Temporal dystrophic remodeling within the intrinsic cardiac nervous system of the streptozotocin-induced diabetic rat model.

INTRODUCTION: The pathogenesis of heart failure (HF) in diabetic individuals, called "diabetic cardiomyopathy", is only partially understood. Alterations in the cardiac autonomic nervous system due to oxidative stress have been implicated. The intrinsic cardiac nervous system (ICNS) is an important regulatory pathway of cardiac autonomic function, however, little is known about the alterations that occur in the ICNS in diabetes. We sought to characterize morphologic changes and the role of oxidative stress within the ICNS of diabetic hearts. Cultured ICNS neuronal cells from the hearts of 3- and 6-month old type 1 diabetic streptozotocin (STZ)-induced diabetic Sprague-Dawley rats and age-matched controls were examined. Confocal microscopy analysis for protein gene product 9.5 (PGP 9.5) and amino acid adducts of (E)-4-hydroxy-2-nonenal (4-HNE) using immunofluorescence was undertaken. Cell morphology was then analyzed in a blinded fashion for features of neuronal dystrophy and the presence of 4-HNE adducts. RESULTS: At 3-months, diabetic ICNS neuronal cells exhibited 30% more neurite swellings per area (p = 0.01), and had a higher proportion with dystrophic appearance (88.1% vs. 50.5%; p = <0.0001), as compared to control neurons. At 6-months, diabetic ICNS neurons exhibited more features of dystrophy as compared to controls (74.3% vs. 62.2%; p = 0.0448), with 50% more neurite branching (p = 0.0015) and 50% less neurite outgrowth (p = <0.001). Analysis of 4-HNE adducts in ICNS neurons of 6-month diabetic rats demonstrated twice the amount of reactive oxygen species (ROS) as compared to controls (p = <0.001). CONCLUSION: Neuronal dystrophy occurs in the ICNS neurons of STZ-induced diabetic rats, and accumulates temporally within the disease process. In addition, findings implicate an increase in ROS within the neuronal processes of ICNS neurons of diabetic rats suggesting an association between oxidative stress and the development of dystrophy in cardiac autonomic neurons.

Differential expression and activity of matrix metalloproteinase-2 and -9 in the calreticulin deficient cells.

Calreticulin is an endoplasmic reticulum protein important in cardiovascular development. Deletion of the calreticulin gene leads to defects in the heart and the formation of omphaloceal. These defects could both be due to changes in the extracellular matrix composition. Matrix metalloproteinases (MMP)-2 and MMP-9 are two of the MMPs which are essential for cardiovascular remodelling and development. Here, we tested the hypothesis that the defects observed in the heart and body wall of the calreticulin null embryos are due to alterations in MMP-2 and MMP-9 activity. Our results demonstrate that there is a significant decrease in the MMP-9 and increase in the MMP-2 activity and expression in the calreticulin deficient cells. We also showed that there is a significant increase in the expression level of membrane type-1 matrix metalloproteinase (MT1-MMP). In contrast, there was no change in the tissue inhibitor of matrix metalloproteinase (TIMP)-1 or -2 in the calreticulin deficient cells as compared to the wild type cells. Interestingly, the inhibition of the MEK kinase pathway using PD98059 attenuated the decrease in the MMP-9 mRNA with no effect on the MMP-2 mRNA level in the calreticulin deficient cells. Furthermore, PI3 kinase inhibitor decreased the expression of both the MMP-2 and MMP-9. This study is the first report on the role of calreticulin in regulating MMP activity.

Upregulation of the alpha1-adrenoceptor-induced phosphoinositide and inotropic response in hypothyroid rat heart.

In this study, we examined changes in the biochemical and inotropic events of the alpha(1)-adrenoceptor signaling pathway in hypothyroid rat hearts. Hypothyroidism was induced by treating experimental animals with 0.05% 6-n-propyl-2-thiouracil (PTU) in drinking water for 7 weeks. A significant decrease of beta- and an increase in alpha(1)-adrenoceptor density as well as an increase in the basal activity of the phosphoinositide (4,5) bisphosphate hydrolyzing phospholipase C was observed in sarcolemmal membranes purified from hypothyroid hearts as compared to age-matched euthyroid controls. Following stimulation with 10 microM phenylephrine (in the presence of 10 microM atenolol), the increase of contractile parameters over baseline values was significantly higher in hypo- than euthyroid hearts, while the opposite occurred under beta-stimulation with 0.1 microM isoproterenol. Interestingly, the increase in phenylephrine-mediated positive inotropy was accompanied by a significant increase in the sarcolemmal phospholipase C activity and in the inositol 1,4,5-trisphosphate content in hypothyroid as compared to euthyroid controls. Our results suggest that cardiac alpha(1)-adrenoceptor and its associated phosphoinositide signaling pathway may act as a reserve for catecholamine inotropic response in hypothyroidism, where the beta-adrenoceptors are compromised.