Thrombospondin-3 augments injury-induced cardiomyopathy by intracellular integrin inhibition and sarcolemmal instability.

Thrombospondins (Thbs) are a family of five secreted matricellular glycoproteins in vertebrates that broadly affect cell-matrix interaction. While Thbs4 is known to protect striated muscle from disease by enhancing sarcolemmal stability through increased integrin and dystroglycan attachment complexes, here we show that Thbs3 antithetically promotes sarcolemmal destabilization by reducing integrin function, augmenting disease-induced decompensation. Deletion of Thbs3 in mice enhances integrin membrane expression and membrane stability, protecting the heart from disease stimuli. Transgene-mediated overexpression of α7β1D integrin in the heart ameliorates the disease predisposing effects of Thbs3 by augmenting sarcolemmal stability. Mechanistically, we show that mutating Thbs3 to contain the conserved RGD integrin binding domain normally found in Thbs4 and Thbs5 now rescues the defective expression of integrins on the sarcolemma. Thus, Thbs proteins mediate the intracellular processing of integrin plasma membrane attachment complexes to regulate the dynamics of cellular remodeling and membrane stability

Periostin as a Heterofunctional Regulator of Cardiac Development and Disease

Periostin (Postn) is a heterofunctional secreted extracellular matrix (ECM) protein comprised of four fasciclin domains that promotes cellular adhesion and movement, as well as collagen fibrillogenesis. Postn is expressed in unique growth centers during embryonic development where it facilitates epithelial-mesenchymal transition (EMT) of select cell populations undergoing reorganization. In the heart, Postn is expressed in the developing valves, cardiac fibroblasts and in regions of the outflow track. In the adult, Postn expression is specifically induced in areas of tissue injury or areas with ongoing cellular re-organization. In the adult heart Postn is induced in the ventricles following myocardial infarction, pressure overload stimulation, or generalized cardiomyopathy. Here we will review the functional consequences associated with Postn induction in both the developing and adult heart. The majority of data collected to date suggest a common function for Postn in both development and disease as a potent inducible regulator of cellular reorganization and extracellular matrix homeostasis, although some alternate and controversial functions have also been ascribed to Postn, the validity of which will be discussed here

Remembering Jeff Robbins: The Father of Cardiac Transgenesis

Jeff graduated from the University of Connecticut with his PhD in 1976 and became an assistant professor in 1978 at the University of Missouri-Columbia. Jeff then moved to Ohio in 1985 to the University of Cincinnati as an associate professor where he stayed until moving his scientific research program across the street to the Cincinnati Children&rsquo;s Hospital in 1993, where he served as a professor in Pediatrics of the University of Cincinnati and as the founding director of the Division of Molecular Cardiovascular Biology. Jeff retired from Cincinnati Children&rsquo;s Hospital and the University of Cincinnati in 2019. &nbsp;</p

Conditional transgenic expression of fibroblast growth factor 9 in the adult mouse heart reduces heart failure mortality after myocardial infarction

BACKGROUND: Fibroblast growth factor 9 (FGF9) is secreted from bone marrow cells, which have been shown to improve systolic function after myocardial infarction (MI) in a clinical trial. FGF9 promotes cardiac vascularization during embryonic development but is only weakly expressed in the adult heart. METHODS AND RESULTS: We used a tetracycline-responsive binary transgene system based on the α-myosin heavy chain promoter to test whether conditional expression of FGF9 in the adult myocardium supports adaptation after MI. In sham-operated mice, transgenic FGF9 stimulated left ventricular hypertrophy with microvessel expansion and preserved systolic and diastolic function. After coronary artery ligation, transgenic FGF9 enhanced hypertrophy of the noninfarcted left ventricular myocardium with increased microvessel density, reduced interstitial fibrosis, attenuated fetal gene expression, and improved systolic function. Heart failure mortality after MI was markedly reduced by transgenic FGF9, whereas rupture rates were not affected. Adenoviral FGF9 gene transfer after MI similarly promoted left ventricular hypertrophy with improved systolic function and reduced heart failure mortality. Mechanistically, FGF9 stimulated proliferation and network formation of endothelial cells but induced no direct hypertrophic effects in neonatal or adult rat cardiomyocytes in vitro. FGF9-stimulated endothelial cell supernatants, however, induced cardiomyocyte hypertrophy via paracrine release of bone morphogenetic protein 6. In accord with this observation, expression of bone morphogenetic protein 6 and phosphorylation of its downstream targets SMAD1/5 were increased in the myocardium of FGF9 transgenic mice. CONCLUSIONS: Conditional expression of FGF9 promotes myocardial vascularization and hypertrophy with enhanced systolic function and reduced heart failure mortality after MI. These observations suggest a previously unrecognized therapeutic potential for FGF9 after MI

