Catecholamine Stimulation Research Articles

Ablation of three major phospho-sites in RyR2 preserves the global adrenergic response but creates an arrhythmogenic substrate.

Ryanodine receptor 2 (RyR2) is one of the first substrates undergoing phosphorylation upon catecholaminergic stimulation. Yet, the role of RyR2 phosphorylation in the adrenergic response remains debated. To date, three residues in RyR2 are known to undergo phosphorylation upon adrenergic stimulation. We generated a model of RyR2 phospho-ablation of all three canonical phospho-sites (RyR2-S2031A/S2808A/S2814A, triple phospho-mutant, TPM) to elucidate the role of phosphorylation at these residues in the adrenergic response. Cardiac structure and function, cellular Ca 2+ dynamics and electrophysiology, and RyR2 channel activity both under basal conditions and under isoproterenol (Iso) stimulation were systematically evaluated. We used echocardiography and electrocardiography in anesthetized mice, single-cell Ca 2+ imaging and whole-cell patch clamp in isolated adult cardiomyocytes, and biochemical assays. Iso stimulation produced normal chronotropic and inotropic responses in TPM mice as well as an increase in the global Ca 2+ transients in isolated cardiomyocytes. Functional studies revealed fewer Ca 2+ sparks in permeabilized TPM myocytes, and reduced RyR2-mediated Ca 2+ leak in intact myocytes under Iso stimulation, suggesting that the canonical sites may regulate RyR2-mediated Ca 2+ leak. TPM mice also displayed increased propensity for arrhythmia. TPM myocytes were prone to develop early afterdepolarizations (EADs), which were abolished by chelating intracellular Ca 2+ with EGTA, indicating that EADs require SR Ca 2+ release. EADs were also blocked by a low concentration of tetrodotoxin, further suggesting reactivation of the sodium current ( I Na ) as the underlying cause. Phosphorylation of the three canonical residues on RyR2 may not be essential for the global adrenergic responses. However, these sites play a vital role in maintaining electrical stability during catecholamine stimulation by fine-tuning RyR2-mediated Ca 2+ leak. These findings underscore the importance of RyR2 phosphorylation and a finite diastolic Ca 2+ leak in maintaining electrical stability during catecholamine stimulation.

Sex- and age-dependent susceptibility to ventricular arrhythmias in the rat heart ex vivo

The incidence of life-threatening ventricular arrhythmias, the most common cause of sudden cardiac death (SCD), depends largely on the arrhythmic substrate that develops in the myocardium during the aging process. There is a large deficit of comparative studies on the development of this substrate in both sexes, with a particular paucity of studies in females. To identify the substrates of arrhythmia, fibrosis, cardiomyocyte hypertrophy, mitochondrial density, oxidative stress, antioxidant defense and intracellular Ca2+ signaling in isolated cardiomyocytes were measured in the hearts of 3- and 24-month-old female and male rats. Arrhythmia susceptibility was assessed in ex vivo perfused hearts after exposure to isoproterenol (ISO) and hydrogen peroxide (H2O2). The number of ventricular premature beats (PVBs), ventricular tachycardia (VT) and ventricular fibrillation (VF) episodes, as well as intrinsic heart rate, QRS and QT duration, were measured in ECG signals recorded from the surfaces of the beating hearts. After ISO administration, VT/VFs were formed only in the hearts of males, mainly older ones. In contrast, H2O2 led to VT/VF formation in the hearts of rats of both sexes but much more frequently in older males. We identified several components of the arrhythmia substrate that develop in the myocardium during the aging process, including high spontaneous ryanodine receptor activity in cardiomyocytes, fibrosis of varying severity in different layers of the myocardium (nonheterogenic fibrosis), and high levels of oxidative stress as measured by nitrated tyrosine levels. All of these elements appeared at a much greater intensity in male individuals during the aging process. On the other hand, in aging females, antioxidant defense at the level of H2O2 detoxification, measured as glutathione peroxidase expression, was weaker than that in males of the same age. We showed that sex has a significant effect on the development of an arrhythmic substrate during aging. This substrate determines the incidence of life-threatening ventricular arrhythmias in the presence of additional stimuli with proarrhythmic potential, such as catecholamine stimulation or oxidative stress, which are constant elements in the pathomechanism of most cardiovascular diseases.

