Martin Vila Petroff

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.

Evidence for an electrogenic Na+-HCO3− symport in rat cardiac myocytes

1 The perforated whole‐cell configuration of patch clamp and the pH fluorescent indicator SNARF were used to determine the electrogenicity of the Na+‐HCO3− cotransport in isolated rat ventricular myocytes. 2 Switching from Hepes buffer to HCO3− buffer at constant extracellular pH (pHo) hyperpolarized the resting membrane potential (RMP) by 2.9 ± 0.4 mV (n= 9, P < 0.05). In the presence of HCO3−, the anion blocker SITS depolarized RMP by 2.6 ± 0.5 mV (n= 5, P < 0.05). No HCO3−‐induced hyperpolarization was observed in the absence of extracellular Na+. The duration of the action potential measured at 50 % of repolarization time (APD50) was 29.2 ± 6.1 % shorter in the presence of HCO3− than in its absence (n= 6, P < 0.05). 3 Quasi‐steady‐state currents were evoked by voltage‐clamped ramps ranging from −130 to +30 mV, during 8 s. The development of a novel component of Na+‐dependent and Cl−‐independent steady‐state outward current was observed in the presence of HCO3−. The reversal potential (Erev) of the Na+‐HCO3− cotransport current (INa,Bic) was measured at four different levels of extracellular Na+. A HCO3−:Na+ ratio compatible with a stoichiometry of 2:1 was detected. INa,Bic was also studied in isolation in standard whole‐cell experiments. Under these conditions, INa,Bic reversed at −96.4 ± 1.9 mV (n= 5), being consistent with the influx of 2 HCO3− ions per Na+ ion through the Na+‐HCO3− cotransporter. 4 In the presence of external HCO3−, after 10 min of depolarizing the membrane potential (Em) with 45 mm extracellular K+, a significant intracellular alkalinization was detected (0.09 ± 0.03 pH units; n= 5, P < 0.05). No changes in pHi were observed when the myocytes were pre‐treated with the anion blocker DIDS (0.001 ± 0.024 pH units; n= 5, n.s.), or when exposed to Na+‐free solutions (0.003 ± 0.037 pH units; n= 6, n.s.). 5 The above results allow us to conclude that the cardiac Na+‐HCO3− cotransport is electrogenic and has an influence on RMP and APD of rat ventricular cells.

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.

