Male and female atria exhibit distinct acute electrophysiological responses to sex steroids.

The electrophysiological properties of the hearts of women and men are different. These differences are at least partly mediated by the actions of circulating estrogens and androgens on the cardiomyocytes. Experimentally, much of our understanding in this field is based on studies focusing on ventricular tissue, with considerably less known in the context of atrial electrophysiology. The aim of this investigation was to compare the electrophysiological properties of male and female atria and assess responses to acute sex steroid exposure. Age-matched adult male and female C57BL/6 mice were anesthetized (4 % isoflurane) and left atria isolated. Atria were loaded with Di-4-ANEPPS voltage sensitive dye and optical mapping performed to assess action potential duration (APD; at 10 %, 20 %, 30 %, 50 %, and 70 % repolarization) and conduction velocity in the presence of 1 nM and 100 nM 17ß-estradiol or testosterone. Male and female left atria demonstrated similar baseline action potential duration and conduction velocity, with significantly greater APD70 spatial heterogeneity evident in females. 17ß-estradiol prolonged action potential duration in both sexes - an effect that was augmented in females. Atrial conduction was slowed in the presence of 100 nM 17ß-estradiol in both males and females. Testosterone prolonged action potential duration in males only and did not modulate conduction velocity in either sex. This study provides novel insights into male and female atrial electrophysiology and its regulation by sex steroids. As systemic sex steroid levels change and intra-cardiac estrogen synthesis capacity increases with aging, these actions may have an increasingly important role in determining atrial arrhythmia vulnerability.

Methods for detection of cardiac glycogen-autophagy.

Glycogen-autophagy ('glycophagy') is a selective autophagy process involved in delivering glycogen to the lysosome for bulk degradation. Glycophagy protein intermediaries include STBD1 as a glycogen tagging receptor, delivering the glycogen cargo into the forming phagosome by partnering with the Atg8 homolog, GABARAPL1. Glycophagy is emerging as a key process of energy metabolism and development of reliable tools for assessment of glycophagy activity is an important priority. Here we show that antibodies raised against the N-terminus of the GABARAPL1 protein (but not the full-length protein) detected a specific endogenous GABARAPL1 immunoblot band at 18kDa. A stable GFP-GABARAPL1 cardiac cell line was used to quantify GABARAPL1 lysosomal flux via measurement of GFP puncta in response to lysosomal inhibition with bafilomycin. Endogenous glycophagy flux was quantified in primary rat ventricular myocytes by the extent of glycogen accumulation with bafilomycin combined with chloroquine treatment (no effect observed with bafilomycin or chloroquine alone). In wild-type isolated mouse hearts, bafilomycin alone and bafilomycin combined with chloroquine (but not chloroquine alone) elicited a significant increase in glycogen content signifying basal glycophagy flux. Collectively, these methodologies provide a comprehensive toolbox for tracking cardiac glycophagy activity to advance research into the role of glycophagy in health and disease.

Distinct functional and molecular profiles between physiological and pathological atrial enlargement offer potential new therapeutic opportunities for atrial fibrillation.

Atrial fibrillation (AF) remains challenging to prevent and treat. A key feature of AF is atrial enlargement. However, not all atrial enlargement progresses to AF. Atrial enlargement in response to physiological stimuli such as exercise is typically benign and reversible. Understanding the differences in atrial function and molecular profile underpinning pathological and physiological atrial remodelling will be critical for identifying new strategies for AF. The discovery of molecular mechanisms responsible for pathological and physiological ventricular hypertrophy has uncovered new drug targets for heart failure. Studies in the atria have been limited in comparison. Here, we characterised mouse atria from (1) a pathological model (cardiomyocyte-specific transgenic (Tg) that develops dilated cardiomyopathy [DCM] and AF due to reduced protective signalling [PI3K]; DCM-dnPI3K), and (2) a physiological model (cardiomyocyte-specific Tg with an enlarged heart due to increased insulin-like growth factor 1 receptor; IGF1R). Both models presented with an increase in atrial mass, but displayed distinct functional, cellular, histological and molecular phenotypes. Atrial enlargement in the DCM-dnPI3K Tg, but not IGF1R Tg, was associated with atrial dysfunction, fibrosis and a heart failure gene expression pattern. Atrial proteomics identified protein networks related to cardiac contractility, sarcomere assembly, metabolism, mitochondria, and extracellular matrix which were differentially regulated in the models; many co-identified in atrial proteomics data sets from human AF. In summary, physiological and pathological atrial enlargement are associated with distinct features, and the proteomic dataset provides a resource to study potential new regulators of atrial biology and function, drug targets and biomarkers for AF.