Effect of catecholamines stimulation via hand cooling and the priming effect on the oxygen consumption kinetics

Introduction
 Priming effect has been shown to accelerate the oxygen consumption (V̇O2) kinetics. This phenomenon could be explained by muscle oxygen (O2) enhanced delivery, motor unit recruitment or intracellular O2 enhanced utilization (Goulding et al. 2023). Catecholamines such as epinephrine (E) and norepinephrine (NE) are known to impact the cardiovascular system as well as the respiratory system and metabolic effects (Zouhal et al. 2008). The aim of this study is two folds. Observing the effect of increased E and NE induced by cold-water hand immersion on V̇O2 kinetic during heavy intensity exercise bout (HCWR). Compare the effect of cold-water hand immersion and previous HCWR on V̇O2 kinetic to see if the sympathetic system could partially explain the priming effects.
 Methods
 Ten healthy active males (mean ± SD, age 23 ± 5 years, body mass 71 ± 6.5 kg) participated in this study. An incremental test was performed on ergometer to prescribe the intensity for the HCWR exercise (∆ 25%). Participants did an 8 min HCWR normal bout (NO), followed by 8 min rest and a second bout (SB) and hand cooling bout (HC) at heavy intensity in a randomized order. For the HC condition, participants put hands in cold water (0°C) for 2 min then directly did the HCWR. Blood samples has been taken at rest, before and directly after the HCWR.
 Results
 Just before exercise, all conditions were different, but HC E (1.095 ± 0.64 nmol/ml) increased by 347% while HC NE (6.98 ± 1.89 nmol/ml) matched values from SB NE (6.89 ± 2.22 nmol/ml). At the end of exercise, no significant difference has been found between NO E (1.44 ± 1.06) and HC E (1.54 ± 1.38 nmol/ml) while SB E (2.35 ± 1.55 nmol/ml) was significantly higher than both conditions but no differences have been found between HC (20.15 ± 8.76 nmol/ml) and SB (20.41 ± 9.74 nmol/ml) NE. V̇O2 kinetic has been sped up for both HC and SB by increasing the absolute primary prime amplitude of HC (33.88 ± 0.96 ml/min/kg),SB (34.60 ± 0.86ml/min/kg) compared to NO (32.75 ± 0.65 ml/min/kg) and reducing the prime amplitude of HC (3.22 ± 0.51 ml/min/kg), SB (2.90 ± 0.64 ml/min/kg) vs NO (3.87 ±0 .56 ml/min/kg) of the slow component of V̇O2.
 Discussion/Conclusion
 Catecholamines stimulation via hand cooling accelerated the V̇O2 kinetics enhancing the primary phase and reducing the slow component. This effect seemed to follow the priming effect but with less intensity. Increased O2 delivery appeared to be a common feature of both conditions, Motor neuron excitability due to increased NE was also a likely hypothesis, as both HC and SB had the same values just before exercise. Finally, intra-muscular enhancement illustrated by improved intracellular partial pressure kinetics and mitochondrial O2 utilization might explain the effects of catecholamines and part of the priming effect. Mitochondrial “warm-up” could be influenced by both catecholamines and priming effect resulting in a better production of adenosine triphosphate.
 References
 Goulding, R. P., Burnley, M., & Wüst, R. C. I. (2023). How priming exercise affects oxygen uptake kinetics: From underpinning mechanisms to endurance performance. Sports Medicine, 53, 959-976. https://doi.org/10.1007/s40279-023-01832-1
 Zouhal, H., Jacob, C., Delamarche, P., & Gratas-Delamarche, A. (2008). Catecholamines and the effects of exercise, training and gender. Sports Medicine 38, 401-423. https://doi.org/10.2165/00007256-200838050-00004

Cardiac monoamine oxidase-A inhibition protects against catecholamine-induced ventricular arrhythmias via enhanced diastolic calcium control.

A mechanistic link between depression and risk of arrhythmias could be attributed to altered catecholamine metabolism in the heart. Monoamine oxidase-A (MAO-A), a key enzyme involved in catecholamine metabolism and longstanding antidepressant target, is highly expressed in the myocardium. The present study aimed to elucidate the functional significance and underlying mechanisms of cardiac MAO-A in arrhythmogenesis. Analysis of the TriNetX database revealed that depressed patients treated with MAO inhibitors had a lower risk of arrhythmias compared with those treated with selective serotonin reuptake inhibitors. This effect was phenocopied in mice with cardiomyocyte-specific MAO-A deficiency (cMAO-Adef), which showed a significant reduction in both incidence and duration of catecholamine stress-induced ventricular tachycardia compared with wild-type mice. Additionally, cMAO-Adef cardiomyocytes exhibited altered Ca2+ handling under catecholamine stimulation, with increased diastolic Ca2+ reuptake, reduced diastolic Ca2+ leak, and diminished systolic Ca2+ release. Mechanistically, cMAO-Adef hearts had reduced catecholamine levels under sympathetic stress, along with reduced levels of reactive oxygen species and protein carbonylation, leading to decreased oxidation of Type II PKA and CaMKII. These changes potentiated phospholamban (PLB) phosphorylation, thereby enhancing diastolic Ca2+ reuptake, while reducing ryanodine receptor 2 (RyR2) phosphorylation to decrease diastolic Ca2+ leak. Consequently, cMAO-Adef hearts exhibited lower diastolic Ca2+ levels and fewer arrhythmogenic Ca2+ waves during sympathetic overstimulation. Cardiac MAO-A inhibition exerts an anti-arrhythmic effect by enhancing diastolic Ca2+ handling under catecholamine stress.

