Hypertrophic Growth Of Cardiomyocytes Research Articles

Abstract 4134695: The Effect and Mechanism of Histone Lactylation In Pathological Cardiac Hypertrophy Induced by Pressure Overload

Introduction: Cardiac hypertrophy is characterized by significant metabolic changes, notably an increase in glycolysis. Histone lactylation, a post-translational modification influenced by the glycolytic state of cells, plays a crucial role in regulating gene transcription. However, the relationship between histone lactylation and cardiac hypertrophy remains unclear. Research Questions: This study aims to elucidate the effects and underlying mechanisms of histone lactylation in the progression of cardiac hypertrophy induced by pressure overload. Methods and Results: We observed an increase in histone lactylation levels in hypertrophic hearts from both humans and mice. To investigate further, male mice underwent transverse aortic constriction (TAC) to induce cardiac hypertrophy, followed by either an intraperitoneal injection of Oxamate (an LDHA inhibitor) to reduce histone lactylation or sodium lactate to increase it. Reducing histone lactylation protected the hearts from TAC-induced hypertrophy and fibrosis, preserving cardiac function, while increased histone lactylation exacerbated cardiac hypertrophy and fibrosis, impairing cardiac function. Cardiomyocyte-specific deletion of LDHA also led to a reduction in cardiac hypertrophy. In vitro experiments showed that inhibiting histone lactylation reduced the expression of hypertrophic markers and the hypertrophic growth of cardiomyocytes stimulated by phenylephrine. Using co-immunoprecipitation, we confirmed that P300 and GCN5 are the transferases mediating histone lactylation in cardiomyocytes. Mechanistic studies using the CUT&Tag technique revealed lactate-dependent histone modification was enriched at the promoter of TGFB2, activating its transcription. Inhibiting cardiac-specific TGFB2 expression through adeno-associated viral delivery of shRNA reduced TAC-induced cardiac hypertrophy. Furthermore, TGFB2 overexpression induced a hypertrophic phenotype in cardiomyocytes and activated the PI3K/AKT/mTOR pathways. This hypertrophic phenotype, induced by TGFB2 overexpression, was suppressed by inhibitors of PI3K or AKT. Conclusion: Our findings reveal a critical role for histone lactylation in the development of pathological cardiac hypertrophy by regulating TGFB2 expression and the PI3K/AKT/mTOR pathway.

TRIM35 triggers cardiac remodeling by regulating SLC7A5-mediated amino acid transport and mTORC1 activation in fibroblasts

BackgroundCardiac maladaptive remodeling is one of the leading causes of heart failure with highly complicated pathogeneses. The E3 ligase tripartite motif containing 35 (TRIM35) has been identified as a crucial regulator governing cellular growth, immune responses, and metabolism. Nonetheless, the role of TRIM35 in fibroblasts in cardiac remodeling remains elusive.MethodsHeart tissues from human donors were used to verify tissue-specific expression of TRIM35. Fibroblast-specific Trim35 gene knockout mice (Trim35cKO) were used to investigate the function of TRIM35 in fibroblasts. Cardiac function, morphology, and molecular changes in the heart tissues were analyzed after transverse aortic constriction (TAC) surgery. The mechanisms by which TRIM35 regulates fibroblast phenotypes were elucidated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and RNA sequencing (RNA-Seq). These findings were further validated through the use of adenoviral and adeno-associated viral transfection systems, as well as the mTORC1 inhibitor Rapamycin.ResultsTRIM35 expression is primarily up-regulated in cardiac fibroblasts in both murine and human fibrotic hearts, and responds to TGF-β1 stimulation. Specific deletion of TRIM35 in cardiac fibroblasts significantly improves cardiac fibrosis and hypertrophy. Consistently, the overexpression of TRIM35 promotes fibroblast proliferation, migration, and differentiation. Through paracrine signaling, it induces hypertrophic growth of cardiomyocytes. Mechanistically, we found that TRIM35 interacts with, ubiquitinates, and up-regulates the amino acid transporter SLC7A5, which enhances amino acid transport and activates the mTORC1 signaling pathway. Furthermore, overexpression of SLC7A5 significantly reverses the reduced cardiac fibrosis and hypertrophy caused by conditional knockout of TRIM35.ConclusionOur findings demonstrate a novel role of fibroblast-TRIM35 in cardiac remodeling and uncover the mechanism underlying SLC7A5-mediated amino acid transport and mTORC1 activation. These results provide a potential novel therapeutic target for treating cardiac remodeling.