Calcium-Calmodulin Kinase II Mediates Digitalis-Induced Arrhythmias

Background —Digitalis-induced Na + accumulation results in an increase in Ca 2+ i via the Na + /Ca 2+ exchanger leading to enhanced SR Ca 2+ load, responsible for the positive inotropic and toxic arrhythmogenic effects of glycosides. Digitalis-induced increase in Ca 2+ i could also activate CaMKII which has been shown to have proarrhythmic effects. Here we investigate whether CaMKII underlies digitalis-induced arrhythmias and the subcellular mechanisms involved. Methods and Results —In paced rat ventricular myocytes (0.5 Hz), 50 μmol/l ouabain increased contraction amplitude by 160 ± 5%. In the absence of electrical stimulation, ouabain promoted spontaneous contractile activity and Ca 2+ waves. Ouabain activated CaMKII (P-CaMKII) which phosphorylated its downstream targets, phospholamban (Thr17) and ryanodine receptor (RyR) (Ser2814). Ouabain-induced spontaneous activity was prevented by inhibiting CaMKII with 2.5 μmol/l KN93 but not by 2.5 μmol/l of the inactive analogue KN92. Similar results were obtained using the CaMKII inhibitor, AIP (1-2.5 μmol/l) and in myocytes from transgenic mice expressing SR-targeted AIP. Consistently, CaMKII overexpression exacerbated ouabain-induced spontaneous contractile activity. Ouabain was associated with an increase in SR Ca 2+ content and Ca 2+ spark frequency, indicative of enhanced SR Ca 2+ leak. KN93 suppressed the ouabain-induced increase in Ca2+ spark frequency without affecting SR Ca 2+ content. Similar results were obtained with digoxin. In vivo , ouabain-induced arrhythmias were prevented by KN93 and absent in SR-AIP mice. Conclusions —These results show for the first time that CaMKII mediates ouabain-induced arrhythmic/toxic effects. We suggest that CaMKII-dependent phosphorylation of the RyR, resulting in Ca 2+ leak from the SR is the underlying mechanism involved.Background— Digitalis-induced Na+ accumulation results in an increase in Ca2+ i via the Na+/Ca2+ exchanger, leading to enhanced sarcoplasmic reticulum (SR) Ca2+ load, responsible for the positive inotropic and toxic arrhythmogenic effects of glycosides. A digitalis-induced increase in Ca2+ i could also activate calcium-calmodulin kinase II (CaMKII), which has been shown to have proarrhythmic effects. Here, we investigate whether CaMKII underlies digitalis-induced arrhythmias and the subcellular mechanisms involved. Methods and Results— In paced rat ventricular myocytes (0.5 Hz), 50 &mgr;mol/L ouabain increased contraction amplitude by 160±5%. In the absence of electric stimulation, ouabain promoted spontaneous contractile activity and Ca2+ waves. Ouabain activated CaMKII (p-CaMKII), which phosphorylated its downstream targets, phospholamban (PLN) (Thr17) and ryanodine receptor (RyR) (Ser2814). Ouabain-induced spontaneous activity was prevented by inhibiting CaMKII with 2.5 &mgr;mol/L KN93 but not by 2.5 &mgr;mol/L of the inactive analog, KN92. Similar results were obtained using the CaMKII inhibitor, autocamtide-2 related inhibitory peptide (AIP) (1 to 2.5 &mgr;mol/L), and in myocytes from transgenic mice expressing SR-targeted AIP. Consistently, CaMKII overexpression exacerbated ouabain-induced spontaneous contractile activity. Ouabain was associated with an increase in SR Ca2+ content and Ca2+ spark frequency, indicative of enhanced SR Ca2+ leak. KN93 suppressed the ouabain-induced increase in Ca2+ spark frequency without affecting SR Ca2+ content. Similar results were obtained with digoxin. In vivo, ouabain-induced arrhythmias were prevented by KN93 and absent in SR-AIP mice. Conclusions— These results show for the first time that CaMKII mediates ouabain-induced arrhythmic/toxic effects. We suggest that CaMKII-dependent phosphorylation of the RyR, resulting in Ca2+ leak from the SR, is the underlying mechanism involved.

