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J Physiol (2003), 553.2, pp. 395-405
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.041954
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ABSTRACT |
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Hyperpolarization-activated inward current (If) and changes in the messenger RNA (mRNA) expression levels of hyperpolarization-activated cyclic nucleotide-gated channel (HCN)2 and HCN4 encoding If channels of the rat heart were studied in control and hypertrophied myocytes isolated from three ventricular regions: the septum (S), the left ventricular free wall (LV) and the right ventricular free wall (RV). Electrophysiological experiments were conducted by ruptured and perforated-patch clamp techniques and quantification of mRNA levels was carried out by quantitative reverse transcriptase polymerase chain reaction. The occurrence, density and maximal specific conductance of If were found to be significantly higher in hypertrophied ventricular myocytes isolated from S and LV than in those isolated from RV or sham-operated rats. Half-maximal activation potential, the slope of the activation curve and the threshold for activation were similar in ventricular myocytes from sham and aortic stenosed rats in the three regions studied. Isoproterenol 1 µmol l-1 increased current size by shifting current activation to more positive potentials in both sham and hypertrophied myocytes. When we studied the mRNA levels of If channel isoforms present in the ventricle, we found a significant increase of HCN2 and HCN4 mRNA levels in hypertrophied myocytes from S and LV but not in RV. We conclude that the occurrence, density and conductance of If is higher in hypertrophied than in control ventricular myocytes, S being the region where all these changes were most evident. These findings are associated with a higher expression of HCN2 and HCN4 mRNA levels in the two regions that developed hypertrophy.
(Received 19 February 2003; accepted after revision 24 September 2003; first published online 26 September 2003)
Corresponding author C. Delgado: Institute of Pharmacology and Toxicology (CSIC-UCM), School of Medicine, Universidad Complutense, 28040 Madrid, Spain. Email: cdelgado{at}med.ucm.es
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INTRODUCTION |
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Cardiac hypertrophy is regarded as a complication common to a number of cardiovascular pathologies (hypertension, valvular disorders, myocardial infarction, cardiomyopathies); it arises as a compensatory mechanism but in the long term may lead to the onset of serious cardiovascular diseases, including heart failure and sudden death (Kannel et al. 1969; Katz, 1991). Many of the modifications that occur in the hypertrophic heart, such as fibrosis, the loss of functional myocytes or the dispersal of the refractory period, give rise to arrythmias (Levy et al. 1987). Alterations of the electrophysiological properties in hypertrophic hearts have also been reported (Hart, 1994; Tomaselli & Marban, 1999), with a longer duration of the action potential in different animal models (Hart, 1994) and in human heart failure (Tomaselli et al. 1994). This seems to be caused by the reduction of the transient outward potassium current (Ito), which in physiological conditions is heterogeneously distributed in the ventricle. The Ito density has been shown to be higher in the epicardium than in the endocardium of several species (Litovsky & Antzelevitch, 1988; Benitah et al. 1993; Nabauer et al. 1996). In the course of hypertrophy development, the decrease of the outward currents and the prolongation of the action potential duration might participate in the genesis of grave arrhythmias by similar mechanisms to those proposed for long-QT syndrome (Antzelevitch & Fish, 2001). In addition, several reports consider that the pacemaker If could also contribute to the appearance of arrhythmias in hypertrophic hearts (Cerbai et al. 1996, 2001; Stilli et al., 2001). If is known to be present not only in regions with primary or secondary automatism (DiFrancesco, 1995), but also in non-automatic regions such as ventricular myocytes isolated from guinea-pigs (Yu et al. 1993), dogs (Yu et al. 1995), rats (Cerbai et al. 1994; Robinson et al. 1997) and humans (Hoppe et al. 1998), the density being higher in hypertrophied rat ventricular myocytes (Cerbai et al. 1996; Stilli et al. 2001) and in human ventricular myocytes from failing hearts (Hoppe et al. 1998; Cerbai et al. 2001). The recent cloning of the pacemaker channels has given rise to a new family of genes known as HCN (hyperpolarization-activated cyclic nucleotide-gated channels) (Gauss et al. 1998; Ludwig et al. 1998; Santoro et al. 1998). At least four isoforms of this family are known in mammals, and three of them represent the molecular correlate of If in the heart (HCN1, HCN2, HCN4). The currents associated with the expression of HCN1, HCN2 and HCN4 are activated by hyperpolarization, but they have different kinetic modes of activation and deactivation. This finding suggests that the different parts of the heart may express these isoforms in different degrees. In the sinoatrial node for example, the most prominently expressed HCN channel is HCN4, and HCN2 and HCN1 are expressed to a lesser extent (Moosmang et al. 2001). Earlier studies (Shi et al. 1999) showed that the ventricles of both neonatal and adult rats express HCN2 and HCN4, although the HCN2/HCN4 mRNA ratio was greater in the adult ventricle. Expression of these two isoforms in cardiac hypertrophy has hardly been explored (Hiramatsu et al. 2002). So far, no studies have been published in which mRNA levels of HCN isoforms and changes of If in cardiac hypertrophy are considered in combination, nor has there been any study of the regional distribution of If in the control and the hypertrophied ventricles. Our aim was to determine the pattern of distribution and the characteristics of the If in the myocytes from control hearts in three anatomical regions of the rat ventricle: the septum (S), the free wall of the left ventricle (LV) and the free wall of the right ventricle (RV), and to compare them with the data from hypertrophied hearts. At the same time, we wished to investigate the possible changes in HCN2 and HCN4 mRNA levels encoding rat If channels in the same hypertrophied myocytes.
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METHODS |
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Hypertrophic model
The animal experimental procedures and care facility were approved by the Bioethical Committee of the Spanish Council for Scientific Research (CSIC).
