Jeffrey J. Saucerman

Modeling β-Adrenergic Control of Cardiac Myocyte Contractility in Silico

The β-adrenergic signaling pathway regulates cardiac myocyte contractility through a combination of feedforward and feedback mechanisms. We used systems analysis to investigate how the components and topology of this signaling network permit neurohormonal control of excitation-contraction coupling in the rat ventricular myocyte. A kinetic model integrating β-adrenergic signaling with excitation-contraction coupling was formulated, and each subsystem was validated with independent biochemical and physiological measurements. Model analysis was used to investigate quantitatively the effects of specific molecular perturbations. 3-Fold overexpression of adenylyl cyclase in the model allowed an 85% higher rate of cyclic AMP synthesis than an equivalent overexpression of β1-adrenergic receptor, and manipulating the affinity of Gsα for adenylyl cyclase was a more potent regulator of cyclic AMP production. The model predicted that less than 40% of adenylyl cyclase molecules may be stimulated under maximal receptor activation, and an experimental protocol is suggested for validating this prediction. The model also predicted that the endogenous heat-stable protein kinase inhibitor may enhance basal cyclic AMP buffering by 68% and increasing the apparent Hill coefficient of protein kinase A activation from 1.0 to 2.0. Finally, phosphorylation of the L-type calcium channel and phospholamban were found sufficient to predict the dominant changes in myocyte contractility, including a 2.6× increase in systolic calcium (inotropy) and a 28% decrease in calcium half-relaxation time (lusitropy). By performing systems analysis, the consequences of molecular perturbations in the β-adrenergic signaling network may be understood within the context of integrative cellular physiology.

Systems analysis of PKA-mediated phosphorylation gradients in live cardiac myocytes.

Compartmentation and dynamics of cAMP and PKA signaling are important determinants of specificity among cAMP’s myriad cellular roles. Both cardiac inotropy and the progression of heart disease are affected by spatiotemporal variations in cAMP/PKA signaling, yet the dynamic patterns of PKA-mediated phosphorylation that influence differential responses to agonists have not been characterized. We performed live-cell imaging and systems modeling of PKA-mediated phosphorylation in neonatal cardiac myocytes in response to G-protein coupled receptor stimuli and UV photolysis of “caged” cAMP. cAMP accumulation was rate-limiting in PKA-mediated phosphorylation downstream of the β-adrenergic receptor. Prostaglandin E1 stimulated higher PKA activity in the cytosol than at the sarcolemma, whereas isoproterenol triggered faster sarcolemmal responses than cytosolic, likely due to restricted cAMP diffusion from submembrane compartments. Localized UV photolysis of caged cAMP triggered gradients of PKA-mediated phosphorylation, enhanced by phosphodiesterase activity and PKA-mediated buffering of cAMP. These findings indicate that combining live-cell FRET imaging and mechanistic computational models can provide quantitative understanding of spatiotemporal signaling.

Proarrhythmic Consequences of a KCNQ1 AKAP-Binding Domain Mutation: Computational Models of Whole Cells and Heterogeneous Tissue

The KCNQ1-G589D gene mutation, associated with a long-QT syndrome, has been shown to disrupt yotiao-mediated targeting of protein kinase A and protein phosphatase-1 to the IKs channel. To investigate how this defect may lead to ventricular arrhythmia during sympathetic stimulation, we use integrative computational models of &bgr;-adrenergic signaling, myocyte excitation-contraction coupling, and action potential propagation in a rabbit ventricular wedge. Paradoxically, we find that the KCNQ1-G589D mutation alone does not prolong the QT interval. But when coupled with &bgr;-adrenergic stimulation in a whole-cell model, the KCNQ1-G589D mutation induced QT prolongation and transient afterdepolarizations, known cellular mechanisms for arrhythmogenesis. These cellular mechanisms amplified tissue heterogeneities in a three-dimensional rabbit ventricular wedge model, elevating transmural dispersion of repolarization and creating other T-wave abnormalities on simulated electrocardiograms. Increasing heart rate protected both single myocyte and the coupled myocardium models from arrhythmic consequences. These findings suggest that the KCNQ1-G589D mutation disrupts a critical link between &bgr;-adrenergic signaling and myocyte electrophysiology, creating both triggers of cardiac arrhythmia and a myocardial substrate vulnerable to such electrical disturbances.