The functional role of m6A demethylase ALKBH5 in cardiomyocyte hypertrophy

Cardiomyocyte hypertrophy is a major outcome of pathological cardiac hypertrophy. The m6A demethylase ALKBH5 is reported to be associated with cardiovascular diseases, whereas the functional role of ALKBH5 in cardiomyocyte hypertrophy remains confused. We engineered Alkbh5 siRNA (siAlkbh5) and Alkbh5 overexpressing plasmid (Alkbh5 OE) to transfect cardiomyocytes. Subsequently, RNA immunoprecipitation (RIP)-qPCR, MeRIP-qPCR analysis and the dual-luciferase reporter assays were applied to elucidate the regulatory mechanism of ALKBH5 on cardiomyocyte hypertrophy. Our study identified ALKBH5 as a new contributor of cardiomyocyte hypertrophy. ALKBH5 showed upregulation in both phenylephrine (PE)-induced cardiomyocyte hypertrophic responses in vitro and transverse aortic constriction (TAC)/high fat diet (HFD)-induced pathological cardiac hypertrophy in vivo. Knockdown or overexpression of ALKBH5 regulated the occurrence of hypertrophic responses, including the increased cardiomyocyte surface areas and elevation of the hypertrophic marker levels, such as brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP). Mechanically, we indicated that ALKBH5 activated JAK2/STAT3 signaling pathway and mediated m6A demethylation on Stat3 mRNA, but not Jak2 mRNA, resulting in the phosphorylation and nuclear translocation of STAT3, which enhances the transcription of hypertrophic genes (e.g., Nppa) and ultimately leads to the emergence of cardiomyocytes hypertrophic growth. Our work highlights the functional role of ALKBH5 in regulating the onset of cardiomyocyte hypertrophy and provides a potential target for hypertrophic heart diseases prevention and treatment.ALKBH5 activated JAK2/STAT3 signaling pathway and mediated m6A demethylation on Stat3 mRNA, but not Jak2 mRNA, resulting in the phosphorylation and nuclear translocation of STAT3, which enhances the transcription of hypertrophic genes (e.g., Nppa) and ultimately leads to the emergence of cardiomyocytes hypertrophic growth.

Dysferlin Enables Tubular Membrane Proliferation in Cardiac Hypertrophy.

Cardiac hypertrophy compensates for increased biomechanical stress of the heart induced by prevalent cardiovascular pathologies but can result in heart failure if left untreated. Here, we hypothesized that the membrane fusion and repair protein dysferlin is critical for the integrity of the transverse-axial tubule (TAT) network inside cardiomyocytes and contributes to the proliferation of TAT endomembranes during pressure overload-induced cardiac hypertrophy. Stimulated emission depletion and electron microscopy were used to localize dysferlin in mouse and human cardiomyocytes. Data-independent acquisition mass spectrometry revealed the cardiac dysferlin interactome and proteomic changes of the heart in dysferlin-knockout mice. After transverse aortic constriction, we compared the hypertrophic response of wild-type versus dysferlin-knockout hearts and studied TAT network remodeling mechanisms inside cardiomyocytes by live-cell membrane imaging. We localized dysferlin in a vesicular compartment in nanometric proximity to contact sites of the TAT network with the sarcoplasmic reticulum, a.k.a. junctional complexes for Ca2+-induced Ca2+ release. Interactome analyses demonstrated a novel protein interaction of dysferlin with the membrane-tethering sarcoplasmic reticulum protein juncophilin-2, a putative interactor of L-type Ca2+ channels and ryanodine receptor Ca2+ release channels in junctional complexes. Although the dysferlin-knockout caused a mild progressive phenotype of dilated cardiomyopathy, global proteome analysis revealed changes preceding systolic failure. Following transverse aortic constriction, dysferlin protein expression was significantly increased in hypertrophied wild-type myocardium, while dysferlin-knockout animals presented markedly reduced left-ventricular hypertrophy. Live-cell membrane imaging showed a profound reorganization of the TAT network in wild-type left-ventricular myocytes after transverse aortic constriction with robust proliferation of axial tubules, which critically depended on the increased expression of dysferlin within newly emerging tubule components. Dysferlin represents a new molecular target in cardiac disease that protects the integrity of tubule-sarcoplasmic reticulum junctional complexes for regulated excitation-contraction coupling and controls TAT network reorganization and tubular membrane proliferation in cardiomyocyte hypertrophy induced by pressure overload.

