Macrophage migration inhibitory factor antagonizes pressure overload-induced cardiac hypertrophy.

Macrophage migration inhibitory factor (MIF) functions as a proinflammatory cytokine when secreted from the cell, but it also exhibits antioxidant properties by virtue of its intrinsic oxidoreductase activity. Since increased production of ROS is implicated in the development of left ventricular hypertrophy, we hypothesized that the redox activity of MIF protects the myocardium when exposed to hemodynamic stress. In a mouse model of myocardial hypertrophy induced by transverse aortic coarctation (TAC) for 10 days, we showed that growth of the MIF-deficient heart was significantly greater by 32% compared with wild-type (WT) TAC hearts and that fibrosis was increased by fourfold (2.62 ± 0.2% vs. 0.6 ± 0.1%). Circulating MIF was increased in TAC animals, and expression of MIF receptor, CD74, was increased in the hypertrophic myocardium. Gene expression analysis showed a 10-fold increase (P < 0.01) in ROS-generating mitochondrial NADPH oxidase and 2- to 3-fold reductions (P < 0.01) in mitochondrial SOD2 and mitochondrial aconitase activities, indicating enhanced oxidative injury in the hypertrophied MIF-deficient ventricle. Hypertrophic signaling pathways showed that phosphorylation of cytosolic glycogen synthase kinase-3α was greater (P < 0.05) at baseline in MIF-deficient hearts than in WT hearts and remained elevated after 10-day TAC. In the hemodynamically stressed MIF-deficient heart, nuclear p21(CIP1) increased sevenfold (P < 0.01), and the cytosolic increase of phospho-p21(CIP1) was significantly greater than in WT TAC hearts. We conclude that MIF antagonizes myocardial hypertrophy and fibrosis in response to hemodynamic stress by maintaining a redox homeostatic phenotype and attenuating stress-induced activation of hypertrophic signaling pathways.

Macrophage migration inhibitory factor provides cardioprotection during ischemia/reperfusion by reducing oxidative stress.

Macrophage migration inhibitory factor (MIF) is a multifunctional protein that exhibits an intrinsic thiol protein oxidoreductase activity and proinflammatory activities. In the present study to examine intracellular MIF redox function, exposure of MIF-deficient cardiac fibroblasts to oxidizing conditions resulted in a 2.3-fold increase (p < 0.001) in intracellular ROS that could be significantly reduced by adenoviral-mediated reexpression of recombinant MIF. In an animal model of myocardial injury by ischemia/reperfusion (I/R), MIF-deficient hearts exhibited higher levels of oxidative stress than did wild-type hearts, as measured by significantly higher oxidized glutathione levels (decreased GSH/GSSG ratio), increased protein oxidation, reduced aconitase activity, and increased mitochondrial injury (increased cytochrome c release). The increased myocardial oxidative stress after I/R was reflected by larger infarct size (INF) in MIF-deficient hearts versus wild-type (WT) hearts (21 ± 6% vs. 8 ± 3% INF/LV; p < 0.05). In vivo hemodynamic measurements showed that left ventricular (LV) contractile function of MIF-deficient hearts subjected to 15-min ischemia failed to recover during reperfusion compared with WT hearts (LV developed pressure and ± dP/dt; p = 0.02). These data represent the first in vivo evidence in support of a cardioprotective role of MIF in the postischemic heart by reducing oxidative stress.

Macrophage migration inhibitory factor induces cardiomyocyte apoptosis.

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that causes cardiac contractile dysfunction, whereas inactivation of MIF improves cardiac function in experimental animal models of sepsis. We used cultured cardiomyocytes to determine whether MIF-induced contractile dysfunction was mediated in part by myocyte apoptosis and to identify MIF-activated intracellular signaling pathways in this process. MIF treatment significantly increased myocyte apoptosis in a dose-dependent manner to 15.5+/-3.9% and 26.0+/-7.1% TUNEL positive nuclei (20 and 30 ng/ml MIF for 24h) vs control (3.7+/-0.9%). This effect was attenuated by inactivation of MIF with the chemical inhibitor, ISO-1. MIF-induced cleavage of caspase 3 and reduction of Bcl-xL/Bax were similarly attenuated by ISO-1 pre-treatment. MIF stimulated the rapid, transient phosphorylation of stress kinases, p38MAPK and JNK. Thus, MIF induces cardiomyocyte apoptosis by activating stress kinases and mitochondria-associated apoptotic mechanisms, whereas inactivation of MIF pro-inflammatory activity improves cardiomyocyte survival.

Thyroid hormone stimulates protein synthesis in the cardiomyocyte by activating the Akt-mTOR and p70S6K pathways.

