Exercise-induced Cardiac Growth Research Articles

Inhibition of Hmbox1 Promotes Cardiomyocyte Survival and Glucose Metabolism Through Gck Activation in Ischemia/Reperfusion Injury.

Exercise-induced physiological cardiac growth regulators may protect the heart from ischemia/reperfusion (I/R) injury. Homeobox-containing 1 (Hmbox1), a homeobox family member, has been identified as a putative transcriptional repressor and is downregulated in the exercised heart. However, its roles in exercise-induced physiological cardiac growth and its potential protective effects against cardiac I/R injury remain largely unexplored. We studied the function of Hmbox1 in exercise-induced physiological cardiac growth in mice after 4 weeks of swimming exercise. Hmbox1 expression was then evaluated in human heart samples from deceased patients with myocardial infarction and in the animal cardiac I/R injury model. Its role in cardiac I/R injury was examined in mice with adeno-associated virus 9 (AAV9) vector-mediated Hmbox1 knockdown and in those with cardiac myocyte-specific Hmbox1 ablation. We performed RNA sequencing, promoter prediction, and binding assays and identified glucokinase (Gck) as a downstream effector of Hmbox1. The effects of Hmbox1 together with Gck were examined in cardiomyocytes to evaluate their cell size, proliferation, apoptosis, mitochondrial respiration, and glycolysis. The function of upstream regulator of Hmbox1, ETS1, was investigated through ETS1 overexpression in cardiac I/R mice in vivo. We demonstrated that Hmbox1 downregulation was required for exercise-induced physiological cardiac growth. Inhibition of Hmbox1 increased cardiomyocyte size in isolated neonatal rat cardiomyocytes and human embryonic stem cell-derived cardiomyocytes but did not affect cardiomyocyte proliferation. Under pathological conditions, Hmbox1 was upregulated in both human and animal postinfarct cardiac tissues. Furthermore, both cardiac myocyte-specific Hmbox1 knockout and AAV9-mediated Hmbox1 knockdown protected against cardiac I/R injury and heart failure. Therapeutic effects were observed when sh-Hmbox1 AAV9 was administered after I/R injury. Inhibition of Hmbox1 activated the Akt/mTOR/P70S6K pathway and transcriptionally upregulated Gck, leading to reduced apoptosis and improved mitochondrial respiration and glycolysis in cardiomyocytes. ETS1 functioned as an upstream negative regulator of Hmbox1 transcription, and its overexpression was protective against cardiac I/R injury. Our studies unravel a new role for the transcriptional repressor Hmbox1 in exercise-induced physiological cardiac growth. They also highlight the therapeutic potential of targeting Hmbox1 to improve myocardial survival and glucose metabolism after I/R injury.

Exercise-induced changes in myocardial glucose utilization during periods of active cardiac growth

Exercise training can promote physiological cardiac growth, which has been suggested to involve changes in glucose metabolism to facilitate hypertrophy of cardiomyocytes. In this study, we used a dietary, in vivo isotope labeling approach to examine how exercise training influences the metabolic fate of carbon derived from dietary glucose in the heart during acute, active, and established phases of exercise-induced cardiac growth. Male and female FVB/NJ mice were subjected to treadmill running for up to 4 weeks and cardiac growth was assessed by gravimetry. Cardiac metabolic responses to exercise were assessed via in vivo tracing of [13C6]-glucose via mass spectrometry and nuclear magnetic resonance. We found that the half-maximal cardiac growth response was achieved by approximately 1 week of daily exercise training, with near maximal growth observed in male mice with 2 weeks of training; however, female mice were recalcitrant to exercise-induced cardiac growth and required a higher daily intensity of exercise training to achieve significant, albeit modest, increases in cardiac mass. We also found that increases in the energy charge of adenylate and guanylate nucleotide pools precede exercise-induced changes in cardiac size and were associated with higher glucose tracer enrichment in the TCA pool and in amino acids (aspartate, glutamate) sourced by TCA intermediates. Our data also indicate that the activity of collateral biosynthetic pathways of glucose metabolism may not be markedly altered by exercise. Overall, this study provides evidence that metabolic remodeling in the form of heightened energy charge and increased TCA cycle activity and cataplerosis precedes cardiac growth caused by exercise training in male mice.

Coordinated Metabolic Responses Facilitate Cardiac Growth in Pregnancy and Exercise.

