Cardioprotective effects of exercise training on myofilament calcium activation in ovariectomized rats.

The risks associated with hormone replacement therapy, especially cardiac diseases in postmenopausal women, have prompted extensive studies for other preventive or therapeutic alternatives. We investigated the cardioprotective effects of exercise training on the changes in cardiac myofilament Ca2+ activation in 10-wk-old ovariectomized rats. The exercise groups were subjected to a 9-wk running program on a motor-driven treadmill 1 wk after surgery. The relationship between pCa (-log molar free Ca2+ concentration) and myofibrillar MgATPase activity of exercise-sham myofibrils or exercise-ovariectomized myofibrils was the same and could not be distinguished from that of sedentary-sham control hearts. In contrast, a significant suppression in maximum MgATPase activity and a leftward shift of pCa50 (half-maximally activating pCa) in the pCa-ATPase activity relationship were detected in sedentary-ovariectomized rats. Exercise training also prevented the shift in myosin heavy chain (MHC) isoforms toward beta-MHC in ovariectomized hearts. The upregulation of beta1-adrenergic receptors in the left ventricular membranes of ovariectomized rat hearts, as measured by receptor binding and immunoblot analyses, was no longer observed in exercise-ovariectomized hearts. Immunoblot analyses of heat shock protein (HSP) 72, an inducible form of HSP70, demonstrated a significant downregulation in ovariectomized hearts. Exercise training in ovariectomized rats completely reversed the expression of HSP72 to the same level as sham controls. Our results clearly indicate the cardioprotective effects of exercise training on changes in cardiac myofilament Ca2+ activation in ovariectomized rats. Alterations in expression of beta1-adrenergic receptors and HSP72 may, in part, play a mechanistic role in the cardioprotective effects.     

Estrogen supplement prevents the calcium hypersensitivity of cardiac myofilaments in ovariectomized rats

Our previous biochemical and mechanical studies have demonstrated an increase in Ca2+ sensitivity of cardiac myofilaments in ovariectomized rats. To test whether the body weight gain associated with ovariectomy contributed some effects to the changes in myofibrillar functions, the relations of pCa (-log Ca2+ molar concentration) to actomyosin adenosine triphosphatase (ATPase) activity of isolated myofibrillar preparations from 10-week pair-fed ovariectomized rats were compared with those from sham-operated controls. Despite similar body weights, the maximum myofibrillar ATPase activity was significantly lower in pair-fed ovariectomized rats as compared to that of sham-operated controls. In addition, the pCa-actomyosin ATPase relationship of pair-fed ovariectomized hearts still demonstrated a significant leftward shift in pCa50 (-log half-maximally Ca2+ activation) from that of sham-operated controls. To find out which hormone was responsible for the observed increase in myofibrillar Ca2+ sensitivity, different sex hormone supplemental regimens were administered to ovariectomized rats. Subcutaneous injection of estrogen (5 microg/rat) or estrogen plus progesterone (1 mg/rat) three times a week could effectively prevent the changes in body weight, heart weight, and uterine weight of the ovariectomized animals. Moreover, supplements of either estrogen or progesterone could prevent a decrease in maximum ATPase activity. In contrast, only the estrogen replacement could abolish the Ca2+ hypersensitivity of the myofilaments in these ovariectomized rats. These results suggest differential cardio-regulatory effects of ovarian sex hormones on the Ca2+ activation of the myofilaments.     

Upregulation of beta 1-adrenergic receptors in ovariectomized rat hearts

Changes in cardiac myofilament Ca(2+) activation have been demonstrated in ovariectomized rats. The underlying mechanisms responsible for these changes, however, are unknown. Accordingly, we measured both density and binding affinity of cardiac beta(1)-adrenergic receptors in sarcolemmal preparations from 10-week ovariectomized rats, pair-fed ovariectomized rats, and sham-operated control rats. Receptor protein content was also measured by immunoblotting. Deprivation of ovarian sex hormones for 10 weeks induced a significant upregulation of beta(1)-adrenergic receptors without affecting binding affinity. The same magnitude of receptor upregulation was also detected in pair-fed ovariectomized hearts. To determine which hormone is responsible for the observed increase in beta(1)-adrenergic receptor density, various sex hormone supplemental regimens were administered to ovariectomized rats. Subcutaneous injection of estrogen (5 microg/rat), progesterone (1 mg/rat), or estrogen plus progesterone three times a week all effectively prevented the upregulation of the beta(1)-adrenoceptors. Western blot analyses using polyclonal antibody of beta(1)-adrenergic receptors revealed the same pattern of changes in the protein content of the receptors in these various groups of experimental hearts as those obtained from the receptor binding assay. These results suggest a possible direct suppressive effect of ovarian sex hormones on the expression of cardiac beta(1)-adrenergic receptors.     

