(Circulation. 2002;106:3421.)
© 2002 American Heart Association, Inc.
Late Breaking Basic Science Abstracts |
Phospholamban Missense Mutation Arg9Cys Causes Dominant Dilated Cardiomyopathy in Man and Mouse
Joachim P. Schmitt, Department of Genetics, Boston, Mass; Mitsuhiro Kamisago, Harvard Medical School, Tokyo, Japan; Michio Asahi, University of Toronto, Toronto, Canada; Guo H. Li, Ferhaan Ahmad, Harvard Medical School, Boston, Mass; David H. MacLennan, University of Toronto, Toronto, Canada; Jonathan Seidman, Harvard Medical School, Boston, Mass; Christine Seidman, Brigham and Women’s Hospital, Boston, Mass. Correspondence to Joachim P. Schmitt, Department of Genetics, Boston, MA 02115. E-mail jschmitt@genetics.med.harvard.edu
Phospholamban (PLN) inhibits the Ca2+-ATPase SERCA2 of the cardiac muscle sarcoplasmatic reticulum (SR). Here, we describe a human kindred with autosomal dominant inheritance of dilated cardiomyopathy and congestive heart failure caused by a Arg9Cys mutation in PLN (PLN-R9C; lod score 4.04;
=0). To understand the underlying mechanisms, we created transgenic mice that over-express PLN-R9C. These animals precisely resemble the human phenotype and die of terminal heart failure at age 4 to 8 months. In vitro assays on Ca2+ transport revealed a 72% reduction in the ability of human PLN-R9C to inhibit Ca2+ transport by SERCA2 (P=0.004). Coexpression of PLN-wild type and PLN-R9C, mimicking the heterozygous condition, demonstrated an almost complete rescue of mutant PLN function by PLN-wild type. Furthermore, mutant PLN-R9C traps protein kinase A, thereby preventing phosphorylation of PLN-wild type. Consequently, PLN-wild type is chronically activated, which inhibits Ca2+ uptake into the SR by SERCA2. These data demonstrate that a human PLN mutation can alter Ca2+ homeostasis, trigger detrimental remodeling processes, and cause dilated cardiomyopathy. Other molecules in this pathway are likely candidate disease genes for heart failure.
Molecular Control of Progenitor Cell Migration to the Infarcted Myocardium
Daniele Torella, Franca Di Meglio, Stefano Chimenti, Igor Jakoniuk, Bernardo Nadal-Ginard, Piero Anversa, Jan Kajstura, Annarosa Leri. From New York Medical College, Valhalla, NY. Correspondence to Annarosa Leri, New York Medical College, Valhalla, NY 10595. E-mail annarosa leri@nymc.edu
In the adult heart, primitive cells are nested in niches mostly located in the left and right atrium. Although undifferentiated cells are stored in the atria, their ability to leave the niches, home into injured myocardium, and promote tissue regeneration is impaired. Primitive and early committed cells express stem cell antigens, c-kit and MDR1, and c-Met and IGF-1 receptors. On this basis, we determined whether HGF and IGF-1, administered locally in the border zone of an acute infarct, changed the phenotype of stem cells and the characteristics of the microenvironment leading to their translocation and cardiac repair. The injection of HGF-IGF-1 in mice 5 hours after infarction resulted in a marked increase in number of primitive cells expressing 
VE3 and 6E4 integrins in the viable myocardium of the border zone and in the dead myocardium of the infarcted region. These changes established by confocal microscopy were evident at 8 hours and increased progressively at 1 and 4 days after coronary occlusion. Also, the number of dividing primitive and progenitor cells increased significantly, as shown by Ki67 labeling of their nuclei. In infarcted-treated mice, immunoprecipitation and Western blot documented physical interaction between 6E4 integrin and c-met. Similarly, complexes between 6E4 integrin and fibronectin, and VE3 integrin and fibrin were identified. Activation of integrin receptors led to the induction of the migratory pathway involving phosphorylation of FAK and paxillin and their physical association. These molecular events increased with time and correlated with the quantitative morphological results. Importantly, growth factors increased the level of the 200-kDa full-length fibronectin, which favors cell locomotion, and decreased the expression of the 40-kDa cleaved peptide that inhibits cell migration. In conclusion, HGF-IGF-1 promoted the replication and translocation of primitive cells to the stressed myocardium bordering and within the infarct. This positive effect was mediated by the enhanced expression of integrin receptors and their extracellular ligand, fibronectin, which together phosphorylated the FAK-paxillin pathway.