Pregnancy and exercise are systemic stressors that promote physiological growth of the heart in response to repetitive volume overload and maintenance of cardiac output. This type of remodeling is distinct from pathological hypertrophy and involves different metabolic mechanisms that facilitate growth; however, it remains unclear how metabolic changes in the heart facilitate growth and if these processes are similar in both pregnancy- and exercise-induced cardiac growth. The ability of the heart to metabolize a myriad of substrates balances cardiac demands for energy provision and anabolism. During pregnancy, coordination of hormonal status with cardiac reductions in glucose oxidation appears important for physiological growth. During exercise, a reduction in cardiac glucose oxidation also appears important for physiological growth, which could facilitate shuttling of glucose-derived carbons into biosynthetic pathways for growth. Understanding the metabolic underpinnings of physiological cardiac growth could provide insight to optimize cardiovascular health and prevent deleterious remodeling, such as that which occurs from postpartum cardiomyopathy and heart failure. This short review highlights the metabolic mechanisms known to facilitate pregnancy-induced and exercise-induced cardiac growth, both of which require changes in cardiac glucose metabolism for the promotion of growth. In addition, we mention important similarities and differences of physiological cardiac growth in these models as well as discuss current limitations in our understanding of metabolic changes that facilitate growth.

Lymphangiogenesis contributes to exercise-induced physiological cardiac growth

BackgroundPromoting cardiac lymphangiogenesis exerts beneficial effects for the heart. Exercise can induce physiological cardiac growth with cardiomyocyte hypertrophy and increased proliferation markers in cardiomyocytes. However, it remains unclear whether and how lymphangiogenesis contributes to exercise-induced physiological cardiac growth. We aimed to investigate the role and mechanism of lymphangiogenesis in exercise-induced physiological cardiac growth. MethodsAdult C57BL6/J mice were subjected to 3 weeks of swimming exercise to induce physiological cardiac growth. Oral treatment with vascular endothelial growth factor receptor 3 (VEGFR3) inhibitor SAR131675 was used to investigate whether cardiac lymphangiogenesis was required for exercise-induced physiological cardiac growth by VEGFR3 activation. Furthermore, human dermal lymphatic endothelial cell (LEC)-conditioned medium was collected to culture isolated neonatal rat cardiomyocytes to determine whether and how LECs could influence cardiomyocyte proliferation and hypertrophy. ResultsSwimming exercise induced physiological cardiac growth accompanied by a remarkable increase of cardiac lymphangiogenesis as evidenced by increased density of lymphatic vessel endothelial hyaluronic acid receptor 1–positive lymphatic vessels in the heart and upregulated LYVE-1 and Podoplanin expressions levels. VEGFR3 was upregulated in the exercised heart, while VEGFR3 inhibitor SAR131675 attenuated exercise-induced physiological cardiac growth as evidenced by blunted myocardial hypertrophy and reduced proliferation marker Ki67 in cardiomyocytes, which was correlated with reduced lymphatic vessel density and downregulated LYVE-1 and Podoplanin in the heart upon exercise. Furthermore, LEC-conditioned medium promoted both hypertrophy and proliferation of cardiomyocytes and contained higher levels of insulin-like growth factor-1 and the extracellular protein Reelin, while LEC-conditioned medium from LECs treated with SAR131675 blocked these effects. Functional rescue assays further demonstrated that protein kinase B (AKT) activation, as well as reduced CCAAT enhancer-binding protein beta (C/EBPβ) and increased CBP/p300-interacting transactivators with E (glutamic acid)/D (aspartic acid)–rich-carboxylterminal domain 4 (CITED4), contributed to the promotive effect of LEC-conditioned medium on cardiomyocyte hypertrophy and proliferation. ConclusionOur findings reveal that cardiac lymphangiogenesis is required for exercise-induced physiological cardiac growth by VEGFR3 activation, and they indicate that LEC-conditioned medium promotes both physiological hypertrophy and proliferation of cardiomyocytes through AKT activation and the C/EBPβ-CITED4 axis. These results highlight the essential roles of cardiac lymphangiogenesis in exercise-induced physiological cardiac growth.

