Molecular and Functional Evidence of HCN4 and Caveolin-3 Interaction During Cardiomyocyte Differentiation from Human Embryonic Stem Cells

Cell culture

All chemicals were obtained from Gibco BRL unless otherwise indicated. All cultures were kept in a humidified incubator at 37°C and maintained with a 5% CO₂ atmosphere. Feeder cells were commercially available human foreskin fibroblasts (HFF) (ATCC). HFF cultures were mitotically inactivated using gamma irradiation (45 Gy), and plated onto 6-well culture dishes (Becton Dickinson AG) at a density of 2.5×10⁴ cells/cm². The feeder cell culture medium comprised of the high glucose Dulbecco's modified Eagle medium (DMEM)+Glutamax with the addition of 50 U/mL penicillin and 50 mg/mL streptomycin and 10% fetal calf serum.

The hESC line H1 from the WiCell Research Institute was cultured as described previously [4]. Briefly, cells were cultured in the stem cell medium consisting of the DMEM/F12 with 2 mM glutamine, 50 U/mL penicillin and 50 mg/mL streptomycin, 1× minimal essential medium (MEM) amino acids, 0.1 mM β-mercaptoethanol (Sigma-Aldrich GmbH), 20% Knock-out Serum Replacement (KSR), and 4 ng/mL of the basic fibroblast growth factor (R&D Systems) on irradiated HFF. After collagenase IV treatment for 10–15 min at 37°C followed by mechanical dissociation, colonies of hESC were passaged once a week at a ratio of 1:3 to 1:5 onto mitotically inactivated HFF.

hF heart cells were isolated from fetal hearts obtained from aborted material (gestational weeks 9–12), upon the validated informed consent procedure and prior approval by the regional ethics committee in Geneva (protocol authorization #02-088, Gyn-Ob 02-007) and the local ethics committee in Florence (protocol #6783-04, AziendaOspedalieraCareggi, 1-02-2002). The investigation conforms to the principles outlined in the Declaration of Helsinki.

For tissue culture, the cardiac material was minced into small pieces and digested in 3–5 mL of a buffer solution (containing [in mmol/L] NaCl, 116; HEPES, 20; NaH₂PO₄, 1; glucose, 5.5; KCl, 5.4; MgSO₄, 0.8; pH 7.35) containing collagenase (Collagenase type IV, Worthington 1 mg/mL) and pancreatin (0.15 mg/mL, Life Technologies), during continuous stirring for 15 min at 37°C in an incubator at humidified atmosphere containing 5% CO₂. The supernatant fraction of cells was collected, while the remaining tissue fragments in the supernatant were repeatedly digested in a fresh enzyme solution. The supernatant was saved every 15 min until all pieces were mostly dissociated. The obtained cells were washed by centrifugation at 140g for 5 min, resuspended in a culture medium composed of knockout (KO)-DMEM with 2 mM glutamine, 50 U/mL penicillin and 50 mg/mL streptomycin, 1×MEM amino acids, 0.1 mM β-mercaptoethanol (Sigma-Aldrich GmbH), 20% fetal bovine serum (FBS) (Hyclone) and seeded onto gelatin-coated glass coverslips.

Embryoid body formation and culture

Differentiation media 1 (DM1) was comprised of the KO-DMEM with 2 mM glutamine, 50 U/mL penicillin and 50 mg/mL streptomycin, 1×MEM amino acids, 0.1 mM β-mercaptoethanol (Sigma-Aldrich GmbH), and 20% KSR. Differentiation media 2 (DM2) was comprised of the KO-DMEM with 2 mM glutamine, 50 U/mL penicillin and 50 mg/mL streptomycin, 1×MEM amino acids, 0.1 mM β-mercaptoethanol (Sigma-Aldrich GmbH), and 20% FBS (Hyclone). Embryoid bodies (EBs) were prepared as previously described [7]. Briefly, 60%–80% confluent hESC cultures at passages 40–70 were treated with collagenase type IV for 15–20 min at 37°C and rinsed once with phosphate-buffered saline (PBS) (Ca²⁺ and Mg²⁺ free). Colonies were scraped gently in DM1, transferred to 15-mL polystyrene tubes, and allowed to sediment by gravity for 5–10 min. A supernatant containing most of the irradiated HFF was discarded. Cell clumps were then transferred to Costar Ultra-Low attachment 6-well plates (Corning) at a 2:1 ratio. After 2 days, the medium was changed with DM1, and then at day 4, ∼3 EB were transferred to each well of a gelatin-coated 24-well plate (Falcon, BD Biosciences). The medium was changed every 2–3 days with DM2. The appearance of beating areas was monitored using an inverted microscope (Eclipse TE300, Nikon) that was equipped with a Coolpix 995 camera (Nikon) to take phase-contrast pictures and movies.

