Luis F. Santana - US grants

The function of cerebral arteries is to provide an adequate supply of blood to the brain. TO achieve this function cerebral arteries must alter their diameter in response to physiological stimuli. Arterial diameter is regulated by the contractile state of vascular smooth muscle cells (VSMCs), which is regulated by the activity of voltage-dependent Ca2+ channels (VDCCs). The current view is that VDCCs constrict cerebral arteries through a direct contribution of Ca2+ or indirectly, by activating Ca2+ release through ryanodine receptors (RYRs) in the sarcoplasmic reticulum RyRs, called "Ca2+ sparks", hyperpolarize VSMCs by activating nearby Ca2+-sensitive potassium (K/ca) channels which close VDCCs, reduces cellular Ca2+ and thus dilates cerebral arteries. This findings underscore the importance of the location, frequency and amplitude of local Ca2+ signals in the function of VSMCs. Recent work by the PI and the collaborator suggest that vasodilators like forskolin and nitric oxide (NO) acting, respectively, through protein kinase A and G alter in a fundamental and previously unsuspected way the communication between VDCCs and RyRs. The goal of this collaborative proposal is to test the hypothesis that activation of protein kinase A and G relax cerebral arteries because they enable local Ca2+ signals from VDCCs to activate K/Ca channels and test this hypothesis the PI and collaborator will perform a series of complementary experiments that involve the use of electrophysiological, confocal microscopy and molecular techniques available in their laboratories. This proposal has three specific aims. 1). To understand the communication between VDCC and RyR, and its modulation by PKA. 2) To understand the communication between VDCC and RyR, and its modulation by PKG. 3). To understand the role of the SR protein phospholamban in the regulation of the communication between VDCCs and RyRs. While the PI will focus on the portions of the proposal involving dissociated VSMCs and gene- transfer, the collaborator will focus on experiments involving intact cerebral arteries. This division of worth will allow the complementary expertise of both investigators ti be applied in a mutually beneficial way. The proposed work should provide new fundamental information on the mechanisms that regulate tone in cerebral arteries and provide insights in the treatment of clinical conditions such as hypertension, stroke, cerebral vasospasm and migraine.

DESCRIPTION (provided by applicant): Arrhythmias are the leading cause of death among patients with heart failure (HF), yet their molecular causes are poorly understood. It has been proposed that an increase in the duration of the action potential (AP) of cardiac myocytes is the major arrhythmogenic event during HF. This prolongation of the AP of failing cardiac myocytes occurs in the absence of known genetic changes that affect membrane currents. Preliminary experiments performed in our laboratory suggest that Na+ (INa) and transient outward K+ (Ito) currents are altered during HF in ways that could prolong the AP of failing cardiac myocytes. Our most recent data suggest that a defect in the post-translational processing of Na+ and Kv4 channels during HF is the cause for these dysfunctional INa and Ito currents. Furthermore, we have evidence that links these changes in Na+ and Kv4 channel currents to the activation of the calcineurin/NFAT genetic pathway in the early stages of HF. The overall goal of this application is to test the hypothesis that the changes in INa and Ito currents observed during HF are the result of poor or incomplete glycosylation of these proteins during post-translational processing, due to the activation of NFAT by calcineurin. To test this hypothesis we will perform a series of complementary experiments that involve the use of molecular, cellular, biochemical, electrophysiological and imaging techniques. This proposal has four specific aims. 1) To investigate the effects of a sustained increase in resting Ca2+ levels on INa and Ito in ventricular myocytes. 2) To investigate the effects of calcineurin-induced NFAT activation on INa and Ito. 3) To investigate the molecular determinants of the changes in INa and Ito during HF. 4) To investigate the effects of calcineurin-induced NFAT activation on INa and Ito in a heterologous expression system. The proposed work should provide fundamental information on the molecular mechanisms underlying arrhythmias during HF and could provide insights in the development of effective therapeutic strategies for this condition.

