Thomas Force

Role of the stress-activated protein kinases in endothelin-induced cardiomyocyte hypertrophy.

The signal transduction pathways governing the hypertrophic response of cardiomyocytes are not well defined. Constitutive activation of the stress-activated protein kinase (SAPK) family of mitogen-activated protein (MAP) kinases or another stress-response MAP kinase, p38, by overexpression of activated mutants of various components of the pathways is sufficient to induce a hypertrophic response in cardiomyocytes, but it is not clear what role these pathways play in the response to physiologically relevant hypertrophic stimuli. To determine the role of the SAPKs in the hypertrophic response, we used adenovirus-mediated gene transfer of SAPK/ERK kinase-1 (KR) [SEK-1(KR)], a dominant inhibitory mutant of SEK-1, the immediate upstream activator of the SAPKs, to block signal transmission down the SAPK pathway in response to the potent hypertrophic agent, endothelin-1 (ET-1). SEK-1(KR) completely inhibited ET-1-induced SAPK activation without affecting activation of the other MAP kinases implicated in the hypertrophic response, p38 and extracellular signal-regulated protein kinases (ERK)-1/ERK-2. Expression of SEK-1(KR) markedly inhibited the ET-1-induced increase in protein synthesis. In contrast, the MAPK/ERK kinase inhibitor, PD98059, which blocks ERK activation, and the p38 inhibitor, SB203580, had no effect on ET-1-induced protein synthesis. ET-1 also induced a significant increase in atrial natriuretic factor mRNA expression as well as in the percentage of cells with highly organized sarcomeres, responses which were also blocked by expression of SEK-1(KR). In summary, inhibiting activation of the SAPK pathway abrogated the hypertrophic response to ET-1. These data are the first demonstration that the SAPKs are necessary for the development of agonist-induced cardiomyocyte hypertrophy, and suggest that in response to ET-1, they transduce critical signals governing the hypertrophic response.

Activation of a human Ste20-like kinase by oxidant stress defines a novel stress response pathway.

Mammalian homologs of the yeast protein kinase, Sterile 20 (Ste20), can be divided into two groups based on their regulation and structure. The first group, which includes PAK1, is regulated by Rac and Cdc42Hs, and activators have been identified. In contrast, very little is known about activators, regulatory mechanisms or physiological roles of the other group, which consists of GC kinase and MST1. We have identified a human Ste20‐like kinase from the GC kinase group, SOK‐1 (Ste20/oxidant stress response kinase‐1), which is activated by oxidant stress. The kinase is activated by autophosphorylation and is markedly inhibited by its non‐catalytic C‐terminal region. SOK‐1 is activated 3‐ to 7‐fold by reactive oxygen intermediates, but is not activated by growth factors, alkylating agents, cytokines or environmental stresses including heat shock and osmolar stress. Although these data place SOK‐1 on a stress response pathway, SOK‐1, unlike GC kinase and PAK1, does not activate either of the stress‐activated MAP kinase cascades (p38 and SAPKs). SOK‐1 is the first mammalian Ste20‐like kinase which is activated by cellular stress, and the activation is relatively specific for oxidant stress. Since SOK‐1 does not activate any of the known MAP kinase cascades, its activation defines a novel stress response pathway which is likely to include a unique stress‐activated MAP kinase cascade.

PLIP, a novel splice variant of Tip60, interacts with group IV cytosolic phospholipase A(2), induces apoptosis, and potentiates prostaglandin production.

