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.

Localized cardiomyocyte lipid accumulation is associated with slowed epicardial conduction in rats.

Transmural action potential duration differences and transmural conduction gradients aid the synchronization of left ventricular repolarization, reducing vulnerability to transmural reentry and arrhythmias. A high-fat diet and the associated accumulation of pericardial adipose tissue are linked with conduction slowing and greater arrhythmia vulnerability. It is predicted that cardiac adiposity may more readily influence epicardial conduction (versus endocardial) and disrupt normal transmural activation/repolarization gradients. The aim of this investigation was to determine whether transmural conduction gradients are modified in a rat model of pericardial adiposity. Adult Sprague-Dawley rats were fed control/high-fat diets for 15 wk. Left ventricular 300 µm tangential slices were generated from the endocardium to the epicardium, and conduction was mapped using microelectrode arrays. Slices were then histologically processed to assess fibrosis and cardiomyocyte lipid status. Conduction velocity was significantly greater in epicardial versus endocardial slices in control rats, supporting the concept of a transmural conduction gradient. High-fat diet feeding increased pericardial adiposity and abolished the transmural conduction gradient. Slowed epicardial conduction in epicardial slices strongly correlated with an increase in cardiomyocyte lipid content, but not fibrosis. The positive transmural conduction gradient reported here represents a physiological property of the ventricular activation sequence that likely protects against reentry. The absence of this gradient, secondary to conduction slowing and cardiomyocyte lipid accumulation, specifically in the epicardium, indicates a novel mechanism by which pericardial adiposity may exacerbate ventricular arrhythmias.

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.

A novel small molecule inhibitor of human Drp1.

Mitochondrial dynamin-related protein 1 (Drp1) is a large GTPase regulator of mitochondrial dynamics and is known to play an important role in numerous pathophysiological processes. Despite being the most widely used Drp1 inhibitor, the specificity of Mdivi-1 towards human Drp1 has not been definitively proven and there have been numerous issues reported with its use including off-target effects. In our hands Mdivi-1 showed varying binding affinities toward human Drp1, potentially impacted by compound aggregation. Herein, we sought to identify a novel small molecule inhibitor of Drp1. From an initial virtual screening, we identified DRP1i27 as a compound which directly bound to the human isoform 3 of Drp1 via surface plasmon resonance and microscale thermophoresis. Importantly, DRP1i27 was found to have a dose-dependent increase in the cellular networks of fused mitochondria but had no effect in Drp1 knock-out cells. Further analogues of this compound were identified and screened, though none displayed greater affinity to human Drp1 isoform 3 than DRP1i27. To date, this is the first small molecule inhibitor shown to directly bind to human Drp1.

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.

Cardiac mechanical efficiency is preserved in primary cardiac hypertrophy despite impaired mechanical function.

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.

Elevated myocardial fructose and sorbitol levels are associated with diastolic dysfunction in diabetic patients, and cardiomyocyte lipid inclusions in vitro.

Diabetes is associated with cardiac metabolic disturbances and increased heart failure risk. Plasma fructose levels are elevated in diabetic patients. A direct role for fructose involvement in diabetic heart pathology has not been investigated. The goals of this study were to clinically evaluate links between myocardial fructose and sorbitol (a polyol pathway fructose precursor) levels with evidence of cardiac dysfunction, and to experimentally assess the cardiomyocyte mechanisms involved in mediating the metabolic effects of elevated fructose. Fructose and sorbitol levels were increased in right atrial appendage tissues of type 2 diabetic patients (2.8- and 1.5-fold increase respectively). Elevated cardiac fructose levels were confirmed in type 2 diabetic rats. Diastolic dysfunction (increased E/e', echocardiography) was significantly correlated with cardiac sorbitol levels. Elevated myocardial mRNA expression of the fructose-specific transporter, Glut5 (43% increase), and the key fructose-metabolizing enzyme, Fructokinase-A (50% increase) was observed in type 2 diabetic rats (Zucker diabetic fatty rat). In neonatal rat ventricular myocytes, fructose increased glycolytic capacity and cytosolic lipid inclusions (28% increase in lipid droplets/cell). This study provides the first evidence that elevated myocardial fructose and sorbitol are associated with diastolic dysfunction in diabetic patients. Experimental evidence suggests that fructose promotes the formation of cardiomyocyte cytosolic lipid inclusions, and may contribute to lipotoxicity in the diabetic heart.