Mechanical loading reveals an intrinsic cardiomyocyte stiffness contribution to diastolic dysfunction in murine cardiometabolic disease.

Cardiometabolic syndromes including diabetes and obesity are associated with occurrence of heart failure with diastolic dysfunction. There are no specific treatments for diastolic dysfunction and therapies to manage symptoms have limited efficacy. Understanding of the cardiomyocyte origins of diastolic dysfunction is an important priority to identify new therapeutics. The investigative goal was to experimentally define in vitro stiffness (stress/strain) properties of isolated cardiomyocytes derived from rodent hearts exhibiting diastolic dysfunction in vivo in response to dietary induction of cardiometabolic disease. Mice fed a High Fat/Sugar Diet (HFSD vs control) for at least 25 weeks exhibited glucose intolerance, obesity and diastolic dysfunction (echo E/e'). Intact paced cardiomyocytes were functionally investigated in three conditions: non-loaded, loaded and stretched. Mean stiffness of HFSD cardiomyocytes was 70% higher than control. The E/e' doppler ratio for the origin hearts was elevated by 35%. A significant relationship was identified between in vitro cardiomyocyte stiffness and in vivo dysfunction severity. With conversion from non-loaded to loaded condition, the decrement in maximal sarcomere lengthening rate was more accentuated in HFSD cardiomyocytes (vs control). With stretch, the Ca 2+ transient decay time course was prolonged. With transition from 2-4Hz pacing, HFSD cardiomyocyte stiffness was further increased, yet diastolic Ca 2+ rise was 50% less than control. Collectively, these findings demonstrate that a component of cardiac diastolic dysfunction in cardiometabolic disease is derived from intrinsic cardiomyocyte mechanical abnormality. Differential responses to load, stretch and pacing suggest that a previously undescribed alteration in myofilament-Ca 2+ interaction contributes to cardiomyocyte stiffness in cardiometabolic disease. KEY POINTS: Understanding cardiomyocyte stiffness components is an important priority for identifying new therapeutics for diastolic dysfunction, a key feature of cardiometabolic disease. In this study cardiac function was measured in vivo (echocardiography) for mice fed a high-fat/sugar diet (HFSD, ≥25weeks) and performance of intact isolated cardiomyocytes derived from the same hearts was measured during pacing under non-loaded, loaded and stretched conditions in vitro . Using a calibrated cardiomyocyte stretch protocol, stiffness (stress/strain) was elevated in HFSD cardiomyocytes in vitro and correlated with diastolic dysfunction (E/e') in vivo . The HFSD cardiomyocyte Ca 2+ transient decay was prolonged in response to stretch, and stiffness was accentuated in response to pacing increase while the rise in diastolic Ca 2+ was attenuated. These findings suggest that stretch-dependent augmentation of the myofilament-Ca 2+ response during diastole partially underlies elevated cardiomyocyte stiffness and diastolic dysfunction of hearts of animals with cardiometabolic disease.

Myocardial glycophagy flux dysregulation and glycogen accumulation characterize diabetic cardiomyopathy.

Diabetic heart disease morbidity and mortality is escalating. No specific therapeutics exist and mechanistic understanding of diabetic cardiomyopathy etiology is lacking. While lipid accumulation is a recognized cardiomyocyte phenotype of diabetes, less is known about glycolytic fuel handling and storage. Based on in vitro studies, we postulated the operation of an autophagy pathway in the myocardium specific for glycogen homeostasis - glycophagy. Here we visualize occurrence of cardiac glycophagy and show that the diabetic myocardium is characterized by marked glycogen elevation and altered cardiomyocyte glycogen localization. We establish that cardiac glycophagy flux is disturbed in diabetes. Glycophagy may represent a potential therapeutic target for alleviating the myocardial impacts of metabolic disruption in diabetic heart disease.

