Samantha Harris

Samantha Harris

Professor, Cellular and Molecular Medicine
Co-Chair, ABBS Program
Professor, Biomedical Engineering
Professor, Physiological Sciences - GIDP
Professor, Physiology
Member of the Graduate Faculty
Professor, BIO5 Institute
Primary Department
Contact
(520) 621-0291

Work Summary

The long-term goal of research in my lab is to understand the molecular mechanisms of muscle contraction. I am especially interested in how contractile proteins of muscle sarcomeres regulate the force and speed of contraction in the heart. The question is important from both basic science and clinical perspectives because mutations in sarcomere proteins of muscle are a leading cause of hypertrophic cardiomyopathy (HCM), the most common cause of sudden cardiac death in the young and a prevalent cause of heart failure in adults. Myosin binding protein-C (MyBP-C) is a muscle regulatory protein that speeds actomyosin cycling kinetics in response to adrenaline (b-adrenergic stimuli) and is one of the two most commonly affected proteins linked to HCM. Currently, the major research focus in my lab is understanding the mechanisms by which cMyBP-C regulates contractile speed and mechanisms by which mutations in cMyBP-C cause disease.

Research Interest

The long-term goal of research in my lab is to understand the molecular mechanisms of muscle contraction. I am especially interested in how contractile proteins of muscle sarcomeres regulate the force and speed of contraction in the heart. The question is important from both basic science and clinical perspectives because mutations in sarcomere proteins of muscle are a leading cause of hypertrophic cardiomyopathy (HCM), the most common cause of sudden cardiac death in the young and a prevalent cause of heart failure in adults. Myosin binding protein-C (MyBP-C) is a muscle regulatory protein that speeds actomyosin cycling kinetics in response to adrenaline (b-adrenergic stimuli) and is one of the two most commonly affected proteins linked to HCM. Currently, the major research focus in my lab is understanding the mechanisms by which cMyBP-C regulates contractile speed and mechanisms by which mutations in cMyBP-C cause disease. In pursuing these interests I have established a variety of approaches to investigate muscle contraction at molecular, cellular, and whole animal levels. Methods include single molecule atomic force microscopy (AFM), mechanical force measurements in permeabilized muscle cells, in vitro motility assays, biochemical enzyme and binding assays, immunofluorescent imaging, knockout/transgenic animal models and the development of a natural large animal model of HCM.

Publications

Karsai, A., Kellermayer, M. S., & Harris, S. P. (2011). Mechanical unfolding of cardiac myosin binding protein-C by atomic force microscopy. Biophysical journal, 101(8), 1968-77.

Cardiac myosin-binding protein-C (cMyBP-C) is a thick-filament-associated protein that performs regulatory and structural roles within cardiac sarcomeres. It is a member of the immunoglobulin (Ig) superfamily of proteins consisting of eight Ig- and three fibronectin (FNIII)-like domains, along with a unique regulatory sequence referred to as the M-domain, whose structure is unknown. Domains near the C-terminus of cMyBP-C bind tightly to myosin and mediate the association of cMyBP-C with thick (myosin-containing) filaments, whereas N-terminal domains, including the regulatory M-domain, bind reversibly to myosin S2 and/or actin. The ability of MyBP-C to bind to both myosin and actin raises the possibility that cMyBP-C cross-links myosin molecules within the thick filament and/or cross-links myosin and thin (actin-containing) filaments together. In either scenario, cMyBP-C could be under mechanical strain. However, the physical properties of cMyBP-C and its behavior under load are completely unknown. Here, we investigated the mechanical properties of recombinant baculovirus-expressed cMyBP-C using atomic force microscopy to assess the stability of individual cMyBP-C molecules in response to stretch. Force-extension curves showed the presence of long extensible segment(s) that became stretched before the unfolding of individual Ig and FNIII domains, which were evident as sawtooth peaks in force spectra. The forces required to unfold the Ig/FNIII domains at a stretch rate of 500 nm/s increased monotonically from ∼30 to ∼150 pN, suggesting a mechanical hierarchy among the different Ig/FNIII domains. Additional experiments using smaller recombinant proteins showed that the regulatory M-domain lacks significant secondary or tertiary structure and is likely an intrinsically disordered region of cMyBP-C. Together, these data indicate that cMyBP-C exhibits complex mechanical behavior under load and contains multiple domains with distinct mechanical properties.

