Multiscale problems require multiscale solutions.
It sounds like an unprovable claim from a generic corporate services ad.
To one international team of researchers, however, it is a hunch that could revolutionize the way doctors diagnose and treat hypertrophic cardiomyopathy (HCM), a prevalent, and often deadly, form of heart disease. HCM is a genetic condition that, confoundingly, affects some people with the mutation very seriously and others hardly at all.
Why HCM shows up so unpredictably and how it progresses upward from a microscopic coding error to a visibly damaged heart are among the questions scientists from the University of Washington, Stanford University, the University of California Santa Barbara, and the Curie Institute in Paris set out to explore five years ago with a $10 million grant from the National Institute of General Medical Sciences (NIGMS). Building off of existing collaborations with UW and UCSB researchers, scientists at the Allen Institute for Cell Science also joined the effort.
Dr. Daniel Bernstein, from the Department of Pediatrics – Cardiology at Stanford University School of Medicine has served as the Principal Investigator for the grant. Other members of the research collaboration include Dr. James Spudich, Dr. Kathleen Ruppel and Dr. Sean Wu (from Stanford) and Dr. Beth Pruitt from USCB. Representing UW are Dr. Mike Regnier, a Professor of Bioengineering and the Director of the UW Center for Translational Muscle Research (CTMR), and Dr. David Mack, an Associate Professor of Rehabilitation Medicine. Regnier and Mack are also members of the UW Institute for Stem Cell and Regenerative Medicine (ISCRM).
While the NIH-funded effort is already producing valuable insights about the mechanics of the human heart, the partnership is also bearing out another premise central to the grant: Multiscale problems don’t just require multiscale solutions. They also require multi-institutional collaborations.
“The idea was to attack a clinically challenging problem by putting together a team to do science that no single lab could do alone,” explains Mack.
Combining their complementary areas of expertise allowed the investigators to trace the pathology of HCM in stem cell models. The chain of events begins when a mutated amino acid causes a protein to have a significant change in structure. As a result, the heart’s contractile machinery enters a hyperactive state, which in turn, leads to metabolic changes at the cell and tissue levels. In effect, the excessive cardiac muscle growth leaves the heart less room to relax and fill, impairing its ability to pump blood.
But not always.
Teaming Up to Study Hypertrophic Cardiomyopathy at Multiple Scales
In some cases, HCM causes few or no symptoms. In other cases, depending on the extent of the decrease of blood flow, the thickened heart muscle that results from the mutation can lead to chest pain, shortness of breath, irregular heartbeats, changes to the heart’s electrical wiring, and even sudden death. While screening can detect HCM, and drugs can be used to ease symptoms and limit risk, there is currently no cure. This variance in severity and the lack of adequate interventions motivated the grant-funded coalition to study HCM at multiple scales.
Now, the first findings from five years of research examining these factors have been published in the journal PNAS. The study describes how crucial capabilities from each university were used to show in unprecedented detail how a mutation in myosin leads to abnormal heart rhythms accompanied by inefficient energy consumption at the cellular level.
Dr. Bernstein from Stanford is the paper’s lead investigator. The first author is Dr. Soah Lee, a former postdoctoral fellow from Dr. Wu’s lab at Stanford who studies how mutations affect transcriptional regulation of proteins. Dr. Ruppel and Dr. Spudich from Stanford, and Dr. Pruitt from UCSB, are also authors. Regnier and Mack are the co-primary investigators from UW.
In total, this multi-lab paper features seven de facto lead authors who shared credit for their equal contributions to the work. In addition to Dr. Lee, these include former postdoctoral fellow Dr. Alison Vander Roest, working with Bernstein, Pruitt, and Spudich; former postdoctoral fellow Dr. Cheavar Blair, working with Pruitt; current PhD student Kerry Kao, working with Regnier; former graduate student Dr. Samantha Bremner, working in the Mack Lab and the lab of ISCRM faculty member Dr. Nate Sniadecki; Dr. Matthew Childers, working in the Regnier and Mack Labs; and Dr. Divya Pathak, postdoctoral fellow working with Spudich and Ruppel.
Many genetic mutations that cause HCM occur in the protein myosin, which binds to another protein, called actin, to power cardiac muscle contractions by converting the molecule ATP into cellular energy. Altering the transfer of power from a myosin head that latches on to actin and pulls along the length of the cardiac muscle cells to create a contraction could mean that the cell requires more fuel (ATP) to perform its life sustaining function (helping our hearts beat).
