Role of cardiac renin-angiotensin system in sarcoplasmic reticulum function and gene expression in the ischemic-reperfused heart

The aim of this study was to explore the possible participation of cardiac renin-angiotensin system (RAS) in the ischemia-reperfusion induced changes in heart function as well as Ca²⁺-handling activities and gene expression of cardiac sarcoplasmic reticulum (SR) proteins. The isolated rat hearts, treated for 10 min without and with 30 M captopril or 100 M losartan, were subjected to 30 min ischemia followed by reperfusion for 60 min and processed for the measurement of SR function and gene expression. Attenuated recovery of the left ventricular developed pressure (LVDP) upon reperfusion of the ischemic heart was accompanied by a marked reduction in SR Ca²⁺-pump ATPase, Ca²⁺-uptake and Ca²⁺-release activities. Northern blot analysis revealed that mRNA levels for SR Ca²⁺-handling proteins such as Ca²⁺-pump ATPase (SERCA2a), ryanodine receptor, calsequestrin and phospholamban were decreased in the ischemia-reperfused heart as compared with the non-ischemic control. Treatment with captopril improved the recovery of LVDP as well as SR Ca²⁺-pump ATPase and Ca²⁺-uptake activities in the postischemic hearts but had no effect on changes in Ca²⁺-release activity due to ischemic-reperfusion. Losartan neither affected the changes in contractile function nor modified alterations in SR Ca²⁺-handling activities. The ischemia-reperfusion induced decrease in mRNA levels for SR Ca²⁺-handling proteins were not affected by treatment with captopril or losartan. The results suggest that the improvement of cardiac function in the ischemic-reperfused heart by captopril is associated with the preservation of SR Ca²⁺-pump activities; however, it is unlikely that this action of captopril is mediated through the modification of cardiac RAS. Furthermore, cardiac RAS does not appear to contribute towards the ischemia-reperfusion induced changes in gene expression for SR Ca²⁺-handling proteins.     

Modulation of mitochondrial contact sites formation in immature rat heart

Creatine phosphokinase-mediated transport of energy from the site of production to the site of consumption is a key process for meeting the energy-demands of reactions in cytosol. The mitochondrial creatine phosphokinase (mCPK) plays an important role in this process, with the enzyme activity localized particularly in the mitochondrial contact sites (MiCS). Earlier studies in adult animals have shown that the formation of MiCS varies in response to the energy demand and the physiological state of the heart, and it is stimulated by an increase in [Ca2+]i. However, there is little known about MiCS formation in juvenile hearts, characterized by metabolism different from adult hearts. In the present study we investigated the modulation of MiCS formation via Ca2+ in hearts of 14-day-old rats. The moderate response of MiCS to various stimuli (elevated extracellular Ca2+, diltiazem, cardiac arrest by Cd2+) may refer to a still increased intracellular Ca2+ concentration, the incomplete development of mitochondrial energy production as well as to persistingly high energy demand of the developing heart.     

Type 1 Diabetic Serum Interferes with Pancreatic β-cell Ca2+ -Handling

The aim of this study was to clarify the frequency of patients with type 1 diabetes that have serum that increases pancreatic β-cell cytoplasmic free Ca²⁺ concentration, [Ca²⁺]_i, and if such an effect is also present in serum from first-degree relatives. We also studied a possible link between the serum effect and ethnic background as well as presence of autoantibodies. Sera obtained from three different countries were investigated as follows: 82 Swedish Caucasians with newly diagnosed type 1 diabetes, 56 Americans with different duration of type 1 diabetes, 117 American first-degree relatives of type 1 diabetic patients with a mixed ethnic background and 31 Caucasian Finnish children with newly diagnosed type 1 diabetes. Changes in [Ca²⁺]_i , upon depolarization, were measured in β-cells incubated overnight with sera from type 1 diabetic patients, first-degree relatives or healthy controls. Our data show that there is a group constituting approximately 30% of type 1 diabetic patients of different gender, age, ethnic background and duration of the disease, as well as first-degree relatives of type 1 diabetic patients, that have sera that interfere with pancreatic β-cell Ca²⁺-handling. This effect on β-cell [Ca²⁺]_i could not be correlated to the presence of autoantibodies. In a defined subgroup of patients with type 1 diabetes and first-degree relatives a defect Ca²⁺-handling may aggravate development of β-cell destruction.     

