Jean-Pierre Benitah

Cardiomyocyte overexpression of neuronal nitric oxide synthase delays transition toward heart failure in response to pressure overload by preserving calcium cycling.

Background— Defects in cardiomyocyte Ca2+ cycling are a signature feature of heart failure (HF) that occurs in response to sustained hemodynamic overload, and they largely account for contractile dysfunction. Neuronal nitric oxide synthase (NOS1) influences myocyte excitation-contraction coupling through modulation of Ca2+ cycling, but the potential relevance of this in HF is unknown. Methods and Results— We generated a transgenic mouse with conditional, cardiomyocyte-specific NOS1 overexpression (double-transgenic [DT]) and studied cardiac remodeling, myocardial Ca2+ handling, and contractility in DT and control mice subjected to transverse aortic constriction (TAC). After TAC, control mice developed eccentric hypertrophy with evolution toward HF as revealed by a significantly reduced fractional shortening. In contrast, DT mice developed a greater increase in wall thickness (P<0.0001 versus control+TAC) and less left ventricular dilatation than control+TAC mice (P<0.0001 for both end-systolic and end-diastolic dimensions). Thus, DT mice displayed concentric hypertrophy with fully preserved fractional shortening (43.7±0.6% versus 30.3±2.6% in control+TAC mice, P<0.05). Isolated cardiomyocytes from DT+TAC mice had greater shortening, intracellular Ca2+ transients, and sarcoplasmic reticulum Ca2+ load (P<0.05 versus control+TAC for all parameters). These effects could be explained, at least in part, through modulation of phospholamban phosphorylation status. Conclusions— Cardiomyocyte NOS1 may be a useful target against cardiac deterioration during chronic pressure-overload–induced HF through modulation of calcium cycling.

Conditional FKBP12.6 Overexpression in Mouse Cardiac Myocytes Prevents Triggered Ventricular Tachycardia Through Specific Alterations in Excitation- Contraction Coupling

Background— Ca2+ release from the sarcoplasmic reticulum via the ryanodine receptor (RyR2) activates cardiac myocyte contraction. An important regulator of RyR2 function is FKBP12.6, which stabilizes RyR2 in the closed state during diastole. &bgr;-Adrenergic stimulation has been suggested to dissociate FKBP12.6 from RyR2, leading to diastolic sarcoplasmic reticulum Ca2+ leakage and ventricular tachycardia (VT). We tested the hypothesis that FKBP12.6 overexpression in cardiac myocytes can reduce susceptibility to VT in stress conditions. Methods and Results— We developed a mouse model with conditional cardiac-specific overexpression of FKBP12.6. Transgenic mouse hearts showed a marked increase in FKBP12.6 binding to RyR2 compared with controls both at baseline and on isoproterenol stimulation (0.2 mg/kg IP). After pretreatment with isoproterenol, burst pacing induced VT in 10 of 23 control mice but in only 1 of 14 transgenic mice (P<0.05). In isolated transgenic myocytes, Ca2+ spark frequency was reduced by 50% (P<0.01), a reduction that persisted under isoproterenol stimulation, whereas the sarcoplasmic reticulum Ca2+ load remained unchanged. In parallel, peak ICa,L density decreased by 15% (P<0.01), and the Ca2+ transient peak amplitude decreased by 30% (P<0.001). A 33.5% prolongation of the caffeine-evoked Ca2+ transient decay was associated with an 18% reduction in the Na+-Ca2+ exchanger protein level (P<0.05). Conclusions— Increased FKBP12.6 binding to RyR2 prevents triggered VT in normal hearts in stress conditions, probably by reducing diastolic sarcoplasmic reticulum Ca2+ leak. This indicates that the FKBP12.6-RyR2 complex is an important candidate target for pharmacological prevention of VT.

Calcium signaling in diabetic cardiomyocytes.

Diabetes mellitus is one of the most common medical conditions. It is associated to medical complications in numerous organs and tissues, of which the heart is one of the most important and most prevalent organs affected by this disease. In fact, cardiovascular complications are the most common cause of death among diabetic patients. At the end of the 19th century, the weakness of the heart in diabetes was noted as part of the general muscular weakness that exists in that disease. However, it was only in the eighties that diabetic cardiomyopathy was recognized, which comprises structural and functional abnormalities in the myocardium in diabetic patients even in the absence of coronary artery disease or hypertension. This disorder has been associated with both type 1 and type 2 diabetes, and is characterized by early-onset diastolic dysfunction and late-onset systolic dysfunction, in which alteration in Ca(2+) signaling is of major importance, since it controls not only contraction, but also excitability (and therefore is involved in rhythmic disorder), enzymatic activity, and gene transcription. Here we attempt to give a brief overview of Ca(2+) fluxes alteration reported on diabetes, and provide some new data on differential modulation of Ca(2+) handling alteration in males and females type 2 diabetic mice to promote further research. Due to space limitations, we apologize for those authors whose important work is not cited.