ABSTRACT The group IV cytosolic phospholipase A2(cPLA2) has been localized to the nucleus (M. R. Sierra-Honigmann, J. R. Bradley, and J. S. Pober, Lab. Investig. 74:684–695, 1996) and is known to translocate from the cytosolic compartment to the nuclear membrane (S. Glover, M. S. de Carvalho, T. Bayburt, M. Jonas, E. Chi, C. C. Leslie, and M. H. Gelb, J. Biol. Chem. 270:15359–15367, 1995; A. R. Schievella, M. K. Regier, W. L. Smith, and L. L. Lin, J. Biol. Chem. 270:30749–30754, 1995). We hypothesized that nuclear proteins interact with cPLA2 and participate in the functional effects of this translocation. We have identified a nuclear protein, cPLA2-interacting protein (PLIP), a splice variant of human Tip60, which interacts with the amino terminal region of cPLA2. Like Tip60, PLIP cDNA includes the MYST domain containing a C2HC zinc finger and well-conserved similarities to acetyltransferases. Both PLIP and Tip60 coimmunoprecipitate and colocalize with cPLA2 within the nuclei of transfected COS cells. A polyclonal antibody raised to PLIP recognizes both PLIP and Tip60. Endogenous Tip60 and/or PLIP in rat mesangial cells is localized to the nucleus in response to serum deprivation. Nuclear localization coincides temporally with apoptosis. PLIP expression, mediated by adenoviral gene transfer, potentiates serum deprivation-induced prostaglandin E2 (PGE2) production and apoptosis in mouse mesangial cells from cPLA2 +/+ mice but not in mesangial cells derived from cPLA2 −/− mice. Thus PLIP, a splice variant of Tip60, interacts with cPLA2 and potentiates cPLA2-mediated PGE2 production and apoptosis.

Frequency of mononuclear diploid cardiomyocytes underlies natural variation in heart regeneration

Adult mammalian cardiomyocyte regeneration after injury is thought to be minimal. Mononuclear diploid cardiomyocytes (MNDCMs), a relatively small subpopulation in the adult heart, may account for the observed degree of regeneration, but this has not been tested. We surveyed 120 inbred mouse strains and found that the frequency of adult mononuclear cardiomyocytes was surprisingly variable (>7-fold). Cardiomyocyte proliferation and heart functional recovery after coronary artery ligation both correlated with pre-injury MNDCM content. Using genome-wide association, we identified Tnni3k as one gene that influences variation in this composition and demonstrated that Tnni3k knockout resulted in elevated MNDCM content and increased cardiomyocyte proliferation after injury. Reciprocally, overexpression of Tnni3k in zebrafish promoted cardiomyocyte polyploidization and compromised heart regeneration. Our results corroborate the relevance of MNDCMs in heart regeneration. Moreover, they imply that intrinsic heart regeneration is not limited nor uniform in all individuals, but rather is a variable trait influenced by multiple genes.

Loss of Adult Cardiac Myocyte GSK-3 Leads to Mitotic Catastrophe Resulting in Fatal Dilated Cardiomyopathy

RATIONALEnCardiac myocyte-specific deletion of either glycogen synthase kinase (GSK)-3α and GSK-3β leads to cardiac protection after myocardial infarction, suggesting that deletion of both isoforms may provide synergistic protection. This is an important consideration because of the fact that all GSK-3-targeted drugs, including the drugs already in clinical trial target both isoforms of GSK-3, and none are isoform specific.nnnOBJECTIVEnTo identify the consequences of combined deletion of cardiac myocyte GSK-3α and GSK-3β in heart function.nnnMETHODS AND RESULTSnWe generated tamoxifen-inducible cardiac myocyte-specific mice lacking both GSK-3 isoforms (double knockout). We unexpectedly found that cardiac myocyte GSK-3 is essential for cardiac homeostasis and overall survival. Serial echocardiographic analysis reveals that within 2 weeks of tamoxifen treatment, double-knockout hearts leads to excessive dilatative remodeling and ventricular dysfunction. Further experimentation with isolated adult cardiac myocytes and fibroblasts from double-knockout implicated cardiac myocytes intrinsic factors responsible for observed phenotype. Mechanistically, loss of GSK-3 in adult cardiac myocytes resulted in induction of mitotic catastrophe, a previously unreported event in cardiac myocytes. Double-knockout cardiac myocytes showed cell cycle progression resulting in increased DNA content and multinucleation. However, increased cell cycle activity was rivaled by marked activation of DNA damage, cell cycle checkpoint activation, and mitotic catastrophe-induced apoptotic cell death. Importantly, mitotic catastrophe was also confirmed in isolated adult cardiac myocytes.nnnCONCLUSIONSnTogether, our findings suggest that cardiac myocyte GSK-3 is required to maintain normal cardiac homeostasis, and its loss is incompatible with life because of cell cycle dysregulation that ultimately results in a severe fatal dilated cardiomyopathy.

