02-0252 R2
ENDOGENOUS ENDOTHELIN-1 CONTRIBUTES TO THE FRANK-STARLING RESPONSE IN HYPERTROPHIC RAT HEARTS
Address for correspondence:
Heikki Ruskoaho, MD, PhD
Department of Pharmacology and Toxicology
Faculty of Medicine, University of Oulu
P.O.Box 5000, FIN-90014, University of Oulu, Finland
Fax: +358-8-5375247; phone: +358-8-5375236
E-Mail:
ONLINE DATA SUPPLEMENT
Isolated Perfused Heart Preparation
The retrograde perfusion system and the Krebs-Henseleit bicarbonate buffer were similar as previously described.1,2 Coronary flow was adjusted to 5 mL/min and 7 mL/min in NTG and dTG rat experiments, respectively, to give perfusion rate of 5.8 mL x gram-1 x min-1. This flow rate results in maximal vasodilatation and thus excludes any secondary effects caused by the vasorelaxation of the coronary arteries induced by the drugs.1 The temperature was set to 37 ºC, and the hearts were stimulated (11 V, 0.5 ms) with a Grass stimulator (model S88, Grass Instruments) at a rate of 300 beats per minute. The isovolumic force of contraction was measured with a balloon inside the left ventricle.2 The following parameters were obtained: peak systolic left ventricular pressure, left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (DP), maximum and minimum values of the first derivative of isovolumic pressure (dP/dtmax, dP/dtmin).
Quantitative Reverse Transcription-PCR
ETA receptor, ET-1, AT1 receptor, angiotensin converting enzyme, phospholamban, Na+-H+-exchanger isoform 1 (NHE-1) and Na+-Ca2+-exchanger (NCX) mRNA levels were measured by real-time quantitative RT-PCR analysis using Taqman chemistry on an ABI 7700 Genetic Analyzer (Applied Biosystems) as described previously.3 Forward and reverse primers and probes for mRNA detection were designed with Primer Express® (Applied Biosystems) program and default settings. Sequences are presented in Table 1.
References
1. Szokodi I, Kinnunen P, Tavi P, Weckström M, Tóth M, Ruskoaho H. Evidence for cAMP-independent mechanisms mediating the effects of adrenomedullin, a new inotropic peptide. Circulation. 1998;97:1062-1070.
2. Szokodi I, Tavi P, Földes G, Voutilainen-Myllylä S, Ilves M, Tokola H, Pikkarainen P, Piuhola J, Rysä J, Tóth M, Ruskoaho H. Apelin, the Novel Endogenous Ligand of the Orphan Receptor APJ, Regulates Cardiac Contractility. Circ Res. Published online August 15, 2002. 10.1161/01.RES.0000033522.37861.69
3. Majalahti-Palviainen T, Hirvinen M, Tervonen V, Ilves M, Ruskoaho H, Vuolteenaho O . Gene structure of a new cardiac peptide hormone: a model for heart- specific gene expression. Endocrinology. 2000;141:731-740.
Table 1 Online. Oligonucleotide Primer and Detection Probe Sequences Used for mRNA Quantitation by RT-PCR
AT1 receptor / GTGGCCAAAGTCACCTGCA / GTGGATGACAGCTGGCAAACT / CATCTGGCTGATGGCTGGCTTGG
ACE / GCACATTCGCAGGAACGTG / GCCCTCCAGTGCCTAGATCC / AAGGTGACTTTGACCCAGGGTCCAAGTT
ET-1 / ATGGACAAGGAGTGTGTCTACTTCTG / GGGACGACGCGCTCG / CACCTGGACATCATCTGGGTCAACACTC
ETA receptor / GGAATGGGAGCTTGCGG / TTTGCCACCTCTCGACGC / TTGCCCTCAGCGAACACCTCAAGC
NHE-1 / TCCAGCTTAACCCTACTGCCA / TGACATCCCCAATTGAGCGT / TCAGAGGTTCTGAACCACCCCGGG
NCX / CTCTTGTTTACCCATGTTGACCATAT / GAGCCAGTACATTCAGTGGTTTCA / TGCAGATACAGAGGCAGAAACAGGAGGAA
PLB / AAGTCTGTCGCCACCGCA / TGGTGGAGGGCCAGGTT / CCTGCACCATGCCAACGCAGC
18S / TGGTTGCAAAGCTGAAACTTAAAG / AGTCAAATTAAGCCGCAGGC / CCTGGTGGTGCCCTTCCGTCA
ACE, angiotensin converting enzyme; ET-1, endothelin-1; NHE-1, Na+-H+ -exchanger isoform 1; NCX, Na+ -Ca2+ exchanger; PLB, phospholamban.