Single acute stress-induced progesterone and ovariectomy alter cardiomyocyte contractile function in female rats

Aim

To assess how ovarian-derived sex hormones (in particular progesterone) modify the effects of single acute stress on the mechanical and biochemical properties of left ventricular cardiomyocytes in the rat.

Methods

Non-ovariectomized (control, n = 8) and ovariectomized (OVX, n = 8) female rats were kept under normal conditions or were exposed to stress (control-S, n = 8 and OVX-S, n = 8). Serum progesterone levels were measured using a chemiluminescent immunoassay. Left ventricular myocardial samples were used for isometric force measurements and protein analysis. Ca²⁺-dependent active force (F_active), Ca²⁺-independent passive force (F_passive), and Ca²⁺-sensitivity of force production were determined in single, mechanically isolated, permeabilized cardiomyocytes. Stress- and ovariectomy-induced alterations in myofilament proteins (myosin-binding protein C [MyBP-C], troponin I [TnI], and titin) were analyzed by sodium dodecyl sulfate gel electrophoresis using protein and phosphoprotein stainings.

Results

Serum progesterone levels were significantly increased in stressed rats (control-S, 35.6 ± 4.8 ng/mL and OVX-S, 21.9 ± 4.0 ng/mL) compared to control (10 ± 2.9 ng/mL) and OVX (2.8 ± 0.5 ng/mL) groups. F_active was higher in the OVX groups (OVX, 25.9 ± 3.4 kN/m² and OVX-S, 26.3 ± 3.0 kN/m²) than in control groups (control, 16.4 ± 1.2 kN/m² and control-S, 14.4 ± 0.9 kN/m²). Regarding the potential molecular mechanisms, F_active correlated with MyBP-C phosphorylation, while myofilament Ca²⁺-sensitivity inversely correlated with serum progesterone levels when the mean values were plotted for all animal groups. F_passive was unaffected by any treatment.Conclusion Stress increases ovary-independent synthesis and release of progesterone, which may regulate Ca²⁺-sensitivity of force production in left ventricular cardiomyocytes. Stress and female hormones differently alter Ca²⁺-dependent cardiomyocyte contractile force production, which may have pathophysiological importance during stress conditions affecting postmenopausal women.The relation between stress, gender, and cardiovascular diseases is well established (1-4). Some of the known risk factors for cardiovascular disease such as smoking, unhealthy diet, and behavioral and psychosocial stress have deleterious effects on the cardiovascular system via activation of the sympathetic nervous system and the hypothalamic-pituitary-adrenal (HPA) axis (5-8). Acute restraint stress is a preferred and widely used method to induce physical stress in animal models (9). Moreover, restraint and immobilization are important as models for psychological stress, which was shown to adversely affect ovarian function (10) and to play a pivotal role in the pathomechmanism of Takotsubo (stress) cardiomyopathy in postmenopausal women (11).Gender is a very important factor in the development of cardiovascular diseases. Premenopausal women have better lipid profile, endothelial function (12), and a lower risk to develop coronary artery disease and myocardial infarction (MI) than men. These advantages of female gender, however, are abolished after menopause, which is associated with increased prevalence of left ventricular (LV) hypertrophy, decreased LV ejection fraction, and LV contractility (13). One of the explanations for the distinct myocardial responses is the cardioprotective effect of female sex hormones (eg, estrogens) (14,15).Progesterone performs several actions on the heart: it exerts an antiarrhythmic effect by accelerating cardiac repolarization (16) and has a preventive role in ischemia-reperfusion injury via reducing inflammatory response (17). It has been shown to inhibit cardiomyocyte apoptosis (18), induce vasodilation, and reduce blood pressure via increasing nitric oxide (NO) levels in normotensive and hypertensive patients (19). Importantly, progesterone is produced by the both ovaries and the adrenal gland: Moreover, the adrenal progesterone content is similar or even larger than that in the ovaries (20). Adrenal progesterone production and secretion increase along with corticosterone regardless of gender and estradiol under stress conditions (21). Progesterone, being an indirect precursor of cortisol (22), increases in response to adrenocorticotrophic hormone (ACTH) stimulation (23).In the heart, there are multiple estrogen hormone receptor types (24). The expression of aromatase in the heart suggests that estrogen may be synthesized also within the cardiomyocyte to exert autocrine/paracrine actions (25). Myocyte contractility seems to be modulated by systemic estrogen levels and altered in cardiomyocytes derived from ovariectomized (OVX) rats (26). In particular, myofilament Ca²⁺-sensitivity is increased in isolated myofibrillar preparations from OVX rats, and restored to the basal levels with estrogen supplementation (27,28).Activation of the sympathetic nervous system plays a central role in the regulation of cardiomyocyte contractile function and myofilament Ca²⁺-sensitivity through beta-adrenergic receptor stimulation, activating the protein kinase A (PKA). PKA-mediated phosphorylation of Ca²⁺-handling and myofilament proteins (myosin binding protein-C [MyBP-C], troponin I [TnI], titin) were shown to alter cardiomyocyte contractile function (29,30). It has been suggested that female cardiomyocytes operate at lower levels of intracellular Ca²⁺ than those of males, particularly under inotropic conditions (31). This difference in Ca²⁺ homeostasis may be related to the fact that estrogen suppresses the L-type Ca²⁺ current (32,33) and may reduce the amount of Ca²⁺ released from the sarcoplasmic reticulum (SR) (34), which was shown to be larger in myocytes from OVX rats (35). Not only cardiomyocyte contraction, but relaxation may also be affected by estrogen via altered Ca²⁺ re-uptake into the SR and modified Ca²⁺ efflux via increased sarcolemmal Na⁺/Ca²⁺ exchange (36). Interestingly, despite similar SR Ca²⁺ content in males and females (37), studies using OVX models report conflicting results concerning changes in the expression and activity of the SR Ca²⁺-ATPase and its regulator protein phospholamban (38-41). Much less is known about the possible effect of progesterone on cardiomyocyte contractile function. We hypothesized that progesterone affected force production of single isolated cardiomyocytes. Therefore, in the present study we aimed to investigate how sex hormones (particularly progesterone) and single acute restraint stress altered cardiomyocyte contractile function and to identify the consequent posttranslational myofilament protein modifications in OVX rats.     