Calreticulin Induces Dilated Cardiomyopathy

Background

Calreticulin, a Ca²⁺-buffering chaperone of the endoplasmic reticulum, is highly expressed in the embryonic heart and is essential for cardiac development. After birth, the calreticulin gene is sharply down regulated in the heart, and thus, adult hearts have negligible levels of calreticulin. In this study we tested the role of calreticulin in the adult heart.

Methodology/Principal Findings

We generated an inducible transgenic mouse in which calreticulin is targeted to the cardiac tissue using a Cre/loxP system and can be up-regulated in adult hearts. Echocardiography analysis of hearts from transgenic mice expressing calreticulin revealed impaired left ventricular systolic and diastolic function and impaired mitral valve function. There was altered expression of Ca²⁺ signaling molecules and the gap junction proteins, Connexin 43 and 45. Sarcoplasmic reticulum associated Ca²⁺-handling proteins (including the cardiac ryanodine receptor, sarco/endoplasmic reticulum Ca²⁺-ATPase, and cardiac calsequestrin) were down-regulated in the transgenic hearts with increased expression of calreticulin.

Conclusions/Significance

We show that in adult heart, up-regulated expression of calreticulin induces cardiomyopathy in vivo leading to heart failure. This is due to an alternation in changes in a subset of Ca²⁺ handling genes, gap junction components and left ventricle remodeling.     

Sarcolemmal Na+-Ca2+ exchange and Ca2+-pump activities in cardiomyopathies due to intracellular Ca2+-overload

In order to identify defects in Na⁺-Ca²⁺ exchange and Ca²⁺-pump systems in cardiomyopathic hearts, the activities of sarcolemmal Na⁺-dependent Ca²⁺ uptake, Na⁺-induced Ca²⁺ release, ATP-dependent Ca²⁺ uptake and Ca²⁺-stimulated ATPase were examined by employing cardiomyopathic hamsters (UM-X7.1) and catecholamine-induced cardiomyopathy produced by injecting isoproterenol into rats. The rates of Na⁺-dependent Ca²⁺ uptake, ATP-dependent Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities of sarcolemmal vesicles from genetically-linked cardiomyopathic as well as catecholamine-induced cardiomyopathic hearts were decreased without any changes in Na⁺-induced Ca²⁺-release. Similar results were obtained in Ca²⁺-paradox when isolated rat hearts were perfused for 5 min with a medium containing 1.25 mM Ca²⁺ following a 5 min perfusion with Ca²⁺-free medium. Although a 2 min reperfusion of the Ca²⁺-free perfused hearts depressed sarcolemmal Ca²⁺-pump activities without any changes in Na⁺-induced Ca²⁺-release, Na⁺-dependent Ca²⁺ uptake was increased. These results indicate that alterations in the sarcolemmal Ca²⁺-efflux mechanisms may play an important role in cardiomyopathies associated with the development of intracellular Ca²⁺ overload.     

Effects of MCC-135 on Ca2+ uptake by sarcoplasmic reticulum and myofilament sensitivity to Ca2+ in isolated ventricular muscles of rats with diabetic cardiomyopathy

Diabetic cardiomyopathy is characterized by delayed cardiac relaxation. Delayed relaxation is suggested to be associated with sarcoplasmic reticulum (SR) dysfunction and/or increase in myofilament sensitivity to Ca²⁺. Although MCC-135, an intracellular Ca²⁺-handling modulator, accelerates the delayed relaxation without inotropic effect in the ventricular muscle isolated from rats with diabetic cardiomyopathy, the underlying mechanism has not been fully understood. We tested the hypotheses that MCC-135 modulates Ca²⁺ uptake by SR and myofilament sensitivity to Ca²⁺. Wistar rats were made diabetic by a single injection of streptozotocin (40 mg/kg i.v.). Seven months later, the left ventricular papillary muscle was isolated and skinned fibers with and without functional SR were prepared by treatment of the papillary muscle with saponin to study SR Ca²⁺ uptake and myofilament sensitivity to Ca²⁺, respectively. In diabetic rats, SR Ca²⁺ uptake was decreased, which was related to decrease in protein level of SR Ca²⁺-ATPase determined by western blot analysis. MCC-135 enhanced SR Ca²⁺ uptake in diabetic rats, but not in normal rats. In diabetic rats, maximum force was decreased but force at diastolic level of Ca²⁺ was increased, without significant change in myofilament sensitivity to Ca²⁺ compared with normal rats. MCC-135 decreased force at any pCa tested (pCa 7.0-4.4), but had no significant effect on myofilament sensitivity to Ca²⁺ in diabetic rats. These results suggest that MCC-135 enhances SR Ca²⁺ uptake and shifts force-pCa curve downward without modulating myofilament sensitivity to Ca²⁺. These effects may contribute to positive lusitropic effect without inotropic effect of MCC-135 observed in the ventricular muscle of diabetic cardiomyopathy.     

