Other articles by Elisabetta Cerbai on PubMed

• Does Recombinant Human Interleukin-11 Exert Direct Electrophysiologic Effects on Single Human Atrial Myocytes? Journal of Cardiovascular Pharmacology. Mar, 2002  |   Pubmed ID: 11862122 Recombinant human interleukin-11 (rhIL-11) treatment given to alleviate side effects of cancer therapy is associated with an increased susceptibility to atrial arrhythmias in elderly patients. To elucidate the mechanism underlying this action, we investigated the direct electrophysiologic effect of rhIL-11 on single human atrial myocytes (HuAM) using the patch-clamp technique. Action potentials (AP) at different driving rates were recorded in the perforated-patch configuration, and L-type calcium current (I(Ca,L)), outward potassium currents (I(to) and I(K)), and the hyperpolarization-activated pacemaker current If were measured in the disrupted whole-cell configuration. At therapeutic concentrations (i.e., 10-100 ng/ml), rhIL-11 did not modify AP parameters and cycle-length dependence of AP duration. I(Ca,L) (measured at 0 mV) was 370 +/- 45 pA in control and 379 +/- 48 pA and 368 +/- 42 pA in the presence of 10 and 50 ng/ml rhIL-11, respectively (p = NS). The amplitude and activation of I(to) were not modified by rhIL-11 (i.e., I(to) was at +60 mV: 2.1 +/- 0.2 nA in control vs. 1.9 +/- 0.2 nA and 2.1 +/- 0.2 nA in the presence of 10 and 50 ng/ml rhIL-11, respectively, p = NS). Similarly, late currents measured at the end of the pulse were unchanged in the presence of 10 or 50 ng/ml of rhIL-11. If activation was not modified by rhIL-11: maximal current was 173 +/- 34 pA in control and 159 +/- 35 pA and 117 +/- 14 pA in the presence of 10 and 50 ng/ml of rhIL-11, respectively; midpoint activation was -99 +/- 3 mV in control and -98 +/- 4 mV and -94 +/- 2 mV in the presence of 10 and 50 ng/ml of rhIL-11, respectively (p = NS). Thus, it is unlikely that direct alterations of membrane potential and currents of HuAM caused by rhIL-11 are the basis for the genesis of atrial arrhythmias observed in patients treated with this agent.
• Functional Expression of the Hyperpolarization-activated, Non-selective Cation Current I(f) in Immortalized HL-1 Cardiomyocytes The Journal of Physiology. Nov, 2002  |   Pubmed ID: 12433951 HL-1 cells are adult mouse atrial myocytes induced to proliferate indefinitely by SV40 large T antigen. These cells beat spontaneously when confluent and express several adult cardiac cell markers including the outward delayed rectifier K(+) channel. Here, we examined the presence of a hyperpolarization-activated I(f) current in HL-1 cells using the whole-cell patch-clamp technique on isolated cells enzymatically dissociated from the culture at confluence. Cell membrane capacitance (C(m)) ranged from 5 to 53 pF. I(f) was detected in about 30% of the cells and its occurrence was independent of the stage of the culture. I(f) maximal slope conductance was 89.7 +/- 0.4 pS pF(-1) (n = 10). I(f) current in HL-1 cells showed typical characteristics of native cardiac I(f) current: activation threshold between -50 and -60 mV, half-maximal activation potential of -83.1 +/- 0.7 mV (n = 50), reversal potential at -20.8 +/- 1.5 mV (n = 10), time-dependent activation by hyperpolarization and blockade by 4 mM Cs(+). In half of the cells tested, activation of adenylyl cyclase by the forskolin analogue L858051 (20 microM) induced both an approximately 6 mV positive shift of the half-activation potential and an approximately 37 % increase in the fully activated I(f) current. RT-PCR analysis of the hyperpolarization-activated, cyclic nucleotide-gated channels (HCN) expressed in HL-1 cells demonstrated major contributions of HCN1 and HCN2 channel isoforms to I(f) current. Cytosolic Ca(2+) oscillations in spontaneously beating HL-1 cells were measured in Fluo-3 AM-loaded cells using a fast-scanning confocal microscope. The oscillation frequency ranged from 1.3 to 5 Hz and the spontaneous activity was stopped in the presence of 4 mM Cs(+). Action potentials from HL-1 cells had a triangular shape, with an overshoot at +15 mV and a maximal diastolic potential of -69 mV, i.e. more negative than the threshold potential for I(f) activation. In conclusion, HL-1 cells display a hyperpolarization-activated I(f) current which might contribute to the spontaneous contractile activity of these cells.
• Selectivity of Different Calcium Antagonists on T- and L-type Calcium Currents in Guinea-pig Ventricular Myocytes Pharmacological Research : the Official Journal of the Italian Pharmacological Society. Dec, 2002  |   Pubmed ID: 12457621 Both L- and T-type calcium channels are present in the heart. In cardiac myocytes L-type calcium channels are blocked by the classical calcium channel blockers, while T-type calcium channels are thought to be insensitive to these drugs and to be selectively blocked by mibefradil. We aimed to compare the T/L calcium channel blocking selectivity of several calcium channel blockers by evaluating their effects on both components evoked in the same cell from a holding potential corresponding to the normal physiological value (-90mV). Currents were recorded in single patch-clamped guinea-pig ventricular myocytes, superfused with a Na(+)- and K(+)-free solution to abolish overlapping currents. Two dihydropyridines (amlodipine and lacidipine), verapamil diltiazem and mibefradil were tested; for each compound concentrations equieffective on L-type Ca(2+) current were used. All calcium channel blockers, at concentrations blocking less than 30% of L-type Ca(2+) current, inhibited a significant amount of T-type Ca(2+) current, varying from 0.8% (diltiazem) to 28% (mibefradil). We calculated for each compound the T/L ratio. As expected, mibefradil showed the highest T selectivity; lacidipine and diltiazem resulted to be L selective. Verapamil and amlodipine were not selective. Thus, the calcium channel blockers can be differentiated on the basis of their T/L selectivity.
• Treatment with Irbesartan Counteracts the Functional Remodeling of Ventricular Myocytes from Hypertensive Rats Journal of Cardiovascular Pharmacology. May, 2003  |   Pubmed ID: 12717113 Changes in electrophysiological (action potential prolongation, decrease in transient outward current I(to), occurrence of the hyperpolarization-activated current I(f)) and contractile properties develop in hypertrophied ventricular myocytes, likely implicated in the increased propensity to arrhythmias. Angiotensin II is a key signal for myocyte hypertrophy; the effect of 8-week treatment with irbesartan, a type 1 angiotensin II receptor (AT(1)) antagonist, on cardiac remodeling was tested. Sixteen-month-old hypertensive rats (SHRs) were treated with irbesartan (20 mg/kg/d) or saline for 8 weeks. At the end of treatment, systolic blood pressure and heart weight to body weight ratio were reduced in irbesartan-treated compared with nontreated SHRs. Electrical and contractile properties were measured in isolated ventricular myocytes, by patch-clamp or video-dimension analysis, respectively. Action potential duration was significantly shorter in irbesartan-treated than in nontreated SHRs (at -60 mV: 119 +/- 24 ms vs 187 +/- 20 ms); correspondingly, maximal I(to) density was larger in irbesartan-treated than in nontreated SHRs (25.4 +/- 2.8 pA/pF vs 18.5 +/- 1.5 pA/pF). Maximal specific conductance of I(f) was lower in irbesartan-treated vs nontreated SHRs (24.8 +/- 3.0 pS/pF vs 35.2 +/- 4.0 pS/pF). Finally, the relaxation rate of shortening in field-stimulated intact myocytes was significantly faster in irbesartan-treated than in nontreated SHRs (7.3 +/- 0.5/s vs 5.7 +/- 0.3/s). Thus, AT(1) blockade with irbesartan, at an oral daily dosage that gave a slight but significant reduction of systolic blood pressure, largely counteracts the development of myocyte hypertrophy and associated functional alterations.
• Prenatal Exposure to Carbon Monoxide Affects Postnatal Cellular Electrophysiological Maturation of the Rat Heart: a Potential Substrate for Arrhythmogenesis in Infancy Circulation. Jan, 2004  |   Pubmed ID: 14718404 Maternal smoking is an independent risk factor for sudden infant death syndrome (SIDS). Carbon monoxide (CO) is a major component of smoke. No information is available about the effect of CO and/or smoking on postnatal maturation of the heart. The aim of this study was to investigate the effect of prenatal exposure to CO on cellular electrophysiological maturation in male Wistar rats.
