Heart Beats and Biophysics

February has been designated Heart Month by the American Heart Association, the CDC, and several other organizations concerned with heart disease and ailments.  The goal of Heart Month is to raise awareness of heart diseases and steps individuals can take to prevent them.  It is also a good time for the Biophysical Society to highlight how advances in basic research contribute to our understanding of these diseases.  BPS member David Eisner, the BHF Professor of Cardiac Physiology at the University of Manchester in the United Kingdom has taken the time to share his lab’s research on heart functioning with us.

eisner picWhat is the connection between your research and heart disease, heart attacks, or heart functioning?

We study calcium signaling in the heart and, specifically, what controls the intracellular concentration of calcium ([Ca2+]i ).  Each heartbeat is initiated by a rise of [Ca2+]i; the greater the rise, the stronger the heart beats.  During exercise, the stronger contraction of the heart results from an increase in the size of the rise of  [Ca2+]i. As well as studying the normal regulation of [Ca2+]i, we are also interested in what happens in disease situations such as  heart failure and cardiac arrhythmias. One of the reasons that the hearts beats more weakly in heart failure is because the rise of [Ca2+]i is smaller than in healthy conditions.  The mechanisms responsible for this are still being unraveled. Many cardiac arrhythmias result from abnormalities of calcium signaling, in particular involving a rise of [Ca2+]i which occurs at the wrong part of the cardiac cycle.  Again, our research aims to understand the origins of these changes.

Why is your research important to those concerned about these diseases?

Understanding cardiac disease requires a much better understanding of the basic physiology of the heart. The fundamental unit of the heart is the cardiac muscle cell (myocyte) and it is at this level that most of our work is focused. We use  patch clamp to measure the movements of calcium across the membranes surrounding these cells and combine this with the use of fluorescent indicators to measure changes of [Ca2+]i. As well as providing information about the normal working of the heart, these sort of  studies will reveal the changes in disease. Furthermore, cellular studies are essential for developing therapies against these conditions. In this context it is important to note that, although enormous progress has been made, the progonosis for someone diagnosed with heart failure is still bleak.

How did you get into this area of research?

When I was at school I wanted to study physics and had never heard of physiology. At university I was taught physiology as the application of physics to the body.  Following undergraduate studies, I did my PhD with Denis Noble in Oxford where I worked with Jon Lederer (now at the University of Maryland) studying the control of contraction in the heart.  I can still remember the sense of immediate gratification when one pushed  a sharp microelectrode into a piece of cardiac muscle  and heard the change of pitch of the audio amplifier.  At that time it was impossible to do electrophysiological recordings on single cells and methods were not available to measure intracellular calcium.  However advances in these areas meant that it became possible to study calcium signaling in the heart.

How long have you been working on it?

Since the early 1980s!   My own research interests began very much at the basic science end of the subject but, over time, together with my long term collaborator Andrew Trafford, we have investigated disease models.

Do you receive public funding for this work? If So, from what agency?

Most of my funding comes from the British Heart Foundation (BHF).   This is a charity supported by the general public.  It is chastening to know that our research is supported by countless volunteers.  The funding environment in the UK  is very different from that in the US with a much smaller fraction of research supported by government funds.

Have you had any surprise findings thus far?

We obtained one very surprising result when we did experiments to increase the opening of the sarcoplasmic reticulum (SR) release channel  (the ryanodine receptor, RyR).  We had confidently expected that this would increase the size of the Ca signal and contraction.  However,  we found that the calcium signal was only increased for a couple of couple of beats before returning to normal levels.   The explanation of this result turned out to be that the increased release of calcium from the SR decreased SR Ca content.   This was the first hint we had of what has turned out to be a much more general phenomenon; the interaction between the various Ca handling pathways results in complicated, emergent behavior which is difficult to predict in advance.  At the simplest level, these results arise from the fact that the cardiac cell is in calcium flux balance.  On each beat the amount of calcium that enters the cell must exactly balance that which leaves.  This highlights the need to study calcium signaling in an integrated way.

What is particularly interesting about the work from the perspective of other researchers?

As a result of our work, others now appreciate that, on each beat, the cell is in calcium flux balance.   This point has to be borne in mind when trying to explain changes in cardiac contractility.

What is particularly interesting about the work from the perspective of the public?

The general public always seem fascinated when they are shown electrical and calcium signals from cardiac cells.  They are amazed by the fact that the heart beats repetitively even when outside the body.  It is a privilege to lecture to the public.  I always think that it is a pity that most people know much more about outer space than about their own bodies.

Do you have a cool image you want to share with the blog post related to this research?

