Different Profile of mRNA Expression in Sinoatrial Node from Streptozotocin-Induced Diabetic Rat

Background Experiments in isolated perfused heart have shown that heart rate is lower and sinoatrial node (SAN) action potential duration is longer in streptozotocin (STZ)–induced diabetic rat compared to controls. In sino-atrial preparations the pacemaker cycle length and sino-atrial conduction time are prolonged in STZ heart. To further clarify the molecular basis of electrical disturbances in the diabetic heart the profile of mRNA encoding a wide variety of proteins associated with the generation and transmission of electrical activity has been evaluated in the SAN of STZ-induced diabetic rat heart. Methodology/Principal Findings Heart rate was measured in isolated perfused heart with an extracellular suction electrode. Expression of mRNA encoding a variety of intercellular proteins, intracellular Ca2+-transport and regulatory proteins, cell membrane transport proteins and calcium, sodium and potassium channel proteins were measured in SAN and right atrial (RA) biopsies using real-time reverse transcription polymerase chain reaction techniques. Heart rate was lower in STZ (203±7 bpm) compared to control (239±11 bpm) rat. Among many differences in the profile of mRNA there are some worthy of particular emphasis. Expression of genes encoding some proteins were significantly downregulated in STZ-SAN: calcium channel, Cacng4 (7-fold); potassium channel, Kcnd2 whilst genes encoding some other proteins were significantly upregulated in STZ-SAN: gap junction, Gjc1; cell membrane transport, Slc8a1, Trpc1, Trpc6 (4-fold); intracellular Ca2+-transport, Ryr3; calcium channel Cacna1g, Cacna1h, Cacnb3; potassium channels, Kcnj5, Kcnk3 and natriuretic peptides, Nppa (5-fold) and Nppb (7-fold). Conclusions/Significance Collectively, 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 SAN of STZ-induced diabetic rat heart. Data from this study will provide a basis for a substantial range of future studies to investigate whether these changes in mRNA translate into changes in electrophysiological function.


Introduction
Cardiovascular complications are common in patients with Type 1 and Type 2 diabetes mellitus and these complications lead to an increased risk of mortality [1]. Although vascular diseases including coronary artery disease and hypertension increase the risk of mortality these patients are also at increased risk of developing cardiac abnormalities that are independent of cardiovascular complications [2]. In addition to mechanical dysfunction disturbances in electrical activity, manifesting as arrhythmias, have also been widely reported in diabetic heart. Patients with diabetes have an increased risk of arrhythmias including QT interval and QRS prolongation which is associated with sudden death [3] Atrial fibrillation, bradyarrhythmias, bundle branch block and atrioventricular block are more prevalent in diabetic patients [4]. In vivo biotelemetry studies performed in experimental models of diabetes have demonstrated disturbances in the electrocardiogram which are variously associated with bradycardia, prolongation of PQ, QRS and QT intervals [5][6][7]. Action potential duration is frequently prolonged in the diabetic heart and prolongation can occur to different extents in different regions of the heart including the SAN [6,8,9]. Very little experimental data is available about the effects of diabetes on SAN electrophysiology. It is known that SAN conduction and pacemaker cycle length are prolonged and that the SAN action potential duration may be prolonged in diabetic heart [10,11]. A variety of ionic currents including L-type and T-type Ca 2+ current, hyperpolarization-activated "funny" current, Na + /Ca 2+ exchange current and various K + currents are important in the generation of the SAN action potential [12]. Sarcoplasmic reticulum (SR) Ca 2+ signaling may also contribute to the generation and decay of the SAN action potential [12]. Disturbances of one or more of these ionic conductances would undoubtedly have implications for the generation and conduction of electrical signals in the SAN which in turn may underlie some of the electrical disturbances that have been frequently reported in diabetic heart [5][6][7]. To further clarify the molecular basis of electrical disturbances in the SAN 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 pacemaker has been evaluated in the STZ-induced diabetic rat heart.

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. Experiments began 10 weeks after STZ treatment. Blood glucose was measured 5 days following STZ treatment to confirm diabetes. Body weight, heart weight and blood glucose were measured immediately prior to experiments. All animal experimentation was carried out in accordance with the Animals (Scientific Procedures) Act 1986 and conforms to the Recommendation from the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals. Approval for this project was obtained from the Animal Ethics Committee, College of Medicine & Health Sciences, United Arab Emirates University.

