Role of angiotensin-signalling in anthracycline-induced cardiotoxicity

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
S Findlay ◽  
J.H Gill ◽  
R Plummer ◽  
C.J Plummer
Keyword(s):  
Gene Expression ◽  
Heart Failure ◽  
Angiotensin Ii ◽  
Late Onset ◽  
Left Ventricular Systolic Dysfunction ◽  
Left Ventricular ◽  
Cardiomyocyte Hypertrophy ◽  
Funding Source ◽  
Study Results

Abstract   Anthracycline chemotherapy remains a key component of cancer treatment regimens in both paediatric and adult patients. A significant issue with their use is the development of anthracycline-induced cardiotoxicity (AIC), with subclinical AIC and clinical heart failure observed in 13.8% and 3.1% of patients, respectively. The major clinical complication of AIC is the development of late-onset cardiotoxicity, occurring several years after drug administration, presenting as life-threatening heart failure (HF). Determining the relationship between subclinical AIC and late-onset HF, strategies for mitigation of AIC, and impacts upon the cancer survivor population remains a complex challenge. Administration of drugs targeting the angiotensin system, specifically angiotensin converting enzyme inhibitors (ACEi), have been reported to reduce AIC in the clinic. Whilst the therapeutic effect of ACEi in management of left ventricular systolic dysfunction and consequent HF is principally through optimisation of cardiac haemodynamics, the mechanism involved with mitigation of late-onset AIC several years after anthracycline exposure are currently unknown. Using a variety of human cardiomyocyte in vitro models we have previously demonstrated induction of cardiomyocyte hypertrophy by angiotensin II and anthracyclines. Importantly, selective blockade of the angiotensin II receptor 1 (ATR1) on cardiomyocytes mitigated the anthracycline-induced hypertrophic response, implicating synergism between AIC and angiotensin signalling in cardiomyocytes. Adult human ventricular cardiac myocyte AC10 cell-line were treated in vitro with a range of clinically relevant doxorubicin doses for clinically appropriate durations, with AT1 receptor gene expression evaluated using semi-quantitative PCR. Our results confirm a positive correlation between clinically-relevant concentration of doxorubicin and induction of genetic expression of ATR1 in AC10 cells, with up to 200% increases in ATR1 expression observed. Maximal doxorubicin-induced gene expression being observed at 8 and 24-hours, respectively. These preliminary results agreeing with clinical exposure parameters for this drug with protein expression studies being optimised to support these gene expression study results. Our preliminary studies also imply patients developing AIC carry a deleted polymorphism within intron 16 of the ACE gene and increased systemic levels of the ACE product angiotensin II, both with a known association to hypertrophic cardiomyopathy. Taken together, these data support our mechanistic hypothesis that a relationship exists between AIC and modulation of the angiotensin signalling pathway in cardiomyocytes, involving structural cellular changes and asymptomatic cardiac hypertrophy. An elevation in angiotensin II levels, potentially through polymorphisms in ACE, could thereby exacerbate anthracycline-induced hypertrophy and promote the development of late-onset anthracycline-induced HF. Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): Cancer Research UK funded PhD

scholarly journals The role of angiotensin signalling in anthracycline-induced cardiotoxicity

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
S Findlay ◽  
C J Plummer ◽  
R Plummer ◽  
J H Gill
Keyword(s):  
Gene Expression ◽  
Heart Failure ◽  
Angiotensin Ii ◽  
Real Time ◽  
Late Onset ◽  
Left Ventricular ◽  
Drug Elimination ◽  
Cell Analysis ◽  
Gene And Protein Expression

