Biology subjects

Clerk, A.

Publications and source records attributed to Clerk, A..

7 recordsLinked to original sources

bioRxiv2026

PKNs are required for cardiac hypertrophy induced by angiotensin II in mice

Of the three PKN isoforms, only homozygous Pkn2 knockout in mice is embryonic lethal; mice with Pkn2 haploinsufficiency or Pkn1/3 double knockout (Pkn1/3DKO) have no overt abnormalities. In adult mice, Pkn1/2 are implicated in development of cardiac disease, but studies of Pkn3 in the heart are lacking. Our hypothesis was that a role for Pkn1/3 in adult hearts would be revealed by pathophysiological stress. Here, we report expression profiles of PKNs in cardiac cells, identifying PKN2 as the dominant isoform with expression of PKN3 largely restricted to endothelial/immune cells. To test the hypothesis, Pkn1/3DKO, heterozygous Pkn2 knockout (Pkn2Het) and wild-type mice were treated with 0.8 mg/kg/d AngII, and cardiac function/dimensions assessed using speckle-tracking echocardiography. Baseline assessment identified minor differences in the left ventricle (LV) basal region in Pkn2Het (not Pkn1/3DKO) vs wild-type mice. AngII induced similar increases in LV end diastolic mass (LVEDM) in wild-type and Pkn1/3DKO mice at 3 d. LVEDM increased further in wild-type mice by 7d, but the initial response in Pkn1/3DKO mice was lost whilst stroke volume and cardiac output declined. Thus, hypertrophy was initiated in Pkn1/3DKO mouse hearts but not sustained. AngII increased cardiac fibrosis in wild-type mice together with mRNAs for cardiomyocyte hypertrophy and matrix proteins; this was suppressed in Pkn1/3DKO mice. Changes in expression of mRNA markers for cardiac non-myocytes suggested this was associated with vascular and immune cell dysfunction. In conclusion, Pkn2 does not substitute for Pkn1/3 in developing cardiac hypertrophy, and Pkn1/3 have an independent role in AngII-induced cardiac hypertrophy.

biochemistry↗

bioRxiv2026

Epidermal growth factor (EGF) receptor family signalling in cardiomyocyte hypertrophy and heart failure

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 perspectivesO_LIAnti-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. C_LIO_LIThe 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. C_LIO_LIThe 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. C_LI

biochemistry↗

bioRxiv2025

The anti-cancer drug trametinib suppresses angiotensin-induced cardiac remodelling in mice but is detrimental to function

Aims. Echocardiography is used widely in preclinical mouse studies, but the emphasis remains the histological/pathological/biochemical changes in the myocardium. Analysis/reporting of cardiac function is generally limited, often relying on M-mode assessment of a single plane across the left ventricle. The aim was to determine if global/segmental endocardial speckle-tracking (strain) has greater potential to assess function by distinguishing between mouse lines and identifying regional effects of different drugs. Methods and results. Echocardiograms from male C57Bl/6J (commercially-available) or C57Bl/6(R) (bred in-house) mice treated with vehicle or angiotensin II (AngII; 0.8 mg/kg/d, 7 d) were analysed. Global strain demonstrated different degrees of hypertrophy induced by AngII in the different lines, and variation in function (e.g. stroke volume/cardiac output were reduced in C57Bl/6J mice only, with no effect on global longitudinal strain). For C57Bl/6J mice, segmental strain (radial/longitudinal peak displacement/velocity/strain/strain rate) and frame-to-frame analysis of radial/longitudinal displacement identified more significant effects of AngII in the basal/mid-regions of the left ventricle. Thus, speckle-tracking distinguished between responses in different mouse lines. The methodology was applied to studies of anti-cancer drugs that inhibit extracellular signal-regulated kinase 1/2 signalling. Dabrafenib and/or trametinib inhibited AngII-induced cardiac hypertrophy, but dabrafenib alone caused abnormalities in endocardial movement whilst, for trametinib with AngII, more abnormalities were detected than with AngII alone. For both, the dominant effect was in the basal/mid-regions of the left ventricle. Conclusion. Echocardiography in preclinical studies can be exploited using endocardial segmental strain for greater insight into how drugs affect cardiac function in different regions of the left ventricle.

physiology↗

bioRxiv2023

Striatin plays a major role in angiotensin II-induced cardiomyocyte and cardiac hypertrophy in mice in vivo.

The three striatins (STRN, STRN3, STRN4) form the core of STRiatin-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 upregulation 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. Clinical perspectivesO_LIBackground. Striatins form the core of STRiatin-Interacting Phosphatase And Kinase (STRIPAK) complexes that regulate crucial cellular processes such as those associated with heart failure. C_LIO_LISummary. The three striatins are expressed in human hearts, with upregulation of STRN and STRN3 in failing hearts, whilst studies in mice indicate that STRN is required in cardiomyocytes for early remodelling of the hypertensive heart. C_LIO_LIPotential significance of results to human health and disease. STRN-based STRIPAKs represent a novel signalling paradigm in the development of pathological cardiac hypertrophy, and modulating this system may provide therapeutic options for managing the cardiac effects of hypertensive heart disease. C_LI

physiology↗

bioRxiv2022

Cardiomyocyte BRAF is a key signalling intermediate in cardiac hypertrophy in mice

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 ERK1/2 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/function were assessed 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 to 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. Clinical perspectivesO_LIBackground. BRAF is a key signalling intermediate that causes cancer and is upregulated in heart failure, but its role in physiological and pathological cardiac hypertrophy remains to be established. C_LIO_LISummary. Cardiomyocyte BRAF is required in male mice for hypertrophy and contributes to interstitial fibrosis in hypertension induced by angiotensin II, but it increases contractility and suppresses fibrosis in physiological hypertrophy induced by 1-adrenergic receptor stimulation with phenylephrine. Differences between males and females are highlighted in the phenylephrine response. C_LIO_LIPotential significance of results to human health and disease. BRAF is a key signalling node in both pathological and physiological hypertrophy: inhibiting BRAF may be beneficial in pathological hypertrophy and the data have implications for repurposing of RAF inhibitors developed for cancer; inhibiting BRAF in physiological hypertrophy may result in increased fibrosis and using RAF inhibitors in this context could be detrimental in the longer term. C_LI

physiology↗

bioRxiv2022

PKN2 deficiency leads both to prenatal congenital cardiomyopathy and defective angiotensin II stress responses.

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

developmental biology↗

bioRxiv2021

Cardiomyocyte BRAF and type 1 RAF inhibitors promote cardiomyocyte and cardiac hypertrophy in mice in vivo

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, if 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 upregulated 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 BRAFV600E 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.

biochemistry↗