Voltage-gated K? channels (Kv) and large-conductance, Ca2+-activated K? (BK) channels control the excitability of vascular smooth muscle. By controlling membrane potential, these channels indirectly regulate Ca 2+channel activity and hence Ca 2+ influx into these cells. Activation of Ca 2+channels causes a global, cellwide increase in intracellular Ca 2+that leads to vasoconstriction. In sharp contrast, we recently found that local, sub-cellular Ca 2+release events through ryanodine receptors (RyR) located in the sarcoplasmic reticulum ("Ca 2?sparks") indirectly relax vascular smooth muscle by activating BK channels and thereby hyperpolarizing smooth muscle cells. Membrane hyperpolarization causes smooth muscle relaxation because it decreases Ca 2?channel opening probability, which decreases intracellular Ca 2+.The experiments outlined in this proposal will investigate the cellular and molecular mechanisms controlling the function of Kv and BK channels in vascular smooth muscle. Our preliminary studies suggest that the Ca+-dependent phosphatase calcineurin modulates the function of Kv and BK channel function in vascular smooth muscle either directly, by controlling the phosphorylation state of these channels, or indirectly, through its control of the transcription factor nuclear factor of activated T cells (NFAT). Furthermore, recent data suggests that the molecular composition and function of BK channels is altered during hypertension. Over the next five years we plan to test four specific hypotheses. First, that activation of calcineurin modifies the communication between BK channels and ryanodine receptors in vascular smooth muscle. Second, that reduced expression of the 131subunit of BK channels during hypertension reduces the sensitivity of these channels to physiological changes in Ca 2?.Third, that activation of NFAT leads to changes in the expression of Kv and BK channels in cerebral vascular smooth muscle. Fourth, that local Ca 2+signals control the nuclear translocation of NFAT in vascular smooth muscle. These experiments will provide new fundamental information on the mechanisms controlling Kv and BK channel function in cerebral vascular smooth muscle and will provide insights into the mechanisms underlying vasospasm, stroke and hypertension.

[unreadable] DESCRIPTION (provided by applicant): Voltage-gated K+ (Kv) currents are differentially distributed across the left ventricular wall. Kv4 currents are larger in left ventricular epicardial (EPI) cells than in endocardial (ENDO) cells. This non-uniform distribution of Kv4 channel function is essential for normal myocardial repolarization. We recently reported that variations in [Ca2+]i are transduced into changes in Kv4 expression through the activation of the Ca2+-sensitive phosphatase calcineurin and the transcription factor NFATc3. This led to our discovery that differential [Ca2+]i/calcineurin/NFATc3 signaling across the left ventricular free wall underlies transmural variations in Kv4 expression. However, the mechanisms underlying regional differences in [Ca2+]j, calcineurin, and NFATc3 signaling are poorly understood. The work proposed in this application employs a series of novel techniques and approaches developed by our group to address these important issues. Our preliminary data suggest that the proposed experiments are not only feasible but will provide new fundamental information regarding Kv channel regulation in the heart. The proposed work addresses three specific hypotheses. First, we will test the hypothesis that regional variations in [Ca2+]j underlie heterogeneous calcineurin activity in the ventricle. Second, we will test the hypothesis that local and global [Ca2+]i signals modulate NFAT translocation and gene expression in ventricular myocytes. We will then use these data to determine how variations in [Ca2+]j signals between ENDO and EPI cells lead to regional differences in NFAT activity. Finally, we will test the hypothesis that calcineurin/NFATc3 signaling is essential for maintaining Kv current heterogeneity in the ventricle. Taken together, this work will provide the first integrated view of calcium signaling, excitability, and contractility in the heart and significantly enhance our understanding of the basic mechanisms, which regulate Kv channel function in health and disease. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Calcium influx via dihydropyridine-sensitive, voltage-gated L-type calcium channels plays a crucial role in the regulation of excitability, contraction, and gene expression in arterial smooth muscle. Our recent discovery that small clusters of L-type calcium channels can operate in a "persistent" gating mode that create sites of nearly continual calcium influx (called "persistent calcium sparklets") in smooth muscle has led to a paradigm shift, whereby calcium influx in these cells is predominantly controlled by this process in combination with rare voltage-dependent openings of individual L-type calcium channels. However, the role of persistent calcium sparklets on the regulation of local and global intracellular calcium as well as the molecular mechanisms underlying the activation and modulation of these calcium influx events in smooth muscle under physiological and pathophysiological conditions is virtually unknown. The proposed work will use new methods developed by our group to define the biophysical properties and functional roles of calcium sparklets in cerebral artery smooth muscle. Our preliminary results suggest that all proposed experiments are feasible and will provide important new information. The proposed work seeks to test three novel hypotheses. In Specific Aim 1, we will test the hypothesis that calcium influx via calcium sparklets contributes to changes in local and global intracellular calcium concentration. The experiments in Specific Aim 2 will test the hypothesis that Cav1.2 channels underlie calcium sparklets in arterial smooth muscle. Finally, in Specific Aim 3, we build on the work in the previous two Specific Aims and test the hypothesis that persistent calcium sparklet activity is increased during hypertension. This work should provide the first integrated view of calcium sparklet-mediated signaling and their role in modulating cerebral arteries function and significantly enhance our understanding of arterial function in health and disease.