Inhibition of Thioredoxin Increases Reactive Oxygen Species and Hypertrophy in the Mouse Heart
Mitsutaka Yamamoto, Chull Hong, Guiping Yang, Jing Liu, Junichi Sadoshima, from UMDNJ, NJMS, Newark, NJ. Eric Holle and Thomas Wagner, from the Oncology Research Institute, Greenville, SC. Correspondence to Mitsutaka Yamamoto, UMDNJ, NJMS, Newark, NJ 07103.
Although thioredoxin (Trx), having a redox-active disulfide/dithiol, is expected to work as an antioxidant in cells, to what extent Trx contributes to overall cellular antioxidant mechanisms is unknown. Trx also works as a cytokine and causes cell growth in cancer cells. However, little is known about the function of Trx in the heart. We generated transgenic mice with cardiac-specific overexpression of dominant negative (DN) mutant (C32S/C35S) of Trx (Tg-DN-Trx), which specifically blocks the function of endogenous Trx. We analyzed 14 nontransgenic mice (NTg) and 16 Tg-DN-Trx (59±2 days vs 61±1 days). Malondialdehyde (MDA) alone and MDA/4-hydroxyalkenals (HAE) complex, markers of lipid oxidation, were significantly increased in hearts from Tg-DN-Trx compared with those from NTg (MDA 89±9 vs 60±2 pmol/mg, P<0.01, MDA/HAE: 151±13 vs 105±11 pmol/mg, P<0.01). Hearts from Tg-DN-Trx showed higher levels of 8-hydroxy-2'-deoxyguanosine, a marker of oxidative DNA damage, suggesting that the level of reactive oxygen species (ROS) is elevated in Tg-DN-Trx. Left ventricular (LV) weight/body weight (LV/BW) was significantly greater in Tg-DN-Trx than in NTg (3.5±0.1 vs 2.8±0.0 mg/g, P<0.01). In echocardiographic measurements, LV wall thickness was significantly increased in Tg-DN-Trx, whereas LV end-diastolic diameter and ejection fraction were similar between the two groups. These results suggest that specific inhibition of Trx causes cardiac hypertrophy with preserved function. To evaluate the role of Trx in cardiac hypertrophy by stress, we applied aortic banding in Tg-DN-Trx and NTg. After two weeks, the pressure gradient was similar in both groups. Although LV/BW was significantly increased in both groups (P<0.01), the % increase was significantly greater in Tg-DN-Trx than in NTg (49±4% vs 31±2%, P<0.01). These results for the first time demonstrated that endogenous Trx plays an essential role in regulating the tissue level of ROS in the heart. Furthermore, distinct from its cytokine actions in other cell types, inhibition of endogenous Trx in the heart stimulates cardiac hypertrophy in basal conditions and in response to pressure overload.
