Antoine Bril

Inhibition of the cardiac electrogenic sodium bicarbonate cotransporter reduces ischemic injury

OBJECTIVE Although it is believed that sodium-driven acid-base transport plays a central role in the development of the reperfusion injury that follows cardiac ischemia, research to date has demonstrated only a role for Na(+)/H(+) exchange (NHE). However, Na(+)-driven HCO(-)(3) transport, which is quantitatively as important as NHE in cardiac cells, has not been examined. METHODS AND RESULTS Here the results show that a neutralizing antibody raised against the human heart electrogenic Na(+)/HCO(3)(-) cotransporter (hhNBC) blocked the recovery of pH after acidic pulse both in HEK-293 cells expressing hhNBC and in rat cardiac myocytes demonstrating the presence of an electrogenic NBC in rat cardiac myocytes similar to hhNBC. Administration of anti-NBC antibody to ischemic-reperfused rat hearts markedly protects systolic and diastolic functions of the heart during reperfusion. Furthermore, using a quantitative real-time RT-PCR (TaqMan) and Western blot analysis we demonstrated that in human cardiomyopathic hearts, mRNA and protein levels of hhNBC increase, whereas mRNA levels of the electroneutral Na(+)/HCO(3)(-) cotransporter (NBCn1) remain unchanged. CONCLUSION Our data provide evidence that inhibition of hhNBC, whose role in cardiac pathologies could be amplified by overexpression, represents a novel therapeutic approach for ischemic heart disease.

Protective Effects of Carvedilol in the Myocardium

Beta blockers have long been used in the treatment of systemic hypertension, where they effectively lower blood pressure and, in so doing, they decrease left ventricular hypertrophy. The sympathetic nervous system is activated in patients with congestive heart failure, and therefore it is logical that beta blockers may also provide benefit in these patients. As such, beta blockers are currently being evaluated in several large clinical trials in congestive heart failure. One particular drug, carvedilol, is a third-generation vasodilating beta blocker that is marketed for the treatment of hypertension. The drug lowers systemic arterial blood pressure without producing reflex tachycardia and preserves renal function. Carvedilol decreases mortality by 65% and decreases hospitalization by 29% in patients with congestive heart failure. The effects of carvedilol in heart failure may result, at least in part, from beta blockade as well as vasodilation, the latter resulting from alpha(1)-adrenoceptor blockade. Interestingly, carvedilol has a number of additional properties that may also provide benefit in these patients. Carvedilol and several of its metabolites are potent antioxidants that may inhibit catecholamine toxicity resulting from the oxidation of norepinephrine and the subsequent formation of toxic intermediates, including the generation of reactive oxygen free radicals in the myocardium. As a result of its antioxidant activity, carvedilol also blocks the expression of several genes involved in myocardial damage and cardiac remodeling, and the drug inhibits free radical-induced activation of transcription factors and programmed cell death (apoptosis). Carvedilol is a novel beta blocker that is highly effective in the treatment of hypertension and congestive heart failure, and combines in one molecule a number of important pharmacologic properties.

Implication of microRNAs in the cardiovascular system.

MicroRNAs (miRNAs) are endogenous, small, noncoding RNAs that regulate about 30% of protein-coding genes of the human genome. Thus far, more than 400 miRNAs have been cloned and sequenced in humans. Their biological importance, initially demonstrated in cancer, viral diseases and developmental processes, was more recently investigated in cardiovascular physiology and pathology. MiRNAs expression is tightly controlled in a tissue-specific and developmental stage-specific manner and some of them are highly and specifically expressed in cardiovascular tissues. Through the regulation of the expression of genes involved in cell growth, contractility and electrical conductance, cardiac miRNAs may play a major role in heart development and function. In vascular cells, miRNAs have been linked to vasculoproliferative conditions such as angiogenesis and neointimal lesion formation. Diagnostic use and therapeutic modulation of individual miRNAs or miRNA clusters in cardiovascular diseases will have to be further explored in the future. Molecules specifically regulating cardiovascular miRNAs, either mimicking or antagonizing miRNAs actions, will hopefully normalize dysfunctional gene networks and constitute a new therapy paradigm of cardiovascular diseases.

Combined potassium and calcium channel antagonistic activities as a basis for neutral frequency dependent increase in action potential duration: comparison between BRL-32872 and azimilide.

