Arrhythmia susceptibility in a rat model of acute atrial dilation.

Atrial fibrillation (AF) is the most common cardiac arrhythmia, associated with an increased risk of stroke and heart failure. Acute AF occurs in response to sudden increases of atrial hemodynamic load, leading to atrial stretch. The mechanisms of stretch-induced AF were investigated in large mammals with controversial results. We optimized an approach to monitor rat atrial electrical activity using a red-shifted voltage sensitive dye (VSD). The methodology includes cauterization of the main ventricular coronary arteries, allowing improved atrial staining by the VSD and appropriate atrial perfusion for long experiments. Next, we developed a rat model of acute biatrial dilation (ABD) through the insertion of latex balloons into both atria, which could be inflated with controlled volumes. A chronic model of atrial dilation (spontaneous hypertensive rats; SHR) was used for comparison. ABD was performed on atria from healthy Wistar-Kyoto (WKY) rats (WKY-ABD). The atria were characterized in terms of arrhythmias susceptibility, action potential duration and conduction velocity. The occurrence of arrhythmias in WKY-ABD was significantly higher compared to non-dilated WKY atria. In WKY-ABD we found a reduction of conduction velocity, similar to that observed in SHR atria, while action potential duration was unchanged. Low-dose caffeine was used to introduce a drop of CV in WKY atria (WKY-caff), quantitatively similar to the one observed after ABD, but no increased arrhythmia susceptibility was observed with caffeine only. In conclusion, CV decrease is not sufficient to promote arrhythmias; enlargement of atrial surface is essential to create a substrate for acute reentry-based arrhythmias.

Selective HCN1 block as a strategy to control oxaliplatin-induced neuropathy.

Chemotherapy-Induced Peripheral Neuropathy (CIPN) is the most frequent adverse effect of pharmacological cancer treatments. The occurrence of neuropathy prevents the administration of fully-effective drug regimen, affects negatively the quality of life of patients, and may lead to therapy discontinuation. CIPN is currently treated with anticonvulsants, antidepressants, opioids and non-opioid analgesics, all of which are flawed by insufficient anti-hyperalgesic efficacy or addictive potential. Understandably, developing new drugs targeting CIPN-specific pathogenic mechanisms would dramatically improve efficacy and tolerability of anti-neuropathic therapies. Neuropathies are associated to aberrant excitability of DRG neurons due to the alteration in the expression or function of a variety of ion channels. In this regard, Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are overexpressed in inflammatory and neuropathic pain states, and HCN blockers have been shown to reduce neuronal excitability and to ameliorate painful states in animal models. However, HCN channels are critical in cardiac action potential, and HCN blockers used so far in pre-clinical models do not discriminate between cardiac and non-cardiac HCN isoforms. In this work, we show an HCN current gain of function in DRG neurons from oxaliplatin-treated rats. Biochemically, we observed a downregulation of HCN2 expression and an upregulation of the HCN regulatory beta-subunit MirP1. Finally, we report the efficacy of the selective HCN1 inhibitor MEL57A in reducing hyperalgesia and allodynia in oxaliplatin-treated rats without cardiac effects. In conclusion, this study strengthens the evidence for a disease-specific role of HCN1 in CIPN, and proposes HCN1-selective inhibitors as new-generation pain medications with the desired efficacy and safety profile.

First Evidence of Cardiac Stem Cells From the Left Ventricular Apical Tip in Patients With Left Ventricular Assist Device Implantation.

BACKGROUND: Recent studies have challenged the dogma that the adult heart is a postmitotic organ and raise the possibility of the existence of resident cardiac stem cells (CSCs). Our study aimed to explore if these CSCs are present in the "ventricular tip" obtained during left ventricular assist device (LVAD) implantation from patients with end-stage heart failure (HF) and the relationship with LV dysfunctional area extent. METHODS: Four consecutive patients with ischemic cardiomyopathy and end-stage HF submitted to LVAD implantation were studied. The explanted "ventricular tip" was used as a sample of apical myocardial tissue for the pathological examination. Patients underwent clinical and echocardiographic examination, both standard transthoracic echocardiography (TTE) and speckle tracking echocardiography (STE), before LVAD implantation. RESULTS: All patients presented severe apical dysfunction, with apical akinesis/diskinesis and very low levels of apical longitudinal strain (-3.5 ± 2.9%). Despite this, the presence of CSCs was demonstrated in pathological myocardial samples of "ventricular tip" in all 4 of the patients. It was found to be a mean of 6 c-kit cells in 10 fields magnification 40×. CONCLUSIONS: Cardiac stem cells can be identified in the LV apical segment of patients who have undergone LVAD implantation despite LV apical fibrosis.

