The antioxidant effect of CAPA on I/R injury
Myocardial infarction usually results from thrombus , vasospasm , routine coronary angioplasty , or open-heart operation . Although restoration of blood flow is the only way to save the myocardium from ischemic injury, reperfusion can exacerbate the myocardial damage caused by ischemia. The mostly recognized cause of reperfusion injury is a burst of free radicals including hydrogen peroxide, hydroxyl radical, and superoxide radical that is formed during reperfusion and plays an important role in the pathophysiological mechanism of I/R injury . Moreover, treatment with antioxidant remedies or agents capable of inducing antioxidant enzymes such as glutathione peroxidase and superoxide dismutase have a cardioprotective effect against I/R injury in experimental animals in vivo and in vitro. Therefore, antioxidant therapy may be an effective process for treating myocardial infarction. However, predicting when myocardial infarction occurs and preventing the infarction with long-term antioxidants use is impractical, and administering medications after ischemia and before reperfusion is a more reasonable way to treat patients with myocardial infarction. Therefore, we administered CAPA 30 min prior to reperfusion to evaluate the cardiac effect of CAPA in I/R. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity of CAPA, as shown by an EC50 of 18.6 ± 3.2 μM (comparable to the EC50 of 15.6 ± 2.0 μM CAPE, data not shown), was in accordance with that seen in other studies of the structure action relationship of CAPE and CAPA [26, 48] and may reduce tissue oxidative stress and increase tissue availability of NO. In addition, our studies in type 2 diabetic mice have shown that CAPA treatment could increase manganese superoxide dismutase in fat tissue , but whether this effect is involved in improving the hemodynamic function of STZ-induced diabetic rats remains unknown.
CAPE administration protected the heart from I/R injury with reduced levels of oxidative stress such as MDA . In our study, CAPA administration reduced MDA to a level comparable to that seen in the control group. An analog of CAPA, dmCAPA, which has no radical-scavenging activity, exhibited no cardioprotective effect on infarct size, suggesting that CAPA, like CAPE, exerts a cardioprotective effect mainly through its antioxidant property. Compared with vehicle treatment, the MPO activity reduced with CAPA administration, but not with dmCAPA. This finding suggests that the cardioprotective effect of CAPA could be partly attributed to the reduced inflammatory response by its antioxidant ability.
NO preservation effect of CAPA on I/R injury
Although the role of NO remains controversial, myocardial I/R injury is exacerbated in the absence of endothelial cell NOS ; and endothelial NOS is able to regulate vascular tension , inhibit platelet aggregation , scavenge ROS, and stimulate endothelial regeneration to protect the heart and blood vessels . CAPA inhibited lipopolysaccharide/interferon-γ (LPS/IFN-γ)-induced inducible NOS (iNOS) expression and NO production , while a CAPA-induced increase in coronary blood flow was prevented by a NOS inhibitor, which suggests that CAPA may enhance coronary blood flow by increasing NO availability or level . In our study, pretreatment with the NOS inhibitor l- NAME eliminated the cardioprotective effect of CAPA on infarct size reduction, suggesting that NO is an important factor responsible for cardioprotection. We propose that the cardioprotective effect of CAPA resulted from its own antioxidant property, which preserves the bioavailable NO in the heart. However, this assumption requires further supported by more direct evidence.
Effect of chronic CAPA treatment on cardiac dysfunction in diabetes
Animals with STZ-induced diabetes showed reduced resting heart rate and pulse pressure . In our study, the heart rate of diabetic rats was significantly lower than that of control rats during the I/R period, while CAPA treatment reversed the reduction after 45 min of ischemia (Figure 6A). The mean blood pressures of the CAPA-treated groups were higher than those of the other two groups when the hearts were subjected to ischemia for 45 min and higher than those of the STZ-diabetic animals after 60 min of reperfusion (Figure 6B). The pressure preservation effect was in accordance with that seen in our earlier study concluding that CAPA may preserve the vasomotor activity in diabetic animals . The maintenance of blood pressure and heart rate by chronic CAPA treatment may contribute to its protective effect against I/R injury in diabetic animals.