Left ventricular (LV) hypertrophy was induced by an aortic stenosis technique, similar to that used previously in our laboratory (Gomez et al. 1997; Martinez et al. 1999). Adult Sprague-Dawley rats weighing 180-200 g underwent overnight fasting before surgery. Anaesthesia was induced by an intraperitoneal injection of sodium pentobarbital (50 mg kg-1). The abdominal aorta was isolated above the left renal artery, and a silver clip (0.3 mm aperture) was placed around it. Slow progressive pressure in the LV developed as the animal grew. Each rat that had been operated on was paired with a sham-operated one of the same weight and age. Established LV hypertrophy was obtained at 7-8 weeks after surgery. Animals were monitored after surgery until recovery from anaesthesia. Postoperative care included antibiotic therapy for 3 days after surgery and regular monitoring of weight.
The total wet weight of the heart was used to estimate the degree of hypertrophy. Only hypertrophied hearts with a heart weight-to-body weight ratio greater than 25 % of control hearts were selected.
Cell isolation
Isolation of single cardiac myocytes was performed as previously described (Martinez et al. 1999). Adult male Sprague-Dawley rats were heparinized (4 i.u. g-1 I.P.) and anaesthetized with sodium pentobarbital (50 mg kg-1). The hearts were removed and mounted on a Langendorff-perfusion apparatus. The ascending aorta was cannulated and a retrograde perfusion was set up. The hearts were successively perfused with the following oxygenated solutions at 35-36 °C: (1) standard nominally Ca2+-free Tyrode solution (3 min), (2) standard nominally Ca2+-free Tyrode solution (15 min (g heart weight)-1) containing 60 i.u. ml-1 of collagenase type II (Worthington) and 1 mg ml-1 bovine serum albumin (BSA, Sigma). The hearts were removed from the Langendorff apparatus, and after removal of atria, the ventricles were carefully dissected into three anatomic parts: the septum (S), the LV free wall (LV) and the right ventricular free wall (RV). Each portion was cut off and gently shaken for 3 min in a standard Tyrode solution containing 0.1 mmol l-1 CaCl2 to disperse the isolated cells. The resulting cell suspensions were filtered through a 250 µm nylon mesh and centrifuged for 4 min at 20 g. Finally, the cell pellets were divided into two fractions, one was suspended in a storage solution containing 0.1 mmol l-1 CaCl2 and 2 mg ml-1 BSA and stored at room temperature until use for electrophysiological experiments (within 5 h of the isolation) and the other fraction was stored immediately at -80 °C for later extraction of the total RNA.
Solutions
The standard nominally Ca2+-free Tyrode solution contained (mmol l-1): 10 Hepes, 10 glucose, 20 taurine, 120 NaCl, 1.2 KH2PO4, 10 KCl and 1.2 MgCl2, with the pH adjusted to 7.2 with NaOH. The storage solution (mmol l-1): 130 NaCl, 5.4 KCl, 0.4 NaH2PO4, 0.5 MgCl2, 25 Hepes, 5 NaHCO3, 22 glucose, with the pH adjusted to 7.4 with NaOH. The If recording solution (mmol l-1): 140 NaCl, 5.4 KCl, 1 MgCl2, 5 BaCl2, 2 CoCl2, 0.5 4-aminopyridine, 10 glucose, 5 Hepes and 1.8 CaCl2, with the pH adjusted to 7.4 with NaOH. In most of the experiments, [K+]o was increased to 20 mmol l-1 and [Na+]o was adjusted equimolarly.
The recording pipettes contained (mmol l-1): 11 EGTA, 130 potassium aspartate, 2 MgCl2, 5 Na2ATP, 5 CaCl2 and 10 Hepes, with the pH adjusted to 7.2 with KOH. Some experiments were performed using the perforated-patch technique, and the pipette internal solution contained (mmol l-1): 130 potassium aspartate, 2 MgCl2, 5 Na2ATP, 1 CaCl2 and 10 Hepes with the pH adjusted to 7.2 with KOH. To obtain perforated-patch configuration, the pipettes also contained 50-150 µg ml-1 amphotericin B (Sigma), which was prepared just before the experiment. The tip of each pipette was filled with recording solution free of amphotericin B. Once the gigaseal was performed, the access resistance stabilized at 8-12 M
within 15-30 min of the formation of the G seal.
In some experiments we investigated the effect of Cs+ on If, adding CsCl (4 mmol l-1) to the recording solution which contained 20 mmol l-1 K+.
To study the 
-adrenergic modulation of If, isoproterenol 1 µmol l-1 (Sigma) was added to the external solution and a perforated-patch clamp was used.
Electrophysiological measurements
If was recorded by ruptured- and perforated-patch clamp techniques. The voltage clamp circuit was provided by an Axopatch-1D amplifier with a 100 M feedback resistance (Headstage CV-4 1/100, Axon Instruments, Foster City, CA, USA) controlled by a computer (IBM-PC AT) equipped with the appropriate software (pClamp, version 6.0, Axon Instruments) and connected to the amplifier by a 125 kHz Labmaster board.
The recording pipettes were made from 1.5-1.6 mm o.d. soft-glass capillary tubing with a microprocessor-based patch pipette puller (P97/PC, Sutter Instruments), and when filled with internal solution, their tip resistance ranged from 0.9 to 1.2 M for the whole-cell configuration and 2 M for the perforated configuration of the patch clamp technique.
The resistances ranged from 3 to 6 M before compensation (60-80 % of the series resistance was compensated) during whole-cell experiments. The temperature used to record If was maintained at 36 ± 0.5 °C.
