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.     

Tuberculous meningitis in children

Summary Ninety cases of tuberculous meningitis have been analysed with special reference to some prognostic factors such as the age of the patient, duration of symptoms before treatment, C.S.F. picture and type of treatment. 78·8 per cent. of cases were males and 84·4 per cent. below the age of 5 years. The peak incidence was below 3 years, 21·1 per cent. of cases occurring below the age of 1 year, the youngest patient being 4 months old. History of contact could be obtained in 27·78 per cent. of cases. X-Ray chest showed evidence of intrathoracic tuberculous foci in 74·4 per cent. of cases, with miliary lesions in 15·5 per cent., hilar lymphadenopathy in 33·3 per cent., bronchopneumonic infiltration in 21·1 per cent., fibrosis in 3·3 per cent. and thickened pleura in 1·1 per cent. of cases. Out of 90 cases, 35 expired, another 35 left the hospital in an unsatisfactory state, and only 20 cases recovered, giving a satisfactory response in 22·2 per cent. of cases. The combined figures for mortality and unsatisfactory results were 82·9 per cent., 77·2 per cent. and 64·3 per cent. between the age groups 0 to 2, 2 to 5 and above 5 years respectively. 84·2 per cent. of cases with coma and/or fits either proved fatal or had severe neurological sequelae. Low cerebrospinal fluid sugar and chlorides and raised proteins affected the prognosis adversely. Treatment under “Category I, II and III” showed satisfactory response in 29·3, 50 and 11·65 per cent. of cases respectively. Steroid therapy did not seem to favourably influence the results in cases picked up at random. From the Department of Pediatrics, Medical College, Amritsar     

多发伤合并休克早期大鼠肺脏中细胞凋亡的实验研究

目的：观察大鼠多发伤合并休克早期肺脏中细胞凋亡的发生情况。方法：采用ＤＮＡ琼脂糖凝胶电泳、原位末端标记法（ＩＳＥＬ），在光镜和透射电镜下检测细胞凋亡，并测定ＤＮＡ片段百分率（ａｐ％）作定量分析。结果：检测到细胞凋亡所特有的ＤＮＡ电泳梯形带，ＩＳＥＬ法亦显示出阳性结果。复苏后６小时，ａｐ％随创伤程度的加重而升高，且与肺通透指数成正相关（ｒ＝０．６２１，Ｐ＜０．００１）。在６处创伤合并休克组，复苏后１小时ａｐ％已显著升高（５．３％±０．６％，Ｐ＜０．０１），３小时即达顶峰（３１．０％±３．０％，Ｐ＜０．０１），以后逐渐下降。形态学显示，细胞凋亡发生于肺脏中的多种细胞（包括肺泡上皮细胞、血管内皮细胞和中性粒细胞）。结论：多发伤合并休克早期大鼠肺脏中发生细胞凋亡，且细胞凋亡可能在早期的肺功能损伤中发挥作用     

Laser-induced plasma spectroscopy (LIPS): use of a geological tool in assessing bone mineral content

Bone may be similar to geological formulations in many ways. Therefore, it may be logical to apply laser-based geological techniques in bone research. The mineral and element oxide composition of bioapatite can be estimated by mathematical models. Laser-induced plasma spectrometry (LIPS) has long been used in geology. This method may provide a possibility to determine the composition and concentration of element oxides forming the inorganic part of bones. In this study, we wished to standardize the LIPS technique and use mathematical calculations and models in order to determine CaO distribution and bone homogeneity using bovine shin bone samples. We used polished slices of five bovine shin bones. A portable LIPS instrument using high-power Nd++YAG laser pulses has been developed (OpLab, Budapest). Analysis of CaO distribution was carried out in a 10?×?10 sampling matrix applying 300-μm sampling intervals. We assessed both cortical and trabecular bone areas. Regions of interest (ROI) were determined under microscope. CaO peaks were identified in the 200–500 nm wavelength range. A mathematical formula was used to calculate the element oxide composition (wt%) of inorganic bone. We also applied two accepted mathematical approaches, the Bartlett’s test and frequency distribution curve-based analysis, to determine the homogeneity of CaO distribution in bones. We were able to standardize the LIPS technique for bone research. CaO concentrations in the cortical and trabecular regions of B1–5 bones were 33.11?±?3.99% (range 24.02–40.43%) and 27.60?±?7.44% (range 3.58–39.51%), respectively. CaO concentrations highly corresponded to those routinely determined by ICP-OES. We were able to graphically demonstrate CaO distribution in both 2D and 3D. We also determined possible interrelations between laser-induced craters and bone structure units, which may reflect the bone structure and may influence the heterogeneity of CaO distributions. By using two different statistical methods, we could confirm if bone samples were homogeneous or not with respect to CaO concentration distribution. LIPS, a technique previously used in geology, may be included in bone research. Assessment of element oxide concentrations in the inorganic part of bone, as well as mathematical calculations may be useful to determine the content of CaO and other element oxides in bone, further analyze bone structure and homogeneity and possibly apply this research to normal, as well as diseased bones.     

