Abdominal aortic aneurysm (AAA) is a common disease among elderly people that, when surgical treatment is inapplicable, results in progressive expansion and rupture of the aorta with high mortality. Although nonsurgical treatment for AAA is much awaited, few options are available because its molecular pathogenesis remains elusive. Here, we identify JNK as a proximal signaling molecule in the pathogenesis of AAA. Human AAA tissue showed a high level of phosphorylated JNK. We show that JNK programs a gene expression pattern in different cell types that cooperatively enhances the degradation of the extracellular matrix while suppressing biosynthetic enzymes of the extracellular matrix. Selective inhibition of JNK in vivo not only prevented the development of AAA but also caused regression of established AAA in two mouse models. Thus, JNK promotes abnormal extracellular matrix metabolism in the tissue of AAA and may represent a therapeutic target.

Although oxidative stress causes activation of c-Jun N-terminal kinase (JNK) and apoptosis in many cell types, how the JNK pathway is connected to the apoptosis pathway is unclear. The molecular mechanism of JNK-mediated apoptosis was investigated in adult rat cardiac myocytes in culture as a model system that is sensitive to oxidative stress. Oxidative stress caused JNK activation, cytochrome c release, and apoptosis without new protein synthesis. Oxidative stress-induced apoptosis was abrogated by dominant negative stress-activated protein kinase/extracellular signal-regulated kinase kinase-1 (SEK1)-mediated inhibition of the JNK pathway, whereas activation of the JNK pathway by constitutively active SEK1 was sufficient to cause apoptosis. Inhibition of caspase-9, an apical caspase in the mitochondrial apoptosis pathway, suppressed oxidative stress-induced apoptosis, whereas inhibition of caspase-8 had no effect, indicating that both the JNK pathway and the mitochondrial apoptosis machinery are central to oxidative stress-induced apoptosis. Both JNK and SEK1 localized on mitochondria where JNK was activated by oxidative stress. Furthermore, active JNK caused the release of apoptogenic factors such as cytochrome c from isolated mitochondria in a cell-free assay. These findings indicate that the JNK pathway is a direct activator of mitochondrial death machinery without other cellular components and provide a molecular linkage from oxidative stress to the mitochondrial apoptosis machinery.

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Apoptosis has been implicated in ischemic heart disease, but its mechanism in cardiomyocytes has not been elucidated. In this study, we investigate the effects of hypoxia and reoxygenation in adult cardiomyocytes and the molecular mechanism involved in cardiomyocyte apoptosis. Morphologically, reoxygenation induced rounding up of the cells, appearance of membrane blebs that were filled with marginated mitochondria, and ultrastructural findings characteristic of apoptosis. Reoxygenation (18 hours of reoxygenation after 6 hours of hypoxia) and prolonged hypoxia (24 hours of hypoxia) resulted in a 59% and 51% decrease in cellular viability, respectively. During reoxygenation, cell death occurred predominantly via apoptosis associated with appearance of cytosolic cytochrome c and activation of caspase-3 and -9. However, nonapoptotic cell death predominated during prolonged hypoxia. Both caspase inhibition and Bcl-2 overexpression during reoxygenation significantly improved cellular viability through inhibition of apoptosis but had minimal effect on hypoxia-induced cell death. Bcl-2 overexpression blocked reoxygenation-induced cytochrome c release and activation of caspase -3 and -9, but caspase inhibition alone did not block cytochrome c release. These results suggest that apoptosis predominates in cardiomyocytes after reoxygenation through a mitochondrion-dependent apoptotic pathway, and Bcl-2 prevents reoxygenation-induced apoptosis by inhibiting cytochrome c release from the mitochondria and prevents activation of caspase-3 and -9.

