Mitochondria play a critical role in mediating both apoptotic and necrotic cell death. The mitochondrial permeability transition (mPT) leads to mitochondrial swelling, outer membrane rupture and the release of apoptotic mediators. The mPT pore is thought to consist of the adenine nucleotide translocator, a voltage-dependent anion channel, and cyclophilin D (the Ppif gene product), a prolyl isomerase located within the mitochondrial matrix. Here we generated mice lacking Ppif and mice overexpressing cyclophilin D in the heart. Ppif null mice are protected from ischaemia/reperfusion-induced cell death in vivo, whereas cyclophilin D-overexpressing mice show mitochondrial swelling and spontaneous cell death. Mitochondria isolated from the livers, hearts and brains of Ppif null mice are resistant to mitochondrial swelling and permeability transition in vitro. Moreover, primary hepatocytes and fibroblasts isolated from Ppif null mice are largely protected from Ca2+-overload and oxidative stress-induced cell death. However, Bcl-2 family member-induced cell death does not depend on cyclophilin D, and Ppif null fibroblasts are not protected from staurosporine or tumour-necrosis factor-alpha-induced death. Thus, cyclophilin D and the mitochondrial permeability transition are required for mediating Ca2+- and oxidative damage-induced cell death, but not Bcl-2 family member-regulated death.

The proinflammatory cytokine tumour necrosis factor-alpha (TNF-alpha) regulates immune responses, inflammation and programmed cell death (apoptosis). The ultimate fate of a cell exposed to TNF-alpha is determined by signal integration between its different effectors, including IkappaB kinase (IKK), c-Jun N-terminal protein kinase (JNK) and caspases. Activation of caspases is required for apoptotic cell death, whereas IKK activation inhibits apoptosis through the transcription factor NF-kappaB, whose target genes include caspase inhibitors. JNK activates the transcription factor c-Jun/AP-1, as well as other targets. However, the role of JNK activation in apoptosis induced by TNF-alpha is less clear. It is unknown whether any crosstalk occurs between IKK and JNK, and, if so, how it affects TNF-alpha-induced apoptosis. We investigated this using murine embryonic fibroblasts that are deficient in either the IKKbeta catalytic subunit of the IKK complex or the RelA/p65 subunit of NF-kappaB. Here we show that in addition to inhibiting caspases, the IKK/NF-kappaB pathway negatively modulates TNF-alpha-mediated JNK activation, partly through NF-kappaB-induced X-chromosome-linked inhibitor of apoptosis (XIAP). This negative crosstalk, which is specific to TNF-alpha signalling and does not affect JNK activation by interleukin-1 (IL-1), contributes to inhibition of apoptosis.

The transcription factor NF-B regulates expression of genes that are involved in inflammation, immune response, viral infection, cell survival, and division. However, the role of NF-B in hypertrophic growth of terminally differentiated cardiomyocytes is unknown. Here we report that NF-B activation is required for hypertrophic growth of cardiomyocytes. In cultured rat primary neonatal ventricular cardiomyocytes, the nuclear translocation of NF-B and its transcriptional activity were stimulated by several hypertrophic agonists, including phenylephrine, endothelin-1, and angiotensin II. The activation of NF-B was inhibited by expression of a ''supersuppressor'' I B␣ mutant that is resistant to stimulationinduced degradation and a dominant negative I B kinase (IKK␤) mutant that can no longer be activated by phosphorylation. Furthermore, treatment with phenylephrine induced I B␣ degradation in an IKK-dependent manner, suggesting that NF-B is a downstream target of the hypertrophic agonists. Importantly, expression of the supersuppressor I B␣ mutant or the dominant negative IKK␤ mutant blocked the hypertrophic agonist-induced expression of the embryonic gene atrial natriuretic factor and enlargement of cardiomyocytes. Conversely, overexpression of NF-B itself induced atrial natriuretic factor expression and cardiomyocyte enlargement. These findings suggest that NF-B plays a critical role in the hypertrophic growth of cardiomyocytes and may serve as a potential target for the intervention of heart disease.

Cardiac hypertrophy is initiated as an adaptive response to sustained overload but progresses pathologically as heart failure ensues1. Here we report that genetic loss of APJ confers resistance to chronic pressure overload by dramatically reducing myocardial hypertrophy and heart failure. In contrast, mice lacking apelin (the endogenous APJ ligand) remain sensitive, suggesting an apelin independent function of APJ. Freshly isolated APJ-null cardiomyocytes exhibit an attenuated response to stretch, indicating that APJ is a mechano-sensor. Activation of APJ by stretch increases cardiomyocyte cell size and induces molecular markers of hypertrophy. Whereas apelin stimulates APJ to activate Gαi and elicits a protective response, stretch signals in an APJ-dependent G-protein-independent fashion to induce hypertrophy. Stretch-mediated hypertrophy is prevented by knockdown of β-arrestins or by pharmacological doses of apelin acting through Gαi. Taken together, our data indicate that APJ is a bifunctional receptor for both mechanical stretch and for the endogenous peptide apelin. By sensing the balance between these stimuli, APJ occupies a pivotal point linking sustained overload to cardiomyocyte hypertrophy.

