Abstract The epidermal growth factor receptor (EGFR) family network comprises 4 receptors (EGFR, ERBB2, ERBB3, ERBB4) and numerous ligands, and is dysregulated in many cancers. Since anti-cancer drugs that target these receptors are cardiotoxic for some patients, it is important to understand the network in cardiac cells. Data from the Human Protein Atlas established that EGFR family members and their ligands are differentially expressed in cardiac cell types. Ligand expression was altered in human failing hearts and may contribute to disease. These ligands stimulated extracellular signal-regulated kinases 1/2 (ERK1/2) and Akt in rat cardiomyocytes but to different degrees. Afatinib (at a concentration to inhibit all EGF family receptors) was used to assess the role of the network in a mouse model of cardiac hypertrophy induced by angiotensin II (AngII). Echocardiography and segmental strain analysis demonstrated that afatinib reduced AngII-induced cardiac hypertrophy and caused cardiac dysfunction. This was associated with loss of cardiomyocyte hypertrophy, enhanced cardiac fibrosis, and reduced expression of Nrg1 . NRG1 binds to ERBB4 in cardiomyocytes which homodimerizes or heterodimerises with ERBB2. The role of ERBB2 in the cardiomyocyte response to NRG1 compared with EGF was dissected using tucatinib (a selective ERBB2 inhibitor) and mRNA expression profiling. Most, but not necessarily all, of the response to NRG1 required ERBB2 signalling; most, but not all, of the response to EGF did not. Thus, the EGFR family network plays an important role in the heart. Understanding this network may identify therapeutic approaches to avoid cardiotoxicity associated with EGFR family anti-cancer drugs. Clinical perspectives Anti-cancer drugs that target the epidermal growth factor receptor (EGFR) family are cardiotoxic for some patients; it is therefore important to understand the network in cardiac cells. The EGFR family and their ligands are differentially expressed in cardiac cells with changes in ligand expression in heart failure; inhibition of all receptors in a mouse model of hypertrophy reduces cardiac hypertrophy and causes cardiac dysfunction with attenuation of cardiomyocyte hypertrophy and enhanced cardiac fibrosis and loss of neuregulin 1 (NRG1); in rat cardiomyocytes, NRG1 signalling to gene expression is largely mediated via ERBB2. The EGFR family network plays an important role in the heart; understanding this network may identify therapeutic approaches to avoid cardiotoxicity associated with anti-cancer drugs targeted against it.

Background The anti-cancer MEK inhibitor trametinib (alone or with the RAF inhibitor dabrafenib) causes cardiac dysfunction or heart failure in some patients. Our hypothesis is that cardiotoxicity is exacerbated by an underlying co-morbidity such as hypertension causing early cardiac dysfunction detectable on echocardiograms. Objectives The objectives were to assess the effects of trametinib on cardiac function in a mouse model of hypertension-induced cardiac hypertrophy and determine if this was moderated by dabrafenib. Methods Male mice were treated with vehicle, trametinib or dabrafenib/trametinib in the absence/presence of angiotensin II (AngII; 0.8 mg/kg/d to increase blood pressure over 7 d). Hearts were imaged using echocardiography. Results Inhibitors alone had a limited effect on mouse hearts over 28 d. Trametinib or trametinib/dabrafenib inhibited cardiac hypertrophy induced by AngII over 7 d, reducing left ventricular (LV) wall thickness and mass. AngII did not significantly affect cardiac function, but the inhibitors caused significant functional deterioration. Segmental analysis revealed variation of contraction around the LV, with selective effects of AngII and trametinib or dabrafenib/trametinib in basal/mid-regional segments. Frame-by-frame analysis of radial (not longitudinal) displacement of the LV endocardial wall demonstrated variation between consecutive cardiac cycles that enabled a high degree of classification according to treatment. Conclusions Trametinib inhibits AngII-induced cardiac hypertrophy in mice but is detrimental to cardiac function, responses that are not moderated by dabrafenib. AngII and MEK/RAF inhibition have regional effects around the LV with greater effects on radial displacement in basal/mid-regional segments. Assessment of such changes may facilitate early identification of developing cardiotoxicity. Competencies in Medical knowledge The anti-cancer drugs trametinib and dabrafenib are cardiotoxic in some patients. These drugs had limited effects themselves on mouse hearts, but suppressed cardiac hypertrophy in a mouse model of hypertension whilst proving detrimental to cardiac function. Segments around the left ventricle were differentially affected by hypertension and inhibitors,with variation between consecutive cardiac cycles. Translational outlook Further research is needed to establish the relationship between hypertension and cardiotoxicity of trametinib in patients, and determine if regional effects in the ventricle wall can predict cardiac dysfunction. If so, a diagnostic algorithm may be a useful tool in identifying developing cardiotoxicity. ![Summary Figure.][1] Summary Figure. Effects of dabrafenib/trametinib on the cardiac response to angiotensin II in mice. Inhibiting ERK1/2 suppresses cardiac fibrosis and cardiomyocyte hypertrophy induced by angiotensin II. This results in a decline in cardiac function, with particular effects on radial wall movement and in different segments of the left ventricle. ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