Group IVA Cytosolic Phospholipase A2 Regulates the G2-to-M Transition by Modulating the Activity of Tumor Suppressor SIRT2.

ABSTRACT The G2-to-M transition (or prophase) checkpoint of the cell cycle is a critical regulator of mitotic entry. SIRT2, a tumor suppressor gene, contributes to the control of this checkpoint by blocking mitotic entry under cellular stress. However, the mechanism underlying both SIRT2 activation and regulation of the G2-to-M transition remains largely unknown. Here, we report the formation of a multiprotein complex at the G2-to-M transition in vitro and in vivo. Group IVA cytosolic phospholipase A2 (cPLA2α) acts as a bridge in this complex to promote binding of SIRT2 to cyclin A-Cdk2. Cyclin A-Cdk2 then phosphorylates SIRT2 at Ser331. This phosphorylation reduces SIRT2 catalytic activity and its binding affinity to centrosomes and mitotic spindles, promoting G2-to-M transition. We show that the inhibitory effect of cPLA2α on SIRT2 activity impacts various cellular processes, including cellular levels of histone H4 acetylated at K16 (Ac-H4K16) and Ac-α-tubulin. This regulatory effect of cPLA2α on SIRT2 defines a novel function of cPLA2α independent of its phospholipase activity and may have implications for the impact of SIRT2-related effects on tumorigenesis and age-related diseases.

Entanglement of GSK-3β, β-catenin and TGF-β1 signaling network to regulate myocardial fibrosis

Nearly every form of the heart disease is associated with myocardial fibrosis, which is characterized by the accumulation of activated cardiac fibroblasts (CFs) and excess deposition of extracellular matrix (ECM). Although, CFs are the primary mediators of myocardial fibrosis in a diseased heart, in the traditional view, activated CFs (myofibroblasts) and resulting fibrosis were simply considered the secondary consequence of the disease, not the cause. Recent studies from our lab and others have challenged this concept by demonstrating that fibroblast activation and fibrosis are not simply the secondary consequence of a diseased heart, but are crucial for mediating various myocardial disease processes. In regards to the mechanism, the vast majority of literature is focused on the direct role of canonical SMAD-2/3-mediated TGF-β signaling to govern the fibrogenic process. Herein, we will discuss the emerging role of the GSK-3β, β-catenin and TGF-β1-SMAD-3 signaling network as a critical regulator of myocardial fibrosis in the diseased heart. The underlying molecular interactions and cross-talk among signaling pathways will be discussed. We will primarily focus on recent in vivo reports demonstrating that CF-specific genetic manipulation can lead to aberrant myocardial fibrosis and sturdy cardiac phenotype. This will allow for a better understanding of the driving role of CFs in the myocardial disease process. We will also review the specificity and limitations of the currently available genetic tools used to study myocardial fibrosis and its associated mechanisms. A better understanding of the GSK-3β, β-catenin and SMAD-3 signaling network may provide a novel therapeutic target for the management of myocardial fibrosis in the diseased heart.

