SR Calcium Cycling Protein Genetic Variations in Human Heart Failure

Our basic studies have recently been extended to the Clinical Arena, in collaboration with our Cardiology Colleagues. We have examined the expression levels of the SR Ca2+-handling proteins in normal and failing human hearts, since their depressed cardiac function has been suggested to reflect abnormalities at the sarcoplasmic reticulum level. Our findings indicate that the levels of SR Ca2+ cycling proteins, such as phospholamban relative to the SR Ca2+-ATPase are increased in human heart failure. This increase may constitute a contributing factor to diastolic dysfunction and depressed contractility exhibited by the failing heart, as indicated by our studies in the transgenic mice. Furthermore, decreased phosphorylation of PLN, without alterations in its protein levels, has been suggested to contribute to the depressed function in human heart failure. The attenuated phospholamban phosphorylation is at least partially due to increased protein phosphatase-1 activity, reflecting inactivation of its Inhibitor-1 protein in human failing hearts. Indeed, both Inhibitor-1 and phospholamban phosphorylation are decreased in human heart failure. Thus, our hypothesis is that: a) the activities of SR Ca2+-ATPase, phospholamban and Inhibitor-1 are major determinants of cardiac function; and b) naturally occurring mutations in the SR Ca2+-ATPase, phospholamban and Inhibitor-1 genes may alter SR Ca2+ cycling and contractility, leading to remodeling and heart failure. In our lab, we are examining whether naturally occurring genetic variations (polymorphisms) may exist in the coding regions of these major sarcoplasmic reticulum Ca2+-handling proteins which may act as "modifiers" of the clinical time course of the disease in patients with heart failure. These studies are done in collaboration with the Cardiologists at the University of Cincinnati and the University of Athens, Greece.

Human SERCA2 Mutations:

We screened a large number of dilated cardiomyopathy patients and identified some mutations in the SR Ca2+-ATPase gene. However, none of these variants changed the amino acid composition, indicating that this gene might be evolutionary conserved in humans.

Human PLN Mutations:

In pursuit of identifying naturally occurring mutation(s) in the human phospholamban gene, a truncation mutation (L39stop) was identified in individuals from two families with inherited dilated cardiomyopathy that resulted in dramatically diminished myocardial phospholamban protein content, consequent loss of phospholamban inhibition of SR Ca2+-ATPase, and the development of heart failure and early mortality in homozygous individuals (Figure 1). These findings demonstrate that, in contrast to mice in which phospholamban-deficiency enhances myocardial inotropy and lusitropy without adverse effects, phospholamban is essential for cardiac health in humans and its absence results in lethal heart failure. Currently, we are assessing the physiological significance of the identified L39stop mutation using a transgenic mouse model harboring the mutation.

We have also extended our studies to screen the 5’ and 3’ UTR of the phospholamban gene for naturally occurring mutations in dilated cardiomyopathy (DCM) patients. A single nucleotide transition was identified in the 5’ UTR of the phospholamban gene (Figure 2). We are further analyzing the functional significance of this mutation.

Human Inhibitor-1 Mutations:

In addition to our ongoing studies in phospholamban, parallel studies on the Inhibitor-1 of protein phosphatase-1, which is the major phospholamban phosphatase in vivo, have been initiated. Thus, our hypothesis is that mutation(s) in the Inhibitor-1 may predispose human subjects to heart failure. To identify such naturally occurring mutation(s) in the human Inhibitor-1 gene, we have screened genomic DNA from DCM patients and identified some genetic variations. Currently, we are studying the significance of these mutations using in vitro and in vivo methods.