and important implications for therapeutic tissue regeneration in general.

Heterogeneous myocyte enhancer factor-2 (Mef2) activation in myocytes predicts focal scarring in hypertrophic cardiomyopathy.

Unknown molecular responses to sarcomere protein gene mutations account for pathologic remodeling in hypertrophic cardiomyopathy (HCM), producing myocyte growth and increased cardiac fibrosis. To determine if hypertrophic signals activated myocyte enhancer factor-2 (Mef2), we studied mice carrying the HCM mutation, myosin heavy-chain Arg403Gln, (MHC(403/+)) and an Mef2-dependent ß-galactosidase reporter transgene. In young, prehypertrophic MHC(403/+) mice the reporter was not activated. In hypertrophic hearts, activation of the Mef2-dependent reporter was remarkably heterogeneous and was observed consistently in myocytes that bordered fibrotic foci with necrotic cells, MHC(403/+) myocytes with Mef2-dependent reporter activation reexpressed the fetal myosin isoform (ßMHC), a molecular marker of hypertrophy, although MHC(403/+) myocytes with or without ßMHC expression were comparably enlarged over WT myocytes. To consider Mef2 roles in severe HCM, we studied homozygous MHC(403/403) mice, which have accelerated remodeling, widespread myocyte necrosis, and neonatal lethality. Levels of phosphorylated class II histone deacetylases that activate Mef2 were substantially increased in MHC(403/403) hearts, but Mef2-dependent reporter activation was patchy. Sequential analyses showed myocytes increased Mef2-dependent reporter activity before death. Our data dissociate myocyte hypertrophy, a consistent response in HCM, from heterogeneous Mef2 activation and reexpression of a fetal gene program. The temporal and spatial relationship of Mef2-dependent gene activation with myocyte necrosis and fibrosis in MHC(403/+) and MHC(403/403) hearts defines Mef2 activation as a molecular signature of stressed HCM myocytes that are poised to die.

Reversible epigenetic modifications of the two cardiac myosin heavy chain genes during changes in expression.

The two genes of the cardiac myosin heavy chain (MHC) locus-alpha-MHC (aMHC) and beta-MHC (bMHC)--are reciprocally regulated in the mouse ventricle during development and in adult conditions such as hypothyroidism and pathological cardiac hypertrophy. Their expressions are under the control of thyroid hormone T3 levels. To gain insights into the epigenetic mechanisms that underlie this inducible and reversible switching of the aMHC and bMHC isoforms, we have investigated the histone modification patterns that occur over the two cardiac MHC promoters during T3-mediated reversible switching of gene expression. Mice fed a diet of propylthiouracil (PTU, an inhibitor of T3 synthesis) for 2 weeks dramatically reduce aMHC mRNA expression and increase bMHC mRNA levels to high levels, while a subsequent withdrawal of PTU diet for 2 weeks completely reverses the T3-mediated changes in MHC expression. Using hearts from mice treated in this way, we carried out chromatin immunoprecipitation-qPCR assays with antibodies against acetylated histone H3 (H3ac) and trimethylated histone (H3K4me3)-two well-documented markers of activation. Our results show that the reexpression of bMHC is associated at the bMHC promoter with increased H3ac but not H3K4me3. In contrast, the silencing of aMHC is associated at its promoter with decreased H3K4me3, but not decreased H3ac. The epigenetic changes at the two MHC promoters are completely reversed when the gene expression returns to initial levels. These data indicate that during reciprocal and inducible gene expression H3ac parallels bMHC isoform expression while H3K4me3 parallels expression of the tightly linked aMHC isoform.

MicroRNA-208a is a regulator of cardiac hypertrophy and conduction in mice.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that have gained status as important regulators of gene expression. Here, we investigated the function and molecular mechanisms of the miR-208 family of miRNAs in adult mouse heart physiology. We found that miR-208a, which is encoded within an intron of alpha-cardiac muscle myosin heavy chain gene (Myh6), was actually a member of a miRNA family that also included miR-208b, which was determined to be encoded within an intron of beta-cardiac muscle myosin heavy chain gene (Myh7). These miRNAs were differentially expressed in the mouse heart, paralleling the expression of their host genes. Transgenic overexpression of miR-208a in the heart was sufficient to induce hypertrophic growth in mice, which resulted in pronounced repression of the miR-208 regulatory targets thyroid hormone-associated protein 1 and myostatin, 2 negative regulators of muscle growth and hypertrophy. Studies of the miR-208a Tg mice indicated that miR-208a expression was sufficient to induce arrhythmias. Furthermore, analysis of mice lacking miR-208a indicated that miR-208a was required for proper cardiac conduction and expression of the cardiac transcription factors homeodomain-only protein and GATA4 and the gap junction protein connexin 40. Together, our studies uncover what we believe are novel miRNA-dependent mechanisms that modulate cardiac hypertrophy and electrical conduction.

Decreased beta-adrenergic responsiveness following hypertrophy occurs only in cardiomyocytes that also re-express beta-myosin heavy chain.

