Increased PHLPP1 expression through ERK-4E-BP1 signaling axis drives nicotine induced oxidative stress related damage of cardiomyocytes.

Nicotine, a key constituent of tobacco/electronic cigarettes causes cardiovascular injury and mortality. Nicotine is known to induce oxidative stress and mitochondrial dysfunction in cardiomyocytes leading to cell death. However, the underlying mechanisms remain unclear. Pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) is a member of metal-dependent protein phosphatase (PPM) family and is known to dephosphorylate several AGC family kinases and thereby regulate a diverse set of cellular functions including cell growth, survival, and death. Our lab has previously demonstrated that PHLPP1 removal reduced cardiomyocyte death and cardiac dysfunction following injury. Here, we present a novel finding that nicotine exposure significantly increased PHLPP1 protein expression in the adolescent rodent heart. Building upon our in vivo finding, we determined the mechanism of PHLPP1 expression in cardiomyocytes. Nicotine significantly increased PHLPP1 protein expression without altering PHLPP2 in cardiomyocytes. In cardiomyocytes, nicotine significantly increased NADPH oxidase 4 (NOX4), which coincided with increased reactive oxygen species (ROS) and increased cardiomyocyte apoptosis which were dependent on PHLPP1 expression. PHLPP1 expression was both necessary and sufficient for nicotine induced mitochondrial dysfunction. Mechanistically, nicotine activated extracellular signal-regulated protein kinases (ERK1/2) and subsequent eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) to increase PHLPP1 protein expression. Inhibition of protein synthesis with cycloheximide (CHX) and 4EGI-1 abolished nicotine induced PHLPP1 protein expression. Moreover, inhibition of ERK1/2 activity by U0126 significantly blocked nicotine induced PHLPP1 expression. Overall, this study reveals a novel mechanism by which nicotine regulates PHLPP1 expression through ERK-4E-BP1 signaling axis to drive cardiomyocyte injury.

PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) regulates G-protein-coupled receptor kinase 5 (GRK5)-induced cardiac hypertrophy in vitro.

PH domain leucine-rich repeat protein phosphatase (PHLPP) is a serine/threonine phosphatase that has been shown to regulate cell growth and survival through dephosphorylation of several members of the AGC family of kinases. G-protein-coupled receptor kinase 5 (GRK5) is an AGC kinase that regulates phenylephrine (PE)-induced cardiac hypertrophy through its noncanonical function of directly targeting proteins to the nucleus to regulate transcription. Here we investigated the possibility that the PHLPP2 isoform can regulate GRK5-induced cardiomyocyte hypertrophy in neonatal rat ventricular myocytes (NRVMs). We show that removal of PHLPP2 by siRNA induces hypertrophic growth of NRVMs as measured by cell size changes at baseline, potentiated PE-induced cell size changes, and re-expression of fetal genes atrial natriuretic factor and brain natriuretic peptide. Endogenous GRK5 and PHLPP2 were found to interact in NRVMs, and PE-induced nuclear accumulation of GRK5 was enhanced upon down-regulation of PHLPP2. Conversely, overexpression of PHLPP2 blocked PE-induced hypertrophic growth, re-expression of fetal genes, and nuclear accumulation of GRK5, which depended on its phosphatase activity. Finally, using siRNA against GRK5, we found that GRK5 was necessary for the hypertrophic response induced by PHLPP2 knockdown. Our findings demonstrate for the first time a novel regulation of GRK5 by the phosphatase PHLPP2, which modulates hypertrophic growth. Understanding the signaling pathways affected by PHLPP2 has potential for new therapeutic targets in the treatment of cardiac hypertrophy and failure.

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 ß-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.

Selective coupling of the S1P3 receptor subtype to S1P-mediated RhoA activation and cardioprotection.

