Female Rat Hearts Research Articles

Protective Effect of Resveratrol against Ischemia-Reperfusion Injury via Enhanced High Energy Compounds and eNOS-SIRT1 Expression in Type 2 Diabetic Female Rat Heart.

Type 2 diabetic women have a high risk of mortality via myocardial infarction even with anti-diabetic treatments. Resveratrol (RSV) is a natural polyphenol, well-known for its antioxidant property, which has also shown interesting positive effects on mitochondrial function. Therefore, we aim to investigate the potential protective effect of 1 mg/kg/day of RSV on high energy compounds, during myocardial ischemia-reperfusion in type 2 diabetic female Goto-Kakizaki (GK) rats. For this purpose, we used 31P magnetic resonance spectroscopy in isolated perfused heart experiments, with a simultaneous measurement of myocardial function and coronary flow. RSV enhanced adenosine triphosphate (ATP) and phosphocreatine (PCr) contents in type 2 diabetic hearts during reperfusion, in combination with better functional recovery. Complementary biochemical analyses showed that RSV increased creatine, total adenine nucleotide heart contents and citrate synthase activity, which could be involved in better mitochondrial functioning. Moreover, improved coronary flow during reperfusion by RSV was associated with increased eNOS, SIRT1, and P-Akt protein expression in GK rat hearts. In conclusion, RSV induced cardioprotection against ischemia-reperfusion injury in type 2 diabetic female rats via increased high energy compound contents and expression of protein involved in NO pathway. Thus, RSV presents high potential to protect the heart of type 2 diabetic women from myocardial infarction.

Increased Expression of Bax and Bcl2 Apoptosis Biomarkers in the Heart of Old Female Rats After Interval Training and Curcumin Consumption

Increased Expression of Bax and Bcl2 Apoptosis Biomarkers in the Heart of Old Female Rats After Interval Training and Curcumin Consumption

Obesity-associated alterations in cardiac connexin-43 and PKC signaling are attenuated by melatonin and omega-3 fatty acids in female rats.

We aimed to explore whether specific high-sucrose intake in older female rats affects myocardial electrical coupling protein, connexin-43 (Cx43), protein kinase C (PKC) signaling, miR-1 and miR-30a expression, and susceptibility of the heart to malignant arrhythmias. Possible benefit of the supplementation with melatonin (40µg/ml/day) and omega-3 polyunsaturated fatty acids (Omacor, 25g/kg of rat chow) was examined as well. Results have shown that 8weeks lasting intake of 30% sucrose solution increased serum cholesterol, triglycerides, body weight, heart weight, and retroperitoneal adipose tissues. It was accompanied by downregulation of cardiac Cx43 and PKCε signaling along with an upregulation of myocardial PKCδ and miR-30a rendering the heart prone to ventricular arrhythmias. There was a clear benefit of melatonin or omega-3 PUFA supplementation due to their antiarrhythmic effects associated with the attenuation of myocardial Cx43, PKC, and miR-30a abnormalities as well as adiposity. The potential impact of these findings may be considerable, and suggests that high-sucrose intake impairs myocardial signaling mediated by Cx43 and PKC contributing to increased susceptibility of the older obese female rat hearts to malignant arrhythmias.

The impact of sex and myocardial ischemic preconditioning on immunohistochemical markers of acute myocardial infarction.

The immunohistochemical detection of dityrosine, troponins I (cTNI) and T (cTnT), and connexin 43 has been proposed as a tool for the diagnosis of myocardial infarction with short survival times. Results of clinical and experimental studies reveal that gender and/or ischemic preconditioning of the heart may have an influence on severity and magnitude of myocardial infarction. To clarify the question, if the above-mentioned markers are influenced by sex or ischemic preconditioning, experiments on isolated rat hearts using the Langendorff technique were performed. Using the hearts of 12 male and 12 female Wistar rats a local ischemia was induced through ligation of the left coronary artery. Furthermore, 12 male rat hearts underwent ischemic preconditioning of the heart by stopping the perfusion of the whole heart for 30min and subsequently reperfusing the heart for another 60min, before inducing local ischemia. The perfusion time after ligation varied from 10 to 60min. A control group was comprised out of 6 male and 2 female rat hearts. These were placed in the Langendorff system for 60min without further manipulation or received ischemic preconditioning without subsequent local ischemia or were excised without being mounted on the Langendorff system at all. All hearts were fixed in formalin and stained immunohistochemically. Depletion of the marker cTnT appeared to be less in females when compared to male hearts, for all other markers tested, no apparent difference in staining results were seen when comparing male and female rat hearts. Male rat hearts with ischemic preconditioning showed no difference compared to male rat hearts without ischemic preconditioning when stained fort dityrosine. Connexin 43 staining was less pronounced in hearts with ischemic preconditioning, whereas cTnI as well as cTnT depletion was more pronounced in preconditioned hearts. The presented findings indicate to some extent the vulnerability of the investigated markers for the influencing factors tested.

