The ALS-associated KIF5A P986L variant is not pathogenic for Drosophila motoneurons.

Amyotrophic lateral sclerosis (ALS) is a devastating paralytic disorder caused by the death of motoneurons. Several mutations in the KIF5A gene have been identified in patients with ALS. Some mutations affect the splicing sites of exon 27 leading to its deletion (Δ27 mutation). KIF5A Δ27 is aggregation-prone and pathogenic for motoneurons due to a toxic gain of function. Another mutation found to be enriched in ALS patients is a proline/leucine substitution at position 986 (P986L mutation). Bioinformatic analyses strongly suggest that this variant is benign. Our study aims to conduct functional studies in Drosophila to classify the KIF5A P986L variant. When expressed in motoneurons, KIF5A P986L does not modify the morphology of larval NMJ or the synaptic transmission. In addition, KIF5A P986L is uniformly distributed in axons and does not disturb mitochondria distribution. Locomotion at larval and adult stages is not affected by KIF5A P986L. Finally, both KIF5A WT and P986L expression in adult motoneurons extend median lifespan compared to control flies. Altogether, our data show that the KIF5A P986L variant is not pathogenic for motoneurons and may represent a hypomorphic allele, although it is not causative for ALS.

ALS-Associated KIF5A Mutation Causes Locomotor Deficits Associated with Cytoplasmic Inclusions, Alterations of Neuromuscular Junctions, and Motor Neuron Loss.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Recently, genome-wide association studies identified KIF5A as a new ALS-causing gene. KIF5A encodes a protein of the kinesin-1 family, allowing the anterograde transport of cargos along the microtubule rails in neurons. In ALS patients, mutations in the KIF5A gene induce exon 27 skipping, resulting in a mutated protein with a new C-terminal region (KIF5A Δ27). To understand how KIF5A Δ27 underpins the disease, we developed an ALS-associated KIF5A Drosophila model. When selectively expressed in motor neurons, KIF5A Δ27 alters larval locomotion as well as morphology and synaptic transmission at neuromuscular junctions in both males and females. We show that the distribution of mitochondria and synaptic vesicles is profoundly disturbed by KIF5A Δ27 expression. That is consistent with the numerous KIF5A Δ27-containing inclusions observed in motor neuron soma and axons. Moreover, KIF5A Δ27 expression leads to motor neuron death and reduces life expectancy. Our in vivo model reveals that a toxic gain of function underlies the pathogenicity of ALS-linked KIF5A mutant.SIGNIFICANCE STATEMENT Understanding how a mutation identified in patients with amyotrophic lateral sclerosis (ALS) causes the disease and the loss of motor neurons is crucial to fight against this disease. To this end, we have created a Drosophila model based on the motor neuron expression of the KIF5A mutant gene, recently identified in ALS patients. KIF5A encodes a kinesin that allows the anterograde transport of cargos. This model recapitulates the main features of ALS, including alterations of locomotion, synaptic neurotransmission, and morphology at neuromuscular junctions, as well as motor neuron death. KIF5A mutant is found in cytoplasmic inclusions, and its pathogenicity is because of a toxic gain of function.

Dystrophin Deficiency Causes Progressive Depletion of Cardiovascular Progenitor Cells in the Heart.

Duchenne muscular dystrophy (DMD) is a devastating condition shortening the lifespan of young men. DMD patients suffer from age-related dilated cardiomyopathy (DCM) that leads to heart failure. Several molecular mechanisms leading to cardiomyocyte death in DMD have been described. However, the pathological progression of DMD-associated DCM remains unclear. In skeletal muscle, a dramatic decrease in stem cells, so-called satellite cells, has been shown in DMD patients. Whether similar dysfunction occurs with cardiac muscle cardiovascular progenitor cells (CVPCs) in DMD remains to be explored. We hypothesized that the number of CVPCs decreases in the dystrophin-deficient heart with age and disease state, contributing to DCM progression. We used the dystrophin-deficient mouse model (mdx) to investigate age-dependent CVPC properties. Using quantitative PCR, flow cytometry, speckle tracking echocardiography, and immunofluorescence, we revealed that young mdx mice exhibit elevated CVPCs. We observed a rapid age-related CVPC depletion, coinciding with the progressive onset of cardiac dysfunction. Moreover, mdx CVPCs displayed increased DNA damage, suggesting impaired cardiac muscle homeostasis. Overall, our results identify the early recruitment of CVPCs in dystrophic hearts and their fast depletion with ageing. This latter depletion may participate in the fibrosis development and the acceleration onset of the cardiomyopathy.