If was elicited by hyperpolarizing pulses (from -60 to -130 mV) from a holding potential of -40 mV. The duration of the step was decreased 200 ms, from 3 s at -60 mV to 1.6 s at -130 mV as the activation of the current became progressively faster. Following the hyperpolarizing steps, the cell was clamped to +20 mV for 400 ms and then to the holding potential. Steps were applied at low frequency (maximum rate, 0.1 Hz) and sampled at 1-2 kHz. The size of the If was measured as the difference between the instantaneous current at the beginning of the hyperpolarizing step and the steady-state current at the end of hyperpolarization.
Current density was calculated from the current amplitude normalized by the membrane capacitance (Cm). Cm was elicited by applying ±10 mV voltage steps from the resting potential and was calculated according to the equation:
Cm = 
cI0/
Em(1 - (I
/I0)),
in which c is the time constant of the membrane capacitance, I0 is the maximum capacitance current, Em is the amplitude of the voltage step and I is the amplitude of the steady-state current.
The specific conductance of If was determined for each cell according to the equation:
g = I/(Vm - Vrev),
where g is the conductance calculated at the membrane potential Vm, I is the current amplitude and Vrev is the reversal potential calculated from the analysis of tail currents.
The maximal specific conductance (gmax) was obtained from a Boltzmann distribution fit according to the equation:
g = gmax/{1 + exp((V50 - Vm)/k)},
where g is the conductance calculated at membrane potential (Vm), V50 is the voltage at half-maximal activation and k is the slope of the curve.
Tail current amplitudes were used to evaluate current reversal potential (Vrev) at two extracellular K+ concentrations, 5.4 and 20 mmol l-1. The protocol to obtain the tail currents consisted of a previous 3 s hyperpolarizing pulse from -35 to -120 mV followed by 13 depolarization pulses from -50 to +10 mV. Tail current amplitudes were plotted as a function of the tail step potentials. Best fit through data points gave a linear relationship to the value of x intercept, the Vrev.
Quantitative RT-PCR
HCN2 and HCN4 mRNA levels were quantified by reverse transcriptase polymerase chain reaction amplification (RT-PCR). The low RNA quantities required make the quantitative RT-PCR technique more suitable for the analysis of mRNA in isolated cells than traditional hybridization techniques, such as Northern blotting or RNase protection assays (Schmittgen, 2001; Walker, 2002).
Total RNA was extracted from isolated myocytes of three or four animals using Trizol solution (Gibco BRL, NY, USA). RNA quantity was determined spectrophotometrically (U 2000 Hitachi) and purity was confirmed by relative absorbance at 260 vs. 280 nm. RNA samples were stored in diethyl pyrocarbonate-treated water at -80 °C. cDNA synthesis was performed with 4 µg of total RNA and M-MLU reverse transcriptase (Invitrogen) in accordance with the manufacturer's instructions, using oligo DT as a primer. cDNA was stored at -20 °C.
Primers were used for HCN2 (forward primer (Tm = 65.0 °C) 5'-GAATCGACTCCGAGGTCTACAAG-3', reverse primer (Tm = 67.5 °C) 5'-AGCAACCGCAGCAGACTGA-3') and HCN4 (forward primer (Tm = 65.6 °C) 5'-GCTGA CCAAG TTGCGTT TTG-3', (reverse primer (Tm = 67.1 °C) 5'-CACGCCG TGCTG GATAAAGT-3'), and 18S rRNA was used as a house-keeping gene to normalize relative mRNA HCN2 and HCN4 levels (forward primer (Tm = 65.5 °C) 5'-GCAATTATTCCCCATGAACGA-3'), (reverse primer (Tm = 65.9 °C) 5'-CAAAGGGCAGGGACT TAAT CAA-3'). Primer sequences were designed using the Primer Express Software (PE Applied Biosystems) from published mRNA sequences (EMBL/GenBank accession numbers, AF247451, AF247453 and M11188, respectively). Polymerase chain reaction amplification and quantification were carried out using SyBr Green PCR kit (PE Applied Biosystems) in a model 7700 Sequence detector (PE Applied Biosystems) and results were analysed using SDS 1.9. software (PE Applied Biosystems).
The absence of genomic contamination in the RNA samples and of DNA contamination in RNA mimics was confirmed with reverse transcriptase-negative controls for each experiment.
Statistical analysis
For statistical comparison of If occurrence in the different groups, the 
2 test was used. For the remaining experiments either Student's t test or one-way analysis of variance tests was adopted. All the data are given as means ± S.E.M. and values of P < 0.05 are taken as statistically significant.
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RESULTS |
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Hypertrophy and cellular size
Our data refer to ventricular cells isolated from 18 control hearts and 17 hypertrophied hearts.
The weights of the animals subjected to the operation of aortic stenosis were not significantly different from those of the control animals who underwent the same surgery but without the application of the silver clip in the abdominal aorta (486.5 ± 13.4 vs. 472.4 ± 9.3 g). However, both the heart weights (1760 ± 40 vs. 2270 ± 60 mg) and the ratio heart weight/body weight (3.72 ± 0.1 vs. 4.7 ± 0.1 mg g-1) were significantly higher (P < 0.001) in the animals subjected to aortic stenosis than in the control animals.
To measure the cellular surface we examined the capacitance of the membrane (Cm) in the two groups of cells under study. The average Cm was significantly higher (P < 0.001) in the group of hypertrophied cells (291.0 ± 23.7 pF, n = 20, in the S cells, 303.8 ± 15.9 pF, n = 26, in LV cells) than in the control cells (197.2 ± 9.1 pF in the S, n = 25; 215.2 ± 17.3 pF in those of LV, n = 17).
On the other hand, we found no significant differences between the measurements of the Cm in the cells taken from the RV, when comparing control animals (192.3 ± 11.6 pF, n = 17) with those subjected to aortic stenosis (217.5 ± 11.4 pF, n = 20), which supports the idea that the RV was not hypertrophied in our experimental model of LVH.
