Regulation Of Cardiac Hypertrophy Research Articles

Baicalin ameliorates angiotensin II-induced cardiac hypertrophy and mitogen-activated protein kinase signaling pathway activation: A target-based network pharmacology approach

Baicalin, a flavonoid glycoside from Scutellaria baicalensis Georgi., exerts anti-hypertensive effects. The present study aimed to assess the cardioprotective role of baicalin and explore its potential mechanisms. Network pharmacology analysis pointed out a total of 477 potential targets of baicalin were obtained from the PharmMapper and SwissTargetPrediction databases, while 11,280 targets were identified associating with hypertensive heart disease from GeneCards database. Based on the above 382 common targets, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed enrichment in the regulation of cardiac hypertrophy, cardiac contraction, cardiac relaxation, as well as the mitogen-activated protein kinase (MAPK) and other signaling pathways. Moreover, baicalin treatment exhibited the amelioration of increased cardiac index and pathological alterations in angiotensin II (Ang II)-infused C57BL/6 mice. Furthermore, baicalin treatment demonstrated a reduction in cell surface area and a down-regulation of hypertrophy markers (including atrial natriuretic peptide and brain natriuretic peptide) in vivo and in vitro. In addition, baicalin treatment led to a decrease in the expression of phosphorylated c-Jun N-terminal kinase (p-JNK)/JNK, phosphorylated p38 (p-p38)/p38, and phosphorylated extracellular signal-regulated kinase (p-ERK)/ERK in the cardiac tissues of Ang II-infused mice and Ang II-stimulated H9c2 cells. These findings highlight the cardioprotective effects of baicalin, as it alleviates hypertensive cardiac injury, cardiac hypertrophy, and the activation of the MAPK pathway.

Abstract We135: Mechanistic Modeling Identifies Novel Regulators of Cardiac Hypertrophy

Introduction: Cardiac hypertrophy is a leading predictor of heart failure. The mechanisms that drive cardiac hypertrophy are poorly understood from a molecular and cellular perspective, due to the lack of knowledge of the governing signaling. Hypothesis and Aims: I hypothesize that there exist genes that regulate hypertrophy beyond the field’s current understanding that can be identified by combining the publicly available, high-throughput International Mouse Phenotyping Consortium (IMPC) database with network modeling. I aim to use a large-scale network model of cardiac hypertrophy to assess the IMPC genes’ effects on hypertrophic phenotypes and validate these findings in vitro to identify novel regulators. Materials & Methods: The IMPC was established by worldwide mouse clinics using in vivo models to perform single gene knockout experiments with primary phenotyping, forming a database with ~9000 genes, ~1000 of which can be associated with cardiac hypertrophy. 1 st neighbors (genes directly interacting with the hypertrophy network model as evidenced by protein-protein interaction databases) were identified and effects on hypertrophy were assessed through the IMPC and network model knockdown simulations. When both methods suggested hypertrophic regulation, a gene was considered a candidate regulator. siRNA experiments are being performed in neonatal rat cardiomyocytes for validation. High-content imaging and qPCR are used to quantify changes in phenotype and confirm gene knockdown. Results: Combining the IMPC with protein-protein interaction data, 37 1 st neighbor genes were identified. Knockdown simulation identified 2 genes (LRIG1, CBL) as negative regulators. 3 genes (VAV2, LEPR, RASA1) were identified as positive regulators. These genes became candidates for in vitro validation given the network-wide hypertrophic effects enacted by their knockdowns. siRNA experiments are being optimized to fully validate modeling findings. Conclusions: Mechanistic modeling identified 5 genes as novel regulators of cardiac hypertrophy. Sensitivity analysis highlighted known influencers of hypertrophy in the knockdown context. siRNA knockdown experiments are being performed to validate the model predictions.

Internalized β2-Adrenergic Receptors Oppose PLC-Dependent Hypertrophic Signaling.

Chronically elevated neurohumoral drive, and particularly elevated adrenergic tone leading to β-adrenergic receptor (β-AR) overstimulation in cardiac myocytes, is a key mechanism involved in the progression of heart failure. β1-AR (β1-adrenergic receptor) and β2-ARs (β2-adrenergic receptor) are the 2 major subtypes of β-ARs present in the human heart; however, they elicit different or even opposite effects on cardiac function and hypertrophy. For example, chronic activation of β1-ARs drives detrimental cardiac remodeling while β2-AR signaling is protective. The underlying molecular mechanisms for cardiac protection through β2-ARs remain unclear. β2-AR signaling mechanisms were studied in isolated neonatal rat ventricular myocytes and adult mouse ventricular myocytes using live cell imaging and Western blotting methods. Isolated myocytes and mice were used to examine the roles of β2-AR signaling mechanisms in the regulation of cardiac hypertrophy. Here, we show that β2-AR activation protects against hypertrophy through inhibition of phospholipaseCε signaling at the Golgi apparatus. The mechanism for β2-AR-mediated phospholipase C inhibition requires internalization of β2-AR, activation of Gi and Gβγ subunit signaling at endosome and ERK (extracellular regulated kinase) activation. This pathway inhibits both angiotensin II and Golgi-β1-AR-mediated stimulation of phosphoinositide hydrolysis at the Golgi apparatus ultimately resulting in decreased PKD (protein kinase D) and histone deacetylase 5 phosphorylation and protection against cardiac hypertrophy. This reveals a mechanism for β2-AR antagonism of the phospholipase Cε pathway that may contribute to the known protective effects of β2-AR signaling on the development of heart failure.