Identification of CRYAB+ KCNN3+ SOX9+ Astrocyte-Like and EGFR+ PDGFRA+ OLIG1+ Oligodendrocyte-Like Tumoral Cells in Diffuse IDH1-Mutant Gliomas and Implication of NOTCH1 Signalling in Their Genesis.

Diffuse grade II IDH-mutant gliomas are slow-growing brain tumors that progress into high-grade gliomas. They present intratumoral cell heterogeneity, and no reliable markers are available to distinguish the different cell subtypes. The molecular mechanisms underlying the formation of this cell diversity is also ill-defined. Here, we report that SOX9 and OLIG1 transcription factors, which specifically label astrocytes and oligodendrocytes in the normal brain, revealed the presence of two largely nonoverlapping tumoral populations in IDH1-mutant oligodendrogliomas and astrocytomas. Astrocyte-like SOX9+ cells additionally stained for APOE, CRYAB, ID4, KCNN3, while oligodendrocyte-like OLIG1+ cells stained for ASCL1, EGFR, IDH1, PDGFRA, PTPRZ1, SOX4, and SOX8. GPR17, an oligodendrocytic marker, was expressed by both cells. These two subpopulations appear to have distinct BMP, NOTCH1, and MAPK active pathways as stainings for BMP4, HEY1, HEY2, p-SMAD1/5 and p-ERK were higher in SOX9+ cells. We used primary cultures and a new cell line to explore the influence of NOTCH1 activation and BMP treatment on the IDH1-mutant glioma cell phenotype. This revealed that NOTCH1 globally reduced oligodendrocytic markers and IDH1 expression while upregulating APOE, CRYAB, HEY1/2, and an electrophysiologically-active Ca2+-activated apamin-sensitive K+ channel (KCNN3/SK3). This was accompanied by a reduction in proliferation. Similar effects of NOTCH1 activation were observed in nontumoral human oligodendrocytic cells, which additionally induced strong SOX9 expression. BMP treatment reduced OLIG1/2 expression and strongly upregulated CRYAB and NOGGIN, a negative regulator of BMP. The presence of astrocyte-like SOX9+ and oligodendrocyte-like OLIG1+ cells in grade II IDH1-mutant gliomas raises new questions about their role in the pathology.

Hypoxic Conditions Promote Rhythmic Contractile Oscillations Mediated by Voltage-Gated Sodium Channels Activation in Human Arteries.

Arterial smooth muscle exhibits rhythmic oscillatory contractions called vasomotion and believed to be a protective mechanism against tissue hypoperfusion or hypoxia. Oscillations of vascular tone depend on voltage and follow oscillations of the membrane potential. Voltage-gated sodium channels (Nav), responsible for the initiation and propagation of action potentials in excitable cells, have also been evidenced both in animal and human vascular smooth muscle cells (SMCs). For example, they contribute to arterial contraction in rats, but their physiopathological relevance has not been established in human vessels. In the present study, we investigated the functional role of Nav in the human artery. Experiments were performed on human uterine arteries obtained after hysterectomy and on SMCs dissociated from these arteries. In SMCs, we recorded a tetrodotoxin (TTX)-sensitive and fast inactivating voltage-dependent INa current. Various Nav genes, encoding α-subunit isoforms sensitive (Nav 1.2; 1.3; 1.7) and resistant (Nav 1.5) to TTX, were detected both in arterial tissue and in SMCs. Nav channels immunostaining showed uniform distribution in SMCs and endothelial cells. On arterial tissue, we recorded variations of isometric tension, ex vivo, in response to various agonists and antagonists. In arterial rings placed under hypoxic conditions, the depolarizing agent KCl and veratridine, a specific Nav channels agonist, both induced a sustained contraction overlaid with rhythmic oscillations of tension. After suppression of sympathetic control either by blocking the release of catecholamine or by antagonizing the target adrenergic response, rhythmic activity persisted while the sustained contraction was abolished. This rhythmic activity of the arteries was suppressed by TTX but, in contrast, only attenuated by antagonists of calcium channels, Na+/Ca2+ exchanger, Na+/K+-ATPase and the cardiac Nav channel. These results highlight the role of Nav as a novel key element in the vasomotion of human arteries. Hypoxia promotes activation of Nav channels involved in the initiation of rhythmic oscillatory contractile activity.

