Abstract
Nitric oxide (NO) is a gaseous signaling molecule that plays a role in cardiac contraction and relaxation. One of the pathways of NO signaling is through S-nitrosylation, which is the covalent attachment of a NO moiety to the thiol side chain of a cysteine residue. One downstream target of NO via S-nitrosylation is the calcium handling protein calcium-calmodulin dependent protein kinase II (CaMKII). There is evidence that the S-nitrosylation of CaMKII promotes calcium mishandling in cardiomyocytes, which is the precursor of cardiac arrhythmias. The role of CaMKII in the whole heart during NO exposure is yet to be understood. Therefore, the aim of this thesis was to determine how CaMKII modification by NO and β-adrenergic stimulation can affect cardiac contractility and arrhythmias.
With the use of echocardiography, cardiac function was compared between wild type (WT) mice and transgenic mice lacking cardiac CaMKIIδ (KO), and loss of CaMKIIδ did not severely alter cardiac structure and function. To determine if NO and CaMKIIδ did affect cardiac function, the hearts were isolated from the mice and treated with different concentrations of a NO donor (GSNO). GSNO reduced contractility measured as left ventricular (LV) developed pressure and increased heart rate in WT mice whereas KO mice showed no response to GSNO treatment. WT mice were susceptible to arrhythmias and the number of arrhythmic events increased with increasing GSNO concentration. There was no change in the number of arrhythmic events in the KO mouse hearts with GSNO treatment compared to baseline. Therefore, CaMKIIδ removal was cardioprotective, as it prevented arrhythmias during acute GSNO treatment.
Having established a role for CaMKII in determining the effects of acute NO treatment on cardiac function, I then focused on a model of chronically elevated NO. Chronic GSNO treatment of WT and KO mice involved a 5-week supplementation with GSNO in drinking water, followed by measurement of cardiac function in isolated hearts from these mice with and without GSNO. Echocardiographic data from WT and KO mice showed no effect of GSNO treatment after 5-week GSNO supplementation. The isolated hearts showed no difference in cardiac function at baseline. Following subsequent GSNO treatment, the LV developed pressure and rates of contraction and relaxation decreased compared to baseline in WT and KO hearts. Both animal models showed a trend towards increased number of arrhythmic events, suggesting that knocking out CaMKIIδ from the heart did not prevent the KO hearts from developing arrhythmias in the chronic treatment compared to the acute treatment.
β-adrenergic stimulation using the β-adrenergic receptor agonist (ISO) enhanced cardiac contractility and arrhythmias in both WT and KO hearts. However, ISO induced an increase in arrhythmic events in WT hearts, but not in KO hearts, suggesting that the presence of CaMKII enhanced the susceptibility of WT hearts to ISO-induced cardiac stress. To determine the effect of NO treatment and β-adrenergic stimulation on cardiac function, WT hearts were treated with ISO and GSNO. There were two groups, ISO-GSNO (ISO before GSNO treatment) and GSNO-ISO (GSNO before ISO treatment). In both groups, ISO increased contraction and relaxation. GSNO did not alter cardiac contractility compared to baseline except in the ISO-GSNO group. ISO-GSNO induced cardiac arrhythmias, and GSNO treatment prolonged the arrhythmias. Interestingly, in the GSNO-ISO group, there was no increase in arrhythmic events compared to baseline. This finding provided evidence of a differential role of NO in the heart.
Overall, the findings in this thesis show, for the first time, CaMKII plays a role in the development of arrhythmias in the whole heart during NO signaling. Additionally, this thesis highlights the dual role of NO in preventing or prolonging cardiac arrhythmias during β-adrenergic stimulation.