Pyroptosis, the most inflammatory form of cell death, is dependent on membrane pore formation governed by the assembly of cleaved Gasdermin D (GSDMD). We hypothesized that regulated necrosis pathways are crucial in the pathophysiology of acute kidney injury (AKI). Mice with an isoleucine‐to‐asparagine loss‐of‐function mutation in the Gasdermin D gene (GSDMD I105N/I105N ) generated by ethylnitrosourea‐mutagenesis were subjected to bilateral renal ischemia–reperfusion injury (IRI) with bio‐molecular readouts performed at 24 h. IRI was also performed in mice pretreated with disulfiram. Whole‐body irradiation followed by syngeneic bone marrow transplantation generated chimeric mice prior to IRI. Mice homozygous for the GSDMD I105N mutation were protected from IRI, demonstrating lower serum creatinine and reduced histological injury, as well as decreased pro‐inflammatory cytokine expression and oxidative stress. Chimeric mice showed that this protection was predominantly governed by mutations in the parenchymal tissue, with a potential contribution from the hematopoietic compartment. Pharmacological inhibition of GSDMD pore formation using disulfiram protected against IRI. Manipulation of GSDMD is an attractive target to mitigate inflammation and cellular death following AKI.

Cardiovascular disease causes vascular dementia and contributes to most clinical dementia. This is embodied in the concept of vascular contributions to cognitive impairment and dementia (VCID). The potent endogenous peptide endothelin-1 (ET1) causes small artery vasoconstriction and fibrosis. ET1 is implicated in microvascular disease and in VCID. There are few experimental animal models relevant to VCID [Hainsworth et al. 2017]. Pigs are higher mammals with a gyrencephalic brain and extensive subcortical white matter. We engineered domestic pigs carrying additional copies of the ET1-encoding gene EDN1 under a Tet-ON promotor, by lentiviral injection into blastocysts. We induced transgene expression for up to 8 days using oral doxycycline in young adults. We studied ET1-overexpressing (n = 6, 3F/3M, mean±SD age 184±61 days) and control animals (n = 5, 3F/2M, 149±41 days). Following doxycycline treatment we observed a wide range of transgene expression at mRNA level. Antibody labelling indicated a spectrum of ET1 abundance in brain and heart tissue. Heart weight was 460±106 g in ET1-overexpressing pigs and 394±25 g in controls. Brain weight: 104±14.4 g in ET1-overexpressing, 101±9.0 in controls. We will report phenotypes relevant to inflammation and blood vessel fibrosis. Adult-onset EDN1 induction in domestic pigs produces ET1 overexpression that is well-tolerated to 8 days. Reference : Hainsworth AH, et al. (2017) BMC Med . 15 (1):16. Translational models for vascular cognitive impairment: a review including larger species.

Patients with chronic kidney disease (CKD) face a high risk of cardiovascular disease. Previous studies reported that endogenous thrombospondin 1 (TSP1) involves right ventricular remodeling and dysfunction. Here we show that a murine model of CKD increased myocardial TSP1 expression and produced left ventricular hypertrophy, fibrosis, and dysfunction. TSP1 knockout mice were protected from these features. In vitro, indoxyl sulfate is driving deleterious changes in cardiomyocyte through the TSP1. In patients with CKD, TSP1 and aryl hydrocarbon receptor were both differentially expressed in the myocardium. Our findings summon large clinical studies to confirm the translational role of TSP1 in patients with CKD.

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.

Hypertensive remodeling involves upregulation of fibrotic players, such as the thrombospondin-1 (TSP1). TSP1, via CD47, is anti-angiogenic; however, whether this involves inactivation of remodeling-linked MAPK cascades is unclear. Dual-specific phosphatases (DUSPs) negatively regulate MAPKs, evoking an added control of remodeling. Here we sought to investigate DUSPs regulated by CD47/hypertension signaling and explore any potential links between DUSP expression and cardiovascular phenotypes including dilated (DCM) vs hypertrophic (HCM) cardiomyopathy, and ejection fraction (EF). Methods: DUSPs were assessed in 1) endothelial cells (ECs) challenged with CD47-activating peptide 7N3 (10μM), and 2) hypertensive C57BL/6J male mice (10 wks) infused vehicle (acidified-PBS, n=10) or angiotensin II (AngII; 0.8 mg/kg/d; n=10). DUSP mRNA or protein expression was assessed by qPCR or immunoblotting, respectively. Statistical tests used a 1-way ANOVA with Holm-Sidak post-test. Human DUSP vs heart failure relationships were explored using a two-sample Mendelian Randomization (MR) framework and tissue-specific expression quantitative trait loci (eQTLs) from 5 cardiovascular tissues: left ventricle, atrial appendage, aorta, coronary artery, and tibial artery; comparing DUSP mRNA levels across DCM, HCM, and EF. Statistical significance is defined at a Bonferroni-corrected threshold of p<0.05. Results: Infusion with Angll increased cardiac and vessel TSP1 levels, but not CD47. In ECs, 7N3 activated ERK and p38 MAPKs, with induction of DUSP1/4/5/6 (all >2-fold; p<0.01). In contrast, CD47 signaling decreased nuclear ERK-regulating DUSP2 levels (p<0.05). Interestingly, the EC DUSP2 response was absent in cardiac tissue homogenates, where DUSP2 was elevated at both the RNA (2.3±0.5-fold, p<0.0001) and protein (3.1±0.4-fold, p<0.001) levels. MR identified that elevated DUSP2 expression in the aorta associates with increased EF and decreased risk of HCM. There are no signals significant (p<0.05/28) in DCM. Conclusion: DUSPs fine-tune ERK1/2 signaling in ECs which contrasts the cardiac response. Computational evidence suggests DUSP2 causes a net downstream response that increases EF and is protective of adverse remodeling preceding HCM.