OBJECTIVE The effects of BRL-32872, azimilide and a selective blocker of the delayed rectifier potassium current, E-4031, were measured at two different basic cycle lengths (BCL), 300 and 1000 ms. Calcium channel antagonists of sarcolemmal (verapamil and nitrendipine) and sarcoplasmic reticulum (ryanodine) membranes were used to investigate whether the inhibition of the calcium current or the calcium release from the sarcoplasmic reticulum could alter the reverse-rate dependence of E-4031 on action potential duration (APD). METHODS Guinea pig isolated papillary muscles were superfused with a Tyrode solution maintained at 37 degrees C and stimulated at a BCL of 300 or 1000 ms. The standard microelectrode technique was used to record action potential parameters and to study the effects of azimilide, BRL-32872 and E-4031. E-4031 was superfused at increasing concentrations (0.01, 0.03, 0.1 and 0.3 microM) in the absence or in the presence of verapamil (0.3 microM), nitrendipine (0.03 microM) or ryanodine (0.1 microM). RESULTS BRL-32872 and azimilide induced a self-limited concentration-dependent increase in APD. The effect of BRL-32872 was not dependent on the stimulation frequency whereas the effect of azimilide was significantly reduced at the shorter BCL. E-4031 induced a concentration-dependent increase in APD at both stimulation BCL. The increase in APD was significantly more pronounced in fibres stimulated at a BCL of 1000 ms than in fibres stimulated at a BCL of 300 ms, characterising the reverse-frequency dependent effect of class III antiarrhythmic agents. The reverse-frequency dependence in action potential prolongation induced by E-4031 was significantly reduced in the presence of a low concentration of verapamil (0.3 microM), nitrendipine (0.03 microM), or ryanodine (0.1 microM. CONCLUSION The results show that BRL-32872, in contrast to azimilide, does not induce the reverse-rate dependency of action potential prolongation typically produced by class III antiarrhythmic agents such as E-4031. Our results also show that reverse-rate dependency induced by E-4031 can be reduced by the simultaneous administration of a low concentration of a calcium channel antagonist or an inhibitor of the release of calcium from the sarcoplasmic reticulum. It is thus suggested that compounds with a suitable balance of potassium and calcium antagonistic activities may have less adverse effects than purely selective potassium channel blockers.

Characterisation of Kv4.3 in HEK293 cells: comparison with the rat ventricular transient outward potassium current

OBJECTIVE The Shal (or Kv4) gene family has been proposed to be responsible for primary subunits of the transient outward potassium current (Ito). More precisely, Kv4.2 and Kv4.3 have been suggested to be the most likely molecular correlates for Ito in rat cells. The purpose of the present study was to compare the properties of the rat Kv4.3 gene product when expressed in a human cell line (HEK293 cells) with that of Ito recorded from rat ventricular cells. METHODS The cDNA encoding the rat Kv4.3 potassium channel was cloned into the pHook2 mammalian expression vector and expressed into HEK293. Patch clamp experiments using the whole cell configuration were used to characterise the electrophysiological parameters of the current induced by Kv4.3 in comparison with the rat ventricular myocyte Ito current. RESULTS The transfection of HEK293 cells with rat Kv4.3 resulted in the expression of a time- and voltage-dependent outward potassium current. The current activated for potentials positive to -40 mV and the steady-state inactivation curve had a midpoint of -47.4 +/- 0.3 mV and a slope of 5.9 +/- 0.2 mV. Rat ventricular Ito current was activated at potentials positive to -20 mV and inactivated with a half-inactivation potential and a Boltzmann factor of -29.1 +/- 0.7 mV and 4.5 +/- 0.5 mV, respectively. The time course of recovery from inactivation of rat Kv4.3 expressed in HEK293 cells and of Ito recorded from native rat ventricular cells were exponentials with time constants of 213.2 +/- 4.1 msec and 23. +/- 1.5 msec, respectively. Pharmacologically, Ito of rat myocytes showed a greater sensitivity to 4-aminopyridine than Kv4.3 since half-maximal effects were obtained with 1.54 +/- 0.13 mM and 0.14 +/- 0.02 mM on Kv4.3 and Ito, respectively. In both Kv4.3 and Ito, 4-aminopyridine appears to bind to the closed state of the channel. Finally, although a higher level of expression was observed in the atria compared to the ventricle, the distribution of the Kv4.3 gene across the ventricles appeared to be homogeneous. CONCLUSION The results of the present study show that Kv4.3 channel may play a major role in the molecular structure of the rat cardiac Ito current. Furthermore, because the distribution of Kv4.3 across the ventricle is homogeneous, the blockade of this channel by specific drugs may not alter the normal heterogeneity of Ito current.

Protective Effect of Angiotensin Converting Enzyme Inhibitors (CEI): Captopril and Perindopril on Vulnerability to Ventricular Fibrillation During Myocardial Ischemia and Reperfusion in Rat

The purpose of the present study was to evaluate the effects of two CEI: captopril and perindopril on reperfusion arrhythmias and noradrenaline 3H (NA3H) release in the isolated rat heart on arrhythmias in pentobarbitone anaesthetized rats subject to left coronary artery ligation. In vitro, in control preparations, reperfusion after 10 min of local ischemia produced by coronary ligation was accompanied by a sudden release of NA3H in coronary flow and long lasting ventricular arrhythmias. Reperfusion arrhythmias were prevented by perfusion medium containing a high dose of CEI and CEI did not change the patterns of NA3H release. In vivo, mortality due to ventricular fibrillation was significantly reduced in rats pretreated with captopril or perindopril 15 min prior to coronary ligation. CEI reduced the severity of ventricular tachycardia (VT) and fibrillation (VF).