• Angiotensin AT2 Receptor: the Younger Sibling Attracts Attention Cardiovascular Research. Apr, 2004  |   Pubmed ID: 15023548
• Restoration of Cardiomyocyte Functional Properties by Angiotensin II Receptor Blockade in Diabetic Rats Diabetes. Jul, 2004  |   Pubmed ID: 15220222 Recent evidence suggests that blockade of the renin-angiotensin system ameliorates diabetes-induced cardiac dysfunction, but the mechanisms involved in this process remain elusive. We investigated the effect of treatment with an angiotensin II receptor blocker, losartan, on the metabolic and electrophysiological properties of cardiomyocytes isolated from streptozotocin-induced diabetic (STZ) rats. Glucose uptake and electrophysiological properties were measured in ventricular cardiomyocytes from normoglycemic and STZ-induced diabetic rats given vehicle or 20 mg x kg(-1) x day(-1) losartan for 8 weeks. Insulin and beta-adrenergic stimulation failed to increase the glucose uptake rate in STZ cardiomyocytes, whereas the alpha-adrenergic effect persisted. Concurrently, a typical prolongation of action potential duration (APD) and a decrease of transient outward current (I(to)) were recorded in patch-clamped STZ myocytes. Treatment with losartan did not affect body weight or glycemia of diabetic or control animals. However, in losartan-treated STZ-induced diabetic rats, beta-adrenergic-mediated enhancement of glucose uptake was completely recovered. APD and I(to) were similar to those measured in losartan-treated control rats. A significant (P < 0.0001) correlation between metabolic and electrophysiological parameters was found in control, diabetic, and losartan-treated diabetic rats. Thus, angiotensin receptor blockade protects the heart from the development of cellular alterations typically associated with diabetes. These data suggest that angiotensin receptor blockers may represent a new therapeutic strategy for diabetic cardiomyopathy.
• Atrial Natriuretic Peptide Modulates the Hyperpolarization-activated Current (If) in Human Atrial Myocytes Cardiovascular Research. Aug, 2004  |   Pubmed ID: 15276478 The relationship between atrial stretching and changes in cell excitability is well documented. Once stretched, human atrial myocytes (HuAM) release atrial natriuretic peptide (hANP). Receptors for hANP (NPR) are coupled to a guanylyl cyclase (GC) activity, and are present on HuAM, but the electrophysiological effects of hANP are largely unknown. We investigated the effect of hANP on If, the hyperpolarization-activated current present in HuAM, and the underlying intracellular pathway.
• N-3 Polyunsaturated Fatty Acids Supplementation Decreases Asymmetric Dimethyl Arginine and Arachidonate Accumulation in Aging Spontaneously Hypertensive Rats European Journal of Nutrition. Sep, 2005  |   Pubmed ID: 15368071 Plasma accumulation of asymmetric dimethyl arginine (ADMA) is considered as a risk factor for endothelial dysfunction and a strong predictor for coronary heart diseases. Eicosapentaenoic (EPA) and docosahexaenoic (DHA) increasing plasma levels have been positively associated with reduced cardiovascular mortality with a mechanism( s) yet unclear. We hypothesised that ADMA reduction might be a part of EPA and DHA beneficial effects on the cardiovascular system.
• Design, Synthesis and Preliminary Biological Evaluation of Zatebradine Analogues As Potential Blockers of the Hyperpolarization-activated Current Bioorganic & Medicinal Chemistry. Feb, 2005  |   Pubmed ID: 15670930 A series of zatebradine analogues, differing in the basic moiety and in the methylene spacer, have been synthesized; their negative chronotropic activity has been determined in guinea pig atria. The most active compounds have been studied for their blocking properties on the hyperpolarization-activated current If (which is one of the main currents underlying automatic activity in the sinus node) measured on ventricular myocytes of old spontaneously-hypertensive rats (SHR) by means of the patch-clamp technique. The majority of the substances were able to block If, with one of them (15) being slightly more potent than zatebradine. Surprisingly one analogue (6), while showing good negative chronotropic activity, was found to inhibit If only at high concentration and to markedly reduce outward currents, suggesting for this substance a different mechanism of action responsible for the negative chronotropic effect.
• Pharmacological Modulation of the Hyperpolarization-activated Current (I F) in Human Atrial Myocytes: Focus on G Protein-coupled Receptors Journal of Molecular and Cellular Cardiology. Mar, 2005  |   Pubmed ID: 15733905 In human atrial myocytes (HuAM) two beta-adrenergic receptors (beta-AR) and four splicing-variants of the serotonin 5-HT(4) receptor are present. Multiple coupling with G stimulatory (G(s)) and G inhibitory (G(i)) proteins has been proposed for both beta(2)-AR and 5-HT((4b)) subtypes, but no functional data exist in HuAM. Serotonin (5-HT) and catecholamines are able to trigger arrhythmias in human atrium, but the underlying cellular mechanisms are not completely understood. The pacemaker current (I(f)) is an inward Na(+)/K(+) current, constitutively present in HuAM and directly modulated by cAMP; I(f) could play a role in triggering human atrial arrhythmias. This study evaluated the different G protein coupling of beta(1)-AR, beta(2)-AR and 5-HT(4) receptors by assessing the modulation of I(f) by selective stimuli.
• [The Pacemaker Current If: a Novel Pharmacological Target for Cardiologists] Italian Heart Journal. Supplement : Official Journal of the Italian Federation of Cardiology. Jul, 2005  |   Pubmed ID: 16082824 The pacemaker current contributes to endow some types of specialized cells (either cardiomyocytes, neurons, or smooth muscle cells) with an intrinsic rhythmic activity. In cardiac cells, this current has been named If for "funny current" by DiFrancesco, who first described it more than 20 years ago. The terminology points to the most peculiar If feature: pacemaker channels activate upon membrane hyperpolarization rather than depolarization, opposite to most voltage-gated channels. Recently, electrophysiological and molecular data demonstrated that f-channels are also present in ventricular cardiomyocytes, and become upregulated in cardiac hypertrophy and failure. Misplaced expression and/or overexpression of f-channels are a consequence of electrophysiological remodeling and, from a clinical point of view, may represent an arrhythmogenic mechanism in heart failure, a condition associated with high risk for sudden cardiac death. Due to its physiological (and pathophysiological) role and to the availability of selective f-channel blockers, If can be considered as a suitable therapeutic target for cardiologists.
• Prenatal Exposure to Carbon Monoxide Temporarily Impairs Maturation of Rat Cardiomyocytes: Electrophysiological Evidence Experimental and Clinical Cardiology. 2005  |   Pubmed ID: 19641682 Maternal smoking is an independent risk factor for sudden infant death syndrome. Carbon monoxide (CO) is a major component of cigarette smoke. No information is available concerning the effect of CO and/or smoking on postnatal maturation of the heart.
• Functional Remodeling in Post-myocardial Infarcted Rats: Focus on Beta-adrenoceptor Subtypes Journal of Molecular and Cellular Cardiology. Feb, 2006  |   Pubmed ID: 16427077 Cellular electrophysiological remodeling of the infarcted heart may lead to the deterioration of cardiac function and/or to arrhythmias. The present study was designed to characterize the functional expression of the hyperpolarization-activated current (I(f)) and its modulation by beta(1)-, beta(2)- and beta(3)-adrenoceptor (AR) subtypes, in patch-clamped ventricular myocytes isolated from the heart of post-myocardial infarcted (PMI) rats and sham-operated control (SHAM) rats. Maximum specific conductance of I(f) was significantly higher in left ventricular myocytes (LVM) from PMI rats compared to right ventricular myocytes from PMI rats as well as LVM and RVM from SHAM rats. All other basic properties of I(f) were similar. beta(1)AR stimulation with noradrenaline caused a rightward shift of V(H) in LVM from PMI rats which was significantly smaller (52.2%) than in LVM from SHAM rats. Incubation with pertussis toxin (PTX) largely restored the effect of beta(1)AR in PMI cells (86.6% vs. SHAM cells), but did not affect beta(1)AR response in SHAM cells. beta(2)AR response was significantly and equally increased by PTX-pretreatment (by 94% in SHAM and 87% in PMI cells). Conversely, beta(3)AR stimulation by the selective agonist SR 58611A caused a leftward shift of the activation curve which was significantly larger in PMI cells than in SHAM cells (P
• I(f) in Non-pacemaker Cells: Role and Pharmacological Implications Pharmacological Research : the Official Journal of the Italian Pharmacological Society. May, 2006  |   Pubmed ID: 16713285 Pacemaker channels play a major role in the generation of sinoatrial rhythmic activity. However, their expression is not confined to specialized myocardial cells, such as primary and subsidiary pacemakers. Electrophysiological and molecular data collected over the last ten years have demonstrated that f-channels are also present in non-pacemaker cardiomyocytes, and become upregulated in cardiac hypertrophy and failure. Mislocalized expression and/or overexpression of f-channels are a consequence of electrophysiological remodeling and, from a clinical point of view, may represent an arrhythmogenic mechanism in heart failure, a condition associated with a high risk for sudden cardiac death. The potential arrhythmogenic role of I(f) and the availability of selective f-channel blockers cause I(f) to be a suitable therapeutic target in heart disease.