The cardiomyocyte origins of diastolic dysfunction: cellular components of myocardial "stiffness".

The impaired ability of the heart to relax and stretch to accommodate venous return is generally understood to represent a state of "diastolic dysfunction" and often described using the all-purpose noun "stiffness." Despite the now common qualitative usage of this term in fields of cardiac patho/physiology, the specific quantitative concept of stiffness as a molecular and biophysical entity with real practical interpretation in healthy and diseased hearts is sometimes obscure. The focus of this review is to characterize the concept of cardiomyocyte stiffness and to develop interpretation of "stiffness" attributes at the cellular and molecular levels. Here, we consider "stiffness"-related terminology interpretation and make links between cardiomyocyte stiffness and aspects of functional and structural cardiac performance. We discuss cross bridge-derived stiffness sources, considering the contributions of diastolic myofilament activation and impaired relaxation. This includes commentary relating to the role of cardiomyocyte Ca2+ flux and Ca2+ levels in diastole, the troponin-tropomyosin complex role as a Ca2+ effector in diastole, the myosin ADP dissociation rate as a modulator of cross bridge attachment and regulation of cross-bridge attachment by myosin binding protein C. We also discuss non-cross bridge-derived stiffness sources, including the titin sarcomeric spring protein, microtubule and intermediate filaments, and cytoskeletal extracellular matrix interactions. As the prevalence of conditions involving diastolic heart failure has escalated, a more sophisticated understanding of the molecular, cellular, and tissue determinants of cardiomyocyte stiffness offers potential to develop imaging and molecular intervention tools.

A gene therapy targeting medium-chain acyl-CoA dehydrogenase (MCAD) did not protect against diabetes-induced cardiac pathology.

Diabetic cardiomyopathy describes heart disease in patients with diabetes who have no other cardiac conditions but have a higher risk of developing heart failure. Specific therapies to treat the diabetic heart are limited. A key mechanism involved in the progression of diabetic cardiomyopathy is dysregulation of cardiac energy metabolism. The aim of this study was to determine if increasing the expression of medium-chain acyl-coenzyme A dehydrogenase (MCAD; encoded by Acadm), a key regulator of fatty acid oxidation, could improve the function of the diabetic heart. Male mice were administered streptozotocin to induce diabetes, which led to diastolic dysfunction 8 weeks post-injection. Mice then received cardiac-selective adeno-associated viral vectors encoding MCAD (rAAV6:MCAD) or control AAV and were followed for 8 weeks. In the non-diabetic heart, rAAV6:MCAD increased MCAD expression (mRNA and protein) and increased Acadl and Acadvl, but an increase in MCAD enzyme activity was not detectable. rAAV6:MCAD delivery in the diabetic heart increased MCAD mRNA expression but did not significantly increase protein, activity, or improve diabetes-induced cardiac pathology or molecular metabolic and lipid markers. The uptake of AAV viral vectors was reduced in the diabetic versus non-diabetic heart, which may have implications for the translation of AAV therapies into the clinic. KEY MESSAGES: The effects of increasing MCAD in the diabetic heart are unknown. Delivery of rAAV6:MCAD increased MCAD mRNA and protein, but not enzyme activity, in the non-diabetic heart. Independent of MCAD enzyme activity, rAAV6:MCAD increased Acadl and Acadvl in the non-diabetic heart. Increasing MCAD cardiac gene expression alone was not sufficient to protect against diabetes-induced cardiac pathology. AAV transduction efficiency was reduced in the diabetic heart, which has clinical implications.

Regulation of mitochondrial calcium uniporter expression and calcium-dependent cell signaling by lncRNA Tug1 in cardiomyocytes.

