Cardiomyocyte Homeodomain-Interacting Protein Kinase 2 Maintains Basal Cardiac Function via Extracellular Signal-Regulated Kinase Signaling.

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.

Cardiomyocyte-GSK-3α promotes mPTP opening and heart failure in mice with chronic pressure overload.

Chronic pressure-overload (PO)- induced cardiomyopathy is one of the leading causes of left ventricular (LV) remodeling and heart failure. The role of the α isoform of glycogen synthase kinase-3 (GSK-3α) in PO-induced cardiac remodeling is unclear and its downstream molecular targets are largely unknown. To investigate the potential roles of GSK-3α, cardiomyocyte-specific GSK-3α conditional knockout (cKO) and control mice underwent trans-aortic constriction (TAC) or sham surgeries. Cardiac function in the cKOs and littermate controls declined equally up to 2 weeks of TAC. At 4 week, cKO animals retained concentric LV remodeling and showed significantly less decline in contractile function both at systole and diastole, vs. controls which remained same until the end of the study (6 wk). Histological analysis confirmed preservation of LV chamber and protection against TAC-induced cellular hypertrophy in the cKO. Consistent with attenuated hypertrophy, significantly lower level of cardiomyocyte apoptosis was observed in the cKO. Mechanistically, GSK-3α was found to regulate mitochondrial permeability transition pore (mPTP) opening and GSK-3α-deficient mitochondria showed delayed mPTP opening in response to Ca2+ overload. Consistently, overexpression of GSK-3α in cardiomyocytes resulted in elevated Bax expression, increased apoptosis, as well as a reduction of maximum respiration capacity and cell viability. Taken together, we show for the first time that GSK-3α regulates mPTP opening under pathological conditions, likely through Bax overexpression. Genetic ablation of cardiomyocyte GSK-3α protects against chronic PO-induced cardiomyopathy and adverse LV remodeling, and preserves contractile function. Selective inhibition of GSK-3α using isoform-specific inhibitors could be a viable therapeutic strategy to limit PO-induced heart failure.

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

RATIONALE: Cardiac myocyte-specific deletion of either glycogen synthase kinase (GSK)-3α and GSK-3ß 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-3ß 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.

Entanglement of GSK-3ß, ß-catenin and TGF-ß1 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-ß signaling to govern the fibrogenic process. Herein, we will discuss the emerging role of the GSK-3ß, ß-catenin and TGF-ß1-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-3ß, ß-catenin and SMAD-3 signaling network may provide a novel therapeutic target for the management of myocardial fibrosis in the diseased heart.

Mitogen-activated protein kinase signaling in the heart: angels versus demons in a heart-breaking tale.

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.

The GSK-3 family as therapeutic target for myocardial diseases.

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-3ß as a regulator of myocardial fibrosis will also be discussed. We will review our recent findings that specific deletion of GSK-3ß 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-3ß 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.

Cardio-Oncology: How New Targeted Cancer Therapies and Precision Medicine Can Inform Cardiovascular Discovery.

Cardio-oncology (the cardiovascular care of cancer patients) has developed as a new translational and clinical field based on the expanding repertoire of mechanism-based cancer therapies. Although these therapies have changed the natural course of many cancers, several may also lead to cardiovascular complications. Many new anticancer drugs approved over the past decade are "targeted" kinase inhibitors that interfere with intracellular signaling contributing to tumor progression. Unexpected cardiovascular and cardiometabolic effects of patient treatment with these inhibitors have provided unique insights into the role of kinases in human cardiovascular biology. Today, an ever-expanding number of cancer therapies targeting novel kinases and other specific cellular and metabolic pathways are being developed and tested in oncology clinical trials. Some of these drugs may affect the cardiovascular system in detrimental ways and others perhaps in beneficial ways. We propose that the numerous ongoing oncology clinical trials are an opportunity for closer collaboration between cardiologists and oncologists to study the cardiovascular and cardiometabolic changes caused by the modulation of these pathways in patients. In this regard, cardio-oncology represents an opportunity and a novel platform for basic and translational investigation and can serve as a potential avenue for optimization of anticancer therapies and for cardiovascular research and drug discovery.