• Tension Generation and Relaxation in Single Myofibrils from Human Atrial and Ventricular Myocardium Pflügers Archiv : European Journal of Physiology. Apr, 2007  |   Pubmed ID: 17123098 Fast solution switching techniques in single myofibrils offer the opportunity to dissect and directly examine the sarcomeric mechanisms responsible for force generation and relaxation. The feasibility of this approach is tested here in human cardiac myofibrils isolated from small samples of atrial and ventricular tissue. At sarcomere lengths between 2.0 and 2.3 mum, resting tensions were significantly higher in ventricular than in atrial myofibrils. The rate constant of active tension generation after maximal Ca(2+) activation (k (ACT)) was markedly faster in atrial than in ventricular myofibrils. In both myofibril types k (ACT) was the same as the rate of tension redevelopment after mechanical perturbations and decreased significantly by decreasing [Ca(2+)] in the activating solution. Upon sudden Ca(2+) removal, active tension fully relaxed. Relaxation kinetics were (1) much faster in atrial than in ventricular myofibrils, (2) unaffected by bepridil, a drug that increases the affinity of troponin for Ca(2+), and (3) strongly accelerated by small increases in inorganic phosphate concentration. The results indicate that myofibril tension activation and relaxation rates reflect apparent cross-bridge kinetics and their Ca(2+) regulation rather than the rates at which thin filaments are switched on or off by Ca(2+) binding or removal. Myofibrils from human hearts retain intact mechanisms for contraction regulation and tension generation and represent a viable experimental model to investigate function and dysfunction of human cardiac sarcomeres.
• Developmental Changes in Cardiomyocytes Differentiated from Human Embryonic Stem Cells: a Molecular and Electrophysiological Approach Stem Cells (Dayton, Ohio). May, 2007  |   Pubmed ID: 17255522 Cardiomyocytes derived from human embryonic stem cells constitute a promising cell source for the regeneration of damaged hearts. The assessment of their in vitro functional properties is mandatory to envisage appropriate cardiac cell-based therapies. In this study, we characterized human embryonic stem cell-derived cardiomyocytes over a 3-month period, using patch-clamp or intracellular recordings to assess their functional maturation and reverse transcriptase-polymerase chain reaction to evaluate the expression of ion channel-encoding subunits. I(to1) and I(K1), the transient outward and inward rectifier potassium currents, were present in cardiomyocytes only, whereas the rapid delayed rectifier potassium current (I(Kr)), pacemaker current (I(f)), and L-type calcium current (I(Ca,L)) could be recorded both in undifferentiated human embryonic stem cells and in cardiomyocytes. Most of the currents underwent developmental maturation in cardiomyocytes, as assessed by modifications in current density (I(to1), I(K1), and I(Ca,L)) and properties (I(f)). Ion-channel mRNAs were always present when the current was recorded. Intracellular recordings in spontaneously beating clusters of cardiomyocytes revealed changes in action potential parameters and in response to pharmacological tools according to time of differentiation. In summary, human embryonic stem cell-derived cardiomyocytes mature over time during in vitro differentiation, approaching an adult phenotype. Disclosure of potential conflicts of interest is found at the end of this article.
• Cardiac Effects of 3-iodothyronamine: a New Aminergic System Modulating Cardiac Function FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. May, 2007  |   Pubmed ID: 17284482 3-Iodothyronamine T1AM is a novel endogenous thyroid hormone derivative that activates the G protein-coupled receptor known as trace anime-associated receptor 1 (TAAR1). In the isolated working rat heart and in rat cardiomyocytes, T1AM produced a reversible, dose-dependent negative inotropic effect (e.g., 27+/-5, 51+/-3, and 65+/-2% decrease in cardiac output at 19, 25, and 38 microM concentration, respectively). An independent negative chronotropic effect was also observed. The hemodynamic effects of T1AM were remarkably increased in the presence of the tyrosine kinase inhibitor genistein, whereas they were attenuated in the presence of the tyrosine phosphatase inhibitor vanadate. No effect was produced by inhibitors of protein kinase A, protein kinase C, calcium-calmodulin kinase II, phosphatidylinositol-3-kinase, or MAP kinases. Tissue cAMP levels were unchanged. In rat ventricular tissue, Western blot experiments with antiphosphotyrosine antibodies showed reduced phosphorylation of microsomal and cytosolic proteins after perfusion with synthetic T1AM; reverse transcriptase-polymerase chain reaction experiments revealed the presence of transcripts for at least 5 TAAR subtypes; specific and saturable binding of [125I]T1AM was observed, with a dissociation constant in the low micromolar range (5 microM); and endogenous T1AM was detectable by tandem mass spectrometry. In conclusion, our findings provide evidence for the existence of a novel aminergic system modulating cardiac function.
• NADPH Oxidase-dependent Redox Signaling in Human Heart Failure: Relationship Between the Left and Right Ventricle Journal of Molecular and Cellular Cardiology. Apr, 2007  |   Pubmed ID: 17346742 Oxidative stress resulting from increased superoxide generation by NADPH oxidase is implicated in the pathophysiology of human heart failure. Downstream targets of NADPH oxidase remain undefined and available information is restricted to the left ventricle (LV). Thus, we aimed to assess the cascade of events triggered by increased NADPH oxidase activity (lipid peroxidation and activation of mitogen-activated protein kinases ERK1/2, JNK and p38) and their mutual relationship in right (RV) and (LV) of end-stage failing human hearts. When compared to control ventricles, diseased RV and LV showed a significant increase in NADPH oxidase superoxide production that positively correlated with p47(phox) membrane translocation (RV: r=0.76, P
• ATP Modulates Cell Proliferation and Elicits Two Different Electrophysiological Responses in Human Mesenchymal Stem Cells Stem Cells (Dayton, Ohio). Jul, 2007  |   Pubmed ID: 17446563 Bone marrow-derived human mesenchymal stem cells (hMSCs) have the potential to differentiate into several cell lines. Extracellular adenosine 5'-triphosphate (ATP) acts as a potent signaling molecule mediating cell-to-cell communication. Particular interest has been focused in recent years on the role of ATP in stem cell proliferation and differentiation. In the present work, we demonstrate that hMSCs at early stages of culture (P0-P5) spontaneously release ATP, which decreases cell proliferation. Increased hMSC proliferation is induced by the unselective P2 antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonate (PPADS) and by the selective P2Y1 antagonist 2'-deoxy-N6-methyladenosine3',5'-bisphosphate (MRS 2179). A functional role of extracellular ATP in modulating ionic conductances with the whole-cell and/or perforated patch-clamp techniques was also investigated. Exogenous ATP increased both the voltage-sensitive outward and inward currents in 47% of cells, whereas, in 31% of cells, only an increase in inward currents was found. Cells responding in this dual manner to ATP presented different resting membrane potentials. Both ATP-induced effects had varying sensitivity to the P2 antagonists PPADS and MRS 2179. Outward ATP-sensitive currents are carried by potassium ions, since they are blocked by cesium replacement and are Ca2+ -dependent because they are eliminated in the presence of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. On the basis of different electrophysiological and pharmacological characteristics, we conclude that outward ATP-sensitive currents are due to Ca2+ -dependent K+ -channel activation following stimulation of P2Y receptors, whereas inward ATP-sensitive currents are mediated by P2X receptor activation. In summary, ATP released in early life stages of hMSCs modulates their proliferation rate and likely acts as one of the early factors determining their cell fate. Disclosure of potential conflicts of interest is found at the end of this article.
• Dopamine Agonists and Valvular Heart Disease The New England Journal of Medicine. Apr, 2007  |   Pubmed ID: 17447276
• Fetal Bovine Serum Enables Cardiac Differentiation of Human Embryonic Stem Cells Differentiation; Research in Biological Diversity. Oct, 2007  |   Pubmed ID: 17459089 During development, cardiac commitment within the mesoderm requires endoderm-secreted factors. Differentiation of embryonic stem cells into the three germ layers in vitro recapitulates developmental processes and can be influenced by supplements added to culture medium. Hence, we investigated the effect of fetal bovine serum (FBS) and KnockOut serum replacement (SR) on germ layers specification and cardiac differentiation of H1 human embryonic stem cells (hESC) within embryoid bodies (EB). At the time of EB formation, FBS triggered an increased apoptosis. As assessed by quantitative PCR on 4-, 10-, and 20-day-old EB, FBS promoted a faster down-regulation of pluripotency marker Oct4 and an increased expression of endodermal (Sox17, alpha-fetoprotein, AFP) and mesodermal genes (Brachyury, CSX). While neuronal and hematopoietic differentiation occurred in both supplements, spontaneously beating cardiomyocytes were only observed in FBS. Action potential (AP) morphology of hESC-derived cardiomyocytes indicated that ventricular cells were present only after 2 months of culture. However, quantification of myosin light chain 2 ventricular (mlc2v)-positive areas revealed that mlc2v-expressing cardiomyocytes could be detected already after 2 weeks of differentiation, but not in all beating clusters. In conclusion, FBS enabled cardiac differentiation of hESC, likely in an endodermal-dependent pathway. Among cardiac cells, ventricular cardiomyocytes differentiated over time, but not as the predominant cardiac cell subtype.