Cardiomyocyte calcium homeostasis is a tightly regulated process. The mitochondrial calcium uniporter (MCU) complex can buffer elevated cytosolic Ca2+ levels and consists of pore-forming proteins including MCU, and various regulatory proteins such as mitochondrial calcium uptake proteins 1 and 2 (MICU1/2). The stoichiometry of these proteins influences the sensitivity to Ca2+ and the activity of the complex. However, the factors that regulate their gene expression remain incompletely understood. Long noncoding RNAs (lncRNAs) regulate gene expression through various mechanisms, and we recently found that the lncRNA Tug1 increased the expression of Mcu and associated genes. To further explore this, we performed antisense LNA knockdown of Tug1 (Tug1 KD) in H9c2 rat cardiomyocytes. Tug1 KD increased MCU protein expression, yet pyruvate dehydrogenase dephosphorylation, which is indicative of mitochondrial Ca2+ uptake, was not enhanced. However, RNA-seq revealed that Tug1 KD increased Mcu along with differential expression of >1,000 genes including many related to Ca2+ regulation pathways in the heart. To understand the effect of this on Ca2+ signaling, we measured phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and its downstream target cAMP Response Element-Binding protein (CREB), a transcription factor known to drive Mcu gene expression. In response to a Ca2+ stimulus, the increase in CaMKII and CREB phosphorylation was attenuated by Tug1 KD. Inhibition of CaMKII, but not CREB, partially prevented the Tug1 KD-mediated increase in Mcu. Together, these data suggest that Tug1 modulates MCU expression via a mechanism involving CaMKII and regulates cardiomyocyte Ca2+ signaling, which could have important implications for cardiac function.NEW & NOTEWORTHY Calcium is essential for signaling, excitation contraction, and energy homeostasis in the heart. Despite this, molecular regulators of these processes are not completely understood. We report that knockdown of lncRNA Tug1 alters the calcium handling transcriptome and increases mitochondrial calcium uniporter expression via a mechanism involving CaMKII. As overexpression of MCU is known to be protective against pathological cardiac remodeling, targeting Tug1 may be a potential strategy for treating cardiovascular disease.

Cellular Mechanisms Mediating Exercise-Induced Protection against Cardiotoxic Anthracycline Cancer Therapy.

Anthracyclines such as doxorubicin are widely used chemotherapy drugs. A common side effect of anthracycline therapy is cardiotoxicity, which can compromise heart function and lead to dilated cardiomyopathy and heart failure. Dexrazoxane and heart failure medications (i.e., beta blockers and drugs targeting the renin-angiotensin system) are prescribed for the primary prevention of cancer therapy-related cardiotoxicity and for the management of cardiac dysfunction and symptoms if they arise during chemotherapy. However, there is a clear need for new therapies to combat the cardiotoxic effects of cancer drugs. Exercise is a cardioprotective stimulus that has recently been shown to improve heart function and prevent functional disability in breast cancer patients undergoing anthracycline chemotherapy. Evidence from preclinical studies supports the use of exercise training to prevent or attenuate the damaging effects of anthracyclines on the cardiovascular system. In this review, we summarise findings from experimental models which provide insight into cellular mechanisms by which exercise may protect the heart from anthracycline-mediated damage, and identify knowledge gaps that require further investigation. Improved understanding of the mechanisms by which exercise protects the heart from anthracyclines may lead to the development of novel therapies to treat cancer therapy-related cardiotoxicity.

Estrogen receptor alpha deficiency in cardiomyocytes reprograms the heart-derived extracellular vesicle proteome and induces obesity in female mice.

