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Background: Cardiac kinases play a critical role in the development of heart failure, and represent potential tractable therapeutic targets. However, only a very small fraction of the cardiac kinome has been investigated. To identify novel cardiac kinases involved in heart failure, we used an integrated transcriptomics and bioinformatics analysis and identified Homeodomain-Interacting Protein Kinase 2 (HIPK2) as a novel candidate kinase. The role of HIPK2 in cardiac biology is unknown. Methods: We used the Expression2Kinase algorithm for the screening of kinase targets. To determine the role of HIPK2 in the heart, we generated cardiomyocyte (CM)-specific HIPK2 knockout and heterozygous mice. Heart function was examined by echocardiography, and related cellular and molecular mechanisms were examined. Adeno-associated virus serotype 9 carrying cardiac-specific constitutively active MEK1 (TnT-MEK1-CA) was administrated to rescue cardiac dysfunction in CM-HIPK2 knockout mice. Results: To our knowledge, this is the first study to define the role of HIPK2 in cardiac biology. Using multiple HIPK2 loss-of-function mouse models, we demonstrated that reduction of HIPK2 in CMs leads to cardiac dysfunction, suggesting a causal role in heart failure. It is important to note that cardiac dysfunction in HIPK2 knockout mice developed with advancing age, but not during development. In addition, CM-HIPK2 knockout mice and CM-HIPK2 heterozygous mice exhibited a gene dose-response relationship of CM-HIPK2 on heart function. HIPK2 expression in the heart was significantly reduced in human end-stage ischemic cardiomyopathy in comparison to nonfailing myocardium, suggesting a clinical relevance of HIPK2 in cardiac biology. In vitro studies with neonatal rat ventricular CMscorroborated the in vivo findings. Specifically, adenovirus-mediated overexpression of HIPK2 suppressed the expression of heart failure markers, NPPA and NPPB , at basal condition and abolished phenylephrine-induced pathological gene expression. An array of mechanistic studies revealed impaired extracellular signal-regulated kinase 1/2 signaling in HIPK2-deficient hearts. An in vivo rescue experiment with adeno-associated virus serotype 9 TnT-MEK1-CA nearly abolished the detrimental phenotype of knockout mice, suggesting that impaired extracellular signal-regulated kinase signaling mediated apoptosis as the key factor driving the detrimental phenotype in CM-HIPK2 knockout mice hearts. Conclusions: Taken together, these findings suggest that CM-HIPK2 is required to maintain normal cardiac function via extracellular signal-regulated kinase signaling.

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, donor’s 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.

Background and rationale Obesity, an independent risk factor for the development of myocardial diseases is a growing healthcare problem worldwide. It's well established that GSK-3β is critical to cardiac pathophysiology. However, the role cardiomyocyte (CM) GSK-3β in diet-induced cardiac dysfunction is unknown. Methods CM-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. Results At 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. Conclusion In summary, these results suggest that cardiac GSK-3β is crucial to protect against obesity-induced adverse ventricular remodeling and cardiac dysfunction.

This chapter explores the signal transduction mechanisms that may be important for cellular volume regulation. When a ligand binds to its receptor on the cell surface, the resulting conformational change in the receptor initiates a series of intracellular events that culminate in a characteristic response by the cell. A large number of agonists bind to a second class of receptors that are linked to guanine nucleotide-binding regulatory proteins. G protein-coupled receptors have a variably sized ligand-binding domain, a common seven-transmembrane-spanning configuration and a variable-length cytoplasmic tail. The best-studied families of the large group of small-molecular-weight G proteins are the ras, rho, and rab families. The rho family, which also includes Rac, has been shown recently to play a critical role in serum and growth factor-induced actin cytoskeletal reorganization. A number of exchange proteins and GTPase-activating proteins have been identified, but whether and how their activity is modulated by activated growth factor receptors remains unclear.

BackgroundOver one million Americans experience myocardial infarction (MI) every year, and the resulting scar and subsequent cardiac fibrosis contribute to heart failure and death. A specialized cell-cell adhesion protein, cadherin-11 (CDH11), contributes to inflammation and fibrosis in rheumatoid arthritis, pulmonary fibrosis, and aortic valve calcification but has not yet been studied in the context of cardiac remodeling after MI. We hypothesized that targeting CDH11 function after MI would reduce inflammation-driven fibrotic remodeling and infarct expansion to improve functional outcomes in mice.MethodsMI was induced by ligation of the left anterior descending artery in transgenic mice with reduced or ablated CDH11, wild type mice receiving bone marrow transplants from Cdh11 transgenic animals, and wild type mice treated with a functional blocking antibody against CDH11 (SYN0012). Cardiac function was measured by echocardiography, expression of cell populations was quantified by flow cytometry, and tissue remodeling by altered histological assessment and transcription of inflammatory and pro-angiogenic genes by qPCR. Co-culture was used to assess interactions between cardiac fibroblasts and macrophages.ResultsMI increased transcription of Cdh11 in non-cardiomyocyte cells. Mice with deletion of Cdh11 and wild type mice receiving bone marrow transplants from Cdh11 transgenic animals had improved cardiac function and dimensions after MI. Animals given SYN0012 had improved cardiac function, reduced tissue remodeling, and altered transcription of inflammatory and proangiogenic genes. Targeting CDH11 also reduced the number of bone marrow-derived myeloid cells and increased pro-angiogenic cells in the heart three days after MI, consistent with a decrease in transcription and expression of IL-6 in the infarct region. Cardiac fibroblast and macrophage interactions led to an increase in IL-6 secretion that was reduced with SYN0012 treatment in vitro.ConclusionsOur findings suggest that CDH11-expressing cells contribute to inflammation-driven fibrotic remodeling after MI, and that targeting CDH11 with a blocking antibody improves cardiac function after MI. This improvement is likely mediated by altered recruitment of bone marrow-derived cells, thereby limiting the macrophage-induced expression of IL-6 by fibroblasts and promoting vascularization.

Rationale: Cardiac 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. Objective: To identify the consequences of combined deletion of cardiac myocyte GSK-3α and GSK-3β in heart function. Methods and Results: We 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. Conclusions: Together, 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.

