Exercise-induced cardiac mitochondrial reorganization and enhancement in spontaneously hypertensive rats.

The myocardium is a highly oxidative tissue in which mitochondria are essential to supply the energy required to maintain pump function. When pathological hypertrophy develops, energy consumption augments and jeopardizes mitochondrial capacity. We explored the cardiac consequences of chronic swimming training, focusing on the mitochondrial network, in spontaneously hypertensive rats (SHR). Male adult SHR were randomized to sedentary or trained (T: 8-week swimming protocol). Blood pressure and echocardiograms were recorded, and hearts were removed at the end of the training period to perform molecular, imaging, or isolated mitochondria studies. Swimming improved cardiac midventricular shortening and decreased the pathological hypertrophic marker atrial natriuretic peptide. Oxidative stress was reduced, and even more interesting, mitochondrial spatial distribution, dynamics, function, and ATP were significantly improved in the myocardium of T rats. In the signaling pathway triggered by training, we detected an increase in the phosphorylation level of both AKT and glycogen synthase kinase-3 ß, key downstream targets of insulin-like growth factor 1 signaling that are crucially involved in mitochondria biogenesis and integrity. Aerobic exercise training emerges as an effective approach to improve pathological cardiac hypertrophy and bioenergetics in hypertension-induced cardiac hypertrophy.

Effects of Ramadan Fasting on Extracorporeal Photopheresis Outcomes.

Although there are many studies on the impact of Ramadan fasting on health in the medical literature, the effects have not been explored in Muslim patients undergoing extracorporeal photopheresis (ECP). This report aimed to describe the potential effects of Ramadan fasting on ECP treatment outcomes. Patients undergoing ECP were prospectively evaluated before and during the month of Ramadan 1443 AH (2022 AD) at the Abu Dhabi Stem Cells Center (ADSCC), United Arab Emirates. The following ECP outcomes were assessed: treatment completion, adverse events reported, body mass index (BMI), and laboratory test results, including complete blood count (CBC), C-reactive protein (CRP), and other systemic immune-inflammatory biomarkers (SIIBs). No statistically significant differences were found in most of the variables analyzed in the three patients who underwent ECP before and during the holy month. Two non-fasting patients were not able to complete the Ramadan ECP schedule, and one fasting patient experienced a vascular access event during his first procedure in Ramadan. These findings suggest that fasting during Ramadan could add further risk factors and develop serious complications related to the ECP treatment. Therefore, we suggest that fasting should be avoided during photopheresis treatment, and we provided recommendations to achieve the best possible clinical outcomes.

"Original Antigenic Sin" in SARS-CoV-2 Vaccination Followed by Infection.

Although the "original antigenic sin" (OAS) effects have been predicted against new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), only a few pieces of evidence are available regarding its impact on the safety and effectiveness of coronavirus disease 2019 (COVID-19) vaccines. This article aims to provide an immunological explanation for the delayed side effects of a SARS-CoV-2 vaccine during an episode of natural infection. We reported a case of a 39-year-old male healthcare worker who complained about pruritus and discomfort around the injection site of an inactivated SARS-CoV-2 vaccine administrated 18, 17, and 13 months earlier. Those symptoms resembled the side effects previously experienced with one of the booster doses, and a sole erythematous papule was also documented. The patient was diagnosed with COVID-19 one or two days after noticing these local signs and symptoms, and high serum titers of immunoglobulin M (IgM) and immunoglobulin E (IgE) were found five weeks after the onset, along with SARS-CoV-2-specific immunoglobulin G (IgG) antibodies. Therefore, the OAS might be a plausible phenomenon to consider in individuals immunized with inactivated vaccines and exposed secondarily to a wild virus with antigenic variations.

Lymphocyte Subsets in Defense Against New Pathogens in Patients with Cancer.