The image at the top of this blog post (work by Jessica Caldwell, Andrew Trafford and colleagues) shows ventricular cells connected together.  The horizontal bands are the transverse tubules which invaginate the cell.  We are currently studying the cellular mechanisms that ensure that the transverse tubule network is laid down in this precise arrangement and why it disappears in heart failure.

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Visualizing Chromosomes, Cell Cycles, and Entropy

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David Goodsell is a legendary figure whose scientific illustrations have inspired many. When I first entered the bacterial chromosome field in 2004 as a theoretical physicist, his illustration was a definite guide to the inner space of Escherichia coli. It conveyed the right sense of scale and the numbers of different types of proteins and biomolecules inside the cell. Goodsell regularly updates his illustrations to incorporate up-to-date information from the scientific literature. A detailed explanation behind his work process can be found in Miniseries: Illustrating the Machinery of Life, Escherichia coli (Biochem Mol Biol Educ. 2009 [37]: 325-332).

In recent years, my lab has been taking multidisciplinary approaches to understand the physical principles that drive organization and segregation of the chromosomes in bacteria. For example, we have revealed the fundamentally “soft” nature of the bacterial chromosome and the entropic forces that can compact it in a crowded intracellular environment (Pelletier et al. Physical manipulation of the Escherichia coli chromosome reveals its soft nature. PNAS Plus. 2012; 109 [40]: E2649–2656), which motivated the work by Shendruk et al. in this issue of Biophysical Journal (108 [4]: February 17, 2015).

In the meantime, Stuart Austin’s group at the National Cancer Institute was tackling what had been considered impossible. They started the measurements and analysis of intracellular positions of dozens of dual genomic loci markers under overlapping cell-cycle conditions. This is a daunting task because, for any given moment, every cell contains several homologous copies of each genomic locus, and deciphering the organization and dynamics of the whole chromosome based on their positional information was something that had never been done before. Nevertheless, Stuart’s group relentlessly pushed their efforts without publishing anything for several years. When the task was done, the end result was simple, elegant, and surprising. The organization of the chromosome during multifork replication was that of a simple branched donut, with the two arms of the chromosome occupying each cell half along the radial axis of the cell. This result could not have been predicted based on our knowledge of the slowest-growing cells, but it encompasses all the previously known results and moves beyond them into something new and general (Youngren et al. The multifork Escherichia coli chromosome is a self-duplicating and self-segregating thermodynamic ring polymer. Genes Dev. 2014 [28]: 71-84).

Because of these radical new findings, I felt that David Goodsell’s famous illustration of E. coli would benefit from revision. Our cover image is the result of 12 revisions. A high-resolution file of Goodsell’s E. coli illustration can be downloaded from my lab web site: http://jun.ucsd.edu.

–Suckjoon Jun

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Bye bye Baltimore

For the first time in days, I woke up in the hotel room with sunlight sifting through the curtain. What a nice day with fresh air and ample daylight! Even for the last day, the schedules are packed with multiple platform talks and poster showing.

I want to feature the Cryo-EM structure talk in the afternoon, as it did cover a great variety of research topics and new progresses. All four speakers are working with very challenging protein systems and getting enough particles for comprehensive structural details is only the last thing on their to-do list. Due to highly dynamic nature of the protein system movements, the inherent heterogeneity of the particle conformations is probably the biggest challenge. To solve the problem involves locking the system in a particular state with different substrates, finding the right conditions for the protein preparation, and also collect particles with different orientations on the surface. I really appreciate that several speakers took the time to explain to the audience the technical difficulties they encounter during the structural solving processes. In a way, the story not only tells about a sophisticated structure, but also shows us countless trial-and-error progress from highly driven scientists.

I really do enjoy the BPS experience, even when I felt rather clueless at some moment.

Bye bye Baltimore, and hopefully see you’all soon in Los Angelos.

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t-loop formation at single molecule level.