The m7G Methyltransferase Mettl1 Drives Cardiac Hypertrophy by Regulating SRSF9-Mediated Splicing of NFATc4.

Cardiac hypertrophy is a key factor driving heart failure (HF), yet its pathogenesis remains incompletely elucidated. Mettl1-catalyzed RNA N7-methylguanosine (m7G) modification has been implicated in ischemic cardiac injury and fibrosis. This study aims to elucidate the role of Mettl1 and the mechanism underlying non-ischemic cardiac hypertrophy and HF. It is found that Mettl1 is upregulated in human failing hearts and hypertrophic murine hearts following transverse aortic constriction (TAC) and Angiotensin II (Ang II) infusion. YY1 acts as a transcriptional factor for Mettl1 during cardiac hypertrophy. Mettl1 knockout alleviates cardiac hypertrophy and dysfunction upon pressure overload from TAC or Ang II stimulation. Conversely, cardiac-specific overexpression of Mettl1 results in cardiac remodeling. Mechanically, Mettl1 increases SRSF9 expression by inducing m7G modification of SRSF9 mRNA, facilitating alternative splicing and stabilization of NFATc4, thereby promoting cardiac hypertrophy. Moreover, the knockdown of SRSF9 protects against TAC- or Mettl1-induced cardiac hypertrophic phenotypes in vivo and in vitro. The study identifies Mettl1 as a crucial regulator of cardiac hypertrophy, providing a novel therapeutic target for HF.

Internalized β2-Adrenergic Receptors Inhibit Subcellular Phospholipase C-Dependent Cardiac Hypertrophic Signaling

Chronic overstimulation of Gs-coupled b-adrenergic receptor (b-AR) signaling by catecholamines, induces pathological cardiac hypertrophy and ultimately heart failure. b1-ARs and b2-ARs are the two major subtypes of b-ARs present in the human heart and mediate the activity of sympathetic nervous system. It is known that b1-ARs and b2-ARs elicit significantly different or even opposite effects on cardiac functions such as contractility and heart failure, though they share overall sequence and structural homology, and both stimulate cAMP production. The molecular mechanisms underlying these distinct biological effects have not been fully elucidated. Previously, our laboratory demonstrated that a cAMP/EPAC/phospholipase Ce (PLCe)-mediated signaling pathway at the Golgi apparatus is important for regulation of cardiac hypertrophy. We also showed that this pathway is selectively stimulated by activation of b1-ARs localized at Golgi apparatus but not those at the plasma membrane (PM). Access to intracellular b1-ARs by catecholamines was mediated by the organic cation transporters (OCTs), and blockade of OCT3 inhibited catecholamine stimulated cardiomyocyte hypertrophy. In my current study using adult ventricular cardiac myocytes, I found that the activation of endosomal b2-ARs opposes stimulation of the prohypertrophic EPAC/PLCe pathway at the Golgi apparatus by either Angiotensin II (ATII), or the activation of Golgi localized b1-ARs by Dobutamine (Dob). b2-AR-dependent inhibition is at the level of PLCe since activation of b2-AR with salmeterol (Sal) blocks direct activation of EPAC/PLCe by the EPAC selective cAMP analog 8-(4-chlorophenylthio)-2′- O-methyl-cAMP (CPTOMe)-AM, and blocks PLCe activation by ATII which activates PLCe through a pathway that does not rely on EPAC. Blockade of internalization of b2-ARs with Dyngo-4A abolished this inhibitory signaling. Downstream of b2-ARs, I found that b2-AR-dependent PLCe inhibition is not mediated by PKA. Rather, blockade of Gi with PTX, or Gbg with gallein, inhibited the ability of b2-ARs to block Golgi PLCe activity. Additionally, inhibition of ERK activation by PD0325901 abolished b2-AR-mediated inhibition of Golgi PLCe activity. This supports a model where b2ARs internalized from the plasma membrane activate Gi releasing Gbg subunits leading to ERK activation and inhibition of PI hydrolysis at the Golgi apparatus, thereby inhibiting hypertrophic signaling by PLCe. In addition, we also showed PKD and HDAC phosphorylation downstream of PLCe signaling are significantly attenuated by b2ARs activation in mouse hearts. Consistent with the results from mice, b2ARs activation by Sal blunted cellular hypertrophy induced by b1-ARs activation by Dob in vitro measured by cell area and the expression of the hypertrophic marker, atrial natriuretic factor (ANF). Importantly, inhibition endosomal Gbg using a highly selective Gbg inhibitor (GRK2CT) fused to an endosomal targeting sequence (FYVE domain) prevent Sal mediated protective signaling in hypertrophy measurements in vitro. This indicates the inhibitory signaling downstream of b2ARs is indeed driven by endosomal Gbg. This study reveals a novel potential mechanism for b2-AR antagonism of the Epac/PLCe pathway that may contribute to the known protective effects of b2AR signaling on the development of heart failure. Elucidation of a mechanism for the anti-hypertrophic versus hypertrophic signaling balance from b1-ARs and b2-ARs gives critical insights into the development of new strategies for treatment of heart failure by targeting bAR subtypes. This work is supported by grant R35 GM127303-01 (A.V.S.). Preprint: https://www.biorxiv.org/content/10.1101/2023.06.07.544153v1. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The crucial relationship between miRNA-27 and CSE/H2S, and the mechanism of action of GLP-1 in myocardial hypertrophy.