Abstract-Two myosin light chain (MLC) kinase (MLCK) proteins, smooth muscle (encoded by mylk1 gene) and skeletal (encoded by mylk2 gene) MLCK, have been shown to be expressed in mammals. Even though phosphorylation of its putative substrate, MLC2, is recognized as a key regulator of cardiac contraction, a MLCK that is preferentially expressed in cardiac muscle has not yet been identified. In this study, we characterized a new kinase encoded by a gene homologous to mylk1 and -2, named cardiac MLCK, which is specifically expressed in the heart in both atrium and ventricle. In fact, expression of cardiac MLCK is highly regulated by the cardiac homeobox protein Nkx2-5 in neonatal cardiomyocytes. The overall structure of cardiac MLCK protein is conserved with skeletal and smooth muscle MLCK; however, the amino terminus is quite unique, without significant homology to other known proteins, and its catalytic activity does not appear to be regulated by Ca 2ϩ /calmodulin in vitro. Cardiac MLCK is phosphorylated and the level of phosphorylation is increased by phenylephrine stimulation accompanied by increased level of MLC2v phosphorylation. Both overexpression and knockdown of cardiac MLCK in cultured cardiomyocytes revealed that cardiac MLCK is likely a new regulator of MLC2 phosphorylation, sarcomere organization, and cardiomyocyte contraction. (Circ Res. 2008;102:571-580.)Key Words: kinase Ⅲ transcription Ⅲ contraction P hosphorylation of both myosin heavy chain and myosin light chain (MLC) affects motor activity and thick filament assembly. 1 In smooth muscle cells, phosphorylation of MLC2 by smooth muscle MLCK is thought to be responsible for the initiation of contraction. 2 In skeletal and cardiac muscles, however, initiation of muscle contraction depends on voltage-gated L-type Ca 2ϩ channels in the plasma membrane and T-tubules. Increased local Ca 2ϩ concentrations allow the sarcoplasmic reticulum to release large amounts of Ca 2ϩ , which bind to troponin C followed by myosin-actin cross-bridge formation. During this process, MLCK potentiates peak tension in skeletal muscle 1,3 and the force and rate of cross-bridge recruitment in cardiac myocytes. 4,5 To date, smooth muscle (encoded by mylk1 gene) and skeletal (encoded by mylk2 gene) MLCKs have been characterized. 3 Mouse skeletal muscle MLCK is predominantly expressed in skeletal muscle, and mouse smooth muscle MLCK is expressed in several tissues but predominantly in smooth muscle. 6,7 Mutations in human skeletal MLCK on human chromosome 20 have been mapped to a disease locus for familial cardiac hypertrophy (Online Mendelian Inheritance in Man no. 606566), suggesting that abnormal function of skeletal MLCK stimulates cardiac hypertrophy. 8 However, the abundance of skeletal MLCK expression in the heart is controversial, 8 -10 and gene-targeted mice for skeletal MLCK appear to have normal cardiac function. 10 Short-form (130-kDa) smooth muscle MLCK is expressed in the heart at lower levels than those detected in smooth muscle-rich organs such as gut, ...

Hypertrophy of mammalian cardiac muscle is mediated, in part, by angiotensin II through an angiotensin II type 1a receptor (AT 1a R)-dependent mechanism. To understand how the level of AT 1a Rs is altered in this pathological state, we studied the expression of an injected AT 1a R promoter-luciferase reporter gene in adult rat hearts subjected to an acute pressure overload by aortic coarctation. This model was validated by demonstrating that coarctation increased expression of the ␣-skeletal actin promoter 1.7-fold whereas the ␣-myosin heavy chain promoter was unaffected. Pressure overload increased expression from the AT 1a R promoter by 1.6-fold compared with controls. Mutations introduced into consensus binding sites for AP-1 or GATA transcription factors abolished the pressure overload response but had no effect on AT 1a R promoter activity in control animals. In extracts from coarcted hearts, but not from control hearts, a Fos-JunB-JunD complex and GATA-4 were detected in association with the AP-1 and GATA sites, respectively. These results establish that the AT 1a R promoter is active in cardiac muscle and its expression is induced by pressure overload, and suggest that this response is mediated, in part, by a functional interaction between AP-1 and GATA-4 transcription factors.Pathological conditions resulting in increased cardiac workload generally are associated with activation of systemic and local renin-angiotensin systems and increased levels of circulating angiotensin II (AngII) (1, 2). However, little is understood about how AngII type 1a receptors (AT 1a R) are modulated under these same pathological conditions. AngII is a potent growth factor that mediates the hypertrophic growth of cardiac muscle cells and is a chemical mediator of stretchinduced cardiomyocyte hypertrophy (3-7). The interaction of AngII with AT 1a R activates a signal transduction cascade that effects the phosphorylation of serum response factor and p62 TCF by pp90 RSK and mitogen-activated protein kinase, respectively, resulting in increased c-fos gene expression (5-7). Hypertrophic stimuli also increase the level of AT1 a R mRNA in cardiomyocytes. A 3-fold increase in AT 1a R mRNA and a 2-fold increase in AT 1a R densities have been reported in spontaneously hypertensive and two kidney one-clip renovascular hypertensive rats with established cardiac hypertrophy (8). It is not known whether this increase in AT 1a R mRNA is mediated by a transcriptional or posttranscriptional mechanism.In this study, we use direct injection of DNA into the heart in conjunction with aortic coarctation (CoA) to study the activity of the AT 1a R promoter in the normal and pressureoverloaded rat heart. The AT 1a R promoter was found to be active in normal adult cardiac muscle, whereas gene expression was increased in response to an acute pressure overload (PO). The induced expression was blocked by mutation of either an AP-1 or a GATA binding site, however, these mutations had no effect on basal expression. Administration of the angiotensin-...

