Blunting of estrogen modulation of cardiac cellular chymase/RAS activity and function in SHR.

The relatively low efficacy of ACE-inhibitors in the treatment of heart failure in women after estrogen loss may be due to their inability to reach the intracellular sites at which angiotensin (Ang) II is generated and/or the existence of cell-specific mechanisms in which ACE is not the essential processing pathway for Ang II formation. We compared the metabolic pathway for Ang II formation in freshly isolated myocytes (CMs) and non-myocytes (NCMs) in cardiac membranes extracted from hearts of gonadal-intact and ovariectomized (OVX) adult WKY and SHR rats. Plasma Ang II levels were higher in WKY vs. SHR (strain effect: WKY: 62 ± 6 pg/ml vs. SHR: 42 ± 9 pg/ml; p < 0.01), independent of OVX. The enzymatic activities of chymase, ACE, and ACE2 were higher in NCMs versus CMs, irrespective of whether assays were performed in cardiac membranes from WKY or SHR or in the presence or absence of OVX. E2 depletion increased chymase activity, but not ACE activity, in both CMs and NCMs. Moreover, cardiac myocyte chymase activity associated with diastolic function in WKYs and cardiac structure in SHRs while no relevant functional and structural relationships between the classic enzymatic pathway of Ang II formation by ACE or the counter-regulatory Ang-(1-7) forming path from Ang II via ACE2 were apparent. The significance of these novel findings is that targeted cell-specific chymase rather than ACE inhibition may have a greater benefit in the management of HF in women after menopause.

Cardiomyocyte-specific deletion of the G protein-coupled estrogen receptor (GPER) leads to left ventricular dysfunction and adverse remodeling: A sex-specific gene profiling analysis.

Activation of G protein-coupled estrogen receptor (GPER) by its agonist, G1, protects the heart from stressors such as pressure-overload, ischemia, a high-salt diet, estrogen loss, and aging, in various male and female animal models. Due to nonspecific effects of G1, the exact functions of cardiac GPER cannot be concluded from studies using systemic G1 administration. Moreover, global knockdown of GPER affects glucose homeostasis, blood pressure, and many other cardiovascular-related systems, thereby confounding interpretation of its direct cardiac actions. We generated a cardiomyocyte-specific GPER knockout (KO) mouse model to specifically investigate the functions of GPER in cardiomyocytes. Compared to wild type mice, cardiomyocyte-specific GPER KO mice exhibited adverse alterations in cardiac structure and impaired systolic and diastolic function, as measured by echocardiography. Gene deletion effects on left ventricular dimensions were more profound in male KO mice compared to female KO mice. Analysis of DNA microarray data from isolated cardiomyocytes of wild type and KO mice revealed sex-based differences in gene expression profiles affecting multiple transcriptional networks. Gene Set Enrichment Analysis (GSEA) revealed that mitochondrial genes are enriched in GPER KO females, whereas inflammatory response genes are enriched in GPER KO males, compared to their wild type counterparts of the same sex. The cardiomyocyte-specific GPER KO mouse model provides us with a powerful tool to study the functions of GPER in cardiomyocytes. The gene expression profiles of the GPER KO mice provide foundational information for further study of the mechanisms underlying sex-specific cardioprotection by GPER.

Activation of GPR30 improves exercise capacity and skeletal muscle strength in senescent female Fischer344 × Brown Norway rats.

The molecular mechanisms of muscle weakness and sarcopenia in postmenopausal women are largely unknown. To determine the effect of a new estrogen receptor, GPR30, in the maintenance of exercise capacity and skeletal muscle function in females, the selective GPR30 agonist, G1 (100 µg/kg/day), or vehicle (V, soybean oil) was administered subcutaneously daily (n = 7 per group) to ovariectomized (OVX) 27-month-old Fischer 344 × Brown Norway (F344BN) female rats. Following 8 weeks of treatment, the exercise capacity (treadmill walk time to exhaustion) was reduced in OVX vs. sham rats (5.1 ± 1.4 vs. 11.0 ± 0.9 min, P < 0.05), and chronic G1 restored exercise capacity (12.9 ± 1.2 min; P < 0.05 vs. OVX-V). Similarly, the peak twitch of electrically stimulated soleus muscles was decreased by 22% in OVX vs. sham rats (P < 0.05), and G1 attenuated this decline (P < 0.05). Western blot analysis showed that chronic G1 treatment attenuated OVX-associated decreases in heat shock protein (HSP) 90, HSP70, and HSP27 expressions. In vitro studies using the L6 myoblast cell line demonstrated that G1 increased mRNA levels of HSPs in cultured cells. Collectively, these data demonstrate that the activation of GPR30 mitigates the adverse effects of estrogen loss on exercise capacity and skeletal muscle contractile function in old F344BN rats. The protective effects of GPR30 might be through its upregulation of heat shock proteins in skeletal muscle.

