WKY Hearts Research Articles

Transforming growth factor-beta 1 in heart development

Defined biochemical stimuli regulating neonatal ventricular myocyte (cardiomyocyte) development have not been established. Since cardiomyocytes stop proliferating during the first 3–5 days of age in the rodent, locally generated ‘anti-proliferative’ and/or differentiation signals can be hypothesized. The transforming growth factor-beta (TGF-β) family of peptides are multifunctional regulators of proliferation and differentiation of many different cell types. We have determined in neonatal and maturing rat hearts that TGF- β 1 gene expression occurs in pups of both normotensive (Wistar Kyoto, WKY) and hypertrophy-prone rats (spontaneously hypertensive, SHR). TGF- β 1 transcript levels were readily apparent in total ventricular RNA from SHR pups within 1 day of age and elevated in 3–7 day old WKY and SHR hearts when cardiomyocyte proliferation indices are diminished. TGF- β 1 transcript levels remain at a ‘relatively’ high level throughout maturation and into adulthood in both strains. Further, TGF- β 1 transcripts were localized to cardiomyocytes of neonatal rat ventricular tissue sections by in situ hybridization. Immunoreactive TGF-β was co-localized to the intracellular compartment of neonatal cardiomyocytes at the light and electron microscopic level. In vitro analysis using primary cultures of fetal and neonatal cardiomyocytes indicated that TGF-βs inhibit mitogen stimulated DNA synthesis and thymidine incorporation. From these data, we propose that locally generated TGF-βs may act as autocrine and/or paracrine regulators of cardiomyocyte proliferation and differentiation as intrinsic components of a multifaceted biochemical regulatory process governing heart development.

Heterogeneous distribution of fatty acid-binding protein in the hearts of Wistar Kyoto and spontaneously hypertensive rats

In the present study we investigated the concentrations of cardiac cytoplasmic fatty acid-binding protein (H-FABP c) in various regions of the left and right ventricles of both Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). To this end, the ventricles of six WKY and six SHR hearts were cut in three slices, which were further dissected in one right ventricular piece and ten left ventricular pieces (five inner layer and five outer layer pieces). After homogenisation, H-FABP c was assessed using an Enzyme Linked Immuno Sorbent Assay (ELISA) of the sandwich type. It was found that, when expressed per gram wet tissue, the overall concentration of H-FABP c tended to be lower in SHR than in WKY hearts (874 ± 53 μg/g and 955 ± 51 μg/g, respectively; 0.1< P<0.2, means ± s.e.m. for n = 6 animals in each group). However, due to a 30–35% higher ventricular heart mass in SHR than in WKY, the total H-FABP c content per heart turned out to be about 20% higher in SHR than in WKY rats. No concentration differences could be detected between right and left ventricles in WKY and SHR but a marked difference between the outer layer and the inner layer of the left ventricular wall was monitored in both groups. In general, the concentration in the outer layer was 5–15% higher than in the corresponding inner layer. These differences reached the level of significance ( P<0.05) in regions close to the basis of the heart.

Intracellular Free Magnesium and High Energy Phosphates in the Perfused Normotensive and Spontaneously Hypertensive Rat Heart A 31P NMR Study

We have employed 31P nuclear magnetic resonance (NMR) spectroscopy to examine the relationship between cytosolic free Mg2+ ([Mg2+]in), intracellular pH, high energy phosphates, and genetic hypertension using the Wistar-Kyoto rat (WKY) as a control and the spontaneously hypertensive rat (SHR) as a model for essential hypertension. The mean systolic blood pressures (measured using the tail cuff method) of control and hypertensive rats (aged 7 to 12 weeks) were 113 +/- 4 mm Hg (mean +/- 2 SE, n = 14) and 162 +/- 5 mm Hg (mean +/- 2 SE, n = 17), respectively. Intracellular free Mg2+ levels were significantly depleted in the isolated Langendorff perfused hypertensive rat hearts (452 +/- 39 mumol/L, mean +/- 2 SE, n = 17) compared to control hearts (756 +/- 52 mumol/L, n = 14); however, intracellular pH did not differ in the SHR hearts (7.02 +/- 0.03, mean +/- 2 SE, n = 7) compared with controls (7.03 +/- 0.03, n = 7). Although we could not demonstrate a statistically significant difference in the levels of P-creatine or ATP, intracellular Pi was two-fold higher (5.71 +/- 2.28 mmol/L v 2.92 +/- 0.66 mmol/L, n = 4) and the phosphorylation potential, [MgATP]/[MgADP][Pi], was correspondingly lower (3.0 X 10(4) +/- 1.6 x 10(4) v 8.3 X 10(4) +/- 1.4 X 10(4) (mol/L)-1, n = 4) in SHR compared to WKY hearts. These data demonstrate free magnesium depletion in heart muscle and indicate an alteration in cardiac bioenergetics in essential hypertension.

