A common pathway to cancer: Oncogenic mutations abolish p53 oscillations.

The tumor suppressor p53 oscillates in response to DNA double-strand breaks, a behavior that has been suggested to be essential to its anti-cancer function. Nearly all human cancers have genetic alterations in the p53 pathway; a number of these alterations have been shown to be oncogenic by experiment. These alterations include somatic mutations and copy number variations as well as germline polymorphisms. Intriguingly, they exhibit a mixed pattern of interactions in tumors, such as co-occurrence, mutual exclusivity, and paradoxically, mutual antagonism. Using a differential equation model of p53-Mdm2 dynamics, we employ Hopf bifurcation analysis to show that these alterations have a common mode of action, to abolish the oscillatory competence of p53, thereby, we suggest, impairing its tumor suppressive function. In this analysis, diverse genetic alterations, widely associated with human cancers clinically, have a unified mechanistic explanation of their role in oncogenesis.

Redundancy and multifunctionality among spinal locomotor networks.

c-Fos is used to identify system-wide neural activation with cellular resolution in vivo. However, c-Fos can only capture neural activation of one event. Targeted recombination in active populations (TRAP) allows the capture of two different c-Fos activation patterns in the same animal. So far, TRAP has only been used to examine brain circuits. This study uses TRAP to investigate spinal circuit activation during resting and stepping, giving novel insights of network activation during these events. The level of colabeled (c-Fos+ and TRAP+) neurons observed after performing two bouts of stepping suggests that there is a probabilistic-like phenomenon that can recruit many combinations of neural populations (synapses) when repetitively generating many step cycles. Between two 30-min bouts of stepping, each consisting of thousands of steps, only ∼20% of the neurons activated from the first bout of stepping were also activated by the second bout. We also show colabeling of interneurons that have been active during stepping and resting. The use of the FosTRAP methodology in the spinal cord provides a new tool to compare the engagement of different populations of spinal interneurons in vivo under different motor tasks or under different conditions.NEW & NOTEWORTHY The results are consistent with there being an extensive amount of redundancy among spinal locomotor circuits. Using the newly developed FosTRAP mouse model, only ∼20% of neurons that were active (labeled by Fos-linked tdTomato expression) during a first bout of 30-min stepping were also labeled for c-Fos during a second bout of stepping. This finding suggests variability of neural networks that enables selection of many combinations of neurons (synapses) when generating each step cycle.

Directing tissue morphogenesis via self-assembly of vascular mesenchymal cells.

Rebuilding injured tissue for regenerative medicine requires technologies to reproduce tissue/biomaterials mimicking the natural morphology. To reconstitute the tissue pattern, current approaches include using scaffolds with specific structure to plate cells, guiding cell spreading, or directly moving cells to desired locations. However, the structural complexity is limited. Also, the artificially-defined patterns are usually disorganized by cellular self-organization in the subsequent tissue development, such as cell migration and cell-cell communication. Here, by working in concert with cellular self-organization rather than against it, we experimentally and mathematically demonstrate a method which directs self-organizing vascular mesenchymal cells (VMCs) to assemble into desired multicellular patterns. Incorporating the inherent chirality of VMCs revealed by interfacing with microengineered substrates and VMCs' spontaneous aggregation, differences in distribution of initial cell plating can be amplified into the formation of striking radial structures or concentric rings, mimicking the cross-sectional structure of liver lobules or osteons, respectively. Furthermore, when co-cultured with VMCs, non-pattern-forming endothelial cells (ECs) tracked along the VMCs and formed a coherent radial or ring pattern in a coordinated manner, indicating that this method is applicable to heterotypical cell organization.

Focal high cell density generates a gradient of patterns in self-organizing vascular mesenchymal cells.

In embryogenesis, structural patterns, such as vascular branching, may form via a reaction-diffusion mechanism in which activator and inhibitor morphogens guide cells into periodic aggregates. We previously found that vascular mesenchymal cells (VMCs) spontaneously aggregate into nodular structures and that morphogen pairs regulate the aggregation into patterns of spots and stripes. To test the effect of a focal change in activator morphogen on VMC pattern formation, we created a focal zone of high cell density by plating a second VMC layer within a cloning ring over a confluent monolayer. After 24 h, the ring was removed and pattern formation monitored by phase-contrast microscopy. At days 2-8, the patterns progressed from uniform distributions to swirl, labyrinthine and spot patterns. Within the focal high-density zone (HDZ) and a narrow halo zone, cells aggregated into spot patterns, whilst in the outermost zone of the plate, cells formed a labyrinthine pattern. The area occupied by aggregates was significantly greater in the outermost zone than in the HDZ or halo. The rate of pattern progression within the HDZ increased as a function of its plating density. Thus, focal differences in cell density may drive pattern formation gradients in tissue architecture, such as vascular branching.

Systems biology of vascular calcification.

