Ca2+ mishandling and mitochondrial dysfunction: a converging road to prediabetic and diabetic cardiomyopathy.

Diabetic cardiomyopathy is defined as the myocardial dysfunction that suffers patients with diabetes mellitus (DM) in the absence of hypertension and structural heart diseases such as valvular or coronary artery dysfunctions. Since the impact of DM on cardiac function is rather silent and slow, early stages of diabetic cardiomyopathy, known as prediabetes, are poorly recognized, and, on many occasions, cardiac illness is diagnosed only after a severe degree of dysfunction was reached. Therefore, exploration and recognition of the initial pathophysiological mechanisms that lead to cardiac dysfunction in diabetic cardiomyopathy are of vital importance for an on-time diagnosis and treatment of the malady. Among the complex and intricate mechanisms involved in diabetic cardiomyopathy, Ca2+ mishandling and mitochondrial dysfunction have been described as pivotal early processes. In the present review, we will focus on these two processes and the molecular pathway that relates these two alterations to the earlier stages and the development of diabetic cardiomyopathy.

CaMKII activation in early diabetic hearts induces altered sarcoplasmic reticulum-mitochondria signaling.

Prediabetic myocardium, induced by fructose-rich diet (FRD), is prone to increased sarcoplasmic reticulum (SR)-Ca2+ leak and arrhythmias due to increased activity of the Ca2+/calmodulin protein kinase II (CaMKII). However, little is known about the role of SR-mitochondria microdomains, mitochondrial structure, and mitochondrial metabolisms. To address this knowledge gap we measured SR-mitochondrial proximity, intracellular Ca2+, and mitochondrial metabolism in wild type (WT) and AC3-I transgenic mice, with myocardial-targeted CaMKII inhibition, fed with control diet (CD) or with FRD. Confocal images showed significantly increased spontaneous Ca2+ release events in FRD vs. CD WT cardiomyocytes. [3H]-Ryanodine binding assay revealed higher [3H]Ry binding in FRD than CD WT hearts. O2 consumption at State 4 and hydrogen peroxide (H2O2) production rate were increased, while respiratory control rate (RCR) and Ca2+ retention capacity (CRC) were decreased in FRD vs. CD WT isolated mitochondria. Transmission Electron Microscopy (TEM) images showed increased proximity at the SR-mitochondria microdomains, associated with increased tethering proteins, Mfn2, Grp75, and VDAC in FRD vs. CD WT. Mitochondria diameter was decrease and roundness and density were increased in FRD vs. CD WT specimens. The fission protein, Drp1 was significantly increased while the fusion protein, Opa1 was unchanged in FRD vs. CD WT hearts. These differences were prevented in AC3-I mice. We conclude that SR-mitochondria microdomains are subject to CaMKII-dependent remodeling, involving SR-Ca2+ leak and mitochondria fission, in prediabetic mice induced by FRD. We speculate that CaMKII hyperactivity induces SR-Ca2+ leak by RyR2 activation which in turn increases mitochondria Ca2+ content due to the enhanced SR-mitochondria tethering, decreasing CRC.

The role of mitochondria in metabolic disease: a special emphasis on heart dysfunction.

Metabolic diseases (MetDs) embrace a series of pathologies characterized by abnormal body glucose usage. The known diseases included in this group are metabolic syndrome, prediabetes and diabetes mellitus types 1 and 2. All of them are chronic pathologies that present metabolic disturbances and are classified as multi-organ diseases. Cardiomyopathy has been extensively described in diabetic patients without overt macrovascular complications. The heart is severely damaged during the progression of the disease; in fact, diabetic cardiomyopathies are the main cause of death in MetDs. Insulin resistance, hyperglycaemia and increased free fatty acid metabolism promote cardiac damage through mitochondria. These organelles supply most of the energy that the heart needs to beat and to control essential cellular functions, including Ca2+ signalling modulation, reactive oxygen species production and apoptotic cell death regulation. Several aspects of common mitochondrial functions have been described as being altered in diabetic cardiomyopathies, including impaired energy metabolism, compromised mitochondrial dynamics, deficiencies in Ca2+ handling, increases in reactive oxygen species production, and a higher probability of mitochondrial permeability transition pore opening. Therefore, the mitochondrial role in MetD-mediated heart dysfunction has been studied extensively to identify potential therapeutic targets for improving cardiac performance. Herein we review the cardiac pathology in metabolic syndrome, prediabetes and diabetes mellitus, focusing on the role of mitochondrial dysfunctions.