The average lifespan of a cell is approximately 50 cycles after which the cells go into senescence, inability to replicate. Early published work clearly suggests that the growing cells have an inherent knowledge of the number of cycles they have divided and this attribute of the cells is very much dependent on the structures on the end of the chromosomes known as the telomeres. These structures at the end of the chromosomes are known to shorten after every cell cycle. Once the telomere reserve has run out, cells stop dividing. Telomeres play important roles in maintaining the stability of linear chromosomes. The telomeric structure allows a cell to distinguish between natural chromosome ends and double-stranded DNA breaks. Telomere dysfunction and associated chromosomal abnormalities have been strongly associated with age-associated degenerative diseases and cancer. Telomere maintenance involves dynamic actions of multiple proteins on a long complex DNA structure. Given the heterogeneity and complexity of telomeres, single-molecule approaches are essential to fully understand the structure-function relationships that govern telomere maintenance. These telomeres form little loops at the end of the chromosomes, which are called the t-loops. These are formed by inserting the ends of the chromosome, which is usually 3’ overhang back into the DNA of the chromosome. Thus, very short telomeres, which is the scenario in old aging cells or sometimes cancer cells, can no longer form t-loops. The exposure of these chromosome ends, 3’ overhangs which cannot be inserted back into the DNA of the chromosome, would alert the cells and thus stop cells from dividing. If we can elucidate the mechanism of this t-loop formation, we can introduce methods to stop shortening of these telomeres and make drugs to stop these processes.
There were two talks this year focusing on the t-loop formation at the single molecule level. While Xi Long talk in the DNA structure session used Magnetic tweezers to understand this, Hong Wang’s talk in the Nanoscale Biophysics session on Saturday talked about these structures , using new technique developed in their lab , DREEM ( Dual Resonance Enhanced Electrostatic force Microscopy). Xi long et al was able to show the melting of telomeric DNA substrates on applying torque and the binding of these substrates with the single stranded oligos but the major drawback in her work being the absence of TRF2 protein from the shelterin complex, which has been shown to be required for the formation of t-loops. Hong et al was able to predict a possible mechanism of the t-loop formation based on the interaction of the telomeric DNA with the shelterin protein, very cool work and technique , actually showing the DNA inside the protein DNA complexes. How cool to be able to see what happens inside the protein DNA complex!!!
Would be looking forward to BPS 2016 for their work to better understand this mechanism…

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Wrap Up & Some Thoughts On Students

With the #BPS15 nearly complete, I would like to share my observations about the meeting, and the students particularly.
As someone who has put on some modestly sized events myself, I have quite a bit of respect for executing one of this scale and quality. There are always bumps and glitches to be found at any event if one looks hard enough, but I can truly say that this event was one of the smoothest I’ve attended in quite some time. We were able to set up and break down our exhibits in record time and the staff was very helpful as well. I wish they all went this smoothly.
What about our exhibition time? One of the things I have noticed as a repeat exhibitor, is the number of academic and student attendees. Some previous exhibit “neighbors” adjoining our booth space had even expressed mild concern at this (?). I believe this is a strength of this meeting!
Our company, founded with academic roots by a university professor over three decades ago, had always strongly embodied those values. Yet I believe that interacting with potential future customers is not only good business, but imperative from an educational, strategic, and demographic standpoint.
That said, it was heartening to hear discussions and explanations of peptide science to these academic.s That, along with the many leads our company gathered during the course of the meeting, combined with the nice interactions with new and old friends, made for a highly successful visit to #bps15! Thanks for reading and see you next time – Bob

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Ceci n’est pas une chaise: a story of the chair experience.

Arguably the most important thing I had to do this week was co-chair a platform. Not that blogging isn’t super important (and you know the people at my own talk, I’m sure were blown away) but in my and my co-chair’s hands was the success of 8 scientists talks and the audience experience that surrounds them.

I had never given a talk at BPS until yesterday, and felt woefully under-qualified to help others do this thing I had never done before myself. To top that off I kept hearing people say, ‘You’re not making any friends by going over time, either as a speaker or a chair.’ The errors just pile up. Rumors fly (‘Did you hear session XYZ has completely phase shifted?’). Everyone is angry. No one gets to ask questions. What if this happens to me?

Fortunately, after only previously knowing one other person to chair a BPS session, this time a handful of my friends were also chairing sessions! I think this is maybe related to the fact that the ‘theme’ of this year’s BPS is basically what I do, and therefore also up-regulated in my friendship circle.

Anyway, I was able to get some friendly advice from people who had chaired the day before me, which was comforting. I learned there would be an IT guy who would take care of both setting up computers and setting up the timer, which was a huge relief. There’s this green light that turns yellow at the twelve minute mark, as a warning for the red light of doom that’ll come at 15 minutes. It was also suggested I get a pad of paper to take notes and jot down question ideas, because it is the chairs responsibility to not only keep the session on time, but also pay attention to the science and have a question handy.

This quickly became the most terrifying aspect of chairing. Especially as I noticed in other sessions how often this extra chair-induced question lubricant was necessary. Furthermore, I am not usually that great at thinking of questions in talks, generally getting my brilliant ideas a few hours after they’re actually useful.

