Research Highlights

Experimentation and Computation Meet to Elucidate Structure-Function Relationships in the Beat of a Heart


Average contact map of residue-residue pairs between NcTnC and NcTnI during 450 ns molecular dynamics simulations.

A normal heart uses the complementary, alternating activities of “systolic” contraction and “diastolic” relaxation to pump blood throughout the body. So scientists are eager to understand the various factors that affect this process, including those that lead to heart disease and failure.

Each pumping cycle is initiated when calcium is released from storage sites in the muscle cells and binds to the protein troponin of the contractile filaments. Troponin is the protein complex that acts as a switch from relaxation to contraction in heart muscle and is the central regulator of the magnitude of contraction.

After a suspected heart attack, for example, doctors look for evidence of troponin, which can be detected from dead cells floating in the blood stream, to confirm that’s what’s happened. In many inherited diseases, troponin mutations can be the culprits that affect the heart’s ability to activate and regulate the relaxation-contraction system. So a long-term scientific goal is to understand how troponin works so researchers can develop more targeted therapeutics to treat dysfunction, first with pharmaceuticals, then with more novel approaches such as reengineering of proteins and cells.

More specifically, troponin consists of three subunits that work together: troponin C (cTnC), troponin I (cTnI), and troponin T. Contraction is initiated by calcium binding to cTnC. The resulting conformational changes lead to the dislocation of cTnI from its inhibitory position on the contractile filaments, allowing the muscle to contract.

cTnC interaction with cTnI is termed C-I interaction. C-I interaction is a critical point for contractile modulation of the normal systolic function of the left ventricle, which drives blood out of the heart into the aorta. It is also critical during adrenergic stimulation (caused by adrenalin), which serves as a primary physiological mechanism that enables the heart to respond to the need for increased circulation.

C-I interaction can be regulated by Protein Kinase A (PKA) phosphorylation of cTnI during adrenergic stimulation. Phosphorylation refers to the addition of a phosphate group to the proteins, which alters their function. When cTnI is phosphorylated, the C-I interaction is weakened, causing a reduction in the calcium affinity of cTnC and an increase in the rates of muscle relaxation.

It is this C-I interaction that has been the topic of several recent interrelated studies – experimental and computational – led by Dr. Michael Regnier and his research group at the University of Washington. He is working with two labs at UCSD led by Dr. J. Andrew McCammon and Dr. Andrew McCulloch to apply an extremely powerful combination of methods – protein biochemistry, measurement of myofibril mechanics/kinetics, and computational modeling – to determine the molecular mechanisms by which the C-I interaction regulates heart muscle contraction and how phosphorylation of myofilament proteins affects contraction and relaxation of normal and diseased cardiac muscle.

The experimental work has just been published in the September 5 issue of Biophysical Journal (see citation [1] at the end of this article.) A second paper (citation [2]), on computational simulations of troponin that parallel the experimental work, is due to be published in October.

Regnier says, “Our collaboration with the National Biomedical Computation Resource has made it possible to conduct these types of studies where experiments provide details about the function of contractile proteins, and molecular dynamics simulations provide detailed molecular structure information to help determine the mechanisms of function.” He adds that this approach can be very powerful as a platform to study normal function and regulation, dysfunction with mutations associated with cardiac diseases such as hypertrophic or dilated cardiomyopathy, and to test targeted therapeutic treatments for these diseases.

McCammon confirms that the two papers are part of a robust collaboration between Regnier’s group and the NBCR groups at UCSD. He explains that a particularly important part was the appointment of Regnier’s postdoc Yuanhua Cheng (second author of the paper in citation [1] and first author of citation [2]) as an official visitor to UCSD, with support from NBCR. “Yuanhua is well plugged in to the experimental work at Washington,” says McCammon, “but she’s also comfortable with computation. Even so, postdocs Pete Kekenes-Huskey and Steffen Lindert from our group were particularly helpful in working with her on the computational aspects.” McCammon provided office space, consulting, and computer access during her visits to UCSD in 2013-14. All these aspects, hallmarks of NBCR collaborations, are critical to success. And, in fact, the success of this project has been underscored by the offer, to Kekenes-Huskey, of an assistant professorship in chemistry at the University of Kentucky.