Determinants of Ca2+ release restitution: Insights from genetically altered animals and mathematical modeling.

Each heartbeat is followed by a refractory period. Recovery from refractoriness is known as Ca2+ release restitution (CRR), and its alterations are potential triggers of Ca2+ arrhythmias. Although the control of CRR has been associated with SR Ca2+ load and RYR2 Ca2+ sensitivity, the relative role of some of the determinants of CRR remains largely undefined. An intriguing point, difficult to dissect and previously neglected, is the possible independent effect of SR Ca2+ content versus the velocity of SR Ca2+ refilling on CRR. To assess these interrogations, we used isolated myocytes with phospholamban (PLN) ablation (PLNKO), knock-in mice with pseudoconstitutive CaMKII phosphorylation of RYR2 S2814 (S2814D), S2814D crossed with PLNKO mice (SDKO), and a previously validated human cardiac myocyte model. Restitution of cytosolic Ca2+ (Fura-2 AM) and L-type calcium current (ICaL; patch-clamp) was evaluated with a two-pulse (S1/S2) protocol. CRR and ICaL restitution increased as a function of the (S2-S1) coupling interval, following an exponential curve. When SR Ca2+ load was increased by increasing extracellular [Ca2+] from 2.0 to 4.0 mM, CRR and ICaL restitution were enhanced, suggesting that ICaL restitution may contribute to the faster CRR observed at 4.0 mM [Ca2+]. In contrast, ICaL restitution did not differ among the different mouse models. For a given SR Ca2+ load, CRR was accelerated in S2814D myocytes versus WT, but not in PLNKO and SDKO myocytes versus WT and S2814D, respectively. The model mimics all experimental data. Moreover, when the PLN ablation-induced decrease in RYR2 expression was corrected, the model revealed that CRR was accelerated in PLNKO and SDKO versus WT and S2814D myocytes, consistent with the enhanced velocity of refilling, SR [Ca2+] recovery, and CRR. We speculate that refilling rate might enhance CRR independently of SR Ca2+ load.

Unbalance Between Sarcoplasmic Reticulum Ca2 + Uptake and Release: A First Step Toward Ca2 + Triggered Arrhythmias and Cardiac Damage.

The present review focusses on the regulation and interplay of cardiac SR Ca2+ handling proteins involved in SR Ca2+ uptake and release, i.e., SERCa2/PLN and RyR2. Both RyR2 and SERCA2a/PLN are highly regulated by post-translational modifications and/or different partners' proteins. These control mechanisms guarantee a precise equilibrium between SR Ca2+ reuptake and release. The review then discusses how disruption of this balance alters SR Ca2+ handling and may constitute a first step toward cardiac damage and malignant arrhythmias. In the last part of the review, this concept is exemplified in different cardiac diseases, like prediabetic and diabetic cardiomyopathy, digitalis intoxication and ischemia-reperfusion injury.

Calcium-calmodulin-dependent protein kinase mediates the intracellular signalling pathways of cardiac apoptosis in mice with impaired glucose tolerance.