Aims Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myelogenous leukaemia (CML). However, cardiotoxicity of these agents remains a serious concern. The underlying mechanism of these adverse cardiac effects is largely unknown. Delineation of the underlying mechanisms of TKIs associated cardiac dysfunction could guide potential prevention strategies, rescue approaches, and future drug design. This study aimed to determine the cardiotoxic potential of approved CML TKIs, define the associated signalling mechanism and identify potential alternatives. Methods and results In this study, we employed a zebrafish transgenic BNP reporter line that expresses luciferase under control of the nppb promoter (nppb:F-Luciferase) to assess the cardiotoxicity of all approved CML TKIs. Our in vivo screen identified ponatinib as the most cardiotoxic agent among the approved CML TKIs. Then using a combination of zebrafish and isolated neonatal rat cardiomyocytes, we delineated the signalling mechanism of ponatinib-induced cardiotoxicity by demonstrating that ponatinib inhibits cardiac prosurvival signalling pathways AKT and extra-cellular-signal-regulated kinase (ERK), and induces cardiomyocyte apoptosis. As a proof of concept, we augmented AKT and ERK signalling by administration of Neuregulin-1β (NRG-1β), and this prevented ponatinib-induced cardiomyocyte apoptosis. We also demonstrate that ponatinib-induced cardiotoxicity is not mediated by inhibition of fibroblast growth factor signalling, a well-known target of ponatinib. Finally, our comparative profiling for the cardiotoxic potential of CML approved TKIs, identified asciminib (ABL001) as a potentially much less cardiotoxic treatment option for CML patients with the T315I mutation. Conclusion Herein, we used a combination of in vivo and in vitro methods to systematically screen CML TKIs for cardiotoxicity, identify novel molecular mechanisms for TKI cardiotoxicity, and identify less cardiotoxic alternatives.

Nicotinamide riboside kinase-2 (NRK-2), a muscle-specific β1 integrin binding protein, predominantly expresses in skeletal muscle with a trace amount expressed in healthy cardiac tissue. NRK-2 expression dramatically increases in mouse and human ischemic heart however, the specific role of NRK-2 in the pathophysiology of ischemic cardiac diseases is unknown. We employed NRK2 knockout (KO) mice to identify the role of NRK-2 in ischemia-induced cardiac remodeling and dysfunction. Following myocardial infarction (MI), or sham surgeries, serial echocardiography was performed in the KO and littermate control mice. Cardiac contractile function rapidly declined and left ventricular interior dimension (LVID) was significantly increased in the ischemic KO vs. control mice at 2 weeks post-MI. An increase in mortality was observed in the KO vs. control group. The KO hearts displayed increased cardiac hypertrophy and heart failure reflected by morphometric analysis. Consistently, histological assessment revealed an extensive and thin scar and dilated LV chamber accompanied with elevated fibrosis in the KOs post-MI. Mechanistically, we observed that loss of NRK-2 enhanced p38α activation following ischemic injury. Consistently, ex vivo studies demonstrated that the gain of NRK-2 function suppresses the p38α as well as fibroblast activation (α-SMA expression) upon TGF-β stimulation, and limits cardiomyocytes death upon hypoxia/re‑oxygenation. Collectively our findings show, for the first time, that NRK-2 plays a critical role in heart failure progression following ischemic injury. NRK-2 deficiency promotes post-MI scar expansion, rapid LV chamber dilatation, cardiac dysfunction and fibrosis possibly due to increased p38α activation.

Visual Abstract, Highlights • SMAD4 is the central intracellular mediator of TGF-β pathway. • CM-specific loss of SMAD4 causes cardiac dysfunction independent of fibrotic remodeling. • Deletion CM-SMAD4 affects CM survival. • CM-SMAD4 loss leads to down-regulation of several ion channels’ genes, resulting in cardiac conduction abnormalities. • CM-SMAD4 deletion alters sarcomere shortening kinetics, in parallel with reduction in cardiac myosin-binding protein C levels. • These results demonstrate a fundamental role for CM-SMAD4–dependent TGF-β signaling in adult heart homeostasis., Summary The role of the transforming growth factor (TGF)-β pathway in myocardial fibrosis is well recognized. However, the precise role of this signaling axis in cardiomyocyte (CM) biology is not defined. In TGF-β signaling, SMAD4 acts as the central intracellular mediator. To investigate the role of TGF-β signaling in CM biology, the authors deleted SMAD4 in adult mouse CMs. We demonstrate that CM-SMAD4–dependent TGF-β signaling is critical for maintaining cardiac function, sarcomere kinetics, ion-channel gene expression, and cardiomyocyte survival. Thus, our findings raise a significant concern regarding the therapeutic approaches that rely on systemic inhibition of the TGF-β pathway for the management of myocardial fibrosis.

Background: Ponatinib, a potent pan-BCR-ABL tyrosine kinase inhibitor (TKI) holds significant promise in the treatment of chronic myelogenous leukemia (CML), although the potential cardiotoxicity of this agent remains a concern. In order to overcome ponatinib associated cardiotoxicity, delineation of linked cardiotoxic mechanisms and potential rescue methods are urgently warranted. Objectives: The objectives of the present study were to delineate the signaling mechanisms responsible for ponatinib-induced cardiotoxicity and to identify the potential rescue strategies. Methods: In this study, we employed direct in vivo drug screening in zebrafish for the prediction of cardiotoxicity. We also exploited neonatal rat ventricular myocytes (NRVMs) to explore cardiotoxic mechanism associated with ponatinib. Results: We observed that ponatinib leads to cardiomyocyte apoptosis, elevation in BNP level, decrease in heart rate and fractional shortening leading to cardiac dysfunction in Zebrafish. Consistently, in cultured rat cardiomyocytes, ponatinib was found most cytotoxic drug among all the approved CML TKIs. Mechanistically, ponatinib inhibits the essential prosurvival AKT and ERK signaling pathway, resulting to cardiomyocyte apoptosis. Interestingly, we evaluated the cardioprotective effects of Neuregulin-1β to limit ponatinib toxicity. Neuregulin-1β treatment significantly protects ponatinib-induced cardiotoxicity by supplementing the essential cardiomyocyte prosurvival AKT/ERK signaling pathways. Additionally, the allosteric tyrosine kinase inhibitor asciminib depicts less pronounced cardiotoxic effects than ponatinib in similar settings. Conclusions: This study will advance our mechanistic understanding of ponatinib-induced cardiotoxicity, accelerate the discovery of cardioprotective strategies to prevent and rescue the ponatinib-induced cardiotoxicity. Furthermore, this study suggests asciminib as an alternate treatment option than ponatinib for patients suffering from CML with T315I “gatekeeper” mutation.

