CRISPR gene editing technology brings new insights into hypertrophic cardiomyopathy

A paper published in the Proceedings of the National Academy of Sciences (PNAS) and by Beth Pratt, Professor of Mechanical Engineering at the University of California, Santa Barbara and Director of the Campus Bioengineering Institute, describes the results of a complex long-term collaboration, including Stanford University researchers at the University of Washington and the University of Kentucky
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This research has given people a new understanding of how genetic mutations cause HCM at the cellular level, and a new perspective on how to prevent HCM

In their paper, the authors explained that more than 1,000 gene mutations that cause HCM have been identified
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Most of them are found in genes encoding body proteins

Before contraction, the head of one of the two myosin molecules entangled is folded over the actin molecule
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When the ATP molecule, known as the "energy currency" of the biological system, binds to the head of myosin, muscle contraction begins

Since HCM is often observed in patients with myocardial myosin mutations, it has been assumed that HCM mutations cause a series of events, which ultimately manifest as damage to the heart itself
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This study tested this idea, focusing on a single mutation, P710R, which significantly reduced the speed of movement in vitro compared with other MYH7 mutations that cause increased movement speed-myosin walks on actin.

The primary research question of the project is to understand how heart disease-related mutations alter heart function at the cellular level
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The research team used CRISPR technology to edit human induced pluripotent stem cell cardiomyocytes (cells responsible for heart contraction) by inserting the P710R mutation
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Pruitt leads UCSB's stem cell bank, where "clean" cell lines with no genetic abnormalities are maintained and replicated for use by university researchers

"You can have 10 people have the same genetic mutation in this protein, and they may have varying degrees of clinical significance because the rest of their genome is different; this is what makes us unique
.
" Pruitt said

This research began about 15 years ago, when Pruitt was still at Stanford University and contributed to this collaborative paper

In this project, once mutations are introduced, Pruitt Laboratory (UCSB) and Bernstein Laboratory (Stanford University) cooperate to detect cells using a traction force microscope.
This detection method can simultaneously observe beating cells and their production.。The force .
Spudich Lab (Stanford University), leading independent research on the same mutant protein using optical traps at the molecular level, medium-light pressure is applied to control the precise position and force between actin "dumbbell" beads as actin myospheres The protein head is along, measuring the power cycle of myosin

In a collaboration with University of Kentucky researcher Kenneth Campbell, these observations were compared with a computational model of how myosin motors interact in cells to generate force
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The results confirmed the key role of myosin "hyper-relaxed state" regulation
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As Pruitt explained, "The head of myosin is in a super-relaxed state many times, which refers to when it is separated from actin
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Any change in the time or strength of the binding of myosin motor to actin Mutations or drugs will change the production of cell power and change the downstream signaling events that drive remodeling, growth or hypertrophy
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"

This study found that the P710R mutation makes the over-relaxation state unstable
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As a result, more myosin heads bind to the actin in the mutant cells, which explains the increase in strength observed in these cells
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For Pruitt, in addition to important scientific discoveries, an important harvest of this work is the value of continuous cooperation
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She said: "The scale covered by this paper is usually not the research topics of any one laboratory or even any two laboratories
.
" "That's why this paper has so many authors, including several students and postdoctoral fellows who work with me.
, James Spuditch and Daniel Bernstein
.

"This is scientifically significant, but also satisfying, because this level of integration makes it possible to test this idea on multiple scales
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Working across these laboratories and these skills in such a wide-ranging, multidisciplinary collaboration It is very interesting to see that the power of molecular measurement and calculation, as well as cell-derived measurements, allows us to genetically engineer and dissect a single mutation," Pruitt said
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"It's really amazing to directly test how a specific mutation causes changes in HCM
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"

As a result of this collaboration, Pruitt added, “We can understand what is happening at the cellular level
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Then we can start to develop models and determine next-generation drug therapies
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In addition to identifying symptoms, we can also study the mechanisms behind the dysfunction, and then Solve these problems at the cellular level before it becomes a disease
.
"

Journal Reference :

1. Alison Schroer Vander Roest, Chao Liu, Makenna M.
   Morck, Kristina Bezold Kooiker, Gwanghyun Jung, Dan Song, Aminah Dawood, Arnav Jhingran, Gaspard Pardon, Sara Ranjbarvaziri, Giovanni Fajardo, Mingming Zhao, K.
   Pruittenneth S.
   Campbell, Beth L.
   Pruittenneth , James A.
   Spudich, Kathleen M.
   Ruppel, Daniel Bernstein.
   Hypertrophic cardiomyopathy β-cardiac myosin mutation (P710R) leads to hypercontractility by disrupting super relaxed state .
   Proceedings of the National Academy of Sciences , 2021; 118 (24): e2025030118 DOI: 10.
   1073/pnas.
   2025030118