Thyroid hormones affect cardiac growth and phenotype; however, the mechanisms by which the hormones induce cardiomyocyte hypertrophy remain uncharacterized. Tri-iodo-L-thyronine (T3) treatment of cultured cardiomyocytes for 24 h resulted in a 41 +/- 5% (p < 0.001) increase in [(3)H]leucine incorporation into total cellular protein. This response was abrogated by the phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin. Co-immunoprecipitation studies showed a direct interaction of cytosol-localized thyroid hormone receptor TRalpha1 and the p85alpha subunit of PI3K. T3 treatment rapidly increased PI3K activity by 52 +/- 3% (p < 0.005), which resulted in increased phosphorylation of downstream kinases Akt and mammalian target of rapamycin (mTOR). This effect was abrogated by pretreatment with wortmannin or LY294002. Phosphorylation of p70(S6K), a known target of mTOR, occurred rapidly following T3 treatment and was inhibited by rapamycin and wortmannin. In contrast, phosphorylation of the p85 variant of S6K in response to T3 was not blocked by LY294002, wortmannin, or rapamycin, thus supporting a T3-activated pathway independent of PI3K and mTOR. 40 S ribosomal protein S6, a target of p70(S6K), and 4E-BP1, a target of mTOR, were both phosphorylated within 15-25 min of T3 treatment and could be inhibited by wortmannin and rapamycin. Thus, rapid T3-mediated activation of PI3K by cytosolic TRalpha1 and subsequent activation of the Akt-mTOR-S6K signaling pathway may underlie one of the mechanisms by which thyroid hormone regulates physiological cardiac growth.

Nuclear localization of protein kinase C-alpha induces thyroid hormone receptor-alpha1 expression in the cardiomyocyte.

Maladaptive cardiac hypertrophy results in phenotypic changes in several genes that are thyroid hormone responsive, suggesting that thyroid hormone receptor (TR) function may be altered by cellular kinases, including protein kinase C (PKC) isozymes that are activated in pathological hypertrophy. To investigate the role of PKC signaling in regulating TR function, cultured neonatal rat ventricular myocytes were transduced with adenovirus (Ad) expressing wild-type (wt) or kinase-inactive (dn) PKC alpha or constitutively active (ca) PKC delta and PKC epsilon. Overexpression of wtPKC alpha, but not caPKC delta or caPKC epsilon, induced a 28-fold increase (P < 0.001) in TR alpha1 protein in the nuclear compartment and a smaller increase in the cytosol. Furthermore, TR alpha1 mRNA was increased 55-fold (P < 0.001). This effect of PKC alpha was dependent on its kinase activity because dnPKC alpha was without effect. Phorbol 12-myristate 13-acetate (PMA) induced nuclear translocation of endogenous PKC alpha and Ad-wtPKC alpha concomitantly with an increase in nuclear TR alpha1 protein. In contrast, PMA-induced nuclear translocation of dnPKC alpha resulted in a decrease of TR alpha1. The increase in TR alpha1 protein in Ad-wtPKC alpha-transduced cardiomyocytes was not the result of a reduced rate of protein degradation, nor was the half-life of TR alpha1 mRNA prolonged, suggesting a PKC alpha-mediated effect on TR alpha transcription. Although phosphorylation of ERK1/2 was increased in Ad-wtPKC alpha-transduced cells, inhibition of phospho-ERK did not change TR alpha1 expression. PKC alpha overexpression in cardiomyocytes caused marked repression of triiodothyronine (T3)-responsive genes, alpha-myosin heavy chain, and the sarcoplasmic reticulum calcium-activated adenosinetriphosphatase SERCA2. Treatment with T3 for 4 h resulted in significant reductions of PKC alpha in nuclear and cytosolic compartments, and decreased TR alpha1 mRNA and protein, with normalization of phenotype. These results implicate PKC alpha as a regulator of TR function and suggest that nuclear localization of PKC alpha may control transcription of the TR alpha gene, and consequently, affect cardiac phenotype.

Ligand-mediated decrease of thyroid hormone receptor-alpha1 in cardiomyocytes by proteosome-dependent degradation and altered mRNA stability.

