Local renin-angiotensin system contributes to hyperthyroidism-induced cardiac hypertrophy

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Local renin–angiotensin system contributes to hyperthyroidism-induced cardiac hypertrophy H Kobori, A Ichihara, Y Miyashita, M Hayashi and T Saruta Department of Internal Medicine, School of Medicine, Keio University, 160–8582, Tokyo, Japan (Requests for offprints should be addressed to T Saruta)

Abstract We have reported previously that thyroid hormone activates the circulating and tissue renin–angiotensin systems without involving the sympathetic nervous system, which contributes to cardiac hypertrophy in hyperthyroidism. This study examined whether the circulating or tissue renin–angiotensin system plays the principal role in hyperthyroidism-induced cardiac hypertrophy. The circulating renin–angiotensin system in Sprague–Dawley rats was fixed by chronic angiotensin II infusion (40 ng/ min, 28 days) via mini-osmotic pumps. Daily i.p. injection of thyroxine (0·1 mg/kg per day, 28 days) was used to mimic hyperthyroidism. Serum free tri-iodothyronine, plasma renin activity, plasma angiotensin II, cardiac renin and cardiac angiotensin II were measured with RIAs. The cardiac expression of renin mRNA was evaluated by semiquantitative reverse transcriptase-polymerase chain

Introduction Cardiac hypertrophy is a serious complication of hyperthyroidism (Shirani et al. 1993). The enhanced hemodynamics produced by increased activity of the sympathetic nervous system (SNS) is a major factor in the cardiac hypertrophy induced by hyperthyroidism (Klein 1990). Increased SNS activity also increases plasma renin activity (PRA) (Hauger-Klevene et al. 1972) following activation of the circulating renin– angiotensin system (RAS). There is evidence that circulating RAS may be involved in the development of cardiac hypertrophy (Morgan & Baker 1991). Angiotensin II (ANGII) exerts a direct physiological effect on the cardiovascular system via specific receptors on the cardiomyocyte plasma membrane that are coupled to guanine nucleotide-binding proteins (Baker et al. 1984, Baker & Singer 1988). The administration of angiotensin I-converting enzyme inhibitors is clinically efficacious in reducing cardiac hypertrophy (Dunn et al. 1984). We therefore hypothesized that activation of the circulating RAS might be involved in the development of the cardiac hypertrophy induced by hyperthyroidism. Journal of Endocrinology (1999) 160, 43–47 0022–0795/99/0160–0043 $10.00/0

reaction. Plasma renin activity and plasma angiotensin II were kept constant in the angiotensin II and angiotensin II+thyroxine groups (0·120·03 and 0·150·03 µg/h per liter, 1265 and 1305 ng/l respectively) (means...). Despite stabilization of the circulating renin–angiotensin system, thyroid hormone induced cardiac hypertrophy (5·00·5 vs 3·50·1 mg/g) in conjunction with the increases in cardiac expression of renin mRNA, cardiac renin and cardiac angiotensin II (742 vs 482%, 6·50·8 vs 3·80·4 ng/h per g, 23130 vs 1492 pg/g respectively). These results indicate that the local renin–angiotensin system plays the primary role in the development of hyperthyroidism-induced cardiac hypertrophy. Journal of Endocrinology (1999) 160, 43–47

However, recent reports have shown that high PRA is present in hyperthyroidism but not hypothyroidism (Hauger-Klevene et al. 1972), and that SNS activity is elevated in both conditions (Polikar et al. 1990). While cardiac hypertrophy is induced by hyperthyroidism, cardiac hypertrophy is not induced by hypothyroidism (Heron & Rakusan 1994). These findings suggest that hyperthyroidism-induced cardiac hypertrophy is caused by factors other than changes in circulating RAS or SNS activity. Expression of the Ren-2 gene in the mouse submandibular gland is stimulated by thyroid hormone (Catanzaro et al. 1985, Karen & Morris 1986). In a pituitary cell line, thyroid hormone has been shown to regulate renin gene promoter activity (Gilbert et al. 1994), suggesting that thyroid hormone may regulate the expression of the tissue renin gene. We previously reported that thyroid hormone activates the circulating and tissue RAS without involving the SNS, and this may account for the cardiac hypertrophy observed in hyperthyroidism (Kobori et al. 1997b). However, it is uncertain which RAS dominates. This study examined the roles circulating or tissue RAS play in hyperthyroidisminduced cardiac hypertrophy.

 1999 Society for Endocrinology Printed in Great Britain

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· Thyroxine-induced cardiac hypertrophy

Materials and Methods Preparation of animals The experiments were approved by the University Committee on Animal Care and Use of Keio University. Fifteen 6-week-old male Sprague–Dawley rats (Charles River, Kanagawa, Japan), 150–200 g, were used. They had free access to standard laboratory chow, containing 110 µmol/g sodium (Oriental Yeast, Tokyo, Japan), and tap water. They were individually housed in a room with a 12 h darkness–light cycle. Animals were divided into a control group, an ANGII group and an ANGII+thyroxine (T4) group. Continuous s.c. administration of ANGII (40 ng/min) for 28 days was given to the ANGII group and the ANGII+T4 group via mini-osmotic pumps (Alzet Model 2004, Alza Corporation, Palo Alto, CA, USA) to fix the circulating RAS. Saline was infused in the same manner in the control group. Daily i.p. injection of T4 (0·1 mg/kg per day) for 28 days was used to mimic hyperthyroidism in the ANGII+T4 group as we have previously described (Kobori et al. 1997a). Systolic blood pressure and heart rate were measured weekly using the tail-cuff method. Body weight was also determined weekly. Rats were killed by decapitation at day 28. Blood was collected, divided between two tubes with or without EDTA, separated into plasma and serum by centrifugation at 4 C and stored at
20 C. After blood was collected, the heart was removed immediately, washed in water free of ribonucleases, weighed, frozen in liquid nitrogen and stored at
20 C until assayed.