Inhibition of programmed necrosis limits infarct size through altered mitochondrial and immune responses in the aged female rat heart.

Both advancing age and estrogen loss exacerbate acute myocardial infarction in the female heart. However, the mechanistic underpinnings of age-related differences in cell death after ischemia-reperfusion (I/R) injury in female subjects and reductions in cardioprotective reserve capacity remain largely unexplored. The aim of the present study was to determine the efficacy of programmed necrosis inhibition on infarct size reduction and preservation of left ventricular (LV) function after I/R injury with female aging. Fischer 344 rats were ovariectomized (OVX) at 15 mo and studied at 24 mo (MO OVX) versus adult rats with intact ovaries (6 mo). After in vivo coronary artery ligation (55-min ischemia and 2- or 6-h reperfusion), necrostatin-1 (Nec-1; 3.5 or 5.7 mg/kg) delivered upon reperfusion significantly reduced infarct size by 37% and improved LV function in the MO OVX group ( P < 0.01). Although age-associated elevations in cyclophilin D and mitochondrial acetylation ( P < 0.001) were unaffected by Nec-1, profound reductions in IL-1, IL-6, and TNF-α ( P < 0.05) as well as cardiac immune cell infiltration were observed in MO OVX but not adult rats. We conclude that chronic inflammation and postmenopausal estrogen deficiency conspire to exacerbate acute infarction through a mechanism involving exaggerated mitochondria-mediated programmed necrosis through receptor-interacting protein 1 signaling. Modulatory effects of programmed necrosis inhibition on proinflammatory cytokine production after I/R reveal a potentially important mechanistic target to restore and preserve cardiac function in the OVX aged female heart. NEW & NOTEWORTHY Myocardial infarct size reduction by inhibition of programmed necrosis in aged female subjects suggests a dominant cell death pathway. Alterations in mitochondrial protein levels and acetylation underscore a mitochondria-dependent mechanism, whereas the profound cytokine reduction in aged subjects alone points to a divergent role for immune modulation of programmed necrosis and viable therapeutic target.

Notch1 Mediates Preconditioning Protection Induced by GPER in Normotensive and Hypertensive Female Rat Hearts.

G protein-coupled estrogen receptor (GPER) is an estrogen receptor expressed in the cardiovascular system. G1, a selective GPER ligand, exerts cardiovascular effects through activation of the PI3K-Akt pathway and Notch signaling in normotensive animals. Here, we investigated whether the G1/GPER interaction is involved in the limitation of infarct size, and improvement of post-ischemic contractile function in female spontaneous hypertensive rat (SHR) hearts. In this model, we also studied Notch signaling and key components of survival pathway, namely PI3K-Akt, nitric oxide synthase (NOS) and mitochondrial K+-ATP (MitoKATP) channels. Rat hearts isolated from female SHR underwent 30 min of global, normothermic ischemia and 120 min of reperfusion. G1 (10 nM) alone or specific inhibitors of GPER, PI3K/NOS and MitoKATP channels co-infused with G1, just before I/R, were studied. The involvement of Notch1 was studied by Western blotting. Infarct size and left ventricular pressure were measured. To confirm endothelial-independent G1-induced protection by Notch signaling, H9c2 cells were studied with specific inhibitor, N-[N-(3,5 difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT, 5 μM), of this signaling. Using DAPT, we confirmed the involvement of G1/Notch signaling in limiting infarct size in heart of normotensive animals. In the hypertensive model, G1-induced reduction in infarct size and improvement of cardiac function were prevented by the inhibition of GPER, PI3K/NOS, and MitoKATP channels. The involvement of Notch was confirmed by western blot in the hypertensive model and by the specific inhibitor in the normotensive model and cardiac cell line. Our results suggest that GPERs play a pivotal role in mediating preconditioning cardioprotection in normotensive and hypertensive conditions. The G1-induced protection involves Notch1 and is able to activate the survival pathway in the presence of comorbidity. Several pathological conditions, including hypertension, reduce the efficacy of ischemic conditioning strategies. However, G1-induced protection can result in significant reduction of I/R injury also female in hypertensive animals. Further studies may ascertain the clinical translation of the present results.