Functional coupling of angiotensin II type 1 receptor with insulin resistance of energy substrate uptakes in immortalized cardiomyocytes (HL-1 cells).

BACKGROUND AND PURPOSE: Increased angiotensin II levels and insulin resistance coexist at the early stages of cardiomyopathies. To determine whether angiotensin II increases insulin resistance in cardiomyocytes, we studied the effect of angiotensin II on basal and insulin-stimulated transport rate of energy substrates in immortalized cardiomyocytes (HL-1 cells). EXPERIMENTAL APPROACH: Glucose and palmitic acid uptakes were measured using [(3)H]2-deoxy-D-glucose and [(14)C]palmitic acid, respectively, in cells exposed or not exposed to angiotensin II (100 nM), angiotensin II plus irbesartan or PD123319, type 1 and 2 receptor antagonists, or PD98059, an inhibitor of ERK1/2 activation. Cell viability, DNA, protein synthesis and surface area were evaluated by the MTT test, [(3)H]thymydine, [(3)H]leucine and morphometric analysis, respectively. Type 1 receptor levels were measured by western blot analysis. KEY RESULTS: Basal uptakes of glucose and palmitic acid by HL-1 cells (0.37+/-0.07 and 7.31+/-0.22 pmol per 10(4)cells per min, respectively) were both stimulated by 100 nM insulin (+91 and +64%, respectively). Cells exposed to angiotensin II remained viable and did not show signs of hypertrophy. In these conditions, the basal palmitic acid uptake of the cells increased (11.41+/-0.46 pmol per 10(4) cells per min) and insulin failed to stimulate the uptake of glucose and fatty acids. Changes in the rate of uptake of energy substrates were prevented or significantly reduced by irbesartan or PD98059. CONCLUSIONS AND IMPLICATIONS: Angiotensin II is a candidate for increasing insulin resistance in cardiomyocytes. Our results suggest a further mechanism for the cardiovascular protection offered by the angiotensin II type 1 receptor blockers.

The properties of the pacemaker current I(F)in human ventricular myocytes are modulated by cardiac disease.

The pacemaker current I(f)is present in ventricular myocytes from the human failing heart where it may contribute to arrhythmogenesis. The role of cardiac disease in the modulation of I(f)expression is still uncertain. We studied the functional expression and properties of I(f)in human ventricular myocytes isolated from control donor hearts or from explanted failing hearts of patients with ischemic and dilated cardiomyopathy. In patch-clamped cells, I(f)was elicited by hyperpolarization. Membrane capacitance (C(m)) was significantly higher in dilated cardiomyopathy than in control or ischemic cardiomyopathy. I(f)was present in all ischemic and dilated cardiomyopathy tested cells and in 76% of control cells. In ischemic and dilated cardiomyopathy, I(f)amplitude measured at -120 mV was significantly greater than in control. However, I(f)density (i.e. current normalized to C(m)) was significantly higher in ischemic cardiomyopathy (2.0+/-0.2 pA/pF) than in dilated cardiomyopathy (1.2+/-0.1 pA/pF) or control (1.0+/-0.1 pA/pF). In diseased hearts, the activation curve was significantly shifted to more positive values compared to control. The slope of the fully-activated I-V relations was greater in ischemic cardiomyopathy than in dilated cardiomyopathy or control (P<0.05) while the intercept with the x -axis (V(rev)) was similar. In conclusion, I(f)is overexpressed in human ventricular myocytes from failing hearts; its functional expression seems related to the etiology of the disease, being higher in ischemic than in dilated cardiomyopathy, and not to the degree of cell hypertrophy.

