Wednesday, February 24, 2010

The new and notable models and Giant Molecules -Cardiac muscle and the failing heart

The cardiac muscle session chaired by Dr. Solaro and brought to the limelight the new key players of muscle contraction. A key observation also included the animal model systems and whole heart studies in vivo, ex vivo and in vitro done to understand the cardiomyopthies in totality as studies traversed from whole heart to the single molecules step by step.  Hence, I enumerate contractile players who are in the focus and models of the same.

Beta -MHC - Myosin Heavy Chain  (220 Kda) occurs in two isoforms in the heart - the alpha MHC and the beta MHC. As the transition from a non-failing to failing heart progresses, the beta MHC isoform takes over and is predominant in the heart. This isoform is slower than the alpha MHC and thus more energetically economical for the heart. Given the energy starved status of the heart, the switch takes over. The human isoform to begin with is predominantly beta. However, in small rodents like mice alpha isoform is predominant. Dr. Susan Lowey highlighted how a single mutation in MHC can severely affect the kinetics of cross bridge cycling in the herat and lead to cardiomyopathies. It is important to keep in mind here that even after PTU treatment of mouse hearts which would convert alpha to beta MHC - the kinetics of contraction are not the same as that found in higher mammals where the beta isoform is always predominant. Hence the need to study higher models like Guinea pigs, rabbits, pigs is very important. My own work in Dr. De Tombe's lab is targeting this issue as I have developed a Guinea pig model of Heart failure.

MyBP-C - Myosin Binding Protein C is a 140 Kda protein found in the contractile apparatus. MyBP-C puts a constraint on the mysoin heads as they bind of actin and regulates the contraction process. In KO models of MyBP-C cardiomyoptahies are known to develop. Dr. Richard Moss gave a very comprehensive talk on the phosphorylation states of the molecule and how they affect the kinetics of cross-bridge cycling. Dr. Sakthivel Saddyappan has worked on various KO mouse models of this protein and shown how important it is in maintaining the physiological function of the heart. Dr. Moss elegantly showed how phosphorylation of MyBP-C alters the kinetics of contraction in the cardiac muscle.

Titin -  The next molecule brought into focus was Titin - a 400 KDa protein. Titin provides a 3 spring system in the contractile machinery to modulate the stiffness of the molecule. Dr. Henk Granzier talked about the how various isoforms of titin alter the passive stiffness of muscle. Dr. Wolfgang Linkes work on the phosphorylation of isoforms of titin by PKG complements the studies shown by Dr. Henk Granzier. In the failing heart a switch takes place from the N2B isoform to the fetal isform N2BA and as this artio alters so does the passive stiffness of the cardiac muscle, leading to impaired relaxation and diastolic failure. Also was shown how PKA and PKC induced phosphorylation of titin alters the passive stiffness.

Also in limelight is Nebulin - the measurement ruler in the muscle, the SERCA pump and Phospholamban system (Dr. Beata Wolska gave a very elegant talk on the workings of the system) which regulate the calcium concentration within the muscle and the Troponin complex - which regulates contraction.

Cheeses, Wines, Fungi, Chocolates, Salami -Ferry Building

Today is the last day of BPS. Lot of us are packing our bags and checking out from the hotels. A must do before you leave the city of SFO, is a visit to the Ferry building. Just walk down the market street towards the Bay bridge and you will reach the Ferry building. From amazing cheeses you can taste and pick at the cowgirl creamery to mouth watering truffle oils to Napa valley wines to delectable chocolates - you got it all at the Ferry building!

So add a small item on your itinerary before you board that plane! You will not regret the visit at all. Not to mention, the view of the Bay bridge is awesome from here and you can have Peet's coffee as you admire the Bridge! The city's romance is unforgettable!

Tuesday, February 23, 2010

Spice up your palate at BPS -Dosas

Spicy, aromatic and delightful fusions of South Indian cuisine tantalized our taste buds at this restaurant tonight. The food is exotic and very tastefully prepared by the chef. The serving style is a delight to the senses. Without being overly priced and offering a most modern ambiance with excellent service, this place is not to be missed out in while you are in San Francisco.

Enjoy a hearty meal before you leave SFO!

The Proteomic Era of Structure and Post-translational modifications

As I attend more and more muscle and cradiac muscle talks at BPS, I realize how much the focus has moved from just studying protein function, to appreciating and studying the much more dynamic changes in proteins via structural studies and post-translational modifications like phosphorylation, methylation, oxidation and ubiquitination. Exchange studies wherein phosphomimetics prepared by single site directed mutagenesis are created and exchanged within the muscle preparations to study these effects. PTM (post-translational modifications) sites are identified using 2-D DIGE studies, mass spectrometry, IEF gels, ProQ diamond staining, phospho-tags. 

The mechanics and kinetics of cross-bridge cycling are then studied by langendorff hearts, intact fiber preparations, single cell studies, single myofibril technique, in-vitro motlity assays, 3- bead assay and exchange studies. Mechanical data are extensively being employed to study the nature of these PTM exchanges to understand how they regulate muscle function. Structural studies using X-ray diifraction, NMR, circular dichroism and mass spectrometry are key tools to really look at the molecular structure and protein folding to understand these phosphomimetics to see what changes within the molecule/s.