The three striatins (STRN, STRN3, STRN4) form the core of STR iatin-Interacting Phosphatase and Kinase (STRIPAK) complexes. These place protein phosphatase 2A (PP2A) in proximity to protein kinases thereby restraining kinase activity and regulating key cellular processes. Our aim was to establish if striatins play a significant role in cardiac remodelling associated with cardiac hypertrophy and heart failure. All striatins were expressed in control human hearts, with up -regulation of STRN and STRN3 in failing hearts. We used mice with global heterozygote gene deletion to assess the roles of STRN and STRN3 in cardiac remodelling induced by angiotensin II (AngII; 7 days). Using echocardiography, we detected no differences in baseline cardiac function or dimensions in STRN +/ - or STRN3 +/ - male mice (8 weeks) compared with wild -type littermates. Heterozygous gene deletion did not affect cardiac function in mice treated with AngII, but the increase in left ventricle mass induced by AngII was inhibited in STRN +/ - (but not STRN3 +/ - ) mice. Histological staining indicated that cardiomyocyte hypertrophy was inhibited. To assess the role of STRN in cardiomyocytes, we converted the STRN knockout line for inducible cardiomyocyte-specific gene deletion. There was no effect of cardiomyocyte STRN knockout on cardiac function or dimensions, but the increase in left ventricle mass induced by AngII was inhibited. This resulted from inhibition of cardiomyocyte hypertrophy and cardiac fibrosis. The data indicate that cardiomyocyte striatin is required for early remodelling of the heart by AngII and identify the striatin-based STRIPAK system as a signalling paradigm in the development of pathological cardiac hypertrophy.

Mammalian cardiomyocytes become terminally-differentiated during the perinatal period. In rodents, cytokinesis ceases after a final division cycle immediately after birth. Nuclear division continues and most cardiomyocytes become binucleated by similar to 11 days. Subsequent growth results from an increase in cardiomyocyte size. The mechanisms involved remain under investigation. Mitogen-activated protein kinases (MAPKs) regulate cell growth/death: extracellular signal-regulated kinases 1/2 (ERK1/2) promote proliferation, whilst c-Jun N-terminal kinases (JNKs) and p38-MAPKs respond to cellular stresses. We assessed their regulation in rat hearts during postnatal development (2, 7, 14, and 28 days, 12 weeks) during which time there was rapid, substantial downregulation of mitosis/cytokinesis genes (Cenpa/e/f, Aurkb, Anln, Cdca8, Orc6) with lesser downregulation of DNA replication genes (Orcs1-5, Mcms2-7). MAPK activation was assessed by immunoblotting for total and phosphorylated (activated) kinases. Total ERK1/2 was downregulated, but not JNKs or p38-MAPKs, whilst phosphorylation of all MAPKs increased relative to total protein albeit transiently for JNKs. These profiles differed from activation of Akt (also involved in cardiomyocyte growth). Dual-specificity phosphatases, upstream MAPK kinase kinases (MAP3Ks), and MAP3K kinases (MAP4Ks) identified in neonatal rat cardiomyocytes by RNASeq were differentially regulated during postnatal cardiac development. The MAP3Ks that we could assess by immunoblotting (RAF kinases and Map3k3) showed greater downregulation of the protein than mRNA. MAP3K2/MAP3K3/MAP4K5 were upregulated in human failing heart samples and may be part of the "foetal gene programme" of re-expressed genes in disease. Thus, MAPKs, along with kinases and phosphatases that regulate them, potentially play a significant role in postnatal remodelling of the heart.a

Cardiac hypertrophy is necessary for the heart to accommodate an increase in workload. Physiological, compensated hypertrophy (e.g. with exercise) is reversible and largely due to cardiomyocyte hypertrophy. Pathological hypertrophy (e.g. with hypertension) is associated with additional features including increased fibrosis and can lead to heart failure. RAF kinases (ARAF/BRAF/RAF1) integrate signals into the extracellular signal-regulated kinase 1/2 cascade, a pathway implicated in cardiac hypertrophy, and activation of BRAF in cardiomyocytes promotes compensated hypertrophy. Here, we used mice with tamoxifen-inducible cardiomyocyte-specific BRAF knockout (CM-BRAFKO) to assess the role of BRAF in hypertension-associated cardiac hypertrophy induced by angiotensin II (AngII; 0.8 mg/kg/d, 7 d) and physiological hypertrophy induced by phenylephrine (40 mg/kg/d, 7 d). Cardiac dimensions/functions were measured by echocardiography with histological assessment of cellular changes. AngII promoted cardiomyocyte hypertrophy and increased fibrosis within the myocardium (interstitial) and around the arterioles (perivascular) in male mice; cardiomyocyte hypertrophy and interstitial (but not perivascular) fibrosis were inhibited in mice with CM-BRAFKO. Phenylephrine had a limited effect on fibrosis but promoted cardiomyocyte hypertrophy and increased contractility in male mice; cardiomyocyte hypertrophy was unaffected in mice with CM-BRAFKO, but the increase in contractility was suppressed and fibrosis increased. Phenylephrine induced a modest hypertrophic response in female mice and, in contrast with the males, tamoxifen-induced loss of cardiomyocyte BRAF reduced cardiomyocyte size, had no effect on fibrosis and increased contractility. The data identify BRAF as a key signalling intermediate in both physiological and pathological hypertrophy in male mice, and highlight the need for independent assessment of gene function in females.

