Robert J. Kelm

A Family of microRNAs Encoded by Myosin Genes Governs Myosin Expression and Muscle Performance

Myosin is the primary regulator of muscle strength and contractility. Here we show that three myosin genes, Myh6, Myh7, and Myh7b, encode related intronic microRNAs (miRNAs), which, in turn, control muscle myosin content, myofiber identity, and muscle performance. Within the adult heart, the Myh6 gene, encoding a fast myosin, coexpresses miR-208a, which regulates the expression of two slow myosins and their intronic miRNAs, Myh7/miR-208b and Myh7b/miR-499, respectively. miR-208b and miR-499 play redundant roles in the specification of muscle fiber identity by activating slow and repressing fast myofiber gene programs. The actions of these miRNAs are mediated in part by a collection of transcriptional repressors of slow myofiber genes. These findings reveal that myosin genes not only encode the major contractile proteins of muscle, but act more broadly to influence muscle function by encoding a network of intronic miRNAs that control muscle gene expression and performance.

Targeted Deletion of MicroRNA-22 Promotes Stress-Induced Cardiac Dilation and Contractile Dysfunction

Background— Delineating the role of microRNAs (miRNAs) in the posttranscriptional gene regulation offers new insights into how the heart adapts to pathological stress. We developed a knockout of miR-22 in mice and investigated its function in the heart. Methods and Results— Here, we show that miR-22–deficient mice are impaired in inotropic and lusitropic response to acute stress by dobutamine. Furthermore, the absence of miR-22 sensitized mice to cardiac decompensation and left ventricular dilation after long-term stimulation by pressure overload. Calcium transient analysis revealed reduced sarcoplasmic reticulum Ca2+ load in association with repressed sarcoplasmic reticulum Ca2+ ATPase activity in mutant myocytes. Genetic ablation of miR-22 also led to a decrease in cardiac expression levels for Serca2a and muscle-restricted genes encoding proteins in the vicinity of the cardiac Z disk/titin cytoskeleton. These phenotypes were attributed in part to inappropriate repression of serum response factor activity in stressed hearts. Global analysis revealed increased expression of the transcriptional/translational repressor purine-rich element binding protein B, a highly conserved miR-22 target implicated in the negative control of muscle expression. Conclusion— These data indicate that miR-22 functions as an integrator of Ca2+ homeostasis and myofibrillar protein content during stress in the heart and shed light on the mechanisms that enhance propensity toward heart failure.

Inhibition of apoptosis and caspase‐3 in vascular smooth muscle cells by plasminogen activator inhibitor type‐1

Increased expression of plasminogen activator inhibitor type 1 (PAI‐1) is associated with decreased apoptosis of neoplastic cells. We sought to determine whether PAI‐1 alters apoptosis in vascular smooth muscle cells (VSMC) and, if so, by what mechanisms. A twofold increase in the expression of PAI‐1 was induced in VSMC from transgenic mice with the use of the SM‐22α gene promoter (SM22‐PAI+). Cultured VSMC from SM22‐PAI+ mice were more resistant to apoptosis induced by tumor necrosis factor plus phorbol myristate acetate or palmitic acid compared with VSMC from negative control littermates. Both wild type (WT) and a stable active mutant form of PAI‐1 (Active) inhibited caspase‐3 amidolytic activity in cell lysates while a serpin‐defective mutant (Mut) PAI‐1 did not. Similarly, both WT and Active PAI‐1 decreased amidolytic activity of purified caspase‐3, whereas Mut PAI‐1 did not. WT but not Mut PAI‐1 decreased the cleavage of poly‐[ADP‐ribose]‐polymerase (PARP), the physiological substrate of caspase‐3. Noncovalent physical interaction between caspase‐3 and PAI‐1 was demonstrable with the use of both qualitative and quantitative in vitro binding assays. High affinity binding was eliminated by mutations that block PAI‐1 serpin activity. Accordingly, attenuated apoptosis resulting from elevated expression of PAI‐1 by VSMC may be attributable, at least in part, to reversible inhibition of caspase‐3 by active PAI‐1.