Ferroptosis contributes to catecholamine-induced cardiotoxicity and pathological remodeling

High levels of circulating catecholamines cause cardiac injury, pathological remodeling, and heart failure, but the underlying mechanisms remain elusive. Here we provide both in vitro and in vivo evidence that excessive β-adrenergic stimulation induces ferroptosis in cardiomyocytes, revealing a novel mechanism for catecholamine-induced cardiotoxicity and remodeling. We found that isoproterenol, a synthetic catecholamine, promoted glutathione depletion and glutathione peroxidase 4 (GPX4) degradation in cardiomyocytes, leading to GPX4 inactivation and enhanced lipid peroxidation. Isoproterenol also promoted heme oxygenase 1 (HO-1) expression by downregulating the transcription suppressor BTB and CNC homology 1 (Bach1), leading to increased labile iron accumulation through heme degradation. Moreover, isoproterenol markedly induced the accumulation of free iron and lipid reactive oxygen species (ROS) in the mitochondria, while targeted inhibition of iron overload and ROS accumulation within mitochondria effectively inhibited ferroptosis in cardiomyocytes. Importantly, isoproterenol administration markedly induced ferroptosis in the myocardium in vivo, associated with elevated non-heme iron accumulation driven by HO-1 upregulation. Strikingly, blockade of ferroptosis with ferrostatin-1 or inhibition of HO-1 activity with zinc protoporphyrin (ZnPP) effectively alleviated cardiac necrosis, pathological remodeling, and heart failure induced by isoproterenol administration. Taken together, our results reveal that catecholamine stimulation primarily induces ferroptotic cell death in cardiomyocyte through GPX4 and Bach1-HO-1 dependent signaling pathways. Targeting ferroptosis may represent a novel therapeutic strategy for catecholamine overload-induced myocardial injury and heart failure.

MiR-150 blunts cardiac dysfunction in mice with cardiomyocyte loss of β1-adrenergic receptor/β-arrestin signaling and controls a unique transcriptome

The β1-adrenergic receptor (β1AR) is found primarily in hearts (mainly in cardiomyocytes [CMs]) and β-arrestin-mediated β1AR signaling elicits cardioprotection through CM survival. We showed that microRNA-150 (miR-150) is upregulated by β-arrestin-mediated β1AR signaling and that CM miR-150 inhibits maladaptive remodeling post-myocardial infarction. Here, we investigate whether miR-150 rescues cardiac dysfunction in mice bearing CM-specific abrogation of β-arrestin-mediated β1AR signaling. Using CM-specific transgenic (TG) mice expressing a mutant β1AR (G protein-coupled receptor kinase [GRK]–β1AR that exhibits impairment in β-arrestin-mediated β1AR signaling), we first generate a novel double TG mouse line overexpressing miR-150. We demonstrate that miR-150 is sufficient to improve cardiac dysfunction in CM-specific GRK–β1AR TG mice following chronic catecholamine stimulation. Our genome-wide circular RNA, long noncoding RNA (lncRNA), and mRNA profiling analyses unveil a subset of cardiac ncRNAs and genes as heretofore unrecognized mechanisms for beneficial actions of β1AR/β-arrestin signaling or miR-150. We further show that lncRNA Gm41664 and GDAP1L1 are direct novel upstream and downstream regulators of miR-150. Lastly, CM protective actions of miR-150 are attributed to repressing pro-apoptotic GDAP1L1 and are mitigated by pro-apoptotic Gm41664. Our findings support the idea that miR-150 contributes significantly to β1AR/β-arrestin-mediated cardioprotection by regulating unique ncRNA and gene signatures in CMs.

Abstract 11967: Microrna-150 Overexpression Blunts Maladaptive Cardiac Remodeling in Mice With Cardiomyocyte-Specific Loss of β-Arrestin-Mediated β 1 -Adrenergic Receptor Signaling

Background and Aims: β-arrestins transduce alternative pathways independent of G protein signaling, a concept known as β-arrestin-biased signaling that is promoted by G protein-coupled receptor kinase (GRK) 5/6 phosphorylation on β-adrenergic receptors (βARs). The β 1 AR is found primarily in the heart (mainly in cardiomyocytes [CMs]) and β-arrestin-mediated β 1 AR signaling elicits cardioprotection by CM survival. We showed that a small noncoding RNA (ncRNA), microRNA-150 (miR-150) is upregulated by the β-blocker carvedilol acting through β-arrestin-mediated β 1 AR signaling, and that CM miR-150 confers cardioprotection (see Figure). Here, we investigate if miR-150 in part rescues maladaptive cardiac remodeling seen in mice with CM-specific abrogation of β-arrestin-mediated β 1 AR signaling. Methods and Results: Using CM-specific transgenic (TG) mice expressing a mutant β 1 AR (i.e. GRK - β 1 AR exhibiting impairment in β-arrestin-mediated β 1 AR signaling and miR-150 activation by carvedilol), we first develop a novel double TG (DTG) mouse line overexpressing miR-150 and CM-specific GRK - β 1 AR. We demonstrate that miR-150 improves maladaptive cardiac remodeling observed in CM-specific GRK - β 1 AR TG mice after chronic catecholamine stimulation. Genome-wide circular RNA, long ncRNA (lncRNA) and mRNA profiling analyses in DTG mouse hearts unveil a subset of cardiac ncRNAs and genes as heretofore unrecognized mechanisms for miR-150’s beneficial effects. We further show that lncRNA Gm41664 and Gdap1l1 have miR-150 binding sites, and that they are direct novel upstream and downstream regulators of miR-150. Lastly, protective roles of miR-150 in CMs are in part attributed to the functional repression of pro-apoptotic Gdap1l1 and are mitigated by pro-apoptotic Gm41664 (see Figure). Conclusions: Our findings support that miR-150 contributes significantly to β 1 AR/β-arrestin-mediated cardioprotection by regulating a unique ncRNA and gene signature in CMs.