Mast Cell Inhibition Attenuates Cardiac Remodeling and Diastolic Dysfunction in Middle-aged, Ovariectomized Fischer 344 × Brown Norway Rats.

The incidence of left ventricular diastolic dysfunction (LVDD) increases in women after menopause, yet the mechanisms are unclear. Because mast cells participate in the pathological processes of various cardiac diseases, we hypothesized that mast cell inhibition would protect against estrogen loss-induced LVDD. The mast cell stabilizer, cromolyn sodium (30 mg·kg·d), or vehicle was administered subcutaneously by osmotic minipump to ovariectomized (OVX) female Fischer 344 × Brown Norway (F344BN) rats starting at 4 weeks after surgery. Eight weeks after OVX, systolic blood pressure increased by 20% in OVX versus sham rats, and this effect was attenuated after 4 weeks of cromolyn treatment. Also, cromolyn mitigated the adverse reductions in myocardial relaxation (e') and increases in left ventricle (LV) filling pressures (E/e'), LV mass, wall thicknesses, and interstitial fibrosis from OVX. Although cardiac mast cell number was increased after OVX, cardiac chymase activity was not overtly altered by estrogen status and tended to decrease by cromolyn. Contrariwise, Ang II content was greater in hearts of OVX versus sham rats, and cromolyn attenuated this effect. Taken together, mast cell inhibition with cromolyn attenuates LV remodeling and LVDD in OVX-Fischer 344 × Brown Norway rats possibly through actions on the heart level and/or through vasodilatory effects at the vascular level.

GPR30 decreases cardiac chymase/angiotensin II by inhibiting local mast cell number.

Chronic activation of the novel estrogen receptor GPR30 by its agonist G1 mitigates the adverse effects of estrogen (E2) loss on cardiac structure and function. Using the ovariectomized (OVX) mRen2.Lewis rat, an E2-sensitive model of diastolic dysfunction, we found that E2 status is inversely correlated with local cardiac angiotensin II (Ang II) levels, likely via Ang I/chymase-mediated production. Since chymase is released from cardiac mast cells during stress (e.g., volume/pressure overload, inflammation), we hypothesized that GPR30-related cardioprotection after E2 loss might occur through its opposing actions on cardiac mast cell proliferation and chymase production. Using real-time quantitative PCR, immunohistochemistry, and immunoblot analysis, we found mast cell number, chymase expression, and cardiac Ang II levels were significantly increased in the hearts of OVX-compared to ovary-intact mRen2.Lewis rats and the GPR30 agonist G1 (50 mg/kg/day, s.c.) administered for 2 weeks limited the adverse effects of estrogen loss. In vitro studies revealed that GPR30 receptors are expressed in the RBL-2H3 mast cell line and G1 inhibits serum-induced cell proliferation in a dose-dependent manner, as determined by cell counting, BrdU incorporation assay, and Ki-67 staining. Using specific antagonists to estrogen receptors, blockage of GPR30, but not ERα or ERß, attenuated the inhibitory effects of estrogen on BrdU incorporation in RBL-2H3 cells. Further study of the mechanism underlying the effect on cell proliferation showed that G1 inhibits cyclin-dependent kinase 1 (CDK1) mRNA and protein expression in RBL-2H3 cells in a dose-dependent manner.

Activation of GPR30 inhibits cardiac fibroblast proliferation.

