Donald R. Menick

Periostin Is Required for Maturation and Extracellular Matrix Stabilization of Noncardiomyocyte Lineages of the Heart

The secreted periostin protein, which marks mesenchymal cells in endocardial cushions following epithelial–mesenchymal transformation and in mature valves following remodeling, is a putative valvulogenesis target molecule. Indeed, periostin is expressed throughout cardiovascular morphogenesis and in all 4 adult mice valves (annulus and leaflets). Additionally, periostin is expressed throughout the fibrous cardiac skeleton and endocardial cushions in the developing heart but is absent from both normal and/or pathological mouse cardiomyocytes. Periostin (perilacZ) knockout mice exhibit viable valve disease, with neonatal lethality in a minority and latent disease with leaflet abnormalities in the viable majority. Surviving perilacZ-null leaflets are truncated, contain ectopic cardiomyocytes and smooth muscle, misexpress the cartilage proteoglycan aggrecan, demonstrate disorganized matrix stratification, and exhibit reduced transforming growth factor-&bgr; signaling. Neonatal perilacZ nulls that die (14%) display additional defects, including leaflet discontinuities, delamination defects, and deposition of acellular extracellular matrix. Assessment of collagen production, 3D lattice formation ability, and transforming growth factor-&bgr; responsiveness indicate periostin-deficient fibroblasts are unable to support normal valvular remodeling and establishment of a mature cardiac skeleton. Furthermore, pediatric stenotic bicuspid aortic valves that have lost normal extracellular matrix trilaminar stratification have greatly reduced periostin. This suggests that loss of periostin results in inappropriate differentiation of mesenchymal cushion cells and valvular abnormalities via a transforming growth factor-&bgr;–dependent pathway during establishment of the mature heart. Thus, perilacZ knockouts provide a new model of viable latent valve disease.

A beta-tubulin leucine cluster involved in microtubule assembly and paclitaxel resistance.

Analysis of β-tubulin alleles from nine paclitaxel-resistant Chinese hamster ovary cell lines revealed an unexpected cluster of mutations affecting Leu-215, Leu-217, and Leu-228. Six of the mutant alleles encode a His, Arg, or Phe substitution at Leu-215; another mutant allele has an Arg substitution at Leu-217; and the final two mutant alleles have substitutions of His or Phe at Leu-228. Using plasmids that allow tetracycline regulated expression, the L215H, L217R, and L228F mutations were introduced into a hemagglutinin antigen-tagged β-tubulin cDNA and transfected into wild-type Chinese hamster ovary cells. In all three cases, low to moderate expression of the transfected mutant gene conferred paclitaxel resistance. Higher levels of expression caused disruption of microtubule assembly, cell cycle arrest at mitosis, and failure to proliferate. Consistent with reduced microtubule stability, cells expressing mutant hemagglutinin β-tubulin had fewer acetylated microtubules than nonexpressing cells in the same population. These data, together with previous studies showing that the paclitaxel-resistant mutant cell lines have less stable microtubules, indicate that the leucine cluster represents an important structural motif for microtubule assembly.

Association of Tyrosine-phosphorylated c-Src with the Cytoskeleton of Hypertrophying Myocardium

Given the central position of the focal adhesion complex, both physically in coupling integrins to the interstitium and biochemically in providing an upstream site for anabolic signal generation, we asked whether the recruitment of non-receptor tyrosine kinases to the cytoskeleton might be a mechanism whereby cellular loading could activate growth regulatory signals responsible for cardiac hypertrophy. Analysis revealed cytoskeletal association of c-Src, FAK, and β3-integrin, but no Fyn, in the pressure-overloaded right ventricle. This association was seen as early as 4 h after right ventricular pressure overloading, increased through 48 h, and reverted to normal in 1 week. Cytoskeletal binding of non-receptor tyrosine kinases was synchronous with tyrosine phosphorylation of several cytoskeletal proteins, including c-Src. Examination of cytoskeleton-bound c-Src revealed that a significant portion of the tyrosine phosphorylation was not at the Tyr-527 site and therefore presumably was at the Tyr-416 site. Thus, these studies strongly suggest that non-receptor tyrosine kinases, in particular c-Src, may play a critical role in hypertrophic growth regulation by their association with cytoskeletal structures, possibly via load activation of integrin-mediated signaling.