Calmodulin Mediates Differential Sensitivity of CaMKII and Calcineurin to Local Ca2+ in Cardiac Myocytes

Calmodulin (CaM) mediates Ca-dependent regulation of numerous pathways in the heart, including CaM-dependent kinase (CaMKII) and calcineurin (CaN), yet the local Ca(2+) signals responsible for their selective activation are unclear. To assess when and where CaM, CaMKII, and CaN may be activated in the cardiac myocyte, we integrated new mechanistic computational models of CaM, CaMKII, and CaN with the Shannon-Bers model of excitation-contraction coupling in the rabbit ventricular myocyte. These models are validated with independent in vitro data. In the intact myocyte, model simulations predict that CaM is highly activated in the dyadic cleft during each beat, but not appreciably in the cytosol. CaMKII-delta(C) was almost insensitive to cytosolic Ca due to relatively low CaM affinity. Dyadic cleft CaMKII exhibits dynamic frequency-dependent responses to Ca, yet autophosphorylates only when local phosphatases are suppressed. In contrast, dyadic cleft CaN in beating myocytes is predicted to be constitutively active, whereas the extremely high affinity of CaN for CaM allows gradual integration of small cytosolic CaM signals. Reversing CaM affinities for CaMKII and CaN also reverses their characteristic local responses. Deactivation of both CaMKII and CaN seems dominated by Ca dissociation from the complex (versus Ca-CaM dissociation from the target). In summary, the different affinities of CaM for CaMKII and CaN determine their sensitivity to local Ca signals in cardiac myocytes.

Synergy between CaMKII Substrates and β-Adrenergic Signaling in Regulation of Cardiac Myocyte Ca2+ Handling

Cardiac excitation-contraction coupling is a highly coordinated process that is controlled by protein kinase signaling pathways, including Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and protein kinase A (PKA). Increased CaMKII expression and activity (as occurs during heart failure) destabilizes EC coupling and may lead to sudden cardiac death. To better understand mechanisms of cardiac CaMKII function, we integrated dynamic CaMKII-dependent regulation of key Ca(2+) handling targets with previously validated models of cardiac EC coupling, Ca(2+)/calmodulin-dependent activation of CaMKII, and β-adrenergic activation of PKA. Model predictions are validated against CaMKII-overexpression data from rabbit ventricular myocytes. The model demonstrates how overall changes to Ca(2+) handling during CaMKII overexpression are explained by interactions between individual CaMKII substrates. CaMKII and PKA activities during β-adrenergic stimulation may synergistically facilitate inotropic responses and contribute to a CaMKII-Ca(2+)-CaMKII feedback loop. CaMKII regulated early frequency-dependent acceleration of relaxation and EC coupling gain (which was highly sarcoplasmic reticulum Ca(2+) load-dependent). Additionally, the model identifies CaMKII-dependent ryanodine receptor hyperphosphorylation as a proarrhythmogenic trigger. In summary, we developed a detailed computational model of CaMKII and PKA signaling in cardiac myocytes that provides unique insights into their regulation of normal and pathological Ca(2+) handling.

Regulation of nuclear PKA revealed by spatiotemporal manipulation of cyclic AMP

Understanding how specific cAMP signals are organized and relayed to their effectors in different compartments of the cell to achieve functional specificity requires molecular tools that allow precise manipulation of cAMP in these compartments. Here we characterize a new method using bicarbonate-activatable and genetically targetable soluble adenylyl cyclase (sAC) to control the location, kinetics and magnitude of the cAMP signal. Using this live-cell cAMP manipulation in conjunction with fluorescence imaging and mechanistic modeling, we uncover the activation of a resident pool of PKA holoenzyme in the nuclei of HEK-293 cells, modifying the existing dogma of cAMP-PKA signaling in the nucleus. Furthermore, we show that phosphodiesterases (PDE) and A-Kinase Anchoring Proteins (AKAP) are critical in shaping nuclear PKA responses. Collectively, our data suggests a new model where AKAP-localized PDEs tune an activation threshold for nuclear PKA holoenzyme, thereby converting spatially distinct second messenger signals to temporally controlled nuclear kinase activity.