Sphingosine-1-phosphate (S1P), a bioactive lysophospholipid, is generated and released at sites of tissue injury in the heart and can act on S1P1, S1P2, and S1P3 receptor subtypes to affect cardiovascular responses. We established that S1P causes little phosphoinositide hydrolysis and does not induce hypertrophy indicating that it does not cause receptor coupling to Gq. We previously demonstrated that S1P confers cardioprotection against ischemia/reperfusion by activating RhoA and its downstream effector PKD. The S1P receptor subtypes and G proteins that regulate RhoA activation and downstream responses in the heart have not been determined. Using siRNA or pertussis toxin to inhibit different G proteins in NRVMs we established that S1P regulates RhoA activation through Gα13 but not Gα12, Gαq, or Gαi. Knockdown of the three major S1P receptors using siRNA demonstrated a requirement for S1P3 in RhoA activation and subsequent phosphorylation of PKD, and this was confirmed in studies using isolated hearts from S1P3 knockout (KO) mice. S1P treatment reduced infarct size induced by ischemia/reperfusion in Langendorff perfused wild-type (WT) hearts and this protection was abolished in the S1P3 KO mouse heart. CYM-51736, an S1P3-specific agonist, also decreased infarct size after ischemia/reperfusion to a degree similar to that achieved by S1P. The finding that S1P3 receptor- and Gα13-mediated RhoA activation is responsible for protection against ischemia/reperfusion suggests that selective targeting of S1P3 receptors could provide therapeutic benefits in ischemic heart disease.

Decline in cellular function of aged mouse c-kit+ cardiac progenitor cells.

KEY POINTS: While autologous stem cell-based therapies are currently being tested on elderly patients, there are limited data on the function of aged stem cells and in particular c-kit+ cardiac progenitor cells (CPCs). We isolated c-kit+ cells from young (3 months) and aged (24 months) C57BL/6 mice to compare their biological properties. Aged CPCs have increased senescence, decreased stemness and reduced capacity to proliferate or to differentiate following dexamethasone (Dex) treatment in vitro, as evidenced by lack of cardiac lineage gene upregulation. Aged CPCs fail to activate mitochondrial biogenesis and increase proteins involved in mitochondrial oxidative phosphorylation in response to Dex. Aged CPCs fail to upregulate paracrine factors that are potentially important for proliferation, survival and angiogenesis in response to Dex. The results highlight marked differences between young and aged CPCs, which may impact future design of autologous stem cell-based therapies. ABSTRACT: Therapeutic use of c-kit+ cardiac progenitor cells (CPCs) is being evaluated for regenerative therapy in older patients with ischaemic heart failure. Our understanding of the biology of these CPCs has, however, largely come from studies of young cells and animal models. In the present study we examined characteristics of CPCs isolated from young (3 months) and aged (24 months) mice that could underlie the diverse outcomes reported for CPC-based therapeutics. We observed morphological differences and altered senescence indicated by increased senescence-associated markers ß-galactosidase and p16 mRNA in aged CPCs. The aged CPCs also proliferated more slowly than their young counterparts and expressed lower levels of the stemness marker LIN28. We subsequently treated the cells with dexamethasone (Dex), routinely used to induce commitment in CPCs, for 7 days and analysed expression of cardiac lineage marker genes. While MEF2C, GATA4, GATA6 and PECAM mRNAs were significantly upregulated in response to Dex treatment in young CPCs, their expression was not increased in aged CPCs. Interestingly, Dex treatment of aged CPCs also failed to increase mitochondrial biogenesis and expression of the mitochondrial proteins Complex III and IV, consistent with a defect in mitochondria complex assembly in the aged CPCs. Dex-treated aged CPCs also had impaired ability to upregulate expression of paracrine factor genes and the conditioned media from these cells had reduced ability to induce angiogenesis in vitro. These findings could impact the design of future CPC-based therapeutic approaches for the treatment of older patients suffering from cardiac injury.

Sphingosine 1-phosphate receptor 3 and RhoA signaling mediate inflammatory gene expression in astrocytes.