Measurement of heart rate and action potentials
Rats were killed using a guillotine. Hearts were then rapidly removed, mounted in Langendorff mode and perfused retrogradely at a constant flow of 8 ml.g heart -1 min -1 and at physiological temperature (36-37°C) with a normal Tyrode containing: 140 mM NaCl; 5 mM KCl; 1 mM MgCl 2 ; 10 mM glucose; 5 mM HEPES; 1.8 mM CaCl 2 and adjusted to pH 7.4 with NaOH and continuously bubbled with oxygen. To measure heart rate action potentials were recorded in spontaneously beating hearts with a purpose built extracellular suction electrode with a tip~2 mm in diameter according to previously described techniques [13]. Recordings were made in the region of the left ventricle. Signals from the electrode were collected at 400 Hz, amplified (ADInstuments, ML136 Bioamp) and conveyed via a Powerlab (ADInstruments, PL410) for display on a PC. Data were analyzed with ADInstruments software version v 4.21 (ADInstruments, Australia).

Expression of mRNA
Expression of genes encoding a range of cardiac muscle proteins was assessed using modifications of previously described techniques [14][15][16]. After sacrifice hearts were removed rapidly from the rats and placed in a dish containing: NaCl 140 mM; KCl 5.4 mM; MgCl 2 1 mM; HEPES 5 mM; D-glucose 5.5 mM; CaCl 2 1.8 mM and adjusted to pH 7.4 with NaOH. The ventricles and the left atrium were removed and the right atrium was opened to expose the SAN and crista terminalis (Fig 1a and 1b). The SAN artery was used to identify the SAN. The SAN was exposed and 2 mm biopsy samples of SAN were carefully collected from 20 STZ (STZ-SAN) and 20 control (CON-SAN) hearts according to previously described techniques [17,18]. Samples of right atrial (RA) tissue were also collected. Once dissected, the samples were immediately placed in RNAlater (AM7021, Life Technologies, Carlsbad, CA, USA) and kept overnight at room temperature to allow thorough penetration of the tissue. Tissue samples were then frozen at -20°C pending further processing. The samples were homogenized at 6500 rpm for 2 runs of 20 seconds each with a 15 second gap (Preceylls 24, Berlin Technology, USA). Isolation of total RNA from the tissue was performed using the SV Total RNA Isolation System (Promega, Madison, USA) according to the manufacturer's instructions. The concentration and purity of the RNA samples was determined by measuring the absorbance at 260 nm and 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 10 x RT Buffer 2.0 μl, 25 x dNTP Mix (100 mM) 0.8 μl, 10 x RT Random Primers 2.0 μl, MultiScribe™ Reverse Transcriptase 1.0 μl, RNase inhibitor 1.0 μl, and Nuclease-free H 2 O (High Capacity cDNA Reverse Transcription Kit (4374966, Applied Biosystems, USA). Reverse transcription was carried out using the following parameter values: 25°C for 10 min, 37°C for 120 min, and 85°C for 5 min on the Veriti thermal cycler (Applied Biosystems, USA). Gene Expression Assays were performed using custom TaqMan Low Density Arrays (Format 32, 4346799, Applied Biosystems, USA). The TaqMan assays are preloaded in each reaction well of the array in triplicate for each RNA sample. As in previous studies 18S ribosomal RNA was used as an endogenous control [19]. Expression of 18S was not significantly different (P>0.05) between the samples of STZ-SAN, CON-SAN, STZ-RA and CON-RA. 100 ng of cDNA (RNA-equivalent) was loaded together with 2 × TaqMan Gene Expression Master Mix (No AmpErase UNG, Applied Biosystems, USA) for a total of 100 μL per port. Two SAN samples were combined and two RA samples were combined for each realtime RT-PCR assay. Real-time RT-PCR was performed in a Fast ABI Prism 7900HT Sequence Detection System (Applied Biosystems, USA). 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 for 1 min. Results were initially analyzed using ABI Prism 7900HT SDS, v2.4. All remaining 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 and the proteins encoded by these genes is shown in Table 1.

Cacnb3
Ca v β3 Voltage-Dependent, Beta 3 Subunit Briefly, 10-20 ug protein was electrophoretically separated onto 7.5% or 12% (depending on the molecular weight of the protein to be separated) polyacrylamide gels and transferred onto nitrocellulose membranes. The expression of the specific proteins was checked by immunoreaction with their specific antibodies by Western blot analysis. β-actin was used as a loading control. The blots were developed using the Pierce Western Blot kit. Densitometric analysis of the protein bands was performed using the Typhoon FLA 9500, GE Healthcare Bio-Sciences AB (Uppsala, Sweden). The ratio of specific protein signal to that of actin control were used to calculate fold change.

Statistics
Results were expressed as the mean ± S.E.M. of 'n' observations. Statistical comparisons were performed using one-way ANOVA and Bonferroni post hoc for multiple comparisons or Independent Samples t-test, as appropriate (SPSS v. 20). P< 0.05 was considered to indicate a significant difference.