Type 1 diabetic Akita mice develop severe cardiac parasympathetic dysfunction that we have previously demonstrated is due at least in part to an abnormality in the response of the end organ to parasympathetic stimulation. Specifically, we had shown that hypoinsulinemia in the diabetic heart results in attenuation of the G-protein coupled inward rectifying K channel (GIRK) which mediates the negative chronotropic response to parasympathetic stimulation due at least in part to decreased expression of the GIRK1 and GIRK4 subunits of the channel. We further demonstrated that the expression of GIRK1 and GIRK4 is under the control of the Sterol Regulatory element Binding Protein (SREBP-1), which is also decreased in response to hypoinsulinemia. Finally, given that hyperactivity of Glycogen Synthase Kinase (GSK)3β, had been demonstrated in the diabetic heart, we demonstrated that treatment of Akita mice with Li+, an inhibitor of GSK3β, increased parasympathetic responsiveness and SREBP-1 levels consistent with the conclusion that GSK3β might regulate IKACh via an effect on SREBP-1. However, inhibitor studies were complicated by lack of specificity for GSK3β. Here we generated an Akita mouse with cardiac specific inducible knockout of GSK3β. Using this mouse, we demonstrate that attenuation of GSK3β expression is associated with an increase in parasympathetic responsiveness measured as an increase in the heart rate response to atropine from 17.3 ± 3.5% (n = 8) prior to 41.2 ± 5.4% (n = 8, P = 0.017), an increase in the duration of carbamylcholine mediated bradycardia from 8.43 ± 1.60 min (n = 7) to 12.71 ± 2.26 min (n = 7, P = 0.028) and an increase in HRV as measured by an increase in the high frequency fraction from 40.78 ± 3.86% to 65.04 ± 5.64 (n = 10, P = 0.005). Furthermore, patch clamp measurements demonstrated a 3-fold increase in acetylcholine stimulated peak IKACh in atrial myocytes from GSK3β deficiency mice compared with control. Finally, western blot analysis of atrial extracts from knockout mice demonstrated increased levels of SREBP-1, GIRK1 and GIRK4 compared with control. Taken together with our prior observations, these data establish a role of increased GSK3β activity in the pathogenesis of parasympathetic dysfunction in type 1 diabetes via the regulation of IKACh and GIRK1/4 expression.

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.

Left ventricular dysfunction is a frequent and potentially severe side effect of many tyrosine kinase inhibitors (TKI). The mode of toxicity is not identified, but may include impairment of mitochondrial or sarcomeric function, autophagy or angiogenesis, either as an on-target or off-target mechanism.We studied concentration-response curves and time courses for nine TKIs in three-dimensional, force generating engineered heart tissue (EHT) from neonatal rat heart cells. We detected a concentration- and time-dependent decline in contractile force for gefitinib, lapatinib, sunitinib, imatinib, sorafenib, vandetanib and lestaurtinib and no decline in contractile force for erlotinib and dasatinib after 96 hours of incubation. The decline in contractile force was associated with an impairment of autophagy (LC3 Western blot) and appearance of autophagolysosomes (transmission electron microscopy).This study demonstrates the feasibility to study TKI-mediated force effects in EHTs and identifies an association between a decline in contractility and inhibition of autophagic flux.

The incidence of drug-induced structural cardiotoxicity, which may lead to heart failure, has been recognized in association with the use of anthracycline anti-cancer drugs for many years, but has also been shown to occur following treatment with the new generation of targeted anti-cancer agents that inhibit one or more receptor or non-receptor tyrosine kinases, serine/threonine kinases as well as several classes of non-oncology agents. A workshop organized by the Medical Research Council Centre for Drug Safety Science (University of Liverpool) on 5 September 2013 and attended by industry, academia and regulatory representatives, was designed to gain a better understanding of the gaps in the field of structural cardiotoxicity that can be addressed through collaborative efforts. Specific recommendations from the workshop for future collaborative activities included: greater efforts to identify predictive (i) preclinical; and (ii) clinical biomarkers of early cardiovascular injury; (iii) improved understanding of comparative physiology/pathophysiology and the clinical predictivity of current preclinical in vivo models; (iv) the identification and use of a set of cardiotoxic reference compounds for comparative profiling in improved animal and human cellular models; (v) more sharing of data (through publication/consortia arrangements) on target-related toxicities; (vi) strategies to develop cardio-protective agents; and (vii) closer interactions between preclinical scientists and clinicians to help ensure best translational efforts.

Glycogen synthase kinase-3 (GSK-3) is one of the few signaling molecules that regulate a truly astonishing number of critical intracellular signaling pathways. It has been implicated in several diseases including heart failure, bipolar disorder, diabetes mellitus, Alzheimer disease, aging, inflammation, and cancer. Furthermore, a recent clinical trial has validated the feasibility of targeting GSK-3 with small molecule inhibitors for human diseases. In the current review, we will focus on its expanding role in the heart, concentrating primarily on recent studies that have used cardiomyocyte- and fibroblast-specific conditional gene deletion in mouse models. We will highlight the role of the GSK-3 isoforms in various pathological conditions including myocardial aging, ischemic injury, myocardial fibrosis, and cardiomyocyte proliferation. We will discuss our recent findings that deletion of GSK-3α specifically in cardiomyocytes attenuates ventricular remodeling and cardiac dysfunction after myocardial infarction by limiting scar expansion and promoting cardiomyocyte proliferation. The recent emergence of GSK-3β as a regulator of myocardial fibrosis will also be discussed. We will review our recent findings that specific deletion of GSK-3β in cardiac fibroblasts leads to fibrogenesis, left ventricular dysfunction, and excessive scarring in the ischemic heart. Finally, we will examine the underlying mechanisms that drive the aberrant myocardial fibrosis in the models in which GSK-3β is specifically deleted in cardiac fibroblasts. We will summarize these recent results and offer explanations, whenever possible, and hypotheses when not. For these studies we will rely heavily on our models and those of others to reconcile some of the apparent inconsistencies in the literature.

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.

Introduction: Previous studies from our group have demonstrated that cardiac myocyte glycogen synthase kinase-3’s (GSK-3) are required to maintain normal cardiac physiology. Adult mice lacking both isoforms of GSK-3 (α and β) in cardiac myocytes exhibit excessive dilatative remodeling and ventricular dysfunction ultimately leading to death. While high fat diet (HFD) induced obesity is associated with increased risk of cardiovascular disease, the specific role of cardiac GSK-3α or GSK-3β in obesity-associated cardiac dysfunction is unknown. Objective: The primary goal of the present study was to investigate the role of cardiomyocyte GSK-3β in cardiac homeostasis in HFD-induced chronic obesity model. Method: Cardiomyocyte specific-GSK-3β knock out (CM-GSK-3βKO) and wild type (WT) mice were fed either a chow (11.5% calories from fat) or high-fat (60% calories from fat) for 24 weeks. Cardiac function was accessed by non-invasive transthoracic echocardiography. Results: HFD significantly increased body weight, lean and fat mass in the WT and CM-GSK-3βKO compared to chow. However, there was no difference in body weights, lean and fat mass between the two genotypes fed either a chow or HFD. Furthermore, ventricular chamber dimensions and cardiac function were comparable between the WT and CM-GSK-3βKO mice fed a chow diet. In contrast, high fat fed CM-GSK-3βKO hearts exhibit significant cardiac hypertrophy (heart weight/tibia length ratio) and ventricular dysfunction (reduced ejection fraction (EF) and fractional shortening (FS)) compared to the WT. Interestingly cardiomyocytes from HF fed CM-GSK-3βKO exhibit structural abnormalities and increased expression of pro-apoptotic protein Bax and reduced expression of Bcl-2, an anti-apoptotic protein. Conclusion: In summary, these data suggests that cardiac GSK-3β is important in the setting of HFD-induced chronic obesity to maintain cardiac function. In the absence of GSK-3β, cardiomyocytes undergo morphometric abnormalities, excessive fat infiltration and apoptosis leading to cardiac dysfunction.