Vascular calcification, a prevalent and progressive disorder, involves numerous interactive, autocrine, paracrine, and endocrine regulatory mechanisms and is thus ideally suited for analysis using a systems approach. This approach focuses on creating quantitative, testable models of complex biological systems that take into consideration the time dimension. They are usually expressed as mathematical models, and because of their time dependence and complexity, they usually require computer simulation to determine predicted outcomes. Here, we provide an example of such a model used to analyze self-organization and mineralization of vascular stem cells, using partial differential equations capable of accurately predicting experimental outcomes. Such systems-based models are useful in many aspects of cardiovascular medicine.

Increased susceptibility of aged hearts to ventricular fibrillation during oxidative stress.

Oxidative stress with hydrogen peroxide (H(2)O(2)) readily promotes early afterdepolarizations (EADs) and triggered activity (TA) in isolated rat and rabbit ventricular myocytes. Here we examined the effects of H(2)O(2) on arrhythmias in intact Langendorff rat and rabbit hearts using dual-membrane voltage and intracellular calcium optical mapping and glass microelectrode recordings. Young adult rat (3-5 mo, N = 25) and rabbit (3-5 mo, N = 6) hearts exhibited no arrhythmias when perfused with H(2)O(2) (0.1-2 mM) for up to 3 h. However, in 33 out of 35 (94%) aged (24-26 mo) rat hearts, 0.1 mM H(2)O(2) caused EAD-mediated TA, leading to ventricular tachycardia (VT) and fibrillation (VF). Aged rabbits (life span, 8-12 yr) were not available, but 4 of 10 middle-aged rabbits (3-5 yr) developed EADs, TA, VT, and VF. These arrhythmias were suppressed by the reducing agent N-acetylcysteine (2 mM) and CaMKII inhibitor KN-93 (1 microM) but not by its inactive form (KN-92, 1 microM). There were no significant differences between action potential duration (APD) or APD restitution slope before or after H(2)O(2) in aged or young adult rat hearts. In histological sections, however, trichrome staining revealed that aged rat hearts exhibited extensive fibrosis, ranging from 10-90%; middle-aged rabbit hearts had less fibrosis (5-35%), whereas young adult rat and rabbit hearts had <4% fibrosis. In aged rat hearts, EADs and TA arose most frequently (70%) from the left ventricular base where fibrosis was intermediate ( approximately 30%). Computer simulations in two-dimensional tissue incorporating variable degrees of fibrosis showed that intermediate (but not mild or severe) fibrosis promoted EADs and TA. We conclude that in aged ventricles exposed to oxidative stress, fibrosis facilitates the ability of cellular EADs to emerge and generate TA, VT, and VF at the tissue level.

Intracellular Ca alternans: coordinated regulation by sarcoplasmic reticulum release, uptake, and leak.

Beat-to-beat alternation in the cardiac intracellular Ca (Ca(i)) transient can drive action potential (AP) duration alternans, creating a highly arrhythmogenic substrate. Although a steep dependence of fractional sarcoplasmic reticulum (SR) Ca release on SR Ca load has been shown experimentally to promote Ca(i) alternans, theoretical studies predict that other factors are also important. Here we present an iterated map analysis of the coordinated effects of SR Ca release, uptake, and leak on the onset of Ca(i) alternans. Predictions were compared to numerical simulations using a physiologically realistic AP model as well as to AP clamp experiments in isolated patch-clamped rabbit ventricular myocytes exposed to 1), the Ca channel agonist BayK8644 (100 nM) to increase SR Ca load and release fraction, 2), overexpression of an adenoviral SERCA2a construct to increase SR Ca uptake, and 3), low-dose FK506 (20 microM) or ryanodine (1 microM) to increase SR Ca leak. Our findings show that SR Ca release, uptake, and leak all have independent direct effects that promote (release and leak) or suppress (uptake) Ca(i) alternans. However, since each factor affects the other by altering SR Ca load, the net balance of their direct and indirect effects determines whether they promote or suppress alternans. Thus, BayK8644 promotes, whereas Ad-SERCA2a overexpression, ryanodine, and FK506 suppress, Ca(i) alternans under AP clamp conditions.

Persistence of motor unit and muscle fiber types in the presence of inactivity.

The clarity of categorizing skeletal muscle fibers in individual motor units into phenotypes based on quantitative single fiber enzyme activities and as a function of neuromuscular activity level was examined. Neuromuscular activity was eliminated in adult cat hindlimb muscles by spinal cord isolation (SI), i.e. complete spinal cord transection at a low thoracic and a high sacral level with bilateral dorsal rhizotomy between the transection sites. One motor unit was isolated via ventral root teasing procedures from the tibialis anterior (TA) muscle of each hindlimb in control and SI cats, and physiologically tested and glycogen depleted through repetitive stimulation; fibers comprising each motor unit were visualized through glycogen staining. Each motor unit was composed of fibers of the same myosin immunohistochemical type. Myofibrillar adenosine triphosphatase, succinate dehydrogenase and alpha-glycerophosphate dehydrogenase activities were determined for a sample of motor unit and non-motor unit fibers, providing a measure of three enzyme activities often used to characterize fiber phenotype within a single unit. Although normal enzyme activities were altered after 6 months of inactivity, the relationships among the three enzymes were largely maintained. These data demonstrate that it is not the diversity in any single enzyme property but the profile of several metabolic pathways that underlies the significance of fiber phenotypes. These profiles must reflect a high level of coordination of expression of selected combinations of genes. Although neuromuscular activity level influences fiber phenotype, the present results demonstrate that activity-independent mechanisms remain important sources of the control of phenotype establishment in the near absence of activity.