Cardiac hypertrophy is the most prevalent compensatory heart disease that ultimately leads to spontaneous heart failure. Mounting evidence suggests that microRNAs (miRs) and endogenous hydrogen sulfide (H2S) play a crucial role in the regulation of cardiac hypertrophy. In this study, we aimed to investigate whether inhibition of miR-27a could protect against cardiac hypertrophy by modulating H2S signaling. We established a model of cardiac hypertrophy by obtaining hypertrophic tissue from mice subjected to transverse aortic constriction (TAC) and from cells treated with angiotensin-II. Molecular alterations in the myocardium were quantified using quantitative real time PCR (qRT-PCR), Western blotting, and ELISA. Morphological changes were characterized by hematoxylin and eosin (HE) staining and Masson's trichrome staining. Functional myocardial changes were assessed using echocardiography. Our results demonstrated that miR-27a levels were elevated, while H2S levels were reduced in TAC mice and myocardial hypertrophy. Further luciferase and target scan assays confirmed that cystathionine-γ-lyase (CSE) was a direct target of miR-27a and was negatively regulated by it. Notably, enhancement of H2S expression in the heart was observed in mice injected with recombinant adeno-associated virus vector 9 (rAAV9)-anti-miR-27a and in cells transfected with a miR-27a inhibitor during cardiac hypertrophy. However, this effect was abolished by co-transfection with CSE siRNA and the miR-27a inhibitor. Conversely, injecting rAAV9-miR-27a yielded opposite results. Interestingly, our findings demonstrated that glucagon-like peptide-1 (GLP-1) agonists could mitigate myocardial damage by down-regulating miR-27a and up-regulating CSE. In summary, our study suggests that inhibition of miR-27a holds therapeutic promise for the treatment of cardiac hypertrophy by increasing H2S levels. Furthermore, our findings unveil a novel mechanism of GLP-1 agonists involving the miR-27a/H2S pathway in the management of cardiac hypertrophy.

Abstract 16957: PRKAG2 Directly Interacts With Myosin Heavy Chains to Modulate Myocyte Electrical and Morphological Changes in Cardiac Hypertrophy

Introduction: Mutations in the PRKAG2 gene cause a complex myocardial disorder characterized by hypertrophic cardiomyopathy (HCM) and conduction disturbances. While prior studies associated PRKAG2 -linked hypertrophy with increased glycogen storage, many HCM phenotypes in the disorder remain unexplained by this effect alone. We aimed to uncover the molecular mechanisms by which PRKAG2 mutations induce myocyte hypertrophy and electrical changes during cardiac development. Methods: We created transgenic zebrafish expressing WT (Tg WT ) and pathological mutant (Tg R299Q ) murine Prkag2 cDNA under a myocardium-specific promoter. Using these models, we studied the impact of Prkag2 variant on myocyte electrical activity, metabolism, and cytoarchitecture across cardiogenesis and in mature hearts. Results: Tg R299Q adult fish showed hypertrophic cardiomyocytes, swollen mitochondria, and shortened sarcomere length compared to Tg WT and WT, mirroring human HCM phenotypes. The glycogen content was elevated in adult hearts but not during early cardiogenesis. Despite the absence of glycogen accumulation at 6-day post fertilization, Tg R299Q hearts showed electrical abnormalities, including a reduced conduction velocity, decreased Ca 2+ amplitude, and prolonged action potential durations in both atria and ventricles, compared to Tg WT and WT. Co-immunoprecipitation and proximity ligation assay revealed physical interactions between Prkag2 and myosin heavy chain (MYH), with the R299Q variant enhancing this interaction and altering the subcellular localization of Prkag2 from mitochondria to sarcomeres. Knockdown of the fish homolog of MYH7 ( vmhcl ) restored the early electrical disturbances caused by Tg R299Q . This abnormal MYH-Prkag2 interaction disturbed mitochondria and sarcomere organization contributing to altered myocyte cytoarchitecture observed in Tg R299Q . Conclusions: Mutant PRKAG2 altered cardiac excitability and Ca 2+ handling during embryogenesis, preceding glycogen accumulation. Enhanced binding between Prkag2 and MYHs contributed to these early changes. Our study revealed a novel link between sarcomere proteins and metabolic regulators in cardiac hypertrophy, suggesting a new therapeutic avenue for treating HCM.

