Nicole H. Purcell

Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death

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-α-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.

MEK1-ERK2 Signaling Pathway Protects Myocardium From Ischemic Injury In Vivo

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+/− 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.

Genetic inhibition of cardiac ERK1/2 promotes stress-induced apoptosis and heart failure but has no effect on hypertrophy in vivo

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.

APJ acts as a dual receptor in cardiac hypertrophy.

Cardiac hypertrophy is initiated as an adaptive response to sustained overload but progresses pathologically as heart failure ensues. Here we report that genetic loss of APJ, a G-protein-coupled receptor, confers resistance to chronic pressure overload by markedly 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 mechanosensor. 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 the endogenous peptide apelin. By sensing the balance between these stimuli, APJ occupies a pivotal point linking sustained overload to cardiomyocyte hypertrophy.

Extracellular Signal-Regulated Kinase 2 Interacts with and Is Negatively Regulated by the LIM-Only Protein FHL2 in Cardiomyocytes

ABSTRACT 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.

DUSP6 (MKP3) Null Mice Show Enhanced ERK1/2 Phosphorylation at Baseline and Increased Myocyte Proliferation in the Heart Affecting Disease Susceptibility

The strength and duration of mitogen-activated protein kinase signaling is regulated through phosphorylation and dephosphorylation by dedicated dual-specificity kinases and phosphatases, respectively. Here we investigated the physiological role that extracellular signal-regulated kinases 1/2 (ERK1/2) dephosphorylation plays in vivo through targeted disruption of the gene encoding dual-specificity phosphatase 6 (Dusp6) in the mouse. Dusp6-/- mice, which were viable, fertile, and otherwise overtly normal, showed an increase in basal ERK1/2 phosphorylation in the heart, spleen, kidney, brain, and fibroblasts, but no change in ERK5, p38, or c-Jun N-terminal kinases activation. However, loss of Dusp6 did not increase or prolong ERK1/2 activation after stimulation, suggesting that its function is more dedicated to basal ERK1/2 signaling tone. In-depth analysis of the physiological effect associated with increased baseline ERK1/2 signaling was performed in cultured mouse embryonic fibroblasts (MEFs) and the heart. Interestingly, mice lacking Dusp6 had larger hearts at every age examined, which was associated with greater rates of myocyte proliferation during embryonic development and in the early postnatal period, resulting in cardiac hypercellularity. This increase in myocyte content in the heart was protective against decompensation and hypertrophic cardiomyopathy following long term pressure overload and myocardial infarction injury in adult mice. Dusp6-/- MEFs also showed reduced apoptosis rates compared with wild-type MEFs. These results demonstrate that ERK1/2 signaling is physiologically restrained by DUSP6 in coordinating cellular development and survival characteristics, directly impacting disease-responsiveness in adulthood.

RhoA protects the mouse heart against ischemia/reperfusion injury.

The small GTPase RhoA serves as a nodal point for signaling through hormones and mechanical stretch. However, the role of RhoA signaling in cardiac pathophysiology is poorly understood. To address this issue, we generated mice with cardiomyocyte-specific conditional expression of low levels of activated RhoA (CA-RhoA mice) and demonstrated that they exhibited no overt cardiomyopathy. When challenged by in vivo or ex vivo ischemia/reperfusion (I/R), however, the CA-RhoA mice exhibited strikingly increased tolerance to injury, which was manifest as reduced myocardial lactate dehydrogenase (LDH) release and infarct size and improved contractile function. PKD was robustly activated in CA-RhoA hearts. The cardioprotection afforded by RhoA was reversed by PKD inhibition. The hypothesis that activated RhoA and PKD serve protective physiological functions during I/R was supported by several lines of evidence. In WT mice, both RhoA and PKD were rapidly activated during I/R, and blocking PKD augmented I/R injury. In addition, cardiac-specific RhoA-knockout mice showed reduced PKD activation after I/R and strikingly decreased tolerance to I/R injury, as shown by increased infarct size and LDH release. Collectively, our findings provide strong support for the concept that RhoA signaling in adult cardiomyocytes promotes survival. They also reveal unexpected roles for PKD as a downstream mediator of RhoA and in cardioprotection against I/R.