Effect of estrogen on global myocardial ischemia-reperfusion injury in female rats

We investigated the effects of estrogen on global myocardial ischemia-reperfusion injury in rats that were ovariectomized (Ovx), sham-operated, or ovariectomized and then given 17beta-estradiol (E(2)beta) supplementation (Ovx+E(2)beta). Hearts were excised, cannulated, perfused with and then immersed in chilled (4 degrees C) cardioplegia solution for 30 min, and then retrogradely perfused with warm (37 degrees C), oxygenated Krebs-Henseleit bicarbonate buffer for 120 min. The coronary flow rate, first derivative of left ventricular pressure, and nitrite production were all significantly lower in Ovx than in sham-operated or Ovx+E(2)beta hearts. However, coronary flow rates or nitrate production were not consistently different throughout the entire reperfusion period. Ca(2+) accumulated more in Ovx rat hearts than in sham-operated or Ovx+E(2)beta hearts, and mitochondrial respiratory function was lower in Ovx hearts than in hearts from the other two groups. Marked interstitial edema and contraction bands were seen in hematoxylin-eosin-stained sections of Ovx rat hearts but not in hearts from either of the other groups. Hematoxylin-basic fuchsin-picric acid-stained sections revealed fewer viable myocytes in hearts from the Ovx group than from the sham or Ovx+E(2)beta group. Transmission electron microscopy demonstrated more severely damaged mitochondria and ultrastructural damage to myocytes in Ovx rat hearts. Our results indicate that estrogen plays a cardioprotective role in global myocardial ischemia-reperfusion injury in female rats.     

Cardioprotection through a PKC-dependent decrease in myofilament ATPase

Activation of myocardial kappa-opioid receptor-protein kinase C (PKC) pathways may improve postischemic contractile function through a myofilament reduction in ATP utilization. To test this, we first examined the effects of PKC inhibitors on kappa-opioid receptor-dependent cardioprotection. The kappa-opioid receptor agonist U50,488H (U50) increased postischemic left ventricular developed pressure and reduced postischemic end-diastolic pressure compared with controls. PKC inhibitors abolished the cardioprotective effects of U50. To determine whether kappa-opioid-PKC-dependent decreases in Ca2+-dependent actomyosin Mg2+-ATPase could account for cardioprotection, we subjected hearts to three separate actomyosin ATPase-lowering protocols. We observed that moderate decreases in myofibrillar ATPase were equally cardioprotective as kappa-opioid receptor stimulation. Immunoblot analysis and confocal microscopy revealed a kappa-opioid-induced increase in myofilament-associated PKC-epsilon, and myofibrillar Ca2+-independent PKC activity was increased after kappa-opioid stimulation. This PKC-myofilament association led to an increase in troponin I and C-protein phosphorylation. Thus we propose PKC-epsilon activation and translocation to the myofilaments causes a decrease in actomyosin ATPase, which contributes to the kappa-opioid receptor-dependent cardioprotective mechanism.     

Increased myocardial stiffness with maintenance of length-dependent calcium activation by female sex hormones in diabetic rats

