Type 2 Diabetes Dysregulates Glucose Metabolism in Cardiac Progenitor Cells.

Type 2 diabetes is associated with increased mortality and progression to heart failure. Recent studies suggest that diabetes also impairs reparative responses after cell therapy. In this study, we examined potential mechanisms by which diabetes affects cardiac progenitor cells (CPCs). CPCs isolated from the diabetic heart showed diminished proliferation, a propensity for cell death, and a pro-adipogenic phenotype. The diabetic CPCs were insulin-resistant, and they showed higher energetic reliance on glycolysis, which was associated with up-regulation of the pro-glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3). In WT CPCs, expression of a mutant form of PFKFB, which mimics PFKFB3 activity and increases glycolytic rate, was sufficient to phenocopy the mitochondrial and proliferative deficiencies found in diabetic cells. Consistent with activation of phosphofructokinase in diabetic cells, stable isotope carbon tracing in diabetic CPCs showed dysregulation of the pentose phosphate and glycero(phospho)lipid synthesis pathways. We describe diabetes-induced dysregulation of carbon partitioning using stable isotope metabolomics-based coupling quotients, which relate relative flux values between metabolic pathways. These findings suggest that diabetes causes an imbalance in glucose carbon allocation by uncoupling biosynthetic pathway activity, which could diminish the efficacy of CPCs for myocardial repair.

Aldo-keto Reductase 1B15 (AKR1B15): a mitochondrial human aldo-keto reductase with activity toward steroids and 3-keto-acyl-CoA conjugates.

Aldo-keto reductases (AKRs) comprise a superfamily of proteins involved in the reduction and oxidation of biogenic and xenobiotic carbonyls. In humans, at least 15 AKR superfamily members have been identified so far. One of these is a newly identified gene locus, AKR1B15, which clusters on chromosome 7 with the other human AKR1B subfamily members (i.e. AKR1B1 and AKR1B10). We show that alternative splicing of the AKR1B15 gene transcript gives rise to two protein isoforms with different N termini: AKR1B15.1 is a 316-amino acid protein with 91% amino acid identity to AKR1B10; AKR1B15.2 has a prolonged N terminus and consists of 344 amino acid residues. The two gene products differ in their expression level, subcellular localization, and activity. In contrast with other AKR enzymes, which are mostly cytosolic, AKR1B15.1 co-localizes with the mitochondria. Kinetic studies show that AKR1B15.1 is predominantly a reductive enzyme that catalyzes the reduction of androgens and estrogens with high positional selectivity (17ß-hydroxysteroid dehydrogenase activity) as well as 3-keto-acyl-CoA conjugates and exhibits strong cofactor selectivity toward NADP(H). In accordance with its substrate spectrum, the enzyme is expressed at the highest levels in steroid-sensitive tissues, namely placenta, testis, and adipose tissue. Placental and adipose expression could be reproduced in the BeWo and SGBS cell lines, respectively. In contrast, AKR1B15.2 localizes to the cytosol and displays no enzymatic activity with the substrates tested. Collectively, these results demonstrate the existence of a novel catalytically active AKR, which is associated with mitochondria and expressed mainly in steroid-sensitive tissues.

Glutamine Regulates Cardiac Progenitor Cell Metabolism and Proliferation.