Results
Body and heart weight were significantly (P<0.01) reduced and heart weight / body weight ratio was increased in STZ compared to control rat. Blood glucose was increased 5-fold in STZ rat compared to control ( Table 2).

Expression of mRNA
Expression of mRNA for cardiac muscle proteins are shown in Fig 3. Expression of Myl1 was significantly (P<0.05) upregulated in STZ-SAN compared to CON-SAN whereas Myl1 was significantly downregulated STZ-RA compared to CON-RA. Expression of mRNA for intercellular   Fig 5a and 5b, respectively. Among the cell membrane transport and Ca 2+ transport proteins expression of Atp2b1, Slc8a1, Trpc1, Trpc6, Casq2 and Itpr1-3 were significantly upregulated and Ryr3 was modestly upregulated in STZ-SAN compared to CON-SAN. Expression of mRNA for hyperpolarization-activated cyclic nucleotide-gated channel proteins are shown in Fig 6. Hcn4 was modestly upregulated in STZ-SAN compared to CON-SAN however, the difference was not significant. Expression of mRNA for calcium channel proteins are shown in Fig 7. Expression of Cacna1g, Cacna1h, Cacna2d and Cacnb3 were upregulated whilst Cacng4

Expression of protein
Western blot techniques were used to compare expression of selected proteins in STZ and control SAN and typical results are shown in Fig 11a. Expression of ANP was significantly reduced whilst Cav3.1 and Ryr3 were increased in STZ compared to control SAN (Fig 11b).