Effects of carbenoxolone on heart rhythm, contractility and intracellular calcium in streptozotocin-induced diabetic rat

Cardiac dysfunction is a frequently reported complication of clinical and experimental diabetes mellitus. Streptozotocin (STZ) – induced diabetes in rat is associated with a variety of cardiac defects including disturbances to heart rhythm and prolonged time-course of cardiac muscle contraction and/or relaxation. The effects of carbenoxolone (CBX), a selective gap junction inhibitor, on heart rhythm and contractility in STZ-induced diabetic rat have been investigated. Heart rate was significantly (P < 0.05) reduced in Langendorff perfused spontaneously beating diabetic rat heart (171±12 BPM) compared to age-matched controls (229± 9 BPM) and further reduced by 10⁻⁵ M CBX in diabetic (20%) and in control (17%) hearts. Action potential durations (APDs), recorded on the epicardial surface of the left ventricle, were prolonged in paced (6 Hz) diabetic compared to control hearts. Perfusion of hearts with CBX caused further prolongation of APDs and to a greater extent in control compared to diabetic heart. Percentage prolongation at 70% from the peak of the action potential amplitude after CBX was 18% in diabetic compared to 48% in control heart. CBX had no significant effect on resting cell length or amplitude of ventricular myocyte shortening in diabetic or control rats. However, resting fura-2 ratio (indicator for intracellular Ca²⁺ concentration) and amplitude of the Ca²⁺ transient were significantly (P < 0.05) reduced by CBX in diabetic rats but not in controls. In conclusion the larger effects of CBX on APD in control ventricle and the normalizing effects of CBX on intracellular Ca²⁺ in ventricular myocytes from diabetic rat suggest that there may be alterations in gap junction electrophysiology in STZ-induced diabetic rat heart.     

Depressed β-adrenergic inotropic responsiveness and intracellular calcium handling abnormalities in Duchenne Muscular Dystrophy patients’ induced pluripotent stem cell–derived cardiomyocytes

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an X-linked disease affecting male and rarely adult heterozygous females, resulting in death by the late 20s to early 30s. Previous studies reported depressed left ventricular function in DMD patients which may result from deranged intracellular Ca²⁺-handling. To decipher the mechanism(s) underlying the depressed LV function, we tested the hypothesis that iPSC-CMs generated from DMD patients feature blunted positive inotropic response to β-adrenergic stimulation. To test the hypothesis, [Ca²⁺]_i transients and contractions were recorded from healthy and DMD-CMs. While in healthy CMs (HC) isoproterenol caused a prominent positive inotropic effect, DMD-CMs displayed a blunted inotropic response. Next, we tested the functionality of the sarcoplasmic reticulum (SR) by measuring caffeine-induced Ca²⁺ release. In contrast to HC, DMD-CMs exhibited reduced caffeine-induced Ca²⁺ signal amplitude and recovery time. In support of the depleted SR Ca²⁺ stores hypothesis, in DMD-CMs the negative inotropic effects of ryanodine and cyclopiazonic acid were smaller than in HC. RNA-seq analyses demonstrated that in DMD CMs the RNA-expression levels of specific subunits of the L-type calcium channel, the β1-adrenergic receptor (ADRβ1) and adenylate cyclase were down-regulated by 3.5-, 2.8- and 3-fold, respectively, which collectively contribute to the depressed β-adrenergic responsiveness.     

