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Highlights • The IGF1–PI3K pathway mediates exercise induced heart growth and protection. • Strategies to mimic the benefits of exercise on the heart are of substantial interest. • Characterizing animal models of altered cardiac PI3K activity can identify new drug targets for heart disease, and biomarkers which distinguish the healthy and diseased heart. • Therapies that increase PI3K activity may provide a promising approach to improve function in the failing heart. • Systemic therapies that reduce PI3K activity, such as some cancer agents, may lead to cardiotoxicity., Heart failure represents the end point of a variety of cardiovascular diseases. It is a growing health burden and a leading cause of death worldwide. To date, limited treatment options exist for the treatment of heart failure, but exercise has been well-established as one of the few safe and effective interventions, leading to improved outcomes in patients. However, a lack of patient adherence remains a significant barrier in the implementation of exercise-based therapy for the treatment of heart failure. The insulin-like growth factor 1 (IGF1)–phosphoinositide 3-kinase (PI3K) pathway has been recognized as perhaps the most critical pathway for mediating exercised-induced heart growth and protection. Here, we discuss how modulating activity of the IGF1–PI3K pathway may be a valuable approach for the development of therapies that mimic the protective effects of exercise on the heart. We outline some of the promising approaches being investigated that utilize PI3K-based therapy for the treatment of heart failure. We discuss the implications for cardiac pathology and cardiotoxicity that arise in a setting of reduced PI3K activity. Finally, we discuss the use of animal models of cardiac health and disease, and genetic mice with increased or decreased cardiac PI3K activity for the discovery of novel drug targets and biomarkers of cardiovascular disease.

Heart growth and pathological changes are accompanied by extracellular matrix‐dependent alterations in integrins and integrin‐associated proteins, suggesting their role in heart development and disease. Most of our knowledge on the involvement of integrins in heart pathology is provided by the in vivo experiments, including cardiac hypertrophy models. However, in vivo studies are limited by the complex organization of heart tissue and fail to discern cell types and particular integrins implicated in hypertrophic signalling. This problem is being addressed by isolated cardiomyocyte primary cultures, which have been successfully used in different in vitro disease models. This review aimed to analyse the general approaches to studying integrins and integrin‐associated signalling pathways in cardiac hypertrophy focusing on the in vitro systems. The lessons learned from culture experiments on the models of hypertrophy induced by stretch, stimulating factors, and/or extracellular matrix components are summarized, demonstrating the major involvement of integrin‐mediated signalling in cardiac hypertrophic response and its apparent crosstalk with signal pathways induced by stretch or hypertrophy stimulating factors. The benefits and perspectives of using cardiomyocyte primary culture as a hypertrophy model are discussed.

Cardiac hypertrophy, defined as an increase in mass of the heart, is a complex process driven by simultaneous changes in hemodynamics, mechanical stimuli, and hormonal inputs. It occurs not only during pre- and post-natal development but also in adults in response to exercise, pregnancy, and a range of cardiovascular diseases. One of the most exciting recent developments in the field of cardiac biomechanics is the advent of computational models that are able to accurately predict patterns of heart growth in many of these settings, particularly in cases where changes in mechanical loading of the heart play an import role. These emerging models may soon be capable of making patient-specific growth predictions that can be used to guide clinical interventions. Here, we review the history and current state of cardiac growth models and highlight three main limitations of current approaches with regard to future clinical application: their inability to predict the regression of heart growth after removal of a mechanical overload, inability to account for evolving hemodynamics, and inability to incorporate known growth effects of drugs and hormones on heart growth. Next, we outline growth mechanics approaches used in other fields of biomechanics and highlight some potential lessons for cardiac growth modeling. Finally, we propose a multiscale modeling approach for future studies that blends tissue-level growth models with cell-level signaling models to incorporate the effects of hormones in the context of pregnancy-induced heart growth.

Hypertrophic cardiomyopathy (HCM) is one of the most commonly inherited heart diseases and the leading cause of sudden cardiac death among adolescents and young adults. Circulating long noncoding RNAs (lncRNAs) have demonstrated potential as diagnostic and therapeutic targets in several cardiovascular diseases. However, the circulating extracellular lncRNA expression profile of patients with HCM remains unclear. Plasma lncRNA expression was evaluated in patients with HCM and healthy controls using a human lncRNA microarray. A weighted correlation network analysis (WGCNA) and linear models for microarray data (Limma) were used. GSE68316 data from cardiac tissue in the Gene Expression Omnibus database were analysed for further validation. Using WGCNA, two modules (referred to as the magenta and the light-yellow module) were identified that were positively associated with HCM. Gene Ontology analysis revealed that lncRNAs in the magenta module targeted ‘heart growth’. Using Limma, a total of 290 lncRNAs were differentially expressed (210 upregulated and 80 downregulated) in the plasma of HCM patients, compared with controls. Moreover, combined WGCNA and Limma analysis demonstrated that 27 hub lncRNAs in the magenta module and 13 hub lncRNAs in the light-yellow module were significantly upregulated, compared with the controls. Moreover, of the 40 differentially expressed hub lncRNAs identified in the two modules, three circulating lncRNAs (lnc-P2RY6-1:1, ENST00000488040 and ENST00000588047) were also significantly upregulated in the HCM cardiac tissue validation dataset. These lncRNAs may serve as biomarkers and therapeutic targets for precise diagnosis and treatment of HCM.

Open-access gene expression data sets provide a useful resource for identifying novel drug targets and biomarkers. The IGF1-PI3K pathway is a critical mediator of physiological cardiac enlargement/hypertrophy and protection. This study arose after mining a gene microarray data set from a previous study that compared heart tissue from cardiac-specific PI3K transgenic mouse models. The top-ranked candidate identified from the microarray data was clusterin. Clusterin has been proposed as a biomarker for multiple diseases including heart failure, and as a cancer drug target. Here, we show that clusterin gene expression is increased in hearts of transgenic mice with increased PI3K and decreased in mice with depressed cardiac PI3K. In vitro, clusterin secretion was elevated in media from neonatal rat ventricular myocytes treated with IGF1. Furthermore, by mining gene expression data from hearts during normal mouse postnatal growth, we also report an increase in clusterin expression with postnatal heart growth. Given we show that clusterin is regulated by the IGF1-PI3K pathway in the heart, and this pathway mediates physiological cardiac hypertrophy and cardioprotection, caution is required when considering clusterin as a biomarker for heart failure and as a cancer target. Mining pre-existing cardiac profiling data sets may be a useful approach to assess whether regulating new drug targets is likely to lead to cardiac damage/toxicity.

Normal organogenesis cannot be recapitulated in vitro for mammalian organs, unlike in species including Drosophila and zebrafish. Available 3D data in the form of ex vivo images only provide discrete snapshots of the development of an organ morphology. Here, we propose a computer-based approach to recreate its continuous evolution in time and space from a set of 3D volumetric images. Our method is based on the remapping of shape data into the space of the coefficients of a spherical harmonics expansion where a smooth interpolation over time is simpler. We tested our approach on mouse limb buds and embryonic hearts. A key advantage of this method is that the resulting 4D trajectory can take advantage of all the available data while also being able to interpolate well through time intervals for which there are little or no data. This allows for a quantitative, data-driven 4D description of mouse limb morphogenesis. We thank Isaac Esteban and Miguel Torres for their invaluable discussions on shape comparison and spherical harmonics; Antoni Matyjaszkiewicz for his help with the webpage of the project; Isabel Romero Calvo for her precious suggestions on the graphical abstract and Carla Manzanas for the media coverage. This research was supported by core funding from EMBL and CRG, and also the ERC advanced grant SIMBIONT (project no. 670555) and a grant from the Fundacion La Marato TV3 (grant 082030). G.D. and M.M. were supported by the ERC advanced grant SIMBIONT (project no. 670555). Sí