Abstract Insulin was discovered over 100 years ago. Whilst the first half century defined many of the physiological effects of insulin, the second emphasised the mechanisms by which it elicits these effects, implicating a vast array of G proteins and their regulators, lipid and protein kinases and counteracting phosphatases, and more. Potential growth-promoting and protective effects of insulin on the heart emerged from studies of carbohydrate metabolism in the 1960s, but the insulin receptors (and the related receptor for insulin-like growth factors 1 and 2) were not defined until the 1980s. A related third receptor, the insulin receptor-related receptor remained an orphan receptor for many years until it was identified as an alkali-sensor. The mechanisms by which these receptors and the plethora of downstream signalling molecules confer cardioprotection remain elusive. Here, we review important aspects of the effects of the three insulin receptor family members in the heart. Metabolic studies are set in the context of what is now known of insulin receptor family signalling and the role of protein kinase B (PKB or Akt), and the relationship between this and cardiomyocyte survival versus death is discussed. PKB/Akt phosphorylates numerous substrates with potential for cardioprotection in the contractile cardiomyocytes and cardiac non-myocytes. Our overall conclusion is that the effects of insulin on glucose metabolism that were initially identified remain highly pertinent in managing cardiomyocyte energetics and preservation of function. This alone provides a high level of cardioprotection in the face of pathophysiological stressors such as ischaemia and myocardial infarction.

The extracellular signal-regulated kinase 1/2 (ERK1/2) cascade promotes cardiomyocyte hypertrophy and is cardioprotective, with the three RAF kinases forming a node for signal integration. Our aims were to determine if BRAF is relevant for human heart failure, whether BRAF promotes cardiomyocyte hypertrophy, and if Type 1 RAF inhibitors developed for cancer (that paradoxically activate ERK1/2 at low concentrations: the 'RAF paradox') may have the same effect. BRAF was up-regulated in heart samples from patients with heart failure compared with normal controls. We assessed the effects of activated BRAF in the heart using mice with tamoxifen-activated Cre for cardiomyocyte-specific knock-in of the activating V600E mutation into the endogenous gene. We used echocardiography to measure cardiac dimensions/function. Cardiomyocyte BRAF(V600E) induced cardiac hypertrophy within 10 d, resulting in increased ejection fraction and fractional shortening over 6 weeks. This was associated with increased cardiomyocyte size without significant fibrosis, consistent with compensated hypertrophy. The experimental Type 1 RAF inhibitor, SB590885, and/or encorafenib (a RAF inhibitor used clinically) increased ERK1/2 phosphorylation in cardiomyocytes, and promoted hypertrophy, consistent with a 'RAF paradox' effect. Both promoted cardiac hypertrophy in mouse hearts in vivo, with increased cardiomyocyte size and no overt fibrosis. In conclusion, BRAF potentially plays an important role in human failing hearts, activation of BRAF is sufficient to induce hypertrophy, and Type 1 RAF inhibitors promote hypertrophy via the 'RAF paradox'. Cardiac hypertrophy resulting from these interventions was not associated with pathological features, suggesting that Type 1 RAF inhibitors may be useful to boost cardiomyocyte function.

The Ser/Thr kinase MAP4K4, like other GCKIV kinases, has N-terminal kinase and C-terminal citron homology (CNH) domains. MAP4K4 can activate c-Jun N-terminal kinases (JNKs), and studies in the heart suggest it links oxidative stress to JNKs and heart failure. In other systems, MAP4K4 is regulated in striatin-interacting phosphatase and kinase (STRIPAK) complexes, in which one of three striatins tethers PP2A adjacent to a kinase to keep it dephosphorylated and inactive. Our aim was to understand how MAP4K4 is regulated in cardiomyocytes. The rat MAP4K4 gene was not properly defined. We identified the first coding exon of the rat gene using 5′-RACE, we cloned the full-length sequence and confirmed alternative-splicing of MAP4K4 in rat cardiomyocytes. We identified an additional α-helix C-terminal to the kinase domain important for kinase activity. In further studies, FLAG-MAP4K4 was expressed in HEK293 cells or cardiomyocytes. The Ser/Thr protein phosphatase inhibitor calyculin A (CalA) induced MAP4K4 hyperphosphorylation, with phosphorylation of the activation loop and extensive phosphorylation of the linker between the kinase and CNH domains. This required kinase activity. MAP4K4 associated with myosin in untreated cardiomyocytes, and this was lost with CalA-treatment. FLAG-MAP4K4 associated with all three striatins in cardiomyocytes, indicative of regulation within STRIPAK complexes and consistent with activation by CalA. Computational analysis suggested the interaction was direct and mediated via coiled-coil domains. Surprisingly, FLAG-MAP4K4 inhibited JNK activation by H2O2 in cardiomyocytes and increased myofibrillar organisation. Our data identify MAP4K4 as a STRIPAK-regulated kinase in cardiomyocytes, and suggest it regulates the cytoskeleton rather than activates JNKs.

