Cardiomyocyte Hypertrophy Research Articles

Abstract P2051: Fibroblast-specific TGF-b Signaling Promotes Interstitial Fibrosis, Cardiomyocyte Hypertrophy And Cardiac Dysfunction In The Diabetic Heart Through Smad3 Activation

Objectives: Patients with diabetes exhibit a high prevalence of HFpEF associated with interstitial fibrosis and cardiomyocyte hypertrophy. However, the cellular and molecular mechanisms involved in diabetic heart disease remain poorly understood. TGF-bs are potent fibrogenic mediators that are activated in diabetic hearts, and act through a single type 2 receptor, TbRII. We hypothesized that fibroblast-specific actions of TGF-bs may mediate fibrosis and dysfunction in diabetic hearts. Methods and Results: To test the hypothesis, we developed lean and db/db type 2 diabetic TbRII KO mice (FTbRII2KO) using the Col1a2 CreER driver. Fibroblast-specific loss of TGF-b signaling attenuated hypertrophy in db/db mouse hearts. Speckle-tracking echocardiography with strain imaging showed that fibroblast-specific TbRII loss improved systolic and diastolic cardiac function in diabetic animals. In contrast, fibroblast TbRII disruption caused a worsening of both cardiac systolic and diastolic function in lean animals. Attenuated dysfunction in diabetic mice lacking fibroblast TGF-b signaling was associated with reduced collagen fiber thickness and with decreased cardiomyocyte size, suggesting paracrine effects of fibroblasts on cardiomyocytes. To study the downstream cascade responsible for the effects of TGF-b, we generated lean and diabetic mice with fibroblast-specific Smad3 loss. In db/db mice, fibroblast-specific Smad3 loss phenocopied the effects of fibroblast-specific TbRII disruption, suggesting that in diabetic cardiac fibroblasts TGF-b acts through Smad3 signaling. The bioinformatic analyses of transcriptomic profiles of cardiac fibroblasts identified thrombospondin 4 as a candidate mediator of Smad3-dependent diabetes-associated activation. Conclusions: Fibroblast-specific TGF-b actions in diabetes promote cardiac dysfunction through effects on collagen deposition and via paracrine actions on cardiomyocytes. The effects of TGF-b on diabetic cardiac fibroblasts are Smad3-dependent. In contrast to the deleterious effects of fibroblast TGFb/Smad3 signaling in diabetic hearts, TGF-b-activated Smad3-dependent signaling is important to maintain homeostatic function and geometry in normal lean animals.

Abstract P2143: New Rock Inhibitor Improves Pulmonary Vascular Resistance And Right Ventricle Function In Animal Model Of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is characterised by increased pulmonary arterial pressure and vascular thickening, which causes RV dysfunction and failure. Current therapies lack efficacy for preventing disease progression and lead to poor survival rate in patients. Rho-activated protein kinases (ROCK) play a key role in smooth muscle contraction and proliferation and cardiomyocyte hypertrophy and survival, which indicate the potential of ROCK inhibitors to treat PAH. This work aims to assess the efficacy of a novel ROCK inhibitor, named LASSBio-2065, on cardiac and vascular function in monocrotaline (MCT)-induced PAH. Intraperitoneal injection of 60 mg/kg MCT induced PAH in Wistar rats, which protocol was approved by the Animal Care and Use Committee at Universidade Federal do Rio de Janeiro. After 2 wks, echocardiography confirmed the PAH and rats were randomly divided in groups and treated with either vehicle or LASSBio-2065 (60 μmol/kg/day, i.p.) for 14 days. At the end of treatment, hemodynamic parameters were evaluated using echocardiography and RV catheterization. Tissues were collected for histologic and morphometric analysis. Data expressed as mean ± SEM was analysed using One-way ANOVA followed by Dunnett post-test. LASSBio-2065 improves pulmonary vascular resistance and RV afterload because the ratio of pulmonary acceleration time-to-total ejection time reduced from 26.7 ± 2.0 to 21.7 ± 1.6 (p< 0.05) and RV output from 64.2 ± 6.1 to 39.1 ± 8.0 (p<0.05). Vascular and cardiac remodelling were prevented by LASSBio-2065, since medial wall thickness of distal pulmonary arterioles reduced and short axis RV-to-LV internal area increased from 85.8 ± 11.4 to 115.2 ± 17.8 (p<0.05). The new ROCK inhibitor also improved the RV diastolic function, restoring both RV end-diastolic pressures increasing from 5.3 ± 0.8 to 11.1 ± 1.9 mmHg (p<0.05) and Tau from 16.9 ± 2.2 to 27.1 ± 2.8 (p<0.05). In conclusion, LASSBio-2065 significantly improved RV function in MCT-induced PAH. In conclusion, the new ROCK inhibitor represents a potential alternative for management of PAH-associated RV dysfunction and remodelling.