Augmented Na, K-ATPase Gene Expression in Spontaneously Hypertensive Rat Hearts

Abnormalities in cardiovascular Na,K-ATPase ion-transport function and regulation may play an important role in the pathogenesis of hypertension. However, it is not known whether these abnormalities are secondary to the effects of hypertension, such as increased pressure, or reflect an intrinsic abnormality in Na,K-ATPase gene expression and regulation. A genetic model of hypertension was used to address this issue. Na,K-ATPase alpha subunit gene expression in hearts was compared between spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Pre-hypertensive, 4-week old SHR hearts exhibited an approximately 4 fold elevation in alpha 1 and 8 fold elevation in alpha 2 mRNA levels compared with age-matched WKY hearts. These SHR mRNA levels remained almost equivalent throughout the development of hypertension at 8 and 16 weeks of age. WKY alpha 1 and alpha 2 mRNA levels exhibited a progressive increase during the same time period. The neonatal alpha 3 mRNA isoform was detected only in pre-hypertensive (4-week) SHR hearts. We conclude that cardiac Na,K-ATPase alpha subunit gene expression is significantly altered in SHR even before the onset of hypertension. These findings suggest that an abnormality in cardiac Na,K-ATPase gene expression constitutes an early, if not primary, event in spontaneous hypertension.

Inositol trisphosphate kinase activity in hypertrophied rat heart

In the present experiment, we demonstrated that IP3 kinase activity was increased in SHRSP heart compared to WKY heart. IP3 kinase activity in the heart was highest in the cytosolic fraction in both SHRSP and WKY. Its activity progressively increased with age in 5- to 20-week SHRSP. The activity reached about three times the level of 5-week-old SHRSP in 40-week-old SHRSP. On the other hand, in WKY it was 1.3-fold at 40 weeks compared with that at 5 weeks. We determined the effect of divalent cations on IP3 kinase activity. Ca2+ stimulated its activity in a dose-dependent manner at 10(-9) to 10(-6) M. In SHRSP it was enhanced about 2.1-fold at 10(-6) M of Ca2+, but in WKY it was 1.5-fold at 1.0(-6) M of Ca2+. Mn2+ also stimulated IP3 kinase activity in both groups of animals, while, Fe2+, Zn2+, and Cu2+ inhibited IP3 kinase activity. In our experiment IP3 kinase activity was increased in SHRSP and its activity was markedly affected by divalent cations. These data suggest that the accumulations of IP3 and IP4 after hormonal stimulation play a physiologic role, possibly by alteration of Ca2+ levels in cardiac tissue.

Response of isolated perfused heart to ischemia after long-term treatment of spontaneously hypertensive rats with diltiazem.

The effects of long-term treatment with diltiazem on the heart in normotensive (WKY) and spontaneously hypertensive rats (SHR) were studied. Diltiazem was added to the drinking fluid (900 mg/liter) and given ad libitum from 19 to 26 weeks of age, whereas tap water was given to the control animals. Although diltiazem did not decrease blood pressure in SHR, it decelerated the increase in their left ventricular weight (p less than 0.01). Hearts were removed and perfused by the working heart technique for 15 min, and then global ischemia was induced for either 10 or 30 min. The ischemic heart was reperfused for 30 min. The extent of recovery of coronary flow after reperfusion, following 30 min of ischemia in the diltiazem-treated SHR, was higher than that in the control SHR (p less than 0.01). The levels of adenosine triphosphate (ATP), creatine phosphate (CrP), and energy charge potential in the SHR heart reperfused after 30 min of ischemia were lower than those in the reperfused WKY heart (p less than 0.01, respectively). Diltiazem improved the restoration of ATP and CrP and prevented the decrease in energy charge potential in SHR after reperfusion following 30 min of ischemia (p less than 0.01, respectively). In conclusion, long-term treatment of SHR with diltiazem may protect the myocardium when myocardial ischemia occurs.

Some enzyme characteristics of spontaneously hypertensive rats myocardium.

