Effects of salt loading and various therapies on cardiac hypertrophy and fibrosis in young spontaneously hypertensive rats.

We investigated the effects of salt loading on blood pressure, cardiac hypertrophy and fibrosis as well as on the effectiveness of various antihypertensive therapies in young spontaneously hypertensive rats (SHR). Twenty-five male SHR were salt-stimulated by drinking 1% NaCl from 3 to 6 months of age. Eighteen of them were treated for the last 2 weeks of salt loading with either the angiotensin-converting enzyme inhibitor captopril, the beta-adrenergic receptor blocker propranolol or the calcium-channel antagonist verapamil. Age-matched male Wistar-Kyoto (WKY) rats and SHR drinking only water served as controls. At the age of 6 months, SHR had significantly elevated blood pressure that was unchanged by salt loading. Relative heart weight was increased in SHR without (3.3) and even more so with salt intake (3.6 vs. 2.4 in WKY). Left ventricular (LV) hypertrophy was accompanied by a 17-fold increase in the expression of mRNA for atrial natriuretic factor (ANF) both in untreated and salt-loaded SHR compared to WKY (p<0.001). Collagen I and III mRNA increased 1.7-1.8-fold in SHR without and with additional salt intake (p<0.01). None of the therapies significantly reduced blood pressure or hypertrophy. Although captopril had no antihypertensive effect, it reduced ANF, collagen I and III mRNA in LV to control level. Less pronounced effects were achieved with verapamil. These findings emphasize the cardioprotective role of captopril which may not be fully expressed in the presence of elevated salt intake.

Norepinephrine-induced changes in cardiac transforming growth factor-beta isoform expression pattern of female and male rats.

Transforming growth factor-beta (TGF-beta) is a ubiquitous growth-regulating protein with an essential role in tissue repair and formation of extracellular matrix (ECM). To better understand the role of different isoforms of TGF-beta in the cardiac remodeling process induced by norepinephrine (NE), the expression of TGF-beta1, TGF-beta2, and TGF-beta3 was studied and compared with the expression of collagen. NE (0.1 mg/kg. h) was intravenously infused in female and male Sprague-Dawley rats for several time periods, and freshly obtained ventricular myocardium after 1 day was dissociated into myocyte and nonmyocyte fractions. Prazosin (0.1 mg/kg x h) and metoprolol (1 mg/kg. h) were used to block alpha- and beta-adrenoceptors, respectively. After NE infusion, the three isoforms of TGF-beta were differentially induced as far as the magnitude and the time course is concerned. The increased expression of TGF-beta2 started earlier with a maximum after 12 hours and was more pronounced (10-fold elevation) than that of the other two isoforms, with a clear specificity for the left ventricle in female hearts. This specificity was also seen in male rats with 16-fold elevation of TGF-beta2 after 1 day of NE-stimulation. The increase of TGF-beta2 was significant only in the myocyte fraction obtained from female as well as from male hearts. The expression of the mRNA of all TGF-beta isoforms of collagen type I and type III, and of the matrix metalloproteinase (MMP)-2 and its inhibitor TIMP-2 was reduced predominantly by alpha-adrenoceptor blockade with prazosin. The increase in TGF-beta isoforms correlated with that of the mRNA expression of collagens, MMP-2 and TIMP-2.

Remodelling of the sarcolemma in diabetic rat hearts: the role of membrane fluidity.

The hyperglycaemia and oxidative stress, that occur in diabetes mellitus, cause impairment of membrane functions in cardiomyocytes. Also reduced sensitivity to Ca-overload was reported in diabetic hearts (D). This enhanced calcium resistance is based on remodelling of the sarcolemmal membranes (SL) with down-regulated, but from the point of view of kinetics relatively well preserved Na,K-ATPase and abnormal Mg- and Ca-ATPase (Mg/Ca-ATPase) activities. It was hypothesised that in these changes may also participate the non-enzymatic glycation of proteins (NEG) and the related free radical formation (FRF), that decrease the membrane fluidity (SLMF), which is in reversal relationship to the fluorescence anisotropy (D 0.235 +/- 0.022; controls (C) 0.185 +/- 0.009; p < 0.001). In order to check the true role of SLMF in hearts of the diabetic rats (streptozotocin, single dose, 45 mg/kg i.v.) animals were treated in a special regimen with resorcylidene aminoguanidine (RAG 4 mg/kg i.m.). The treatment with RAG eliminated completely the diabetes-induced decrease in the SLMF (C 0.185 +/- 0.009; D + RAG 0.167 +/- 0.013; p < 0.001) as well as in NEG (fructosamine microg x mg(-1) of protein: C 2.68 +/- 0.14; D 4.48 +/- 0.85; D + RAG 2.57 +/- 0.14; p < 0.001), and FRF in the SL (malondialdehyde: C 5.3 +/- 0.3; D 8.63 +/- 0.2; D + RAG 5.61 +/- 0.53 micromol x g(-1); p < 0.05). Nevertheless, the SL ATPase activity in diabetic animals was not considerably influenced by RAG (increase in D + RAG vs. D 3.3%, p > 0.05). On the other hand, RAG increased considerably the vulnerability of the diabetic heart to overload with external Ca2+ (C 100% of hearts failed, D 83.3%, D + RAG 46.7% of hearts survived). So we may conclude, that: (i) The NEG and FRF caused alterations in SLMF, that accompanied the diabetes-induced remodelling of SL, also seem to participate in the protection of diabetic heart against Ca2+-overload; (ii) Although, the changes in SLMF were shown to influence considerably the ATPase activities in cells of diverse tissues, they seem to be little responsible for changes in ATPases-mediated processes in the SL of chronic diabetic hearts.