Lipolysis defect in people with obesity who undergo metabolic surgery.

ObjectiveCross‐sectional studies demonstrate that catecholamine stimulation of fat cell lipolysis is blunted in obesity. We investigated whether this defect persists after substantial weight loss has been induced by metabolic surgery, and whether it is related to the outcome.Design/MethodsPatients with obesity not able to successfully reduce body weight by conventional means (n = 126) were investigated before and 5 years after Roux‐en‐Y gastric bypass surgery (RYGB). They were compared with propensity‐score matched subjects selected from a control group (n = 1017), and with the entire group after adjustment for age, sex, body mass index (BMI), fat cell volume and other clinical parameters. Catecholamine‐stimulated lipolysis (glycerol release) was investigated in isolated fat cells using noradrenaline (natural hormone) or isoprenaline (synthetic beta‐adrenoceptor agonist).ResultsFollowing RYGB, BMI was reduced from 39.9 (37.5–43.5) (median and interquartile range) to 29.5 (26.7–31.9) kg/m2 (p < 0.0001). The post‐RYGB patients had about 50% lower lipolysis rates compared with the matched and total series of controls (p < 0.0005). Nordrenaline activation of lipolysis at baseline was associated with the RYGB effect; those with high lipolysis activation (upper tertile) lost 30%–45% more in body weight, BMI or fat mass than those with low (bottom tertile) initial lipolysis activation (p < 0.0007).ConclusionPatients with obesity requiring metabolic surgery have impaired ability of catecholamines to stimulate lipolysis, which remains despite long‐term normalization of body weight by RYGB. Furthermore, preoperative variations in the ability of catecholamines to activate lipolysis may predict the long‐term reduction in body weight and fat mass.

Comparison of visceral fat lipolysis adaptation to high-intensity interval training in obesity-prone and obesity-resistant rats

Background/objectivesVisceral obesity is one of the key features of metabolic syndrome. High-intensity interval training (HIIT) could effectively reduce visceral fat, but its effects show strong heterogeneity in populations with different degrees of obesity. The mechanism may be related to the differential adaptation to training between obesity phenotypes, namely obesity prone (OP) and obesity resistant (OR). The aim of the present study was to compare adaptive changes of visceral adipose lipolysis adaptation to HIIT between OP and OR animals and further explore the upstream pathway.MethodsOP and OR Sprague Dawley rats were established after feeding a high-fat diet for 6 weeks; they were then divided into HIIT (H-OP and H-OR) and control (C-OP and C-OR) groups. After 12 weeks of HIIT or a sedentary lifestyle, animals were fasted for 12 h and then sacrificed for histology as well as gene and protein analysis. Visceral adipocytes were isolated without fasting for catecholamine stimulation and β3-adrenergic receptor (β3-AR) blockade in vitro to evaluate the role of upstream pathways.ResultsAfter training, there were no differences in weight loss or food intake between OP and OR rats (P > 0.05). However, the visceral fat mass, adipocyte volume, serum triglycerides and liver lipids of OP rats decreased by more than those of OR rats (P < 0.05). Meanwhile, the cell lipolytic capacity and the increase in the expression of β3-AR were higher in the OP compared with OR groups (P < 0.05). Although training did not increase sympathetic nervous system activity (P > 0.05), the cell sensitivity to catecholamine increased significantly in the OP compared with OR groups (P < 0.05). Following blocking β3-AR, the increased sensitivity disappeared.ConclusionWith HIIT, OP rats lost more visceral fat than OR rats, which was related to stronger adaptive changes in lipolysis. Increased β3-AR expression mediated this adaptation.

Metabolic Response in Endothelial Cells to Catecholamine Stimulation Associated with Increased Vascular Permeability.