The incidence of left ventricular diastolic dysfunction significantly increases in postmenopausal women suggesting the association between estrogen loss and diastolic dysfunction. The in vivo activation of G protein-coupled estrogen receptor (GPR30) attenuates the adverse effects of estrogen loss on cardiac fibrosis and diastolic dysfunction in mRen2.Lewis rats. This study was designed to address the effects of GPR30 on cardiac fibroblast proliferation in rats. The expression of GPR30 in cardiac fibroblasts isolated from adult Sprague-Dawley rats was confirmed by RT-PCR, Western blot analysis, and immunofluorescence staining. Results from BrdU incorporation assays, cell counting, carboxyfluorescein diacetate succinimidyl ester labeling in conjunction with flow cytometry, and Ki-67 staining showed that treatment with G1, a specific agonist of GPR30, inhibited cardiac fibroblast proliferation in a dose-dependent manner, which was associated with decreases in CDK1 and cyclin B1 protein expressions. In the GPR30-KO cells, BrdU incorporation, and CDK1 and cyclin B1 expressions significantly increased when compared to GPR30-intact cells. G1 had no effect on BrdU incorporation, CDK1 and cyclin B1 mRNA levels in GPR30-KO cells. In vivo studies showed increases in CDK1 and cyclin B1 mRNA levels, Ki-67-positive cells, and the immunohistochemistry staining of vimentin, a fibroblast marker, in the left ventricles from ovariectomized mRen2.Lewis rats versus hearts from ovary-intact littermates; 2 weeks of G1 treatment attenuated these adverse effects of estrogen loss. This study demonstrates that GPR30 is expressed in rat cardiac fibroblasts, and activation of GPR30 limits proliferation of these cells likely via suppression of the cell cycle proteins, cyclin B1, and CDK1.

Sevoflurane modulation of Ca2+ regulation in skeletal muscle sarcoplasmic reticulum vesicles from young and mature rabbits.

INTRODUCTION: Developmental differences in splice variants of the two key sarcoplasmic reticulum (SR) calcium regulatory proteins, ryanodine (RyR1), and sarcoendoplasmic reticulum calcium pump (SERCA1) have been linked to various neuromuscular disorders, but not malignant hyperthermia (MH). However, it is unclear whether an age-related difference in volatile anesthetic-mediated SR calcium function exists that could add to our current understanding of the clinical presentation of MH syndrome and provide insight into molecular mechanisms for general anesthesia that may have other physiologic and/or pathophysiologic significance. Therefore, the effects of sevoflurane on intracellular calcium regulation in isolated SR membrane vesicles from the skeletal muscle of healthy young rabbits were compared to their adult counterpart using an established in vitro model with the assumption that exposure to sevoflurane would elicit a weaker response in the young SR. METHODS: Through dual wavelength spectroscopy of Ca(2+): Arsenazo III difference absorbance, the effects of sevoflurane on SR Ca(2+) uptake rate and release in heavy and light fraction SR membrane vesicles isolated from the white muscle of anesthetized, postweaned (age = 6 weeks, n = 5) and adult (age = 6 months, n = 5) male New Zealand rabbits were examined. RESULTS: The adult group showed a 50% increase in Ca(2+) uptake rate from control at both subclinical and clinically relevant anesthetic concentrations, whereas in the SR from the younger animals, Ca(2+) uptake rate was not altered by any concentration of sevoflurane. The sensitivity of both the low and high affinity Ca(2+)-binding sites on RyR1 was increased by sevoflurane to the same extent in the SR vesicles from the young and mature adult rabbits. Interestingly, a greater potency of sevoflurane for the high affinity-binding site was identified, and this was independent of age. CONCLUSIONS: These findings suggest that the sensitivity of the SR to sevoflurane-mediated Ca(2+) uptake may be increased with maturity, while an analogous developmental effect on RyR1 is less probable. Nonetheless, this study shows for the first time that a potent inhalational agent such as sevoflurane can influence the high affinity SR calcium-binding site by lowering the extraluminal concentration of calcium necessary to trigger calcium release. While this may not be of consequence when inhaled anesthetics are administered to normal children or adults, it may have life-threatening consequences in carriers of RyR1 mutations.

Effects of short-term treadmill exercise training or growth hormone supplementation on diastolic function and exercise tolerance in old rats.

Whether the lusitropic potential of short-term exercise in aged rats is linked to an augmentation in the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis and an alteration in the cardiac renin angiotensin system (RAS) is unknown. Old (28-month-old) male, Fischer 344xBrown Norway rats were randomized to 4 weeks of GH supplementation (300 microg subcutaneous, twice daily) or 4 weeks of treadmill running, or were used as sedentary controls. Six-month-old rats, sedentary or exercised, were used as young controls. Training improved exercise capacity in old animals. Exercise and GH attenuated age-related declines in myocardial relaxation despite an exercise-induced suppression of IGF-1. The regulatory protein, sarcoplasmic Ca2+ adenosine triphosphatase (SERCA2), increased with exercise but not GH. Among aged rats, the cardiac RAS was not altered by training or GH. Thus, the signaling pathway underlying the lusitropic benefit of short-term habitual exercise in the aged rat may be distinct from GH-mediated benefits and independent of the cardiac RAS.