IRE1α Mediates the Hypertrophic Growth of Cardiomyocytes Through Facilitating the Formation of Initiation Complex to Promote the Translation of TOP-Motif Transcripts.

Cardiomyocyte growth is coupled with active protein synthesis, which is one of the basic biological processes in living cells. However, it is unclear whether the unfolded protein response transducers and effectors directly take part in the control of protein synthesis. The connection between critical functions of the unfolded protein response in cellular physiology and requirements of multiple processes for cell growth prompted us to investigate the role of the unfolded protein response in cell growth and underlying molecular mechanisms. Cardiomyocyte-specific inositol-requiring enzyme 1α (IRE1α) knockout and overexpression mouse models were generated to explore its function in vivo. Neonatal rat ventricular myocytes were isolated and cultured to evaluate the role of IRE1α in cardiomyocyte growth in vitro. Mass spectrometry was conducted to identify novel interacting proteins of IRE1α. Ribosome sequencing and polysome profiling were performed to determine the molecular basis for the function of IRE1α in translational control. We show that IRE1α is required for cell growth in neonatal rat ventricular myocytes under prohypertrophy treatment and in HEK293 cells in response to serum stimulation. At the molecular level, IRE1α directly interacts with eIF4G and eIF3, 2 critical components of the translation initiation complex. We demonstrate that IRE1α facilitates the formation of the translation initiation complex around the endoplasmic reticulum and preferentially initiates the translation of transcripts with 5' terminal oligopyrimidine motifs. We then reveal that IRE1α plays an important role in determining the selectivity and translation of these transcripts. We next show that IRE1α stimulates the translation of epidermal growth factor receptor through an unannotated terminal oligopyrimidine motif in its 5' untranslated region. We further demonstrate a physiological role of IRE1α-governed protein translation by showing that IRE1α is essential for cardiomyocyte growth and cardiac functional maintenance under hemodynamic stress in vivo. These studies suggest a noncanonical, essential role of IRE1α in orchestrating protein synthesis, which may have important implications in cardiac hypertrophy in response to pressure overload and general cell growth under other physiological and pathological conditions.

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.

Poly (ADP-ribose) polymerases 16 triggers pathological cardiac hypertrophy via activating IRE1α–sXBP1–GATA4 pathway