Abstract 13218: CITED4 is Required for Exercise-Induced Cardiac Growth and Cardiomyogenesis in Mice

Introduction: CITED4 (CBP/p300-interacting transactivators with E [glutamic acid]/D [aspartic acid]-rich-carboxylterminal domain 4) is a transcriptional regulator which limits pathologic remodeling after ischemic and pressure overload models of cardiac stress. It is also upregulated in the heart with exercise, an effective intervention which induces physiological hypertrophy and cardiomyogenesis in adult mice. We hypothesized that CITED4 is required for exercise-induced cardiac growth and cardiomyogenesis. Methods and Results: Eight- to ten-week-old mice with cardiomyocyte-specific CITED4 knockout (C4KO) and littermate controls underwent 8 weeks of voluntary wheel running in comparison to sedentary controls. 5-Ethynyl-2′-deoxyuridine (EdU) was injected subcutaneously every two days for the first two weeks. There were no significant differences in cardiac size and function at baseline between C4KO and control mice. In control mice, running increased heart weight to tibial length ratio (HW/TL, 6.26±0.25 vs 7.37±0.75, p=0.01, n=5-9 per group), heart weight to body weight ratio (HW/BW, 4.08±0.40 vs 4.93±0.48, p=0.01), and cardiomyocyte (CM) size without affecting fractional shortening (FS), consistent with physiologic remodeling. There was also a nonsignificant trend to increased EdU+ CMs in the control exercised mice (2.96±2.34% vs 5.23±6.28%, p=0.44), suggestive of previously documented exercise-induced cardiomyogenesis. In contrast, running did not change HW/TL and CM size in C4KO mice (HW/TL, 6.44±0.48 vs 6.66±0.29, p=0.22, n= 0-13 per group) but reduced %FS (58.78±3.27 vs 53.67±4.59, p=0.03) and increased chamber size (LVIDs, 1.32±0.13 vs 1.50±0.17, p=0.032), suggesting maladaptive cardiac remodeling with exercise, confirming prior findings observed in a more extreme swim-exercise model. Rather than the expected increase, hearts from exercised C4KO mice had fewer EdU+ CMs compared to sedentary C4KO mice (3.76±2.60% vs 2.11±0.76%, p=0.05) and reduced expression of cell cycle genes Cdkn1b, Cyclin B1, Cdkn2a, and Ki-67. Conclusions: CITED4 is required for exercise-induced cardiomyogenesis, CM hypertrophy, and adaptive remodeling. CITED4 deficiency leads to maladaptive cardiac remodeling with physiologic stress.

Long Noncoding RNA Cardiac Physiological Hypertrophy-Associated Regulator Induces Cardiac Physiological Hypertrophy and Promotes Functional Recovery After Myocardial Ischemia-Reperfusion Injury.

The benefits of exercise training in the cardiovascular system have been well accepted; however, the underlying mechanism remains to be explored. Here, we report the initial functional characterization of an exercise-induced cardiac physiological hypertrophy-associated novel long noncoding RNA (lncRNA). Using lncRNA microarray profiling, we identified lncRNAs in contributing the modulation of exercise-induced cardiac growth that we termed cardiac physiological hypertrophy-associated regulator (CPhar). Mice with adeno-associated virus serotype 9 driving CPhar overexpression and knockdown were used in in vivo experiments. Swim training was used to induce physiological cardiac hypertrophy in mice, and ischemia reperfusion injury surgery was conducted to investigate the protective effects of CPhar in mice. To investigate the mechanisms of CPhar's function, we performed various analyses including quantitative reverse transcription polymerase chain reaction, Western blot, histology, cardiac function (by echocardiography), functional rescue experiments, mass spectrometry, in vitro RNA transcription, RNA pulldown, RNA immunoprecipitation, chromatin immunoprecipitation assay, luciferase reporter assay, and coimmunoprecipitation assays. We screened the lncRNAs in contributing the modulation of exercise-induced cardiac growth through lncRNA microarray profiling and found that CPhar was increased with exercise and was necessary for exercise-induced physiological cardiac growth. The gain and loss of function of CPhar regulated the expression of proliferation markers, hypertrophy, and apoptosis in cultured neonatal mouse cardiomyocytes. Overexpression of CPhar prevented myocardial ischemia reperfusion injury and cardiac dysfunction in vivo. We identified DDX17 (DEAD-Box Helicase 17) as a binding partner of CPhar in regulating CPhar downstream factor ATF7 (activating transcription factor 7) by sequestering C/EBPβ (CCAAT/enhancer binding protein beta). Our study of this lncRNA CPhar provides new insights into the regulation of exercise-induced cardiac physiological growth, demonstrating the cardioprotective role of CPhar in the heart, and expanding our mechanistic understanding of lncRNA function, as well.