Han et al. study the effect of primary cardiac hypertrophy on cardiac mechano-energetics. Using isolated cardiac tissues, they show that despite impaired contractile performance, cardiac mechanical efficiency is preserved in hypertrophic hearts., Increased heart size is a major risk factor for heart failure and premature mortality. Although abnormal heart growth subsequent to hypertension often accompanies disturbances in mechano-energetics and cardiac efficiency, it remains uncertain whether hypertrophy is their primary driver. In this study, we aimed to investigate the direct association between cardiac hypertrophy and cardiac mechano-energetics using isolated left-ventricular trabeculae from a rat model of primary cardiac hypertrophy and its control. We evaluated energy expenditure (heat output) and mechanical performance (force length work production) simultaneously at a range of preloads and afterloads in a microcalorimeter, we determined energy expenditure related to cross-bridge cycling and Ca2+ cycling (activation heat), and we quantified energy efficiency. Rats with cardiac hypertrophy exhibited increased cardiomyocyte length and width. Their trabeculae showed mechanical impairment, evidenced by lower force production, extent and kinetics of shortening, and work output. Lower force was associated with lower energy expenditure related to Ca2+ cycling and to cross-bridge cycling. However, despite these changes, both mechanical and cross-bridge energy efficiency were unchanged. Our results show that cardiac hypertrophy is associated with impaired contractile performance and with preservation of energy efficiency. These findings provide direction for future investigations targeting metabolic and Ca2+ disturbances underlying cardiac mechanical and energetic impairment in primary cardiac hypertrophy.

Left ventricular hypertrophy (LVH) is a major risk factor for cardiovascular morbidity and mortality. Pathological LVH engages transcriptional programs including reactivation of canonical fetal genes and those inducing fibrosis. Histone lysine demethylases (KDMs) are emerging regulators of transcriptional reprogramming in cancer, though their potential role in abnormal heart growth and fibrosis remains little understood. Here, we investigate gain and loss of function of an H3K9me2 specific demethylase, Kdm3a, and show it promotes LVH and fibrosis in response to pressure-overload. Cardiomyocyte KDM3A activates Timp1 transcription with pro-fibrotic activity. By contrast, a pan-KDM inhibitor, JIB-04, suppresses pressure overload-induced LVH and fibrosis. JIB-04 inhibits KDM3A and suppresses the transcription of fibrotic genes that overlap with genes downregulated in Kdm3a-KO mice versus WT controls. Our study provides genetic and biochemical evidence for a pro-hypertrophic function of KDM3A and proof-of principle for pharmacological targeting of KDMs as an effective strategy to counter LVH and pathological fibrosis., Histone lysine demethylases (KDMs) can mediate transcriptional reprogramming in disease states. Here the authors show that KDM3A promotes left ventricular hypertrophy and cardiac fibrosis by activating the transcription of Timp1, and that pharmacological inhibition of KDM3A attenuates cardiac remodeling induced by pressure overload.

OBJECTIVES We evaluated the outcomes of biventricular repair after initial hybrid palliation performed in small infants with various forms of left ventricle hypoplasia. METHODS Between September 2010 and January 2020, a total of 27 patients had biventricular repair after hybrid palliation at a median age of 11 days. Indications for the hybrid approach included growth promotion of the left ventricle outflow tract and/or the aortic valve in 14 patients and that of the left ventricle in 13 patients. Seven reinterventions and 7 reoperations were performed during the interstage period. Significant growth of left ventricle parameters was noted during the median interstage period of 62 days. Sixteen subjects had aortic arch repair, ventricular septal defect closure and relief of subaortic stenosis; 5 patients had the Ross–Konno procedure; 5 patients underwent the Yasui procedure; and 1 patient had unbalanced atrioventricular septal defect and aortic arch repair. RESULTS Twenty-three patients (85.2%) are alive at a median follow-up of 3.3 years. Two and 3 patients died early and late after achieving biventricular circulation, respectively. There were 22 reinterventions and 15 reoperations after biventricular repair. CONCLUSIONS Hybrid palliation can stimulate left heart growth in some patients with left ventricle hypoplasia. More patients may eventually achieve biventricular circulation than was initially thought. Additional interventions and operations are foreseeable. Despite ventricular rehabilitation, some patients with borderline left ventricles may develop restrictive physiology.

Heart failure is often preceded by pathological cardiac hypertrophy, a thickening of the heart musculature driven by complex gene regulatory and signaling processes. The Drosophila heart has great potential as a genetic model for deciphering the underlying mechanisms of cardiac hypertrophy. However, current methods for evaluating hypertrophy of the Drosophila heart are laborious and difficult to carry out reproducibly. Here we demonstrate that micro-computerized tomography (microCT) is an accessible, highly reproducible method for non-destructive, quantitative analysis of Drosophila heart morphology and size. To validate our microCT approach for analyzing Drosophila cardiac hypertrophy, we show that expression of constitutively active Ras (Ras85DV12), previously shown to cause hypertrophy of the fly heart, results in significant thickening of both adult and larval heart walls when measured from microCT images. We then show using microCT analysis that genetic upregulation of store-operated Ca2+ entry (SOCE) driven by expression of constitutively active Stim (StimCA) or Orai (OraiCA) proteins also results in significant hypertrophy of the Drosophila heart, through a process that specifically depends on Orai Ca2+ influx channels. Intravital imaging of heart contractility revealed significantly reduced end diastolic dimensions in StimCA and OraiCA expressing hearts, consistent with the hypertrophic phenotype. These results demonstrate that increased SOCE activity is an important driver of hypertrophic cardiomyocyte growth, and demonstrate how microCT analysis combined with tractable genetic tools in Drosophila can be used to delineate molecular signaling processes that underlie cardiac hypertrophy and heart failure.NEW AND NOTEWORTHYGenetic analysis of cardiac hypertrophy in Drosophila holds immense potential for the discovery of new therapeutic targets to prevent and treat heart failure. However, this potential has been hindered by a lack of rapid and effective methods for analysis of heart size in flies. Here we demonstrate that analysis of the Drosophila heart with micro-computerized tomography yields accurate and highly reproducible heart size measurements that can be used to efficiently analyze heart growth and cardiac hypertrophy in Drosophila.

The ubiquitous NF-Y gene regulates the expression of different genes in various signaling pathways. However, the function of NF-Y in zebrafish heart development is largely unknown. Previously we identified a same group of cell cycle related gene cluster (CCRG) was downregulated in the embryonic hearts with impeded growth due to various stresses. The promoter regions of these CCRG genes shared a most common motif for NF-Y. Chromatin immunoprecipitation experiment demonstrated that the binding of NF-Y to its motif was real on the CCRG candidate gene promoters. Knockdown of embryonic NF-Y by morpholinos led to a small heart, mimicking the abnormal heart phenotype caused by other stresses. In parallel the expression of certain CCRG candidate genes was reduced in the NF-Y A morphant hearts exposed to malignant environments. Absence of NF-Y A also led to undermine cardiomyocyte proliferation and hence less total number of caridomyocytes per heart. Trans-AM Elisa experiment also found that in the presence of the stresses such as TCDD and TNNT2 MO, the binding capacity of NF-Y A subunit to its core motif was reduced. We conclude that NF-Y sustains proper cardiomyocyte proliferation in the heart, thus it plays a positive role in promoting early zebrafish heart growth.