BackgroundPressure overload-induced pathological cardiac hypertrophy is an independent predecessor of heart failure (HF), which remains the leading cause of worldwide mortality. However, current evidence on the molecular determinants of pathological cardiac hypertrophy is still inadequacy. This study aims to elucidate the role and mechanisms of Poly (ADP-ribose) polymerases 16 (PARP16) in the pathogenesis of pathological cardiac hypertrophy.MethodsGain and loss of function approaches were used to demonstrate the effects of genetic overexpression or deletion of PARP16 on cardiomyocyte hypertrophic growth in vitro. Ablation of PARP16 by transducing the myocardium with serotype 9 adeno-associated virus (AAV9)-encoding PARP16 shRNA were then subjected to transverse aortic construction (TAC) to investigate the effect of PARP16 on pathological cardiac hypertrophy in vivo. Co-immunoprecipitation (IP) and western blot assay were used to detect the mechanisms of PARP16 in regulating cardiac hypertrophic development.ResultsPARP16 deficiency rescued cardiac dysfunction and ameliorated TAC-induced cardiac hypertrophy and fibrosis in vivo, as well as phenylephrine (PE)-induced cardiomyocyte hypertrophic responses in vitro. Whereas overexpression of PARP16 exacerbated hypertrophic responses including the augmented cardiomyocyte surface area and upregulation of the fetal gene expressions. Mechanistically, PARP16 interacted with IRE1α and ADP-ribosylated IRE1α and then mediated the hypertrophic responses through activating the IRE1α–sXBP1–GATA4 pathway.ConclusionsCollectively, our results implicated that PARP16 is a contributor to pathological cardiac hypertrophy at least in part via activating the IRE1α–sXBP1–GATA4 pathway, and may be regarded as a new potential target for exploring effective therapeutic interventions of pathological cardiac hypertrophy and heart failure.

Inhibition of HIPK2 protects stress-induced pathological cardiac remodeling.

SummaryBackgroundHomeodomain-Interacting Protein Kinase 2 (HIPK2) has been reported to maintain basal cardiac function, however, its role in pathological cardiac remodeling remains unclear.MethodsHIPK2 inhibitors (tBID and PKI1H) treated mice and two lines of HIPK2−/− mice were subjected to transverse aortic constriction (TAC). HIPK2 knockdown were performed in neonatal rat cardiomyocytes (NRCMs), neonatal rat cardiac fibroblasts (NRCFs), and human embryonic stem cell-derived cardiomyocytes (hESC-CMs). Microarray analysis was used to screen HIPK2 targets. Overexpression of early growth response 3 (EGR3) and C-type lectin receptor 4D (CLEC4D) were performed in NRCMs, while an activator of Smad3 was used in NRCFs, to rescue the effects of HIPK2 knockdown. Finally, the effects of EGR3 and CLEC4D knockdown by AAV9 in TAC were determined.FindingsHIPK2 was elevated in TAC mice model, as well as cardiomyocyte hypertrophy and NRCFs fibrosis model. Pharmacological and genetic inhibition of HIPK2 improved cardiac function and suppressed cardiac hypertrophy and fibrosis induced by TAC. In vitro, HIPK2 inhibition prevented cardiomyocyte hypertrophic growth and NRCFs proliferation and differentiation. At the mechanistic level, we identified EGR3 and CLEC4D as new targets of HIPK2, which were regulated by ERK1/2-CREB and mediated the protective function of HIPK2 inhibition in cardiomyocytes. Meanwhile, inhibition of phosphorylation of Smad3 was responsible for the suppression of cardiac fibroblasts proliferation and differentiation by HIPK2 inhibition. Finally, we found that inhibition of EGR3 or CLEC4D protected against TAC.InterpretationHIPK2 inhibition protects against pathological cardiac remodeling by reducing EGR3 and CLEC4D with ERK1/2-CREB inhibition in cardiomyocytes, and by suppressing the phosphorylation of Smad3 in cardiac fibroblasts.FundingThis work was supported by the grants from National Key Research and Development Project (2018YFE0113500 to J.X.), National Natural Science Foundation of China (82020108002 and 81911540486 to J.X., 81400647 to MJ Xu), the grant from Science and Technology Commission of Shanghai Municipality (21XD1421300 and 20DZ2255400 to J.X.), the “Dawn” Program of Shanghai Education Commission (19SG34 to J.X.), and Shanghai Sailing Program (21YF1413200 to Q.Z.).