• Characterization of a Novel SCN5A Mutation Associated with Brugada Syndrome Reveals Involvement of DIIIS4-S5 Linker in Slow Inactivation Cardiovascular Research. Dec, 2007  |   Pubmed ID: 17854786 Mutations in SCN5A, the gene encoding the alpha-subunit of the cardiac sodium channel (Na(v)1.5), have been associated with various inherited arrhythmia syndromes, including Brugada syndrome (BrS). Here, we report the functional consequences of a novel missense SCN5A mutation, G1319V, identified in a BrS patient. The G1319V mutation is located in the loop connecting transmembrane segments 4 and 5 in domain III (DIIIS4-S5), a region so far considered to be exclusively involved in fast inactivation.
• I(f) Channels As a Therapeutic Target in Heart Disease Future Cardiology. Nov, 2007  |   Pubmed ID: 19804286 In the normal heart, impulses are generated from the sinoatrial node. It is generally accepted that the pacemaker current, I(f), plays a major role in the spontaneous rhythmic activity. Recently, several electrophysiological and molecular data demonstrate that I(f) channels are present in embryonic and post-natal ventricular myocytes and undergo a downregulation during maturation. Interestingly, the I(f) current is re-expressed in some pathological conditions such as cardiac hypertrophy and heart failure. In these conditions, the overexpression of f-channels is a consequence of electrophysiological remodeling and may represent an arrhythmogenic mechanism in heart failure, a condition associated with high risk for sudden cardiac death. For its physiological and pathophysiological role and the availability of selective f-channel blockers, I(f) may be a suitable therapeutic target in heart failure.
• Expression of the Hyperpolarization-activated Current, I(f), in Cultured Adult Rat Ventricular Cardiomyocytes and Its Modulation by Hypertrophic Factors Pharmacological Research : the Official Journal of the Italian Pharmacological Society. Feb, 2008  |   Pubmed ID: 18255311 The hyperpolarization-activated, cyclic nucleotide-gated (HCN) current, I(f), is typically overexpressed in myocytes from hypertrophied and failing hearts, where it may play an arrhythmogenic role. Signaling pathways activated by agonists such as angiotensin-II, endothelin-1 and phenylephrine, via G protein-coupled receptors (GPCR), promote myocardial hypertrophy, but their effect on cellular electrophysiological remodeling, particularly I(f) expression is largely unknown. Thus, I(f) expression was measured by patch-clamp and quantitative RT-PCR measurement in cultured adult rat ventricular cardiomyocytes (VCM) exposed to different culture conditions, that is, in the absence or presence of: fetal bovine serum (FBS, 5%), 0.1 microM angiotensin-II, 0.1 microM endothelin-1 or 20 microM phenylephrine. Membrane capacitance (C(m)) was used to estimate cell size and current density in patch-clamped VCM. At 8 days of culture, about 60% of VCM showed I(f). In serum-free medium, I(f) density was increased by phenylephrine (2.28+/-0.51 vs. 0.84+/-0.30 pA/pF in CTR, p
• Do Amniotic Fluid-derived Stem Cells Differentiate into Neurons in Vitro? Nature Biotechnology. Mar, 2008  |   Pubmed ID: 18327228
• Holt-oram Syndrome and Atrial Fibrillation: Opening the (T)-box Circulation Research. Jun, 2008  |   Pubmed ID: 18535267
• Molecular Basis of Funny Current (If) in Normal and Failing Human Heart Journal of Molecular and Cellular Cardiology. Aug, 2008  |   Pubmed ID: 18556018 I(f) overexpression has been functionally demonstrated in ventricular myocytes from failing human hearts. Altered expression of I(f)-channels as a consequence of electrophysiological remodeling may represent an arrhythmogenic mechanism in heart failure; however, the molecular basis of I(f) overexpression in human cardiac disease is unknown. HCN1, 2 and 4 subtypes, which encode I(f)-channels, have been identified in the heart. The present study was designed to characterize HCN isoform expression in failing and non-failing hearts. Ventricular and atrial samples were obtained from normal or failing hearts explanted from patients with end-stage ischemic cardiomyopathy. I(f) was recorded in patch-clamped left ventricular myocytes. mRNA and protein expression of HCN subunits were measured in both atria and ventricles of control and diseased hearts. HCN2 and HCN4 were detected in human myocardium. Both mRNA and protein levels of HCN2/4 were significantly augmented in failing ventricles (p
• Role of Potassium Currents in Cardiac Arrhythmias Europace : European Pacing, Arrhythmias, and Cardiac Electrophysiology : Journal of the Working Groups on Cardiac Pacing, Arrhythmias, and Cardiac Cellular Electrophysiology of the European Society of Cardiology. Oct, 2008  |   Pubmed ID: 18653669 Abnormal excitability of myocardial cells may give rise to ectopic beats and initiate re-entry around an anatomical or functional obstacle. As K(+) currents control the repolarization process of the cardiac action potential (AP), the K(+) channel function determines membrane potential and refractoriness of the myocardium. Both gain and loss of the K(+) channel function can lead to arrhythmia. The former because abbreviation of the active potential duration (APD) shortens refractoriness and wave length, and thereby facilitates re-entry and the latter because excessive prolongation of APD may lead to torsades de pointes (TdP) arrhythmia and sudden cardiac death. The pro-arrhythmic consequences of malfunctioning K(+) channels in ventricular and atrial tissue are discussed in the light of three pathophysiologically relevant aspects: genetic background, drug action, and disease-induced remodelling. In the ventricles, loss-of-function mutations in the genes encoding for K(+) channels and many drugs (mainly hERG channel blockers) are related to hereditary and acquired long-QT syndrome, respectively, that put individuals at high risk for developing TdP arrhythmias and life-threatening ventricular fibrillation. Similarly, down-regulation of K(+) channels in heart failure also increases the risk for sudden cardiac death. Mutations and polymorphisms in genes encoding for atrial K(+) channels can be associated with gain-of-function and shortened, or with loss-of-function and prolonged APs. The block of atrial K(+) channels becomes a particular therapeutic challenge when trying to ameliorate atrial fibrillation (AF). This arrhythmia has a strong tendency to cause electrical remodelling, which affects many K(+) channels. Atrial-selective drugs for the treatment of AF without affecting the ventricles could target structures such as I(Kur) or constitutively active I(K,ACh) channels.
• Beating to Time: Calcium Clocks, Voltage Clocks, and Cardiac Pacemaker Activity American Journal of Physiology. Heart and Circulatory Physiology. Mar, 2009  |   Pubmed ID: 19151259
• Electrophysiologic Changes in Heart Failure: Focus on Pacemaker Channels Canadian Journal of Physiology and Pharmacology. Feb, 2009  |   Pubmed ID: 19234571 Heart failure is a common clinical syndrome occurring as a result of cardiac overload, injury, and a complex interplay among genetic, neurohormonal, inflammatory, and biochemical factors. Occurrence of arrhythmias in heart failure is largely a consequence of disease-induced electrical remodeling of cardiac myocytes, a phenomenon consisting of alterations of ion channels and the ion-transport function that predispose patients to develop lethal arrhythmias. In most cases, the mechanism is the rapid onset of a ventricular tachyarrhythmia progressing to ventricular fibrillation and hemodynamic compromise. This paper highlights some of the important changes in ion channel expression and function that underlie electrical remodeling of the failing heart. Particular attention will be focused on the presence, features, and pharmacologic modulation of f channels expressed in ventricular cardiac myocytes.
• Modulation of Cardiac Ionic Homeostasis by 3-iodothyronamine Journal of Cellular and Molecular Medicine. Sep, 2009  |   Pubmed ID: 19298522 3-iodothyronamine (T(1)AM) is a novel endogenous relative of thyroid hormone, able to interact with trace amine-associated receptors, a class of plasma membrane G protein-coupled receptors, and to produce a negative inotropic and chronotropic effect. In the isolated rat heart 20-25 microM T(1)AM decreased cardiac contractility, but oxygen consumption and glucose uptake were either unchanged or disproportionately high when compared to mechanical work. In adult rat cardiomyocytes acute exposure to 20 microM T(1)AM decreased the amplitude and duration of the calcium transient. In patch clamped cardiomyocytes sarcolemmal calcium current density was unchanged while current facilitation by membrane depolarization was abolished consistent with reduced sarcoplasmic reticulum (SR) calcium release. In addition, T(1)AM decreased transient outward current (I(to)) and I(K1) background current. SR studies involving 20 microM T(1)AM revealed a significant decrease in ryanodine binding due to reduced B(max), no significant change in the rate constant of calcium-induced calcium release, a significant increase in calcium leak measured under conditions promoting channel closure, and no effect on oxalate-supported calcium uptake. Based on these observations we conclude T(1)AM affects calcium and potassium homeostasis and suggest its negative inotropic action is due to a diminished pool of SR calcium as a result of increased diastolic leak through the ryanodine receptor, while increased action potential duration is accounted for by inhibition of I(to) and I(K1) currents.
