Cdc42 is an antihypertrophic molecular switch in the mouse heart

To improve contractile function, the myocardium undergoes hypertrophic growth without myocyte proliferation in response to both pathologic and physiologic stimulation. Various membrane-bound receptors and intermediate signal transduction pathways regulate the induction of cardiac hypertrophy, but the cardioprotective regulatory pathways or effectors that antagonize cardiac hypertrophy remain poorly understood. Here we identify the small GTPase Cdc42 as a signaling intermediate that restrained the cardiac growth response to physiologic and pathologic stimuli. Cdc42 was specifically activated in the heart after pressure overload and in cultured cardiomyocytes by multiple agonists. Mice with a heart-specific deletion of Cdc42 developed greater cardiac hypertrophy at 2 and 8 weeks of stimulation and transitioned more quickly into heart failure than did wild-type controls. These mice also displayed greater cardiac hypertrophy in response to neuroendocrine agonist infusion for 2 weeks and, more remarkably, enhanced exercise-induced hypertrophy and sudden death. These pathologies were associated with an inability to activate JNK following stimulation through a MEKK1/MKK4/MKK7 pathway, resulting in greater cardiac nuclear factor of activated T cells (NFAT) activity. Restoration of cardiac JNK signaling with an Mkk7 heart-specific transgene reversed the enhanced growth effect. These results identify what we believe to be a novel antihypertrophic and protective cardiac signaling pathway, whereby Cdc42-dependent JNK activation antagonizes calcineurin-NFAT activity to reduce hypertrophy and prevent transition to heart failure.     

Activation of Mst1 causes dilated cardiomyopathy by stimulating apoptosis without compensatory ventricular myocyte hypertrophy

Activation 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.     

The role of the Grb2-p38 MAPK signaling pathway in cardiac hypertrophy and fibrosis

Cardiac hypertrophy is a common response to pressure overload and is associated with increased mortality. Mechanical stress in the heart can result in the integrin-mediated activation of focal adhesion kinase and the subsequent recruitment of the Grb2 adapter molecule. Grb2, in turn, can activate MAPK cascades via an interaction with the Ras guanine nucleotide exchange factor SOS and with other signaling intermediates. We analyzed the role of the Grb2 adapter protein and p38 MAPK in cardiac hypertrophy. Mice with haploinsufficiency of the Grb2 gene (Grb2(+/-) mice) appear normal at birth but have defective T cell signaling. In response to pressure overload, cardiac p38 MAPK and JNK activation was inhibited and cardiac hypertrophy and fibrosis was blocked in Grb2(+/-) mice. Next, transgenic mice with cardiac-specific expression of dominant negative forms of p38alpha (DN-p38alpha) and p38beta (DN-p38beta) MAPK were examined. DN-p38alpha and DN-p38beta mice developed cardiac hypertrophy but were resistant to cardiac fibrosis in response to pressure overload. These results establish that Grb2 action is essential for cardiac hypertrophy and fibrosis in response to pressure overload, and that different signaling pathways downstream of Grb2 regulate fibrosis, fetal gene induction, and cardiomyocyte growth.     

Inhibition of p38 alpha MAPK rescues cardiomyopathy induced by overexpressed beta 2-adrenergic receptor, but not beta 1-adrenergic receptor

We examined the role of p38alpha MAPK in mediating cardiomyopathy in mice overexpressing beta(1)-adrenergic receptor (beta(1)-AR) or beta(2)-AR by mating them with dominant-negative p38alpha (DNp38alpha) MAPK mice. Both beta(1)-AR and beta(2)-AR Tg mice had enhanced LV ejection fraction (LVEF) as young adults and developed similar cardiomyopathy at 11-15 months, characterized by reduced LVEF, myocyte hypertrophy, fibrosis, and apoptosis. We inhibited p38alpha MAPK by mating beta(1)-AR Tg and beta(2)-AR Tg mice with DNp38alpha MAPK mice, which rescued the depressed LVEF and reduced apoptosis and fibrosis in bigenic beta(2)-AR x DNp38alpha MAPK mice, but not bigenic beta(1)-AR x DNp38alpha MAPK mice, and failed to reduce myocyte hypertrophy in either group. G(salpha) was increased in both beta(1)-AR Tg and beta(2)-AR Tg mice and was still present in bigenic beta(1)-AR x DNp38alpha MAPK mice, but not bigenic beta(2)-AR x DNp38alpha MAPK mice. This suggests that p38alpha MAPK is one critical downstream signal for the development of cardiomyopathy following chronic beta(2)-AR stimulation, but other kinases may be more important in ameliorating the adverse effects of chronic beta(1)-AR stimulation.     

