Franck Aimond

Akt regulates L-type Ca 2+ channel activity by modulating Ca vα1 protein stability

The insulin IGF-1–PI3K–Akt signaling pathway has been suggested to improve cardiac inotropism and increase Ca2+ handling through the effects of the protein kinase Akt. However, the underlying molecular mechanisms remain largely unknown. In this study, we provide evidence for an unanticipated regulatory function of Akt controlling L-type Ca2+ channel (LTCC) protein density. The pore-forming channel subunit Cavα1 contains highly conserved PEST sequences (signals for rapid protein degradation), and in-frame deletion of these PEST sequences results in increased Cavα1 protein levels. Our findings show that Akt-dependent phosphorylation of Cavβ2, the LTCC chaperone for Cavα1, antagonizes Cavα1 protein degradation by preventing Cavα1 PEST sequence recognition, leading to increased LTCC density and the consequent modulation of Ca2+ channel function. This novel mechanism by which Akt modulates LTCC stability could profoundly influence cardiac myocyte Ca2+ entry, Ca2+ handling, and contractility.

Functional evidence for an active role of B-type natriuretic peptide in cardiac remodelling and pro-arrhythmogenicity

AIMSnDuring heart failure (HF), the left ventricle (LV) releases B-type natriuretic peptide (BNP), possibly contributing to adverse cardiovascular events including ventricular arrhythmias (VAs) and LV remodelling. We investigated the cardiac effects of chronic BNP elevation in healthy mice and compared the results with a model of HF after myocardial infarction (PMI mice).nnnMETHODS AND RESULTSnHealthy mice were exposed to circulating BNP levels (BNP-Sham) similar to those measured in PMI mice. Telemetric surface electrocardiograms showed that in contrast with fibrotic PMI mice, electrical conduction was not affected in BNP-Sham mice. VAs were observed in both BNP-Sham and PMI but not in Sham mice. Analysis of heart rate variability indicated that chronic BNP infusion increased cardiac sympathetic tone. At the cellular level, BNP reduced Ca(2+) transients and impaired Ca(2+) reuptake in the sarcoplasmic reticulum, in line with blunted SR Ca(2+) ATPase 2a and S100A1 expression. BNP increased Ca(2+) spark frequency, reflecting Ca(2+) leak through ryanodine receptors, elevated diastolic Ca(2+), and promoted spontaneous Ca(2+) waves. Similar effects were observed in PMI mice. Most of these effects were reduced in BNP-Sham and PMI mice by the selective β1-adrenergic blocker metoprolol.nnnCONCLUSIONnElevated BNP levels, by inducing sympathetic overdrive and altering Ca(2+) handling, promote adverse cardiac remodelling and VAs, which could account in part for the progression of HF after MI. The early use of β-blockers to prevent the deleterious effects of chronic BNP exposure may be beneficial in HF.

Trpm4 Gene Invalidation Leads to Cardiac Hypertrophy and Electrophysiological Alterations

Rationale TRPM4 is a non-selective Ca2+-activated cation channel expressed in the heart, particularly in the atria or conduction tissue. Mutations in the Trpm4 gene were recently associated with several human conduction disorders such as Brugada syndrome. TRPM4 channel has also been implicated at the ventricular level, in inotropism or in arrhythmia genesis due to stresses such as ß-adrenergic stimulation, ischemia-reperfusion, and hypoxia re-oxygenation. However, the physiological role of the TRPM4 channel in the healthy heart remains unclear. Objectives We aimed to investigate the role of the TRPM4 channel on whole cardiac function with a Trpm4 gene knock-out mouse (Trpm4 -/-) model. Methods and Results Morpho-functional analysis revealed left ventricular (LV) eccentric hypertrophy in Trpm4 -/- mice, with an increase in both wall thickness and chamber size in the adult mouse (aged 32 weeks) when compared to Trpm4+/+ littermate controls. Immunofluorescence on frozen heart cryosections and qPCR analysis showed no fibrosis or cellular hypertrophy. Instead, cardiomyocytes in Trpm4-/- mice were smaller than Trpm4+/+with a higher density. Immunofluorescent labeling for phospho-histone H3, a mitosis marker, showed that the number of mitotic myocytes was increased 3-fold in the Trpm4-/-neonatal stage, suggesting hyperplasia. Adult Trpm4 -/- mice presented multilevel conduction blocks, as attested by PR and QRS lengthening in surface ECGs and confirmed by intracardiac exploration. Trpm4-/-mice also exhibited Luciani-Wenckebach atrioventricular blocks, which were reduced following atropine infusion, suggesting paroxysmal parasympathetic overdrive. In addition, Trpm4 -/- mice exhibited shorter action potentials in atrial cells. This shortening was unrelated to modifications of the voltage-gated Ca2+ or K+ currents involved in the repolarizing phase. Conclusions TRPM4 has pleiotropic roles in the heart, including the regulation of conduction and cellular electrical activity which impact heart development.

