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1.
Life Sci ; 312: 121219, 2023 Jan 01.
Article in English | MEDLINE | ID: mdl-36435222

ABSTRACT

Two alkalinizing mechanisms coexist in cardiac myocytes to maintain intracellular pH: sodium/bicarbonate cotransporter (electroneutral isoform NBCn1 and electrogenic isoform NBCe1) and sodium/proton exchanger (NHE1). Dysfunction of these transporters has previously been reported to be responsible for the development of cardiovascular diseases. The aim of this study was to evaluate the contribution of the downregulation of the NBCe1 to the development of cardiac hypertrophy. To specifically reduce NBCe1 expression, we cloned shRNA into a cardiotropic adeno-associated vector (AAV9-shNBCe1). After 28 days of being injected with AAV9-shNBCe1, the expression and the activity of NBCe1 in the rat heart were reduced. Strikingly, downregulation of NBCe1 causes significant hypertrophic heart growth, lengthening of the action potential in isolated myocytes, an increase in the duration of the QT interval and an increase in the frequency of Ca2+ waves without any significant changes in Ca2+ transients. An increased compensatory expression of NBCn1 and NHE1 was also observed. We conclude that reduction of NBCe1 is sufficient to induce cardiac hypertrophy and modify the electrical features of the rat heart.


Subject(s)
Bicarbonates , Sodium-Bicarbonate Symporters , Rats , Animals , Sodium-Bicarbonate Symporters/genetics , Sodium-Bicarbonate Symporters/metabolism , Bicarbonates/metabolism , Cardiomegaly/genetics , Cardiomegaly/metabolism , Sodium/metabolism , Protein Isoforms/metabolism , Hydrogen-Ion Concentration
2.
J Physiol ; 594(11): 3005-30, 2016 06 01.
Article in English | MEDLINE | ID: mdl-26695843

ABSTRACT

KEY POINTS: Mice with Ca(2+) -calmodulin-dependent protein kinase (CaMKII) constitutive pseudo-phosphorylation of the ryanodine receptor RyR2 at Ser2814 (S2814D(+/+) mice) exhibit a higher open probability of RyR2, higher sarcoplasmic reticulum (SR) Ca(2+) leak in diastole and increased propensity to arrhythmias under stress conditions. We generated phospholamban (PLN)-deficient S2814D(+/+) knock-in mice by crossing two colonies, S2814D(+/+) and PLNKO mice, to test the hypothesis that PLN ablation can prevent the propensity to arrhythmias of S2814D(+/+) mice. PLN ablation partially rescues the altered intracellular Ca(2+) dynamics of S2814D(+/+) hearts and myocytes, but enhances SR Ca(2+) sparks and leak on confocal microscopy. PLN ablation diminishes ventricular arrhythmias promoted by CaMKII phosphorylation of S2814 on RyR2. PLN ablation aborts the arrhythmogenic SR Ca(2+) waves of S2814D(+/+) and transforms them into non-propagating events. A mathematical human myocyte model replicates these results and predicts the increase in SR Ca(2+) uptake required to prevent the arrhythmias induced by a CaMKII-dependent leaky RyR2. ABSTRACT: Mice with constitutive pseudo-phosphorylation at Ser2814-RyR2 (S2814D(+/+) ) have increased propensity to arrhythmias under ß-adrenergic stress conditions. Although abnormal Ca(2+) release from the sarcoplasmic reticulum (SR) has been linked to arrhythmogenesis, the role played by SR Ca(2+) uptake remains controversial. We tested the hypothesis that an increase in SR Ca(2+) uptake is able to rescue the increased arrhythmia propensity of S2814D(+/+) mice. We generated phospholamban (PLN)-deficient/S2814D(+/+) knock-in mice by crossing two colonies, S2814D(+/+) and PLNKO mice (SD(+/+) /KO). SD(+/+) /KO myocytes exhibited both increased SR Ca(2+) uptake seen in PLN knock-out (PLNKO) myocytes and diminished SR Ca(2+) load (relative to PLNKO), a characteristic of S2814D(+/+) myocytes. Ventricular arrhythmias evoked by catecholaminergic challenge (caffeine/adrenaline) in S2814D(+/+) mice in vivo or programmed electric stimulation and high extracellular Ca(2+) in S2814D(+) /(-) hearts ex vivo were significantly diminished by PLN ablation. At the myocyte level, PLN ablation converted the arrhythmogenic Ca(2+) waves evoked by high extracellular Ca(2+) provocation in S2814D(+/+) mice into non-propagated Ca(2+) mini-waves on confocal microscopy. Myocyte Ca(2+) waves, typical of S2814D(+/+) mice, could be evoked in SD(+/+) /KO cells by partially inhibiting SERCA2a. A mathematical human myocyte model replicated these results and allowed for predicting the increase in SR Ca(2+) uptake required to prevent the arrhythmias induced by a Ca(2+) -calmodulin-dependent protein kinase (CaMKII)-dependent leaky RyR2. Our results demonstrate that increasing SR Ca(2+) uptake by PLN ablation can prevent the arrhythmic events triggered by SR Ca(2+) leak due to CaMKII-dependent phosphorylation of the RyR2-S2814 site and underscore the benefits of increasing SERCA2a activity on SR Ca(2+) -triggered arrhythmias.