Sorafenib cardiotoxicity increases mortality after myocardial infarction.

RATIONALE: Sorafenib is an effective treatment for renal cell carcinoma, but recent clinical reports have documented its cardiotoxicity through an unknown mechanism. OBJECTIVE: Determining the mechanism of sorafenib-mediated cardiotoxicity. METHODS AND RESULTS: Mice treated with sorafenib or vehicle for 3 weeks underwent induced myocardial infarction (MI) after 1 week of treatment. Sorafenib markedly decreased 2-week survival relative to vehicle-treated controls, but echocardiography at 1 and 2 weeks post MI detected no differences in cardiac function. Sorafenib-treated hearts had significantly smaller diastolic and systolic volumes and reduced heart weights. High doses of sorafenib induced necrotic death of isolated myocytes in vitro, but lower doses did not induce myocyte death or affect inotropy. Histological analysis documented increased myocyte cross-sectional area despite smaller heart sizes after sorafenib treatment, further suggesting myocyte loss. Sorafenib caused apoptotic cell death of cardiac- and bone-derived c-kit+ stem cells in vitro and decreased the number of BrdU+ (5-bromo-2'-deoxyuridine+) myocytes detected at the infarct border zone in fixed tissues. Sorafenib had no effect on infarct size, fibrosis, or post-MI neovascularization. When sorafenib-treated animals received metoprolol treatment post MI, the sorafenib-induced increase in post-MI mortality was eliminated, cardiac function was improved, and myocyte loss was ameliorated. CONCLUSIONS: Sorafenib cardiotoxicity results from myocyte necrosis rather than from any direct effect on myocyte function. Surviving myocytes undergo pathological hypertrophy. Inhibition of c-kit+ stem cell proliferation by inducing apoptosis exacerbates damage by decreasing endogenous cardiac repair. In the setting of MI, which also causes large-scale cell loss, sorafenib cardiotoxicity dramatically increases mortality.

Molecular mechanisms of cardiotoxicity of tyrosine kinase inhibition.

Cancer therapy has progressed remarkably in recent years. In no area has this been more apparent than in the development of "targeted therapies", particularly those using drugs that inhibit the activity of certain tyrosine kinases, activating mutations or amplifications of which are causal, or strongly contributory, to tumorigenesis. However, some of these therapies have been associated with toxicity to the heart. Here we summarize what is known about the cardiotoxicity of cancer drugs that target tyrosine kinases. We focus on basic mechanisms through which interruption of specific signalling pathways leads to cardiomyocyte dysfunction and/or death, and contrast this with therapeutic responses in cancer cells.

Cardiac fibroblast glycogen synthase kinase-3ß regulates ventricular remodeling and dysfunction in ischemic heart.

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-3ß (GSK-3ß) is phosphorylated (inhibited) in fibrotic tissues from ischemic human and mouse heart. Using 2 fibroblast-specific GSK-3ß knockout mouse models, we show that deletion of GSK-3ß in cardiac fibroblasts leads to fibrogenesis, left ventricular dysfunction, and excessive scarring in the ischemic heart. Deletion of GSK-3ß induces a profibrotic myofibroblast phenotype in isolated cardiac fibroblasts, in post-myocardial infarction hearts, and in mouse embryonic fibroblasts deleted for GSK-3ß. Mechanistically, GSK-3ß inhibits profibrotic transforming growth factor-ß1/SMAD-3 signaling via interactions with SMAD-3. Moreover, deletion of GSK-3ß 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-3ß in myocardial fibrotic disorders and establish critical roles of cardiac fibroblasts in remodeling and ventricular dysfunction.

Emerging paradigms in cardiomyopathies associated with cancer therapies.

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.

Prevention of liver cancer cachexia-induced cardiac wasting and heart failure.

