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Doctoral Student Supervision (Jan 2008 - May 2019)
During myocardial infarction, the heart enters into an ischemic state that, if untreated, leads to cell death. To avoid this, reperfusion must be instituted. However, this causes a type of cardiac damage known as myocardial ischemia-reperfusion (I/R) injury. Concomitant diseases such as diabetes not only increase the risk of myocardial infarction, but also intensify susceptibility to I/R injury. Over-activation of Rho kinase (ROCK) has been reported to contribute to I/R injury; however, it is unknown whether ROCK1, ROCK2 or both isoforms, is/are responsible for this effect. Furthermore, it has not been determined if the cardioprotective activity of ROCK inhibition is maintained under diabetic conditions. Here, we evaluated the contribution of ROCK2 to myocardial I/R injury, as well as its role during diabetes. The induction of I/R injury impaired cardiac function in wild-type (WT) but not heterozygous ROCK2-knockdown (ROCK2+/-) mice. Infarct size was also lower in ROCK2+/- mice than in their WT counterparts, while an I/R-induced increase in ROCK activity was detected only in WT hearts. The cardioprotection observed in ROCK2+/- mice was associated with increased Akt and GSK-3β phosphorylation and a reduction of I/R-induced cytokine production. Compared to their ROCK2-expressing counterparts, cardiac-specific ROCK2 knockout (cROCK2-/-) mice subjected to I/R also had a smaller infarct size, suggesting that the deleterious effects of I/R are at least partially regulated by cardiomyocyte ROCK2. However, the induction of diabetes by streptozotocin treatment abrogated the protective effect of cardiomyocyte-specific ROCK2 deletion. ROCK activity was comparable in control and diabetic post-I/R cROCK2-/- hearts, but the increase in Akt and GSK-3β phosphorylation was no longer detected under diabetic conditions. Furthermore, cytokine production in non-diabetic cROCK2-/- mice was higher than in their ROCK2 expressing counterparts, and this was normalized in diabetic conditions.Overall, these results show that ROCK2 plays a significant role in the development of myocardial I/R injury, and that its inhibition is associated with activation of the pro-survival RISK-pathway. Moreover, the cardioprotection obtained by the cardiac-specific deletion of this isoform suggests that I/R-induced ROCK2 activation takes place in the cardiomyocyte. However, this cardioprotective effect is lost during diabetes, possibly due to impaired Akt and GSK-3β phosphorylation.
Diabetic patients have an increased risk of heart failure and sudden death, attributed in part to the development of diabetic cardiomyopathy, defined as ventricular dysfunction independent of hypertension and coronary artery disease. The mechanisms contributing to diabetic cardiomyopathy are not completely understood, but over-activation of the RhoA/ROCK pathway has been identified as a contributor. This research further investigated the roles of cardiomyocyte RhoA and of ROCK2 in the development of diabetic cardiomyopathy. In study one, the effects of heterozygous deletion of ROCK2 (ROCK2+/-) on cardiac function in a CD1 mouse model of type 1 diabetes induced by streptozotocin (STZ) were analyzed, since homozygous ROCK2 deletion is embryonically lethal. Thirteen weeks after diabetes induction, global cardiac function was unchanged in diabetic compared to non-diabetic mice. However, cardiomyocytes isolated from wild-type diabetic mice exhibited arrhythmic Ca²⁺ transients associated with increased ryanodine receptor 2 and CAMKII phosphorylation. These observations were attenuated in ROCK2+/- animals, suggesting that inhibition of ROCK2 may protect against arrhythmogenesis in the diabetic heart.The purpose of study two was to compare the development and progression of cardiac dysfunction in C57BL/6 mice, the strain of mice used in the final study, made diabetic or insulin resistant by dietary intervention and/or STZ treatment. Mice made diabetic with STZ showed the earliest and most severe signs of cardiac dysfunction compared to other models investigated, establishing this as an appropriate model. Given that RhoA is expressed in many different cell types in the heart, the purpose of study three was to analyze the role of cardiomyocyte RhoA in the development of diabetic cardiomyopathy, using mice with inducible cardiac-specific knockdown of RhoA (RhoA-/-). Hearts from diabetic RhoA-/- mice were protected against the development of contractile dysfunction. This was associated with prevention of cardiomyocyte fibrosis, hypertrophy and apoptosis, and with normalization of signaling through the TGF-β pathway, including Smad2 and 3 phosphorylation, and Smad7 expression.Overall, the results demonstrate that deletion of ROCK2 and of cardiomyocyte RhoA protect the diabetic heart. Inhibition of this pathway may be an important therapeutic avenue to decrease the risk of heart failure and sudden death in diabetes.
