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.

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.

Introduction The ERK1/2 cascade, a key pathway involved in cardiac remodelling, is regulated by RAF kinases. Small molecule inhibitors of RAF have been developed due to activating oncogenic mutations, however paradoxical activity has been seen in early generations of inhibitors. Therefore, ‘paradox breaker’ inhibitors (e.g. PLX8394) have been developed and are undergoing clinical trials. Here, we investigated the effects of PLX8394 on vascular ERK1/2 signalling in vitro and on hypertensive cardiac remodelling in vivo. Methods Murine endothelial cells (ECs) or human cardiac fibroblasts (HCFs) were incubated with PLX8394 and effects on RAF-ERK1/2 pathway activity determined by western blotting, with effects on cell migration and proliferation assessed via wound healing and BrdU assays. For in vivo characterisation, PLX8394 (5mg/kg/d) was infused with/without angiotensin-II (AngII; 0.8mg/kg/d) for 7 days by osmotic minipumps in male wildtype C57Bl/6J mice (n=8–11/group). Cardiac function/dimensions were assessed using echocardiography; effects on cardiac morphology were assessed by histological staining. mRNA expression was assessed by qPCR. Statistical tests used 1-way ANOVA with Holm-Sidak’s post-test. Results PLX8394 (5 min; 1uM) activated ERK1/2 (n=3; p=0.018) pathway via CRAF (n=3; p=0.047) in ECs with no change seen in BRAF activity. This was accompanied by increased BrdU incorporation (n=6; p=0.0002; p=0.0009) but significantly inhibited migration (n=6; p<0.0001; p<0.0001) both at baseline and with AngII (100nM), respectively. In HCFs however, PLX8394 had no effect on baseline or AngII migration (n=4; p=0.99; p=0.98) or BrdU incorporation (n=6; p=0.95; p=0.65). In vivo, PLX8394 did not alter the AngII-induced cardiac hypertrophy with maintained wall thickness to internal diameter ratio (p=0.45). While PLX8394 was able to significantly reduce cardiomyocyte cross sectional area (p=0.0068), no changes were seen in Myh7, Nppa or Nppb mRNAs. Moreover, PLX8394 did not significantly alter the perivascular (p=0.69) or interstitial (p=0.052) fibrotic area with no changes in mRNA expression of collagens1–4. Conclusion PLX8394, despite development as a cancer cell ‘paradox breaker’, activates ERK1/2 signalling in ECs, but not HCFs. In vivo, PLX8394 had minimal effect on hypertensive cardiac fibrotic remodelling despite reducing myocyte hypertrophy, likely reflecting a cell-type dependent response. Thus, paradox-breaker RAF inhibitors, currently in clinical trials for RAF-mutant cancers, may have limited viability as hypertension therapies.

The protein kinase PKN2 is required for embryonic development and PKN2 knockout mice die as a result of failure in the expansion of mesoderm, cardiac development and neural tube closure. In the adult, cardiomyocyte PKN2 and PKN1 (in combination) are required for cardiac adaptation to pressure-overload. The specific role of PKN2 in con-tractile cardiomyocytes during development and its role in the adult heart remain to be fully established. We used mice with cardiomyocyte-directed knockout of PKN2 or global PKN2 haploinsufficiency to assess cardiac development and function using high reso-lution episcopic microscopy, MRI, micro-CT and echocardiography. Biochemical and histological changes were also assessed. Cardiomyocyte-directed PKN2 knockout embryos displayed striking abnormalities in the compact myocardium, with frequent myo-cardial clefts and diverticula, ventricular septal defects and abnormal heart shape. The sub-Mendelian homozygous knockout survivors developed cardiac failure. RNASeq data showed up-regulation of PKN2 in patients with dilated cardiomyopathy, suggesting an involvement in adult heart disease. Given the rarity of homozygous survivors with cardio-myocyte-specific deletion of PKN2, the requirement for PKN2 in adult mice was explored using the constitutive heterozygous PKN2 knockout. Cardiac hypertrophy resulting from hypertension induced by angiotensin II was reduced in these haploinsufficient PKN2 mice relative to wild-type littermates, with suppression of cardiomyocyte hypertrophy and cardiac fibrosis. It is concluded that cardiomyocyte PKN2 is essential for heart develop-ment and the formation of compact myocardium and is also required for cardiac hyper-trophy in hypertension. Thus, PKN signalling may offer therapeutic options for managing congenital and adult heart diseases.