The collaboration began through Regnier’s connection with McCulloch. “We have similar research interests and wanted to work together on modeling,” says Regnier. McCulloch invited him to a workshop at UCSD at which they explored how to incorporate more biology into some of the questions NBCR was investigating. “Since changes in troponin could potentially affect all the scales at which NBCR researchers were working,” says Regnier, “it was a natural choice to focus on.”

As a result of this engagement, Regnier became a co-investigator with his “driving biomedical project” in the NBCR renewal grant submitted earlier this year and recently awarded. The goal of the project: to build a molecular dynamics model of whole troponin and scaling tools to model the effects of troponin mutations associated with genetic cardiac diseases at the protein, myofilament, and cell levels.

Looking at the project more broadly, McCulloch says that Regnier’s project has been a great driver for new “cross-core” innovation in NBCR, especially towards the goal of developing multi-scale computational models that integrate from the level of molecular defects to whole-organ disease phenotypes. “This project has high importance,” he says, “because it is helping elucidate how genetic defects in genes encoding for contractile proteins in heart muscle can give rise to familial heart diseases such as hypertrophic cardiomyopathy.” Because much more common heart diseases like ventricular hypertrophy and heart failure share common features with these relatively rare genetic diseases, the researchers are also learning much about the molecular mechanisms of disease and potential new drug targets and therapeutic strategies.

Indeed, NBCR Director Rommie Amaro commented, “The collaboration with the Regnier group has been a key driver for our technology development. It is one of NBCR’s flagship projects because it allows us to develop new tools and modeling schemes that unite studies from a single protein molecule all the way up to models of whole organs. In this regard, the collaboration with the Regnier group is unique and particularly important to our success as a resource.”

When asked about the most exciting aspects of the project, Regnier responds enthusiastically, “This is the first time we have been able to put together an entire working model of the troponin complex to study regulation by phosphorylation. What that means is that we’ve combined computational, mechanical, and biochemical approaches to couple experimentation with computational modeling to demonstrate the relationship between structure and function.” The bottom line, he says, is that, in future, they will be able to integrate this information into larger computational models at the cell and organ level to see how single amino-acid level mutations can affect the function of the whole organ.

Additional studies of a hypertrophic cardiomyopathy mutation in TnI (R145G) have been conducted, with one paper in review and another in preparation. These new studies of mutation in troponin protein subunits will build on the framework of the normal structure-function analysis.

“Speaking of excitement, these studies provided a bit in their own right,” says Regnier. “With modeling, we found the structural reasons for why phosphorylation increases relaxation. But with the mutation, the binding wasn’t occurring and the model showed why. The postdoc was concerned about these results when, in fact, to her credit they were absolutely correct.”


  1. Rao, Vijay, Yuanhua Cheng, Steffen Lindert, Dan Wang, Lucas Oxenford, Andrew D. McCulloch, J. Andrew McCammon, and Michael Regnier, PKA Phosphorylation of Cardiac Troponin I Modulates Activation and Relaxation Kinetics of Ventricular Myofibrils, Biophysical Journal, Vol. 107, September 5, 2014, pp. 1-9,
  2. Cheng, Yuanhua, Steffen Lindert, Peter Kekenes-Huskey, Vijay S. Rao, R. John Solaro, Paul R. Rosevear, Rommie Amaro, Andrew D. McCulloch, J. Andrew McCammon, and Michael Regnier, Computational Studies of the S23D/S24D Troponin I Mutation on Cardiac Troponin Structural Dynamics, Biophysical Journal (in press; publication planned for October 2014).

Figure 1: Average contact map of residue-residue pairs between NcTnC and NcTnI during 450 ns molecular dynamics simulations for (A) wild type and (B) cTnI-S23D/S24D complexes. The blue end of the spectrum (value 0) reflects no contact between residue-residue pair, while the red end of the spectrum (value 1) represents 100% contact. (C) Difference contact map of residue-residue pairs between NcTnC and NcTnI mostly affected upon introducing the S23D/S24D mutations. Green (value 0) reflects no difference between the two systems, the red end of the spectrum (values above 0) reflects more contacts in the cTnI-S23D/S24D cTn system, and blue (values below 0) indicates more contacts in the wild-type model.