Sukanya V. Subramanian

Oxygen Sensing by Primary Cardiac Fibroblasts: A Key Role of p21Waf1/Cip1/Sdi1

Abstract— In mammalian organs under normoxic conditions, O2 concentration ranges from 12% to <0.5%, with O2 ≈14% in arterial blood and <10% in the myocardium. During mild hypoxia, myocardial O2 drops to ≈1% to 3% or lower. In response to chronic moderate hypoxia, cells adjust their normoxia set point such that reoxygenation-dependent relative elevation of Po2 results in perceived hyperoxia. We hypothesized that O2, even in marginal relative excess of the Po2 to which cardiac cells are adjusted, results in activation of specific signal transduction pathways that alter the phenotype and function of these cells. To test this hypothesis, cardiac fibroblasts (CFs) isolated from adult murine ventricle were cultured in 10% or 21% O2 (hyperoxia relative to the Po2 to which cells are adjusted in vivo) and were compared with those cultured in 3% O2 (mild hypoxia). Compared with cells cultured in 3% O2, cells that were cultured in 10% or 21% O2 demonstrated remarkable reversible G2/M arrest and a phenotype indicative of differentiation to myofibroblasts. These effects were independent of NADPH oxidase function. CFs exposed to high O2 exhibited higher levels of reactive oxygen species production. The molecular signature response to perceived hyperoxia included (1) induction of p21, cyclin D1, cyclin D2, cyclin G1, Fos-related antigen-2, and transforming growth factor-&bgr;1, (2) lowered telomerase activity, and (3) activation of transforming growth factor-&bgr;1 and p38 mitogen-activated protein kinase. CFs deficient in p21 were resistant to such O2 sensitivity. This study raises the vital broad-based issue of controlling ambient O2 during the culture of primary cells isolated from organs.

Reprogramming of vascular smooth muscle α-actin gene expression as an early indicator of dysfunctional remodeling following heart transplant

OBJECTIVE Chronic rejection in cardiac allografts depletes vascular smooth muscle (VSM) alpha-actin from the coronary arterial smooth muscle bed while promoting its abnormal accumulation in cardiomyocytes and myofibroblasts. The objective was to determine if the newly discovered TEF1, MSY1, Puralpha and Purbeta VSM alpha-actin transcriptional reprogramming proteins (TRPs) were associated with development of chronic rejection histopathology in accepted murine cardiac allografts. METHODS A mouse heterotopic cardiac transplant model was employed using H2 locus-mismatched mouse strains (DBA/2 or FVB/N to C57BL/6). Recipients were immunosuppressed to promote long-term allograft acceptance and emergence of chronic rejection. Explanted grafts and isolated heart cells were evaluated for changes in the DNA-binding activity and subcellular distribution of VSM alpha-actin transcriptional regulatory proteins. RESULTS The DNA-binding activity of all four TRPs was high in the developing mouse ventricle, minimal in adult donor hearts and increased substantially within 30 days after transplantation. Immunohistologic analysis revealed nuclear localization of Purbeta and MSY1 particularly in fibrotic areas of the allograft myocardium demonstrating extravascular accumulation of VSM alpha-actin. Cardiomyocytes isolated from adult, non-transplanted mouse hearts not only exhibited less VSM alpha-actin expression and lower levels of TRPs compared to isolated cardiac fibroblasts or neonatal cardiomyocytes, but also contained a novel size variant of the MSY1 protein. CONCLUSION Accumulation of TRPs in cardiac allografts, particularly within the fibroblast-enriched myocardial interstitium, was consistent with their potential role in VSM alpha-actin gene reprogramming, fibrosis and dysfunctional remodeling following transplant. These nuclear protein markers could help stage peri-transplant cellular events that precede formation of graft-destructive fibrosis and coronary vasculopathy during chronic rejection.

Vascular smooth muscle alpha-actin expression as an indicator of parenchymal cell reprogramming in cardiac allografts.

BACKGROUND In addition to transplant-associated vascular sclerosis, cardiac allografts also may be vulnerable to a previously unrecognized aspect of remodeling involving reactivation of fetal structural genes in the adult heart. METHODS Vascular smooth muscle (VSM) alpha-actin is encoded by a gene that normally is repressed in the ventricle during late gestation. Immunohistochemical analysis of accepted mouse cardiac allografts was performed to determine whether this fetal actin was reexpressed after transplant. RESULTS VSM alpha-actin was detected within 30 days after transplant throughout the allograft myocardium, where it frequently exhibited a distinct periodicity suggestive of protein localization in sarcomeres. By 90 days after transplant, VSM alpha-actin filaments specifically accumulated in the left ventricular endocardium. Donor hearts and isografts did not express myocardial VSM alpha-actin, indicating that fetal gene activation was linked to chronic rejection. CONCLUSION The results indicate that chronic rejection is associated with fetal muscle gene activation, which may facilitate parenchymal cell remodeling and impair graft function.