P5399microRNA-486 mediates exercise-induced cardiac growth and prevents cardiac ischemia-reperfusion injury

Abstract Background Molecules mediating exercise-induced physiological cardiac growth may be beneficial to prevent cardiac injury and remodeling. MicroRNA-486 (miR-486) is a muscle-enriched microRNA which was found to be increased in exercised heart. However, the role of miR-486 in exercise-induced cardiac growth is largely unknown. Purpose We aimed to study the function of miR-486 in exercise-induced cardiac growth, and to investigate its potential role in protection against cardiac ischemia/reperfusion (I/R) injury. Methods Male C57BL/6 adult mice were subjected to 3-week swimming exercise to induce physiological cardiac growth. Knockdown of miR-486 was performed by intramyocardial injection of miR-486 sponge to evaluate the role of miR-486 in exercise-induced cardiac growth. Moreover, intramyocardial injection of miR-486 overexpression lentivirus was performed to investigate whether miR-486 could reduce infarct size in mice suffered from acute I/R injury (30 min ligation and 24 hr reperfusion of left anterior descending coronary artery). Meanwhile, the functional role of miR-486 was evaluated in neonatal rat cardiomyocytes (NRCMs) treated with oxygen glucose deprivation/reperfusion (OGDR) using Tunel staining. Finally, function-rescue assays were performed to identify target genes of miR-486. Results Exercise significantly upregulated miR-486 in the heart, which was reduced by intramyocardial injection of miR-486 sponge. Exercise induced-cardiac growth was abolished in mice with intramyocardial injection of miR-486 sponge, as evidenced by reduced heart weight/body weight ratio and heart weight/tibia length ratio compared to control exercised-mice, suggesting a potential role of miR-486 in mediating exercise-induced cardiac growth. Meanwhile, we found that intramyocardial injection of miR-486 overexpression lentivirus was able to reduce the infarct size as determined by TTC staining. Moreover, in OGDR-induced apoptosis of NRCMs, miR-486 mimics was able to reduce cardiomyocyte apoptosis, while miR-486 inhibitor performed the opposite. Finally, PTEN and FoxO1 were identified as target genes of miR-486 in the control of cardiomyocyte apoptosis. Conclusions miR-486 mediates exercise-induced cardiac growth and protects against cardiac I/R injury. Increasing miR-486 might serve as a potential target to reduce myocardial apoptosis upon I/R injury. Acknowledgement/Funding This work was supported by the grants from National Natural Science Foundation of China 81722008, 91639101 and 81570362 to JJ Xiao, 81770401 to Y Bei

P5395Exercise-induced miR-17-3p Prevents Pathological Myocardial Dysfunction

Abstract Background Exercise is beneficial for pathological myocardial dysfunction and heart failure. We previously reported that miR-17-3p contributed to exercise-induced cardiac growth and protected against cardiac ischemia/reperfusion injury. However, if exercise-induced miR-17-3p can prevent pathological myocardial dysfunction is undetermined. Purpose To investigate if exercise-induced miR-17-3p can prevent pathological myocardial dysfunction. Methods The miR-17-3p expression was examined in phenylephrine (PE, 50μmol/L, 48 h) and angiotensin II (Ang II, 1μmol/L, 48 h) treated primary neonatal rat ventricular cardiomyocytes (NRCM), and the myocardium of thoracic aortic constriction (TAC, 4weeks) or angiotensin II (1.3 mg/kg/day, 4weeks) induced cardiac hypertrophy murine model. miR-17-3p transgenic mice were generated and subjected to TAC or Ang II to investigate the effect of miR-17-3p overexpression in attenuating pathological cardiac dysfunction by echocardiography. Cell area and hypertrophic genes were determined by Wheat Germ Agglutinin (WGA) staining and qRT-PCRs. In addition, cell area and hypertrophic genes (ANP, BNP, β-MHC) were determined by immunolabeling and qRT-PCR in NRCM after the transfection of miR-17-3p mimic or inhibitor. Furthermore, functional rescue assays were performed to identify phosphatase and tensin homolog (PTEN) as a target gene of miR-17-3p, and myocyte enhancer factor 2C (MEF2C) as an upstream regulator of miR-17-3p in pathological cardiac hypertrophy. Results The expression of miR-17-3p was significantly decreased in the heart from TAC or Ang II mouse model, and in PE or Ang II-induced cardiomyocyte hypertrophy model. miR-17-3p overexpression mice displayed improved cardiac function and reduced cardiac hypertrophy after TAC or Ang II treatment in vivo. miR-17-3p mimic significantly attenuated PE or Ang II-induced cardiomyocyte hypertrophy in vitro. Based on functional rescue experiments, PTEN was identified as a direct target of miR-17-3p that mediated its protective effects in cardiac hypertrophy. Moreover, MEF2C was identified as a negative upstream regulator of miR-17-3p involved in the control of cardiac hypertrophy. Proposed mechanism of miR-17-3p Conclusion Exercise-induced miR-17-3p can prevent pathological myocardial dysfunction by targeting PTEN. MEF2C was an upstream regulator of miR-17-3p. Targeting MEF2C/miR-17-3p/ PTEN represents a novel therapeutic strategy for pathological myocardial dysfunction. Acknowledgement/Funding The grants from National Natural Science Foundation of China (81722008, 91639101 and 81570362 to JJ Xiao)