The role of canonical transforming growth factor-β (TGF-β) pathway is well recognized in fibroblast biology and fibrosis in diseased hearts. However, its role in cardiomyocyte (CM) biology is not clear. SMAD4 is the central intracellular mediator of canonical TGF-β signaling. Herein, we investigate the role of SMAD4 in CM-biology and cardiac pathophysiology. SMAD4 homozygous floxed ( SMAD4 fl/fl ) and heterozygous floxed ( SMAD4 fl/- ) mice were crossed with α-myosin heavy chain (Mer-Cre-Mer) to create conditional CM-specific SMAD4-KO ( SMAD4 fl/fl Cre +/- ) and SMAD4 haploinsufficiency ( SMAD4 fl/- Cre +/- ), respectively. At 10 Wks of age, mice were subjected to well established tamoxifen diet protocol for 2 Wks. Echocardiographic analysis at 4 Wks post-tamoxifen treatment reveals that CM-specific loss of SMAD4 leads to dilatative ventricular remodeling as reflected by significantly increased LVIDs. This dilatative remodeling was associated with severe ventricular dysfunction as reflected by significantly reduced ejection fraction (EF) and fractional shortening (FS). Both HW/BW and LW/BW were significantly elevated in SMAD4 KO mice, suggesting pathological hypertrophy and heart failure in KOs. Analysis of Masson trichrome stained heart sections reveals a marked increase in fibrosis in the KO hearts. Q-PCR analysis showed the re-expression of fetal gene program (ANP, BNP), further confirming pathological remodeling in the KO hearts. As the heart functions of heterozygous mice and littermate controls were comparable at baseline, we stress them with TAC surgery. Consistent with KO findings, at 4 Wks post-TAC, heterozygous mice demonstrated characteristic heart failure phenotype. Taken together, these findings suggest that SMAD4 is required to maintain basal cardiac function and to prevent adverse ventricular remodeling in a pressure-overloaded heart.

Background The amino acid response ( AAR ) is an evolutionarily conserved protective mechanism activated by amino acid deficiency through a key kinase, general control nonderepressible 2. In addition to mobilizing amino acids, the AAR broadly affects gene and protein expression in a variety of pathways and elicits antifibrotic, autophagic, and anti‐inflammatory activities. However, little is known regarding its role in cardiac stress. Our aim was to investigate the effects of halofuginone, a prolyl‐ tRNA synthetase inhibitor, on the AAR pathway in cardiac fibroblasts, cardiomyocytes, and in mouse models of cardiac stress and failure. Methods and Results Consistent with its ability to inhibit prolyl‐ tRNA synthetase, halofuginone elicited a general control nonderepressible 2–dependent activation of the AAR pathway in cardiac fibroblasts as evidenced by activation of known AAR target genes, broad regulation of the transcriptome and proteome, and reversal by l ‐proline supplementation. Halofuginone was examined in 3 mouse models of cardiac stress: angiotensin II /phenylephrine, transverse aortic constriction, and acute ischemia reperfusion injury. It activated the AAR pathway in the heart, improved survival, pulmonary congestion, left ventricle remodeling/fibrosis, and left ventricular function, and rescued ischemic myocardium. In human cardiac fibroblasts, halofuginone profoundly reduced collagen deposition in a general control nonderepressible 2–dependent manner and suppressed the extracellular matrix proteome. In human induced pluripotent stem cell–derived cardiomyocytes, halofuginone blocked gene expression associated with endothelin‐1‐mediated activation of pathologic hypertrophy and restored autophagy in a general control nonderepressible 2/ eIF 2α‐dependent manner. Conclusions Halofuginone activated the AAR pathway in the heart and attenuated the structural and functional effects of cardiac stress.

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.

Heart failure is a life-threatening disease and addressing it should be considered a global health priority. At present, approximately 26 million people worldwide are living with heart failure. The outlook for such patients is poor, with survival rates worse than those for bowel, breast or prostate cancer. Furthermore, heart failure places great stresses on patients, caregivers and healthcare systems. Demands on healthcare services, in particular, are predicted to increase dramatically over the next decade as patient numbers rise owing to ageing populations, detrimental lifestyle changes and improved survival of those who go on to develop heart failure as the final stage of another disease. It is time to ease the strain on healthcare systems through clear policy initiatives that prioritize heart failure prevention and champion equity of care for all. Despite the burdens that heart failure imposes on society, awareness of the disease is poor. As a result, many premature deaths occur. This is in spite of the fact that most types of heart failure are preventable and that a healthy lifestyle can reduce risk. Even after heart failure has developed, premature deaths could be prevented if people were taught to recognize the symptoms and seek immediate medical attention. Public awareness campaigns focusing on these messages have great potential to improve outcomes for patients with heart failure and ultimately to save lives. Compliance with clinical practice guidelines is also associated with improved outcomes for patients with heart failure. However, in many countries, there is considerable variation in how closely physicians follow guideline recommendations. To promote equity of care, improvements should be encouraged through the use of hospital performance measures and incentives appropriate to the locality. To this end, policies should promote the research required to establish an evidence base for performance measures that reflect improved outcomes for patients. Continuing research is essential if we are to address unmet needs in caring for patients with heart failure. New therapies are required for patients with types of heart failure for which current treatments relieve symptoms but do not address the disease. More affordable therapies are desperately needed in the economically developing world. International collaborative research focusing on the causes and treatment of heart failure worldwide has the potential to benefit tens of millions of people. Change at the policy level has the power to drive improvements in prevention and care that will save lives. It is time to make a difference across the globe by confronting the problem of heart failure. A call to action: policy recommendations We urge policymakers at local, national and international levels to collaborate and act on the following recommendations. Promote heart failure prevention Support the development and implementation of public awareness programmes about heart failure. These should define heart failure in simple and accessible language, explain how to recognize the symptoms and emphasize that most types of heart failure are preventable. Highlight the need for healthcare professionals across all clinical disciplines to identify patients with illnesses that increase the risk of heart failure and to prescribe preventive medications. Prioritize the elimination of infectious diseases in parts of the world where they still cause heart failure. Improve heart failure awareness amongst healthcare professionals Encourage the development and use of heart failure education programmes for all appropriate healthcare professionals. These should aim to improve the prevention, diagnosis, treatment and long-term management of heart failure and raise awareness of clinical practice guidelines. Ensure equity of care for all patients with heart failure Provide a healthcare system that delivers timely access to diagnostic services and treatment of heart failure, as well as a seamless transition to long-term management. Ensure that the best available and most appropriate care is consistently provided to all patients with heart failure through efficient use of resources. Support and empower patients and their caregivers Provide resources for the education and practical support of patients with heart failure and their families or other caregivers, empowering them to engage proactively in long-term care. Promote heart failure research Fund and encourage international collaborative research to improve understanding of the patterns, causes and effects of modern day heart failure and how the disease can be prevented across the globe. Fund and encourage research into new and more affordable therapies and medical devices for all types of heart failure. Fund and encourage research into evidence-based healthcare performance measures that reflect improved clinical outcomes for patients with heart failure.