Electrophysiologic effects of lercanidipine on repolarizing potassium currents.

Blockade of cardiac repolarizing potassium channels by drugs may result in QT-interval prolongation, eventually degenerating into "torsades de pointes," a life-threatening arrhythmia. Lercanidipine (LER) is a recently introduced lipophilic calcium antagonist with no cardiodepressant activity and long-lasting antihypertensive action. Its chemical structure is characterized by the presence of a diphenylpropylaminoalkyl group, which is present in some of the drugs that have been reported to cause QT-interval prolongation. Our previous data demonstrated that LER blocks L-type calcium channels without affecting sodium current; however, no data are available concerning its effects on cardiac potassium channels. Transient outward (I(to)), delayed rectifier (I(K)), background currents, and action potential (AP) profile were measured from patch-clamped ventricular myocytes isolated from rat, guinea pig, or human hearts using enzymatic dissociation procedures. LER did not affect I(K) (and I(Kr)) density and activation curve in guinea pig myocytes; the reversal potential of the background current (I(K1)) and its slope were not changed by the drug. Maximal diastolic potential (MDP) and duration of the AP measured at -60 mV (APD(-60)) were not significantly changed. I(to) density and activation curves measured in rat myocytes were similar in the absence and presence of 1 or 10 microM LER. Finally, the effect of LER was tested in human ventricular myocytes: superfusion with 1 microM LER did not affect MDP and APD(-60). I(to) density and the midpoint of activation and inactivation curves were similar in the absence and presence of LER. In conclusion, our data demonstrate that LER does not affect repolarizing potassium currents and action potential profile recorded from guinea pig, rat, and human ventricular myocytes. It is unlikely that LER could cause QT prolongation in vivo.

Semicarbazide-sensitive amine oxidases in heart and bovine serum.

In guinea pig dorsal skin the semicarbazide-sensitive amine oxidase (SSAO) is localised in fibroblasts. Fibroblasts in culture lose the ability to express this enzymatic activity with doublings, thus suggesting that the SSAO expression needs some factors which are not present in the 10% bovine serum culture medium. Fresh bovine serum of adult animals contains two SSAO activities, one with high affinity for benzylamine and one with lower affinity. The enzyme with lower affinity for benzylamine was identified as spermine oxidase, the oxidation of [14C]-benzylamine was inhibited by semicarbazide, alpha-aminoguanidine and B24, a specific inhibitor of benzylamine oxidase and spermine oxidase, both SSAO enzymes. The enzymatic activity of bovine serum was partially purified, the kinetic properties and sensitivity to inhibitors studied. A mathematical procedure for the analysis of the kinetics resulting from the activity of two enzymes acting on the same substrate seems to give better results than the methods previously described.

Isolated cardiac cells for electropharmacological studies.

Single cardiac myocytes provide a model widely used to characterize the electrophysiological properties of drugs and to identify new therapeutic targets. This review focuses on isolation procedures to obtain single cardiac myocytes from several mammal species, including humans, and on patch-clamp technique as a useful method to investigate the molecular mechanism of druy actions.

Long-term treatment of spontaneously hypertensive rats with losartan and electrophysiological remodeling of cardiac myocytes.

