Altered profile of mRNA expression in atrioventricular node of streptozotocin‑induced diabetic rats.

Prolonged action potential duration, reduced action potential firing rate, upstroke velocity and rate of diastolic depolarization have been demonstrated in atrioventricular node (AVN) cells from streptozotocin (STZ)‑induced diabetic rats. To further clarify the molecular basis of these electrical disturbances, the mRNA profiles encoding a variety of proteins associated with the generation and conduction of electrical activity in the AVN, were evaluated in the STZ‑induced diabetic rat heart. Expression of mRNA was measured in AVN biopsies using reverse transcription‑quantitative polymerase chain reaction techniques. Notable differences in mRNA expression included upregulation of genes encoding membrane and intracellular Ca2+ transport, including solute carrier family 8 member A1, transient receptor potential channel 1, ryanodine receptor 2/3, hyperpolarization‑activated cyclic‑nucleotide 2 and 3, calcium channel voltage‑dependent, β2 subunit and sodium channels 3a, 4a, 7a and 3b. In addition to this, potassium channels potassium voltage‑gated channel subfamily A member 4, potassium channel calcium activated intermediate/small conductance subfamily N α member 2, potassium voltage‑gated channel subfamily J members 3, 5, and 11, potassium channel subfamily K members 1, 2, 3 and natriuretic peptide B (BNP) were upregulated in AVN of STZ heart, compared with controls. Alterations in gene expression were associated with upregulation of various proteins including the inwardly rectifying, potassium channel Kir3.4, NCX1 and BNP. The present study demonstrated notable differences in the profile of mRNA encoding proteins associated with the generation, conduction and regulation of electrical signals in the AVN of the STZ‑induced diabetic rat heart. These data will provide a basis for a substantial range of future studies to investigate whether variations in mRNA translate into alterations in electrophysiological function.

Introduction

Diabetes mellitus (DM) is a serious global health problem and there is clear evidence of the negative influence of diabetes on the prevalence, severity, and prognosis of cardiovascular disease (1). Disorders of the vasculature, particularly coronary artery disease and hypertension, increase the incidence of mortality in individuals with DM (2). Diabetic patients are at greater risk of developing heart problems that are independent of vascular dysfunction which is indicative of a distinct diabetic cardiomyopathy (3,4). Impaired contractile function including a reduction in amplitude and depressed time course of contraction and relaxation of ventricular myocytes have been demonstrated in experimental models of DM including the streptozotocin (STZ)-induced diabetic rat (5–10). These changes in contractility have been partly attributed to alterations in Ca2+ transport including elevated diastolic Ca2+ and depression in amplitude and prolonged time course of the intracellular Ca2+ transient (6–8,11,12). Mechanisms underlying the alterations in Ca2+ transient include impaired sarcoplasmic reticulum (SR) Ca2+ transport and suppressed L-type Ca2+ current and Na+/Ca2+ exchange current (5–7,11–15). DM also has profound effects on the electrical conduction system of the heart which may give rise to arhythmogenic activity. Prolongation of the QT interval and QRS complex correlate with an increased incidence of sudden cardiac death in diabetic patients (16,17). Atrial fibrillation is prevalent and there is a higher incidence of atrioventricular block in diabetic patients compared to healthy controls (17–20).

Previous in vivo biotelemetry and isolated perfused heart studies have demonstrated reduced heart rate in the STZ rat (21–23). Slowing of electrical conduction has also been demonstrated in diabetic rat myocardium (24). Various experimental studies in animal models of DM have variously demonstrated changes in ion channel activity including depressed L-type calcium current, transient outward potassium current, rapid and slow delayed potassium rectifier currents all of which can result in a prolongation of action potential duration and reduced heart rate (23,25–33). DM can increase the duration of the sinoatrial node (SAN) action potential and prolong sino-atrial node conduction time and pacemaker cycle length which is associated with alterations in intercellular gap junctional coupling (23,34). Previous studies in STZ rat have demonstrated a variety of changes in mRNA, and in some cases proteins, that are important to the generation of action potentials in the SAN (35). Increased duration of the action potential in STZ-induced diabetic rat AVN has been attributed to a leftward shift in the zero current potential under voltage clamp, a reduction in peak L-type Ca2+ current density and reduced amplitude of delayed rectifier and hyperpolarization-activated currents (32). L-type calcium channels are fundamental to normal activity in the atrioventricular node (AVN) region and L-type calcium current contributes to the late stages of the pacemaker potential and generation of the action potential upstroke, and is responsible for the timing of conduction velocity through the AVN, thereby contributing to PR interval duration.