CDC-like kinase 4 deficiency contributes to pathological cardiac hypertrophy by modulating NEXN phosphorylation

Kinase-catalyzed phosphorylation plays a crucial role in pathological cardiac hypertrophy. Here, we show that CDC-like kinase 4 (CLK4) is a critical regulator of cardiomyocyte hypertrophy and heart failure. Knockdown of Clk4 leads to pathological cardiomyocyte hypertrophy, while overexpression of Clk4 confers resistance to phenylephrine-induced cardiomyocyte hypertrophy. Cardiac-specific Clk4-knockout mice manifest pathological myocardial hypertrophy with progressive left ventricular systolic dysfunction and heart dilation. Further investigation identifies nexilin (NEXN) as the direct substrate of CLK4, and overexpression of a phosphorylation-mimic mutant of NEXN is sufficient to reverse the hypertrophic growth of cardiomyocytes induced by Clk4 knockdown. Importantly, restoring phosphorylation of NEXN ameliorates myocardial hypertrophy in mice with cardiac-specific Clk4 deletion. We conclude that CLK4 regulates cardiac function through phosphorylation of NEXN, and its deficiency may lead to pathological cardiac hypertrophy. CLK4 is a potential intervention target for the prevention and treatment of heart failure.

RNF207 exacerbates pathological cardiac hypertrophy via post-translational modification of TAB1.

The heart undergoes pathological remodelling, featured by the hypertrophic growth of cardiomyocytes and increased cardiac fibrosis, under biomechanical stress such as haemodynamic overload. Ring Finger Protein 207 (RNF207) is an E3 ubiquitin ligase that is predominantly expressed in the heart, but its function remains elusive. In this study, we aimed to explore the role of RNF207 in the development of pathological cardiac hypertrophy and dysfunction. Transverse aortic constriction (TAC) surgery was performed on mice to induce cardiac hypertrophy. Cardiac function and remodelling were evaluated by echocardiography, histological assessment, and molecular analyses. Our data indicated that RNF207 overexpression (OE) exacerbated cardiac hypertrophy, fibrosis, and systolic dysfunction. In contrast, TAC-induced cardiac remodelling was profoundly blunted in RNF207 knockdown (KD) hearts. In line with the in vivo findings, RNF207 OE augmented, whereas RNF207 KD alleviated, phenylephrine-induced cardiomyocyte hypertrophy in vitro. Mechanistically, we demonstrated that RNF207 elicited detrimental effects by promoting K63-linked ubiquitination of TAK1-binding protein 1 (TAB1), which triggered the autophosphorylation of transforming growth factor-β activated kinase 1 (TAK1) and the activation of downstream p38 and c-Jun N-terminal kinase (JNK)1/2 signalling pathways. In the TAB1-KD cardiomyocytes, RNF207-OE-induced cell hypertrophy was significantly attenuated, indicating that RNF207-induced hypertrophy is, at least in part, TAB1-dependent. This study demonstrates that RNF207 exacerbates pressure overload-induced cardiac hypertrophy and dysfunction via post-translational modification of TAB1.

Loss of m6A Methyltransferase METTL5 Promotes Cardiac Hypertrophy Through Epitranscriptomic Control of SUZ12 Expression.

Enhancement of protein synthesis from mRNA translation is one of the key steps supporting cardiomyocyte hypertrophy during cardiac remodeling. The methyltransferase-like5 (METTL5), which catalyzes m6A modification of 18S rRNA at position A1832, has been shown to regulate the efficiency of mRNA translation during the differentiation of ES cells and the growth of cancer cells. It remains unknown whether and how METTL5 regulates cardiac hypertrophy. In this study, we have generated a mouse model, METTL5-cKO, with cardiac-specific depletion of METTL5 in vivo. Loss function of METTL5 promotes pressure overload-induced cardiomyocyte hypertrophy and adverse remodeling. The regulatory function of METTL5 in hypertrophic growth of cardiomyocytes was further confirmed with both gain- and loss-of-function approaches in primary cardiomyocytes. Mechanically, METTL5 can modulate the mRNA translation of SUZ12, a core component of PRC2 complex, and further regulate the transcriptomic shift during cardiac hypertrophy. Altogether, our study may uncover an important translational regulator of cardiac hypertrophy through m6A modification.