Diabetes mellitus leads to a unique pathological entity termed diabetic cardiomyopathy, the mechanisms of which have not been fully defined. In diabetic rat hearts, RhoA expression is increased and the RhoA/ROCK pathway is activated, while ROCK inhibition acutely improves contractile function of diabetic hearts. The mechanisms underlying this improvement and those responsible for the detrimental activation of RhoA/ROCK were investigated here. Nitric oxide (NO) has been reported to upregulate RhoA expression in smooth muscle, and previous reports showed that iNOS expression is increased in diabetic rat hearts. In the first part of this thesis, the hypothesis that in diabetic cardiomyopathy, iNOS induction is responsible for increased RhoA expression was investigated. The results demonstrate that increased NO production from iNOS induction leads to RhoA upregulation in the diabetic heart and in isolated cardiomyocytes, contributing to the RhoA/ROCK mediated contractile dysfunction by increasing the total pool of RhoA available for activation.In diabetic rat hearts, PKCβ₂ activation induces iNOS expression, leading to increased nitrosative/oxidative stress. This suggests that PKCβ2 might positively regulate RhoA expression, although in preliminary experiments inhibition of ROCK itself reduced RhoA expression. Therefore, in the second part, the hypothesis that PKCβ₂/iNOS and RhoA/ROCK interact together to form a positive feedback loop was tested. The results show that RhoA/ROCK overactivation is sustained by a positive feedback loop that involves PKCβ₂ activation and iNOS induction. This feedback loop requires an intact actin cytoskeleton and plays a key role in elevating superoxide production in diabetic rat hearts.In the third part, the hypothesis that ROCK inhibition augments contraction by improving Ca²⁺ signaling was tested. Inhibition of ROCK improved contractile function and abolished the diabetes-induced delayed aftercontractions in isolated cardiomyocytes, in association with an improvement in Ca²⁺ transients. Overall, the results show that in diabetic cardiomyopathy, overactivation of RhoA/ROCK contributes to contractile dysfunction by sustaining PKCβ2 activation, iNOS induction and superoxide production via a positive feedback loop that leads to impaired intracellular Ca²⁺ homeostasis. Inhibition of ROCK disrupts the loop, resulting in decreased oxidative stress, and improved Ca²⁺ handling and cardiomyocyte contraction, suggesting that ROCK inhibition might be a novel approach in treating diabetic cardiomyopathy.
The metabolic syndrome is a cluster of cardiovascular risk factors and is a global health concern. The most accepted and unifying hypothesis proposes that insulin resistance is the major common underlying abnormality that describes the metabolic syndrome and links it to the development of cardiovascular disease. The fructose-fed rat is an animal model that exhibits several features observed in the metabolic syndrome including insulin resistance, hyperinsulinemia, hypertriglyceridemia and hypertension. This animal model is used to study the relationship between these metabolic disturbances and hypertension. Numerous mechanisms have been proposed to mediate the link between insulin resistance and hypertension. The objectives of this thesis were to further investigate the underlying mechanisms that have been proposed to contribute to the development of hypertension in fructose-fed rats through the use of various pharmacological agents. We demonstrated that chronic treatment with bosentan, a dual endothelin receptor antagonist, L-158,809, an angiotensin receptor antagonist, prazosin, an α₁-adrenoceptor antagonist or etanercept, a soluble recombinant fusion protein consisting of the extracellular ligand binding domain of tumor necrosis factor receptor type 2, prevented the development of fructose-induced hypertension without affecting insulin levels or insulin sensitivity. These results suggest that increased production and/or activity of the endothelin system, renin angiotensin system or sympathetic nervous system contribute to the development of hypertension through insulin-independent mechanisms. Both the endothelin system and renin angiotensin system are crucial players in the development of fructose-induced hypertension, with endothelin-1 contributing its effects through modulation of angiotensin II. Overactivation of the sympathetic nervous system contributed to the development of hypertension, but does not appear to be an initial, precipitating mediator. Chronic etanercept treatment prevented the development of hypertension by improving vascular function and restoring endothelial nitric oxide synthase expression. Therefore, the pathogenesis of hypertension in fructose-fed rats is complex in nature and involves numerous pathways that do not necessarily function independently from one another.
Cardiovascular complications of diabetes are related in part to abnormal vascular function, a manifestation of the many changes induced in the arterial wall by the metabolic abnormalities accompanying diabetes and insulin resistance. We investigated the biochemical and functional consequences of diabetes and metabolic abnormalities, particularly insulin resistance, on vascular function, using two different animal models, the streptozotocin (STZ)-diabetic rat model of type 1 diabetes and the high fructose diet-fed rat model of insulin resistance and hypertension (FHR). In STZ diabetic rats, we found that complex biochemical interactions led to changes in hemodynamic variables. Specifically, we found that increased activation of PKCβ2 leads to induction of inducible nitric oxide synthase (iNOS), which results in increased production of both nitric oxide and peroxynitrite, causing pressor hypo-responsiveness, depressed cardiac function, mean arterial blood pressure and heart rate and impaired endothelial function in STZ-diabetic rats. Further, we found that hyperglycemia-induced activation of PKCβ2 is antecedent to increases in oxidative stress, activation of ERK1/2, NF-κB, and iNOS expression, and the protective effects of PKCβ or iNOS inhibition in STZ diabetic rats are associated with inhibition of iNOS-mediated peroxynitrite formation.In contrast to STZ diabetic rats, endothelial dysfunction in FHR is associated with hypertension. In FHR, we investigated the role of matrix metalloproteinases (MMP) and epidermal growth factor receptor (EGFR) transactivation, a novel pathway that has been proposed to link all the pathological features of hypertension including endothelial dysfunction, enhanced vascular tone and hypertrophic growth of cardiovascular tissue. We found that in normal arteries, MMP-EGFR pathway modulates vascular tone, at least in part, via activation of PI3-kinase and mitochondrial ATP synthesis. In FHR arteries, inhibition of MMPs by doxycycline improved endothelial function while EGFR inhibition by AG1478 promoted vasorelaxation. Further, in insulin resistant vascular smooth muscle cells and arteries from FHR, pharmacological or siRNA inhibition of MMP-EGFR signaling normalized the increased expression and activity of contractile proteins (MLCK, MLC II) and their transcriptional activators (P90RSK and SRF) in addition to the prevention of hypertension in FHR. Our data suggests that the MMP-EGFR pathway could be a potential target in the treatment of hypertension.