GW29-e1644 FNDC5 Is Necessary for Exercise-Induced Cardiac Growth and Protects Against Ischemia Reperfusion Injury Remodeling

To explore the function of FNDC5 in physiological cardiac hypertrophy, as well as clarify the relative molecular mechanism, and then put forward a new treatment strategy of myocardial ischemia-reperfusion injury based on FNDC5. 1. FNDC5 expression was compared in control and exercise training mice

Understanding Key Mechanisms of Exercise-Induced Cardiac Protection to Mitigate Disease: Current Knowledge and Emerging Concepts.

The benefits of exercise on the heart are well recognized, and clinical studies have demonstrated that exercise is an intervention that can improve cardiac function in heart failure patients. This has led to significant research into understanding the key mechanisms responsible for exercise-induced cardiac protection. Here, we summarize molecular mechanisms that regulate exercise-induced cardiac myocyte growth and proliferation. We discuss in detail the effects of exercise on other cardiac cells, organelles, and systems that have received less or little attention and require further investigation. This includes cardiac excitation and contraction, mitochondrial adaptations, cellular stress responses to promote survival (heat shock response, ubiquitin-proteasome system, autophagy-lysosomal system, endoplasmic reticulum unfolded protein response, DNA damage response), extracellular matrix, inflammatory response, and organ-to-organ crosstalk. We summarize therapeutic strategies targeting known regulators of exercise-induced protection and the challenges translating findings from bench to bedside. We conclude that technological advancements that allow for in-depth profiling of the genome, transcriptome, proteome and metabolome, combined with animal and human studies, provide new opportunities for comprehensively defining the signaling and regulatory aspects of cell/organelle functions that underpin the protective properties of exercise. This is likely to lead to the identification of novel biomarkers and therapeutic targets for heart disease.

MiR-17-3p Contributes to Exercise-Induced Cardiac Growth and Protects against Myocardial Ischemia-Reperfusion Injury.

Limited microRNAs (miRNAs, miRs) have been reported to be necessary for exercise-induced cardiac growth and essential for protection against pathological cardiac remodeling. Here we determined members of the miR-17-92 cluster and their passenger miRNAs expressions in two distinct murine exercise models and found that miR-17-3p was increased in both. miR-17-3p promoted cardiomyocyte hypertrophy, proliferation, and survival. TIMP-3 was identified as a direct target gene of miR-17-3p whereas PTEN was indirectly inhibited by miR-17-3p. Inhibition of miR-17-3p in vivo attenuated exercise-induced cardiac growth including cardiomyocyte hypertrophy and expression of markers of myocyte proliferation. Importantly, mice injected with miR-17-3p agomir were protected from adverse remodeling after cardiac ischemia/reperfusion injury. Collectively, these data suggest that miR-17-3p contributes to exercise-induced cardiac growth and protects against adverse ventricular remodeling. miR-17-3p may represent a novel therapeutic target to promote functional recovery after cardiac ischemia/reperfusion.

The Non-cardiomyocyte Cells of the Heart. Their Possible Roles in Exercise-Induced Cardiac Regeneration and Remodeling.