EDTA, 1·6 mmol/l dimercaprol, 3·4 mmol/l 8hydroxyquinoline sulfate, 0·2 mmol/l phenylmethylsulfonyl fluoride and 5 mmol/l ammonium acetate. The homogenate was frozen and thawed four times, centrifuged at 20 000 g for 30 min at 4 C and the supernatant removed. An aliquot of the supernatant was diluted 1:10. In addition, 0·5 ml plasma obtained from nephrectomized male rats was added to the same volume of diluted solution as a substrate for the enzymatic reaction. Renin activity was determined as in our previous study (Ichihara et al. 1995) using the Renin-Riabead assay. The cardiac level of renin was calculated using the formula: cardiac level of renin (ng of angiotensin I/h per g of heart)=renin activity (ng of angiotensin I/h per ml)dilution rate (102=20)volume of the buffer (10 ml)/weight of the aliquot of the heart assayed (g). The second piece of each chamber was used for determination of the cardiac ANGII level as described previously (Kobori et al. 1997b). In brief, the heart was thawed and homogenized with a Polytron in 10 ml buffer that contained 0·1 mol/l HCl, which would inactivate endogenous tissue proteases. The homogenate was centrifuged at 20 000 g for 30 min at 4 C and 1 ml of the supernatant was immediately applied to an octadecasilyl-silica solid phase extraction column (Sep-Pak Plus C18 cartridge, Millipore, Bedford, MA, USA). The concentration of ANGII in the sample was determined as described above. The cardiac level of ANGII was calculated using the formula: cardiac level of ANGII (pg/g of heart)=ANGII concentration (pg/ml)volume of the buffer (10 ml)/weight of the aliquot of the heart assayed (g).

Hormone measurements in serum and plasma A commercially available RIA kit (Amarex-MAB Free T3, Ortho-Clinical Diagnostics, Tokyo, Japan) was used to determine the serum level of free tri-iodothyronine (T3). One half of the plasma was used to determine PRA with a commercially available RIA kit, according to the manufacturer’s instructions (Renin-Riabead, Dainabot, Tokyo, Japan). The remaining plasma was used to determine the level of ANGII with a commercially available RIA kit, according to the manufacturer’s instructions (Angiotensin II Radioimmunoassay Kit, Nichols Institute Diagnostics, San Juan Capistrano, CA, USA). Hormone measurements in cardiac tissue Frozen hearts were dissected into the four chambers. One-third of each chamber was used for each of the following measurements. The first portion of each chamber was used to measure the cardiac level of renin as described previously (Kobori et al. 1997b). In brief, the heart was thawed and homogenized with a Polytron (Kinematica, Littau, Switzerland) in 10 ml buffer containing 2·6 mmol/l Journal of Endocrinology (1999) 160, 43–47

Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) Semiquantitative RT-PCR was carried out as previously described (Kobori et al. 1997a,b, Ichihara et al. 1998). In brief, total RNA was extracted from the last piece of each heart chamber according to the manufacturer’s instructions using the Total RNA Separator Kit (Clontech, Palo Alto, CA, USA). The extracted RNA was suspended in ribonuclease-free water and quantified by measuring the absorbance at 260 nm. Total RNA from each heart was reverse transcribed using the GeneAmp RNA PCR Core Kit (Perkin Elmer, Norwalk, CT, USA) according to the manufacturer’s instructions. Oligonucleotide primers were designed from the published cDNA sequences of renin (Tada et al. 1988) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Tso et al. 1985). GAPDH was used as an internal standard. The sequences of the renin primers were sense 5 -TGCCACCTTGTTGTGTGAGG-3 (exon 7 bases 851–870) and antisense 5 -ACCCGATGCGATTGTTA TGCCG-3 (exon 9 bases 1203–1224). The sequences of

Thyroxine-induced cardiac hypertrophy ·

H KOBORI

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Table 1 Changes in parameters produced by ANGII and T4 treatment. The data are expressed as mean S.E.M., n=5

Parameters/groups Serum free T3 (ng/l) Heart weight/body weight (mg/g) Systolic blood pressure (mmHg) Heart rate (beats/min) PRA (ìg angiotensin I/h/l) Plasma ANGII (ng/l) Renin/GAPDH mRNA (% ratio) Cardiac renin (ng angiotensin I/h/g) Cardiac ANGII (pg/g)

Control

ANGII

ANGII+T4

2·10·1 2·90·2 1063 32421 5·481·17 373 612 10·00·8 8313

2·30·2 3·50·1* 1766* 34812 0·120·03* 1265* 482* 3·80·4* 1492*

6·60·3† 5·00·5† 1804 44613† 0·150·03 1305 742† 6·50·8† 23130†

*P