New role of TRPM4 channel in the cardiac excitation-contraction coupling in response to physiological and pathological hypertrophy in mouse.

The transient receptor potential Melastatin 4 (TRPM4) channel is a calcium-activated non-selective cation channel expressed widely. In the heart, using a knock-out mouse model, the TRPM4 channel has been shown to be involved in multiple processes, including ß-adrenergic regulation, cardiac conduction, action potential duration and hypertrophic adaptations. This channel was recently shown to be involved in stress-induced cardiac arrhythmias in a mouse model overexpressing TRPM4 in ventricular cardiomyocytes. However, the link between TRPM4 channel expression in ventricular cardiomyocytes, the hypertrophic response to stress and/or cellular arrhythmias has yet to be elucidated. In this present study, we induced pathological hypertrophy in response to myocardial infarction using a mouse model of Trpm4 gene invalidation, and demonstrate that TRPM4 is essential for survival. We also demonstrate that the TRPM4 is required to activate both the Akt and Calcineurin pathways. Finally, using two hypertrophy models, either a physiological response to endurance training or a pathological response to myocardial infarction, we show that TRPM4 plays a role in regulating transient calcium amplitudes and leads to the development of cellular arrhythmias potentially in cooperation with the Sodium-calcium exchange (NCX). Here, we report two functions of the TRPM4 channel: first its role in adaptive hypertrophy, and second its association with NCX could mediate transient calcium amplitudes which trigger cellular arrhythmias.

How Degeneration of Cells Surrounding Motoneurons Contributes to Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by the progressive degeneration of upper and lower motoneurons. Despite motoneuron death being recognized as the cardinal event of the disease, the loss of glial cells and interneurons in the brain and spinal cord accompanies and even precedes motoneuron elimination. In this review, we provide striking evidence that the degeneration of astrocytes and oligodendrocytes, in addition to inhibitory and modulatory interneurons, disrupt the functionally coherent environment of motoneurons. We discuss the extent to which the degeneration of glial cells and interneurons also contributes to the decline of the motor system. This pathogenic cellular network therefore represents a novel strategic field of therapeutic investigation.

Mechanisms of artemether toxicity on single cardiomyocytes and protective effect of nanoencapsulation.

BACKGROUND AND PURPOSE: The artemisinin derivative, artemether, has antimalarial activity with potential neurotoxic and cardiotoxic effects. Artemether in nanocapsules (NC-ATM) is more efficient than free artemether for reducing parasitaemia and increasing survival of Plasmodium berghei-infected mice. NCs also prevent prolongation of the QT interval of the ECG. Here, we assessed cellular cardiotoxicity of artemether and how this toxicity was prevented by nanoencapsulation. EXPERIMENTAL APPROACH: Mice were treated with NC-ATM orally (120 mg·kg-1 twice daily) for 4 days. Other mice received free artemether, blank NCs, and vehicle for comparison. We measured single-cell contraction, intracellular Ca2+ transient using fluorescent Indo-1AM Ca2+ dye, and electrical activity using the patch-clamp technique in freshly isolated left ventricular myocytes. The acute effect of free artemether was also tested on cardiomyocytes of untreated animals. KEY RESULTS: Artemether prolonged action potentials (AP) upon acute exposure (at 0.1, 1, and 10 µM) of cardiomyocytes from untreated mice or after in vivo treatment. This prolongation was unrelated to blockade of K+ currents, increased Ca2+ currents or promotion of a sustained Na+ current. AP lengthening was abolished by the NCX inhibitor SEA-0400. Artemether promoted irregular Ca2+ transients during pacing and spontaneous Ca2+ events during resting periods. NC-ATM prevented all effects. Blank NCs had no effects compared with vehicle. CONCLUSION AND IMPLICATIONS: Artemether induced NCX-dependent AP lengthening (explaining QTc prolongation) and disrupted Ca2+ handling, both effects increasing pro-arrhythmogenic risks. NCs prevented these adverse effects, providing a safe alternative to the use of artemether alone, especially to treat malaria.

