The defects identified in the mechanical activity of the hearts from type 1 diabetic animals include alteration of Ca 2؉ signaling via changes in critical processes that regulate intracellular Ca 2؉ concentration. These defects result partially from a dysfunction of cardiac ryanodine receptor calcium release channel (RyR2). The present study was designed to determine whether the properties of the Ca 2؉ sparks might provide insight into the role of RyR2 in the altered Ca 2؉ signaling in cardiomyocytes from diabetic animals when they were analyzed together with Ca 2؉ transients. Basal Ca 2؉ level as well as Ca 2؉ -spark frequency of cardiomyoctes isolated from 5-week streptozotocin (STZ)-induced diabetic rats significantly increased with respect to aged-matched control rats. Ca 2؉ transients exhibited significantly reduced amplitude and prolonged time courses as well as depressed Ca 2؉ loading of sarcoplasmic reticulum in diabetic rats. Spatio-temporal properties of the Ca 2؉ sparks in cardiomyocytes isolated from diabetic rats were also significantly altered to being almost parallel to the changes of Ca 2؉ transients. In addition, RyR2 from diabetic rat hearts were hyperphosphorylated and protein levels of both RyR2 and FKBP12.6 depleted. These data show that STZ-induced diabetic rat hearts exhibit altered local Ca 2؉ signaling with increased basal Ca 2؉ level.

Red blood cells (RBC) play an important role in the balance between generation and scavenging of nitric oxide (NO) and hence its local bioavailability and influence on vasomotor control. Previous studies have reported increased NO levels in RBC suspensions subsequent to exposure to shear forces; the present study was designed to further investigate changes in intracellular NO concentration and possible mechanisms involved for RBC exposed to well-controlled shear forces. Attached human RBC were subjected to shear stresses up to 0.1 Pa in a parallel-plate flow channel; fluorescent methods were used to monitor changes in intracellular NO and calcium concentrations. Intracellular NO concentration, estimated by the fluorescence level of 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM), increased sharply within 30 s following the application of shear stress between 0.013 to 0.1 Pa. This increase was only partially prevented by the absence of L-arginine and by the presence of L-N-acetyl-methyl-arginine (L-NAME), strongly suggesting that this response was in part related to the activation of NO-synthase (NOS) enzyme. The increase in intracellular NO concentration under shear stress was also inhibited by calcium chelation in the suspending medium, indicating the role of calcium entry for NOS activation. Increases of intracellular calcium concentrations under the same shearing conditions were demonstrated by monitoring Fluo-3/AM fluorescence in RBC exposed to shear stress. Serine 1177 phosphorylated NOS protein, the activated form of the enzyme determined by immunohistochemistry, was found to be significantly increased following the exposure of RBC to 0.1 Pa shear stress for 1 min. These data confirm that RBC possess a NOS enzyme that is actively synthesizing NO and activated by effective shear forces. The data also suggest that there may be additional (e.g., non-enzymatic) NO generating mechanisms in RBC that are also enhanced under shear stress.

Background and purpose: Reactive oxygen and nitrogen species play an important role in the development of diabetic cardiomyopathy. They can activate matrix metalloproteinases (MMPs), and MMP-2 in particular is known to mediate early consequences of oxidative stress injury in the heart. Therefore, we investigated the role of MMP-2 and the effect of the MMP inhibitor doxycycline on the changes of heart function caused by diabetes. Experimental approach: Using streptozotocin-induced diabetic rats, we evaluated the effect of doxycycline on both mechanical and electrical function of isolated hearts, papillary muscle and cardiomyocytes. Key results: Doxycycline abolished the diabetes-induced depression in left ventricular developed pressure and the rates of changes in developed pressure in isolated hearts and normalized the prolongation of the action potential in papillary muscles. In cardiomyocytes isolated from doxycycline-treated diabetic rats, the altered kinetic parameters of Ca 2 þ transients, depressed Ca 2 þ loading of sarcoplasmic reticulum and basal intracellular Ca 2 þ level, and the spatio-temporal properties of Ca 2 þ sparks were significantly restored. Gelatin zymography and western blot data indicated that the diabetes-induced alterations in MMP-2 activity and protein level, level of tissue inhibitor of matrix metalloproteinase-4 and loss of troponin I were restored to control levels with doxycycline. Conclusions and implications: Our data suggest that these beneficial effects of doxycycline on the mechanical, electrical and biochemical properties of the diabetic rat heart appear, at least in part, to be related to inhibition of MMP activity, implying a role for MMPs in the development of diabetic cardiomyopathy.