KEY POINTS: Spontaneous sarcoplasmic reticulum (SR) Ca2+ release events increased in fructose-rich diet mouse (FRD) myocytes vs. control diet (CD) mice, in the absence of significant changes in SR Ca2+ load. In HEK293 cells, hyperglycaemia significantly enhanced [3 H]ryanodine binding and Ca2+ /calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2-S2814 residue vs. normoglycaemia. These increases were prevented by CaMKII inhibition. FRD significantly augmented cardiac apoptosis in WT vs. CD-WT mice, which was prevented by co-treatment with the reactive oxygen species scavenger Tempol. Oxidative stress was also increased in FRD-SR-autocamide inhibitory peptide (AIP) mice, expressing the SR-targeted CaMKII inhibitor AIP, without any significant enhancement of apoptosis vs. CD-SR-AIP mice. FRD produced mitochondrial swelling and membrane depolarization in FRD-WT mice but not in FRD-S2814A mice, in which the CaMKII site on ryanodine receptor 2 was ablated. FRD decreased mitochondrial area, mean Feret diameter and the mean distance between SR and the outer mitochondrial membrane vs. CD hearts. This remodelling was prevented in AC3I mice, with cardiac-targeted CaMKII inhibition. ABSTRACT: The impact of cardiac apoptosis in pre-diabetic stages of diabetic cardiomyopathy is unknown. We show that myocytes from fructose-rich diet (FRD) animals exhibit arrhythmias produced by exacerbated Ca2+ /calmodulin-protein kinase (CaMKII) activity, ryanodine receptor 2 (RyR2) phosphorylation and sarcoplasmic reticulum (SR) Ca2+ leak. We tested the hypothesis that this mechanism also underlies cardiac apoptosis in pre-diabetes. We generated a pre-diabetic model in FRD mice. FRD mice showed an increase in oxidative stress, hypertrophy and systolic dysfunction. FRD myocytes exhibited enhanced SR Ca2+ spontaneous events in the absence of SR Ca2+ load alterations vs. control-diet (CD) myocytes. In HEK293 cells, hyperglycaemia significantly enhanced [3 H]ryanodine binding and CaMKII phosphorylation of RyR2-S2814 residue vs. normoglycaemia. CaMKII inhibition prevented hyperglycaemia-induced alterations. FRD also evoked cardiac apoptosis in WT mice vs. CD-WT mice. Co-treatment with the reactive oxygen species scavenger Tempol prevented FRD-induced apoptosis in WT mice. In contrast, FRD enhanced oxidative stress but not apoptosis in FRD-SR-AIP mice, in which a CaMKII inhibitor is targeted to the SR. FRD produced mitochondrial membrane depolarization in WT mice but not in S2814A mice, in which the CaMKII phosphorylation site on RyR2 was ablated. Furthermore, FRD decreased mitochondrial area, mean Feret diameter and mean SR-mitochondrial distance vs. CD-WT hearts. This remodelling was prevented in AC3I mice, with cardiac-targeted CaMKII inhibition. CaMKII phosphorylation of RyR2, SR Ca2+ leak and mitochondrial membrane depolarization are critically involved in the apoptotic pathway of the pre-diabetic heart. The FRD-induced decrease in SR-mitochondrial distance is likely to additionally favour Ca2+ transit between the two organelles.

Ryanodine receptor phosphorylation by CaMKII promotes spontaneous Ca(2+) release events in a rodent model of early stage diabetes: The arrhythmogenic substrate.

BACKGROUND: Heart failure and arrhythmias occur more frequently in patients with type 2 diabetes (T2DM) than in the general population. T2DM is preceded by a prediabetic condition marked by elevated reactive oxygen species (ROS) and subclinical cardiovascular defects. Although multifunctional Ca2+ calmodulin-dependent protein kinase II (CaMKII) is ROS-activated and CaMKII hyperactivity promotes cardiac diseases, a link between prediabetes and CaMKII in the heart is unprecedented. OBJECTIVES: To prove the hypothesis that increased ROS and CaMKII activity contribute to heart failure and arrhythmogenic mechanisms in early stage diabetes. METHODS-RESULTS: Echocardiography, electrocardiography, biochemical and intracellular Ca2+ (Ca2+i) determinations were performed in fructose-rich diet-induced impaired glucose tolerance, a prediabetes model, in rodents. Fructose-rich diet rats showed decreased contractility and hypertrophy associated with increased CaMKII activity, ROS production, oxidized CaMKII and enhanced CaMKII-dependent ryanodine receptor (RyR2) phosphorylation compared to rats fed with control diet. Isolated cardiomyocytes from fructose-rich diet showed increased spontaneous Ca2+i release events associated with spontaneous contractions, which were prevented by KN-93, a CaMKII inhibitor, or addition of Tempol, a ROS scavenger, to the diet. Moreover, fructose-rich diet myocytes showed increased diastolic Ca2+ during the burst of spontaneous Ca2+i release events. Mice treated with Tempol or with sarcoplasmic reticulum-targeted CaMKII-inhibition by transgenic expression of the CaMKII inhibitory peptide AIP, were protected from fructose-rich diet-induced spontaneous Ca2+i release events, spontaneous contractions and arrhythmogenesis in vivo, despite ROS increases. CONCLUSIONS: RyR2 phosphorylation by ROS-activated CaMKII, contributes to impaired glucose tolerance-induced arrhythmogenic mechanisms, suggesting that CaMKII inhibition could prevent prediabetic cardiovascular complications and/or evolution.