BACKGROUND: Sphingosine 1-phosphate (S1P) signals through G protein-coupled receptors to elicit a wide range of cellular responses. In CNS injury and disease, the blood-brain barrier is compromised, causing leakage of S1P from blood into the brain. S1P can also be locally generated through the enzyme sphingosine kinase-1 (Sphk1). Our previous studies demonstrated that S1P activates inflammation in murine astrocytes. The S1P1 receptor subtype has been most associated with CNS disease, particularly multiple sclerosis. S1P3 is most highly expressed and upregulated on astrocytes, however, thus we explored the involvement of this receptor in inflammatory astrocytic responses. METHODS: Astrocytes isolated from wild-type (WT) or S1P3 knockout (KO) mice were treated with S1P3 selective drugs or transfected with short interfering RNA to determine which receptor subtypes mediate S1P-stimulated inflammatory responses. Interleukin-6 (IL-6), and vascular endothelial growth factor A (VEGFa) messenger RNA (mRNA) and cyclooxygenase-2 (COX-2) mRNA and protein were assessed by q-PCR and Western blotting. Activation of RhoA was measured using SRE.L luciferase and RhoA implicated in S1P signaling by knockdown of Gα12/13 proteins or by inhibiting RhoA activation with C3 exoenzyme. Inflammation was simulated by in vitro scratch injury of cultured astrocytes. RESULTS: S1P3 was highly expressed in astrocytes and further upregulated in response to simulated inflammation. Studies using S1P3 knockdown and S1P3 KO astrocytes demonstrated that S1P3 mediates activation of RhoA and induction of COX-2, IL-6, and VEGFa mRNA, with some contribution from S1P2. S1P induces expression of all of these genes through coupling to the Gα12/13 proteins which activate RhoA. Studies using S1P3 selective agonists/antagonists as well as Fingolimod (FTY720) confirmed that stimulation of S1P3 induces COX-2 expression in astrocytes. Simulated inflammation increased expression of Sphk1 and consequently activated S1P3, demonstrating an autocrine pathway through which S1P is formed and released from astrocytes to regulate COX-2 expression. CONCLUSIONS: S1P3, through its ability to activate RhoA and its upregulation in astrocytes, plays a unique role in inducing inflammatory responses and should be considered as a potentially important therapeutic target for CNS disease progression.

Phospholipase C epsilon links G protein-coupled receptor activation to inflammatory astrocytic responses.

Neuroinflammation plays a major role in the pathophysiology of diseases of the central nervous system, and the role of astroglial cells in this process is increasingly recognized. Thrombin and the lysophospholipids lysophosphatidic acid and sphingosine 1-phosphate (S1P) are generated during injury and can activate G protein-coupled receptors (GPCRs) on astrocytes. We postulated that GPCRs that couple to Ras homolog gene family, member A (RhoA) induce inflammatory gene expression in astrocytes through the small GTPase responsive phospholipase Cε (PLCε). Using primary astrocytes from wild-type and PLCε knockout mice, we demonstrate that 1-h treatment with thrombin or S1P increases cyclooxygenase 2 (COX-2) mRNA levels ∼10-fold and that this requires PLCε. Interleukin-6 and interleukin-1ß mRNA levels are also increased in a PLCε-dependent manner. Thrombin, lysophosphatidic acid, and S1P increase COX-2 protein expression through a mechanism involving RhoA, catalytically active PLCε, sustained activation of protein kinase D (PKD), and nuclear translocation of NF-κB. Endogenous ligands that are released from astrocytes in an in vitro wounding assay also induce COX-2 expression through a PLCε- and NF-κB-dependent pathway. Additionally, in vivo stab wound injury activates PKD and induces COX-2 and other inflammatory genes in WT but not in PLCε knockout mouse brain. Thus, PLCε links GPCRs to sustained PKD activation, providing a means for GPCR ligands that couple to RhoA to induce NF-κB signaling and promote neuroinflammation.

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

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 after in vivo I/R and define early cardiomyocyte-autonomous events regulated by CaMKIIδ using cardiac-specific knockout mice. METHODS AND RESULTS: Wild-type and CaMKIIδ 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. 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 nuclear factor-κB (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 leads to IκB kinase phosphorylation and concomitant increases in nuclear p65. Experiments using an IκB kinase 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 after 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.

Collagen XIV is important for growth and structural integrity of the myocardium.

Collagen XIV is a fibril-associated collagen with an interrupted triple helix (FACIT). Previous studies have shown that this collagen type regulates early stages of fibrillogenesis in connective tissues of high mechanical demand. Mice null for Collagen XIV are viable, however formation of the interstitial collagen network is defective in tendons and skin leading to reduced biomechanical function. The assembly of a tightly regulated collagen network is also required in the heart, not only for structural support but also for controlling cellular processes. Collagen XIV is highly expressed in the embryonic heart, notably within the cardiac interstitium of the developing myocardium, however its role has not been elucidated. To test this, we examined cardiac phenotypes in embryonic and adult mice devoid of Collagen XIV. From as early as E11.5, Col14a1(-/-) mice exhibit significant perturbations in mRNA levels of many other collagen types and remodeling enzymes (MMPs, TIMPs) within the ventricular myocardium. By post natal stages, collagen fibril organization is in disarray and the adult heart displays defects in ventricular morphogenesis. In addition to the extracellular matrix, Col14a1(-/-) mice exhibit increased cardiomyocyte proliferation at post natal, but not E11.5 stages, leading to increased cell number, yet cell size is decreased by 3 months of age. In contrast to myocytes, the number of cardiac fibroblasts is reduced after birth associated with increased apoptosis. As a result of these molecular and cellular changes during embryonic development and post natal maturation, cardiac function is diminished in Col14a1(-/-) mice from 3 months of age; associated with dilation in the absence of hypertrophy, and reduced ejection fraction. Further, Col14a1 deficiency leads to a greater increase in left ventricular wall thickening in response to pathological pressure overload compared to wild type animals. Collectively, these studies identify a new role for type XIV collagen in the formation of the cardiac interstitium during embryonic development, and highlight the importance of the collagen network for myocardial cell survival, and function of the working myocardium after birth.