Project Summary The work outlined in this application stems from our recent discovery that dihydropyridine-sensitive, voltage- gated CaV1.2 and CaV1.3 channels form clusters that undergo dynamic allosteric interactions, which allow cooperative gating of these channels in cardiac myocytes. The significance of these findings is underscored by our demonstration that coupled activation of these channels modulates pace-making activity in sinoatrial node (SAN) cells (CaV1.2 and CaV1.3) and contraction in ventricular myocytes (CaV1.2) under physiological and pathological conditions. The experiments proposed in this application test a novel model for the regulation of CaV1.2 and CaV1.3 channel activity in SAN and ventricular myocytes. In this model, CaV1.2 and CaV1.3 channels undergo reciprocal physical and functional interactions that are initiated by increases in intracellular Ca2+ concentration ([Ca2+]i). During the action potential, channel-to-channel coupling is initiated when membrane depolarization opens CaV1.2 and CaV1.3 channels, allowing a small amount of Ca2+ to enter the cell. The incoming Ca2+ binds to calmodulin (CaM), thereby promoting physical coupling of adjacent channels via the pre-IQ domains located in the C-tails of the channels. Physical contact increases the activity of adjoined channels. As individual channels within a cluster inactivate and close, [Ca2+]i decreases and coupling fades, but persists longer than the current that evoked it, serving as a type of `molecular memory'. A new concept in our model is that the overall activity of CaV1.2 and CaV1.3 channels within a cluster depends on the number of channels that couple and the duration of these interactions. The project will test the physiological and pathological implications of this model in three specific aims. Specific aim 1 tests the hypothesis that coupling between CaV1.2 and CaV1.3 channels in SAN cells regulates pace-making activity. Specific aim 2 tests the hypothesis that persistent CaV1.2 channel coupling in ventricular myocytes induces long-term potentiation of Ca2+ currents and increases contractility. Specific aim 3 tests the hypothesis that long-QT syndrome CaM mutants increase the probability of arrhythmogenesis by altering functional coupling between CaV1.2 channels. Diverse, state-of-the-art methods, including patch-clamp electrophysiology, optical clamping, optogenetics and confocal, TIRF, and super-resolution microscopy, will be used to achieve these aims.

DESCRIPTION (provided by applicant): The experiments in this application will test the hypothesis that clusters of voltage-gated L-type CaV1.2 channels are capable of undergoing coordinated openings (coupled gating), amplifying Ca2+ influx into arterial smooth muscle. A key discovery is that Ca2+ influx via coupled CaV1.2 channels plays a critical role in excitation-contraction (EC) coupling and excitation-transcription (ET) coupling in arterial myocytes. Preliminary data suggest that association of CaV1.2 channels with the anchoring protein AKAP150 is necessary for coupled gating activity in these cells. The significance of these findings is underscored by the observation that the frequency of coupled CaV1.2 gating events increases in arterial smooth muscle during hypertension and that loss of AKAP150 protects against coupled gating and hypertension. The project has two specific aims designed to investigate the mechanisms and physiological implications of these findings. Specific aim 1 is to test the hypothesis that coupled gating of CaV1.2 channels amplifies Ca2+ influx in arterial myocytes. Specific aim 2 is to test the hypothesis that AKAP150 is required for increased coupled CaV1.2 channel activity and the induction of arterial dysfunction during the development of hypertension. The methods that will be used to achieve these aims include patch-clamp electrophysiology, optical clamping, light- and chemically-induced dynamic targeting of kinases to cellular membranes, light-induced activation of adrenergic signaling (i.e., optogenetics), confocal, and TIRF microscopy. Experiments will involve new transgenic, knock in, and knock out mice. This work will generate fundamental information on the mechanisms by which AKAP150 and CaV1.2 channels control of excitability, gene expression, and EC coupling in vascular smooth muscle under physiological and pathological conditions.