CaMKII-dependent phosphorylation of cardiac ryanodine receptors regulates cell death in cardiac ischemia/reperfusion injury.

Ca(2+)-calmodulin kinase II (CaMKII) activation is deleterious in cardiac ischemia/reperfusion (I/R). Moreover, inhibition of CaMKII-dependent phosphorylations at the sarcoplasmic reticulum (SR) prevents CaMKII-induced I/R damage. However, the downstream targets of CaMKII at the SR level, responsible for this detrimental effect, remain unclear. In the present study we aimed to dissect the role of the two main substrates of CaMKII at the SR level, phospholamban (PLN) and ryanodine receptors (RyR2), in CaMKII-dependent I/R injury. In mouse hearts subjected to global I/R (45/120min), phosphorylation of the primary CaMKII sites, S2814 on cardiac RyR2 and of T17 on PLN, significantly increased at the onset of reperfusion whereas PKA-dependent phosphorylation of RyR2 and PLN did not change. Similar results were obtained in vivo, in mice subjected to regional myocardial I/R (1/24h). Knock-in mice with an inactivated serine 2814 phosphorylation site on RyR2 (S2814A) significantly improved post-ischemic mechanical recovery, reduced infarct size and decreased apoptosis. Conversely, knock-in mice, in which CaMKII site of RyR2 is constitutively activated (S2814D), significantly increased infarct size and exacerbated apoptosis. In S2814A and S2814D mice subjected to regional myocardial ischemia, infarct size was also decreased and increased respectively. Transgenic mice with double-mutant non-phosphorylatable PLN (S16A/T17A) in the PLN knockout background (PLNDM) also showed significantly increased post-ischemic cardiac damage. This effect cannot be attributed to PKA-dependent PLN phosphorylation and was not due to the enhanced L-type Ca(2+) current, present in these mice. Our results reveal a major role for the phosphorylation of S2814 site on RyR2 in CaMKII-dependent I/R cardiac damage. In contrast, they showed that CaMKII-dependent increase in PLN phosphorylation during reperfusion opposes rather than contributes to I/R damage.

Increased Na⁺/Ca²âº exchanger expression/activity constitutes a point of inflection in the progression to heart failure of hypertensive rats.

UNLABELLED: Spontaneously hypertensive rat (SHR) constitutes a genetic model widely used to study the natural evolution of hypertensive heart disease. Ca²âº-handling alterations are known to occur in SHR. However, the putative modifications of Ca²âº-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 Ca²âº, Ca²âº-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 Ca²âº 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-Ca²âº transient decay and an increase in Na⁺/Ca²âº exchanger (NCX) abundance (p<0.05) with no changes in SERCA2a expression/activity. An increased Ca²âº-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 Ca²âº efflux from the cell, constitutes a primary alteration of Ca²âº-handling proteins in the evolution to HF. 3. No changes in SERCA2a expression/activity are observed when HF signs become evident.

Early apoptosis in different models of cardiac hypertrophy induced by high renin-angiotensin system activity involves CaMKII.

