David A. Saint

Validation of a quantitative method for real time PCR kinetics

Real time RT-PCR is the most sensitive method for quantitation of gene expression levels. The accuracy can be dependent on the mathematical model on which the quantitative methods are based. The generally accepted mathematical model assumes that amplification efficiencies are equal at the exponential phase of the reactions for the same amplicon. However, no methods are available to test the assumptions regarding amplification efficiency before one starts the real time PCR quantitation. Here we further develop and test the validity of a new mathematical model which dynamically fits real time PCR data with good correlation (R(2)=0.9995+/-0.002, n=50). The method is capable of measuring cycle-by-cycle PCR amplification efficiencies and demonstrates that these change dynamically. Validation of the method revealed the intrinsic relationship between the initial amount of gene transcript and kinetic parameters. A new quantitative method is proposed which represents a simple but accurate quantitative method.

Hypoxia increases persistent sodium current in rat ventricular myocytes.

1. A persistent inward current activated by depolarization was recorded using the whole‐cell, tight seal technique in rat isolated cardiac myocytes. The amplitude of the inward current increased when cells were exposed to a solution with low oxygen tension. 2. The persistent inward current had the characteristics of the persistent Na+ current described previously in rat ventricular myocytes: it was activated at negative potentials (‐70 mV), reversed close to the equilibrium potential for Na+ (ENa), was blocked by TTX and was resistant to inactivation. 3. Persistent single Na+ channel currents activated by long (200‐400 ms) depolarizations were recorded in cell‐attached patches on isolated ventricular myocytes. Hypoxia increased the frequency of opening of the persistent Na+ channels. 4. Persistent Na+ channels recorded during hypoxia had characteristics similar to those of persistent Na+ channels recorded at normal oxygen tensions. They had a null potential at ENa, their amplitude varied with [Na+], they were resistant to inactivation and their mean open time increased with increasing depolarization. 5. The persistent Na+ channels in cell‐attached patches were blocked by TTX (50 microM) in the patch pipette and by lidocaine (100 microM). 6. It was concluded that hypoxia increases the open probability of TTX‐sensitive, inactivation‐resistant Na+ channels. The voltage dependence of these channels, and their greatly increased activity during hypoxia, suggest that they may play an important role in the generation of arrhythmias during hypoxia.

Atrial remodeling in obstructive sleep apnea: implications for atrial fibrillation.

BACKGROUND There is a known association between obstructive sleep apnea (OSA) and atrial fibrillation (AF); however, how OSA affects the atrial myocardium is not well described. OBJECTIVE To determine whether patients with OSA have an abnormal atrial substrate. METHODS Forty patients undergoing ablation of paroxysmal AF and in sinus rhythm (20 with OSA [apnea-hypopnea index ≥ 15] and 20 reference patients with no OSA [apnea-hypopnea index < 15] by polysomnography) were studied. Multipolar catheters were positioned at the lateral right atrium (RA), coronary sinus, crista terminalis, and RA septum to determine the effective refractory period at 5 sites, conduction time along linear catheters at the RA and the coronary sinus, conduction at the crista terminalis, and sinus node function (corrected sinus node recovery time). Biatrial electroanatomic maps were created to determine the voltage, conduction, and distribution of complex electrograms (duration ≥ 50 ms). RESULTS The groups had no differences in the prevalence of established risk factors for AF. Patients with OSA had the following compared with those without OSA: no difference in effective refractory period (P = .9), prolonged conduction times along the coronary sinus and RA (P = .02), greater number (P = .003) and duration (P = .03) of complex electrograms along the crista terminalis, longer P-wave duration (P = .01), longer corrected sinus node recovery time (P = .02), lower atrial voltage (RA, P <.001; left atrium, P <.001), slower atrial conduction velocity (RA, P = .001; left atrium, P = .02), and more widespread complex electrograms in both atria (RA, P = .02; left atrium, P = .01). CONCLUSION OSA is associated with significant atrial remodeling characterized by atrial enlargement, reduction in voltage, site-specific and widespread conduction abnormalities, and longer sinus node recovery. These features may in part explain the association between OSA and AF.

A persistent sodium current in rat ventricular myocytes.