If occurrence
This study was carried out on cells isolated from the S, the LV and the RV, of both control and hypertrophied rats. The presence of a hyperpolarization-activated time-dependent increasing inward current (If) was confirmed in each cell by application of hyperpolarizing pulses from -60 to -130 mV (holding potential (Vh) = -40 mV) in 10 mV steps and with variable duration from 3 s at -60 mV to 1.6 s at -130 mV. When If was present it became larger and activated more rapidly with progressively more negative potentials. In most cells, recordings were obtained first at 5.4 mM [K+]o and then at 20 mM [K+]o to verify current amplitude increase with increasing [K+]o.
Figure 1 shows If occurrence in the three regions, and their modulation with hypertrophy. In S, the occurrence was 53 % in control and 86.4 % in the hypertrophied cells (P < 0.01). In the LV, the percentage of control cells with If was 34 %, rising to 65.9 % in the hypertrophied cells (P < 0.01). RV, which showed no development of hypertrophy, is the region in which the percentages were similar in the two groups: 56.3 % in the control and 61.1 % in the 'hypertrophied' group (n.s.).
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Figure 1. Occurrence of the hyperpolarization-activated current (If) in ventricular cells isolated from different regions of control and hypertrophied rat hearts Bar graphs illustrate percentage of cells in which If was present in three regions of the ventricle: the septum (S), the left ventricle (LV) and the right ventricle (RV). Open bars show values of If occurrence obtained in cells isolated from control hearts and filled bars show values of If occurrence obtained in cells isolated from hypertrophied hearts. ** P < 0.01 control vs. hypertrophy. | ||
These results show that the presence of hypertrophy increases the probability of finding If, S being the region where this probability was higher.
Density and conductance of If
Figure 2A illustrates families of current traces obtained in three control and three hypertrophied cells from the different ventricular regions of our study, using an If recording solution containing [K+]o 20 mmol l-1. From a Vh of -40 mV, a family of hyperpolarization steps (from -60 to -130 mV) in 10 mV increments elicited a time-dependent inward current that increased with more negative potentials. The amplitude of the current was always greater in the hypertrophied cells from the S and LV regions whereas it was similar in the RV cells of both groups.
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Figure 2. If in ventricular cells from different regions of control and hypertrophied rat hearts A, If tracing recorded in three cells from control and three cells from hypertrophied hearts isolated from S (a), LV (b) and RV (c). Membrane capacitance (Cm) values of control cells were 210, 196 and 145 pF for S, LV and RV, respectively, and Cm values of hypertrophied cells were 476, 359 and 258 pF for S, LV and RV, respectively. If was elicited by hyperpolarizing pulses (from -60 to -130 mV) from a holding potential of -40 mV. The duration of the step was decreased from 3 to 1.6 s. Steps were applied at 0.1 Hz. Recording solution contained 20 mmol l-1 [K+]o. B, mean If density values obtained at -130 mV in ventricular cells from different regions (S, LV and RV) of control (open bars) and hypertrophied rat hearts (filled bars). * P < 0.05, ** P < 0.01 control vs. hypertrophy. | ||
The histograms in Fig. 2B illustrate the average current densities at -130 mV in cells from S, LV and RV of the control and hypertrophied animals. These densities were significantly higher in the regions that had developed hypertrophy. The average values, expressed as picoamps per picofarad (pA pF-1), control vs. hypertrophy, were: -2.9 ± 0.7, n = 17 vs. -6.8 ± 1.6, n = 13 in S (P < 0.01); -2.0 ± 0.4, n = 10 vs. -4.0 ± 0.7, n = 8 in LV (P < 0.05). In RV, no significant differences in density at -130 mV were found between the cells from control (-2.1 ± 0.5 pA pF-1, n = 15) and those from the hypertrophied hearts (-2.1 ± 0.5 pA pF-1, n = 15).
Figure 3 shows the voltage dependence of If activation in control and hypertrophied cells from the different regions. Recording solution containing [K+]o 20 mmol l-1. Mean current specific conductance (g), normalized to Cm, was plotted as a function of the hyperpolarizing step potential and it was significantly greater in hypertrophied cells (from -80 mV in S, panel A, and from -100 mV in LV, panel B) but not in cells isolated from RV (panel C). Maximal specific conductance (gmax) in hypertrophied cells was roughly double that observed in control cells whereas in RV, gmax values were similar in both groups (Table 1). Activation curves were fitted by a Boltzmann function and showed similar values of V50 and k (Table 1).
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Figure 3. Voltage dependence of If in ventricular cells from different regions of control and hypertrophied rat hearts A, B and C, the specific conductance (g), normalized to Cm, was plotted against step potential in S (A), LV (B) and RV (C). The recording solution contained 20 mmol l-1 [K+]o. Each point represents the mean ± S.E.M. value of g (pS pF-1) obtained in ventricular cells from control (open circles) and hypertrophied rat hearts (filled circles). Activation curves were fitted to the data points by use of a Bolzmann distribution. * P < 0.05, ** P < 0.01 control vs. hypertrophy. | ||
Effect of extracellular concentration of K+ and Cs+
Another characteristic of the If is its permeability to monovalent cations, i.e. K+ and Na+. It is also sensitive to blocking by Cs+ but insensitive to moderate concentrations of Ba2+ (Noma et al. 1983; DiFrancesco et al. 1986). Figure 4A shows original current traces of If obtained in one hypertrophied cell from S (Cm = 292 pF). If was elicited by hyperpolarizing pulses (from -60 to -130 mV) from a Vh of -40 mV. The duration of the step was decreased from 3 to 1.6 s. Following the hyperpolarizing steps, the cell was clamped to +20 mV for 400 ms and then to the holding potential. Figure 4A illustrates the amplitude of If traces using an If recording solution containing [K+]o 5.4 mmol l-1. Figure 4B shows that the current amplitude increased when [K+]o was increased from 5.4 to 20 mmol l-1. Extracellular addition of Cs+ 4 mmol l-1 (Fig. 4C) almost suppressed the time-dependent part of the inward current. Similar results were obtained in another eight experiments (4 control and 4 hypertrophied cells).