This work was supported by NIH grant (RO1HL073402-16) to G.O, AHA grant (18CDA34110356) to X.L, 5T32HL134633 to W.M, FPU grant from the Spanish Ministry of Education, Culture and Sports and EMBO ShortTerm Fellowship to E.C.C, Leducq TNE-17CVD and RD16/0011/0019 (ISCIII) from the Spanish Ministry of Science, Innovation, and Universities to M.T, NIH T32 GM008061 to C.L, HL63762, and NS093382 to J.H. We thank Dr. Gabriele M. Rune and Dr. Bianka Brunne for the Reln +/− strain. RNAseq work was supported by the Northwestern University NUSeq Core Facility. We thank the Robert H. Lurie Cancer Center Flow Cytometry facility supported by NCI CCSG P30 CA060553 for their invaluable assistance. Flow Cytometry Cell Sorting was performed on a BD FACSAria SORP system and BD FACSymphony S6 SORP system, purchased through the support of NIH 1S10OD011996-01 and 1S10OD026814-01. Imaging work was performed at the Northwestern University Center for Advanced Microscopy supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. Spinning disk confocal microscopy was performed on an Andor XDI Revolution microscope, purchased through the support of NCRR 1S10 RR031680-01. Proteomics services were performed by the Northwestern Proteomics Core Facility supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center, instrumentation award (S10OD025194) from NIH Office of Director, and the National Resource for Translational and Developmental Proteomics supported by P41 GM108569. We thank the George M. O’Brien Kidney Research Core Center (NU GoKidney, supported by a P30 DK114857 award from NIDDK) for the use of the Echocardiography machine. The myocardial infarction surgeries were performed by the comprehensive Transplant Center Microsurgery Core, partially supported by NIH NIAID P01AI112522. We thank Dr. Jing Jin and Dr. Pan Liu for help with the ELISA reagents and data analysis, Dr. Ruihua Ma for help with DNA polyploidy analysis, Ao Shi for the Mef2c antibodies, Dr. Michael Dellinger for the Prox1CreERT2 mice and Dr. Hossein Ardehali for the MhcCre mice. We specially thank Dr. Beatriz Sosa-Pineda for advice and valuable suggestions and Dr. Pilar Ruiz-Lozano for shearing her expertise in the preparation of the collagen patches. Recent studies have suggested that lymphatics help to restore heart function after cardiac injury1-6. Here we report that lymphatics promote cardiac growth, repair and cardioprotection in mice. We show that a lymphoangiocrine signal produced by lymphatic endothelial cells (LECs) controls the proliferation and survival of cardiomyocytes during heart development, improves neonatal cardiac regeneration and is cardioprotective after myocardial infarction. Embryos that lack LECs develop smaller hearts as a consequence of reduced cardiomyocyte proliferation and increased cardiomyocyte apoptosis. Culturing primary mouse cardiomyocytes in LEC-conditioned medium increases cardiomyocyte proliferation and survival, which indicates that LECs produce lymphoangiocrine signals that control cardiomyocyte homeostasis. Characterization of the LEC secretome identified the extracellular protein reelin (RELN) as a key component of this process. Moreover, we report that LEC-specific Reln-null mouse embryos develop smaller hearts, that RELN is required for efficient heart repair and function after neonatal myocardial infarction, and that cardiac delivery of RELN using collagen patches improves heart function in adult mice after myocardial infarction by a cardioprotective effect. These results highlight a lymphoangiocrine role of LECs during cardiac development and injury response, and identify RELN as an important mediator of this function. Sí

Pregnancy stands at the interface of mechanics and biology. The growing fetus continuously loads the maternal organs as circulating hormone levels surge, leading to significant changes in mechanical and hormonal cues during pregnancy. In response to these cues, maternal soft tissues undergo remarkable growth and remodeling to support both mother and baby for a healthy pregnancy. We focus here on the maternal left ventricle, which increases its cardiac output and mass during pregnancy. The objective of this study is to build a multiscale cardiac growth model for pregnancy to understand how mechanical and hormonal cues interact to drive this growth process. Towards this objective, we coupled a cell signaling network model that predicts cell-level hypertrophy in response to hormones and stretch, to a compartmental model of the rat heart and circulation that predicts organ-level growth in response to hemodynamic changes. Since pregnancy is associated with a volume overloaded state and elevated hormones, we first calibrated the coupled, multiscale model to data from experimental volume overload (VO) and hormonal infusion of angiotensin 2 (AngII), estrogen (E2), and progesterone (P4). We then validated the ability of our model to capture interactions between inputs by comparing model predictions against published observations for the combinations of VO+E2 and AngII+E2. Finally, we simulated pregnancy-induced changes in hormones and hemodynamics to predict heart growth during pregnancy. Our multiscale model produced realistic heart growth consistent with experimental data. Overall, our analysis suggests that much of heart growth during pregnancy is driven by the early rise in P4, particularly during the first half of gestation. We conclude with suggestions for future experimental studies that will provide a better understanding of how hormonal and mechanical cues interact to drive pregnancy-induced heart growth.

Endothelial cells (ECs) line the inner surface of all blood and lymphatic vessels throughout the body, making endothelium one of the largest tissues. In addition to its transport function, endothelium is now appreciated as a dynamic organ actively participating in angiogenesis, permeability and vascular tone regulation, as well as in the development and regeneration of tissues. The identification of endothelial-derived secreted factors, angiocrines, has revealed non-angiogenic mechanisms of endothelial cells in both physiological and pathological tissue remodeling. In the heart, ECs play a variety of important roles during cardiac development as well as in growth, homeostasis and regeneration of the adult heart. To date, several angiocrines affecting cardiomyocyte growth in response to physiological or pathological stimuli have been identified. In this review, we discuss the effects of angiogenesis and EC-mediated signaling in the regulation of cardiac hypertrophy. Identification of the molecular and metabolic signals from ECs during physiological and pathological cardiac growth could provide novel therapeutic targets to treat heart failure, as endothelium is emerging as one of the potential target organs in cardiovascular and metabolic diseases.

Aims: Animal models have been used to show that there are critical molecular mechanisms that can be activated to induce myocardial repair at specific times in development. For example, specific miRNAs are critical for regulating the response to myocardial infarction (MI) and improving the response to injury. Manipulating these miRNAs in small animal models provides beneficial effects post-MI; however it is not known if these miRNAs are regulated similarly in large mammals. Studying a large animal where the timing of heart development in relation to birth is similar to humans may provide insights to better understand the capacity to repair a developing mammalian heart and its application to the adult heart.Methods: We used a sheep model of MI that included permanent ligation of the left anterior descending (LAD) coronary artery. Surgery was performed on fetuses (at105 days gestation when all cardiomyocytes are mononucleated and proliferative) and adolescent sheep (at 6 months of age when all cardiomyocytes contribute to heart growth by hypertrophy). A microarray was utilized to determine the expression of known miRNAs within the damaged and undamaged tissue regions in fetal and adolescent hearts after MI. Results: 73 miRNAs were up-regulated and 58 miRNAs were down-regulated significantly within the fetal infarct compared to remote cardiac samples. From adolescent hearts 69 non-redundant miRNAs were up-regulated and 63 miRNAs were down-regulated significantly in the infarct area compared to remote samples. Opposite differential expression profiles of 10 miRNAs within tissue regions (Infarct area, Border zone and Remote area of the left ventricle) occurred between the fetuses and adolescent sheep. These included miR-558 and miR-1538, which when suppressed using LNA anti-miRNAs in cell culture, increased cardiomyoblast proliferation. Conclusion: There were significant differences in miRNA responses in fetal and adolescent sheep hearts following a MI, suggesting that the modulation of novel miRNA expression may have therapeutic potential, by promoting proliferation or repair in a damaged heart. Refereed/Peer-reviewed

Background Pregnancy complications such as preterm birth and fetal growth restriction are associated with altered prenatal and postnatal cardiac development. We studied whether there were changes related specifically to pregnancy hypertension. Methods and Results Left and right ventricular volumes, mass, and function were assessed at birth and 3 months of age by echocardiography in 134 term‐born infants. Fifty‐four had been born to mothers who had normotensive pregnancy and 80 had a diagnosis of preeclampsia or pregnancy‐induced hypertension. Differences between groups were interpreted, taking into account severity of pregnancy disorder, sex, body size, and blood pressure. Left and right ventricular mass indexed to body surface area (LVMI and RVMI) were similar in both groups at birth (LVMI 20.9±3.7 versus 20.6±4.0 g/m2, P=0.64, RVMI 17.5±3.7 versus 18.1±4.7 g/m2, P=0.57). However, right ventricular end diastolic volume index was significantly smaller in those born to hypertensive pregnancy (16.8±5.3 versus 12.7±4.7 mL/m2, P=0.001), persisting at 3 months of age (16.4±3.2 versus 14.4±4.8 mL/m2, P=0.04). By 3 months of age these infants also had significantly greater LVMI and RVMI (LVMI 24.9±4.6 versus 26.8±4.9 g/m2, P=0.04; RVMI 17.1±4.2 versus 21.1±3.9 g/m2, P