1. The tight seal, whole‐cell, voltage‐clamp technique was used to record currents from single ventricular myocytes acutely dissociated from adult rat hearts. Subtraction of currents recorded in the presence and absence of tetrodotoxin (TTX, 50 microM) revealed a small, persistent, inward current following a much larger, transient, inward current. 2. Both currents were sodium currents because they reversed close to the sodium equilibrium potential and were depressed when choline was substituted for extracellular sodium. 3. The persistent sodium current could be recorded when the transient current had been inactivated with conditioning depolarization. Only slight inactivation of the persistent current occurred during depolarizing pulses lasting up to 900 ms. 4. A lower concentration of TTX (0.1 microM) blocked the persistent sodium current while having little effect on the transient sodium current. 5. The persistent sodium current was activated at more negative potentials than the transient sodium current. It cannot have been a window current because it was recorded at positive potentials when the transient current was completely inactivated. 6. Because the persistent and transient sodium currents had a different voltage dependence and sensitivity to TTX, it was concluded that different channels are responsible for the two currents.

Inhibition of cardiac sodium currents in adult rat myocytes by n-3 polyunsaturated fatty acids.

1 The acute effects of n‐3 polyunsaturated fatty acids were determined on whole‐cell sodium currents recorded in isolated adult rat ventricular myocytes using patch clamp techniques. 2 The n‐3 polyunsaturated fatty acids docosahexaenoic acid (22:6, n‐3), eicosapentaenoic acid (20:5, n‐3) and α‐linolenic acid (18:3, n‐3) dose‐dependently blocked the whole‐cell sodium currents evoked by a voltage step to −30 mV from a holding potential of −90 mV with EC50 values of 6.0 ± 1.2, 16.2 ± 1.3 and 26.6 ± 1.3 μM, respectively. 3 Docosahexaenoic acid, eicosapentaenoic acid and α‐linolenic acid at 25 μM shifted the voltage dependence of activation of the sodium current to more positive potentials by 9.2 ± 2.0, 10.1 ± 1.1 and 8.3 ± 0.9 mV, respectively, and shifted the voltage dependence of inactivation to more negative potentials by 22.3 ± 0.9, 17.1 ± 3.7 and 20.5 ± 1.0 mV, respectively. In addition, the membrane fluidising agent benzyl alcohol (10 mM) shifted the voltage dependence of activation to more positive potentials by 7.8 ± 2.5 mV and shifted the voltage dependence of inactivation to more negative potentials (by −24.6 ± 3.6 mV). 4 Linoleic acid (18:2, n‐6), oleic acid (18:1, n‐9) and stearic acid (18:0) were either ineffective or much less potent at blocking the sodium current or changing the voltage dependence of the sodium current compared with the n‐3 fatty acids tested. 5 Docosahexaenoic acid, eicosapentaenoic acid, α‐linolenic acid and benzyl alcohol significantly increased sarcolemmal membrane fluidity as measured by fluorescence anisotropy (steady‐state, rss, values of 0.199 ± 0.004, 0.204 ± 0.006, 0.213 ± 0.005 and 0.214 ± 0.009, respectively, compared with 0.239 ± 0.002 for control), whereas stearic, oleic and linoleic acids did not alter fluidity (the rss was not significantly different from control). 6 The potency of the n‐3 fatty acids docosahexaenoic acid, eicosapentaenoic acid and α‐linolenic acid to block cardiac sodium currents is correlated with their ability to produce an increase in membrane fluidity.

The cardiac persistent sodium current: an appealing therapeutic target?

The sodium current in the heart is not a single current with a mono‐exponential decay but rather a mixture of currents with different kinetics. It is not clear whether these arise from distinct populations of channels, or from modulation of a single population. A very slowly inactivating component, [INa(P)] INa(P) is usually about 1% of the size of the peak transient current [INa(T)], but is enhanced by hypoxia. It contributes to Na+ loading and cellular damage in ischaemia and re‐perfusion, and perhaps to ischaemic arrhythmias. Class I antiarrhythmic agents such as flecainide, lidocaine and mexiletine generally block INA(P) more potently than block of INa(T) and have been used clinically to treat LQT3 syndrome, which arises because mutations in SCN5A produce defective inactivation of the cardiac sodium channel. The same approach may be useful in some pathological situations, such as ischaemic arrhythmias or diastolic dysfunction, and newer agents are being developed with this goal. For example, ranolazine blocks INa(P) about 10 times more potently than INa(T) and has shown promise in the treatment of angina. Alternatively, the combination of INa(P) block with K+ channel block may provide protection from the induction of Torsades de Pointe when these agents are used to treat atrial arrhythmias (eg Vernakalant). In all of these scenarios, an understanding of the role of INa(P) in cardiac pathophysiology, the mechanisms by which it may affect cardiac electrophysiology and the potential side effects of blocking INa(P) in the heart and elsewhere will become increasingly important.

