Signal transduction of mechanical stress in myocytes and fibroblasts derived from neonatal rat ventricles.

BACKGROUND: In cardiomyocytes and cardiac fibroblasts, stretch induces a growth response. METHODS: To investigate which signal transduction pathways are involved in the stretch-induced growth response of cardiomyocytes and cardiac fibroblasts, we used a model of mechanical stress in which cells are submitted to biaxial cyclic stretch. RESULTS: In stretched cardiomyocytes major bands of tyrosine-phosphorylated (P-Tyr) proteins of 58, 49, and 27 kDa were detected and minor bands of 65 and 40 kDa. Furthermore, major bands of serine/threonine phosphorylated (P-Ser/Thr) proteins of 46, 42, and 21 kDa were detected. Phosphorylation of the 40 kDa P-Tyr protein increased significantly upon stretch. In cardiac fibroblasts major bands of P-Tyr proteins of 63, 53, and 23 kDa were detected and minor bands of 72 and 39 kDa. In addition, major bands of P-Ser/Thr proteins of 51, 47, and 23 kDa were detected and minor bands of 54 and 33 kDa. Phosphorylation of the 54 and 33 kDa P-Ser/Thr proteins increased significantly upon stretch. Phosphorylated JNK 1 and JNK 2 activities were not detected in fibroblasts. In cardiomyocytes levels of phosphorylated JNK 1 and 2 were very low, but tend to increase upon stretch. Phosphorylated p38 MAPK could not be identified in both cell types. The intensity of phosphorylation of paxillin increased upon stretch in both cell types, although the increases were only significantly different in stretched fibroblasts. Finally, stretch increased PLC activity in cardiomyocytes as well as in fibroblasts. CONCLUSION: Our findings are in favour of mechanotransduction of the stretch signal via integrins and focal adhesion components such as FAK, Src kinase, PLC and paxillin. The activation of the last two focal adhesion components by stretch of cardiomyocytes and fibroblasts is demonstrated in this article.

Cyclic stretch induces the release of growth promoting factors from cultured neonatal cardiomyocytes and cardiac fibroblasts.

Growth factors and hormones may play an autocrine/paracrine role in mechanical stress-induced cardiac hypertrophy. Using an in vitro model of mechanical stress, i.e. stretch of cardiomyocytes and cardiac fibroblasts, we tested the involvement of growth factors and hormones in this process. We found that conditioned medium (CM) derived from 4 h cyclicly (1 Hz) stretched cardiomyocytes increased the rate of protein synthesis in static cardiomyocytes by 8 +/- 3%. Moreover, CM derived from 2 h stretched fibroblasts increased the rate of protein synthesis in static fibroblasts as well as in static cardiomyocytes by 8 +/- 2 and 6 +/- 2%, respectively. Analysis of CM using size-exclusion HPLC showed that cardiomyocytes and fibroblasts released at least three factors with MW < or = 10 kD, their quantities being time-dependently increased by stretch. Subsequent analyses using immunoassays revealed that cardiomyocytes released atrial natriuretic peptide (ANP) and transforming growth factor-beta1 (TGFbeta1) being increased by 45 +/- 17 and 21 +/- 4% upon 4 h of stretch, respectively. Fibroblasts released TGFbeta1 and very low quantity of endothelin-1 (ET-1). The release of TGFbeta1 was significantly increased by 18 +/- 4% after 24 h of stretch in fibroblasts. Both cell types released no detectable amount of angiotensin II (Ang II). In conclusion, upon cyclic stretch cardiomyocytes and fibroblasts secrete growth factors and hormones which induce growth responses in cardiomyocytes and fibroblasts in an autocrine/paracrine way. TGFbeta secreted by cardiomyocytes and fibroblasts, and ANP secreted by cardiomyocytes are likely candidates. We found no evidence for the involvement of Ang II and ET-1 in autocrine/paracrine mechanisms between cardiac cell types.

Risk of overestimation of free malondialdehyde in perfused rat hearts due to homogenization artifacts.