Abstract   Anthracyclines (e.g. epirubicin, doxorubicin, daunorubicin) are widely used for the treatment of adult and paediatric cancers. Despite their therapeutic efficacy, anthracyclines are associated with both acute and late-onset cardiac toxicities. Meta-analyses report an overt cardiotoxicity incidence of 6.3%, whilst sub-clinical cardiotoxicity incidence is 17.9% (1). Angiotensin converting enzyme (ACE) inhibitors are used to treat anthracycline-induced cardiotoxicity (AIC) (2) and despite their efficacy being well studied for the treatment of heart failure, hypertension and post-acute coronary syndromes, their mechanism(s) for treating and preventing AIC remain unknown. Using in vitro cardiomyocytes, we evaluated the angiotensin signalling mechanisms stimulated by doxorubicin chemotherapy, applying quantitative PCR, immunofluorescence and real-time cell analysis technologies. In vitro adult human ventricular cardiomyocytes (AC10 cell line) treated with clinically relevant sub-toxic concentrations of doxorubicin, demonstrate a dose and time-dependent increase in angiotensin II type-1 receptor (AT1R) gene expression. Maximal AT1R expression was observed after 24 hours' exposure at 250 nanomolar (nM), with qPCR recording up to 13-fold increases in expression relative to control (figure 1). Consistent with gene expression studies, doxorubicin also induced expression of AT1R at the protein level, with immunofluorescence imaging displaying up-regulation of AT1R in association with doxorubicin concentrations up to 500nM (figure 2). Western blot results also support the induction of AT1R, however no relationship was observed between either doxorubicin concentration or drug exposure time. Cellular growth and morphological changes of cardiomyocytes in response to clinically relevant doses of doxorubicin treatment were evaluated with real-time cell analysis using impedance-based xCELLigence technology. During the early phases of doxorubicin exposure, an increase in cell size was observed, whilst experiments modelling the pharmacokinetics and serial half-lives of doxorubicin demonstrated reversibility of doxorubicin-induced cardiomyocyte injury following drug elimination. These data support the mechanistic hypothesis that a relationship exists between AIC and modulation of the angiotensin signalling pathway in cardiomyocytes. We demonstrate that cardiomyocyte exposure to doxorubicin induces AT1R gene and protein expression, whilst doxorubicin-induced cardiomyocyte injury displays reversibility following drug elimination. Genetic polymorphisms within the ACE gene have been associated with cardiomyopathy and left ventricular hypertrophy. Our research now provides the platform to ascertain whether the ACE genotype contributes to heart failure from AIC, and whether an elevation in pro-hypertrophic angiotensin II levels could exacerbate anthracycline-induced hypertrophy and promote the development of late-onset anthracycline-induced heart failure. FUNDunding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Cancer Research UK PhD research grant

Sialyltransferase7A promotes angiotensin II-induced cardiomyocyte hypertrophy via HIF-1α-TAK1 signalling pathway

2019 ◽  
Vol 116 (1) ◽  
pp. 114-126 ◽  
Author(s):  
Xiaoying Yan ◽  
Ran Zhao ◽  
Xiaorong Feng ◽  
Jingzhou Mu ◽  
Ying Li ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Cardiac Hypertrophy ◽  
Transforming Growth Factor ◽  
Troponin T ◽  
Hypoxia Inducible Factor ◽  
Transforming Growth Factor Β ◽  
Left Ventricular ◽  
Cardiomyocyte Hypertrophy ◽  
Ang Ii

Abstract Aims Sialylation is up-regulated during the development of cardiac hypertrophy. Sialyltransferase7A (Siat7A) mRNA is consistently over-expressed in the hypertrophic left ventricle of hypertensive rats independently of genetic background. The aims of this study were: (i) to detect the Siat7A protein levels and its roles in the pathological cardiomyocyte hypertrophy; (ii) to elucidate the effect of sialylation mediated by Siat7A on the transforming-growth-factor-β-activated kinase (TAK1) expression and activity in cardiomyocyte hypertrophy; and (iii) to clarify hypoxia-inducible factor 1 (HIF-1) expression was regulated by Siat7A and transactivated TAK1 expression in cardiomyocyte hypertrophy. Methods and results Siat7A protein level was increased in hypertrophic cardiomyocytes of human and rats subjected to chronic infusion of angiotensin II (ANG II). Delivery of adeno-associated viral (AAV9) bearing shRNA against rat Siat7A into the left ventricular wall inhibited ventricular hypertrophy. Cardiac-specific Siat7A overexpression via intravenous injection of an AAV9 vector encoding Siat7A under the cardiac troponin T (cTNT) promoter aggravated cardiac hypertrophy in ANG II-treated rats. In vitro, Siat7A knockdown inhibited the induction of Sialyl-Tn (sTn) antigen and cardiomyocyte hypertrophy stimulated by ANG II. Mechanistically, ANG II induced the activation of TAK1-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling in parallel to up-regulation of Siat7A in hypertrophic cardiomyocytes. Siat7A knockdown inhibited activation of TAK1-NF-κB pathway. Interestingly, HIF-1α expression was increased in cardiomyocytes stimulated by ANG II but decreased after Siat7A knockdown. HIF-1α knockdown efficiently decreased TAK1 expression. ChIP and luciferase assays showed that HIF-1α transactivated the TAK1 promoter region (nt −1285 to −1274 bp) in the cardiomyocytes following ANG II stimulus. Conclusion Siat7A was up-regulated in hypertrophic myocardium and promoted cardiomyocyte hypertrophy via activation of the HIF-1α-TAK1-NF-κB pathway.