Sanglifehrin A Has No Protective Effects in Non‐reperfused Myocardial Infarction: The Role of Mitochondria

BackgroundExcessive reactive oxygen species (ROS) production and mitochondrial permeability transition pore (mPTP) formation due to mitochondrial dysfunction is one of the hallmarks of heart failure. The mPTP plays a critical role in pathogenesis of myocardial infarction (MI)/ischemia‐reperfusion (IR). Cyclopilin D (CypD) activation leads to mPTP formation thereby stimulating excessive ROS production and subsequent protein oxidation. Inhibition of CypD has been linked to cardioprotection through inhibition of mPTP formation and therefore mitochondrial dysfunction. However, failure of recent CIRCUS clinical trials indicates the necessity to further study the efficacy of CypD inhibition during MI/IR injury.HypothesisInhibition of CypD by sanglifehrin A (SfA) protects the heart from reperfused but not non‐reperfused MI by preserving mitochondrial and cardiac function.MethodsMI was performed in female rats (200–250 g) by coronary artery ligation (CAL) for 30 min followed by 2‐ and 28‐ day post‐surgery with or without reperfusion. The animals were divided in the following groups: i) Sham (S, no MI); ii) Sham+SfA (SS); iii) MI (I, no reperfusion); iv) I+SfA (IS); v) MI+reperfusion (IR); and vi) IR+SfA (IRS). SfA (5 mg/kg, IV) was administered 25 min after the start of sham surgery or MI. Cardiac and left ventricular mitochondrial functions were analyzed at 2‐ and 28‐days post‐surgery.ResultsSfA significantly improved ejection fraction (EF%) after 2 (28±2 vs. 53±3) and 28 (35±3 vs. 44±2) days in reperfused hearts. SfA administration to permanent occluded hearts has no effect on EF% at 2 (36±4 vs. 40±4) and 28 (32±3 vs. 40±4) days. Left ventricular respiratory control index (RCI) was significantly reduced (7.5±0.5 vs. 5.6±0.3) in animals subjected to IR when compared to controls after 2 days but not after 28 days. Formation of the mPTP was the same in all groups at 2 and 28 days. An increase in ROS production was observed in permanent occluded hearts (10.0±0.8 vs 7.3±0.6) after the addition of 1 mM calcium, and SfA significantly reduced protein oxidation in reperfused hearts (16.2±1.4 vs 21.8±2.1) at 2 days. SfA significantly reduced ROS production, at 1 mM calcium, in all groups at 28 days although no differences were detected in protein oxidation levels. A 14% increase in respirasome content was observed in IR hearts at 2 days and a 13% in permanent occluded hearts at 28 days. Menstrual cycle affected post‐ischemic recovery, where cycles with high estrogen levels positively correlated (r2=0.48) with an increase in EF% in reperfused MI hearts.ConclusionCypD inhibition has cardioprotective effects in reperfused infarcted hearts but it has no beneficial effects in hearts with non‐reperfused MI. Hearts of female rats presumably develop compensatory mechanisms post‐MI that preserve mitochondrial function in response to oxidative stress.Support or Funding InformationSupported by the American Physiological Society Porter Physiology Development Fellowship and the National Institutes of Health (Grants SC1HL118669, SC1GM128210)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Effects of Short‐Term Versus Long‐Term Exercise on Reducing Infarct Size After Ischemia‐Reperfusion in Male and Female Rat Hearts