Stimulation of local renin-angiotensin system and increased levels of oxidants characterize the diabetic heart. Downregulation of ANG II type 1 receptors (AT1) and enhancement in PKC activity in the heart point out the role of AT1 blockers in diabetes. The purpose of this study was to evaluate a potential role of an AT1 blocker, candesartan, on abnormal Ca 2ϩ release mechanisms and its relationship with PKC in the cardiomyocytes from streptozotocin-induced diabetic rats. Cardiomyocytes were isolated enzymatically and then incubated with either candesartan or a nonspecific PKC inhibitor bisindolylmaleimide I (BIM) for 6 -8 h at 37°C. Both candesartan and BIM applied on diabetic cardiomyocytes significantly restored the altered kinetic parameters of Ca 2ϩ transients, as well as depressed Ca 2ϩ loading of sarcoplasmic reticulum, basal Ca 2ϩ level, and spatiotemporal properties of the Ca 2ϩ sparks. In addition, candesartan and BIM significantly antagonized the hyperphosphorylation of cardiac ryanodine receptor (RyR2) and restored the depleted protein levels of both RyR2 and FK506 binding protein 12.6 (FKBP12.6). Furthermore, candesartan and BIM also reduced the increased PKC levels and oxidized protein thiol level in membrane fraction of diabetic rat cardiomyocytes. Taken together, these data demonstrate that AT 1 receptor blockade protects cardiomyocytes from development of cellular alterations typically associated with Ca 2ϩ release mechanisms in diabetes mellitus. Prevention of these alterations by candesartan may present a useful pharmacological strategy for the treatment of diabetic cardiomyopathy.heart; candesartan; Type 1 diabetes; thiol oxidation CHRONIC DIABETES ALTERS the structure and function of the human heart, and individuals with diabetes mellitus usually develop a specific cardiac dysfunction known as diabetic cardiomyopathy (37). Several mechanisms involved in the development of cardiomyopathy have been postulated, including alterations in intracellular ion homeostasis and glucose metabolism and enhanced oxidative stress. Although alteration of Ca 2ϩ signaling via changes in critical processes that regulate intracellular Ca 2ϩ has become a hallmark of this type of cardiomyopathy, controversies, currently going on, relate to specific alterations in Ca 2ϩ signaling pathways contributing to the cardiac defects in diabetes (7,20). Recently, we reported that these defects result partially from altered local Ca 2ϩ signaling due to a dysfunction of cardiac PKA-mediated ryanodine receptor Ca 2ϩ release channel (RyR2) (51).Several mechanisms have been proposed to explain how all of the pathologies involved in the progression of diabetic cardiomyopathy might result from hyperglycemia. Increased PKC isoform expression and increased polyol pathway flux are two main hypotheses presented to describe how hyperglycemia might cause all of the diabetic complications (6). Furthermore, it has been demonstrated that hyperglycemia activates the local renin-angiotensin system (RAS) and enhanced RAS activity in diabetes (3...

Blockage of AT1 receptor increased strength, mass and trabecular connections of ovariectomized rat femurs. Therefore, it is tempting to speculate that drugs, including AT1 receptor blockers, may be used for the treatment of osteoporosis or reduction of its detrimental effects in the future.