One of the discoveries revealed in the study is that mutations at the protein level can cause more myosin heads to become available for binding to actin, which can lead to hypertension and, subsequently, hypercontractility of the walls of the heart. As the cells work harder, and mitochondrial genes become upregulated, the increase in activity can lead to excessive cardiac muscle growth, which can impair electrical signaling and blood flow, leading to symptoms associated with the disease.
“There are billions of cells in your muscles and trillions of myosin molecules inside those cells,” says Regnier. “That means small changes can have huge consequences if a significant portion of those myosin heads requires even one percent more energy.”
Combining Areas of Expertise and Analytical Tools
The investigation used a variety of analytical tools to study how HCM progresses from the point of origin to the point of manifestation.
The Mack Lab, in partnership with the Pruitt Lab, used a genetically engineered stem cell line generated by the Allen Institute for Cell Science to create a model for studying HCM at the cellular and tissue levels. Mack praises Dr. Brock Roberts from the Allen Institute for producing the lines through a self-devised CRISPR-editing technique that gave the researchers a “matched set” of different genetic mutations, which allow for apple-to-apples comparisons in future studies.
Dr. Bremner, the former graduate student in the Mack Lab and Sniadecki labs, used the Allen cells to create engineered heart tissues (EHTs) to examine the impact of the disease as millions of cardiac cells contract in unison to generate force.
Ruwanthi (Ru) Gunawardane, PhD is the Executive Director of the Allen Institute for Cell Science. She speaks to her team’s enthusiasm for the HCM research. “The Allen Institute for Cell Science team is thrilled to be part of this important collaborative effort by creating the collection of gene-edited stem cell lines used to conduct this important work. We are confident that the research done by these groups, collectively, will not only deepen our understanding of the disease but also pave the way for developing therapies that make a significant difference in the lives of patients affected by this condition.”
Mack also credits the Pruitt Lab with bringing a specific skill set to the table: traction force microscopy – or the ability to measure force exerted by individual cells. Understanding the relative contribution of each heart cell and then evaluating how they work together is at the center of this multiscale approach.
Going smaller, the Regnier Lab studied the mutation at the protein level, using Molecular Dynamics simulations to determine how the single amino acid change altered the structure throughout the myosin motor and then used experimental approaches to demonstrate how this affected function at the level of sub-cellular contractile organelles called myofibrils.
Meanwhile, in Palo Alto, the Ruppel and Spudich Lab in the Department of Biochemistry and the Bernstein Lab for Cardiovascular Research zoomed in to the molecular level to better understand the interplay between protein shape and function, metabolism and muscle contraction, and the cascading consequences of HCM mutations.
Improving Prevention, Diagnoses, and Management of HCM
Regnier and Mack both stressed the important roles that trainees from each partner institution played in the research. “Just at UW, we had more than a dozen postdocs, graduate students and even undergraduates all working together brilliantly to pull this off,” says Regnier. “And it’s a golden opportunity for them to learn early in their careers how to collaborate effectively.”
Ultimately, Mack and Regnier emphasize the significance of a multidisciplinary team of muscle experts, biophysicists, engineers, and geneticists joining forces to understand the roots of a widespread health challenge and to explore and test new strategies to manage the excessive muscle growth at different points in the progression of the disease.
“There are billions of cells in your muscles and trillions of myosin molecules inside those cells,” says Regnier. “That means small changes can have huge consequences if a significant portion of those myosin heads requires even one percent more energy.”
One question for further study will be why the mutations affect individuals with the genetic abnormality so differently. In the course of the research, the team made seven different mutations in different parts of the protein and found that each mutation had its own pathology, and that the severity varied widely. The researchers are still learning why this is the case. One theory is that scattered locations of the mutation may help explain the unpredictable penetrance.
What is clear is the importance of a collaborative, multiscale approach.
“The paper highlights beautifully the information you can get from each of these scales of analysis,” says Mack. “Seeing how the different data types all fit together allows us to ask where to intervene therapeutically and to potentially design a drug that will work in concert across those different levels. We hope this becomes a road map for how other heart diseases will be studied.”
“For decades we have been studying the end stages of the disease,” adds Regnier. “We are now using these incredible tools to study the initiation of the disease process. That’s where we need to be to improve prevention, diagnoses, and early management, all of which contribute to better outcomes for patients.”