In order to ascertain the pathogenesis of myocardial cell vulnerability in spontaneously hypertensive rats (SHR), several enzyme activities were examined by using subcellular fractions of myocardium and compared to those in Wistar-Kyoto rats (WKY). In the normotensive WKY heart, both 5'-nucleotidase and Na+/K(+)-ATPase, which are plasma membrane associated enzymes, increased with age. But in the SHR heart, both enzymes were lower at 16 weeks than they were at 10 weeks of age. Moreover, at 16 weeks of age they were lower in SHR than in WKY. On the other hand, NADP(+)-isocitrate dehydrogenase activity, a mitochondria associated enzyme, was higher in SHR than in WKY at 6 weeks, but lower at 10 and again at 16 weeks of age. The activities of both acid phosphatase and N-acetyl-beta-glucosaminidase, which are lysosomal enzymes, decreased with age in SHR but not in WKY. These results suggest that an enzymatic alteration in the plasma membrane and mitochondria may be one of important factors behind myocardial vulnerability in the SHR heart.

Study of the pathogenesis of cardiac hypertrophy. Biochemical differences of cultured heart cells from normotensive and spontaneously hypertensive rats.

To elucidate the metabolic properties of the heart of spontaneously hypertensive rats (SHR), we examined the uptakes of 3H-uridine (3H) and 14C-leucine (14C) into single cultured heart cells, as well as the total protein content and cell size of heart cells in the presence and absence of stimulation, and compared the findings with those obtained with Wistar Kyoto rat (WKY) cells. As a result, (1) Spontaneous uptakes of 3H and 14C into WKY heart cells increased rapidly from day 2 to days 5-6, and then decreased gradually. In contrast, in SHR heart cells a constant increase in the uptakes of 3H and 14C was observed until the 10th day. The uptakes of both 3H and 14C into 10-day-old SHR heart cells were significantly higher than those into WKY heart cells (p less than 0.05). (2) The total protein content of SHR heart cells cultured for 4 or 5 days was significantly less than that of WKY heart cells, but there was no significant difference between cells cultured for more than 6 days. As for cell size, no significant difference was observed regardless of how long the cells were cultured. However, the increment in SHR heart cell total protein and cell size during cultivation appeared to be higher than they were in WKY heart cells. (3) Stimulation of heart cells with 10(-8) - 10(-5) M L(-)-isoproterenol (ISO) for 1 hour increased the uptake of 3H into heart cells from both kinds of rats, with no significant difference between the two.(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneously occurring hypertrophic cardiomyopathy in the rat. II. Distribution of, and correlations between, various cardiac abnormalities in the WKY/NCrj and its related strains.

We performed gross-anatomic and histologic examinations quantitatively on 18 male and 30 female WKY/NCrj rats, and their 299 first generation off spring. The results were analyzed in comparison with those obtained from 3 other strains of rats. The offspring of the WKY rats showed various cardiac abnormalities quite similar to those in their parents. The cardiac abnormalities in the offspring of the WKY rats were: increased heart weight (in about 10%), left or right atrial hypertrophy (50%), disproportionate septal thickening (50%), small left ventricular cavity (40%), right ventricular hypertrophy (40%), abnormal fiber arrangement greater than 5% in either of the 3 ventricular walls (90%), myocardial fibrosis (70%), and abnormal location of arteries in the ventricular septum (30%). ECG findings of the WHY-F rats such as high QRS complexes and high T waves and axis deviation showed a high sensitivity and specificity in the diagnosis of marked cardiac hypertrophy. Another conspicuous histologic finding observed in the WKY hearts was that the continuity of the latitudinal fiber bundle of the ventricular septum with that of the left ventricular free wall, an important functioning unit for pressure generation in the left ventricle, was markedly disturbed in the area of junction between the 2 walls; the smaller the continuity, the greater the cardiac hypertrophy; the disadvantage of the discontinuity for the pressure generation may be related to the development of cardiac hypertrophy. Also examined were two strains of Wistar rats with the same origin as the WKY/NCrj. These rats also had gross-anatomic and histologic abnormalities of the heart quite similar to those in the 2 generations of WKY/NCrj rats, suggesting that a common factor related to the development of the abnormalities, probably a genetic predisposition, has been transmitted in these 3 strains.

Spontaneously occurring hypertrophic cardiomyopathy in the rat. I. Pathologic features.