Autologous transplantation of cardiac progenitor cells (CPCs) alleviates myocardial dysfunction in the damaged heart; however, the mechanisms that contribute to their reparative qualities remain poorly understood. In this study, we examined CPC metabolism to elucidate the metabolic pathways that regulate their proliferative capacity. In complete growth medium, undifferentiated CPCs isolated from adult mouse heart proliferated rapidly (Td = 13.8 hours). CPCs expressed the Glut1 transporter and their glycolytic rate was increased by high extracellular glucose (Glc) concentration, in the absence of insulin. Although high Glc concentrations did not stimulate proliferation, glutamine (Gln) increased CPC doubling time and promoted survival under conditions of oxidative stress. In comparison with Glc, pyruvate (Pyr) or BSA-palmitate, Gln, when provided as the sole metabolic substrate, increased ATP-linked and uncoupled respiration. Although fatty acids were not used as respiratory substrates when present as a sole carbon source, Gln-induced respiration was doubled in the presence of BSA-palmitate, suggesting that Gln stimulates fatty acid oxidation. Additionally, Gln promoted rapid phosphorylation of the mTORC1 substrate, p70S6k, as well as retinoblastoma protein, followed by induction of cyclin D1 and cdk4. Inhibition of either mTORC1 or glutaminolysis was sufficient to diminish CPC proliferation, and provision of cell permeable α-ketoglutarate in the absence of Gln increased both respiration and cell proliferation, indicating a key role of Gln anaplerosis in cell growth. These findings suggest that Gln, by enhancing mitochondrial function and stimulating mTORC1, increases CPC proliferation, and that interventions to increase Gln uptake or oxidation may improve CPC therapy.

Bioenergetic differences between MCF-7 and T47D breast cancer cells and their regulation by oestradiol and tamoxifen.

Oestrogen receptor α (ERα+) breast tumours rely on mitochondria (mt) to generate ATP. The goal of the present study was to determine how oestradiol (E2) and 4-hydroxytamoxifen (4-OHT) affect cellular bioenergetic function in MCF-7 and T47D ERα+ breast cancer cells in serum-replete compared with dextran-coated charcoal (DCC)-stripped foetal bovine serum (FBS)-containing medium ('serum-starved'). Serum-starvation reduced oxygen consumption rate (OCR), extracellular acidification rate (ECAR), ATP-linked OCR and maximum mt capacity, reflecting lower ATP demand and mt respiration. Cellular respiratory stateapparent was unchanged by serum deprivation. 4-OHT reduced OCR independent of serum status. Despite having a higher mt DNA/nuclear DNA ratio than MCF-7 cells, T47D cells have a lower OCR and ATP levels and higher proton leak. T47D express higher nuclear respiratory factor-1 (NRF-1) and NRF-1-regulated, nuclear-encoded mitochondrial transcription factor TFAM and cytochrome c, but lower levels of cytochrome c oxidase, subunit IV, isoform 1 (COX4, COX4I1). Mitochondrial reserve capacity, reflecting tolerance to cellular stress, was higher in serum-starved T47D cells and was increased by 4-OHT, but was decreased by 4-OHT in MCF-7 cells. These data demonstrate critical differences in cellular energetics and responses to 4-OHT in these two ERα+ cell lines, likely reflecting cancer cell avoidance of apoptosis.

High glucose induces mitochondrial dysfunction independently of protein O-GlcNAcylation.

Diabetes is characterized by hyperglycaemia and perturbations in intermediary metabolism. In particular, diabetes can augment flux through accessory pathways of glucose metabolism, such as the hexosamine biosynthetic pathway (HBP), which produces the sugar donor for the ß-O-linked-N-acetylglucosamine (O-GlcNAc) post-translational modification of proteins. Diabetes also promotes mitochondrial dysfunction. Nevertheless, the relationships among diabetes, hyperglycaemia, mitochondrial dysfunction and O-GlcNAc modifications remain unclear. In the present study, we tested whether high-glucose-induced increases in O-GlcNAc modifications directly regulate mitochondrial function in isolated cardiomyocytes. Augmentation of O-GlcNAcylation with high glucose (33 mM) was associated with diminished basal and maximal cardiomyocyte respiration, a decreased mitochondrial reserve capacity and lower Complex II-dependent respiration (P<0.05); however, pharmacological or genetic modulation of O-GlcNAc modifications under normal or high glucose conditions showed few significant effects on mitochondrial respiration, suggesting that O-GlcNAc does not play a major role in regulating cardiomyocyte mitochondrial function. Furthermore, an osmotic control recapitulated high-glucose-induced changes to mitochondrial metabolism (P<0.05) without increasing O-GlcNAcylation. Thus, increased O-GlcNAcylation is neither sufficient nor necessary for high-glucose-induced suppression of mitochondrial metabolism in isolated cardiomyocytes.