OBJECTIVE: Cardiac hypertrophy due to pressure overload is associated with several cellular electrophysiological alterations such as prolongation of action potential duration (APD), decrease in transient outward current (Ito) and occurrence of the pacemaker current I(f). These alterations may play a role in sudden arrhythmic death, which is a major risk factor in myocardial hypertrophy and failure. Since angiotensin II is a key signal for myocyte hypertrophy, we tested if an 8-week treatment of old spontaneously hypertensive rats (SHR) with the antagonist of type-1 angiotensin II receptor (AT1), losartan (10 mg/kg/day), was able to influence the cellular electrophysiologic remodeling associated with cardiac hypertrophy. METHODS: Left ventricular myocytes were isolated from control (CTR) or losartan-treated (LOS) 18-month old SHR. Patch-clamped LVM were superfused with a normal Tyrode's solution (to measure action potential) or appropriately modified Tyrode's solution (to measure Ito and I(f)). RESULTS: Heart weight to body weight ratio (HW/BW) was significantly smaller in LOS (5.69 +/- 0.25 mg/g) than in CTR rats (6.67 +/- 0.37 mg/g; P < 0.05). Membrane capacitance, an index of cell size, was significantly reduced in LOS (342 +/- 12, n = 92) vs. CTR (422 +/- 14 pF, n = 96, P < 0.001). APD was significantly shorter in LOS than in CTR (at -60 mV: 197 +/- 23 vs. 277 +/- 19 ms, n = 28, P < 0.001); this effect was paralleled by a larger maximum Ito density in the LOS group (LOS: 15.1 +/- 1.4 pA/pF, CTR: 10.0 +/- 0.8 pA/pF) (n = 27, P < 0.02). I(f), elicited by hyperpolarizing steps (range: -60 to -130 mV), was consistently recorded in SHR cells; however, its maximal specific conductance was significantly lower in LOS than in CTR rats (28.6 +/- 3.6 vs. 54.2 +/- 8.0 pS/pF, n = 55, P < 0.001). Voltage of half-maximal activation (V1/2) of both Ito and I(f) was unchanged by the treatment. CONCLUSIONS: AT1 receptor blockade with losartan prevents the development of myocyte hypertrophy and associated electrophysiological alterations in old SHR.

Influence of postnatal-development on I(f) occurrence and properties in neonatal rat ventricular myocytes.

OBJECTIVE: I(f) is a hyperpolarization-activated current, which plays a key role in determining the spontaneous rate of cardiac pacemaker cells. We have previously shown that I(f) is also expressed in left ventricular myocytes isolated from spontaneously hypertensive rats; in these cells, its occurrence and density is linearly related with the severity of myocardial hypertrophy. Since hypertrophy induces a re-expression of genes encoding fetal proteins, we investigated changes in I(f) properties during post-natal development. METHODS: Fresh ventricular myocytes were enzymatically isolated from the heart of 1-2- to 28-day-old Wistar rats. The whole-cell configuration of the patch-clamp technique was employed to record the action potential and I(f). RESULTS: Membrane capacitance, an index of cell size, progressively increased from 13 +/- 1 pF at 1-2 days to 66 +/- 4 pF at 28 days of age (p < 0.01). At 1-2 days, a cesium-sensitive hyperpolarization-activated inward current (I(f)) was recorded in the majority of tested cells (n = 51). The midpoint of the activation curve (V1/2) was -78 +/- 2 mV (n = 32), and specific current conductance of fully activated I(f) (gf.max) was 60 +/- 11 pS/pF. Reversal potential (Vrev) measured by tail-current analysis was -24 +/- 3 mV (n = 8). Reduction of extracellular Na+ from 140 to 35 mM or extracellular K+ from 25 to 5.4 mM caused a shift of -12 +/- 1 mV (n = 3) or -11 +/- 2 mV (n = 5) of Vrev, respectively. Occurrence of I(f) decreased with aging, being present in 64%, 48% and 32% of cells at 10, 15 and 28 days, respectively. When present, I(f) density was significantly smaller than at 1-2 days (p < 0.05), reaching a value of 8 +/- 2 pS/pF at 28 days. However, V1/2 did not change in the older rats, being -80 +/- 2, -83 +/- 4 and -85 +/- 3 mV at 10, 15 and 28 days, respectively. Vrev at 10 and 15 days was -27 and -28 mV, respectively, thus suggesting that channel selectivity did not change. CONCLUSIONS: The pacemaker current, I(f), is expressed in ventricular myocytes from neonatal rats and progressively disappears; when present, it shows electrophysiological properties similar to I(f) re-expressed in hypertrophied adult rat ventricular myocytes. Thus, it is likely that the occurrence of I(f) in ventricular myocytes of hypertrophied and failing hearts is due to the re-expression of a fetal gene.