Ca2+, pH and the regulation of cardiac myofilament force and ATPase activity

When the (pH_i) surrounding myofilaments of striated muscle is reduced there is an inhibition of both the actin-myosin reaction as well as the Ca²⁺-sensitivity of the myofilaments. Although the mechanism for the effect of acidic pH on Ca²⁺-sensitivity has been controversial, we have evidence for the hypothesis that acidic pH reduces the affinity of troponin C (TNC) for Ca²⁺. This effect of acidic pH depends not only on a direct effect of protons on Ca²⁺-binding to TNC, but also upon neighboring thin filament proteins, especially TNI, the inhibitory component of the TN complex. Using flourescent probes that report Ca²⁺-binding to the regulatory sites of skeletal and cardiac TNC, we have shown, for example, that acidic pH directly decreases the Ca²⁺-affinity of TNC, but only by a relatively small amount. However, with TNC in whole TN or in the TNI-TNC complex, there is about a 2-fold enhancement of the effects of acidic pH on Ca²⁺-binding to TNC. Acidic pH decreases the affinity of skeletal TNI for skeletal TNC, and also influences the micro-environment of a probe postioned at Cys-133 of TNI, a region of interaction with TNC. Other evidence that the effects of acidic pH on Ca²⁺-TNC activation of myofilaments are influenced by TNI comes from studies with developing hearts. In contrast, to the case with the adult preparations, Ca²⁺-activation of detergent extracted fibers prepared from dog or rat hearts in the peri-natal period are weakly affected by a drop in pH from 7.0 to 6.5. This difference in the effect of acidic (pH_i) appears to be due to a difference in the isoform population of TNI, and not to differences in isotype population or amount of TNC.     

Alterations in Ca2+-channels during the development of diabetic cardiomyopathy

In order to examine the status of Ca²⁺ channels in heart sarcolemma during the development of diabetes, rats were injected intravenously with 65 mg/kg streptozotocin and hearts were removed 1, 3 and 8 weeks later. Crude membranes from the ventricular muscle were prepared and the specific binding of ³H-nitrendipine was studied by employing different concentrations of this Ca ²⁺-antagonist. A significant decrease in both dissociation constant and maximal number of 3H-nitrendipine binding was observed in 3 and 8 weeks diabetic preparations. No such alterations were evident in diabetic brain membranes. Treatment of diabetic animals with insulin prevented the occurrence of these changes in the myocardium. The altered ³H-nitrendipine binding characteristics in diabetic heart membranes may not be due to the high levels of circulating catecholamines in this experimental model because no such changes were seen upon injecting a high dose (40 mg/kg) of isoproterenol in rats for 24 hr. The reduced number of ³H-nitrendipine binding sites may decrease Ca²⁺-influx through voltage sensitive Ca²⁺ channels and partly explain the depressed cardiac contractile force development in chronic diabetes whereas the increased affinity of Ca²⁺ channels may partly explain the increased sensitivity of diabetic heart to Ca ²⁺.     

Cytosolic Ca2+ concentration during Ca2+ depletion of isolated rat hearts

Reperfusion of isolated mammalian hearts with a Ca²⁺-containing solution after a short Ca²⁺-free period at 37°:C results in massive influx of Ca²⁺ into the cells and irreversible cell damage: the Ca²⁺paradox. Information about the free intracellular, cytosolic [Ca²⁺] ([Ca²⁺]_i) during Ca²⁺ depletion is essential to assess the possibility of Ca²⁺ influx through reversed Na⁺/Ca²⁺ exchange upon Ca²⁺ repletion. Furthermore, the increase in end-diastolic pressure often seen during Ca²⁺-free perfusion of intact hearts may be similar to that seen during ischemia and caused by liberation of Ca²⁺ from intracellular stores. Therefore, in this study, we measured [Ca²⁺]i during Ca²⁺- free perfusion of isolated rat hearts. To this end, the fluorescent indicator Indo-1 was loaded into isolated Langendorff-perfused hearts and Ca²⁺-transients were recorded. Ca²⁺-transients disappeared within 1 min of Ca²⁺ depletion. Systolic [Ca²⁺]i during control perfusion was 268±54 nM. Diastolic [Ca²⁺]i during control perfusion was 114±34 nM and decreased to 53±19 nM after 10 min of Ca²⁺ depletion. Left ventricular end-diastolic pressure (LVEDP) significantly increased from 13±4 mmHg during control perfusion after Indo-1 AM loading to 31±5 mmHg after 10 min Ca²⁺ depletion. Left ventricular developed pressure did not recover during Ca²⁺ repletion, indicating a full Ca²⁺ paradox. These results show that LVEDP increased during Ca²⁺ depletion despite a decrease in [Ca²⁺]i, and is therefore not comparable to the contracture seen during ischemia. Furthermore, calculation of the driving force for the Na⁺/Ca²⁺ exchanger showed that reversed Na⁺/Ca²⁺ exchange during Ca²⁺ repletion is not able to increase [Ca²⁺]i to cytotoxic levels.     

Remodelling of Ca2+ transport in cancer: how it contributes to cancer hallmarks?