PDGF-mediated autophagy regulates vascular smooth muscle cell phenotype and resistance to oxidative stress.

Vascular injury and chronic arterial diseases result in exposure of VSMCs (vascular smooth muscle cells) to increased concentrations of growth factors. The mechanisms by which growth factors trigger VSMC phenotype transitions remain unclear. Because cellular reprogramming initiated by growth factors requires not only the induction of genes involved in cell proliferation, but also the removal of contractile proteins, we hypothesized that autophagy is an essential modulator of VSMC phenotype. Treatment of VSMCs with PDGF (platelet-derived growth factor)-BB resulted in decreased expression of the contractile phenotype markers calponin and α-smooth muscle actin and up-regulation of the synthetic phenotype markers osteopontin and vimentin. Autophagy, as assessed by LC3 (microtubule-associated protein light chain 3 α; also known as MAP1LC3A)-II abundance, LC3 puncta formation and electron microscopy, was activated by PDGF exposure. Inhibition of autophagy with 3-methyladenine, spautin-1 or bafilomycin stabilized the contractile phenotype. In particular, spautin-1 stabilized α-smooth muscle cell actin and calponin in PDGF-treated cells and prevented actin filament disorganization, diminished production of extracellular matrix, and abrogated VSMC hyperproliferation and migration. Treatment of cells with PDGF prevented protein damage and cell death caused by exposure to the lipid peroxidation product 4-hydroxynonenal. The results of the present study demonstrate a distinct form of autophagy induced by PDGF that is essential for attaining the synthetic phenotype and for survival under the conditions of high oxidative stress found to occur in vascular lesions.

COVID-19-Induced Complete Heart Block: Case Series and Literature Review.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has led to the emergence of a wide range of complications, including those affecting the cardiovascular system. In this case series, we present four patients who developed complete atrioventricular block, a serious and potentially life-threatening heart rhythm disorder, during the course of their coronavirus disease 2019 (COVID-19) illness. The mechanisms by which SARS-CoV-2 may lead to arrhythmias are not fully understood but may involve direct infection and damage to heart tissue, as well as inflammation and cytokine storms. The extent and duration of complete heart block varied among these cases, highlighting the need for further research to understand the spectrum of disease and to improve mortality and morbidity in future waves of SARS-CoV-2 infections. We hope that this case series will draw attention to this serious complication of COVID-19 and inspire further research to improve management and outcomes for affected patients.

Verapamil stereoisomers induce antiproliferative effects in vascular smooth muscle cells via autophagy.

Calcium channel blockers (CCBs) are important in the management of hypertension and limit restenosis. Although CCB efficacy could derive from decreased blood pressure, other mechanisms independent of CCB activity also can contribute to antiproliferative action. To understand mechanisms of CCB-mediated antiproliferation, we studied two structurally dissimilar CCBs, diltiazem and verapamil, in cultured rat vascular smooth muscle cells (VSMC). To elucidate CCB-independent effects, pure stereoisomers of verapamil (R-verapamil, inactive VR; S-verapamil, active, VS) were used. The effects of CCB exposure on cell viability (MTT reduction), cell proliferation ((3)H-thymidine incorporation), VSMC morphology by light and transmission electron microscopy (TEM) and autophagy (LC3I/II, ATG5) were measured. In general, verapamil, VR or VS treatment alone (80 µM) appreciably enhanced MTT absorbance although higher concentrations (VR or VS) slightly decreased MTT absorbance. Diltiazem (140 µM) markedly decreased MTT absorbance (40%) at 120 h. VR or VS treatment inhibited (3)H-thymidine incorporation (24h) and induced cytological alterations (i.e., karyokinesis, enhanced perinuclear MTT deposition, accumulated perinuclear "vacuoles"). TEM revealed perinuclear "vacuoles" to be aggregates of highly laminated and electron-dense vesicles resembling autophagosomes and lysosomes, respectively. Increased autophagosome activity was confirmed by a concentration-dependent increase in LC3-II formation by Western blotting and by increased perinuclear LC3-GFP(+) puncta in verapamil-treated VSMC. Verapamil stereoisomers appeared to decrease perinuclear mitochondrial density. These observations indicate that antiproliferative effects of verapamil stereoisomers are produced by enhanced mitochondrial damage and upregulated autophagy in VSMC. These effects are independent of CCB activity indicating a distinct mechanism of action that could be targeted for more efficacious anti-atherosclerotic and anti-restenosis therapy.