• From in Vivo Plasma Composition to in Vitro Cardiac Electrophysiology and in Silico Virtual Heart: the Extracellular Calcium Enigma Philosophical Transactions. Series A, Mathematical, Physical, and Engineering Sciences. Jun, 2009  |   Pubmed ID: 19414453 In spite of its potential impact on simulation results, the problem of setting the appropriate Ca(2+) concentration ([Ca(2+)](o)) in computational cardiac models has not yet been properly considered. Usually [Ca(2+)](o) values are derived from in vitro electrophysiology. Unfortunately, [Ca(2+)](o) in the experiments is set significantly far (1.8 or 2 mM) from the physiological [Ca(2+)] in blood (1.0-1.3 mM). We analysed the inconsistency of [Ca(2+)](o) among in vivo, in vitro and in silico studies and the dependence of cardiac action potential (AP) duration (APD) on [Ca(2+)](o). Laboratory measurements confirmed the difference between standard extracellular solutions and normal blood [Ca(2+)]. Experimental data on human atrial cardiomyocytes confirmed literature data, demonstrating an inverse relationship between APD and [Ca(2+)](o). Sensitivity analysis of APD on [Ca(2+)](o) for five of the most used cardiac cell models was performed. Most of the models responded with AP prolongation to increases in [Ca(2+)](o), i.e. opposite to the AP shortening observed in vitro and in vivo. Modifications to the Ten Tusscher-Panfilov model were implemented to demonstrate that qualitative consistency among in vivo, in vitro and in silico studies can be achieved. The Courtemanche atrial model was used to test the effect of changing [Ca(2+)](o) on quantitative predictions about the effect of K(+) current blockade. The present analysis suggests that (i) [Ca(2+)](o) in cardiac AP models should be changed from 1.8 to 2 mM to approximately 1.15 mM in order to reproduce in vivo conditions, (ii) the sensitivity to [Ca(2+)](o) of ventricular AP models should be improved in order to simulate real conditions, (iii) modifications to the formulation of Ca(2+)-dependent I(CaL) inactivation can make models more suitable to analyse AP when [Ca(2+)](o) is set to lower physiological values, and (iv) it could be misleading to use non-physiological high [Ca(2+)](o) when the quantitative analysis of in vivo pathophysiological mechanisms is the ultimate aim of simulation.
• Identifying Needs and Opportunities for Advancing Translational Research in Cardiovascular Disease Cardiovascular Research. Aug, 2009  |   Pubmed ID: 19502281
• Losartan Counteracts the Hyper-reactivity to Angiotensin II and ROCK1 Over-activation in Aortas Isolated from Streptozotocin-injected Diabetic Rats Cardiovascular Diabetology. 2009  |   Pubmed ID: 19545435 In streptozotocin-injected rats (STZ-rats), we previously demonstrated a role for angiotensin II (AT-II) in cardiac remodelling and insulin resistance partially counteracted by in vivo treatment with losartan, an AT-II receptor antagonist.We now aimed to investigate the effect of treating diabetic STZ-rats with losartan on diabetes vascular response to vasoconstrictors.
• Oleuropein Aglycon Prevents Cytotoxic Amyloid Aggregation of Human Amylin The Journal of Nutritional Biochemistry. Aug, 2010  |   Pubmed ID: 19616928 Pancreatic amyloid deposits of amylin are a hallmark of Type II diabetes and considerable evidence indicates that amylin oligomers are cytotoxic to beta-cells. Many efforts are presently spent to find out naturally occurring molecules, or to design synthetic ones, able to hinder amylin aggregation or to protect cells against aggregate cytotoxicity. In this context, a protective effect of some polyphenols against amyloid cytotoxicity was reported. Actually dietary polyphenols are endowed with multiple health benefits, and extra virgin olive oil is attracting increasing interest as a source of these substances. Here, we investigated the effects on amylin aggregation and cytotoxicity of the secoiridoid oleuropein aglycon, the main phenolic component of extra virgin olive oil. We found that oleuropein, when present during the aggregation of amylin, consistently prevented its cytotoxicity to RIN-5F pancreatic beta-cells, as determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide test and caspase-3 activity assay. A lack of interaction with the cell membrane of amylin aggregates grown in the presence of oleuropein was shown by fluorescence microscopy and synthetic lipid vesicle permeabilization. Moreover, our ThT assay, circular dichroism analysis and electron microscopy images suggested that oleuropein interferes with amylin aggregation, resulting in a different path skipping the formation of toxic pre-fibrillar aggregates. These results provide a molecular basis for some of the benefits potentially coming from extra virgin olive oil consumption and pave the way to further studies on the possible pharmacological use of oleuropein to prevent or to slow down the progression of type II diabetes.
• Enhanced ROS Production by NADPH Oxidase is Correlated to Changes in Antioxidant Enzyme Activity in Human Heart Failure Biochimica Et Biophysica Acta. Mar, 2010  |   Pubmed ID: 19892017 In pathological conditions, the balance between reactive oxygen species (ROS) and antioxidants may shift toward a relative increase of ROS, resulting in oxidative stress. Conflicting data are available on antioxidant defenses in human failing heart and they are limited to the left ventricle. Thus, we aimed to investigate and compare the source of oxidant and antioxidant enzyme activities in the right (RV) and left (LV) ventricles of human failing hearts. We found a significant increase in superoxide production only by NADPH oxidase in both failing ventricles, more marked in RV. Despite unchanged mRNA or protein expression, catalase (CAT) and glutathione peroxidase (GPx) activities were increased, and their increases reflected the levels of Tyr phosphorylation of the respective enzyme. Manganese superoxide dismutase (Mn-SOD) activity appeared unchanged. The increase in NADPH oxidase-dependent superoxide production positively correlated with the activation of both CAT and GPx. However, the slope of the linear correlation (m) was steeper in LV than in RV for GPx (LV: m=2.416; RV: m=1.485) and CAT (LV: m=1.007; RV: m=0.354). Accordingly, malondialdehyde levels, an indirect index of oxidative stress, were significantly higher in the RV than LV. We conclude that in human failing RV and LV, oxidative stress is associated with activation of antioxidant enzyme activity. This activation is likely due to post-translational modifications and more evident in LV. Overall, these findings suggest a reduced protection of RV against oxidative stress and its potential contribution to the progression toward overt heart failure.
• Cardiac and Electrophysiological Effects of Primary and Refined Extracts from Leonurus Cardiaca L. (Ph.Eur.) Planta Medica. Apr, 2010  |   Pubmed ID: 19918711 Although several antiarrhythmic drugs of chemical origin are in clinical use since decades, their application is often limited by their adverse effects and especially by their inherited proarrhythmic risk, which can lead to a significantly increased mortality in patients receiving these compounds. On the other hand, aqueous extracts from the aerial parts of the European Lamiaceae Leonurus cardiaca (Ph.Eur.) have been used for centuries as a remedy against tachyarrhythmia and other cardiac disorders. Nevertheless, a scientific basis for the claim of direct cardiac electrophysiological, antiarrhythmic, or functional effects of Leonurus cardiacae herba (LCH) preparations has not been established until now. In order to enrich the active constituents from the primary extract which was tested as the most cardioactive, namely the aqueous Soxhlet extract, and to eliminate undesired substances such as the dichloromethanic fraction or potassium, a bioassay guided fractionation procedure was applied, resulting in the development of a Leonurus cardiaca refined extract (LCRE) which was characterised together with Leonurus crude extracts by a newly developed gradient elution HPLC fingerprint analysis for separation and quantification of six major phenolics as well as by qNMR for determining the stachydrine content. This refined extract was applied intracoronarily in isolated rabbit hearts perfused according to the Langendorff technique. Mapping experiments with 256 electrodes on the heart surface showed a reduction of left ventricular pressure and an increase of relative coronary flow at concentrations of 1.0 and 2.0 mg/mL LCRE. Furthermore, the PQ-interval was prolonged and both the basic cycle length and the activation recovery interval increased. In addition, voltage-clamp measurements were performed on the following cell models in order to characterise the electrophysiological profile of LCRE: neonatal rat ventricular cardiomyocytes to investigate the effect on I(Na) and I(Ca.L), sinoatrial node cells and ventricular myocytes isolated from adult guinea pigs to test effects on I(f) and action potential (AP) duration, as well as HERG-transfected HEK 293 cells to analyse the influence on the I (K.r). In these voltage clamp experiments LCRE exerted a calcium-antagonistic activity by I(Ca.L) blockade, reduced the repolarising current I(K.r), and prolonged the AP-duration, while I(Na) was not affected. Although LCRE displayed only weak effects on the I(f) amplitude and voltage dependence, it significantly prolonged the activation time constant of I(f). Thus, LCRE acts on multiple electrophysiological targets, specifically I(Ca.L), I(K.r), and I(f), observed both at whole organ and single cell level.