The STZ-induced diabetic animals underwent hemodynamic changes in cardiac function measured by PV loops [55–57]. In our study, the dP/dt max, dP/dt min, ejection fraction, ESPVR, EDPVR, and PRSW values were decreased and the LVEDP value was increased in the STZ-vehicle group; in agreement with the well-established model of STZ-induced diabetic cardiomyopathy. Compared with the STZ-vehicle group, CAPA treatment preserved the basal cardiac function in terms of PRSW, ESPVR, and ejection fraction, but slight preservation of PRSW and stroke work occurred after I/R injury. CAPA treatment did not reverse any parameters of hemodynamic function after reperfusion; however, the ameliorations of these parameters at baseline may contribute to the reduction of infarct size after I/R.
The underlying mechanisms of CAPA against cardiac dysfunction in diabetes
There are several mechanisms involved in the development of diabetic cardiomyopathy, including increased oxidative stress, and activation of the renin-angiotensin system .
In the STZ-induced diabetic rats subjected to I/R injury, hyperglycemia, an independent risk factor, worsens cardiac performance, cell survival, and tissue injury following myocardial I/R via increased oxidant production and reduced antioxidant defenses [2, 3, 59]. In our study, CAPA ameliorated the I/R injury, but this protective effect disappeared when the –OH functional groups were substituted with methoxyl groups, and the antioxidant activity of CAPA may contribute to the protective effect on the I/R injury and cardiac dysfunction in diabetes.
Intracellular angiotensin II was proven to have a significant role in the pathological process in AT1a receptor-deficient diabetic mice. The inhibition of intracellular angiotensin II level by a renin or angiotensin-converting-enzyme inhibitor prevented the development of cardiac dysfunction in these diabetic mice . AT-1 receptor antagonists attenuate cardiac failure by decreasing cardiac inflammation and normalizing matrix metalloproteinase activity, leading to the alleviation of cardiac fibrosis in STZ-induced diabetic cardiomyopathy . The inhibition of Rho-kinase protects the cerebral barrier from ischemia-evoked injury by modulating endothelial cell oxidative stress and tight junctions  and protects the structure and function of cardiac mitochondria from diabetes by attenuating oxidative stress . Acute Rho-kinase inhibition improves coronary dysfunction in vivo in the early diabetic microcirculation , while long-term Rho-kinase inhibition ameliorates myocardial hypertrophy, apoptosis, fibrosis, and subsequent cardiac remodeling in diabetes . The Rho-associated protein kinase-mediated signaling pathway is known to be involved in the vascular effects of angiotensin II , and renin-angiotensin system blockade is beneficial to the cardiovascular system and a known treatment for diabetic cardiomyopathy . In our earlier study, we found that CAPA decreased plasma angiotensin II level in mice with abdominal aortic banding-induced ventricular hypertrophy , implicating that the angiotensin II-lowering activity of CAPA may protect the cardiovascular function in diabetes. The level of iNOS is associated with the induction of RhoA expression in the hearts of diabetic rats , while CAPA inhibits LPS/IFN-γ-induced iNOS expression , implicating that the inhibition of iNOS expression may contribute to its protection against diabetic cardiomyopathy.
Recent evidence shows that an increased infarct size is associated with low levels of myocardial heme oxygenase-1 (HO-1) during I/R in diabetic rats [2, 3]. The nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway regulates the oxidative stress response, and altered Nrf2 responses may contribute to the observed selective cytotoxicity of electrophilic compounds . Nrf2 is a transcription factor that regulates the expression of many detoxification or antioxidant enzymes . CAPE stimulates ho-1 gene activity by promoting inactivation of the Nrf2–Keap1 complex, leading to increased Nrf2 binding to the resident ho-1 AREs, and the induction of HO-1 by CAPE requires Nrf2/ARE pathway activation . In HO-1 activation, CAPA was as effective as CAPE at inducing HO-1 mRNA (nine-fold over vehicle control) as determined by reverse transcription–polymerase chain reaction , and CAPA can induce HO-1 mRNA expression in rat primary cultured microglia . These data indicate that CAPA treatment may mitigate infarction in diabetic rats by inducing HO-1 activity.