Supravalvular aortic stenosis is an autosomal-dominant disease of elastin (Eln) insufficiency caused by loss-of-function mutations or gene deletion. Recently, we have modeled this disease in mice (Eln+/–) and found that Eln haploinsufficiency results in unexpected changes in cardiovascular hemodynamics and arterial wall structure. Eln+/– animals were found to be stably hypertensive from birth, with a mean arterial pressure 25–30 mmHg higher than their wild-type counterparts. The animals have only moderate cardiac hypertrophy and live a normal life span with no overt signs of degenerative vascular disease. Examination of arterial mechanical properties showed that the inner diameters of Eln+/– arteries were generally smaller than wild-type arteries at any given intravascular pressure. Because the Eln+/– mouse is hypertensive, however, the effective arterial working diameter is comparable to that of the normotensive wild-type animal. Physiological studies indicate a role for the renin-angiotensin system in maintaining the hypertensive state. The association of hypertension with elastin haploinsufficiency in humans and mice strongly suggests that elastin and other proteins of the elastic fiber should be considered as causal genes for essential hypertension.
Gilles Faury, Mylène Pezet, Russell H. Knutsen, Walter A. Boyle, Scott P. Heximer, Sean E. McLean, Robert K. Minkes, Kendall J. Blumer, Attila Kovacs, Daniel P. Kelly, Dean Y. Li, Barry Starcher, Robert P. Mecham
Atherosclerosis is now generally accepted as a chronic inflammatory condition. The transcription factor NF-κB is a key regulator of inflammation, immune responses, cell survival, and cell proliferation. To investigate the role of NF-κB activation in macrophages during atherogenesis, we used LDL receptor–deficient mice with a macrophage-restricted deletion of IκB kinase 2 (IKK2), which is essential for NF-κB activation by proinflammatory signals. These mice showed increased atherosclerosis as quantified by lesion area measurements. In addition, the lesions were more advanced and showed more necrosis and increased cell number in early lesions. Southern blotting revealed that deletion of IKK2 was approximately 65% in macrophages, coinciding with a reduction of 50% in NF-κB activation, as compared with controls. In both groups, the expression of differentiation markers, uptake of bacteria, and endocytosis of modified LDL was similar. Upon stimulation with LPS, production of TNF was reduced by approximately 50% in IKK2-deleted macrophages. Interestingly, we also found a major reduction in the anti-inflammatory cytokine IL-10. Our data show that inhibition of the NF-κB pathway in macrophages leads to more severe atherosclerosis in mice, possibly by affecting the pro- and anti-inflammatory balance that controls the development of atherosclerosis.
Edwin Kanters, Manolis Pasparakis, Marion J.J. Gijbels, Monique N. Vergouwe, Iris Partouns-Hendriks, Remond J.A. Fijneman, Björn E. Clausen, Irmgard Förster, Mark M. Kockx, Klaus Rajewsky, Georg Kraal, Marten H. Hofker, Menno P.J. de Winther
β-Adrenergic receptor (βAR) downregulation and desensitization are hallmarks of the failing heart. However, whether abnormalities in βAR function are mechanistically linked to the cause of heart failure is not known. We hypothesized that downregulation of cardiac βARs can be prevented through inhibition of PI3K activity within the receptor complex, because PI3K is necessary for βAR internalization. Here we show that in genetically modified mice, disrupting the recruitment of PI3K to agonist-activated βARs in vivo prevents receptor downregulation in response to chronic catecholamine administration and ameliorates the development of heart failure with pressure overload. Disruption of PI3K/βAR colocalization is required to preserve βAR signaling, since deletion of a single PI3K isoform (PI3Kγ knockout) is insufficient to prevent the recruitment of other PI3K isoforms and subsequent βAR downregulation with catecholamine stress. These data demonstrate a specific role for receptor-localized PI3K in the regulation of βAR turnover and show that abnormalities in βAR function are associated with the development of heart failure. Thus, a strategy that blocks the membrane translocation of PI3K and leads to the inhibition of βAR-localized PI3K activity represents a novel therapeutic approach to restore normal βAR signaling and preserve cardiac function in the pressure overloaded failing heart.