INTRODUCTION: Immunity in cancer patients is modified both by the cancer itself and by oncospecific treatments. Whether a patient's adaptive immunity is impaired depends on their levels of naive lymphocytes and other cell populations. During the COVID-19 pandemic, cancer patients are at greater risk of progressing to severe forms of the disease and have higher mortality rates than individuals without cancer, particularly while they are receiving cancer-specific therapies. An individual's protection against infection, their response to vaccines, and even the tests that determine the humoral immune response to SARS-CoV-2, depend on lymphocyte populations, meriting their study. OBJECTIVE: Estimate blood concentrations of lymphocytes involved in the immune response to new pathogens in cancer patients. METHODS: We carried out an analytical study of 218 cancer patients; 124 women and 94 men, 26-93 years of age, who were treated at the National Oncology and Radiobiology Institute in Havana, Cuba, March-June, 2020. Patients were divided into five groups: (1) those with controlled disease who were not undergoing cancer-specific treatment; (2) those undergoing debulking surgery; (3) patients undergoing chemotherapy; (4) patients undergoing radiation therapy and (5) patients currently battling infection. We evaluated the following peripheral blood lymphocyte subpopulations via flow cytometry: B lymphocytes (total, naive, transitional, memory, plasmablasts and plasma cells); T lymphocytes (total, helper, cytotoxic and their respective naive, activated, central memory and effector memory subsets); and total, secretory and cytotoxic natural killer cells and T natural killer cells. We also estimated neutrophil/lymphocyte ratios. Lymphocyte concentrations were associated with controlled disease and standard cancer therapy. For variables that did not fall within a normal distribution, ranges were set by medians and 2.5-97.5 percentiles. The two-tailed Mann-Whitney U test was used to measure the effect of sex and to compare lymphocyte populations. We calculated odds ratios to estimate lymphopenia risk. RESULTS: All cancer patients had lower values of naive helper and cytotoxic T lymphocyte populations, naive B lymphocytes, and natural killer cells than normal reference medians. Naive helper T cells were the most affected subpopulation. Memory B cells, plasmablasts, plasma cells, activated T helper cells, and cytotoxic central memory T cells were increased. Patients undergoing treatment had lower levels of naive lymphocytes than untreated patients, particularly during radiation therapy. The risk of B lymphopenia was higher in patients in treatment. The odds ratio for B lymphopenia was 8.0 in patients who underwent surgery, 12.9 in those undergoing chemotherapy, and 13.9 in patients in radiotherapy. CONCLUSIONS: Cancer and conventional cancer therapies significantly affect peripheral blood B lymphocyte levels, particularly transitional T helper lymphocytes, reducing the immune system's ability to trigger primary immune responses against new antigens.

In vivo Overexpression of Electrogenic Sodium/Bicarbonate Cotransporter (NBCe1) by AAV9 Modifies the Cardiac Action Potential and the QT Interval in Mice.

Cardiac cells depend on specific sarcolemmal ion transporters to assure the correct intracellular pH regulation. The sodium/bicarbonate cotransporter (NBC) is one of the major alkalinizing mechanisms. In the heart two different NBC isoforms have been described: the electroneutral NBCn1 (1Na+:1 HCO 3 - ) and the electrogenic NBCe1 (1Na+:2 HCO 3 - ). NBCe1 generates an anionic repolarizing current that modulates the action potential duration (APD). In addition to regulating the pH, the NBC is a source of sodium influx. It has been postulated that NBC could play a role in the development of hypertrophy. The aim of this research was to study the contribution of NBCe1 in heart electrophysiology and in the development of heart hypertrophy in an in vivo mouse model with overexpression of NBCe1. Heart NBCe1 overexpression was achieved by a recombinant cardiotropic adeno-associated virus (AAV9) and was evidenced by western-blot and qPCR. AAV9-mCherry was used as a transduction control. NBCe1 overexpression fails to increase heart growth. Patch clamp and electrocardiogram were performed. We observed a reduction on both, ventricular myocytes APD and electrocardiogram QT interval corrected by cardiac rate, emphasizing for the first time NBCe1 relevance for the electrical activity of the heart.

Interplay between SERCA, 4E-BP, and eIF4E in the Drosophila heart.