Disruption to endothelial cell homeostasis results in an extensive variety of human pathologies that are particularly relevant to major trauma. Circulating catecholamines, such as adrenaline and noradrenaline, activate endothelial adrenergic receptors triggering a potent response in endothelial function. The regulation of the endothelial cell metabolism is distinct and profoundly important to endothelium homeostasis. However, a precise catalogue of the metabolic alterations caused by sustained high catecholamine levels that results in endothelial dysfunction is still underexplored. Here, we uncover a set of up to 46 metabolites that exhibit a dose–response relationship to adrenaline-noradrenaline equimolar treatment. The identified metabolites align with the glutathione-ascorbate cycle and the nitric oxide biosynthesis pathway. Certain key metabolites, such as arginine and reduced glutathione, displayed a differential response to treatment in early (4 h) compared to late (24 h) stages of sustained stimulation, indicative of homeostatic metabolic feedback loops. Furthermore, we quantified an increase in the glucose consumption and aerobic respiration in endothelial cells upon catecholamine stimulation. Our results indicate that oxidative stress and nitric oxide metabolic pathways are downstream consequences of endothelial cell stimulation with sustained high levels of catecholamines. A precise understanding of the metabolic response in endothelial cells to pathological levels of catecholamines will facilitate the identification of more efficient clinical interventions in trauma patients.

Loss-of-function mutations in the co-chaperone protein BAG5 cause dilated cardiomyopathy requiring heart transplantation.

Dilated cardiomyopathy (DCM) is a major cause of heart failure, characterized by ventricular dilatation and systolic dysfunction. Familial DCM is reportedly caused by mutations in more than 50 genes, requiring precise disease stratification based on genetic information. However, the underlying genetic causes of 60 to 80% of familial DCM cases remain unknown. Here, we identified that homozygous truncating mutations in the gene encoding Bcl-2–associated athanogene (BAG) co-chaperone 5 (BAG5) caused inherited DCM in five patients among four unrelated families with complete penetrance. BAG5 acts as a nucleotide exchange factor for heat shock cognate 71 kDa protein (HSC70), promoting adenosine diphosphate release and activating HSC70-mediated protein folding. Bag5 mutant knock-in mice exhibited ventricular dilatation, arrhythmogenicity, and poor prognosis under catecholamine stimulation, recapitulating the human DCM phenotype, and administration of an adeno-associated virus 9 vector carrying the wild-type BAG5 gene could fully ameliorate these DCM phenotypes. Immunocytochemical analysis revealed that BAG5 localized to junctional membrane complexes (JMCs), critical microdomains for calcium handling. Bag5-mutant mouse cardiomyocytes exhibited decreased abundance of functional JMC proteins under catecholamine stimulation, disrupted JMC structure, and calcium handling abnormalities. We also identified heterozygous truncating mutations in three patients with tachycardia-induced cardiomyopathy, a reversible DCM subtype associated with abnormal calcium homeostasis. Our study suggests that loss-of-function mutations in BAG5 can cause DCM, that BAG5 may be a target for genetic testing in cases of DCM, and that gene therapy may potentially be a treatment for this disease.

Abstract 115: Cell-type-specific Gene Expression And Transcriptional Networks Reveal Adamts2 As A Powerful Regulator Of Cardiac Homeostasis During Heart Failure

Background: Heart failure (HF) is a highly heterogeneous disorder characterized by the interactions of multiple genetic and environmental factors as well as the interaction of different cell types in the heart. Although reductionistic approaches have successfully identified many genes involved in HF, heritability studies suggest that many genes have resisted discovery through these approaches. By utilizing cell-type-specific gene expression paired with transcriptomic data from a large cohort of mice, we sought to identify important drivers of HF using a systems genetics approach. Methods and Results: Mice from 93 unique inbred lines of the Hybrid Mouse Diversity Panel were given 30 ug/g/day of isoproterenol for three weeks via osmotic minipump to induce heart failure. Transcriptomes were generated from these mice and the weighted Maximal Information Component Analysis (wMICA) algorithm was applied to generate transcriptomic gene networks. Cardiomyocytes and Fibroblasts were isolated from both control and isoproterenol-treated adult C57BL/6J hearts using a Langendorff apparatus (n=3 per sex/treatment) and transcriptomes were generated. Significantly differentially expressed genes were identified using DESEQ2 and used to query the wMICA-derived network, identifying the gene Adamts2 as a potential regulator of cardiac hypertrophy. Follow-up in vitro and in vivo work has demonstrated that Adamts2 knockdown significantly blunts the hypertrophic effect of isoproterenol on cardiomyocytes while simultaneously reducing fibroblast proliferation and increasing apoptosis as measured by TUNEL staining. Careful examination of the gene network reveals evidence of paracrine signaling between cardiomyocytes and fibroblasts and suggests a key trans-cell-type role of Adamts2 in the regulation of HF after catecholamine stimulation. Conclusion: Co-expression network algorithms combined with cell-type-specific transcriptomics identified Adamts2 as a driver of HF. Adamts2 plays an important role via paracrine signaling in the proliferative response of fibroblasts and the hypertrophic response of cardiomyocytes to catecholamines. Further mechanistic analysis of Adamts2 will further reveal its role in the progression of heart failure.