Integrin Activation and Focal Complex Formation in Cardiac Hypertrophy

Cardiac hypertrophy is characterized by both remodeling of the extracellular matrix (ECM) and hypertrophic growth of the cardiocytes. Here we show increased expression and cytoskeletal association of the ECM proteins fibronectin and vitronectin in pressure-overloaded feline myocardium. These changes are accompanied by cytoskeletal binding and phosphorylation of focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, c-Src at Tyr-416, recruitment of the adapter proteins p130Cas, Shc, and Nck, and activation of the extracellular-regulated kinases ERK1/2. A synthetic peptide containing the Arg-Gly-Asp (RGD) motif of fibronectin and vitronectin was used to stimulate adult feline cardiomyocytes cultured on laminin or within a type-I collagen matrix. Whereas cardiocytes under both conditions showed RGD-stimulated ERK1/2 activation, only collagen-embedded cells exhibited cytoskeletal assembly of FAK, c-Src, Nck, and Shc. In RGD-stimulated collagen-embedded cells, FAK was phosphorylated only at Tyr-397 and c-Src association occurred without Tyr-416 phosphorylation and p130Cas association. Therefore, c-Src activation is not required for its cytoskeletal binding but may be important for additional phosphorylation of FAK. Overall, our study suggests that multiple signaling pathways originate in pressure-overloaded heart following integrin engagement with ECM proteins, including focal complex formation and ERK1/2 activation, and many of these pathways can be activated in cardiomyocytes via RGD-stimulated integrin activation.

The Role of GATA, CArG, E-box, and a Novel Element in the Regulation of Cardiac Expression of the Na+-Ca2+ Exchanger Gene

The cardiac Na+-Ca2+ exchanger (NCX1) is the principal Ca2+ efflux mechanism in cardiocytes. The exchanger is up-regulated in both cardiac hypertrophy and failure. In this report, we identify the cis-acting elements that control cardiac expression and α-adrenergic up-regulation of the exchanger gene. Deletion analysis revealed that a minimal cardiac promoter fragment from −184 to +172 is sufficient for cardiac expression and α-adrenergic stimulation. Mutational analysis revealed that both the CArG element at −80 and the GATA element at −50 were required for cardiac expression. Gel mobility shift assay supershift analysis demonstrated that the serum response factor binds to the CArG element and GATA-4 binds to the GATA element. Point mutations in the −172 E-box demonstrated that it was required for α-adrenergic induction. In addition, deletion analysis revealed one or more enhancer elements in the first intron (+103 to +134) that are essential for phenylephrine up-regulation but bear no homology to any known transcription element. Therefore, this work demonstrates that SRF and GATA-4 are critical for NCX1 expression in neonatal cardiomyocytes and that the −172 E-box in addition to a novel enhancer element(s) are required for phenylephrine up-regulation of NCX1 and may mediate its hypertrophic up-regulation.

CLONING OF CARDIAC, KIDNEY, AND BRAIN PROMOTERS OF THE FELINE NCX1 GENE

The Na+-Ca2+ exchanger (NCX1) plays a major role in calcium efflux and therefore in the control and regulation of intracellular calcium in the heart. The exchanger has been shown to be regulated at several levels including transcription. NCX1 mRNA levels are up-regulated in both cardiac hypertrophy and failure. In this work, the 5′-end of the ncx1 gene has been cloned to study the mechanisms that mediate hypertrophic stimulation and cardiac expression. The feline ncx1 gene has three exons that encode 5′-untranslated sequences that are under the control of three tissue-specific promoters. The cardiac promoter drives expression in cardiocytes, but not in mouse L cells. Although it contains at least one enhancer (−2000 to −1250 base pairs (bp)) and one or more negative elements (−1250 to −250 bp), a minimum promoter (−250 to +200 bp) is sufficient for cardiac expression and α-adrenergic stimulation.

Inhibition of Histone Deacetylase Protects the Retina from Ischemic Injury

PURPOSE. The pathogenesis of retinal ischemia results from a series of events involving changes in gene expression and inflammatory cytokines. Protein acetylation is an essential mechanism in regulating transcriptional and inflammatory events. The purpose of this study was to investigate the neuroprotective action of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) in a retinal ischemic model. METHODS. To investigate whether HDAC inhibition can reduce ischemic injury, rats were treated with TSA (2.5 mg/kg intraperitoneally) twice daily on days 0, 1, 2, and 3. Seven days after ischemic injury, morphometric and electroretinographic (ERG) analyses were used to assess retinal structure and function. Western blot and immunohistochemical analyses were used to evaluate TSA-induced changes in histone-H3 acetylation and MMP secretion. RESULTS. In vehicle-treated animals, ERG a- and b-waves from ischemic eyes were significantly reduced compared with contralateral responses. In addition, histologic examination of these eyes revealed significant degeneration of inner retinal layers. In rats treated with TSA, amplitudes of ERG a- and b-waves from ischemic eyes were significantly increased, and normal inner retina morphology was preserved. Ischemia also increased the levels of retinal TNF-alpha, which was blocked by TSA treatment. In astrocyte cultures, the addition of TNF-alpha (10 ng/mL) stimulated the secretion of MMP-1 and MMP-3, which were blocked by TSA (100 nM). CONCLUSIONS. These studies provide the first evidence that suppressing HDAC activity can protect the retina from ischemic injury. This neuroprotective response is associated with the suppression of retinal TNF-alpha expression and signaling. The use of HDAC inhibitors may provide a novel treatment for ischemic retinal injury.