Appropriate cardiac performance depends on a tightly controlled handling of Ca2+ in a broad range of species, from invertebrates to mammals. The role of the Ca2+ ATPase, SERCA, in Ca2+ handling is pivotal, and its activity is regulated, inter alia, by interacting with distinct proteins. Herein, we give evidence that 4E binding protein (4E-BP) is a novel regulator of SERCA activity in Drosophila melanogaster during cardiac function. Flies over-expressing 4E-BP showed improved cardiac performance in young individuals associated with incremented SERCA activity. Moreover, we demonstrate that SERCA interacts with translation initiation factors eIF4E-1, eIF4E-2 and eIF4E-4 in a yeast two-hybrid assay. The specific identification of eIF4E-4 in cardiac tissue leads us to propose that the interaction of elF4E-4 with SERCA may be the basis of the cardiac effects observed in 4E-BP over-expressing flies associated with incremented SERCA activity.

Discordant Ca2+ release in cardiac myocytes: characterization and susceptibility to pharmacological RyR2 modulation.

Systolic Ca2+ transients are shaped by the concerted summation of Ca2+ sparks across cardiomyocytes. At high pacing rates, alterations of excitation-contraction coupling manifest as pro-arrhythmic Ca2+ alternans that can be classified as concordant or discordant. Discordance is ascribed to out-of-phase alternation of local Ca2+ release across the cell, although the triggers and consequences of this phenomenon remain unclear. Rat ventricular cardiomyocytes were paced at increasing rates. A discordance index (SD of local alternans ratios) was developed to quantify discordance in confocal recordings of Ca2+ transients. Index values were significantly increased by rapid pacing, and negatively correlated with Ca2+ transient amplitude change, indicating that discordance is an important contributor to the negative Ca2+ transient-frequency relationship. In addition, the largest local calcium transient in two consecutive transients was measured to build a potential "best release" profile, which quantitatively confirmed discordance-induced Ca2+ release impairment (DICRI). Diastolic Ca2+ homeostasis was also observed to be disrupted by discordance, as late Ca2+ release events elicited instability of resting Ca2+ levels. Finally, the effects of two RyR2 inhibitors (VK-II-86 and dantrolene) were tested. While both compounds inhibited Ca2+ wave generation, only VK-II-86 augmented subcellular discordance. Discordant Ca2+ release is a quantifiable phenomenon, sensitive to pacing frequency, and impairs both systolic and diastolic Ca2+ homeostasis. Interestingly, RyR2 inhibition can induce discordance, which should be considered when evaluating pharmacological RyR2 modulators for clinical use.

Implications of Low-grade Inflammation in SARS-CoV-2 Immunopathology.

INTRODUCTION: Advanced age and chronic disease comorbidities are indicators of poor prognosis in COVID-19 clinical progression. Fatal outcomes in patients with these characteristics are due to a dysfunctional immune response. Understanding COVID-19's immunopathogenesis helps in designing strategies to prevent and mitigate complications during treatment. OBJECTIVE: Describe the main immunopathogenic alterations of COVID-19 in patients of advanced age or with chronic non-communicable diseases. DATA ACQUISITION: We carried out a bibliographic search of primary references in PubMed, Elsevier, Science Direct and SciELO. A total of 270 articles met our initial search criteria. Duplicate articles or those unrelated to at least one chronic comorbidity, senescence or inflammation and those that studied only patient clinical characteristics, laboratory tests or treatments were excluded. Finally, our selection included 124 articles for analysis: 10 meta-analyses, 24 original research articles, 67 review articles, 9 editorials, 9 comments, 3 books and 2 websites. DEVELOPMENT: Hypertension and diabetes mellitus are the most common comorbidities in COVID-19 patients. Risk of developing severe manifestations of the disease, including death, is increased in senescent and obese patients and those with cardiovascular disease, cancer or chronic obstructive pulmonary disease. Low-grade chronic inflammation is characteristic of all these conditions, reflected in a pro-inflammatory state, endothelial dysfunction, and changes to innate immunity; mainly of the monocyte-macrophage system with changes in polarization, inflammation, cytotoxicity and altered antigenic presentation. In the case of SARS-CoV-2 infection, mechanisms involved in acute inflammation overlap with the patient's pro-inflammatory state, causing immune system dysfunction. SARS-CoV-2 infection amplifies already-existing alterations, causing failures in the immune system's control mechanisms. The resulting cytokine storm causes an uncontrolled systemic inflammatory response marked by high serum levels of inflammatory biomarkers and a pro-inflammatory cytokine profile with decompensation of underlying diseases. In asthma, chronic eosinophilic inflammation protects against infection by producing a reduced interferon-mediated response and a reduced number of ACE2 receptors. CONCLUSIONS: Low-grade chronic inflammation present in advanced age and chronic diseases-but not in bronchial asthma-produces a pro-inflammatory state that triggers a dysregulated immune response, favoring development of severe forms of COVID-19 and increasing lethality.