So when the time came, I was a bit riled up, but luckily my co-chair turned out to be a relatively senior guy who seemed to know what he was doing, and I relaxed quickly. According to a grad student witness, the best part of the whole session happened before it even started: my co-chair’s phone alarm accidentally went off with a jazzy little tune, and I instinctively did a little dance, apparently visible to the audience because they laughed.

The first few talks went pretty smoothly: things were pretty much on time, and I was able to think of good questions! Then somehow we started slipping minute by minute later off schedule. Maybe because the talks were pretty cool ( phosphorylation near drug binding sites, green and black tea polyphenol’s effect on amyloid-beta formation in alzheimers, finding ligands that increase the probability of a particular protein-protein interaction, etc.), and I was concentrating on question duties. I think by the time we hit the fourth speaker and I was introducing the speakers instead of my co-chair (as part of our splitting-of-duties agreement), we were starting a full 7 minutes late. Then we had some technical difficulties. One of the speakers had to reboot their computer so it would be able to connect to the projector! Utter disaster.

What do we do? Do we stop letting people ask questions? Should I be making wild hand gestures in conjunction with the lights? But all the speakers are being really great about being on time, it’s just me with the questions and transitions that has been a little off. The little green light is surprisingly misleading, as it only relates to the speaker’s internal timing, not to the overall fact that we had already started seven minutes late!

Luckily it didn’t really matter that much. We’re here to do science. The talks were good. The questions were good. We started encouraging people to keep their questions quick, and overlapped questions with next speaker setup a little bit more, and I think ultimately we ended on time, with my talk at the end. At the end of it all, I think it all wrapped up well. I even had someone come up to me and say ‘Nice job chairing,’ then I must have made a surprised face or something, cause they followed up with, ‘Oh yeah, and nice job on the talk, too!’

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The best seat in the house for watching HIV entry

One of my major reasons for attending BPS this year was to expand my knowledge in a field that isn’t very important at all for the work that I do in my day to day.  My work involves designing molecules that can alter protein function and hopefully “drug” an interaction or protein conformation that is useful therapeutically.  The readouts for whether we are successful are pragmatic ones — we look at cell viability, downstream effects, preservation or desolation of certain cellular pathways as needed.  What we generally don’t concern ourself with is confirming with mechanistic insight how exactly the molecules we make do what they do.  So I decided to go learn more about biophysical techniques for looking at protein dynamics and allostery — the best place to do that was BPS.

Well I’ve learned a lot, and have a lot more to learn from all the papers and techniques that others have suggested I look into.  One of the most fascinating examples of a study hoping to shed light on protein dynamics of therapeutic importance was presented yesterday by James Munro.  Professor Munro used single molecule FRET to monitor conformational changes in the HIV envelope protein gp120 as it interacted with receptors on the host cell surface.  Only one envelope protein on each HIV virion was dually labeled, with a FRET donor at one relatively “fixed” location, and an acceptor at one of three locations on nearby loops of gp120.  FRET is a very powerful technique, and smFRET is even better since it gives conformational trajectories that can give valuable information about the kinetics being observed. However with two labels, the FRET readout is one dimensional — only one coordinate is generated, with points along a line of FRET efficiency indicating the distance between two points on a protein surface.  Can something as complex as HIV envelope binding and entry be observed usefully along a single coordinate?

The answer, as published in Science last year, is yes.  With the choice of a coordinate indicating the distance between the V1/V1 loop region and the V5 loop in the outer domain, Munro and his coworkers were able to observe three distinct conformations accessed by the envelope protein: a highly occupied low-FRET “ground” state indicating prefusion envelope protein, a high-FRET state indicating the envelope protein bound to its receptor CD4, and an intermediate-FRET state indicating binding to both CD4 and the HIV coreceptor.  Many experiments with both laboratory and clinically-derived HIV strains with a variety of ligands confirmed this result.  The smFRET kinetics also supported this view, as fitting the traces to a three-state Markov model showed many transitions from unbound to CD4-bound, and transitions from CD4-bound to CD4+Coreceptor-bound, but very rarely transitions from unbound directly to the doubly-bound conformation.

This choice of coordinate was not a lucky guess, it was guided by existing low-resolution structures of the envelope protein during membrane fusion, and even so, likely was the result of many grad student/postdoc-years of trial and error.  What this study does show is that even complicated and dynamic processes like HIV membrane fusion can often be monitored and deep information gleaned from a very clever choice of one coordinate.

I’ve often spend time choosing a coordinate to succinctly show the transitions in a molecular dynamics system of interest, and seeing someone not only choose the right coordinate, but get a working smFRET experiment working along it for such a cool system was a lot of fun.

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