Background Approximately 1 in 6 adolescents report regular binge alcohol consumption, and we hypothesize it affects heart growth during this period. Methods and Results Adolescent, genetically diverse, male Wistar rats were gavaged with water or ethanol once per day for 6 days. In vivo structure and function were assessed before and after exposure. Binge alcohol exposure in adolescence significantly impaired normal cardiac growth but did not affect whole‐body growth during adolescence, therefore this pathology was specific to the heart. Binge rats also exhibited signs of accelerated pathological growth (concentric cellular hypertrophy and thickening of the myocardial wall), suggesting a global reorientation from physiologic to pathologic growth. Binge rats compensated for their smaller filling volumes by increasing systolic function and sympathetic stimulation. Consequently, binge alcohol exposure increased PKA (protein kinase A) phosphorylation of troponin I, inducing myofilament calcium desensitization. Binge alcohol also impaired in vivo relaxation and increased titin‐based cellular stiffness due to titin phosphorylation by PKCα (protein kinase C α). Mechanistically, alcohol inhibited extracellular signal‐related kinase activity, a nodal signaling kinase activating physiology hypertrophy. Thus, binge alcohol exposure depressed genes involved in growth. These cardiac structural alterations from binge alcohol exposure persisted through adolescence even after cessation of ethanol exposure. Conclusions Alcohol negatively impacts function in the adult heart, but the adolescent heart is substantially more sensitive to its effects. This difference is likely because adolescent binge alcohol impedes the normal rapid physiological growth and reorients it towards pathological hypertrophy. Many adolescents regularly binge alcohol, and here we report a novel pathological consequence as well as mechanisms involved.

Protein kinase A (PKA) activity is pivotal for proper functioning of the human heart, and its dysregulation has been implicated in a variety of cardiac pathologies. PKA regulatory subunit 1α (R1α, encoded by the PRKAR1A gene) is highly expressed in the heart, and controls PKA kinase activity by sequestering PKA catalytic subunits. Patients with PRKAR1A mutations are often diagnosed with Carney complex (CNC) in early adulthood, and may die later in life from cardiac complications such as heart failure. However, it remains unknown whether PRKAR1A deficiency interferes with normal heart development. Here, we showed that left ventricular mass was reduced in young CNC patients with PRKAR1A mutations or deletions. Cardiac‐specific heterozygous ablation of PRKAR1A in mice increased cardiac PKA activity, and reduced heart weight and cardiomyocyte size without altering contractile function at 3 months of age. Silencing of PRKAR1A, or stimulation with the PKA activator forskolin completely abolished α1‐adrenergic receptor‐mediated cardiomyocyte hypertrophy. Mechanistically, depletion of PRKAR1A provoked PKA‐dependent inactivating phosphorylation of Drp1 at S637, leading to impaired mitochondrial fission. Pharmacologic inhibition of Drp1 with Mdivi 1 diminished hypertrophic growth of cardiomyocytes. In conclusion, PRKAR1A deficiency suppresses cardiomyocyte hypertrophy and impedes heart growth, likely through inhibiting Drp1‐mediated mitochondrial fission. These findings provide a potential novel mechanism for the cardiac manifestations associated with CNC., PRKAR1A mutations in humans are known to cause Carney complex, an autosomal dominant genetic disease often associated with premature death. Here, we report that PRKAR1A deficiency suppresses cardiomyocyte hypertrophy and reduces heart size, likely through PKA‐dependent inhibition of Drp1‐mediated mitochondrial fission.

Background/Aims: Obesity is a risk factor associated with cardiometabolic complications. Recently, we reported that miRNA-22 deletion attenuated high-fat diet-induced adiposity and prevented dyslipidemia without affecting cardiac hypertrophy in male mice. In this study, we examined the impact of miRNA-22 in obesogenic diet-induced cardiovascular and metabolic disorders in females. Methods: Wild type (WT) and miRNA-22 knockout (miRNA-22 KO) females were fed a control or an obesogenic diet. Body weight gain, adiposity, glucose tolerance, insulin tolerance, and plasma levels of total cholesterol and triglycerides were measured. Cardiac and white adipose tissue remodeling was assessed by histological analyses. Echocardiography was used to evaluate cardiac function and morphology. RNA-sequencing analysis was employed to characterize mRNA expression profiles in female hearts. Results: Loss of miRNA-22 attenuated body weight gain, adiposity, and prevented obesogenic diet-induced insulin resistance and dyslipidemia in females. WT obese females developed cardiac hypertrophy. Interestingly, miRNA-22 KO females displayed cardiac hypertrophy without left ventricular dysfunction and myocardial fibrosis. Both miRNA-22 deletion and obesogenic diet changed mRNA expression profiles in female hearts. Enrichment analysis revealed that genes associated with regulation of the force of heart contraction, protein folding and fatty acid oxidation were enriched in hearts of WT obese females. In addition, genes related to thyroid hormone responses, heart growth and PI3K signaling were enriched in hearts of miRNA-22 KO females. Interestingly, miRNA-22 KO obese females exhibited reduced mRNA levels of Yap1, Egfr and Tgfbr1 compared to their respective controls. Conclusion: This study reveals that miRNA-22 deletion induces cardiac hypertrophy in females without affecting myocardial function. In addition, our findings suggest miRNA-22 as a potential therapeutic target to treat obesity-related metabolic disorders in females.

Aims The Hippo signalling pathway regulates multiple cellular processes during organ development and maintenance by modulating activity of the transcriptional cofactor Yap. Core components of this pathway are required for neonatal mouse heart regeneration, however, investigations to date have typically focused on expression and activity in cardiomyocytes. Due to the regenerative capacity of zebrafish and the fact that global loss of Yap is not fully embryonic lethal in zebrafish, we leveraged a yap null mutant to investigate the impact of constitutive Yap deletion during zebrafish heart regeneration. Methods and results Following cryoinjury in adult hearts, myocyte proliferation was not decreased in yap mutants, contrary to expectations based on mouse data. Experiments in larval zebrafish (Danio rerio) revealed that deletion of either Yap or Taz had a modest effect on heart growth, reducing gross organ size, while their combined deletion was synergistic; thus, Yap and Taz share some overlapping roles in zebrafish heart development. Surprisingly, adult yap mutants exhibited decreased collagen composition at 7 days post-injury, suggesting a critical role for Yap in scar formation during heart regeneration. siRNA-mediated Yap knockdown in primary rat (Rattus norvegicus) cardiac cells revealed a fibroblast-specific role for Yap in controlling the expression of cytoskeletal and myofibroblast activation genes, as well as pro-inflammatory cyto/chemokines. Corroborating these RNAseq data, we observed increased macrophage infiltration in the scars of yap mutants at 7 days post-injury. Conclusion These results suggest that Yap deletion has minimal effect on myocyte proliferation in adults, but significantly influences scar formation and immune cell infiltration during zebrafish heart regeneration. Collectively, these data suggest an unexpected role for Yap in matrix formation and macrophage recruitment during heart regeneration.