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 H(2)O(2). 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.

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-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.

On the PHLPPside: Emerging roles of PHLPP phosphatases in the heart.

PH domain leucine-rich repeat protein phosphatase (PHLPP) is a family of enzymes made up of two isoforms (PHLPP1 and PHLPP2), whose actions modulate intracellular activity via the dephosphorylation of specific serine/threonine (Ser/Thr) residues on proteins such as Akt. Recent data generated in our lab, supported by findings from others, implicates the divergent roles of PHLPP1 and PHLPP2 in maintaining cellular homeostasis since dysregulation of these enzymes has been linked to various pathological states including cardiovascular disease, diabetes, ischemia/reperfusion injury, musculoskeletal disease, and cancer. Therefore, development of therapies to modulate specific isoforms of PHLPP could prove to be therapeutically beneficial in several diseases especially those targeting the cardiovascular system. This review is intended to provide a comprehensive summary of current literature detailing the role of the PHLPP isoforms in the development and progression of heart disease.

Activation of nuclear factor-kappaB is necessary for myotrophin-induced cardiac hypertrophy.

The transcription factor nuclear factor-kappaB (NF-kappaB) regulates expression of a variety of genes involved in immune responses, inflammation, proliferation, and programmed cell death (apoptosis). Here, we show that in rat neonatal ventricular cardiomyocytes, activation of NF-kappaB is involved in the hypertrophic response induced by myotrophin, a hypertrophic activator identified from spontaneously hypertensive rat heart and cardiomyopathic human hearts. Myotrophin treatment stimulated NF-kappaB nuclear translocation and transcriptional activity, accompanied by IkappaB-alpha phosphorylation and degradation. Consistently, myotrophin-induced NF-kappaB activation was enhanced by wild-type IkappaB kinase (IKK) beta and abolished by the dominant-negative IKKbeta or a general PKC inhibitor, calphostin C. Importantly, myotrophin-induced expression of two hypertrophic genes (atrial natriuretic factor [ANF] and c-myc) and also enhanced protein synthesis were partially inhibited by a potent NF-kappaB inhibitor, pyrrolidine dithio-carbamate (PDTC), and calphostin C. Expression of the dominant-negative form of IkappaB-alpha or IKKbeta also partially inhibited the transcriptional activity of ANF induced by myotrophin. These findings suggest that the PKC-IKK-NF-kappaB pathway may play a critical role in mediating the myotrophin-induced hypertrophic response in cardiomyocytes.

Extracellular signal-regulated kinase 2 interacts with and is negatively regulated by the LIM-only protein FHL2 in cardiomyocytes.

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.

Sphingosine 1-phosphate elicits RhoA-dependent proliferation and MRTF-A mediated gene induction in CPCs.

Although c-kit(+) cardiac progenitor cells (CPCs) are currently used in clinical trials there remain considerable gaps in our understanding of the molecular mechanisms underlying their proliferation and differentiation. G-protein coupled receptors (GPCRs) play an important role in regulating these processes in mammalian cell types thus we assessed GPCR mRNA expression in c-kit(+) cells isolated from adult mouse hearts. Our data provide the first comprehensive overview of the distribution of this fundamental class of cardiac receptors in CPCs and reveal notable distinctions from that of adult cardiomyocytes. We focused on GPCRs that couple to RhoA activation in particular those for sphingosine-1-phosphate (S1P). The S1P2 and S1P3 receptors are the most abundant S1P receptor subtypes in mouse and human CPCs while cardiomyocytes express predominantly S1P1 receptors. Treatment of CPCs with S1P, as with thrombin and serum, increased proliferation through a pathway requiring RhoA signaling, as evidenced by significant attenuation when Rho was inhibited by treatment with C3 toxin. Further analysis demonstrated that both S1P- and serum-induced proliferation are regulated through the S1P2 and S1P3 receptor subtypes which couple to Gα12/13 to elicit RhoA activation. The transcriptional co-activator MRTF-A was activated by S1P as assessed by its nuclear accumulation and induction of a RhoA/MRTF-A luciferase reporter. In addition S1P treatment increased expression of cardiac lineage markers Mef2C and GATA4 and the smooth muscle marker GATA6 through activation of MRTF-A. In conclusion, we delineate an S1P-regulated signaling pathway in CPCs that introduces the possibility of targeting S1P2/3 receptors, Gα12/13 or RhoA to influence the proliferation and commitment of c-kit(+) CPCs and improve the response of the myocardium following injury.