Dysregulation of estrogen receptor alpha (ERα) has been linked with increased metabolic and cardiovascular disease risk. Here, we generate and characterize cardiomyocyte-specific ERα knockout (ERαHKO) mice to assess the role of ERα in the heart. The most striking phenotype was obesity in female ERαHKO but not male ERαHKO mice. Female ERαHKO mice showed cardiac dysfunction, mild glucose and insulin intolerance and reduced ERα gene expression in skeletal muscle and white adipose tissue. Transcriptomic, proteomic, lipidomic and metabolomic analyses revealed evidence of contractile and/or metabolic dysregulation in heart, skeletal muscle and white adipose tissue. We show that heart-derived extracellular vesicles from female ERαHKO mice contain a distinct proteome associated with lipid and metabolic regulation, and have the capacity to metabolically reprogram the target skeletal myocyte proteome with functional impacts on glycolytic capacity and reserve. This multi-omics study uncovers a cardiac-initiated and sex-specific cardiometabolic phenotype regulated by ERα and provides insights into extracellular vesicle-mediated interorgan communication.

New Perspectives on Sex Steroid and Mineralocorticoid Receptor Signaling in Cardiac Ischemic Injury.

The global burden of ischemic heart disease is burgeoning for both men and women. Although advances have been made, the need for new sex-specific therapies targeting key differences in cardiovascular disease outcomes in men and women remains. Mineralocorticoid receptor directed treatments have been successfully used for blood pressure control and heart failure management and represent a potentially valuable therapeutic option for ischemic cardiac events. Clinical and experimental data indicate that mineralocorticoid excess or inappropriate mineralocorticoid receptor (MR) activation exacerbates ischemic damage, and many of the intracellular response pathways activated in ischemia and subsequent reperfusion are regulated by MR. In experimental contexts, where MR are abrogated genetically or mineralocorticoid signaling is suppressed pharmacologically, ischemic injury is alleviated, and reperfusion recovery is enhanced. In the chronic setting, mineralocorticoid signaling induces fibrosis, oxidative stress, and inflammation, which can predispose to ischemic events and exacerbate post-myocardial infarct pathologies. Whilst a range of cardiac cell types are involved in mineralocorticoid-mediated regulation of cardiac function, cardiomyocyte-specific MR signaling pathways are key. Selective inhibition of cardiomyocyte MR signaling improves electromechanical resilience during ischemia and enhances contractile recovery in reperfusion. Emerging evidence suggests that the MR also contribute to sex-specific aspects of ischemic vulnerability. Indeed, MR interactions with sex steroid receptors may differentially regulate myocardial nitric oxide bioavailability in males and females, potentially determining sex-specific post-ischemic outcomes. There is hence considerable impetus for exploration of MR directed, cell specific therapies for both women and men in order to improve ischemic heart disease outcomes.

Glycogen-autophagy: Molecular machinery and cellular mechanisms of glycophagy.

Autophagy is an essential cellular process involving degradation of superfluous or defective macromolecules and organelles as a form of homeostatic recycling. Initially proposed to be a "bulk" degradation pathway, a more nuanced appreciation of selective autophagy pathways has developed in the literature in recent years. As a glycogen-selective autophagy process, "glycophagy" is emerging as a key metabolic route of transport and delivery of glycolytic fuel substrate. Study of glycophagy is at an early stage. Enhanced understanding of this major noncanonical pathway of glycogen flux will provide important opportunities for new insights into cellular energy metabolism. In addition, glycogen metabolic mishandling is centrally involved in the pathophysiology of several metabolic diseases in a wide range of tissues, including the liver, skeletal muscle, cardiac muscle, and brain. Thus, advances in this exciting new field are of broad multidisciplinary interest relevant to many cell types and metabolic states. Here, we review the current evidence of glycophagy involvement in homeostatic cellular metabolic processes and of molecular mediators participating in glycophagy flux. We integrate information from a variety of settings including cell lines, primary cell culture systems, ex vivo tissue preparations, genetic disease models, and clinical glycogen disease states.

Protein phosphatase 2A in the healthy and failing heart: New insights and therapeutic opportunities.