Project Summary Dihydropyridine-sensitive, L-type Cav1.2 and delayed rectifier Kv2.1 channels play critical roles in the regulation of excitability and contraction in arterial smooth muscle. A salient feature of Cav1.2 channels is that they form clusters within which they undergo dynamic, reciprocal interactions that allow functional coupling of adjacent channels and thus amplification of Ca2+ signaling, which is critical to the development of myogenic tone. At present, however, the mechanisms controlling Cav1.2 clustering are unknown. The Trimmer and Santana labs have joined forces to address this fundamental issue. New preliminary data from our labs suggest a novel model that represents a paradigm shift relative to the generally accepted canonical role of Kv2.1, and K+ channels in general, as acting solely as K+ conducting electrical determinants of the intrinsic membrane properties of arterial myocytes. In this model, the Kv2.1 channel has a physical role to increase clustering and thus cooperative gating of Cav1.2 channels. Our data indicate that the balance between the separable electrical and structural roles of Kv2.1 channels fine tunes membrane potential, Cav1.2 clustering, functional coupling of these channels, and hence Ca2+ influx, myogenic tone, and, ultimately, blood pressure. A key finding that underscores the significance of our work is that Kv2.1 expression varies with sex, leading to significant differences in Ca2+ influx and myogenic tone between female and male arterial myocytes. The combination of our complementary skill sets allows us to implement a multi-scale systems approach that involves the use of cellular, molecular, biophysical, imaging, gene editing and whole-animal approaches to rigorously investigate the mechanisms controlling Kv2.1 and Cav1.2 organization, and how they impact cell, organ, and whole-body functions under physiological conditions. The project has three specific aims. Aim 1 is to determine the impact of altered Kv2.1 expression levels on clustering and activity of Cav1.2 channels, and myogenic tone in arterial smooth muscle, and on blood pressure. Aim 2 is to define the mechanisms underlying Kv2.1-mediated regulation of Cav1.2 function. Finally, Aim 3 is to use novel genetic models to define the cell autonomous role of Kv2.1, and its separable conducting and non-conducting functions, in regulating Cav1.2 function, and the myogenic response in arterial smooth muscle cells, and systemic blood pressure. The proposed studies have the potential of transforming our understanding of how ion channels are organized in vascular smooth muscle, and provide insights into how arterial diameter and blood pressure are differentially regulated in females versus males.

Project Summary The function of arteries is to deliver oxygen and nutrients necessary to sustain the function and survival of every cell in the body. Arterial diameter, a key determinant of blood flow, is finely tuned by the contractile state of the smooth muscle cells lining the walls of these vessels. To date, however, models of myogenic control of arterial smooth muscle tone have largely been based on data from male myocytes. Yet, recent data from our team suggest that sex-specific features that determine the operation of arterial myocytes are attributable to differences in the spatial organization of signaling complexes formed by the PKC-anchoring protein AKAP150 and CaV1.2 channels, and the manner in which they regulate smooth muscle contractility. Our findings challenge the general applicability of a model for electrical and pharmacological control of the myogenic response based on data from male myocytes and support the view that myogenic responses are differentially regulated in male and female arteries. In this project, we implement a multi-scale systems approach that includes super-resolution imaging, electrophysiology, and computational approaches to rigorously investigate the mechanisms controlling blood flow through the male and female pial-parenchymal circulatory unit under physiological and pathological conditions. Specific aim 1 is to establish the fundamental sex-specific mechanisms controlling Ca2+ influx via CaV1.2 channels in vascular smooth muscle. Specific aim 2 is to determine the impact of the physical organization of type 1 AngII receptors (AngIIR1), AKAP150, PKC?, and CaV1.2 channels on myogenic tone in male and female arterial smooth muscle. Finally, in specific aim 3 we will investigate whether changes in the number and molecular organization of CaV1.2 and AngIIR1/AKAP150/PKC? signaling units differentially alter Ca2+ influx, arterial wall [Ca2+]i, and myogenic tone in male and female smooth muscle during the development of hypertension. This work will lead to the first model of vascular smooth muscle function that incorporates sex- specific variations in protein organization, electrical activity, and Ca2+ signaling during health and disease, which could inform the development of rational strategies for the treatment of hypertension in male and female.