BackgroundInjury due to myocardial infarction (MI) is largely irreversible. Once an infarct has occurred, the clinical goal becomes limiting remodeling, preserving left ventricular function, and preventing heart failure. Although traditional approaches (e.g., β-blockers) partially preserve left ventricular function, novel strategies are needed to limit ventricular remodeling post-MI.ObjectivesThe aim of this study was to determine the role of glycogen synthase kinase–3α (GSK-3α) in post-MI remodeling.MethodsMice with cardiomyocyte-specific conditional deletion of Gsk3α and littermate controls underwent sham or MI surgery. Heart function was assessed using serial M-mode echocardiography.ResultsGsk3α deletion in the heart markedly limits remodeling and preserves left ventricular function post-MI. This is due at least in part to dramatic thinning and expansion of the scar in the control hearts, which was less in the heart of knockout (KO) mice. In contrast, the border zone in the KO mice demonstrated a much thicker scar, and there were more viable cardiomyocytes within the scar/border zone. This was associated with less apoptosis and more proliferation of cardiomyocytes in the KO mice. Mechanistically, reduced apoptosis was due, at least in part, to a marked decrease in the Bax/Bcl-2 ratio, and increased cardiomyocyte proliferation was mediated through cyclin E1 and E2F-1 in the hearts of the KO mice.ConclusionsTaken together, these findings show that reducing GSK-3α expression in cardiomyocytes limits ventricular remodeling and preserves cardiac function post-MI. Specifically targeting GSK-3α could be a novel strategy to limit adverse remodeling and heart failure.

Background— Myocardial infarction–induced remodeling includes chamber dilatation, contractile dysfunction, and fibrosis. Of these, fibrosis is the least understood. After myocardial infarction, activated cardiac fibroblasts deposit extracellular matrix. Current therapies to prevent fibrosis are inadequate, and new molecular targets are needed. Methods and Results— Herein we report that glycogen synthase kinase-3β (GSK-3β) is phosphorylated (inhibited) in fibrotic tissues from ischemic human and mouse heart. Using 2 fibroblast-specific GSK-3β knockout mouse models, we show that deletion of GSK-3β in cardiac fibroblasts leads to fibrogenesis, left ventricular dysfunction, and excessive scarring in the ischemic heart. Deletion of GSK-3β induces a profibrotic myofibroblast phenotype in isolated cardiac fibroblasts, in post–myocardial infarction hearts, and in mouse embryonic fibroblasts deleted for GSK-3β. Mechanistically, GSK-3β inhibits profibrotic transforming growth factor-β1/SMAD-3 signaling via interactions with SMAD-3. Moreover, deletion of GSK-3β resulted in the significant increase of SMAD-3 transcriptional activity. This pathway is central to the pathology because a small-molecule inhibitor of SMAD-3 largely prevented fibrosis and limited left ventricular remodeling. Conclusions— These studies support targeting GSK-3β in myocardial fibrotic disorders and establish critical roles of cardiac fibroblasts in remodeling and ventricular dysfunction.

Background Imatinib mesylate is a selective tyrosine-kinase inhibitor used in the treatment of multiple cancers, most notably chronic myelogenous leukemia (CML). There is evidence that imatinib can induce cardiotoxicity in cancer patients. Our hypothesis is that imatinib alters calcium regulatory mechanisms and can contribute to development of pathological cardiac hypertrophy.

The cardiovascular care of cancer patients (cardio-oncology) has emerged as a new discipline in clinical medicine, given recent advances in cancer therapy, and is driven by the cardiovascular complications that occur as a direct result of cancer therapy. Traditional therapies such as anthracyclines and radiation have been recognized for years to have cardiovascular complications. Less expected were the cardiovascular effects of targeted cancer therapies, which were initially thought to be specific to cancer cells and would spare any adverse effects on the heart. Cancers are typically driven by mutations, translocations, or overexpression of protein kinases. The majority of these mutated kinases are tyrosine kinases, though serine/threonine kinases also play key roles in some malignancies. Several agents were developed to target these kinases, but many more are in development. Major successes have been largely restricted to agents targeting human epidermal growth factor receptor-2 (mutated or overexpressed in breast cancer), BCR-ABL (chronic myelogenous leukemia and some cases of acute lymphoblastic leukemia), and c-Kit (gastrointestinal stromal tumor). Other agents targeting more complex malignancies, such as advanced solid tumors, have had successes, but have not extended life to the degree seen with chronic myelogenous leukemia. Years before the first targeted therapy, Judah Folkman correctly proposed that to address solid tumors one had to target the inherent neoangiogenesis. Unfortunately, emerging evidence confirms that angiogenesis inhibitors cause cardiac complications, including hypertension, thrombosis, and heart failure. And therein lies the catch-22. Nevertheless, cardio-oncology has the potential to be transformative as the human cardiomyopathies that arise from targeted therapies can provide insights into the normal function of the heart.

Aging is regulated by conserved signaling pathways. The glycogen synthase kinase-3 (GSK-3) family of serine/threonine kinases regulates several of these pathways, but the role of GSK-3 in aging is unknown. Herein, we demonstrate premature death and acceleration of age-related pathologies in the Gsk3a global KO mouse. KO mice developed cardiac hypertrophy and contractile dysfunction as well as sarcomere disruption and striking sarcopenia in cardiac and skeletal muscle, a classical finding in aging. We also observed severe vacuolar degeneration of myofibers and large tubular aggregates in skeletal muscle, consistent with impaired clearance of insoluble cellular debris. Other organ systems, including gut, liver, and the skeletal system, also demonstrated age-related pathologies. Mechanistically, we found marked activation of mTORC1 and associated suppression of autophagy markers in KO mice. Loss of GSK-3α, either by pharmacologic inhibition or Gsk3a gene deletion, suppressed autophagy in fibroblasts. mTOR inhibition rescued this effect and reversed the established pathologies in the striated muscle of the KO mouse. Thus, GSK-3α is a critical regulator of mTORC1, autophagy, and aging. In its absence, aging/senescence is accelerated in multiple tissues. Strategies to maintain GSK-3α activity and/or inhibit mTOR in the elderly could retard the appearance of age-related pathologies.

