CaM kinase II regulates cardiac hemoglobin expression through histone phosphorylation upon sympathetic activation.

Sympathetic activation of ß-adrenoreceptors (ß-AR) represents a hallmark in the development of heart failure (HF). However, little is known about the underlying mechanisms of gene regulation. In human ventricular myocardium from patients with end-stage HF, we found high levels of phosphorylated histone 3 at serine-28 (H3S28p). H3S28p was increased by inhibition of the catecholamine-sensitive protein phosphatase 1 and decreased by ß-blocker pretreatment. By a series of in vitro and in vivo experiments, we show that the ß-AR downstream protein kinase CaM kinase II (CaMKII) directly binds and phosphorylates H3S28. Whereas, in CaMKII-deficient myocytes, acute catecholaminergic stimulation resulted in some degree of H3S28p, sustained catecholaminergic stimulation almost entirely failed to induce H3S28p. Genome-wide analysis of CaMKII-mediated H3S28p in response to chronic ß-AR stress by chromatin immunoprecipitation followed by massive genomic sequencing led to the identification of CaMKII-dependent H3S28p target genes. Forty percent of differentially H3S28p-enriched genomic regions were associated with differential, mostly increased expression of the nearest genes, pointing to CaMKII-dependent H3S28p as an activating histone mark. Remarkably, the adult hemoglobin genes showed an H3S28p enrichment close to their transcriptional start or end sites, which was associated with increased messenger RNA and protein expression. In summary, we demonstrate that chronic ß-AR activation leads to CaMKII-mediated H3S28p in cardiomyocytes. Thus, H3S28p-dependent changes may play an unexpected role for cardiac hemoglobin regulation in the context of sympathetic activation. These data also imply that CaMKII may be a yet unrecognized stress-responsive regulator of hematopoesis.

Diabetes changes gene expression but not DNA methylation in cardiac cells.

BACKGROUND: Diabetes mellitus is a worldwide epidemic that causes high mortality due to cardiovascular complications, in particular heart failure. Diabetes is associated with profound pathophysiological changes in the heart. The aim of this study was to investigate the impact of diabetes on gene expression and DNA methylation in cardiac cells. METHODS AND RESULTS: Transcriptome analysis of heart tissue from mice with streptozotocin-induced diabetes revealed only 39 genes regulated, whereas cell type-specific analysis of the diabetic heart was more sensitive and more specific than heart tissue analysis and revealed a total of 3205 differentially regulated genes in five cell types. Whole genome DNA methylation analysis with basepair resolution of distinct cardiac cell types identified highly specific DNA methylation signatures of genic and regulatory regions. Interestingly, despite marked changes in gene expression, DNA methylation remained stable in streptozotocin-induced diabetes. Integrated analysis of cell type-specific gene expression enabled us to assign the particular contribution of single cell types to the pathophysiology of the diabetic heart. Finally, analysis of gene regulation revealed ligand-receptor pairs as potential mediators of heterocellular interaction in the diabetic heart, with fibroblasts and monocytes showing the highest degree of interaction. CONCLUSION: In summary, cell type-specific analysis reveals differentially regulated gene programs that are associated with distinct biological processes in diabetes. Interestingly, despite these changes in gene expression, cell type-specific DNA methylation signatures of genic and regulatory regions remain stable in diabetes. Analysis of heterocellular interactions in the diabetic heart suggest that the interplay between fibroblasts and monocytes is of pivotal importance.

Maternal, Fetal, and Neonatal Outcomes of Gestation in Women with and Without Brucella Infection.