Cell isolation from EBs and fetal ventricles used for electrophysiological recordings

Beating EBs were dissected using a microscalpel under an inverted microscope and rinsed with PBS containing 0.9 mM CaCl₂. hF ventricles were minced into small pieces in PBS supplemented with 0.9 mM CaCl₂. Digestion was performed with collagenase B (Roche Applied Science) in PBS (1–2 mg/mL) supplemented with 30 μM CaCl₂ for 15–20 min at 37°C. Dissociated cells were plated on gelatin-coated dishes in the DM2 medium and used within 2 days.

Isolation of single human atrial or ventricular myocytes

For all experiments involving human tissue, each investigation conforms to the principles outlined in the Declaration of Helsinki and had been approved by the local ethics committee in Florence (No. 2006/0023797). Atrial CM were isolated from human atrial appendages routinely excised from the heart of patients undergoing a coronary bypass surgery. Ventricular cells were isolated from the septal tissue of a patient affected by hypertrophic cardiomyopathy and undergoing septalmyectomy. The specimens were transported to the laboratory in a cold transport saline solution, cut into small blocks, and then digested in a tissue dissociation vessel at 37°C, using collagenase type IV (Sigma) and protease type XXIV (Sigma) [8]. After isolation, the cells were collected in tubes with the Kraft-Bruhe solution (see “Solutions and chemicals”) for ½ h. Cells were centrifuged at 700 rpm for 5 min and the pellet was resuspended in the Tyrode solution (see “Solutions and chemicals”) for electrophysiological recordings or in the new Kraft-Bruhe solution for immunocytochemistry.

Immunocytochemistry

Beating clusters derived from hESC and hF heart cells were grown on gelatin-coated coverslips. Adult atrial and ventricular CM were seeded on laminin-coated dishes in the Kraft-Bruhe solution. Before staining, coverslips were rinsed once with PBS, fixed for 15 min with 4% paraformaldeyde, rinsed 3 times with PBS, and permeabilized with 0.3% Triton X-100 in PBS for 10 min. Cells were then rinsed 3 times with PBS for 5 min and blocked with 1% bovine serum albumin (BSA) in PBS for 10 min. Primary antibodies at appropriate dilutions were incubated for 1 h at 37°C in PBS containing 0.1% Tween-20 and 1% BSA. Before incubation with secondary antibodies, cells were blocked again for 10 min with 10% BSA in PBS. Secondary antibodies at appropriate dilutions were incubated for 1 h at 37°C in PBS containing 0.1% Tween-20 and 1% BSA. Cells were then washed with 0.1% Tween-20 in PBS containing 4′,6-diamidino-2-phenylindole (DAPI) for 10 min. Finally, cells were washed 2 more times with 0.1% Tween 20 in PBS for 10 min, and then mounted onto glass slides with Mowiol and left overnight in the dark at room temperature before observation under the microscope. Atrial and ventricular CM were mounted with the HardSet Mounting Medium containing DAPI (Vectashield) The following primary antibodies were used: mouse monoclonal anti-Oct-4 (Santa Cruz Biotechnology); mouse monoclonal anti-α-actinin (Sigma); mouse anti-Caveolin-3 (BD Biosciences); rabbit anti-HCN-4 (Alomone), rat anti-HCN-4 (Abcam); goat anti-Nkx-2.5 (Santa Cruz). The following secondary antibodies were used: goat anti-mouse Alexa 568 (Molecular Probes); goat anti-rabbit Alexa 568 (Molecular Probes); rabbit anti-goat IgG-FITC (Sigma); goat anti-mouse Alexa Fluor 594 (Invitrogen) and goat anti-rat Alexa Fluor 488 (Invitrogen) for atrial cells; anti-mouse Fi2000 490 (Vector Laboratories) and goat anti-rat Alexa Fluor 594 (Invitrogen) for ventricular cells. Images were visualized with a Zeiss axiophot microscope (Carl Zeiss) equipped with an Axiocam camera (Carl Zeiss); confocal images were obtained using the LSM 510 laser scanning confocal microscope (Carl Zeiss). For atrial and ventricular cells, images were obtained with a Leica TCSSP5 and a Leica SP2-AOBS confocal microscope. 3D images were created using the image analysis program, Imaris.