Raf kinases signal via extracellular signal-regulated kinases 1/2 (ERK1/2) to drive cell division. Since activating mutations in BRAF (B-Raf proto-oncogene, serine/threonine kinase) are highly oncogenic, BRAF inhibitors including dabrafenib have been developed for cancer. Inhibitors of ERK1/2 signalling used for cancer are cardiotoxic in some patients, raising the question of whether dabrafenib is cardiotoxic. In the heart, ERK1/2 signalling promotes not only cardiomyocyte hypertrophy and is cardioprotective but also promotes fibrosis. Our hypothesis is that ERK1/2 signalling is not required in a non-stressed heart but is required for cardiac remodelling. Thus, dabrafenib may affect the heart in the context of, for example, hypertension. In experiments with cardiomyocytes, cardiac fibroblasts and perfused rat hearts, dabrafenib inhibited ERK1/2 signalling. We assessed the effects of dabrafenib (3 mg/kg/d) on male C57BL/6J mouse hearts in vivo. Dabrafenib alone had no overt effects on cardiac function/dimensions (assessed by echocardiography) or cardiac architecture. In mice treated with 0.8 mg/kg/d angiotensin II (AngII) to induce hypertension, dabrafenib inhibited ERK1/2 signalling and suppressed cardiac hypertrophy in both acute (up to 7 d) and chronic (28 d) settings, preserving ejection fraction. At the cellular level, dabrafenib inhibited AngII-induced cardiomyocyte hypertrophy, reduced expression of hypertrophic gene markers and almost completely eliminated the increase in cardiac fibrosis both in interstitial and perivascular regions. Dabrafenib is not overtly cardiotoxic. Moreover, it inhibits maladaptive hypertrophy resulting from AngII-induced hypertension. Thus, Raf is a potential therapeutic target for hypertensive heart disease and drugs such as dabrafenib, developed for cancer, may be used for this purpose.

Insulin and insulin-like growth factor stimulate protein synthesis and cardioprotection in the heart, acting through their receptors (INSRs, IGF1Rs) and signalling via protein kinase B (PKB, also known as Akt). Protein synthesis is increased in hearts perfused at alkaline pHo to the same extent as with insulin. Moreover, α1-adrenergic receptor (α1-AR) agonists (e.g. phenylephrine) increase protein synthesis in cardiomyocytes, activating PKB/Akt. In both cases, the mechanisms are not understood. Our aim was to determine if insulin receptor-related receptors (INSRRs, activated in kidney by alkaline pH) may account for the effects of alkaline pHo on cardiac protein synthesis, and establish if α1-ARs signal through the insulin receptor family. Alkaline pHo activated PKB/Akt signalling to the same degree as insulin in perfused adult male rat hearts. INSRRs were expressed in rat hearts and, by immunoblotting for phosphorylation (activation) of INSRRs/INSRs/IGF1Rs, we established that INSRRs, together with INSRs/IGF1Rs, are activated by alkaline pHo. The INSRR/INSR/IGF1R kinase inhibitor, linsitinib, prevented PKB/Akt activation by alkaline pHo, indicating that INSRRs/INSRs/IGF1Rs are required. Activation of PKB/Akt in cardiomyocytes by α1-AR agonists was also inhibited by linsitinib. Furthermore, linsitinib inhibited cardiomyocyte hypertrophy induced by α1-ARs in cultured cells, reduced the initial cardiac adaptation (24 h) to phenylephrine in vivo (assessed by echocardiography) and increased cardiac fibrosis over 4 days. We conclude that INSRRs are expressed in the heart and, together with INSRs/IGF1Rs, the insulin receptor family provide a potent system for promoting protein synthesis and cardioprotection. Moreover, this system is required for adaptive hypertrophy induced by α1-ARs.