mTORC1-independent Raptor prevents hepatic steatosis by stabilizing PHLPP2.

Mechanistic target of rapamycin complex 1 (mTORC1), defined by the presence of Raptor, is an evolutionarily conserved and nutrient-sensitive regulator of cellular growth and other metabolic processes. To date, all known functions of Raptor involve its scaffolding mTOR kinase with substrate. Here we report that mTORC1-independent ('free') Raptor negatively regulates hepatic Akt activity and lipogenesis. Free Raptor levels in liver decline with age and in obesity; restoration of free Raptor levels reduces liver triglyceride content, through reduced ß-TrCP-mediated degradation of the Akt phosphatase, PHLPP2. Commensurately, forced PHLPP2 expression ameliorates hepatic steatosis in diet-induced obese mice. These data suggest that the balance of free and mTORC1-associated Raptor governs hepatic lipid accumulation, and uncover the potentially therapeutic role of PHLPP2 activators in non-alcoholic fatty liver disease.

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.

Physiological activation of Akt by PHLPP1 deletion protects against pathological hypertrophy.

AIMS: To examine the role of physiological Akt signalling in pathological hypertrophy through analysis of PHLPP1 (PH domain leucine-rich repeat protein phosphatase) knock-out (KO) mice. METHODS AND RESULTS: To investigate the in vivo requirement for 'physiological' control of Akt activation in cardiac growth, we examined the effect of deleting the Akt phosphatase, PHLPP, on the induction of cardiac hypertrophy. Basal Akt phosphorylation increased nearly two-fold in the cardiomyocytes from PHLPP1 KO mice and physiological hypertrophy induced by swimming exercise was accentuated as assessed by increased heart size and myocyte cell area. In contrast, the development of pathophysiological hypertrophy induced by pressure overload and assessed by increases in heart size, myocyte cell area, and hypertrophic gene expression was attenuated. This attenuation coincided with decreased fibrosis and cell death in the KO mice. Cast moulding revealed increased capillary density basally in the KO hearts, which was further elevated relative to wild-type mouse hearts in response to pressure overload. In vitro studies with isolated myocytes in co-culture also demonstrated that PHLPP1 deletion in cardiomyocytes can enhance endothelial tube formation. Expression of the pro-angiogenic factor VEGF was also elevated basally and accentuated in response to transverse aortic constriction in hearts from KO mice. CONCLUSION: Our data suggest that enhancing Akt activity by inhibiting its PHLPP1-mediated dephosphorylation promotes processes associated with physiological hypertrophy that may be beneficial in attenuating the development of pathological hypertrophy.

PHLPP1 gene deletion protects the brain from ischemic injury.

A recently discovered protein phosphatase PHLPP (PH domain Leucine-rich repeat Protein Phosphatase) has been shown to dephosphorylate Akt on its hydrophobic motif (Ser473) thereby decreasing Akt kinase activity. We generated PHLPP1 knockout (KO) mice and used them to explore the ability of enhanced in vivo Akt signaling to protect the brain against ischemic insult. Brains from KO mice subjected to middle cerebral artery occlusion (MCAO) for 2 hours showed significantly greater increases in Akt activity and less neurovascular damage after reperfusion than wild-type (WT) mice. Remarkably, infarct volume in the PHLPP1 KO was significantly reduced compared with WT (12.7±2.7% versus 22.9±3.1%) and this was prevented by Akt inhibition. Astrocytes from KO mice and neurons in which PHLPP1 was downregulated showed enhanced Akt activation and diminished cell death in response to oxygen-glucose deprivation. Thus, deletion of PHLPP1 can enhance Akt activation in neurons and astrocytes, and can significantly increase cell survival and diminish infarct size after MCAO. Inhibition of PHLPP could be a therapeutic approach to minimize damage after focal ischemia.

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.