Explaining why so many cases of cardiac arrest strike in the morning

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Michael Woods
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202-872-6293 American Chemical Society
Explaining why so many cases of cardiac arrest strike in the morning INDIANAPOLIS, Sept. 8, 2013 — Evidence from people with heart disease strongly supports the existence of the molecular link first discovered in laboratory mice between the body's natural circadian rhythms and cardiac arrest or sudden cardiac death (SCD) — the No. 1 cause of death in heart attacks, a scientist said here today.

The research, which offers the most focused explanation ever for SCD's predilection for the morning hours, was part of the 246th National Meeting & Exposition of the American Chemical Society (ACS), the world's largest scientific society. The meeting, which features almost 7,000 reports on discoveries in science and other topics, continues through Thursday in the Indiana Convention Center and downtown hotels.

Mukesh Jain, M.D., who reported on the research, said that it pinpoints a previously unrecognized factor in the electrical storm that makes the heart's main pumping chambers suddenly begin to beat erratically in a way that stops the flow of blood to the brain and body. Termed ventricular fibrillation, the condition causes SCD, in which the victim instantly becomes unconscious and dies unless CPR or a defibrillator is available to shock the heart back into its steady beat.

"Sudden cardiac death due to this electrical instability causes an estimated 325,000 deaths annually in the United States alone," Jain explained. He is with Case Western Reserve University in Cleveland. "That includes the 3 out of 4 heart disease deaths in people aged 35-44. In all too many cases, there is no second chance. The first event is the last event. Our research points the way toward possible ways of easing that toll — new drugs that could reduce that risk, for example."

One of the deepest mysteries about SCD has been its timing. Health experts have known for more than 30 years that the erratic heartbeat responsible for SCD strikes most often at certain times of the day. The peak risk hours range from 6 a.m. to 10 a.m., with a smaller peak in the late afternoon. Scientists long suspected a link between SCD and the 24-hour body clock, located in the brain. It governs 24-hour cycles of sleep and wakefulness called circadian rhythms that coordinate a range of body functions with the outside environment.

Jain's group discovered a protein called KLF15 that helps regulate the heart's electrical activity, and occurs in the body in levels that change like clockwork throughout the day. KLF15 helps form channels that allow substances to enter and exit heart cells in ways critical to maintaining a normal, steady heartbeat.

They first discovered that patients with heart failure have lower levels of KLF15. Then, they established in laboratory mice that KLF15 is the molecular link between SCD and the circadian rhythm. And mice with low levels of the protein have the same heart problems as people with SCD. "It turns out that people with low KLF15 levels are the ones that are most susceptible to these sudden death episodes that occur in the early morning hours," said Jain. "So, we think that if we could in some way boost KLF15 levels in patients with heart problems, maybe we can reduce the occurrence of these arrhythmias and SCD."

Jain's group is currently studying drugs that boost KLF15 levels. The protein also has effects on other body processes, however. "If we can find out how these compounds are boosting KLF15 levels, then maybe we can make much more targeted and specific therapies for the heart that would prevent SCD, but leave the other KLF15-related processes alone," he explained. Other groups are working on developing genetic tests to identify people who have mutations in the KLF15 gene and would likely be at higher risk of SCD, he noted.

### A press conference on this topic will be held Sunday, Sept. 8, at 8 a.m. in the ACS Press Center, Room 211, in the Indiana Convention Center. Reporters can attend in person or access live audio and video of the event and ask questions at http://www.ustream.tv/channel/acslive.

Jain acknowledged funding from the National Institutes of Health and the American Heart Association.

The presentation was part of the symposium "Beyond Jet Lag: Targeting Aberrant Circadian Rhythm To Attack Diseases from Diabetes To Depression." Abstracts appear below.

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Abstracts

KLF15 links the circadian clock to arrhythmogenesis Mukesh K. Jain, susan.dowhan@UHhospitals.org, DOM Division of Cardiovascular Medicine, Case Western Reserve University, Cleveland, Ohio 44106, United States

Sudden cardiac death (SCD) secondary to ventricular arrhythmia is the most common cause of mortality from cardiovascular disease worldwide. Despite decades of investigation, a thorough understanding of triggers and effective pharmacologic treatments for SCD are lacking and the primary treatment remains mechanical defibrillation. This sobering reality has led to the view that a complete re-examination of the fundamental mechanisms underlying the development of SCD is needed to provide a foundation for the development of novel, effective therapies.