A decrease in peak early diastolic filling velocity in postmenopausal women implies a sex hormone-related diastolic dysfunction. The regulatory effect of female sex hormones on cardiac distensibility therefore was evaluated in ovariectomized rats by determining the sarcomere length-passive tension relationship of ventricular skinned fiber preparations. Diabetes also was induced in the rat to assess the protective significance of female sex hormones on diastolic function. While ovariectomy had no effect on myocardial stiffness, collagen content, or titin ratio, a significant increase in myocardial stiffness was observed in diabetic rat only when female sex hormones were intact. The increased stiffness in diabetic-sham rats was accompanied by an elevated collagen content resulting from increases in the levels of procollagen and Smad2. Surprisingly, the increased myocardial stiffness in diabetic-sham rats was accompanied by a shift toward a more compliant N2BA of cardiac titin isoforms. The pCa-active tension relationship was analyzed at fixed sarcomere lengths of 2.0 and 2.3 μm to determine the magnitude of changes in myofilament Ca(2+) sensitivity between the two sarcomere lengths. Interestingly, high expression of N2BA titin was associated with a suppressed magnitude of changes in myofilament Ca(2+) sensitivity only in the diabetic-ovariectomized condition. Estrogen supplementation in diabetic-ovariectomized rats partially increased myocardial stiffness but completely reversed the change in myofilament Ca(2+) sensitivity. These results indicate a restrictive adaptation of myocardium governed by female sex hormones to maintain myofilament activity in compensation to the pathophysiological induction of cardiac dilatation by the diabetic condition.     

Time dependent response of cardiac myofibrillar ATPase activity to exercise

1. The purpose of the present study was to investigate the time course of run training effects on the Ca2+ kinetics of the cardiac myofibrillar ATPase activity in female Sprague-Dawley rats. 2. The cardiac myofibrillar ATPase activity was measured at varying Ca2+ levels, and the Hill-n and pCa50 were measured in the hearts of rats after 3, 6 and 9 weeks of running training with a training program that began with an initially high intensity (HINT) and a training program with a more progressive increase in intensity (PROG). 3. After 3 and 6 weeks of training cardiac myofibrillar ATPase activity in the hearts of the trained rats in both training programs was elevated by 28-40% over the control group (P less than 0.05) at a pCa5 but was not different from the control groups after 9 weeks of training (P greater than or equal to 0.05). 4. Also the Ca2+ co-operativity as measured by the Hill-n was elevated in the hearts of the trained rats after 6 and 9 weeks of training when compared to control groups suggesting changes in the regulatory proteins of the myofibrils of hearts from trained rats. 5. The elevations in cardiac myofibrillar ATPase activity suggest that the myocardium responded to the training stimulus in a phasic manner. 6. The regression of cardiac myofibrillar ATPase in the late weeks of training might be related to a reduction or a loss of a specific training stimulus for the myocardium.     

Gender differences in the expression of heat shock proteins: the effect of estrogen

The heat shock proteins (HSPs) are an important family of endogenous, protective proteins that are found in all tissues. In the heart, HSP72, the inducible form of HSP70, has been the most intensely studied. It is well established that HSP72 is induced with ischemia and is cardioprotective. Overexpression of other HSPs also is protective against cardiac injury. Recently, we observed that 17beta-estradiol increases levels of HSPs in male rat cardiac myocytes. We hypothesized that there were gender differences in HSP72 expression in the heart secondary to estrogen. To test this hypothesis, we examined cardiac levels of HSP72 by ELISA in male and female Sprague-Dawley rats. In addition, three other HSPs were assessed by Western blot (HSP27, HSP60, and HSP90). To determine whether estrogen status affected HSP72 expression in other muscles or tissues, two other muscle tissues, slow twitch muscle (soleus muscle) and fast twitch muscle (gastrocnemius muscle), were studied as well as two other organs, the kidney and liver. Because HSP72 is cardioprotective, and females are known to have less cardiovascular disease premenopause, the effects of ovariectomy were examined. We report that female Sprague-Dawley rat hearts have twice as much HSP72 as male hearts. Ovariectomy reduced the level of HSP72 in female hearts, and this could be prevented by estrogen replacement therapy. These data show that the expression of cardiac HSP72 is greater in female rats than in male rats, due to upregulation by estrogen.     

Changes in myocardial contractile protein ATPases in chronically adrenalectomized rats with and without glucocorticoid replacement

Studies were conducted to examine the effects of chronic adrenalectomy (Adx) and adrenalectomy plus glucocorticoid replacement therapy on rat cardiac contractile protein ATPase activities. The Ca2+-dependent Mg-ATPase activity of myofibrils isolated from rat ventricles 3 weeks postadrenalectomy (Adx) was significantly decreased at all pCa2+ concentrations (P less than 0.01), compared to sham-operated (SO) rats. Similarly, Ca2+-, K+-EDTA, and actin-activated myosin ATPase activities of Adx rat hearts were markedly decreased below that of SO rats (P less than 0.01). Dexamethasone administration to Adx rats prevented the decrease of Ca2+- and K+-ATPase activities of myosin, but not of myofibrillar Ca2+-dependent Mg-ATPase or actin-activated myosin Mg-ATPase activities. These studies suggest that glucocorticoid insufficiency induced by adrenalectomy results in altered myocardial contractile protein ATPase activity which may underlie impaired cardiac performance.     