Summary: Exercise-induced heart growth provides protection against cardiovascular disease, whereas disease-induced heart growth leads to heart failure. These distinct forms of growth are associated with different molecular profiles (e.g., mRNAs, non-coding RNAs, and proteins), and targeting differentially regulated genes has therapeutic potential for heart failure. The effects of exercise on the cardiac and circulating lipidomes in comparison to disease are unclear. Lipidomic profiling was performed on hearts and plasma of mice subjected to swim endurance training or a cardiac disease model (moderate or severe pressure overload). Several sphingolipid species and phospholipids containing omega-3/6 fatty acids were distinctly altered in heart and/or plasma with exercise versus pressure overload. A subset of lipids was validated in an independent mouse model with heart failure and atrial fibrillation. This study highlights the adaptations that occur to lipid profiles in response to endurance training versus pathology and provides a resource to investigate potential therapeutic targets and biomarkers. : Tham et al. utilized a HPLC-ESI/MS/MS targeted lipidomics approach to show distinct remodeling of the cardiac and plasma lipidomes in response to exercise in comparison to heart disease stimuli. Differentially altered lipids were validated in a model with heart failure and atrial fibrillation and represent potential biomarkers and drug targets. Keywords: lipids, heart, exercise, physiological hypertrophy, pathological hypertrophy, atrial fibrillation, phospholipids, sphingolipids, biomarkers, treatment

OBJECTIVES Fetal endoscopic tracheal occlusion (FETO) is offered to fetuses with congenital diaphragmatic hernia (CDH) and severe lung hypoplasia to promote lung growth and may secondarily affect left heart growth. The effects of FETO on left heart hypoplasia (LHH) are not described post-CDH repair. METHODS A retrospective analysis was performed for fetuses with left-sided CDH who underwent FETO and severity-matched controls from 2007 to 2016 at our institution. Echocardiographic, ultrasound, and MRI data were reviewed. Left heart dimensions were assessed prenatally and postnatally. Primary clinical outcome evaluated was death. RESULTS Twelve FETO patients and 18 controls were identified. Fetal LHH was noted in both groups and worsened after FETO. Postnatal mitral valve dimensions were larger in the FETO group pre-CDH repair (P = .03). Post-CDH repair, mitral valve and left ventricular dimensions were not significantly different between groups (P = .79 and P = .63 respectively) while FETO aortic valve dimensions were smaller (P = .04). Extracorporeal membrane oxygenation use was lower in the FETO group. No associations were found between left heart dimensions and outcomes. CONCLUSION Although increased lung growth was seen after FETO, fetal LHH persisted with relative normalization seen post-repair. Persistent LHH post-FETO could be secondary to a small contribution of pulmonary venous return to the fetal left heart and increased intrathoracic pressures post-FETO.

Mammalian cardiac myocytes (CMs) stop proliferating soon after birth and subsequent heart growth comes from hypertrophy, limiting the adult heart's regenerative potential after injury. The molecular events that mediate CM cell cycle exit are poorly understood. To determine the epigenetic mechanisms limiting CM cycling in adult CMs (ACMs) and whether trimethylation of lysine 9 of histone H3 (H3K9me3), a histone modification associated with repressed chromatin, is required for the silencing of cell cycle genes, we developed a transgenic mouse model where H3K9me3 is specifically removed in CMs by overexpression of histone demethylase, KDM4D. Although H3K9me3 is found across the genome, its loss in CMs preferentially disrupts cell cycle gene silencing. KDM4D binds directly to cell cycle genes and reduces H3K9me3 levels at these promotors. Loss of H3K9me3 preferentially leads to increased cell cycle gene expression resulting in enhanced CM cycling. Heart mass was increased in KDM4D overexpressing mice by postnatal day 14 (P14) and continued to increase until 9-weeks of age. ACM number, but not size, was significantly increased in KDM4D expressing hearts, suggesting CM hyperplasia accounts for the increased heart mass. Inducing KDM4D after normal development specifically in ACMs resulted in increased cell cycle gene expression and cycling. We demonstrated that H3K9me3 is required for CM cell cycle exit and terminal differentiation in ACMs. Depletion of H3K9me3 in adult hearts prevents and reverses permanent cell cycle exit and allows hyperplastic growth in adult hearts in vivo.

Objective Fetal aortic stenosis may progress to hypoplastic left heart syndrome. Fetal valvuloplasty (FV) has been proposed to improve left heart hemo-dynamics and maintain biventricular (BV) circulation. The aim of this study was to assess FV efficacy by comparing survival and postnatal circulation between fetuses that underwent FV and those that did not. Methods This was a retrospective multicenter study of fetuses with aortic stenosis that underwent FV between 2005 and 2012, compared with contemporaneously enrolled natural history (NH) cases sharing similar characteristics at presentation but not undergoing FV. Main outcome measures were overall survival, BV-circulation survival and survival after birth. Secondary outcomes were hemodynamic change and left heart growth. A propensity score model was created including 54/67 FV and 60/147 NH fetuses. Analyses were performed using logistic, Cox or linear regression models with inverse probability of treatment weighting (IPTW) restricted to fetuses with a propensity score of 0.14-0.9, to create a final cohort for analysis of 42 FV and 29 NH cases. Results FV was technically successful in 59/67 fetuses at a median age of 26 (21-34) weeks. There were 7/72 10%) procedure-related losses, and 22/53 (42%) FV babies were delivered at < 37 weeks. IPTW demonstrated improved survival of liveborn infants following FV (hazard ratio, 0.38; 95% CI, 0.23-0.64; P = 0.0001), after adjusting for circulation and postnatal surgical center. Similar proportions had BV circulation (36% for the FV cohort and 38% for the NH cohort) and survival was similar between final circulations. Successful FV cases showed improved hemodynamic response and less deterioration of left heart growth compared with NH cases (P

SUMMARY Understanding the mechanisms that control human cardiomyocyte proliferation might be applicable to regenerative medicine. We screened a whole genome collection of human miRNAs, identifying 96 to be capable of increasing proliferation (DNA synthesis and cytokinesis) of human iPSC-derived cardiomyocytes. Chemical screening and computational approaches indicated that most of these miRNAs (67) target different components of the Hippo pathway and that their activity depends on the nuclear translocation of the Hippo transcriptional effector YAP. 53 of the 67 miRNAs are present in human iPSC cardiomyocytes, yet anti-miRNA screening revealed that none are individually essential for basal proliferation of hiPSC cardiomyocytes despite the importance of YAP for proliferation. We propose a model in which multiple endogenous miRNAs redundantly suppress Hippo signaling to sustain the cell cycle of immature cardiomyocytes., In Brief Diez-Cuñado et al. show that a large set of miRNAs are individually sufficient to promote cell division of immature human cardiomyocytes. Most of these miRNAs act by repressing Hippo and activating YAP, defining Hippo/YAP as a central node controlling cardiomyocyte proliferation and as a promising target to achieve therapeutic cardiac regeneration.

Preterm birth (delivery prior to 37 completed weeks of gestation) is the leading cause of perinatal deaths worldwide. Preterm infants are born when their hearts are structurally and functionally immature; as a result, maladaptive cardiac remodelling occurs in the neonatal period which may lead to cardiac dysfunction later in life. Hypotension is a common co-morbidity of preterm birth in the neonatal period; however, hypertension often manifests in adulthood. Adults born preterm exhibit altered heart growth, which may in part be linked to their elevation in blood pressure. Clinical interventions used to facilitate lung function in preterm infants, including antenatal glucocorticoids and mechanical ventilation, may also impact cardiac growth early in life, with lifelong implications for cardiac structure and function.