Biological processes that oscillate with a 24-hour periodicity are termed circadian. All cells have a circadian clock that synchronizes changes in gene expression with rhythmic patterns of daily life, i.e. eating or sleeping. The observation that SCD exhibits a peak during early morning, is increased in shift workers suggests that circadian influences may be operative. However, a direct link between the circadian clock, metabolism, and cardiac electrical activity was lacking. In this regard, recent studies from our laboratory identified the first molecular link between endogenous circadian rhythms and SCD. Specifically, we reported that a specific cardiac ion channel that controls myocyte repolarization exhibited circadian oscillation under the control of the clock-dependent oscillator KLF15. Alterations in KLF15 levels in vivo rendered animals susceptible to SCD. Finally, the observation that KLF15 is altered in subjects with heart failure and Brugada syndrome suggests that the study of this pathway may have implications for human disease.

Synthetic REV-ERB ligands: Tools for probing the chemical biology of the circadian clock Thomas P Burris, tburris@scripps.edu, Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, FL 33458, United States

Physiological processes including metabolism and behavior are governed in a circadian rhythm. This 24h rhythm is maintained by a cell autonomous transcriptional/translational feedback loop composed of the transcription factors BMAL1 and CLOCK and their target genes, PER and CRY. The nuclear receptor REV-ERB also plays an important regulatory role in maintaining the circadian oscillator by direct regulation of the Bmal1 and Clock genes. Appropriate oscillations in this molecular clock are required for normal physiological function and behavior. In fact, abnormal clock function has been associated with a range of disorders including metabolic diseases, sleep disorders, mental disorders and cancer. Using a chemical biology approach, we recently demonstrated that synthetic compounds that modulate the circadian expression of clock genes also alters metabolic processes. We demonstrated that synthetic REV-ERB agonists increase the metabolic rate of mice leading to decreased fat mass. Diet induced obese mice also display weight loss when the REV-ERB agonists is administered and, additionally, demonstrate improved plasma lipid profiles. Here, I will describe recent results examining the effects of REV-ERB agonists in additional models of metabolic disorders as well as models of sleep and anxiety. In summary, our data indicate that drugs that modulate clock activity, REV-ERB agonists in particular, may have utility in treatment of human diseases.

Characterization of ligands targeting RAR-related orphan receptor alpha (RORα) Timothy I Richardson1, richardson_timothy_i@lilly.com, Christian A Clarke1, Michael J Genin1, Joseph T Brozinick1, Mark R Wade1, Christine C Cheng1, Chahrzad Montrose-Rafizadeh1, Robert J Barr1, Richard W Zink1, Wayne P Bocchinfuso1, Jerry Bernard1, Shane Atwell1, Zhanna Druzina1, Margaret Kearins1, David K Clawson1, Leah M Helvering1, Laurie Burris1, Keith R Stayrook1, Keith A Wafford1, Janelle L Lauer2, Scott Novick2, Michael J Chalmers2, Patrick R Griffin2, Jeffrey A Dodge1. (1) Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, IN 46285, United States, (2) Molecular Therapeutics, The Scripps Research Institute, Jupiter, FL 33458, United States

All animals have evolved complex systems of gene regulation in response to environmental cues such as the 24hr light-dark cycle, which has influenced many aspects of animal behavior and physiology including feeding and metabolism. In mammals, the central clock, located in the Suprachiasmatic Nucleus (SCN) of the hypothalamus, is entrained by light. In turn, the SCN entrains peripheral clocks that are maintained, even in the absence of a light-dark cycle, by a web of interacting gene transcription feedback loops, tightly controlling gene expression with 24hr periodicity. Two transcription factors, CLOCK and BMAL1, activate gene expression of Per (Period) and Cry (Cryptochrome). The protein products of Per and Cry form a heterodimeric complex that translocates back into the nucleus and inhibits BMAL1 and CLOCK expression, forming a negative feedback loop. Superimposed upon this transcriptional loop are two nuclear hormone receptors, RORα and Rev-erbα, which are ligand dependent transcription factors that have recently been deorphanized. The endogenous ligand for RAR-related orphan receptor alpha (RORα) has been identified as 7α-hydroxycholesterol while Rev-erbα requires heme. Both receptors are constitutive, RORα as an activator and Rev-erbα as a repressor, and they compete for the same binding elements near the transcription start sites of metabolically important genes. Here we describe synthetic ligands for RORα, co-crystal structures of these ligands bound to the ligand binding domain, and protein hydrogen deuterium exchange (HDX) studies of ligand binding. Using these compounds, we identified RORα target genes and demonstrated activity in vitro and in vivo.