MAPK signaling pathways function as critical regulators of cellular differentiation, proliferation, stress responsiveness, and apoptosis. One branch of the MAPK signaling pathway that culminates in ERK1/2 activation is hypothesized to regulate the growth and adaptation of the heart to both physiologic and pathologic stimuli, given its known activation in response to virtually every stress-and agonist-induced hypertrophic stimulus examined to date. Here we investigated the requirement of ERK1/2 signaling in mediating the cardiac hypertrophic growth response in Erk1 ؊/؊ and Erk2 ؉/؊ mice, as well as in transgenic mice with inducible expression of an ERK1/2-inactivating phosphatase in the heart, dual-specificity phosphatase 6. Although inducible expression of dual-specificity phosphatase 6 in the heart eliminated ERK1/2 phosphorylation at baseline and after stimulation without affecting any other MAPK, it did not diminish the hypertrophic response to pressure overload stimulation, neuroendocrine agonist infusion, or exercise. Similarly, Erk1 ؊/؊ and Erk2 ؉/؊ mice showed no reduction in pathologic or physiologic stimulus-induced cardiac growth in vivo. However, blockade or deletion of cardiac ERK1/2 did predispose the heart to decompensation and failure after long-term pressure overload in conjunction with an increase in myocyte TUNEL. Thus, ERK1/2 signaling is not required for mediating physiologic or pathologic cardiac hypertrophy in vivo, although it does play a protective role in response to pathologic stimuli.cardiomyopathy ͉ MAPK ͉ signaling

Background-Myocardial infarction causes a rapid and largely irreversible loss of cardiac myocytes that can lead to sudden death, ventricular dilation, and heart failure. Members of the mitogen-activated protein kinase (MAPK) signaling cascade have been implicated as important effectors of cardiac myocyte cell death in response to diverse stimuli, including ischemia-reperfusion injury. Specifically, activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) has been associated with cardioprotection, likely through antagonism of apoptotic regulatory pathways. Methods and Results-To establish a causal relationship between ERK1/2 signaling and cardioprotection, we analyzed Erk1 nullizygous gene-targeted mice, Erk2 heterozygous gene-targeted mice, and transgenic mice with activated MEK1-ERK1/2 signaling in the heart. Although MEK1 transgenic mice were largely resistant to ischemia-reperfusion injury, Erk2 ϩ/Ϫ gene-targeted mice showed enhanced infarction areas, DNA laddering, and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick-end labeling (TUNEL) compared with littermate controls. In contrast, enhanced MEK1-ERK1/2 signaling protected hearts from DNA laddering, TUNEL, and preserved hemodynamic function assessed by pressure-volume loop recordings after ischemia-reperfusion injury. Conclusions-These data are the first to demonstrate that ERK2 signaling is required to protect the myocardium from ischemia-reperfusion injury in vivo. Key Words: ischemia Ⅲ cardiac output Ⅲ mitogen-activated protein kinases Ⅲ infarction Ⅲ signal transduction T he mitogen-activated protein kinases (MAPK) constitute an essential signal transduction cascade that occupies a central position in cell growth, differentiation, apoptosis, and transformation. 1 Although it is somewhat oversimplified, the MAPK signaling pathway consists of a sequence of successively acting kinases that ultimately result in the dual phosphorylation and activation of the terminal kinases p38, c-Jun N-terminal kinase (JNKs), and extracellular signal-regulated kinase (ERKs). 2 The MAPK signaling cascade is initiated in cardiac myocytes by G-protein-coupled receptors, receptor tyrosine kinases, cardiotrophin-1 (gp130 receptor), and stress stimuli. 3 The major upstream activators of ERK1/2 are 2 MAP kinase kinases (MAPKK), MEK1 and MEK2, which directly phosphorylate the dual site on ERK kinases (Thr-Glu-Tyr). 2 Within the heart, members of the MAPK cascade have been implicated in regulating myocyte survival after ischemia-reperfusion injury, oxidative stress, and anthracycline exposure. Indeed, a number of studies support the hypothesis that the MEK1-ERK1/2 branch of the MAPK pathway is cardioprotective by directly antagonizing myocyte apoptosis. 4 Although MEK1-ERK1/2 signaling is thought to protect the myocardium from apoptotic insults, definitive genetic data demonstrating a necessary function for this pathway in vivo have not been reported. Methods Murine In Vivo Ischemia-Reperfusion and Pressure-Volume AcquisitionAdult mice (aged 8 to 10 week...