OBJECTIVES: The aims of the present study were to determine (1) whether free malondialdehyde (MDA) was artifactually formed during homogenization of myocardial tissue and (2) whether free MDA was increased in reperfused rat hearts. METHODS: Groups of isolated buffer-perfused rat hearts were subjected to control perfusion, or 20 min of ischemia, or 20 min of ischemia followed by 5 or 30 min of reperfusion. The hearts were subsequently assayed for free MDA by ion-pairing high-performance liquid chromatography following homogenization in the absence or presence of the antioxidant butylated hydroxytoluene (0.01%). RESULTS: Tissue homogenates prepared in the absence of butylated hydroxytoluene contained significantly higher (P < 0.001) free MDA levels than tissue homogenates from the same hearts prepared in the presence of butylated hydroxytoluene. Free MDA levels of tissue homogenates prepared in the presence of butylated hydroxytoluene were below the detection limit (20 pmol/mg protein) in 27 of 30 tissue homogenates, irrespective of the perfusion protocol. Control experiments showed that the presence of butylated hydroxytoluene did not interfere with the detection of free MDA. CONCLUSIONS: These results indicate that free MDA was formed artifactually during tissue homogenization in the absence of butylated hydroxytoluene. Furthermore, free MDA could not be detected in perfused rat hearts after control perfusion, or 20 min of ischemia, or 20 min of ischemia followed by 5 or 30 min of reperfusion.

Postischemic injury in isolated rat hearts is not aggravated by prior depletion of myocardial glutathione.

The aim of this study was to test the hypothesis that a decreased myocardial concentration of reduced glutathione (GSH) during ischemia renders the myocardium more susceptible to injury by reactive oxygen species generated during early reperfusion. To this end, rats were pretreated with L-buthionine-S,R-sulfoximine (2 mmol/kg), which depleted myocardial GSH by 55%. Isolated buffer-perfused hearts were subjected to 30 min of either hypothermic or normothermic no-flow ischemia followed by reperfusion. Prior depletion of myocardial GSH did not lead to oxidative stress during reperfusion, as myocardial concentration of glutathione disulfide (GSSG) was not increased after 5 and 30 min of reperfusion. In addition, prior depletion of GSH did not exacerbate myocardial enzyme release, nor did it impair the recoveries of tissue ATP, coronary flow rate and left ventricular developed pressure during reperfusion after either hypothermic or normothermic ischemia. Even administration of the prooxidant cumene hydroperoxide (20 microM) to postischemic GSH-depleted hearts during the first 10 min of reperfusion did not aggravate postischemic injury, although this prooxidant load induced oxidative stress, as indicated by an increased myocardial concentration of GSSG. These results do not support the hypothesis that a reduced myocardial concentration of GSH during ischemia increases the susceptibility to injury mediated by reactive oxygen species generated during reperfusion. Apparently, myocardial tissue possesses a large excess of GSH compared to the quantity of reactive oxygen species generated upon reperfusion.

Glutathione disulfide as an index of oxidative stress during postischemic reperfusion in isolated rat hearts.

The objectives of this study were to determine 1) whether reactive oxygen species generated upon postischemic reperfusion lead to oxidative stress in rat hearts, and 2) whether an exogenous prooxidant present in the early phase of reperfusion causes additional injury. Isolated buffer-perfused rat hearts were subjected to 30 min of hypothermic no-flow ischemia followed by 30 min of reperfusion. Increased myocardial content of glutathione disulfide (GSSG) and increased active transport of GSSG were used as indices of oxidative stress. To impose a prooxidant load, cumene hydroperoxide (20 microM) was administered during the first 10 min of reperfusion to a separate group of postischemic hearts. Reperfusion after 30 min of hypothermic ischemia resulted in a recovery of myocardial ATP from 28% at end-ischemia to 50-60%, a release of 5% of total myocardial LDH, and an almost complete recovery of both coronary flow rate and left ventricular developed pressure. After 5 and 30 min of reperfusion, neither myocardial content of GSSG nor active transport of GSSG were increased. These indices were increased, however, if cumene hydroperoxide was administered during early reperfusion. After stopping the administration of cumene hydroperoxide, myocardial GSSG content returned to control values and GSH content increased, indicating an unimpaired glutathione reductase reaction. Despite the induction of oxidative stress, reperfusion with cumene hydroperoxide did not cause additional metabolic, structural, or functional injury when compared to reperfusion without cumene hydroperoxide. We conclude that reactive oxygen species generated upon postischemic reperfusion did not lead to oxidative stress in isolated rat hearts. Moreover, even a superimposed prooxidant load during early reperfusion did not cause additional injury.