Smoking flies: testing the effect of tobacco cigarettes on heart function of Drosophila melanogaster.

Studies about the relationship between substances consumed by humans and their impact on health, in animal models, have been a challenge due to differences between species in the animal kingdom. However, the homology of certain genes has allowed extrapolation of certain knowledge obtained in animals. Drosophila melanogaster, studied for decades, has been widely used as model for human diseases as well as to study responses associated with the consumption of several substances. In the present work we explore the impact of tobacco consumption on a model of 'smoking flies'. Throughout these experiments, we aim to provide information about the effects of tobacco consumption on cardiac physiology. We assessed intracellular calcium handling, a phenomenon underlying cardiac contraction and relaxation. Flies chronically exposed to tobacco smoke exhibited an increased heart rate and alterations in the dynamics of the transient increase of intracellular calcium in myocardial cells. These effects were also evident under acute exposure to nicotine of the heart, in a semi-intact preparation. Moreover, the alpha 1 and 7 subunits of the nicotinic receptors are involved in the heart response to tobacco and nicotine under chronic (in the intact fly) as well as acute exposure (in the semi-intact preparation). The present data elucidate the implication of the intracellular cardiac pathways affected by nicotine on the heart tissue. Based on the probed genetic and physiological similarity between the fly and human heart, cardiac effects exerted by tobacco smoke in Drosophila advances our understanding of the impact of it in the human heart. Additionally, it may also provide information on how nicotine-like substances, e.g. neonicotinoids used as insecticides, affect cardiac function.This article has an associated First Person interview with the first author of the paper.

A low-cost Portable Device to Deliver Smoke, Volatile or Vaporized Substances to Drosophila melanogaster, Useful for Research and/or Educational Assays.

Drosophila melanogaster has been used to test drugs of abuse, substances with potential benefits for medical purposes, as well as contaminants and hazardous volatile compounds. This model has also been used for the characterization of behavioral changes, physiopathological consequences, and subcellular mechanisms of the use of cocaine, methamphetamines, ethanol, nicotine, cannabinoids, toluene, and other airborne volatile organic compounds. When testing these substances, routes of administration are important to define. Admixing the test compounds with water or food is one suitable option in many cases, but the inhalation route is especially suitable when the administration of one or more volatile compounds is desired. One advantage of the administration of substances via the inhalation route is its rapid exchange and distribution throughout the cuticle and the tracheal system. In addition, this route allows treating a large group of individuals simultaneously. Moreover, the inhalation route is frequently used to administer different drugs to humans. A good model system shares physiology and molecular pathways with humans, and D. melanogaster possesses almost 75% homologous genes associated with human diseases. Methodologies to deliver the abovementioned substances usually include customized devices. Herein, we focus on the development of a low-cost customized device useful to deliver smoke or vaporizable compounds to D. melanogaster. This approach might be applied for acute or chronic exposure to vaporized substances. In particular, our device was utilized for testing cigarette smoke and vaporized cannabis extract on cardiac performance of adult individuals during chronic treatment. We are describing how to set up this low-cost portable device, useful for research and/or educational assays, taking advantage of the amenability of D. melanogaster to test different compounds in relatively short periods, and especially including a large number of individuals at the same time. Graphic abstract: Custom-made device useful for inhalation pathway assays in Drosophila melanogaster.

Determinants of Ca2+ release restitution: Insights from genetically altered animals and mathematical modeling.