Cancer involves defects in the mechanisms underlying cell proliferation, death and migration. Calcium ions are central to these phenomena, serving as major signalling agents with spatial localization, magnitude and temporal characteristics of calcium signals ultimately determining cell''s fate. Cellular Ca²⁺ signalling is determined by the concerted action of a molecular Ca²⁺-handling toolkit which includes: active energy-dependent Ca²⁺ transporters, Ca²⁺-permeable ion channels, Ca²⁺-binding and storage proteins, Ca²⁺-dependent effectors. In cancer, because of mutations, aberrant expression, regulation and/or subcellular targeting of Ca²⁺-handling/transport protein(s) normal relationships among extracellular, cytosolic, endoplasmic reticulum and mitochondrial Ca²⁺ concentrations or spatio-temporal patterns of Ca²⁺ signalling become distorted. This causes deregulation of Ca²⁺-dependent effectors that control signalling pathways determining cell''s behaviour in a way to promote pathophysiological cancer hallmarks such as enhanced proliferation, survival and invasion. Despite the progress in our understanding of Ca²⁺ homeostasis remodelling in cancer cells as well as in identification of the key Ca²⁺-transport molecules promoting certain malignant phenotypes, there is still a lot of work to be done to transform fundamental findings and concepts into new Ca²⁺ transport-targeting tools for cancer diagnosis and treatment.     

Increased Na+/Ca2+ Exchanger Expression/Activity Constitutes a Point of Inflection in the Progression to Heart Failure of Hypertensive Rats

Spontaneously hypertensive rat (SHR) constitutes a genetic model widely used to study the natural evolution of hypertensive heart disease. Ca²⁺-handling alterations are known to occur in SHR. However, the putative modifications of Ca²⁺-handling proteins during the progression to heart failure (HF) are not well established. Moreover, the role of apoptosis in SHR is controversial. We investigated intracellular Ca²⁺, Ca²⁺-handling proteins and apoptosis in SHR vs. control Wistar rats (W) from 3 to 15 months (mo). Changes associated with the transition to HF (i.e. lung edema and decrease in midwall fractional shortening), occurred at 15 mo in 38% of SHR (SHRF). In SHRF, twitch and caffeine-induced Ca²⁺ transients, significantly decreased relative to 6/9 mo and 15 mo without HF signs. This decrease occurred in association with a decrease in the time constant of caffeine-Ca²⁺ transient decay and an increase in Na⁺/Ca²⁺ exchanger (NCX) abundance (p<0.05) with no changes in SERCA2a expression/activity. An increased Ca²⁺-calmodulin-kinase II activity, associated with an enhancement of apoptosis (TUNEL and Bax/Bcl2) was observed in SHR relative to W from 3 to 15 mo. Conclusions: 1. Apoptosis is an early and persistent event that may contribute to hypertrophic remodeling but would not participate in the contractile impairment of SHRF. 2. The increase in NCX expression/activity, associated with an increase in Ca²⁺ efflux from the cell, constitutes a primary alteration of Ca²⁺-handling proteins in the evolution to HF. 3. No changes in SERCA2a expression/activity are observed when HF signs become evident.     

Remodelling of the sarcolemma in diabetic rat hearts: The role of membrane fluidity

The hyperglycaemia and oxidative stress, that occur in diabetes mellitus, cause impairment of membrane functions in cardiomyocytes. Also reduced sensitivity to Ca-overload was reported in diabetic hearts (D). This enhanced calcium resistance is based on remodelling of the sarcolemmal membranes (SL) with down-regulated, but from the point of view of kinetics relatively well preserved Na,K-ATPase and abnormal Mg- and Ca-ATPase (Mg/Ca-ATPase) activities. It was hypothesised that in these changes may also participate the non-enzymatic glycation of proteins (NEG) and the related free radical formation (FRF), that decrease the membrane fluidity (SLMF), which is in reversal relationship to the fluorescence anisotropy (D 0.235 ± 0.022; controls (C) 0.185 ± 0.009; p < 0.001). In order to check the true role of SLMF in hearts of the diabetic rats (streptozotocin, single dose, 45 mg/kg i.v.) animals were treated in a special regimen with resorcylidene aminoguanidine (RAG, 4 mg/kg i.m.). The treatment with RAG eliminated completely the diabetes-induced decrease in the SLMF (C 0.185 ± 0.009; D + RAG 0.167 ± 0.013; p < 0.001rpar; as well as in NEG (fructosamine g.mg^–1 of protein: C 2.68 ± 0.14; D 4.48 ± 0.85; D + RAG 2.57 ± 0.14; p < 0.001), and FRF in the SL (malondialdehyde: C 5.3 ± 0.3; D 8.63 ± 0.2; D + RAG 5.61 ± 0.53 mol.g^–1; p < 0.05). Nevertheless, the SL ATPase activity in diabetic animals was not considerably influenced by RAG (increase in D + RAG vs. D 3.3%, p > 0.05). On the other hand, RAG increased considerably the vulnerability of the diabetic heart to overload with external Ca²⁺ (C 100% of hearts failed, D 83.3%, D + RAG 46.7% of hearts survived). So we may conclude, that: (i) The NEG and FRF caused alterations in SLMF, that accompanied the diabetes-induced remodelling of SL, also seem to participate in the protection of diabetic heart against Ca²⁺-overload; (ii) Although, the changes in SLMF were shown to influence considerably the ATPase activities in cells of diverse tissues, they seem to be little responsible for changes in ATPases-mediated processes in the SL of chronic diabetic hearts.     