β-Adrenergic blockade combined with subcutaneous B-type natriuretic peptide: a promising approach to reduce ventricular arrhythmia in heart failure?

Aims Clinical studies failed to prove convincingly efficiency of intravenous infusion of neseritide during heart failure and evidence suggested a pro-adrenergic action of B-type natriuretic peptide (BNP). However, subcutaneous BNP therapy was recently proposed in heart failure, thus raising new perspectives over what was considered as a promising treatment. We tested the efficiency of a combination of oral β1-adrenergic receptor blocker metoprolol and subcutaneous BNP infusion in decompensated heart failure. Methods and results The effects of metoprolol or/and BNP were studied on cardiac remodelling, excitation–contraction coupling and arrhythmias in an experimental mouse model of ischaemic heart failure following postmyocardial infarction. We determined the cellular and molecular mechanisms involved in anti-remodelling and antiarrhythmic actions. As major findings, the combination was more effective than metoprolol alone in reversing cardiac remodelling and preventing ventricular arrhythmia. The association of the two molecules improved cardiac function, reduced hypertrophy and fibrosis, and corrected the heart rate, sympatho-vagal balance (low frequencies/high frequencies) and ECG parameters (P to R wave interval (PR), QRS duration, QTc intervals). It also improved altered Ca2+ cycling by normalising Ca2+-handling protein levels (S100A1, SERCA2a, RyR2), and prevented pro-arrhythmogenic Ca2+ waves derived from abnormal Ca2+ sparks in ventricular cardiomyocytes. Altogether these effects accounted for decreased occurrence of ventricular arrhythmias. Conclusions Association of subcutaneous BNP and oral metoprolol appeared to be more effective than metoprolol alone. Breaking the deleterious loop linking BNP and sympathetic overdrive in heart failure could unmask the efficiency of BNP against deleterious damages in heart failure and bring a new potential approach against lethal arrhythmia during heart failure.

ACE inhibition prevents diastolic Ca2+ overload and loss of myofilament Ca2+ sensitivity after myocardial infarction.

Prevention of adverse cardiac remodeling after myocardial infarction (MI) remains a therapeutic challenge. Angiotensin-converting enzyme inhibitors (ACE-I) are a well-established first-line treatment. ACE-I delay fibrosis, but little is known about their molecular effects on cardiomyocytes. We investigated the effects of the ACE-I delapril on cardiomyocytes in a mouse model of heart failure (HF) after MI. Mice were randomly assigned to three groups: Sham, MI, and MI-D (6 weeks of treatment with a non-hypotensive dose of delapril started 24h after MI). Echocardiography and pressure-volume loops revealed that MI induced hypertrophy and dilation, and altered both contraction and relaxation of the left ventricle. At the cellular level, MI cardiomyocytes exhibited reduced contraction, slowed relaxation, increased diastolic Ca2+ levels, decreased Ca2+-transient amplitude, and diminished Ca2+ sensitivity of myofilaments. In MI-D mice, however, both mortality and cardiac remodeling were decreased when compared to non-treated MI mice. Delapril maintained cardiomyocyte contraction and relaxation, prevented diastolic Ca2+ overload and retained the normal Ca2+ sensitivity of contractile proteins. Delapril maintained SERCA2a activity through normalization of P-PLB/PLB (for both Ser16- PLB and Thr17-PLB) and PLB/SERCA2a ratios in cardiomyocytes, favoring normal reuptake of Ca2+ in the sarcoplasmic reticulum. In addition, delapril prevented defective cTnI function by normalizing the expression of PKC, enhanced in MI mice. In conclusion, early therapy with delapril after MI preserved the normal contraction/relaxation cycle of surviving cardiomyocytes with multiple direct effects on key intracellular mechanisms contributing to preserve cardiac function.