Immunohistochemistry

Beating clusters derived from hESC and hF heart (∼12 weeks) were embedded in paraffin and sectioned into 3-μM slices and placed on glass slides, as reported elsewhere. Slices were deparaffinized, and then rehydrated and unmasking of the epitopes was performed using a pressure cooker in the citrate buffer. After blocking with 10% BSA in PBS for 1 h, primary antibodies at appropriate dilutions were incubated for 1 h at room temperature, and then stained as per the DakoEnVision™ Detection Systems Peroxidase/DAB Kit (see manufacturer's instructions).

RNA extraction and real-time PCR (qPCR)

Total RNA was extracted from cells with the TRIZOL reagent (Invitrogen AG) according to the manufacturer's instructions. 1 μg of total RNA was converted to cDNA with M-MLV Reverse Transcriptase (Invitrogen) using random primers in a 20 μL reaction incubated at 25°C for 10 min followed by 50 min at 37°C and 15 min at 70°C. Volume was adjusted to 50 μL. qPCR was performed with 1 μL of cDNA in a total volume of 25 μL using SYBR green with the ABI Prism 7900 HT detection system (Applied Biosystems). Primers sequences used were as follows:

GusB-F: CCACCAGGGACCATCCAAT; R: AGTCAAAA TATGTGTTCTGGACAAAGTAA

Patch-clamp recordings

The experimental setup for patch-clamp recordings and data acquisition was similar to that described previously (Sartiani L, 2007 [4]; Bettiol E, 2007 [9]). Isolated cells were superfused by means of a temperature-controlled (37°C) microsuperfusor, allowing rapid changes of the solution. Patch-clamp pipettes, prepared from glass capillary tubes (Harvard Apparatus Ltd., www.harvardapparatus.com) by means of a 2-stage horizontal puller (P-87; Sutter Instrument) had a resistance of 2–3 MΩ when filled with the internal solution (see “Solutions and chemicals”). Series resistance (Rs) and membrane capacitance (Cm) were compensated to minimize the capacitive transient and routinely checked during the experiment. Only cells showing a stable Cm and Rs were included in the analysis. I_f current was measured in the whole-cell configuration by using appropriate external and pipette solutions (see “Solutions and chemicals”). From a holding potential of −40 mV, I_f was elicited by hyperpolarizing steps to −60/−140 mV and the current amplitude was measured as the difference between the instantaneous current at the beginning of the hyperpolarizing step and the steady-state current recorded at the end of the step.

Specific conductance was determined following the equation: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}\begin{align*}{\rm Gf} = {\rm I} / ({\rm V_m} - {\rm V}_{\rm rev})\end{align*}\end{document}

where Gf (pS pF⁻¹) is the conductance calculated at the membrane potential Vm (mV), I (pA pF⁻¹) the current density, and Vrev (mV), the reversal potential of the current. I_f specific conductance was normalized with respect to maximal conductance and values, expressed as mean±SEM, were plotted versus membrane potential. Activation data were fitted by a Boltzmann function expressed by the equation: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}\begin{align*}{\rm g} = {\rm gmax} / \{1 + {\rm exp} [({\rm V}_{1 / 2} - {\rm V_m}) / {\rm k}] \} ,\end{align*}\end{document}

where g is the conductance calculated at membrane potential Vm (mV), V_1/2 (mV) the fitted potential for half-maximal activation, and k (mV) the slope factor describing the steepness of the activation curve.