Each heartbeat is followed by a refractory period. Recovery from refractoriness is known as Ca2+ release restitution (CRR), and its alterations are potential triggers of Ca2+ arrhythmias. Although the control of CRR has been associated with SR Ca2+ load and RYR2 Ca2+ sensitivity, the relative role of some of the determinants of CRR remains largely undefined. An intriguing point, difficult to dissect and previously neglected, is the possible independent effect of SR Ca2+ content versus the velocity of SR Ca2+ refilling on CRR. To assess these interrogations, we used isolated myocytes with phospholamban (PLN) ablation (PLNKO), knock-in mice with pseudoconstitutive CaMKII phosphorylation of RYR2 S2814 (S2814D), S2814D crossed with PLNKO mice (SDKO), and a previously validated human cardiac myocyte model. Restitution of cytosolic Ca2+ (Fura-2 AM) and L-type calcium current (ICaL; patch-clamp) was evaluated with a two-pulse (S1/S2) protocol. CRR and ICaL restitution increased as a function of the (S2-S1) coupling interval, following an exponential curve. When SR Ca2+ load was increased by increasing extracellular [Ca2+] from 2.0 to 4.0 mM, CRR and ICaL restitution were enhanced, suggesting that ICaL restitution may contribute to the faster CRR observed at 4.0 mM [Ca2+]. In contrast, ICaL restitution did not differ among the different mouse models. For a given SR Ca2+ load, CRR was accelerated in S2814D myocytes versus WT, but not in PLNKO and SDKO myocytes versus WT and S2814D, respectively. The model mimics all experimental data. Moreover, when the PLN ablation-induced decrease in RYR2 expression was corrected, the model revealed that CRR was accelerated in PLNKO and SDKO versus WT and S2814D myocytes, consistent with the enhanced velocity of refilling, SR [Ca2+] recovery, and CRR. We speculate that refilling rate might enhance CRR independently of SR Ca2+ load.

Normal Values of T, B and NK Lymphocyte Subpopulations in Peripheral Blood of Healthy Cuban Adults.

INTRODUCTION Quantification of lymphocyte subpopulations is useful for evaluating immune response in states of health and disease, including immunodeficiencies, autoimmunity, infections and cancer. Studies have found that concentrations and proportions of different cell subpopulations vary with geographic location, age, sex and ethnicity. Knowing the normal values of these cells and their variation in healthy populations will contribute to improved clinical practice and scientific research. OBJECTIVE Estimate normal absolute concentrations and percentages of the most abundant lymphocyte subpopulations in peripheral blood and their relation to sex and age. METHODS A cross-sectional analysis was conducted in 129 healthy adults, 61 men and 68 women aged 18-80 years; 89 aged <50 years and 40 ≥50 years. We included individuals who agreed to participate by written informed consent. Exclusion criteria were chronic disease, or use of tobacco, alcohol or medications that can alter immune system cell numbers and functions. Through dual platform flow cytometry, we determined absolute and percentage values for T lymphocyte subsets CD3+, CD3+/CD4+T, CD3+/CD8+T, CD19+ B cells and CD3-/CD56+ natural killer cells in peripheral blood, using an 8-color flow cytometer. We estimated medians and the 2.5 and 97.5 percentiles and calculated the Pearson correlation coefficient to evaluate associations. Significance tests were also used to compare groups. The significance threshold was p = 0.05 in all cases. RESULTS Ranges of absolute values and percentages (%) were: total lymphocytes: 1200-3475 cells/µL (20.2-49.3); CD3+ T cells: 880-2623 cells/µL (56.5-84.7); CD3+/CD4+ T cells: 479-1792 cells/µL (30.3-55.7); CD3+/CD8+ T cells: 248-1101 cells/µL (13.2-42.9); CD19+ B cells: 114-1491 cells/µL (5.4-49.5); CD3-/CD56+ natural killer cells: 70-652 cells/µL (3.7-28.0); and the CD4+:CD8+ index: 0.80-3.92. Absolute numbers--but not percentages--of lymphocytes and CD3+ T cells were higher in those <50 years (p = 0.025 and 0.020, respectively). Absolute values and relative percentages of CD3+/CD8+ and relative values of CD3+/ CD4+ T cells were significantly higher in the younger subgroup (p = 0.004 and p = 0.047). Age was not associated significantly with B lymphocytes or natural killer cells. Absolute and relative values ​​of CD3+/CD4+ T lymphocytes were significantly higher in women (p = 0.009 and 0.036, respectively). CONCLUSIONS. Absolute numbers of total lymphocytes and T and CD3+/CD8+ T lymphocytes are higher in younger individuals. In percentage values, CD3+/CD4+ T lymphocytes are lower in older persons. Absolute and percentage values ​​of CD3+/CD4+ T phenotype are higher in women. These differences justify adjusting clinical analyses to different values ​​by age and sex. KEYWORDS T lymphocytes, B lymphocytes, normal values, flow cytometry, age, sex, Cuba.