Modifying L-type calcium current kinetics: consequences for cardiac excitation and arrhythmia dynamics.

The L-type Ca current (I(Ca,L)), essential for normal cardiac function, also regulates dynamic action potential (AP) properties that promote ventricular fibrillation. Blocking I(Ca,L) can prevent ventricular fibrillation, but only at levels suppressing contractility. We speculated that, instead of blocking I(Ca,L), modifying its shape by altering kinetic features could produce equivalent anti-fibrillatory effects without depressing contractility. To test this concept experimentally, we overexpressed a mutant Ca-insensitive calmodulin (CaM(1234)) in rabbit ventricular myocytes to inhibit Ca-dependent I(Ca,L) inactivation, combined with the ATP-sensitive K current agonist pinacidil or I(Ca,L) blocker verapamil to maintain AP duration (APD) near control levels. Cell shortening was enhanced in pinacidil-treated myocytes, but depressed in verapamil-treated myocytes. Both combinations flattened APD restitution slope and prevented APD alternans, similar to I(Ca,L) blockade. To predict the arrhythmogenic consequences, we simulated the cellular effects using a new AP model, which reproduced flattening of APD restitution slope and prevention of APD/Ca(i) transient alternans but maintained a normal Ca(i) transient. In simulated two-dimensional cardiac tissue, these changes prevented the arrhythmogenic spatially discordant APD/Ca(i) transient alternans and spiral wave breakup. These findings provide a proof-of-concept test that I(Ca,L) can be targeted to increase dynamic wave stability without depressing contractility, which may have promise as an antifibrillatory strategy.

Functional characterization of atrial electrograms in sinus rhythm delineates sites of parasympathetic innervation in patients with paroxysmal atrial fibrillation.

OBJECTIVES: This study sought to characterize left atrial (LA) sinus rhythm electrogram (EGM) patterns and their relationship to parasympathetic responses during atrial fibrillation (AF) ablation. BACKGROUND: The mechanistic basis of fractionated LA EGMs in patients with paroxysmal AF is not well understood. METHODS: We analyzed 1,662 LA ablation sites from 30 patients who underwent catheter ablation for paroxysmal AF. Pre-ablation EGM characteristics (number of deflections, amplitude, and duration) were measured in sinus rhythm. Parasympathetic responses during radiofrequency application (increase of atrial-His interval by > or =10 ms or decrease of sinus rate by > or =20%) were assessed at all sites. We also prospectively studied the effect of adenosine, a pharmacological agent mimicking acetylcholine signaling in myocytes, on LA EGMs. Finally, we performed mathematical simulations of atrial tissue to delineate possible mechanisms of fractionated EGMs in sinus rhythm. RESULTS: A specific pattern of pre-ablation sinus rhythm EGM (deflections > or =4, amplitude > or =0.7 mV, and duration > or =40 ms) was strongly associated with parasympathetic responses (sensitivity 72%, specificity 91%). The sites associated with these responses were found to be located mainly in the posterior wall of the LA. Adenosine administration and mathematical simulation of the effect of acetylcholine were able to reproduce a similar EGM pattern. CONCLUSIONS: Parasympathetic activation during AF ablation is associated with the presence of pre-ablation high-amplitude fractionated EGMs in sinus rhythm. Local acetylcholine release could potentially explain this phenomenon.

Understanding biological complexity: lessons from the past.

Advances in molecular biology now permit complex biological systems to be tracked at an exquisite level of detail. The information flow is so great, however, that using intuition alone to draw connections is unrealistic. Thus, the need to integrate mathematical biology with experimental biology is greater than ever. To achieve this integration, obstacles that have traditionally prevented effective communication between theoreticians and experimentalists must be overcome, so that experimentalists learn the language of mathematics and dynamical modeling and theorists learn the language of biology. Fifty years ago Alan Hodgkin and Andrew Huxley published their quantitative model of the nerve action potential; in the same year, Alan Turing published his work on pattern formation in activator-inhibitor systems. These classic studies illustrate two ends of the spectrum in mathematical biology: the detailed model approach and the minimal model approach. When combined, they are highly synergistic in analyzing the mechanisms underlying the behavior of complex biological systems. Their effective integration will be essential for unraveling the physical basis of the mysteries of life.