Abstract P1166: O-GlcNAcylation Regulates Protein Synthesis In Cardiomyocytes Through Effects On The Translational Regulator Eif2α

The regulated and reversible modification of Ser/Thr residues by O-linked N-Acetylglucosamine (O-GlcNAc) is termed O-GlcNAcylation. O-GlcNAc cycling on and off proteins is regulated by two conserved enzymes, the ‘writer’ O-GlcNAc Transferase (OGT) and the ‘eraser’ O-GlcNAcase (OGA). Crucially, O-GlcNAcylation is implicated in nutrient sensing, cell growth and stress adaptation. Despite these significant roles, the precise pathways impacted by O-GlcNAcylation and what key substrate proteins are involved remain incompletely understood. A hallmark of cardiomyocyte hypertrophy is the rapid production of new proteins that assemble into the contractile machinery of the growing myocyte. eIF2α is a rate-limiting factor in translation initiation. Interestingly, phosphorylation of eIF2α causes the arrest of general mRNA translation and favors the translation of stress-adaptive mRNAs. In the present work we investigated whether altering O-GlcNAcylation affects nascent protein synthesis (NPS) in hypertrophic cardiomyocytes. NPS was significantly elevated by the agonist phenylephrine, and this response was suppressed by OSMI-1 (OGT inhibitor). Moreover, we found that OGT-inhibited cells had increased phosphorylation of eIF2α. Additionally, we found increased protein levels of the stress adaptive factors Atf4 and Chop, whose mRNA translation is favored when eIF2α is phosphorylated. ISRIB, a small molecule designed to suppress the effects of phospho-eIF2α in altering translation, significantly prevented OSMI-1-induced accumulation of Atf4. Furthermore, we used metabolic labeling of glycans in cells, coupled with alkyne-azide ‘click’ chemistry and streptavidin pull-down, to show that eIF2α can be O-GlcNAcylated in cardiomyocytes. Furthermore, protein enrichment followed by mass spectrometry identified potential sites of O-GlcNAcylation on eIF2α. Taken together, these results highlight a novel role of O-GlcNAcylation on cardiomyocyte protein synthesis through the regulation of eIF2α. Future investigations will characterize the upstream mechanisms that promote eIF2α phosphorylation in OGT-inhibited cells and investigate the implications of direct eIF2α O-GlcNAcylation on hypertrophic protein synthesis in cardiomyocytes.

Abstract P2084: Adipose Tissues Thermogenesis Provides Cardioprotection Against Diastolic Dysfunction Developed In An Obesity-Induced HFpEF Model

Background: Heart failure with preserved ejection fraction (HFpEF) is a major source of morbidity/ mortality in the United States. A systemic inflammatory state established through underlying comorbidities, such as obesity, is thought to develop cardiac dysfunction in HFpEF. Clinical observations associate adiposity with diastolic dysfunction. Herein we demonstrate that adipose tissue is a direct source of diastolic dysfunction and reveal a cardioprotective role of adipose tissue thermogenesis in obesity related HFpEF. Methods and Results: Mice were treated with a β3-AR agonist, CL-316243 (CL), to induce thermogenesis, followed by HFpEF treatment consisting of HFD and L-NAME. Echocardiography revealed that this protected against HFpEF-induced diastolic dysfunction. Whole heart extracts and AT were then evaluated for the signature of β3-AR signaling. In the heart, no signature of β3-AR signaling was observed. In AT, though, various aspects of β3-AR induced thermogenesis were observed. Histological analysis revelated that CL treatment conferred resistance against obesity-induced whitening of interscapular brown adipose tissue (iBAT). Gene and protein expression changes of thermogenic regulators and lipid metabolism enzymes suggested greater thermogenic activity in CL-treated mice. Accordingly, indirect calorimetry showed enhanced energy expenditure, while regression analysis revealed that AT uniquely contributed to this. Transplantation experiments involving CL-treated donor AT and wild-type donors followed by echocardiographic measurements confirmed that the effects of CL were mediated through AT. This approach was complemented with a genetic model we previously used ( Ucp1 -Cre ERT2 ; Cdkn2a fl/fl ) to promote the expansion of beige AT. Here, beige AT expansion protected against diastolic dysfunction and cardiomyocyte hypertrophy developed during HFpEF. Conclusion: Our study indicates enhanced energy expenditure in AT is cardioprotective in obesity related HFpEF. Furthermore, identifying obese AT as a direct source of diastolic dysfunction highlights the therapeutic potential of targeting AT metabolism as a relevant source of diastolic dysfunction.

Abstract P1173: Deciphering CITED4-dependent Signaling In The Heart Through Nascent Proteomics And Secretomics