Previous studies have demonstrated increased action potential duration associated with a reduced action potential firing rate that is associated with reductions in L-type calcium current, delayed rectifier and hyperpolarization-activated currents in AVN cells from STZ-induced diabetic rat (32,33). Modification of ion channel properties either by altered trafficking and expression, or post-translational modification of channel gating properties, can therefore have a significant impact on AVN function, and result in clinical AVN abnormalities. To further clarify the molecular basis of electrical disturbances in the AVN of diabetic heart the profile of mRNA that encodes a wide variety of proteins that are associated with the generation and conduction of electrical activity in the AVN has been evaluated in the STZ-induced diabetic rat heart.

Materials and methods

Experimental protocol

Forty male Wistar rats aged 8 weeks were divided into 2 subgroups. All animals received normal rat chow and drinking water ad libitum. One subgroup of rats received STZ/citrate buffer (60 mg/kg, intraperitoneal) whilst the other subgroup received citrate buffer alone. Blood glucose was measured 5 days following STZ treatment to confirm diabetes. Experiments began 12 weeks after STZ treatment. Body weight, heart weight and blood glucose were measured immediately prior to experiments. Approval for this study was obtained from the Animal Ethics Committee, College of Medicine and Health Sciences, United Arab Emirates University.

Expression of mRNA

Expression of genes encoding a wide range of cardiac proteins was assessed using previously described techniques with small modifications (35). After animals were sacrificed the hearts were removed rapidly and placed in a dish containing: NaCl 140 mM; KCl 5.4 mM; MgCl2 1 mM; HEPES 5 mM; D-glucose 5.5 mM; CaCl2 1.8 mM and adjusted to pH 7.4 with NaOH. Hearts were dissected and 2 mm biopsy samples of AVN were carefully collected from 20 STZ and 20 control hearts as illustrated in Fig. 1 and according to previously described techniques (36–38). Immediately after removal AVN samples were immersed in RNAlater (AM7021; Life Technologies, Carlsbad, CA, USA) and stored overnight at room temperature to allow thorough penetration of the tissue. AVN samples were then frozen at −20°C pending further processing. Samples were homogenized at 6,500 rpm for 2 runs of 20 sec each with a 15 sec gap (Preceylls 24; Bertin Technologies, Raleigh, NC, USA). The SV Total RNA Isolation system (Promega, Madison, WI, USA) was used to isolate total RNA. The concentration and purity of the RNA was determined by measuring the ratio of absorbance at 260 nm and 280 nm (ND-1000; NanoDrop). A two-step RT-PCR procedure was used to generate cDNA. Total RNA (500 ng) was converted into cDNA in a 25 µl PCR reaction with 10x RT Buffer 2.0 µl, 25x dNTP Mix (100 mM) 0.8 µl, 10x RT Random Primers 2.0 µl, MultiScribe™ Reverse Transcriptase 1.0 µl, RNase inhibitor 1.0 µl, and nuclease-free H2O (High Capacity cDNA Reverse Transcription kit (4374966; Applied Biosystems, Foster, CA, USA). Reverse transcription was carried out using the following protocol: 25°C for 10 min, 37°C for 120 min, and 85°C for 5 min on the Veriti thermal cycler (Applied Biosystems). Gene Expression Assays were performed using custom TaqMan Low Density Arrays (Format 32, 4346799; Applied Biosystems). The TaqMan assays are pre-loaded in each reaction well of the array in triplicate for each RNA sample. As in previous experiments in heart 18S RNA was used as an endogenous control (39,40). Expression of 18S was not significantly different (P>0.05) between AVN samples collected from STZ and control hearts. cDNA (RNA-equivalent) (100 ng) was loaded together with 2x TaqMan Gene Expression Master Mix (No AmpErase UNG; Applied Biosystems) for a total of 100 µl per port. Two AVN samples were combined for each real-time RT-PCR assay. Real-time RT-PCR was performed in a Fast ABI Prism 7900HT Sequence Detection system (Applied Biosystems). The PCR thermal cycling parameters were run in standard mode as follows: 50°C for 2 min, 94.5°C for 10 min, followed by 40 cycles of 97°C for 30 sec and 59.7°C for 1 min. Results were initially analyzed using ABI Prism 7900HT SDS, v2.4. Calculations and statistical analysis were performed by the SDS RQ Manager 1.1.4 software using the 2−ΔΔCt method with a relative quantification RQmin/RQmax confidence set at 95%. A list of the target genes, proteins and protein descriptions are shown in Table I.