NO triggers RGS4 degradation to coordinate angiogenesis and cardiomyocyte growth

Myocardial hypertrophy is an adaptation to increased hemodynamic demands. An increase in heart tissue must be matched by a corresponding expansion of the coronary vasculature to maintain and adequate supply of oxygen and nutrients for the heart. The physiological mechanisms that underlie the coordination of angiogenesis and cardiomyocyte growth are unknown. We report that induction of myocardial angiogenesis promotes cardiomyocyte growth and cardiac hypertrophy through a novel NO-dependent mechanism. We used transgenic, conditional overexpression of placental growth factor (PlGF) in murine cardiac tissues to stimulate myocardial angiogenesis and increase endothelial-derived NO release. NO production, in turn, induced myocardial hypertrophy by promoting proteasomal degradation of regulator of G protein signaling type 4 (RGS4), thus relieving the repression of the Gβγ/PI3Kγ/AKT/mTORC1 pathway that stimulates cardiomyocyte growth. This hypertrophic response was prevented by concomitant transgenic expression of RGS4 in cardiomyocytes. NOS inhibitor L-NAME also significantly attenuated RGS4 degradation, and reduced activation of AKT/mTORC1 signaling and induction of myocardial hypertrophy in PlGF transgenic mice, while conditional cardiac-specific PlGF expression in eNOS knockout mice did not induce myocardial hypertrophy. These findings describe a novel NO/RGS4/Gβγ/PI3Kγ/AKT mechanism that couples cardiac vessel growth with myocyte growth and heart size.     

Alterations in the cardiac inflammatory response to burn trauma in mice lacking a functional Toll-like receptor 4 gene

Our group and others have previously shown that Toll-like receptor 4 (TLR-4) inactivation prevents burn-induced myocardial contractile dysfunction; however, the molecular mechanisms that are involved in this cardioprotection are not well defined. This present study examines the involvement of TLR-4 in the cardiac inflammatory response to thermal insult. C3H/HeJ (TLR-4 mutant mice) and C3H/HeN wild-type (WT) mice were subjected to either a sham burn or 40% full-thickness burn injury and were fluid resuscitated with lactated Ringer using the Parkland formula. Mice (n = 7-9 per group) were killed at 2, 4, or 24 h postsham or burn, and heart tissue was harvested. Immunoblotting was performed to evaluate phosphorylated p38 mitogen-activated protein kinase (MAPK), nuclear p50, and cytoplasmic p50. Nuclear factor-kappaB was also characterized via electrophoretic mobility shift assay. Systemic and cardiac myocyte secretion of TNF-alpha, IL-1 beta, IL-6, and IL-10 were measured by enzyme-linked immunosorbent assay. Burn injury in WT mice promoted myocardial inflammatory signaling that included increased expression of phosphorylated p38 MAPK, nuclear p50, and increased cardiac myocyte secretion of cytokines. Systemic cytokines were also increased in WT animals, although not to the extent of the myocardial cytokine expression. Toll-like receptor 4 inactivation resulted in an attenuation of several burn-induced responses, including phosphorylation of p38 MAPK, nuclear translocation of nuclear factor-kappaB, and cytokine secretion. These data suggest that burn injury initiates an inflammatory response via Toll/IL-1 signaling in the heart, which contributes to cardiac injury and contractile dysfunction.     

Selective activation and functional significance of p38alpha mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils

Activation of leukocytes by proinflammatory stimuli selectively initiates intracellular signal transduction via sequential phosphorylation of kinases. Lipopolysaccharide (LPS) stimulation of human neutrophils is known to result in activation of p38 mitogen-activated protein kinase (MAPk); however, the upstream activator(s) of p38 MAPk is unknown, and consequences of p38 MAPk activation remain largely undefined. We investigated the MAPk kinase (MKK) that activates p38 MAPk in response to LPS, the p38 MAPk isoforms that are activated as part of this pathway, and the functional responses affected by p38 MAPk activation. Although MKK3, MKK4, and MKK6 all activated p38 MAPk in experimental models, only MKK3 was found to activate recombinant p38 MAPk in LPS-treated neutrophils. Of p38 MAPk isoforms studied, only p38alpha and p38delta were detected in neutrophils. LPS stimulation selectively activated p38alpha. Specific inhibitors of p38alpha MAPk blocked LPS-induced adhesion, nuclear factor-kappa B (NF-kappaB) activation, and synthesis of tumor necrosis factor-alpha (TNF-alpha). Inhibition of p38alpha MAPk resulted in a transient decrease in TNF-alpha mRNA accumulation but persistent loss of TNF-alpha synthesis. These findings support a pathway by which LPS stimulation of neutrophils results in activation of MKK3, which in turn activates p38alpha MAPk, ultimately regulating adhesion, NF-kappaB activation, enhanced gene expression of TNF-alpha, and regulation of TNF-alpha synthesis.     

Cardiac-specific overexpression of AT1 receptor mutant lacking G alpha q/G alpha i coupling causes hypertrophy and bradycardia in transgenic mice

Ang II type 1 (AT1) receptors activate both conventional heterotrimeric G protein-dependent and unconventional G protein-independent mechanisms. We investigated how these different mechanisms activated by AT1 receptors affect growth and death of cardiac myocytes in vivo. Transgenic mice with cardiac-specific overexpression of WT AT1 receptor (AT1-WT; Tg-WT mice) or an AT1 receptor second intracellular loop mutant (AT1-i2m; Tg-i2m mice) selectively activating G(alpha)q/G(alpha)i-independent mechanisms were studied. Tg-i2m mice developed more severe cardiac hypertrophy and bradycardia coupled with lower cardiac function than Tg-WT mice. In contrast, Tg-WT mice exhibited more severe fibrosis and apoptosis than Tg-i2m mice. Chronic Ang II infusion induced greater cardiac hypertrophy in Tg-i2m compared with Tg-WT mice whereas acute Ang II administration caused an increase in heart rate in Tg-WT but not in Tg-i2m mice. Membrane translocation of PKCepsilon, cytoplasmic translocation of G(alpha)q, and nuclear localization of phospho-ERKs were observed only in Tg-WT mice while activation of Src and cytoplasmic accumulation of phospho-ERKs were greater in Tg-i2m mice, consistent with the notion that G(alpha)q/G(alpha)i-independent mechanisms are activated in Tg-i2m mice. Cultured myocytes expressing AT1-i2m exhibited a left and upward shift of the Ang II dose-response curve of hypertrophy compared with those expressing AT1-WT. Thus, the AT1 receptor mediates downstream signaling mechanisms through G(alpha)q/G(alpha)i-dependent and -independent mechanisms, which induce hypertrophy with a distinct phenotype.     

Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis

The Raf/MEK/extracellular signal-regulated kinase (ERK) signaling pathway regulates diverse cellular processes such as proliferation, differentiation, and apoptosis and is implicated as an important contributor to the pathogenesis of cardiac hypertrophy and heart failure. To examine the in vivo role of Raf-1 in the heart, we generated cardiac muscle-specific Raf-1-knockout (Raf CKO) mice with Cre-loxP-mediated recombination. The mice demonstrated left ventricular systolic dysfunction and heart dilatation without cardiac hypertrophy or lethality. The Raf CKO mice showed a significant increase in the number of apoptotic cardiomyocytes. The expression level and activation of MEK1/2 or ERK showed no difference, but the kinase activity of apoptosis signal-regulating kinase 1 (ASK1), JNK, or p38 increased significantly compared with that in controls. The ablation of ASK1 rescued heart dysfunction and dilatation as well as cardiac fibrosis. These results indicate that Raf-1 promotes cardiomyocyte survival through a MEK/ERK-independent mechanism.     