Objective: Neuregulin-1 (Nrg1) promotes cardiomyocyte hypertrophy, survival, and cell cycle activity through ErbB signaling. It is investigated in clinical trials as a cardioprotective agent; however, its therapeutic use might be limited due to its pro-neoplastic potential for non-cardiomyocytes that makes targeted approaches necessary. High-throughput screening of hypertrophic agonists found that Nrg1 induces CITED4 (C4) expression. C4 is also upregulated in both physiological and pathological cardiac growth and necessary to prevent adverse remodeling in vivo. However, how C4 regulates Nrg1-signaling in the heart is yet unknown. Methods: We combined pulsed-SILAC labeling, click-chemistry and mass spectrometry that allows to capture immediate changes in the proteome and secretome after Nrg1 (12h and 24h) stimulation in siRNA-mediated C4-knockdown (C4KD) and control (ctr) NRVM and complemented this with established cell- and molecular biology techniques to validate and investigate cellular function and molecular signaling. Results: We confirmed dose-dependent C4 mRNA upregulation in response to Nrg1 stimulation in NRVM. Nrg1 downstream signaling through AKT was hindered in C4KD. Computational analysis comparing the nascent proteome after Nrg1 stimulation in C4KD and ctr NRVM revealed that C4 significantly regulates proteins responsible for translation, RNA processing and energy derivation. Consistent with previously observed development of cardiac fibrosis in C4 knockout mice in vivo , we found significant upregulation of TGFb2 in C4KD NRVM. We next confirmed the upregulation of TGFb2 and its downstream effectors in C4KD and ctr, while adenoviral overexpression of C4 led to reduced TGFb2 expression. Additionally, we found TGFb2 secretion increased from C4KD NRVM with Nrg1 stimulation in the nascent secretome. Conditioned media from C4KD NRVM led to an increased pro-fibrotic response in cardiac fibroblasts. In conclusion, nascent proteomics and secretomics help to elucidate C4-dependent Nrg1-signaling, which may be an important contribution toward our goal to identify targetable cardioprotective pathways in the heart.

Abstract P1140: Acute Mechanosensitive Adaptation To Phenylephrine In Cardiomyocytes And Fibroblasts

Mechanosensitive pathways are fine tuned to the physiological environment of the cell. Adrenergic stimulation induces cell type specific responses in the heart, including cardiomyocyte hypertrophy and fibroblast proliferation. We hypothesized acute induction of hypertrophy by phenylephrine (PHE) would activate mechanosensitive pathways in fibroblasts and myocytes, revealing novel molecular features of early hypertrophic adaptation. Male mice were subcutaneously injected with 20 mg/kg of PHE or PBS every other day for one week. Echocardiography revealed significant cardiac hypertrophy with increased ejection fraction (72.45% + 6.15 PHE v 56.32 + 5.32 PBS, p<0.0001) and LV wall thickness (2.25 mm + 0.29 PHE v 1.62 + 0.13 PBS, p<0.0001), along with diastolic impairment, as measured by elevated E/e’ ratio (37.25 + 7.29 PHE v 23.67 + 3.62 PBS, p<0.0001). Interestingly, histological analysis showed increased fibrotic deposition and cardiomyocyte cross-sectional area was preferential increased in fibrotic areas (265.5 um 2 + 67.5 PHE fibrotic v 195.1 + 47.46 PHE non-fibrotic v 165.6 + 46.96 PBS). While cardiac fibroblasts from PHE-treated animals did not show alpha smooth muscle actin staining, a traditional marker for activated myofibroblasts, fibroblasts from PHE-treated mice showed increased f-actin stress fibers and nuclear localization of Mrtfa, a mechanosensitive transcriptional coactivator. We performed RNA-seq on isolated cardiomyocytes, demonstrating activation of myocyte-specific responses to increased pressure ( Des, Nppa ), as well as activation of fibrotic genes ( Tgfbr1, Tgfbr2, Tgfb2 ) in myocytes . We sought to investigate mechanosensitive pathways initiated by PHE treatment in cardiomyocytes and identified transcriptional changes in Ankrd1, a Titin binding protein which translocates to the nucleus upon stress. We demonstrate a PHE induced increase in Ankrd1 protein expression and nuclear localization (nuclear area occupancy, as measured by microscopy, 17.04% + 8.76 PHE v 11.91% + 9.134 PBS, p=0.002) in cardiomyocytes. Together these findings demonstrate distinct acute phenotyptic adaptation to adrenergic stress in myocytes and fibroblasts, revealing cell type-specific mechanosensitive signaling mechanisms.

Abstract P3037: MicroRNA-205 Is Required For Normal Cell Cycle And Cardiac Growth In The Neonatal Mouse Heart.

After birth, most newborn mouse cardiomyocytes (CM) undergo a final cycle of cell division in the absence of cytokinesis, which leads to binucleation. The majority of subsequent increases in cardiac mass are attributable to cardiomyocyte hypertrophy, as a negligible number of new CM are created after birth. Mice with a heart-specific deletion of Dicer, a crucial enzyme necessary for microRNA maturation, die by the fifth postnatal day in spite of having normal cardiac morphology and function at birth. Despite the significance of this phenomenon, little is known about the molecular/genetic foundation for the change from hyperplastic to hypertrophic-based myocardial expansion that occurs in the neonatal mouse heart. We hypothesize that a microRNA is required for the normal transition from fetal to adult heart during the neonatal period. Heart microRNA profiles (3 arrays/time point) were measured from RNA was isolated from E19, 1, 3, 5, 7, 10, and 35-day-old mouse hearts (9 hearts/time point pooled). Bioinformatic analysis identified microRNA-205 as a possible participant in the cardiac transitional program. We observed a transient 20-fold increase in miR205 expression between postnatal days 1 and 5, with levels returning to baseline by day 10. In-situ hybridization revealed miR-205 expression is restricted to the heart's epicardium.We next generated mice with a CM-specific deletion of miR205 using MHCcre targeting of a floxed-miR205 allele. Mice lacking miR-205 in their CM appear normal at birth. Elevated cell cycle markers (Ki67 & phospho-pH3) were detected at postnatal day-14 with 20% larger hearts at postnatal day 14, and adult hearts measuring up to 50% larger than controls. In contrast, a CM-specific miR205 over-expression mouse model resulted in more CM being present at both 5-day and 14-day post-birth. Interestingly, no difference in cell-cycle markers or heart size was observed. miR205 directly targets YAP within the evolutionarily conserved hippo pathway, which regulates organ size. YAP protein expression is significantly increased in hearts lacking miR205. Both of the miR205-heart models present with altered Hippo signaling kinetics after birth. We conclude that miR205 regulates postnatal heart size by directly modulating the Hippo pathway.