Hypertension is a major public health concern and poses a significant risk for sudden cardiac death (SCD). However, the characterisation of human tissues tends to be macroscopic, with little appreciation for the quantification of the pathological remodelling responsible for the advancement of the disease. While the components of hypertensive remodelling are well established, the timeline and comparative quantification of pathological changes in hypertension have not been shown before. Here, we sought to identify the phasing of cardiac remodelling with hypertension using post-mortem tissue from SCD patients with early and advanced hypertensive heart disease (HHD). In order to study and quantify the progression of phenotypic changes, human specimens were contrasted to a well-described angiotensin-II-mediated hypertensive mouse model. While cardiomyocyte hypertrophy is an early adaptive response in the mouse that stabilises in established hypertension and declines as the disease progresses, this finding did not translate to the human setting. In contrast, optimising fibrosis quantification methods and applying them to each setting identified perivascular fibrosis as the prevailing possible cause for overall disease progression. Indeed, assessing myocardial inflammation highlights CD45+ inflammatory cell infiltration that precedes fibrosis and is an early-phase event in response to elevated arterial pressures that may underscore perivascular remodelling. Along with aetiology insight, we highlight cross-species comparison for quantification of cardiac remodelling in human hypertension. As such, this platform could assist with the development of therapies specific to the disease phase rather than targeting global components of hypertension, such as blood pressure lowering.

Introduction: The ERK1/2 cascade, activated by RAF kinases, is a key pathway for cardiac remodelling and cytoprotection. Due to activating oncogenic mutations in BRAF, small molecule RAF inhibitors have been developed. However, owing to inhibitor resistance or paradoxical ERK1/2 pathway activation, a new generation of ‘paradox breaking’ drugs are currently in clinical trials. Our previous work characterised RAF targeting in hypertension, where it is instrumental in driving cardiac remodelling. Here we explored whether use of the RAF paradox breakers (e.g. PLX8394) are viable modalities for targeting RAF in hypertension. Hypothesis: We hypothesize that RAF ‘paradox breakers’ will protect cardiac function by inhibiting maladaptive hypertensive remodelling. Methods & Results: To assess the role of RAF inhibition in cardiac adaptation to hypertension in vivo , C57Bl/6J mice were treated with angiotensin-II (AngII; 0.8mg/kg/d, 7d n=8;) without/with 5mg/kg/d PLX8394 (n=11). PLX8394 alone had no detrimental effect on cardiac function or remodelling. AngII promoted cardiac hypertrophy, by increasing left ventricular (LV) wall thickness (WT) and decreasing LV internal diameter (ID; assessed by echocardiography). PLX8394 had no impact on AngII-induced hypertrophy (WT:ID, p=0.45), or abundance of AngII-induced hypertrophic Nppa (p=0.77) or Nppb (p=0.49) mRNAs. Additionally, PLX8394 did not affect AngII-induced perivascular fibrosis (p=0.69; picrosirius red histology) or moderate Col1a1 (p=0.22) and Col3a1 (p=0.82) mRNAs. However, PLX8394 use in AngII-hypertension was pro-inflammatory, with increased pro-inflammatory cytokines ( Il1b , p=0.02; Il6 , p=0.008) and the inflammatory cell marker CD45 (p=0.001) mRNAs; a response echoed in hearts labelled by immunohistochemistry for CD45 (p=0.02). Moreover, western blotting revealed elevated PARP cleavage (p=0.005), indicating increased apoptosis with PLX8394 use. Conclusion: Despite no adverse effect on cardiac function or global hypertensive remodelling at 7 days, RAF inhibition by PLX8394 appears to be pro-inflammatory and pro-apoptotic. Thus, long-term use of RAF ‘paradox breakers’ in hypertensive individuals may be cardiotoxic and promote adverse cardiac events.