The expression of mRNA of cytokines and of extracellular matrix proteins in triiodothyronine-treated rat hearts.

In various models of cardiac hypertrophy, e.g. treatment of rats with norepinephrine infusion or pressure overload, increased expression of cytokines together with increase in extracellular matrix proteins (ECMP) was reported. In this study the effect of triiodothyronine (T3) on the expression of mRNA for cytokines and ECMP was investigated. Female Sprague-Dawley rats were treated daily with T3 in a dose of 0.2 mg x kg(-1) of body weight s.c. Changes in the left (LV) and right (RV) ventricular function were measured 6, 24, 48, 72 h and 7 and 14 days after the first T3-injection using Millar ultraminiature pressure catheter transducers. RNA was isolated from LV and RV tissue, and the expression of cytokines and ECMP was measured using the ribonuclease protection assay. T3-treatment induced a significant increase in LV dP/dtmax and RV dP/dtmax, (p < 0.05) 24 h after the first injection of T3 together with an increase in heart rate (p < 0.01). The RV systolic pressure increased 48 h after the first T3 injection, whereas the LV systolic pressure remained unchanged. After 48 h the heart weight to body weight ratio was increased (p < 0.01). Hypertrophy of the RV was more prominent than that of the LV (155.9 vs. 137.7%). In all groups the expression of mRNA for interleukins (IL) IL-6, IL-1beta, IL-1alpha and tumour necrosis factor (TNF)-alpha in both ventricles did not change (p > 0.05). There was a significant increase in the mRNA for colligin 24 h after the T3 injection in both LV (p < 0.01) and RV (p < 0.05). This was followed by an increase in the mRNA for collagen I and III 72 h after the first T3-dose (p < 0.05 in RV; p < 0.01 in LV). At this point, the mRNA for tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) was increased (p < 0.01) in the LV only. Moreover, after 7 days also the mRNA for matrix metalloproteinase (MMP)-2 increased (p < 0.01) in the LV. Both, TIMP-2 and MMP-2 were increased in the RV only after 14 days (p < 0.05). The gelatinase activity of MMP-2, however, was unchanged in both ventricles. The T3-induced cardiac hypertrophy was not accompanied by fibrosis as measured by the Sirius red staining after 14-days of T3-treatment. The moderate increase in mRNA for ECMP and MMP may be attributed more to the increasing mass of the ventricles with the accompanying remodelling of the ECM than to increased fibrosis.

Regulation of mitochondrial contact sites in neonatal, juvenile and diabetic hearts.

Mitochondrial contact sites (MiCS) are dynamic structures involved in high capacity transport of energy from mitochondria into the cytosole. Previous studies revealed that in normal conditions the actual number of MiCS is in correlation with the energy requirements of the heart, particularly with those for its contractile work. Although the detailed mechanisms of signalling between the processes of energy utilisation and MiCS formation in the heart are not yet elucidated, it is known that intracellular Ca2+ transients are intimately involved in this crosstalk. The present study is devoted to investigation of Ca2+-linked MiCS formation in healthy adult hearts and in hearts with modified Ca2+-handling such as in developing, in juvenile and diabetic myocardium. Experiments were performed on hearts of healthy rats on the 22nd embryonal day, 1st, 4th, 7th and 14th postnatal days as well as on adult hearts. Diabetic hearts were investigated on the 8th day after streptozotocin injection (45 mg x kg(-1) iv.) to adult rats. Intracellular Ca2+ movements were affected by modulation of Ca2+ concentration in perfusion solution (1.6 or 2.2 mmol l(-1) in isolated, Langendorff-perfused hearts, by calcium paradox (CaP) or by replacing of Ca2+ by Cd2+ ions. Elevation of extracellular Ca2+ was reflected by 30.1, 10.4 and 24.1% increase in intracellular free Ca2+ concentration in healthy adult, diabetic and 14-day old hearts respectively. In developing hearts the amount of MiCS was culminating on the 4th postnatal day. In adult hearts, elevated calcium in the perfusion solution, CaP as well as diabetes led to a significant increase in the amounts of MiCS formed (58.1, 77.2 and 86.5% respectively; p < 0.05). Diabetic and 14-day old hearts naturally exhibited amounts of MiCS comparable to those obtained by Ca2+-stimulation of MiCS formation in adult healthy hearts. In contrast to healthy controls, perfusion of diabetic and 14-day old hearts with elevated Ca2+ as well as induction of CaP exerted little influence on MiCS formation (4.4 and 8.2% for elevated Ca2+; 2.9 and 10.7% for CaP; p > 0.05). A replacement of Ca2+ by Cd2+ ions lowered the amount of MiCS in healthy adult and diabetic hearts (61 and 52.2%; p < 0.05). In conclusion, during development, the formation of MiCS may be influenced by both, permanent stimulation by Ca2+-signalling and the availability of mCPK. In healthy adult hearts the amount of MiCS is modulated by intracellular Ca2+ transients in response to changes in extracellular Ca2+ concentration. In diabetic hearts the modulation of MiCS formation is naturally attenuated, apparently as a consequence of persisting alterations in Ca2+-handling.