Ischemic heart disease is the leading cause of death in both males and females worldwide, with myocardial infarction due to ischemia‐reperfusion (IR) injury being the most common manifestation. Exercise is known to protect the myocardium against IR injury, but the volume of exercise sufficient to confer cardioprotection is not well understood. A single bout of exercise has been shown to confer protection against IR injury, but it is not known whether longer term exercise training (weeks) adds additional cardioprotective benefit. In addition, it is unclear to what extent sex differences contribute to cardioprotection. To address these questions, we studied female and male Sprague‐Dawley rats (3 mo of age) randomly assigned to a 5 day exercise training (EX5d) group (n=20), an 8 week exercise training (EX8w) group (n=20), or a sedentary (Sed) control group (n=10). EX5d rats ran for 5 consecutive days, while EX8w animals ran 5 days a week for 8 weeks. The speed was gradually increased until animals were running 21–23 m/min for 60 min. Maximal running capacity improved in all groups as a result of training. EX5d animals improved maximal distance run by 31.9 ± 9% (pre vs post) while EX8w animals improved by 118.5 ± 15.6% (p&lt;0.01, EX5d vs EX8w). At least 24 h after the last training session, animals were anesthetized and the left anterior descending coronary artery was occluded via suture to induce regional ischemia for 45 min, followed by 24 h of reperfusion. After 24 hours, occlusion was reestablished and 0.5% Evans blue dye was injected into the aorta and perfused through the heart. Hearts were then sectioned, placed in a 0.1% triphenyltetrazolium chloride solution and incubated at 37° C for 10 minutes, then flash frozen in OCT. Infarct areas were quantified using ImageJ software, and infarct values calculated as a percentage of the zone at risk (ZAR). Exercise training led to a significant reduction in infarct size, with Sed, 5d, and 8w animals having infarct sizes of 37.2 ± 3, 27.5 ± 2, and 21.4 ± 1% of the ZAR, respectively (p&lt;0.01, Sed vs EX5d and Sed vs EX8w). Hearts from Sed females had significantly smaller infarcts than those of Sed males (41.8 ± 3 and 32.6 ± 1% of the ZAR, respectively). This sex difference persisted in response to exercise training, with females exhibiting smaller infarct sizes following 5 days of exercise training compared to males (25.1 ± 1 and 29.9 ± 1% of the ZAR, in females and males respectively (p&lt;0.05)), as well as 8 weeks of training (19.4 ± 1 and 23.5 ± 1% of the ZAR, respectively (p&lt;0.05)). When comparing within‐sex differences in cardioprotection, 8 weeks of exercise did not lead to significant infarct size reduction compared to 5 days of exercise in males (23.5 vs 29.9% of the ZAR). However, hearts from EX8w females had significantly smaller infarct sizes when compared to EX5d females (19.4 vs 25.1% of the ZAR, respectively; p&lt;0.01). These results show that 1) long‐term exercise training can augment infarct sparing against IR injury when compared to short duration exercise, and 2) this effect is sex‐dependent.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tolerance to ischemia-reperfusion injury in a metabolic syndrome model: Effect of gender?

Metabolic syndrome can predict type 2 diabetes and cardiovascular complications, such as myocardial infarction. However, few studies explore gender differences in this context. The aim of this study was to compare the effects of a high-fat-high-sucrose diet (HFHSD) on the sensitivity to ischemia-reperfusion injury of male and female Wistar rat hearts. Male and female Wistar rats were subjected to HFHSD or normal diet (ND) for 5 months. Then, rats underwent an intraperitoneal glucose tolerance test (IPGTT) to determine glycemic status, and ex vivo experiments on the isolated perfused heart were performed to study simultaneously cardiac function and energy metabolism with 31 P MRS during an ischemia-reperfusion injury. Finally, hearts were freeze-clamped for biochemical assays. After 5 months of HFHSD, body weight was increased in males ( P < 0.001 vs. males ND) but not in females and fat percent was higher in both HFHSD groups (respectively, P < 0.01 and P < 0.05 in males and females HFHSD vs. controls). IPGTT showed a significant glucose intolerance in males and females HFHSD ( P < 0.001 vs. controls) which was more pronounced in females ( P < 0.05 vs. males HFHSD at T15 min). HFHSD increased fasting blood glucose in males and females compared with controls ( P < 0.05) but increased plasma free fatty acids only in females ( P < 0.05 vs. control females). Heart weight to tibia length ratio was higher with HFHSD only in males ( P < 0.001 vs. control males). Finally, in males and females HFHSD, we found impaired myocardial function (respectively, P < 0.001 and P < 0.05 vs. controls) and impaired coronary flow (respectively, P < 0.01 and P < 0.05 vs. controls) after ischemia with no difference between males and females. Despite gender differences in tolerance to glucose, body weight and heart weight to tibia length ratio, we found no effect of gender in tolerance to ischemia-reperfusion injury in our model of metabolic syndrome.

Toll-like receptor expression and apoptosis morphological patterns in female rat hearts with takotsubo syndrome induced by isoprenaline

AimsToll-like receptors (TLR) and apoptosis were indicated as important factors in heart failure. Our aim was to characterize the morphological pattern of apoptosis, TLR2, TLR4, and TLR6 expression in female rat hearts in the model of takotsubo syndrome (TTS). Main methods60 Sprague-Dawley female rats were treated with a single dose of 150 mg/kg b.wt. of isoprenaline (ISO) or 0.9% NaCl (controls). Hearts were collected 24, 48, 72 h and 7 days post-ISO injection. 32/60 hearts were used in immunohistopathological studies and 28/60 in real time. Key findingsApoptosis was observed 24 h post-ISO in cardiomyocytes, 24, 48, 72 h and 7 days post-ISO in infiltrating inflammatory cells, 7 days post-ISO in endothelial cells of vessels. Diffuse TLR4CD68 (CD68, a macrophage marker) and TLR6CD68 positive cells and TLR2, TLR4, TLR6 mononuclear cells were observed in both acute and recovery phase of TTS. In the foci located in the neighborhood of damaged (necrotic/apoptotic) cardiomyocytes in TTS, high (strong) protein expression of TLR2 (TLR2high) was observed: 24, 48, 72 h post-ISO; TLR4high – 48 and 72 h post-ISO; TLR6high – 48 h post-ISO. Whereas in cardiomyocytes of remote myocardium: TLR2high – 72 h post-ISO; TLR4high – 24 and 72 h post-ISO; TLR6high – 24 h post-ISO. TLR2 mRNA was down-regulated 48 and 72 h post-ISO whereas TLR4 up-regulated 7 days post-ISO. SignificanceThe expression pattern of apoptosis and TLR differs in the course of TTS in comparison with the control rats. We hypothesize that innate immunity and apoptosis may play a crucial role in TTS pathophysiology.