Augmentation of Proliferation of Vascular Smooth Muscle Cells by Plasminogen Activator Inhibitor Type 1

Objective—Proliferation of vascular smooth muscle cells (VSMCs) contributes to restenosis after coronary intervention. We have shown previously that increased expression of plasminogen activator inhibitor type 1 (PAI-1) limits VSMC apoptosis. Because apoptosis and proliferation appear to be linked, we sought to determine whether increased PAI-1 would affect VSMC proliferation. Methods and Results—VSMCs were explanted from control and transgenic mice (SM22-PAI+) in which VSMC expression of PAI-1 was increased. Increased growth of SM22-PAI+-VSMCs (2.3±0.4-fold) reflected, at least partially, increased proliferation. Greater expression of FLICE-like inhibitory protein (FLIP; 2.7-fold) and its cleaved active form were seen in SM22-PAI+-VSMCs. The balance between caspase-8 and FLIP favored proliferation in SM22-PAI+-VSMCs. Increased expression of NF-&kgr;B and activation of extracellular signal-regulated kinase (ERK) were demonstrated in SM22-PAI+-VSMCs (fold=NF-&kgr;B=2.2±0.1, fold=phosphorylated-ERK=1.6±0.1). Results were confirmed when expression of PAI-1 was increased by transfection. Inhibition of NF-&kgr;B and ERK attenuated proliferation in SM22-PAI+-VSMCs. Increased expression of PAI-1 promoted proliferation when VSMCs were exposed to tumor necrosis factor (TNF). Conclusions—Increased expression of PAI-1 is associated with greater activity of FLIP that promotes VSMC proliferation through NF-&kgr;B and ERK. Thus, when vascular wall expression of PAI-1 is increased, restenosis after coronary intervention is likely to be potentiated by greater proliferation of VSMC and resistance to apoptosis.

The HDAC inhibitors trichostatin A and suberoylanilide hydroxamic acid exhibit multiple modalities of benefit for the vascular pathobiology of sickle transgenic mice

The vascular pathobiology of sickle cell anemia involves inflammation, coagulation, vascular stasis, reperfusion injury, iron-based oxidative biochemistry, deficient nitric oxide (NO) bioavailability, and red cell sickling. These disparate pathobiologies intersect and overlap, so it is probable that multimodality therapy will be necessary for this disease. We have, therefore, tested a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), for efficacy in reducing endothelial activation. We found that pulmonary vascular endothelial VCAM-1 and tissue factor (TF) expression (both are indicators of endothelial activation) are powerfully and significantly inhibited by TSA. This is seen both with pretreatment before the inducing stress of hypoxia/reoxygenation (NY1DD sickle transgenic mouse), and upon longer-term therapy after endothelial activation has already occurred (hBERK1 sickle mouse at ambient air). In addition, TSA prevented vascular stasis in sickle mice, it exhibited activity as an iron chelator, and it induced expression of the antisickling hemoglobin, hemoglobin F. Notably, the TSA analog SAHA (suberoylanilide hydroxaminc acid) that is already approved for human clinical use exhibits the same spectrum of biologic effects as TSA. We suggest that SAHA possibly could provide true, multimodality, salubrious effects for prevention and treatment of the chronic vasculopathy of sickle cell anemia.

Nuclear factor-kappa B (NFκB) component p50 in blood mononuclear cells regulates endothelial tissue factor expression in sickle transgenic mice: implications for the coagulopathy of sickle cell disease

Sickle cell anemia is accompanied by the activation of coagulation and thrombosis. We have studied the abnormal expression of tissue factor (TF) by the pulmonary vein endothelium of the mild-phenotype NY1DD sickle transgenic. As detected by immunofluorescence microscopy, this occurs only after the NY1DD mouse is exposed to hypoxia/reoxygenation (H/R), which actually causes ischemia/reperfusion in the sickle cell disease-but not the normal-mouse model. We tested the hypothesis that the nuclear factor-kappa B (NFkappaB)-activating inflammation that develops in post-H/R NY1DD mice is responsible for this phenotype switch. Various NFkappaB inhibitors (including p50-specific andrographolide) demonstrated that endothelial TF positivity is NFkappaB dependent. Several systemic inflammatory stimulators (tumor necrosis factor [TNFalpha], lipopolysaccharide, thioglycollate, and carageenan) given to control mice showed that the inflammatory promotion of TF expression by only pulmonary vein endothelium is not specific to the sickle cell disease model. We bred the NFkappaB(p50)-/- state into the NY1DD mouse. Combined with marrow transplantation, this allowed the creation of NY1DD mice that were NFkappaB(p50)-/- only in peripheral blood cells (and marrow) versus only in vessel walls (and tissues). This process revealed that endothelial TF expression in the NY1DD mouse is highly dependent on NFkappaB(p50) in peripheral blood mononuclear cells-but not in the vessel wall. In confirmation, the infusion of post-H/R sickle mouse blood mononuclear cells into naïve NY1DD mice stimulated endothelial TF expression; the infusion of such cells from unstimulated sickle cell disease mice at ambient air did not stimulate TF expression. We conclude that peripheral blood mononuclear cells indirectly promote endothelial TF expression via a NFkappaB(p50)-dependent mechanism. This approach may be relevant to the role of coagulopathy in clinical sickle cell disease.

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.