• Electrophysiological Characterization of Isolated Human Atrial Myocytes Exposed to Tegaserod Basic & Clinical Pharmacology & Toxicology. May, 2010  |   Pubmed ID: 20050846 Tegaserod (Teg), a 5-hydroxytryptamine type-4 (5-HT(4)) receptor partial agonist, represents a novel treatment for irritable bowel syndrome with constipation and chronic constipation. Cardiovascular safety data from pooled clinical studies showed a signal suggestive of increased occurrence of ischaemic cardiovascular events in patients exposed to Teg versus placebo. Thereafter, marketing of Teg was suspended in the USA and other countries. The clinical data did not demonstrate a causative effect but raised questions of whether a non-recognized effect on the heart was present. Our aim was to evaluate for arrhythmogenic potential of Teg on human cardiomyocytes. Cells isolated from human atrial specimens during cardiac surgery were used to assess the effects of Teg (1, 10, 30 and 100 nM) on action potential and I(f) (funny current) by patch-clamp technique. Results showed that Teg (at all concentrations tested) did not significantly affect action potential characteristics of atrial myocytes when driven at different rates (0.2, 0.5 and 1 Hz). In contrast, 5HT significantly prolonged action potential duration (1 and 10 nM) and caused cell un-excitability (100 nM). Teg, at the highest concentration tested (100 nM, corresponding to 10 times C(max), produced by the recommended dose of 6 mg b.i.d.) increased the I(f) amplitude and caused a shift of its activation curve. This effect of a high concentration of Teg is not considered clinically relevant. When evaluated on single human atrial cells, Teg does not appear to exhibit arrhythmogenic properties, as it did not affect the action potential profile.
• Italy's Stem-cell Challenge Gaining Momentum Nature. Feb, 2010  |   Pubmed ID: 20148012
• Effects of Chronic Atrial Fibrillation on Active and Passive Force Generation in Human Atrial Myofibrils Circulation Research. Jul, 2010  |   Pubmed ID: 20466979 Chronic atrial fibrillation (cAF) is associated with atrial contractile dysfunction. Sarcomere remodeling may contribute to this contractile disorder.
• Prenatal Exposure to Carbon Monoxide Delays Postnatal Cardiac Maturation Laboratory Investigation; a Journal of Technical Methods and Pathology. Nov, 2010  |   Pubmed ID: 20644522 Prenatal exposure to toxicants, such as maternal smoking, may impair cardiovascular autonomic maturation in infants. We recently showed that exposure of pregnant rats to a mild concentration of carbon monoxide (CO), a component of cigarette smoke, delays postnatal electrophysiological maturation of ventricular myocytes from newborns rats, likely predisposing to life-threatening arrhythmias. To get a comprehensive view of developmental molecular abnormalities induced, at cardiac level, by prenatal CO exposure, we used microarray analysis approach on the rat heart at 4, 7 and 20 days postnatal life. The relationship between molecular and functional alterations was investigated by assessing the ventricular expression of f-current, an electrophysiological marker of immature cardiac phenotype. Rats were prenatally exposed to 0 (CTR) or 150 p.p.m. CO and mRNA obtained from ventricular samples. Differential analysis and biological pathway analysis of microarray data were performed by using Newton's approach and the GENMAPP/MAPPFinder, respectively. The real-time RT-PCR reactions were performed by TaqMan probe-based chemistry. Freshly isolated patch-clamped ventricular cardiomyocytes were used to measure I(f). Genes and pathways controlling cell cycle and excitation-contraction coupling were significantly modified in CO-exposed rats. The higher effect was observed in cardiomyocytes harvested from 7-day-old rats, in which mRNA expression for crucial sarcomeric proteins (myosin and actin subunits, troponin I), transporters (Ca(2+) transporting ATPase) and enzymes (aldolase) were significantly downregulated. Accordingly, the molecular and functional expression of f-channels, which represents a marker of fetal ventricular phenotype, was transiently greater in CO-exposed rats (+200%) than in control ones. In conclusion, our study provides new insights into the molecular and functional mechanisms underlying cardiac maturation and its impairment by prenatal exposure to toxic components of smoking, such as CO.
• Acetaminophen, Via Its Reactive Metabolite N-acetyl-p-benzo-quinoneimine and Transient Receptor Potential Ankyrin-1 Stimulation, Causes Neurogenic Inflammation in the Airways and Other Tissues in Rodents FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Dec, 2010  |   Pubmed ID: 20720158 Acetaminophen [N-acetyl-p-aminophenol (APAP)] is the most common antipyretic/analgesic medicine worldwide. If APAP is overdosed, its metabolite, N-acetyl-p-benzo-quinoneimine (NAPQI), causes liver damage. However, epidemiological evidence has associated previous use of therapeutic APAP doses with the risk of chronic obstructive pulmonary disease (COPD) and asthma. The transient receptor potential ankyrin-1 (TRPA1) channel is expressed by peptidergic primary sensory neurons. Because NAPQI, like other TRPA1 activators, is an electrophilic molecule, we hypothesized that APAP, via NAPQI, stimulates TRPA1, thus causing airway neurogenic inflammation. NAPQI selectively excites human recombinant and native (neuroblastoma cells) TRPA1. TRPA1 activation by NAPQI releases proinflammatory neuropeptides (substance P and calcitonin gene-related peptide) from sensory nerve terminals in rodent airways, thereby causing neurogenic edema and neutrophilia. Single or repeated administration of therapeutic (15-60 mg/kg) APAP doses to mice produces detectable levels of NAPQI in the lung, and increases neutrophil numbers, myeloperoxidase activity, and cytokine and chemokine levels in the airways or skin. Inflammatory responses evoked by NAPQI and APAP are abated by TRPA1 antagonism or are absent in TRPA1-deficient mice. This novel pathway, distinguished from the tissue-damaging effect of NAPQI, may contribute to the risk of COPD and asthma associated with therapeutic APAP use.
• Design, Synthesis, and Preliminary Biological Evaluation of New Isoform-selective F-current Blockers Journal of Medicinal Chemistry. Sep, 2010  |   Pubmed ID: 20795648 New I(f) blockers have been designed and tested on HEK293 cells stably expressing the HCN1, HCN2, and HCN4 channels to find compounds able to discriminate among the channel isoforms. Among the synthesized compounds, the cis-butene derivative (R)-5 shows some preference for HCN2 while the pseudodimeric product (R)-6 shows selectivity for HCN1. These compounds can be important pharmacological tools to study the channels in native tissues and may be useful to design safe drugs.
• Nitric Oxide/reactive Oxygen Species Generation and Nitroso/redox Imbalance in Heart Failure: from Molecular Mechanisms to Therapeutic Implications Antioxidants & Redox Signaling. Jan, 2011  |   Pubmed ID: 20624031 Adaptation of the heart to intrinsic and external stress involves complex modifications at the molecular and cellular levels that lead to tissue remodeling, functional and metabolic alterations, and finally to failure depending upon the nature, intensity, and chronicity of the stress. Reactive oxygen species (ROS) have long been considered as merely harmful entities, but their role as second messengers has gradually emerged. At the same time, our comprehension of the multifaceted role of nitric oxide (NO) and the related reactive nitrogen species (RNS) has been upgraded. The tight interlay between ROS and RNS suggests that their imbalance may implicate the impairment in physiological NO/redox-based signaling that contributes to the failing of the cardiovascular system. This review initially provides basic concepts on the role of nitroso/oxidative stress in the pathophysiology of heart failure with a particular focus on sources of ROS/RNS, their downstream targets, and endogenous modulators. Then, the role of NO/redox regulation of cardiomyocyte function, including calcium homeostasis, electrogenesis, and insulin signaling pathways, is described. Finally, an overview of old and emerging therapeutic opportunities in heart failure is presented, focusing on modulation of NO/redox mechanisms and discussing benefits and limitations.
• Heart Rate Reduction with Ivabradine Prevents the Global Phenotype of Left Ventricular Remodeling American Journal of Physiology. Heart and Circulatory Physiology. Jan, 2011  |   Pubmed ID: 20952661 The aim of this study was to investigate the effect of chronic heart rate (HR) reduction with the hyperpolarization-activated current inhibitor ivabradine on the global phenotype of left ventricular (LV) remodeling in a ligated rat model. Seven days after coronary artery ligation, Wistar rats received ivabradine (10 mg · kg(-1) · day(-1) administered in drinking water) [myocardial infarction + ivabradine (MI+IVA), n = 22] or vehicle only (drinking water) (MI, n = 20) for 90 days. A sham group (n = 20) was included for model validation. MI+IVA rats had 12% lower HR (P < 0.01), improved LV volumes, 15% higher LV ejection fraction (LVEF, P < 0.01) than MI rats, and 33% reductions in both plasma atrial natriuretic peptide (ANP, P = 0.052) and cardiac hydroxyproline. Using patch-clamp, action potential duration was reduced and transient outward current density increased (P < 0.05). Cardiac energy metabolism was also improved (+33% creatine phosphate, P < 0.001; +15% ATP; and +9% energy charge, P < 0.05). Significant correlations were found between HR and parameters of cardiac metabolism, ANP, and LVEF (all P < 0.05). The HR-reducing properties of ivabradine prevent changes in the global phenotype of LV remodeling in the rat, optimize energy consumption, and avoid electrophysiological and structural remodeling.
