Evidence for a lack of inotropic and chronotropic effects of glucagon and glucagon receptors in the human heart

Tissue collection

Human cardiac tissues were obtained from the nonfailing hearts of multiorgan donors that were not used for transplantation and from the explanted hearts of patients with end-stage myocardial failure who underwent heart transplantation at the Hospital CSV Arrixaca of Murcia (Spain). Nonfailing hearts used in this study were in asystole for 83 ± 23 min (n = 5) before explantation, while organs other than the heart were retrieved for transplantation. Failing hearts were not in asystole before explantation. The characteristics of the heart donors are described in Table 1. Immediately after explantation, parts of the free wall of the atrial (right, RA and left, LA) and ventricular (right, RV and left, LV) myocardium, as well as from the portion of the right atrium attached to the entrance of the superior vena cava, were dissected. After being removed, these tissues were placed in oxygenated cool Tyrode solution containing 136.9 mmol/L NaCl, 5.0 mmol/L KCl, 1.8 mmol/L CaCl2, 1.5 mmol/L MgCl2, 0.4 mmol/L H2PO4, 11.9 mmol/L NaHCO3, and 5.0 mmol/L dextrose and transported to the laboratory (approximately 15 min). To investigate the inotropic effects of glucagon, RA, LA, RV and LV tissues were dissected to yield trabecular strips measuring 4–6 mm in length and 0.4–0.8 mm in diameter. The chronotropic effects of glucagon were investigated in SN tissue obtained from the right atrial wall where the crista terminalis meets the superior vena cava. To better find the SN region, we followed the SN artery trajectory by carefully removing the epicardium and subepicardial connective tissue from the external surface of the right atrium. The atrial wall, to which the SN artery connects, contains the SN [20]. This portion of the atrial wall, as well as the attached pectinate muscle, was dissected. Contraction of the pectinate muscle when stimulated by SN tissue was recorded as the measure of the SN rate. In addition to functional identification, we aimed to identify SN tissue by determining the level of the hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channel, which is predominantly located in SN tissue [21].

Table 1 Characteristics of the heart donors

We also evaluated the inotropic and chronotropic effects of glucagon on rat hearts. For this purpose, Sprague–Dawley rats (250–300 g, both sexes) were stunned and exsanguinated. The chest was opened, and the heart was rapidly removed and placed in Tyrode solution saturated with 95% O2 and 5% CO2. The right atrium and the free wall of the right ventricle were excised, and strips of the right ventricle (1.5 mm wide, 10 mm long, and 1 mm thick) were obtained. All procedures were performed in the presence of Tyrode solution.

Sample setup and construction of concentration–response curves

To investigate inotropic effects, trabeculae from human myocardium (RA, LA, RV and LV) or strips of the rat right ventricle were mounted vertically between two platinum electrodes in a 30 ml double-walled organ bath containing Tyrode solution at 37 °C, pH 7.4, and 95% O2 plus 5% CO2. The samples were electrically stimulated (Grass SD-9 stimulator) at a frequency of 1 Hz for 3 ms with supramaximal (threshold + 25%) voltage. A length–force curve was obtained, and the tissues were left at the length associated with the maximum developed force [6, 15].

The chronotropic effect of glucagon was studied in human SN tissue that was obtained as previously described, as well as in isolated rat right atria. The tissues were mounted vertically in a 30 ml double-walled glass chamber filled with Tyrode solution, gassed continuously with 95% O2 and 5% CO2, and maintained at pH 7.4 and 37 °C. A preload tension of 0.5 gm. was applied. Under these conditions, human SN samples (Additional file 1), as well as rat right atria, started beating spontaneously. Contractions were measured using a Grass FT-03 force‒displacement transducer (Quincy, MA, USA) and displayed on a computer screen using a Stemtech amplifier (Stemtech Inc., Houston, Texas) and ACODAS software (DATAQ Instruments, Inc., Akron, Ohio). The tissues were allowed to equilibrate for 45–60 min in Tyrode solution.

After equilibration, cumulative concentration‒response curves to glucagon (Novo Nordisk Pharma S.A. Madrid, Spain), were determined in human and rat tissues by increasing the concentration stepwise by 0.5 log units.

