Evolution of the Cardiac Electrophysiological System and Regulation of Ion Channel Gene Expression
The Graduate School, Stony Brook University: Stony Brook, NY.
There are two basic ways by which biological systems can evolve, either by changes in the sequence of protein coding regions (structural evolution) or by changes in gene regulatory function (regulatory evolution). The relative importance of regulatory versus structural evolution for the evolution of different biological systems is a subject of controversy. The primacy of regulatory evolution in the diversification of morphological traits has been promoted by many evolutionary developmental biologists. For physiological traits, however, the role of regulatory evolution has received less attention or has been considered to be relatively unimportant. To address this issue for electrophysiological systems, I examined the importance of regulatory and structural evolution in the evolution of the electrophysiological function of cardiac myocytes in mammals. The enormous variation in mammalian body size is an important factor in the evolutionary success of this class of animals and appropriate scaling of cardiovascular system function is critical in supporting this morphological diversity. Scaling of cardiac electrophysiology with body mass requires large changes in the ventricular action potential duration and heart rate in mammals. These changes in cellular electrophysiological function are produced by systematic and coordinated changes in the expression of multiple ion channel and transporter genes. Two related phenomena were first studied: the change in action potential morphology in small mammals and the scaling of action potential duration across mammalian phylogeny. In general, the functional properties of the ion channels involved in ventricular action potential repolarization were found to be relatively invariant. In contrast, there were large changes in the expression levels of multiple ion channel and transporter genes. For the Kv2.1 and Kv4.2 potassium channel genes, which are primary determinants of the action potential morphology in small mammals, the functional properties of the proximal promoter regions were found to vary in concordance with species dependent differences in mRNA expression, suggesting that evolution of cis-regulatory elements is the primary determinant of this trait. Scaling of action potential duration was found to be a complex phenomenon, involving changes in the expression of a large number of channels and transporters. Given the static nature of mammalian ion channel coding sequences and gene number, regulatory evolution is concluded as the primary mechanisms for the evolution of most mammalian electrophysiological systems. Expression of one important potassium current, the transient outward current (Ito), changes significantly during mammalian evolution. Changes in Ito expression are determined, in part, by variation in the expression of an obligatory auxiliary subunit encoded by the KChIP2 gene. Expression of the KChIP2 gene is restricted to electrically excitable cells, primarily cardiac myocytes and a subset of neurons. Transcription in both heart and brain is initiated from the same CpG island promoter, which belongs to a large and poorly understood class of promoters in mammals. Species-dependent variation of KChIP2 expression in heart is mediated by the evolution of the cis-regulatory function of this gene. Surprisingly, the major locus of evolutionary change for KChIP2 gene expression in heart lies within the CpG island core promoter. It was demonstrated that CpG island promoters are not simply permissive for gene expression but can also contribute to tissue-selective expression and, as such, can function as an important locus for the evolution of cis-regulatory function. More generally, evolution of the cis-regulatory function of voltage-gated ion channel genes appears to be an effective and efficient way to modify channel expression levels in order to optimize electrophysiological function. In addition, appropriate regulation of ion channel expression is critical for the maintenance of both electrical stability and normal contractile function in the heart. Mechanisms that contribute to maintaining expression of functional ion channels at relatively constant levels following perturbations of channel biosynthesis are likely to contribute significantly to the stability of electrophysiological systems in some pathological conditions. In order to examine the robustness of L-type calcium current expression, the response to changes in Ca2+ channel Cav1.2 gene dosage was studied in adult mice. Using a cardiac-specific inducible Cre recombinase system, Cav1.2 mRNA was reduced to 11 ?? 1% of control values in homozygous floxed mice and the mice died rapidly (11.9 ?? 3 days) after induction of gene deletion. For these mice, no effective compensatory changes in ion channel gene expression were triggered following deletion of both Cav1.2 alleles, despite the dramatic decay in cardiac function. In contrast to the homozygote knockout mice, following knockout of only one Cav1.2 allele, cardiac function remained unchanged, as did survival. Cav1.2 mRNA expression in the left ventricle of heterozygous knockout mice was reduced to 58 ?? 3% of control values and there was a 21 ?? 2% reduction in Cav1.2 protein expression. There was no significant reduction in L-type Ca2+ current density in these mice. The results are consistent with a model of L-type calcium channel biosynthesis in which there are one or more saturated steps, which act to buffer changes in both total Cav1.2 protein and L-type current expression.