Popis: |
The failing human heart is characterized by an inability to pump blood at a rate commensurate with the demands of the body. Heart failure is a complex syndrome in which a number of cellular and intracellular processes become altered, progressively leading to poor cardiac function. Over the past two decades, it has become clear that the maintenance of normal intracellular calcium homeostasis by the sarcoplasmic reticulum (SR) is critically important; indeed, depressed SR calcium cycling is a hallmark of heart failure. Thus, efforts have been made to characterize the mechanisms responsible for maintaining normal SR calcium cycling, and to understand why SR calcium cycling becomes depressed in the context of heart failure. With a clear understanding of the causes of poor SR calcium cycling, novel therapeutic approaches directed at correcting calcium homeostasis may be developed.In this dissertation, the roles of Histidine-Rich Calcium Binding Protein (HRC) and Protein Phosphatase Inhibitor-1 (I-1) in regulating SR calcium cycling were investigated. HRC is an intraluminar SR Ca2+-binding protein which appears to play a role in SR calcium release, reuptake, and storage. I-1, in contrast, is a regulator of the phosphorylation status of several proteins, and plays a role in regulating the overall rate and amplitude of SR calcium cycling. To elucidate the role of HRC, its expression was altered by genetic manipulation. Studies revealed that HRC interacts with SERCA in a calcium-dependent manner, and may influence the maximum SR calcium-uptake velocity and cardiac function.Using recombinant inhibitor-1 wild-type and mutated proteins, a novel phosphorylation site on inhibitor-1 was identified: Threonine-75. This site was phosphorylated in vitro by protein kinase C-α independently and to the same extent as serine-67, the previously known protein kinase C-α site. Generation of specific antibodies for the phosphorylated and dephosphorylated threonine-75 revealed that this site is phosphorylated in both canine and rat cardiac tissue. Adenoviral-mediated expression of the constitutively phosphorylated threonine-75 inhibitor-1 in isolated cardiomyocytes was associated with specific stimulation of type-1 protein phosphatase activity and marked reduction in the affinity of SERCA for calcium, resulting in depressed contractility. Thus, phosphorylation of inhibitor-1 at threonine-75 represents a new mechanism of cardiac contractility regulation, partially through the alteration of sarcoplasmic reticulum calcium transport activity.Given these results, the functional consequences of constitutive phosphorylation of Ser-67 were examined to determine whether these two “neighboring” sites were equivalent in function. Interestingly, constitutive phosphorylation of Ser-67 also led to depressed cardiomyocyte mechanical function and SR calcium transport rates. Similar results were also obtained in cardiomyocytes expressing an I-1 form in which both sites were constitutively phosphorylated. These studies established that phosphorylation of either Ser-67 or Thr-75 leads to depressed cardiomyocyte function.Since constitutive phosphorylation of either Ser-67 or Thr-75 resulted in depressed cardiomyocyte function, it was next investigated whether PKA stimulation could reverse or correct these deficits. Though improvements in SR calcium uptake and cardiomyocyte mechanical function were observed following forskolin treatment, both of these parameters remained depressed compared to control cells. Thus, it appeared that constitutive phosphorylation of I-1 at Ser-67 or Thr-75 prohibits the cells from attaining the same contractile state as cells expressing I-1 WT.In sum, these studies provide further insights into the regulatory mechanisms involved in SR calcium cycling, and form the basis for many future studies. As the roles of HRC and I-1 in regulating SR calcium cycling are clarified, a better understanding of the pathogenesis of heart failure will be obtained. |