Rationale Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been implicated as a maladaptive mediator of cardiac ischemic injury. We hypothesized that the inflammatory response associated with in vivo ischemia/reperfusion (I/R) is initiated through CaMKII signaling. Objective To assess the contribution of CaMKIIδ to the development of inflammation, infarct and ventricular dysfunction following in vivo I/R and define early cardiomyocyte-autonomous events regulated by CaMKIIδ using cardiac-specific knockout (KO) mice. Methods and Results Wild-type (WT) and CaMKIIδ KO mice were subjected to in vivo I/R by occlusion of the left anterior descending (LAD) artery for 1-hr followed by reperfusion for various times. CaMKIIδ deletion protected the heart against I/R damage as evidenced by decreased infarct size, attenuated apoptosis and improved functional recovery. CaMKIIδ deletion also attenuated I/R induced inflammation and upregulation of NF-κB target genes. Further studies demonstrated that I/R rapidly increases CaMKII activity, leading to NF-κB activation within minutes of reperfusion. Experiments using cyclosporine A and cardiac-specific CaMKIIδ knockout mice indicate that NF-κB activation is initiated independent of necrosis and within cardiomyocytes. Expression of activated CaMKII in cardiomyocytes lead to I kappa B kinase (IKK) phosphorylation and concomitant increases in nuclear p65. Experiments using an IKK inhibitor support the conclusion that this is a proximal site of CaMKII-mediated NF-κB activation. Conclusions This is the first study demonstrating that CaMKIIδ mediates NF-κB activation in cardiomyocytes following in vivo I/R and suggests that CaMKIIδ serves to trigger, as well as to sustain subsequent changes in inflammatory gene expression that contribute to myocardial I/R damage.

The mitogen-activated protein kinase (MAPK) signaling pathway regulates diverse biologic functions including cell growth, differentiation, proliferation, and apoptosis. The extracellular signal-regulated kinases (ERKs) constitute one branch of the MAPK pathway that has been implicated in the regulation of cardiac differentiated growth, although the downstream mechanisms whereby ERK signaling affects this process are not well characterized. Here we performed a yeast two-hybrid screen with ERK2 bait and a cardiac cDNA library to identify novel proteins involved in regulating ERK signaling in cardiomyocytes. This screen identified the LIM-only factor FHL2 as an ERK interacting protein in both yeast and mammalian cells. In vivo, FHL2 and ERK2 colocalized in the cytoplasm at the level of the Z-line, and interestingly, FHL2 interacted more efficiently with the activated form of ERK2 than with the dephosphorylated form. ERK2 also interacted with FHL1 and FHL3 but not with the muscle LIM protein. Moreover, at least two LIM domains in FHL2 were required to mediate efficient interaction with ERK2. The interaction between ERK2 and FHL2 did not influence ERK1/2 activation, nor was FHL2 directly phosphorylated by ERK2. However, FHL2 inhibited the ability of activated ERK2 to reside within the nucleus, thus blocking ERK-dependent transcriptional responsiveness of ELK-1, GATA4, and the atrial natriuretic factor promoter. Finally, FHL2 partially antagonized the cardiac hypertrophic response induced by activated MEK-1, GATA4, and phenylephrine agonist stimulation. Collectively, these results suggest that FHL2 serves a repressor function in cardiomyocytes through its ability to inhibit ERK1/2 transcriptional coupling.Mitogen-activated protein kinases (MAPKs) and stress-activated protein kinases consist of an amplification cascade of successively acting kinases that culminate in the dual phosphorylation and activation of terminal effector kinases that subsequently phosphorylate diverse effector proteins. In mammalian cells, the MAPK signaling cascade is comprised of at least three major branches (named for the terminal effector kinases) including p38, c-Jun N-terminal kinases (JNKs), and extracellular signal-regulated kinases (ERKs) (reviewed in reference 38). In cardiomyocytes, the MAPK signaling cascade is initiated by both cell stretching and by diverse neuroendocrine factors mediated through G protein-coupled receptors and receptor tyrosine kinases (3, 32). Once activated, p38, JNKs, and ERKs each phosphorylate a wide array of intracellular targets, including diverse transcription factors resulting in the reprogramming of gene expression. The major upstream activators of ERK1/2 are two MAPK kinases, MEK1 and MEK2, which directly phosphorylate a dual acceptor motif in ERKs (Thr-Glu-Tyr).In cardiomyocytes, MEK1/2-ERK1/2 have been implicated as important transducers of the hypertrophic growth response both in cell culture-based studies and within the intact heart. For example, ERK1/2 are activated in cultured cardiomyocytes by ca...