Abstract P1019: High-throughput Generation And Functional Characterization Of Myosin Heavy Chain 7 Variants In Genome-edited Cardiomyocytes

Hypertrophic cardiomyopathy (HCM), an inheritable cardiac condition characterized by idiopathic left ventricular hypertrophy, can cause sudden cardiac death or heart failure. Autosomal-dominant missense mutations in MYH7 (encoding the sarcomeric βMHC protein) account for 33% of genotype-positive HCM cases. While pathogenic MYH7 variants are clinically-actionable, 75% of MYH7 missense variants in ClinVar are variants of unknown significance (VUS). Gene-editing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can help determine the function of MYH7 VUS, however, the discovery of new variants by genetic testing outpaces the ability to gene-edit hiPSCs. Thus, we developed a novel method called CR ISPR a On- T arget E diting R etrieval (CRaTER) which increases the success of gene-editing by 25-fold compared to current methods. We leveraged CRaTER to enrich for hiPSCs edited with a library of MYH7 missense variants and recovered 113 different heterozygous variants. As deleterious mutations can reduce protein stability, we flow sorted MYH7 variant hiPSC-CMs into bins based on differing βMHC abundance followed by next generation sequencing of each bin to calculate variant abundance scores. This assay identified 31 MYH7 missense variants as functionally abnormal (lower abundance scores than synonymous controls), including all known pathogenic variants in the library. This βMHC depletion phenotype was validated in clonal hiPSC-CMs and supported by western blotting of myocardium from a patient with a pathogenic MYH7 missense mutation. Furthermore, we rescued βMHC protein levels by genetically ablating the gene encoding for Muscle RING-finger protein-1, an E3 protein ligase that targets βMHC for degradation, and found that the cardiomyocyte hypertrophy induced by the pathogenic MYH7 E848G HCM variant was significantly blunted. In summary, CRaTER enables the generation of gene-edited hiPSC lines at unprecedented scale. Functional analysis of MYH7 variant hiPSC-CMs reveals a novel correlation between loss of βMHC protein and clinical pathogenicity that can be leveraged for clinical variant interpretation. Interventions that prevent the loss of βMHC may potentially be therapeutic for MYH7 -associated HCM.

Paeoniflorin alleviates AngII-induced cardiac hypertrophy in H9c2 cells by regulating oxidative stress and Nrf2 signaling pathway

Cardiac hypertrophy is frequently associated with ventricular dysfunction and heart failure. Paeoniflorin, has been widely used to treat cardiovascular dysfunction-related diseases. However, the underlying mechanism has been unclear. Here, we investigated the potential inhibitory effects and mechanism of paeoniflorin on oxidative stress of cardiac hypertrophy induced by angiotensin II (AngII) in vitro. Using MTS assay, qRT-PCR, WGA staining assay, and western blot, different dosages (50–400 μM) of paeoniflorin were utilized to examine the antihypertrophy effects on H9c2 cells. Western blot examination revealed the presence of apoptosis-related proteins Bax, Bcl2, and Cytc, antioxidative stress-related proteins Nrf2, HO-1, SOD, and CAT, and mitophagy-related proteins PINK1 and Parkin. qRT-PCR was used to detect the mRNA expression of Bax, Bcl2, Nrf2, and HO-1. TUNEL, caspase3/9 enzyme viability, and MDA, T-AOC, and superoxide levels were all evaluated using commercial kits.The fluorescent probes DCFH-DA and JC-1 were employed to measure cellular ROS and MMP levels. Nrf2 siRNA was utilized to investigate Nrf2's role in paeoniflorin-treated cardiac hypertrophy. Paeoniflorin dramatically reduced cell section area (CSA) and hypertrophic marker (ANP, BNP) expression while inhibiting oxidative stress by modulating ROS and MDA, CAT, SOD, and T-AOC levels. Furthermore, in AngII-induced cardiomyocyte hypertrophy, paeoniflorin restores H9c2 apoptosis by restoring Bax, Bcl-2 Cyt-C, Caspase 3, and Caspase 9 levels. Paeoniflorin also restored Nrf2/HO-1 and PINK1/Parkin expression, and its anti-AngII activities were mediated by Nrf2, which was regulated by Nrf2 knockdown. In conclusion, Our data confirm that paeoniflorin alleviates cardiac hypertrophy through modulating oxidative stress and Nrf2 signaling pathway in vitro.

SGLT2 inhibitor-pretreated macrophage transplantation improves adverse ventricular remodeling after acute myocardial infarction.