Figure 1.

Dissection of the atrioventricular node junction in a typical control heart showing the location where tissue samples were collected. CT, crista terminalis; RA, right atrium; TV, tricuspid valve; RV, right ventricle; VS, ventricular septum; AS, atrial septum; CS, coronary sinus; SAN, sinoatrial node; AVN, atrioventricular node; Ao, Aorta.

Table I.

Target genes and proteins.

Genes	Proteins	Protein descriptions
Intercellular proteins
Gja1	Cx43	Connexin43
Gja5	Cx40	Connexin40
Gjc1	Cx45	Connexin45
Gjd3	Cx31.9	Connexin31.9
Cell membrane transport
Atp1a1	Na/K ATPase, α1	ATPase, Na+/K+ transporting, α1 polypeptide
Atp1a2	Na/K ATPase, α2	ATPase, Na+/K+ transporting, α2 polypeptide
Atp1a3	Na/K ATPase, α3	ATPase, Na+/K+ transporting, α3 polypeptide
Atp1b1	Na/K ATPase, β1	ATPase, Na+/K+ transporting, β1 polypeptide
Atp2b1	Na/K ATPase, β2	ATPase, Ca++ transporting, plasma membrane 1
Slc8a1	NCX1	Solute carrier family 8 (sodium/calcium exchanger), member 1
Trpc1	TRPC1	Transient receptor potential channel 1
Trpc3	TRPC3	Transient receptor potential channel 3
Trpc4	TRPC4	Transient receptor potential channel 4
Trpc6	TRPC6	Transient receptor potential channel 6
Intracellular Ca2+ transport and Ca2+ regulation
Atp2a2	SERCA2	Sarcoplasmic/endoplasmic reticulum calcium ATPase 2
Calm1	Calm1	Calmodulin1
Calm3	Calm3	Calmodulin3
Casq2	Casq2	Calsequestrin 2
Itpr1	IP3R1	Inositol 1,4,5-trisphosphate receptor, type 1
Itpr2	IP3R2	Inositol 1,4,5-trisphosphate receptor, type 2
Itpr3	IP3R3	Inositol 1,4,5-trisphosphate receptor, type 3
Ryr2	RYR2	Ryanodine receptor 2
Ryr3	RYR3	Ryanodine receptor 3
Pln	PLB	Phospholamban
Hyperpolarization-activated cyclic nucleotide-gated channels
Hcn1	HCN1	Hyperpolarization-activated cyclic nucleotide-gated channels 1
Hcn2	HCN2	Hyperpolarization-activated cyclic nucleotide-gated channels 2
Hcn3	HCN3	Hyperpolarization-activated cyclic nucleotide-gated channels 3
Hcn4	HCN4	Hyperpolarization-activated cyclic nucleotide-gated channels 4
Calcium channels
Cacna1c	Cav1.2	Voltage-dependent, L type, α1C subunit
Cacna1d	Cav1.3	Voltage-dependent, L type, α1D subunit
Cacna1g	Cav3.1	Voltage-dependent, T type, α1G subunit
Cacna1h	Cav3.2	Voltage-dependent, T type, α1H subunit
Cacna2d1	Cavα2δ1	Voltage-dependent, α2/δ subunit 1
Cacna2d2	Cavα2δ2	Voltage-dependent, α2/δ subunit 2
Cacna2d3	Cavα2δ3	Voltage-dependent, α2/δ subunit 3
Cacnb1	Cavβ1	Voltage-dependent, β1 subunit
Cacnb2	Cavβ2	Voltage-dependent, β2 subunit
Cacnb3	Cavβ3	Voltage-dependent, β3 subunit
Cacng4	Cavγ4	Voltage-dependent, γ subunit 4
Cacng7	Cavγ7	Voltage-dependent, γ subunit 7
Sodium channels
Scn1a	Nav1.1	Voltage gated, type Iα subunit
Scn3a	Nav1.3	Voltage gated, type IIIα subunit
Scn4a	Nav1.4	Voltage gated, type IVα subunit
Scn5a	Nav1.5	Voltage gated, type V, α subunit
Scn7a	Nav2.1	Voltage gated, type VII, α subunit
Scn8a	Nav1.6	Voltage gated, type VIII, α subunit
Scn1b	Navβ1	Voltage gated, type I, β subunit
Scn2b	Navβ2	Voltage gated, type II, β subunit
Scn3b	Navβ3	Voltage gated, type III, β subunit
Potassium channels
Kcna1	Kv1.1	Voltage gated shaker related subfamily A, member 1
Kcna2	Kv1.2	Voltage gated shaker related subfamily A, member 2