AIMS: Symptoms of cancer cachexia (CC) include fatigue, shortness of breath, and impaired exercise capacity, which are also hallmark symptoms of heart failure (HF). Herein, we evaluate the effects of drugs commonly used to treat HF (bisoprolol, imidapril, spironolactone) on development of cardiac wasting, HF, and death in the rat hepatoma CC model (AH-130). METHODS AND RESULTS: Tumour-bearing rats showed a progressive loss of body weight and left-ventricular (LV) mass that was associated with a progressive deterioration in cardiac function. Strikingly, bisoprolol and spironolactone significantly reduced wasting of LV mass, attenuated cardiac dysfunction, and improved survival. In contrast, imidapril had no beneficial effect. Several key anabolic and catabolic pathways were dysregulated in the cachectic hearts and, in addition, we found enhanced fibrosis that was corrected by treatment with spironolactone. Finally, we found cardiac wasting and fibrotic remodelling in patients who died as a result of CC. In living cancer patients, with and without cachexia, serum levels of brain natriuretic peptide and aldosterone were elevated. CONCLUSION: Systemic effects of tumours lead not only to CC but also to cardiac wasting, associated with LV-dysfunction, fibrotic remodelling, and increased mortality. These adverse effects of the tumour on the heart and on survival can be mitigated by treatment with either the ß-blocker bisoprolol or the aldosterone antagonist spironolactone. We suggest that clinical trials employing these agents be considered to attempt to limit this devastating complication of cancer.

Differential activation of cultured neonatal cardiomyocytes by plasmalemmal versus intracellular G protein-coupled receptor 55.

The L-α-lysophosphatidylinositol (LPI)-sensitive receptor GPR55 is coupled to Ca(2+) signaling. Low levels of GPR55 expression in the heart have been reported. Similar to other G protein-coupled receptors involved in cardiac function, GPR55 may be expressed both at the sarcolemma and intracellularly. Thus, to explore the role of GPR55 in cardiomyocytes, we used calcium and voltage imaging and extracellular administration or intracellular microinjection of GPR55 ligands. We provide the first evidence that, in cultured neonatal ventricular myocytes, LPI triggers distinct signaling pathways via GPR55, depending on receptor localization. GPR55 activation at the sarcolemma elicits, on one hand, Ca(2+) entry via L-type Ca(2+) channels and, on the other, inositol 1,4,5-trisphosphate-dependent Ca(2+) release. The latter signal is further amplified by Ca(2+)-induced Ca(2+) release via ryanodine receptors. Conversely, activation of GPR55 at the membrane of intracellular organelles promotes Ca(2+) release from acidic-like Ca(2+) stores via the endolysosomal NAADP-sensitive two-pore channels. This response is similarly enhanced by Ca(2+)-induced Ca(2+) release via ryanodine receptors. Extracellularly applied LPI produces Ca(2+)-independent membrane depolarization, whereas the Ca(2+) signal induced by intracellular microinjection of LPI converges to hyperpolarization of the sarcolemma. Collectively, our findings point to GPR55 as a novel G protein-coupled receptor regulating cardiac function at two cellular sites. This work may serve as a platform for future studies exploring the potential of GPR55 as a therapeutic target in cardiac disorders.

Troponin I-interacting protein kinase: a novel cardiac-specific kinase, emerging as a molecular target for the treatment of cardiac disease.