DMD Pluripotent Stem Cell Derived Cardiac Cells Recapitulate in vitro Human Cardiac Pathophysiology.

Duchenne muscular dystrophy (DMD) is a severe genetic disorder characterized by the lack of functional dystrophin. DMD is associated with progressive dilated cardiomyopathy, eventually leading to heart failure as the main cause of death in DMD patients. Although several molecular mechanisms leading to the DMD cardiomyocyte (DMD-CM) death were described, mostly in mouse model, no suitable human CM model was until recently available together with proper clarification of the DMD-CM phenotype and delay in cardiac symptoms manifestation. We obtained several independent dystrophin-deficient human pluripotent stem cell (hPSC) lines from DMD patients and CRISPR/Cas9-generated DMD gene mutation. We differentiated DMD-hPSC into cardiac cells (CC) creating a human DMD-CC disease model. We observed that mutation-carrying cells were less prone to differentiate into CCs. DMD-CCs demonstrated an enhanced cell death rate in time. Furthermore, ion channel expression was altered in terms of potassium (Kir2.1 overexpression) and calcium handling (dihydropyridine receptor overexpression). DMD-CCs exhibited increased time of calcium transient rising compared to aged-matched control, suggesting mishandling of calcium release. We observed mechanical impairment (hypocontractility), bradycardia, increased heart rate variability, and blunted ß-adrenergic response connected with remodeling of ß-adrenergic receptors expression in DMD-CCs. Overall, these results indicated that our DMD-CC models are functionally affected by dystrophin-deficiency associated and recapitulate functional defects and cardiac wasting observed in the disease. It offers an accurate tool to study human cardiomyopathy progression and test therapies in vitro.

Biocompatible modified water as a non-pharmaceutical approach to prevent metabolic syndrome features in obesogenic diet-fed mice.

The prevalence of metabolic syndrome (MetS), elevating cardiovascular risks, is increasing worldwide, with no available global therapeutic options. The intake of plain, mineral or biocompatible modified waters was shown to prevent some MetS features. This study was designed to analyze, in mice fed a high fat and sucrose diet (HFSD), the effects on MetS features of the daily intake of a reverse osmosed, weakly remineralized, water (OW) and of an OW dynamized by a physical processing (ODW), compared to tap water (TW). The HFSD was effective at inducing major features of MetS such as obesity, hepatic steatosis and inflammation, blood dyslipidemia, systemic glucose intolerance and muscle insulin resistance. Compared to TW, OW intake decreased hepatic fibrosis and inflammation, and mitigated hepatic steatosis and dyslipidemia. ODW intake further improved skeletal muscle insulin sensitivity and systemic glucose tolerance. This study highlights the deleterious metabolic impacts of the daily intake of TW, in combination with a high energy diet, and its possible involvement in MetS prevalence increase. In addition, it demonstrates that biocompatible modified water may be promising non-pharmaceutical, cost-effective tools for nutritional approaches in the treatment of MetS.

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.

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 (2.0 mg/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.

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 Ca(2+) 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 Ca(2+) 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.

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 ß1-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 Ca(2+) cycling by normalising Ca(2+)-handling protein levels (S100A1, SERCA2a, RyR2), and prevented pro-arrhythmogenic Ca(2+) waves derived from abnormal Ca(2+) 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.

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

AIMS: During 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). METHODS AND RESULTS: Healthy 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 ß1-adrenergic blocker metoprolol. CONCLUSION: Elevated 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 ß-blockers to prevent the deleterious effects of chronic BNP exposure may be beneficial in HF.

New insights into sexual dimorphism during progression of heart failure and rhythm disorders.