Rho exchange factors in the cardiovascular system

Increasing evidence has accumulated to implicate overactivation of Rho protein as a common component for the pathogenesis of several cardiovascular disorders including hypertension, coronary and cerebral vasospasm, atherosclerosis, and diabetes. Recent advances in Rho protein signaling research indicate that the Rho exchange factors (Rho GEFs) which activate Rho proteins by catalyzing the exchange of GDP for GTP are major regulators of Rho protein activity. In addition, linkage analysis and association studies have recently identified Rho GEFs as susceptibility genes for cardiovascular diseases. All of these data are converging to suggest that as upstream activators of Rho proteins, Rho GEFs expressed in cardiovascular cells are good candidate targets for the treatment of cardiovascular disorders.

Neuropeptide AF and FF Modulation of Adipocyte Metabolism PRIMARY INSIGHTS FROM FUNCTIONAL GENOMICS AND EFFECTS ON β-ADRENERGIC RESPONSIVENESS

The presence of a neuropeptide AF and FF receptor (NPFF-R2) mRNA in human adipose tissue (Elshourbagy, N. A., Ames, R. S., Fitzgerald, L. R., Foley, J. J., Chambers, J. K., Szekeres, P. G., Evans, N. A., Schmidt, D. B., Buckley, P. T., Dytko, G. M., Murdock, P. R., Tan, K. B., Shabon, U., Nuthulaganti, P., Wang, D. Y., Wilson, S., Bergsma, D. J., and Sarau, H. M. (2000) J. Biol. Chem. 275, 25965–25971) suggested these peptides, principally recognized for their pain modulating effects, may also impact on adipocyte metabolism, an aspect that has not been explored previously. Our aim was thus to obtain more insights into the actions of these peptides on adipocytes, an approach initially undertaken with a functional genomic assay. First we showed that 3T3-L1 adipocytes express both NPFF-R1 andNPFF-R2 transcripts, and that NPAF binds adipocyte membranes with a nanomolar affinity as assessed by surface plasmon resonance technology. Then, and following a 24-h treatment with NPFF or NPAF (1 μm), we have measured using real-time quantitative reverse transcriptase-PCR the mRNA steady state levels of already well characterized genes involved in key pathways of adipose metabolism. Among the 45 genes tested, few were modulated by NPFF (∼10%) and a larger number by NPAF (∼27%). Interestingly, NPAF increased the mRNA levels of β2- and β3-adrenergic receptors (AR), and to a lesser extent those of β1-ARs. These variations in catecholamine receptor mRNAs correlated with a clear induction in the density of β2- and β3-AR proteins, and in the potency of β-AR subtype-selective agonists to stimulate adenylyl cyclase activity. Altogether, these data show that NPFF-R1 and NPFF-R2 are functionally present in adipocytes and suggest that besides their well described pain modulation effects, NPAF and to a lesser extent NPFF, may have a global impact on body energy storage and utilization.

Biophysical interaction between phospholamban and protein phosphatase 1 regulatory subunit GM

Regulation of the sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA 2a) depends on the phosphorylation state of phospholamban (PLB). When PLB is phosphorylated, its inhibitory effect towards SERCA 2a is relieved, leading to an enhanced myocardial performance. This process is reversed by a sarcoplasmic reticulum (SR)‐associated type 1 protein phosphatase (PP1) composed of a catalytic subunit PP1C and a regulatory subunit GM. Human GM and PLB have been produced in an in vitro transcription/translation system and used for co‐immunoprecipitation and biosensor experiments. The detected interaction between the two partners suggests that cardiac PP1 is targeted to PLB via GM and we believe that this process occurs with the identified transmembrane domains of the two proteins. Thus, the interaction between PLB and GM may represent a specific way to modulate the SR function in human cardiac muscle.

RhoA guanine exchange factor expression profile in arteries: evidence for a Rho kinase-dependent negative feedback in angiotensin II-dependent hypertension

Sustained overactivation of RhoA is a common component for the pathogenesis of several cardiovascular disorders, including hypertension. Although activity of Rho proteins depends on Rho exchange factors (Rho-GEFs), the identity of Rho-GEFs expressed in vascular smooth muscle cells (VSMC) and participating in the control of Rho protein activity and Rho-dependent functions remains unknown. To address this question, we analyzed by quantitative RT-PCR the expression profile of 28 RhoA-GEFs in arteries of normotensive (saline-treated) and hypertensive (ANG II-treated) rats. Sixteen RhoA-GEFs were downregulated in mesenteric arteries of hypertensive rats, among which nine are also downregulated in cultured VSMC stimulated by ANG II (100 nM, 48 h), suggesting a direct effect of ANG II. Inhibition of type 1 ANG II receptors (losartan, 1 μM) or Rho kinase (fasudil, 10 μM) prevented ANG II-induced RhoA-GEF downregulation. Functionally, ANG II-induced downregulation of RhoA-GEFs is associated with decreased Rho kinase activation in response to endothelin-1, norepinephrine, and U-46619. This work thus identifies a group of RhoA-GEFs that controls RhoA and RhoA-dependent functions in VSMC, and a negative feedback of RhoA/Rho kinase activity on the expression of these RhoA-GEFs that may play an adaptative role to limit RhoA/Rho kinase activation.