BACKGROUND: Maternal, fetal, and neonatal complications of brucellosis in pregnant women are probably higher than those in the general population. This comparative study aimed to survey the mentioned complications in pregnant women with positive and negative Brucella serologic tests. STUDY DESIGN: This is a prospective cohort study. METHODS: In this study, 2160 pregnant women residing in the rural area of Hamadan province were screened for Brucella infection by agglutination test. Then, 106 (4.90%) pregnant women with a positive test (exposed group) were compared with 210 subjects (non-exposed group) who were randomly selected from more than 2000 pregnant women with a negative serological test in terms of maternal, fetal, and neonatal outcomes from October 2018 to March 2020. Data were analyzed by SPSS 20 software at a 95% confidence level. RESULTS: The mean age of mothers in both exposed and unexposed groups was 27.84±6.13 and 38.71±6.85 years, respectively. Past medical history of brucellosis, animal contact, and the consumption of unpasteurized dairy products were reported to be 14 (13.2%), 63 (59.4%), and 82 (77.4%), respectively, in the exposed group. The mentioned measures were 3 (1.5%), 109 (51.9%), and 54 (26.9%) in the unexposed group, respectively. Among exposed and unexposed groups, the incidence of abortion was 9 (8.6%) and 5 (2.4%) with P=0.005, intrauterine fetal death was 2 (1.9%) and zero with P=0.211, low birth weight was 10 (10.6%) and 7 (3.4%) with P=0.012, and premature birth was 15 (15.2%) and 18 (8.8%) with P=0.066, respectively. CONCLUSION: Brucella infection in pregnant women appears to be associated with the risk of miscarriage, low birth weight, and premature birth.

The Prevalence of Human Immunodeficiency Virus Infection in Patients with Sexually Transmitted Diseases.

BACKGROUND: Human immunodeficiency virus (HIV) transmission pattern in Iran has been changed from injection drug to sexual contact. Lack of accurate assessment of HIV in people with sexually transmitted diseases (STDs) in Iran prompted us to conduct this study to determine the frequency of HIV infection in these patients. MATERIALS AND METHODS: In this cross-sectional study which conducted in 2016-2017, overall, 190 patients with STDs referring to two hospitals of Hamadan were enrolled in the study. All of the patients were examined for HIV in the first visit by rapid test and then 1 and 4 months later by the 4th generation ELISA. A questionnaire including demographic data, clinical manifestations, and high-risk behaviors was completed for all of the referring people. The collected data were analyzed using appropriate statistical tests. RESULTS: Of 190 patients, 126 (66.3%) were female with a mean age of 34.1 ± 10.1 years and 64 (33.7%) were male with a mean age of 30.8 ± 7.8 years. One hundred twenty-eight (67.4%) got married, 73 (38.4%) and 76 (40%) had a diploma and postgraduate education, respectively, 32 (16.8%) mentioned the history of unsafe sex, and 23 (12.1%) had used condoms continuously during sexual contacts. The most common STDs were reported genital warts, 107 patients (56.3%), vaginal discharge (28, 14.7%), and genital ulcer (33, 17.4%). Two (1%) patients were positive for HIV at the first visit. CONCLUSION: Patients with STDs should be considered as an important source of HIV transmission, so clinicians should pay more attention to screening these patients for HIV infection.

Comparison of Quantiferon-TB Gold and TST tests in the diagnosis of latent tuberculosis infection among HIV infected patients in Hamadan, west of Iran.

INTRODUCTION: Human immunodeficiency virus (HIV) infection increases the susceptibility of patients for latent tuberculosis infection (LTBI) and reactivtion tuberculosis. This study aimed to compare the Quantiferon-TB gold-in tube test (QFT) with tuberculin skin test (TST) in the diagnosis of LTBI in HIV infected patients. METHODOLOGY: This comparative study of 89 patients with HIV in the Behavioral Diseases Counseling Center in Hamadan was carried out from July 2015 to November 2016. After obtaining consent from the patients, all demographic data, clinical manifestations, and laboratory results (CD4 count, TST and QFT) were entered into the questionnaires. The CD4 count is usually routinely performed using flow cytometry at the Behavioral Counselling Center. Quantiferon-TB test was done by using Qiagen - Quantiferon-2 plate kit ELISA. RESULTS: Totally, 89 HIV infected patients with the mean age of 39.55 ± 10.31 years old were enrolled in the study. Sixty patients (67.42%) were male. The mean duration of HIV infection was 4.44 ± 3.88 years and the mean of CD4 count was 388.65 ± 260.66 cells/µL . Twenty patients had LTBI based on TST. Considering the QFT intermediate results as a positive test, the percent agreement of QFT and TST was 59.55%, which was not statistically significant (P = 0.2387). CONCLUSIONS: According to the results, there was no significant percent agreement between QFT and TST for detecting LTBI in HIV infected patients. However, by decreasing CD4 counts, there was a significant relation between TST positive and LTBI in HIV patients.

