Issue published May 15, 2003

A ctivation of mammalian sterile 20–like kinase 1 (Mst1) by genotoxic compounds is known to stimulate apoptosis in some cell types. The importance of Mst1 in cell death caused by clinically relevant pathologic stimuli is unknown, however. In this study, we show that Mst1 is a prominent myelin basic protein kinase activated by proapoptotic stimuli in cardiac myocytes and that Mst1 causes cardiac myocyte apoptosis in vitro in a kinase activity–dependent manner. In vivo, cardiac-specific overexpression of Mst1 in transgenic mice results in activation of caspases, increased apoptosis, and dilated cardiomyopathy. Surprisingly, however, Mst1 prevents compensatory cardiac myocyte elongation or hypertrophy despite increased wall stress, thereby obscuring the use of the Frank-Starling mechanism, a fundamental mechanism by which the heart maintains cardiac output in response to increased mechanical load at the single myocyte level. Furthermore, Mst1 is activated by ischemia/reperfusion in the mouse heart in vivo. Suppression of endogenous Mst1 by cardiac-specific overexpression of dominant-negative Mst1 in transgenic mice prevents myocyte death by pathologic insults. These results show that Mst1 works as both an essential initiator of apoptosis and an inhibitor of hypertrophy in cardiac myocytes, resulting in a previously unrecognized form of cardiomyopathy.

T he MAPKs are important transducers of growth and stress stimuli in virtually all eukaryotic cell types. In the mammalian heart, MAPK signaling pathways have been hypothesized to regulate myocyte growth in response to developmental signals or physiologic and pathologic stimuli. Here we generated cardiac-specific transgenic mice expressing dominant-negative mutants of p38α, MKK3, or MKK6. Remarkably, attenuation of cardiac p38 activity produced a progressive growth response and myopathy in the heart that correlated with the degree of enzymatic inhibition. Moreover, dominant-negative p38α, MKK3, and MKK6 transgenic mice each showed enhanced cardiac hypertrophy following aortic banding, Ang II infusion, isoproterenol infusion, or phenylephrine infusion for 14 days. A mechanism underlying this enhanced-growth profile was suggested by the observation that dominant-negative p38α directly augmented nuclear factor of activated T cells (NFAT) transcriptional activity and its nuclear translocation. In vivo, NFAT-dependent luciferase reporter transgenic mice showed enhanced activation in the presence of the dominant-negative p38α transgene before and after the onset of cardiac hypertrophy. More significantly, genetic disruption of the calcineurin Aβ gene rescued hypertrophic cardiomyopathy and depressed functional capacity observed in p38-inhibited mice. Collectively, these observations indicate that reduced p38 signaling in the heart promotes myocyte growth through a mechanism involving enhanced calcineurin-NFAT signaling.

I t is established that mutations in viral antigenic epitopes, or antigenic drifts, allow viruses to escape recognition by both Ab’s and T lymphocytes. It is unclear, however, whether tumor cells can escape immune recognition via antigenic drift. Here we show that adoptive therapy with both monoclonal and polyclonal transgenic CTLs, specific for a natural tumor antigen, P1A, selects for multiple mutations in the P1A antigenic epitope. These mutations severely diminish T cell recognition of the tumor antigen by a variety of mechanisms, including modulation of MHC:peptide interaction and TCR binding to MHC:peptide complex. These results provide the first evidence for tumor evasion of T cell recognition by antigenic drift, and thus have important implications for the strategy of tumor immunotherapy.

H eart failure is a common, lethal condition whose pathogenesis is poorly understood. Recent studies have identified low levels of myocyte apoptosis (80–250 myocytes per 10⁵ nuclei) in failing human hearts. It remains unclear, however, whether this cell death is a coincidental finding, a protective process, or a causal component in pathogenesis. Using transgenic mice that express a conditionally active caspase exclusively in the myocardium, we demonstrate that very low levels of myocyte apoptosis (23 myocytes per 10⁵ nuclei, compared with 1.5 myocytes per 10⁵ nuclei in controls) are sufficient to cause a lethal, dilated cardiomyopathy. Interestingly, these levels are four- to tenfold lower than those observed in failing human hearts. Conversely, inhibition of cardiac myocyte death in this murine model largely prevents the development of cardiac dilation and contractile dysfunction, the hallmarks of heart failure. To our knowledge, these data provide the first direct evidence that myocyte apoptosis may be a causal mechanism of heart failure, and they suggest that inhibition of this cell death process may constitute the basis for novel therapies.