Protein phosphatases have emerged as critical regulators of phosphoprotein homeostasis in settings of health and disease. Protein phosphatase 2A (PP2A) encompasses a large subfamily of enzymes that remove phosphate groups from serine/threonine residues within phosphoproteins. The heterogeneity in PP2A structure, which arises from the grouping of different catalytic, scaffolding and regulatory subunit isoforms, creates distinct populations of catalytically active enzymes (i.e. holoenzymes) that localise to different parts of the cell. This structural complexity, combined with other regulatory mechanisms, such as interaction of PP2A heterotrimers with accessory proteins and post-translational modification of the catalytic and/or regulatory subunits, enables PP2A holoenzymes to target phosphoprotein substrates in a highly specific manner. In this review, we summarise the roles of PP2A in cardiac physiology and disease. PP2A modulates numerous processes that are vital for heart function including calcium handling, contractility, ß-adrenergic signalling, metabolism and transcription. Dysregulation of PP2A has been observed in human cardiac disease settings, including heart failure and atrial fibrillation. Efforts are underway, particularly in the cancer field, to develop therapeutics targeting PP2A activity. The development of small molecule activators of PP2A (SMAPs) and other compounds that selectively target specific PP2A holoenzymes (e.g. PP2A/B56α and PP2A/B56ε) will improve understanding of the function of different PP2A species in the heart, and may lead to the development of therapeutics for normalising aberrant protein phosphorylation in settings of cardiac remodelling and dysfunction.

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay.

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to >10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in >1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A protocol for rapid and parallel isolation of myocytes and non-myocytes from multiple mouse hearts.

This protocol features parallel isolation of myocytes and non-myocytes from murine hearts. It was designed with considerations for (1) time required to extract cardiac cells, (2) cell viability, and (3) protocol scalability. Here, a peristaltic pump and 3D-printed elements are combined to perfuse the heart with enzymes to dissociate cells. Myocytes and non-myocytes extracted using this protocol are separated by centrifugation and/or fluorescence-activated cell sorting for use in downstream applications including single-cell omics or other bio-molecular analyses. For complete details on the use and execution of this protocol, please refer to McLellan et al. (2020).

FoxO1 is required for physiological cardiac hypertrophy induced by exercise but not by constitutively active PI3K.

The insulin-like growth factor 1 receptor (IGF1R) and phosphoinositide 3-kinase p110α (PI3K) are critical regulators of exercise-induced physiological cardiac hypertrophy and provide protection in experimental models of pathological remodeling and heart failure. Forkhead box class O1 (FoxO1) is a transcription factor that regulates cardiomyocyte hypertrophy downstream of IGF1R/PI3K activation in vitro, but its role in physiological hypertrophy in vivo was unknown. We generated cardiomyocyte-specific FoxO1 knockout (cKO) mice and assessed the phenotype under basal conditions and settings of physiological hypertrophy induced by 1) swim training or 2) cardiac-specific transgenic expression of constitutively active PI3K (caPI3KTg+). Under basal conditions, male and female cKO mice displayed mild interstitial fibrosis compared with control (CON) littermates, but no other signs of cardiac pathology were present. In response to exercise training, female CON mice displayed an increase (∼21%) in heart weight normalized to tibia length vs. untrained mice. Exercise-induced hypertrophy was blunted in cKO mice. Exercise increased cardiac Akt phosphorylation and IGF1R expression but was comparable between genotypes. However, differences in Foxo3a, Hsp70, and autophagy markers were identified in hearts of exercised cKO mice. Deletion of FoxO1 did not reduce cardiac hypertrophy in male or female caPI3KTg+ mice. Cardiac Akt and FoxO1 protein expressions were significantly reduced in hearts of caPI3KTg+ mice, which may represent a negative feedback mechanism from chronic caPI3K, and negate any further effect of reducing FoxO1 in the cKO. In summary, FoxO1 contributes to exercise-induced hypertrophy. This has important implications when one is considering FoxO1 as a target for treating the diseased heart.NEW & NOTEWORTHY Regulators of exercise-induced physiological cardiac hypertrophy and protection are considered promising targets for the treatment of heart failure. Unlike pathological hypertrophy, the transcriptional regulation of physiological hypertrophy has remained largely elusive. To our knowledge, this is the first study to show that the transcription factor FoxO1 is a critical mediator of exercise-induced cardiac hypertrophy. Given that exercise-induced hypertrophy is protective, this finding has important implications when one is considering FoxO1 as a target for treating the diseased heart.