Luis Alberto Gonano

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.

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.

Aldosterone stimulates the cardiac sodium/bicarbonate cotransporter via activation of the g protein-coupled receptor gpr30

Some cardiac non-genomic effects of aldosterone (Ald) are reported to be mediated through activation of the classic mineralocorticoid receptor (MR). However, in the last years, it was proposed that activation of the novel G protein-coupled receptor GPR30 mediates certain non-genomic effects of Ald. The aim of this study was to elucidate if the sodium/bicarbonate cotransporter (NBC) is stimulated by Ald and if the activation of GPR30 mediates this effect. NBC activity was evaluated in rat cardiomyocytes perfused with HCO3(-)/CO2 solution in the continuous presence of HOE642 (sodium/hydrogen exchanger blocker) during recovery from acidosis using intracellular fluorescence measurements. Ald enhanced NBC activity (% of ΔJHCO3(-); control: 100±5.82%, n=7 vs Ald: 151.88±11.02%, n=5; P<0.05), which was prevented by G15 (GPR30 blocker, 90.53±7.81%, n=7). Further evidence for the involvement of GPR30 was provided by G1 (GPR30 agonist), which stimulated NBC (185.13±18.28%, n=6; P<0.05) and this effect was abrogated by G15 (124.19±10.96%, n=5). Ald- and G1-induced NBC stimulation was abolished by the reactive oxygen species (ROS) scavenger MPG and by the NADPH oxidase inhibitor apocynin. In addition, G15 prevented Ald- and G1-induced ROS production. Pre-incubation of myocytes with wortmannin (PI3K-AKT pathway blocker) prevented Ald- or G1-induced NBC stimulation. In summary, Ald stimulates NBC by GPR30 activation, ROS production and AKT stimulation.

Subcellular Mechanisms Underlying Digitalis-Induced Arrhythmias: Role of Calcium/Calmodulin-Dependent Kinase II (CaMKII) in the Transition from an Inotropic to an Arrhythmogenic Effect

Cardiotonic glycosides or digitalis are positive inotropes used in clinical practice for the treatment of heart failure, which also exist as endogenous ligands of the Na(+)/K(+) ATPase. An increase in the intracellular Ca2+ content mediates their positive inotropic effect, but has also been proposed as a trigger of life-threatening arrhythmias. Although the mechanisms involved in the positive inotropic effect of these compounds have been extensively studied, those underlying their arrhythmogenic action remain ill defined. Recent evidence has placed posttranslational modifications of the ryanodine receptor (RyR2), leading to arrhythmogenic Ca2+ release, in the centre of the storm. In this review we will examine, in depth, the mechanisms that generate the arrhythmogenic substrate, focussing on the role played by the RyR2 and how its CaMKII-dependent regulation may shift the balance from an inotropic to an arrhythmogenic Ca2+ release. Finally, we will provide evidence suggesting that stabilising RyR2 function could result in a potential new strategy to prevent cardiotonic glycoside-induced arrhythmias that could lead to a safer and more extensive use of these compounds.

Calcium/Calmodulin Protein Kinase II-Dependent Ryanodine Receptor Phosphorylation Mediates Cardiac Contractile Dysfunction Associated With Sepsis.