Responses of hypertrophied myocytes to reactive species: implications for glycolysis and electrophile metabolism.

During cardiac remodelling, the heart generates higher levels of reactive species; yet an intermediate 'compensatory' stage of hypertrophy is associated with a greater ability to withstand oxidative stress. The mechanisms underlying this protected myocardial phenotype are poorly understood. We examined how a cellular model of hypertrophy deals with electrophilic insults, such as would occur upon ischaemia or in the failing heart. For this, we measured energetics in control and PE (phenylephrine)-treated NRCMs (neonatal rat cardiomyocytes) under basal conditions and when stressed with HNE (4-hydroxynonenal). PE treatment caused hypertrophy as indicated by augmented atrial natriuretic peptide and increased cellular protein content. Hypertrophied myocytes demonstrated a 2.5-fold increase in ATP-linked oxygen consumption and a robust augmentation of oligomycin-stimulated glycolytic flux and lactate production. Hypertrophied myocytes displayed a protected phenotype that was resistant to HNE-induced cell death and a unique bioenergetic response characterized by a delayed and abrogated rate of oxygen consumption and a 2-fold increase in glycolysis upon HNE exposure. This augmentation of glycolytic flux was not due to increased glucose uptake, suggesting that electrophile stress results in utilization of intracellular glycogen stores to support the increased energy demand. Hypertrophied myocytes also had an increased propensity to oxidize HNE to 4-hydroxynonenoic acid and sustained less protein damage due to acute HNE insults. Inhibition of aldehyde dehydrogenase resulted in bioenergetic collapse when myocytes were challenged with HNE. The integration of electrophile metabolism with glycolytic and mitochondrial energy production appears to be important for maintaining myocyte homoeostasis under conditions of increased oxidative stress.

Pneumopericardium, Epidural Pneumatosis, and Muscular Emphysema: Rare Complications of Spontaneous Pneumomediastinum Due to Refractory Hyperemesis Gravidarum.

Various complications of hyperemesis gravidarum and pneumomediastinum have been documented in the literature. Commonly, these cases resolve spontaneously or with the use of antiemetics and supportive care. In rare instances, these symptoms persist into the second trimester and are associated with an increased risk of complications. Herein, we present a case of a healthy 20-year-old female with a history of marijuana use who presented with intractable nausea and vomiting and was found to have multiple rare complications of spontaneous pneumomediastinum.

Zieve Syndrome in a Patient With Hepatitis C.

Zieve syndrome is a very rare syndrome that presents as a triad of hemolytic anemia, jaundice, and transient hyperlipidemia in patients with alcoholic liver disease. Herein, we present a case of a 30-year-old female with alcoholic liver disease and chronic hepatitis C. She presented with altered mental status and profound jaundice and was subsequently found to have acute hemolytic anemia due to Zieve syndrome. All other causes of hemolytic anemia were ruled out. She abstained from alcohol and received blood transfusions as needed, leading to the improvement of her anemia. This case highlights the need for more medical education about Zieve syndrome as the under-recognition of the disease can lead to unnecessary treatments. We review the existing literature to explain the epidemiology, pathogenesis, diagnosis, and treatment of Zieve syndrome. This case represents a rare presentation of Zieve syndrome in a patient with hepatitis C, and we have hypothesized a possible role of chronic hepatitis C infection in its pathophysiology.