Solutions and chemicals

The Kraft-Bruhe solution (mM) comprised glucose 20, creatine 5, taurine 5, EGTA 0.5, succinic acid 5, K₂-ATP 2, pyruvic acid 5, β-hydroxy-butyric acid 5, KCl 85, K₂HPO₄ 3H₂O 30, MgSO₄ 7H₂O 5, pH 7,1 with KOH.

The normal Tyrode solution (mM) comprised NaCl 140; KCl 5.4; CaCl₂ 1.8; MgCl₂ 1.2; D-glucose 10; Hepes 5, pH 7.35 with NaOH.

The modified Tyrode solution for I_f current was obtained from the normal Tyrode solution supplemented with (mM) MnCl₂ 2, BaCl₂ 2, and 4-aminopyridine 0.5 to eliminate Ca²⁺ current (T- and L-type), inward rectifier K⁺ current, I_K1, and transient outward K⁺ current, Ito, respectively. KCl was increased to 25 mM to amplify I_f. The pipette solution (mM) comprised K⁺ aspartate 130; Na₂GTP 0.1; Na₂ATP 5; MgCl₂ 2; EGTA 11; CaCl₂ 5 (pCa 6.9); Hepes 10, pH 7.2 with KOH.

To deplete cholesterol, freshly isolated human atrial CM were resuspended in the Tyrode solution containing 200 μM CaCl₂ and incubated with 2 mM methyl-β-cyclodextrin (MβCD) (Sigma) for 90 min [10,11].

HCN4 and Cav3 colocalize in hAV and hAA CM, but not in hF CM

As shown in Fig. 1A, B, HCN4, and Cav3 appear to be expressed across the whole cell surface membrane of hAA- and hAV-CM; colocalization can be inferred by the merging of the green and red channels in the same focal plane as shown by the yellow marking (right panels of A and B). HCN4 and Cav3 expression patterns in hF heart cells (hF-CM, ∼12 weeks of age), were characterized by a spotted appearance across the cell membrane of both HCN4 and Cav3 (Fig. 1C; n=5 independent experiments). Simultaneous expression of Cav3 and HCN4 in the same cells was inconsistent and colocalization was not observed in >99% of the cells examined, either when studied in individual cells (Fig. 1C) or in CM clusters (not shown).

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FIG. 1.

Expression of hyperpolarization-activated cyclic nucleotide-gated channel (HCN)4 and Caveolin-3 (Cav3) in human adult and fetal cardiomyocytes (CM). Human adult ventricular (hAV) (A) and atrial (hAA) (B) CM expressing HCN4 (green) and Cav3 (red). Colocalization is observed upon merging (yellow). (C) Human fetal (hF) CM expressing HCN4 (green) and Cav3 (red). No colocalization is observed upon merging. Color images available online at www.liebertpub.com/scd

Immunocytochemical analysis of hESC, hESC-CM, and HCN4-Cav3 colocalization

As expected from the previous demonstration of their functional presence [4], expression of the HCN4 protein was clearly detected in undifferentiated hESC; expression is evident at the cell surface and characterized by a low-density pattern of expression with a spotted appearance across the membrane of the whole cell (Fig. 2A). Moreover, the expression of HCN4 in hESC, as examined using the 3D image reconstruction, clearly showed that the channels were localized mainly on the cell surface (Fig. 2B).Cav3 was absent in undifferentiated hESC.

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FIG. 2.