Jeffrey J. Nienaber, Hideo Tachibana, Sathyamangla V. Naga Prasad, Giovanni Esposito, Dianqing Wu, Lan Mao, Howard A. Rockman
Erythropoietin (EPO) has been shown to protect neurons from ischemic stroke, but can also increase thrombotic events and mortality rates in patients with ischemic heart disease. We reasoned that benefits of EPO might be offset by increases in hematocrit and evaluated the direct effects of EPO in the ischemic heart. We show that preconditioning with EPO protects H9c2 myoblasts in vitro and cardiomyocytes in vivo against ischemic injury. EPO treatment leads to significantly improved cardiac function following myocardial infarction. This protection is associated with mitigation of myocyte apoptosis, translating into more viable myocardium and less ventricular dysfunction. EPO-mediated myocyte survival appears to involve Akt activation. Importantly, cardioprotective effects of EPO were seen without an increase in hematocrit (eliminating oxygen delivery as an etiologic factor in myocyte survival and function), demonstrating that EPO can directly protect the ischemic and infarcted heart.
Cyrus J. Parsa, Akio Matsumoto, Jihee Kim, Ryan U. Riel, Laura S. Pascal, G. Brant Walton, Richard B. Thompson, Jason A. Petrofski, Brian H. Annex, Jonathan S. Stamler, Walter J. Koch
Activation of multiple pathways is associated with cardiac hypertrophy and heart failure. Repression of antihypertrophic pathways has rarely been demonstrated to cause cardiac hypertrophy in vivo. Hop is an unusual homeodomain protein that is expressed by embryonic and postnatal cardiac myocytes. Unlike other homeodomain proteins, Hop does not bind DNA. Rather, it modulates cardiac growth and proliferation by inhibiting the transcriptional activity of serum response factor (SRF) in cardiomyocytes. Here we show that Hop can inhibit SRF-dependent transcriptional activation by recruiting histone deacetylase (HDAC) activity and can form a complex that includes HDAC2. Transgenic mice that overexpress Hop develop severe cardiac hypertrophy, cardiac fibrosis, and premature death. A mutant form of Hop, which does not recruit HDAC activity, does not induce hypertrophy. Treatment of Hop transgenic mice with trichostatin A, an HDAC inhibitor, prevents hypertrophy. In addition, trichostatin A also attenuates hypertrophy induced by infusion of isoproterenol. Thus, chromatin remodeling and repression of otherwise active transcriptional processes can result in hypertrophy and heart failure, and this process can be blocked with chemical HDAC inhibitors.
Hyun Kook, John J. Lepore, Aaron D. Gitler, Min Min Lu, Wendy Wing-Man Yung, Joel Mackay, Rong Zhou, Victor Ferrari, Peter Gruber, Jonathan A. Epstein
Hypertension is a clinical syndrome characterized by increased vascular tone. However, the molecular mechanisms underlying vascular dysfunction during acquired hypertension remain unresolved. Localized intracellular Ca2+ release events through ryanodine receptors (Ca2+ sparks) in the sarcoplasmic reticulum are tightly coupled to the activation of large-conductance, Ca2+-activated K+ (BK) channels to provide a hyperpolarizing influence that opposes vasoconstriction. In this study we tested the hypothesis that a reduction in Ca2+ spark–BK channel coupling underlies vascular smooth muscle dysfunction during acquired hypertension. We found that in hypertension, expression of the β1 subunit was decreased relative to the pore-forming α subunit of the BK channel. Consequently, the BK channels were functionally uncoupled from Ca2+ sparks. Consistent with this, the contribution of BK channels to vascular tone was reduced during hypertension. We conclude that downregulation of the β1 subunit of the BK channel contributes to vascular dysfunction in hypertension. These results support the novel concept that changes in BK channel subunit composition regulate arterial smooth muscle function.
Gregory C. Amberg, Adrian D. Bonev, Charles F. Rossow, Mark T. Nelson, Luis F. Santana
Increased production of reactive oxygen species and loss of endothelial NO bioactivity are key features of vascular disease states such as diabetes mellitus. Tetrahydrobiopterin (BH4) is a required cofactor for eNOS activity; pharmacologic studies suggest that BH4 may mediate some of the adverse effects of diabetes on eNOS function. We have now investigated the importance and mechanisms of BH4 availability in vivo using a novel transgenic mouse model with endothelial-targeted overexpression of the rate-limiting enzyme in BH4 synthesis, guanosine triphosphate–cyclohydrolase I (GTPCH). Transgenic (GCH-Tg) mice demonstrated selective augmentation of endothelial BH4 levels. In WT mice, induction of diabetes with streptozotocin (STZ) increased vascular oxidative stress, resulting in oxidative loss of BH4, forming BH2 and biopterin. Endothelial cell superoxide production in diabetes was increased, and NO-mediated endothelium-dependent vasodilatation was impaired. In diabetic GCH-Tg mice, superoxide production from the endothelium was markedly reduced compared with that of WT mice, endothelial BH4 levels were maintained despite some oxidative loss of BH4, and NO-mediated vasodilatation was preserved. These findings indicate that BH4 is an important mediator of eNOS regulation in diabetes and is a rational therapeutic target to restore NO-mediated endothelial function in diabetes and other vascular disease states.