Coronary artery disease is the leading cause of death and disability worldwide. In patients with acute coronary syndromes, timely and effective myocardial reperfusion by percutaneous coronary intervention is the primary treatment of choice to minimize the ischemic injury and limit the size of the myocardial infarction (MI). However, reperfusion can itself promote cardiomyocyte death, which leads to cardiac dysfunction via reperfusion injury. The molecular mechanisms of ischemia-reperfusion (IR) injury are not completely understood and new drug targets are needed. Recently, we reported that cardiac troponin I-interacting protein kinase (TNNI3K), a cardiomyocyte-specific kinase, promotes IR injury via profound oxidative stress, thereby promoting cardiomyocyte death. By using novel genetic animal models and newly developed small-molecule TNNI3K inhibitors, we demonstrated that TNNI3K-mediated IR injury occurs through impaired mitochondrial function and is in part dependent on p38 MAPK. Here we discuss the emerging role of TNNI3K as a promising new drug target to limit IR-induced myocardial injury. We will also examine the underlying mechanisms that drive the profoundly reduced infarct size in mice in whichTNNI3Kis specifically deleted in cardiomyocytes. Because TNNI3K is a cardiac-specific kinase, it could be an ideal molecular target, as inhibiting it would have little or no effect on other organ systems, a serious problem associated with the use of kinase inhibitors targeting kinases that are more widely expressed.

Glycogen synthase kinase-3α limits ischemic injury, cardiac rupture, post-myocardial infarction remodeling and death.

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-3ß is protective.

A novel preclinical strategy for identifying cardiotoxic kinase inhibitors and mechanisms of cardiotoxicity.

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.

Wnt signaling exerts an antiproliferative effect on adult cardiac progenitor cells through IGFBP3.

RATIONALE: Recent work in animal models and humans has demonstrated the presence of organ-specific progenitor cells required for the regenerative capacity of the adult heart. In response to tissue injury, progenitor cells differentiate into specialized cells, while their numbers are maintained through mechanisms of self-renewal. The molecular cues that dictate the self-renewal of adult progenitor cells in the heart, however, remain unclear. OBJECTIVE: We investigate the role of canonical Wnt signaling on adult cardiac side population (CSP) cells under physiological and disease conditions. METHODS AND RESULTS: CSP cells isolated from C57BL/6J mice were used to study the effects of canonical Wnt signaling on their proliferative capacity. The proliferative capacity of CSP cells was also tested after injection of recombinant Wnt3a protein (r-Wnt3a) in the left ventricular free wall. Wnt signaling was found to decrease the proliferation of adult CSP cells, both in vitro and in vivo, through suppression of cell cycle progression. Wnt stimulation exerted its antiproliferative effects through a previously unappreciated activation of insulin-like growth factor binding protein 3 (IGFBP3), which requires intact IGF binding site for its action. Moreover, injection of r-Wnt3a after myocardial infarction in mice showed that Wnt signaling limits CSP cell renewal, blocks endogenous cardiac regeneration and impairs cardiac performance, highlighting the importance of progenitor cells in maintaining tissue function after injury. CONCLUSIONS: Our study identifies canonical Wnt signaling and the novel downstream mediator, IGFBP3, as key regulators of adult cardiac progenitor self-renewal in physiological and pathological states.

Cardiotoxicity of the cancer therapeutic agent imatinib mesylate.

Imatinib mesylate (Gleevec) is a small-molecule inhibitor of the fusion protein Bcr-Abl, the causal agent in chronic myelogenous leukemia. Here we report ten individuals who developed severe congestive heart failure while on imatinib and we show that imatinib-treated mice develop left ventricular contractile dysfunction. Transmission electron micrographs from humans and mice treated with imatinib show mitochondrial abnormalities and accumulation of membrane whorls in both vacuoles and the sarco- (endo-) plasmic reticulum, findings suggestive of a toxic myopathy. With imatinib treatment, cardiomyocytes in culture show activation of the endoplasmic reticulum (ER) stress response, collapse of the mitochondrial membrane potential, release of cytochrome c into the cytosol, reduction in cellular ATP content and cell death. Retroviral gene transfer of an imatinib-resistant mutant of c-Abl, alleviation of ER stress or inhibition of Jun amino-terminal kinases, which are activated as a consequence of ER stress, largely rescues cardiomyocytes from imatinib-induced death. Thus, cardiotoxicity is an unanticipated side effect of inhibition of c-Abl by imatinib.

CCM3/PDCD10 stabilizes GCKIII proteins to promote Golgi assembly and cell orientation.

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, 14-3-3zeta. 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.