Macrophages play an important role in the progression of acute myocardial infarction (AMI). Studies have shown that sodium-dependent glucose transporter 2 inhibitor (SGLT2i) after AMI could increase the proportion of M2 type/M1 macrophages and reduces adverse ventricular remodeling (AVR) post AMI. This study aimed to investigate whether SGLT2i-pretreated macrophage transplantation could reduce AVR after AMI and the underlying mechanisms. C57BL/6 mice were used to establish an AMI model by ligating the coronary arteries. Dynamic observation of transplanted bone marrow-derived macrophages (BMDMs) was performed using an in vivo imaging system (IVIS). Cardiac function was assessed using echocardiography. The fibrosis ratio was measured using Masson's trichrome staining. Cardiomyocyte apoptosis was measured using the TUNEL assay. Macrophage subtypes were measured using flow cytometry. We detected the expression of inflammatory factors in the myocardium and serum using enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). IVIS revealed that transplanted SGLT2i-pretreated BMDMs were present in the infarcted myocardium for 7 d. Flow cytometry revealed that SGLT2i-pretreated BMDMs promoted the conversion of native-derived macrophages to the M2-type. SGLT2i-pretreated BMDMs also reduced inflammatory factors (IL-6, TNFα, and IL-1β) in the infarcted myocardium and serum. At 28 d post-AMI, SGLT2i-pretreated BMDMs increased cardiac function and vascular density, but reduced cardiomyocyte hypertrophy. SGLT2i-pretreated BMDMs could reduce cardiomyocyte apoptosis and fibrotic area ratio. In conclusion, transplanted SGLT2i-pretreated BMDMs were present in the infarcted myocardium for 7 d and improved AVR by reducing inflammation and modulating the conversion of native mice-derived macrophages to M2-type macrophages.

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.

LncRNA-encoded peptides: unveiling their significance in cardiovascular physiology and pathology - current research insights.

Long non-coding RNAs (lncRNAs), which are RNA transcripts exceeding 200 nucleotides, were believed to lack any protein-coding capacity. But advancements in -omics technology have revealed that some lncRNAs have small open reading frames (sORFs) that can be translated by ribosomes to encode peptides, some of which have important biological functions. These encoded peptides subserve important biological functions by interacting with their targets to modulate transcriptional or signaling axes, thereby enhancing or suppressing CVD occurrence and progression. In this review, we summarize what is known about the research strategy of lncRNA-encoded peptides, mainly comprising predictive websites/tools and experimental methods that have been widely used for prediction, identification, and validation. More importantly, we have compiled a list of lncRNA- encoded peptides, with a focus on those that play significant roles in cardiovascular physiology and pathology, including RNO-sORF6/RNO-sORF7/RNO-sORF8, DOWRF, and NLN etc. Additionally, we have outlined the functions and mechanisms of these peptides in cardiovascular physiology and pathology, such as cardiomyocyte hypertrophy, myocardial contraction, myocardial infarction and vascular remodeling. Finally, an overview of the existing challenges and potential future developments in the realm of lncRNA-encoded peptides was provided, with consideration given to prospective avenues for further research. Given that many lncRNA encoded peptides have not been functionally annotated yet, their application in CVD diagnosis and treatment still requires further research.

Tripartite motif‑containing 14 may aggravate cardiac hypertrophy via the AKT signalling pathway in neonatal rat cardiomyocytes and transgenic mice.

Tripartite motif‑containing 14 (TRIM14) is an E3 ubiquitin ligase that primarily participates in the natural immune response and in tumour development via ubiquitination. However, the role of TRIM14 in cardiac hypertrophy is not currently clear. The present study examined the role of TRIM14 in cardiac hypertrophy and its potential molecular mechanism. TRIM14 was overexpressed in neonatal rat cardiomyocytes using adenovirus and cardiomyocyte hypertrophy was induced using phenylephrine (PE). Cardiomyocyte hypertrophy was assessed by measuring cardiomyocyte surface area and markers of hypertrophy. In addition, TRIM14‑transgenic (TRIM14‑TG) mice were created and cardiac hypertrophy was induced using transverse aortic constriction (TAC). Cardiac function, heart weight‑to‑body weight ratio (HW/BW), cardiomyocyte cross‑sectional area, cardiac fibrosis and hypertrophic markers were further examined. The expression of AKT signalling pathway‑related proteins was detected. TRIM14 overexpression in cardiomyocytes promoted PE‑induced increases in cardiomyocyte surface area and hypertrophic markers. TRIM14‑TG mice developed worse cardiac function, greater HW/BW, cross‑sectional area and cardiac fibrosis, and higher levels of hypertrophic markers in response to TAC. TRIM14 overexpression also increased the phosphorylation levels of AKT, GSK‑3β, mTOR and p70S6K invivo and invitro. To the best our knowledge, the present study was the first to reveal that overexpression of TRIM14 aggravated cardiac hypertrophy invivo and invitro, which may be related to activation of the AKT signalling pathway.

Indoleamine 2,3-Dioxygenase 1 Deletion-Mediated Kynurenine Insufficiency Inhibits Pathological Cardiac Hypertrophy.