Sustained anti-obesity effects of life-style change and anti-inflammatory interventions after conditional inactivation of the activin receptor ALK7.

Life-style change and anti-inflammatory interventions have only transient effects in obesity. It is not clear how benefits obtained by these treatments can be maintained longer term, especially during sustained high caloric intake. Constitutive ablation of the activin receptor ALK7 in adipose tissue enhances catecholamine signaling and lipolysis in adipocytes, and protects mice from diet-induced obesity. Here, we investigated the consequences of conditional ALK7 ablation in adipocytes of adult mice with pre-existing obesity. Although ALK7 deletion had little effect on its own, it synergized strongly with a transient switch to low-fat diet (life-style change) or anti-inflammatory treatment (Na-salicylate), resulting in enhanced lipolysis, increased energy expenditure, and reduced adipose tissue mass and body weight gain, even under sustained high caloric intake. By themselves, diet-switch and salicylate had only a temporary effect on weight gain. Mechanistically, combination of ALK7 ablation with either treatment strongly enhanced the levels of β3-AR, the main adrenergic receptor for catecholamine stimulation of lipolysis, and C/EBPα, an upstream regulator of β3-AR expression. These results suggest that inhibition of ALK7 can be combined with simple interventions to produce longer-lasting benefits in obesity.

Sepsis as a Pan-Endocrine Illness-Endocrine Disorders in Septic Patients.

Sepsis is defined as “life-threatening organ dysfunction caused by a dysregulated host response to infection”. One of the elements of dysregulated host response is an endocrine system disorder. Changes in its functioning in the course of sepsis affect almost all hormonal axes. In sepsis, a function disturbance of the hypothalamic–pituitary–adrenal axis has been described, in the range of which the most important seems to be hypercortisolemia in the acute phase. Imbalance in the hypothalamic–pituitary–thyroid axis is also described. The most typical manifestation is a triiodothyronine concentration decrease and reverse triiodothyronine concentration increase. In the somatotropic axis, a change in the secretion pattern of growth hormone and peripheral resistance to this hormone has been described. In the hypothalamic–pituitary–gonadal axis, the reduction in testosterone concentration in men and the stress-induced “hypothalamic amenorrhea” in women have been described. Catecholamine and β-adrenergic stimulation disorders have also been reported. Disorders in the endocrine system are part of the “dysregulated host response to infection”. They may also affect other components of this dysregulated response, such as metabolism. Hormonal changes occurring in the course of sepsis require further research, not only in order to explore their potential significance in therapy, but also due to their promising prognostic value.

Post-exercise Effects and Long-Term Training Adaptations of Hormone Sensitive Lipase Lipolysis Induced by High-Intensity Interval Training in Adipose Tissue of Mice.

Although studies have proven that high-intensity interval training (HIIT) shows a comparable effect to moderate-intensity continuous training (MICT) on reducing body fat, especially visceral fat, the mechanism is still unclear. Since MICT consumes more fat during exercise, the mechanism of HIIT weight loss may be related to post-exercise effects, long-term adaptive changes, and hormone sensitive lipase (HSL). The objective of this study was to compare the post-effects of acute exercise, long-term adaptive changes on HSL activity, and catecholamine-induced lipolysis between HIIT and MICT. Following a 14-week high-fat diet (HFD), obese female C57Bl/6 mice were divided into acute exercise groups (one time training, sacrificed at rest and 0, 1, and 12 h after exercise, n = 49), -L groups (12-week long-term training, 12-h fasting, n = 21), and -C groups (12-week training, primary adipocytes were isolated and stimulated by catecholamine in vitro, n = 18). MICT or HIIT treadmill protocols (running distance matched) were carried out during training. Comparison of acute exercise effects by two-way ANOVA showed no time × group interaction effect, however, a significant increase in HSL-Ser563 (at 0 and 1 h) and Ser660 phosphorylation (at 0, 1, and 12 h) in inguinal (subcutaneous) fat was only observed in HIIT mice (p < 0.05 vs. rest), but not in MICT mice. The periuterine (visceral) fat HSL expression and phosphorylation of HIIT mice was similar to or lower than MICT mice. After long-term training, 12-h fasting significantly increased periuterine fat Ser563 phosphorylation in HIIT mice (p < 0.05), but there was no change in MICT mice. Under stimulation of catecholamine in vitro, isolated primary adipocytes from periuterine fat of long-term HIIT mice showed a higher Ser563 increase than that found in MICT mice (p < 0.05). The quantity of triglyceride (TG) lipid bonds (representing lipolysis level) was significantly lower after HIIT than MICT (p < 0.05). The results indicate that (1) acute HIIT can induce an increase of HSL phosphorylation in subcutaneous fat lasting at least 12 h, implying longer post-exercise lipolysis than MICT and (2) long-time HIIT has a better effect on improving catecholamine resistance of visceral adipocytes caused by a HFD, which allows fat to be mobilized more easily when stimulated.

Intracellular β1-Adrenergic Receptors and Organic Cation Transporter 3 Mediate Phospholamban Phosphorylation to Enhance Cardiac Contractility.