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Figure 4. Effects of extracellular K+ and Cs+ on If A, B and C, original current traces of a single hypertrophied cell isolated from S with Cm = 292 pF and superfused with [K+]o 5.4 mmol l-1 (A), followed by [K+]o 20 mmol l-1 (B) and CsCl 4 mmol l-1 (C). If was elicited by hyperpolarizing pulses (from -60 to -130 mV) from a holding potential of -40 mV. The duration of the step was decreased from 3 to 1.6 s. Following the hyperpolarizing steps, the cell was clamped to +20 mV for 400 ms and then to the holding potential. | ||
Table 2 illustrates average values of If density at -130 mV, threshold for activation (Vth) and reversal potential (Vrev) obtained in the same cell when [K+]o was increased from 5.4 to 20 mmol l-1. If density increased when [K+]o was increased and this effect was similar in control and hypertrophied cells, independently of the region. Average values of Vth showed no significant differences either between regions or between the control and the hypertrophied cells. Tail current recordings were used to evaluate Vrev of If. Figure 5A and B shows an example of tail current obtained in one control cell from S when [K+]o was changed from 5.4 mmol l-1 (panel A) to 20 mmol l-1 (panel B). After a hyperpolarizing pulse to -120 mV, the clamp potential was changed in the range from -50 to +10 mV and the tail current amplitudes were measured. For the sake of clarity we show only the traces obtained at -50, -40, -30, -20 and -10 mV. We next determined the reversal potential of If in the same cell. Figure 5C shows tail current amplitudes plotted as a function of step potential at 5.4 mmol l-1 (open circles) and 20 mmol l-1 (filled circles) [K+]o. Best fit through data points gave a linear relationship with Vrev of -40.1 mV at 5.4 mmol l-1 and -27.6 mV at 20 mmol l-1 [K+]o. The slope of the current-voltage (I-V) relationship was steeper at 20 mmol l-1 [K+]o.
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Figure 5. Reversal potential of If A and B, representative tail currents obtained in one control cell from Septum. Tail currents were obtained upon repolarization to various test voltages (-50 to 10 mV). For clarity, only 5 step potentials are shown. Tail currents were obtained at 5.4 and 20 mmol l-1 [K+]o. C, tail current amplitudes are plotted as a function of test potential to obtain the reversal potential (Vrev) of If at the two [K+]o. Best fit through data points gave a linear relationship to the value of the x intercept, the Vrev. In this representative cell from S, Vrev was -40.1 mV at 5.4 mmol l-1 and -27.6 mV at 20 mmol l-1 [K+]o. | ||
Mean values of Vrev are shown in Table 2 and were similar in the three regions or between the control and hypertrophied cells.
-Adrenergic modulation of If
Several studies have demonstrated that If can be modulated by -adrenergic stimulation (DiFrancesco, 1993; Cerbai et al. 1996); however, when the ruptured-patch configuration is used, the effect of -adrenergic agonists on If is erratic and slight (Zhou & Lipsius, 1993). To avoid this problem, we used the perforated-patch configuration of the patch clamp technique to preserve cells from the loss of some cytosolic second messengers due to intracellular dialysis (Horn & Marty, 1988). In this group of experiments only cells from S were used.
Figure 6A illustrates If recordings obtained from a hypertrophied cell isolated from S (Cm = 288 pF). Records were obtained at different potentials (-60, -70, -80, -90, -100 mV) from a Vh of -40 mV in the absence and in the presence of 1 µmol l-1 isoproterenol (Iso). Figure 6B and C shows activation curves calculated from Bolzmann fits of normalized If conductances in six control (panel B) and six hypertrophied cells from S (panel C). Iso shifted V50 to more positive potentials with similar values in both groups: 14.2 ± 1.0 mV in control (-93.6 ± 5.4 mV before and -80.0 ± 5.8 mV after Iso, n = 6) and 15.2 ± 4.1 mV in hypertrophied cells (-95.8 ± 4.9 mV before and -80.7 ± 3.4 mV after Iso, n = 6).
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Figure 6. A, original If traces obtained in one hypertrophied cell from S (Cm = 288 pF) using the perforated-patch clamp technique. Records were obtained at -60, -70, -80, -90 and -100 mV in the absence and in the presence of isoproterenol (Iso) 1 µmol l-1. Iso shifted the half-maximal potential by 12.4 mV. B and C, activation curves calculated by Boltzmann fits of normalized current conductance in control (A) and hypertrophied cells (B) in the absence (open circles) and in the presence (filled circles) of isoproterenol 1 µmol l-1. Isoproterenol shifted the potential of half-maximal activation by 14.2 ± 1.0 mV in control (-93.6 ± 5.4 mV before and -80.0 ± 5.8 mV after Iso n = 6) and of 15.2 ± 4.1 mV in hypertrophy (-95.8 ± 4.9 mV before and -80.7 ± 3.4 mV, after Iso n = 6). The recording solution contained 20 mmol l-1 [K+]o. | ||
Expression levels of HCN2 and HCN4 mRNA
Quantitative RT-PCR analysis of HCN2 and HCN4 mRNA levels is illustrated in Fig. 7. Panels A and B show that HCN2 and HCN4 mRNA levels were significantly higher in S and LV cells isolated from hypertrophied hearts (n = 5) compared with control hearts (n = 5) (P < 0.05). The presence of HCN1 mRNA was also analysed, but this isoform was not detected either in control or in hypertrophied rat ventricular myocytes (data not shown). Fig. 7C illustrates the efficiency of RT-PCR amplification of HCN2, HCN4 and house-keeping 18S rRNA gene expression. This was tested on serial dilutions of rat ventricular myocytes cDNA. Co-amplification of HCN2, HCN4 and 18S rRNA resulted in a linear response of similar slope, demonstrating identical amplification efficiency over the range tested.