Introduction: Epidermal growth factor (EGF) receptors (EGFRs: ERBB1-4) are activated by a family of ligands (e.g. EGF, Hb-EGF, EREG, TGFa), signaling through ERK1/2 and Akt to promote cell division and cancer. Antibody-based inhibition of ERBB2 in breast cancer can cause heart failure, but the role of other receptors and EGFR ligands in the heart, and potential cardiotoxicity of generic EGFR inhibitors is unclear. Hypothesis: We hypothesize that EGFR ligands play an important role in cardiac adaptation to hypertension, acting through EGFRs to promote adaptive remodelling. Methods & Results: EGF ligand/receptor mRNA expression was assessed in human failing hearts and normal controls (n=12/8). EGFRs were expressed at similar levels, but ligand expression differed with significant up- or downregulation of EGF/Hb-EGF vs EREG/TGFa, respectively, in failing hearts (p<0.05). EGF potently activated ERK1/2 and Akt (assessed by immunoblotting) in neonatal rat cardiomyocytes, leading to hypertrophy (p<0.05, n=4). The anti-cancer drug afatinib inhibits EGFRs. To assess the role of EGF signaling in cardiac adaptation to hypertension in vivo , C57Bl/6J mice (n=6) were treated with 0.8 mg/kg/d angiotensin II (AngII; 7d) ± 0.45 mg/kg/d afatinib. AngII promoted cardiac hypertrophy with increased left ventricular (LV) wall thickness (WT) and decreased LV internal diameter (ID; assessed by echocardiography). Afatinib enhanced AngII-induced hypertrophy with significantly increased WT:ID ratios (1.30-fold and 1.54-fold in diastole and systole, respectively; p<0.05) but inhibited AngII-induced increases in Nppb mRNA expression and cardiomyocyte cross-sectional area (208.80±9.78 vs 161.10±3.87μm 2 ; p<0.05). In contrast, Col1a1 mRNA expression was enhanced by afatinib, along with interstitial and perivascular fibrosis (3.21±0.38 vs 5.61±0.46, 0.98±0.06 vs 1.45±0.18 % area; p<0.05). Conclusion: EGFR signaling is modulated in human heart failure, promotes cardiomyocyte hypertrophy and is required for cardiac adaptation to hypertension. Since EGFR inhibition in hypertension prevents adaptive cardiomyocyte hypertrophy whilst promoting fibrosis, EGFR inhibitors are likely to cause cardiac dysfunction and be cardiotoxic in hypertensive patients.

The function of the heart is to provide the tissues of the body with sufficient oxygenated blood and metabolites to meet the moment-to-moment needs as dictated by physical activity and postural and emotional changes. Cardiac myocytes are the contractile cells of the heart and constitute the bulk of heart mass. There are differences between the myocytes of the ventricles, the atria, and the conduction system: ventricular myocytes are elongated cells, packed with myofibrils (the contractile apparatus) and mitochondria (for ATP production). Myofibrils are repeating units (sarcomeres) made up of thin actin filaments anchored at the Z-discs at either end of the sarcomere, and thick myosin filaments which interdigitate and interact with the thin filaments. Contraction results from sarcomere shortening produced by the ATP-dependent movement of the thin and thick filaments relative to one another. Transverse (T-) tubules facilitate extracellular Ca...

Systemic hypertension increases cardiac workload causing cardiomyocyte hypertrophy and increased cardiac fibrosis. An underlying feature is increased production of reactive oxygen species. Redox-sensitive ASK1 (apoptosis signal-regulating kinase 1) activates stress-regulated protein kinases (p38-MAPK [mitogen-activated protein kinases] and JNKs [c-Jun N-terminal kinases]) and promotes fibrosis in various tissues. Here, we determined the specificity of ASK1 signaling in the heart, with the hypothesis that ASK1 inhibitors may be used to manage fibrosis in hypertensive heart disease. Using immunoblotting, we established that moderate levels of H 2 O 2 activate ASK1 in neonatal rat cardiomyocytes and perfused rat hearts. ASK1 was activated during ischemia in adult rat hearts, but not on reperfusion, consistent with activation by moderate (not high) reactive oxygen species levels. In contrast, IL (interleukin)-1β activated an alternative kinase, TAK1 (transforming growth factor–activated kinase 1). ASK1 was not activated by IL1β in cardiomyocytes and activation in perfused hearts was due to increased reactive oxygen species. Selonsertib (ASK1 inhibitor) prevented activation of p38-MAPKs (but not JNKs) by oxidative stresses in cultured cardiomyocytes and perfused hearts. In vivo (C57Bl/6J mice with osmotic minipumps for drug delivery), selonsertib (4 mg/[kg·d]) alone did not affect cardiac function/dimensions (assessed by echocardiography). However, it suppressed hypertension-induced cardiac hypertrophy resulting from angiotensin II (0.8 mg/[kg·d], 7d), with inhibition of Nppa/Nppb mRNA upregulation, reduced cardiomyocyte hypertrophy and, notably, significant reductions in interstitial and perivascular fibrosis. Our data identify a specific reactive oxygen species→ASK1→p38-MAPK pathway in the heart and establish that ASK1 inhibitors protect the heart from hypertension-induced cardiac remodeling. Thus, targeting the ASK1→p38-MAPK nexus has potential therapeutic viability as a treatment for hypertensive heart disease.