PHLPP-1 Negatively Regulates Akt Activity and Survival in the Heart

Rationale The recently discovered PHLPP-1 (PH domain leucine-rich repeat protein phosphatase-1) selectively dephosphorylates Akt at Ser473 and terminates Akt signaling in cancer cells. The regulatory role of PHLPP-1 in the heart has not been considered. Objective To test the hypothesis that blockade/inhibition of PHLPP-1 could constitute a novel way to enhance Akt signals and provide cardioprotection. Methods and Results PHLPP-1 is expressed in neonatal rat ventricular myocytes (NRVMs) and in adult mouse ventricular myocytes (AMVMs). PHLPP-1 knockdown by small interfering RNA significantly enhances phosphorylation of Akt (p-Akt) at Ser473, but not at Thr308, in NRVMs stimulated with leukemia inhibitory factor (LIF). The increased phosphorylation is accompanied by greater Akt catalytic activity. PHLPP-1 knockdown enhances LIF-mediated cardioprotection against doxorubicin and also protects cardiomyocytes against H2O2. Direct Akt effects at mitochondria have been implicated in cardioprotection and mitochondria/cytosol fractionation revealed a significant enrichment of PHLPP-1 at mitochondria. The ability of PHLPP-1 knockdown to potentiate LIF-mediated increases in p-Akt at mitochondria and an accompanying increase in mitochondrial hexokinase-II was demonstrated. We generated PHLPP-1 knockout (KO) mice and demonstrate that AMVMs isolated from KO mice show potentiated p-Akt at Ser473 in response to agonists. When isolated perfused hearts are subjected to ischemia/reperfusion, p-Akt in whole-heart homogenates and in the mitochondrial fraction is significantly increased. Additionally in PHLPP-1 KO hearts, the increase in p-Akt elicited by ischemia/reperfusion is potentiated and, concomitantly, infarct size is significantly reduced. Conclusions These results implicate PHLPP-1 as an endogenous negative regulator of Akt activity and cell survival in the heart.

Ca2+/Calmodulin-dependent protein kinase II δ mediates myocardial ischemia/reperfusion injury through nuclear factor-κB.

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 CaMKII&dgr; to the development of inflammation, infarct, and ventricular dysfunction after in vivo I/R and define early cardiomyocyte–autonomous events regulated by CaMKII&dgr; using cardiac-specific knockout mice. Methods and Results: Wild-type and CaMKII&dgr; knockout mice were subjected to in vivo I/R by occlusion of the left anterior descending artery for 1 hour followed by reperfusion for various times. CaMKII&dgr; deletion protected the heart against I/R damage as evidenced by decreased infarct size, attenuated apoptosis, and improved functional recovery. CaMKII&dgr; deletion also attenuated I/R-induced inflammation and upregulation of nuclear factor-&kgr;B (NF-&kgr;B) target genes. Further studies demonstrated that I/R rapidly increases CaMKII activity, leading to NF-&kgr;B activation within minutes of reperfusion. Experiments using cyclosporine A and cardiac-specific CaMKII&dgr; knockout mice indicate that NF-&kgr;B activation is initiated independent of necrosis and within cardiomyocytes. Expression of activated CaMKII in cardiomyocytes leads to I&kgr;B kinase phosphorylation and concomitant increases in nuclear p65. Experiments using an I&kgr;B kinase inhibitor support the conclusion that this is a proximal site of CaMKII-mediated NF-&kgr;B activation. Conclusions: This is the first study demonstrating that CaMKII&dgr; mediates NF-&kgr;B activation in cardiomyocytes after in vivo I/R and suggests that CaMKII&dgr; serves to trigger, as well as to sustain subsequent changes in inflammatory gene expression that contribute to myocardial I/R damage.Rationale: Ca 2+ /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.

Is Nuclear Factor κB an Attractive Therapeutic Target for Treating Cardiac Hypertrophy

Cardiovascular disease is the leading cause of morbidity and mortality in the United States. In most instances, heart failure is the final consequence of many underlying disease etiologies such as long-standing hypertension, coronary heart disease, valvular insufficiency, arrhythmia, viral myocarditis, and mutations in sarcomere-encoding genes. A compensatory enlargement of the myocardium, or hypertrophy, typically accompanies many of the predisposing insults discussed above and is a leading predictor for the development of more serious and life-threatening disease.1,2 Given the strong association between the presentation of pathological cardiac hypertrophy and a progressive deterioration in myocardial function, investigators have focused on understanding the molecular mechanisms that initiate the compensatory growth response or that precipitate the transition to heart failure. A number of signal transduction pathways are thought to play pivotal roles in mediating the hypertrophic growth of the myocardium in response to pathological stimuli.3 In this issue of Circulation , Cook et al4 focus their attention on the nuclear factor (NF)–κB signaling pathway as a potentially important intracellular mediator of cardiac hypertrophy. See p 664 NF-κB is a transcription factor that can directly regulate the expression of immediate-early genes and genes involved in the stress response following a variety of physiological or pathological stimuli. Traditionally, the NF-κB pathway has been implicated as a pivotal intracellular mediator of the inflammatory response associated with septic shock, ischemia-reperfusion injury, acute respiratory distress syndrome, viral-induced cytotoxic effects, and cytokine-mediated chronic disease states.5 These disease associations have made NF-κB an attractive target for pharmacological inhibition with the goal of preserving end organ function following acute injury or resulting from chronic inflammatory disorders. Recent studies by several investigators have also implicated NF-κB activation as a causal event in the cardiac hypertrophic response, as modeled in cultured cardiac myocytes.6–9 This linkage between NF-κB and myocyte …