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Figure 7. HCN2 and HCN4 mRNA levels in ventricular cells from the different regions of control and hypertrophied rat hearts A and B, bar graphs illustrate HCN2 and HCN4 mRNA levels obtained using quantitative RT-PCR technique. HCN2 and HCN4 mRNA levels were significantly higher in the two regions that developed hypertrophy (S and LV) (filled bars) compared to control cells (open bars; * P < 0.05). C, the efficiency of RT-PCR amplification of HCN2, HCN4 and 18S rRNA gene expression was tested on serial dilutions of rat ventricular myocyte cDNA. Co-amplification of HCN2, HCN4 and 18S rRNA resulted in curves showing a similar slope, demonstrating identical amplification efficiency over the range tested. | ||
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DISCUSSION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The present study indicates that the probability of finding If in the population of rat ventricular cells increases with the development of left ventricular hypertrophy, S being the region of the ventricle in which this probability is higher. Our results also demonstrate that the density and conductance of If is greater in those regions that developed hypertrophy, S being the region where these differences were most evident. Moreover, although previous studies have demonstrated an increase in If density using several models of cardiac hypertrophy (Cerbai et al. 1994, 1996; Stilli et al. 2001) and heart failure (Cerbai et al. 1997; Hoppe et al. 1998), this is the first analysis of If changes and mRNA expression levels of HCN2 and HCN4 isoforms in the same hypertrophied ventricular myocyte, which means that we are the first to demonstrate that the upregulation of If is related to the upregulation of HCN2 and HCN4 mRNA levels in the hypertrophied ventricle.
The HCN gene family representing the molecular correlate of the If channels has recently been cloned and shows structural characteristics similar to voltage-gated K+ channels. These characteristics include six transmembrane domains (S1-S6), a pore loop between the segment S5 and S6, and a cyclic nucleotide-binding domain in the C-terminal region of the polypeptide (Gauss et al. 1998; Ludwig et al. 1998; Santoro et al. 1998). Four members of the HCN gene family are currently known to exist in mammals (HCN1-4). In the sinoatrial node the most prominently expressed HCN isoform is HCN4 with HCN2 and HCN1 being expressed to a lesser extent (Moosmang et al. 2001). In both neonatal and adult rat ventricle only HCN2 and HCN4 are present (Shi et al. 1999). The currents produced by expression of HCN1, HCN2 and HCN4 isoforms are activated by hyperpolarization but have different relatives rates of activation and deactivation. The fastest kinetics are associated with HCN1 and the most negative threshold for activation and half-maximal voltage of the activation curves is linked to HCN2 (Accili et al. 2002). The results shown in the present study demonstrate for the first time that the increase in If density and conductance associated with left ventricular hypertrophy was related to an increase in HCN2 and HCN4 mRNA levels in hypertrophied ventricular myocytes. Recently, one study has been published showing similar changes in channel mRNA levels of HCN2 and HCN4 in cardiac hypertrophy induced by abdominal aortic banding (Hiramatsu et al. 2002), but the authors did not measure the protein levels of these ion channels, nor did they evaluate functional changes in corresponding currents.
In recent years, many papers have been published on the electric heterogeneity within the ventricular wall (Antzelevitch & Fish, 2001). At present it is recognized that the ventricular wall is made up of three different types of cells: ventricular epicardial cells, ventricular mid-myocardial cells (known as 'M') and ventricular endocardial cells. It has been demonstrated that these cells show differences in their electrophysiological and pharmacological profiles (Antzelevitch & Fish, 2001). In the present study we determine for the first time the regional distribution of If in the adult ventricle. The three regions used reflect three anatomical parts of the rat ventricle: the left ventricular free wall (LV), the right ventricular free wall (RV) and the interventricular septum (S). The first two have characteristics of the epicardial cells, since they have a prominent Ito whereas the S cells reflect those of the endocardial cells with a much smaller Ito (Benitah et al. 1993). No evidence is available regarding M cells in the rat heart (Shipsey et al. 1997). The results of our study showed that If density was not regionally distributed in the normal or in the hypertrophied ventricle, but it was significantly increased in hypertrophied cells, this being more pronounced in the S region. One possible explanation for this finding would be that the overload on the LV is higher in those layers adjacent to the ventricular cavity, S being the region where this pressure would be higher.
The results obtained with physiological [K+]o (5.4 mmol l-1) indicate that the threshold potential for activation varied between -80 and -90 mV, and was similar in the control and in the hypertrophied cells (Table 2), which means that the If would not be activated in the range of diastolic potential in the rat (Gomez et al. 1997). In addition, ventricular cells of the rat show prominent inward rectifier K+ channels (IK1; Wahler, 1992) which in these conditions would maintain the balance towards hyperpolarization. The role played by the If in the healthy ventricle is not clear, although it could be a mechanism that in a limited situation might protect the ventricular cell from excessive hyperpolarization. Regarding the hypertrophied ventricle, our data also indicate (Table 2) that, although the presence of hypertrophy raises the density and the incidence of If, it does not modify the threshold of If activation in relation to that of the control cells. However, the shift to the right of the activation curves by isoproterenol that we observed in our experiments could mean that, in conditions in which sympathetic activity is increased, the hypertrophic cells of the ventricle, particularly those of the S region, could promote diastolic depolarizations in the ventricular myocytes, thus contributing to the appearance of certain types of arrhythmia (Barbieri et al. 1994). It should be noted that this would require a lowering of the IK1 (Opthof, 1998). In fact some studies have demonstrated a fall in the density of IK1 in heart failure (Koumi et al. 1994), in ischaemic cardiomyopathy in humans (Koumi et al. 1995) and in failing canine hearts (Li et al. 2002). Moreover, there is experimental evidence indicating that specific suppression of Kir2-encoded inward-rectifier K+ channels, can give rise to pacemaker activity in ventricular myocytes from guinea-pig (Miake et al. 2002).
Isoproterenol is known to shift the curves of If activation towards more positive potentials without modifying the maximum conductance or the current-voltage relation of the channel (DiFrancesco, 1993). The shift in the activation curves is the result of the modulation of adenylate cyclase by isoproterenol and the consequent changes of the intracellular levels of cAMP, whose increase induce a shift of the activation curves towards more positive potentials. The shift of the If activation in our experiments (calculated as the algebraic sum of the voltage shift induced by isoproterenol in each experiment) was similar in the two groups, 14.2 ± 1.0 mV in the control cells and 15.2 ± 4.1 mV in the hypertrophied cells. However, in spite of the closeness of the shifts of the activation curves, the higher occurrence and greater density and conductance of If in the hypertrophied cells and particularly in the S region, might suggest the participation of this region in the arrhythmogenesis associated with left ventricular hypertrophy.
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REFERENCES |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
| Accili EA, Proenza C, Baruscotti M & DiFrancesco D (2002). From funny current to HCN channels: 20 years of excitation. News Physiol Sci 17, 32-37 | [Abstract/Full Text] |
| Antzelevitch C , & Fish J (2001). Electrical heterogeneity within the ventricular wall. Basic Res Cardiol 96, 517-527 | [Medline] |
| Barbieri M, Varani K, Cerbai E, Guerra L, Li Q, Borea PA & Mugelli A (1994). Electrophysiological basis for the enhanced cardiac arrhythmogenic effect of isoprenaline in aged spontaneously hypertensive rats. J Mol Cell Cardiol 26, 849-860 | [Medline] |
| Benitah JP, Gomez AM, Bailly P, Da Ponte JP, Berson G, Delgado C & Lorente P (1993). Heterogeneity of the early outward current in ventricular cells isolated from normal and hypertrophied rat hearts. J Physiol 469, 111-138 | [Abstract] |
| Cerbai E, Barbieri M & Mugelli A (1994). Characterization of the hyperpolarization-activated current, I(f), in ventricular myocytes isolated from hypertensive rats. J Physiol 481, 585-591 | [Abstract] |
| Cerbai E, Barbieri M & Mugelli A (1996). Occurrence and properties of the hyperpolarization-activated current If in ventricular myocytes from normotensive and hypertensive rats during aging. Circulation 94, 1674-1681 | [Abstract/Full Text] |
| Cerbai E, Pino R, Porciatti F, Sani G, Toscano M, Maccherini M, Giunti G & Mugelli A (1997). Characterization of the hyperpolarization-activated current, I(f), in ventricular myocytes from human failing heart. Circulation 95, 568-571 | [Abstract/Full Text] |
| Cerbai E, Sartiani L, DePaoli P, Pino R, Maccherini M, Bizzarri F, Diciolla F, Davoli G, Sani G & Mugelli A (2001). The properties of the pacemaker current I(F) in human ventricular myocytes are modulated by cardiac disease. J Mol Cell Cardiol 33, 441-448 | [Medline] |
| DiFrancesco D, (1993). Pacemaker mechanisms in cardiac tissue. Annu Rev Physiol 55, 455-472 | [Medline] |
| DiFrancesco D, (1995). The onset and autonomic regulation of cardiac pacemaker activity: relevance of the f current. Cardiovasc Res 29, 449-456 | [Medline] |
| DiFrancesco D, Ferroni A, Mazzanti M & Tromba C (1986). Properties of the hyperpolarizing-activated current (if) in cells isolated from the rabbit sino-atrial node. J Physiol 377, 61-88 | [Abstract] |
| Gauss R, Seifert R & Kaupp UB (1998). Molecular identification of a hyperpolarization-activated channel in sea urchin sperm. Nature 393, 583-587 | [Medline] |
| Gomez AM, Benitah JP, Henzel D, Vinet A, Lorente P & Delgado C (1997). Modulation of electrical heterogeneity by compensated hypertrophy in rat left ventricle. Am J Physiol 272, H1078-1086 | [Medline] |
| Hart G, (1994). Cellular electrophysiology in cardiac hypertrophy and failure. Cardiovasc Res 28, 933-946 | [Medline] |
| Hiramatsu M, Furukawa T, Sawanobori T & Hiraoka M (2002). Ion channel remodeling in cardiac hypertrophy is prevented by blood pressure reduction without affecting heart weight increase in rats with abdominal aortic banding. J Cardiovasc Pharmacol 39, 866-874 | [Medline] |
| Hoppe UC, Jansen E, Sudkamp M & Beuckelmann DJ (1998). Hyperpolarization-activated inward current in ventricular myocytes from normal and failing human hearts. Circulation 97, 55-65 | [Abstract/Full Text] |
| Horn R , & Marty A (1988). Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol 92, 145-159 | [Abstract] |
| Kannel WB, Gordon T & Offutt D (1969). Left ventricular hypertrophy by electrocardiogram. Prevalence, incidence, and mortality in the Framingham study. Ann Intern Med 71, 89-105 | |
| Katz AM, (1991). The cardiomyopathy of overload: a hypothesis. J Cardiovasc Pharmacol 18 suppl. 2, S68-71 | |
| Koumi S, Arentzen CE, Backer CL & Wasserstrom JA (1994). Alterations in muscarinic K+ channel response to acetylcholine and to G protein-mediated activation in atrial myocytes isolated from failing human hearts. Circulation 90, 2213-2224 | [Abstract] |
| Koumi S, Backer CL & Arentzen CE (1995). Characterization of inwardly rectifying K+ channel in human cardiac myocytes. Alterations in channel behavior in myocytes isolated from patients with idiopathic dilated cardiomyopathy. Circulation 92, 164-174 | [Abstract/Full Text] |
| Levy D, Anderson KM, Savage DD, Balkus SA, Kannel WB & Castelli WP (1987). Risk of ventricular arrhythmias in left ventricular hypertrophy: the Framingham Heart Study. Am J Cardiol 60, 560-565 | [Medline] |
| Li G-R, Lau C-P, Ducharme A, Tardif J-C & Nattel S (2002). Transmural action potential and ionic current remodeling in ventricles of failing canine hearts. Am J Physiol Heart Circ Physiol 283, H1031-1041 | [Abstract/Full Text] |
| Litovsky SH , & Antzelevitch C (1988). Transient outward current prominent in canine ventricular epicardium but not endocardium. Circ Res 62, 116-126 | [Abstract] |
| Ludwig A, Zong X, Jeglitsch M, Hofmann F & Biel M (1998). A family of hyperpolarization-activated mammalian cation channels. Nature 393, 587-591 | [Medline] |
| Martinez ML, Heredia MP & Delgado C (1999). Expression of T-type Ca(2+) channels in ventricular cells from hypertrophied rat hearts. J Mol Cell Cardiol 31, 1617-1625 | [Medline] |
| Miake J, Marban E & Nuss HB (2002). Biological pacemaker created by gene transfer. Nature 419, 132-133 | |
| Moosmang S, Stieber J, Zong X, Biel M, Hofmann F & Ludwig A (2001). Cellular expression and functional characterization of four hyperpolarization-activated pacemaker channels in cardiac and neuronal tissues. Eur J Biochem 268, 1646-1652 | [Abstract/Full Text] |
| Nabauer M, Beuckelmann DJ, Uberfuhr P & Steinbeck G (1996). Regional differences in current density and rate-dependent properties of the transient outward current in subepicardial and subendocardial myocytes of human left ventricle. Circulation 93, 168-177 | [Abstract/Full Text] |
| Noma A, Morad M & Irisawa H (1983). Does the 'pacemaker current' generate the diastolic depolarization in the rabbit SA node cells? Pflugers Arch 397, 190-194 | [Medline] |
| Robinson RB, Yu H, Chang F & Cohen IS (1997). Developmental change in the voltage-dependence of the pacemaker current, if, in rat ventricle cells. Pflugers Arch 433, 533-535 | [Medline] |
| Santoro B, Liu DT, Yao H, Bartsch D, Kandel ER, Siegelbaum SA & Tibbs GR (1998). Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell 93, 717-729 | [Medline] |
| Schmittgen TD, (2001). Real-time quantitative PCR. Methods 25, 383-385 | [Medline] |
| Shi W, Wymore R, Yu H, Wu J, Wymore RT, Pan Z, Robinson RB, Dixon JE, McKinnon D & Cohen IS (1999). Distribution and prevalence of hyperpolarization-activated cation channel (HCN) mRNA expression in cardiac tissues. Circ Res 85, e1-6 | [Medline] |
| Shipsey SJ, Bryant SM & Hart G (1997). Effects of hypertrophy on regional action potential characteristics in the rat left ventricle: a cellular basis for T-wave inversion? Circulation 96, 2061-2068 | [Abstract/Full Text] |
| Tomaselli GF, Beuckelmann DJ, Calkins HG, Berger RD, Kessler PD, Lawrence JH, Kass D, Feldman AM & Marban E (1994). Sudden cardiac death in heart failure. The role of abnormal repolarization. Circulation 90, 2534-2539 | [Abstract] |
| Tomaselli GF , & Marban E (1999). Electrophysiological remodeling in hypertrophy and heart failure. Cardiovasc Res 42, 270-283 | [Medline] |
| Wahler GM, (1992). Developmental increases in the inwardly rectifying potassium current of rat ventricular myocytes. Am J Physiol 262, C1266-1272 | [Medline] |
| Walker NJ, (2002). Tech. Sight. A technique whose time has come. Science 296, 557-559 | |
| Yu H, Chang F & Cohen IS (1993). Pacemaker current exists in ventricular myocytes. Circ Res 72, 232-236 | [Abstract] |
| Yu H, Chang F & Cohen IS (1995). Pacemaker current i(f) in adult canine cardiac ventricular myocytes. J Physiol 485, 469-483 | [Abstract] |
| Zhou Z , & Lipsius SL (1993). Effect of isoprenaline on I(f) current in latent pacemaker cells isolated from cat right atrium: ruptured vs. perforated patch whole-cell recording methods. Pflugers Arch 423, 442-447 | [Medline] |
Acknowledgements
This study was supported by the Ministry of Science and Technology of Spain (SAF99-0068 and BFI2002-00536). We are grateful to M. Bas, F. Ortego and M. L. Hidalgo for their excellent technical assistance. The authors are grateful to Dr José Manuel Rodríguez Peña and to the Centre of Genomics and Proteomics of the Complutense University of Madrid. M.F and J.B. are graduate research fellows of the Ministry of Science and Technology of Spain.
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