Systematic quantification of histologic ventricular fibrosis in isolated mitral valve prolapse and sudden cardiac death.

BACKGROUND: Cardiac fibrosis in mitral valve prolapse (MVP) is implicated in the development of sudden cardiac death (SCD); however, the pattern remains poorly characterized. OBJECTIVE: The purpose of this study was to systematically quantify left and right ventricular fibrosis in individuals with isolated MVP and SCD (iMVP-SCD), whereby other potential causes of death are excluded, compared to a control cohort. METHODS: Individuals with iMVP-SCD were identified from the Victorian Institute of Forensic Medicine, Australia, and matched for age, sex, and body mass index to control cases with noncardiac death. Cardiac tissue sections were analyzed to determine collagen deposition in the left ventricular free wall (anterior, lateral, and posterior portions), interventricular septum, and right ventricle. Within the iMVP-SCD cases, the endocardial-to-epicardial distribution of fibrosis within the left ventricle was specifically characterized. RESULTS: Seventeen cases with iMVP-SCD were matched 1:1 with 17 controls, yielding 149 samples and 1788 histologic regions. The iMVP-SCD group had increased left ventricular (anterior, lateral, and posterior; all P <.001) and interventricular septum fibrosis (P <.001), but similar amounts of right ventricular fibrosis (P = .62) compared to controls. In iMVP-SCD, left ventricular fibrosis was significantly higher in the lateral and posterior walls compared to the anterior wall and interventricular septum (all P <.001). Within the lateral and posterior walls, iMVP-SCD cases had a significant endocardial-to-epicardial gradient of cardiac fibrosis (P <.01) similar to other known conditions that cause cardiac remodeling. CONCLUSION: Our study indicates that nonuniform left ventricular remodeling with both localized and generalized left ventricular fibrosis is important in the pathogenesis of SCD in individuals with MVP.

Epicardial Adipose Tissue Accumulation Confers Atrial Conduction Abnormality.

BACKGROUND: Clinical studies have reported that epicardial adipose tissue (EpAT) accumulation associates with the progression of atrial fibrillation (AF) pathology and adversely affects AF management. The role of local cardiac EpAT deposition in disease progression is unclear, and the electrophysiological, cellular, and molecular mechanisms involved remain poorly defined. OBJECTIVES: The purpose of this study was to identify the underlying mechanisms by which EpAT influences the atrial substrate for AF. METHODS: Patients without AF undergoing coronary artery bypass surgery were recruited. Computed tomography and high-density epicardial electrophysiological mapping of the anterior right atrium were utilized to quantify EpAT volumes and to assess association with the electrophysiological substrate in situ. Excised right atrial appendages were analyzed histologically to characterize EpAT infiltration, fibrosis, and gap junction localization. Co-culture experiments were used to evaluate the paracrine effects of EpAT on cardiomyocyte electrophysiology. Proteomic analyses were applied to identify molecular mediators of cellular electrophysiological disturbance. RESULTS: Higher local EpAT volume clinically correlated with slowed conduction, greater electrogram fractionation, increased fibrosis, and lateralization of cardiomyocyte connexin-40. In addition, atrial conduction heterogeneity was increased with more extensive myocardial EpAT infiltration. Cardiomyocyte culture studies using multielectrode arrays showed that cardiac adipose tissue-secreted factors slowed conduction velocity and contained proteins with capacity to disrupt intermyocyte electromechanical integrity. CONCLUSIONS: These findings indicate that atrial pathophysiology is critically dependent on local EpAT accumulation and infiltration. In addition to myocardial architecture disruption, this effect can be attributed to an EpAT-cardiomyocyte paracrine axis. The focal adhesion group proteins are identified as new disease candidates potentially contributing to arrhythmogenic atrial substrate.

New perspectives on the role of Drp1 isoforms in regulating mitochondrial pathophysiology.