Chronic loss of inhibitor-1 diminishes cardiac RyR2 phosphorylation despite exaggerated CaMKII activity.

Inhibitor-1 (I-1) modulates protein phosphatase 1 (PP1) activity and thereby counteracts the phosphorylation by kinases. I-1 is downregulated and deactivated in failing hearts, but whether its role is beneficial or detrimental remains controversial, and opposing therapeutic strategies have been proposed. Overactivity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) with hyperphosphorylation of ryanodine receptors (RyR2) at the CaMKII-site is recognized to be central for heart failure and arrhythmias. Using an I-1-deficient mouse line as well as transfected cell lines, we investigated the effects of acute and chronic modulation of I-1 on CaMKII activity and RyR2 phosphorylation. We demonstrate that I-1 acutely modulates CaMKII by regulating PP1 activity. However, while ablation of I-1 should thus limit CaMKII-activation, we unexpectedly found exaggerated CaMKII-activation under ß-adrenergic stress upon chronic loss of I-1 in knockout mice. We unraveled that this is due to chronic upregulation of the exchange protein activated by cAMP (EPAC) leading to augmented CaMKII activation, and using computational modeling validated that an increase in EPAC expression can indeed explain our experimental findings. Interestingly, at the level of RyR2, the increase in PP1 activity more than outweighed the increase in CaMKII activity, resulting in reduced RyR phosphorylation at Ser-2814. Exaggerated CaMKII activation due to counterregulatory mechanisms upon loss of I-1 is an important caveat with respect to suggested therapeutic I-1-inhibition, as CaMKII overactivity has been heavily implicated in several cardiac pathologies.

The poorly membrane permeable antipsychotic drugs amisulpride and sulpiride are substrates of the organic cation transporters from the SLC22 family.

Variations in influx transport at the blood-brain barrier might affect the concentration of psychotropic drugs at their site of action and as a consequence might alter therapy response. Furthermore, influx transporters in organs such as the gut, liver and kidney may influence absorption, distribution, and elimination. Here, we analyzed 30 commonly used psychotropic drugs using a parallel artificial membrane permeability assay. Amisulpride and sulpiride showed the lowest membrane permeability (P e < 1.5 × 10(-6) cm/s) and will require influx transport to penetrate the blood-brain barrier and other physiological barriers. We then studied the uptake of amisulpride and sulpiride by the organic cation transporters of the SLC22 family OCT1, OCT2, OCT3, OCTN1, and OCTN2 Amisulpride was found to be transported by all five transporters studied. In contrast, sulpiride was only transported by OCT1 and OCT2. OCT1 showed the highest transport ability both for amisulpride (CLint = 1.9 ml/min/mg protein) and sulpiride (CLint = 4.2 ml/min/mg protein) and polymorphisms in OCT1 significantly reduced the uptake of both drugs. Furthermore, we observed carrier-mediated uptake that was inhibitable by known OCT inhibitors in the immortalized human brain microvascular endothelial cell line hCMEC/D3. In conclusion, this study demonstrates that amisulpride and sulpiride are substrates of organic cation transporters of the SLC22 family. SLC22 transporters may play an important role in the distribution of amisulpride and sulpiride, including their ability to penetrate the blood-brain barrier.