Obesity has reached epidemic proportions worldwide and is associated with increased risk of cardiovascular and metabolic disease, resulting in enhanced morbidity and mortality. Indeed, the primary cause of mortality in patients with diabetes is cardiovascular disease, accounting for 50-80% of deaths. Numerous studies have implicated GSK-3β in the pathogenesis of insulin resistance, metabolic syndrome and diabetes. However, these studies are primarily relies on non-isoform specific inhibitors. We and others have reported not only isoform-specific functions of GSK-3 but also their distinct roles in various tissues. At present, all available inhibitors of GSK-3 are non-isoform-specific; therefore it’s impossible to assess the isoform-specific functions by employing these agents. The primary goal of present study was to determine the role of cardiac GSK-3β in obesity-induced metabolic perturbations and cardiac dysfunction. An oral glucose tolerance test (GTT) was performed on cardiomyocyte-specific GSK-3β KO (GSK-3β KO) and controls. At baseline, GSK-3β KO and controls displays a comparable lean mass, fat mass and oral glucose tolerance. To determine the functional role of cardiac GSK-3β in obesity-induced glucose intolerance, GSK-3β KO and controls were subjected to HFD for 24 wks and an oral GTT assay was performed on fasting mice (6h). The hyperglycemic response was significantly increased in the obese GSK-3β KO mice in comparison to obese WT mice. Importantly, body weight and food consumption were comparable between groups confirming that observed glucose intolerance in GSK-3β KO were not confounded by variable body mass composition. Furthermore, HFD leads to accelerated cardiac dysfunction in GSK-3β KO hearts compared to controls as reflected by significantly reduced ejection fraction (EF) and functional shortening. In summary, these data suggest that cardiac-specific deletion of GSK-3β lead to systemic glucose intolerance and cardiac dysfunction. These findings suggest that cardiac GSK-3β is required for the maintenance of global metabolic homeostasis and cardiac function in diabetic hearts and strategies to maintain GSK-3β activity may lead to therapeutic benefits for the disease.

Chronic pressure-overload (PO) induced-dilated cardiomyopathy (DCM) is one of the leading causes of left ventricular (LV) remodeling and heart failure. The role of glycogen synthase kinase-3α (GSK-3α) in PO-induced remodeling is not clear and existing dataset with global transgenic and knockout (KO) models show opposing roles. We sought to identify the specific role of GSK-3α in PO-induced dilatative cardiac remodeling. To better understand the role of GSK-3α, we employed cardiomyocyte-specific GSK3A ( GSK3A fl/fl MerCreMer ) KO mice. Post-tamoxifen treatment, the GSK-3α KO and littermate control mice underwent sham or trans-aortic constriction (TAC) surgery. Heart function was assessed at 0, 2, 4 and 6 week post-TAC using serial M-mode echocardiography. Cardiac function in the KOs and littermate controls declines equally up to 2 weeks of TAC. At 4 week, KO hearts underwent further hypertrophy, retaining concentric LV remodeling and preserved contractile function both at systole and diastole. In contrast, wild-type LV showed significant chamber dilatation with an impaired contractility. Significantly reduced LV chamber dilatation [LVIDd(mm); 5.4±0.4 vs. 4.9±0.4, P =0.01] and preserved contractile function [LVEF(%); 22.2±12.6 vs. 40.0±18.7, P =0.02] remains same in the KO mice until the end of the study (6 wk). Furthermore, LV posterior wall thickness in the KO hearts, both at systole and diastole, were significantly greater in comparison to the controls. Consistent with preserved LV dimension, significantly less mortality was observed in the KO vs. control group during the remodeling phase. Histological analysis of heart sections further revealed better preserved LV chamber and protection against TAC-induced cellular hypertrophy in the GSK-3α KOs. Moreover, KO hearts showed significantly less fibrosis accompanied with low level of cardiomyocyte apoptosis post-6 wk of TAC. Taken together, these observations show that cardiomyocyte-specific deletion of GSK-3α protects against chronic PO-induced adverse LV remodeling and preserves contractile function. Inhibiting specifically GSK-3α using isoform-specific inhibitor could be a viable therapeutic strategy to limit the PO-induced DCM, adverse remodeling and heart failure.

Heart failure is the leading cause of mortality, morbidity and healthcare expenditures worldwide. Numerous studies from our lab and others have implicated GSK-3 as a promising therapeutic target for cardiovascular diseases. Recently, we reported that cardiomyocyte-specific deletion of GSK3α limits adverse remodeling and preserves cardiac function, post-MI. However, the role of cardiac fibroblast GSK3α (CF-GSK-3α) in myocardial biology is unknown. To determine the role of CF-GSK-3α in MI-induced fibrotic remodeling, we created CF-GSK-3α KO mice in which Cre recombinase is driven by Postn (periostin) promoter. WT and CF-GSK-3α-KO mice were subjected to MI or sham surgery at 2 months age and cardiac function were monitored by serial echocardiography. Interestingly, at four weeks post-MI, CF-GSK-3α-KO mice showed preserved chamber dimensions and LV functions as reflected by preserved ejection fraction and fractional shortening as compared to littermate controls. The preserved LV dimensions and cardiac function were remained significantly better up to the end of the study i.e., 8 weeks post-MI. At 8 weeks post MI, hearts were excised, and Masson trichrome staining was performed. LV scar circumference was measured and expressed as a percentage of total area of LV myocardium. Scar tissue percent circumference was significantly reduced in CF-GSK-3α-KO hearts. Furthermore, GSK3α KO heart scars were thicker with much higher numbers of viable cardiomyocytes as compared to WT. To gain the mechanistic insights of observed GSK-3α mediated fibrotic remodeling, we examined profibrotic TGFβ signaling and myofibroblast transformation. WT and GSK-3α KO mouse embryonic fibroblasts (MEFs) were treated with TGF-β1 (10 ng/mL) for 1 hour, and phosphorylation of SMAD-3 (Ser423/25) was determined. Indeed, TGF-β1 induced activation of SMAD3 was significantly reduced in GSK3α KO cells. Consistently, deletion of GSK-3α leads to reduced myofibroblast transformation as reflected by significantly reduced expression of α-SMA in GSK-3α KO cells. These findings suggest that CF-specific deletion of GSK3α is protective in ischemic heart and GSK3α could be a novel therapeutic target for management of adverse fibrotic remodeling in the diseased heart.

Cardiac 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.To identify the consequences of combined deletion of cardiac myocyte GSK-3α and GSK-3β in heart function.We 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.Together, 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.