Endotoxin depresses heart rate variability in mice: cytokine and steroid effects

Heart rate variability (HRV) falls in humans with sepsis, but the mechanism is not well understood. We utilized a mouse model of endotoxemia to test the hypothesis that cytokines play a role in abnormal HRV during sepsis. Adult male C57BL/6 mice underwent surgical implantation of probes to continuously monitor electrocardiogram and temperature or blood pressure via radiotelemetry. Administration of high-dose LPS (Escherichia coli LPS, 10 mg/kg, n = 10) caused a biphasic response characterized by an early decrease in temperature and heart rate at 1 h in some mice, followed by a prolonged period of depressed HRV in all mice. Further studies showed that LPS doses as low as 0.01 mg/kg evoked a significant decrease in HRV. With high-dose LPS, the initial drops in temperature and HR were temporally correlated with peak expression of TNFalpha 1 h post-LPS, whereas maximal depression in HRV coincided with peak levels of multiple other cytokines 3-9 h post-LPS. Neither hypotension nor hypothermia explained the HRV response. Pretreatment with dexamethasone prior to LPS significantly blunted expression of 7 of the 10 cytokines studied and shortened the duration of depressed HRV by about half. Interestingly, dexamethasone treatment alone caused a dramatic increase in both low- and high-frequency HRV. Administration of recombinant TNFalpha caused a biphasic response in HR and HRV similar to that caused by LPS. Understanding the role of cytokines in abnormal HRV during sepsis could lead to improved strategies for detecting life-threatening nosocomial infections in intensive care unit patients.

A Novel MitoTimer Reporter Gene for Mitochondrial Content, Structure, Stress, and Damage in Vivo

Background: Mitochondrial health is difficult to assess in vivo. Results: We have generated a reporter gene, MitoTimer, which targets mitochondria, and fluoresces green and shifts to red when oxidized, for assessment of mitochondrial content, structure, stress, and damage under physiological and pathological conditions. Conclusion: MitoTimer is useful for assessment of mitochondrial health in vivo. Significance: MitoTimer could advance mitochondrial research in multiple disciplines. Mitochondrial dysfunction plays important roles in many diseases, but there is no satisfactory method to assess mitochondrial health in vivo. Here, we engineered a MitoTimer reporter gene from the existing Timer reporter gene. MitoTimer encodes a mitochondria-targeted green fluorescent protein when newly synthesized, which shifts irreversibly to red fluorescence when oxidized. Confocal microscopy confirmed targeting of the MitoTimer protein to mitochondria in cultured cells, Caenorhabditis elegans touch receptor neurons, Drosophila melanogaster heart and indirect flight muscle, and mouse skeletal muscle. A ratiometric algorithm revealed that conditions that cause mitochondrial stress led to a significant shift toward red fluorescence as well as accumulation of pure red fluorescent puncta of damaged mitochondria targeted for mitophagy. Long term voluntary exercise resulted in a significant fluorescence shift toward green, in mice and D. melanogaster, as well as significantly improved structure and increased content in mouse FDB muscle. In contrast, high-fat feeding in mice resulted in a significant shift toward red fluorescence and accumulation of pure red puncta in skeletal muscle, which were completely ameliorated by voluntary wheel running. Hence, MitoTimer allows for robust analysis of multiple parameters of mitochondrial health under both physiological and pathological conditions and will be highly useful for future research of mitochondrial health in multiple disciplines in vivo.

Cardiac β‐Adrenergic Signaling

Abstract:  β‐adrenergic signaling plays a central role in the neurohumoral regulation of the heart and the progression of heart failure. Initially thought to be a simple linear cascade, this complex network is now recognized to utilize cross‐talk with numerous other pathways, spatial compartmentation, and feedback control to coordinate cardiac electrophysiology, contractility, and adaptive remodeling. Here, we review recent basic insights and novel quantitative approaches that are leading to a more comprehensive understanding of β‐adrenergic signaling and thus motivate new therapeutic strategies for cardiac disease.beta-adrenergic signaling plays a central role in the neurohumoral regulation of the heart and the progression of heart failure. Initially thought to be a simple linear cascade, this complex network is now recognized to utilize cross-talk with numerous other pathways, spatial compartmentation, and feedback control to coordinate cardiac electrophysiology, contractility, and adaptive remodeling. Here, we review recent basic insights and novel quantitative approaches that are leading to a more comprehensive understanding of beta-adrenergic signaling and thus motivate new therapeutic strategies for cardiac disease.

Computational Models Reduce Complexity and Accelerate Insight Into Cardiac Signaling Networks

Cardiac signaling networks exhibit considerable complexity in size and connectivity. The intrinsic complexity of these networks complicates the interpretation of experimental findings. This motivates new methods for investigating the mechanisms regulating cardiac signaling networks and the consequences these networks have on cardiac physiology and disease. Next-generation experimental techniques are also generating a wealth of genomic and proteomic data that can be difficult to analyze or interpret. Computational models are poised to play a key role in addressing these challenges. Computational models have a long history in contributing to the understanding of cardiac physiology and are useful for identifying biological mechanisms, inferring multiscale consequences to cell signaling activities and reducing the complexity of large data sets. Models also integrate well with experimental studies to explain experimental observations and generate new hypotheses. Here, we review the contributions computational modeling approaches have made to the analysis of cardiac signaling networks and forecast opportunities for computational models to accelerate cardiac signaling research.