In contrast to mammals, the zebrafish maintains its cardiomyocyte proliferation capacity throughout adulthood. However, neither the molecular mechanisms that orchestrate the proliferation of cardiomyocytes during developmental heart growth nor in the context of regeneration in the adult are sufficiently defined yet. We identified in a forward genetic N-ethyl-N-nitrosourea (ENU) mutagenesis screen the recessive, embryonic-lethal zebrafish mutant baldrian (bal), which shows severely impaired developmental heart growth due to diminished cardiomyocyte proliferation. By positional cloning, we identified a missense mutation in the zebrafish histone deacetylase 1 (hdac1) gene leading to severe protein instability and the loss of Hdac1 function in vivo. Hdac1 inhibition significantly reduces cardiomyocyte proliferation, indicating a role of Hdac1 during developmental heart growth in zebrafish. To evaluate whether developmental and regenerative Hdac1-associated mechanisms of cardiomyocyte proliferation are conserved, we analyzed regenerative cardiomyocyte proliferation after Hdac1 inhibition at the wound border zone in cryoinjured adult zebrafish hearts and we found that Hdac1 is also essential to orchestrate regenerative cardiomyocyte proliferation in the adult vertebrate heart. In summary, our findings suggest an important and conserved role of Histone deacetylase 1 (Hdac1) in developmental and adult regenerative cardiomyocyte proliferation in the vertebrate heart., Author summary Heart disease is one of the most common causes of death in all developed countries. While zebrafish cardiomyocytes are able to proliferate throughout adulthood, mammalian cardiomyocytes lose this ability during early development, and therefore are not capable to replace and renew cardiomyocytes after injury. The underlying mechanisms of cardiomyocyte proliferation are still not completely resolved. Understanding how zebrafish cardiomyocytes preserve their proliferating state, would be a valuable information to foster cardiac regeneration, e.g. after myocardial infarction in patients. Knowledge of the signaling pathways that need to be activated, or deactivated in order to induce cardiomyocyte proliferation after acute or chronic injury will pave the way for the development of genetic and/or pharmacological treatment options. In an ENU-mutagenesis screen, we identified the zebrafish mutant baldrian, which shows reduced embryonic cardiomyocyte proliferation. As genetic cause of the observed phenotype, we identified a missense mutation in the hdac1 gene. By treatment of heart-injured adult fish with the HDAC1 inhibitor Mocetinostat, we were able to show a reduced rate of cardiomyocyte proliferation also in the adult zebrafish heart in vivo, suggesting a role of Hdac1 in embryonic heart growth and adult regenerative cardiomyocyte proliferation in zebrafish.

HIGHLIGHTS: Urine proteomics allows the identification of the pathways modulated in neonates. Up-regulated pathways in preterm include immunity, metabolism and oxidative stress. Some of these pathways seem to be modulated by the nutritional support. AGT and RBP4 might be related to the development of cardiovascular diseases. ABSTRACT: Preterm birth has been associated with an increased risk of cardiovascular diseases (CVD) in adulthood. The goal of our study was to give new molecular insights on the relationship between prematurity and CVD risk and to identify putative biomarkers that would facilitate the development of effective screening and therapeutic strategies. In this sense, mass spectrometry (MS)-based proteomics was applied to the characterization of urine protein profile. GeLC–MS/MS analysis of urine (desalted and concentrated with a 10-kDa filter) followed by bioinformatics was applied for the characterization of preterm and full-term neonates. Urine proteome profiling retrieved 434 unique proteins, from which 126 were common to both groups, 37 were unique to preterm and 58 to full-term neonates. Protein-protein interaction analysis for unique proteins and common ones present in significant distinct levels retrieved immune system, metabolism, defense systems and tissue remodeling as the most representative clusters in preterm neonates. Metabolic adaptation along with the up-regulation of heart growth (identified by angiotensinogen and retinol-binding protein 4) may account for an increased CVD risk in preterm neonates. These proteins may have predictive value of CVD in adulthood of this specific group of neonates. The follow-up of urinary proteome dynamics of preterm and full-term neonates will be crucial for the validation of this hypothesis.

Recently, PSEN1 has been reported to have mutations in dilated cardiomyopathy pedigrees. However, the function and mechanism of PSEN1 in cardiomyopathy remains unresolved. Here, we established four types of genetically modified mice to determine the function of PSEN1 in cardiac development and pathology. PSEN1 null mutation resulted in perinatal death, retardation of heart growth, ventricular dilatation, septum defects, and valvular thickening. PSEN1 knockout in adults led to decreased muscle fibers, widened sarcomere Z lines and reduced lengths of sarcomeres in cardiomyocytes. Cardiovascular loss of function of PSEN1 induced by Sm22a-Cre or Myh6-Cre/ER/tamoxifen also resulted in severe ultrastructural abnormalities, such as relaxed gap junctions between neighboring cardiomyocytes. Functionally, cardiovascular deletion of PSEN1 caused spontaneous mortality from birth to adulthood and led to diastolic heart dysfunction, including decreased volume of the left ventricle at the end-systolic and end-diastolic stages. Additionally, in a myocardial ischemia model, deletion of PSEN1 in the cardiovascular system first protected mice by inducing adaptive hypertrophy but ultimately resulted in severe heart failure. Furthermore, a collection of genes was abnormally expressed in the hearts of cardiac-specific PSEN1 knockout mice. They were enriched in cell proliferation, calcium regulation, and so on. Taken together, dynamic regulation and abnormal function of PSEN1 underlie the pathogenesis of cardiovascular diseases due to ultrastructural abnormality of cardiomyocytes.

The Drosophila heart provides a simple model to examine the remodelling of muscle insertions with growth, extracellular matrix (ECM) turnover, and fibrosis. Between hatching and pupation, the Drosophila heart increases in length five-fold. If major cardiac ECM components are secreted remotely, how is ECM "self assembly" regulated? We explored whether ECM proteases were required to maintain the morphology of a growing heart while the cardiac ECM expanded. An increase in expression of Drosophila's single tissue inhibitor of metalloproteinase (TIMP), or reduced function of metalloproteinase MMP2, resulted in fibrosis and ectopic deposition of two ECM Collagens; type-IV and fibrillar Pericardin. Significant accumulations of Collagen-IV (Viking) developed on the pericardium and in the lumen of the heart. Congenital defects in Pericardin deposition misdirected further assembly in the larva. Reduced metalloproteinase activity during growth also increased Pericardin fibre accumulation in ECM suspending the heart. Although MMP2 expression was required to remodel and position cardiomyocyte cell junctions, reduced MMP function did not impair expansion of the heart. A previous study revealed that MMP2 negatively regulates the size of the luminal cell surface in the embryonic heart. Cardiomyocytes align at the midline, but do not adhere to enclose a heart lumen in MMP2 mutant embryos. Nevertheless, these embryos hatch and produce viable larvae with bifurcated hearts, indicating a secondary pathway to lumen formation between ipsilateral cardiomyocytes. MMP-mediated remodelling of the ECM is required for organogenesis, and to prevent assembly of excess or ectopic ECM protein during growth. MMPs are not essential for normal growth of the Drosophila heart.

AimsCardiomyocyte (CM) cell cycle arrest, decline of mononucleated-diploid CMs, sarcomeric maturation, and extracellular matrix remodeling are implicated in loss of cardiac regenerative potential in mice after birth. Recent studies show a 3-day neonatal regenerative capacity in pig hearts similar to mice, but postnatal pig CM growth dynamics are unknown. We examined cardiac maturation in postnatal pigs and mice, to determine the relative timing of developmental events underlying heart growth and regenerative potential in large and small mammals.Methods and ResultsLeft ventricular tissue from White Yorkshire-Landrace pigs at postnatal day (P)0 to 6 months (6mo) was analyzed to span birth, weaning, and adolescence in pigs, compared to similar physiological timepoints in mice. Collagen remodeling increases by P7 in postnatal pigs, but sarcomeric and gap junctional maturation only occur at 2mo. Also, there is no postnatal transition to beta-oxidation metabolism in pig hearts. Mononucleated CMs, predominant at birth, persist to 2mo in swine, with over 50% incidence of mononucleated-diploid CMs at P7-P15. Extensive multinucleation with 4-16 nuclei per CM occurs beyond P30. Pigs also exhibit increased CM length relative to multinucleation, preceding increase in CM width at 2mo-6mo. Further, robust CM mitotic nuclear pHH3 activity and cardiac cell cycle gene expression is apparent in pig left ventricles up to 2mo. By contrast, in mice, these maturational events occur concurrently in the first two postnatal weeks alongside loss of cardiac regenerative capacity.ConclusionsCardiac maturation occurs over a 6mo postnatal period in pigs, despite a similar early-neonatal heart regenerative window as mice. Postnatal pig CM growth includes increase in CM length alongside multinucleation, with CM cell cycle arrest and loss of mononucleated-diploid CMs occurring at 2mo-6mo. These CM characteristics are important to consider for pig preclinical studies and may offer opportunities to study aspects of heart regeneration unavailable in other models.