Objectives: Sepsis is associated with cardiac contractile dysfunction attributed to alterations in Ca2+ handling. We examined the subcellular mechanisms involved in sarcoplasmic reticulum Ca2+ loss that mediate altered Ca2+ handling and contractile dysfunction associated with sepsis. Design: Randomized controlled trial. Setting: Research laboratory Subjects: Male wild type and transgenic mice Interventions: We induced sepsis in mice using the colon ascendens stent peritonitis model. Measurements and Main Results: Twenty-four hours after colon ascendens stent peritonitis surgery, we observed that wild type mice had significantly elevated proinflammatory cytokine levels, reduced ejection fraction, and fractional shortening (ejection fraction %, 54.76 ± 0.67; fractional shortening %, 27.53 ± 0.50) compared with sham controls (ejection fraction %, 73.57 ± 0.20; fractional shortening %, 46.75 ± 0.38). At the cardiac myocyte level, colon ascendens stent peritonitis cells showed reduced cell shortening, Ca2+ transient amplitude and sarcoplasmic reticulum Ca2+ content compared with sham cardiomyocytes. Colon ascendens stent peritonitis hearts showed a significant increase in oxidation-dependent calcium and calmodulin-dependent protein kinase II activity, which could be prevented by pretreating animals with the antioxidant tempol. Pharmacologic inhibition of calcium and calmodulin-dependent protein kinase II with 2.5 µM of KN93 prevented the decrease in cell shortening, Ca2+ transient amplitude, and sarcoplasmic reticulum Ca2+ content in colon ascendens stent peritonitis myocytes. Contractile function was also preserved in colon ascendens stent peritonitis myocytes isolated from transgenic mice expressing a calcium and calmodulin-dependent protein kinase II inhibitory peptide (AC3-I) and in colon ascendens stent peritonitis myocytes isolated from mutant mice that have the ryanodine receptor 2 calcium and calmodulin-dependent protein kinase II-dependent phosphorylation site (serine 2814) mutated to alanine (S2814A). Furthermore, colon ascendens stent peritonitis S2814A mice showed preserved ejection fraction and fractional shortening (ejection fraction %, 73.06 ± 6.31; fractional shortening %, 42.33 ± 5.70) compared with sham S2814A mice (ejection fraction %, 71.60 ± 4.02; fractional shortening %, 39.63 ± 3.23). Conclusions: Results indicate that oxidation and subsequent activation of calcium and calmodulin-dependent protein kinase II has a causal role in the contractile dysfunction associated with sepsis. Calcium and calmodulin-dependent protein kinase II, through phosphorylation of the ryanodine receptor would lead to Ca2+ leak from the sarcoplasmic reticulum, reducing sarcoplasmic reticulum Ca2+ content, Ca2+ transient amplitude and contractility. Development of organ-specific calcium and calmodulin-dependent protein kinase II inhibitors may result in a beneficial therapeutic strategy to ameliorate contractile dysfunction associated with sepsis.

Non-β-Blocking Carvedilol Analog, VK-II-86, Prevents Ouabain-Induced Cardiotoxicity

BACKGROUND It has been shown that carvedilol and its non β-blocking analog, VK-II-86, inhibit spontaneous Ca2+ release from the sarcoplasmic reticulum (SR). The aim of this study is to determine whether carvedilol and VK-II-86 suppress ouabain-induced arrhythmogenic Ca2+ waves and apoptosis in cardiac myocytes. Methods and Results: Rat cardiac myocytes were exposed to toxic doses of ouabain (50 µmol/L). Cell length (contraction) was monitored in electrically stimulated and non-stimulated conditions. Ouabain treatment increased contractility, frequency of spontaneous contractions and apoptosis compared to control cells. Carvedilol (1 µmol/L) or VK-II-86 (1 µmol/L) did not affect ouabain-induced inotropy, but significantly reduced the frequency of Ca2+ waves, spontaneous contractions and cell death evoked by ouabain treatment. This antiarrhythmic effect was not associated with a reduction in Ca2+ calmodulin-dependent protein kinase II (CaMKII) activity, phospholamban and ryanodine receptor phosphorylation or SR Ca2+ load. Similar results could be replicated in human cardiomyocytes derived from stem cells and in a mathematical model of human myocytes. CONCLUSIONS Carvedilol and VK-II-86 are effective to prevent ouabain-induced apoptosis and spontaneous contractions indicative of arrhythmogenic activity without affecting inotropy and demonstrated to be effective in human models, thus emerging as a therapeutic tool for the prevention of digitalis-induced arrhythmias and cardiac toxicity.