scholarly journals Cardiac fibroblasts acquire properties of matrifibrocytes in vitro and in mice with pressure overload-induced congestive heart failure

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
K.M Herum ◽  
G Gilles ◽  
A Romaine ◽  
A.O Melleby ◽  
G Christensen ◽  
...  
Keyword(s):  
Heart Failure ◽  
Congestive Heart Failure ◽  
Cardiac Fibroblasts ◽  
Pressure Overload ◽  
Left Ventricular Pressure ◽  
Left Ventricular ◽  
Funding Source ◽  
High Stiffness

Abstract Introduction Activation of cardiac fibroblasts (CFB) is a key step in development of fibrosis in the heart. It was recently shown that, in addition to the well-studied myofibroblast (myoFB) phenotype, activated cardiac fibroblasts can adopt a newly defined matrifibrocyte phenotype, characterized by expression of extracellular matrix (ECM) genes associated with bone, cartilage and tendon development. However, it is unknown whether matrifibrocytes exists in the pressure-overloaded fibrotic and failing heart, and whether substrate stiffness drives differentiation. Hypothesis Matrifibrocyte differentiation occurs in vitro during culturing of primary cardiac fibroblasts, and in vivo in response to left ventricular pressure overload. Methods Left ventricular pressure overload induced by o-ring aortic banding (ORAB) induced cardiac phenotypes of concentric hypertrophic remodelling and congestive heart failure. Primary CFB from adult mice were cultured on plastic or soft polyacrylamide hydrogels (4.5 kPa) for various times. mRNA expression of phenotypic markers were measured by RT-PCR. Presence of smooth muscle α-actin (SMA) fibers was determined by immunocytochemistry. Results ECM genes normally expressed in bone and cartilage (COMP, CILP-2, OPG and SCX) were upregulated in hypertrophic left ventricles of mice with congestive heart failure. The myoFB marker acta2 was increased 2 weeks after ORAB, returned to baseline at 4 weeks and increased again at 20 weeks when the left ventricle was dilating and failing, indicating that the myoFB phenotype is not permanent. In vitro, primary CFB upregulated bone/cartilage-associated ECM genes after 12 days of culturing on plastic. Acta2 mRNA and SMA protein levels peaked after 9 days in culture whereafter they declined, indicating a shift in phenotype. Culturing primary CFB on soft (4.5 kPa) hydrogels delayed, but did not prevent, myoFB differentiation while expression of bone/cartilage ECM genes was absent or low, indicating that high stiffness is a driver of the matrifibrocyte phenotype. Blockers of mechanotransduction, SB431542 (TGFβRI inhibitor), Y27623 (ROCK inhibitor) and cyclosporine A (calcineurin inhibitor), completely inhibited myoFB differentiation but upregulated several matrifibrocyte markers, indicating that distinct signaling pathways regulate myoFB and matrifibrocyte differentiation. Removing inhibitors re-induced myofibroblast markers in cells on plastic but not on soft gels consistent with high stiffness promoting myofibroblast differentiation. Conclusion Primary cardiac fibroblasts acquire characteristics of matrifibrocytes in vitro when cultured for long time on plastic and in vivo in left ventricles of mice with pressure overload-induced congestive heart failure. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): Marie Sklodowska-Curie Individual Fellowship

scholarly journals m6A RNA methylation contributes to translational control in heart failure progression

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
E Buchholz ◽  
T Berulava ◽  
V Elerdashvili ◽  
T Pena ◽  
D Lbik ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heart Failure ◽  
Cardiac Function ◽  
Human Tissue ◽  
Left Ventricular ◽  
Funding Source ◽  
Rna Methylation ◽  
Heart Failure Progression ◽  
M6a Rna Methylation ◽  
Epigenetic Processes