Postnatal mammalian cardiomyocytes undergo a major transition from hyperplasia (increases in cell numbers) to hypertrophy (expansion in cell size). This process is accompanied by rapid mitochondrial biogenesis and metabolic switches to meet the demand of increased cardiac output. Although most mitochondrial components express ubiquitously, recent transcriptomic and proteomic analyses have discovered numerous tissue-specific mitochondrial proteins whose physiological functions are largely unknown. Here we report that a highly evolutionarily conserved mitochondrial protein Coq10a is predominantly expressed in mammalian cardiac and skeletal muscles, and is highly up-regulated around birth in a thyroid hormone-dependent manner. Deletion ofCoq10aby CRISPR/Cas9 leads to enhanced cardiac growth after birth. Surprisingly, adultCoq10amutant mice maintain the hypertrophic heart phenotype with increased levels of coenzyme Q (CoQ) per cardiomyocyte, preserved cardiac contractile function and mitochondrial respiration, which contrasts with reported mice and humans with mutations in other Coq family genes. Further RNA-seq analysis and mitochondrial characterization suggest an increase of mitochondrial biogenesis in theCoq10amutant heart as a possible consequence of Peroxisome proliferator-activated receptor Gamma Coactivator 1-alpha (PGC1α) activation, consistent with a recent intriguing report that CoQ may function as a natural ligand and partial agonist of Peroxisome Proliferator-Activated Receptor (PPAR) α/γ. Taken together, our study reveals a previously unknown function of a novel striated muscle-enriched mitochondrial protein Coq10a in regulating postnatal heart growth.

Obesity and cardiovascular diseases are worldwide public health issues. In this review, we discussed the participation of ROCK protein in cardiac hypertrophy, mainly through the modulation of leptin and insulin signaling pathways. Leptin plays a role in cardiovascular disease development and, through the Rho-associated protein kinase (ROCK), promotes cardiac hypertrophy. ROCK protein, is regulated by small Rho-GTPases and has two isoforms with high homology. ROCK is able to activate the MAP kinase (MAPK) pathway and modulate insulin signaling in the heart, participating in cardiac hypertrophy development of concentric and eccentric left ventricle growth. Although different types of stimulus can lead to morphologically antagonistic heart growth, physical exercise promotes improvements in hemodynamic function, emerging as a promising non-pharmacological tool to improve overall health. Leptin can activate ROCK in a pathological way, increasing MAPK activity and decreasing insulin signaling via insulin receptor substrate 1 (IRS1) serine 307 residue phosphorylation, phosphatase and tensin homolog, and protein kinase Cβ2. In turn, physical exercise decreases leptin levels and positively modulates insulin signaling as well as increases ROCK-dependent IRS1 (Ser632/635) phosphorylation, improving phosphatidylinositol 3-kinase/protein kinase B axis and promoting physiologic heart growth. Currently, there is a lack of studies about differences in ROCK isoforms, especially during exercise and/or obesity. However, the understanding of its biological function and the complex mechanism underlying the distinct types of cardiac hypertrophy development can be a useful tool in the improvement and treatment of cardiovascular outcomes.

Doxorubicin (DOX) is one of the most effective chemotherapeutic drugs; however, the incidence of cardiotoxicity compromises its therapeutic index. Oxidative stress and apoptosis are believed to be involved in DOX-induced cardiotoxicity. Chitosan oligosaccharides (COS), the enzymatic hydrolysates of chitosan, have been reported to possess diverse biological activities including antioxidant and anti-apoptotic properties. The objective of the present study was to investigate the potential role of COS against DOX-induced cardiotoxicity, and the effects of COS on apoptosis and oxidative stress in rats and H9C2 cells. Furthermore, we also shed light on the involved pathways during the whole process. For this purpose, first, we demonstrated that COS exhibited a significant protective effect on cardiac tissue by not only inducing a decrease in body and heart growth but also ameliorated oxidative damage and ECG alterations in DOX-treated rats. Second, we found that COS reversed the decrease of cell viability induced by DOX, reduced the intracellular reactive oxygen species (ROS), increased the mitochondrial membrane potential (MMP) and Bcl-2/Bax ratio. COS treatment also results in reduced caspase-3 and caspase-9 expressions, and an increase in the phosphorylation of MAPKs (mitogen-activated protein kinases) in DOX-exposed H9C2 cells. Additionally, cellular homeostasis was re-established via stabilization of MAPK mediated nuclear factor erythroid 2-related factor 2/antioxidant-response element (Nrf2/ARE) signaling and transcription of downstream cytoprotective genes. In summary, these findings suggest that COS could be a potential candidate for the prevention and treatment of DOX-induced cardiotoxicity.

Cardiac remodeling results from both physiological and pathological stimuli. Compared with mammalian hearts, fish hearts show a broader array of remodeling changes in response to environmental influences, providing exceptional models for dissecting the molecular and cellular bases of cardiac remodeling. We recently characterized a form of pathological remodeling in juvenile pink salmon (Oncorhynchus gorbuscha) in response to crude oil exposure during embryonic cardiogenesis. In the absence of overt pathology (cardiomyocyte death or inflammatory infiltrate), cardiac ventricles in exposed fish showed altered shape, reduced thickness of compact myocardium and hypertrophic changes in spongy, trabeculated myocardium. Here, we used RNA sequencing to characterize molecular pathways underlying these defects. In juvenile ventricular cardiomyocytes, antecedent embryonic oil exposure led to dose-dependent upregulation of genes involved in innate immunity and two NKX homeobox transcription factors not previously associated with cardiomyocytes, nkx2.3 and nkx3.3 Absent from mammalian genomes, the latter is largely uncharacterized. In zebrafish embryos, nkx3.3 demonstrated a potent effect on cardiac morphogenesis, equivalent to that of nkx2.5, the primary transcription factor associated with ventricular cardiomyocyte identity. The role of nkx3.3 in heart growth is potentially linked to the unique regenerative capacity of fish and amphibians. Moreover, these findings support a cardiomyocyte-intrinsic role for innate immune response genes in pathological hypertrophy. This study demonstrates how an expanding mechanistic understanding of environmental pollution impacts - i.e. the chemical perturbation of biological systems - can ultimately yield new insights into fundamental biological processes.

Background Activating transcription 3 (ATF3) is a member of the basic leucine zipper family of transcription factors. ATF3 is an immediate early gene expressed following various cellular stresses. ATF3 acts through binding to cyclic AMP response elements found in the promoters of key regulatory proteins that determine cell fate. In the heart, multiple cardiac stresses result in chronic ATF3 expression. Transgenic mice with ATF3 expression in cardiomyocytes clearly demonstrate that ATF3 serves a leading role in heart hypertrophy, cardiac fibrosis, cardiac dysfunction and death. In contrast, the use of ATF3 whole body knockout mice resulted non-conclusive results. The heart is composed of various cell types such as cardiomyocytes, fibroblasts, endothelial and immune cells. The question that we addressed in this study is whether ablation of ATF3 in unique cell types in the heart results in diverse cardiac phenotypes. Methods ATF3-flox mice were crossed with αMHC and Postn specific promoters directing CRE expression and thus ATF3 ablation in cardiomyocytes and myofibroblast cells. Mice were challenged with transverse aortic constriction (TAC) for eight weeks and heart function, ventricle weight, hypertrophic markers, fibrosis markers and ATF3 expression were assessed by qRT-PCR. Results The results of the study show that ATF3 deletion in cardiomyocytes followed by TAC resulted in reduced heart growth and dampened fibrosis response while ATF3 ablation in myofibroblasts displayed a reduced hypertrophic gene program. Conclusions TAC-operation results in increased ATF3 expression in both myofibroblasts and cardiomyocytes that promotes a hypertrophic program and fibrotic cardiac growth, respectively.