Androgens Regulate the Macrophage Expression of Atherosclerosis-Related Genes in a Gender-Dependent Manner: Molecular Insights Into Sex Differences in Atherosclerosis
Martin K. Ng, from the Royal Prince Alfred Hospital, Sydney, NSW, Australia; Carmel Quinn, from the Centre for Thrombosis and Vascular Research, Sydney, Australia; Shirley Nakhla, from the Heart Research Institute, Sydney, Australia; Jane A. McCrohon, from the Royal Prince Alfred Hospital, Sydney, Australia; Wendy Jessup, from the Centre for Thrombosis and Vascular Biology, Sydney, Australia; David J. Handelsman, from the ANZAC Research Institute, Sydney, Australia; David S. Celermajer, from the Royal Prince Alfred Hospital, Sydney, Australia; Alison K. Death, Department of Medicine, University of Sydney, Sydney, Australia. Correspondence to Martin K.C. Ng, Royal Prince Alfred Hospital, Sydney, NSW, Australia 2088. E-mail luminous@amaze.net.au
Men have an earlier onset and higher incidence of coronary disease than women. Androgen exposure produces a significant increase in foam cell formation in male (M) but not in female (F) macrophages, and M macrophages express more than 4-fold more androgen receptor mRNA than F. Androgen exposure may therefore have sex-specific and potentially pro-atherogenic effects on macrophage gene expression. Methods: Utilizing cDNA arrays of 588 genes, we compared the effects of androgen treatment (dihydrotestosterone - 40 nmol/l) with vehicle control on human monocyte-derived macrophages isolated from young healthy male and female donors (n=4 hybridizations; 2M, 2F). An at least 2-fold increase in expression relative to control in both arrays for each sex was regarded as significant. Differential expression of atherosclerosis-related genes was confirmed by real time reverse transcriptase-polymerase chain reaction (RT-PCR) in 5 M and 5 F donors. Functional confirmation of lysosomal acid lipase (LAL) upregulation was performed by lipoprotein degradation studies with 125I-Acetylated(Ac) LDL. Results: In M macrophages, androgen treatment produced differential upregulation of 27 genes concentrated in 5 functional classes: (1) lipoprotein processing; (2) cell surface adhesion; (3) extracellular signaling; (4) coagulation and fibrinolysis; and (5) transport protein genes. By contrast, none of 588 genes were upregulated in F macrophages. By RT-PCR, we confirmed the sex-specific upregulation of 6 of these atherosclerosis-related genes: acyl coA:cholesterol acyl transferase, LAL, caveolin 2, CD40, VEGF-165 receptor, and tissue factor pathway inhibitor. Functionally, androgen-treated male macrophages showed increased rates of lysosomal AcLDL degradation by 45–75% after 15–20 hours of 125I-AcLDL incubation (P=0.001), consistent with increased LAL activity. Conclusions: Androgens increase expression of atherosclerosis-related genes in macrophages in a strikingly sex-dependent manner. These findings may contribute to the male predisposition to atherosclerosis.
Genomic and Proteomic Alterations of Cardiac Troponin T, a Novel Mechanism for the Transition From Hypertrophy to Heart Failure
Lin Yan, Christophe Depre, Hui Ge, Hong Li, Junichi Sadoshima, Stephen F. Vatner, Dorothy E. Vatner, from the UMDNJ-New Jersey Medical School, Newark, NJ. Correspondence to Lin Yan, UMDNJ-New Jersey Medical School, Newark, NJ 07103. E-mail yanl2@umdnj.edu
The molecular mechanisms that precipitate the transition from left ventricular hypertrophy (LVH) to heart failure (HF) remain largely unknown, in part because of a lack of relevant chronic models that reproduce the clinical condition. We utilized a canine model in which HF occurred spontaneously or was induced by rapid pacing in the setting of severe, chronic (2-year) LVH. We examined potential alterations in sarcomeric proteins, which are recognized to play a role in familial genetic LVH, but less is known about their role in HF induced by altered load. Genomic subtraction hybridization demonstrated downregulation of cardiac troponin T (cTnT) mRNA expression in HF versus LVH, which was confirmed by qPCR (P<0.01). By combining 2D gel electrophoresis with in gel proteolysis, peptide mapping and sequencing by mass spectrometry, and sequence database searches for protein identification, we found a dramatic alteration of cTnT protein expression. As shown in the Figure below, 10 protein spots were identified as cTnT; 7 of them with molecular weights (35–40 kDa) and pI (5.3–5.8) in LVH. The intensity of these spots clearly decreased in HF consistent with the mRNA data and 3 new spots with lower MW (30–32 kDa) and more acidic pI (5.0–5.2) were only observed in HF. By mass spectrometry, these three cTnT fragments in HF corresponded to specific degradation of the protein near its C-terminus. Therefore, changes of both transcriptional and posttranslational levels are involved in the alteration of cTnT expression during the transition from LVH to HF.
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