Nuclear factor κB (NF-κB) activation is a deleterious molecular mechanism that drives acute kidney injury (AKI) and manifests in transplanted kidneys as delayed graft function. The TNFAIP3 gene encodes A20, a cytoplasmic ubiquitin ligase and a master negative regulator of the NF- κB signaling pathway. Common population-specific TNFAIP3 coding variants that reduce A20's enzyme function and increase NF- κB activation have been linked to heightened protective immunity and autoimmune disease, but have not been investigated in AKI. Here, we functionally identified a series of unique human TNFAIP3 coding variants linked to the autoimmune genome-wide association studies single nucleotide polymorphisms of F127C; namely F127C;R22Q, F127C;G281E, F127C;W448C and F127C;N449K that reduce A20's anti-inflammatory function in an NF- κB reporter assay. To investigate the impact of TNFAIP3 hypomorphic coding variants in AKI we tested a mouse Tnfaip3 hypomorph in a model of ischemia reperfusion injury (IRI). The mouse Tnfaip3 coding variant I325N increases NF- κB activation without overt inflammatory disease, providing an immune boost as I325N mice exhibit enhanced innate immunity to a bacterial challenge. Surprisingly, despite exhibiting increased intra-kidney NF- κB activation with inflammation in IRI, the kidney of I325N mice was protected. The I325N variant influenced the outcome of IRI by changing the dynamic expression of multiple cytoprotective mechanisms, particularly by increasing NF- κB-dependent anti-apoptotic factors BCL-2, BCL-XL, c-FLIP and A20, altering the active redox state of the kidney with a reduction of superoxide levels and the enzyme super oxide dismutase-1, and enhancing cellular protective mechanisms including increased Foxp3+ T cells. Thus, TNFAIP3 gene variants represent a kidney and population-specific molecular factor that can dictate the course of IRI.

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.

Background: Cerebral small vessel disease (SVD) is common in older people and causes lacunar stroke and vascular cognitive impairment. Risk factors include old age, hypertension and variants in the genes COL4A1/COL4A2 encoding collagen alpha-1(IV) and alpha-2(IV), here termed collagen-IV, which are core components of the basement membrane. We tested the hypothesis that increased vascular collagen-IV associates with clinical hypertension and with SVD in older persons and with chronic hypertension in young and aged primates and genetically hypertensive rats. Methods: We quantified vascular collagen-IV immunolabeling in small arteries in a cohort of older persons with minimal Alzheimer pathology (N=52; 21F/31M, age 82.8±6.95 years). We also studied archive tissue from young (age range 6.2–8.3 years) and older (17.0–22.7 years) primates ( M mulatta ) and compared chronically hypertensive animals (18 months aortic stenosis) with normotensives. We also compared genetically hypertensive and normotensive rats (aged 10–12 months). Results: Collagen-IV immunolabeling in cerebral small arteries of older persons was negatively associated with radiological SVD severity (ρ: −0.427, P =0.005) but was not related to history of hypertension. General linear models confirmed the negative association of lower collagen-IV with radiological SVD ( P <0.017), including age as a covariate and either clinical hypertension ( P <0.030) or neuropathological SVD diagnosis ( P <0.022) as fixed factors. Reduced vascular collagen-IV was accompanied by accumulation of fibrillar collagens (types I and III) as indicated by immunogold electron microscopy. In young and aged primates, brain collagen-IV was elevated in older normotensive relative to young normotensive animals ( P =0.029) but was not associated with hypertension. Genetically hypertensive rats did not differ from normotensive rats in terms of arterial collagen-IV. Conclusions: Our cross-species data provide novel insight into sporadic SVD pathogenesis, supporting insufficient (rather than excessive) arterial collagen-IV in SVD, accompanied by matrix remodeling with elevated fibrillar collagen deposition. They also indicate that hypertension, a major risk factor for SVD, does not act by causing accumulation of brain vascular collagen-IV.

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.

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.

mitochondria was examined in the endothelium in intact blood vessels. In controls, TRPV4 activation with GSK1016790A(GSK) generated repetitive Ca2+ oscillations that required Ca2+ influx. When the Dy m was depolarised, by the uncoupler carbonyl cyanide m-chlorophenyl hydrazine (CCCP) or the complex I inhibitor rotenone, TRPV4 activation generated a much larger Ca2+ rise and propagating multicellular Ca2+ waves. The ATP synthase inhibitor oligo-mycin did not potentiate TRPV4 mediated Ca2+ influx. GSK-evoked Ca2+ waves, that occurred when mitochondria were depolarised, persisted in a Ca2+ free extracellular solu-tion i.e. were independent of Ca2+ influx. These signals were blocked by the TRPV4 channel blocker HC067047 (HC067), the SERCA inhibitor cyclopiazonic acid, the phos-pholipase C (PLC) blocker U73122 and the inositol triphos-phate receptor (IP3R) blocker caffeine. These observations suggest that TRPV4 may directly activate Ca2+ release from the internal store. The large propagating waves were inhib-ited by the pannexin blocker probenecid and the extracellular ATP blockers suramin and apyrase. These results highlight a previously unknown role of mitochondria in shaping TRPV4 mediated Ca2+ signalling and show that TRPV4 may trigger ATP release via a pannexin hemichannel when mitochondria are depolarised.

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.