S ystemic lupus erythematosus (SLE) is a CD4⁺ T cell–dependent, immune complex–mediated, autoimmune disease that primarily affects women of childbearing age. Generation of high-titer affinity-matured IgG autoantibodies, specific for double-stranded DNA and other nuclear antigens, coincides with disease progression. Current forms of treatment of SLE including glucocorticosteroids are often inadequate and induce severe side effects. Immunological approaches for treating SLE in mice using anti-CD4 mAb’s or CTLA4-Ig and anti-CD154 mAb’s have proven to be effective. However, like steroid treatment, these regimens induce global immunosuppression, and their withdrawal allows for disease progression. In this report we show that lupus-prone NZB × NZW F₁ mice given three injections of anti-CD137 (4-1BB) mAb’s between 26 and 35 weeks of age reversed acute disease, blocked chronic disease, and extended the mice’s lifespan from 10 months to more than 2 years. Autoantibody production in recipients was rapidly suppressed without inducing immunosuppression. Successful treatment could be traced to the fact that NZB × NZW F₁ mice, regardless of their age or disease status, could not maintain pathogenic IgG autoantibody production in the absence of continuous CD4⁺ T cell help. Our data support the hypothesis that CD137-mediated signaling anergized CD4⁺ T cells during priming at the DC interface.

C hronic hypoxia induces pulmonary vascular remodeling, leading to pulmonary hypertension, right ventricular hypertrophy, and heart failure. Heterozygous deficiency of hypoxia-inducible factor–1α (HIF-1α), which mediates the cellular response to hypoxia by increasing expression of genes involved in erythropoiesis and angiogenesis, has been previously shown to delay hypoxia-induced pulmonary hypertension. HIF-2α is a homologue of HIF-1α and is abundantly expressed in the lung, but its role in pulmonary hypertension remains unknown. Therefore, we analyzed the pulmonary response of WT and viable heterozygous HIF-2α–deficient (Hif2α^+/–) mice after exposure to 10% O₂ for 4 weeks. In contrast to WT mice, Hif2α^+/– mice were fully protected against pulmonary hypertension and right ventricular hypertrophy, unveiling a critical role of HIF-2α in hypoxia-induced pulmonary vascular remodeling. Pulmonary expression levels of endothelin-1 and plasma catecholamine levels were increased threefold and 12-fold respectively in WT but not in Hif2α^+/– mice after hypoxia, suggesting that HIF-2α–mediated upregulation of these vasoconstrictors contributes to the development of hypoxic pulmonary vascular remodeling.

T he inward rectifier current I_K1 is tightly regulated regionally within the heart, downregulated in heart failure, and genetically suppressed in Andersen syndrome. We used in vivo viral gene transfer to dissect the role of I_K1 in cardiac repolarization and maintenance of the resting membrane potential (RMP) in guinea pig ventricular myocytes. Kir2.1 overexpression boosted Ba²⁺-sensitive I_K1 by more than 100% (at –50mV), significantly shortened action potential durations (APDs), accelerated phase 3 repolarization, and hyperpolarized RMP compared with control cells (nongreen cells from the same hearts and green cells from GFP-transduced hearts). The dominant-negative Kir2.1AAA reduced I_K1 by 50–90%; those cells with less than 80% reduction of I_K1 exhibited prolonged APDs, decelerated phase 3 repolarization, and depolarization of the RMP. Further reduction of I_K1 resulted in a pacemaker phenotype, as previously described. ECGs revealed a 7.7% ± 0.9% shortening of the heart rate–corrected QT interval (QTc interval) in Kir2.1-transduced animals (n = 4) and a 16.7% ± 1.8% prolongation of the QTc interval (n = 3) in Kir2.1AAA-transduced animals 72 hours after gene delivery compared with immediate postoperative recordings. Thus, I_K1 is essential for establishing the distinctive electrical phenotype of the ventricular myocyte: rapid terminal repolarization to a stable and polarized resting potential. Additionally, the long-QT phenotype seen in Andersen syndrome is a direct consequence of dominant-negative suppression of Kir2 channel function.