Binding of the transcriptional co-activator YAP with the transcription factor TEAD stimulates growth of the heart and other organs. YAP overexpression potently stimulates fetal cardiomyocyte (CM) proliferation, but YAP's mitogenic potency declines post-natally. While investigating factors that limit YAP's postnatal mitogenic activity, we found that the CM-enriched TEAD1 binding protein VGLL4 inhibits CM proliferation by inhibiting TEAD1-YAP interaction and by targeting TEAD1 for degradation. Importantly, VGLL4 acetylation at lysine 225 negatively regulated its binding to TEAD1. This developmentally regulated acetylation event critically governs postnatal heart growth, since overexpression of an acetylation-refractory VGLL4 mutant enhanced TEAD1 degradation, limited neonatal CM proliferation, and caused CM necrosis. Our study defines an acetylation-mediated, VGLL4-dependent switch that regulates TEAD stability and YAP-TEAD activity. These insights may improve targeted modulation of TEAD-YAP activity in applications from cardiac regeneration to cancer.

The study of the mechanisms leading to cardiac hypertrophy is essential to better understand cardiac development and regeneration. Pathological conditions such as ischemia or pressure overload can induce a release of extracellular nucleotides within the heart. We recently investigated the potential role of nucleotide P2Y receptors in cardiac development. We showed that adult P2Y4-null mice displayed microcardia resulting from defective cardiac angiogenesis. Here we show that loss of another P2Y subtype called P2Y6, a UDP receptor, was associated with a macrocardia phenotype and amplified pathological cardiac hypertrophy. Cardiomyocyte proliferation and size were increased in vivo in hearts of P2Y6-null neonates, resulting in enhanced postnatal heart growth. We then observed that loss of P2Y6 receptor enhanced pathological cardiac hypertrophy induced after isoproterenol injection. We identified an inhibitory effect of UDP on in vitro isoproterenol-induced cardiomyocyte hyperplasia and hypertrophy. The present study identifies mouse P2Y6 receptor as a regulator of cardiac development and cardiomyocyte function. P2Y6 receptor could constitute a therapeutic target to regulate cardiac hypertrophy., SCOPUS: ar.j, info:eu-repo/semantics/published

The heart either hypertrophies or dilates in response to familial mutations in genes encoding sarcomeric proteins, which are responsible for contraction and pumping. These mutations typically alter calcium-dependent tension generation within the sarcomeres, but how this translates into the spectrum of hypertrophic versus dilated cardiomyopathy is unknown. By generating a series of cardiac-specific mouse models that permit the systematic tuning of sarcomeric tension generation and calcium fluxing, we identify a significant relationship between the magnitude of tension developed over time and heart growth. When formulated into a computational model, the integral of myofilament tension development predicts hypertrophic and dilated cardiomyopathies in mice associated with essentially any sarcomeric gene mutations, but also accurately predicts human cardiac phenotypes from data generated in induced-pluripotent-stem-cell-derived myocytes from familial cardiomyopathy patients. This tension-based model also has the potential to inform pharmacologic treatment options in cardiomyopathy patients.

Aims Lipid phosphate phosphatase-3 (LPP3) is expressed at high levels in endothelial cells (ECs). Although LPP3 is known to hydrolyse the phosphate group from lysolipids such as spingosine-1-phosphate and its structural homologues, the function of Lpp3 in ECs is not completely understood. In this study, we investigated how tyrosine-protein kinase receptor ( TEK or Tie2) promoter–dependent deletion of Lpp3 alters EC activities. Methods and results Lpp3fl/fl mice were crossed with the tg.Tie2Cre transgenic line. Vasculogenesis occurred normally in embryos with Tie2Cre -mediated deletion of Lpp3 (called Lpp3ECKO ), but embryonic lethality occurred in two waves, the first wave between E8.5 and E10.5, while the second between E11.5 and E13.5. Lethality in Lpp3ECKO embryos after E11.5 was accompanied by vascular leakage and haemorrhage, which likely resulted in insufficient cardiovascular development. Analyses of haematoxylin- and eosin-stained heart sections from E11.5 Lpp3ECKO embryos showed insufficient heart growth associated with decreased trabeculation, reduced growth of the compact wall, and absence of cardiac cushions. Staining followed by microscopic analyses of Lpp3ECKO embryos revealed the presence of apoptotic ECs. Furthermore, Lpp3 -deficient ECs showed decreased gene expression and protein levels of Cyclin-D1 , VE-cadherin , Fibronectin , Klf2 , and Klf4 . To determine the underlying mechanisms of vascular leakage and barrier disruption, we performed knockdown and rescue experiments in cultured ECs. LPP3 knockdown decreased transendothelial electrical resistance and increased permeability. Re-expression of β-catenin cDNA in LPP3 -knockdown ECs partially restored the effect of the LPP3 loss, whereas re-expression of p120ctn cDNA did not. Conclusion These findings demonstrate the essential roles of LPP3 in the maturation of EC barrier integrity and normal cardiovascular development.

More people die every year from ischemic heart disease than any other disease. Because the human heart lacks sufficient ability to replenish the damaged cardiac muscles, extensive research has been devoted toward understanding the homeostatic and regenerative potential of the heart and to develop regenerative therapies for heart disease. Here, we discuss recent advances in the understanding of mechanisms governing heart growth during homeostasis or injury, including those from observational studies in humans and experimental research in animal models of cardiac regeneration. We also discuss how progress in stem cell biology and cellular reprogramming has enabled exciting new strategies for cardiac regeneration.

Objective Myocardial infarction (MI) remains a leading cause of morbidity and mortality worldwide. Exercise training could improve cardiac function following MI. However, the mechanisms are still not well-known. Neuregulin 1 (NRG1)plays an important role in heart development and regeneration.In this study, we investigated the effect of NRG1 on cardiac regeneration in a zebrafish model, detected whether exercise could improve cardiac function through regulating NRG1 expression in infarcted heart and explore the possible role of up-regulation of NRG1 in skeletal muscle play in the cardioprotective effects in rats with MI. Methods Transgenic zebrafish line, cmlc2:CreERandβ-act2:BSNrg1,wereusedto study the effect of NRG1 on heart growth and regeneration after injury. PCNA was detected by immunofluorescence staining andmRNAexpression of gata4, nkx2.5, tbx5, smyd1b, hsp90α and murf were tested by RT-PCR.Sprague-Dawley rats were used to establish MI model and underwent fourweeks of exercise training (ET) or pAAV-{dMCK promoter}rNRG1-eGFP intervention.AG1478 was used asan inhibitor of NRG1/ErbBs signaling pathway. Cardiac function and structure,cardiomyocyte proliferation and NRG1 expression were detected in the heart or skeletal. Results Cardiac-specific overexpression of NRG1 induced cardiac hypertrophy and cardiomyocyte proliferation, regulated the mRNA expression of gata4, nkx2.5, tbx5, smyd1b, hsp90α andmurf in uninjuriedzebrafish, and promote cardiac repair and regeneration after injury in the zebrafish.Exercise activated NRG1/ErbBs signaling pathway, improved cardiac remodeling and heart function, enhanced cardiomyocyte proliferation, reduced cardiomyocyte apoptosis, ROS level and MuRF1 protein expression in rats with MI. BlockingErbB signaling attenuated the ET-induced cardioprotection effects in rat with MI.up-regulation of NRG1 expression in skeletal muscle could increase the protein level of NRG1 in serum and infarcted heart, improve cardiomyocyte proliferation and reduce the level of cardiac fibrosis, finally promote cardiac function. Conclusions Up-regulation of NRG1 expression in the heart or skeletal musclemay be one of the underlying mechanisms of thebeneficial effects of exercise training following MI.