Nicholas J. Alp, Shafi Mussa, Jeffrey Khoo, Shijie Cai, Tomasz Guzik, Andrew Jefferson, Nicky Goh, Kirk A. Rockett, Keith M. Channon
The thin filament protein cardiac troponin T (cTnT) is an important regulator of myofilament activation. Here we report a significant change in cardiac energetics in transgenic mice bearing the missense mutation R92Q within the tropomyosin-binding domain of cTnT, a mutation associated with a clinically severe form of familial hypertrophic cardiomyopathy. This functional domain of cTnT has recently been shown to be a crucial modulator of contractile function despite the fact that it does not directly interact with the ATP hydrolysis site in the myosin head. Simultaneous measurements of cardiac energetics using 31P NMR spectroscopy and contractile performance of the intact beating heart revealed both a decrease in the free energy of ATP hydrolysis available to support contractile work and a marked inability to increase contractile performance upon acute inotropic challenge in hearts from R92Q mice. These results show that alterations in thin filament protein structure and function can lead to significant defects in myocardial energetics and contractile reserve.
Maryam M. Javadpour, Jil C. Tardiff, Ilka Pinz, Joanne S. Ingwall
Ectopic calcification is a frequent complication of many degenerative diseases. Here we identify the serum protein α2–Heremans-Schmid glycoprotein (Ahsg, also known as fetuin-A) as an important inhibitor of ectopic calcification acting on the systemic level. Ahsg-deficient mice are phenotypically normal, but develop severe calcification of various organs on a mineral and vitamin D–rich diet and on a normal diet when the deficiency is combined with a DBA/2 genetic background. This phenotype is not associated with apparent changes in calcium and phosphate homeostasis, but with a decreased inhibitory activity of the Ahsg-deficient extracellular fluid on mineral formation. The same underlying principle may contribute to many calcifying disorders including calciphylaxis, a syndrome of severe systemic calcification in patients with chronic renal failure. Taken together, our data demonstrate a critical role of Ahsg as an inhibitor of unwanted mineralization and provide a novel therapeutic concept to prevent ectopic calcification accompanying various diseases.
Cora Schäfer, Alexander Heiss, Anke Schwarz, Ralf Westenfeld, Markus Ketteler, Jürgen Floege, Werner Müller-Esterl, Thorsten Schinke, Willi Jahnen-Dechent
Hepatic lipase (HL) has a well-established role in lipoprotein metabolism. However, its role in atherosclerosis is poorly understood. Here we demonstrate that HL deficiency raises the proatherogenic apoB-containing lipoprotein levels in plasma but reduces atherosclerosis in lecithin cholesterol acyltransferase (LCAT) transgenic (Tg) mice, similar to results previously observed with HL-deficient apoE-KO mice. These findings suggest that HL has functions that modify atherogenic risk that are separate from its role in lipoprotein metabolism. We used bone marrow transplantation (BMT) to generate apoE-KO and apoE-KO × HL-KO mice, as well as LCAT-Tg and LCAT-Tg × HL-KO mice, chimeric for macrophage HL gene expression. Using in situ RNA hybridization, we demonstrated localized production of HL by donor macrophages in the artery wall. We found that expression of HL by macrophages enhances early aortic lesion formation in both apoE-KO and LCAT-Tg mice, without changing the plasma lipid profile, lipoprotein lipid composition, or HL and lipoprotein lipase activities. HL does, however, enhance oxidized LDL uptake by peritoneal macrophages. These combined data demonstrate that macrophage-derived HL significantly contributes to early aortic lesion formation in two independent mouse models and identify a novel mechanism, separable from the role of HL in plasma lipoprotein metabolism, by which HL modulates atherogenic risk in vivo.
Zengxuan Nong, Herminia González-Navarro, Marcelo Amar, Lita Freeman, Catherine Knapper, Edward B. Neufeld, Beverly J. Paigen, Robert F. Hoyt, Jamila Fruchart-Najib, Silvia Santamarina-Fojo