Mechanisms Underlying Heterogeneous Ca2+ Sparklet Activity in Arterial Smooth Muscle

In arterial smooth muscle, single or small clusters of Ca2+ channels operate in a high probability mode, creating sites of nearly continual Ca2+ influx (called “persistent Ca2+ sparklet” sites). Persistent Ca2+ sparklet activity varies regionally within any given cell. At present, the molecular identity of the Ca2+ channels underlying Ca2+ sparklets and the mechanisms that give rise to their spatial heterogeneity remain unclear. Here, we used total internal reflection fluorescence (TIRF) microscopy to directly investigate these issues. We found that tsA-201 cells expressing L-type Cavα1.2 channels recapitulated the general features of Ca2+ sparklets in cerebral arterial myocytes, including amplitude of quantal event, voltage dependencies, gating modalities, and pharmacology. Furthermore, PKCα activity was required for basal persistent Ca2+ sparklet activity in arterial myocytes and tsA-201 cells. In arterial myocytes, inhibition of protein phosphatase 2A (PP2A) and 2B (PP2B; calcineurin) increased Ca2+ influx by evoking new persistent Ca2+ sparklet sites and by increasing the activity of previously active sites. The actions of PP2A and PP2B inhibition on Ca2+ sparklets required PKC activity, indicating that these phosphatases opposed PKC-mediated phosphorylation. Together, these data unequivocally demonstrate that persistent Ca2+ sparklet activity is a fundamental property of L-type Ca2+ channels when associated with PKC. Our findings support a novel model in which the gating modality of L-type Ca2+ channels vary regionally within a cell depending on the relative activities of nearby PKCα, PP2A, and PP2B

PKCα regulates the hypertrophic growth of cardiomyocytes through extracellular signal–regulated kinase1/2 (ERK1/2)

Members of the protein kinase C (PKC) isozyme family are important signal transducers in virtually every mammalian cell type. Within the heart, PKC isozymes are thought to participate in a signaling network that programs developmental and pathological cardiomyocyte hypertrophic growth. To investigate the function of PKC signaling in regulating cardiomyocyte growth, adenoviral-mediated gene transfer of wild-type and dominant negative mutants of PKCα, βII, δ, and ɛ (only wild-type ζ) was performed in cultured neonatal rat cardiomyocytes. Overexpression of wild-type PKCα, βII, δ, and ɛ revealed distinct subcellular localizations upon activation suggesting unique functions of each isozyme in cardiomyocytes. Indeed, overexpression of wild-type PKCα, but not βII, δ, ɛ, or ζ induced hypertrophic growth of cardiomyocytes characterized by increased cell surface area, increased [3H]-leucine incorporation, and increased expression of the hypertrophic marker gene atrial natriuretic factor. In contrast, expression of dominant negative PKCα, βII, δ, and ɛ revealed a necessary role for PKCα as a mediator of agonist-induced cardiomyocyte hypertrophy, whereas dominant negative PKCɛ reduced cellular viability. A mechanism whereby PKCα might regulate hypertrophy was suggested by the observations that wild-type PKCα induced extracellular signal–regulated kinase1/2 (ERK1/2), that dominant negative PKCα inhibited PMA-induced ERK1/2 activation, and that dominant negative MEK1 (up-stream of ERK1/2) inhibited wild-type PKCα–induced hypertrophic growth. These results implicate PKCα as a necessary mediator of cardiomyocyte hypertrophic growth, in part, through a ERK1/2-dependent signaling pathway

Deletion of periostin reduces muscular dystrophy and fibrosis in mice by modulating the transforming growth factor-β pathway