Kcna3	Kv1.3	Voltage gated shaker related subfamily A, member 3
Kcna4	Kv1.4	Voltage gated shaker related subfamily A, member 4
Kcna5	Kv1.5	Voltage gated shaker related subfamily A, member 5
Kcna6	Kv1.6	Voltage gated shaker related subfamily A, member 6
Kcnb1	Kv2.1	Voltage gated shab related subfamily B, member 1
Kcnd1	Kv4.1	Voltage gated shal related subfamily D, member 1
Kcnd2	Kv4.2	Voltage gated shal related subfamily D, member 2
Kcnd3	Kv4.3	Voltage gated shal related subfamily D, member 3
Kcnh2	ERG-1	Ether-a-go-go-related protein 1
Kcnip2	KChIP2	Kv channel interacting protein 2
Kcnn1	SK1	Ca++ activated intermediate/small conductance subfamily n α, member 1
Kcnn2	SK2	Ca++ activated intermediate/small conductance subfamily n α, member 2
Kcnn3	SK3	Ca++ activated intermediate/small conductance subfamily n α, member 3
Kcnq1	Kv7.1	Voltage gated KQT-like subfamily Q, member 1
Kcnj2	Kir2.1	Inwardly rectifying subfamily J, member 2
Kcnj3	Kir3.1	Inwardly rectifying subfamily J, member 3
Kcnj5	Kir3.4	Inwardly rectifying subfamily J, member 5
Kcnj8	Kir6.1	Inwardly rectifying subfamily J, member 8
Kcnj11	Kir6.2	Inwardly rectifying subfamily J, member 11
Kcnj12	Kir2.2	Inwardly rectifying subfamily J, member 12
Kcnj14	Kir2.4	Inwardly rectifying subfamily J, member 14
Kcnk1	TWIK1	Two pore domain subfamily K, member 1
Kcnk2	TREK1	Two pore domain subfamily K, member 2
Kcnk3	K2P3.1	Two pore domain subfamily K, member 3
Kcnk5	K2P5.1	Two pore domain subfamily K, member 5
Kcnk6	TWIK2	Two pore domain subfamily K, member 6
Miscellaneous proteins
Abcc8	SUR1	ATP-binding cassette transporter sub-family C member 8
Abcc9	SUR2	ATP-binding cassette, sub-family C member 9
Nppa	ANP	Atrial natriuretic peptide
Nppb	BNP	Brain natriuretic peptide
Pias3	KChAP	Protein inhibitor of activated STAT, 3

Expression of protein

Protein expression was measured using previously described SDS-PAGE and western blotting techniques with small modifications (35). AVN from STZ and control rats were carefully dissected, rinsed with ice-cold saline and homogenised in RIPA buffer (Tris 50 mM; NaCl 150 mM; Triton X 1%; sodium deoxylate 0.5%; SDS 0.1% adjusted to pH 7.4 and finally addition of PMSF 0.1 mM-Sigma, P7626) at 6,500 rpm for 2 runs of 20 sec each with a 15 sec gap (Preceylls 24; Bertin Technologies). Protein concentration was measured with Bio-Rad reagent. The supernatant was used for SDS-PAGE and western blotting. Protein (40 µg) was electrophoretically separated onto 8 or 10% (depending on the molecular weight of the protein to be separated) polyacrylamide gels and transferred onto nitrocellulose membranes. Expression of the specific proteins was confirmed by immunoreaction with their specific antibodies by western blot analysis. β-actin was used as a loading control. Blots were developed using the Pierce Western Blot kit. Images were obtained using the Typhoon FLA 9500, GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Quantitation of protein was performed using the method described in the following website: http://lukemiller.org/index.php/2010/11/analyzing-gels-and-western-blots-with-image-j/.