The objective of this study was to establish whether 1) hyperactivity of renin-angiotensin-aldosterone system (RAAS) produces apoptosis in early stages of cardiac disease; and 2) Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) is involved in these apoptotic events. Two models of hypertrophy were used at an early stage of cardiac disease: spontaneously hypertensive rats (SHR) and isoproterenol-treated rats (Iso-rats). At 4 mo, SHR showed blood pressure, aldosterone serum levels, used as RAAS activity index, and left ventricular mass index, used as hypertrophy index, above control values by 84.2 ± 2.6 mmHg, 211.2 ± 25.8%, and 8.6 ± 1.1 mg/mm, respectively. There was also an increase in apoptotis (Bax-to-Bcl-2 ratio and terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling positive cells) associated with an enhancement of CaMKII activity with respect to age-matched controls (phosphorylated-CaMKII, 98.7 ± 14.1 above control). Similar results were observed in 4-mo-old Iso-rats. Cardiac function studied by echocardiography remained unaltered in all groups. Enalapril treatment significantly prevented hypertrophy, apoptosis, and CaMKII activity. Moreover, intracellular Ca(2+) handling in isolated myocytes was similar between SHR, Iso-rats, and their aged-matched controls. However, SHR and Iso-rats showed a significant increase in superoxide anion generation (lucigenin) and lipid peroxidation (thiobarbituric acid reactive substance). In transgenic mice with targeted cardiomyocyte expression of a CaMKII inhibitory peptide (AC3-I) or a scrambled control peptide (AC3-C), Iso treatment increased thiobarbituric acid reactive substance in both strains, whereas it increased CaMKII activity and apoptosis only in AC3-C mice. Endogenous increases in RAAS activity induce ROS and CaMKII-dependent apoptosis in vivo. CaMKII activation could not be associated with intracellular Ca(2+) increments and was directly related to the increase in oxidative stress.

Na+/K+-ATPase inhibition by ouabain induces CaMKII-dependent apoptosis in adult rat cardiac myocytes.

The positive inotropic effect produced by Na(+)/K(+)-ATPase inhibition has been used for the treatment of heart failure for over 200 years. Recently, administration of toxic doses of ouabain has been shown to induce cardiac myocyte apoptosis. However, whether prolonged administration of non-toxic doses of ouabain can also promote cardiac myocyte cell death has never been explored. The aim of this study was to assess whether non-toxic doses of ouabain can induce myocyte apoptosis and if so, to examine the underlying mechanisms. For this purpose, cardiac myocytes from rat and cat, two species with different sensitivity to digitalis, were cultured for 24h in the presence or absence of 2 microM (rat) and 25 nm-2 microM ouabain (cat). Cell viability and apoptosis assays showed that ouabain produced, in the rat, a 43+/-5% decrease in cell viability due to apoptosis (enhanced caspase-3 activity, increased Bax/Bcl-2 and TUNEL-positive nuclei) and necrosis (LDH release and trypan blue staining). Similar results were obtained with 25 nM ouabain in the cat. Ouabain-induced reduction in cell viability was prevented by the NCX inhibitor KB-R7943 and by the CaMKII inhibitors, KN93 and AIP. Furthermore, CaMKII overexpression exacerbated ouabain-induced cell mortality which in contrast was reduced in transgenic mice with chronic CaMKII inhibition. However, KN93 failed to affect ouabain-induced inotropy. In addition, whereas ERK(1/2) inhibition with PD-98059 had no effect on cell mortality, PI3K inhibition with wortmannin, exacerbated myocyte death. We conclude that ouabain triggers an apoptotic cascade that involves NCX and CaMKII as a downstream effector. Ouabain simultaneously activates an antiapoptotic cascade involving PI3K/AKT which is however, insufficient to completely repress apoptosis. The finding that KN93 prevents ouabain-induced apoptosis without affecting inotropy suggests the potential use of CaMKII inhibitors as an adjunct to digitalis treatment for cardiovascular disease.

Angiotensin II-induced oxidative stress resets the Ca2+ dependence of Ca2+-calmodulin protein kinase II and promotes a death pathway conserved across different species.