The muscular dystrophies are broadly classified as muscle wasting diseases with myofiber dropout due to cellular necrosis, inflammation, alterations in extracellular matrix composition, and fatty cell replacement. These events transpire and progress despite ongoing myofiber regeneration from endogenous satellite cells. The degeneration/regeneration response to muscle injury/disease is modulated by the proinflammatory cytokine transforming growth factor-β (TGF-β), which can also profoundly influence extracellular matrix composition through increased secretion of profibrotic proteins, such as the matricellular protein periostin. Here we show that up-regulation and secretion of periostin is pathological and enhances disease in the δ-sarcoglycan null (Sgcd(−/−)) mouse model of muscular dystrophy (MD). Indeed, MD mice lacking the Postn gene showed dramatic improvement in skeletal muscle structure and function. Mechanistically, Postn gene deletion altered TGF-β signaling so that it now enhanced tissue regeneration with reduced levels of fibrosis. Systemic antagonism of TGF-β with a neutralizing monoclonal antibody mitigated the beneficial effects of Postn deletion in vivo. These data suggest that periostin functions as a disease determinant in MD by promoting/allowing the pathological effects of TGF-β, suggesting that inhibition of periostin could represent a unique treatment approach

Regulation of angiogenesis by a non-canonical Wnt-Flt1 pathway in myeloid cells

Myeloid cells are a feature of most tissues. Here we show that during development, retinal myeloid cells (RMCs) produce Wnt ligands to regulate blood vessel branching. In the mouse retina, where angiogenesis occurs postnatally, somatic deletion in RMCs of the Wnt ligand transporter Wntless results in increased angiogenesis in the deeper layers. We also show that mutation of Wnt5a and Wnt11 results in increased angiogenesis and that these ligands elicit RMC responses via a non-canonical Wnt pathway. Using cultured myeloid-like cells and RMC somatic deletion of Flt1, we show that an effector of Wnt-dependent suppression of angiogenesis by RMCs is Flt1, a naturally occurring inhibitor of vascular endothelial growth factor (VEGF). These findings indicate that resident myeloid cells can use a non-canonical, Wnt-Flt1 pathway to suppress angiogenic branching

Rcan1 negatively regulates FcεRI-mediated signaling and mast cell function

Aggregation of the high affinity IgE receptor (FcεRI) activates a cascade of signaling events leading to mast cell activation. Subsequently, inhibitory signals are engaged for turning off activating signals. We identified that regulator of calcineurin (Rcan) 1 serves as a negative regulator for turning off FcεRI-mediated mast cell activation. FcεRI-induced Rcan1 expression was identified by suppression subtractive hybridization and verified by real-time quantitative polymerase chain reaction and Western blotting. Deficiency of Rcan1 led to increased calcineurin activity, increased nuclear factor of activated T cells and nuclear factor κB activation, increased cytokine production, and enhanced immunoglobulin E–mediated late-phase cutaneous reactions. Forced expression of Rcan1 in wild-type or Rcan1-deficient mast cells reduced FcεRI-mediated cytokine production. Rcan1 deficiency also led to increased FcεRI-mediated mast cell degranulation and enhanced passive cutaneous anaphylaxis. Analysis of the Rcan1 promoter identified a functional Egr1 binding site. Biochemical and genetic evidence suggested that Egr1 controls Rcan1 expression. Our results identified Rcan1 as a novel inhibitory signal in FcεRI-induced mast cell activation and established a new link of Egr1 and Rcan1 in FcεRI signaling

Calreticulin signals upstream of calcineurin and MEF2C in a critical Ca2+-dependent signaling cascade

We uncovered a new pathway of interplay between calreticulin and myocyte-enhancer factor (MEF) 2C, a cardiac-specific transcription factor. We establish that calreticulin works upstream of calcineurin and MEF2C in a Ca2+-dependent signal transduction cascade that links the endoplasmic reticulum and the nucleus during cardiac development. In the absence of calreticulin, translocation of MEF2C to the nucleus is compromised. This defect is reversed by calreticulin itself or by a constitutively active form of calcineurin. Furthermore, we show that expression of the calreticulin gene itself is regulated by MEF2C in vitro and in vivo and that, in turn, increased expression of calreticulin affects MEF2C transcriptional activity. The present findings provide a clear molecular explanation for the embryonic lethality observed in calreticulin-deficient mice and emphasize the importance of calreticulin in the early stages of cardiac development. Our study illustrates the existence of a positive feedback mechanism that ensures an adequate supply of releasable Ca2+ is maintained within the cell for activation of calcineurin and, subsequently, for proper functioning of MEF2C