CaMKII Mediates Digitalis-Induced Arrhythmias

Background —Digitalis-induced Na + accumulation results in an increase in Ca 2+ i via the Na + /Ca 2+ exchanger leading to enhanced SR Ca 2+ load, responsible for the positive inotropic and toxic arrhythmogenic effects of glycosides. Digitalis-induced increase in Ca 2+ i could also activate CaMKII which has been shown to have proarrhythmic effects. Here we investigate whether CaMKII underlies digitalis-induced arrhythmias and the subcellular mechanisms involved. Methods and Results —In paced rat ventricular myocytes (0.5 Hz), 50 μmol/l ouabain increased contraction amplitude by 160 ± 5%. In the absence of electrical stimulation, ouabain promoted spontaneous contractile activity and Ca 2+ waves. Ouabain activated CaMKII (P-CaMKII) which phosphorylated its downstream targets, phospholamban (Thr17) and ryanodine receptor (RyR) (Ser2814). Ouabain-induced spontaneous activity was prevented by inhibiting CaMKII with 2.5 μmol/l KN93 but not by 2.5 μmol/l of the inactive analogue KN92. Similar results were obtained using the CaMKII inhibitor, AIP (1-2.5 μmol/l) and in myocytes from transgenic mice expressing SR-targeted AIP. Consistently, CaMKII overexpression exacerbated ouabain-induced spontaneous contractile activity. Ouabain was associated with an increase in SR Ca 2+ content and Ca 2+ spark frequency, indicative of enhanced SR Ca 2+ leak. KN93 suppressed the ouabain-induced increase in Ca2+ spark frequency without affecting SR Ca 2+ content. Similar results were obtained with digoxin. In vivo , ouabain-induced arrhythmias were prevented by KN93 and absent in SR-AIP mice. Conclusions —These results show for the first time that CaMKII mediates ouabain-induced arrhythmic/toxic effects. We suggest that CaMKII-dependent phosphorylation of the RyR, resulting in Ca 2+ leak from the SR is the underlying mechanism involved.Background— Digitalis-induced Na+ accumulation results in an increase in Ca2+ i via the Na+/Ca2+ exchanger, leading to enhanced sarcoplasmic reticulum (SR) Ca2+ load, responsible for the positive inotropic and toxic arrhythmogenic effects of glycosides. A digitalis-induced increase in Ca2+ i could also activate calcium-calmodulin kinase II (CaMKII), which has been shown to have proarrhythmic effects. Here, we investigate whether CaMKII underlies digitalis-induced arrhythmias and the subcellular mechanisms involved. Methods and Results— In paced rat ventricular myocytes (0.5 Hz), 50 &mgr;mol/L ouabain increased contraction amplitude by 160±5%. In the absence of electric stimulation, ouabain promoted spontaneous contractile activity and Ca2+ waves. Ouabain activated CaMKII (p-CaMKII), which phosphorylated its downstream targets, phospholamban (PLN) (Thr17) and ryanodine receptor (RyR) (Ser2814). Ouabain-induced spontaneous activity was prevented by inhibiting CaMKII with 2.5 &mgr;mol/L KN93 but not by 2.5 &mgr;mol/L of the inactive analog, KN92. Similar results were obtained using the CaMKII inhibitor, autocamtide-2 related inhibitory peptide (AIP) (1 to 2.5 &mgr;mol/L), and in myocytes from transgenic mice expressing SR-targeted AIP. Consistently, CaMKII overexpression exacerbated ouabain-induced spontaneous contractile activity. Ouabain was associated with an increase in SR Ca2+ content and Ca2+ spark frequency, indicative of enhanced SR Ca2+ leak. KN93 suppressed the ouabain-induced increase in Ca2+ spark frequency without affecting SR Ca2+ content. Similar results were obtained with digoxin. In vivo, ouabain-induced arrhythmias were prevented by KN93 and absent in SR-AIP mice. Conclusions— These results show for the first time that CaMKII mediates ouabain-induced arrhythmic/toxic effects. We suggest that CaMKII-dependent phosphorylation of the RyR, resulting in Ca2+ leak from the SR, is the underlying mechanism involved.

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.

The electrogenic Na+/HCO3− cotransport modulates resting membrane potential and action potential duration in cat ventricular myocytes

Perforated whole‐cell configuration of patch clamp was used to determine the contribution of the electrogenic Na+/HCO3− cotransport (NBC) on the shape of the action potential in cat ventricular myocytes. Switching from Hepes to HCO3− buffer at constant extracellular pH (pHo) hyperpolarized resting membrane potential (RMP) by 2.67 ± 0.42 mV (n= 9, P < 0.05). The duration of action potential measured at 50% of repolarization time (APD50) was 35.8 ± 6.8% shorter in the presence of HCO3− than in its absence (n= 9, P < 0.05). The anion blocker SITS prevented and reversed the HCO3−‐induced hyperpolarization and shortening of APD. In addition, no HCO3−‐induced hyperpolarization and APD shortening was observed in the absence of extracellular Na+. Quasi‐steady‐state currents were evoked by 8 s duration voltage‐clamped ramps ranging from −130 to +30 mV. A novel component of SITS‐sensitive current was observed in the presence of HCO3−. The HCO3−‐sensitive current reversed at −87 ± 5 mV (n= 7), a value close to the expected reversal potential of an electrogenic Na+/HCO3− cotransport with a HCO3−:Na+ stoichiometry ratio of 2: 1. The above results allow us to conclude that the cardiac electrogenic Na+/HCO3− cotransport has a relevant influence on RMP and APD of cat ventricular cells.

