Quantification of GLUT4 Gene Expression in Human Atrial Myocardium of Hypertensive Patients and the Effect of Experimental Ischaemia Thereupon

Journal of Clinical and Basic Cardiology 2007; 10 (1-4): 1-6

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Keywords: GLUT4,  Hypertonie,  Ischämie,  Kardiologie,  Myokard,  cardiology,  heart,  hypertension,  ischaemia

Introduction: Transmembrane glucose transport and thus cellular high-energy metabolism of the cardiovascular system depend largely on the insulin-responsive GLUT4 isoform of the trans-membrane glucose transport molecule. GLUT4 plays a key role in the development of myocardial and vascular stiffness, in the context of osmolarity, compartmental water distribution and homeostasis. In hypertensive rats, expression of GLUT4 mRNA as well as the amount of protein in the membrane was found to be decreased. In the context of hypertension and ischaemia, no reports can be found on GLUT4 in human myocardium. Myocardial ischaemia increases glucose uptake through translocation of GLUT1 and GLUT4 from an intracellular compartment to sarcolemma. This appears to be a beneficial effect during ischaemia and possibly recovery. Insulin and ischaemia have additive effects in the increase of in vivo glucose utilisation and augment glucose transporter translocation. Delivery of glucose to the glycolytic pathway appears to be a major controlling site of glycolysis in low-flow ischaemia. While many experimental studies suggest that an increase in glucose uptake and metabolism by the ischaemic myocardium help protect myocardial cells from irreversible injury, little or nothing is known in this context about human cardiac trans-membrane glucose transport, SLC2A4-expression and its regulation. Methods: We investigated tissue samples (60–150 mg) from the right atrial auricle from patients with arterial hypertension subjected to cardiac surgery, which were snap-frozen in liquid nitrogen and stored at –70 °C until homogenisation. RNA-Isolation and cDNA transcription: Total RNA was extracted by the TRIZOL® method (Invitrogen Corp., Carlsbad, CA, USA) and further purified using RNeasy Mini Kit (QIAGEN Inc., Hilden, GER). RNA was transcribed into DIG-labelled cDNA. For reverse transcription of isolated RNA we used the High Capacity cDNA Archive Kit (Applied Biosystems) and the Thermocycler MyCyclerTM from Biorad. Real-time PCR was performed using the LightCycler® 2.0 System (Roche). Furthermore, in human cardiac tissue (right auricle), using microarray technique, we first looked at general changes in expression profiles during simulated myocardial ischaemia and then at the behaviour of SLC2A4 (GLUT4, solute carrier family 2 [facilitated glucose transporter], member 4) as well as its regulator gene SLC2A4RG. Then, using real-time PCR (Light Cycler), we quantified GLUT4 mRNA expression changes under ischaemic and control conditions in normotensive and hypertensive patients. Results: We showed in 28 patients that GLUT4 mRNA was significantly less expressed in patients with arterial hypertension compared to controls. Using the microarray technique, we found that both the expression of GLUT4 gene (SLC2A4) and its regulator gene remain practically unchanged in experimental ischaemia. In real-time PCR (Light Cycler), the mean ratio for GLUT4 gene expression compared to the housekeeping gene G6PDH was under well oxygenated conditions –0.0052 ± 0.0203 and under N2-simulated ischaemia 0.0179 ± 0.0196 (n = 8; ± SEM). No statistically significant difference could be found between the two groups, however, results show a trend towards a slight increase in expression. Summary: Our experiments show that GLUT4 mRNA expression is in fact decreased in arterial hypertension compared to normal controls. From these data, one can deduce that arterial hypertension is genuinely associated with decreased GLUT4 expression. However, no significant changes are seen in the expression of the GLUT4 gene as well as in its regulatory gene after 30 minutes of N2-mediated experimental ischaemia. Similarly, biological processes involved in glucose metabolism are not significantly de-regulated, as are others. This, as well as a slight trend towards up-regulation, can be interpreted as an attempt of the myocyte to maintain the energy metabolism stable under hypoxic conditions.