RATIONALE: Angiotensin (Ang) II-induced apoptosis was reported to be mediated by different signaling molecules. Whether these molecules are either interconnected in a single pathway or constitute different and alternative cascades by which Ang II exerts its apoptotic action, is not known. OBJECTIVE: To investigate in cultured myocytes from adult cat and rat, 2 species in which Ang II has opposite inotropic effects, the signaling cascade involved in Ang II-induced apoptosis. METHODS AND RESULTS: Ang II (1 micromol/L) reduced cat/rat myocytes viability by approximately 40%, in part, because of apoptosis (TUNEL/caspase-3 activity). In both species, apoptosis was associated with reactive oxygen species (ROS) production, Ca(2+)/calmodulin-dependent protein kinase (CaMK)II, and p38 mitogen-activated protein kinase (p38MAPK) activation and was prevented by the ROS scavenger MPG (2-mercaptopropionylglycine) or the NADPH oxidase inhibitor DPI (diphenyleneiodonium) by CaMKII inhibitors (KN-93 and AIP [autocamtide 2-related inhibitory peptide]) or in transgenic mice expressing a CaMKII inhibitory peptide and by the p38MAPK inhibitor, SB202190. Furthermore, p38MAPK overexpression exacerbated Ang II-induced cell mortality. Moreover, although KN-93 did not affect Ang II-induced ROS production, it prevented p38MAPK activation. Results further show that CaMKII can be activated by Ang II or H(2)O(2), even in the presence of the Ca(2+) chelator BAPTA-AM, in myocytes and in EGTA-Ca(2+)-free solutions in the presence of the calmodulin inhibitor W-7 in in vitro experiments. CONCLUSIONS: (1) The Ang II-induced apoptotic cascade converges in both species, in a common pathway mediated by ROS-dependent CaMKII activation which results in p38MAPK activation and apoptosis. (2) In the presence of Ang II or ROS, CaMKII may be activated at subdiastolic Ca(2+) concentrations, suggesting a new mechanism by which ROS reset the Ca(2+) dependence of CaMKII to extremely low Ca(2+) levels.

Changes in islet plasma membrane calcium-ATPase activity and isoform expression induced by insulin resistance.

We studied the effect of insulin resistance (IR) induced by administration of a fructose-rich diet (FRD) to normal Wistar rats for 21days, upon islet plasma membrane calcium ATPases (PMCAs) and insulin secretion. FRD rats showed significantly higher triglyceride and insulin levels, insulin:glucose ratio and HOMA-IR index than controls. FRD islets released significantly more insulin in response to glucose and showed (a) marked changes in PMCA isoform protein content (decreased PMCA 2 and increased PMCA 3), (b) a decrease in total PMCAs activity, and (c) higher levels of cytosolic calcium [Ca(2+)](i). The lower PMCAs activity with the resultant increase in [Ca(2+)](i) would favor the compensatory greater release of insulin necessary to cope with the IR state present in FRD rats and to maintain normal glucose homeostasis. Thus, changes in PMCAs activity and isoform expression play a modulatory role upon insulin secretion during long-term adaptation to an increased hormone demand.

Angiotensin II: a regulator of cardiomyocyte function and survival.

Angiotensin II (Ang II) has been recognized as an important myocardial inotropic modulator, but the subtleties of the signalling pathways involved remain to be fully elucidated. The inotropic effect of Ang II reflects the net outcome of competing positive and negative signalling mechanisms. In pathophysiological states such as heart failure, characterized by chronic exposure to elevated Ang II, the balance of inotropic influences could be shifted to unmask negative inotropism linked with p38 MAPK activation. Coincident with loss of inotropic balance, Ang II-mediated morphologic remodelling of the heart and apoptosis are observed and accepted to play a crucial role in contractile dysfunction and transition to heart failure. Both Ca2+-dependent and independent pathways appear to be important, and we highlight Ang II mediated CaMKII activation as a potential key integrator of these two pathways in apoptosis induction in the failing heart. To identify new therapeutic molecular targets in heart failure, further work is required to clearly establish the signalling events involved in Ang II inotropic and apoptotic signalling and the potential link between them.

Multiple alterations in Ca2+ handling determine the negative staircase in a cellular heart failure model.