Lysophosphatidylcholine (1-acyl-sn-glycero-3-phosphocholine, LPC) is the most abundant glycerol-based lysophospholipid present in cell membranes and oxidized lipoproteins. It has been proposed that LPC contributes to the altered vaso-reactivity associated with various cardiovascular diseases in which elevated LPC levels were identified. However, the contribution of LPC in regulating vascular resistance has not been completely elucidated, as the majority of previous studies have used either large blood vessels or isolated cells. Therefore, our study aimed to investigate the vasoactive effects and the underlying mechanisms of LPC in small arteries/arterioles that are crucial in the determination of vascular resistance and the maintenance of organ function.The unique finding of our investigation is that LPC possesses biphasic effects on both peripheral arterial resistance and coronary circulation, and even ventricular function. Specifically, in the isolated perfused rat mesenteric arterial bed, both endothelium-derived relaxing factors and thromboxane A₂ (TxA₂, a vasoconstricor) are diminished by LPC perfusion. However, LPC washout stimulates a rebound overproduction of TxA₂, which results in an enhanced contractile response to alpha1-adrenoceptor stimulation.Our study next found that sustained perfusion of hearts with LPC augmented coronary perfusion pressure and reduced left ventricular developed pressure. These effects were exaggerated when LPC was removed from the perfusate. Furthermore, LPC selectively potentiated the receptor-coupled vasoconstrictor response of isolated rat septal coronary artery to U-46619, a TxA₂ mimetic. Interestingly, when LPC was washed out, the potentiation to U-46619 was even more pronounced. Both the immediate and residual effects of LPC were endothelium-dependent. Endothelium-derived hyperpolarizing factor was likely the sole mediator responsible for the direct effects of LPC on U-46619-vasoconstriction, whereas the augmented vasoconstrictor responses following LPC washout may in part be related to an increase in endothelin-1, and a striking reduction in the bioavailability of nitric oxide.Our data suggest that simply reducing LPC levels to normal may not be sufficient to reverse the adverse consequences of this lysolipid accumulation in vasculature. Further understanding of the residual effects of LPC will enable the identification of more effective treatment targets for LPC-related diseases.
Master's Student Supervision (2010 - 2018)
No abstract available.
Hypertension is the most common cardiovascular complication associated with diabetes mellitus. The RhoA/ROCK pathway has been implicated in the regulation of vascular smooth muscle contractile responses associated with hypertension and diabetes. Here we wished to determine the role of the RhoA/ROCK pathway in vascular smooth muscle contractile responses in different types of diabetic models and its contribution to diabetes-associated hypertension. Our results suggest that the involvement of ROCK may be more important in U-46619- than in PE-induced contractile responses in endothelium denuded mesenteric resistance arteries from control rats. On the other hand, as found in our previous work and other reports, PE-induced contractile responses were attenuated by inhibition of ROCK in rat superior mesenteric arteries, suggesting that ROCK is likely to play an essential role in PE-induced contraction in superior mesenteric arteries. Blood pressure in GK rats was normalized by one-week treatment with fasudil, a known ROCK inhibitor, suggesting that the elevated blood pressure in GK rats may be due to enhanced ROCK pathway. However, agonist-induced contractile responses in GK mesenteric resistance arteries were not augmented compared to control arteries, and moreover, expression of ROCK/RhoA as well as activity of ROCK were similar between control and GK arteries. The specific causes of the discrepancy remain unknown. In the presence of L-NAME, ROCK inhibitor significantly attenuated contraction induced by PE or U-46619 in mesenteric resistance arteries from GK rats, suggesting that agonist-induced contractile responses in mesenteric resistance arteries from GK rats were likely to be ROCK-mediated. This was further supported by Western blotting results, in that phosphorylation of MYPT was significantly increased by U-46619. Interestingly, U-46619-induced contraction in mesenteric resistance arteries from control rats was no longer sensitive to inhibition of ROCK in the presence of L-NAME, although the reasons were unclear.In conclusion, results from the GK rat study do not support the hypothesis that activation of the RhoA/ROCK pathway contributes to the development of diabetes-associated hypertension in GK rats by enhancing vascular smooth muscle contractile responses. To further investigate this study, a more ROCK selective ROCK inhibitor is needed.