CRISPR gene-editing technology leads to new insights into hypertrophic cardiomyopathy – sciencedaily

An article published in the Proceedings of the National Academy of Sciences (PNAS) and co-authored by Beth Pruitt, professor of mechanical engineering at UC Santa Barbara and director of the Institute for BioEngineering on campus, describes the results of a complex, long-term collaboration that included researchers from the Stanford University, the University of Washington, and the University of Kentucky. The study led to a new understanding of how genetic mutations occur at the cellular level to cause HCM, and new insights into how to prevent it.

In their article, the authors explain that more than a thousand genetic mutations that cause HCM have been identified. The majority of them are found in the genes that code for sarcomere proteins, the structural building blocks of heart muscle responsible for the generation and regulation of contraction and relaxation. About a third of mutations are located in cardiac beta myosin, the main protein that causes heart cells to contract. Contraction of heart muscle, and all other muscles in our body, results from a process in which the motor protein myosin “walks” along a chain of actin molecules, a process known as cycle of cross bridges. During this process, chemical energy in the form of ATP is converted into mechanical energy, ultimately leading to cardiac contraction.

Before a contraction, the head of one strand of an intertwined two-stranded myosin molecule is folded back against an actin molecule. Muscle contraction is initiated when an ATP molecule, known as the “energy currency” of biological systems, binds to the myosin head. The myosin head and attached ATP then break away from actin, initiating the hydrolysis of ATP, which turns into ADP plus a phosphate group. This process releases energy which “arms” the myosin protein into a high energy state and changes the shape of the myosin so that it is ready to crawl along the actin. At this point, the phosphate is released from the myosin, which causes the myosin to push on the actin and release the phosphate, which causes the myosin to walk to the next actin chain and contract the muscle. . All of this, involving millions of myosin heads passing through actin in microsecond steps, must occur at the right rate in order to maintain heart health.

Since HCM is often seen in patients with cardiac myosin beta protein mutations, it has been hypothesized that HCM mutations cause a cascade of events that ultimately manifest themselves through damage to the heart itself. This study put that idea to the test, focusing on a single mutation, P710R, which dramatically decreased the rate of motility in vitro – the rate at which the myosin motor runs on actin – unlike d ‘other MYH7 mutations, which led to increased motility. speed.

The overall research question for this project was to learn how a mutation linked to heart disease in patients alters heart function at the cellular level.

The team used CRISPR technology to modify human-induced pluripotent stem cell cardiomyocytes (cells responsible for cardiac contraction) by inserting the P710R mutation. Pruitt runs the UCSB stem cell bank, where “clean” cell lines, showing no genetic abnormalities, are stored and reproduced for university researchers. These clean lines and without mutation constitute a perfect reference for the comparison with the cells in order to see very precisely the effects of the P710R mutation. For example, the research team is now testing the effects of different heart disease-related mutations in the same genetic background.

“You can have ten people with the same genetic mutation in that protein, and they can have different degrees of clinical significance, because the rest of their genome is different; that’s what makes us individuals,” Pruitt said. “These lines allow us to examine what is the result of the genetic mutation. By comparing the effect of different mutations, we can begin to distinguish how these changes lead to HCM. This allows us to take a close look at how and why cells adapt to mutation that way, and get data and relate it to the thickness of the heart wall and all the other things that happen downstream. “

This research began almost 15 years ago, while Pruitt was still at Stanford, and has led to this collaborative article. Now, CRISPR technology allows researchers to design cells expressing specific mutations linked to heart disease, then assess molecular and functional changes to determine the cellular impact of individual mutations that have been identified in patients with HCM. These studies will provide a mechanistic understanding of how individual mutations at the molecular level translate to HCM in patients.

In this project, once the mutation was introduced, the cells were tested in a collaboration between the Pruitt Laboratory (UCSB) and the Bernstein Laboratory (Stanford University), using tensile force microscopy, a test that allows the simultaneous observation of a beating cell and the force it generates. The Spudich lab (Stanford) conducted separate studies on the same mutated protein at the molecular level using an optical trap, in which light pressure is applied to precisely control the location and strength of a ” dumbbell “actin held between the beads as the myosin heads walk. along actin, to measure the potency cycle of myosin. The test found that the P710R mutation reduced the step size of the myosin motor (i.e., the length of each step) and the rate at which myosin was released from actin.

In a collaboration with University of Kentucky researcher Kenneth Campbell, these observations were then compared to a computer model of how myosin motors interact in the cell to generate force. The results confirmed a key role for the regulation of what is called the “super-relaxed state” of myosin. As Pruitt explained, “Myosin buds spend a lot of time in a super relaxed state, referring to when they are untied from actin. Any mutation or drug that changes the length or strength with which myosin motors bind to actin will change cell strength producing and changing downstream signaling events that result in remodeling and growth or hypertrophy. . “

The P710R mutation in this study was shown to destabilize the super-relaxed state. As a result, more myosin heads are bound to actin in the cells that harbor the mutation, which explains the increase in strength seen in those cells.

For Pruitt, a key element of the work, beyond important scientific discoveries, is the value of sustained collaboration. “The scales covered by the paper are generally not researched in one lab or even two labs,” she said. “This is why the article has so many authors, including several students and post-docs working with me, James Spudich and Daniel Bernstein.

“It’s important from a scientific point of view but also satisfying insofar as this level of integration makes it possible to test this idea on several scales. It’s been fun to work through these labs and skills on such an extensive multidisciplinary collaboration, and to see the power of molecular measurements and computation, and cell-derived measurements that allow us to genetically modify and dissect a only mutation, ”said Pruitt. “It’s really phenomenal, to directly test how a particular mutation introduces changes that lead to HCM.”

As a result of this collaboration, Pruitt added, “We can understand what’s going on at the cellular level. Then we can start developing models and identifying next-generation drug therapies. Instead of just identifying the symptoms, we can look at the mechanisms behind the dysfunctions and then tackle those at the cellular level before they turn into disease. “