The alpha(1A/C)- and alpha(1B)-adrenergic receptors are required for physiological cardiac hypertrophy in the double-knockout mouse

Catecholamines and alpha(1)-adrenergic receptors (alpha(1)-ARs) cause cardiac hypertrophy in cultured myocytes and transgenic mice, but heart size is normal in single KOs of the main alpha(1)-AR subtypes, alpha(1A/C) and alpha(1B). Here we tested whether alpha(1)-ARs are required for developmental cardiac hypertrophy by generating alpha(1A/C) and alpha(1B) double KO (ABKO) mice, which had no cardiac alpha(1)-AR binding. In male ABKO mice, heart growth after weaning was 40% less than in WT, and the smaller heart was due to smaller myocytes. Body and other organ weights were unchanged, indicating a specific effect on the heart. Blood pressure in ABKO mice was the same as in WT, showing that the smaller heart was not due to decreased load. Contractile function was normal by echocardiography in awake mice, but the smaller heart and a slower heart rate reduced cardiac output. alpha(1)-AR stimulation did not activate extracellular signal-regulated kinase (Erk) and downstream kinases in ABKO myocytes, and basal Erk activity was lower in the intact ABKO heart. In female ABKO mice, heart size was normal, even after ovariectomy. Male ABKO mice had reduced exercise capacity and increased mortality with pressure overload. Thus, alpha(1)-ARs in male mice are required for the physiological hypertrophy of normal postnatal cardiac development and for an adaptive response to cardiac stress.     

Inhibition of norepinephrine-induced cardiac hypertrophy in s100beta transgenic mice.

We have recently reported that the Ca2+-binding protein S100beta was induced in rat heart after infarction and forced expression of S100beta in neonatal rat cardiac myocyte cultures inhibited alpha1-adrenergic induction of beta myosin heavy chain (MHC) and skeletal alpha-actin (skACT). We now extend this work by showing that S100beta is induced in hearts of human subjects after myocardial infarction. Furthermore, to determine whether overexpression of S100beta was sufficient to inhibit in vivo hypertrophy, transgenic mice containing multiple copies of the human gene under the control of its own promoter, and CD1 control mice were treated with norepinephrine (NE) (1.5 mg/kg) or vehicle, intraperitoneally twice daily for 15 d. In CD1, NE produced an increase in left ventricular/body weight ratio, ventricular wall thickness, induction of skACT, atrial natriuretic factor, betaMHC, and downregulation of alphaMHC. In transgenic mice, NE induced S100beta transgene mRNA and protein, but provoked neither hypertrophy nor regulated cardiac-specific gene expression. NE induced hypertrophy in cultured CD1 but not S100beta transgenic myocytes, confirming that the effects of S100beta on cardiac mass reflected myocyte-specific responses. These transgenic studies complement in vitro data and support the hypothesis that S100beta acts as an intrinsic negative regulator of the myocardial hypertrophic response.     

Dominant-negative effect of a mutant cardiac troponin T on cardiac structure and function in transgenic mice.

Hypertrophic cardiomyopathy (HCM) is a disease of sarcomeric proteins. The mechanism by which mutant sarcomeric proteins cause HCM is unknown. The leading hypothesis proposes that mutant sarcomeric proteins exert a dominant-negative effect on myocyte structure and function. To test this, we produced transgenic mice expressing low levels of normal or mutant human cardiac troponin T (cTnT). We constructed normal (cTnT-Arg92) and mutant (cTnT-Gln92) transgenes, driven by a murine cTnT promoter, and produced three normal and five mutant transgenic lines, which were identified by PCR and Southern blotting. Expression levels of the transgene proteins, detected using a specific antibody, ranged from 1 to 10% of the total cTnT pool. M-mode and Doppler echocardiography showed normal left ventricular dimensions and systolic function, but diastolic dysfunction in the mutant mice evidenced by a 50% reduction in the E/A ratio of mitral inflow velocities. Histological examination showed cardiac myocyte disarray in the mutant mice, which amounted to 1-15% of the total myocardium, and a twofold increase in the myocardial interstitial collagen content. Thus, the mutant cTnT-Gln92, responsible for human HCM, exerted a dominant-negative effect on cardiac structure and function leading to disarray, increased collagen synthesis, and diastolic dysfunction in transgenic mice.     

Dilated cardiomyopathy and impaired cardiac hypertrophic response to angiotensin II in mice lacking FGF-2