Reduced Collagen Deposition in Infarcted Myocardium Facilitates Induced Pluripotent Stem Cell Engraftment and Angiomyogenesis for Improvement of Left Ventricular Function

OBJECTIVES The purpose of this study was to assess the effect of scar tissue composition on engraftment of progenitor cells into infarcted myocardium. BACKGROUND Scar tissue formation after myocardial infarction creates a barrier that severely compromises tissue regeneration, limiting potential functional recovery. METHODS In vitro: A tricell patch (Tri-P) was created from peritoneum seeded and cultured with induced pluripotent stem cell-derived cardiomyocytes, endothelial cells, and mouse embryonic fibroblasts. The expression of fibrosis-related molecules from mouse embryonic fibroblasts and infarcted heart was measured by Western blot and quantitative reverse transcriptase polymerase chain reaction. In vivo: A Tri-P was affixed over the entire infarcted area 7 days after myocardial infarction in mice overexpressing adenylyl cyclase 6 (AC6). Engraftment efficiency of progenitor cells in hearts of AC6 mice was compared with that of control wild-type (WT) mice using a combination of in vivo bioluminescence imaging, post-mortem ex vivo tissue analysis, and the number of green fluorescent protein-positive cells. Echocardiography of left ventricular (LV) function was performed weekly. Hearts were harvested for analysis 4 weeks after Tri-P application. Mouse embryonic fibroblasts were stimulated with forskolin before an anoxia/reoxygenation protocol. Fibrosis-related molecules were analyzed. RESULTS In AC6 mice, infarcted hearts treated with Tri-P showed significantly higher bioluminescence imaging intensity and numbers of green fluorescent protein-positive cells than in WT mice. LV function improved progressively in AC6 mice from weeks 2 to 4 and was associated with reduced LV fibrosis. CONCLUSIONS Application of a Tri-P in AC6 mice resulted in significantly higher induced pluripotent stem cell engraftment accompanied by angiomyogenesis in the infarcted area and improvement in LV function.

Mutations in DCHS1 cause mitral valve prolapse.

Mitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery. Despite a clear heritable component, the genetic aetiology leading to non-syndromic MVP has remained elusive. Four affected individuals from a large multigenerational family segregating non-syndromic MVP underwent capture sequencing of the linked interval on chromosome 11. We report a missense mutation in the DCHS1 gene, the human homologue of the Drosophila cell polarity gene dachsous (ds), that segregates with MVP in the family. Morpholino knockdown of the zebrafish homologue dachsous1b resulted in a cardiac atrioventricular canal defect that could be rescued by wild-type human DCHS1, but not by DCHS1 messenger RNA with the familial mutation. Further genetic studies identified two additional families in which a second deleterious DCHS1 mutation segregates with MVP. Both DCHS1 mutations reduce protein stability as demonstrated in zebrafish, cultured cells and, notably, in mitral valve interstitial cells (MVICs) obtained during mitral valve repair surgery of a proband. Dchs1+/− mice had prolapse of thickened mitral leaflets, which could be traced back to developmental errors in valve morphogenesis. DCHS1 deficiency in MVP patient MVICs, as well as in Dchs1+/− mouse MVICs, result in altered migration and cellular patterning, supporting these processes as aetiological underpinnings for the disease. Understanding the role of DCHS1 in mitral valve development and MVP pathogenesis holds potential for therapeutic insights for this very common disease.

β-Adrenergic Receptor Stimulated Ncx1 Upregulation is Mediated via a CaMKII/AP-1 Signaling Pathway in Adult Cardiomyocytes

The Na(+)-Ca(2+) exchanger gene (Ncx1) is upregulated in hypertrophy and is often found elevated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. beta-Adrenergic receptor (beta-AR) signaling plays an important role in the regulation of calcium homeostasis in the cardiomyocyte, but chronic activation in periods of cardiac stress contributes to heart failure by mechanisms which include Ncx1 upregulation. Here, using a Ca(2+)/calmodulin-dependent protein kinase II (CaMKIIdelta(c)) null mouse, we demonstrate that beta-AR-stimulated Ncx1 upregulation is dependent on CaMKII. beta-AR-stimulated Ncx1 expression is mediated by activator protein 1 (AP-1) factors and is independent of cAMP-response element-binding protein (CREB) activation. The MAP kinases (ERK1/2, JNK and p38) are not required for AP-1 factor activation. Chromatin immunoprecipitation demonstrates that beta-AR stimulation activates the ordered recruitment of JunB homodimers, which then are replaced by c-Jun homodimers binding to the proximal AP-1 elements of the endogenous Ncx1 promoter. In conclusion, this work has provided insight into the intracellular signaling pathways and transcription factors regulating Ncx1 gene expression in a chronically beta-AR-stimulated heart.