Aberrant amino acid metabolism is implicated in cardiac hypertrophy, while the involvement of tryptophan metabolism in pathological cardiac hypertrophy remains elusive. Herein, we aimed to investigate the effect and potential mechanism of IDO1 (indoleamine 2,3-dioxygenase) and its metabolite kynurenine (Kyn) on pathological cardiac hypertrophy. Transverse aortic constriction was performed to induce cardiac hypertrophy in IDO1-knockout (KO) mice and AAV9-cTNT-shIDO1 mice. Liquid chromatography-mass spectrometry was used to detect the metabolites of tryptophan-Kyn pathway. Chromatin immunoprecipitation assay and dual luciferase assay were used to validate the binding of protein and DNA. IDO1 expression was upregulated in both human and murine hypertrophic myocardium, alongside with increased IDO1 activity and Kyn content in transverse aortic constriction-induced mice's hearts using liquid chromatography-mass spectrometry analysis. Myocardial remodeling and heart function were significantly improved in transverse aortic constriction-induced IDO1-KO mice, but were greatly exacerbated with subcutaneous Kyn administration. IDO1 inhibition or Kyn addition confirmed the alleviation or aggravation of hypertrophy in cardiomyocyte treated with isoprenaline, respectively. Mechanistically, IDO1 and metabolite Kyn contributed to pathological hypertrophy via the AhR (aryl hydrocarbon receptor)-GATA4 (GATA binding protein 4) axis. This study demonstrated that IDO1 deficiency and consequent Kyn insufficiency can protect against pathological cardiac hypertrophy by decreasing GATA4 expression in an AhR-dependent manner.

Transmembrane protein 117 knockdown protects against angiotensin-II-induced cardiac hypertrophy

Mitochondrial dysfunction plays a critical role in the pathogenesis of pathological cardiac hypertrophy. Transmembrane protein 117 modulate mitochondrial membrane potential that may be involved in the regulation of oxidative stress and mitochondrial function. However, its role in the development of angiotensin II (Ang-II)-induced cardiac hypertrophy is unclear. Cardiac-specific TMEM117-knockout and control mice were subjected to cardiac hypertrophy induced by Ang-II infusion. Small-interfering RNAs against TMEM117 or adenovirus-based plasmids encoding TMEM117 were delivered into left ventricles of mice or incubated with neonatal murine ventricular myocytes (NMVMs) before Ang-II stimulation. We found that TMEM117 was upregulated in hypertrophic hearts and cardiomyocytes and TMEM117 deficiency attenuated Ang-II-induced cardiac hypertrophy in vivo. Consistently, the in vitro data demonstrated that Ang-II-induced cardiomyocyte hypertrophy significantly alleviated by TMEM117 knockdown. Conversely, overexpression of TMEM117 exacerbated cardiac hypertrophy and dysfunction. An Ang II-induced increase in cardiac (cardiomyocyte) oxidative stress was alleviated by cardiac-specific knockout (knockdown) of TMEM117 and was worsened by TMEM117 supplementation (overexpression). In addition, TMEM117 knockout decreased endoplasmic reticulum stress induced by Ang-II, which was reversed by TMEM117 supplementation. Furthermore, TMEM117 deficiency mitigated mitochondrial injury in hypertrophic hearts and cardiomyocyte, which was abolished by TMEM117 supplementation (overexpression). Taken together, these findings suggest that upregulation of TMEM117 contributes to the development of cardiac hypertrophy and the downregulation of TMEM117 may be a new therapeutic strategy for the prevention and treatment of cardiac hypertrophy.

Loss of USP22 alleviates cardiac hypertrophy induced by pressure overload through HiF1-α-TAK1 signaling pathway

Ubiquitin-specific protease 22 (USP22) is a member of the ubiquitin specific protease family (ubiquitin-specific protease, USPs), the largest subfamily of deubiquitinating enzymes, and plays an important role in the treatment of tumors. USP22 is also expressed in the heart. However, the role of USP22 in heart disease remains unclear. In this study, we found that USP22 was elevated in hypertrophic mouse hearts and in angiotensin II (Ang II)-induced cardiomyocytes. The inhibition of USP22 expression with adenovirus significantly rescued hypertrophic phenotype and cardiac dysfunction induced by pressure overloaded. Consistent with in vivo study, silencing by USP22 shRNA expression in vitro had similar results. Molecular analysis revealed that transforming growth factor-β-activating protein 1 (TAK1)-(JNK1/2)/P38 signaling pathway and HIF-1α was activated in the Ang II-induced hypertrophic cardiomyocytes, whereas HIF-1α expression was decreased after the inhibition of USP22. Inhibition of HIF-1α expression reduces TAK1 expression. Co-immunoprecipitation and ubiquitination studies revealed the regulatory mechanism between USP22 and HIF1α.Under hypertrophic stress conditions, USP22 enhances the stability of HIF-1α through its deubiquitination activity, which further activates the TAK1-(JNK1/2)/P38 signaling pathway to lead to cardiac hypertrophy. Inhibition of HIF-1α expression further potentiates the in vivo pathological effects caused by USP22 deficiency. In summary, this study suggests that USP22, through HIF-1α-TAK1-(JNK1/2)/P38 signaling pathway, may be potential targets for inhibiting pathological cardiac hypertrophy induced by pressure overload.