Regulation of cardiac sarcolemmal Ca2+ channels and Ca2+ transporters by thyroid hormone

In order to examine the regulatory role of thyroid hormone on sarcolemmal Ca²⁺-channels, Na⁺–Ca²⁺ exchange and Ca²⁺-pump as well as heart function, the effects of hypothyroidism and hyperthyroidism on rat heart performance and sarcolemmal Ca²⁺-handling were studied. Hyperthyroid rats showed higher values for heart rate (HR), maximal rates of ventricular pressure development+(dP/dt)max and pressure fall–(dP/dt)max, but shorter time to peak ventricular pressure (TPVP) and contraction time (CT) when compared with euthyroid rats. The left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP), as well as aortic systolic and diastolic pressures (ASP and ADP, respectively) were not significantly altered. Hypothyroid rats exhibited decreased values of LVSP, HR, ASP, ADP, +(dP/dt)max and –(dP/dt)max but higher CT when compared with euthyroid rats; the values of LVEDP and TPVP were not changed. Studies with isolated-perfused hearts showed that while hypothyroidism did not modulate the inotropic response to extracellular Ca²⁺ and Ca²⁺ channel blocker verapamil, hyperthyroidism increased sensitivity to Ca²⁺ and decreased sensitivity to verapamil in comparison to euthyroid hearts. Studies of [³H]-nitrendipine binding with purified cardiac sarcolemmal membrane revealed decreased number of high affinity binding sites (B_max) without any change in the dissociation constant for receptor-ligand complex (K_d) in the hyperthyroid group when compared with euthyroid sarcolemma; hypothyroidism had no effect on these parameters. The activities of sarcolemmal Ca²⁺-stimulated ATPase, ATP-dependent Ca²⁺ uptake and ouabain-sensitive Na⁺–K⁺ ATPase were decreased whereas the Mg²⁺-ATPase activity was increased in hypothyroid hearts. On the other hand, sarcolemmal membranes from hyperthyroid samples exhibited increased ouabain-sensitive Na⁺–K⁺ ATPase activity, whereas Ca²⁺-stimulated ATPase, ATP-dependent Ca²⁺ uptake, and Mg²⁺-ATPase activities were unchanged. The V_max and K_a for Ca²⁺ of cardiac sarcolemmal Na⁺–Ca²⁺ exchange were not altered in both hyperthyroid and hypothyroid states. These results indicate that the status of sarcolemmal Ca²⁺-transport processes is regulated by thyroid hormones and the modification of Ca²⁺-fluxes across the sarcolemmal membrane may play a crucial role in the development of thyroid state-dependent contractile changes in the heart.     

?-Blocker Timolol Prevents Arrhythmogenic Ca2+ Release and Normalizes Ca2+ and Zn2+ Dyshomeostasis in Hyperglycemic Rat Heart