Discussion
Spontaneous heart rate was lower in STZ compared to control hearts. Previous in vivo biotelemetry experiments have also demonstrated reduced heart rate in STZ rat [5,21]. Prolongation of SAN action potentials in isolated perfused heart and prolonged pacemaker cycle length and sino-atrial conduction time in isolated sino-atrial preparations have been demonstrated in STZ rat heart [9,10]. Collectively, data from various isolated preparations suggest that the reduced heart rate in STZ rat could, at least in part, may be attributed to intrinsic defects in the electrophysiological function of the SAN. An early step in elucidating the mechanisms that underlie low heart rate in STZ rat is to study the profile of genes associated with proteins that are involved in the generation and propagation of the SAN action potential. Of particular interest were genes encoding Gja7 (2-fold), Trpc1 (3-fold), Trpc6 (4-fold), Ryr3 (2-fold), Cacna1g (2-fold), Cacnb3 (3-fold), Kcnj5 (3-fold), Nppa (5-fold) and Nppb (7-fold) which were upregulated and Cacng4 (3-fold) which was downregulated in STZ-SAN compared to CON-SAN. Gja7 (Cx45) was upregulated in STZ-SAN compared to CON-SAN. Cx45 protein was also moderately increased in STZ-SAN compared to CON-SAN. Connexin proteins, pore forming subunits of gap junctions, play an important role in ensuring efficient cell-to-cell communication and the maintenance of cardiac rhythmicity [22]. At least five connexins (Cx30.2, Cx37, Cx40, Cx43 and Cx45) are prominently expressed in the heart and each shows regional and cell type specific expression [22,23]. Gja7 (Cx45) is expressed in the SAN and previous studies have demonstrated upregulation of genes encoding connexin proteins, and in particular Cx45, in STZ-SAN [10,[24][25][26]. It is known that action potential duration and SAN conduction and pacemaker cycle length can be prolonged in diabetic heart and this may be associated with remodeling of connexin proteins [10,11].
Trpc1 (TRPC1) and Trpc6 (TRPC6) were upregulated in STZ-SAN compared to CON-SAN. The transient receptor potential (TRP) channels are a large family of non-selective and nonvoltage gated ion channels that are widely expressed in human tissue including the heart and vasculature [27,28]. TRPC1 and TRPC6 are mechano-sensitive, non-selective cation channels that are expressed in mouse ventricular muscle [29]. In the cardiovascular system the TRPC family has been found to play a role in vascular and cardiac disease [30]. Upregulation of TRPC channels is involved in the development of cardiac hypertrophy and heart failure [27,31,32]. TRPC6 is induced in heart hypertrophy and inhibition of TRPC6 has been shown to suppress agonist-induced hypertrophic responses [33][34][35]. Whilst the physiological role of the TRP channels in the SAN is unclear, upregulation of Trpc1 and Trpc6 in STZ-SAN if associated with altered entry of Na + or Ca 2+ through non-selective TRP channels might have implications for the generation of the pacemaker or action potential in diabetic SAN cells [31,36,37].
Cacna1g (Ca v 3.1) was upregulated in STZ-SAN compared to CON-SAN however, expression of Cacna1g was not altered in STZ-RA compared to CON-RA. Ca v 3.1 protein was also increased in STZ-SAN compared to CON-SAN. Previous studies have reported upregulation of Cacna1g in ventricle from the Goto-Kakizaki rat, an experimental model of type 2 diabetes mellitus, and the Zucker diabetic fatty rat [38,39]. The protein encoded by Cacna1g represents the alpha 1G subunit, also known as Ca v 3.1, of the T-type calcium channel. In the heart T-type Ca 2+ channels are found in the SAN and conduction cells [40]. In mice disruption of the gene mRNA Expression in Sinoatrial Node of Diabetic Rat encoding Ca v 3.1channels abolishes T-type Ca 2+ current in isolated cells from the SAN and atrioventricular node without affecting L-type Ca 2+ current. Inactivation of Cacna1g slowed the heart rate in vivo and prolonged the SAN recovery time and slowed pacemaker activity of individual SAN cells through a reduction of the slope of the diastolic depolarization [41]. Upregulation of Cacna1g (Ca v 3.1) might be expected to increase T-type Ca 2+ current and hence, the slope of the pacemaker potential and heart rate in STZ rat.
Cacnb3 (Ca v β3) was upregulated in STZ-SAN compared to CON-SAN. Cacnb3 (Ca v β3) encodes expression of the beta 3 subunit (Ca v β3) of the L-type Ca 2+ channel. Previous studies have demonstrated that overexpression of the beta subunits (1)(2)(3)(4) in adult cultured heart cells increased whole-cell L-type Ca 2+ current density [42]. In the SAN L-type Ca 2+ current contributes to the pacemaker potential and generates the upstroke of the action potential therefore upregulation of Cacnb3 (Ca v β3) might be expected to increase L-type Ca 2+ current, the slope of the pacemaker potential and hence heart rate.
Ryr3 (RYR3) was upregulated in STZ-SAN compared to CON-SAN. RY3 protein was also increased in STZ-SAN compared to CON-SAN. Various studies have shown that sub-sarcolemmal diastolic Ca 2+ release from the sarcoplasmic reticulum (SR) may contribute to the  mRNA Expression in Sinoatrial Node of Diabetic Rat generation of electrical activity in SAN cells [43,44]. Upregulation of the SR Ca 2+ release channel (ryandodine receptor) would be expected to facilitate increased release of Ca 2+ from the SR and hence, might influence spontaneous electrical activity in the SAN cell.
Cacng4 (Ca v γ4) was downregulated (3-fold) in STZ-SAN compared to CON-SAN however, expression of Cacng4 was not altered in STZ-RA compared to CON-RA. The cardiac voltagegated L-type Ca 2+ channel is the Ca 2+ channel that is required for excitation-contraction coupling and that also contributes to the plateau phase of the cardiac action potential and pacemaker activity in nodal cells [45,46]. The protein encoded by Cacng4 (Ca v γ4) represents one of the gamma subunits of the L-type Ca 2+ channel. It has been reported that the gamma subunits expressed in heart (gamma 4, 6, 7 and 8) form macromolecular complexes with Cav1.2 and can differentially modulate its function [45]. Downregulation of Cacng4 might have implications for the modulation and hence, function of the L-type Ca 2+ channel and L-type Ca 2+ current which in turn are important for generation of the pacemaker potential and the upstroke of the SAN action potential.
Nppa (ANP) and Nppb (BNP) were upregulated in STZ-SAN compared to CON-SAN however, neither Nppa or Nppb were significantly altered in STZ-RA compared to CON-RA. ANP protein was also significantly reduced in STZ-SAN compared to CON-SAN. 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 [47]. 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 [47][48][49]. Previous studies have demonstrated the expression of ANP, albeit at low levels, in the SAN node [50,51]. ANP plays a key role in cardiac electrophysiology, modulating the autonomic nervous system and regulating the function of various cardiac ion channels [52]. 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 [Ca 2+ ] i in ventricular myocytes from STZ-induced diabetic rat [53,54]. 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 [55][56][57]. Upregulation of Nppa and Nppb in the SAN may be associated with mechanisms that compensate for the low heart rate seen in the STZ-induced diabetic heart [53,54]. Interestingly, expression of Nppa and Nppb were increased whilst ANP protein was reduced in STZ-SAN compared to CON-SAN. Previous studies have demonstrated increased ANP and BNP levels in plasma and atria in STZ rats compared to controls [53,54].
Collectively, 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 SAN of STZ-induced diabetic rat heart. Data from this study will provide a basis for a substantial range of studies to investigate whether these changes in mRNA translate into changes in electrophysiological function.