Palmitoylation-dependent regulation of cardiomyocyte Rac1 signaling activity and minor effects on cardiac hypertrophy

S-palmitoylation is a reversible lipid modification catalyzed by 23 S-acyltransferases with a conserved zinc finger aspartate-histidine-histidine-cysteine (zDHHC) domain that facilitates targeting of proteins to specific intracellular membranes. Here we performed a gain-of-function screen in the mouse and identified the Golgi-localized enzymes zDHHC3 and zDHHC7 as regulators of cardiac hypertrophy. Cardiomyocyte-specific transgenic mice overexpressing zDHHC3 show cardiac disease, and S-acyl proteomics identified the small GTPase Rac1 as a novel substrate of zDHHC3. Notably, cardiomyopathy and congestive heart failure in zDHHC3 transgenic mice is preceded by enhanced Rac1 S-palmitoylation, membrane localization, activity, downstream hypertrophic signaling, and concomitant induction of all Rho family small GTPases whereas mice overexpressing an enzymatically dead zDHHC3 mutant show no discernible effect. However, loss of Rac1 or other identified zDHHC3 targets Gαq/11 or galectin-1 does not diminish zDHHC3-induced cardiomyopathy, suggesting multiple effectors and pathways promoting decompensation with sustained zDHHC3 activity. Genetic deletion of Zdhhc3 in combination with Zdhhc7 reduces cardiac hypertrophy during the early response to pressure overload stimulation but not over longer time periods. Indeed, cardiac hypertrophy in response to 2weeks of angiotensin-II infusion is not diminished by Zdhhc3/7 deletion, again suggesting other S-acyltransferases or signaling mechanisms compensate to promote hypertrophic signaling. Taken together, these data indicate that the activity of zDHHC3 and zDHHC7 at the cardiomyocyte Golgi promote Rac1 signaling and maladaptive cardiac remodeling, but redundant signaling effectors compensate to maintain cardiac hypertrophy with sustained pathological stimulation in the absence of zDHHC3/7.

DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1

Pathological cardiac hypertrophy involves multiple regulators and several signal transduction pathways. Currently, the mechanisms of it are not well understood. Differentially expressed in FDCP 6 homolog (DEF6) was reported to participate in immunity, bone remodeling, and cancers. The effects of DEF6 on pathological cardiac hypertrophy, however, have not yet been fully characterized. We initially determined the expression profile of DEF6 and found that DEF6 was upregulated in hypertrophic hearts and cardiomyocytes. Our in vivo results revealed that DEF6 deficiency in mice alleviated transverse aortic constriction (TAC)-induced cardiac hypertrophy, fibrosis, dilation and dysfunction of left ventricle. Conversely, cardiomyocyte-specific DEF6-overexpression aggravated the hypertrophic phenotype in mice under chronic pressure overload. Similar to the animal experiments, the in vitro data showed that adenovirus-mediated knockdown of DEF6 remarkably inhibited phenylephrine (PE)-induced cardiomyocyte hypertrophy, whereas DEF6 overexpression exerted the opposite effects. Mechanistically, exploration of the signal pathways showed that the mitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2)-extracellular signal-regulated kinase 1/2 (ERK1/2) cascade might be involved in the prohypertrophic effect of DEF6. Coimmunoprecipitation and GST (glutathione S-transferase) pulldown analyses demonstrated that DEF6 can directly interact with small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), and the Rac1 activity assay revealed that the activity of Rac1 is altered with DEF6 expression in TAC-cardiac hypertrophy and PE-triggered cardiomyocyte hypertrophy. In the end, western blot and rescue experiments using Rac1 inhibitor NSC23766 and the constitutively active mutant Rac1(G12V) verified the requirement of Rac1 and MEK1/2-ERK1/2 activation for DEF6-mediated pathological cardiac hypertrophy. Our study substantiates that DEF6 acts as a deleterious regulator of cardiac hypertrophy by activating the Rac1 and MEK1/2-ERK1/2 signaling pathways, and suggests that DEF6 may be a potential treatment target for heart failure.

Cardiac-specific BACH1 ablation attenuates pathological cardiac hypertrophy by inhibiting the Ang II type 1 receptor expression and the Ca2+/CaMKII pathway.