Cells with regulatory function of the innate and adaptive immune system in primary Sjögren's syndrome

The aim of the present study was to describe subsets of cells with regulatory properties in primary Sjögren's syndrome (pSS), and to correlate these cell populations with clinical symptoms. Among the 32 investigated patients, 23 had extraglandular manifestations (EGMs), while nine had only glandular symptoms. Twenty healthy individuals served as controls. The percentages of natural killer (NK), natural killer T cells (NK T), interleukin (IL)‐10 producing T regulatory type 1 (Tr1) cells and CD4⁺CD25⁺ regulatory T cells (T_reg) cells were determined by flow cytometry and serum cytokine levels of IL‐4, IL‐6, IL‐10, tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ were evaluated by enzyme‐linked immunosorbent assay (ELISA). Functional tests were carried out to assess the suppressor properties of T_reg cells in patients and controls. Peripheral NK, NK T and Tr1 cell percentages were elevated in pSS, while CD4⁺CD25⁺ T_reg cells showed reduced frequencies in patients compared to controls. In pSS, elevated percentages of NK T, Tr1 and CD4⁺CD25⁺ T_reg cells were observed in patients with EGMs, when compared to patients with sicca symptoms only. CD4⁺CD25⁺ T_reg cell percentages showed a negative correlation with sialometry values. The in vitro functional assay demonstrated lower suppression activity of CD4⁺CD25⁺ T_reg cells in patients compared to controls. Serum IL‐6 and TNF‐α levels were elevated, while IL‐10 was decreased in patients compared to controls. Negative correlation was found between IL‐10 levels and the percentages of Tr1 cells. Changes in the investigated subsets of regulatory cells in pSS may contribute to the development and progression of the disease.     

Risk of recurrence in major central nervous system malformations between 1976 and 2005

OBJECTIVE: The goal of the current publication is to review isolated central nervous system malformations (CSMs) using a database in excess of 75 000 cases, with special regard to the risk of recurrence of these malformations alone or in combination. METHODS: In the period between 1 January 1976 and 31 December 2005, among the 75 320 documented cases, consultations were requested due to earlier isolated CSMs in the patients' histories in 3030 cases (4.2%). Processing the data we only considered disorders of genetic origin, and that was why we excluded the cases due to intrauterine infection. Monogenically inherited malformations were also excluded from the analysis. The diagnosis of the malformations was based on the prenatal diagnosis of ultrasonography as well as the findings of the foetopathological examination. RESULTS: In 65% of the cases, the couples sought counselling because of malformation in a previous pregnancy. In these cases, the risk of recurrence was thought to be 5.2%, while in the case of two affected children this figure stood at 21.9%. Analysing the values for the risk of recurrence in 5-year periods, neural tube defects (NTDs) (particularly anencephaly and spina bifida) showed a detectable decrease, which could be attributed to a growing use of folic acid supplementation around the time of conception and during pregnancy. CONCLUSION: There is a clear decrease of risk of recurrence of NTDs, while in the case of the other CSMs in this study, there is no noteworthy chronological change in their risk of recurrence.     