For each lane the protein level was normalized to that of β-actin. The ratio of specific protein signal to that of the β-actin control was used to calculate fold-change.

Statistical analysis

Results were expressed as the mean ± SEM. of ‘n’ observations. Statistical comparisons were performed using independent sample t-test (SPSS vs. 20). P≤0.05 was considered to indicate a statistically significant difference.

Results

General characteristics: Bodyweight and heart weight were reduced and heart weight/bodyweight ratio was increased in STZ rats compared to controls. Blood glucose was elevated 5-fold in STZ rats compared to controls (Table II).

Table II.

General characteristics of streptozotocin-induced diabetic rats.

Characteristics	Control	Streptozotocin
Bodyweight (g)	401.56±9.68	246.44±13.28[a]
Heart weight (g)	1.37±0.03	1.08±0.04[a]
Heart weight/bodyweight (mg/g)	3.42±0.06	4.47±0.12[a]
Blood glucose (mg/dl)	99.88±2.07	514.69±18.86[a]

Data are presented as the mean ± standard error of the mean, n=16 hearts,

P<0.01.

Expression of genes encoding intercellular proteins is shown in Fig. 2. There were no significant (P>0.05) differences in the expression of mRNA in AVN from STZ compared to control heart. Expression of genes encoding various cell membrane transport and intracellular Ca2+ transport and regulatory proteins are shown in Figs. 3 and 4, respectively. mRNA for Atp1b1 (2-fold), Atp2b1 (2-fold), Slc8a1 (6-fold), Trpc1 (7-fold), Trpc3 (2-fold), Casq2 (2-fold), Ryr2 (3-fold), Ryr3 (4-fold) were all significantly (P<0.05) upregulated in AVN from STZ compared to control heart. Expression of genes encoding the hyperpolarization-activated cyclic nucleotide-gated channel proteins are shown in Fig. 5. mRNA for Hcn2 (2-fold) and Hcn3 (9-fold) were significantly upregulated in AVN from STZ compared to control heart. Expression of genes encoding calcium channel proteins are shown in Fig. 6. mRNA for Cacnb2 (2-fold) was upregulated in AVN from STZ compared to control heart. Expression of genes encoding sodium channel proteins are shown in Fig. 7. mRNA for Scn3a (3-fold), Scn4a (3-fold), Scn7a (2-fold) and Scn3b (7-fold) were upregulated in AVN from STZ compared to control heart. Expression of genes encoding potassium channel proteins are shown in Fig. 8. mRNA for Kcna4 (3-fold), Kcnh2 (4-fold), Kcnn2 (9-fold), Kcnj3 (2-fold), Kcnj5 (5-fold), Kcnj11 (2-fold), Kcnk1 (2-fold), Kcnk2 (2-fold) and Kcnk3 (3-fold) were upregulated in AVN from STZ compared to control heart. Expression of genes encoding various miscellaneous proteins are shown in Fig. 9. mRNA for Abcc9 (2-fold), Nppb (3-fold) and Pias3 (8-fold) were upregulated in AVN from STZ compared to Control heart.

Figure 2.

Expression of genes encoding various intercellular proteins. Data are mean ± SEM, n=6–8 samples from STZ and control rat each containing samples from 2 hearts.

Figure 3.

Expression of genes encoding various cell membrane transport proteins. Data are mean ± SEM, n=5–8 samples from STZ and control rat each containing samples from 2 hearts. *P<0.05, **P<0.01 vs. CON-AVN.

Figure 4.

Expression of genes encoding various intracellular Ca2+ transport and Ca2+ regulation proteins. Data are mean ± SEM, n=7–8 samples from STZ and control rat each containing samples from 2 hearts. *P<0.05, **P<0.01 vs. CON-AVN.

Figure 5.

Expression of genes encoding various hyperpolarization-activated cyclic-nucleotide-gated channels. Data are mean ± SEM, n=5–8 samples from STZ and control rat each containing samples from 2 hearts. *P<0.05 vs. CON-AVN.