Progressive diastolic dysfunction in the female mRen(2). Lewis rat: influence of salt and ovarian hormones.

This study determined the contribution of chronic salt loading and early loss of ovarian hormones on diastolic function in the hypertensive female mRen(2). Lewis rat, a monogenetic strain that expresses the mouse renin-2 gene in various tissues. Estrogen-intact mRen2 rats fed a high salt (HS) (8% sodium chloride) diet exhibited early diastolic dysfunction when compared to normal salt-fed (NS) (1% sodium chloride) rats. In contrast, ovariectomized (OVX) rats on either NS or HS diets showed impaired relaxation with evidence of elevated left ventricular filling pressures (E/e') or pseudonormalization. This more advanced stage of diastolic dysfunction was associated with increases in interstitial cardiac fibrosis and high circulating levels of aldosterone, two factors leading to reduced ventricular compliance. These findings may explain the preponderance of diastolic dysfunction and diastolic heart failure in postmenopausal women and provide a potential animal model for evaluating prevention and treatment interventions for this disorder.

G protein-coupled estrogen receptor (GPER) deficiency induces cardiac remodeling through oxidative stress.

Oxidative stress has been implicated in the unfavorable changes in cardiac function and remodeling that occur after ovarian estrogen loss. Using ovariectomized rat models, we previously reported that the cardioprotective actions of estrogen are mediated by the G protein-coupled estrogen receptor (GPER). Here, in 9-month-old, female cardiomyocyte-specific GPER knockout (KO) mice vs sex- and age-matched wild-type (WT) mice, we found increased cardiac oxidative stress and oxidant damage, measured as a decreased ratio of reduced glutathione to oxidized glutathione, increased 4-hydroxynonenal and 8-hydroxy-2'-deoxyguanosine (8-oxo-DG) staining, and increased expression of oxidative stress-related genes. GPER KO mice also displayed increased heart weight, cardiac collagen deposition, and Doppler-derived filling pressure, and decreased percent fractional shortening and early mitral annular velocity compared with WT controls. Treatment of GPER KO mice for 8 weeks with phosphonium [10-(4,5-dimethoxy-2-methyl 3,6-dioxo-1,4-cyclohexadien-1-yl)decyl] triphenyl-,mesylate (MitoQ), a mitochondria-targeted antioxidant, significantly attenuated these measures of cardiac dysfunction, and MitoQ decreased 8-oxo-DG intensity compared with treatment with an inactive comparator compound, (1-decyl)triphenylphosphonium bromide (P <0.05). A real-time polymerase chain reaction array analysis of 84 oxidative stress and antioxidant defense genes revealed that MitoQ attenuates the increase in NADPH oxidase 4 and prostaglandin-endoperoxide synthase 2 and the decrease in uncoupling protein 3 and glutathione S-transferase kappa 1 seen in GPER KO mice. Our findings suggest that the cardioprotective effects of GPER include an antioxidant role and that targeted strategies to limit oxidative stress after early noncancerous surgical extirpation of ovaries or menopause may help limit alterations in cardiac structure and function related to estrogen loss.

Self- vs proxy-reported mobility using the mobility assessment tool-short form in elderly preoperative patients.

BACKGROUND: Mobility is fundamental to maintenance of an independent lifestyle and can predict clinical outcomes after health events among older individuals. However, certain clinical situations do not accommodate physical or self-assessments. This investigation examines whether proxy-reported assessments of function using the Mobility Assessment Tool-short (MAT-sf) form is a reliable alternative. METHODS: Sixty-six older persons (≥ age 70) and their proxies were enrolled. Proxies rated patients' mobility using the MAT-sf as did the patients. RESULTS: The mean age of patients was 78.4 yr. (±6.2); 44% were female and 86% were white. Spouses made up 55% of the proxies, while 39% were children/in-laws. The correlation coefficient between patient and proxy MAT-sf scores was 0.81 (p < 0.01); a comparison of the slope of the regression line relating patient- and proxy-reported MAT-sf to a line of identity showed disagreement (p < 0.01), with proxy reports underreporting patient responses by 8.3% in lower mobility patients. The intra-class correlation characterizing agreement between repeated proxy reports 0.81. CONCLUSION: Proxy reports of mobility in older patients have good reliability. However, in patients with poor mobility, the proxies tend to report a lower mobility than the patients.