Agrobacterium tumefaciens -mediated transformation of the zygomycete fungus Backusella lamprospora

The Agrobacterium -mediated transformation was adapted to Backusella lamprospora, a zygomycete fungus closely related to Mucor. The transforming plasmid contained the hygromycin B resistance (hph) and the green fluorescent protein (gfp) genes under the control of the regulator regions of the Mucor circinelloides gpd1 gene. The presence of the hph and gfp genes in the transformants was detected by PCR. The introduced genes could also be amplified directly from the spores of the transformants. The transformation efficiency was investigated by fluorescence microscopy of the transformed spores. A gradual decrease in the hygromycin B resistance was observed during several cultivation cycles: the growth of the transformants on the selection medium became slower, and the detection of the introduced gene became more difficult.     

Development of Arterial Blood Supply in Experimental Liver Metastases

In this study, we present a mechanism for the development of arterial blood supply in experimental liver metastases. To analyze the arterialization process of experimental liver metastases, we elucidated a few key questions regarding the blood supply of hepatic lobules in mice. The microvasculature of the mouse liver is characterized by numerous arterioportal anastomoses and arterial terminations at the base of the lobules. These terminations supply one hepatic microcirculatory subunit per lobule, which we call an arterial hepatic microcirculatory subunit (aHMS). The process of arterialization can be divided into the following steps: 1) distortion of the aHMS by metastasis; 2) initial fusion of the sinusoids of the aHMS at the tumor parenchyma interface; 3) fusion of the sinusoids located at the base of the aHMSs, which leads to the disruption of the vascular sphincter (burst pipe); 4) incorporation of the dilated artery and the fused sinusoids into the tumor; and 5) further development of the tumor vasculature (arterial tree) by proliferation, remodeling, and continuous incorporation of fused sinusoids at the tumor–parenchyma interface. This process leads to the inevitable arterialization of liver metastases above the 2000- to 2500-μm size, regardless of the origin and growth pattern of the tumor.It is widely accepted that hepatic metastases and tumors are predominantly supplied by arterial blood, a notion that serves as the basis for hepatic arterial chemotherapy and chemoembolization.^{1,2,3,4,5,6,7} The most cited article on this field dates back to the 1950s.¹ Since then numerous papers have been published using human and experimental materials and different methods such as corrosion casting, confocal and electron microscopy, angiography, radiolabeled microspheres, and in vivo microscopy, have been used to study the blood supply of liver metastases.^{2,3,4,5,6,7,8,9,10,11,12,13,14,15,16} A large proportion of these articles have confirmed the original observation of Breedis and Young,¹ but no mechanism for the development of the arterial blood supply in metastases has ever been presented.^2,3,4,5,6,7 On the other hand, numerous papers, including ours, have emphasized the contribution of the portal vein, either directly or through the sinusoids in the blood supply of hepatic metastases.^{8,9,10,11,12,13,14} This apparent contradiction might result from the observed continuity of the sinusoidal with the tumor vasculature and the presumption that blood flows in an “outside-in” direction from the sinusoids toward the tumor vasculature. Most of the studies dealing with the blood supply of metastases have neglected the importance of arterioportal anastomoses and other interspecies differences in the hepatic microcirculation, which could lead to seriously biased results. According to the observations of Yamamoto et al¹⁵ there are extensive arterioportal anastomoses throughout the vascular tree in rats, whereas a separate arterial and portal tree, without direct arterioportal communication, can be observed in hamster and human liver. Opinions about the presence of arterioportal anastomoses in mice are controversial^10,16; therefore, we have addressed this question first.The classic lobule can be divided into several conical hepatic microcirculatory subunits (HMSs) supplied by a single inlet portal venule. Hepatic arterioles terminate either on the inlet venules or directly on sinusoids. The number of these terminations within a lobule is species-dependent. The blood flow through the inlet venules and terminal arterioles is regulated by sphincters.¹⁷ The most detailed studies on microcirculation of the liver and vessel architecture of liver metastases were performed by corrosion casting. However, in these studies the livers were completely filled with uncolored resin, which made analyzing the three-dimensional organization of the deep interlobular vessels difficult.^10,14,15In the present study, we used a two color corrosion casting technique to analyze the blood supply in liver metastases of experimental tumors in mice. A special filling method was used to prevent the mixing of the “portal and arterial resin” upstream of the hepatic sinusoids. This technique enabled us to analyze separately the contribution of the two vascular systems to the blood supply of liver metastases and to establish the steps of the arterialization process.     