Figure 6.

Expression of genes encoding various calcium channel proteins. Data are mean ± SEM, n=5–9 samples from STZ and control rat each containing samples from 2 hearts. **P<0.01 vs. CON-AVN.

Figure 7.

Expression of genes encoding various sodium channel proteins. Data are mean ± SEM, n=5–9 samples from STZ and control rat each containing samples from 2 hearts. *P<0.05, **P<0.01 vs. CON-AVN.

Figure 8.

(A and B) Expression of genes encoding various potassium channel proteins. Data are mean ± SEM, n=5–8 samples from STZ and control rat each containing samples from 2 hearts. *P<0.05, **P<0.01 vs. CON-AVN.

Figure 9.

Expression of genes encoding miscellaneous cardiac proteins. Data are mean ± SEM, n=6–8 samples from STZ and control rat each containing samples from 2 hearts. *P<0.05, **P<0.01 vs. CON-AVN.

Expression of proteins

Representative Western blots comparing various proteins from STZ and control AVN are shown in Fig. 10A. The protein/actin ratio for the different proteins are shown in Fig. 10B. Expression of Kir3.4, NCX1 and BNP were significantly upregulated and SK2, ERG-1, HCN3 and Navβ3 were not significantly altered in AVN from STZ compared to control heart.

Figure 10.

(A) Typical western blot comparing expression of various proteins from STZ and control AVN. β-actin which was used as the loading control is also shown in each blot. The blots shown are representative of 6 individual samples from STZ and control rats. (B) Protein/β-actin ratios for the different proteins. Data are mean ± SEM, n=6 samples from STZ and control rat each containing 3 pooled AVNs from a total of 18 hearts AVN. *P<0.05, **P<0.01 vs. CON-AVN.

Discussion

Diabetes in the STZ-induced diabetic rat was characterized by reduced bodyweight and heart weight and increased heart weight/bodyweight ratio and a 5-fold increase in blood glucose. Major findings of this study included: i) upregulation of Slc8a1 mRNA and NCX1 protein; ii) upregulation of Trpc1 and Trpc3 mRNA; iii) upregulation of Ryr2 and Ryr3 mRNA; iv) upregulation of Hcn2 and Hcn3; v) upregulation of Cacnb2 mRNA; vi) upregulation of Scn3a/4a/7a and Scn3b; vii) upregulation of Kcna4, Kcnh2, Kcnn2, Kcnj3/11 and Kcnk2/3 mRNA and Kcnj5 mRNA and Kir3.4 protein; and viii) upregulation of Nppb mRNA and BNP protein in AVN from STZ compared to control heart.

Slc8a1 mRNA and NCX1 protein were upregulated in AVN from STZ rat heart. Upregulation of Slc8a1 has also recently been reported in the SAN from STZ-induced diabetic rat (35). The Slc8 gene family encodes the Na+/Ca2+ exchanger (NCX). Altered expression and regulation of NCX proteins contribute to abnormal Ca2+ homeostasis in heart failure, arrhythmia, hypertension and diabetes (41). Impaired Ca2+ homeostasis, due to depressed SR Ca2+ ATPase and NCX activity and NCX current have been demonstrated in ventricular myocytes from STZ-induced diabetic heart (6,42,43). The functional importance of NCX current in rabbit and mouse AVN cells has been demonstrated and depressed L-type Ca2+ current has been reported in AVN cells from STZ rat heart (32,33,44–46). Upregulation of Slc8a1 mRNA and NCX1 protein in the AVN from STZ heart may provide a compensatory pathway to facilitate Ca2+ influx and/or Ca2+ efflux from the cell which may be required for the generation and recovery of action potentials in AVN cells.