The non-cardiomyocyte cellular microenvironment of the heart includes diverse types of cells of mesenchymal origin. During development, the majority of these cells derive from the epicardium, while a subset derives from the endothelium/endocardium and neural crest derived mesenchyme. This subset includes cardiac fibroblasts and telocytes, the latter of which are a controversial type of "connecting cell" that support resident cardiac progenitors in the postnatal heart. Smooth muscle cells, pericytes, and endothelial cells are also present, in addition to adipocytes, which accumulate as epicardial adipose connective tissue. Furthermore, the heart harbors many cells of hematopoietic origin, such as mast cells, macrophages, and other immune cell populations. Most of these control immune reactions and inflammation. All of the above-mentioned non-cardiomyocyte cells of the heart contribute to this organ's well-orchestrated physiology. These cells also contribute to regeneration as a result of injury or age, in addition to tissue remodeling triggered by chronic disease or increased physical activity (exercise-induced cardiac growth). These processes in the heart, the most important vital organ in the human body, are not only fascinating from a scientific standpoint, but they are also clinically important. It is well-known that regular exercise can help prevent many cardiovascular diseases. However, the precise mechanisms underpinning myocardial remodeling triggered by physical activity are still unknown. Surprisingly, exercise-induced adaptation mechanisms are often identical or very similar to tissue remodeling caused by pathological conditions, such as hypertension, cardiac hypertrophy, and cardiac fibrosis. This review provides a summary of our current knowledge regarding the cardiac cellular microenvironment, focusing on the clinical applications this information to the study of heart remodeling during regular physical exercise.

C/EBPB-CITED4 in Exercised Heart.

C/EBPB is a crucial transcription factor, participating in a variety of biological processes including cell proliferation, differentiation and development. In the cardiovascular system, C/EBPB-CITED4 signaling is known as a signaling pathway mediating exercise-induced cardiac growth. After its exact role in exercised heart firstly reported in 2010, more and more evidence confirmed that. MicroRNA (e.g. miR-222) and many molecules (e.g. Alpha-lipoic acid) can regulate this pathway and then involve in the cardiac protection effect induced by endurance exercise training. In addition, in cardiac growth during pregnancy, C/EBPB is also a required regulator. This chapter will give an introduction of the C/EBPB-CITED4 signaling and the regulatory network based on this signaling pathway in exercised heart.

TCTAP A-038 miR-17-3p Contributes to Exercise-Induced Cardiac Growth and Protects Against Myocardial Ischemia-Reperfusion Injury

TCTAP A-038 miR-17-3p Contributes to Exercise-Induced Cardiac Growth and Protects Against Myocardial Ischemia-Reperfusion Injury

Telocytes in exercise-induced cardiac growth.

Exercise can induce physiological cardiac growth, which is featured by enlarged cardiomyocyte cell size and formation of new cardiomyocytes. Telocytes (TCs) are a recently identified distinct interstitial cell type, existing in many tissues and organs including heart. TCs have been shown to form a tandem with cardiac stem/progenitor cells in cardiac stem cell niches, participating in cardiac regeneration and repair. Although exercise‐induced cardiac growth has been confirmed as an important way to promote cardiac regeneration and repair, the response of cardiac TCs to exercise is still unclear. In this study, 4 weeks of swimming training was used to induce robust healthy cardiac growth. Exercise can induce an increase in cardiomyocyte cell size and formation of new cardiomyocytes as determined by Wheat Germ Lectin and EdU staining respectively. TCs were identified by three immunofluorescence stainings including double labelling for CD34/vimentin, CD34/platelet‐derived growth factor (PDGF) receptor‐α and CD34/PDGF receptor‐β. We found that cardiac TCs were significantly increased in exercised heart, suggesting that TCs might help control the activity of cardiac stem/progenitor cells, cardiomyocytes or endothelial cells. Adding cardiac TCs might help promote cardiac regeneration and renewal.

Exercise as a platform for pharmacotherapy development in cardiac diseases.

