Publications by authors named "Makenna M Morck"

4 Publications

  • Page 1 of 1

Hypertrophic cardiomyopathy β-cardiac myosin mutation (P710R) leads to hypercontractility by disrupting super relaxed state.

Proc Natl Acad Sci U S A 2021 Jun;118(24)

Department of Pediatrics (Cardiology), Stanford University School of Medicine, Palo Alto, CA 94304;

Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1,000 mutations, many in β-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreased in vitro motility velocity and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super relaxed state in longer, two-headed myosin constructs, freeing more heads to generate force. Micropatterned human induced pluripotent derived stem cell (hiPSC)-cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrated the measured molecular changes to predict the measured traction forces. These results confirm a key role for regulation of the super relaxed state in driving hypercontractility in HCM with the P710R mutation and demonstrate the value of a multiscale approach in revealing key mechanisms of disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2025030118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8214707PMC
June 2021

The hypertrophic cardiomyopathy mutations R403Q and R663H increase the number of myosin heads available to interact with actin.

Sci Adv 2020 04 3;6(14):eaax0069. Epub 2020 Apr 3.

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA.

Hypertrophic cardiomyopathy (HCM) mutations in β-cardiac myosin and myosin binding protein-C (MyBP-C) lead to hypercontractility of the heart, an early hallmark of HCM. We show that hypercontractility caused by the HCM-causing mutation R663H cannot be explained by changes in fundamental myosin contractile parameters, much like the HCM-causing mutation R403Q. Using enzymatic assays with purified human β-cardiac myosin, we provide evidence that both mutations cause hypercontractility by increasing the number of functionally accessible myosin heads. We also demonstrate that the myosin mutation R403Q, but not R663H, ablates the binding of myosin with the C0-C7 fragment of MyBP-C. Furthermore, addition of C0-C7 decreases the wild-type myosin basal ATPase single turnover rate, while the mutants do not show a similar reduction. These data suggest that a primary mechanism of action for these mutations is to increase the number of myosin heads functionally available for interaction with actin, which could contribute to hypercontractility.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.aax0069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7124958PMC
April 2020

Deciphering the super relaxed state of human β-cardiac myosin and the mode of action of mavacamten from myosin molecules to muscle fibers.

Proc Natl Acad Sci U S A 2018 08 13;115(35):E8143-E8152. Epub 2018 Aug 13.

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305;

Mutations in β-cardiac myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. However, measurements of the intrinsic force, velocity, and ATPase activity of myosin have not provided a consistent mechanism to link mutations to muscle pathology. An alternative model posits that mutations in myosin affect the stability of a sequestered, super relaxed state (SRX) of the protein with very slow ATP hydrolysis and thereby change the number of myosin heads accessible to actin. Here we show that purified human β-cardiac myosin exists partly in an SRX and may in part correspond to a folded-back conformation of myosin heads observed in muscle fibers around the thick filament backbone. Mutations that cause hypertrophic cardiomyopathy destabilize this state, while the small molecule mavacamten promotes it. These findings provide a biochemical and structural link between the genetics and physiology of cardiomyopathy with implications for therapeutic strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1809540115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126717PMC
August 2018

Recurrent RNA motifs as scaffolds for genetically encodable small-molecule biosensors.

Nat Chem Biol 2017 03 16;13(3):295-301. Epub 2017 Jan 16.

Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado USA.

Allosteric RNA devices are increasingly being viewed as important tools capable of monitoring enzyme evolution, optimizing engineered metabolic pathways, facilitating gene discovery and regulators of nucleic acid-based therapeutics. A key bottleneck in the development of these platforms is the availability of small-molecule-binding RNA aptamers that robustly function in the cellular environment. Although aptamers can be raised against nearly any desired target through in vitro selection, many cannot easily be integrated into devices or do not reliably function in a cellular context. Here, we describe a new approach using secondary- and tertiary-structural scaffolds derived from biologically active riboswitches and small ribozymes. When applied to the neurotransmitter precursors 5-hydroxytryptophan and 3,4-dihydroxyphenylalanine, this approach yielded easily identifiable and characterizable aptamers predisposed for coupling to readout domains to allow engineering of nucleic acid-sensory devices that function in vitro and in the cellular context.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nchembio.2278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5310984PMC
March 2017
-->