Julieta Palomeque

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 &mgr;mol/L) reduced cat/rat myocytes viability by ≈40%, in part, because of apoptosis (TUNEL/caspase-3 activity). In both species, apoptosis was associated with reactive oxygen species (ROS) production, Ca2+/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 H2O2, even in the presence of the Ca2+ chelator BAPTA-AM, in myocytes and in EGTA-Ca2+–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 Ca2+ concentrations, suggesting a new mechanism by which ROS reset the Ca2+ dependence of CaMKII to extremely low Ca2+ levels.

Increased intracellular Ca2+ and SR Ca2+ load contribute to arrhythmias after acidosis in rat heart. Role of Ca2+/calmodulin-dependent protein kinase II.

Returning to normal pH after acidosis, similar to reperfusion after ischemia, is prone to arrhythmias. The type and mechanisms of these arrhythmias have never been explored and were the aim of the present work. Langendorff-perfused rat/mice hearts and rat-isolated myocytes were subjected to respiratory acidosis and then returned to normal pH. Monophasic action potentials and left ventricular developed pressure were recorded. The removal of acidosis provoked ectopic beats that were blunted by 1 muM of the CaMKII inhibitor KN-93, 1 muM thapsigargin, to inhibit sarcoplasmic reticulum (SR) Ca(2+) uptake, and 30 nM ryanodine or 45 muM dantrolene, to inhibit SR Ca(2+) release and were not observed in a transgenic mouse model with inhibition of CaMKII targeted to the SR. Acidosis increased the phosphorylation of Thr(17) site of phospholamban (PT-PLN) and SR Ca(2+) load. Both effects were precluded by KN-93. The return to normal pH was associated with an increase in SR Ca(2+) leak, when compared with that of control or with acidosis at the same SR Ca(2+) content. Ca(2+) leak occurred without changes in the phosphorylation of ryanodine receptors type 2 (RyR2) and was blunted by KN-93. Experiments in planar lipid bilayers confirmed the reversible inhibitory effect of acidosis on RyR2. Ectopic activity was triggered by membrane depolarizations (delayed afterdepolarizations), primarily occurring in epicardium and were prevented by KN-93. The results reveal that arrhythmias after acidosis are dependent on CaMKII activation and are associated with an increase in SR Ca(2+) load, which appears to be mainly due to the increase in PT-PLN.

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 Ca2+–calmodulin‐dependent protein kinase (CaMKII)‐dependent phosphorylation of the Thr17 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 Ser16 and Thr17 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 Ca2+ 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 Thr17 phosphorylation. However, for a similar relaxant effect produced by isoprenaline, the phosphorylation of PLN (Ser16 and Thr17) was significantly higher in the presence of the β‐agonist. Moreover, the time course of Thr17 phosphorylation was significantly delayed with respect to the onset of FDAR. In contrast, the time course of Ser16 phosphorylation, the first residue that becomes phosphorylated with isoprenaline, was temporally associated with IDAR. Furthermore, KN‐93 significantly decreased the phosphorylation of Thr17 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 Ca2+ uptake and myocardial relaxation.

Subcellular mechanisms of the positive inotropic effect of angiotensin II in cat myocardium

1 Cat ventricular myocytes loaded with [Ca2+]i‐ and pHi‐sensitive probes were used to examine the subcellular mechanism(s) of the Ang II‐induced positive inotropic effect. Ang II (1 μM) produced parallel increases in contraction and Ca2+ transient amplitudes and a slowly developing intracellular alkalisation. Maximal increases in contraction amplitude and Ca2+ transient amplitude were 163 ± 22 and 43 ± 8 %, respectively, and occurred between 5 and 7 min after Ang II administration, whereas pHi increase (0·06 ± 0·03 pH units) became significant only 15 min after the addition of Ang II. Furthermore, the inotropic effect of Ang II was preserved in the presence of Na+‐H+ exchanger blockade. These results indicate that the positive inotropic effect of Ang II is independent of changes in pHi. 2 Similar increases in contractility produced by either elevating extracellular [Ca2+] or by Ang II application produced similar increases in peak systolic Ca2+ indicating that an increase in myofilament responsiveness to Ca2+ does not participate in the Ang II‐induced positive inotropic effect. 3 Ang II significantly increased the L‐type Ca2+ current, as assessed by using the perforated patch‐clamp technique (peak current recorded at 0 mV: ‐1·88 ± 0·16 pA pF−1 in control vs. ‐3·03 ± 0·20 pA pF−1 after 6‐8 min of administration of Ang II to the bath solution). 4 The positive inotropic effect of Ang II was not modified in the presence of either KB‐R7943, a specific blocker of the Na+‐Ca2+ exchanger, or ryanodine plus thapsigargin, used to block the sarcoplasmic reticulum function. 5 The above results allow us to conclude that in the cat ventricle the Ang II‐induced positive inotropic effect is due to an increase in the intracellular Ca2+ transient, an enhancement of the L‐type Ca2+ current being the dominant mechanism underlying this increase.

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.

KChIP2 attenuates cardiac hypertrophy through regulation of Ito and intracellular calcium signaling

Recent evidence shows that the auxiliary subunit KChIP2, which assembles with pore-forming Kv4-subunits, represents a new potential regulator of the cardiac calcium-independent transient outward potassium current (I(to)) density. In hypertrophy and heart failure, KChIP2 expression has been found to be significantly decreased. Our aim was to examine the role of KChIP2 in cardiac hypertrophy and the effect of restoring its expression on electrical remodeling and cardiac mechanical function using a combination of molecular, biochemical and gene targeting approaches. KChIP2 overexpression through gene transfer of Ad.KChIP2 in neonatal cardiomyocytes resulted in a significant increase in I(to)-channel forming Kv4.2 and Kv4.3 protein levels. In vivo gene transfer of KChIP2 in aortic banded adult rats showed that, compared to sham-operated or Ad.beta-gal-transduced hearts, KChIP2 significantly attenuated the developed left ventricular hypertrophy, robustly increased I(to) densities, shortened action potential duration, and significantly altered myocyte mechanics by shortening contraction amplitudes and maximal rates of contraction and relaxation velocities and decreasing Ca(2+) transients. Interestingly, blocking I(to) with 4-aminopyridine in KChIP2-overexpressing adult cardiomyocytes significantly increased the Ca(2+) transients to control levels. One-day-old rat pups intracardially transduced with KChIP2 for two months then subjected to aortic banding for 6-8 weeks (to induce hypertrophy) showed similar echocardiographic, electrical and mechanical remodeling parameters. In addition, in cultured adult cardiomyocytes, KChIP2 overexpression increased the expression of Ca(2+)-ATPase (SERCA2a) and sodium calcium exchanger but had no effect on ryanodine receptor 2 or phospholamban expression. In neonatal myocytes, KChIP2 notably reversed Ang II-induced hypertrophic changes in protein synthesis and MAP-kinase activation. It also significantly decreased calcineurin expression, NFATc1 expression and nuclear translocation and its downstream target, MCiP1.4. Altogether, these data show that KChIP2 can attenuate cardiac hypertrophy possibly through modulation of intracellular calcium concentration and calcineurin/NFAT pathway.

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 μm), 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.

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.