Divergent Effects of Cyclophilin-D Inhibition on the Female Rat Heart: Acute Versus Chronic Post-Myocardial Infarction

Background/Aims: The mitochondrial permeability transition pore opening plays a critical role in the pathogenesis of myocardial infarction. Inhibition of cyclophilin-D (CyP-D), a key regulator of the mitochondrial permeability transition pore, has been shown to exert cardioprotective effects against ischemia-reperfusion injury on various animal models, mostly in males. However, failure of recent clinical trials requires a detailed elucidation of the cardioprotective efficacy of CyP-D inhibition. The aim of this study was to examine whether cardioprotective effects of sanglifehrin A, a potent inhibitor of CyP-D, on post-infarcted hearts depends on reperfusion. Methods: Acute or chronic myocardial infarction was induced by coronary artery ligation with/without subsequent reperfusion for 2 and 28 days in female Sprague-Dawley rats. Cardiac function was estimated by echocardiography. Oxygen consumption rates, ROS production, permeability transition pore opening, protein carbonylation and respiratory supercomplexes were analyzed in isolated cardiac mitochondria. Results: Sanglifehrin A significantly improved cardiac function of reperfused hearts at 2 days but failed to protect after 28 days. No protection was observed in non-reperfused post-infarcted hearts. The respiratory control index of mitochondria was significantly reduced in reperfused infarcted hearts at 2-days with no effect at 28-days post-infarction on reperfused and non-reperfused hearts. Likewise, only a minor increase in reactive oxygen species production was observed at 2-days in non-reperfused post-infarcted hearts. Conclusion: This study demonstrates that CyP-D inhibition exerts cardioprotective effects in reperfused but not in non-reperfused infarcted hearts of female rats, and the effects are observed only during acute post-infarction injury.

Ursodeoxycholic acid prevents ventricular conduction slowing and arrhythmia by restoring T-type calcium current in fetuses during cholestasis.

BackgroundIncreased maternal serum bile acid concentrations in intrahepatic cholestasis of pregnancy (ICP) are associated with fetal cardiac arrhythmias. Ursodeoxycholic acid (UDCA) has been shown to demonstrate anti-arrhythmic properties via preventing ICP-associated cardiac conduction slowing and development of reentrant arrhythmias, although the cellular mechanism is still being elucidated.MethodsHigh-resolution fluorescent optical mapping of electrical activity and electrocardiogram measurements were used to characterize effects of UDCA on one-day-old neonatal and adult female Langendorff-perfused rat hearts. ICP was modelled by perfusion of taurocholic acid (TC, 400μM). Whole-cell calcium currents were recorded from neonatal rat and human fetal cardiomyocytes.ResultsTC significantly prolonged the PR interval by 11.0±3.5% (P<0.05) and slowed ventricular conduction velocity (CV) by 38.9±5.1% (P<0.05) exclusively in neonatal and not in maternal hearts. A similar CV decline was observed with the selective T-type calcium current (ICa,T) blocker mibefradil 1μM (23.0±6.2%, P<0.05), but not with the L-type calcium current (ICa,L) blocker nifedipine 1μM (6.9±6.6%, NS). The sodium channel blocker lidocaine (30μM) reduced CV by 60.4±4.5% (P<0.05). UDCA co-treatment was protective against CV slowing induced by TC and mibefradil, but not against lidocaine. UDCA prevented the TC-induced reduction in the ICa,T density in both isolated human fetal (−10.2±1.5 versus −5.5±0.9 pA/pF, P<0.05) and neonatal rat ventricular myocytes (−22.3±1.1 versus −9.6±0.8 pA/pF, P<0.0001), whereas UDCA had limited efficacy on the ICa,L.ConclusionOur findings demonstrate that ICa,T plays a significant role in ICP-associated fetal cardiac conduction slowing and arrhythmogenesis, and is an important component of the fetus-specific anti-arrhythmic activity of UDCA.