The Timing of Onset of Mechanical Systole and Diastole in Reference to the QRS-T Complex: a Study to Determine Performance Criteria for a Non-Invasive Diastolic Timed Vibration Massage System in Treatment of Potentially Unstable Cardiac Disorders

Our institution is in development of a low frequency, non-invasive Diastolic Timed Vibrator (DTV) for use in emergency treatment of ST Elevation Myocardial Infarction (STEMI). It is preferable to avoid vibration emissions during the IsoVolumetric Contraction Period (IVCP) and at least the majority of mechanical systole thereafter, as systolic vibration may cause a negative inotropic effect in the ischemic heart. Furthermore diastolic vibration should preferably include the IsoVolumetric Relaxation Period (IVRP) which has been shown in clinical studies to improve cardiac performance and enhance coronary flow. Electrocardiographic (ECG) monitoring can be used to enable diastolic tracking, however, the timing of the phases of the cardiac cycle in relation to the ECG waveform must first be verified. The objective of this study was therefore to determine timing of onset of mechanical systole and diastole in reference to the QRS-T Complex. One hundred and twenty-three adult echocardiographic studies were assessed for the point of mitral and aortic valve closure in relation to the QRS complex and T wave in a representative population. We found that onset of mechanical systole occurred on and usually shortly after the peak of a first dominant QRS complex deflection, and onset of diastole occurred at the earliest on and most commonly beyond the peak or midpoint of the T wave. A DTV should ideally be able to stop vibrating on or before the peak of the first dominant deflection of a QRS complex, and begin vibrating near the peak of the T wave. Given early detection of ventricular depolarization can occur 10–20 ms prior to R wave peak, it is proposed that a DTV should preferably be able to stop vibrating within 10 ms of a triggered stop command. Onset of vibration during peak of T wave could be approximated by a rate adapted Q-T interval regression equation, and then fine tuned by manual adjustment during therapy.     

Percutaneous retrieval of a biliary stent after migration and ileal perforation

We present a case of a migrated biliary stent that resulted in a distal small bowel perforation, abscess formation and high grade partial small bowel obstruction in a medically stable patient without signs of sepsis or diffuse peritonitis. We performed a percutaneous drainage of the abscess followed by percutaneous retrieval of the stent. The entero-peritoneal fistula closed spontaneously with a drain in place. We conclude, migrated biliary stents associated with perforation distal to the Ligament of Trietz (LOT), may be treated by percutaneous drainage of the abscess and retrieval of the stent from the peritoneal cavity, even when associated with a large intra-abdominal abscess.     

Investigation of Thr715Pro P-selectin gene polymorphism and soluble P-selectin levels in type 2 diabetes mellitus

Increased levels of soluble P-selectin (sP-selectin) have been shown in a number of different disorders, e.g. diabetes mellitus (DM) and cardiovascular disease (CVD). Several studies have attempted to demonstrate the association of the most intensively examined variant of P-selectin gene polymorphism (Thr715Pro) with sP-selectin levels in healthy subjects and in CVD, but contradictory data have been reported. To clarify the effect of Pro715 allele on the sP-selectin levels in type 2 DM, we analysed this polymorphism in diabetic patients and compared these data with sP-selectin levels. Type 2 DM patients (n = 119), 48 BMImatched non diabetic individuals - consisting mostly of overweight subjects - and 57 healthy volunteers were included in the study. TheThr715Pro polymorphism was analysed by PCR-RFLP, while sP-selectin levels were measured by ELISA. Significantly elevated sP-selectin levels were found in both DM and in overweight subjects compared to healthy controls. We confirmed previous reports that in healthy Pro715 allele carriers lower sPselectin levels could be measured; however, this difference was only significant in case of lean subjects. No significant difference was detected in sP-selectin level among DM and overweight individuals according to this genotype. However, significant difference was observed in sP-selectin levels in older DM patients compared to younger ones, but these levels were not accounted for by the Thr715Pro polymorphism. We suggest that in type 2 DM individuals, the significantly elevated sP-selectin levels are not due to the Thr715Pro P-selectin gene polymorphism.