Inhalation of marijuana affects Drosophila heart function.

We investigated the effect of inhalation of vaporized marijuana on cardiac function in Drosophila melanogaster, a suitable genetic model for studying human diseases. Adult flies were exposed to marijuana for variable time periods and the effects on cardiac function were studied. Short treatment protocol incremented heart-rate variability. Contractility was augmented only under prolonged exposure to cannabis and it was associated with incremented calcium transient within cardiomyocytes. Neither the activity of the major proteins responsible for calcium handling nor the calcium load of the sarcoplasmic reticulum were affected by the cannabis treatment. The observed changes manifested in the cardiomyocytes even in the absence of the canonical cannabinoid receptors described in mammals. Our results are the first evidence of the in vivo impact of phytocannabinoids in D. melanogaster. By providing a simple and affordable platform prior to mammalian models, this characterization of cardiac function under marijuana exposure opens new paths for conducting genetic screenings using vaporized compounds.This article has an associated First Person interview with the first author of the paper.

Unbalance Between Sarcoplasmic Reticulum Ca2 + Uptake and Release: A First Step Toward Ca2 + Triggered Arrhythmias and Cardiac Damage.

The present review focusses on the regulation and interplay of cardiac SR Ca2+ handling proteins involved in SR Ca2+ uptake and release, i.e., SERCa2/PLN and RyR2. Both RyR2 and SERCA2a/PLN are highly regulated by post-translational modifications and/or different partners' proteins. These control mechanisms guarantee a precise equilibrium between SR Ca2+ reuptake and release. The review then discusses how disruption of this balance alters SR Ca2+ handling and may constitute a first step toward cardiac damage and malignant arrhythmias. In the last part of the review, this concept is exemplified in different cardiac diseases, like prediabetic and diabetic cardiomyopathy, digitalis intoxication and ischemia-reperfusion injury.

Ablation of phospholamban rescues reperfusion arrhythmias but exacerbates myocardium infarction in hearts with Ca2+/calmodulin kinase II constitutive phosphorylation of ryanodine receptors.

AIMS: Abnormal Ca2+ release from the sarcoplasmic reticulum (SR), associated with Ca2+-calmodulin kinase II (CaMKII)-dependent phosphorylation of RyR2 at Ser2814, has consistently been linked to arrhythmogenesis and ischaemia/reperfusion (I/R)-induced cell death. In contrast, the role played by SR Ca2+ uptake under these stress conditions remains controversial. We tested the hypothesis that an increase in SR Ca2+ uptake is able to attenuate reperfusion arrhythmias and cardiac injury elicited by increased RyR2-Ser2814 phosphorylation. METHODS AND RESULTS: We used WT mice, which have been previously shown to exhibit a transient increase in RyR2-Ser2814 phosphorylation at the onset of reperfusion; mice with constitutive pseudo-phosphorylation of RyR2 at Ser2814 (S2814D) to exacerbate CaMKII-dependent reperfusion arrhythmias and cardiac damage, and phospholamban (PLN)-deficient-S2814D knock-in (SDKO) mice resulting from crossbreeding S2814D with phospholamban knockout deficient (PLNKO) mice. At baseline, S2814D and SDKO mice had structurally normal hearts. Moreover none of the strains were arrhythmic before ischaemia. Upon cardiac I/R, WT, and S2814D hearts exhibited abundant arrhythmias that were prevented by PLN ablation. In contrast, PLN ablation increased infarct size compared with WT and S2814D hearts. Mechanistically, the enhanced SR Ca2+ sequestration evoked by PLN ablation in SDKO hearts prevented arrhythmogenic events upon reperfusion by fragmenting SR Ca2+ waves into non-propagated and non-arrhythmogenic events (mini-waves). Conversely, the increase in SR Ca2+ sequestration did not reduce but rather exacerbated I/R-induced SR Ca2+ leak, as well as mitochondrial alterations, which were greatly avoided by inhibition of RyR2. These results indicate that the increase in SR Ca2+ uptake is ineffective in preventing the enhanced SR Ca2+ leak of PLN ablated myocytes from either entering into nearby mitochondria and/or activating additional CaMKII pathways, contributing to cardiac damage. CONCLUSION: Our results demonstrate that increasing SR Ca2+ uptake by PLN ablation can prevent the arrhythmic events triggered by CaMKII-dependent phosphorylation of RyR2-induced SR Ca2+ leak. These findings underscore the benefits of increasing SERCA2a activity in the face of SR Ca2+ triggered arrhythmias. However, enhanced SERCA2a cannot prevent but rather exacerbates I/R cardiac injury.