Defective cardiac mechanical activity in diabetes results from alterations in intracellular Ca²⁺ handling, in part, due to increased oxidative stress. Beta-blockers demonstrate marked beneficial effects in heart dysfunction with scavenging free radicals and/or acting as an antioxidant. The aim of this study was to address how β-blocker timolol-treatment of diabetic rats exerts cardioprotection. Timolol-treatment (12-week), one-week following diabetes induction, prevented diabetes-induced depressed left ventricular basal contractile activity, prolonged cellular electrical activity, and attenuated the increase in isolated-cardiomyocyte size without hyperglycemic effect. Both in vivo and in vitro timolol-treatment of diabetic cardiomyocytes prevented the altered kinetic parameters of Ca²⁺ transients and reduced Ca²⁺ loading of sarcoplasmic reticulum (SR), basal intracellular free Ca²⁺ and Zn²⁺ ([Ca²⁺]_i and [Zn²⁺]_i), and spatio-temporal properties of the Ca²⁺ sparks, significantly. Timolol also antagonized hyperphosphorylation of cardiac ryanodine receptor (RyR2), and significantly restored depleted protein levels of both RyR2 and calstabin2. Western blot analysis demonstrated that timolol-treatment also significantly normalized depressed levels of some [Ca²⁺]_i-handling regulators, such as Na⁺/Ca²⁺ exchanger (NCX) and phospho-phospholamban (pPLN) to PLN ratio. Incubation of diabetic cardiomyocytes with 4-mM glutathione exerted similar beneficial effects on RyR2-macromolecular complex and basal levels of both [Ca²⁺]_i and [Zn²⁺]_i, increased intracellular Zn²⁺ hyperphosphorylated RyR2 in a concentration-dependent manner. Timolol also led to a balanced oxidant/antioxidant level in both heart and circulation and prevented altered cellular redox state of the heart. We thus report, for the first time, that the preventing effect of timolol, directly targeting heart, seems to be associated with a normalization of macromolecular complex of RyR2 and some Ca²⁺ handling regulators, and prevention of Ca²⁺ leak, and thereby normalization of both [Ca²⁺]_i and [Zn²⁺]_i homeostasis in diabetic rat heart, at least in part by controlling the cellular redox status of hyperglycemic cardiomyocytes.     

Relationship between mechanical dysfunction and depression of sarcolemmal Ca2+-pump activity in hearts perfused with oxygen free radicals

Although in vitro studies have shown that oxygen free radicals depress the sarcolemmal Ca²⁺-pump activity and thereby may cause the occurrence of intracellular Ca²⁺ overload for the genesis of contractile failure, the exact relationship between changes in sarcolemmal Ca²⁺-pump activity and cardiac function due to these radicals is not clear. In this study we examined the effects of oxygen radicals on sarcolemmal Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities as well as contractile force development by employing isolated rat heart preparations. When hearts were perfused with medium containing xanthine plus xanthine oxidase, the sarcolemmal Ca²⁺-stimulated ATPase activity and ATP-dependent Ca²⁺ accumulation were depressed within 1 min whereas the developed contractile force, rate of contraction and rate of relaxation were increased at 1 min and decreased over 3–20 min of perfusion. The resting tension started increasing at 2 min of perfusion with xanthine plus xanthine oxidase. Catalase showed protective effects against these alterations in heart function and sarcolemmal Ca²⁺-pump activities upon perfusion with xanthine plus xanthine oxidase whereas superoxide dismutase did not exert such effects. The combination of catalase and superoxide dismutase did not produce greater effects in comparison to catalase alone. These results are consistent with the view that the depression of heart sarcolemmal Ca²⁺ pump activities may result in myocardial dysfunction due to the formation of hydrogen peroxide and/or hydroxyl radicals upon perfusing the hearts with xanthine plus xanthine oxidase.     

Mechanisms that may be involved in calcium tolerance of the diabetic heart

In diabetes the hearts exhibit impaired membrane functions, but also increased tolerance to Ca²⁺ (iCaT) However, neither the true meaning nor the molecular mechanisms of these changes are fully understood. The present study is devoted to elucidation of molecular alterations, particularly those induced by non-enzymatic glycation of proteins, that may be responsible for iCaT of the rat hearts in the stage of fully developed, but still compensated diabetic cardiomyopathy (DH). Insulin-dependent diabetes (DIA) was induced by a single i.v. dose of streptozotocin (45 mg.kg^-1). Beginning with the subsequent day, animals obtained 6 U insulin daily. Glucose, triglycerides, cholesterol and glycohemoglobin were investigated in blood. ATPase activities, the kinetics of activation of (Na,K)-ATPase by Na⁺ and K⁺, further the fluorescence anisotropy of diphenyl-hexatriene as well as the order parameters of membranes in isolated heart sarcolemma (SL) were also investigated. In addition, the degree of glycation and glycation-related potency for radical generation in SL proteins were determined by investigating their fructosamine content. In order to study calcium tolerance of DH in a 'transparent' model, hearts were subjected to calcium paradox (Ca-Pa, 3 min of Ca²⁺ depletion; 10 min of Ca²⁺ repletion). In this model of Ca²⁺-overload, Ca²⁺ ions enter the cardiac cells in a way that is not mediated by receptors. Results revealed that more than 83% of the isolated perfused DH recovered, while the non-DIA control hearts all failed after Ca-Pa. DH exhibited well preserved SL ATPase activities and kinetics of (Na,K)-ATPase activation by Na⁺, even after the Ca-Pa. This was considered as a reason for their iCaT. Pretreatment and administration of resorcylidene aminoguanidine (RAG 4 or 8 mg.kg^-1) during the disease prevented partially the pathobiochemical effects of DIA-induced glycation of SL proteins. DIAinduced perturbations in anisotropy and order parameters of SL were completely prevented by administration of RAG (4 mg.kg^-1). Although, the latter treatment exerted little influence on the (Na,K)-ATPase activity, it decreased the calcium tolerance of the DH. Results are supporting our hypothesis that the glycation-induced enhancement in free radical formation and protein crosslinking in SL may participate in adaptive mechanisms that may be also considered as 'positive' and are responsible for iCaT of the DH.     