Protective effect and mechanism of AKAP1 on myocardial injury induced by highland hypobaric hypoxia

Objective: To investigate the protective effect and its possible mechanism of A-kinase anchored protein 1 (AKAP1) on the myocardial injury induced by highland hypobaric hypoxia. Methods: From January 2021 to May 2022, male C57BL/6 SPF grade mice were divided into wild type control (WT) group and highland hypobaric hypoxia (HH) group with 6 mice in each group. HH group simulated 6000 m altitude with low pressure oxygen chamber for 4 weeks to build the model. Primary myocardial cells of SD rats were divided into normoxia control group and hypoxia experimental group (n=3). Cell models were constructed in a three-gas hypoxia incubator with 1% oxygen concentration for 24 h. AKAP1 protein and mRNA expression in myocardial tissue and cells were detected by western blotting, immunohistochemistry and quantitative real-time polymerase chain reaction (qPCR). After myocardial point injection of the AKAP1 or the control adenovirus, the mice were divided into 3 groups (n=6) : WT group, highland hypobaric hypoxia overexpression control group (HH+Ad-Ctrl group) and highland hypobaric hypoxia overexpression experimental group (HH+Ad-AKAP1 group). The cardiac function of mice was detected by noninvasive M-type ultrasonic cardiomotive, myocardial fibrosis was detected by Masson and Sirius Red staining, and cardiomyocyte hypertrophy was detected by wheat germ agglutinin. After the expression of AKAP1 in primary cardiomyocytes was downregulated by siRNA and upregulated by adenovirus, the cells were divided into three groups (n=3) : normoxia control group, hypoxia interference control group (hypoxia+siCtrl group), hypoxia AKAP1 knockdown group (hypoxia+siAKAP1 group) ; normoxia control group, hypoxia overexpression control group (hypoxia+Ad-Ctrl group), hypoxia AKAP1 overexpression group (hypoxia+Ad-AKAP1 group). Apoptosis was detected by flow cytometry, AKAP1, apoptosis-related protein and mRNA expression levels were detected by western blotting and qPCR, mitochondrial membrane potential was detected by JC-1 staining, and mitochondrial reactive oxygen specie (ROS) level was detected by MitoSOX. Results: The expression of AKAP1 in cardiac muscle of HH group was lower than that in the WT group, and the expression of AKAP1 in hypoxia experimental group was lower than that in normoxia control group (P<0.01). Compared with WT group, the left ventricular ejection fraction and fraction shortening of left ventricle in HH+Ad-Ctrl group were decreased (P<0.01), myocardial fibrosis and hypertrophy were aggravated (P<0.01), and the expression of B-cell lymphoma-2 (BCL-2) was decreased, the expressions of BCL-2-associated X protein (BAX), Caspase 3 and Caspase 9 were increased (P<0.01). After AKAP1 overexpression, compared with HH+Ad-Ctrl group, the left ventricular ejection fraction and left ventricular fraction shortening were increased in HH+Ad-AKAP1 group (P<0.01), myocardial fibrosis and hypertrophy were reduced (P<0.01), and the expression of BCL-2 was increased, the expressions of BAX, Caspase 3 and Caspase 9 were decreased (P<0.01). Compared with normoxia control group, the expression of BCL-2 in hypoxia+siCtrl group was decreased, the expressions of BAX, Caspase 3, Caspase 9 were increased, the apoptosis level was increased (P<0.01), the mitochondrial membrane potential was decreased and the production of ROS was increased (P<0.01). After AKAP1 knockdown, compared with hypoxia+siCtrl group, the expression of BCL-2 in hypoxia+siAKAP1 group was decreased, the expressions of BAX, Caspase 3, Caspase 9 were increased, the apoptosis level was increased (P<0.01), mitochondrial membrane potential was decreased, and the production of ROS was increased (P<0.01). After AKAP1 overexpression, compared with hypoxia+Ad-Ctrl group, the expression of BCL-2 in hypoxia+Ad-AKAP1 group was increased, the expressions of BAX, Caspase 3 and Caspase 9 were decreased (P<0.05), the apoptosis level was decreased (P<0.01), and the mitochondrial membrane potential was enhanced, and the production of ROS was decreased (P<0.01) . Conclusion: The downregulation of AKAP1 in cardiomyocytes under highland hypobaric hypoxia may lead to the decrease of mitochondrial membrane potential and the increase of ROS generation, leading to the apoptosis of cardiomyocytes, and thus aggravating the myocardial injury at highland hypobaric hypoxia.

Macrophage DCLK1 promotes obesity-induced cardiomyopathy via activating RIP2/TAK1 signaling pathway

