Abstract
Cardiovascular disease is a major cause of morbidity and mortality in New Zealand. Elucidating its pathogenesis will help us better treat those who are affected by it. One protein that has been implicated in cardiovascular pathology is Calcium (Ca2+)/Calmodulin-dependent protein Kinase II (CaMKII). As its name suggests, it is activated by Ca2+-bound calmodulin. In a healthy heart, CaMKII plays a role in regulating Ca2+-handling proteins in the cardiomyocyte, such as Ryanodine Receptor and Phospholamban. CaMKII has the ability to become autonomously activated so that it can exert its effects even when the Ca2+ concentration decreases. However, if CaMKII is activated when it does not need to be, it can have pathological effects. It can be activated pathologically by hyperglycemia, oxidative stress and hyperadrenergic states, all of which are seen in cardiovascular pathology such as heart failure or diabetic cardiomyopathy. Alpha-adrenergic receptor signaling can also activate CaMKII, but little is known about the effects of alpha-adrenergic stimulation on the heart.
The aim of this project was the determine the functional effects of the alpha-adrenergic agonist Methoxamine on isolated wild-type mouse hearts, and in isolated CaMKII knockout mouse heart, in incremental doses to observe differences in the responses between the two groups to determine the role of CaMKII in the functional response to alpha-adrenergic stimulus in the heart.
The technique used to address these aims was the isolated heart set-up, which is also known as the Langendorff setup. The mouse hearts were surgically removed from the animal, with part of the aorta intact, and placed in an arresting buffer with a high potassium chloride concentration to ionically arrest the heart. The heart was then cannulated via the aorta, and perfused with a Krebs-Henseleit Buffer (KHB) that had all the necessary ions and nutrients to allow the heart to contract outside the body. The stability of the heart was determined via measurements such as coronary flow rate, perfusion pressure, and temperature. A fluid-filled balloon inserted into the ventricle allowed measurement of contractile and relaxation parameters. Increasing doses of alpha-adrenergic receptor agonist, Methoxamine, were added to the KHB perfusate. Three doses were used; 10-8, 10-6, and 10-4mmol/L.
I found that in response to alpha-adrenergic stimulation, there was a significant increase in the rate of contraction (1.384-fold increase; p= 0.0114) and the rate of relaxation (1.435-fold increase; p= 0.0066) in the healthy, wild-type mouse hearts, but not in the CaMKII knockout mouse hearts (rate of contraction- 1.167-fold change; p=0.5080; rate of relaxation- 1.156-fold change; p= 0.5862). There was a significant increase in the total developed pressure in the heart during systole in the healthy mouse hearts in response to alpha-adrenergic stimulation (1.407-fold increase; p= 0.0206), but this increase was not seen in the CaMKII knockout mice (1.099-fold change; p= 0.8750). There was also a significant reduction in the minimum pressure reached by the heart, and the end-diastolic pressure in the healthy wild-type mouse hearts in response to alpha-adrenergic stimulation. Again, this change was not seen in the CaMKII knockout mouse hearts.
The results show that CaMKII may be responsible for the regulation of some contractile and relaxation parameters in the heart. By phosphorylation of its target protein, Ryanodine Receptor, CaMKII can increase the amount and rate of Ca2+ release into the cytoplasm of the cardiomyocyte which would allow for faster and more robust contractions. Furthermore, by phosphorylating Phospholamban, CaMKII can increase the rate and amount of Ca2+ cleared from the cytoplasm of the cardiomyocyte which would allow for faster and better relaxation. The results also tell us that CaMKII may be party of the alpha-adrenergic signaling pathway.
Future research into the involvement of alpha-adrenergic signaling in cardiovascular pathology, and whether inhibiting CaMKII attenuates or completely ablates disease progression will allow us to target CaMKII or the alpha-adrenergic signaling pathway as a therapeutic for cardiovascular disease.