Trpc1 and Trpc3 were upregulated in AVN from STZ rat heart. Upregulation of Trpc1 has also recently been reported in the SAN from STZ-induced diabetic rat (35). The transient receptor potential channels (TRPCs) are a large family of non-selective and non-voltage-gated ion channels that convey signaling information linked to a broad range of sensory inputs including neurohormonal and mechanical load stimulation (47). TRPC1 is a mechano-sensitive, non-selective cation channel which is expressed in ventricle and atrium in a variety of mammalian species including rat (48–50). TRPC1 functions in Ca2+ influx, and its upregulation is involved in the development of cardiac hypertrophy (51). TRPC1 is also expressed in mouse SAN and in single pacemaker cells and mouse SAN may exhibit store-operated Ca2+ channel (SOCC) activity which may suggest that SOCCs are involved in regulating pacemaker firing rate (52). Upregulation of Trpc1 may be a consequence of hemodynamic disturbances in the diabetic heart which in turn stimulates mechano-sensitive TRPC channels thereby providing an alternative entry pathway for Ca2+ in the face of depressed L-type Ca2+ current in AVN cells and reduced heart rate in STZ-induced diabetic heart (32,33). Previous studies have reported elevation of mean arterial pressure in STZ-induced diabetic rats which may in turn lead to hypertrophy of the heart which would be consistent with the increase in heart weight to body weight ratio, which in turn might elicit an effect on the mechano-sensitive channels (53).

Ryr2 and Ryr3 were upregulated in AVN from STZ rat heart. The Ryr family of genes encode proteins that form the SR Ca2+ release channel. SR Ca2+ cycling is important for the genesis of spontaneous activity in the AVN (44,54–56). Whilst little is known about the effects of diabetes on SR Ca2+ signaling in the AVN, previous studies have demonstrated depressed SR Ca2+ loading, Ca2+ uptake/release and Ca2+ leak in ventricular myocytes from STZ-induced diabetic heart (6,12,15,57). These disturbances in SR Ca2+ signaling have been variously attributed to structural and/or functional defects of the RYR2 receptors or Ca2+-ATPase (SERCA) pump proteins (12,15,58–61). It was also interesting to note upregulation of Casq2, an SR Ca2+ binding protein, in AVN from STZ rat. Upregulation of Ryr2 may facilitate release of Ca2+ from the SR which in turn might compensate for depressed L-type Ca2+ current in AVN from STZ compared to control heart (32,33).

Hcn2 and Hcn3 were upregulated in AVN from STZ rat heart. Upregulation of Hcn3 was only associated with a small increase in HCN3 protein in AVN from STZ compared to control heart. The funny current which is conducted through the hyperpolarization-activated cyclic nucleotide channels plays a key role in the generation of the pacemaker current and hence, rhythmicity of the heart. Previous studies have demonstrated reduced action potential firing rates accompanied by a reduction in the amplitude of funny current, L-type Ca2+ current and delayed rectifier current in AVN cells from STZ-induced diabetic heart (32,33). In the longer term, as DM progresses, upregulation of Hcn2 and/or Hcn3, if accompanied by an increase in HCN2 and HCN3 protein, might result in an increase in the conductance of funny current and steepening of the slope of the pacemaker potential, which in turn would increase heart rate.

Cacnb2 was upregulated in AVN from STZ rat heart. Cacnb2 codes for the auxiliary β-subunit (Cavβ) which is an important modulator of Ca2+ channel activity. Expression of the β-subunit is required for normal function of cardiac L-type Ca2+ channels. Cavβ binds to the α1 pore-forming subunit of L-type Ca2+ channels and augments L-type Ca2+ current by facilitating channel opening and increasing the number of channels in the membrane (62). Several cardiovascular diseases including hypertension, heart failure and sudden cardiac death have been linked to Cacnb2 (63). Upregulation of Cacnb2 may facilitate L-type Ca2+ channel opening and provide a compensatory pathway for depressed L-type Ca2+ current in AVN cells from STZ compared to control heart (32,33). Interestingly, there were no significant changes in the expression of Cacna1c and Cacna1d suggesting that at this stage of diabetes (12 weeks after STZ treatment), whilst other changes in gene expression are taking place, changes to the α-1C and α-1D are not yet evident.

Scn3b and Scn4a were upregulated in AVN from STZ rat heart. Sodium channel SCN5a (Nav1.5) is regulated by four sodium channel auxiliary β subunits (SCN1-4b). Mutations in SCN3b have been associated with ventricular and atrial arrhythmias and altered electrophysiological properties of the sodium channel including reduced peak sodium current (64–66). Sodium channel SCN4A (Nav1.4) encodes the α-subunit of the voltage-gated sodium channel Nav1.4 and studies suggest the involvement of SCN4A variants in the pathophysiological mechanisms underlying arrhythmias in some patients with Brugada syndrome (67,68). The role of SCN3B and SCN4A in AVN remains to be clarified.