Biodegradable Polymeric Nanocapsules Prevent Cardiotoxicity of Anti-Trypanosomal Lychnopholide

Chagas disease is a neglected parasitic disease caused by the protozoan Trypanosoma cruzi. New antitrypanosomal options are desirable to prevent complications, including a high rate of cardiomyopathy. Recently, a natural substance, lychnopholide, has shown therapeutic potential, especially when encapsulated in biodegradable polymeric nanocapsules. However, little is known regarding possible adverse effects of lychnopholide. Here we show that repeated-dose intravenous administration of free lychnopholide (2.0u2009mg/kg/day) for 20 days caused cardiopathy and mortality in healthy C57BL/6 mice. Echocardiography revealed concentric left ventricular hypertrophy with preserved ejection fraction, diastolic dysfunction and chamber dilatation at end-stage. Single cardiomyocytes presented altered contractility and Ca2+ handling, with spontaneous Ca2+ waves in diastole. Acute in vitro lychnopholide application on cardiomyocytes from healthy mice also induced Ca2+ handling alterations with abnormal RyR2-mediated diastolic Ca2+ release. Strikingly, the encapsulation of lychnopholide prevented the cardiac alterations induced in vivo by the free form repeated doses. Nanocapsules alone had no adverse cardiac effects. Altogether, our data establish lychnopholide presented in nanocapsule form more firmly as a promising new drug candidate to cure Chagas disease with minimal cardiotoxicity. Our study also highlights the potential of nanotechnology not only to improve the efficacy of a drug but also to protect against its adverse effects.

ACE Inhibitor Delapril Prevents Ca(2+)-Dependent Blunting of IK1 and Ventricular Arrhythmia in Ischemic Heart Disease.

Angiotensin-converting enzyme inhibitors (ACE-I) improve clinical outcome in patients with myocardial infarction (MI) and chronic heart failure. We investigated potential anti-arrhythmic (AA) benefits in a mouse model of ischemic HF. We hypothesized that normalization of diastolic calcium (Ca(2+)) by ACE-I may prevent Ca(2+)-dependent reduction of inward rectifying K(+) current (IK1) and occurrence of arrhythmias after MI. Mice were randomly assigned to three groups: Sham, MI, and MI-D (6 weeks of treatment with ACE-I delapril started 24h after MI). Electrophysiological analyses showed that delapril attenuates MI-induced prolongations of electrocardiogram parameters (QRS complex, QT, QTc intervals) and conduction time from His bundle to ventricular activation. Delapril improved the sympatho-vagal balance (LF/HF) and reduced atrio-ventricular blocks and ventricular arrhythmia. Investigations in cardiomyocytes showed that delapril prevented the decrease of IK1 measured by patch-clamp technique. IK1 reduction was related to intracellular Ca(2+) overload. This reduction was not observed when intracellular free-Ca(2+) was maintained low. Conversely, increasing intracellular free-Ca(2+) in Sham following application of SERCA2a inhibitor thapsigargin reduced IK1. Thapsigargin had no effect in MI animals and abolished the benefits of delapril on IK1 in MI-D mice. Delapril prevented both the prolongation of action potential late repolarization and the depolarization of resting membrane potential, two phenomena known to trigger abnormal electrical activities, promoted by MI. In conclusion, early chronic therapy with delapril after MI prevented Ca(2+)-dependent reduction of IK1. This mechanism may significantly contribute to the antiarrhythmic benefits of ACE-I in patients at risk for sudden cardiac death.

The TRPM4 channel is functionally important for the beneficial cardiac remodeling induced by endurance training