Subject(s)
Arrhythmias, Cardiac/metabolism , Calcium-Binding Proteins/deficiency , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Action Potentials/physiology , Animals , Arrhythmias, Cardiac/genetics , Arrhythmias, Cardiac/physiopathology , Calcium/metabolism , Calcium-Binding Proteins/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Gene Knock-In Techniques , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/physiology , Phosphorylation/physiology , Ryanodine Receptor Calcium Release Channel/genetics
3.
Br J Pharmacol ; 171(18): 4207-21, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24628066

ABSTRACT

BACKGROUND AND PURPOSE: Despite the importance of mitochondrial Ca(2+) to metabolic regulation and cell physiology, little is known about the mechanisms that regulate Ca(2+) entry into the mitochondria. Accordingly, we established a system to determine the role of the mitochondrial Ca(2+) uniporter in an isolated heart model, at baseline and during increased workload following ß-adrenoceptor stimulation. EXPERIMENTAL APPROACH: Cardiac contractility, oxygen consumption and intracellular Ca(2+) transients were measured in ex vivo perfused murine hearts. Ru360 and spermine were used to modify mitochondrial Ca(2+) uniporter activity. Changes in mitochondrial Ca(2+) content and energetic phosphate metabolite levels were determined. KEY RESULTS: The addition of Ru360 , a selective inhibitor of the mitochondrial Ca(2+) uniporter, induced progressively and sustained negative inotropic effects that were dose-dependent with an EC50 of 7 µM. Treatment with spermine, a uniporter agonist, showed a positive inotropic effect that was blocked by Ru360 . Inotropic stimulation with isoprenaline elevated oxygen consumption (2.7-fold), Ca(2+) -dependent activation of pyruvate dehydrogenase (5-fold) and mitochondrial Ca(2+) content (2.5-fold). However, in Ru360 -treated hearts, this parameter was attenuated. In addition, ß-adrenoceptor stimulation in the presence of Ru360 did not affect intracellular Ca(2+) handling, PKA or Ca(2+) /calmodulin-dependent PK signalling. CONCLUSIONS AND IMPLICATIONS: Inhibition of the mitochondrial Ca(2+) uniporter decreases ß-adrenoceptor response, uncoupling between workload and production of energetic metabolites. Our results support the hypothesis that the coupling of workload and energy supply is partly dependent on mitochondrial Ca(2+) uniporter activity.


Subject(s)
Calcium Channels/physiology , Calcium/physiology , Heart/physiology , Mitochondria, Heart/physiology , Receptors, Adrenergic, beta/physiology , Adrenergic beta-Agonists/pharmacology , Animals , Calcium-Binding Proteins/metabolism , Cardiotonic Agents/pharmacology , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Glutathione/metabolism , Heart/drug effects , Isoproterenol/pharmacology , Male , Mice , Mitochondria, Heart/drug effects , Myocytes, Cardiac/metabolism , Oxidative Stress/drug effects , Oxygen Consumption , Rats, Wistar , Ruthenium Compounds/pharmacology , Spermine/pharmacology
4.
J Mol Cell Cardiol ; 51(6): 936-44, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21888910

ABSTRACT

To explore whether CaMKII-dependent phosphorylation events mediate reperfusion arrhythmias, Langendorff perfused hearts were submitted to global ischemia/reperfusion. Epicardial monophasic or transmembrane action potentials and contractility were recorded. In rat hearts, reperfusion significantly increased the number of premature beats (PBs) relative to pre-ischemic values. This arrhythmic pattern was associated with a significant increase in CaMKII-dependent phosphorylation of Ser2814 on Ca(2+)-release channels (RyR2) and Thr17 on phospholamban (PLN) at the sarcoplasmic reticulum (SR). These phenomena could be prevented by the CaMKII-inhibitor KN-93. In transgenic mice with targeted inhibition of CaMKII at the SR membranes (SR-AIP), PBs were significantly decreased from 31±6 to 5±1 beats/3min with a virtually complete disappearance of early-afterdepolarizations (EADs). In mice with genetic mutation of the CaMKII phosphorylation site on RyR2 (RyR2-S2814A), PBs decreased by 51.0±14.7%. In contrast, the number of PBs upon reperfusion did not change in transgenic mice with ablation of both PLN phosphorylation sites (PLN-DM). The experiments in SR-AIP mice, in which the CaMKII inhibitor peptide is anchored in the SR membrane but also inhibits CaMKII regulation of L-type Ca(2+) channels, indicated a critical role of CaMKII-dependent phosphorylation of SR proteins and/or L-type Ca(2+) channels in reperfusion arrhythmias. The experiments in RyR2-S2814A further indicate that up to 60% of PBs related to CaMKII are dependent on the phosphorylation of RyR2-Ser2814 site and could be ascribed to delayed-afterdepolarizations (DADs). Moreover, phosphorylation of PLN-Thr17 and L-type Ca(2+) channels might contribute to reperfusion-induced PBs, by increasing SR Ca(2+) content and Ca(2+) influx.


Subject(s)
Arrhythmias, Cardiac/enzymology , Arrhythmias, Cardiac/etiology , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Myocardial Reperfusion Injury/complications , Myocardial Reperfusion Injury/enzymology , Signal Transduction , Action Potentials , Amino Acid Substitution , Animals , Arrhythmias, Cardiac/genetics , Arrhythmias, Cardiac/prevention & control , Benzylamines/pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Type 2/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Heart/drug effects , Heart/physiopathology , Male , Mice , Mice, Transgenic , Mutation , Myocardial Reperfusion Injury/genetics , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Rats , Rats, Wistar , Sarcoplasmic Reticulum/drug effects , Sarcoplasmic Reticulum/metabolism , Sulfonamides/pharmacology
8.
Bull Pan Am Health Organ ; 13(3): 253-6, 1979.
Article in English | MEDLINE | ID: mdl-519078
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