Effects of streptozotocin-induced diabetes mellitus on some bone turnover markers in the vertebrae of ovary-intact and ovariectomized adult rats.

Diabetes mellitus and estrogen deficit are known causes of osteopenia in animal models as well as in humans. In the present work, the combined effect of ovariectomy and diabetes was investigated. Diabetes was induced in ovary-intact and ovariectomized female Wistar rats with a single injection (50 mg/kg body weight, i.p.) of streptozotocin. The rats were administered insulin (I) daily or 17-beta estradiol (E2) on alternate days for a period of 35 days and sacrificed. Serum calcium (Ca2+), phosphorus (P), alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), vertebral ALP, collagen, and glycosaminoglycans were estimated. The levels of serum Ca2+ and P increased in diabetic rats, but decreased after I or E2 treatments. Serum ALP and TRAP activity increased in the ovary-intact and ovariectomized diabetic rats. Vertebral ALP activity increased in ovariectomized diabetic rats, but decreased in diabetic rats, which were treated with I or E2. In the vertebrae, TRAP activity was elevated as a result of diabetes, but this was prevented by insulin or estradiol. Diabetes induced a decrease in total collagen in the vertebrae, while I or E2 treatment induced an increase. The levels of chondroitin sulphate and heparan sulphate decreased significantly in the vertebrae of both ovary-intact and ovariectomized diabetic rats, while hyaluronic acid increased. In conclusion, diabetes and ovariectomy each seem to affect the process of matrix formation and mineralization in the bone, and this is aggravated by the combination of diabetes and ovariectomy. The effects of I and E2 were similar, and both hormones reversed the changes brought about by diabetes.     

Modification of myosin protein and gene expression in failing hearts due to myocardial infarction by enalapril or losartan

The effects of enalapril, an angiotensin converting enzyme (ACE) inhibitor, and losartan, an angiotensin II receptor type I antagonist, were investigated on alterations in myofibrillar ATPase activity as well as myosin heavy chain (MHC) content and gene expression in failing hearts following myocardial infarction (MI). Three weeks after ligation of the left coronary artery, rats were treated with or without enalapril (10 mg/kg/day), and/or losartan (20 mg/kg/day) for 5 weeks. The infarcted animals exhibited an increase in left ventricle (LV) end diastolic pressure and depressed rates of LV pressure development as well as pressure decay. LV myofibrillar Ca2+ -stimulated ATPase activity was decreased in the infarcted hearts compared with controls, MHC alpha-isoform content was significantly decreased whereas that of MHC beta-isoform was markedly increased. The level of MHC alpha-isoform mRNA was decreased whereas that of MHC beta-isoform was increased in the viable infarcted LV. Treatment of animal with enalapril, losartan, or combination of enalapril and losartan partially prevented the MI induced changes in LV function, myofibrillar Ca2+ -stimulated ATPase activity, MHC protein expression and MHC gene expression. The results suggest that the beneficial effects of the renin-angiotensin system blockade in heart failure are associated with partial prevention of myofibrillar remodeling.     

Renin-angiotensin blockade attenuates cardiac myofibrillar remodelling in chronic diabetes

Previous studies have shown that the renin-angiotensin system (RAS) is activated in diabetes and this may contribute to the subcellular remodelling and heart dysfunction in this disease. Therefore, we examined the effects of RAS blockade by enalapril, an angiotensin-converting enzyme inhibitor, and losartan, an angiotensin receptor AT1 antagonist, on cardiac function, myofibrillar and myosin ATPase activity as well as myosin heavy chain (MHC) isozyme expression in diabetic hearts. Diabetes was induced in rats by a single injection of streptozotocin (65 mg/kg; i.v.) and these animals were treated with and without enalapril (10 mg/kg/day; oral) or losartan (20 mg/kg/day; oral) for 8 weeks. Enalapril or losartan prevented the depressions in left ventricular rate of pressure development, rate of pressure decay and ventricular weight seen in diabetic animals. Both drugs also attenuated the decrease in myofibrillar Ca2+-ATPase, Mg2+-ATPase and myosin ATPase activity seen in diabetic rats. The diabetes-induced increase in beta-MHC content and gene expression as well as the decrease in alpha-MHC content and mRNA levels were also prevented by enalapril and losartan. These results suggest the occurrence of myofibrillar remodelling in diabetic cardiomyopathy and provide evidence that the beneficial effects of RAS blockade in diabetes may be associated with attenuation of myofibrillar remodelling in the heart.     