Ca2+ Fluxes Involvement in Gene Expression During Cardiac Hypertrophy

Cardiac hypertrophy arises as a response of the heart to many different pathological stimuli that challenge its work. Regardless of the initial pathologic cause, cardiac hypertrophy shares some characteristics resulting from a genetic reprogramming of several proteins. Recent studies point to Ca2+ as a key signaling element in the initiation of this genetic reprogramming. In fact, besides its important role in excitation-contraction coupling, Ca2+ regulates cardiac growth by activation of Ca2+-dependent transcription factors. This mechanism has been termed excitation-transcription (ET) coupling. Some information about cardiac ET coupling is being gathered from the analysis of cardiac hypertrophy development, where two Ca2+ dependent enzymes are key actors: the Ca2+/calmodulin kinase II (CaMKII) and the phosphatase calcineurin, both activated by Ca2+/Calmodulin. In this review we focus on some neurohormonal signaling pathways involved in cardiac hypertrophy, which could be ascribed as activators of ET coupling, for instance, adrenergic stimulation and the renin-angiotensin-aldosterone system. β-adrenergic receptor (β-AR) produces cAMP, which directly, (through cAMP response element) or indirectly (through activating Epac) induces cardiac hypertrophy. α1 AR and angiotensin receptor type 1 are Gq protein coupled receptors, which when activated, stimulate phospholipase C producing inositol 1,4,5 triphosphate (IP3) and diacylglycerol (DAG). IP3 promotes elevation of [Ca2+] in the nucleus, activating CaMKII/MEF2 (myocyte enhancer factor 2) pathway and may indirectly induce Ca2+ entry through transient receptor potential channels (TRPC). Other TRPC channels are activated by DAG. Ca2+ entry activates calcineurin/NFAT hypertrophic signaling. By promoting L-type Ca2+ channel expression, aldosterone may also have an important role in the genetic reprogramming during hypertrophy.

The other side of cardiac Ca2+ signaling: transcriptional control

Ca2+ is probably the most versatile signal transduction element used by all cell types. In the heart, it is essential to activate cellular contraction in each heartbeat. Nevertheless Ca2+ is not only a key element in excitation-contraction coupling (EC coupling), but it is also a pivotal second messenger in cardiac signal transduction, being able to control processes such as excitability, metabolism, and transcriptional regulation. Regarding the latter, Ca2+ activates Ca2+-dependent transcription factors by a process called excitation-transcription coupling (ET coupling). ET coupling is an integrated process by which the common signaling pathways that regulate EC coupling activate transcription factors. Although ET coupling has been extensively studied in neurons and other cell types, less is known in cardiac muscle. Some hints have been found in studies on the development of cardiac hypertrophy, where two Ca2+-dependent enzymes are key actors: Ca2+/Calmodulin kinase II (CaMKII) and phosphatase calcineurin, both of which are activated by the complex Ca2+/Calmodulin. The question now is how ET coupling occurs in cardiomyocytes, where intracellular Ca2+ is continuously oscillating. In this focused review, we will draw attention to location of Ca2+ signaling: intranuclear ([Ca2+]n) or cytoplasmic ([Ca2+]c), and the specific ionic channels involved in the activation of cardiac ET coupling. Specifically, we will highlight the role of the 1,4,5 inositol triphosphate receptors (IP3Rs) in the elevation of [Ca2+]n levels, which are important to locally activate CaMKII, and the role of transient receptor potential channels canonical (TRPCs) in [Ca2+]c, needed to activate calcineurin (Cn).