Expression of HCN4 and Cav3 in undifferentiated human embryonic stem cell (hESC). (A) Undifferentiated hESC expressing HCN4 (green), but not Cav3. (B) 3D reconstruction of a stack of confocal images showing HCN4 expression (red) in a single undifferentiated hES cell, showing clearly the cell surface (sarcolemmal) localization of the ion channel. Scale bar=10 μm. Color images available online at www.liebertpub.com/scd

Since both HCN4 and Cav3 appear to be expressed and colocalized across the whole-cell sarcolemma only in hAA- and hAV-CM (Fig. 1A, B), but not in undifferentiated hESC or hF-CM, we sought the time-course of this process in hESC-CM at different stages of maturation. To examine the expression and localization of HCN4 and Cav3 at the single cardiomyocyte level, whole beating hESC-CM clusters were fixed and stained immunocytochemically.

At d30, HCN4 had mainly a spotted appearance in hESC-CM (Fig. 3A, white arrow), similar to what was observed in undifferentiated hESC (Fig. 2A); however, the expression pattern varied within the cluster, as can be seen in Fig. 3A (arrows). Low expression of Cav3 was seen in ∼20% of the cells of the cluster, localized also at the cell surface membrane (n=3 independent experiments). HCN4 and Cav3 were expressed in the same cells (Fig. 3A), but colocalization was absent, as suggested by the lack of yellow upon merging. A similar staining pattern was seen in hESC-CM at d60; however, Cav3 was expressed in ∼50% of cells and with a greater density (n=3 independent experiments). Again, HCN4 and Cav3 were observed to be expressed in the same cells. Finally, and more interestingly, HCN4 and Cav3 were not only expressed in the same cells, but also colocalized in hESC-CM at d110; colocalization was never detected at any of the earlier stages of maturation. This pattern was observed in n=3 independent experiments. Expression of α-actinin was concurrently examined in HCN4-positive cells to confirm the cardiomyocytic nature of these cells (Fig. 3B). As HCN2 is also expressed in the heart and could contribute to f-current, we also examined its expression in relation to Cav3 at the same time points as were examined for HCN4. Expression of this HCN isoform did not change over time nor did it ever colocalize with Cav3 at any time point tested (Supplementary Fig. 1; Supplementary Data are available online at www.liebertonline.com/scd).

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FIG. 3.

Expression of Cav3, HCN4, and α-actinin in d30, d60, and d110 hESC-CM. (A) Cav3 and HCN4 double staining (d30: white arrow=low density; yellow arrow=high density) (B) α-actinin and HCN4 double staining to verify expression of HCN4 in cardiac-specific cells. Scale bar=10 μm. Color images available online at www.liebertpub.com/scd

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Immunohistochemical analysis of HCN4-Cav3 expression in hESC-CM and fetal heart

To preserve tissue structural integrity and the spatial relationship among cells, immunohistochemical staining was performed at d60 and d110 on consecutive sections to examine the expression of HCN4 and Cav3, as well as α-actinin. First, α-actinin was expressed in the beating clusters at both d60 and d110, which can be seen clearly in Fig. 4. Cav3 was also expressed at d60 and d110 through the cardiomyocyte (α-actinin-positive) portions of the clusters. Conversely, we could detect differential HCN4 expression within a single beating cluster, with some areas appearing to be more highly enriched than others. A general higher expression of HCN4 is observed at d60 in comparison to d110, where only a small section of the cluster contains cells expressing HCN4 in high density, as clearly shown in Fig. 4. Expression was compared to that found in fetal heart slices, where a clear-cut expression of each protein examined was observed (see Supplementary Fig. 2).

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FIG. 4.

Immunohistochemical (IHC) staining of whole hESC-CM clusters. IHC staining of hESC-CM at d60 and d110 with α-actinin, Cav3, and HCN4. Color images available online at www.liebertpub.com/scd

Transcriptional regulation of HCN4 and Cav3 in undifferentiated hESC and hESC-CM

As protein expression is regulated by different mechanisms in the cell, we assessed whether HCN4 and Cav3 are regulated at the transcriptional level during maturation. Thus, we manually isolated beating clusters of hESC-CM from hESC-derived EBs, at 3 time points (d30, d60, d110) and measured the mRNA expression of different HCN isoforms in these cells. Overall, mRNA expression of HCN and Cav3 suggests major transcriptional regulation. In fact, we observed that HCN4 mRNA was highly expressed in undifferentiated hESC and in early CM (d30), in agreement with previous data from ours [4] and other laboratories [12,13], and decreased over time (d60 and d110) (Fig. 5). HCN1 and HCN2 were also expressed in undifferentiated hESC (d0); HCN1 also decreased over time upon maturation, whereas HCN2 expression remained relatively constant (Supplementary Fig. 3). Cav3 on the other hand, was not expressed in undifferentiated hESC (d0); it was clearly expressed at d60, and dramatically increased at d110, in agreement with our ICC findings.