Reactive oxygen species (ROS) play a key role in development of heart failure but, at a cellular level, their effects range from cytoprotection to induction of cell death. Understanding how this is regulated is crucial to develop novel strategies to ameliorate only the detrimental effects. Here, we revisited the fundamental hypothesis that the level of ROS per se is a key factor in the cellular response by applying different concentrations of H2O2 to cardiomyocytes. High concentrations rapidly reduced intracellular ATP and inhibited protein synthesis. This was associated with activation of AMPK which phosphorylated and inhibited Raptor, a crucial component of mTOR complex-1 that regulates protein synthesis. Inhibition of protein synthesis by high concentrations of H2O2 prevents synthesis of immediate early gene products required for downstream gene expression, and such mRNAs (many encoding proteins required to deal with oxidant stress) were only induced by lower concentrations. Lower concentrations of H2O2 promoted mTOR phosphorylation, associated with differential recruitment of some mRNAs to the polysomes for translation. Some of the upregulated genes induced by low H2O2 levels are cytoprotective. We identified p21Cip1/WAF1 as one such protein, and preventing its upregulation enhanced the rate of cardiomyocyte apoptosis. The data support the concept of a "redox rheostat" in which different degrees of ROS influence cell energetics and intracellular signalling pathways to regulate mRNA and protein expression. This sliding scale determines cell fate, modulating survival vs death.

Introduction: Akt signalling was delineated in the context of insulin stimulation and is cardioprotective. Akt is activated by insulin or insulin-like growth factor 1 (IGF1), acting through their receptors (IRs, IGF1Rs). Other receptors including α 1 -adrenergic receptors (α 1 ARs) activate Akt, but the mechanism is unclear. Hypothesis: We hypothesise that α 1 -ARs activate Akt via insulin receptor family receptors (InsRFs). Our aims were to establish if this is so, and if linsitinib [a highly selective inhibitor of InsRFs developed for cancer] affects cardiac responses to α 1 -AR agonists. Methods: Rat neonatal cardiomyocytes were exposed to or adult rat hearts perfused with α 1 AR agonists [50 nM A61603; 100 μM phenylephrine (PE)] with/without linsitinib. Effects on signalling were assessed by immunoblotting. Protein synthesis was assessed using 3 H-Phe. Effects of linsitinib (2 mg/kg/d) on cardiac function/hypertrophy induced by PE (40 mg/kg/d, 4 d) in mice (male C57Bl6; 10 wks) in vivo were determined. Echocardiography was used to assess cardiac function and pulmonary/aortic flow. Results: We identified a third InsRF in heart, the insulin receptor-related receptor (IRR). IRRs are alkali-sensitive and required for pH regulation in kidney. Alkaline pH (pH 9.0) activated IRRs and Akt, and increased protein synthesis in perfused hearts, being activated in a complex with IR/IGF1Rs. α 1 ARs activated Akt in cardiomyocytes and perfused hearts and increased protein synthesis. Linsitinib inhibited activation of Akt and the increase in protein synthesis induced by alkaline pH or α 1 AR agonists. Thus, α 1 ARs signal via InsRFs to Akt and protein synthesis. In perfused hearts, linsitinib enhanced the increase in developed pressure induced by A61603 and, in vivo, linsitinib enhanced PE-induced left ventricular (LV) hypertrophy with increased LV wall thickness and decreased internal diameter. Aortic flow was compromised in the presence of PE/linsitinib. Conclusions: Our data demonstrate a novel signalling paradigm in which α 1 -ARs signal through one or more InsRFs in cardiomyocytes to activate Akt and increase protein synthesis. However, functionally, the InsRF signal appears to mitigate some of the hypertrophic effects of α 1 -AR signalling in the whole heart.