COVID-19 and the cardiovascular system: an update.

As COVID-19 continues to cause an increasing number of deaths worldwide, it is important that providers stay abreast with new research related to the pathophysiology of COVID-19 disease presentation states and clinical management. It is now well recognized that COVID-19 affects extrapulmonary organs, particularly the cardiovascular system. For example, cardiogenic shock has been increasingly observed in patients with COVID-19, owing to the various mechanisms involved and the affinity of the SARS-CoV-2 virus to cells comprising the cardiovascular system. In this review, we have briefly discussed the link between the cardiovascular system and COVID-19 infection, focusing on underlying mechanisms including but not limited to cytokine storm, direct virus-induced myocarditis, and ST-elevation myocardial infarction leading to cardiogenic shock. We have highlighted the cardiovascular risk factors associated with disease prognostication in COVID-19 patients. We have also briefly discussed vasopressors and inotropes used for treating shock and presented their mechanism of action, contraindications, and side effects in the hopes of providing a quick reference to help the provider optimize management of COVID-19 patients presenting with cardiovascular complications such as shock.

Induction of Hypopituitarism Following Ipilimumab/Nivolumab Therapy Followed by Radiation in the Treatment of Metastatic Scalp Melanoma.

Description Immune checkpoint inhibitors (ICI) are antagonistic antibodies that block specific immune checkpoint molecules, such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death protein (PD-1) and its ligand PD-L1. With FDA approval, the use of these checkpoint inhibitors has led to long-lasting tumor responses. However, by stimulating the immune system, checkpoint inhibitors can cause immune-related adverse events involving the endocrine organs, among others. Pituitary dysfunction (hypophysitis) leading to secondary adrenal insufficiency, or primary adrenal insufficiency caused by immune checkpoint inhibitors, have been documented. In this report, we present a case of a 70-year-old man with scalp melanoma with metastasis to the neck lymph nodes who remained asymptomatic 2 weeks after treatment completion with ipilimumab/nivolumab. However, he became lethargic, lost his appetite and was unable to perform activities of daily living following the initiation of radiation therapy to his neck. After hospitalization, he was found to have hyponatremia, hypocortisolism and hypopituitarism. He was treated with hydrocortisone, which lead to significant symptom improvement. His case suggests a dual-hit mechanism of injury to the pituitary caused by combined ICI and radiation therapies. Following ICI therapy, he was on the verge of pituitary dysfunction that fully materialized following a second insult. We suggest that for patients treated with ICIs, particularly with ipilimumab/nivolumab, a washout period may be considered before starting other forms of therapies, such as radiation therapy to the head/neck regions. Otherwise, prophylactic low dose corticosteroids may be initiated in cases where radiation therapy must not be delayed.

Starvation Ketoacidosis Induced by Ketogenic Diet and Consumption of Ketone Supplement.

The ever-changing landscape of dieting and its correlation with health outcomes have continued to evolve with time. New diets appear and disappear just as quickly as they gain notoriety. This is a rare case of a 67-year-old female with a history of type II diabetes who presented with generalized weakness, nausea, and vomiting, and was found to have severe anion gap metabolic acidosis. In an effort to lose weight, she was combining a ketogenic diet with prolonged fasting and exogenous ketone supplement use that she purchased online. The patient reported drinking an exogenous ketone ester supplement that contained 30 g of D-beta hydroxybutyrate (BHB) per serving, three times per day. This case is unique in that the patient was initially thought to be in diabetic ketoacidosis upon arrival, but after further investigation into her initial labs, medication, and social history, the underlying factor for hospitalization became evident; that is, a combination of a ketogenic diet with prolonged fasting and exogenous BHB-induced ketoacidosis in the setting of type II diabetes. Thus, this case highlights the importance of thorough history taking, the dangers of over-the-counter supplement consumption, and the risks consumers inherit with trend dieting.