Mitochondria are dynamic organelles constantly undergoing fusion and fission. A concerted balance between the process of mitochondrial fusion and fission is required to maintain cellular health under different physiological conditions. Mutation and dysregulation of Drp1, the major driver of mitochondrial fission, has been associated with various neurological, oncological and cardiovascular disorders. Moreover, when subjected to pathological insults, mitochondria often undergo excessive fission, generating fragmented and dysfunctional mitochondria leading to cell death. Therefore, manipulating mitochondrial fission by targeting Drp1 has been an appealing therapeutic approach for cytoprotection. However, studies have been inconsistent. Studies employing Drp1 constructs representing alternate Drp1 isoforms, have demonstrated differing impacts of these isoforms on mitochondrial fission and cell death. Furthermore, there are distinct expression patterns of Drp1 isoforms in different tissues, suggesting idiosyncratic engagement in specific cellular functions. In this review, we will discuss these inherent variations among human Drp1 isoforms and how they could affect Drp1-mediated mitochondrial fission and cell death.

Cardiomyocyte functional screening: interrogating comparative electrophysiology of high-throughput model cell systems.

Cardiac arrhythmias of both atrial and ventricular origin are an important feature of cardiovascular disease. Novel antiarrhythmic therapies are required to overcome current drug limitations related to effectiveness and pro-arrhythmia risk in some contexts. Cardiomyocyte culture models provide a high-throughput platform for screening antiarrhythmic compounds, but comparative information about electrophysiological properties of commonly used types of cardiomyocyte preparations is lacking. Standardization of cultured cardiomyocyte microelectrode array (MEA) experimentation is required for its application as a high-throughput platform for antiarrhythmic drug development. The aim of this study was to directly compare the electrophysiological properties and responses to isoproterenol of three commonly used cardiac cultures. Neonatal rat ventricular myocytes (NRVMs), immortalized atrial HL-1 cells, and custom-generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were cultured on microelectrode arrays for 48-120 h. Extracellular field potentials were recorded, and conduction velocity was mapped in the presence/absence of the ß-adrenoceptor agonist isoproterenol (1 µM). Field potential amplitude and conduction velocity were greatest in NRVMs and did not differ in cardiomyocytes isolated from male/female hearts. Both NRVMs and hiPSC-CMs exhibited longer field potential durations with rate dependence and were responsive to isoproterenol. In contrast, HL-1 cells exhibited slower conduction and shorter field potential durations and did not respond to 1 µM isoproterenol. This is the first study to compare the intrinsic electrophysiologic properties of cultured cardiomyocyte preparations commonly used for in vitro electrophysiology assessment. These findings offer important comparative data to inform methodological approaches in the use of MEA and other techniques relating to cardiomyocyte functional screening investigations of particular relevance to arrhythmogenesis.

Mitochondrial Assays Using Cardiac Stem Cells.

Ischemic heart disease is the leading cause of death worldwide. Stem cell therapy to repair and regenerate the infarcted myocardium is a promising approach to address this unmet medical need. However, the poor survival of transplanted cells in the hostile ischemic myocardium has been a major hurdle in achieving an effective cell therapy against myocardial infarction. As such, novel strategies to promote the survival of transplanted cells are highly sought after. Mitochondria are intimately involved in cell survival and have been the main organelles being targeted for cytoprotection. Mitochondrial morphology is linked to mitochondrial function and cell viability. Therefore, quantitative methodologies to obtain reliable and reproducible results of mitochondrial morphology and function are essential for identifying and developing new cytoprotective strategies to enhance the survival of stem cells post-transplantation. Here, we describe methods for assessing mitochondrial morphology, mitochondrial membrane potential, and mitochondrial reactive oxygen species production.

Impact of Estrogens on the Regulation of White, Beige, and Brown Adipose Tissue Depots.

As adipose tissue depots are active endocrine organs, they secrete a variety of hormones (including estrogens from white adipose) and inflammatory mediators, which have important implications in numerous obesity-associated diseases. Adipose tissues are broadly characterized as consisting of white, beige, and brown depot types. The endocrine, metabolic, and inflammatory profiles of adipose are depot dependent and influenced by the estrogenic and androgenic status of the adipose tissue. Estrogen receptors mediate both the genomic and nongenomic actions of estrogens and are expressed in the brain, heart, and other peripheral tissues. All three known estrogen receptor α (ERα) and estrogen receptor ß (ERß), and the G-protein coupled estrogen receptor (GPER/GPR30) are expressed in white adipose and can modulate adipose mass. Expression of each receptor is dependent on depot location, adipose cell type, and estrogen levels. Estrogen receptor expression profiles in beige and brown adipocytes are less well established. This review will discuss the effects of estrogens on the differential deposition of the major adipose tissues and the impact of estrogens within white adipose depots. © 2019 American Physiological Society. Compr Physiol 9:457-475, 2019.