Serum response factor neutralizes Purα- and Purβ-mediated repression of the fetal vascular smooth muscle α-actin gene in stressed adult cardiomyocytes

Mouse hearts subjected to repeated transplant surgery and ischemia-reperfusion injury develop substantial interstitial and perivascular fibrosis that was spatially associated with dysfunctional activation of fetal smooth muscle alpha-actin (SM alpha A) gene expression in graft ventricular cardiomyocytes. Compared with cardiac fibroblasts in which nuclear levels of the Sp1 and Smad 2/3 transcriptional-activating proteins increased markedly after transplant injury, the most abundant SM alpha A gene-activating protein in cardiomyocyte nuclei was serum response factor (SRF). Additionally, cardiac intercalated discs in heart grafts contained substantial deposits of Pur alpha, an mRNA-binding protein and known negative modulator of SRF-activated SM alpha A gene transcription. Activation of fetal SM alpha A gene expression in perfusion-isolated adult cardiomyocytes was linked to elevated binding of a novel protein complex consisting of SRF and Pur alpha to a purine-rich DNA element in the SM alpha A promoter called SPUR, previously shown to be required for induction of SM alpha A gene transcription in injury-activated myofibroblasts. Increased SRF binding to SPUR DNA plus one of two nearby CArG box consensus elements was observed in SM alpha A-positive cardiomyocytes in parallel with enhanced Pur alpha:SPUR protein:protein interaction. The data suggest that de novo activation of the normally silent SM alpha A gene in reprogrammed adult cardiomyocytes is linked to elevated interaction of SRF with fetal-specific CArG and injury-activated SPUR elements in the SM alpha A promoter as well as the appearance of novel Pur alpha protein complexes in both the nuclear and cytosolic compartments of these cells.

Y-box binding protein-1 implicated in translational control of fetal myocardial gene expression after cardiac transplant.

Peri-transplant surgical trauma and ischemia/reperfusion injury in accepted murine heterotopic heart grafts has been associated with myofibroblast differentiation, cardiac fibrosis and biomechanical-stress activation of the fetal myocardial smooth muscle α-actin (SMαA) gene. The wound-healing agonists, transforming growth factor β1 and thrombin, are known to coordinate SMαA mRNA transcription and translation in activated myofibroblasts by altering the subcellular localization and mRNA-binding affinity of the Y-box binding protein-1 (YB-1) cold-shock domain (CSD) protein that governs a variety of cellular responses to metabolic stress. YB-1 accumulated in polyribosome-enriched regions of the sarcoplasm proximal to cardiac intercalated discs in accepted heart grafts. YB-1 binding to a purine-rich motif in exon 3 of SMαA mRNA that regulates translational efficiency increased substantially in perfusion-isolated, rod-shaped adult rat cardiomyocytes during phenotypic de-differentiation in the presence of serum-derived growth factors. Cardiomyocyte de-differentiation was accompanied by the loss of a 60 kDa YB-1 variant that was highly expressed in both adult myocardium and freshly isolated myocytes and replacement with the 50 kDa form of YB-1 (p50) typically expressed in myofibroblasts that demonstrated sequence-specific interaction with SMαA mRNA. Accumulation of p50 YB-1 in reprogrammed, de-differentiated myocytes was associated with a 10-fold increase in SMαA protein expression. Endomyocardial biopsies collected from patients up to 14 years after heart transplant showed variable yet coordinately elevated expression of SMαA and p50 YB-1 protein and demonstrable p50 YB-1:SMαA mRNA interaction. The p60 YB-1 variant in human heart graft samples, but neither mouse p60 nor mouse or human p50, reacted with an antibody specific for the phosphoserine 102 modification in the YB-1 CSD. Modulation of YB-1 subcellular compartmentalization and mRNA-binding activity may be linked with reprogramming of contractile protein gene expression in ventricular cardiomyocytes that could contribute to maladaptive remodeling in accepted, long-term heart grafts.