Kcna4, Kcnh2, Kcnj3/5/11, Kcnk2/3 and Kcnn2 were all upregulated in AVN from STZ rat heart. Upregulation of Kcnj5 and Kcnk3 have also recently been reported in the SAN from STZ-induced diabetic rat (35). Kir3.4 protein encoded by Kcnj5 was upregulated and ERG-1 encoded by Kcnh2 was unaltered in AVN from STZ compared to control heart. At this stage of diabetes (12 weeks after STZ treatment) alterations in gene expression might not translate into alterations in protein expression. Kcna4 encodes the Kv1.4 protein which forms the channel that carries transient outward current which makes a major contribution to the repolarizing current and termination of the cardiac action potential. Transient outward current has been widely demonstrated in different regions of the heart including ventricular myocytes and AVN cells (69–71). Kcnh2 encodes ERG-1 protein which is the α subunit of a potassium ion channel that mediates the repolarizing rapid delayed rectifier current (IKr) current in the cardiac action potential. Several studies have characterized the electrophysiological properties of IKr in AVN cells from various species including rabbit and mouse (54,71–73). IKr plays a role in both action potential repolarization and pacemaker depolarization (74,75). The Kcnj5 gene encodes the G-protein-activated inwardly rectifying potassium channel 4 and loss of the Kir3.4 gene strongly reduces cholinergic regulation of pacemaker activity in SAN cells and delays recovery of heart rate after stress, physical exercise or pharmacological β-adrenergic stimulation (76). Upregulation of Kcnj5 and Kir3.4 protein might partly underlie the slow heart rate in STZ rat heart (21–23). Kcnn2 encodes the small calcium-activated potassium channel member SK2. SK2 mRNA has been detected in a variety of organs including heart and within the heart expression of SK channels are more abundant in the atria and pacemaking tissues compared with the ventricles (77,78). Calcium-activated potassium channels are present in a variety of cells and serve to integrate changes in intracellular Ca2+ with changes in K+ conductance and membrane potential (79). Overexpression of SK2 channels result in shortening of the spontaneous action potentials of AVN cells and an increase in firing frequency whilst genetic knockout of SK2 channels result in the delay in atrial myocyte repolarization and atrial arrhythmias (80,81). Recent studies have demonstrated down-regulation of SK2 and prolonged action potentials in STZ-induced diabetic atria (82).

Nppb and BNP protein were upregulated in AVN from STZ rat heart. Upregulation of Nppa and Nppb have recently been reported in the SAN from STZ-induced diabetic rat (35). The natriuretic peptides are a family of related peptides that include atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) that are secreted from the cardiac atria and ventricles (83). ANP and BNP decrease blood pressure and cardiac hypertrophy and BNP acts locally to reduce ventricular fibrosis and they are both involved in the pathogenic mechanisms leading to major cardiovascular diseases, including heart failure, coronary heart diseases, hypertension and left ventricular hypertrophy (83–85). Previous studies have demonstrated increases in ANP and BNP in blood plasma and atrial tissues and varying effects of ANP and BNP on the amplitude and kinetics of shortening and intracellular Ca2+ in ventricular myocytes from STZ-induced diabetic rat (86,87). BNP has been shown to increase heart rate and electrical conduction velocity in isolated hearts and in the SAN and also increase spontaneous action potential frequency in isolated SAN myocytes (88). Upregulation of Nppb and BNP protein in the AVN may be associated with mechanisms that compensate for the low heart rate seen in the STZ-induced diabetic heart or alternatively be a consequence of the hypertrophy (21–23,86,87).

The SAN and AVN contribute to the generation and orderly propagation of electrical signals in the heart and it is interesting that the expression of genes that encode a variety of proteins involved in cardiac electrical transmission are similarly altered in the SAN and AVN of STZ-induced diabetic rat (35). This study has demonstrated differences in the profile of mRNA encoding a variety of proteins that are associated with the generation, conduction and regulation of electrical signals in the AVN of STZ-induced diabetic rat heart. Data from this study will provide a basis for a substantial range of future studies to investigate whether changes in mRNA translate into changes in electrophysiological function.