Dlx5 is a positive regulator of chondrocyte differentiation during endochondral ossification

The process of endochondral ossification in which the bones of the limb are formed after generation of cartilage models is dependent on a precisely regulated program of chondrocyte maturation. Here, we show that the homeobox-containing gene Dlx5 is expressed at the onset of chondrocyte maturation during the conversion of immature proliferating chondrocytes into postmitotic hypertrophying chondrocytes, a critical step in the maturation process. Moreover, retroviral misexpression of Dlx5 during differentiation of the skeletal elements of the chick limb in vivo results in the formation of severely shortened skeletal elements that contain excessive numbers of hypertrophying chondrocytes which extend into ectopic regions, including sites normally occupied by immature chondrocytes. The expansion in the extent of hypertrophic maturation detectable histologically is accompanied by expanded and upregulated domains of expression of molecular markers of chondrocyte maturation, particularly type X collagen and osteopontin, and by expansion of mineralized cartilage matrix, which is characteristic of terminal hypertrophic differentiation. Furthermore, Dlx5 misexpression markedly reduces chondrocyte proliferation concomitant with promoting hypertrophic maturation. Taken together, these results indicate that Dlx5 is a positive regulator of chondrocyte maturation and suggest that it regulates the process at least in part by promoting conversion of immature proliferating chondrocytes into hypertrophying chondrocytes. Retroviral misexpression of Dlx5 also enhances formation of periosteal bone, which is derived from the Dlx5-expressing perichondrium that surrounds the diaphyses of the cartilage models. This suggests that Dlx5 may be involved in regulating osteoblast differentiation, as well as chondrocyte maturation, during endochondral ossification.     

A novel cell-based system for evaluating skeletal muscle cell hypertrophy-inducing agents

Skeletal muscle is a tissue that adapts to increased use by increasing contractile protein gene expression and ultimately skeletal muscle mass (hypertrophy). To identify hypertrophy-inducing agents that may be potentially useful in the treatment of age-related muscle loss (sarcopenia) and to better understand hypertrophy signal transduction pathways, we have created a skeletal muscle cell-based hypertrophy-responsive system. This system was created by permanently modifying the relatively undifferentiated C2C12 cell line so that it contains the beta-myosin heavy chain (beta-MHC) gene promoter and enhancer regions fused to a luciferase reporter gene. This cell line responds, by increasing luciferase expression, to a variety of skeletal muscle hypertrophy-inducing agents, including insulin, insulin-like growth factor I, testosterone, and the beta-adrenergic receptor agonist isoproterenol, in both the undifferentiated and differentiated states. This cell-based system should be useful for identifying novel hypertrophy-inducing agents as well as understanding hypertrophy signal transduction.     

Increased insulin-like growth factor-I gene expression precedes left ventricular cardiomyocyte hypertrophy in a rapidly-hypertrophying rat model system

Chronic pressure overload leads to an increase in the size, i.e. hypertrophy, of cardiomyocytes in the heart. However, the molecular mechanisms underlying this hypertrophy are not understood. Insulin-like growth factor-I (IGF-I) synthesized locally in the heart is known to be associated with the hypertrophic process. So far, however, cardiac IGF-I gene expression in the widely used rat model system has only been shown to be increased when the hypertrophy induced by pressure-overload was already established. Therefore, the question of whether IGF-I serves as an initiating or early-enhancing factor for the cardiac hypertrophy remains unanswered. Here, cardiac hypertension and hypertrophy were rapidly induced in the rat by complete constriction of the abdominal aorta between the origins of the renal arteries. Carotid arterial systolic blood pressure remained unchanged in sham rats but increased rapidly in the pressure-overloaded constricted rats with a sustained hypertension established by 3 days. Hypertrophy of left ventricular (LV) cardiomyocytes in constricted rats also occurred by 3 days. However, this hypertrophy was preceded by increases in LV IGF-I mRNA and protein which occurred within 1 day. These results support the hypothesis that cardiac-synthesized IGF-I is an initiating or early-enhancing factor for hypertrophy of LV cardiomyocytes.     

Compensated hypertrophy of cardiac ventricles in aged transgenic FVB/N mice overexpressing calsequestrin

Cardiac-specific overexpression of murine cardiac calsequestrin results in depressed contractile parameters and hypertrophy in transgenic mice. To determine the long-term consequences of calsequestrin overexpression, the cardiac phenotype of young (2–3-months old) and aged (17 months old) transgenic FVB/N mice was characterized. Ventricular/body weight ratios, which were increased in young transgenics compared with wild-types, were unaltered with age. Left atria of aged transgenics exhibited enlargement and mineralization, but their ventricles did not display fibrosis, mineralization and other injuries. Although echocardiography suggested a time-dependent change in ventricular geometry and loading conditions in vivo, as well as an age-dependent reduction of left ventricular fractional shortening in transgenic mice, Langendorff-perfused hearts of young and aged transgenics indicated that there were no age-related reductions of contractile parameters (±dP/dt). Furthermore, neither genotype nor age altered lung/body weight ratios. Thus, our findings suggest that left ventricular performance in calsequestrin overexpressing mice becomes apparently depressed with age, but this depression is not associated with progressive reduction of left ventricular contractility and heart failure.     