Ryanodine Receptor Channelopathies: The New Kid in the Arrhythmia Neighborhood

Some of these cardiac diseases are acquired as cardiac hypertrophy, which develops as an adaptation of the heart to diseases that challenge the heart work chronically. Cardiac hypertrophy often degenerates in heart failure (HF), the final outcome of most cardiovascular diseases. Chronic HF prevalence is increasing in western countries, with only 25% of men and 38% of women surviving 5 years after the onset of clinical signs. Quality of life is hampered by the reduced pump function, which can also lead to death. However, half of deceases in HF patients are sudden due to cardiac arrhythmia. During cardiac pathology, altered activity of the cardiac, type 2, ryanodine receptor (RyR2) may generate arrhythmia and sudden death. This risk is high in HF where there is a profound remodeling of Ca2+ cycling, and alterations in transmembrane Ca2+ influx, Ca2+ release or/and sarcoplasmic reticulum (SR) Ca2+-load underlie systolic dysfunction (Gomez et al., 1997; Benitah JP, 2002). Thus, when dealing with HF and poor cardiac outcomes, it is a need to better understand the mechanisms of cardiac arrhythmia in order to efficiently treat these patients. However, a large number of inherited arrhythmogenic syndromes that cause sudden death have been characterised. Some are associated with structural heart disease, such as familial hypertrophic cardiomyopathy and arrythmogenic right ventricular cardiomyopathy type 2 (ARVD2). Others do not produce structural heart disease and so are difficult to detect. Most of these cardiomyopathies are due to mutations in plasmalemmal cardiac ion channels, mainly the Na+ channel and several K+ channels (Lehnart et al., 2007). These mutations promote arrhythmogenesis by altering the action potential (AP) duration, which therefore may enhance the propensity of arrhythmic activity via the development of early after depolarizations (EADs). However, the recent finding of mutations in the Ca2+ release channel (RyR2) associated with catecholaminergic polymorphic ventricular tachycardia

RyR2R420Q catecholaminergic polymorphic ventricular tachycardia mutation induces bradycardia by disturbing the coupled clock pacemaker mechanism

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a lethal genetic arrhythmia that manifests syncope or sudden death in children and young adults under stress conditions. CPVT patients often present bradycardia and sino-atrial node (SAN) dysfunction. However, the mechanism remains unclear. We analyzed SAN function in two CPVT families and in a novel knock-in (KI) mouse model carrying the RyR2R420Q mutation. Humans and KI mice presented slower resting heart rate. Accordingly, the rate of spontaneous intracellular Ca2+ ([Ca2+]i) transients was slower in KI mouse SAN preparations than in WT, without any significant alteration in the funny current (If ). The L-type Ca2+ current was reduced in KI SAN cells in a [Ca2+]i-dependent way, suggesting that bradycardia was due to disrupted crosstalk between the voltage and Ca2+ clock, and the mechanisms of pacemaking was induced by aberrant spontaneous RyR2- dependent Ca2+ release. This finding was consistent with a higher Ca2+ leak during diastolic periods produced by long-lasting Ca2+ sparks in KI SAN cells. Our results uncover a mechanism for the CPVT-causing RyR2 N-terminal mutation R420Q, and they highlight the fact that enhancing the Ca2+ clock may slow the heart rhythm by disturbing the coupling between Ca2+ and voltage clocks.

0015 : Epac signalling in doxorubicin-induced cardiotoxicity: a novel implication in death pathways

The main mechanisms underlying doxorubicin (Dox)-induced cardiotoxicity involve classically reactive oxygen species generation, DNA intercalation and topoisomerase II (TopII) inhibition which lead ultimately to cardiomyocyte death. However, new signalling pathways are now emerging. β-adrenergic signalling with Epac (exchange protein directly activated by cAMP) implication could be worth investigating as Epac activates small G proteins (Rac1, Rho) known to be involved in dox-induced cardiotoxicity. Therefore, we have investigated the Epac role in Dox cardiotoxicity in both in vivo (C57Bl63/ Knock-out Epac1 mice, iv injections, 12mg/kg cumulative dose) and in vitro model (primary culture of neonatal rat cardiomyocytes (NRVM), 24h, Dox 1μM). In vivo, long-term follow up showed the development of dilated cardiomyopathy (DCM) 15 weeks post treatment associated with Ca2+ homeostasis dysfunction in Dox-treated mice. We also observed a time-dependent modulation of Epac1, Epac2, Rho A and Rac1 expression with alterated, compensated and final decompensated phase at, 2, 6 and 15 weeks respectively. In vitro, Dox treatments led to a global alteration of Epac signalling: Dox treatment led to a reduced Epac 1 / 2 expressions while Epac1 activity is increased. Dox also decreased Rap1 and Rac1 expression and activities. Moreover, the inhibition of Epac1 (ESI09, CE3F4, shEpac1), but not Epac2, prevented Doxinduced DNA-TopIIβ cleavable complex/DNA damage in part by TopIIβ downregulation. We also reported that Dox induces cell death in cardiomyocytes through activation of the mitochondrial caspase dependent apoptotic pathway which as again prevented by specific EPAC1 inhibition. These results were confirmed in vivo as Dox-induced cardiotoxicity was prevented in Epac1 KO as evidenced by lack of DCM, alteration of cardiac function and calcium homeostasis (15 weeks). In conclusion, Epac could be a valuable therapeutic target to prevent Anthacyclines-induced cardiomyopathy. The author hereby declares no conflict of interest