Tri-iodo-L-thyronine (T3) is essential for maintaining normal cardiac contractile function by regulating transcription of numerous T3-responsive genes. Both hormone availability and relative amounts of nuclear thyroid hormone receptor isoforms (TRalpha1, TRbeta1) determine T3 effectiveness. Cultured neonatal rat ventricular myocytes grown in T3-depleted medium expressed predominantly TRalpha1 protein, but within 4 h of T3 treatment, TRbeta1 protein increased significantly, whereas TRalpha1 was decreased by 46 +/- 5%. Using replication-defective adenoviruses to overexpress TRalpha1 in cardiomyocytes, we studied the mechanisms by which T3 mediated the decrease in TRalpha1 protein. Inhibitors of the proteosome pathway resulted in an accumulation of ubiquitylated TRalpha1 in the nucleus and prevented T3-induced degradation of ubiquitylated TRalpha1, suggesting that T3 induced proteosome-mediated degradation of TRalpha1; however, TR ubiquitylation was T3 independent. TRalpha1 transcriptional activity, measured using transient transfection of a thyroid hormone-responsive element (TRE) reporter plasmid, was T3 dose dependent and inversely proportional to nuclear TRalpha1 content, with 10 nM T3 having maximum effect. Quantitative RT-PCR showed that both endogenous and adenovirus-expressed TRalpha1 mRNAs were significantly decreased to 54 +/- 11 and 25 +/- 5%, respectively, within 4 h of T3 treatment. Measurements of TRalpha1 mRNA half-life in actinomycin D-treated cardiomyocytes showed that T3 treatment significantly decreased TRalpha1 mRNA half-life from 4 h to less than 2 h, whereas it had no effect of TRbeta1 mRNA half-life. These data support a role for both the proteosome degradation pathway and altered mRNA stability in T3-induced decrease of nuclear TRalpha1 in the cardiomyocyte and provide novel cellular targets for therapeutic development.

Altered myocardial Ca2+ cycling after left ventricular assist device support in the failing human heart.

OBJECTIVES: The objective of the present study was to determine whether improved contractility after left ventricular assist device (LVAD) support reflects altered myocyte calcium cycling and changes in calcium-handling proteins. BACKGROUND: Previous reports demonstrate that LVAD support induces sustained unloading of the heart with regression of pathologic hypertrophy and improvements in contractile performance. METHODS: In the human myocardium of subjects with heart failure (HF), with non-failing hearts (NF), and with LVAD-supported failing hearts (HF-LVAD), intracellular calcium ([Ca(2+)](i)) transients were measured in isolated myocytes at 0.5 Hz, and frequency-dependent force generation was measured in multicellular preparations (trabeculae). Abundance of sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (SERCA), Na(+)/Ca(2+) exchanger (NCX), and phospholamban was assessed by Western analysis. RESULTS: Compared with NF myocytes, HF myocytes exhibited a slowed terminal decay of the Ca(2+) transient (DT(terminal), 376 +/- 18 ms vs. 270 +/- 21 ms, HF vs. NF, p < 0.0008), and HF-LVAD myocytes exhibited a DT(terminal) that was much shorter than that observed in HF myocytes (278 +/- 10 ms, HF vs. HF-LVAD, p < 0.0001). Trabeculae from HF showed a negative force-frequency relationship, compared with a positive relationship in NF, whereas a neutral relationship was observed in HF-LVAD. Although decreased SERCA abundance in HF was not altered by LVAD support, improvements in [Ca(2+)](i) transients and frequency-dependent contractile function were associated with a significant decrease in NCX abundance and activity from HF to HF-LVAD. CONCLUSIONS: Improvement in rate-dependent contractility in LVAD-supported failing human hearts is associated with a faster decay of the myocyte calcium transient. These improvements reflect decreases in NCX abundance and transport capacity without significant changes in SERCA after LVAD support. Our results suggest that reverse remodeling may involve selective, rather than global, normalization of the pathologic patterns associated with the failing heart.

Role of USF1 phosphorylation on cardiac alpha-myosin heavy chain promoter activity.

Contractile activity of the cardiac myocyte is required for maintaining cell mass and phenotype, including expression of the cardiac-specific alpha-myosin heavy chain (alpha-MHC) gene. An E-box hemodynamic response element (HME) located at position -47 within the alpha-MHC promoter is both necessary and sufficient to confer contractile responsiveness to the gene and has been shown to bind upstream stimulatory factor-1 (USF1). When studied in spontaneously contracting cardiac myocytes, there is enhanced binding of USF1 to the HME compared with quiescent cells, which correlates with a threefold increase in alpha-MHC promoter activity. A molecular mechanism by which contractile function modulates alpha-MHC transcriptional activity may involve signaling via phosphorylation of USF1. The present studies showed that purified rat USF1 was phosphorylated in vitro by protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) but not casein kinase II. Phosphorylated USF1 by either PKC or PKA had increased DNA binding activity to the HME. PKC-mediated phosphorylation also leads to the formation of USF1 multimers as assessed by gel shift assay. Analysis of in vivo phosphorylated nuclear proteins from cultured ventricular myocytes showed that USF1 was phosphorylated, and resolution by two-dimensional gel electrophoresis identified at least two distinct phosphorylated USF1 molecules. These results suggest that endogenous kinases can covalently modify USF1 and provide a potential molecular mechanism by which the contractile stimulus mediates changes in myocyte gene transcription.