BACH1 is upregulated in hypertrophic hearts, but its function in cardiac hypertrophy remains largely unknown. This research investigates the function and mechanisms of BACH1 in the regulation of cardiac hypertrophy. Male cardiac-specific BACH1 knockout mice or cardiac-specific BACH1 transgenic (BACH1-Tg) mice and their respective wild-type littermates developed cardiac hypertrophy induced by angiotensin II (Ang II) or transverse aortic constriction (TAC). Cardiac-specific BACH1 knockout in mice protected the hearts against Ang II- and TAC-induced cardiac hypertrophy and fibrosis, and preserved cardiac function. Conversely, cardiac-specific BACH1 overexpression markedly exaggerated cardiac hypertrophy and fibrosis and reduced cardiac function in mice with Ang II- and TAC-induced hypertrophy. Mechanistically, BACH1 silencing attenuated Ang II- and norepinephrine-stimulated calcium/calmodulin-dependent protein kinase II (CaMKII) signaling, the expression of hypertrophic genes, and hypertrophic growth of cardiomyocytes. Ang II stimulation promoted the nuclear localization of BACH1, facilitated the recruitment of BACH1 to the Ang II type 1 receptor (AT1R) gene promoter, and then increased the expression of AT1R. Inhibition of BACH1 attenuated Ang II-stimulated AT1R expression, cytosolic Ca2+ levels, and CaMKII activation in cardiomyocytes, whereas overexpression of BACH1 led to the opposite effects. The increased expression of hypertrophic genes induced by BACH1 overexpression upon Ang II stimulation was suppressed by CaMKII inhibitor KN93. The AT1R antagonist, losartan, significantly attenuated BACH1-mediated CaMKII activation and cardiomyocyte hypertrophy under Ang II stimulation in vitro. Similarly, Ang II-induced myocardial pathological hypertrophy, cardiac fibrosis, and dysfunction in BACH1-Tg mice were blunted by treatment with losartan. This study elucidates a novel important role of BACH1 in pathological cardiac hypertrophy by regulating the AT1R expression and the Ca2+/CaMKII pathway, and highlights potential therapeutic target in pathological cardiac hypertrophy. Transcription factor BACH1 is increased in failing human myocardium and hypertrophic human hearts. We revealed that cardiomyocyte-specific BACH1 ablation in mice protected the hearts against Ang II- and pressure overload-induced cardiac hypertrophy and fibrosis, and preserved cardiac function. BACH1 activated AT1R promoter activity under Ang II stimulation, and BACH1-mediated facilitation of cardiac hypertrophy under Ang II stimulation was regulated at least in part by AT1R expression. Moreover, BACH1 silencing attenuated the norepinephrine-stimulated CaMKII signaling pathway and hypertrophic growth of cardiomyocytes. Our findings suggest that BACH1 may pose a promising therapeutic target in pathological cardiac hypertrophy and heart failure.

Circ-sh3rf3/GATA-4/miR-29a regulatory axis in fibroblast-myofibroblast differentiation and myocardial fibrosis.

The transdifferentiation from cardiac fibroblasts to myofibroblasts is an important event in the initiation of cardiac fibrosis. However, the underlying mechanism is not fully understood. Circ-sh3rf3 (circular RNA SH3 domain containing Ring Finger 3) is a novel circular RNA which was induced in hypertrophied ventricles by isoproterenol hydrochloride, and our work has established that it is a potential regulator in cardiac hypertrophy, but whether circ-sh3rf3 plays a role in cardiac fibrosis remains unclear, especially in the conversion of cardiac fibroblasts into myofibroblasts. Here, we found that circ-sh3rf3 was down-regulated in isoproterenol-treated rat cardiac fibroblasts and cardiomyocytes as well as during fibroblast differentiation into myofibroblasts. We further confirmed that circ-sh3rf3 could interact with GATA-4 proteins and reduce the expression of GATA-4, which in turn abolishes GATA-4 repression of miR-29a expression and thus up-regulates miR-29a expression, thereby inhibiting fibroblast-myofibroblast differentiation and myocardial fibrosis. Our work has established a novel Circ-sh3rf3/GATA-4/miR-29a regulatory cascade in fibroblast-myofibroblast differentiation and myocardial fibrosis, which provides a new therapeutic target for myocardial fibrosis.

Crosstalk between PML and p53 in response to TGF-β1: A new mechanism of cardiac fibroblast activation.

Cardiac fibrosis is a common pathological cardiac remodeling in a variety of heart diseases, characterized by the activation of cardiac fibroblasts. Our previous study uncovered that promyelocytic leukemia protein (PML)-associated SUMO processes is a new regulator of cardiac hypertrophy and heart failure. The present study aimed to explore the role of PML in cardiac fibroblasts activation. Here we found that PML is significantly upregulated in cardiac fibrotic tissue and activated cardiac fibroblasts treated with transforming growth factor-β1 (TGF-β1). Gain- and loss-of-function experiments showed that PML impacted cardiac fibroblasts activation after TGF-β1 treatment. Further study demonstrated that p53 acts as the transcriptional regulator of PML, and participated in TGF-β1 induced the increase of PML expression and PML nuclear bodies (PML-NBs) formation. Knockdown or pharmacological inhibition of p53 produced inhibitory effects on the activation of cardiac fibroblasts. We further found that PML also may stabilize p53 through inhibiting its ubiquitin-mediated proteasomal degradation in cardiac fibroblasts. Collectively, this study suggests that PML crosstalk with p53 regulates cardiac fibroblasts activation, which provides a novel therapeutic strategy for cardiac fibrosis.