Introduction RAF kinases activate the ERK1/2 cascade, a key pathway involved in cardiac remodelling and cytoprotection. Since activating mutations in BRAF cause cancer, small molecule inhibitors of RAF have been developed. However, a paradoxical effect is observed with some inhibitors which activate rather than inhibit ERK1/2. Therefore, ‘paradox breaker’ inhibitors have been developed as new-generation cancer therapies void of this effect (e.g. PLX8394). Here, we determined the effects of PLX8394 on endothelial cell (EC) ERK1/2 signalling and the heart in vivo. Methods Murine ECs were incubated with PLX8394 and effects on ERK1/2 activity determined by western blotting for the phosphorylated (i.e. activated) kinases. Effects on gene expression were determined by qPCR. The effects of PLX8394 on the heart in vivo were determined by infusing male wildtype C57Bl/6J mice (10-12wks, n= 6/group) with PLX8394 (5mg/kg/d, 7d) using osmotic minipumps. Cardiac function/dimensions were assessed using echocardiography; effects on cardiac morphology were assessed by histological staining. mRNA expression was assessed by qPCR. Statistical tests used 1-way ANOVA with Holm-Sidak’s post-test (in vitro studies) and unpaired t-tests (in vivo studies). Results PLX8394 activated ERK1/2 in ECs in a time (7.4±2.3-fold at 5 min; p=0.0365; n=5) and concentration (>1uM; p=0.0625; n=3) dependent manner. This was associated with significant increases in expression of mRNAs encoding the immediate early gene Fos (6.1±2.6-fold; p<0.0001; n=4) and the vasoconstrictor peptide endothelin-1 (Edn1) (2.7±0.9-fold; p=0.0038; n=4). In vivo, PLX8394 decreased cardiac output (p=0.0092), predominantly through reduced stroke volume (p=0.0103). Structurally, PLX8394 promoted cardiac hypertrophy, with increased diastolic left ventricular (LV) posterior wall thickness (p=0.0425) and decreased LV internal diameter (p=0.0463) at 7 d. Cardiac hypertrophy resulted from increased cardiomyocyte cross-sectional area (p=0.0002) despite no changes in Myh7, Nppa or Nppb mRNAs. Moreover, PLX8394-induced cardiac remodelling was not due to increased fibrosis, with no change in mRNA expression of collagens1-4 and using histological assessment. Conclusion Despite being developed as a ‘paradox breaker’ for cancer, PLX8394 promoted ERK1/2 signalling in murine ECs and cardiac remodelling in vivo. These preliminary findings suggest that such inhibitors, currently in Phase 3 trials for RAF-mutant cancers, have potential to modulate cardiac function in patients. Conflict of Interest N/A

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.

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.

Hypertensive heart disease refers to changes in the myocardium that result from hypertension. The relationship between hypertensive heart disease and sudden cardiac death is well established, but there are few pathological studies. We examined the clinical and pathological features of hypertensive heart disease in sudden cardiac death victims from a national cardiovascular pathology registry. We investigated 5239 cases of sudden cardiac death between 1994 and 2018. Hearts were examined by two expert cardiac pathologists. Diagnostic criteria included history of hypertension, increased heart weight and left ventricular wall thickness in the absence of other causes. Collagen was quantified using picrosirius red staining and imaging software. Of 75 sudden cardiac death cases due to hypertensive heart disease (age at death: 54 ± 16 years; 56% males), 56 (75%) reported no prior cardiac symptoms. Thirty-four (45%) recorded a BMI ≥ 30. Only two (2.7%) had hypertensive heart disease diagnosed antemortem. Four (5%) were diagnosed clinically with hypertrophic cardiomyopathy, but lacked myocyte disarray at autopsy. All hearts showed concentric left ventricular hypertrophy and myocyte hypertrophy. Fibrosis was identified microscopically in 59 cases (81%). The posterior left ventricular wall showed the greatest increase in the percentage of collagen in hypertensive diseased hearts compared to controls (25.2% vs 17.9%, p = 0.034). Most sudden deaths due to hypertensive heart disease occur without prior cardiac symptoms; thus, clinical risk stratification is challenging. Hypertensive heart disease can be misdiagnosed in life as hypertrophic cardiomyopathy which has major implications for relatives. Pathologists require a history of hypertension and histology for a definitive diagnosis of hypertensive heart disease.