Introduction: Raf kinases lie upstream of ERK1/2 with BRaf having the highest activity. ERK1/2 promote hypertrophy/cardioprotection, but the role of BRaf in the adult heart is unclear. BRaf mutations cause cancer and Raf inhibitors (Rafi) are used but, paradoxically, they can activate ERK1/2 and the cardiac consequences remain to be established. Hypothesis: We hypothesise that BRaf is critical in regulating ERK1/2 signaling in cardiomyocytes and Rafi affect cardiac function. Methods: We studied 8 wk wild-type male C57Bl/6 mice, or mice with cardiomyocyte-specific tamoxifen-inducible knock-in of activating BRaf(V600E) or BRaf knockout (BRafKO). V600E and BRafKO mice received vehicle or tamoxifen (40mg/kg; 6-12 mice per group). Mice were also treated with 0.8 mg/kg/d AngII, 0.5 mg/kg/d SB590885 (SB), 3 mg/kg/d dabrafenib (Dab) or vehicle delivered by osmotic minipumps. Echocardiography was performed and data for each mouse were normalized to the mean of 2 baseline control scans. Kinase activities and mRNA expression were assessed by immunoblotting and qPCR. Results: Knockin of BRaf(V600E) activated ERK1/2 and increased expression of ERK1/2 dependent genes (24h). By 7 d, there was increased ejection fraction (24%) and cardiac output (17%) with increased systolic left ventricular (LV) posterior wall (PW) thickness (13%). Cardiac function normalised by 10 d, but there was significant hypertrophy with increased LVPW in systole/diastole (11-19%) and decreased LV internal diameter. BRafKO alone did not affect cardiac function/dimensions or changes in function induced by AngII, but loss of BRaf inhibited the increase in systolic/diastolic LVPW (30% increase with AngII alone vs 9% with AngII/BRafKO). SB (type I Rafi), but not Dab (type 1.5 Rafi) activated cardiomyocyte ERK1/2 and induced hypertrophy in cultured neonatal rat ventricular myocytes. Consistent with this, SB (not Dab) promoted cardiac hypertrophy in vivo with increased LVPW (3d), but Dab (not SB) inhibited AngII-induced hypertrophy (7 d). Conclusions: Activation of cardiomyocyte BRaf promotes cardiac hypertrophy and cardiomyocyte BRaf is required for AngII-induced hypertrophy. However, Rafi developed for cancer have differential effects on the heart according to their mode of action.

BackgroundThe α1-adrenergic receptor (α1AR) agonist phenylephrine (PE), acts via ERK1/2 to phosphorylate (i.e. activate) p90 ribosomal S6 kinases (RSKs). PE promotes compensated hypertrophy and increases cardiomyocyte size. ERK1/2 and RSKs phosphorylate transcription factors producing changes in gene expression. In cardiomyocytes, potent ERK1/2 signalling causes nuclear-localisation of activated RSK1/RSK2 isoforms. This promotes cardiomyocyte growth, but hypertrophy is not sustained. Lesser ERK1/2 activation by PE causes nuclear activation of only RSK2, and this signalling is associated with a different gene expression profile. Our hypothesis is that activation of nuclear-localised RSK1 in addition to RSK2 promotes changes in gene expression that are not compatible with compensated hypertrophy induced by α1ARs. Our aim was to investigate if expression of nuclear-localised RSK1 is detrimental to the hypertrophic response induced in vivo by PE.Methods/resultsNuclear-localised (NL) RSK1 or RSK2 were expressed in cultured cells using adenoviral vectors. Immunostaining confirmed expression of NL-RSKs was confined to nuclei of HEK293, SVEC (endothelial cells) or neonatal rat cardiomyocytes. By immunoblotting with antibodies for phosphorylated (i.e. activated) RSKs, we confirmed that NL-RSKs were inactive in serum-starved cells, and activated following treatment with epidermal growth factor. We generated transgenic mice for tamoxifen-inducible, cardiomyocyte-specific expression of NL-RSK1 by inserting the transgene in the ROSA26 locus, separated from the promoter by a LoxP-flanked stop cassette. NL-RSK1 mice were crossed with mice with tamoxifen (Tam) inducible CRE under control of the α myosin heavy chain promoter, producing double heterozygotes and wild-type (WT) littermates for experiments (n=9 per group). Echocardiography was used to assess cardiac function/dimensions at baseline and following treatment. There was no significant difference between transgenic mice and WT litter mates up to 10 weeks of age in any of the variables studied. Tam-treatment (7 d) significantly increased diastolic (d) or systolic (s) left ventricular (LV) posterior wall (PW) thickness (16%) in transgenic, not wild-type (WT) mice indicating that enhanced NL-RSK1 alone promotes hypertrophy. PE (40 mg/kg/d, 3 d) increased LVPW~99% in WT or transgenic mice with corresponding decreases in LV internal diameters, but the degree of hypertrophy was significantly reduced (~45%) in transgenic mice pretreated (4 d) with Tam.ConclusionsIncreased nuclear-localised RSK1 in cardiomyocytes promotes cardiac hypertrophy, but compromises the compensated hypertrophic response to PE. Selective inhibition of RSK1 (rather than RSK2) may preserve the compensated hypertrophic state and, thus, reduce heart failure progression.