The gross anatomic and microscopic appearance of the hearts of young and adult WKY/NCrj rats was examined in comparison with that of normotensive Wistar and SHR/NCrj rats. In a substantial number of the WKY rats, the heart weight and thickness of ventricular septum were much greater than those of the Wistar and SHR rats. The ventricular septum to left ventricular free wall thickness ratio was greater than 1.3 in about one sixth of the WKY rats. In most of the hypertrophied WKY hearts, the transverse area of the left ventricular cavity was smaller in relation to the wall area than in the Wistar and SHR rat hearts, although in a few it was greater. Abnormal fiber arrangement, myocyte hypertrophy, and myocardial fibrosis were far more prominent in the hypertrophied myocardium of the WKY rats compared with the Wistar or SHR rats. Intramural arteries with marked wall thickening existed frequently in the hypertrophied and dilated hearts. Electron microscopic examination revealed marked disarrangement of bundles of myofilaments and widened Z-bands in the hypertrophied myocardium. Blood pressure was not elevated in the rats with cardiac hypertrophy. These findings show that a disease of the myocardium with the pathologic features similar to those of hypertrophic cardiomyopathy in man occurs spontaneously in rats.

Comparative Study of the Left Ventricular Transmembrane Action Potential of Prenatal WKY and SHR Heart

Comparative Study of the Left Ventricular Transmembrane Action Potential of Prenatal WKY and SHR Heart

Decrease in Na+,K+-ATPase activity and [3H]ouabain binding sites in sarcolemma prepared from hearts of spontaneously hypertensive rats.

Na+,K+-ATPase activity, phosphorylation, and [3H]ouabain binding in sarcolemma isolated from spontaneously hypertensive rat (SHR) hearts were compared to the same parameters in sarcolemma from normotensive rat (WKY) hearts. Sarcolemma prepared from SHR heart contained significantly less ouabain-inhibitable ATPase activity than sarcolemma from WKY heart. No significant differences in sarcolemmal protein content or recovery were noted between the two groups. The numbers of phosphorylation sites and ouabain binding sites were lower for SHR hearts than for WKY hearts. The KD values for ouabain binding were the same (0.30 muM) in cardiac sarcolemma of SHR and WKY. The I50 values for inhibition by ouabain of Na+,K+-ATPase were also the same for both groups (SHR = 49 microM; WKY = 44 microM). These data suggest that the decrease of cardiac sarcolemmal Na+,K+-ATPase activity in SHR hearts is due to a decrease in the number of active sites.

Myocardium in hypertrophy: Oxygen consumption by isolated cardiac myocytes and working hearts from spontaneously hypertensive rats

Oxygen consumption was measured on suspensions of calcium tolerant myocytes obtained from hearts of Spontaneously Hypertensive Rats (SHR) and normotensive Wistar Kyoto Rats (WKY). Oxygen consumptions of the isolated cells were not significantly different from each other either in the presence or absence of added calcium (1.5 mM). Additionally, there was excellent agreement between the oxygen consumption of the isolated cells and estimates of basal oxygen consumption obtained from linear regression analysis of the relationship between work and myocardial oxygen utilization in isolated perfused working hearts. At any given workload there was no significant difference in oxygen consumption between SHR hearts and WKY hearts. The mechanical performance of the SHR hearts was lower compared to that of the WKY hearts at low preloads. At high preloads and high afterloads the SHR hearts developed higher pressures than did hearts obtained from WKY rats. The data suggest that: (a) basal oxygen consumption of the two hearts are similar and (b) the contractile defects in the SHR heart are not the result of hypoxia.

Hydralazine: effect on contraction mechanics of WKY and SHR rat heart muscle.

Chronic hydralazine treatment (2 weeks) in 22-week-old normotensive (WKY) and spontaneously hypertensive rats (SHR) significantly lowered the systolic blood pressure in both groups. Left ventricular papillary muscles from nontreated and treated WKY and SHR were placed in an isometric myography, and contractile indices monitored. Nontreated WKY and SHR were not statistically different comparing: tension, maximum contraction and relaxation rates, time to maximum tension, total contraction time, or passive and active length-tension curves. Hydralazine-treated WKY and SHR had significantly reduced tension and maximum rates of tension development and relaxation; passive length-tension characteristics were not altered. Stressing the papillary muscles with increased frequency of electrical stimulation (0.1-2 Hz) did not differentiate the various groups. Significant (p less than 0.05) alteration with isoproterenol (10(-9)-10(-5) M) occurred with the hydralazine-treated WKY, which responded with a greater increase in relaxation rate than the hydralazine-treated SHR. It is suggested that the clinically very useful drug, hydralazine, causes a distinct contractile state alteration in rat myocardium after treatment sufficient to lower SHR blood pressure to a normal range.