A bdominal aortic aneurysm (AAA) is an age-related disease that occurs in ≤9% of adults aged of ≥65 years and is responsible for a significant number of deaths in Western countries.1 Although aggressive management of hypertension and hyperlipidemia is recommended in patients with AAA, these strategies have only little effect on the progression of AAA. Surgery is the only treatment for patients with AAA, but this is costly and associated with high morbidity and mortality.2 Endovascular repair is associated with lower postoperative mortality rates as compared to open surgical repair; however, there are no significant differences in total mortality or AAA-related mortality in the long term, with a higher incidence of reintervention in endovascular repair.3 When surgical treatment is not possible, AAAs typically progress to rupture with high mortality primarily because no effective nonsurgical treatment is currently available. Thus, a better understanding of the underlying mechanisms involved in AAA formation may help in identifying new therapeutic targets that could suppress AAA progression and reduce the risk of rupture.© 2014 American Heart Association, Inc. Objective-Abdominal aortic aneurysm (AAA) is considered a chronic inflammatory disease; however, the molecular basis underlying the sterile inflammatory response involved in the process of AAA remains unclear. We previously showed that the inflammasome, which regulates the caspase-1-dependent interleukin-1β production, mediates the sterile cardiovascular inflammatory responses. Therefore, we hypothesized that the inflammasome is a key mediator of initial inflammation in AAA formation. Approach and Results-Apoptosis-associated speck-like protein containing a caspase recruitment domain is highly expressed in adventitial macrophages in human and murine AAA tissues. Using an established mouse model of AAA induced by continuous infusion of angiotensin II in Apoe -/-mice, NLR family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1 deficiency in Apoe -/-mice were shown to decrease the incidence, maximal diameter, and severity of AAA along with adventitial fibrosis and inflammatory responses significantly, such as inflammatory cell infiltration and cytokine expression in the vessel wall. NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1 deficiency in Apoe -/-mice also reduced elastic lamina degradation and metalloproteinase activation in the early phase of AAA formation. Furthermore, angiotensin II stimulated generation of mitochondria-derived reactive oxygen species in the adventitial macrophages, and this mitochondria-derived reactive oxygen species generation was inhibited by NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1 deficiency. In vitro experiments revealed that angiotensin II stimulated the NLRP3 inflammasome activation and subsequent interleukin-1β release in macrophages,...

h Mitochondria are dynamic organelles, and their fusion and fission regulate cellular signaling, development, and mitochondrial homeostasis, including mitochondrial DNA (mtDNA) distribution. Cardiac myocytes have a specialized cytoplasmic structure where large mitochondria are aligned into tightly packed myofibril bundles; however, recent studies have revealed that mitochondrial dynamics also plays an important role in the formation and maintenance of cardiomyocytes. Here, we precisely analyzed the role of mitochondrial fission in vivo. The mitochondrial fission GTPase, Drp1, is highly expressed in the developing neonatal heart, and muscle-specific Drp1 knockout (Drp1-KO) mice showed neonatal lethality due to dilated cardiomyopathy. The Drp1 ablation in heart and primary cultured cardiomyocytes resulted in severe mtDNA nucleoid clustering and led to mosaic deficiency of mitochondrial respiration. The functional and structural alteration of mitochondria also led to immature myofibril assembly and defective cardiomyocyte hypertrophy. Thus, the dynamics of mtDNA nucleoids regulated by mitochondrial fission is required for neonatal cardiomyocyte development by promoting homogeneous distribution of active mitochondria throughout the cardiomyocytes.