Background— The molecular pathways that regulate the extent of ischemic injury and post–myocardial infarction (MI) remodeling are not well understood. We recently demonstrated that glycogen synthase kinase-3α (GSK-3α) is critical to the heart's response to pressure overload. However, the role, if any, of GSK-3α in regulating ischemic injury and its consequences is not known. Methods and Results— MI was induced in wild-type (WT) versus GSK-3α (−/−) (KO) littermates by left anterior descending coronary artery ligation. Pre-MI, WT, and KO hearts had comparable chamber dimensions and ventricular function, but as early as 1 week post-MI, KO mice had significantly more left ventricular dilatation and dysfunction than WT mice. KO mice also had increased mortality during the first 10 days post-MI (43% versus 22%; P =0.04), and postmortem examination confirmed cardiac rupture as the cause of most of the deaths. In the mice that survived the first 10 days, left ventricular dilatation and dysfunction remained worse in the KO mice throughout the study (8 weeks). Hypertrophy, fibrosis, and heart failure were all increased in the KO mice. Given the early deaths due to rupture and the significant reduction in left ventricular function evident as early as 1 week post-MI, we examined infarct size following a 48-hour coronary artery ligation and found it to be increased in the KO mice. This was accompanied by increased apoptosis in the border zone of the MI. This increased susceptibility to ischemic injury–induced apoptosis was also seen in cardiomyocytes isolated from the KO mice that were exposed to hypoxia. Finally, Bax translocation to the mitochondria and cytochrome C release into the cytosol were increased in the KO mice. Conclusion— GSK-3α confers resistance to ischemic injury, at least in part, via limiting apoptosis. Loss of GSK-3α promotes ischemic injury, increases risk of cardiac rupture, accentuates post-MI remodeling and left ventricular dysfunction, and increases the progression to heart failure. These findings are in striking contrast to multiple previous reports in which deletion or inhibition of GSK-3β is protective.

Rationale: Despite intense interest in strategies to predict which kinase inhibitor (KI) cancer therapeutics may be associated with cardiotoxicity, current approaches are inadequate. Sorafenib is a KI of concern because it inhibits growth factor receptors and Raf-1/B-Raf, kinases that are upstream of extracellular signal-regulated kinases (ERKs) and signal cardiomyocyte survival in the setting of stress. Objectives: To explore the potential use of zebrafish as a preclinical model to predict cardiotoxicity and to determine whether sorafenib has associated cardiotoxicity, and, if so, define the mechanisms. Methods and Results: We find that the zebrafish model is readily able to discriminate a KI with little or no cardiotoxicity (gefitinib) from one with demonstrated cardiotoxicity (sunitinib). Sorafenib, like sunitinib, leads to cardiomyocyte apoptosis, a reduction in total myocyte number per heart, contractile dysfunction, and ventricular dilatation in zebrafish. In cultured rat cardiomyocytes, sorafenib induces cell death. This can be rescued by adenovirus-mediated gene transfer of constitutively active MEK1, which restores ERK activity even in the presence of sorafenib. Whereas growth factor–induced activation of ERKs requires Raf, α-adrenergic agonist-induced activation of ERKs does not require it. Consequently, activation of α-adrenergic signaling markedly decreases sorafenib-induced cell death. Consistent with these in vitro data, inhibition of α-adrenergic signaling with the receptor antagonist prazosin worsens sorafenib-induced cardiomyopathy in zebrafish. Conclusions: Zebrafish may be a valuable preclinical tool to predict cardiotoxicity. The α-adrenergic signaling pathway is an important modulator of sorafenib cardiotoxicity in vitro and in vivo and appears to act through a here-to-fore unrecognized signaling pathway downstream of α-adrenergic activation that bypasses Raf to activate ERKs.

Studies with sPLA(2) Group X, and cPLA(2) α gene-targeted mice suggest that absence of sPLA(2) Group X results in protection from ischemia/reperfusion (I/R) injury in the heart, and absence of cPLA(2) α Group IV is protective in the brain. Although latter studies might suggest a similar deleterious role for cPLA(2) α in I/R injury in the heart, the pathophysiology of stroke is intricately related to excitotoxicity and cannot necessarily be extrapolated to the heart. We report here that unlike findings in the brain, cPLA(2) α((-/-)) mice have exaggerated injury following I/R in vivo. In contrast, there is no difference in injury induced by simulated ischemia in cardiomyocytes isolated from cPLA(2) α((-/-)) versus cPLA(2) α((+/+)) mice. This suggests that cPLA(2) α does not have an important cardiomyocyte autonomous effect on ischemic injury. Prostaglandin E(2) (PGE(2) ) levels are significantly reduced in the hearts of the cPLA(2) α((-/-)) mice, and the enhanced injury is ameliorated by treatment with the PGE analog, misoprostol. We demonstrate that cPLA(2) α is cardioprotective in vivo, and this is likely via cPLA(2) α-mediated production of cardioprotective eicosanoids. These studies are the first to identify a protective role for cPLA(2) in I/R injury in any organ and raise concerns over long-term inhibition of cPLA(2).

Targeted therapeutics, particularly those that inhibit the activity of protein kinases that are mutated and/or overexpressed in cancer, have revolutionized the treatment of some cancers and improved survival rates in many others. Although these agents dominate drug development in cancer, significant toxicities, including cardiotoxicity, have emerged. In this Review, we examine the underlying mechanisms that result in on-target or off-target cardiotoxicities of small molecule kinase inhibitors. We also discuss how well the various preclinical safety models and strategies might predict clinical cardiotoxicity. It is hoped that a thorough understanding of the mechanisms underlying cardiotoxicity will lead to the development of safe, effective drugs and consequently, fewer costly surprises as agents progress through clinical trials.

pre-clinicalreductions in mortality and morbidity being achieved among cancer patients with current therapies represent a major achievement. However, given their mechanisms of action, many anti-cancer agents may have significant potential for cardiovascular side effects, including the induction of heart failure. The magnitude of this problem remains unclear and is not readily apparent from current clinical trials of emerging targeted agents, which generally under-represent older patients and those with significant co-morbidities. The risk of adverse events may also increase when novel agents, which frequently modulate survival pathways, are used in combination with each other or with other conventional cytotoxic chemotherapeutics. The extent to which survival and growth pathways in the tumour cell (which we seek to inhibit) coincide with those in cardiovascular cells (which we seek to preserve) is an open question but one that will become ever more important with the development of new cancer therapies that target intracellular signalling pathways. It remains unclear whether potential cardiovascular problems can be predicted from analyses of such basic signalling mechanisms and what pre-clinical evaluation should be undertaken. The screening of patients, optimization of therapeutic schemes, monitoring of cardiovascular function during treatment, and the management of cardiovascular side effects are likely to become increasingly important in cancer patients. This paper summarizes the deliberations of a cross-disciplinary workshop organized by the Heart Failure Association of the European Society of Cardiology (held in Brussels in May 2009), which brought together clinicians working in cardiology and oncology and those involved in basic, translational, and pharmaceutical science.