MiR-208a in Cardiac Hypertrophy and Remodeling.

Various stresses, including pressure overload and myocardial stretch, can trigger cardiac remodeling and result in heart diseases. The disorders are associated with high risk of morbidity and mortality and are among the major health problems in the world. MicroRNAs, a class of ~22nt-long small non-coding RNAs, have been found to participate in regulating heart development and function. One of them, miR-208a, a cardiac-specific microRNA, plays key role(s) in modulating gene expression in the heart, and is involved in a broad array of processes in cardiac pathogenesis. Genetic deletion or pharmacological inhibition of miR-208a in rodents attenuated stress-induced cardiac hypertrophy and remodeling. Transgenic expression of miR-208a in the heart was sufficient to cause hypertrophic growth of cardiomyocytes. miR-208a is also a key regulator of cardiac conduction system, either deletion or transgenic expression of miR-208a disturbed heart electrophysiology and could induce arrhythmias. In addition, miR-208a appeared to assist in regulating the expression of fast- and slow-twitch myofiber genes in the heart. Notably, this heart-specific miRNA could also modulate the “endocrine” function of cardiac muscle and govern the systemic energy homeostasis in the whole body. Despite of the critical roles, the underlying regulatory networks involving miR-208a are still elusive. Here, we summarize the progress made in understanding the function and mechanisms of this important miRNA in the heart, and propose several topics to be resolved as well as the hypothetical answers. We speculate that miR-208a may play diverse and even opposite roles by being involved in distinct molecular networks depending on the contexts. A deeper understanding of the precise mechanisms of its action under the conditions of cardiac homeostasis and diseases is needed. The clinical implications of miR-208a are also discussed.

Piezo1-Mediated Mechanotransduction Promotes Cardiac Hypertrophy by Impairing Calcium Homeostasis to Activate Calpain/Calcineurin Signaling.

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Cyclin D1: A Golden Gene in Cancer, Cardiotoxicity, and Cardioprotection

Objective: Cyclin D1 is an essential protein that acts as a mitogenic sensor. In this manuscript, we discuss the importance of cyclin D1 in oncology and cardio-oncology, and we challenge the prognostic and therapeutic response values of cyclin D1 to figure out if it can be a beneficial marker. We also discuss the agents and microRNAs that can be used as a potential therapeutic approach via regulating cyclin D1 expression in oncology and cardio-oncology. Discussion: Clinical significance of cyclin D1 is defined not only in several cancers such as breast cancer, melanoma, and glioblastoma but also in cardiomyocyte regeneration and cardiac hypertrophic growth. Several studies have indicated that the injection of cardiotoxic agents such as doxorubicin (DOX) induces damage to the cardiac system and increases cyclin D expression at single injection, which might be related to DXO-mediated damage in the adult heart. However, cyclin D1 overexpression leads to hypertrophic growth of cardiomyocytes, and cyclin-dependent kinase (CDK)) inhibitors such as p16 do not inhibit the hypertrophic growth of cardiomyocytes. Thus, the reaction is CDK-independent. Conclusions: Cyclin D1 overexpression is positively correlated with tumor progression, treatment response, cardiotoxicity, and poor prognosis. Cyclin D1 expression has an important role in cardiac hypertrophy, and it can be a promising marker in monitoring cardiomyocyte treatment responses, cardioprotection, and cardiotoxicity. Finally, cyclin D1 plays an important role in hypertrophic growth of cardiomyocytes via a novel mechanism. Given all these pieces of evidence, cyclin D1 can be introduced as a favorable biomarker in future cardiology and cardio-oncology.