Inflammatory and mitochondrial gene expression data in GPER-deficient cardiomyocytes from male and female mice.

We previously showed that cardiomyocyte-specific G protein-coupled estrogen receptor (GPER) gene deletion leads to sex-specific adverse effects on cardiac structure and function; alterations which may be due to distinct differences in mitochondrial and inflammatory processes between sexes. Here, we provide the results of Gene Set Enrichment Analysis (GSEA) based on the DNA microarray data from GPER-knockout versus GPER-intact (intact) cardiomyocytes. This article contains complete data on the mitochondrial and inflammatory response-related gene expression changes that were significant in GPER knockout versus intact cardiomyocytes from adult male and female mice. The data are supplemental to our original research article "Cardiomyocyte-specific deletion of the G protein-coupled estrogen receptor (GPER) leads to left ventricular dysfunction and adverse remodeling: a sex-specific gene profiling" (Wang et al., 2016) [1]. Data have been deposited to the Gene Expression Omnibus (GEO) database repository with the dataset identifier GSE86843.

Effect of Age, Estrogen Status, and Late-Life GPER Activation on Cardiac Structure and Function in the Fischer344×Brown Norway Female Rat.

Age-associated changes in cardiac structure and function, together with estrogen loss, contribute to the progression of heart failure with preserved ejection fraction in older women. To investigate the effects of aging and estrogen loss on the development of its precursor, asymptomatic left ventricular diastolic dysfunction, echocardiograms were performed in 10 middle-aged (20 months) and 30 old-aged (30 months) female Fischer344×Brown-Norway rats, 4 and 8 weeks after ovariectomy (OVX) and sham procedures (gonads left intact). The cardioprotective potential of administering chronic G1, the selective agonist to the new G-protein-coupled estrogen receptor (GPER), was further evaluated in old rats (Old-OVX+G1) versus age-matched, vehicle-treated OVX and gonadal intact rats. Advanced age and estrogen loss led to decreases in myocardial relaxation and elevations in filling pressure, in part, due to reductions in phosphorylated phospholamban and increases in cardiac collagen deposition. Eight weeks of G-protein-coupled estrogen receptor activation in Old-OVX+G1 rats reversed the adverse effects of age and estrogen loss on myocardial relaxation through increases in sarcoplasmic reticulum Ca2+ ATPase expression and reductions in interstitial fibrosis. These findings may explain the preponderance of heart failure with preserved ejection fraction in older postmenopausal women and provide a promising, late-life therapeutic target to reverse or halt the progression of left ventricular diastolic dysfunction.

Characterization of the cardiac renin angiotensin system in oophorectomized and estrogen-replete mRen2.Lewis rats.

The cardioprotective effects of estrogen are well recognized, but the mechanisms remain poorly understood. Accumulating evidence suggests that the local cardiac renin-angiotensin system (RAS) is involved in the development and progression of cardiac hypertrophy, remodeling, and heart failure. Estrogen attenuates the effects of an activated circulating RAS; however, its role in regulating the cardiac RAS is unclear. Bilateral oophorectomy (OVX; n = 17) or sham-operation (Sham; n = 13) was performed in 4-week-old, female mRen2.Lewis rats. At 11 weeks of age, the rats were randomized and received either 17 ß-estradiol (E2, 36 µg/pellet, 60-day release, n = 8) or vehicle (OVX-V, n = 9) for 4 weeks. The rats were sacrificed, and blood and hearts were used to determine protein and/or gene expression of circulating and tissue RAS components. E2 treatment minimized the rise in circulating angiotensin (Ang) II and aldosterone produced by loss of ovarian estrogens. Chronic E2 also attenuated OVX-associated increases in cardiac Ang II, Ang-(1-7) content, chymase gene expression, and mast cell number. Neither OVX nor OVX+E2 altered cardiac expression or activity of renin, angiotensinogen, angiotensin-converting enzyme (ACE), and Ang II type 1 receptor (AT1R). E2 treatment in OVX rats significantly decreased gene expression of MMP-9, ACE2, and Ang-(1-7) mas receptor, in comparison to sham-operated and OVX littermates. E2 treatment appears to inhibit upsurges in cardiac Ang II expression in the OVX-mRen2 rat, possibly by reducing chymase-dependent Ang II formation. Further studies are warranted to determine whether an E2-mediated reduction in cardiac chymase directly contributes to this response in OVX rats.