Abstract Background/Introduction Heart failure, characterized by reduced cardiac function and left ventricular dilatation, is a leading cause of hospital admission and mortality. Among increased apoptosis and fibrosis, the progression of heart failure is accompanied by changes in gene expression. There is increasing evidence, that also epigenetic processes such as DNA and histone modifications, long non-coding RNAs and transcription factors orchestrate aberrant gene expression in heart failure. Among these epigenetic processes, N6-methyladenosine (m6A) is the most prevalent modification found in all classes of RNA. Such m6A patterns in for example mRNA can have influence on various mechanisms such as splicing, transport, storage or decay of mRNAs. Due to its reversible and dynamic nature regulated via methyltransferases (mainly the METTL3/METTL14/WTAP-complex) and demethylases (mainly FTO and ALkBH5) it adds a new layer of epigenetic regulation. Purpose Changes in epigenetic processes are important mechanisms in heart failure progression. We aimed to elucidate the potential role of m6A methylation in heart failure development. Methods We analysed m6A methylation in different stages of heart failure progression in mouse and human tissue via methylated RNA immunoprecipitation (meRIP) followed by next generation sequencing (NGS). With polysome fractionation followed by NGS, we studied a potential link between polysomal occupancy and m6a RNA methylation. Results We found that approximately one quarter of all RNA transcripts in healthy mouse and human tissue carry m6A RNA methylation. During progression to heart failure we found that changes in m6A methylation exceed changes in gene expression in both, mouse and human. RNAs with altered m6A levels were mainly linked to metabolic and regulatory pathways, whereas changes in expression represented changes in structural plasticity. Furthermore, we found a link between m6A RNA methylation and altered RNA translation. Interestingly, transcripts with unchanged expression level but a differential change in their methylation level also showed differential polysomal occupancy. We could show a corresponding change in protein level, which points to a potential new mechanism of transcription-independent modulation of translation. The importance of m6A methylation was furthermore confirmed in a cardiomyocyte specific knock-out of the RNA demethylase FTO in mice where it lead to impaired cardiac function compared to control mice. Conclusions We could show that the m6A landscape is altered in heart hypertrophy and heart failure. Methylation changes exceed expression changes in disease progression and lead to changes in protein abundance, which uncovers a new transcription-independent mechanism of translation regulation. Therefore, our data suggest that targeting epitranscriptomic mechansims, such as m6A methylation, might be a an interesting approach for thereapeutic interventions. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): SFB 1002 Modulatory Units in Heart Failure

scholarly journals The CREB leucine zipper regulates CREB phosphorylation, cardiomyopathy, and lethality in a transgenic model of heart failure

2007 ◽  
Vol 293 (3) ◽  
pp. H1877-H1882 ◽  
Author(s):  
Gordon S. Huggins ◽  
John J. Lepore ◽  
Sarah Greytak ◽  
Richard Patten ◽  
Rachel McNamee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heart Failure ◽  
Leucine Zipper ◽  
Contractile Function ◽  
Camp Response Element Binding ◽  
Left Ventricular ◽  
Littermate Control ◽  
Creb Phosphorylation

Signaling through cAMP plays an important role in heart failure. Phosphorylation of cAMP response element binding protein (CREB) at serine-133 regulates gene expression in the heart. We examined the functional significance of CREB-S133 phosphorylation by comparing transgenic models in which a phosphorylation resistant CREB-S133A mutant containing either an intact or a mutated leucine zipper domain (CREB-S133A-LZ) was expressed in the heart. In vitro, CREB-S133A retained the ability to interact with wild-type CREB, whereas CREB-S133A-LZ did not. In vivo, CREB-S133A and CREB-S133A-LZ were expressed at comparable levels in the heart; however, CREB-S133A markedly suppressed the phosphorylation of endogenous CREB, whereas CREB-S133A-LZ had no effect. The one-year survival of mice from two CREB-S133A-LZ transgenic lines was equivalent to nontransgenic littermate control mice (NTG), whereas transgenic CREB-S133A mice died with heart failure at a median 30 wk of age ( P < 0.0001). CREB-S133A mice had an altered gene expression characteristic of the failing heart, whereas CREB-S133A-LZ mice did not. Left ventricular contractile function was substantially reduced in CREB-S133A mice versus NTG mice and only modestly reduced in CREB-S133A-LZ mice ( P < 0.02). When considered in light of other studies, these findings indicate that overexpression of the CREB leucine zipper is required for both inhibition of endogenous CREB phosphorylation and cardiomyopathy in this murine model of heart failure.

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