• The Effect of Losartan Treatment on the Response of Diabetic Cardiomyocytes to ATP Depletion Pharmacological Research : the Official Journal of the Italian Pharmacological Society. Mar, 2011  |   Pubmed ID: 21078391 The present work aimed to investigate the effect of losartan treatment of healthy and diabetic rats on cardiomyocyte response to ATP depletion. Cells were isolated from normoglycemic (N) and streptozotocin-injected (55 mg/kg) rats (D) treated or not treated with losartan (20 mg/kg/day in the drinking water; NL and DL, respectively) for 3 weeks. In each group of cells, enzyme activities such as glucose-6-phosphate (G6PDH) and glycerol-3-phosphate dehydrogenases (G3PDH), lactate/pyruvate, glycogen levels and citrate synthase were measured as an index of glycolytic dysregulation and mitochondrial mass, respectively. Cells were then challenged with NaCN (2 mM) in glucose-free Tyrode solution (metabolic intoxication, MI), a protocol to study ischemia at cell level. Under these conditions, the time to contractile failure up to rigor-type hyper-contracture in field-stimulated cells and K(ATP) current activation by patch-clamp recordings were measured. In comparison with N and NL, D cells presented higher G6PDH and cytoplasmic G3PDH activities, lactate/pyruvate, glycogen content but similar levels of citrate synthase, and decay time of contraction. When subjected to MI, D cells showed delayed activation of the K(ATP) current (25.7±7.1 min; p
• Growth Factor-induced Mobilization of Cardiac Progenitor Cells Reduces the Risk of Arrhythmias, in a Rat Model of Chronic Myocardial Infarction PloS One. 2011  |   Pubmed ID: 21445273 Heart repair by stem cell treatment may involve life-threatening arrhythmias. Cardiac progenitor cells (CPCs) appear best suited for reconstituting lost myocardium without posing arrhythmic risks, being commissioned towards cardiac phenotype. In this study we tested the hypothesis that mobilization of CPCs through locally delivered Hepatocyte Growth Factor and Insulin-Like Growth Factor-1 to heal chronic myocardial infarction (MI), lowers the proneness to arrhythmias. We used 133 adult male Wistar rats either with one-month old MI and treated with growth factors (GFs, n = 60) or vehicle (V, n = 55), or sham operated (n = 18). In selected groups of animals, prior to and two weeks after GF/V delivery, we evaluated stress-induced ventricular arrhythmias by telemetry-ECG, cardiac mechanics by echocardiography, and ventricular excitability, conduction velocity and refractoriness by epicardial multiple-lead recording. Invasive hemodynamic measurements were performed before sacrifice and eventually the hearts were subjected to anatomical, morphometric, immunohistochemical, and molecular biology analyses. When compared with untreated MI, GFs decreased stress-induced arrhythmias and concurrently prolonged the effective refractory period (ERP) without affecting neither the duration of ventricular repolarization, as suggested by measurements of QTc interval and mRNA levels for K-channel α-subunits Kv4.2 and Kv4.3, nor the dispersion of refractoriness. Further, markers of cardiomyocyte reactive hypertrophy, including mRNA levels for K-channel α-subunit Kv1.4 and β-subunit KChIP2, interstitial fibrosis and negative structural remodeling were significantly reduced in peri-infarcted/remote ventricular myocardium. Finally, analyses of BrdU incorporation and distribution of connexin43 and N-cadherin indicated that cytokines generated new vessels and electromechanically-connected myocytes and abolished the correlation of infarct size with deterioration of mechanical function. In conclusion, local injection of GFs ameliorates electromechanical competence in chronic MI. Reduced arrhythmogenesis is attributable to prolongation of ERP resulting from improved intercellular coupling via increased expression of connexin43, and attenuation of unfavorable remodeling.
• Electrophysiological Effects of Ivabradine in Dog and Human Cardiac Preparations: Potential Antiarrhythmic Actions European Journal of Pharmacology. Oct, 2011  |   Pubmed ID: 21821019 Ivabradine is a novel antianginal agent which inhibits the pacemaker current. The effects of ivabradine on maximum rate of depolarization (V(max)), repolarization and spontaneous depolarization have not yet been reported in human isolated cardiac preparations. The same applies to large animals close to human in heart size and spontaneous frequency. Using microelectrode technique action potential characteristics and by applying patch-clamp technique ionic currents were studied. Ivabradine exerted concentration-dependent (0.1-10 μM) decrease in the amplitude of spontaneous diastolic depolarization and reduction in spontaneous rate of firing of action potentials and produced a concentration- and frequency-dependent V(max) block in dog Purkinje fibers while action potential duration measured at 50% of repolarization was shortened. In the presence of ivabradine, at 400 ms cycle length, V(max) block developed with an onset kinetic rate constant of 13.9 ± 3.2 beat(-1) in dog ventricular muscle. In addition to a fast recovery of V(max) from inactivation (τ=41-46 ms) observed in control, a second slow component for recovery of V(max) was expressed (offset kinetics of V(max) block) having a time constant of 8.76 ± 1.34 s. In dog after attenuation of the repolarization reserve ivabradine moderately but significantly lengthened the repolarization. In human, significant prolongation of repolarization was only observed at 10 μM ivabradine. Ivabradine in addition to the Class V antiarrhythmic effect also has Class I/C and Class III antiarrhythmic properties, which can be advantageous in the treatment of patients with ischemic heart disease liable to disturbances of cardiac rhythm.
• Novel Blockers of Hyperpolarization-activated Current with Isoform Selectivity in Recombinant Cells and Native Tissue British Journal of Pharmacology. May, 2012  |   Pubmed ID: 22091830 BACKGROUND AND PURPOSE Selective hyperpolarization activated, cyclic nucleotide-gated channel (HCN) blockers represent an important therapeutic goal due to the wide distribution and multiple functions of these proteins, representing the molecular correlate of f- and h-current (I(f) or I(h) ). Recently, new compounds able to block differentially the homomeric HCN isoforms expressed in HEK293 have been synthesized. In the present work, the electrophysiological and pharmacological properties of these new HCN blockers were characterized and their activities evaluated on native channels. EXPERIMENTAL APPROACH HEK293 cells expressing mHCN1, mHCN2 and hHCN4 isoforms were used to verify channel blockade. Selected compounds were tested on native guinea pig sinoatrial node cells and neurons from mouse dorsal root ganglion (DRG) by patch-clamp recordings and on dog Purkinje fibres by intracellular recordings. KEY RESULTS In HEK293 cells, EC18 was found to be significantly selective for HCN4 and MEL57A for HCN1 at physiological membrane potential. When tested on guinea pig sinoatrial node cells, EC18 (10 µM) maintained its activity, reducing I(f) by 67% at -120 mV, while MEL57A (3 µM) reduced I(f) by 18%. In contrast, in mouse DRG neurons, only MEL57A (30 and 100 µM) significantly reduced I(h) by 60% at -80 mV. In dog cardiac Purkinje fibres, EC18, but not MEL57A, reduced the amplitude and slowed the slope of the spontaneous diastolic depolarization. CONCLUSIONS Our results have identified novel and highly selective HCN isoform blockers, EC18 and MEL57A; the selectivity found in recombinant system was maintained in various tissues expressing different HCN isoforms.
• Action Potential Propagation in Transverse-axial Tubular System is Impaired in Heart Failure Proceedings of the National Academy of Sciences of the United States of America. Apr, 2012  |   Pubmed ID: 22451916 The plasma membrane of cardiac myocytes presents complex invaginations known as the transverse-axial tubular system (TATS). Despite TATS's crucial role in excitation-contraction coupling and morphological alterations found in pathological settings, TATS's electrical activity has never been directly investigated in remodeled tubular networks. Here we develop an ultrafast random access multiphoton microscope that, in combination with a customly synthesized voltage-sensitive dye, is used to simultaneously measure action potentials (APs) at multiple sites within the sarcolemma with submillisecond temporal and submicrometer spatial resolution in real time. We find that the tight electrical coupling between different sarcolemmal domains is guaranteed only within an intact tubular system. In fact, acute detachment by osmotic shock of most tubules from the surface sarcolemma prevents AP propagation not only in the disconnected tubules, but also in some of the tubules that remain connected with the surface. This indicates that a structural disorganization of the tubular system worsens the electrical coupling between the TATS and the surface. The pathological implications of this finding are investigated in failing hearts. We find that AP propagation into the pathologically remodeled TATS frequently fails and may be followed by local spontaneous electrical activity. Our findings provide insight on the relationship between abnormal TATS and asynchronous calcium release, a major determinant of cardiac contractile dysfunction and arrhythmias.