Septic Arthritis Masquerading as a Flare of Rheumatoid Arthritis: A Not So Straightforward Diagnosis.

A typical presentation of septic arthritis (SA) includes pain, swelling, and erythema in the affected joint. Often, patients complain of inability to bear weight on the affected limb. However, some patients may present with subtle symptoms of pain and no fever or obvious swelling of the affected limb thus making the initial suspicion of SA low. Especially, patients with rheumatoid arthritis (RA) may present with polyarticular joint pain and initial synovial fluid analysis from an infected joint not consistent with overt septic arthritis. In such situations, the diagnosis of septic arthritis could be missed on delayed. In this case report, we present a 79-year-old female with a history of RA who presents with polyarticular pain, most notably in her right knee. SA was not initially suspected because of her history of RA and her current presentation with polyarticular pain. The initial synovial analysis did not suggest SA as well. However, cultures of synovial fluid from her right knee confirmed SA. Thus, we have highlighted that physicians should have a high suspicion for SA when addressing joint pain in RA patients.

Physicians at the crossroad of prognosis and faith: practical help communicating with patients and families during the COVID-19 pandemic.

Physicians will ultimately face the necessary but unpleasant task of caring for a dying patient at some point in their careers. Communicating with patients or their families during such dire times is very important especially when the patient or family members have unrealistic expectations. Herein, we have highlighted practical suggestions which if applied can prevent unnecessary draining encounters with patients and families; for example, incorporating ancillary staff such as palliative and pastoral care into the care team. We have also proposed a new concept of 'physician optimism'. Based on this concept, the physician can be classified as a pessimist or an optimist with realistic or unrealistic expectations and communicate to patients with or without requisite empathy. To ensure the best outcomes, we conclude that physicians must be realistic optimists who always communicate with empathy. Unrealistic optimism, no matter how well-intentioned, is deceiving to patients and their families will never allow a culture of trust.

Third-Degree Heart Block Associated With Saddle Pulmonary Embolism: A Rare Sequelae of COVID-19-Induced Hypercoagulable State.

The pathophysiology of coronavirus disease 2019 (COVID-19) involves multi-organ dysfunction, particularly involving the respiratory, cardiovascular and hematological systems. This dysfunction is partly due to systemic inflammation causing a wide array of pathological sequelae thus posing a significant challenge to management despite the advances in treatment made thus far. In this report, we present a COVID-19 patient who developed a transient complete heart block and was temporarily paced as a complication of a saddle pulmonary embolus (PE). The mechanism of complete heart block is unclear, may be related to strain, ischemia, or vagal response. We believe that this is a unique sequence of events in a COVID-19 patient and, to our knowledge, is the first of its kind to be reported.

COVID-19 Coagulopathy: Current knowledge and guidelines on anticoagulation.

COVID-19-associated coagulopathy (CAC) is a feature of COVID-19 that can lead to various thrombotic complications and death. In this review, we briefly highlight possible etiologies, including direct cytotoxicity caused by the SARS-CoV-2 virus, and the activation of proinflammatory molecules such as cytokines, underlying coagulopathy. Endothelial dysfunction has been highlighted as pivotal, irrespective of the mechanism involved in CAC. Specific features of CAC distinguishing it from disseminated intravascular coagulopathy and sepsis or ARDS-associated coagulopathy have been discussed. We have also highlighted some hematological parameters, such as elevated d-dimers and partial prothrombin and prothrombin times prolongation, which can guide the use of anticoagulation in critically ill patients. We conclude by highlighting the importance of prophylactic anticoagulation in all COVID-19 hospitalized patients and reiterate the need for institution-specific guidelines for anticoagulation COVID-19 patients since individual institutions have different patient populations.