L-type Ca2+ channels (LTCCs) play a fundamental role in brain neurons as mediators of diverse Ca2+ signaling events. LTCCs on neuronal somata play a unique and crucial role in regulating Ca2+-dependent gene expression. A salient feature of LTCCs is that their activity is regulated by clustering through cooperative gating of clustered channels. Their clustering also localizes them to specialized Ca2+ signaling microdomains within which they functionally couple to Ca2+-dependent proteins that transduce the impact of LTCC-mediated Ca2+ entry to specific Ca2+ signaling pathways. Through their canonical function as K+ conducting voltage-gated channels, somatic Kv2.1 channels play critical roles in the regulation of action potentials, with a subsequent impact on LTCC activity. The general consensus is that the functions of LTCCs and Kv2.1 channels in neurons are otherwise largely independent from one another. Our recent work challenges this view. We discovered a novel and unexpected nonconducting role for Kv2.1 in physically regulating the organization of neuronal LTCCs, enhancing their activity and impacting their localization in specific microdomains. These exciting new results lead to a novel model that in brain neurons, Kv2.1 plays dual roles, one as a canonical K+ channel shaping the intrinsic membrane properties of neurons, and the other a nonconducting physical role to cluster LTCCs to enhance their activity and localize them in Ca2+ signaling microdomains. The combination of the complementary backgrounds and skill sets of the Timmer and Santana labs allows us to implement a multi-scale systems approach that involves the use of cellular, molecular, biophysical, imaging, gene editing and whole-animal approaches to rigorously investigate the molecular mechanisms whereby Kv2.1 impacts LTCC organization, and the consequences to LTCC function and neuronal signaling. The project has three specific aims, which are to determine how selectively eliminating 1) Kv2.1 expression, 2) Kv2.1 clustering, and 3) the ability of Kv2.1 to enhance LTCC clustering impacts somatic LTCC localization and function, Ca2+-induced Ca2+ release or sparks, and LTCC-dependent transcript factor activation. The proposed studies have the potential of transforming our understanding of how neuronal ion channels are regulated and how this impacts Ca2+ signaling in health and when altered in disease.

PROJECT SUMMARY: A major factor plaguing drug development is that there is no drug-screening tool that can distinguish between drugs that will induce cardiac arrhythmias from chemically similar safe drugs. The current approaches rely on substitute markers such as action potential duration or QT interval prolongation on the ECG. There is an urgent need to identify a new approach that can predict actual proarrhythmia from the drug chemistry rather than relying on surrogate indicators. We have brought together an expert team to innovate at the interfaces of experimental and computational modeling disciplines and develop an in silico simulation pipeline to predict cardiotoxicity over multiple temporal and spatial scales from the atom to the cardiac rhythm. An essential and unique aspect of our approach is that we propose to utilize atomistic scale simulation to predict the transition rates of ion channels and adrenergic receptors and how they are modified by drug interaction. We hypothesize that it is the subtleties of these interactions that are likely to be the critical determinants of drug associated safety or proarrhythmia. In the last award period, we successfully developed an unprecedented linkage: We connected the highly disparate space and time scales of ion channel structure and function. We utilized atomistic simulation to compute drug kinetic rates were directly used as parameters in a hERG function model. The model components were then integrated into predictive models at the cell and tissue scales to expose fundamental arrhythmia vulnerability mechanisms and complex interactions underlying emergent behaviors. Human clinical data were used for model validation and showed excellent agreement, demonstrating feasibility of this new approach for cardiotoxicity prediction. In this renewal application we propose to hugely extend this approach to include prediction of the interaction of cardiac channel gating and drug interaction as well as the inclusion of adrenergic receptor interactions with drugs. Another essential aspect of safety pharmacology is the development of new approaches to allow more efficient drug design, screening and prediction of cardiotoxicity. Therefore, we will seek to develop, extend and apply a variety of machine learning and deep learning approaches to improve drug discovery by predicting proarrhythmia from the drug chemistry with an efficient process that identify drug congeners via machine learning to maximize therapy and minimize side effects. Finally, we propose to classify drugs into categories based on proarrhythmia risk in normal and diseased virtual tissue settings. The multiscale model for prediction of cardiopharmacology that we will develop in this application will be applied to projects demonstrating its usefulness for efficacy or toxicity of drug treatments in the complex physiological system of the heart.