Acute Rho-kinase inhibition improves coronary dysfunction in vivo, in the early diabetic microcirculation.

OBJECTIVES: Activation of RhoA/Rho-kinase (ROCK) is increasingly implicated in acute vasospasm and chronic vasoconstriction in major organ systems. Therefore we aimed to ascertain whether an increase in ROCK activity plays a role in the deterioration of coronary vascular function in early stage diabetes. METHODS: Synchrotron radiation microangiography was used to determine in vivo coronary responses in diabetic (3 weeks post streptozotocin 65 mg/kg ip) and vehicle treated male Sprague-Dawley rats (n = 8 and 6). Changes in vessel number and calibre during vasodilator stimulation before and after blockade of nitric oxide synthase and cyclooxygenase were compared between rats. Acute responses to ROCK inhibitor, fasudil (10 mg/kg iv) was evaluated. Further, perivascular and myocardial fibrosis, arterial intimal thickening were assessed by histology, and capillary density, nitrotyrosine and ROCK1/2 expressions were evaluated by immunohistochemical staining. RESULTS: Diabetic rats had significantly elevated plasma glucose (P < 0.001 vs control), but did not differ in fibrotic scores, media to lumen ratio, capillary density or baseline visible vessel number or calibre. Responses to acetylcholine and sodium nitroprusside stimulation were similar between groups. However, in comparison to control rats the diabetic rats showed more segmental constrictions during blockade, which were not completely alleviated by acetylcholine, but were alleviated by fasudil. Further, second order vessel branches in diabetic rats were significantly more dilated relative to baseline (37% vs 12% increase, P < 0.05) after fasudil treatment compared to control rats, while visible vessel number increased in both groups. ROCK2 expression was borderline greater in diabetic rat hearts (P < 0.053). CONCLUSIONS: We found that ahead of the reported decline in coronary endothelial vasodilator function in diabetic rats there was moderate elevation in ROCK expression, more widespread segmental constriction when nitric oxide and prostacyclin production were inhibited and notably, increased calibre in second and third order small arteries-arterioles following ROCK inhibition. Based on nitrotyrosine staining oxidative stress was not significantly elevated in early diabetic rats. We conclude that tonic ROCK mediated vasoconstriction contributes to coronary vasomotor tone in early diabetes.

Myosin heads are displaced from actin filaments in the in situ beating rat heart in early diabetes.

Diabetes is independently associated with a specific cardiomyopathy, characterized by impaired cardiac muscle relaxation and force development. Using synchrotron radiation small-angle x-ray scattering, this study investigated in the in situ heart and in real-time whether changes in cross-bridge disposition and myosin interfilament spacing underlie the early development of diabetic cardiomyopathy. Experiments were conducted using anesthetized Sprague-Dawley rats 3 weeks after treatment with either vehicle (control) or streptozotocin (diabetic). Diffraction patterns were recorded during baseline and dobutamine infusions simultaneous with ventricular pressure-volumetry. From these diffraction patterns myosin mass transfer to actin filaments was assessed as the change in intensity ratio (I(1,0)/I(1,1)). In diabetic hearts cross-bridge disposition was most notably abnormal in the diastolic phase (p < 0.05) and to a lesser extent the systolic phase (p < 0.05). In diabetic rats only, there was a transmural gradient of contractile depression. Elevated diabetic end-diastolic intensity ratios were correlated with the suppression of diastolic function (p < 0.05). Furthermore, the expected increase in myosin head transfer by dobutamine was significantly blunted in diabetic animals (p < 0.05). Interfilament spacing did not differ between groups. We reveal that impaired cross-bridge disposition and radial transfer may thus underlie the early decline in ventricular function observed in diabetic cardiomyopathy.

Dynamic synchrotron imaging of diabetic rat coronary microcirculation in vivo.

OBJECTIVE: In diabetes, long-term micro- and macrovascular damage often underlies the functional decline in the cardiovascular system. However, it remains unclear whether early-stage diabetes is associated with in vivo functional impairment in the coronary microvasculature. Synchrotron imaging allows us to detect and quantify regional differences in resistance microvessel caliber in vivo, even under conditions of high heart rate. METHODS AND RESULTS: Synchrotron cine-angiograms of the coronary vasculature were recorded using anesthetized Sprague-Dawley rats 3 weeks after treatment with vehicle or streptozotocin (diabetic). In the early diabetic state, in the presence of nitric oxide and prostacyclin, vessel diameters were smaller (P<0.01) and endothelium-dependent vessel recruitment was already depressed (P<0.05). Endothelium-dependent and -independent vasodilatory responses in individual coronary vessels were not different in vivo. Inhibition of NO and PGI(2) production in diabetes uncovered early localized impairment in dilation. Diabetic animals displayed focal stenoses and segmental constrictions during nitric oxide synthase/cyclooxygenase blockade, which persisted during acetylcholine infusion (P<0.05), and a strong trend toward loss of visible microvessels. CONCLUSIONS: Synchrotron imaging provides a novel method to investigate coronary microvascular function in vivo at all levels of the arterial tree. Furthermore, we have shown that early-stage diabetes is associated with localized coronary microvascular endothelial dysfunction.