The cardiac translational landscape reveals that micropeptides are new players involved in cardiomyocyte hypertrophy

Hypertrophic growth of cardiomyocytes is one of the major compensatory responses in the heart after physiological or pathological stimulation. Protein synthesis enhancement, which is mediated by the translation of messenger RNAs, is one of the main features of cardiomyocyte hypertrophy. Although the transcriptome shift caused by cardiac hypertrophy induced by different stimuli has been extensively investigated, translatome dynamics in this cellular process has been less studied. Here, we generated a nucleotide-resolution translatome as well as transcriptome data from isolated primary cardiomyocytes undergoing hypertrophy. More than 10,000 open reading frames (ORFs) were detected from the deep sequencing of ribosome-protected fragments (Ribo-seq), which orchestrated the shift of the translatome in hypertrophied cardiomyocytes. Our data suggest that rather than increase the translational rate of ribosomes, the increased efficiency of protein synthesis in cardiomyocyte hypertrophy was attributable to an increased quantity of ribosomes. In addition, more than 100 uncharacterized short ORFs (sORFs) were detected in long noncoding RNA genes from Ribo-seq with potential of micropeptide coding. In a random test of 15 candidates, the coding potential of 11 sORFs was experimentally supported. Three micropeptides were identified to regulate cardiomyocyte hypertrophy by modulating the activities of oxidative phosphorylation, the calcium signaling pathway, and the mitogen-activated protein kinase (MAPK) pathway. Our study provides a genome-wide overview of the translational controls behind cardiomyocyte hypertrophy and demonstrates an unrecognized role of micropeptides in cardiomyocyte biology.

Matrix Metalloproteinases Repress Hypertrophic Growth in Cardiac Myocytes

PurposeMatrix metalloproteinases (MMPs) are identified as modulators of the extracellular matrix in heart failure progression. However, evidence for intracellular effects of MMPs is emerging. Pro- and anti-hypertrophic cardiac effects are described. This may be due to the various sources of different MMPs in the heart tissue. Therefore, the aim of the present study was to determine the role of MMPs in hypertrophic growth of isolated rat ventricular cardiac myocytes.MethodsCardiomyocytes were isolated form ventricular tissues of the rat hearts by collagenase perfusion. RT-qPCR, western blots, and zymography were used for expression and MMP activity analysis. Cross-sectional area and the rate of protein synthesis were determined as parameters for hypertrophic growth.ResultsMMP-1, MMP-2, MMP-3, MMP-9 and MMP-14 mRNAs were detected in cardiomyocytes, and protein expression of MMP-2, MMP-9, and MMP-14 was identified. Hypertrophic stimulation of cardiomyocytes did not enhance, but interestingly decreased expression of MMPs, indicating that downregulation of MMPs may promote hypertrophic growth. Indeed, the nonselective MMP inhibitors TAPI-0 or TIMP2 and the MMP-2-selective ARP-100 enhanced hypertrophic growth. Furthermore, TAPI-0 increased phosphorylation and thus activation of extracellular signaling kinase (ERK) and Akt (protein kinase B), as well as inhibition of glycogen synthase 3β (GSK3β). Abrogation of MEK/ERK- or phosphatidylinositol-3-kinase(PI3K)/Akt/GSK3β-signaling with PD98059 or LY290042, respectively, inhibited hypertrophic growth under TAPI-0.ConclusionMMPs’ inhibition promotes hypertrophic growth in cardiomyocytes in vitro. Therefore, MMPs in the healthy heart may be important players to repress cardiac hypertrophy.

β-Catenin Regulates Cardiac Energy Metabolism in Sedentary and Trained Mice.

The role of canonical Wnt signaling in metabolic regulation and development of physiological cardiac hypertrophy remains largely unknown. To explore the function of β-catenin in the regulation of cardiac metabolism and physiological cardiac hypertrophy development, we used mice heterozygous for cardiac-specific β-catenin knockout that were subjected to a swimming training model. β-Catenin haploinsufficient mice subjected to endurance training displayed a decreased β-catenin transcriptional activity, attenuated cardiomyocytes hypertrophic growth, and enhanced activation of AMP-activated protein kinase (AMPK), phosphoinositide-3-kinase–Akt (Pi3K–Akt), and mitogen-activated protein kinase/extracellular signal-regulated kinases 1/2 (MAPK/Erk1/2) signaling pathways compared to trained wild type mice. We further observed an increased level of proteins involved in glucose aerobic metabolism and β-oxidation along with perturbed activity of mitochondrial oxidative phosphorylation complexes (OXPHOS) in trained β-catenin haploinsufficient mice. Taken together, Wnt/β-catenin signaling appears to govern metabolic regulatory programs, sustaining metabolic plasticity in adult hearts during the adaptation to endurance training.