FGF-2 has been implicated in the cardiac response to hypertrophic stimuli. Angiotensin II (Ang II) contributes to maintain elevated blood pressure in hypertensive individuals and exerts direct trophic effects on cardiac cells. However, the role of FGF-2 in Ang II-induced cardiac hypertrophy has not been established. Therefore, mice deficient in FGF-2 expression were studied using a model of Ang II-dependent hypertension and cardiac hypertrophy. Echocardiographic measurements show the presence of dilated cardiomyopathy in normotensive mice lacking FGF-2. Moreover, hypertensive mice without FGF-2 developed no compensatory cardiac hypertrophy. In wild-type mice, hypertrophy was associated with a stimulation of the c-Jun N-terminal kinase, the extracellular signal regulated kinase, and the p38 kinase pathways. In contrast, mitogen-activated protein kinase (MAPK) activation was markedly attenuated in FGF-2-deficient mice. In vitro, FGF-2 of fibroblast origin was demonstrated to be essential in the paracrine stimulation of MAPK activation in cardiomyocytes. Indeed, fibroblasts lacking FGF-2 expression have a defective capacity for releasing growth factors to induce hypertrophic responses in cardiomyocytes. Therefore, these results identify the cardiac fibroblast population as a primary integrator of hypertrophic stimuli in the heart, and suggest that FGF-2 is a crucial mediator of cardiac hypertrophy via autocrine/paracrine actions on cardiac cells.     

Dominant-negative c-Jun inhibits rat cardiac hypertrophy induced by angiotensin II and hypertension

Cardiac activator protein-1 (AP-1), composed of c-Jun, is significantly activated by hypertension or angiotensin II (AngII). This study was undertaken to elucidate whether c-Jun could be the potential target for treatment of cardiac hypertrophy. We constructed recombinant adenovirus carrying dominant-negative mutant of c-Jun (Ad.DN-c-Jun). Using catheter-based technique of adenoviral gene transfer, we achieved global myocardial transduction of DN-c-Jun in rats, to specifically inhibit cardiac AP-1. (1) AngII (200 ng/kg/min) infusion in rats caused cardiac hypertrophy, increased cardiac p70S6 kinase activity by 1.3-fold (P<0.05) and enhanced the gene expression of cardiac hypertrophic markers. Ad.DN-c-Jun, which was transferred to the heart 2 days before AngII infusion, prevented cardiac hypertrophy (P<0.01), decreased p70S6 kinase phosphorylation (P<0.05), and suppressed cardiac gene expression of brain natriuretic peptide, collagen I, III, and IV, monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) (P<0.01). (2) In genetically hypertensive rats with cardiac hypertrophy, cardiac gene transfer of Ad.DN-c-Jun, without affecting hypertension, regressed cardiac hypertrophy (P<0.05), and suppressed p70S6 kinase phosphorylation by 20% (P<0.05) and suppressed the enhanced expression of collagen I, III, and IV, MCP-1 and PAI-1. These results provided the first evidence that in vivo blockade of cardiac c-Jun inhibits pathologic cardiac hypertrophy.     

Plasma membrane Ca2+-ATPase isoform 4 antagonizes cardiac hypertrophy in association with calcineurin inhibition in rodents

How Ca²⁺-dependent signaling effectors are regulated in cardiomyocytes, given the extreme cytoplasmic Ca²⁺ concentration changes that underlie contraction, remains unknown. Cardiomyocyte plasma membrane Ca²⁺-ATPase (PMCA) extrudes Ca²⁺ but has little effect on excitation-contraction coupling, suggesting its potential role in controlling Ca²⁺-dependent signaling effectors such as calcineurin. We generated cardiac-specific inducible PMCA4b transgenic mice that displayed normal global Ca²⁺ transient and cellular contraction levels and reduced cardiac hypertrophy following transverse aortic constriction (TAC) or phenylephrine/Ang II infusion, but showed no reduction in exercise-induced hypertrophy. Transgenic mice were protected from decompensation and fibrosis following long-term TAC. The PMCA4b transgene reduced the hypertrophic augmentation associated with transient receptor potential canonical 3 channel overexpression, but not that associated with activated calcineurin. Furthermore, Pmca4 gene–targeted mice showed increased cardiac hypertrophy and heart failure events after TAC. Physical associations between PMCA4b and calcineurin were enhanced by TAC and by agonist stimulation of cultured neonatal cardiomyocytes. PMCA4b reduced calcineurin nuclear factor of activated T cell–luciferase activity after TAC and in cultured neonatal cardiomyocytes after agonist stimulation. PMCA4b overexpression inhibited cultured cardiomyocyte hypertrophy following agonist stimulation, but much less so in a Ca²⁺ pumping–deficient PMCA4b mutant. Thus, Pmca4b likely reduces the local Ca²⁺ signals involved in reactive cardiomyocyte hypertrophy via calcineurin regulation.