Cardiac hypertrophy (CH) is an adaptive process that exists in two distinct forms and allows the heart to adequately respond to an organism’s needs. The first form of CH is physiological, adaptive and reversible. The second is pathological, irreversible and associated with fibrosis and cardiomyocyte death. CH involves multiple molecular mechanisms that are still not completely defined but it is now accepted that physiological CH is associated more with the PI3-K/Akt pathway while the main signaling cascade activated in pathological CH involves the Calcineurin-NFAT pathway. It was recently demonstrated that the TRPM4 channel may act as a negative regulator of pathological CH by regulating calcium entry and thus the Cn-NFAT pathway. In this study, we examined if the TRPM4 channel is involved in the physiological CH process. We evaluated the effects of 4 weeks endurance training on the hearts of Trpm4+/+ and Trpm4−/− mice. We identified an elevated functional expression of the TRPM4 channel in cardiomyocytes after endurance training suggesting a potential role for the channel in physiological CH. We then observed that Trpm4+/+ mice displayed left ventricular hypertrophy after endurance training associated with enhanced cardiac function. By contrast, Trpm4−/− mice did not develop these adaptions. While Trpm4−/− mice did not develop gross cardiac hypertrophy, the cardiomyocyte surface area was larger and associated with an increase of Tunel positive cells. Endurance training in Trpm4+/+ mice did not increase DNA fragmentation in the heart. Endurance training in Trpm4+/+ mice was associated with activation of the classical physiological CH Akt pathway while Trpm4−/− favored the Calcineurin pathway. Calcium studies demonstrated that TRPM4 channel negatively regulates calcium entry providing support for activation of the Cn-NFAT pathway in Trpm4−/− mice. In conclusion, we provide evidence for the functional expression of TRPM4 channel in response to endurance training. This expression may help to maintain the balance between physiological and pathological hypertrophy.

Antagonism of Nav channels and α1-adrenergic receptors contributes to vascular smooth muscle effects of ranolazine

Ranolazine is a recently developed drug used for the treatment of patients with chronic stable angina. It is a selective inhibitor of the persistent cardiac Na+ current (INa), and is known to reduce the Na+-dependent Ca2+ overload that occurs in cardiomyocytes during ischemia. Vascular effects of ranolazine, such as vasorelaxation,have been reported and may involve multiple pathways. As voltage-gated Na+ channels (Nav) present in arteries play a role in contraction, we hypothesized that ranolazine could target these channels. We studied the effects of ranolazine in vitro on cultured aortic smooth muscle cells (SMC) and ex vivo on rat aortas in conditions known to specifically activate or promote INa. We observed that in the presence of the Nav channel agonist veratridine, ranolazine inhibited INa and intracellular Ca2+ calcium increase in SMC, and arterial vasoconstriction. In arterial SMC, ranolazine inhibited the activity of tetrodotoxin-sensitive voltage-gated Nav channels and thus antagonized contraction promoted by low KCl depolarization. Furthermore, the vasorelaxant effects of ranolazine, also observed in human arteries and independent of the endothelium, involved antagonization of the α1-adrenergic receptor. Combined α1-adrenergic antagonization and inhibition of SMCs Nav channels could be involved in the vascular effects of ranolazine.

Early calcium handling imbalance in pressure overload-induced heart failure with nearly normal left ventricular ejection fraction

Heart failure with preserved ejection fraction (HFpEF) is a common clinical syndrome associated with high morbidity and mortality. Therapeutic options are limited due to a lack of knowledge of the pathology and its evolution. We investigated the cellular phenotype and Ca2+ handling in hearts recapitulating HFpEF criteria. HFpEF was induced in a portion of male Wistar rats four weeks after abdominal aortic banding. These animals had nearly normal ejection fraction and presented elevated blood pressure, lung congestion, concentric hypertrophy, increased LV mass, wall stiffness, impaired active relaxation and passive filling of the left ventricle, enlarged left atrium, and cardiomyocyte hypertrophy. Left ventricular cell contraction was stronger and the Ca2+ transient larger. Ca2+ cycling was modified with a RyR2 mediated Ca2+ leak from the sarcoplasmic reticulum and impaired Ca2+ extrusion through the Sodium/Calcium exchanger (NCX), which promoted an increase in diastolic Ca2+. The Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2a) and NCX protein levels were unchanged. The phospholamban (PLN) to SERCA2a ratio was augmented in favor of an inhibitory effect on the SERCA2a activity. Conversely, PLN phosphorylation at the calmodulin-dependent kinase II (CaMKII)-specific site (PLN-Thr17), which promotes SERCA2A activity, was increased as well, suggesting an adaptive compensation of Ca2+ cycling. Altogether our findings show that cardiac remodeling in hearts with a HFpEF status differs from that known for heart failure with reduced ejection fraction. These data also underscore the interdependence between systolic and diastolic adaptations of Ca2+ cycling with complex compensative interactions between Ca2+ handling partner and regulatory proteins.