Chronobiology of inflammation Timothy M Willson, tim.m.willson@gsk.com, Chemical Biology, GlaxoSmithKline, Research Triangle Park, NC 27709, United States

We are interested in applying the principles of chronobiology to the development of anti-inflammatory drugs through optimization of their time of day dosing. Asthma and rheumatoid arthritis are known to display circadian symptomatology, with increased morbidity in the early morning. To shed light on the molecular basis for these observations, we have identified components in the lung and inflammatory signaling pathways that are under circadian control. The application of these insights to the design of chronotherapeutics will be discussed.

Succeeding in an unprecedented CNS target space: Discovery of selective casein kinase (CK1δ/ε) inhibitors for the treatment of circadian rhythm disorder Travis T Wager1, travis.t.wager@pfizer.com, Michael Marconi2, Kevin Walton2, Stefan Steyn3, Cheng Chang3, Angela Doran3, Jenifer Bradley2, Ramalakshmi Chandrasekaran1, David Gebhard2, Matthew Griffor1, Jianke Li1, Wayne McDonald1, Scot Mente1, Vanessa Paradis4, Subbu Subramanyam4, Terri Swanson2, Todd Wisialowski5, William Angus5, Michael Green1, Todd Butler4, Paul Galatsis1, John Knafels4, Jeffrey Ohren4, Kenneth Dirico4, George DeMarco5. (1) Worldwide Medicinal Chemistry, Pfizer, Cambridge, MA 02139, United States, (2) Neuroscience Research Unit, Pfizer, Groton, CT 06340, United States, (3) Pharmacokinetics, Dynamics and Metabolism, Pfizer, Groton, CT 06340, United States, (4) Medicinal Chemistry, Pfizer, Groton, CT 06340, United States, (5) Pfizer Worldwide Research and Development, Pfizer, United States

CK1 delta (CK1δ) and CK1 epsilon (CK1ε) are closely related members of a family of seven mammalian serine/threonine protein kinases previously known as casein kinases. The CK1δ and CK1ε isoforms are highly expressed in the suprachiasmatic nucleus (SCN) where they form an essential component of the mammalian biological clock. Selective inhibitors of this class of Kinases will provide tools to study the role of circadian clock in CNS disorders such as: morning lark, bipolar or unipolar depression. A challenge with targeting Kinase inhibitors for chronic CNS indications is a seemingly insurmountable task because of the need to achieve good brain penetration for efficacy and high selectivity for safety. Through a combination of structure-based design, computational chemistry, and innovative medicinal chemistry we have identified a series of highly selective, brain penetrant CK1δ inhibitors which demonstrated phase shift in mouse and cynomolgus monkey models of circadian rhythm. These inhibitors also provided excellent therapeutic index in exploratory toxicology studies. This presentation will highlight the discovery of such compounds as well as general guidelines for targeting kinases for CNS disorders.

Transcriptional architecture of the circadian clock in mammals Joseph S Takahashi, Joseph.takahashi@utsouthwestern.edu, HHMI, Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75390-9111, United States

The circadian clock mechanism in animals involves an autoregulatory transcriptional feedback loop in which CLOCK and BMAL1 activate the transcription of the Period and Cryptochrome genes. The PERIOD and CRYPTOCHROME proteins then feedback and repress their own transcription by interaction with CLOCK and BMAL1. Recently, we have focused on the biochemical mechanisms of the core circadian transcriptional regulators and have used structural biology and genomics to study the CLOCK:BMAL1 complex and its genomic targets. Using x-ray crystallography, we have solved the three-dimensional structure of the CLOCK:BMAL1 heterodimeric complex. In addition, we have interrogated on a genome-wide level the cis-acting regulatory elements (cistrome) of the entire CLOCK:BMAL1 transcriptional feedback loop. This has revealed a global circadian regulation of transcription factor occupancy, RNA polymerase II recruitment and initiation, nascent transcription and chromatin remodelling. In addition, we have used cell-based circadian rhythms to screen for small molecules that perturb the clock system.