Differential effects of late-life initiation of low-dose enalapril and losartan on diastolic function in senescent Fischer 344 x Brown Norway male rats.

No proven pharmacological therapies to delay or reverse age-related diastolic dysfunction exist. We hypothesized that late-life low-dose (non-blood-pressure-lowering) angiotensin-converting enzyme inhibition vs. angiotensin II receptor blockade would be equally efficacious at mitigating diastolic dysfunction in the senescent Fischer 344 × Brown Norway rat. Enalapril (10 mg/kg/day; n = 9) initiated at 24 months of age and continued for 6 months, increased myocardial relaxation (e'), reduced Doppler-derived indices of filling pressure (E/e'), favorably lowered the ratio of phospholamban-SERCA2 and reduced oxidative stress markers, Rac1 and nitrotyrosine, in aged hearts. Treatment with losartan (15 mg/kg/day; n = 9) similarly mitigated signs of cardiac oxidative stress, but impairments in diastolic function persisted when compared with untreated rats (n = 7). Our findings favor the idea that the lusitropic benefit of low-dose angiotensin-converting enzyme inhibitor initiated late in life may be related to an antioxidant-mediated modulation of SERCA2, resulting in improved relaxation rather than via overt effects on cardiac structure or blood pressure.

Activation of GPR30 attenuates diastolic dysfunction and left ventricle remodelling in oophorectomized mRen2.Lewis rats.

AIMS: GPR30 is a novel oestrogen receptor expressed in various tissues, including the heart. We determined the role of GPR30 in the maintenance of left ventricular (LV) structure and diastolic function after the surgical loss of ovarian hormones in the female mRen2.Lewis rat, a model emulating the cardiac phenotype of the post-menopausal woman. METHODS AND RESULTS: Bilateral oophorectomy (OVX) or sham surgery was performed in study rats; the selective GPR30 agonist, G-1 (50 µg/kg/day), or vehicle was given subcutaneously to OVX rats from 13-15 weeks of age. Similar to the cardiac phenotype of sham rats, G-1 preserved diastolic function and structure relative to vehicle-treated OVX littermates independent of changes in blood pressure. G-1 limited the OVX-induced increase in LV filling pressure, LV mass, wall thickness, interstitial collagen deposition, atrial natriuretic factor and brain natriuretic peptide mRNA levels, and cardiac NAD(P)H oxidase 4 (NOX4) expression. In vitro studies showed that G-1 inhibited angiotensin II-induced hypertrophy in H9c2 cardiomyocytes, evidenced by reductions in cell size, protein content per cell, and atrial natriuretic factor mRNA levels. The GPR30 antagonist, G15, inhibited the protective effects of both oestradiol and G-1 on this hypertrophy. CONCLUSION: These data show that the GPR30 agonist G-1 mitigates the adverse effects of oestrogen loss on LV remodelling and the development of diastolic dysfunction in the study rats. This expands our knowledge of the sex-specific mechanisms underlying diastolic dysfunction and provides a potential therapeutic target for reducing the progression of this cardiovascular disease process in post-menopausal women.

Effects of short-term GH supplementation and treadmill exercise training on physical performance and skeletal muscle apoptosis in old rats.