Morphine is a substrate of the organic cation transporter OCT1 and polymorphisms in OCT1 gene affect morphine pharmacokinetics after codeine administration.

We investigated whether morphine and its pro-drug codeine are substrates of the highly genetically polymorphic organic cation transporter OCT1 and whether OCT1 polymorphisms may affect morphine and codeine pharmacokinetics in humans. Morphine showed low transporter-independent membrane permeability (0.5 × 10⁻6 cm/s). Morphine uptake was increased up to 4-fold in HEK293 cells overexpressing human OCT1. The increase was concentration-dependent and followed Michaelis-Menten kinetics (KM = 3.4 µM, VMAX = 27 pmol/min/mg protein). OCT1-mediated morphine uptake was abolished by common loss-of-function polymorphisms in the OCT1 gene and was strongly inhibited by drug-drug interactions with irinotecan, verapamil and ondansetron. Morphine uptake in primary human hepatocytes was strongly reduced by MPP⁺, an inhibitor of organic cation transporters, and morphine was not a substrate of OCT3, the other organic cation transporter expressed in human hepatocytes. In concordance with the in vitro data, morphine plasma concentrations in healthy volunteers were significantly dependent on OCT1 polymorphisms. After codeine administration, the mean AUC of morphine was 56% higher in carriers of loss-of-function OCT1 polymorphisms compared to non-carriers (P = 0.005). The difference remained significant after adjustment for CYP2D6 genotype (P = 0.03). Codeine itself had high transporter-independent membrane permeability (8.2 × 10⁻6 cm/s). Codeine uptake in HEK293 cells was not affected by OCT1 overexpression and OCT1 polymorphisms did not affect codeine AUCs. In conclusion, OCT1 plays an important role in the hepatocellular uptake of morphine. Carriers of loss-of-function OCT1 polymorphisms may be at higher risk of adverse effects after codeine administration, especially if they are also ultra-rapid CYP2D6 metabolizers.

The prototypic pharmacogenetic drug debrisoquine is a substrate of the genetically polymorphic organic cation transporter OCT1.

Debrisoquine is a probe drug for in vivo phenotyping of human CYP2D6 metabolic activity. However, debrisoquine is positively charged under physiological conditions and it is unclear how it enters the hepatocytes to undergo CYP2D6 metabolism. We analysed whether debrisoquine is a substrate of the hepatic organic cation transporter OCT1 and whether drug-drug interactions at OCT1, or polymorphisms in OCT1 gene, affect debrisoquine uptake. Debrisoquine showed low carrier-independent membrane permeability (P(e) of 0.01×10⁻6 cm/s in artificial PAMPA membranes) and strongly inhibited the uptake of the model OCT1 substrate MPP+ (IC50 of 6.2 ± 0.8 µM). Debrisoquine uptake was significantly increased in HEK293 cells overexpressing OCT1 compared to control cells. The OCT1-mediated uptake of debrisoquine followed Michaelis-Menten kinetics (K(M) of 5.9 ± 1.5 µM and V(max) of 41.9 ± 4.5pmol/min/mg protein) and was inhibited by known OCT1 inhibitors and by commonly used drugs. OCT1-mediated debrisoquine uptake was reduced or missing in cells expressing loss-of-function OCT1 isoforms. Deletion of Met420 or substitution of Arg61Cys or Gly401Ser reduced V(max) by 48, 63 and 91%, respectively, but did not affect the K(M). The OCT1 isoforms carrying Cys88Arg or Gly465Arg substitutions completely lacked OCT1-mediated debrisoquine uptake. In conclusion, debrisoquine is a substrate of OCT1 and has the potential to be used as a phenotyping marker for OCT1 activity. Moreover, variations in debrisoquine metabolic phenotypes and their associations with diseases may be due not only to genetic variations CYP2D6, but also in OCT1.