BACKGROUND: The flat or negative force frequency relationship (FFR) is a hallmark of the failing heart. Either decreases in SERCA2a expression, increases in Na(+)/Ca(2+) exchanger (NCX) expression or elevated Na(+)(i) have been independently proposed as mediators of the negative FFR. METHODS AND RESULTS: To determine whether each one of these mechanisms is sufficient to account for the negative FFR of the failing heart or on the contrary, various mechanisms, acting in concert are required. SERCA2a was pharmacologically inhibited with thapsigargin (TG) or cyclopiazonic acid (CPA) or by using siRNA technology; Na(+)(i) was increased with either ouabain (Oua) or monensin and NCX protein was overexpressed by gene transfer (Ad.NCX), to mimic in nonfailing cat myocytes the phenotype of the failing heart and examine their effect on the FFR. The positive FFR of healthy myocytes remained unaffected after either SERCA2a inhibition, Na(+)(i) elevation, or NCX overexpression. However, the combination of TG + Oua, Oua + Ad.NCX, or TG + Ad.NCX, converted the positive FFR to negative. Moreover, the FFR became negative at lower frequencies, when the 3 interventions were combined. CONCLUSIONS: Ca(2+) handling has to be altered at several levels to explain the negative FFR of the failing heart. These anomalies in Ca(2+) homeostasis acting in synergy have additive effects.

Angiotensin II-induced negative inotropy in rat ventricular myocytes: role of reactive oxygen species and p38 MAPK.

The octapeptide angiotensin II (ANG II) can modulate cardiac contractility and is increased in heart failure, where contractile function is impaired. In rat cardiac myocytes, 1 microM of ANG II produces a negative inotropic effect (NIE) (24.6 +/- 5% reduction). However, the subcellular signaling involved in this effect remains elusive. We examined the mechanisms and signaling events involved in the reduction in contractile function induced by the peptide in indo-1-loaded rat cardiomyocytes. The results showed that the NIE of ANG II was not associated with a parallel decrease in the intracellular Ca2+ transient, indicating that a decrease in myofilament responsiveness to Ca2+ underlies the reduction in contractility. We assessed the role of PKC, tyrosine kinases, reactive oxygen species (ROS), and mitogen-activated protein kinases (MAPKs) in the NIE of the peptide. Pretreatment of cells with the NAD(P)H oxidase inhibitor diphenyleneiodonium chloride or with the superoxide scavenger 4,5-dihydroxy-1,3-benzene-disulfonic acid did not affect the ANG II-induced NIE. Moreover, ANG II-induced ROS production, after 20 min of incubation with the peptide, could not be detected with the use of either the fluorophore 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorecein diacetate or lucigenin-enhanced chemiluminescence. In contrast, the ANG II-induced NIE was abrogated by the inhibitors of PKC (calphostin C), tyrosine kinase (genistein), and p38 MAPK (SB-202190). Furthermore, the NIE was significantly exacerbated (60 +/- 10% reduction) by p38 MAPK overexpression. These results exclude the participation of ROS in the NIE of the peptide and point to PKC and tyrosine kinase as upstream mediators. Furthermore, they reveal p38 MAPK as the putative effector of the reduction in myofilament responsiveness to Ca2+ and the decrease in contractility induced by the peptide.

Frequency-dependent acceleration of relaxation in mammalian heart: a property not relying on phospholamban and SERCA2a phosphorylation.

An increase in stimulation frequency causes an acceleration of myocardial relaxation (FDAR). Several mechanisms have been postulated to explain this effect, among which is the Ca(2+)-calmodulin-dependent protein kinase (CaMKII)-dependent phosphorylation of the Thr(17) site of phospholamban (PLN). To gain further insights into the mechanisms of FDAR, we studied the FDAR and the phosphorylation of PLN residues in perfused rat hearts, cat papillary muscles and isolated cat myocytes. This allowed us to sweep over a wide range of frequencies, in species with either positive or negative force-frequency relationships, as well as to explore the FDAR under isometric (or isovolumic) and isotonic conditions. Results were compared with those produced by isoprenaline, an intervention known to accelerate relaxation (IDAR) via PLN phosphorylation. While IDAR occurs tightly associated with a significant increase in the phosphorylation of Ser(16) and Thr(17) of PLN, FDAR occurs without significant changes in the phosphorylation of PLN residues in the intact heart and cat papillary muscles. Moreover, in intact hearts, FDAR was not associated with any significant change in the CaMKII-dependent phosphorylation of sarcoplasmic/endoplasmic Ca(2+) ATPase (SERCA2a), and was not affected by the presence of the CaMKII inhibitor, KN-93. In isolated myocytes, FDAR occurred associated with an increase in Thr(17) phosphorylation. However, for a similar relaxant effect produced by isoprenaline, the phosphorylation of PLN (Ser(16) and Thr(17)) was significantly higher in the presence of the beta-agonist. Moreover, the time course of Thr(17) phosphorylation was significantly delayed with respect to the onset of FDAR. In contrast, the time course of Ser(16) phosphorylation, the first residue that becomes phosphorylated with isoprenaline, was temporally associated with IDAR. Furthermore, KN-93 significantly decreased the phosphorylation of Thr(17) that was evoked by increasing the stimulation frequency, but failed to affect FDAR. Taken together, the results provide direct evidence indicating that CaMKII phosphorylation pathways are not involved in FDAR and that FDAR and IDAR do not share a common underlying mechanism. More likely, a CaMKII-independent mechanism could be involved, whereby increasing stimulation frequency would disrupt the SERCA2a-PLN interaction, leading to an increase in SR Ca(2+) uptake and myocardial relaxation.