RCAN1 deficiency aggravates sepsis-induced cardiac remodeling and dysfunction by accelerating mitochondrial pathological fission.

Cardiac dysfunction and remodeling are serious complications of sepsis and are the main causes of death in sepsis. RCAN1 is a feedback regulator of cardiac hypertrophy. Here, we aim to investigate the role of RCAN1 in septic cardiomyopathy. Mice were randomly divided into control-WT, control-RCAN1-/-, LPS-induced WT and LPS-induced RCAN1-/- groups, some with Midiv-1 or KN93 treatment. The protein levels of RCAN1, p-ERK1/2, NFAT3, Drp1, p-Drp1, p-CaMKII in mouse hearts or cultured cardiomyocytes were determined by Western blotting. Myocardial function was assessed by echocardiography. Cardiac hypertrophy and fibrosis were detected by H&E and Masson's trichrome staining. Mitochondrial morphology was examined by transmission electron microscope. Serum level of LDH was detected by ELISA. Our data show that RCAN1 was downregulated in septic mouse heart and LPS-induced cardiomyocytes. RCAN1-/- mice showed a severe impairment of cardiac function, and increased myocardial hypertrophy and fibrosis. The protein levels of NFAT3 and p-ERK1/2 were significantly increased in the heart tissues of RCAN1-/- mice. Further, RCAN1 deficiency aggravated sepsis-induced cardiac mitochondrial injury as indicated by increased ROS production, pathological fission and the loss of mitochondrial membrane potential. Inhibition of fission with Mdivi-1 reversed LPS-induced cardiac hypertrophy, fibrosis and dysfunction in RCAN1-/- mice. Moreover, RCAN1 depletion promoted mitochondrial translocation of CaMKII, which enhanced fission and septic hypertrophy, while inhibition of CaMKII with KN93 reduced excessive fission, improved LPS-mediated cardiac remodeling and dysfunction in RCAN1-/- mice. Our finding demonstrated that RCAN1 deficiency aggravated mitochondrial injury and septic cardiomyopathy through activating CaMKII. RCAN1 serves as a novel therapeutic target for treatment of sepsis-related cardiac remodeling and dysfunction.

Doxorubicin-induced upregulation of follistatin-like 3 (FSTL3): a new therapeutic target

Abstract Introduction Doxorubicin (DOX) is among the most used anticancer drugs with associated cardiotoxicity. Follistatin-like 3 (FSTL3), a secreted member of the follistatins family that can selectively bind to members of the TGF-β superfamily, is involved in regulation of cardiac hypertrophy and heart failure. FSTL3 is also upregulated in breast and colorectal cancer tumours, is also an unfavourable prognostic indicator for various cancers. Purpose We aim to determine the dual role of FSTL3 in prevention of DOX-induced cardiotoxicity and synergistic anti-cancer effects. Methods Human cardiomyocytes (HCMs) were treated with DOX at 1uM (EC50) for 72 hours. Cell viability was assessed via CellTiter-Glo®. Secreted FSTL3 levels, as measured by ELISA (R&D systems). FSTL3 and TGF-β mRNA levels were measured by qPCR. Co-treatment of DOX with human anti-FSTL3 antibodies (Aviva Systems Biology) at 10ug/mL were introduced for 72hrs treatment. Results Secreted FSTL3 levels were significantly increased in DOX-treated HCMs at 72hrs compared to control (n=5, p<0.001). Consistently, FSTL3 and TGF-β mRNA levels, in collected HCMs were significantly increased in DOX-treated cells. Co-treatment of DOX with human anti-FSTL3 antibodies at 10ug/mL significantly improved HCM viability compared to IgG control group. Conversely, anti-FSTL3 antibodies provided synergistic anti-cancer effects with DOX: MCF-7 breast cancer cells were significantly reduced when co-treated with DOX and anti-FSTL3 antibody vs. IgG controls. Conclusion We show, for the first time, that: 1) FSTL3 is secreted directly from HCMs; 2) FSTL3 levels (both circulating and mRNA) is markedly elevated with DOX treatment; 3) neutralisation of FSTL3 in DOX-treated HCMs, restored HCM viability; and 4) exhibit synergistic anti-cancer effects with DOX. Taken together, FSTL3 is a potential target for dual anti-cancer and cardioprotective effects. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Heart Foundation of Australia Future Leader FellowshipsNSW Ministry of Health EMC FellowshipNSW Ministry of Health Translational Research Grant

G-protein coupled receptor 30 attenuates myocardial hypertrophy by reducing oxidative stress and apoptosis in Ang II-treated mice