Estimating Normal Values of Rare T-Lymphocyte Populations in Peripheral Blood of Healthy Cuban Adults.

INTRODUCTION Flow cytometry allows immunophenotypic characterization of important lymphocyte subpopulations for diagnosis of diseases such as cancer, autoimmune diseases, immunodeficiencies and some infections. Normal values of rare lymphoid cells in blood, quantified by cytometry, vary among different populations; so it is indispensable to obtain normal national values that can be used in clinical practice. OBJECTIVE Characterize distribution of rare T-lymphocyte populations in peripheral blood, specifically double-positive T, natural killer T and activated T lymphocytes, as well as their relationship to sex and age. METHODS A cross-sectional study was carried out in 129 adults (68 women, 61 men) aged >18 years, without chronic diseases or unhealthy habits, who signed informed consent. Peripheral blood was collected for immunophenotyping of lymphocyte subpopulations with monoclonal antibodies specific for CD4+CD8+ double-positive T cells, CD3+CD56+ natural killer T cells, and CD3+CD25+HLA-DR+ activated T cells. An eight-color flow cytometer (Beckman Coulter Gallios) was used. The analytic strategy was modified, associating variables of interest in a single graphic, using conventional monoclonal labeling antibodies. Medians and minimum and maximum percentiles (2.5 and 97.5, respectively) were used as descriptive statistics, stratified by sex, for cell counts and percentages. A linear regression model was applied to assess age effects and a two-tailed Mann-Whitney U test for independent samples was used to assess sex differences. The significance threshold was set as p ≤0.05. RESULTS Median percentages of total lymphocytes: natural killer T cells 6.3% (1.4%-23%) in men and 4.7% (0.8%-11.3%) in women (p = 0.003); activated T cells 1.0% (0.2%-2.2%) in men and 1.2% (0.4%-3.1%) in women, without statistical significance; and double positives 0.8% (0.1%-4.2%) in men and 0.9% (0.3-5.1) in women, also without statistical significance. Median cell counts (cells/mL) were: natural killer T cells, 126 (27-580) in men and 105 (20-279) in women (p = 0.023); activated T cells: 20 (4-46) in men and 25 (7-75) in women, (p = 0.013) and double-positive T cells: 17 (2-85) in men and 21 (7-154) in women, without statistical significance. Sex influenced natural killer T cells, but age did not. CONCLUSIONS Age does not affect counts and percentages of rare T lymphocyte subpopulations in the blood of healthy Cuban adults. Sex differences found for some phenotypes suggest the need for different reference values for women and men.

Ryanodine receptor phosphorylation by CaMKII promotes spontaneous Ca(2+) release events in a rodent model of early stage diabetes: The arrhythmogenic substrate.