Background Stress-regulated mitogen-activated protein kinases (SR-MAPKs; i.e. p38-MAPKs and JNKs) are activated by various stresses in the heart, causing cardiac dysfunction and cardiomyocyte death. Consequently, pathway components are potential therapeutic targets for heart failure. Cardiac ischemia is associated with increased oxidative stress that is further enhanced on reperfusion (I/R). Oxidative stress (e.g. H2O2) activates SR-MAPKs in cardiomyocytes and perfused hearts. However, ischemia selectively activates p38-MAPKs, with activation of JNKs by I/R. SR-MAPKs are activated by specific MAPK kinases (MKKs), but upstream MAP3Ks that initiate the signal in different conditions are not well-defined. The MAP3K, ASK1, is an oxidative stress-responsive activator of SR-MAPKs and is required for cardiac hypertrophy, but its specific role in ischemia or I/R is not clear. Our aims were to determine if ASK1 signals to SR-MAPKs in the heart during ischemia and/or I/R, and establish downstream consequences of ASK1 signalling. Methods/results We confirmed that H2O2 activated SR-MAPKs in rat neonatal cardiomyocytes by immunoblotting with antibodies to the phosphorylated (activated) kinases. The concentration-dependence for ASK1 phosphorylation by H2O2 was bell-shaped, with maximal activation at 1 mM and reduced activation >1 mM H2O2. Rat hearts were perfused ex vivo and subjected to ischemia (15 min) or I/R (15/45 min). ASK1 was activated only during ischemia, consistent with moderate levels of oxidative stress being required. We did not detect activated ASK1 with I/R. Selonsertib (1 µM), a highly selective ASK1 inhibitor, suppressed activation of p38-MAPK (not JNKs) by H2O2 in cardiomyocytes or perfused hearts, or hearts subjected to ischemia or I/R. Thus, ASK1 signals selectively to p38-MAPK. The effects of 4 mg/kg/d selonsertib on mouse hearts (male C57Bl6; 10 weeks) in vivo were assessed with/without 0.8 mg/kg/d angiotensin II (AngII), a hypertensive model associated with increased oxidative stress (7 d; n=6–8). Echocardiography was used to assess cardiac function/dimensions. Selonsertib alone had no effect on any of the variables studied. AngII promoted cardiac hypertrophy with increased diastolic and systolic left ventricular posterior wall thickness. This was significantly inhibited by selonsertib indicating ASK1 is required in AngII-induced cardiac hypertrophy. Conclusions ASK1 is an oxidative-stress responsive MAP3K that signals selectively to p38-MAPK in cardiomyocytes in the context of moderate stress during ischaemia. High level oxidative stress as occurs on reperfusion does not activate ASK1 to a significant degree. Moreover, ASK1 activation in hypertension contributes to the hypertrophic response. By dissecting upstream activators of p38-MAPK and JNKs, it will become apparent which may be targeted under specific conditions to manipulate p38-MAPK and/or JNK signalling for the management of heart failure.

Introduction: ERK1/2 promote hypertrophy and are protective in the heart, but cause cancer in dividing cells. Raf kinases lie upstream of ERK1/2 and Raf inhibitors (e.g. SB590885 (SB), dabrafenib (Dab)) are in development/use for cancer. Paradoxically, in cancer cells, low concentrations of SB/Dab stimulate (rather than inhibit) ERK1/2. Hypothesis: Our hypothesis is that the heart is primed for Raf paradox signaling. Raf inhibitors have potential to activate ERK1/2 in cardiomyocytes and promote cardiac hypertrophy. Methods: Neonatal rat ventricular cardiomyocytes (NRVMs) were exposed to inhibitors. Dab or SB (3 or 0.5 mg/kg/d) were studied in 12 wk male C57Bl6 mice in vivo in the presence of angiotensin II (AngII, 0.8 mg/kg/d) (n=6-11) using osmotic minipumps. Effects were compared with vehicle controls. Echocardiography was performed (Vevo2100). M-mode images (short axis view) were analyzed; data for each mouse were normalized to the mean of 2 baseline controls. Kinase activities were assessed by immunoblotting or in vitro kinase assays. Results: SB (0.1 μM) or Dab (1 μM) activated ERK1/2 (2.3±0.1 fold; n=4) in NRVMs consistent with Raf paradox signaling. An explanation is that Raf kinases dimerise and submaximal inhibitor concentrations bind one Raf protomer, locking it in an active conformation but activating the partner. In accord with this, 0.1 μM SB increased Raf activities. High SB concentrations (1-10 μM) initially inhibited ERK1/2 in NRVMs, but ERK1/2 were then activated (1 - 24 h) and promoted hypertrophy. In vivo (24 h), Dab and SB activated the ERK1/2 cascade, increasing ANF (17.3 ± 3.1 fold) and BNP (4.5 ± 0.8 fold) mRNA (n=4/5). Over 3 d, Dab and SB increased fractional shortening in the presence of AngII (1.22±0.06; 1.17±0.08), relative to AngII alone (0.95±0.04), increased systolic left ventricular (LV) wall thickness, and reduced systolic LV volume and internal diameter (0.83±0.03 cf 0.97±0.02 for AngII alone). Conclusions: The heart is primed for Raf paradox signaling and Raf inhibitors activate ERK1/2 in cardiomyocytes, promoting hypertrophy. In vivo, Raf inhibitors enhance ERK1/2 signaling and hypertrophy in the context of hypertension, and cardiac hypertrophy may be increased in hypertensive cancer patients receiving Raf inhibitors.