Computational simulations of mitral regurgitation quantification using the flow convergence method: Comparison of hemispheric and hemielliptic formulae

Mitral regurgitation results from the incomplete closure of the mitral valve, and the noninvasive diagnosis of this disease remains an important clinical goal. In this study, steady flow computer simulations were used to evaluate flow convergence method for flow rate estimation. The hemispheric and hemielliptic formulae were compared for accuracy in the presence of complicating factors such, as ventricular confinement, orifice shape, and aortic outflow. Results showed that in the absence of aortic outflow and ventricular confinement, there was a plateau zone where the hemispheric formula approximated the true flow rate, independent of orifice shape. However, in the presence of complicating factors such as aortic outflow and ventricular confinement, there was no clear zone where the hemispheric formula could be applied. The hemielliptic formula, however, worked in, all cases, regardless of chamber size or magnitude of aortic outflow. Therefore, application of the hemielliptic formula shold be considered in future clinical studies.     

Relationship between neuronal vulnerability and potassium-chloride cotransporter 2 immunoreactivity in hippocampus following transient forebrain ischemia

Cation chloride cotransporters have been reported to be expressed in neurons in the hippocampus and to regulate intracellular Cl(-) concentration. The neuron-specific K-Cl cotransporter 2 (KCC2) is necessary for maintaining the low intracellular chloride concentration required for the hyperpolarizing actions of GABA. In this study we examined the vulnerability of KCC2-containing neurons as well as the changes in the pattern of KCC2 distribution in the rat hippocampus following 15 min ischemia induced by four-vessel occlusion. Immunostaining for the 72 kDa heat shock protein (HSP-72) was used to investigate the extent of damage in neuronal populations previously shown to be vulnerable to ischemia. At 6-24 h after ischemia, when the pyramidal cells in the CA1 (subfield of cornu Ammonis) region showed no morphological signs of damage, a small rise of KCC2 immunoreactivity was already observed. After 2 days, when the CA1 pyramidal cells started to degenerate, a progressive downregulation of the KCC2 protein was visible. Interestingly, in the same areas, the parvalbumin containing interneurons showed no signs of ischemic damage, and KCC2 immunoreactivity was retained on their membrane surface. In CA1 pyramidal cells, the reduction in KCC2 expression may lead to an elevation of intracellular Cl(-) concentration, which causes a shift in equilibrium potential toward more positive levels. Consequently, the reduction of the inhibitory action of GABA through downregulation of KCC2 function may be involved in the pathomechanisms of delayed neuronal death in the CA1 subfield.     

Sexually transmissible infections and prostate cancer risk

BACKGROUND: Sexually transmissible infections (STI) have been variably associated with increased risks of prostate cancer, largely in case-control studies. METHODS: In the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial, we examined risk of prostate cancer in relation to serum antibodies to Chlamydia trachomatis, human papillomavirus-16 and -18, herpes simplex virus-2, cytomegalovirus, and human herpesvirus-8 in 868 cases (765 Whites and 103 Blacks) and 1,283 controls matched by race, age, time since initial screening, and year of blood draw; all blood samples were collected at least 1 year before prostate cancer diagnosis, except for 43 Black cases. We also assessed risk associated with self-reported history of syphilis and gonorrhea. RESULTS: Prevalences of the 7 STIs among controls were weakly correlated, and all were more frequent among Blacks than Whites, except for human herpesvirus-8. Among Whites, prostate cancer risk was not significantly associated with the individual infections or with their number (P(trend) = 0.1); however, men with one or more STI had slightly higher risk (odds ratio, 1.3; 95% confidence interval, 1.0-1.6). Among Blacks, excess risk was associated with IgA antibody to C. trachomatis (odds ratio, 2.1; 95% confidence interval, 1.2-3.6). CONCLUSION: This large prospective study of prostate cancer shows no consistent association with specific STIs and a borderline association with any versus none. Whether a shared response or correlated infection not directly measured underlies the weak association requires further study.     

Familial occurrence of bilateral renal agenesis

The 58 cases of bilateral renal agenesis (Potter syndrome) registered in the Genetic Counselling unit of our institute in the last 12 years are reviewed. The only familial recurrent case which has been prenatally diagnosed is described in detail. A urinary bladder anomaly like that of the subsequent third child has not been previously reported. The authors analyze the possible inheritance patterns. They suggest the malformation is a genetically heterogeneous entity. They emphasize that nowadays the birth of a newborn with bilateral renal agenesis can be prevented in all cases.