Insulin improves postischemic recovery of function through PI3K in isolated working rat heart

Insulin improves contractile function after ischemia, but does not increase glucose uptake in the isolated working rat heart. We tested the hypothesis that the positive inotropic effect of insulin is independent of the signaling pathway responsible for insulin-stimulated glucose uptake. We inhibited this pathway at the level of phosphatidyl inositol 3-kinase (PI3K) with wortmannin. Hearts were perfused for 70 min at physiological workload with Krebs-Henseleit buffer containing [2-³H] glucose (5 mM, 0.05 Ci/ml) and oleate (0.4 mM, 1% BSA) in the presence (WM, n = 5) or absence (control, n = 7) of wortmannin (WM, 3 mol/L). After 20 min, hearts were subjected to 15 min of total global ischemia followed by 35 min of reperfusion. Insulin (1 mU/ml) was added at the beginning of reperfusion (WM + insulin n = 8, insulin n = 8). Cardiac power before ischemia was 8.1 ± 0.7 mW. Recovery of contractile function after ischemia was significantly increased in the presence of insulin (73.5 ± 8.9% vs. 38.5 ± 6.7%, p < 0.01). The addition of wortmannin completely abolished the effect of insulin on recovery (32.6 ± 6.4%). Glucose uptake was 1.84 ± 0.32 mol/min/g dry before ischemia and was slightly elevated during reperfusion (2.68 ± 0.35 mol/min/g dry, n.s.). Insulin did not affect postischemic glucose uptake. In the presence of wortmannin, glucose uptake was lowest during reperfusion (n.s.). The results suggest that PI3K is involved in the insulin-induced improvement in postischemic recovery of contractile function. This effect of insulin is independent of its effect on glucose uptake.     

Protein kinase C isoform-selective signals that lead to cardiac hypertrophy and the progression of heart failure

Protein kinase C isoforms comprise a family of structurally related serine/threonine kinases that are activated by second messenger molecules formed via receptor-dependent activation of phospholipase C. Cardiomyocytes co-express multiple protein kinase C isoforms which play key roles in a spectrum of adaptive and maladaptive cardiac responses. This chapter focuses on the structural features, modes of activation, and distinct cellular actions of individual PKC isoforms in the heart. Particular emphasis is placed on progress that comes from studies in molecular models of PKC isoform overexpression or gene deletion in mice. Recent studies that distinguish the functional properties of novel PKC isoforms (PKC and PKC) from each other, and from the actions of the conventional PKC isoforms, and suggest that these proteins may play a particularly significant role in pathways leading to cardiac growth and/or cardioprotection also are considered.     

The effect of early treatment by cerivastatin on the serum level of C-reactive protein,interleukin-6, and interleukin-8 in patients with unstable angina and non-Q-wave myocadial infarction

The aim of our study was to evaluate whether a single dose of cerivastatin at the time of admission of patients with unstable angina pectoris (UAP) or non-Q-wave myocardial infarction (NQMI) can influence the serum level of C-reactive protein (CRP), interleukin-6 (IL-6) and interleukin-8 (IL-8) 24 h later. Forty-four patients with rest chest pain and subendocardial ischemia on ECG were randomized to receive cerivastatin 0.3 mg at the time of admission (group C+) to standard therapy or to remain just on standard therapy (group C–). Blood samples for determination of troponin I (TI), CRP, IL-6 and IL-8 were collected at admission (entry level) and 24 h later (final level). Patients with non-physiological baseline levels of TI, as well as patients with progression to Q wave MI were excluded. All baseline, clinical and demographic data and final values of TI were comparable in the two groups. In patients treated with cerivastatin (group C+, n = 13) we observed decrease in the CRP level (–6.73 ± 3.93 mg/L); on the other hand, in group C– (n = 17) the CRP level increased (+7.92 ± 2.77 mg/L, p = 0.004). Similar differences were observed also in IL-6: in group C+ the level was significantly reduced as compared with the increase in group C– (–0.76 ± 0.52 vs. 4.58 ± 1.49 ng/L, p = 0.005). The level of IL-8 was not affected. Our results suggest that early treatment with cerivastatin can decrease the serum level of CRP and IL-6 in patients with UAP/NQMI; this might positively influence their prognosis. Nevertheless, further studies are needed to support this hypothesis.     

Differential cytokine expression in myocytes and non-myocytes after myocardial infarction in rats

The proinflammatory cytokines interleukin (IL)-1 and IL-6 are increased after acute myocardial infarction (MI). Moreover, serum IL-6 level is elevated after MI, but has also been associated with heart failure. In the present study, heart function was monitored in a rat model of chronic MI. Cytokine expression in the infarcted and non-infarcted myocardium as well as in hearts of sham-operated controls was measured by the ribonuclease-protection assay. To identify the cells contributing to the increased cytokine expression, we further analyzed myocytes and non-myocytes isolated in the acute phase as well as during congestive heart failure (CHF) after MI. There was a strong induction in cytokine expression in the myocytes of the infarct area 6 h after MI. In the non-infarcted myocardium, cytokine expression increased only slightly in the non-myocytes after 6 h. This was not different from sham-operated controls and may, therefore, be induced by stress and catecholamines. In CHF, however, cytokine expression level in myocytes was normal. It increased slightly but significantly in the non-myocytes 4 and 8 weeks after MI. In conclusion, we suggest that pro-inflammatory cytokines, produced by the ischemic myocytes may be involved in the initiation of wound healing of the necrotic area, whereas the effect of pro-inflammatory cytokines in CHF, if any, seems not to be crucial.