Maintained contractile reserve in a transgenic mouse model of myocardial stunning

Cardiac excitation-contraction (E-C) coupling is impaired at the myofilament level in the reversible postischemic dysfunction known as "stunned" myocardium. We characterized tension development and calcium cycling in intact isolated trabeculae from transgenic (TG) mice expressing the major proteolytic degradation fragment of troponin I (TnI) found in stunned myocardium (TnI(1-193)) and determined the ATPase activity of myofibrils extracted from TG and non-TG mouse hearts. The phenotype of these mice at baseline recapitulates that of stunning. Here, we address the question of whether contractile reserve is preserved in these mice, as it is in genuine stunned myocardium. During twitch contractions, calcium cycling was normal, whereas tension was greatly reduced, compared with non-TG controls. A decrease in maximum Ca2+-activated tension and Ca2+ desensitization of the myofilaments accounted for this contractile dysfunction. The decrease in maximum tension was paralleled by an equivalent decrease in maximum Ca2+-activated myofibrillar ATPase activity. Exposure to high calcium or isoproterenol recruited a sizable contractile reserve in TG muscles, which was proportionately similar to that in control muscles but scaled downward in amplitude. These results suggest that calcium regulatory pathways and beta-adrenergic signal transduction remain intact in isolated trabeculae from stunned TG mice, further recapitulating key features of genuine stunned myocardium.     

Mg-ATPase and CA2+ activated myosin ATPase activity in ventricular myofibrils from non-failing and diseased human hearts – effects of calcium sensitizing agents MCI-154, DPI 201–106, and caffeine

We investigated the effects of two purported calcium sensitizing agents, MCI-154 and DPI 201–106, and a known calcium sensitizer caffeine on Mg-ATPase (myofibrillar ATPase) and myosin ATPase activity of left ventricular myofibrils isolated from non-failing, idiopathic (IDCM) and ischemic cardiomyopathic (ISCM) human hearts (i.e. failing hearts). The myofibrillar ATPase activity of non-failing myofibrils was higher than that of diseased myofibrils. MCI-154 increased myofibrillar ATPase Ca²⁺ sensitivity in myofibrils from non-failing and failing human hearts. Effects of caffeine similarly increased Ca²⁺ sensitivity. Effects of DPI 201–106 were, however, different. Only at the 10^–6 M concentration was a significant increase in myofibrillar ATPase calcium sensitivity seen in myofibrils from non-failing human hearts. In contrast, in myofibrils from failing hearts, DPI 201–106 caused a concentration-dependent increase in myofibrillar ATPase Ca²⁺ sensitivity. Myosin ATPase activity in failing myocardium was also decreased. In the presence of MCI-154, myosin ATPase activity increased by 11, 19, and 24% for non-failing, IDCM, and ISCM hearts, respectively. DPI 201–106 caused an increase in the enzymatic activity of less than 5% for all preparations, and caffeine induced an increase of 4, 11, and 10% in non-failing, IDCM and ISCM hearts, respectively. The mechanism of restoring the myofibrillar Ca²⁺ sensitivity and myosin enzymatic activity in diseased human hearts is most likely due to enhancement of the Ca²⁺ activation of the contractile apparatus induced by these agents. We propose that myosin light chain-related regulation may play a complementary role to the troponin-related regulation of myocardial contractility.     

Phosphorylation of C-protein, troponin I and phospholamban in isolated rabbit hearts.