β1ARs (β1-adrenoceptors) exist at intracellular membranes and OCT3 (organic cation transporter 3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular β1AR in cardiac contractility remains to be elucidated. Test localization and function of intracellular β1AR on cardiac contractility. Membrane fractionation, super-resolution imaging, proximity ligation, coimmunoprecipitation, and single-molecule pull-down demonstrated a pool of β1ARs in mouse hearts that were associated with sarco/endoplasmic reticulum Ca2+-ATPase at the sarcoplasmic reticulum (SR). Local PKA (protein kinase A) activation was measured using a PKA biosensor targeted at either the plasma membrane (PM) or SR. Compared with wild-type, myocytes lacking OCT3 (OCT3-KO [OCT3 knockout]) responded identically to the membrane-permeant βAR agonist isoproterenol in PKA activation at both PM and SR. The same was true at the PM for membrane-impermeant norepinephrine, but the SR response to norepinephrine was suppressed in OCT3-KO myocytes. This differential effect was recapitulated in phosphorylation of the SR-pump regulator phospholamban. Similarly, OCT3-KO selectively suppressed calcium transients and contraction responses to norepinephrine but not isoproterenol. Furthermore, sotalol, a membrane-impermeant βAR-blocker, suppressed isoproterenol-induced PKA activation at the PM but permitted PKA activation at the SR, phospholamban phosphorylation, and contractility. Moreover, pretreatment with sotalol in OCT3-KO myocytes prevented norepinephrine-induced PKA activation at both PM and the SR and contractility. Functional β1ARs exists at the SR and is critical for PKA-mediated phosphorylation of phospholamban and cardiac contractility upon catecholamine stimulation. Activation of these intracellular β1ARs requires catecholamine transport via OCT3.

β2-AR activation promotes cleavage and nuclear translocation of Her2 and metastatic potential of cancer cells.

Prolonged hypersecretion of catecholamine induced by chronic stress may correlate with malignant progression of cancer. β2‐adrenergic receptor (β2‐AR) overexpressed in certain cancer cells may translate the signals from neuroendocrine system to malignant signals by interacting with oncoproteins, such as Her2. In the present study, we demonstrate that catecholamine stimulation activates the expression and proteolytic activity of ADAM10 by modulating the expression of miR‐199a‐5p and SIRT1 and also confirm that catecholamine induction triggers the activities of γ‐secretase, leading to shedding of Her2 extracellular domain (ECD) by ADAM10 and subsequent intramembranous cleavage of Her2 intracellular domain (ICD) by presenilin‐dependent γ‐secretase, nuclear translocation of Her2 ICD, and enhanced transcription of tumor metastasis‐associated gene COX‐2. Chronic stimulation of catecholamine strongly promotes the invasive activities of cancer cells in vitro and spontaneous tumor lung metastasis in mice. Furthermore, nuclear localization of Her2 was significantly correlated with overexpression of β2‐AR in human breast cancer tissues, indicating that catecholamine‐induced β2‐AR activation plays decisive roles in tumor metastasis. Our data also reveal that an unknown mechanism by which the regulated intramembrane proteolysis (RIP) initiated by β2‐AR‐mediated signaling controls a novel Her2‐mediated signaling transduction.

UNCOMMON AMONGST COMMON: ANGIOTENSIN II-INDUCED PERIPHERAL ISCHEMIA IN REFRACTORY SEPTIC SHOCK

SESSION TITLE: Medical Student/Resident Critical Care Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: Angiotensin (AT) II, a naturally occurring hormone, modulates blood pressure through direct vasoconstriction of the circulatory system and through stimulation of various catecholamines and vasopressin. Derived from the hydrolysis of AT I via the AT-converting enzyme (ACE) in the pulmonary & renal endothelium, AT II has been studied for cases of vasodilatory shock since the 1930’. Most recently, the ATHOS-3 trial demonstrated AT II’s utility as a rescue vasopressor agent, highlighting its catecholamine-sparing effects. CASE PRESENTATION: 34-year-old African American male presented with 3-day history of dyspnea, productive cough and an episode of hemoptysis. Upon presentation, he was febrile, tachycardic, hypotensive and hypoxic prompting endotracheal intubation and vasopressor support. Laboratory studies were significant for leukocytosis with bandemia, lactic acidosis, acute kidney injury, transaminitis & positive for influenza A. Despite aggressive critical care management with maximum ventilator requirements, broad-spectrum antibiotics, renal replacement therapy & proning, his overall condition continued to worsen. AT II was initiated on Day 2 of ICU admission for refractory hypotension secondary to septic shock. Approximately 48-72 hours after the initiation of AT II, he developed skin discoloration and blistering in all extremities secondary to both arterial and venous thromboemboli despite prophylaxis. The lesions ultimately progressed leading to gangrenous necrosis extending proximally to the torso. Unfortunately, on day 7 of ICU admission, patient suffered a cardiac arrest and expired despite ACLS efforts. DISCUSSION: In our patient, we exhibited similar results to those described in the ATHOS-3 trials. Administration of the AT II resulted in an overall improvement in the patient’s mean arterial pressure, systemic vascular resistance and reduced norepinephrine requirements. We also observed a concomitant improvement in serum lactate, indicating improvement in overall organ perfusion. There are no absolute contraindications to AT II administration, but ATHOS-3 did report an increased incidence of both venous and arterial thromboembolic events in patients who received AT II. Although AT II has been FDA approved for the management of refractory septic shock, more studies are necessary to define those populations most at risk of the adverse effects. CONCLUSIONS: AT II is an important vasopressor agent used in refractory shock; however, more studies are needed to understand the adverse effects in populations most at risk. Reference #1: National Center for Biotechnology Information. PubChem Database. Angiotensin II, CID=172198, https://pubchem.ncbi.nlm.nih.gov/compound/Angiotensin-II (accessed on Mar. 26, 2020) DISCLOSURES: No relevant relationships by Krupa Gandhi, source=Web Response No relevant relationships by Mohit Mody, source=Web Response