Moderate exercise is an effective and economic way to prevent and treat cardiovascular diseases. Unlike pathological cardiac growth, exercise-induced cardiac growth, excluding extreme strenuous exercise, does not cause cardiac cell death, fibrosis, and cardiac dysfunction. The balanced cardiomyogenesis (cardiomyocyte hypertrophy and hyperplasia) and neo-angiogenesis are essential determinants for exercise-induced cardiac growth. In particular, exercise leads to physiological cardiac growth through regulating the IGF-1-PI3K-Akt, nitric oxide (NO), C/EBPβ, and PGC-1α signaling pathways, which might be novel therapeutic targets for cardiac diseases. The formation of new cardiomyocytes in response to exercise suggests that exercise might be a useful tool to enhance cardiac regenerative capacity. Exercise also exerts its protective effects against cardiac aging and cardiac metabolic derangement. Moreover, growing evidence reveals the regulation of cardiac and circulating microRNAs in response to exercise. A better understanding of the mechanisms underlying exercise-induced cardioprotection will lead to the development of innovative pharmacotherapies for cardiac diseases.

Abstract 220: miR-486 Mediates the Benefits of Exercise in Attenuating Cardiac Fibrosis

MicroRNAs (miRNAs, miRs), a novel group of small non-coding RNAs, play important roles in cardiac fibrosis. Exercise-induced physiological cardiac growth is associated with hypertrophy and proliferation of cardiomyocytes. In addition, exercise has been shown to inhibit cardiac fibrosis. However, relative little is known about whether exercise could attenuating cardiac fibrosis via targeting miRNA. miR-486 is a muscle enriched miRNAs, however, its role in heart is relative unclear. The current study aimed to investigate the role of miR-486 in exercise-induced cardiac growth in a 3-week swimming training murine model as well as in the function of cardiac fibroblasts and production of extracellular matrix (ECM) using neonatal rat cardiac fibroblasts in primary culture. Our data showed that exercised mice displayed increased about three-fold expression of miR-486 in hearts as measured by microarray analysis and qRT-PCRs. EdU proliferation assays demonstrated that miR-486 mimics decreased (5.90%±0.57% vs 4.02%±0.27% in nc-mimics vs miR-486-mimics, respectively), while miR-486 inhibitor increased the proliferation of cardiac fibroblasts in vitro (5.87%±0.16% vs 9.60%±0.58% in nc-inhibitor vs miR-486-inhibitor, respectively). Although downregulation of miR-486 had no regulatory effect on α-sma and collagen-1 gene expression in cardiac fibroblasts, overexpression of miR-486 significantly reduced the mRNA level of α-sma (1.01±0.08 vs 0.28±0.04 in nc-mimics vs miR-486-mimics, respectively) and collagen-1(1.02±0.12 vs 0.58±0.09 in nc-mimics vs miR-486-mimics, respectively), indicative of attenuated activation of fibroblasts and reduced production of ECM. These data reveal that miR-486 is essentially involved in the proliferation and activation of cardiac fibroblasts, and might be a key regulator mediating the benefit of exercise in preventing cardiac fibrosis.

Abstract 112: An Exercise-induced MicroRNA Pathway That Protects Against Apoptosis and Pathological Cardiac Dysfunction

Background: Exercise induces physiological cardiac growth and protects against adverse cardiac remodeling. microRNAs (miRNA) are important regulators in cardiovascular pathology and disease. Less is known about miRNA roles in the cardiac effects of exercise, and we investigated their roles in exercise-induced cardiac growth. Methods and Results: Based on cardiac miRNA profiling in two exercise models (swimming, running) and functional screening in neonatal rat cardiomyocytes (NRCMs), miR-222 was selected for further study. miR-222 was upregulated ~2-fold in both exercise models, pathological hypertrophic and failing hearts (p<0.05). We found that ERK, JNK and p38 signal pathways are involved in FBS increasing miR-222 in NRCMs. miR-222 increased NRCM proliferation and size (p<0.01) and reduced NRCM apoptosis induced by serum starvation with or without doxorubicin. Four potential miR-222 targets ( p27 , Hipk1 , Hipk2 , and Hmbox-1 ) were identified and confirmed as direct targets. siRNA knockdown (KD) of p27 or Hipk2 inhibited NRCM apoptosis. To investigate miR-222’s cardiac role in vivo , we made transgenic mice with cardiac-specific, inducible (TgI-miR-222) or constitutive (TgC-miR-222) expression of miR-222. RT-PCR showed that miR-222 is increased ~6-fold in TgI-miR-222 hearts after induction and ~10-fold in TgC-miR-222 hearts. Surprisingly, both transgenic lines have normal heart size and function, and the 4 miR-222 targets are unaffected at baseline. After ischemia-reperfusion injury (IRI), TgI-miR-222 had no difference in initial infarct size or dysfunction but were protected against adverse remodeling over the next six weeks with better function (p<0.01), less cardiac fibrosis (68%, p<0.05), and reduced CM apoptosis (300%, p<0.05). Two weeks after TAC, TgC-miR-222 had less increase in cardiac mass (HW/TL, p<0.05), better cardiac function ([[Unable to Display Character: ∆]]32%, p<0.01), reduced expression of markers of fibrosis ( Col1a1 , Col3a1 , Mmp2, p<0.05 ) and apoptosis ( Caspase 1, 3, 8, p<0.05). Four months after TAC, TgC-miR-222 had improved survival (p<0.05) compared to wide type controls. Conclusion: Cardiac miR-222 is upregulated by exercise and protects against adverse remodeling and cardiac dysfunction after IRI or TAC.