Comparison of myocardial changes between pressure induced hypertrophy and normal growth in the rat heart.

To evaluate differences in tissue composition between hearts with pressure overload hypertrophy and normal hearts of comparable weight, 30 rat hearts with aortic constriction of 4, 10 and 30 days, and nine hearts of sham operated controls were studied. Surgery was performed at age 70 days. Morphometric analysis of myocardial tissue sections revealed (1) myocyte hypertrophy in left ventricular myocardium of hypertrophic hearts was proportional to heart weight, and in normal growth myocyte volume increased in proportion to heart weight; (2) myocyte number in left ventricular myocardium was identical in hypertrophic and normal hearts; (3) non-muscle cell proliferation was proportional to heart weight identically in hypertrophic and normal hearts; (4) volume fractions of myocytes were significantly lower in hypertrophic hearts [0.76(SD 0.05)] than in normal hearts [0.82(0.04)]; (5) volume fractions of all nuclei, myocyte nuclei and non-myocyte nuclei were similar in hypertrophic and normal hearts; (6) measured ventricular DNA content increased with heart weight identically in hypertrophic and normal hearts, and equalled DNA content calculated using the data on tissue composition. Neither right ventricular weight nor right ventricular DNA content were affected by the presence of left ventricular hypertrophy. We conclude that left ventricular hypertrophy due to aortic constriction in the rat resulted in changes of myocardial tissue composition similar to the changes associated with normal growth. Tissue composition of hypertrophic rat hearts corresponds strikingly to that of normal rat hearts with comparable heart weight, although myocardial changes in hypertrophy develop considerably faster than in normal growth.

Myocardial (iso)enzyme activities, DNA concentration and nuclear polyploidy in hearts of patients operated upon for congenital heart disease, and in normal and hypertrophic adult human hearts at autopsy.

To investigate biochemical characteristics of hypertrophic myocardium of young and adult humans, we analysed myocardial biopsies obtained from 28 mainly young patients undergoing cardiac surgery for congenital heart disease and 41 autopsied hearts from 18 adult normal and 23 hypertrophic human subjects. Myocardial activities of the enzymes creatine kinase and lactate dehydrogenase were independent of age during childhood, but decreased significantly with hypertrophy at adult age. Myocyte nuclei showed increased polyploidization during childhood which was progressive with age, and in the adult stage polyploidization was correlated with heart weight. Nevertheless myocardial DNA concentration fell under both conditions, which is to be ascribed to the 'diluting' effect of myocyte hypertrophy. Before an age of 8 years DNA concentration in the child heart material studied has reached the value found in adult nonhypertrophic hearts, although at that time polyploidization of myocyte nuclei in child hearts was only half the value found in adult non-hypertrophic hearts. Biochemical measurement of DNA concentration in peroperatively taken myocardial biopsies may contribute to the in vivo diagnosis of ventricular hypertrophy in quantitative terms, in combination with radiology, echocardiography and histology.

Assessment of hypertrophy in myocardial biopsies taken during correction of congenital heart disease.

Myocardial biopsies were obtained from 27 patients undergoing corrective cardiac surgery for congenital heart disease. Normal hearts of 18 autopsied patients were used as reference. The biopsy material was assessed for desoxyribonucleic acid (DNA) concentration and ploidy profile of cell nuclei in order to quantitate myocardial hypertrophy at the time of operation. DNA-concentration decreased significantly with age (r = -0.76; p less than 0.001). Ploidy profile of myocardial nuclei correlated with age: the relative number of diploid nuclei decreased (r = -0.67; p less than 0.001), the relative numbers of tetraploid and octoploid nuclei increased with age (r = 0.58; p less than 0.01 and r = 0.77; p less than 0.001 respectively). At 8 years of age the patients with congenital heart disease reached myocardial DNA-concentrations comparable with those in normal adult hearts. At higher age the patients with congenital heart disease exceeded normal adult values for myocardial DNA-concentration. These findings are interpreted to represent rapid development of hypertrophy with an early onset, reaching at 8 years of age values observed in normal adult hearts. Quantitation of myocardial hypertrophy by DNA-concentration and ploidy profile of nuclei may offer a means to explain some of the factors of influence on the outcome of corrective cardiac surgery for congenital heart disease in relation to its timing. Our data stress the need for preventing irreversible myocardial damage by timely (surgical) therapy.