To ascertain the role of PDE in regulating glucagon responses, a second cumulative concentration‒response curve was obtained for glucagon in the presence of the nonselective PDE inhibitor 3- isobutyl-1-methylxanthine (IBMX) [14] (Sigma/Aldrich, Madrid, Spain). The samples were washed and left to stabilize during an additional period of 30 min, and then 30 μM IBMX was applied. This concentration of IBMX effectively inhibited the predominant PDE activity in human and rat hearts [22, 23]. IBMX was left in contact with the tissue for 15 min before the construction of a second concentration–response curve for glucagon. Drugs were added to the organ bath in volumes less than or equal to 0.1 ml. The experiments of contractility (human and rat tissues) and chronotropism (rat right atrial and human SN tissue) were finalized with the addition of 9 mM CaCl2 and 10 µM noradrenaline (Sigma/Aldrich, Madrid, Spain), respectively, to determine the response capacity of the samples. Changes in contractile force are expressed in mN and as percentages with respect to the basal control contraction amplitude. The frequency is expressed as beats min−1, and the results are expressed as differences with respect to the basal rate.

Real-time PCR

Samples from the right and left atria, right and left ventricles and SNs were carefully dissected. Quantitative PCR was performed using QuantStudio 5 (Applied Biosystems, Thermo Fisher Scientific, Massachusetts, USA). Total mRNA was extracted from heart tissue using TriPure Isolation Reagent (Roche, Paris, France), and 1 μg of RNA was used for the reverse transcription reaction (iScript cDNA Synthesis kit. Bio-Rad, CA, USA). The reactions were carried out in a final volume of 5 μl containing 300 nM primers and 1 μl of cDNA using a SYBR Premix Ex Taq (Tli RNaseH Plus) kit (Takara BioInc., Göteborg, Sweden). The samples were subjected to the following conditions: 30 s at 95 °C, 40 cycles (10 s at 95 °C, 30 s at 60 °C), and a melting curve at 60–95 °C with a slope of 0.1 °C/s. The reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the endogenous control for quantification (KiCqStart Primers, Merck, Darmstadt, Germany). The resulting values are expressed as the relative levels with respect to the control levels (2−ΔΔCT). Human liver tissue (“Biobanco Región de Murcia”, national register number B.0000859) was used as a positive control for glucagon receptor gene expression.

Specific primers for gene level analysis, as well as accession numbers and amplicon lengths, are shown (Additional file 2: Table S2).

Tissue samples for western blotting

Total protein was extracted from heart samples (right and left atria, right and left ventricle, and SN). Briefly, 100 mg of frozen tissues was disrupted in a polytron homogenizer using radioimmunoprecipitation assay lysis buffer with protease and phosphatase inhibitor cocktails and quantified with a Bradford assay using bovine serum albumin as a standard (Protein Assay Kit, Bio-Rad, Hercules, CA). Total protein extracts (30 µg) were mixed with 5 × sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 0.005% bromophenol blue) and resolved by SDS–polyacrylamide gel electrophoresis on 10% acrylamide gels. Proteins were detected immunologically following electrotransfer to polyvinylidene fluoride membranes (Millipore, Bedford, MA) that were activated with methanol. The membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline (PBS) and 0.1% Tween-20 for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: anti-glucagon receptor (rabbit polyclonal, 1:500 dilution, MyBioSource Inc. San Diego, CA) and anti-HCN1 (rabbit polyclonal, 1:1000 dilution, Sigma/Aldrich, Madrid, Spain). The blots were washed three times for 10 min each in PBS and 0.1% Tween-20 and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibodies (1/5000 dilution) (Arigo Biolaboratories, Hsinchu City, Taiwan) for 1 h at room temperature. The blots were developed using a peroxidase reaction with an enhanced chemiluminescent immunoblotting detection system (ECL-Plus, GE Healthcare, Little-Chalfont, Buckinghamshire, UK). Antibodies were accepted when they exhibited a single predominant band at the expected molecular weights. α-Tubulin (mouse monoclonal, 1:10,000 dilution, Proteintech, Deansgate, Manchester, UK) was used as the loading control. Band intensity was determined by densitometry using the program Image Quant TL Plus (General Electric, USA).

Statistical analysis

The results are expressed as the mean values ± standard errors. Concentration‒response curves were fitted with a nonlinear regression sigmoidal concentration‒response curve, variable slope, and –log EC50, and the maximal effect (Emax) values were estimated from the concentration‒response curves depicted. Concentration‒response curves and statistical analyses were performed with GraphPad 5 Software, Inc. (San Diego, CA, USA). Statistical significance was determined by paired or unpaired Student`s t test, Wilcoxon test and parametric binomial analysis as appropriate. A P value ≤ 0.05 was considered statistically significant.

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