MiR-222 Is Necessary for Exercise-Induced Cardiac Growth and Protects against Pathological Cardiac Remodeling

Exercise induces physiological cardiac growth and protects the heart against pathological remodeling. Recent work suggests exercise also enhances the heart's capacity for repair, which could be important for regenerative therapies. While microRNAs are important in certain cardiac pathologies, less is known about their functional roles in exercise-induced cardiac phenotypes. We profiled cardiac microRNA expression in two distinct models of exercise and found microRNA-222 (miR-222) was upregulated in both. Downstream miR-222 targets modulating cardiomyocyte phenotypes were identified, including HIPK1 and HMBOX1. Inhibition of miR-222 in vivo completely blocked cardiac and cardiomyocyte growth in response to exercise while reducing markers of cardiomyocyte proliferation. Importantly, mice with inducible cardiomyocyte miR-222 expression were resistant to adverse cardiac remodeling and dysfunction after ischemic injury. These studies implicate miR-222 as necessary for exercise-induced cardiomyocyte growth and proliferation in the adult mammalian heart and show that it is sufficient to protect the heart against adverse remodeling.

Abstract 238: miR-222 is Necessary for Exercise-induced Cardiac Growth and Protects Against Pathological Cardiac Remodeling

Exercise induces cardiac growth, protects against adverse remodeling, and may also induce a regenerative response. Since microRNAs (miRNA) play important roles in cardiovascular disease, we investigated their role in the cardiac exercise response. We used the TaqMan rodent miRNA array to profile cardiac miRNA expressed at three weeks in two exercise models (swimming, running) compared to sedentary controls. Sixteen concordantly regulated miRNAs were identified and validated in both models, and examined for functional effects in neonatal rat ventricular cardiomyocytes (CMs). miR-222 was upregulated ~two-fold in both models and increased CM size (22%, p<0.01) and proliferation markers (EdU and Ki67, p<0.01). Bioinformatic and expression analyses identified four potential miR-222 targets (p27, Hipk1, Hipk2, and Hmbox-1) in CMs. These were confirmed as direct targets by luciferase assays, site-directed mutagenesis, and immunoblotting. siRNA knockdown (KD) of p27 or Hipk1 induced neonatal CM proliferation, while siRNA KD of Hmbox-1 increased CM size. To examine miR-222’s role in vivo, LNA-antimiR-222 was injected via tail vein or subcutaneously and shown to reduce cardiac miR-222 levels to 1.5% (p<0.01). Untreated animals subjected to three weeks of swimming had the expected increase in heart size (15% in HW/TL, p<0.05), CM size (26%, p<0.05), and markers of CM proliferation (Ki67 and pHH3, p<0.05). Increases in heart and CM size were unaffected by control LNA-antimiR but completely blocked by LNA-antimiR-222, while CM proliferation markers decreased (60%, p<0.05). To see if miR-222 is sufficient to mediate the benefits of exercise, we made transgenic mice with cardiac-specific, regulated expression of miR-222 (Tg-miR-222). Tg-miR-222 have normal heart size and function at baseline. After ischemia-reperfusion injury (IRI), Tg-miR-222 had similar initial dysfunction but were protected against adverse remodeling over the next six weeks with better function (p<0.01), less cardiac fibrosis (68%, p<0.05), and increased CM proliferation markers (63%, p<0.05). Conclusion: Cardiac miR-222 is upregulated by exercise, necessary for exercise-induced cardiac growth, and protects against pathological cardiac remodeling after IRI.