Muscle mechanics - the emerging techniques of note II

Many new techniques were brought to the forefront with the muscle mechanics sessions we have had thus far. Having detailed some in my previous article, I detail some more emergent techniques having a great impact in the field.

Single Myofibril Technique - It was developed in Dr. Poggesis lab in Italy and has been a focus of our lab (Dr. De Tombe) as well (check Ryan Matejas poster on Wednesday). My work heavily employs this technique as well. It enables us to measure kinetics of activation/relaxation in the muscle within milliseconds. The forces recorded are of the order of nano newtons and the muscle preparation itself is approx. 20-80 um long to 2-4 um in diameter. The technique uses phase contrast microscopy. The myofibrils (a single skinned string of sarcomeres ) are bound to the coverslip glass by electrostatic interactions. We pick them up between two glass needles. One needle is stiff and doe snot move while the second needle has an L-shaped tip of about 7um and is compliant. As the solutions are switched from relaxing to activating using a double barreled perfusion pipette, the muscle contracts, pulls on the compliant needle thereby making it move and the movement is then detected by video microscopy. The forces and kinetics are then calculated. 

Exchange Mass Spectroscopy - Deuterium water exchange is been exploited to study the structure of various molecules (myosin, troponins, tropomysoin) in the contractile apparatus in various conformations (calcium free, calcium bound, phosphorylated, methylated, binary complexes, ternary complexes). The idea behind the technique is simply to replace the hydrogen ions in the amino acid backbone by the deuterium labeled isoform of hydrogen and then capture the mass difference  in form of shifted m/z ratio of peptide/protein peaks by mass spectrometry. A qualitative as well as quantitative analysis ensues.

In Vitro Motility assay - This technique enables us to measure the kinetics of sliding velocity of myosins over the thin filament. The major advantage of this technique is that  one can  play around with a lot of the contractile proteins individually and study their effects very specifically. The  myosin heads are bound on the coverslip. As thin filament molecules (actin, actin with Tropomysoin, actin with tropomysoin and troponin) are injected onto the coverslips they start sliding under unloaded conditions. You could change the isotonic strength of the solutions, the ATP/ADP ratios, phosphomimetic protein modifications and study these changes very easily as the velocity of thin filamnet sliding varies. The movement is captured by video microscopy.

Flourescence Resonance Energy Transfer (FRET) - Intermolecular interactions are studied as two molecules in close proximity to each other are labeled with two different flourophores. The preparation is excited at two different wavelengths and the signal is captured. As the molecules interact based on the experimental conditions, conformational changes might take place which can be recorded using FRET by measuring the distance changes between the two flourescent signals under a confocal microscope. Currently FRET is being used in Dr. Seth Robias lab to take it to the next level wherein intramolecular changes can be recorded by measuring the distances between the signals.

STED - Developed in Dr. Lehnharts lab, STED is an emission microscopy based technique wherein a higher resolution of imaging is obtained in contrast to confocal microscopy. For molecules like t-tubules where sizes are less than 300nm, confocal does not capture all the nuances of the structure as the wavelength it uses is within the same range of 300nm. However with STED you get sharper images and better resolution. A doughnut shape around the area of excitation by laser is exploited in STED. A major advantage of STED is that it can capture structural dimensions in the z-axis as well and reveal in-depth details.

Muscle Mechanics - the emerging techniques of note - I

The Monday morning muscle sessions started with two awesome talks on the relay loop of the myosin and its function in stabilizing the myofibril structure assembly and force generation. While the morning session talks were super interesting, of note were the new emerging techniques that were talked about.

Gravitational Force Spectroscopy (GFS) - a new technique developed in the Root lab, essentially exploits a two microsphere (small glass beads) system wherein the larger of the two micro-spheres is anchored and the smaller one is free floating. The distance between the centers of these two microspheres is at its minimum. As a myosin molecule tethers itself between the microspheres, gravitational force is exploited such that it is either parallel or perpendicular to the orientation of the molecule. As the free floating microsphere rotates based off the gravity, one can calculate the min. and max. distance between the centers of the two microspheres and thereby calculate the flexibility of the myosin tethered. It is a novel and neat technique, with lots of potential for experiments contrasting diseased and control states of various molecules and post translational modifications altering flexibility.

3- Optical bead Assay - Highlighted in the last two talks of the session was the 3-optical bead assay. The gist of the technique - mount two anchored polystyrene glass beads on two opposite ends on a coverslip. Motion sensors are attached to each of these beads. Actin filaments attach these two beads together. A third bead is positioned in between these two beads and a myosin head is tethered to it. As the myosin head forms a bond with the actin filament, the anchored beads move, the distance is sensed by the motion sensors and the step size of force generation by one head is revealed. Now if you attach small loads to each of the two beads attached to the actin filament, you get the working stroke  of the muscle under loaded conditions!

The other notable techniques that are emerging are the single myofibril technique, FRET, mass spectroscopy and cryo- EM. which i would cover in part II of this article later in the day. Have to rush to career workshops now!

Monday, February 22, 2010

Students go Hungry, BPS Grad Student Breakfast runs out

Bright and early students wake up and jaunt up to Room 301 at 7:30 am to get to know fellow graduate students and have a hearty breakfast hosted by BPS for graduate students. Lo and behold they run out of food in less than 10 minutes. BPS calculations went off by far! Students were not happy about the situation.
I am headed to the muscle mechanics sessions now!

Until later updates - have a good day!