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors that mediate the effects of cardiac diseases. GPR30, also named G-protein-coupled estrogen receptor, shows beneficial effect on female patients with heart failure. This research aimed to probe the role and mechanism of GPR30 in myocardial hypertrophy. The model of cardiac hypertrophy was induced by infusion of angiotensin (Ang) II in mice, and was induced by Ang II treatment in neonatal rat cardiomyocyte (NRCM). The mouse model of myocardial hypertrophy was induced by angiotensin (Ang) Ⅱ, and the neonatal rat cardiomyocyte (NRCM) was induced by Ang Ⅱ treatment. GPR30 agonist G1 reduced cardiac hypertrophy induced by Ang II in mice, and reduced cardiac atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) induced by Ang II. Ang Ⅱ treatment of myocardial fibrosis in mice was suppressed after administration of G1. GPR30 deficiency produced the opposite results. Oxidative stress and apoptosis were enhanced in the mice heart induced by Ang II, which were suppressed by G1 administration, but were further exacerbated after GPR30 deficiency. The outcomes demonstrated that GPR30 participated in the regulation of cardiac hypertrophy and fibrosis. Activation of GPR30 ameliorated cardiac hypertrophy and fibrosis by reducing oxidative stress and apoptosis.

Protections of transcription factor BACH2 and natural product myricetin against pathological cardiac hypertrophy and dysfunction.

Pathological hypertrophic myocardium under consistent adverse stimuli eventually can cause heart failure. This study aims to explore the role of BACH2, a member of the basic region leucine zipper transcription factor family, in cardiac hypertrophy and failure. Transverse aortic constriction surgery was operated to induce cardiac hypertrophy and failure in mice. BACH2 was overexpressed in mice through tail vein injection of AAV9-Bach2. Mice with systemic or cardiac-specific knockdown of Bach2 were adopted. Neonatal rat ventricular myocytes (NRVMs) were isolated and infected with lentivirus to overexpress Bach2 or transfected with siRNA to knock down Bach2. Our data showed that overexpression of BACH2 ameliorated TAC-induced cardiac hypertrophy and failure in mice and decreased isoproterenol (ISO)-triggered myocyte hypertrophy in NRVMs. Systemic or cardiac-specific knockdown of Bach2 worsened the cardiac hypertrophy and failure phenotype in mice. Further assays showed that BACH2 bound to the promotor region of Akap6 at the -600 to -587 site and repressed its expression, which functioned as a crucial scaffold for cardiac hypertrophy and failure signaling pathways. Small molecular natural product library screening suggested that myricetin could up-regulate expression of Bach2 and simultaneously suppress the transcriptional levels of hypertrophic marker genes Bnp and Myh7. Further studies showed that myricetin exerted a BACH2-dependent protective effect against cardiac hypertrophy in vivo and in vitro. Taken together, our findings demonstrated that BACH2 plays a crucial role in the regulation of cardiac hypertrophy and failure and can be a potential therapeutic target in the future.

MALAT1 regulates hypertrophy of cardiomyocytes by modulating the miR-181a/HMGB2 pathway.

Noncoding RNAs are important for the regulation of cardiac hypertrophy. The function of MALAT1 (a long noncoding mRNA), miR-181a, and HMGB2, their contribution to cardiac hypertrophy, and the regulatory relationship between them during this process remain unknown. In the present study, we treated primary cardiomyocytes with angiotensin II (Ang II) to mimic cardiac hypertrophy. MALAT1 expression was significantly downregulated in Ang II-treated cardiomyocytes compared with control cardiomyocytes. Ang II-induced cardiac hypertrophy was suppressed by overexpression of MALAT1 and promoted by genetic knockdown of MALAT1. A dual-luciferase reporter assay demonstrated that MALAT1 acted as a sponge for miR-181a and inhibited its expression during cardiac hypertrophy. Cardiac hypertrophy was suppressed by overexpression of an miR-181a inhibitor and enhanced by overexpression of an miR-181a mimic. HMGB2 was downregulated during cardiac hypertrophy and was identified as a target of miR-181a by bioinformatics analysis and a dual-luciferase reporter assay. miR-181a overexpression decreased the mRNA and protein levels of HMGB2. Rescue experiments indicated that MALAT1 overexpression reversed the effect of miR-181a on HMGB2 expression. In summary, the results of the present study show that MALAT1 acts as a sponge for miR-181a and thereby regulates expression of HMGB2 and development of cardiac hypertrophy. The novel MALAT1/miR-181a/HMGB2 axis might play a crucial role in cardiac hypertrophy and serve as a new therapeutic target.

Identification of a long noncoding RNA Gm17501 as a novel negative regulator of cardiac hypertrophy

Pathological cardiac hypertrophy is an independent risk factor for the development of heart failure. Long noncoding RNAs (lncRNAs), an emerging class of non-protein-coding transcripts, are involved in regulation of multiple cardiac diseases through diverse molecular mechanism, whereas the role of cytoplasmic lncRNAs in regulating cardiac hypertrophy remains unclear. In this study, we identified a novel and functional long noncoding RNA Gm17501, which was predominantly expressed in the cytoplasm of cardiomyocytes. The expression level of lncRNA Gm17501 was altered in cardiac hypertrophy induced by pressure overload and phenylephrine treatment. Moreover, lncRNA Gm17501 expression was decreased in the heart tissue of patients with heart failure. Silencing lncRNA Gm17501 aggravated cardiac hypertrophy under pathological stress. Inhibition of lncRNA Gm17501 did not alter the expression of nearby genes but decreased mRNA level of calcium handling proteins which were involved in cardiac contraction. Therefore, the cytoplasmic lncRNA Gm17501 might protect cardiomyocytes against hypertrophy, possibly by maintaining calcium signaling pathway.