Age and ischemia differentially impact mitochondrial ultrastructure and function in a novel model of age-associated estrogen deficiency in the female rat heart.

Altered mitochondrial respiration, morphology, and quality control collectively contribute to mitochondrial dysfunction in the aged heart. Because myocardial infarction remains the leading cause of death in aged women, the present study utilized a novel rodent model to recapitulate human menopause to interrogate the combination of age and estrogen deficiency on mitochondrial ultrastructure and function with cardiac ischemia/reperfusion (I/R) injury. Female F344 rats were ovariectomized (OVX) at 15months and studied at 24months (MO OVX; n = 40) vs adult ovary intact (6months; n=41). Temporal declines in estrogen concomitant with increased visceral adipose tissue were observed in MO OVX vs adult. Following in vivo coronary artery ligation or sham surgery, state 3 mitochondrial respiration was selectively reduced by age in subsarcolemmal mitochondria (SSM) and by I/R in interfibrillar mitochondria (IFM); left ventricular maximum dP/dt was reduced in MO OVX (p<0.05). Elevated cyclophilin D and exacerbated I/R-induced mitochondrial acetylation in MO OVX suggest permeability transition pore involvement and reduced protection vs adult (p<0.05). Mitochondrial morphology by TEM revealed an altered time course of autophagy coordinate with attenuated Drp1 and LC3BII protein levels with age-associated estrogen loss (p<0.05). Here, reductions in both SSM and IFM function may play an additive role in enhanced susceptibility to regional I/R injury in aged estrogen-deficient female hearts. Moreover, novel insight into altered cardiac mitochondrial quality control garnered here begins to unravel the potentially important regulatory role of mitochondrial dynamics on sustaining respiratory function in the aged female heart.

Evidence of Altered Mitochondrial Protein Expression After Chronic Ethanol Consumption in the Aged Estrogen-Deficient Female Rat Heart.

Estrogen loss has been implicated to increase the risk of alcoholic cardiomyopathy in postmenopausal women. The purpose of this study was to identify novel mitochondrial protein targets for the treatment of alcoholic cardiomyopathy in aged women using a state-of-the-art proteomic approach. We hypothesized that chronic ethanol (EtOH) ingestion exacerbates maladaptive mitochondrial protein expression in the aged female heart. Adult (3months) and aged (18months) F344 ovary-intact or ovariectomized (OVX) rats were randomly assigned an EtOH or control Lieber-DeCarli "all-liquid" diet for 20weeks. Proteomic analyses were conducted in mitochondria isolated from left ventricles using isobaric tags for relative and absolute quantification (iTRAQ) 8plex labeling and mass spectrometry (n=3 to 5/group). After EtOH, significant differences (false discovery rate <5%) were observed in electron transport chain components (NADH dehydrogenase [ubiquinone] flavoprotein 2) as well as proteins involved in lipid metabolism (2,4 dienoyl-CoA reductase) and cellular defense (catalase), suggesting a possible link to congestive heart failure. Directional changes in protein levels were confirmed by Western blotting. Additionally, EtOH significantly reduced state 3 mitochondrial respiration in all groups, yet only reduced respiratory control index in the aged OVX rat heart (p<0.05). Collectively, the data reveal that EtOH-induced changes in the mitochondrial proteome exacerbate cardiac dysfunction in aged and estrogen-deficient hearts, but not in adult. In conclusion, iTRAQ is a powerful tool for investigating new mitochondrial targets of alcoholic cardiomyopathy.

Physical training promotes similar effects to the blockade of angiotensin-converting enzyme on the cardiac morphology and function in old female rats subjected to premature ovarian failure.

We investigated the effects of angiotensin-converting enzyme (ACE) inhibition and aerobic physical training on the heart of old female rats (82-wk-old) submitted to premature ovarian failure (10-wk.-old). We used different approaches: morphology and function by echocardiography, reactivity of the coronary bed and left ventricular contractibility (Langendorff Technique). Female Wistar ovariectomized (OVX) rats (n=42) were assigned to one of four groups: OVX, vehicle treated only; OVX-EM, Enalapril Maleate only (EM, 10mg·kg-1·d-1); OVX-T, aerobic trained only; and OVX-EMT, treated with Enalapril Maleate and aerobic trained. Both Enalapril Maleate treatment and aerobic training were done in the last 20weeks of the experimental protocol. When compared to the OVX group, the OVX-EM group showed lower values of wall thickness and left ventricular (LV) mass, lower values of coronary bed reactivity and reduced maximum response of LV contractility to dobutamine, while the OVX-T group showed lower values of LV wall thickness, increase in end-systolic volume, reduced maximum response of LV contractility to dobutamine, and left intraventricular pressure due to increased flow. The combination of treatments (EM and aerobic physical training) did not promote additional important effects on the parameters evaluated. Our results suggest similar beneficial effects of physical training and EM treatment on the morphology and cardiac function in old female rats submitted to premature ovarian failure. Although the causes of these benefits are still unknown, both treatments have promoted a decrease in cardiac contractility, and the reduced β1-adrenergic sensitivity suggests that both treatments may attenuate the sympathetic effect on the heart.