Pacing staircase phenomenon in the heart: from Bodwitch to the XXI century.

The frequency of pacing is a fundamental physiological modulator of myocardial function. When the pacing rate increases there is normally an increase in contractility (a positive force-frequency relationship). However in small rodents, fish and end-stage failing myocardium, the force-frequency response has been found to be flat or even negative. The positive staircase is understood to be related with the increase in the intracellular Ca(2+) transient, mainly due to an enhanced sarcoplasmic reticulum Ca(2+) content at higher stimulation frequencies, resulting from an increase in Ca(2+) influx per unit time and reduced Ca(2+) efflux between beats. However, additional mechanisms, such as increased activity of Ca(2+)/calmodulin-dependent protein kinase or enhanced myofilament responsiveness to Ca(2+) may also play a role. Although an increase in contraction frequency has been shown to be associated with an increase in intracellular Na(+), several studies have shown a temporal dissociation between the increase in Na(i)(+) and the increase in force evoked by changes in pacing frequency. The way in which the Na(+)/Ca(2+) exchanger contributes to contraction frequency inotropy is still not well understood. The aim of this review is to examine the contribution of the fundamental components of cardiac excitation-contraction coupling to frequency inotropy in healthy and failing hearts.

Na(+)-Ca2+ exchange function underlying contraction frequency inotropy in the cat myocardium.

In most mammalian species, an increase in stimulation frequency (ISF) produces an increase in contractility (treppe phenomenon), which results from larger Ca2+ transients at higher frequencies, due to an increase in sarcoplasmic reticulum Ca2+ load and release. The present study attempts to elucidate the contribution of the Na(+)-Ca2+ exchanger (NCX) to this phenomenon. Isolated cat ventricular myocytes, loaded with [Ca2+]i- and [Na+]i-sensitive probes, were used to determine whether the contribution of the NCX to the positive inotropic effect of ISF is due to an increase in Ca2+ influx (reverse mode) and/or a decrease in Ca2+ efflux (forward mode) via the NCX, due to frequency-induced [Na+]i elevation, or whether it was due to the reduced time for the NCX to extrude Ca2+. The results showed that the positive intropic effect produced by ISF was temporally dissociated from the increase in [Na+]i and was not modified by KB-R7943 (1 or 5 microM), a specific blocker of the reverse mode of the NCX. Whereas the ISF from 10 to 30 beats min(-1) (bpm) did not affect the forward mode of the NCX (assessed by the time to half-relaxation of the caffeine-induced Ca2+ transient), the ISF to 50 bpm produced a significant reduction of the activity of the forward mode of the NCX, which occurred in association with an increase in [Na+]i (from 4.33+/-0.40 to 7.25+/-0.50 mM). However, both changes became significant well after the maximal positive inotropic effect had been reached. In contrast, the positive inotropic effect produced by ISF from 10 to 50 bpm was associated with an increase in diastolic [Ca2+]i, which occurred in spite of a significant increase in the relaxation rate and at a time at which no increases in [Na+]i were detected. The contribution of the NCX to stimulus frequency inotropy would therefore depend on a decrease in NCX-mediated Ca2+ efflux due to the reduced diastolic interval between beats and not on [Na+]i-dependent mechanisms.