Dietary fish oil protects against stretch-induced vulnerability to atrial fibrillation in a rabbit model.

Introduction: Dietary fish oil is thought to reduce sudden cardiac death by suppressing ventricular arrhythmias but little is known about its impact on atrial arrhythmias. We examined the effect of dietary fish oil on the rabbit model of stretch‐induced vulnerability to atrial fibrillation (AF).

The Role of the Persistent Na+ Current During Cardiac Ischemia and Hypoxia

Introduction: Inadequate or zero cardiac perfusion, if prolonged beyond about 10 minutes, can result in irreversible cell damage. Paradoxically, much of this damage occurs when perfusion is restored, and this appears to be linked to an uncontrolled rise in intracellular calcium. This article reviews the causes of this rise in calcium.

Effects of lignocaine and quinidine on the persistent sodium current in rat ventricular myocytes.

1 The effects of the Class 1 antiarrhythmic agents lignocaine and quinidine on action potentials, and on sodium currents and potassium currents activated by depolarization, were examined in rat isolated ventricular myocytes by the whole cell, tight seal recording technique. 2 Tetrodotoxin and lignocaine shortened, whereas quinidine prolonged, the duration of the plateau phase of action potentials. 3 At low concentrations, lignocaine and quinidine blocked a persistent sodium current that was resistant to inactivation but they had only a small effect on the transient sodium current. At higher concentrations, they also blocked the transient sodium current. 4 Quinidine, but not tetrodotoxin or lignocaine, depressed potassium currents activated by depolarization and this could account for the prolongation of the plateau phase caused by quinidine. 5 It is suggested that block of the persistent sodium current may be responsible, at least in part, for the antiarrhythmic action of lignocaine and quinidine.

GENE EXPRESSION OF STRETCH‐ACTIVATED CHANNELS AND MECHANOELECTRIC FEEDBACK IN THE HEART

1 Mechanoelectric feedback (MEF) in the heart is the process by which mechanical forces on the myocardium can change its electrical properties. Mechanoelectric feedback has been demonstrated in many animal models, ranging from isolated cells, through isolated hearts to whole animals. In humans, MEF has been demonstrated directly in both the atria and the ventricles. It seems likely that MEF provides either the trigger or the substrate for some types of clinically important arrhythmias. 2 Mechanoelectric feedback may arise because of the presence of stretch‐sensitive (or mechano‐sensitive) ion channels in the cell membrane of the cardiac myocytes. Two types have been demonstrated: (i) a non‐specific cation channel (stretch‐activated channel (SAC); conductance of approximately 25 pS); and (ii) a potassium channel with a conductance of approximately 100 pS. The gene coding for the SAC has not yet been identified. The gene for the potassium channel is likely to be TREK, a member of the tandem pore potassium channel gene family. We have recorded stretch‐sensitive potassium channels in rat isolated myocytes that have the properties of TREK channels expressed in heterologous systems. 3 It has been shown that TREK mRNA is expressed heterogeneously in the rat ventricular wall, with 17‐fold more expression in endocardial compared with epicardial cells. This difference is reflected in the TREK currents recorded from endocardial and epicardial cells using whole‐cell patch‐clamp techniques, although the difference in current density was less pronounced (approximately threefold). Consistent with this, we show here that when the ventricle is stretched by inflation of an intraventricular balloon in a Langendorff perfused rat isolated heart, action potential shortening was more pronounced in the endocardium (30% shortening at 40 mmHg) compared with that in the epicardium (10% shortening at the same pressure). 4 Computer models of the mechanics of the (pig) heart show pronounced spatial variations in strain in the myocardium with large transmural differences (in the left ventricle in particular) and also large differences between the base and apex of the ventricle. 5 The importance of MEF and the non‐homogeneous gene expression and strain distribution for arrhythmias is discussed.