Maternal low-protein diet in female rat heart: possible protective effect of estradiol.

Several studies have shown that maternal low-protein (LP) diet induces detrimental effects in cardiovascular system and oxidative stress in male animals. Additional studies suggested that female has lower incidence of cardiovascular disease. However until present data, the possible effects of estradiol on the undernutrition during gestational and lactation periods are not discussed. The present study was conducted to evaluate the effects of a maternal LP diet during gestational and lactation period on oxidative balance in the female rat hearts ventricles at two ages. Dams were fed with normal protein (NP) or a LP diet during the gestational and lactation period, and their female offspring were divided into age groups (22 or 122 days, corresponding to a low or high estrogen level) composing four experimental groups. Evaluating the nutritional effect showed an increase in oxidative stress biomarkers and decrease in enzymatic defense in LP-22D compared with NP-22D. In contrast, no changes were observed in malondialdehyde and carbonyls, but an increase in glutathione-S-transferase (GST) activity in the LP-122D compared with NP-122D. The global oxy-score in the LP-22D group indicated a predominance of oxidative damage when compared with NP-22D, while in LP-122D group the global oxy-score was restored to NP-122D levels. Evaluating the estradiol effect, our data show a significant decrease in oxidative stress with increase in CAT and GST activity, associated with increase in intracellular thiols. Our data suggest that in situation with low levels of estradiol, hypoproteic diet during gestation and lactation period has detrimental effects on heart, however when estradiol levels raise, the detrimental effects induced are mitigated.

Swimming exercise reverses aging-related contractile abnormalities of female heart by improving structural alterations.

The objective of this study was to examine the effect of swimming exercise on aging-related Ca2+ handling alterations and structural abnormalities of female rat heart. For this purpose, 4-month and 24-month old female rats were used and divided into three following groups: sedentary young (SY), sedentary old (SO), and exercised old (Ex-O). Swimming exercise was performed for 8 weeks (60 min/day, 5 days/week). Myocyte shortening, L-type Ca2+ currents and associated Ca2+ transients were measured from ventricular myocytes at 36 ± 1°C. NOX-4 levels, aconitase activity, glutathione measurements and ultrastructural examination by electron microscopy were conducted in heart tissue. Swimming exercise reversed the reduced shortening and slowed kinetics of aged cardiomyocytes. Although the current density was similar for all groups, Ca2+ transients were higher in SO and Ex-O myocytes with respect to the SY group. Caffeine-induced Ca2+ transients and the integrated NCX current were lower in cardiomyocytes of SY rats compared with other groups, suggesting an increased sarcoplasmic reticulum Ca2+ content in an aged heart. Aging led to upregulated cardiac NOX-4 along with declined aconitase activity. Although it did not reverse these oxidative parameters, swimming exercise achieved a significant increase in glutathione levels and improved structural alterations of old rats' hearts. We conclude that swimming exercise upregulates antioxidant defense capacity and improves structural abnormalities of senescent female rat heart, although it does not change Ca2+ handling alterations further. Thereby, it improves contractile function of aged myocardium by mitigating detrimental effects of oxidative stress.

Progesterone Protects Against Bisphenol A-Induced Arrhythmias in Female Rat Cardiac Myocytes via Rapid Signaling.