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.

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.

OBJECTIVE:Endocarditis is increasing in incidence due to growing numbers of cardiac interventions, valve replacements and immunosuppressants. It can be difficult to diagnose clinically, has high mortality and can present as sudden cardiac death (SCD) with few/subtle preceding symptoms. True incidence of SCD related to endocarditis is unknown. METHODS:Retrospective analysis of UK national database of 6000 cases of SCD, 1994-2020, for "endocarditis" as cause of death. RESULTS:Of 30 cases (0.50%), 19(63%) were male and mean age was 36.2 ± 20.1 years. Postmortem examination showed the aortic valve was solely affected in 13 (43%), mitral in 9 (30%), tricuspid in 2(6.7%) and pulmonary in 1 (3.3%). Three cases (10%) had more than one valve affected and 2 (6.7%) were nonvalvular affecting the ascending aorta. Vegetations ranged from small easily missed irregularities to large fungating masses. Ten (33%) patients developed aortic abscesses, 2 of which had aneurysms, 13 (43%) had coronary artery septic emboli with micro-abscesses and myocardial microinfarction, and 2 (6.7%) were healed endocarditis with perforation and regurgitation with ventricular remodeling. Thirteen (43%) had an identifiable underlying valve abnormality or replacement, most common being a bicuspid aortic valve (7; 54%). CONCLUSIONS:This study highlights that although rare, endocarditis is an important cause of SCD in those with normal valves, valvular disease and valve replacement surgery. Absence of a premortem diagnosis in 70% of our cohort highlights the need for detailed analysis of the heart and cardiac valves at autopsy. Gross appearance of vegetations varies widely and can be missed. Awareness of associated cardiac complications is required for elucidation of the cause of death and will provide valuable lessons for clinicians.

Congenital heart disease is a rare but important finding in adults who experience sudden death. Examination of the congenitally malformed heart has historically been considered esoteric and best left to those with expertise. The Cardiac Risk in the Young cardiovascular pathology laboratory based at St George's University of London has now received over 6,000 cases. Of these, 21 congenitally malformed hearts were retained for research and educational purposes. Hearts were assessed using sequential segmental analysis, and causes of death were adjudicated based on thorough macroscopic examination and histology. Congenital malformations that were encountered included atrial septal defects, ventricular septal defects, tetralogy of Fallot, and transposition of the great arteries in both its regular and congenitally corrected variants. Findings also included hearts with mirror‐imaged and isomeric atrial appendages. Direct causes of death included myocardial fibrosis, pulmonary hypertension, and hemorrhage. A small but notable proportion did not reveal a substrate for arrhythmia, raising the question of whether the terminal event was due to the congenital heart disease itself, or an underlying channelopathy. Here, we demonstrate the value of simple sequential segmental analysis in describing and categorizing the cases, with the concept of the “morphological method” serving to identify the distinguishing features of the cardiac components. Clin. Anat. 33:394–404, 2020. © 2019 Wiley Periodicals, Inc.

We appreciate the interest in commotio cordis expressed by Drs. Lupariello and Di Vella from Turin, Italy. As this entity emerged as a new cause of unexpected sudden death in young people (athletes and nonathletes alike), the criteria and definition for such events has been paramount to victims, families, researchers, and the legal community.