CaMKII‐dependent ryanodine receptor phosphorylation mediates sepsis‐induced cardiomyocyte apoptosis

Sepsis is associated with cardiac dysfunction, which is at least in part due to cardiomyocyte apoptosis. However, the underlying mechanisms are far from being understood. Using the colon ascendens stent peritonitis mouse model of sepsis (CASP), we examined the subcellular mechanisms that mediate sepsis‐induced apoptosis. Wild‐type (WT) CASP mice hearts showed an increase in apoptosis respect to WT‐Sham. CASP transgenic mice expressing a CaMKII inhibitory peptide (AC3‐I) were protected against sepsis‐induced apoptosis. Dantrolene, used to reduce ryanodine receptor (RyR) diastolic sarcoplasmic reticulum (SR) Ca²⁺ release, prevented apoptosis in WT‐CASP. To examine whether CaMKII‐dependent RyR2 phosphorylation mediates diastolic Ca²⁺ release and apoptosis in sepsis, we evaluated apoptosis in mutant mice hearts that have the CaMKII phosphorylation site of RyR2 (Serine 2814) mutated to Alanine (S2814A). S2814A CASP mice did not show increased apoptosis. Consistent with RyR2 phosphorylation‐dependent enhancement in diastolic SR Ca²⁺ release leading to mitochondrial Ca²⁺ overload, mitochondrial Ca²⁺ retention capacity was reduced in mitochondria isolated from WT‐CASP compared to Sham and this reduction was absent in mitochondria from CASP S2814A or dantrolene‐treated mice. We conclude that in sepsis, CaMKII‐dependent RyR2 phosphorylation results in diastolic Ca²⁺ release from SR which leads to mitochondrial Ca²⁺ overload and apoptosis.     

Halothane alters contractility and Ca2+ transport in ventricular myocytes from streptozotocin-induced diabetic rats

General anaesthetics have previously been shown to have profound effects on myocardial function. Moreover, many patients suffering from diabetes mellitus are anaesthetised during surgery. This study investigated compromised functioning of cardiac myocytes from streptozotocin (STZ)-induced diabetic rats and the additive effects of halothane on these dysfunctions. Ventricular myocytes were isolated from 8 to 12 weeks STZ-treated rats. Contraction and intracellular free calcium concentration ([Ca²⁺] _i ) were measured in electrically field-stimulated (1 Hz) fura-2-AM-loaded cells using a video-edge detection system and a fluorescence photometry system, respectively. L-type Ca²⁺ current was measured in whole cell, voltage-clamp mode. Halothane significantly (p < 0.01) depressed the amplitude and the time course of the Ca²⁺ transients in a similar manner in myocytes from control and STZ-treated rats. However, the effect of halothane on the amplitude of shortening and L-type Ca²⁺ current was more pronounced in myocytes from STZ-treated animals compared to age-matched controls. Myofilament sensitivity to Ca²⁺ was significantly (p < 0.01) increased in myocytes from STZ-treated rats compared to control. However, in the presence of halothane the myofilament sensitivity to Ca²⁺ was significantly (p < 0.05) reduced to a greater extent in myocytes from STZ-treated rats compared to controls. In conclusion, these results show that contractility, Ca²⁺ transport and myofilament sensitivity were all altered in myocytes from STZ-treated rats and these processes were further altered in the presence of halothane suggesting that hearts from STZ-induced diabetic rats are sensitive to halothane. (Mol Cell Biochem 261: 251–261, 2004)