Functionality of beta-adrenergic receptors in patients with cirrhosis treated chronically with non-selective beta-blockers.

In patients with cirrhosis, beta-adrenoceptors expressed on peripheral blood mononuclear cells have a reduced response to catecholamine stimulation. This study aimed to determine if chronic treatment with beta-blockers influences these changes. Blood samples were collected from patients with cirrhosis treated in outpatient clinics. Differences in cyclic AMP production before and after stimulation of mononuclear cells with epinephrine and/or N-Formylmethionine-leucyl-phenylalanine (fMLP) was used as a marker of beta-adrenoceptors activity in patients treated (N = 19) versus not treated (N = 55) with beta-blockers. In addition, we studied the gene expression of different types of adrenoceptors and possible associations with the activity of beta-adrenoceptors, the serum concentrations of catecholamines and cytokines, and the presence of bacterial antigens such as DNA or gram-negative bacterial endotoxin in patients' blood. The increase in intracellular cAMP concentrations after stimulation of adrenergic receptors with epinephrine was significantly higher in samples from patients treated with beta-blockers. Older patients showed lower responses to epinephrine stimulus, while the response increased linearly with the duration of the beta-blocker treatment. mRNA expression levels of adrenoceptors β1, β2, β3 and α1-A, B and D showed no significant differences according to treatment with beta-blockers. Neither serum cytokines nor catecholamines levels were significantly associated with the intracellular production of cAMP after adrenergic stimulation. Chronic treatment with beta-blockers in patients with cirrhosis enables beta-adrenoceptors to respond to catecholamine stimulation irrespective of the degree of systemic adrenergic or immune activations of the patient at the time of sampling.

Abstract 464: Dynamic Regulation of the Epidermal Growth Factor Receptor Protein Interactome via β1-adrenergic Receptor-mediated Transactivation in Cardiomyocytes

Heart Failure (HF) is a chronic, progressive condition wherein the heart cannot perfuse adequate amounts of blood and oxygen to meet the demands of metabolizing tissues. Gold standard HF therapeutics currently consist of a small repertoire of neurohormone modulators that still result in ~50% mortality within 5 years. Our ongoing goal is to develop new signaling modalities as refined HF therapeutics. We have shown that β1-adrenergic receptor (β1AR) stimulation rapidly induces epidermal growth factor receptor (EGFR)-dependent protein signaling responses in various regions of cardiomyocytes (CM). We hypothesize that differences in the subcellular localization and protein interactomes of EGFR following differential ligand stimulation modulate CM functions in distinct manners. To test this, we infected neonatal rat ventricular myocytes (NRVM) with adenoviruses encoding HA-β1AR and Flag-EGFR, stimulated them with vehicle, isoproterenol or EGF for 5 min and performed mass spectroscopy analysis of the Flag-EGFR immunoprecipitates. While hundreds of protein interactions were identified, one of the highly enriched subcellular groups belonged to mitochondrial proteins, for which there is no information regarding the role of EGFR in their regulation in CM. Via fractionation and digestion analyses, we have confirmed EGFR localization to the outer mitochondrial membrane in CM and have begun to assess the impact of EGFR on mitochondrial function. Transmission electron microcopy analysis of mitochondrial morphology show that CM-specific EGFR knockdown (CM-EGFR-KD) increases mitochondrial circularity and mitochondria/area without changes in other parameters including perimeter or cristae density, suggesting altered bioenergetic potential. Indeed, extracellular flux (Seahorse) analysis of CM-EGFR-KD versus control CM revealed that CM-EGFR-KD increased maximal and reserve capacities and a trend toward increased ATP production. Elucidating the mechanism(s) of EGFR localization to and association with proteins at CM mitochondria, as well as its impact on mitochondrial function normally or in response to catecholamine stimulation will provide insight into the potential utility of targeting mitochondrial EGFR to improve HF outcomes.