Abstract MP161: A Calcineurin-hoxb13 Axis Regulates Growth Mode of Mammalian Cardiomyocytes

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. Our group recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes1,2. We recently also showed that Meis1, a TALE family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest3. Here we report that Hoxb13 acts as a Meis1 cofactor in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can both extend the postnatal window of cardiomyocyte proliferation and reactivate cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1/Hoxb13 double knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and an improvement in left ventricular systolic function following myocardial infarction both by echocardiography and MRI. ChIP-seq analysis demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204 (S204), resulting in its nuclear localization and cell cycle arrest. Collectively, these results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes.

Genomic Binding Patterns of Forkhead Box Protein O1 Reveal Its Unique Role in Cardiac Hypertrophy.

Cardiac hypertrophic growth is mediated by robust changes in gene expression and changes that underlie the increase in cardiomyocyte size. The former is regulated by RNA polymerase II (pol II) de novo recruitment or loss; the latter involves incremental increases in the transcriptional elongation activity of pol II that is preassembled at the transcription start site. The differential regulation of these distinct processes by transcription factors remains unknown. Forkhead box protein O1 (FoxO1) is an insulin-sensitive transcription factor that is also regulated by hypertrophic stimuli in the heart. However, the scope of its gene regulation remains unexplored. To address this, we performed FoxO1 chromatin immunoprecipitation-deep sequencing in mouse hearts after 7 days of isoproterenol injections (3 mg·kg-1·mg-1), transverse aortic constriction, or vehicle injection/sham surgery. Our data demonstrate increases in FoxO1 chromatin binding during cardiac hypertrophic growth, which positively correlate with extent of hypertrophy. To assess the role of FoxO1 on pol II dynamics and gene expression, the FoxO1 chromatin immunoprecipitation-deep sequencing results were aligned with those of pol II chromatin immunoprecipitation-deep sequencing across the chromosomal coordinates of sham- or transverse aortic constriction-operated mouse hearts. This uncovered that FoxO1 binds to the promoters of 60% of cardiac-expressed genes at baseline and 91% after transverse aortic constriction. FoxO1 binding is increased in genes regulated by pol II de novo recruitment, loss, or pause-release. In vitro, endothelin-1- and, in vivo, pressure overload-induced cardiomyocyte hypertrophic growth is prevented with FoxO1 knockdown or deletion, which was accompanied by reductions in inducible genes, including Comtd1 in vitro and Fstl1 and Uck2 in vivo. Together, our data suggest that FoxO1 may mediate cardiac hypertrophic growth via regulation of pol II de novo recruitment and pause-release; the latter represents the majority (59%) of FoxO1-bound, pol II-regulated genes after pressure overload. These findings demonstrate the breadth of transcriptional regulation by FoxO1 during cardiac hypertrophy, information that is essential for its therapeutic targeting.

A calcineurin-Hoxb13 axis regulates growth mode of mammalian cardiomyocytes.

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. We recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes1,2 and that Meis1, a three amino acid loop extension (TALE) family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest3. Here we report that Hoxb13 acts as a cofactor of Meis1 in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can extend the postnatal window of cardiomyocyte proliferation and reactivate the cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1-Hoxb13 double-knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and improved left ventricular systolic function following myocardial infarction, as demonstrated by echocardiography and magnetic resonance imaging. Chromatin immunoprecipitation with sequencing demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204, resulting in its nuclear localization and cell cycle arrest. These results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes.