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Molecular basis of funny current (If) in normal and failing human heart
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Journal of Molecular and Cellular Cardiology 45 (2008) 289 – 299 www.elsevier.com/locate/yjmcc
Original article
Molecular basis of funny current (If) in normal and failing human heart Francesca Stillitano a,1 , Giuseppe Lonardo a,b,1 , Stephen Zicha b , Andras Varro c,d , Elisabetta Cerbai a , Alessandro Mugelli a,⁎, Stanley Nattel b b
a Centro Interuniversitario di Medicina Molecolare e Biofisica Applicata (CIMMBA), University of Florence, Viale Pieraccini 6, 50139 Florence, Italy Montreal Heart Institute Research Center and Department of Medicine, Université de Montréal, 5000 Belanger St. E. H1T 1C8 Montreal, Quebec, Canada c Division of Cardiovascular Pharmacology, Hungarian Academy of Sciences, University of Szeged, Hungary d Department of Pharmacology and Pharmacotherapy, University of Szeged, Hungary
Received 28 March 2008; received in revised form 28 April 2008; accepted 30 April 2008 Available online 11 May 2008
Abstract If overexpression has been functionally demonstrated in ventricular myocytes from failing human hearts. Altered expression of If-channels as a consequence of electrophysiological remodeling may represent an arrhythmogenic mechanism in heart failure; however, the molecular basis of If overexpression in human cardiac disease is unknown. HCN1, 2 and 4 subtypes, which encode If-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. If 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 b 0.01 for mRNA, p b 0.05 for protein). These results are consistent with the electrophysiological data showing that, in failing ventricular myocytes, If is of larger amplitude and activates at less negative potential. Changes in mRNA and protein expression of both HCN2/4 isoforms in atrial specimens from patients with heart failure mirrored those observed in ventricles ( p b 0.001 for mRNA, p b 0.05 for protein). No diseasedependent alteration was detected for MiRP1, the putative β-subunit of the If-channel. In conclusion, HCN4 is the predominant channel subtype in normal human heart, and its expression is further amplified by disease. HCN upregulation likely contributes to increased If and may play a role in ventricular and atrial arrhythmogenesis in heart failure. © 2008 Elsevier Inc. All rights reserved. Keywords: Hyperpolarization-activated Cyclin Nucleotide gated channel; Ischemic cardiomyopathy; Human heart failure; Gene expression; Electrophysiology; Arrhythmias
1. Introduction The heart failure burden is still very high. The syndrome has an annual incidence ranging from 1 to 5 per 1000, increasing with age up to 40 cases per 1000 in people older than 75 years [1]. Recent estimates suggest annual mortalities for NYHA Class II, III and IV patients of 10%, 20% and 60%, respectively [2]. Mortality after 8 years is ~ 80% and half of the deaths are sudden and unexpected [3]. Sudden deaths are generally thought to be due to ventricular arrhythmias [4]. Studies on possible underlying arrhythmogenic mechanisms have focused largely on repolarization abnormal⁎ Corresponding author. Tel.: +39 055 4271264; fax: +39 055 4271280. E-mail address: [email protected] (A. Mugelli). 1 These authors contributed equally to this paper. 0022-2828/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2008.04.013
ities, and investigated the ionic basis of abnormal prolongation of the action potential duration [5]. However, other abnormalities in ventricular myocyte electrophysiology may contribute to enhanced electrical instability or trigger life-threatening arrhythmias in heart failure [4]. Recently, several studies have addressed the pathophysiological role of the hyperpolarizationactivated current (If) in this setting [6–8]. If is a mixed Na+/K+ inward current, activated by hyperpolarization, and encoded by a family of genes termed Hyperpolarization-activated Cyclic Nucleotide-gated channel (HCN) genes [9]. Originally regarded as a hallmark of pacemaker cells in the heart, If has been identified in atrial and ventricular myocytes from both animal models and humans [10]. An arrhythmogenic role for If in cardiac hypertrophy and failure has been inferred, based on the presence of this current in ventricular myocytes isolated from severely hypertrophied rat hearts [11] and from human hearts
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explanted because of terminal heart failure [12]. Indeed, the functional expression of If seems to be differentially modulated by disease, since its density is larger in human ventricular myocytes (HuVMs) isolated from the hearts of patients with ischemic than in those with dilated cardiomyopathy [6]. So far, the molecular basis of If overexpression in human heart failure is largely unknown. A 3-fold increase of mRNA coding for HCN4 was reported in end-stage failing human hearts [13] and a ~2-fold increase of HCN4 protein was reported in the right atrium of a canine heart failure model [14], but no information is available regarding the relative proportion of diverse HCN isoforms in the human heart. In fact, HCN subtypes expressed in rat ventricular myocytes and HEK 293 cells have characteristic kinetic properties [15] and different sensitivity to cAMP has been described [16]. The present study aimed to analyze, in healthy and diseased human cardiac tissues, the profile of HCN isoform expression, in terms of mRNA and protein, and to correlate these data with electrophysiological properties (activation kinetics and response to β−AR stimulation) of If in HuVMs. 2. Methods 2.1. Patients Ventricular samples were obtained from undiseased hearts of organ donors whose hearts were explanted to obtain pulmonary and aortic valves for transplant surgery (University of Szeged). Before cardiac explantation, these organ donor patients did not receive medication except dobutamine, furosemide, and plasma expanders. All experimental protocols were approved by the Albert Szent-Gyorgyi Medical University Ehtical review Board (No. 51-57/1997 OEj). Failing heart tissues for biochemical studies were obtained from patients undergoing transplantation for end-stage ischemic cardiomyopathy (Montreal Heart Institute). Atrial appendages were obtained from patients undergoing cardiac surgery for coronary artery bypass graft with conserved ventricular function and from ICM explanted hearts. Characteristics of patients are reported in Table 1. Samples were quickly frozen in liquid nitrogen and stored at − 80 °C, or used for isolation of single HuVMs as previously described [12]. Informed consent for the use of tissue samples was obtained from patients undergoing cardiac surgery. The investigation conforms with the principles outlined in the “Declaration of Helsinki” of the World Medical Association [17] and was approved by the local ethical committee. 2.2. Cell isolation and If recording The isolation procedure has been described elsewhere [6]. After digestion of a portion of the left ventricle, cells were dissociated from small transmural specimens including epicardium, midmyocardium, and endocardium. Following clamp steps to hyperpolarized membrane potential (usually −120 mV), an If like current was recorded in most of the tested cells. If was considered to be present in a given cell when hyperpolarization elicited a time-dependent increasing inward current which could be blocked by adding 4 mM CsCl. To establish the activation
Table 1 Characteristics of the patients from whom atrial (A) or ventricular (V) samples were obtained
Total number (m/f) Age (mean ± sem) EF (%)
A (Ctrl)
A (ICM)
V (Ctrl)
V (ICM)
26 (16/10) 54.5 ± 2.3 51.8 ± 2.3 (13) normal a (13)
4 (3/1) 57.4 ± 4.5 22.6 ± 5.6
17 (9/8) 47 ± 3 n.a. b
13 (12/1) 56.7 ± 2.3 20.8 ± 4.2
5 2 3 5 3 6
– – – –
16 11 6 13 4 6
Medication (number of patients) Diuretics 1 Digoxin 2 Antiarrhythmics 9 ACE-I 4 Β-blockers 3 Anticoagulants 9
EF: Ejection Fraction; ACE-I: angiotensin converting enzyme inhibitors. Ctrl: control i.e. patients without cardiac dysfunction; ICM: patients with ischemic cardiomyopathy. a The ejection fraction was reported as normal without specifying percentage value, in the clinical records of 10 patients. b No pre-mortem cardiac function data were available for control ventricles. All hearts were from patients with presumed normal hearts that could not be transplanted for technical reasons.
curve, If was evoked by hyperpolarizing steps (range: −50 to − 140 mV) from a holding potential of −40 mV. The duration of pulses was progressively reduced from 3200 ms (at − 50 mV) to 1600 ms (at − 140 mV). Fitting was carried out by using the Clampfit program (pClamp vers. 9, Molecular Devices Inc.). Amplitude was automatically calculated as the difference between the value at the beginning of the hyperpolarizing step and the value extrapolated to the steady state. All experiments were performed at 36 °C. 2.3. RNA isolation Total mRNA was isolated as previously described [18]. Briefly, tissue specimens were homogenized in TRIzol reagent (Gibco BRL). Genomic DNA was eliminated by incubating in DNase I. RNA was quantified by spectrophotometry. All A260/ A280 nanometer ratios were above 1.8. Integrity was evaluated by ethidium bromide staining on a denaturing agarose gel. RNA samples were stored at −80 °C in RNAsecure resuspension solution (Ambion). 2.4. PCR primers and construction of RNA mimics Methods for RNA mimic construction has been previously described [14]. Briefly, gene-specific primers for RT-PCR were designed according to previously cloned sequences from regions with minimal homology among HCNs and specificity verified by comparison with the GenBank database with the use of BLAST and FASTA (see Table 2). Resulting PCR products were sequenced to ensure subunit-specificity. 2.5. Competitive RT-PCR and TaqMan real time PCR Competitive RT-PCR was performed as described [14], by using RNA mimic samples of serial 10-fold dilutions, added to
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Table 2 Competitive RT-PCR primers
HCN-1 HCN-2 HCN-4
Primers
Position
Product size (bp)
T (°C)
GeneBank
F-tggtggctacaatgccttta R-ttcctccgggacctcgtt F-cgttaccagggcaagatgtttg R-gttgtccacgctcagcgaat F-gtactcctacgcgctcttca R-gctctcctcgtcgaagatct
1406–1725
320
55
AF488549
1519–1911
393
55
NM_001194
1392–1704
313
55
AJ238850s
Real-time PCR primers and probes
HCN-4
GAPDH
Primer
Sequence
Forward Probe Reverse Forward Probe Reverse
5′-gagcaggagagggttaagtcagc-3′ 5′-ccacccctacagcgacttcagattttactgg-3′ 5′-cagcaacagcatcgtcaggt-3′ 5′-cttcaccaccatggagaaggc-3′ 5′-cctggccaaggtcatccatgacaacttt-3′ 5′-ggcatggactgtggtcatgag-3′
reaction mixtures containing 1 µg total RNA. First-strand cDNAs were synthesized at 42 °C (1 h) and used as template in PCR reactions. PCR conditions were 93 °C for 3 min followed by 30 cycles of 30 s at 93 °C, 30 s at 55 °C and 30 s at 72 °C. A final 72 °C extension step was performed for 5 min. Expression levels of HCN4 and MiRP1 genes were further investigated using real-time quantitative RT-PCR and TaqMan® probe-based chemistry. Primers and probes specific for human HCN4 and GAPDH were designed using PRIMER EXPRESS software (PE Applied Biosytems) (see Table 2). Resulting PCR products were sequenced to ensure subunit-specificity. Primers and probe for MiRP1 were obtained from ABI (Foster City, California) TaqMan Gene Expression Assay catalogue. This assay comes in a 20× reaction mix, spans an exon-exon junction, and is optimized to give approximately 100% efficiency. The real-time RT-PCR reactions were performed using TaqMan Universal PCR Master Mix (Applied BioSystems) in a 20 µl reaction volume containing 50 ng of cDNA. All reactions were performed in triplicate and included a negative control. PCR reactions were carried out using an ABI Prism 7500 Sequence Detection System (Applied Biosystems). Cycling conditions were: 2 min at 50 °C, 10 min at 95 °C, and 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Relative quantification of mRNA levels was obtained by the 7500 system software which uses the comparative method (ΔCT). 2.6. Western blot Membrane proteins were extracted according to previously described methods [18,19]. Equal amounts (100 μg/sample) of ventricular and atrial membrane proteins were separated on 7.5% SDS-PAGE gels and transferred to polyvinylidine difluoride (PVDF) membranes. Membranes were then incubated with the following antibodies: polyclonal anti-HCN1 and anti-HCN2 (Alomone), monoclonal anti-HCN4 (Abcam), polyclonal antiMiRP1 (Santa Cruz Biotechnology, Santa Cruz, California). Immunoreactive bands were detected by Immobilon Western Chemiluminescence HRP Substrate (Millipore) and quantified by densitometry analysis using an ImageQuant 350 imager and
ImageQuant TL-1 software (GE Healthcare). Anti-actin and/or anti-GAPDH antibody (Sigma, Santa Cruz) were used to control for equal protein loading and to normalize ion channel protein band intensity. All Western blot target bands are expressed quantitatively by normalization to the control band on the same lane. Stably transfected mHCN2 and hHCN4 HEK293 cell lines (kindly provided by M. Biel) were used as positive controls for detection of HCN2 and HCN4. Rat brain was used as a positive control for detection of the HCN1 isoform. 2.7. Drugs (-)-Isoproterenol (Sigma-Aldrich) was dissolved in distilled water to get a stock solution with a final concentration of 10 mM. The stock solution, containing ascorbic acid (1 mg/ml), was then diluted with Tyrode's solution to get the final isoproterenol concentration (1 µM). 2.8. Data analysis and statistics 2.8.1. Patch-clamp experiments Activation curves, obtained by plotting specific conductance of If (gf) vs. membrane potential, were analyzed as described previously [20]. Time constants for If activation and deactivation were estimated by fitting macroscopic and tail currents with a mono-exponential function. For estimation of opening and closing rates, the bell-shaped distribution of averaged time constant for current activation and deactivation was fitted to tau = 1 / (α0 · e− Vm / Vo + ß0 · eVm / Vo) where the best fits of α0 and ß0 represent the opening and closing rates at zero voltage, respectively, and with Vm: transmembrane potential and V0: reversal potential. Data analysis and fitting were performed by using the programs Clampfit (pClamp9, Molecular Devices Inc.) and Origin 4.1 (MicroCal Software Inc.). 2.8.2. RT-PCR and Western blot experiments Each determination was performed on an individual heart. For competitive RT-PCR, products were visualized under UV light with ethidium bromide staining in 1.5% agarose gels. The images
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were captured with a Nighthawk camera, and band density was determined with Quantity One software. A DNA ladder (100 ng) was used to determine the size and quantity of DNA bands and to create a standard curve in each experiment for absolute quantification. Natural logarithm plots LN([target] / [mimic]) versus LN([mimic]) were fitted by linear regression to determine the absolute concentration of target mRNA as previously described [18]. Relative mRNA quantification performed with real-time PCR was calculated by the comparative ΔCt method [21]. Western blot band intensities are expressed as OD units corresponding to densitometric band intensity following background subtraction, divided by Actin/Gapdh-signal intensity for the same sample. Data are expressed as mean ± SEM. Statistical comparisons were performed with unpaired Student's t tests. A 2-tailed probability b 0.05 was taken to indicate statistical significance.
3. Results 3.1. Characterization of If in HuVMs Our previous results [6] demonstrated a 2-fold increase of If amplitude in HuVM from ICM patients compared to control hearts. To get deeper insights into the molecular mechanisms underlying this phenomenon, we first analysed the kinetic properties of If in HuVM and the effects of β-AR stimulation. Fig. 1 shows representative experiments performed in isolated myocytes from control (Ctrl) and ICM hearts in the absence and presence of 1 µM isoproterenol (A). If was evoked by a twopulse protocol: from a holding potential of − 30 mV, the cell was hyperpolarized to − 70 mV and then to −110 mV. In all tested cells, exposure to 1 µM isoproterenol was able to increase the current amplitude during the first pulse but not during the second
Fig. 1. Effect of isoproterenol on If in HuVM from control and deseased hearts. Analysis of If kinetic properties. Panel A: Typical If recordings in isolated myocytes from control (Ctrl) and diseased (ICM) hearts in the absence (○) and presence (●) of 1 µM isoproterenol, evoked by a two-step pulse. Related activation curves are shown in panel B. Panel C: Normalized and superimposed If recordings, evoked by a step potential at − 110 mV, in Ctrl and ICM human ventricular cardiomyocytes: activation kinetic is faster in ICM than Ctrl. Panel D reports mean time constant values (tau) for Ctrl and ICM as function of test potential: the bell-shaped curve shifts toward less negative potentials, as indicated by the lines drawn from the peak of curves (midpoint voltage of activation curves). Closed and open triangles: mean ± SEM values for Ctrl cells, as estimated from If activation and deactivation, respectively; closed and open squares: mean ± SEM values for ICM cells, as estimated from If activation and deactivation, respectively. ⁎p b 0.05; ⁎⁎p b 0.01.
F. Stillitano et al. / Journal of Molecular and Cellular Cardiology 45 (2008) 289–299 Table 3 Estimated values for activation and deactivation time constant of If
Control ICM
α0 (s− 1)
β0 (s− 1)
V0 (mV)
0.0010 0.0021
37.3 21.25
− 14.7 − 14.2
α0 and ß0 represent the opening and closing rates at zero voltage, respectively, and V0 the reversal potential of If. Time constants and reversal potentials were estimated by fitting macroscopic and tail currents with a mono-exponential function as described in the Methods section.
one, suggesting a shift of the activation curve of If rather than a real increase in its amplitude. Indeed, isoproterenol produced a significant (p b 0.05) positive shift of the activation potential midpoint (ΔVh): ΔVh[Control] = 11.5 ± 2.6 mV (n = 3; from − 80.0 ± 0.8 to − 71.5 ± 0.3 mV), and ΔVh[ICM] = 12.6 ± 3.3 mV (n = 9; from − 72.4 ± 0.4 to − 62.3 ± 0.6 mV); averaged activation curves are shown in Fig. 1B. This result strongly suggests the prevalence of cAMP-regulated isoforms (i.e. HCN2 and HCN4) in both normal and failing hearts. Panel C in Fig. 1 shows that, after normalizing and superimposing current tracings, If recorded in ICM was faster than in control. This was a consistent finding, as shown by panel D: at − 90 mV, the activation constant (tau) was significantly faster in ICM (768 ± 97 ms, n = 39), than in control (1674 ± 260, n = 29; p b 0.01). However, plotting activation and deactivation kinetics as a function of voltage steps (panel D) demonstrated that kinetics ranked between similar values in the
293
two groups. Fitting values as described in the Methods section gave typical bell-shaped curves: for ICM cells, the curve was indeed shifted toward less negative values compared to control cells. As expected, peaks of bell-shaped curves lay near the midpoint voltage of activation curves as previously documented [6], which are indicated in Fig. 1 by dotted (control, − 80.6 mV) and dashed lines (ICM, − 71 mV). Table 3 summarizes the values deduced by best-fitting of kinetic parameters of If recorded in controls and cardiomyopathic cells, using the equation given in Methods. V0 estimated by fitting of activation-deactivation constants was consistent with the reversal potential of If previously measured by tail current analysis [6]. Current density, measured at − 90 mV, was significantly larger in ICM (17.0 ± 1.6 pA/pF, n = 41) than in control (7.6 ± 1.0 pA/pF, n = 30) (p b 0.001). On the whole, electrophysiological results allowed us to hypothesise that If expression changes in the failing human ventricle and that these differences may be attributable to qualitative and/or quantitative changes in the expression of different HCN isoforms. Therefore, we directly investigated whether molecular analyses substantiate this hypothesis. 3.2. HCN mRNA expression in human ventricle Panels A and C in Fig. 2 show examples of gels for HCN2 and HCN4 obtained from ventricular tissue of ICM (VICM) and control (VCtrl) hearts: the competition between mimic and target
Fig. 2. mRNA expression of HCN2 and HCN4 in human ventricle. Panels A and C: Representative gels for HCN2 and HCN4 competitive RT-PCR on control (VCtrl) and diseased (VICM) ventricles; for each lane (1–5) is reported the mRNA amount of mimic gene used, whilst the mRNA of specific gene is always constant (1 μg of total mRNA extracted). These conditions are valid for all RT-PCR gels shown in this paper. Panels B and D: the histograms show the HCN2 and HCN4 mRNA amounts as mean ± SEM obtained in control (VCtrl, n = 4 patients) and diseased (VICM, n = 9 patients for HCN2 and n = 8 patients for HCN4); ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001.
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mRNA supports the specificity of the method. The bar graphs on the right in Fig. 2 summarize the results obtained by competitive RT-PCR in control (VCtrl) and ICM (VICM) hearts for HCN2 (panel B) and HCN4 (panel D). The average absolute amounts of HCN2 mRNA (expressed as attomol/μg total mRNA) were markedly increased in ICM compared to control (5.5 ± 1.8; n = 4 and 0.6 ± 0.1; n = 9 respectively; p b 0.01). Likewise, a significant increase (p b 0.001) in mRNA levels for HCN4 was observed in ICM (32.4 ± 7.9; n = 4) compared to control (4.6 ± 1.1, n = 8). Since upregulation of HCN4 has been indicated as a hallmark of altered gene expression in human heart failure [13], mRNA levels were quantitatively assessed by using real-time PCR analysis. Again, a significant increase of HCN4 gene expression in ICM hearts compared to control was found (control = 0.97 ± 0.05 n = 6; ICM = 3.0 ± 0.77 n = 9, p b 0.05).
3.3. HCN protein levels in human ventricle Typical Western blots for HCN2 and HCN4 proteins are shown in Fig. 3 (panel A and C respectively). Consistent with data in the literature [19], a band of ~ 100 kDa was observed for HCN2 protein. This result was further validated by Western blot analysis in HEK293 cells expressing selectively mHCN2 (+CHCN2) or hHCN4 (+CHCN4). The positive-control experiments with HCN2 and HCN4 expressing HEK293 cells suggest that HCN subunits in HEK293 cells exist in two isoforms, a predominant isoform with a molecular mass being in the range of the theoretical molecular mass and a larger isoform with a higher molecular mass, as previously demonstrated in the literature [19]. To investigate whether the size difference is due to N-linked glycosylation, we treated membrane fractions containing HCN channels with N-glycosidase F (Data not
Fig. 3. Protein expression of HCN2 and HCN4 in human ventricle. Panel A: Representative Western blots for HCN2 protein, obtained in human ventricle from control (VCtrl, grey line) and diseased (VICM, black line) hearts. M indicates size marker (Santa Cruz Biotechnology), +CHCN2 indicates stably transfected mHCN2 HEK293 cell line. Two bands are visible, likely corresponding to unglycosylated (lower molecular mass) and N-glycosylated (higher molecular mass) forms of the protein (see text for details). Bottom: ACTIN signals on the same samples. Panel B: Bar graph shows the averaged values for HCN2 protein in control (VCtrl, n = 6) and diseased (VICM, n = 5) patients; ⁎p b 0.05. Panel C: Representative Western blots for HCN4 protein, obtained in human left ventricle from control (VCtrl, grey line) and diseased (VICM, black line) hearts. M indicates size marker (Santa Cruz Biotechnology), +CHCN4 represents stably transfected hHCN4 HEK293 cell lines; two bands are visible, likely corresponding to unglycosylated (lower molecular mass) and N-glycosylated (higher molecular mass) forms of the protein (see text for details). Bottom: ACTIN signals on the same samples. Panel D: Average values for HCN4 protein in control (VCtrl, n = 7 patients) and diseased (VICM, n = 6 patients); ⁎p b 0.05.
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shown). After incubation with this enzyme antibodies directed against HCN2 or HCN4 detected virtually only the smaller band, whereas the larger band almost completely disappeared. We have referred to the lower molecular mass bands in order to identify and quantify HCN2 and HCN4 protein in human atrial and ventricular tissue. The protein amount (normalized with respect to actin and expressed in arbitrary OD units) was significantly larger in ICM (2.4 ± 0.36, n = 5) than in control (1.3 ± 0.2; p b 0.05 n = 6) (Fig. 3B), paralleling changes in mRNA levels. Western blots for HCN4, carried out in control (VCtrl) and diseased (VICM) ventricular samples, are illustrated in Fig. 3C. As for HCN2, protein changes paralleled those observed for mRNA, with HCN4 being significantly higher in ICM (7.3 ± 1.7, n = 6) than in control samples (2.9 ± 0.8, n = 7) (p b 0.05) (Fig. 3D).
As shown in Fig. 4, mRNA levels for HCN2 were significantly increased in atrial tissue of ICM patients (AICM) (1.28 ± 0.25 n = 4) compared to those observed in atrial specimens from patients with normal ventricular function (ACtrl) (0.06 ± 0.02, n = 7), p b 0.0001 (A, B). Likewise, a greater expression of mRNA HCN4 was found in ICM (AICM) (33.5 ± 2.5, n = 4) compared to control (ACtrl) (8.5 ± 3.4 n = 8), p b 0.01 (C, D). As for the ventricle, protein expression tested by Western blotting showed the presence of bands at the expected molecular masses for HCN4 and HCN2 (130 and 97 kDA, respectively), further confirmed by the corresponding bands obtained in lysates from stably-expressing cell lines (CHCN4 and CHCN2). Protein levels mirrored mRNA levels for both HCN2 (ICM: 6.7 ± 1.42 n = 4; control: 3.5 ± 0.4 n = 6; p b 0.05) and HCN4 (ICM: 4.78 ± 0.8, n = 5; control: 1.46 ± 0.5 n = 6; p b 0.01) (Fig. 5).
3.4. Changes in HCN expression in human atrium parallel those in ventricle
3.5. HCN1 mRNA and protein determination
Finally, we investigated whether altered HCN expression is exclusive of ventricular remodeling, or similar abnormalities also occur in the atria of heart failure patients. To date, few data exist on atrial HCN expression in humans [22].
At variance with the ventricular tissue, a clear-cut band corresponding to HCN1 mRNA was detected in atrial samples (Fig. 6A); no difference exists between failing (AICM: 188.50 ± 59.07 n = 4) and non-failing patients (ACtrl126.60 ± 35.83 n = 8) (Fig. 6B).
Fig. 4. mRNA expression of HCN2 and HCN4 in human right atrium. Panels A and C: Representative gels for HCN2 and HCN4 competitive RT-PCR on control (ACtrl) and diseased (AICM) atrium; for each lane (1–5) is reported the mRNA amount of mimic gene used, whilst the mRNA of specific gene is always constant (1 μg of total mRNA extracted). These conditions are valid for all RT-PCR gels shown in this paper. Panels B and D: the histograms show the HCN2 (⁎⁎⁎p b 0.001) and HCN4 (⁎⁎⁎p b 0.001) mRNA amounts as mean ± SEM obtained in control (ACtrl, n = 7 patients for HCN2 and n = 8 patients for HCN4) and diseased (AICM, n = 4 patients) atrium.
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human specimens. An example of a Western blot is shown in Fig. 6D; the expected size is 25 kDa and the MiRP1 band is indicated by the arrow, as confirmed by the pre-incubation with the control antigen. Neither regional- nor disease-dependent differences were observed in the human samples (Fig. 6E). However, the quality of the blots was suboptimal and they contained several bands in addition to the band we presumed to be MiRP1 based on molecular mass and suppression by preincubation with control antigen. We were unable to perform competitive RT-PCR because of difficulty obtaining acceptable probes. Because of the technical limitations in protein quantification and difficulties with competitive RT-PCR, we sought to correlate our protein data with gene-expression measurements for MiRP1 in atrial and ventricular samples by TaqMan RealTime PCR. The results demonstrate that at the mRNA level, MiRP1 expression is near the detection limit (Ct ~ 40). As shown in Fig. 6F, MiRP1 transcript expression was very weak compared to GAPDH, and there were no tissue or disease related differences. The validity of GAPDH as a disease-independent control is supported by the data in Fig. 6G, which show that GAPDH transcript expression was unaffected by ICM. 4. Discussion
Fig. 5. Protein expression of HCN2 and HCN4 in human right atrium. Panel A: An example of Western blot for HCN2 in human atrium from control (grey line) and diseased (black line) hearts. M indicates size marker (Santa Cruz Biotechnology), +CHCN2 indicates stably transfected mHCN2 HEK293 cell line, +CHCN4 indicates stably transfected hHCN4 HEK293 cell lines; for both cell lines, two bands are visible, likely corresponding to unglycosylated (lower molecular mass) and N-glycosylated (higher molecular mass) forms of the HCN2 and HCN4 protein, respectively. Bottom: ACTIN signals on the same samples. Panel B: The graph reports the obtained values as mean ± SEM for HCN2 protein in control (ACtrl, n = 6 patients) and diseased (AICM, n = 4 patients); ⁎p b 0.05. Panel C: Typical Western blot for HCN4 in human atrium from control (grey line) and diseased (black line) hearts. Bottom: ACTIN signals on the same samples. Panel D: The graph reports the averaged values for HCN4 protein in human atrium from control (ACtrl, n = 6 patients) and diseased (AICM, n = 5 patients); ⁎⁎p b 0.01.
However, as shown in Fig. 6C, we could not detect any HCN1 protein band in atrial samples, similar to ventricle. 3.6. MiRP1 mRNA and protein determination Recent data suggest that the expression of MiRP1 (the putative β subunit of the If-channel) shows regional differences, being larger in atrial versus ventricular tissue in the rabbit [23], and undergoes changes in the failing heart [24]. Therefore, we analysed the expression of MiRP1 in atrial and ventricular
The major novelty of our study consists in the definition of the molecular mechanisms underlying changes in If expression and properties in the diseased human heart. Indeed, this is the first study comparing the mRNA and protein expression of HCN subunits in the human atria and ventricle under normal and heart failure conditions. Our results demonstrate that in human heart failure, an upregulation of ventricular HCN2 and HCN4 underlies the increase in functional If current; moreover, a similar phenomenon occurs in the atria of explanted hearts, where molecular changes mirror those observed in the left ventricle. The molecular profile of HCN isoform expression is in agreement with electrophysiological properties of If, i.e., activation kinetics and response to β-AR stimulation, detected in single myocytes from normal and diseased human hearts. Overall, these findings are of particular relevance in view of the potential proarrhythmic role of overexpression of If, which may contribute to increased ventricular (and atrial) propensity to arrhythmias in heart failure. Previous and present electrophysiological data show that, in ICM myocytes, If is not only of larger amplitude [6] but also activates at a less negative potential (− 71 mV) compared to cells from controls (− 81 mV). This difference can be appreciated also by comparing bell-shaped curves describing activation and deactivation kinetics: the ICM curve was shifted rightward with respect to control by approximately 10 mV. Such a difference may explain why current elicited by hyperpolarization to − 90 mV was apparently faster in ICM than in control cells: indeed, according to the allosteric model of the f-channel [25], the more negative the voltage step (relative to midpoint activation), the faster is activation kinetics. This shift could be due to changes in the relative proportion of different HCN isoforms, since heterologous re-expression of human HCN4 gives a current whose Vh is less negative (−81 mV) than that showed by HCN2 in similar conditions (−92 mV) [26].
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Fig. 6. Expression of HCN1 and MiRP1 in human myocardium. Panel A: Examples of gels of RT-PCR products for HCN1 in human atrium (A) and ventricle (V) from control (Ctrl) hearts; in the ventricle only the mimic gene was detected. Panel B: The graph summarizes the values as mean ± SEM for HCN1 mRNA in control (ACtrl) and diseased (AICM) atrium. Panel C: Typical Western blot for HCN1 protein in rat brain (RB, used as positive control), in human ventricle (V) and atrium (A) from control (Ctrl) and diseased (ICM) hearts; the effect of pre-incubation with control antigen (CA) is also shown. Panel D: Representative Western blot for MiRP1 protein in human ventricle (V) and atrium (A) from control (Ctrl) and diseased (ICM) hearts; the specificity of the MiRP1 protein band was confirmed by pre-incubation of the same membrane with control antigen (CA); the same loading control (GAPDH) applies for both conditions. Panel E: The graph summarizes the amount of protein as mean ± SEM. Panel F: Mean ± SEM MiRP1 mRNA expression relative to GAPDH in human ventricle (V) and atrium (A) from control (Ctrl) and diseased (ICM) hearts. Panel G: Mean ± SEM GAPDH mRNA expression relative to 18S-rRNA in control and diseased ventricles.
Moreover, since both isoforms (HCN2 and HCN4) are modulated by cAMP, in contrast with HCN1, these results imply that channel sensitivity to ligands activating the cAMP pathway (such as catecholamines) should be similar in diseased and normal heart. Indeed, isoproterenol produced a significant positive shift of the midpoint activation potential similar in both diseased and control cells. A deeper comprehension of the molecular mechanisms underlying If overexpression and changes in voltage-dependence of activation cannot be obtained by solely applying electrophysiological techniques, which are unable to reveal the isoform composition of native channels. Therefore, we aimed to assay mRNA and protein levels for specific HCN isoforms [27,28]. As men-
tioned above, heterologously re-expressed HCN isoforms differ significantly in their biophysical properties. The kinetics of HCN2 are slower than those of HCN1, and faster than those of HCN4; also, HCN2 has a more negative activation threshold than either HCN1 or HCN4 [26]. The sensitivity to cAMP varies among different isoforms, with HCN1 being much less responsive than either HCN2 or HCN4 [29]. Heterogeneity of If-channels may be produced by the combination of diverse mechanisms such as a switch of the subunit composition and stoichiometry [30,31], up- and downregulation of auxiliary subunits such as MiRP1 [32], and the influence of cellular environment [15]. However, our present results do not suggest changes in the relative proportion of different HCN isoforms, or important modifications in the
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expression of the auxiliary subunit MiRP1. Indeed, other mechanisms might be responsible for the altered biophysical properties of If in ICM myocytes, such as: 1) influence of the cellular environment; 2) altered function and/or expression of other auxiliary subunit(s), not yet identified; 3) lipid raft disruption altering HCN4 subunit localization and electrophysiological properties [33]. These hypotheses need further investigation. In agreement with previous studies, [34] our results demonstrate that HCN1 mRNA and protein are absent in human ventricles. On the other hand, HCN1 mRNA was detected in atrial specimens; but no protein was found in atrial Western blots, likely because of low expression. HCN2 expression is stronger in ventricles than atria and HCN4 mRNA is expressed ~ 10-fold more strongly than HCN2. The predominance of HCN4 supports our hypothesis on the nature of native If current in human ventricle, as deduced from If properties. The If activation time constant at − 90 mV averaged 1674 ± 260 ms in normal HuVM, a value similar to that reported for heterologously re-expressed HCN4 (1271 ms) and slower than that of HCN2 (278 ms) [15]. Although competitive RT-PCR experiments using RNA mimics as standards may have some inaccuracy, the consistency of these results with those obtained by Western blots and electrophysiological measurements allows us to infer that mRNA and protein expression of HCN2 and HCN4 increased in both atria and ventricles from failing hearts. In addition, quantitative PCR provided qualitatively similar results to competitive RTPCR for HCN4. Since the MiRP1 subunit can modify If for a given level of HCN expression [32], we quantified MiRP1 protein and mRNA expression and found them to be unaffected by heart failure. Thus, If gain of function in heart failure is mainly supported by increased transcription and synthesis of channel alpha subunits (HCN2/4), although post-translational mechanisms cannot presently be ruled out. Electrical heterogeneity exists within the human ventricular wall, likely due to the differential expression of specific ion channel genes [35]. Our samples contained epicardial, midmyocardial and endocardial cells; thus, possible transmural differences in HCN overexpression were not evaluated in the present study. A higher If expression has been detected in the left compared to the right ventricle in rat myocytes [36,37], but to our knowledge, no information exists on transmural variation of If within the left ventricle. It is worth noting, however, that mRNA transcript for HCN did not seem to vary within the left ventricular wall in the rat or canine heart, at variance with sodium (SCN5A) and potassium (Kcnd2) channels [38]. Another original finding of our work is the demonstration that a similar pattern of HCN overexpression occurs in the atria of heart failure patients. It is worth noting that all these patients were in sinus rhythm. Thus, alterations of systemic and local neurohormonal environment may similarly affect ventricular and atrial HCN patterns, by selectively inducing the overexpression of HCN2/4 isoforms at both mRNA and protein level. No difference in mRNA expression of HCN1 was observed between normal and heart failure atria. HCN1 protein was absent in atrial tissue in both conditions, suggesting that post-transcriptional mechanisms reduce protein expression to undetectable levels or that the mRNA is not transcribed. Similar results have been obtained in cultured
adult rat ventricular myocytes [39]. Taken together, these findings may allow hypothesising that environmental factors, such as hypertrophic factors, specifically regulate mRNA transcription of some isoforms (HCN 2/4) and not others (HCN1). The upregulation of HCN2 and HCN4 in heart failure patients and the associated increased contribution of If to spontaneous cellular activity [40] might be implicated in ectopic rhythm formation. Heart failure causes downregulation of a variety of potassium channels, produces spatially-heterogeneous abnormalities in connexin expression and function, and induces tissue fibrosis that impairs impulse propagation [41]. The resulting enhanced dispersion of repolarization and spatial disturbances in conduction properties provide a substrate for reentry arrhythmias [41]. The increased pacemaker current expression and function seen in both the atria and ventricles in heart failure may provide ectopic triggers that initiate clinically significant arrhythmias including atrial fibrillation and potentially life-threatening ventricular tachyarrhythmias. In conclusion, a combination of functional and molecular approaches, as used here, might allow for clarification of the mechanisms by which pathology (e.g., heart failure) affects channel expression related to cardiac cellular pacemaker function, thus modifying the electrophysiological properties of myocytes to cause susceptibility to cardiac arrhythmias. Acknowledgments The financial support of Minister of Education, University and Research (PRIN 2006), Telethon (Grant GGP05093) and Ente Cassa di Risparmio di Firenze, Italy and of the Canadian Institutes of Health Research, is gratefully acknowledged. We wish to acknowledge the support of the Normacor Partners (contract LSH M/CT/2006/018676) and particularly Prof. Martin Biel (University of Munich, Germany) for providing stably transfected HCN2 and HCN4 HEK293 cells. References [1] Cowie MR, Mosterd A, Wood DA, Deckers JW, Poole-Wilson PA, Sutton GC, et al. The epidemiology of heart failure. Eur Heart J 1997;18:208–25. [2] Guyatt GH, Devereaux PJ. A review of heart failure treatment. Mt.Sinai. J Med 2004;71:47–54. [3] Thom T, Haase N, Rosamond W, Howard VJ, Rumsfeld J, Manolio T, et al. Heart Disease and Stroke Statistics—2006 Update: a report from the american heart association statistics committee and stroke statistics subcommittee. Circulation 2006;113:e85–151. [4] Tomaselli GF, Zipes DP. What causes sudden death in heart failure? Circ Res 2004;95:754–63. [5] Tomaselli GF, Beuckelmann DJ, Calkins HG, Berger RD, Kessler PD, Lawrence JH, et al. Sudden cardiac death in heart failure. The role of abnormal repolarization. Circulation 1994;90:2534–9. [6] Cerbai E, Sartiani L, DePaoli P, Pino R, Maccherini M, Bizzarri F, et al. The properties of the pacemaker current I-F in human ventricular myocytes are modulated by cardiac disease. J Mol Cell Cardiol 2001;33:441–8. [7] Hoppe UC, Jansen E, Sudkamp M, Beuckelmann DJ. Hyperpolarizationactivated inward current in ventricular myocytes from normal and failing human hearts. Circulation 1998;97:55–65. [8] Sridhar A, Dech SJ, Lacombe VA, Elton TS, McCune SA, Altschuld RA, et al. Abnormal diastolic currents in ventricular myocytes from spontaneous hypertensive heart failure rats. Am J Physiol Heart Circ Physiol 2006;291:H2192–8.
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Journal of Molecular and Cellular Cardiology 45 (2008) 289 – 299 www.elsevier.com/locate/yjmcc
Original article
Molecular basis of funny current (If) in normal and failing human heart Francesca Stillitano a,1 , Giuseppe Lonardo a,b,1 , Stephen Zicha b , Andras Varro c,d , Elisabetta Cerbai a , Alessandro Mugelli a,⁎, Stanley Nattel b b
a Centro Interuniversitario di Medicina Molecolare e Biofisica Applicata (CIMMBA), University of Florence, Viale Pieraccini 6, 50139 Florence, Italy Montreal Heart Institute Research Center and Department of Medicine, Université de Montréal, 5000 Belanger St. E. H1T 1C8 Montreal, Quebec, Canada c Division of Cardiovascular Pharmacology, Hungarian Academy of Sciences, University of Szeged, Hungary d Department of Pharmacology and Pharmacotherapy, University of Szeged, Hungary
Received 28 March 2008; received in revised form 28 April 2008; accepted 30 April 2008 Available online 11 May 2008
Abstract If overexpression has been functionally demonstrated in ventricular myocytes from failing human hearts. Altered expression of If-channels as a consequence of electrophysiological remodeling may represent an arrhythmogenic mechanism in heart failure; however, the molecular basis of If overexpression in human cardiac disease is unknown. HCN1, 2 and 4 subtypes, which encode If-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. If 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 b 0.01 for mRNA, p b 0.05 for protein). These results are consistent with the electrophysiological data showing that, in failing ventricular myocytes, If is of larger amplitude and activates at less negative potential. Changes in mRNA and protein expression of both HCN2/4 isoforms in atrial specimens from patients with heart failure mirrored those observed in ventricles ( p b 0.001 for mRNA, p b 0.05 for protein). No diseasedependent alteration was detected for MiRP1, the putative β-subunit of the If-channel. In conclusion, HCN4 is the predominant channel subtype in normal human heart, and its expression is further amplified by disease. HCN upregulation likely contributes to increased If and may play a role in ventricular and atrial arrhythmogenesis in heart failure. © 2008 Elsevier Inc. All rights reserved. Keywords: Hyperpolarization-activated Cyclin Nucleotide gated channel; Ischemic cardiomyopathy; Human heart failure; Gene expression; Electrophysiology; Arrhythmias
1. Introduction The heart failure burden is still very high. The syndrome has an annual incidence ranging from 1 to 5 per 1000, increasing with age up to 40 cases per 1000 in people older than 75 years [1]. Recent estimates suggest annual mortalities for NYHA Class II, III and IV patients of 10%, 20% and 60%, respectively [2]. Mortality after 8 years is ~ 80% and half of the deaths are sudden and unexpected [3]. Sudden deaths are generally thought to be due to ventricular arrhythmias [4]. Studies on possible underlying arrhythmogenic mechanisms have focused largely on repolarization abnormal⁎ Corresponding author. Tel.: +39 055 4271264; fax: +39 055 4271280. E-mail address: [email protected] (A. Mugelli). 1 These authors contributed equally to this paper. 0022-2828/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2008.04.013
ities, and investigated the ionic basis of abnormal prolongation of the action potential duration [5]. However, other abnormalities in ventricular myocyte electrophysiology may contribute to enhanced electrical instability or trigger life-threatening arrhythmias in heart failure [4]. Recently, several studies have addressed the pathophysiological role of the hyperpolarizationactivated current (If) in this setting [6–8]. If is a mixed Na+/K+ inward current, activated by hyperpolarization, and encoded by a family of genes termed Hyperpolarization-activated Cyclic Nucleotide-gated channel (HCN) genes [9]. Originally regarded as a hallmark of pacemaker cells in the heart, If has been identified in atrial and ventricular myocytes from both animal models and humans [10]. An arrhythmogenic role for If in cardiac hypertrophy and failure has been inferred, based on the presence of this current in ventricular myocytes isolated from severely hypertrophied rat hearts [11] and from human hearts
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explanted because of terminal heart failure [12]. Indeed, the functional expression of If seems to be differentially modulated by disease, since its density is larger in human ventricular myocytes (HuVMs) isolated from the hearts of patients with ischemic than in those with dilated cardiomyopathy [6]. So far, the molecular basis of If overexpression in human heart failure is largely unknown. A 3-fold increase of mRNA coding for HCN4 was reported in end-stage failing human hearts [13] and a ~2-fold increase of HCN4 protein was reported in the right atrium of a canine heart failure model [14], but no information is available regarding the relative proportion of diverse HCN isoforms in the human heart. In fact, HCN subtypes expressed in rat ventricular myocytes and HEK 293 cells have characteristic kinetic properties [15] and different sensitivity to cAMP has been described [16]. The present study aimed to analyze, in healthy and diseased human cardiac tissues, the profile of HCN isoform expression, in terms of mRNA and protein, and to correlate these data with electrophysiological properties (activation kinetics and response to β−AR stimulation) of If in HuVMs. 2. Methods 2.1. Patients Ventricular samples were obtained from undiseased hearts of organ donors whose hearts were explanted to obtain pulmonary and aortic valves for transplant surgery (University of Szeged). Before cardiac explantation, these organ donor patients did not receive medication except dobutamine, furosemide, and plasma expanders. All experimental protocols were approved by the Albert Szent-Gyorgyi Medical University Ehtical review Board (No. 51-57/1997 OEj). Failing heart tissues for biochemical studies were obtained from patients undergoing transplantation for end-stage ischemic cardiomyopathy (Montreal Heart Institute). Atrial appendages were obtained from patients undergoing cardiac surgery for coronary artery bypass graft with conserved ventricular function and from ICM explanted hearts. Characteristics of patients are reported in Table 1. Samples were quickly frozen in liquid nitrogen and stored at − 80 °C, or used for isolation of single HuVMs as previously described [12]. Informed consent for the use of tissue samples was obtained from patients undergoing cardiac surgery. The investigation conforms with the principles outlined in the “Declaration of Helsinki” of the World Medical Association [17] and was approved by the local ethical committee. 2.2. Cell isolation and If recording The isolation procedure has been described elsewhere [6]. After digestion of a portion of the left ventricle, cells were dissociated from small transmural specimens including epicardium, midmyocardium, and endocardium. Following clamp steps to hyperpolarized membrane potential (usually −120 mV), an If like current was recorded in most of the tested cells. If was considered to be present in a given cell when hyperpolarization elicited a time-dependent increasing inward current which could be blocked by adding 4 mM CsCl. To establish the activation
Table 1 Characteristics of the patients from whom atrial (A) or ventricular (V) samples were obtained
Total number (m/f) Age (mean ± sem) EF (%)
A (Ctrl)
A (ICM)
V (Ctrl)
V (ICM)
26 (16/10) 54.5 ± 2.3 51.8 ± 2.3 (13) normal a (13)
4 (3/1) 57.4 ± 4.5 22.6 ± 5.6
17 (9/8) 47 ± 3 n.a. b
13 (12/1) 56.7 ± 2.3 20.8 ± 4.2
5 2 3 5 3 6
– – – –
16 11 6 13 4 6
Medication (number of patients) Diuretics 1 Digoxin 2 Antiarrhythmics 9 ACE-I 4 Β-blockers 3 Anticoagulants 9
EF: Ejection Fraction; ACE-I: angiotensin converting enzyme inhibitors. Ctrl: control i.e. patients without cardiac dysfunction; ICM: patients with ischemic cardiomyopathy. a The ejection fraction was reported as normal without specifying percentage value, in the clinical records of 10 patients. b No pre-mortem cardiac function data were available for control ventricles. All hearts were from patients with presumed normal hearts that could not be transplanted for technical reasons.
curve, If was evoked by hyperpolarizing steps (range: −50 to − 140 mV) from a holding potential of −40 mV. The duration of pulses was progressively reduced from 3200 ms (at − 50 mV) to 1600 ms (at − 140 mV). Fitting was carried out by using the Clampfit program (pClamp vers. 9, Molecular Devices Inc.). Amplitude was automatically calculated as the difference between the value at the beginning of the hyperpolarizing step and the value extrapolated to the steady state. All experiments were performed at 36 °C. 2.3. RNA isolation Total mRNA was isolated as previously described [18]. Briefly, tissue specimens were homogenized in TRIzol reagent (Gibco BRL). Genomic DNA was eliminated by incubating in DNase I. RNA was quantified by spectrophotometry. All A260/ A280 nanometer ratios were above 1.8. Integrity was evaluated by ethidium bromide staining on a denaturing agarose gel. RNA samples were stored at −80 °C in RNAsecure resuspension solution (Ambion). 2.4. PCR primers and construction of RNA mimics Methods for RNA mimic construction has been previously described [14]. Briefly, gene-specific primers for RT-PCR were designed according to previously cloned sequences from regions with minimal homology among HCNs and specificity verified by comparison with the GenBank database with the use of BLAST and FASTA (see Table 2). Resulting PCR products were sequenced to ensure subunit-specificity. 2.5. Competitive RT-PCR and TaqMan real time PCR Competitive RT-PCR was performed as described [14], by using RNA mimic samples of serial 10-fold dilutions, added to
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Table 2 Competitive RT-PCR primers
HCN-1 HCN-2 HCN-4
Primers
Position
Product size (bp)
T (°C)
GeneBank
F-tggtggctacaatgccttta R-ttcctccgggacctcgtt F-cgttaccagggcaagatgtttg R-gttgtccacgctcagcgaat F-gtactcctacgcgctcttca R-gctctcctcgtcgaagatct
1406–1725
320
55
AF488549
1519–1911
393
55
NM_001194
1392–1704
313
55
AJ238850s
Real-time PCR primers and probes
HCN-4
GAPDH
Primer
Sequence
Forward Probe Reverse Forward Probe Reverse
5′-gagcaggagagggttaagtcagc-3′ 5′-ccacccctacagcgacttcagattttactgg-3′ 5′-cagcaacagcatcgtcaggt-3′ 5′-cttcaccaccatggagaaggc-3′ 5′-cctggccaaggtcatccatgacaacttt-3′ 5′-ggcatggactgtggtcatgag-3′
reaction mixtures containing 1 µg total RNA. First-strand cDNAs were synthesized at 42 °C (1 h) and used as template in PCR reactions. PCR conditions were 93 °C for 3 min followed by 30 cycles of 30 s at 93 °C, 30 s at 55 °C and 30 s at 72 °C. A final 72 °C extension step was performed for 5 min. Expression levels of HCN4 and MiRP1 genes were further investigated using real-time quantitative RT-PCR and TaqMan® probe-based chemistry. Primers and probes specific for human HCN4 and GAPDH were designed using PRIMER EXPRESS software (PE Applied Biosytems) (see Table 2). Resulting PCR products were sequenced to ensure subunit-specificity. Primers and probe for MiRP1 were obtained from ABI (Foster City, California) TaqMan Gene Expression Assay catalogue. This assay comes in a 20× reaction mix, spans an exon-exon junction, and is optimized to give approximately 100% efficiency. The real-time RT-PCR reactions were performed using TaqMan Universal PCR Master Mix (Applied BioSystems) in a 20 µl reaction volume containing 50 ng of cDNA. All reactions were performed in triplicate and included a negative control. PCR reactions were carried out using an ABI Prism 7500 Sequence Detection System (Applied Biosystems). Cycling conditions were: 2 min at 50 °C, 10 min at 95 °C, and 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Relative quantification of mRNA levels was obtained by the 7500 system software which uses the comparative method (ΔCT). 2.6. Western blot Membrane proteins were extracted according to previously described methods [18,19]. Equal amounts (100 μg/sample) of ventricular and atrial membrane proteins were separated on 7.5% SDS-PAGE gels and transferred to polyvinylidine difluoride (PVDF) membranes. Membranes were then incubated with the following antibodies: polyclonal anti-HCN1 and anti-HCN2 (Alomone), monoclonal anti-HCN4 (Abcam), polyclonal antiMiRP1 (Santa Cruz Biotechnology, Santa Cruz, California). Immunoreactive bands were detected by Immobilon Western Chemiluminescence HRP Substrate (Millipore) and quantified by densitometry analysis using an ImageQuant 350 imager and
ImageQuant TL-1 software (GE Healthcare). Anti-actin and/or anti-GAPDH antibody (Sigma, Santa Cruz) were used to control for equal protein loading and to normalize ion channel protein band intensity. All Western blot target bands are expressed quantitatively by normalization to the control band on the same lane. Stably transfected mHCN2 and hHCN4 HEK293 cell lines (kindly provided by M. Biel) were used as positive controls for detection of HCN2 and HCN4. Rat brain was used as a positive control for detection of the HCN1 isoform. 2.7. Drugs (-)-Isoproterenol (Sigma-Aldrich) was dissolved in distilled water to get a stock solution with a final concentration of 10 mM. The stock solution, containing ascorbic acid (1 mg/ml), was then diluted with Tyrode's solution to get the final isoproterenol concentration (1 µM). 2.8. Data analysis and statistics 2.8.1. Patch-clamp experiments Activation curves, obtained by plotting specific conductance of If (gf) vs. membrane potential, were analyzed as described previously [20]. Time constants for If activation and deactivation were estimated by fitting macroscopic and tail currents with a mono-exponential function. For estimation of opening and closing rates, the bell-shaped distribution of averaged time constant for current activation and deactivation was fitted to tau = 1 / (α0 · e− Vm / Vo + ß0 · eVm / Vo) where the best fits of α0 and ß0 represent the opening and closing rates at zero voltage, respectively, and with Vm: transmembrane potential and V0: reversal potential. Data analysis and fitting were performed by using the programs Clampfit (pClamp9, Molecular Devices Inc.) and Origin 4.1 (MicroCal Software Inc.). 2.8.2. RT-PCR and Western blot experiments Each determination was performed on an individual heart. For competitive RT-PCR, products were visualized under UV light with ethidium bromide staining in 1.5% agarose gels. The images
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were captured with a Nighthawk camera, and band density was determined with Quantity One software. A DNA ladder (100 ng) was used to determine the size and quantity of DNA bands and to create a standard curve in each experiment for absolute quantification. Natural logarithm plots LN([target] / [mimic]) versus LN([mimic]) were fitted by linear regression to determine the absolute concentration of target mRNA as previously described [18]. Relative mRNA quantification performed with real-time PCR was calculated by the comparative ΔCt method [21]. Western blot band intensities are expressed as OD units corresponding to densitometric band intensity following background subtraction, divided by Actin/Gapdh-signal intensity for the same sample. Data are expressed as mean ± SEM. Statistical comparisons were performed with unpaired Student's t tests. A 2-tailed probability b 0.05 was taken to indicate statistical significance.
3. Results 3.1. Characterization of If in HuVMs Our previous results [6] demonstrated a 2-fold increase of If amplitude in HuVM from ICM patients compared to control hearts. To get deeper insights into the molecular mechanisms underlying this phenomenon, we first analysed the kinetic properties of If in HuVM and the effects of β-AR stimulation. Fig. 1 shows representative experiments performed in isolated myocytes from control (Ctrl) and ICM hearts in the absence and presence of 1 µM isoproterenol (A). If was evoked by a twopulse protocol: from a holding potential of − 30 mV, the cell was hyperpolarized to − 70 mV and then to −110 mV. In all tested cells, exposure to 1 µM isoproterenol was able to increase the current amplitude during the first pulse but not during the second
Fig. 1. Effect of isoproterenol on If in HuVM from control and deseased hearts. Analysis of If kinetic properties. Panel A: Typical If recordings in isolated myocytes from control (Ctrl) and diseased (ICM) hearts in the absence (○) and presence (●) of 1 µM isoproterenol, evoked by a two-step pulse. Related activation curves are shown in panel B. Panel C: Normalized and superimposed If recordings, evoked by a step potential at − 110 mV, in Ctrl and ICM human ventricular cardiomyocytes: activation kinetic is faster in ICM than Ctrl. Panel D reports mean time constant values (tau) for Ctrl and ICM as function of test potential: the bell-shaped curve shifts toward less negative potentials, as indicated by the lines drawn from the peak of curves (midpoint voltage of activation curves). Closed and open triangles: mean ± SEM values for Ctrl cells, as estimated from If activation and deactivation, respectively; closed and open squares: mean ± SEM values for ICM cells, as estimated from If activation and deactivation, respectively. ⁎p b 0.05; ⁎⁎p b 0.01.
F. Stillitano et al. / Journal of Molecular and Cellular Cardiology 45 (2008) 289–299 Table 3 Estimated values for activation and deactivation time constant of If
Control ICM
α0 (s− 1)
β0 (s− 1)
V0 (mV)
0.0010 0.0021
37.3 21.25
− 14.7 − 14.2
α0 and ß0 represent the opening and closing rates at zero voltage, respectively, and V0 the reversal potential of If. Time constants and reversal potentials were estimated by fitting macroscopic and tail currents with a mono-exponential function as described in the Methods section.
one, suggesting a shift of the activation curve of If rather than a real increase in its amplitude. Indeed, isoproterenol produced a significant (p b 0.05) positive shift of the activation potential midpoint (ΔVh): ΔVh[Control] = 11.5 ± 2.6 mV (n = 3; from − 80.0 ± 0.8 to − 71.5 ± 0.3 mV), and ΔVh[ICM] = 12.6 ± 3.3 mV (n = 9; from − 72.4 ± 0.4 to − 62.3 ± 0.6 mV); averaged activation curves are shown in Fig. 1B. This result strongly suggests the prevalence of cAMP-regulated isoforms (i.e. HCN2 and HCN4) in both normal and failing hearts. Panel C in Fig. 1 shows that, after normalizing and superimposing current tracings, If recorded in ICM was faster than in control. This was a consistent finding, as shown by panel D: at − 90 mV, the activation constant (tau) was significantly faster in ICM (768 ± 97 ms, n = 39), than in control (1674 ± 260, n = 29; p b 0.01). However, plotting activation and deactivation kinetics as a function of voltage steps (panel D) demonstrated that kinetics ranked between similar values in the
293
two groups. Fitting values as described in the Methods section gave typical bell-shaped curves: for ICM cells, the curve was indeed shifted toward less negative values compared to control cells. As expected, peaks of bell-shaped curves lay near the midpoint voltage of activation curves as previously documented [6], which are indicated in Fig. 1 by dotted (control, − 80.6 mV) and dashed lines (ICM, − 71 mV). Table 3 summarizes the values deduced by best-fitting of kinetic parameters of If recorded in controls and cardiomyopathic cells, using the equation given in Methods. V0 estimated by fitting of activation-deactivation constants was consistent with the reversal potential of If previously measured by tail current analysis [6]. Current density, measured at − 90 mV, was significantly larger in ICM (17.0 ± 1.6 pA/pF, n = 41) than in control (7.6 ± 1.0 pA/pF, n = 30) (p b 0.001). On the whole, electrophysiological results allowed us to hypothesise that If expression changes in the failing human ventricle and that these differences may be attributable to qualitative and/or quantitative changes in the expression of different HCN isoforms. Therefore, we directly investigated whether molecular analyses substantiate this hypothesis. 3.2. HCN mRNA expression in human ventricle Panels A and C in Fig. 2 show examples of gels for HCN2 and HCN4 obtained from ventricular tissue of ICM (VICM) and control (VCtrl) hearts: the competition between mimic and target
Fig. 2. mRNA expression of HCN2 and HCN4 in human ventricle. Panels A and C: Representative gels for HCN2 and HCN4 competitive RT-PCR on control (VCtrl) and diseased (VICM) ventricles; for each lane (1–5) is reported the mRNA amount of mimic gene used, whilst the mRNA of specific gene is always constant (1 μg of total mRNA extracted). These conditions are valid for all RT-PCR gels shown in this paper. Panels B and D: the histograms show the HCN2 and HCN4 mRNA amounts as mean ± SEM obtained in control (VCtrl, n = 4 patients) and diseased (VICM, n = 9 patients for HCN2 and n = 8 patients for HCN4); ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001.
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mRNA supports the specificity of the method. The bar graphs on the right in Fig. 2 summarize the results obtained by competitive RT-PCR in control (VCtrl) and ICM (VICM) hearts for HCN2 (panel B) and HCN4 (panel D). The average absolute amounts of HCN2 mRNA (expressed as attomol/μg total mRNA) were markedly increased in ICM compared to control (5.5 ± 1.8; n = 4 and 0.6 ± 0.1; n = 9 respectively; p b 0.01). Likewise, a significant increase (p b 0.001) in mRNA levels for HCN4 was observed in ICM (32.4 ± 7.9; n = 4) compared to control (4.6 ± 1.1, n = 8). Since upregulation of HCN4 has been indicated as a hallmark of altered gene expression in human heart failure [13], mRNA levels were quantitatively assessed by using real-time PCR analysis. Again, a significant increase of HCN4 gene expression in ICM hearts compared to control was found (control = 0.97 ± 0.05 n = 6; ICM = 3.0 ± 0.77 n = 9, p b 0.05).
3.3. HCN protein levels in human ventricle Typical Western blots for HCN2 and HCN4 proteins are shown in Fig. 3 (panel A and C respectively). Consistent with data in the literature [19], a band of ~ 100 kDa was observed for HCN2 protein. This result was further validated by Western blot analysis in HEK293 cells expressing selectively mHCN2 (+CHCN2) or hHCN4 (+CHCN4). The positive-control experiments with HCN2 and HCN4 expressing HEK293 cells suggest that HCN subunits in HEK293 cells exist in two isoforms, a predominant isoform with a molecular mass being in the range of the theoretical molecular mass and a larger isoform with a higher molecular mass, as previously demonstrated in the literature [19]. To investigate whether the size difference is due to N-linked glycosylation, we treated membrane fractions containing HCN channels with N-glycosidase F (Data not
Fig. 3. Protein expression of HCN2 and HCN4 in human ventricle. Panel A: Representative Western blots for HCN2 protein, obtained in human ventricle from control (VCtrl, grey line) and diseased (VICM, black line) hearts. M indicates size marker (Santa Cruz Biotechnology), +CHCN2 indicates stably transfected mHCN2 HEK293 cell line. Two bands are visible, likely corresponding to unglycosylated (lower molecular mass) and N-glycosylated (higher molecular mass) forms of the protein (see text for details). Bottom: ACTIN signals on the same samples. Panel B: Bar graph shows the averaged values for HCN2 protein in control (VCtrl, n = 6) and diseased (VICM, n = 5) patients; ⁎p b 0.05. Panel C: Representative Western blots for HCN4 protein, obtained in human left ventricle from control (VCtrl, grey line) and diseased (VICM, black line) hearts. M indicates size marker (Santa Cruz Biotechnology), +CHCN4 represents stably transfected hHCN4 HEK293 cell lines; two bands are visible, likely corresponding to unglycosylated (lower molecular mass) and N-glycosylated (higher molecular mass) forms of the protein (see text for details). Bottom: ACTIN signals on the same samples. Panel D: Average values for HCN4 protein in control (VCtrl, n = 7 patients) and diseased (VICM, n = 6 patients); ⁎p b 0.05.
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shown). After incubation with this enzyme antibodies directed against HCN2 or HCN4 detected virtually only the smaller band, whereas the larger band almost completely disappeared. We have referred to the lower molecular mass bands in order to identify and quantify HCN2 and HCN4 protein in human atrial and ventricular tissue. The protein amount (normalized with respect to actin and expressed in arbitrary OD units) was significantly larger in ICM (2.4 ± 0.36, n = 5) than in control (1.3 ± 0.2; p b 0.05 n = 6) (Fig. 3B), paralleling changes in mRNA levels. Western blots for HCN4, carried out in control (VCtrl) and diseased (VICM) ventricular samples, are illustrated in Fig. 3C. As for HCN2, protein changes paralleled those observed for mRNA, with HCN4 being significantly higher in ICM (7.3 ± 1.7, n = 6) than in control samples (2.9 ± 0.8, n = 7) (p b 0.05) (Fig. 3D).
As shown in Fig. 4, mRNA levels for HCN2 were significantly increased in atrial tissue of ICM patients (AICM) (1.28 ± 0.25 n = 4) compared to those observed in atrial specimens from patients with normal ventricular function (ACtrl) (0.06 ± 0.02, n = 7), p b 0.0001 (A, B). Likewise, a greater expression of mRNA HCN4 was found in ICM (AICM) (33.5 ± 2.5, n = 4) compared to control (ACtrl) (8.5 ± 3.4 n = 8), p b 0.01 (C, D). As for the ventricle, protein expression tested by Western blotting showed the presence of bands at the expected molecular masses for HCN4 and HCN2 (130 and 97 kDA, respectively), further confirmed by the corresponding bands obtained in lysates from stably-expressing cell lines (CHCN4 and CHCN2). Protein levels mirrored mRNA levels for both HCN2 (ICM: 6.7 ± 1.42 n = 4; control: 3.5 ± 0.4 n = 6; p b 0.05) and HCN4 (ICM: 4.78 ± 0.8, n = 5; control: 1.46 ± 0.5 n = 6; p b 0.01) (Fig. 5).
3.4. Changes in HCN expression in human atrium parallel those in ventricle
3.5. HCN1 mRNA and protein determination
Finally, we investigated whether altered HCN expression is exclusive of ventricular remodeling, or similar abnormalities also occur in the atria of heart failure patients. To date, few data exist on atrial HCN expression in humans [22].
At variance with the ventricular tissue, a clear-cut band corresponding to HCN1 mRNA was detected in atrial samples (Fig. 6A); no difference exists between failing (AICM: 188.50 ± 59.07 n = 4) and non-failing patients (ACtrl126.60 ± 35.83 n = 8) (Fig. 6B).
Fig. 4. mRNA expression of HCN2 and HCN4 in human right atrium. Panels A and C: Representative gels for HCN2 and HCN4 competitive RT-PCR on control (ACtrl) and diseased (AICM) atrium; for each lane (1–5) is reported the mRNA amount of mimic gene used, whilst the mRNA of specific gene is always constant (1 μg of total mRNA extracted). These conditions are valid for all RT-PCR gels shown in this paper. Panels B and D: the histograms show the HCN2 (⁎⁎⁎p b 0.001) and HCN4 (⁎⁎⁎p b 0.001) mRNA amounts as mean ± SEM obtained in control (ACtrl, n = 7 patients for HCN2 and n = 8 patients for HCN4) and diseased (AICM, n = 4 patients) atrium.
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human specimens. An example of a Western blot is shown in Fig. 6D; the expected size is 25 kDa and the MiRP1 band is indicated by the arrow, as confirmed by the pre-incubation with the control antigen. Neither regional- nor disease-dependent differences were observed in the human samples (Fig. 6E). However, the quality of the blots was suboptimal and they contained several bands in addition to the band we presumed to be MiRP1 based on molecular mass and suppression by preincubation with control antigen. We were unable to perform competitive RT-PCR because of difficulty obtaining acceptable probes. Because of the technical limitations in protein quantification and difficulties with competitive RT-PCR, we sought to correlate our protein data with gene-expression measurements for MiRP1 in atrial and ventricular samples by TaqMan RealTime PCR. The results demonstrate that at the mRNA level, MiRP1 expression is near the detection limit (Ct ~ 40). As shown in Fig. 6F, MiRP1 transcript expression was very weak compared to GAPDH, and there were no tissue or disease related differences. The validity of GAPDH as a disease-independent control is supported by the data in Fig. 6G, which show that GAPDH transcript expression was unaffected by ICM. 4. Discussion
Fig. 5. Protein expression of HCN2 and HCN4 in human right atrium. Panel A: An example of Western blot for HCN2 in human atrium from control (grey line) and diseased (black line) hearts. M indicates size marker (Santa Cruz Biotechnology), +CHCN2 indicates stably transfected mHCN2 HEK293 cell line, +CHCN4 indicates stably transfected hHCN4 HEK293 cell lines; for both cell lines, two bands are visible, likely corresponding to unglycosylated (lower molecular mass) and N-glycosylated (higher molecular mass) forms of the HCN2 and HCN4 protein, respectively. Bottom: ACTIN signals on the same samples. Panel B: The graph reports the obtained values as mean ± SEM for HCN2 protein in control (ACtrl, n = 6 patients) and diseased (AICM, n = 4 patients); ⁎p b 0.05. Panel C: Typical Western blot for HCN4 in human atrium from control (grey line) and diseased (black line) hearts. Bottom: ACTIN signals on the same samples. Panel D: The graph reports the averaged values for HCN4 protein in human atrium from control (ACtrl, n = 6 patients) and diseased (AICM, n = 5 patients); ⁎⁎p b 0.01.
However, as shown in Fig. 6C, we could not detect any HCN1 protein band in atrial samples, similar to ventricle. 3.6. MiRP1 mRNA and protein determination Recent data suggest that the expression of MiRP1 (the putative β subunit of the If-channel) shows regional differences, being larger in atrial versus ventricular tissue in the rabbit [23], and undergoes changes in the failing heart [24]. Therefore, we analysed the expression of MiRP1 in atrial and ventricular
The major novelty of our study consists in the definition of the molecular mechanisms underlying changes in If expression and properties in the diseased human heart. Indeed, this is the first study comparing the mRNA and protein expression of HCN subunits in the human atria and ventricle under normal and heart failure conditions. Our results demonstrate that in human heart failure, an upregulation of ventricular HCN2 and HCN4 underlies the increase in functional If current; moreover, a similar phenomenon occurs in the atria of explanted hearts, where molecular changes mirror those observed in the left ventricle. The molecular profile of HCN isoform expression is in agreement with electrophysiological properties of If, i.e., activation kinetics and response to β-AR stimulation, detected in single myocytes from normal and diseased human hearts. Overall, these findings are of particular relevance in view of the potential proarrhythmic role of overexpression of If, which may contribute to increased ventricular (and atrial) propensity to arrhythmias in heart failure. Previous and present electrophysiological data show that, in ICM myocytes, If is not only of larger amplitude [6] but also activates at a less negative potential (− 71 mV) compared to cells from controls (− 81 mV). This difference can be appreciated also by comparing bell-shaped curves describing activation and deactivation kinetics: the ICM curve was shifted rightward with respect to control by approximately 10 mV. Such a difference may explain why current elicited by hyperpolarization to − 90 mV was apparently faster in ICM than in control cells: indeed, according to the allosteric model of the f-channel [25], the more negative the voltage step (relative to midpoint activation), the faster is activation kinetics. This shift could be due to changes in the relative proportion of different HCN isoforms, since heterologous re-expression of human HCN4 gives a current whose Vh is less negative (−81 mV) than that showed by HCN2 in similar conditions (−92 mV) [26].
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Fig. 6. Expression of HCN1 and MiRP1 in human myocardium. Panel A: Examples of gels of RT-PCR products for HCN1 in human atrium (A) and ventricle (V) from control (Ctrl) hearts; in the ventricle only the mimic gene was detected. Panel B: The graph summarizes the values as mean ± SEM for HCN1 mRNA in control (ACtrl) and diseased (AICM) atrium. Panel C: Typical Western blot for HCN1 protein in rat brain (RB, used as positive control), in human ventricle (V) and atrium (A) from control (Ctrl) and diseased (ICM) hearts; the effect of pre-incubation with control antigen (CA) is also shown. Panel D: Representative Western blot for MiRP1 protein in human ventricle (V) and atrium (A) from control (Ctrl) and diseased (ICM) hearts; the specificity of the MiRP1 protein band was confirmed by pre-incubation of the same membrane with control antigen (CA); the same loading control (GAPDH) applies for both conditions. Panel E: The graph summarizes the amount of protein as mean ± SEM. Panel F: Mean ± SEM MiRP1 mRNA expression relative to GAPDH in human ventricle (V) and atrium (A) from control (Ctrl) and diseased (ICM) hearts. Panel G: Mean ± SEM GAPDH mRNA expression relative to 18S-rRNA in control and diseased ventricles.
Moreover, since both isoforms (HCN2 and HCN4) are modulated by cAMP, in contrast with HCN1, these results imply that channel sensitivity to ligands activating the cAMP pathway (such as catecholamines) should be similar in diseased and normal heart. Indeed, isoproterenol produced a significant positive shift of the midpoint activation potential similar in both diseased and control cells. A deeper comprehension of the molecular mechanisms underlying If overexpression and changes in voltage-dependence of activation cannot be obtained by solely applying electrophysiological techniques, which are unable to reveal the isoform composition of native channels. Therefore, we aimed to assay mRNA and protein levels for specific HCN isoforms [27,28]. As men-
tioned above, heterologously re-expressed HCN isoforms differ significantly in their biophysical properties. The kinetics of HCN2 are slower than those of HCN1, and faster than those of HCN4; also, HCN2 has a more negative activation threshold than either HCN1 or HCN4 [26]. The sensitivity to cAMP varies among different isoforms, with HCN1 being much less responsive than either HCN2 or HCN4 [29]. Heterogeneity of If-channels may be produced by the combination of diverse mechanisms such as a switch of the subunit composition and stoichiometry [30,31], up- and downregulation of auxiliary subunits such as MiRP1 [32], and the influence of cellular environment [15]. However, our present results do not suggest changes in the relative proportion of different HCN isoforms, or important modifications in the
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expression of the auxiliary subunit MiRP1. Indeed, other mechanisms might be responsible for the altered biophysical properties of If in ICM myocytes, such as: 1) influence of the cellular environment; 2) altered function and/or expression of other auxiliary subunit(s), not yet identified; 3) lipid raft disruption altering HCN4 subunit localization and electrophysiological properties [33]. These hypotheses need further investigation. In agreement with previous studies, [34] our results demonstrate that HCN1 mRNA and protein are absent in human ventricles. On the other hand, HCN1 mRNA was detected in atrial specimens; but no protein was found in atrial Western blots, likely because of low expression. HCN2 expression is stronger in ventricles than atria and HCN4 mRNA is expressed ~ 10-fold more strongly than HCN2. The predominance of HCN4 supports our hypothesis on the nature of native If current in human ventricle, as deduced from If properties. The If activation time constant at − 90 mV averaged 1674 ± 260 ms in normal HuVM, a value similar to that reported for heterologously re-expressed HCN4 (1271 ms) and slower than that of HCN2 (278 ms) [15]. Although competitive RT-PCR experiments using RNA mimics as standards may have some inaccuracy, the consistency of these results with those obtained by Western blots and electrophysiological measurements allows us to infer that mRNA and protein expression of HCN2 and HCN4 increased in both atria and ventricles from failing hearts. In addition, quantitative PCR provided qualitatively similar results to competitive RTPCR for HCN4. Since the MiRP1 subunit can modify If for a given level of HCN expression [32], we quantified MiRP1 protein and mRNA expression and found them to be unaffected by heart failure. Thus, If gain of function in heart failure is mainly supported by increased transcription and synthesis of channel alpha subunits (HCN2/4), although post-translational mechanisms cannot presently be ruled out. Electrical heterogeneity exists within the human ventricular wall, likely due to the differential expression of specific ion channel genes [35]. Our samples contained epicardial, midmyocardial and endocardial cells; thus, possible transmural differences in HCN overexpression were not evaluated in the present study. A higher If expression has been detected in the left compared to the right ventricle in rat myocytes [36,37], but to our knowledge, no information exists on transmural variation of If within the left ventricle. It is worth noting, however, that mRNA transcript for HCN did not seem to vary within the left ventricular wall in the rat or canine heart, at variance with sodium (SCN5A) and potassium (Kcnd2) channels [38]. Another original finding of our work is the demonstration that a similar pattern of HCN overexpression occurs in the atria of heart failure patients. It is worth noting that all these patients were in sinus rhythm. Thus, alterations of systemic and local neurohormonal environment may similarly affect ventricular and atrial HCN patterns, by selectively inducing the overexpression of HCN2/4 isoforms at both mRNA and protein level. No difference in mRNA expression of HCN1 was observed between normal and heart failure atria. HCN1 protein was absent in atrial tissue in both conditions, suggesting that post-transcriptional mechanisms reduce protein expression to undetectable levels or that the mRNA is not transcribed. Similar results have been obtained in cultured
adult rat ventricular myocytes [39]. Taken together, these findings may allow hypothesising that environmental factors, such as hypertrophic factors, specifically regulate mRNA transcription of some isoforms (HCN 2/4) and not others (HCN1). The upregulation of HCN2 and HCN4 in heart failure patients and the associated increased contribution of If to spontaneous cellular activity [40] might be implicated in ectopic rhythm formation. Heart failure causes downregulation of a variety of potassium channels, produces spatially-heterogeneous abnormalities in connexin expression and function, and induces tissue fibrosis that impairs impulse propagation [41]. The resulting enhanced dispersion of repolarization and spatial disturbances in conduction properties provide a substrate for reentry arrhythmias [41]. The increased pacemaker current expression and function seen in both the atria and ventricles in heart failure may provide ectopic triggers that initiate clinically significant arrhythmias including atrial fibrillation and potentially life-threatening ventricular tachyarrhythmias. In conclusion, a combination of functional and molecular approaches, as used here, might allow for clarification of the mechanisms by which pathology (e.g., heart failure) affects channel expression related to cardiac cellular pacemaker function, thus modifying the electrophysiological properties of myocytes to cause susceptibility to cardiac arrhythmias. Acknowledgments The financial support of Minister of Education, University and Research (PRIN 2006), Telethon (Grant GGP05093) and Ente Cassa di Risparmio di Firenze, Italy and of the Canadian Institutes of Health Research, is gratefully acknowledged. We wish to acknowledge the support of the Normacor Partners (contract LSH M/CT/2006/018676) and particularly Prof. Martin Biel (University of Munich, Germany) for providing stably transfected HCN2 and HCN4 HEK293 cells. References [1] Cowie MR, Mosterd A, Wood DA, Deckers JW, Poole-Wilson PA, Sutton GC, et al. The epidemiology of heart failure. Eur Heart J 1997;18:208–25. [2] Guyatt GH, Devereaux PJ. A review of heart failure treatment. Mt.Sinai. J Med 2004;71:47–54. [3] Thom T, Haase N, Rosamond W, Howard VJ, Rumsfeld J, Manolio T, et al. Heart Disease and Stroke Statistics—2006 Update: a report from the american heart association statistics committee and stroke statistics subcommittee. Circulation 2006;113:e85–151. [4] Tomaselli GF, Zipes DP. What causes sudden death in heart failure? Circ Res 2004;95:754–63. [5] Tomaselli GF, Beuckelmann DJ, Calkins HG, Berger RD, Kessler PD, Lawrence JH, et al. Sudden cardiac death in heart failure. The role of abnormal repolarization. Circulation 1994;90:2534–9. [6] Cerbai E, Sartiani L, DePaoli P, Pino R, Maccherini M, Bizzarri F, et al. The properties of the pacemaker current I-F in human ventricular myocytes are modulated by cardiac disease. J Mol Cell Cardiol 2001;33:441–8. [7] Hoppe UC, Jansen E, Sudkamp M, Beuckelmann DJ. Hyperpolarizationactivated inward current in ventricular myocytes from normal and failing human hearts. Circulation 1998;97:55–65. [8] Sridhar A, Dech SJ, Lacombe VA, Elton TS, McCune SA, Altschuld RA, et al. Abnormal diastolic currents in ventricular myocytes from spontaneous hypertensive heart failure rats. Am J Physiol Heart Circ Physiol 2006;291:H2192–8.
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