Phosphorylation of myofibrillar and sacroplasmic-reticulum (SR) proteins was studied in Langendorff-perfused rabbit hearts subjected to various inotropic interventions. Stimulation of hearts with isoprenaline resulted in the phosphorylation of both troponin I (TnI) and C-protein in myofibrils and phospholamban in SR. Phosphorylation of phospholamban could be reversed by a 15 min perfusion with drug-free buffer, after a 1 minute pulse perfusion with isoprenaline, at which time the mechanical effects of isoprenaline stimulation had also been reversed. However, both TnI and C-protein remained phosphorylated at this time. Moreover, the inhibition of Ca2+ activation of the Mg2+-dependent ATPase (Mg-ATPase) activity associated with myofibrillar phosphorylation persisted in myofibrils prepared from hearts frozen after 15 min of washout of isoprenaline. To assess the contribution of C-protein phosphorylation in the decrease of Ca2+ activation of the myofibrillar Mg-ATPase activity, we reconstituted a regulated actomyosin system in which only C-protein was phosphorylated. In this system, C-protein phosphorylation did not contribute to the decrease in Ca2+ activation of Mg-ATPase activity, indicating that TnI phosphorylation is responsible for the diminished sensitivity of the myofibrils to Ca2+. These observations support the hypothesis that phospholamban phosphorylation plays a more dominant role than TnI or C-protein phosphorylation in the mechanical response of the mammalian heart to beta-adrenergic stimulation.     

Role of regulatory proteins (troponin-tropomyosin) in pathologic states

In vertebrate striated muscle, troponon-tropomyosin is responsible, in part, not only for transducing the effect of calcium on contractile protein activation, but also for inhibiting actin and myosin interaction when calcium is absent. The regulatory troponin (Tn) complex displays several molecular and calcium binding variations in cardiac muscles of different species and undergoes genetic changes with development and in various pathologic states.Extensive reviews on the role of tropomyosin (Tm) and Tn in the regulation of striated muscle contraction have been published describing the molecular mechanisms involved in contractile protein regulation. In our studies, we have found an increase in Mg²⁺ ATPase activity in cardiac myofibrils from dystrophic hamsters and in rats with chronic coronary artery narrowing. The abnormalities in myofibrillar ATPase activity from cardiomyopathic hamsters were largely corrected by recombining the preparations with a TnTm, complex isolated from normal hamsters indicating that the TnTm, may play a major role in altered myocardial function. We have also observed down regulation of Ca²⁺ Mg²⁺ ATPase of myofibrils from hypertrophic guinea pig hearts, myocardial infarcted rats and diabetic-hypertensive rat hearts. In myosin from diabetic rats, this abnormality was substantially corrected by adding troponin-tropomyosin complex from control hearts. All of these disease models are associated with decreased ATPase activities of pure myosin and in the case of rat and hamster models, shifts of myosin, heavy chain from alpha to beta predominate.In summary, there are three main troponin subunit components which might alter myofibrillar function however, very few direct links of molecular alterations in the regulatory proteins to physiologic and pathologic function have been demonstrated so far.     

Protein synthesis, myosin ATPase activity and myofibrillar protein composition in hearts from tumour-bearing rats and mice.

Growing rats and adult weight-stable mice bearing a transplantable methylcholanthrene-induced sarcoma were compared with animals with various states of malnutrition. Heart protein synthesis was measured in vivo. Myocardial RNA, myofibrillar protein composition and the Ca2+-activated ATPase activity in heavy chains of native myosin were measured. 'Fingerprints' were made from myosin by trypsin treatment to evaluate possible structural changes in the protein. Cardiac protein-synthesis rate was decreased by 20% in growing tumour-bearing rats, by 35% in protein-malnourished (rats) and by 47% in starved rats, compared with freely fed controls (P less than 0.05). Adult tumour-bearing mice showed no significant decrease in myocardial protein synthesis. Pair-weighed control mice had significantly depressed heart protein synthesis. Protein translational efficiency was maintained in both tumour-bearing rats and mice, but was decreased in several groups of malnourished control animals. The Ca2+-activated myosin ATPase activity was decreased in all groups of malnourished animals, including tumour-bearing mice and rats, without any evidence of a change in cardiac isomyosin composition. We conclude that loss of cardiac muscle mass in tumour disease is communicated by both depressed synthesis and increased degradation largely owing to anorexia and host malnutrition. Increased adrenergic sensitivity in hearts from tumour-bearing and malnourished animals is not communicated by increased Ca2+-activated ATPase activity. This may be down-regulated in all groups with malnutrition, without any observable alterations in the isomyosin profile.     

