Catherine Proenza, PhD - US grants

Hyperpolarization-activated "pacemaker" currents are important for rhythmic firing in the mammalian heart and brain. This proposal addresses two previously undescribed features of pacemaker currents, (1) a voltage-independent instantaneous current, and (2) inactivation of mammalian currents. The molecular mechanisms responsible for these two current properties will be probed experimentally in heterologously- expressed pacemaker channels by a combination of molecular biology, chemical modification, and patch clamp electrophysiology. The presence of an instantaneous current would suggest that a background current accompanies expression of pacemaker channels in the heart and brain, and would raise the question of whether similar mechanisms exist in other ion channels. The hypotheses that cAMP-dependent inactivation existing in mammalian pacemarker currents and is altered by intracellular factors suggest a novel mechanism for modulation of these currents. Determination of the molecular mechanism for an instantaneous current and/or inactivation of mammalian pacemaker currents would advance our understanding of the control and modulation of spontaneous rhythmic activity in the heart and brain. Description of these channel behavior may also present opportunities for development of state-dependent pharmacological agents that act on pacemaker channels.

[unreadable] DESCRIPTION (provided by applicant): Each beat of the heart begins as a spontaneous electrical depolarization of specialized pacemaker cells in the sinoatrial node. The sympathetic nervous system increases heart rate primarily by releasing norepinephrine from nerves that innervate the sinoatrial node. Within the sinoatrial myocytes, this aspect of the sympathetic fight-or-flight response requires communication between the 2 adrenergic receptors (2ARs) that respond to the norepinephrine and the ion channels that collectively control the timing and shape of action potentials. The long-term goal of this project is to understand the molecular machinery that produces and regulates pacemaker activity in sinoatrial myocytes. Experiments outlined in this proposal focus on some aspects of signaling between 2ARs and hyperpolarization-activated, cyclic nucleotide sensitive (HCN, or pacemaker) ion channels. HCN channels are activated by 2ARs and are thought to be critical both for setting the resting heart rate and for mediating the positive chronotropic effect of 2 agonists. However, the biophysical mechanisms for HCN channel involvement in pacemaking, and the functional and physical relationships between HCN channels and 2 adrenergic receptors are poorly understood. The working hypotheses to be tested in this project are that a leak current produced by HCN channels is critical for pacemaker activity in sinoatrial myocytes, and that sympathetic regulation of pacemaking requires a macromolecular signaling complex that contains 2ARs and HCN channels. These questions will be addressed using expressed HCN channels, acutely isolated murine sinoatrial myocytes and cultured sinoatrial myocytes. The principle techniques to be employed are patch clamp electrophysiology, confocal immunofluorescent microscopy, and immunoaffinity chromatography. There are three specific aims: (1) To understand the biophysical mechanisms for HCN channel activity during diastole, (2) To describe the functional relationships between 2ARs and HCN channels that control firing rate in sinoatrial myocytes, and (3) To determine the subcellular localization and physical interactions of proteins that participate in sympathetic control of pacemaking. [unreadable] [unreadable] [unreadable]

The long term goals of this project are to understand the molecular and biophysical mechanisms for the regulation of cardiac pacemaking in sinoatrial node myocytes (SAMs) across the gamut of physiological conditions. SAMs function as cardiac pacemaker cells by firing spontaneous action potentials (APs). As in other excitable cells, the precise shape of sinoatrial APs reflects the composite activity of the unique complement of ion channels and transporters on the plasma membrane. AP waveforms are not static; they vary in response to short- and long-term changes in physiological context. In principle, differences in AP waveforms should lend insight into the changes in ionic currents that underlie cellular electrophysiological responses. However, our ability to decode the causal relationships between ionic currents and AP shape remains an elusive goal in all excitable cells. This gap in understanding is caused by a lack of information about AP waveforms and currents in different physiological contexts and by difficulties inherent to the study of interrelated systems using conventional research approaches. The present proposal addresses these general questions by focusing on the mechanisms by which aging slows cardiac pacemaking. Proposed experiments follow from work in prior funding periods and new preliminary data which show that aging slows pacemaking in part by decreasing the spontaneous AP firing rate of SAMs in association with changes in a limited subset of AP waveform parameters and reductions in the funny current (If) and voltage-gated Ca2+ currents (ICa,L and ICa,T). They also address the prior observation that age-dependent reductions in pacemaker activity and If in SAMs can be reversed by high concentrations of exogenous cAMP via a cAMP-mimetic mechanism. Proposed experiments will use new research tools developed during the current funding period (1) to define age-dependent changes in the relative contributions of currents active during different phases of the AP in SAMs, (2) to test the ability of different currents, singly and in combination, to transform the AP phenotype of young and aged SAMs, and (3) to test the hypothesis that age-dependent reduction in a novel If regulatory protein is responsible for the hyperpolarizing shift in voltage-dependence and resulting slowing of AP firing rate in SAMs and heart rate in mice. Results of these studies will experimentally define for the first time causal links between individual ionic currents and AP waveform parameters in SAMs that are responsible for cardiac pacemaking in general and will reveal how these mechanisms are changed during normal aging.