Obesity increases the risk for cardiovascular diseases and induces cardiomyopathy. Chronic inflammation plays a significant role in obesity-induced cardiomyopathy and may provide new therapeutic targets for this disease. Doublecortin-like kinase 1 (DCLK1) is an important target for cancer therapy and the role of DCLK1 in obesity and cardiovascular diseases is unclear. Herein, we showed that DCLK1 was overexpressed in the cardiac tissue of obese mice and investigated the role of DCLK1 in obesity-induced cardiomyopathy. We generated DCLK1-deleted mice and showed that macrophage-specific DCLK1 knockout, rather than cardiomyocyte-specific DCLK1 knockout, prevented high-fat diet (HFD)-induced heart dysfunction, cardiac hypertrophy, and fibrosis. RNA sequencing analysis showed that DCLK1 deficiency exerted cardioprotective effects by suppressing RIP2/TAK1 activation and inflammatory responses in macrophages. Upon HFD/palmitate (PA) challenge, macrophage DCLK1 mediates RIP2/TAK1 phosphorylation and subsequent inflammatory cytokine release, which further promotes hypertrophy in cardiomyocytes and fibrogenesis in fibroblasts. Finally, a pharmacological inhibitor of DCLK1 significantly protects hearts in HFD-fed mice. Our study demonstrates a novel role and a pro-inflammatory mechanism of macrophage DCLK1 in obesity-induced cardiomyopathy and identifies DCLK1 as a new therapeutic target for the treatment of this disease.Upon HFD/PA challenge, DCLK1 induces RIP2/TAK1-mediated inflammatory response in macrophages, which subsequently promotes cardiac hypertrophy and fibrosis. Macrophage-specific DCLK1 deletion or pharmacological inhibition of DCLK1 protects hearts in HFD-fed mice.

Dysregulated Rbfox2 produces aberrant splicing of CaV1.2 calcium channel in diabetes-induced cardiac hypertrophy

BackgroundL-type Ca2+ channel CaV1.2 is essential for cardiomyocyte excitation, contraction and gene transcription in the heart, and abnormal functions of cardiac CaV1.2 channels are presented in diabetic cardiomyopathy. However, the underlying mechanisms are largely unclear. The functions of CaV1.2 channels are subtly modulated by splicing factor-mediated alternative splicing (AS), but whether and how CaV1.2 channels are alternatively spliced in diabetic heart remains unknown.MethodsDiabetic rat models were established by using high-fat diet in combination with low dose streptozotocin. Cardiac function and morphology were assessed by echocardiography and HE staining, respectively. Isolated neonatal rat ventricular myocytes (NRVMs) were used as a cell-based model. Cardiac CaV1.2 channel functions were measured by whole-cell patch clamp, and intracellular Ca2+ concentration was monitored by using Fluo-4 AM.ResultsWe find that diabetic rats develop diastolic dysfunction and cardiac hypertrophy accompanied by an increased CaV1.2 channel with alternative exon 9* (CaV1.2E9*), but unchanged that with alternative exon 8/8a or exon 33. The splicing factor Rbfox2 expression is also increased in diabetic heart, presumably because of dominate-negative (DN) isoform. Unexpectedly, high glucose cannot induce the aberrant expressions of CaV1.2 exon 9* and Rbfox2. But glycated serum (GS), the mimic of advanced glycation end-products (AGEs), upregulates CaV1.2E9* channels proportion and downregulates Rbfox2 expression in NRVMs. By whole-cell patch clamp, we find GS application hyperpolarizes the current-voltage curve and window currents of cardiac CaV1.2 channels. Moreover, GS treatment raises K+-triggered intracellular Ca2+ concentration ([Ca2+]i), enlarges cell surface area of NRVMs and induces hypertrophic genes transcription. Consistently, siRNA-mediated knockdown of Rbfox2 in NRVMs upregulates CaV1.2E9* channel, shifts CaV1.2 window currents to hyperpolarization, increases [Ca2+]i and induces cardiomyocyte hypertrophy.ConclusionsAGEs, not glucose, dysregulates Rbfox2 which thereby increases CaV1.2E9* channels and hyperpolarizes channel window currents. These make the channels open at greater negative potentials and lead to increased [Ca2+]i in cardiomyocytes, and finally induce cardiomyocyte hypertrophy in diabetes. Our work elucidates the underlying mechanisms for CaV1.2 channel regulation in diabetic heart, and targeting Rbfox2 to reset the aberrantly spliced CaV1.2 channel might be a promising therapeutic approach in diabetes-induced cardiac hypertrophy.

Virtual drug screen reveals context-dependent inhibition of cardiomyocyte hypertrophy.

Pathological cardiomyocyte hypertrophy is a response to cardiac stress that typically leads to heart failure. Despite being a primary contributor to pathological cardiac remodelling, the therapeutic space that targets hypertrophy is limited. Here, we apply a network model to virtually screen for FDA-approved drugs that induce or suppress cardiomyocyte hypertrophy. A logic-based differential equation model of cardiomyocyte signalling was used to predict drugs that modulate hypertrophy. These predictions were validated against curated experiments from the prior literature. The actions of midostaurin were validated in new experiments using TGFβ- and noradrenaline (NE)-induced hypertrophy in neonatal rat cardiomyocytes. Model predictions were validated in 60 out of 70 independent experiments from the literature and identify 38 inhibitors of hypertrophy. We additionally predict that the efficacy of drugs that inhibit cardiomyocyte hypertrophy is often context dependent. We predicted that midostaurin inhibits cardiomyocyte hypertrophy induced by TGFβ, but not noradrenaline, exhibiting context dependence. We further validated this prediction by cellular experiments. Network analysis predicted critical roles for the PI3K and RAS pathways in the activity of celecoxib and midostaurin, respectively. We further investigated the polypharmacology and combinatorial pharmacology of drugs. Brigatinib and irbesartan in combination were predicted to synergistically inhibit cardiomyocyte hypertrophy. This study provides a well-validated platform for investigating the efficacy of drugs on cardiomyocyte hypertrophy and identifies midostaurin for consideration as an antihypertrophic drug.