On the road to discovering a new actor of cardiac pathological hypertrophy

Cardiac hypertrophy develops in response to hemodynamic overload, which can occur in hypertension and aortic stenosis. Maintenance of these pathological conditions eventually evolves into heart failure (HF) and death. O-GlcNAcylation, a post-translational modification of proteins, has been shown to drastically increase during cardiac hypertrophy development and to mediate maladaptive remodeling implicated in the progression of HF. Using mass spectrometry analysis, our lab recently identified several O-GlcNAcylated cardiac proteins that could be involved in such remodeling. Among them, O-GlcNAcylated protein 1 (OGP-1) looks to be a promising candidate. We investigated the role of OGP-1 in cardiac hypertrophy development. Change in OGP-1 expression was followed in neonatal rat cardiomyocytes (NRVM) treated with the pro-hypertrophic agent phenylephrine (PE) for 24 h. The same was done in vivo in two different hypertrophic mouse models, the first induced by Angiotensin II (Ang II) infusion for 5 to 14 days and the second promoted by transverse aortic constriction (TAC) for 2 to 6 weeks. The expression of OGP-1 was also evaluated in human hearts biopsies from different degree of cardiac remodeling. NRVM size was evaluated using α-actinin staining that labels sarcomeric Z-line. Pro-hypertrophic markers (ANP, BNP and β-MHC) were analyzed by quantitative PCR. The effect of OGP-1 overexpression and siRNA-mediated silencing was finally performed in NRVM. A significant decrease in OGP-1 expression was detected in both in vitro and in vivo rodent hypertrophic models. To evaluate the putative implication of OGP-1 in the regulation of cardiac hypertrophy, we repressed OGP-1 expression using siRNA in NRVM. We showed that OGP-1 knockdown induced a basal increase in cell size, similar to that found in PE-treated control cardiomyocytes, suggesting that OGP-1 repression may be sufficient to induce cardiomyocyte hypertrophy. Unexpectedly, the increase in cell size was not related to classical pro-hypertrophic pathways, ANP, BNP and β-MHC expression being unaffected. The other way round, OGP-1 overexpression prevented PE-induced NRVM hypertrophy. Our results underlie OGP-1 as a potential anti-hypertrophic protein and an interesting future target for the treatment of cardiac hypertrophy. Further investigations aim to identify the precise function of OGP-1 in the regulation of cardiac hypertrophy.

Epigenetic Reader Bromodomain Containing Protein 2 Facilitates Pathological Cardiac Hypertrophy via Regulating the Expression of Citrate Cycle Genes.

The bromodomain and extra-terminal domain proteins (BETs) family serve as epigenetic “readers”, which recognize the acetylated histones and recruit transcriptional regulator complexes to chromatin, eventually regulating gene transcription. Accumulating evidences demonstrate that pan BET inhibitors (BETi) confer protection against pathological cardiac hypertrophy, a precursor progress for developing heart failure. However, the roles of BET family members, except BRD4, remain unknown in pathological cardiac hypertrophy. The present study identified BRD2 as a novel regulator in cardiac hypertrophy, with a distinct mechanism from BRD4. BRD2 expression was elevated in cardiac hypertrophy induced by β-adrenergic agonist isoprenaline (ISO) in vivo and in vitro. Overexpression of BRD2 upregulated the expression of hypertrophic biomarkers and increased cell surface area, whereas BRD2 knockdown restrained ISO-induced cardiomyocyte hypertrophy. In vivo, rats received intramyocardial injection of adeno-associated virus (AAV) encoding siBRD2 significantly reversed ISO-induced pathological cardiac hypertrophy, cardiac fibrosis, and cardiac function dysregulation. The bioinformatic analysis of whole-genome sequence data demonstrated that a majority of metabolic genes, in particular those involved in TCA cycle, were under regulation by BRD2. Real-time PCR results confirmed that the expressions of TCA cycle genes were upregulated by BRD2, but were downregulated by BRD2 silencing in ISO-treated cardiomyocytes. Results of mitochondrial oxygen consumption rate (OCR) and ATP production measurement demonstrated that BRD2 augmented cardiac metabolism during cardiac hypertrophy. In conclusion, the present study revealed that BRD2 could facilitate cardiac hypertrophy through upregulating TCA cycle genes. Strategies targeting inhibition of BRD2 might suggest therapeutic potential for pathological cardiac hypertrophy and heart failure.