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FIG. 5.

Transcriptional regulation of HCN4 and Cav3 in undifferentiated hESC and hESC-CM. Real time PCR data for the expression of HCN4 and Cav3 in hESC-CM over time (*P<0.05).

Functional consequences of developmental changes in HCN4/Cav3 expression and localization

Figure 6 (A–E) shows typical I_f recordings obtained from hESC-CM at d60 and d110, hF-, hAA-, and hAV-CM. Currents exhibited different activation kinetics likely reflecting diverse isoform distribution, as previously reported by our and other laboratories [4,14]. When examining the voltage dependence (Fig. 6F), the potential of half-maximal activation (I_f V_1/2) for hAA-CM and hAV-CM were shifted leftward by about −20 and −10 mV, respectively, in comparison to hF-CM (hAA-CM:V_1/2=−101.5±4.1 mV, n=3; hAV-CM:V_1/2=−94.4±1.7 mV, n=6; and hF-CMV_1/2=82.3±1.96, n=11). Interestingly, a similar difference of I_f voltage dependence was detected between hESC-CM at d100 (−96.8±5.9 mV, n=6) and hESC-CM at d60 (−83.2±6.9 mV, n=8). This modification turns into different I_f fractional activations at physiological resting potentials (e.g., −90 mV, for ventricular cells). In fact, in hESC-CM at d60, 65%±12% (n=8) of the current was activated with respect to 30%±0.14% (n=6, P<0.05) in hESC-CM at d100, a value resembling what was observed in hAA-CM (0.34±0.07, n=3) and hAV-CM (0.39±0.06, n=6).

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FIG. 6.

Cardiomyocyte electrophysiological recordings. (A–E) Representative I_f current traces in hESC-CM (60 and 100 days), hF-CM and hAA- and hAV-CM. (F) Activation curves of I_f current for each cell type (*P<0.05). (G) Effects of membrane cholesterol depletion on activation properties of f-channels in hAA-CM (n=3) and hF-CM (n=6). (H) Bar graph showing mean potential (±SEM) of half maximal activation of f-current (I_f V_1/2) in hAA-CM and hF-CM, treated or not with methyl-β-cyclodextrin (MβCD). ***P<0.001.

Caveolae disruption changes the properties of I_f current expressed in human adult CM

Previous data suggest that HCN4-Cav3 colocalization has relevant functional consequences in rodent SAN cells [2], namely, on voltage-dependent activation. We tested whether such property also holds true for Cav3 expressed in human CM. Therefore, we disrupted the caveolar compartment of CM by depleting membrane cholesterol with MβCD and examined their activation properties. Using isolated hAA CM, which represent the best eligible cell population in our experimental setting, we found that hAA-CM incubated with or without MβCD, resulted in a shift of +25 mV of I_f V_1/2 (from −101.5±4.1 to −75.9±2.7 mV, n=3, P<0.001) (Fig. 6G). Of note, the latter value was not statistically different from that of hF-CM (−82.3±1.96 mV, n=11; Fig. 6 F–H). Finally, we assessed the consequence of treatment with MβCD on hF-CM and demonstrated that voltage-dependence of f-channel activation remained completely unaffected by the treatment (V_1/2 –83.1±2.1 mV; Fig. 6G, H).Therefore, disruption of caveolae, and thus, disruption of Cav3-f-channel colocalization, resulted in the regression of I_fvoltage dependence toward values measured in immature fetal CM (hF-CMs), also becoming similar to that of hESC-CM at d60.