T he cardiac pacemaker current I_f is a major determinant of diastolic depolarization in sinus nodal cells and has a key role in heartbeat generation. Therefore, we hypothesized that some forms of “idiopathic” sinus node dysfunction (SND) are related to inherited dysfunctions of cardiac pacemaker ion channels. In a candidate gene approach, a heterozygous 1-bp deletion (1631delC) in exon 5 of the human HCN4 gene was detected in a patient with idiopathic SND. The mutant HCN4 protein (HCN4-573X) had a truncated C-terminus and lacked the cyclic nucleotide–binding domain. COS-7 cells transiently transfected with HCN4-573X cDNA indicated normal intracellular trafficking and membrane integration of HCN4-573X subunits. Patch-clamp experiments showed that HCN4-573X channels mediated I_f-like currents that were insensitive to increased cellular cAMP levels. Coexpression experiments showed a dominant-negative effect of HCN4-573X subunits on wild-type subunits. These data indicate that the cardiac I_f channels are functionally expressed but with altered biophysical properties. Taken together, the clinical, genetic, and in vitro data provide a likely explanation for the patient’s sinus bradycardia and the chronotropic incompetence.

T he absence of immune defects that occurs in the syndrome of long-term nonprogressive (LTNP) HIV infection offers insights into the pathophysiology of HIV-induced immune disease. The (H[F/S]RIG)₂ domain of viral protein R (Vpr) induces apoptosis and may contribute to HIV-induced T cell depletion. We demonstrate a higher frequency of R77Q Vpr mutations in patients with LTNP than in patients with progressive disease. In addition, T cell infections using vesicular stomatitis virus G (VSV-G) pseudotyped HIV-1 Vpr R77Q result in less (P = 0.01) T cell death than infections using wild-type Vpr, despite similar levels of viral replication. Wild-type Vpr-associated events, including procaspase-8 and -3 cleavage, loss of mitochondrial transmembrane potential (Δψ_m), and DNA fragmentation factor activation are attenuated by R77Q Vpr. These data highlight the pathophysiologic role of Vpr in HIV-induced immune disease and suggest a novel mechanism of LTNP.

R ecent evidence suggests the existence of a hepatoportal vein glucose sensor, whose activation leads to enhanced glucose use in skeletal muscle, heart, and brown adipose tissue. The mechanism leading to this increase in whole body glucose clearance is not known, but previous data suggest that it is insulin independent. Here, we sought to further determine the portal sensor signaling pathway by selectively evaluating its dependence on muscle GLUT4, insulin receptor, and the evolutionarily conserved sensor of metabolic stress, AMP-activated protein kinase (AMPK). We demonstrate that the increase in muscle glucose use was suppressed in mice lacking the expression of GLUT4 in the organ muscle. In contrast, glucose use was stimulated normally in mice with muscle-specific inactivation of the insulin receptor gene, confirming independence from insulin-signaling pathways. Most importantly, the muscle glucose use in response to activation of the hepatoportal vein glucose sensor was completely dependent on the activity of AMPK, because enhanced hexose disposal was prevented by expression of a dominant negative AMPK in muscle. These data demonstrate that the portal sensor induces glucose use and development of hypoglycemia independently of insulin action, but by a mechanism that requires activation of the AMPK and the presence of GLUT4.

P revious studies established that IL-5–producing CD4⁺ T cells play a pivotal role in allergic respiratory inflammation. It was also reported that CD4⁺ T cells express higher levels of CD44 in the airway than in peripheral blood of patients with allergic respiratory diseases. We have used experimental pulmonary eosinophilia induced in mice by Ascaris suum (Asc) extract to investigate the role of CD44 in the development of allergic respiratory inflammation. Intraperitoneal administration of anti-CD44 mAb prevented both lymphocyte and eosinophil accumulation in the lung. Anti-CD44 mAb also blocked antigen-induced elevation of Th2 cytokines as well as chemokines (CCL11, CCL17) in bronchoalveolar lavage fluid (BALF). Treatment with anti-CD44 mAb inhibited the increased levels of hyaluronic acid (HA) and leukotriene concentrations in BALF that typically result from antigen challenge. Anti-CD44 mAb also blocked antigen-induced airway hyperresponsiveness. An anti-CD44 mAb (IM7) inhibited the HA-binding ability of splenocytes associated with decreased levels of CD44. Soluble CD44 levels in serum were increased in Asc-challenged IM7–treated mice, but not in KM201-treated mice, compared with Asc-challenged rat IgG–treated mice. Ab’s that block CD44-HA binding reduced allergic respiratory inflammation by preventing lymphocyte and eosinophil accumulation in the lung. Thus, CD44 may be critical for development of allergic respiratory inflammation.