Bisphenol A (BPA) is an estrogenic endocrine-disrupting chemical (EDC) that has a range of potential adverse health effects. Previously we showed that acute exposure to BPA promoted arrhythmias in female rat hearts through estrogen receptor rapid signaling. Progesterone (P4) and estrogen have antagonistic or complementary actions in a number of tissues and systems. In the current study, we examined the influence and possible protective effect of P4 on the rapid cardiac actions of BPA in female rat cardiac myocytes. Preincubation with physiological concentration (1 nM) of P4 abolished BPA-induced triggered activities in female cardiac myocytes. Further, P4 abrogated BPA-induced alterations in Ca2+ handling, including elevated sarcoplasmic reticulum Ca2+ leak and Ca2+ load. Key to the inhibitory effect of P4 is its blockade of BPA-induced increase in the phosphorylation of phospholamban. At myocyte and protein levels, these inhibitory actions of P4 were blocked by pretreatment with the nuclear P4 receptor (nPR) antagonist RU486. Analysis using membrane-impermeable bovine serum albumin-conjugated P4 suggested that the actions of P4 were mediated by membrane-initiated signaling. Inhibitory G (Gi) protein and phophoinositide-3 kinase (PI3K), but not tyrosine protein kinase activation, were involved in the observed effects of P4. In conclusion, P4 exerts an acute protective effect against BPA-induced arrhythmogenesis in female cardiac myocytes through nPR and the Gi/PI3K signaling pathway. Our findings highlight the importance of considering the impact of EDCs in the context of native hormonals and may provide potential therapeutic strategies for protection against the cardiac toxicities associated with BPA exposure.

Reduced up-regulation of the nitric oxide pathway and impaired endothelial and smooth muscle functions in the female type 2 diabetic goto-kakizaki rat heart

BackgroundType 2 diabetes is associated with greater relative risk of cardiovascular diseases in women than in men, which is not well understood. Consequently, we have investigated if male and female displayed differences in cardiac function, energy metabolism, and endothelial function which could contribute to increased cardiovascular complications in type 2 diabetic female.MethodsMale and female Control and type 2 diabetic Goto-Kakizaki (GK) isolated rat hearts were perfused during 28 min with a physiological buffer before freeze-clamping for biochemical assays. High energy phosphate compounds and intracellular pH were followed using 31P magnetic resonance spectroscopy with simultaneous measurement of contractile function. Nitric oxide (NO) pathway and endothelium-dependent and independent vasodilatations were measured as indexes of endothelial function. Results were analyzed via two-way ANOVA, p < 0.05 was considered as statistically significant.ResultsMyocardial function was impaired in male and female diabetic versus Control groups (p < 0.05) without modification of energy metabolism. Coronary flow was decreased in both diabetic versus Control groups but to a higher extent in female GK versus male GK rat hearts (p < 0.05). NO production was up-regulated in diabetic groups but to a less extent in female GK rat hearts (p < 0.05). Endothelium-dependent and independent vasodilatations were impaired in female GK rat compared with male GK (p < 0.05) and female Control (p < 0.05) rat hearts.ConclusionsWe reported here an endothelial damage characterized by a reduced up-regulation of the NO pathway and impaired endothelial and smooth muscle functions, and coronary flow rates in the female GK rat hearts while energy metabolism was normal. Whether these results are related to the higher risk of cardiovascular complications among type 2 diabetic female needs to be further elicited in the future.

Intramyocardially Transplanted Neonatal Cardiomyocytes (NCMs) Show Structural and Electrophysiological Maturation and Integration and Dose-Dependently Stabilize Function of Infarcted Rat Hearts

Cardiac cell replacement therapy is a promising therapy to improve cardiac function in heart failure. Persistence, structural and functional maturation, and integration of transplanted cardiomyocytes into recipients' hearts are crucial for a safe and efficient replacement of lost cells. We studied histology, electrophysiology, and quantity of intramyocardially transplanted rat neonatal cardiomyocytes (NCMs) and performed a detailed functional study with repeated invasive (pressure–volume catheter) and noninvasive (echocardiography) analyses of infarcted female rat hearts including pharmacological stress before and 3 weeks after intramyocardial injection of 5 × 106 (low NCM) or 25 × 106 (high NCM) syngeneic male NCMs or medium as placebo (Ctrl). Quantitative real-time polymerase chain reaction (PCR) for Y-chromosome confirmed a fivefold higher persisting male cell number in high NCM versus low NCM after 3 weeks. Sharp electrode measurements within viable slices of recipient hearts demonstrated that transplanted NCMs integrate into host myocardium and mature to an almost adult phenotype, which might be facilitated through gap junctions between host myocardium and transplanted NCMs as indicated by connexin43 in histology. Ejection fraction of recipient hearts was severely impaired after ligation of left anterior descending (LAD; pressure–volume catheter: 39.2 ± 3.6%, echocardiography: 39.9 ± 1.4%). Repeated analyses revealed a significant further decline within 3 weeks in Ctrl and a dose-dependent stabilization in cell-treated groups. Consistently, stabilized cardiac function/morphology in cell-treated groups was seen in stroke volume, cardiac output, ventricle length, and wall thickness. Our findings confirm that cardiac cell replacement is a promising therapy for ischemic heart disease since immature cardiomyocytes persist, integrate, and mature after intramyocardial transplantation, and they dose-dependently stabilize cardiac function after myocardial infarction.