• Mathematical Modelling of the Action Potential of Human Embryonic Stem Cell Derived Cardiomyocytes Biomedical Engineering Online. 2012  |   Pubmed ID: 22929020 Human embryonic stem cell derived cardiomyocytes (hESC-CMs) hold high potential for basic and applied cardiovascular research. The development of a reliable simulation platform able to mimic the functional properties of hESC-CMs would be of considerable value to perform preliminary test complementing in vitro experimentations.
• Late Sodium Current Inhibition Reverses Electromechanical Dysfunction in Human Hypertrophic Cardiomyopathy Circulation. Feb, 2013  |   Pubmed ID: 23271797 Hypertrophic cardiomyopathy (HCM), the most common mendelian heart disorder, remains an orphan of disease-specific pharmacological treatment because of the limited understanding of cellular mechanisms underlying arrhythmogenicity and diastolic dysfunction.
• Molecular and Functional Evidence of HCN4 and Caveolin-3 Interaction During Cardiomyocyte Differentiation from Human Embryonic Stem Cells Stem Cells and Development. Jun, 2013  |   Pubmed ID: 23311301 Maturation of human embryonic stem cell-derived cardiomyocytes (hESC-CM) is accompanied by changes in ion channel expression, with relevant electrophysiological consequences. In rodent CM, the properties of hyperpolarization-activated cyclic nucleotide-gated channel (HCN)4, a major f-channel isoform, depends on the association with caveolin-3 (Cav3). To date, no information exists on changes in Cav3 expression and its associative relationship with HCN4 upon hESC-CM maturation. We hypothesize that Cav3 expression and its compartmentalization with HCN4 channels during hESC-CM maturation accounts for the progression of f-current properties toward adult phenotypes. To address this, hESC were differentiated into spontaneously beating CM and examined at ∼30, ∼60, and ∼110 days of differentiation. Human adult and fetal CM served as references. HCN4 and Cav3 expression and localization were analyzed by real time PCR and immunocyto/histochemistry. F-current was measured in patch-clamped single cells. HCN4 and Cav3 colocalize in adult human atrial and ventricular CM, but not in fetal CM. Proteins and mRNA for Cav3 were not detected in undifferentiated hESC, but expression increased during hESC-CM maturation. At 110 days, HCN4 appeared to be colocalized with Cav3. Voltage-dependent activation of the f-current was significantly more positive in fetal CM and 60-day hESC-CM (midpoint activation, V1/2, ∼ -82 mV) than in 110-day hESC-CM or adult CM (V1/2∼-100 mV). In the latter cells, caveolae disruption reversed voltage dependence toward a more positive or an immature phenotype, with V1/2 at -75 mV, while in fetal CM voltage dependence was not affected. Our data show, for the first time, a developmental change in HCN4-Cav3 association in hESC-CM. Cav3 expression and its association with ionic channels likely represent a crucial step of cardiac maturation.
• Chronic Atrial Fibrillation Alters the Functional Properties of If in the Human Atrium Journal of Cardiovascular Electrophysiology. Dec, 2013  |   Pubmed ID: 23869794 Despite the evidence that the hyperpolarization-activated current (If) is highly modulated in human cardiomyopathies, no definite data exist in chronic atrial fibrillation (cAF). We investigated the expression, function, and modulation of If in human cAF.
• Human Amniotic Fluid Stem Cell Differentiation Along Smooth Muscle Lineage FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Aug, 2013  |   Pubmed ID: 23995291 Functional smooth muscle engineering requires isolation and expansion of smooth muscle cells (SMCs), and this process is particularly challenging for visceral smooth muscle tissue where progenitor cells have not been clearly identified. Herein we showed for the first time that efficient SMCs can be obtained from human amniotic fluid stem cells (hAFSCs). Clonal lines were generated from c-kit(+) hAFSCs. Differentiation toward SM lineage (SMhAFSCs) was obtained using a medium conditioned by PDGF-BB and TGF-β1. Molecular assays revealed higher level of α smooth muscle actin (α-SMA), desmin, calponin, and smoothelin in SMhAFSCs when compared to hAFSCs. Ultrastructural analysis demonstrated that SMhAFSCs also presented in the cytoplasm increased intermediate filaments, dense bodies, and glycogen deposits like SMCs. SMhAFSC metabolism evaluated via mass spectrometry showed higher glucose oxidation and an enhanced response to mitogenic stimuli in comparison to hAFSCs. Patch clamp of transduced hAFSCs with lentiviral vectors encoding ZsGreen under the control of the α-SMA promoter was performed demonstrating that SMhAFSCs retained a smooth muscle cell-like electrophysiological fingerprint. Eventually SMhAFSCs contractility was evident both at single cell level and on a collagen gel. In conclusion, we showed here that hAFSCs under selective culture conditions are able to give rise to functional SMCs.-Ghionzoli, M., Repele, A., Sartiani, L., Costanzi, G., Parenti, A., Spinelli, V., David, A. L., Garriboli, M., Totonelli, G., Tian, J., Andreadis, S. T., Cerbai, E., Mugelli, A., Messineo, A., Pierro, A., Eaton, S., De Coppi, P. Human amniotic fluid stem cell differentiation along smooth muscle lineage.
• Response to Letter Regarding Article, "Late Sodium Current Inhibition Reverses Electromechanical Dysfunction in Human Hypertrophic Cardiomyopathy" Circulation. Sep, 2013  |   Pubmed ID: 24002721
• Altered Calcium Regulation in Isolated Cardiomyocytes from Egr-1 Knock-out Mice Canadian Journal of Physiology and Pharmacology. Dec, 2013  |   Pubmed ID: 24289086 Early growth response-1 one gene (Egr-1), one of the immediate early response genes, plays an important role in the adaptive response of the myocardium to hypertrophic stimuli. We aimed to investigate the effects of Egr-1 deletion on cardiac function. Egr-1 knock-out (Egr-1(-/-)) homozygous mice were employed to evaluate the electrophysiological and molecular properties of left ventricular cardiomyocytes (VCM) by using patch-clamp technique, intracellular calcium measurements, real-time PCR, and Western blot. Action potential was prolonged and diastolic potential was positive-shifted in VCMs isolated from Egr-1(-/-) mice, in comparison with those from their wild-type (WT) littermates. The calcium content of the sarcoplasmic reticulum was reduced and the decay time for steady-state calcium transient slowed down. Serca2, Ryr, L-type Ca(2+)-channel, and PLB mRNA expression were reduced in Egr-1(-/-) mice compared with the controls. Moreover, Serca2 protein was reduced, while the amount of Ncx1 protein was increased in Egr-1(-/-) hearts compared with those of the WT littermates. Furthermore, genes involved in heart development (GATA-4, TGF-β) and in Egr-1 regulation (Nab1, Nab2) were down regulated in Egr-1(-/-) mice. These results suggest that Egr-1 plays a pivotal role in regulating excitation-contraction coupling in cardiac myocytes.
• Regulation of Intracellular Na(+) in Health and Disease: Pathophysiological Mechanisms and Implications for Treatment Global Cardiology Science & Practice. 2013  |   Pubmed ID: 24689024 Transmembrane sodium (Na(+)) fluxes and intracellular sodium homeostasis are central players in the physiology of the cardiac myocyte, since they are crucial for both cell excitability and for the regulation of the intracellular calcium concentration. Furthermore, Na(+) fluxes across the membrane of mitochondria affect the concentration of protons and calcium in the matrix, regulating mitochondrial function. In this review we first analyze the main molecular determinants of sodium fluxes across the sarcolemma and the mitochondrial membrane and describe their role in the physiology of the healthy myocyte. In particular we focus on the interplay between intracellular Ca(2+) and Na(+). A large part of the review is dedicated to discuss the changes of Na(+) fluxes and intracellular Na(+) concentration([Na(+)]i) occurring in cardiac disease; we specifically focus on heart failure and hypertrophic cardiomyopathy, where increased intracellular [Na(+)]i is an established determinant of myocardial dysfunction. We review experimental evidence attributing the increase of [Na(+)]i to either decreased Na(+) efflux (e.g. via the Na(+)/K(+) pump) or increased Na(+) influx into the myocyte (e.g. via Na(+) channels). In particular, we focus on the role of the "late sodium current" (INaL), a sustained component of the fast Na(+) current of cardiac myocytes, which is abnormally enhanced in cardiac diseases and contributes to both electrical and contractile dysfunction. We analyze the pathophysiological role of INaL enhancement in heart failure and hypertrophic cardiomyopathy and the consequences of its pharmacological modulation, highlighting the clinical implications. The central role of Na(+) fluxes and intracellular Na(+) physiology and pathophysiology of cardiac myocytes has been highlighted by a large number of recent works. The possibility of modulating Na(+) inward fluxes and [Na(+)]i with specific INaL inhibitors, such as ranolazine, has made Na(+)a novel suitable target for cardiac therapy, potentially capable of addressing arrhythmogenesis and diastolic dysfunction in severe conditions such as heart failure and hypertrophic cardiomyopathy.