T he Toll-like receptors (TLRs) are recently discovered germline-encoded receptors on APCs that are critically important in innate immune recognition of microbial pathogens. However, their role in solid-organ transplantation is unknown. To explore this role, we employed a skin allograft model using mice with targeted deletion of the universal TLR signal adaptor protein, MyD88. We report that minor antigen–mismatched (HY-mismatched) allograft rejection cannot occur in the absence of MyD88 signaling. Furthermore, we show that the inability to reject these allografts results from a reduced number of mature DCs in draining lymph nodes, leading to impaired generation of anti–graft-reactive T cells and impaired Th1 immunity. Hence, this work demonstrates that TLRs can be activated in a transplant setting and not solely by infections. These results link innate immunity to the initiation of the adaptive alloimmune response.

C ardiovascular diseases remain the leading cause of death in the United States. Two factors associated with a decreased risk of developing cardiovascular disease are elevated HDL levels and sex — specifically, a decreased risk is found in premenopausal women. HDL and estrogen stimulate eNOS and the production of nitric oxide, which has numerous protective effects in the vascular system including vasodilation, antiadhesion, and anti-inflammatory effects. We tested the hypothesis that HDL binds to its receptor, scavenger receptor class B type I (SR-BI), and delivers estrogen to eNOS, thereby stimulating the enzyme. HDL isolated from women stimulated eNOS, whereas HDL isolated from men had minimal activity. Studies with ovariectomized and ovariectomized/estrogen replacement mouse models demonstrated that HDL-associated estradiol stimulation of eNOS is SR-BI dependent. Furthermore, female HDL, but not male HDL, promoted the relaxation of muscle strips isolated from C57BL/6 mice but not SR-BI null mice. Finally, HDL isolated from premenopausal women or postmenopausal women receiving estradiol replacement therapy stimulated eNOS, whereas HDL isolated from postmenopausal women did not stimulate eNOS. We conclude that HDL-associated estrodial is capable of the stimulating eNOS. These studies establish a new paradigm for examining the cardiovascular effects of HDL and estrogen.

T he pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D), have been reported to bind lipopolysaccharide (LPS), opsonize microorganisms, and enhance the clearance of lung pathogens. In this study, we examined the effect of SP-A and SP-D on the growth and viability of Gram-negative bacteria. The pulmonary clearance of Escherichia coli K12 was reduced in SP-A–null mice and was increased in SP-D–overexpressing mice, compared with strain-matched wild-type controls. Purified SP-A and SP-D inhibited bacterial synthetic functions of several, but not all, strains of E. coli, Klebsiella pneumoniae, and Enterobacter aerogenes. In general, rough E. coli strains were more susceptible than smooth strains, and collectin-mediated growth inhibition was partially blocked by coincubation with rough LPS vesicles. Although both SP-A and SP-D agglutinated E. coli K12 in a calcium-dependent manner, microbial growth inhibition was independent of bacterial aggregation. At least part of the antimicrobial activity of SP-A and SP-D was localized to their C-terminal domains using truncated recombinant proteins. Incubation of E. coli K12 with SP-A or SP-D increased bacterial permeability. Deletion of the E. coli OmpA gene from a collectin-resistant smooth E. coli strain enhanced SP-A and SP-D–mediated growth inhibition. These data indicate that SP-A and SP-D are antimicrobial proteins that directly inhibit the proliferation of Gram-negative bacteria in a macrophage- and aggregation-independent manner by increasing the permeability of the microbial cell membrane.