Expression and distribution of voltage-gated ion channels in ferret sinoatrial node

Abstract

Spontaneous diastolic depolarization in the sinoatrial (SA) node enables it to serve as pacemaker of the heart. The variable cell morphology within the SA node predicts that ion channel expression would be heterogeneous and different from that in the atrium. To evaluate ion channel heterogeneity within the SA node, we used fluorescent in situ hybridization to examine ion channel expression in the ferret SA node region and atrial appendage. SA nodal cells were distinguished from surrounding cardiac myocytes by expression of the slow (SA node) and cardiac (surrounding tissue) forms of troponin I. Nerve cells in the sections were identified by detection of GAP-43 and cytoskeletal middle neurofilament. Transcript expression was characterized for the 4 hyperpolarization-activated cation channels, 6 voltage-gated Na(+) channels, 3 voltage-gated Ca(2+) channels, 24 voltage-gated K(+) channel α-subunits, and 3 ancillary subunits. To ensure that transcript expression was representative of protein expression, immunofluorescence was used to verify localization patterns of voltage-dependent K(+) channels. Colocalizations were performed to observe any preferential patterns. Some overlapping and nonoverlapping binding patterns were observed. Measurement of different cation channel transcripts showed heterogeneous expression with many different patterns of expression, attesting to the complexity of electrical activity in the SA node. This study provides insight into the possible role ion channel heterogeneity plays in SA node pacemaker activity.

Fig. 1.

Identification and examination of the ferret sinoatrial (SA) node. A: photograph of the ferret heart showing the aorta, superior vena cava (SVC), inferior vena cava (IVC), and right atrial (RA) appendages. Black arrows show the location of incisions that exposed the SA nodal region. B: endocardium of the medial RA appendage. The SA node is located near the crista terminalis (CT) and is marked by the box. The pink line shows the path of sections, which were perpendicular to the crista terminalis. C: light microscopic (LM) view of the section. Scale bar, 125 μm. D: enlarged image of the SA node and the proximity of the central part of the SA node to the nodal arteries, marked with red arrows. Scale bar, 50 μm. Growth-associated protein 43 (GAP-43) localization in this section is shown in Fig. 2F.

Fig. 2.

Localization of SA node and neuronal markers. The data in A–E were acquired by fluorescence in situ hybridization (FISH); F was an immunofluorescence (IF) localization. Each cross section through SA node was made at 5-μm thickness. A and B: troponin I mRNA [cardiac (TnI_C) and slow twitch (TnI_S)] FISH signals obtained by using TnI_S antisense probe to mark the SA nodal region and TnI_C antisense probe to identify atrial myocytes in this region. C, an overlay of TnI_S (red) and TnI_C (green) from A and B, respectively, shows that these transcripts are not coexpressed. D: FISH negative control using a TnI_S sense probe; the exposure time on the photograph was identical to that in A. All FISH experiments were done simultaneously with sense and antisense probes. All sections included in this report had sense probe negative controls similar to that shown in D. E: distribution of cytoskeletal middle neurofilament protein. F: distribution of the neuronal marker GAP-43 in the SA node.

Fig. 3.

Analysis of ion channel mRNA expression by FISH in cross sections of ferret SA node. TnI_C (A; marker for atrial myocytes); TnI_S (B; SA nodal cell marker); hyperpolarization-activated cation channels HCN1 (C), HCN2 (D), HCN3 (E), and HCN4 (F); voltage-gated Ca²⁺ channels Cav1.2 (G), Cav3.1 (H), and Cav3.2 (I); and voltage-gated Na⁺ channels Nav1.1 (J), Nav1.3 (K), Nav1.4 (L), Nav1.5 (M), Nav1.8 (N), and Nav1.9 (O).

Fig. 4.

Colocalization of hyperpolarization-activated cation channels and K⁺ channels. Images are overlays from other figures with false color added to allow comparison of expression patterns. Individual proteins or transcripts are red (first listed) or green. All images are from FISH, except F–H, which are IF. Coexpression appears as yellow. TnI_S with HCN1 (A), HCN1 with HCN2 (B), HCN1 with HCN3 (C), HCN1 with HCN4 (D), TnI_S with TnI_C (E), Kv1.4 with Kv4.2 (F), Kv1.4 with Kv4.3 (G), Kv4.2 with Kv4.3 (H), minK with Kv11.1 (I), minK with Kv7.1 (J), and Kv11.2 with Kv11.3 (K) are shown. The images in A–E were created by merging Fig. 3, A–F, and those in G–K by merging Fig. 6, R–V.

Fig. 5.

Colocalization of voltage-gated Na⁺ channels and voltage-gated Ca²⁺ channels. Images presented from Fig. 3 with false color added as in Fig. 4. All images are from FISH. TnI_S with Nav1.1 (A), Nav1.1 with HCN4 (B), TnI_S with Nav1.5 (C), TnI_C with Nav1.5 (D), TnI_S with Cav1.2 (E), Cav1.2 with HCN4 (F), Cav3.1 with HCN4 (G), Cav3.2 with HCN4 (H), Cav3.1 with Cav1.2 (I), Cav3.1 with Cav3.2 (J), and Nav1.1 with Cav1.2 (K) are shown.

Fig. 6.

Analysis of voltage-gated K⁺ channel subunit mRNA expression by FISH in cross sections of ferret SA node. In all panels green fluorescence in each section indicates the presence of specific mRNA expression of different K⁺ and associated channel antisense probes. Note the differential and distinct pattern of expression for each channel type. Channel types: Kv1.1–6 (A–F), Kv2.1 (G) and Kv2.2 (H), Kv5.1 (I), Kv6.1 (J), Kv3.1–4 (K–N), Kv4.1–3 (O–Q), Kv7.1 (R), minK (S), Kv11.1–3 (T–V), Kv10.1 (W), HCN1 (X).

Fig. 7.

Cav1.2 and Cav3.1 are coexpressed in the SA node. FISH on Cav1.2, Cav3.1, and anti-GAP-43 antibody was performed as described in materials and methods. Bars, 25 μm. A: GAP-43 (red) overlaid with Cav1.2 (green) shows considerable expression of Cav1.2 both in the SA node and in the surrounding neurons. B: GAP-43 with Cav3.1 shows that the Ca²⁺ channel is excluded from the neurons but is expressed in the central nodal area. C: Cav1.2 with Cav3.1 shows that the channels are expressed in the same cells in the central SA nodal tissue.

Fig. 8.

Comparison of mRNA and protein expression of K⁺ channel distribution in ferret SA node. FISH was performed with antisense oligonucleotide probes labeled with rhodamine (red), IF was performed as described in materials and methods with antibodies labeled with Alexa 488 (green). The overlaid images shown are Kv1.4 (A), Kv1.5 (B), Kv4.2 (C), Kv4.3 (D), Kv1.1 (E), Kv1.2 (F), Kv2.1 (G), and Kv11.1 (H). Note the significant difference in expression level of Kv1.4 and Kv1.5 mRNA and protein expression.