BACKGROUND: Heart failure and arrhythmias occur more frequently in patients with type 2 diabetes (T2DM) than in the general population. T2DM is preceded by a prediabetic condition marked by elevated reactive oxygen species (ROS) and subclinical cardiovascular defects. Although multifunctional Ca2+ calmodulin-dependent protein kinase II (CaMKII) is ROS-activated and CaMKII hyperactivity promotes cardiac diseases, a link between prediabetes and CaMKII in the heart is unprecedented. OBJECTIVES: To prove the hypothesis that increased ROS and CaMKII activity contribute to heart failure and arrhythmogenic mechanisms in early stage diabetes. METHODS-RESULTS: Echocardiography, electrocardiography, biochemical and intracellular Ca2+ (Ca2+i) determinations were performed in fructose-rich diet-induced impaired glucose tolerance, a prediabetes model, in rodents. Fructose-rich diet rats showed decreased contractility and hypertrophy associated with increased CaMKII activity, ROS production, oxidized CaMKII and enhanced CaMKII-dependent ryanodine receptor (RyR2) phosphorylation compared to rats fed with control diet. Isolated cardiomyocytes from fructose-rich diet showed increased spontaneous Ca2+i release events associated with spontaneous contractions, which were prevented by KN-93, a CaMKII inhibitor, or addition of Tempol, a ROS scavenger, to the diet. Moreover, fructose-rich diet myocytes showed increased diastolic Ca2+ during the burst of spontaneous Ca2+i release events. Mice treated with Tempol or with sarcoplasmic reticulum-targeted CaMKII-inhibition by transgenic expression of the CaMKII inhibitory peptide AIP, were protected from fructose-rich diet-induced spontaneous Ca2+i release events, spontaneous contractions and arrhythmogenesis in vivo, despite ROS increases. CONCLUSIONS: RyR2 phosphorylation by ROS-activated CaMKII, contributes to impaired glucose tolerance-induced arrhythmogenic mechanisms, suggesting that CaMKII inhibition could prevent prediabetic cardiovascular complications and/or evolution.

Chasing cardiac physiology and pathology down the CaMKII cascade.

Calcium dynamics is central in cardiac physiology, as the key event leading to the excitation-contraction coupling (ECC) and relaxation processes. The primary function of Ca(2+) in the heart is the control of mechanical activity developed by the myofibril contractile apparatus. This key role of Ca(2+) signaling explains the subtle and critical control of important events of ECC and relaxation, such as Ca(2+) influx and SR Ca(2+) release and uptake. The multifunctional Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) is a signaling molecule that regulates a diverse array of proteins involved not only in ECC and relaxation but also in cell death, transcriptional activation of hypertrophy, inflammation, and arrhythmias. CaMKII activity is triggered by an increase in intracellular Ca(2+) levels. This activity can be sustained, creating molecular memory after the decline in Ca(2+) concentration, by autophosphorylation of the enzyme, as well as by oxidation, glycosylation, and nitrosylation at different sites of the regulatory domain of the kinase. CaMKII activity is enhanced in several cardiac diseases, altering the signaling pathways by which CaMKII regulates the different fundamental proteins involved in functional and transcriptional cardiac processes. Dysregulation of these pathways constitutes a central mechanism of various cardiac disease phenomena, like apoptosis and necrosis during ischemia/reperfusion injury, digitalis exposure, post-acidosis and heart failure arrhythmias, or cardiac hypertrophy. Here we summarize significant aspects of the molecular physiology of CaMKII and provide a conceptual framework for understanding the role of the CaMKII cascade on Ca(2+) regulation and dysregulation in cardiac health and disease.

Aging and CaMKII alter intracellular Ca2+ transients and heart rhythm in Drosophila melanogaster.

Aging is associated to disrupted contractility and rhythmicity, among other cardiovascular alterations. Drosophila melanogaster shows a pattern of aging similar to human beings and recapitulates the arrhythmogenic conditions found in the human heart. Moreover, the kinase CaMKII has been characterized as an important regulator of heart function and an arrhythmogenic molecule that participate in Ca2+ handling. Using a genetically engineered expressed Ca2+ indicator, we report changes in cardiac Ca2+ handling at two different ages. Aging prolonged relaxation, reduced spontaneous heart rate (HR) and increased the occurrence of arrhythmias, ectopic beats and asystoles. Alignment between Drosophila melanogaster and human CaMKII showed a high degree of conservation and indicates that relevant phosphorylation sites in humans are also present in the fruit fly. Inhibition of CaMKII by KN-93 (CaMKII-specific inhibitor), reduced HR without significant changes in other parameters. By contrast, overexpression of CaMKII increased HR and reduced arrhythmias. Moreover, it increased fluorescence amplitude, maximal rate of rise of fluorescence and reduced time to peak fluorescence. These results suggest that CaMKII in Drosophila melanogaster acts directly on heart function and that increasing CaMKII expression levels could be beneficial to improve contractility.