AIMS: Cardiac hypertrophy is associated with a reduction in the contractile response to beta-adrenergic stimulation, and with re-expression of foetal genes such as beta-myosin heavy chain (MHC). However, whether these two markers of pathology develop concordantly in the same individual cells or independently in different cells is not known. METHODS AND RESULTS: To answer this question, we examined the beta-adrenergic response of individual beta-MHC expressing and non-expressing myocytes from hypertrophic hearts, using a previously generated mouse model (YFP/beta-MHC) in which a yellow fluorescent protein (YFP) is fused to the native beta-MHC protein allowing easy identification of beta-MHC expressing cells. Yellow fluorescent protein/beta-MHC mice were submitted to 4 weeks of transverse aortic constriction (TAC), and the contractile parameters of isolated individual myocytes in response to the beta-adrenergic agonist isoproterenol were assessed. Our results demonstrate that the decrease in isoproterenol-induced cell shortening that develops in TAC hearts occurs only in those hypertrophic myocytes that re-express beta-MHC. Hypertrophic myocytes that do not express beta-MHC have contractility indices indistinguishable from non-TAC controls. CONCLUSION: These data show that the reduction of beta-adrenergic response occurs only in subsets, rather than in all myocytes, and is coincident with re-expression of beta-MHC.

Discordant on/off switching of gene expression in myocytes during cardiac hypertrophy in vivo.

To determine whether the expression of cardiac genes changes in a graded manner or by on/off switching when cardiac myocytes change genetic programs in living animals, we have studied two indicator genes that change their expression oppositely in mouse binucleate ventricular cardiomyocytes during development and in response to cardiac hypertrophy. One is a single-copy transgene controlled by an alpha-myosin heavy chain (aMHC) promoter and coding for CFP. The other is the endogenous beta-myosin heavy chain (bMHC) gene modified to code for a YFP-bMHC fusion protein. Using high-resolution confocal microscopy, we determined the expression of the two indicator genes in individual cardiomyocytes perinatally and after inducing cardiac hypertrophy by transverse aortic constriction. Our results provide strong evidence that the cardiac genes respond by switching their expression in an on/off rather than graded manner, and that responding genes within a single cell and within the two nuclei of cardiomyocytes do not necessarily switch concordantly.

Fibrosis, not cell size, delineates beta-myosin heavy chain reexpression during cardiac hypertrophy and normal aging in vivo.

Reexpression of the fetally expressed beta-myosin heavy chain (beta-MHC) gene is a well documented marker of pathological cardiac hypertrophy and normal aging in many experimental models. To gain insights into factors affecting this reexpression of beta-MHC within the complex anatomical structure of the heart, we investigated the spatial pattern of its expression at the level of single cells during aging and hypertrophy. We generated mice that express yellow fluorescent protein fused to the N terminus of the beta-MHC and examined its expression pattern during normal aging and in mice with hypertrophy induced by constitutive expression of a renin transgene. The localization of fibrosis within the hearts also was determined by using a fluorescent lectin. The results show that reexpression of beta-MHC occurs in discrete subsets of myocytes within the subendocardium rather than uniformly throughout the heart, that beta-MHC induction is not an obligatory consequence of cellular hypertrophy, and that beta-MHC-expressing cells in the normal aging heart and the hypertrophic heart are distributed predominantly in clusters within and surrounding foci of fibrosis. We conclude that beta-MHC gene expression in the normal aging adult and hypertrophic mouse heart is a marker of fibrosis rather than of cellular hypertrophy.

Three-dimensional diffusion tensor microscopy of fixed mouse hearts.

The relative utility of 3D, microscopic resolution assessments of fixed mouse myocardial structure via diffusion tensor imaging is demonstrated in this study. Isotropic 100-microm resolution fiber orientation mapping within 5.5 degrees accuracy was achieved in 9.1 hr scan time. Preliminary characterization of the diffusion tensor primary eigenvector reveals a smooth and largely linear angular rotation across the left ventricular wall. Moreover, a higher level of structural hierarchy is evident from the organized secondary and tertiary eigenvector fields. These findings are consistent with the known myocardial fiber and laminar structures reported in the literature and suggest an essential role of diffusion tensor microscopy in developing quantitative atlases for studying the structure-function relationships of mouse hearts.

Basic residues within the Kruppel zinc finger DNA binding domains are the critical nuclear localization determinants of EKLF/KLF-1.

EKLF/KLF-1 is an erythroid-restricted transcription factor essential for expression of the adult beta-globin gene. EKLF/KLF-1 is a 358-amino acid nuclear protein with an amino-terminal proline-rich domain and a carboxyl-terminal DNA binding domain. The nuclear localization signal (NLS) of EKLF/KLF-1 has not been empirically determined. We generated a series of epitope-tagged deletion and point mutants and assessed their subcellular localization. Our results delimit the NLS to the 83-amino acid (amino acids 276-358) DNA binding domain that consists of three Kruppel zinc fingers. All three zinc fingers are necessary for efficient nuclear localization; deletion of any individual finger results in cytoplasmic accumulation. Fusion of the three zinc fingers to green fluorescent protein (GFP) targeted GFP to the nucleus, demonstrating that the zinc finger domain is sufficient for nuclear localization. EKLF/KLF-1 containing histidine to alanine mutations that disrupt the structure of all three fingers retains appropriate nuclear localization, indicating that neither the tertiary structure of the zinc fingers nor specific DNA binding are necessary for nuclear localization. We demonstrate that basic residues within the fingers are the critical determinants for nuclear localization; mutations of these basic residues to alanine resulted in cytoplasmic mislocalization. The basic residues of all mammalian Kruppel zinc fingers are highly conserved; therefore we propose that these basic residues are a common NLS shared by all Kruppel family members.