Effects of isoproterenol treatment of isolated perfused rat hearts on myofibrillar phosphorylation and ATPase activity

Perfusion of isolated rat hearts with isoproterenol resulted in increases in the level of protein-bound phosphate of the myofibrils. After perfusion of the hearts with 32P, followed by SDS-polyacrylamide gel electrophoresis of the purified myofibrils, four major 32P-containing protein bands were identified. Most of the increased 32P incorporation produced by isoproterenol was localized on the troponin I and myosin light chain bands, and, to lesser extent, on the M-protein band. ATPase activity was tested in the purified myofibrils. No changes in Ca2+ requirement for activation were found after isoproterenol perfusion. However, maximal ATPase activity was markedly reduced in the myofibrils obtained from isoproterenol-treated hearts. It would appear that the myofibrillar protein phosphorylation induced by isoproterenol perfusion results in a decrease in actomyosin ATPase activity.     

Effect of estrogen on calcium-handling proteins, beta-adrenergic receptors, and function in rat heart

Regulation of cellular Ca(2+) cycling is central to myocardial contractile function. Loss of Ca(2+) regulation is associated with cardiac dysfunction and pathology. Estrogen has been shown to modify contractile function and to confer cardioprotection. Therefore, we investigated the effect of estrogen on expression of rat heart myocardial Ca(2+)-handling proteins and beta-adrenergic receptor (beta(1)-AR) and examined functional correlates. Female rats were sham-operated (SHAM) or ovariectomized. Two weeks after ovariectomy rats were injected (i.p.) daily with estradiol benozoate (OVX+EB) or sesame oil (OVX) for 2 weeks. Protein abundance was measured by immunoblotting and mRNA was quantified by real-time RT-PCR. OVX significantly decreased estrogen and progesterone levels and EB replacement returned both estrogen and progesterone to physiological levels. OVX induced a 75% reduction of uterine weight and a gain in body weight. Replacement restored weights to SHAM level. OVX increased and estrogen-replacement normalized abundance of beta(1)-AR and L-type Ca(2+) channel (Cav1.2) protein. OVX decreased sodium-Ca(2+) exchange protein (NCX) and estrogen restored protein abundance to SHAM levels. Sarcoplasmic reticular ATPase (SERCA), phospholamban (PLB), and ryanodine receptor (RyR) abundance was not altered by hormone status. Levels of mRNA encoding for beta(1)-AR, Cav1.2, and NCX were not influenced by OVX or estrogen replacement. OVX had no effect on SERCA and PLB mRNA level but estrogen replacement elicited a significant increase compared to OVX and SHAM. Estrogen-dependent changes in Ca(2+)-handling proteins and beta(1)-AR are theoretically consistent reduced myocellular Ca(2+) load. However, hormone-dependent alterations in protein were not associated with changes in contractile function.     

Renin–angiotensin blockade attenuates cardiac myofibrillar remodelling in chronic diabetes

Previous studies have shown that the renin–angiotensin system (RAS) is activated in diabetes and this may contribute to the subcellular remodelling and heart dysfunction in this disease. Therefore, we examined the effects of RAS blockade by enalapril, an angiotensin-converting enzyme inhibitor, and losartan, an angiotensin receptor AT₁ antagonist, on cardiac function, myofibrillar and myosin ATPase activity as well as myosin heavy chain (MHC) isozyme expression in diabetic hearts. Diabetes was induced in rats by a single injection of streptozotocin (65 mg/kg; i.v.) and these animals were treated with and without enalapril (10 mg/kg/day; oral) or losartan (20 mg/kg/day; oral) for 8 weeks. Enalapril or losartan prevented the depressions in left ventricular rate of pressure development, rate of pressure decay and ventricular weight seen in diabetic animals. Both drugs also attenuated the decrease in myofibrillar Ca²⁺-ATPase, Mg²⁺-ATPase and myosin ATPase activity seen in diabetic rats. The diabetes-induced increase in -MHC content and gene expression as well as the decrease in -MHC content and mRNA levels were also prevented by enalapril and losartan. These results suggest the occurrence of myofibrillar remodelling in diabetic cardiomyopathy and provide evidence that the beneficial effects of RAS blockade in diabetes may be associated with attenuation of myofibrillar remodelling in the heart. (Mol Cell Biochem 261: 271–278, 2004)