Cardiomyocyte-specific deletion of GSK-3β leads to cardiac dysfunction in a diet induced obesity model

BACKGROUND AND RATIONALEnObesity, an independent risk factor for the development of myocardial diseases is a growing healthcare problem worldwide. Its well established that GSK-3β is critical to cardiac pathophysiology. However, the role cardiomyocyte (CM) GSK-3β in diet-induced cardiac dysfunction is unknown.nnnMETHODSnCM-specific GSK-3β knockout (CM-GSK-3β-KO) and littermate controls (WT) mice were fed either a control diet (CD) or high-fat diet (HFD) for 55weeks. Cardiac function was assessed by transthoracic echocardiography.nnnRESULTSnAt baseline, body weights and cardiac function were comparable between the WT and CM-GSK-3β-KOs. However, HFD-fed CM-GSK-3β-KO mice developed severe cardiac dysfunction. Consistently, both heart weight/tibia length and lung weight/tibia length were significantly elevated in the HFD-fed CM-GSK-3β-KO mice. The impaired cardiac function and adverse ventricular remodeling in the CM-GSK-3β-KOs were independent of body weight or the lean/fat mass composition as HFD-fed CM-GSK-3β-KO and controls demonstrated comparable body weight and body masses. At the molecular level, on a CD, CM-GSK-3α compensated for the loss of CM-GSK-3β, as evident by significantly reduced GSK-3αs21 phosphorylation (activation) resulting in a preserved canonical β-catenin ubiquitination pathway and cardiac function. However, this protective compensatory mechanism is lost with HFD, leading to excessive accumulation of β-catenin in HFD-fed CM-GSK-3β-KO hearts, resulting in adverse ventricular remodeling and cardiac dysfunction.nnnCONCLUSIONnIn summary, these results suggest that cardiac GSK-3β is crucial to protect against obesity-induced adverse ventricular remodeling and cardiac dysfunction.

Inhibition of GSK-3 to induce cardiomyocyte proliferation-a recipe for in situ cardiac regeneration

With an estimated 38 million current patients, heart failure (HF) is a leading cause of morbidity and mortality worldwide. Although the aetiology differs, HF is largely a disease of cardiomyocyte (CM) death or dysfunction. Due to the famously limited amount of regenerative capacity of the myocardium, the only viable option for advanced HF patients is cardiac transplantation; however, donors hearts are in very short supply. Thus, novel regenerative strategies are urgently needed to reconstitute the injured hearts. Emerging data from our lab and others have elucidated that CM-specific deletion of glycogen synthase kinase (GSK)-3 family of kinases induces CM proliferation, and the degree of proliferation is amplified in the setting of cardiac stress. If this proliferation is sufficiently robust, one could induce meaningful regeneration without the need for delivering exogenous cells to the injured myocardium (i.e. cardiac regeneration in situ). Herein, we will discuss the emerging role of the GSK-3s in CM proliferation and differentiation, including their potential implications in cardiac regeneration. The underlying molecular interactions and cross-talk among signalling pathways will be discussed. We will also review the specificity and limitations of the available small molecule inhibitors targeting GSK-3 and their potential applications to stimulate the endogenous cardiac regenerative responses to repair the injured heart.

Response by Zhou et al to Letter Regarding Article, “Loss of Adult Cardiac Myocyte GSK-3 Leads to Mitotic Catastrophe Resulting in Fatal Dilated Cardiomyopathy”

We thank Drs Karlstaedt and Taegtmeyer for their favorable comments on our recent report demonstrating mitotic catastrophe as a key mechanism of fatal dilated cardiomyopathy in glycogen synthase kinase 3 (GSK-3)–deficient hearts (conditional GSK-3α/β double knockout [DKO]).1 Considering the complex biology of GSK-3 in regulating numerous biological processes and cellular functions, we completely agree with Drs Karlstaedt and Taegtmeyer that there may be additional layer(s) of complexity to be elucidated for the complete understanding of the molecular basis of the observed phenotype. Indeed, our focus on cell cycle regulation and mitotic catastrophe was guided by unbiased microarray analysis.nnIt is well established that GSK-3 is a central regulator of nutrient and energy homeostasis. In response to the comments of Karlstaedt et al regarding metabolism, now we have examined the metabolic consequences of the complete loss of GSK-3 in adult hearts. As expected, an increased Periodic acid–Schiff–diastase positivity demonstrated enhanced glycogen deposition in the DKO hearts (Online Figure IA). …