Transforming Growth Factor β1-mediated Activation of the Smooth Muscle α-Actin Gene in Human Pulmonary Myofibroblasts Is Inhibited by Tumor Necrosis Factor-α via Mitogen-activated Protein Kinase Kinase 1-dependent Induction of the Egr-1 Transcriptional Repressor

Transforming growth factor (TGF) beta1 is a mediator of myofibroblast differentiation in healing wounds in which it activates transcription of the smooth muscle alpha-actin (SMalphaA) gene via dynamic interplay of nuclear activators and repressors. Targeting components of TGFbeta1 signaling may be an effective strategy for controlling myofibroblasts in chronic fibrotic diseases. We examined the ability of proinflammatory tumor necrosis factor (TNF)-alpha to antagonize TGFbeta1-mediated human pulmonary myofibroblast differentiation. TNF-alpha abrogated TGFbeta1-induced SMalphaA gene expression at the level of transcription without disrupting phosphorylation of regulatory Smads. Intact mitogen-activated protein kinase kinase (Mek)-extracellular signal-regulated kinase (Erk) kinase signaling was required for myofibroblast repression by TNF-alpha via induction of the early growth response factor-1 (Egr-1) DNA-binding protein. Egr-1 bound to the GC-rich SPUR activation element in the SMalphaA promoter and potently suppressed Smad3- and TGFbeta1-mediated transcription. Reduction in Smad binding to the SMalphaA promoter in TNF-alpha-treated myofibroblasts was accompanied by an increase in Egr-1 and YB-1 repressor binding, suggesting that the molecular mechanism underlying repression may involve competitive interplay between Egr-1, YB-1, and Smads. The ability of TNF-alpha to attenuate myofibroblast differentiation via modulation of a Mek1/Erk/Egr-1 regulatory axis may be useful in designing new therapeutic targets to offset destructive tissue remodeling in chronic fibrotic disease.

Endothelial nitric oxide synthase and nitric oxide regulate endothelial tissue factor expression in vivo in the sickle transgenic mouse

Activation of the coagulation system is a characteristic feature of sickle cell anemia, which also includes clinical thrombosis. The sickle transgenic mouse abnormally expresses tissue factor (TF) on the pulmonary vein endothelium. Knowing that this aberrancy is stimulated by inflammation, we sought to determine whether nitric oxide (NO) contributes to regulation of endothelial TF expression in the sickle mouse model. We used the NY1DD sickle mouse, which exhibits a low‐TF to high‐TF phenotype switch on exposure to hypoxia/reoxygenation. Manipulations of NO biology, such as breathing NO or addition of arginine or L‐NAME (N‐nitro‐L‐arginine‐methyl‐ester) to the diet, caused significant modulations of TF expression. This was also seen in hBERK1 sickle mice, which have a different genetic background and already have high‐TF even at ambient air. Study of NY1DD animals bred to overexpress endothelial nitric oxide synthase (eNOS; eNOS‐Tg) or to have an eNOS knockout state (one eNOS−/− animal and several eNOS+/− animals) demonstrated that eNOS modulates endothelial TF expression in vivo by down‐regulating it. Thus, the biodeficiency of NO characteristic of patients with sickle cell anemia may heighten risk for activation of the coagulation system. Am. J. Hematol., 2010.

Structure/Function Analysis of Mouse Purβ, a Single-stranded DNA-binding Repressor of Vascular Smooth Muscle α-Actin Gene Transcription

Plasticity of smooth muscle α-actin gene expression in fibroblasts and vascular smooth muscle cells is mediated by opposing effects of transcriptional activators and repressors. Among these factors, three single-stranded DNA-binding proteins, Purα, Purβ, and MSY1, have been implicated as coregulators of a cryptic 5′-enhancer module. In this study, a molecular analysis of Purβ, the least well characterized member of this group, was conducted. Southwestern and Northwestern blotting of purified Purβ deletion mutants using smooth muscle α-actin-derived probes mapped the minimal single-stranded DNA/RNA-binding domain to a conserved region spanning amino acids 37–263. Quantitative binding assays indicated that the relative affinity and specificity of Purβ for single-stranded DNA were influenced by purine/pyrimidine content; by non-conserved regions outside amino acids 37–263; and by cell-derived proteins, specifically MSY1. When overexpressed in A7r5 vascular smooth muscle cells, Purβ (but not Purα) inhibited transcription of a smooth muscle-specific mouse α-actin promoter transgene. Structural domains required for Purβ repressor activity included the minimal DNA-binding region and a C-terminal domain required for stabilizing high affinity protein and nucleic acid interactions. Purβ inhibitory activity in transfected A7r5 cells was potentiated by MSY1, but antagonized by serum response factor, reinforcing the idea that interplay among activators and repressors may account for phenotypic changes in smooth muscle α-actin-expressing cell types.