Neurohormonal imbalance is a key determinant of the progression of heart failure (HF), which results in an elevated risk of mortality. A better understanding of mechanisms involved may influence treatment strategies. The incidence and prevalence of HF are lower in women. We explored sexual dimorphism in the progression of HF using a mice model of neurohormonal-dependent HF. Male and female mice overexpressing the human beta2-adrenergic receptor (TG4 strain) develop HF. We compared TG4 animals with age-matched wild-type controls. Cardiac function was studied in vivo by echocardiography and electrocardiography. Histological studies were performed. Conduction parameters were assessed by intracardiac electrophysiological exploration, as was the occurrence of spontaneous and inducible arrhythmias. The patch-clamp technique was used to determine the cellular electrophysiological profile. The role of hormonal status in HF progression was investigated by surgical gonadectomy. High mortality rate was observed in TG4 mice with a dramatic difference between males and females. Male TG4 mice exhibited intraventricular conduction abnormalities, as measured by infrahisian interval and QRS durations potentially determining reentrant circuits and increasing susceptibility to arrhythmia. The severity of HF was correlated with the degree of fibrosis, which was modulated by the gonadal hormones. Action potentials recorded from male and female left ventricular cardiomyocytes were indistinguishable, although both sexes exhibited delayed repolarization when compared with their wild-type counterparts. In conclusion, female TG4 mice were better protected than males against cardiac remodeling and rhythm disorders. A link between fibrosis, conduction time, and mortality was established in relation with sex hormones.

Akt regulates L-type Ca2+ channel activity by modulating Cavalpha1 protein stability.

The insulin IGF-1-PI3K-Akt signaling pathway has been suggested to improve cardiac inotropism and increase Ca(2+) 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 Ca(2+) channel (LTCC) protein density. The pore-forming channel subunit Ca(v)alpha1 contains highly conserved PEST sequences (signals for rapid protein degradation), and in-frame deletion of these PEST sequences results in increased Ca(v)alpha1 protein levels. Our findings show that Akt-dependent phosphorylation of Ca(v)beta2, the LTCC chaperone for Ca(v)alpha1, antagonizes Ca(v)alpha1 protein degradation by preventing Ca(v)alpha1 PEST sequence recognition, leading to increased LTCC density and the consequent modulation of Ca(2+) channel function. This novel mechanism by which Akt modulates LTCC stability could profoundly influence cardiac myocyte Ca(2+) entry, Ca(2+) handling, and contractility.

PPARalpha-mediated remodeling of repolarizing voltage-gated K+ (Kv) channels in a mouse model of metabolic cardiomyopathy.

Diabetes is associated with increased risk of diastolic dysfunction, heart failure, QT prolongation and rhythm disturbances independent of age, hypertension or coronary artery disease. Although these observations suggest electrical remodeling in the heart with diabetes, the relationship between the metabolic and the functional derangements is poorly understood. Exploiting a mouse model (MHC-PPARalpha) with cardiac-specific overexpression of the peroxisome proliferator-activated receptor alpha (PPARalpha), a key driver of diabetes-related lipid metabolic dysregulation, the experiments here were aimed at examining directly the link(s) between alterations in cardiac fatty acid metabolism and the functioning of repolarizing, voltage-gated K(+) (Kv) channels. Electrophysiological experiments on left (LV) and right (RV) ventricular myocytes isolated from young (5-6 week) MHC-PPARalpha mice revealed marked K(+) current remodeling: I(to,f) densities are significantly (P<0.01) lower, whereas I(ss) densities are significantly (P<0.001) higher in MHC-PPARalpha, compared with age-matched wild type (WT), LV and RV myocytes. Consistent with the observed reductions in I(to,f) density, expression of the KCND2 (Kv4.2) transcript is significantly (P<0.001) lower in MHC-PPARalpha, compared with WT, ventricles. Western blot analyses revealed that expression of the Kv accessory protein, KChIP2, is also reduced in MHC-PPARalpha ventricles in parallel with the decrease in Kv4.2. Although the properties of the endogenous and the "augmented" I(ss) suggest a role(s) for two pore domain K(+) channel (K2P) pore-forming subunits, the expression levels of KCNK2 (TREK1), KCNK3 (TASK1) and KCNK5 (TASK2) in MHC-PPARalpha and WT ventricles are not significantly different. The molecular mechanisms underlying I(to,f) and I(ss) remodeling in MHC-PPARalpha ventricular myocytes, therefore, are distinct.