Growth hormone (GH) supplementation at old age has been shown to improve body composition, although its effect on muscle performance is still debated. On the other hand, resistance training increases muscle mass and strength even when initiated at advanced age. In the present study, we investigated the effects of short-term GH supplementation and exercise training on physical performance and skeletal muscle apoptosis in aged rats. Old (28 mo) male Fischer 344 x Brown Norway rats were randomized to 4 wk of GH supplementation (300 mug subcutaneous, twice daily) or 4 wk of treadmill running or used as sedentary controls. Eight-month-old rats, sedentary or exercised, were used as young controls. Exercise training improved exercise capacity and muscle strength in old animals. In soleus muscle, age and exercise were not associated with significant changes in the extent of apoptosis. However, we detected an age-related increase of cleaved caspase-8 (+98%), cleaved caspase-3 (+136%), and apoptotic DNA fragmentation (+203%) in the extensor digitorum longus muscle of old sedentary rats, which was attenuated by exercise. GH administration neither ameliorated physical performance nor attenuated apoptosis in extensor digitorum longus and was associated with increased apoptosis in soleus muscle (+206% vs. old controls). Our findings indicate that a short-term program of exercise training started at advanced age reverses age-related skeletal muscle apoptosis and represents an effective strategy to improve physical performance. In contrast, short-term administration of GH late in life does not provide any protection against functional decline or muscle aging and may even accelerate apoptosis in slow-twitch muscles, such as the soleus.

Developmental differences in spinal cyclooxygenase 1 expression after surgical incision.

BACKGROUND: Systemic administration of a cyclooxygenase 1 (COX-1) inhibitor reduces hypersensitivity to mechanical stimuli after incisional paw surgery in 4-week-old, but not 2-week-old, animals. The purpose of the current study was to test whether this developmental difference was reflected by differences in COX-1 expression in the spinal cord after surgery. METHODS: Rats 2 and 4 weeks of age, paralleling infant and child human neurologic developmental stages, were used. A paw incision was made under general anesthesia and the withdrawal thresholds were measured before and after systemic and intrathecal administration of a COX-1 selective inhibitor (SC560). Immunohistochemistry was used to assess COX-1 protein in the spinal cord, and real-time polymerase chain reaction was used to quantify gene expression of COX-1 mRNA. RESULTS: Systemic and intrathecal administration of SC560 produced an increase in withdrawal threshold in the 4-week-old, but not in the 2-week-old, animals. Intrathecal SC560 increased withdrawal thresholds in the 4-week-old animals at a dose 100-fold less than with systemic administration. Cyclooxygenase 1 protein in the spinal cord was increased ipsilateral to surgery in the 4-week-old, but not in the 2-week-old, animals. Cyclooxygenase 1 mRNA was increased in the 4-week-old animals in the spinal cord ipsilateral to surgery relative to the contralateral side of the spinal cord, but not in the 2-week-old animals. CONCLUSIONS: These results suggest that developmental differences in COX-1 expression in the spinal cord likely explain the lack of efficacy of COX-1 inhibitors in the 2-week-old rats. Whether this reflects a deficit in factors that stimulate COX-1 expression or a difference in response to these factors is not addressed, but should similar deficits occur in humans, COX-1 inhibitors may exhibit reduced efficacy in infants.

Effects of moderate and deep hypothermia on Ca2+ signaling in rat ventricular myocytes.

BACKGROUND/AIMS: We investigated whether the degree of hypothermia determines the impairment in cardiac muscle function upon rewarming and whether the sarcoplasmic reticulum Ca2+ release channel, RyR(2), contributes to hypothermia-induced changes in myoplasmic [Ca2+]. METHODS: Tension measurements using rat papillary muscle and calcium transients (Fluorescent Ca2+ indicator Fura 2-AM) in rat ventricular myocytes were compared during deep (10 degrees C-16 degrees C) and moderate hypothermic (28 degrees C) myocardial temperatures. In a second experiment, myocytes were pretreated with dantrolene, an RyR(2) antagonist; calcium transients were determined at control temperatures (32 degrees C), 16 degrees C, and upon rewarming (32 degrees C). RESULTS: Papillary muscle contractility and myocyte calcium transients were significantly reduced during and after rewarming from 16 degrees C. At 28 degrees C, papillary muscle isometric tension was potentiated and calcium transients were unaffected. After rewarming from 28 degrees C, excitation-contraction coupling was maintained as isometric tension returned to 90% of control values. After rewarming from 16 degrees C, myocytes pretreated with dantrolene had return of calcium transients to 89% of control values while myocytes not treated with dantrolene recovered to only 50% of their control values. CONCLUSION: We conclude that deep hypothermia, as opposed to moderate hypothermia of the myocardium, disrupts excitation-contraction coupling at cellular and tissue levels. Our finding of preserved calcium transients in dantrolene-pretreated myocytes exposed to deep hypothermia suggests a potential role for the RyR(2) channel in post-hypothermia reductions in cardiac function.