Role of CaMKII and ROS in rapid pacing-induced apoptosis

Tachycardia promotes cell death and cardiac remodeling, leading to congestive heart failure. However, the underlying mechanism of tachycardia- or rapid pacing (RP)-induced cell death remains unknown. Myocyte loss by apoptosis is recognized as a critical factor in the progression to heart failure and simulation of tachycardia by RP has been shown to increase the intracellular levels of at least two potentially proapoptotic molecules, Ca(2+) and reactive oxygen species (ROS). However, whether these molecules mediate tachycardia- or RP-induced cell death has yet to be determined. The aim of this study was to examine the subcellular mechanisms underlying RP-induced apoptosis. For this purpose rat ventricular myocytes were maintained quiescent or paced at 0.5, 5 and 8Hz for 1hr. RP at 5 and 8Hz decreased myocyte viability by 58±3% and 75±6% (n=24), respectively, compared to cells maintained at 0.5Hz, and increased caspase-3 activity and Bax/Bcl-2 ratio, indicative of apoptosis. RP-induced cell death and apoptosis were prevented when pacing protocols were conducted in the presence of either the ROS scavenger, MPG, or nifedipine to reduce Ca(2+) entry or the CaMKII inhibitors, KN93 and AIP. Consistently, myocytes from transgenic mice expressing a CaMKII inhibitory peptide (AC3-I) were protected against RP-induced cell death. Interestingly, tetracaine and carvedilol used to reduce ryanodine receptor (RyR) diastolic Ca(2+) release, and ruthenium red used to prevent Ca(2+) entry into the mitochondria prevented RP-induced cell death, whereas PI3K inhibition with Wortmannin exacerbated pacing-induced cell mortality. We conclude that CaMKII activation and ROS production are involved in RP-induced apoptosis. Particularly, our results suggest that CaMKII-dependent posttranslational modifications of the cardiac ryanodine receptor (RyR) leading to enhanced diastolic Ca(2+) release and mitochondrial Ca(2+) overload could be the underlying mechanism involved. We further show that RP simultaneously activates a protective cascade involving PI3K/AKT signaling which is however, insufficient to completely suppress apoptosis.

Cardiac CaMKIIδ splice variants exhibit target signaling specificity and confer sex-selective arrhythmogenic actions in the ischemic-reperfused heart

BACKGROUND Ischemia-related arrhythmic incidence is generally lower in females (vs males), though risk is selectively increased in women with underlying cardiopathology. Ca(2+)/calmodulin dependent kinase II (CaMKII) has been implicated in ischemia/reperfusion arrhythmias, yet the role of CaMKII in the ischemic female heart has not been determined. The aim of this study was to define the role and molecular mechanism of CaMKII activation in reperfusion arrhythmias in male/female hearts. METHODS AND RESULTS Male and female rat hearts and cardiomyocytes were subjected to multiple arrhythmogenic challenges. An increased capacity to upregulate autophosphorylated CaMKII (P-CaMKII) in Ca(2+)-challenged female hearts was associated with an enhanced ability to maintain diastolic function. In ischemia/reperfusion, female hearts (vs male) exhibited less arrhythmias (59 ± 18 vs 548 ± 9, s, p<0.05), yet had augmented P-CaMKII (2.69 ± 0.30 vs 1.50 ± 0.14, rel. units, p<0.05) and downstream phosphorylation of phospholamban (1.71 ± 0.42 vs 0.90 ± 0.10, p<0.05). In contrast, hypertrophic female hearts had more reperfusion arrhythmias and lower phospholamban phosphorylation. Isolated myocyte experiments (fura-2) confirmed Ca(2+)-handling arrhythmogenic involvement. Molecular analysis showed target specificity of CaMKII was determined by post-translational modification, with CaMKIIδB and CaMKIIδC splice variants selectively co-localized with autophosphorylation and oxidative modifications of CaMKII respectively. CONCLUSIONS This study provides new mechanistic evidence that CaMKIIδ splice variants are selectively susceptible to autophosphorylation/oxidation, and that augmented generation of P-CaMKIIδB(Thr287) is associated with arrhythmia suppression in the female heart. Collectively these findings indicate that therapeutic approaches based on selective CaMKII splice form targeting may have potential benefit, and that sex-selective CaMKII intervention strategies may be valid.