Among the myriad of intracellular signaling networks that govern the cardiac development and pathogenesis, mitogen-activated protein kinases (MAPKs) are prominent players that have been the focus of extensive investigations in the past decades. The four best characterized MAPK subfamilies, ERK1/2, JNK, p38, and ERK5, are the targets of pharmacological and genetic manipulations to uncover their roles in cardiac development, function, and diseases. However, information reported in the literature from these efforts has not yet resulted in a clear view about the roles of specific MAPK pathways in heart. Rather, controversies from contradictive results have led to a perception that MAPKs are ambiguous characters in heart with both protective and detrimental effects. The primary object of this review is to provide a comprehensive overview of the current progress, in an effort to highlight the areas where consensus is established verses the ones where controversy remains. MAPKs in cardiac development, cardiac hypertrophy, ischemia/reperfusion injury, and pathological remodeling are the main focuses of this review as these represent the most critical issues for evaluating MAPKs as viable targets of therapeutic development. The studies presented in this review will help to reveal the major challenges in the field and the limitations of current approaches and point to a critical need in future studies to gain better understanding of the fundamental mechanisms of MAPK function and regulation in the heart.

Hypertrophic cardiomyopathy (HCM) is a myocardial disorder characterized by left ventricular (LV) hypertrophy without dilatation and without apparent cause (ie, it occurs in the absence of severe hypertension, aortic stenosis, or other cardiac or systemic diseases that might cause LV hypertrophy). Numerous excellent reviews and consensus documents provide a wealth of additional background.1–8 HCM is the leading cause of sudden death in young people and leads to significant disability in survivors. It is caused by mutations in genes that encode components of the sarcomere. Cardiomyocyte and cardiac hypertrophy, myocyte disarray, interstitial and replacement fibrosis, and dysplastic intramyocardial arterioles characterize the pathology of HCM. Clinical manifestations include impaired diastolic function, heart failure, tachyarrhythmia (both atrial and ventricular), and sudden death. At present, there is a lack of understanding of how the mutations in genes encoding sarcomere proteins lead to the phenotypes described above. Current therapeutic approaches have focused on the prevention of sudden death, with implantable cardioverter defibrillator placement in high-risk patients. But medical therapies have largely focused on alleviating symptoms of the disease, not on altering its natural history. The present Working Group of the National Heart, Lung, and Blood Institute brought together clinical, translational, and basic scientists with the overarching goal of identifying novel strategies to prevent the phenotypic expression of disease. Herein, we identify research initiatives that we hope will lead to novel therapeutic approaches for patients with HCM. The epidemiology of HCM suggests that it is present in ≈1 in 500 adults.9 Because of the delay in phenotypic expression of the disease, HCM is not commonly recognized clinically in young children, but when it is, it is much more frequently recognized in males.10 This is likely due to greater penetrance in young males.11,12 HCM is underdiagnosed clinically in blacks and in women, yet …

Over the last 40 years targeting G protein-coupled receptors and their ligands has had a major impact on the treatment of cardiovascular disease. However, the last decade has seen little progress and focus has shifted, particularly in the field of cancer biology, to downstream kinases. This review focuses on the kinases within the heart that become active during myocardial infarction and heart failure and contribute to cardiac dysfunction, with a special emphasis on p38 mitogen-activated protein kinase (MAPK).

Rationale : Numerous studies have proposed that glycogen synthase kinase (GSK)-3β is a central regulator of the hypertrophic response of cardiomyocytes. However, all of this work has relied on overexpression of GSK-3β, expression of constitutively active mutants, or small molecule inhibitors with documented off-target effects. Genetic loss of function approaches have not been used in the adult mouse because germ-line deletion of GSK-3β is embryonic-lethal. Objective : This study was designed to define the role played by GSK-3β in pressure overload (PO)-induced hypertrophy and remodeling following myocardial infarction (MI). Methods and Results : We used a mouse model that allows inducible, cardiomyocyte-specific deletion of GSK-3β in the adult knockout. Surprisingly, we find that knockout mice exposed to PO induced by thoracic aortic constriction exhibit a normal hypertrophic response. Thus, in contrast to virtually all prior published studies, GSK-3β appears to play at most a minor role in the hypertrophic response to PO stress. However, GSK-3β does regulate post-MI remodeling because the GSK-3β knockouts had less left ventricular dilatation and better-preserved left ventricular function at up to 8 weeks post-MI despite demonstrating significantly more hypertrophy in the remote myocardium. Deletion of GSK-3β also led to increased cardiomyocyte proliferation following PO and MI. Conclusions : Deletion of GSK-3β protects against post-MI remodeling and promotes stress-induced cardiomyocyte proliferation in the adult heart. These studies suggest that inhibition of GSK-3β could be a strategy to both prevent remodeling and to promote cardiac regeneration in pathological states.

Mutations in CCM3/PDCD10 result in cerebral cavernous malformations (CCMs), a major cause of cerebral hemorrhage. Despite intense interest in CCMs, very little is known about the function of CCM3. Here, we report that CCM3 is located on the Golgi apparatus, forming a complex with proteins of the germinal center kinase III (GCKIII) family and GM130, a Golgi-resident protein. Cells depleted of CCM3 show a disassembled Golgi apparatus. Furthermore, in wound-healing assays, CCM3-depleted cells cannot reorient the Golgi and centrosome properly, and demonstrate impaired migration. Golgi disassembly after either depletion of CCM3 or dissociation of CCM3 from the GM130-GCKIII complex is the result of destabilization of GCKIII proteins and dephosphorylation of their substrate, 14-3-3ζ. Significantly, the phenotype induced by CCM3 depletion can be reverted by expression of wild-type CCM3, but not by disease-associated mutants. Our findings suggest that Golgi dysfunction and the ensuing abnormalities of cell orientation and migration resulting from CCM3 mutations contribute to CCM pathogenesis.

SOK1 is a Ste20 protein kinase of the germinal center kinase (GCK) family that has been shown to be activated by oxidant stress and chemical anoxia, a cell culture model of ischemia. More recently, it has been shown to be localized to the Golgi apparatus, where it functions in a signaling pathway required for cell migration and polarization. Herein, we demonstrate that SOK1 regulates cell death after chemical anoxia, as its down-regulation by RNA interference enhances cell survival. Furthermore, expression of SOK1 elicits apoptotic cell death by activating the intrinsic pathway. We also find that a cleaved form of SOK1 translocates from the Golgi to the nucleus after chemical anoxia and that this translocation is dependent on both caspase activity and on amino acids 275-292, located immediately C-terminal to the SOK1 kinase domain. Furthermore, SOK1 entry into the nucleus is important for the cell death response since SOK1 mutants unable to enter the nucleus do not induce cell death. In summary, SOK1 is necessary to induce cell death and can induce death when overexpressed. Furthermore, SOK1 appears to play distinctly different roles in stressed versus non-stressed cells, regulating cell death in the former.