L-Type Ca2+ Current in Cardiac Arrhythmias

Cardiac arrhythmias result from the confluence of structural and functional changes in the heart and genetic predisposition, reflecting an interaction between a susceptible substrate (e.g. an anatomically defined circuit, a myocardial scar, fibrosis or a monogenic arrhythmia syndrome) and a specific electrophysiological triggering event. Such triggered activities arises from delayed afterdepolarizations (DADs) or early afterdepolarizations (EADs), in which action potential prolongation and aberrant Ca2+ fluxes are a recurrent theme. Ca2+ channels in cardiomyocytes provide the main influx pathway for Ca2+. Three types of high threshold Ca2+ channels are expressed in heart: two L-type channels, Cav1.2 and Cav1.3 and a P-type channel, Cav2.1. The Cav2.1 channel protein is expressed at a very low level the in heart (Starr et al., 1991) while Cav1.3 is mainly expressed in fetal hearts and only in adult sinoatrial and atrioventricular nodes and atrial tissues of adult (Lipscombe et al., 2004; Qu et al., 2005). We will focus attention on the Cav1.2 L-type Ca2+ channel (LTCC) which is the main player in electrical activity and excitation-contraction coupling (EC coupling) in the ventricular cardiomyocyte. The LTCC of cardiomyocytes is a complex multimeric molecular sarcolemmal ensemble that during an action potential (AP) allows Ca2+ to flow down its electrochemical gradient into the cardiac cell. LTCCs are mostly localized in the transverse tubular system of cardiomyocytes (Wibo et al., 1991; Kawai et al., 1999; Brette et al., 2004). Activation of LTCC generates a Ca2+ current (ICaL) through the sarcolemma large enough to be involved in AP overshoot and in the control of AP duration (APD) in different cardiac cells types (Bers, 2001). ICaL serves as a trigger for Ca2+ release from the sarcoplasmic reticulum (SR) during the excitation-contraction coupling by a mechanism known as calcium-induced calcium release (CICR, Fabiato & Fabiato, 1975; Bers, 2001). LTCC activation can also play a role in transcription mechanisms in cardiomyocytes (Atar et al., 1995; Brette et al., 2006). Several hormones and neuromediators modulate the activity of LTCC via complex intracellular signaling pathways and, as well, several intracellular molecules and the cytoskeleton can influence LTCC activity (Benitah et al., 2010). However, intracellular Ca2+ concentration is strictly controlled in normal cells by different mechanisms (Bers, 2001) since a Ca2+ overload can have deleterious effects including arrhythmias and myocardial remodeling via a genetic reprogramming of the cardiac cell (Benitah et al., 2003).

Transcriptional Up-Regulation by Aldosterone of the Cardiac Cav1.2 Encoding Gene CACNA1C

Although the effect of aldosterone beyond the epithelia is now well recognized, specific action through the mineralocorticoid receptor (MR) in heart is still matter of debate. In the past decade, we have accumulated evidence, both ex vivo and in vivo, that modulation of Ca2+ signaling, especially Ca2+ influx via L-type Ca2+ channel (Cav1.2), is a central factor in the cardiac action of aldosterone. Even if our results established that a specific transcriptional upregulation is involved in the effect of aldosterone on Cav1.2, it cannot be concluded whether this genomic effect is direct or indirect. The effect of the mineralocorticoid, aldosterone, on the regulation of the cardiac Cav1.2 expression was investigated in primary cultures of neonatal rat ventricular myocytes (nRVM). By use of quantitative real time RT-PCR, we found that aldosterone increased the Cav1.2 mRNA amount in time- and dose-dependent manner. This up-regulation appeared as early as 1hour of incubation even with 1 nM aldosterone. Inspection of the upstream promoter region from rat Cav1.2 encoding gene, revealed one putative hormone response element, GRE. The activity of the promoter-luciferase reporter gene constructs in transfected nRVMs showed similar time- and dose-dependent stimulation with aldosterone. We will examine the intracellular trafficking of MR in nRVMs using a fusion protein of green fluorescent protein and human MR. Selectivity of these effects will be tested as well as electrophoretic mobility shift assays to identify the region of the cacna1c promoter that can bind MRs. The data will provide new insights in the molecular mechanisms underlying the regulation of Cav1.2 channel expression by mineralocorticoids.