McNamara, J. W., Li, A., Smith, N. J., Lal, S., Graham, R. M., Kooiker, K. B., van Dijk, S. J., Remedios, C. G., Harris, S. P., & Cooke, R. (2016). Ablation of cardiac myosin binding protein-C disrupts the super-relaxed state of myosin in murine cardiomyocytes. Journal of molecular and cellular cardiology, 94, 65-71.

Cardiac myosin binding protein-C (cMyBP-C) is a structural and regulatory component of cardiac thick filaments. It is observed in electron micrographs as seven to nine transverse stripes in the central portion of each half of the A band. Its C-terminus binds tightly to the myosin rod and contributes to thick filament structure, while the N-terminus can bind both myosin S2 and actin, influencing their structure and function. Mutations in the MYBPC3 gene (encoding cMyBP-C) are commonly associated with hypertrophic cardiomyopathy (HCM). In cardiac cells there exists a population of myosin heads in the super-relaxed (SRX) state, which are bound to the thick filament core with a highly inhibited ATPase activity. This report examines the role cMyBP-C plays in regulating the population of the SRX state of cardiac myosin by using an assay that measures single ATP turnover of myosin. We report a significant decrease in the proportion of myosin heads in the SRX state in homozygous cMyBP-C knockout mice, however heterozygous cMyBP-C knockout mice do not significantly differ from the wild type. A smaller, non-significant decrease is observed when thoracic aortic constriction is used to induce cardiac hypertrophy in mutation negative mice. These results support the proposal that cMyBP-C stabilises the thick filament and that the loss of cMyBP-C results in an untethering of myosin heads. This results in an increased myosin ATP turnover, further consolidating the relationship between thick filament structure and the myosin ATPase.

Shaffer, J. F., Kensler, R. W., & Harris, S. P. (2009). The myosin-binding protein C motif binds to F-actin in a phosphorylation-sensitive manner. The Journal of biological chemistry, 284(18), 12318-27.

Cardiac myosin-binding protein C (cMyBP-C) is a regulatory protein expressed in cardiac sarcomeres that is known to interact with myosin, titin, and actin. cMyBP-C modulates actomyosin interactions in a phosphorylation-dependent way, but it is unclear whether interactions with myosin, titin, or actin are required for these effects. Here we show using cosedimentation binding assays, that the 4 N-terminal domains of murine cMyBP-C (i.e. C0-C1-m-C2) bind to F-actin with a dissociation constant (K(d)) of approximately 10 microm and a molar binding ratio (B(max)) near 1.0, indicating 1:1 (mol/mol) binding to actin. Electron microscopy and light scattering analyses show that these domains cross-link F-actin filaments, implying multiple sites of interaction with actin. Phosphorylation of the MyBP-C regulatory motif, or m-domain, reduced binding to actin (reduced B(max)) and eliminated actin cross-linking. These results suggest that the N terminus of cMyBP-C interacts with F-actin through multiple distinct binding sites and that binding at one or more sites is reduced by phosphorylation. Reversible interactions with actin could contribute to effects of cMyBP-C to increase cross-bridge cycling.

Kittleson, M. D., Meurs, K. M., & Harris, S. P. (2015). The genetic basis of hypertrophic cardiomyopathy in cats and humans. Journal of veterinary cardiology : the official journal of the European Society of Veterinary Cardiology, 17 Suppl 1, S53-73.

Mutations in genes that encode for muscle sarcomeric proteins have been identified in humans and two breeds of domestic cats with hypertrophic cardiomyopathy (HCM). This article reviews the history, genetics, and pathogenesis of HCM in the two species in order to give veterinarians a perspective on the genetics of HCM. Hypertrophic cardiomyopathy in people is a genetic disease that has been called a disease of the sarcomere because the preponderance of mutations identified that cause HCM are in genes that encode for sarcomeric proteins (Maron and Maron, 2013). Sarcomeres are the basic contractile units of muscle and thus sarcomeric proteins are responsible for the strength, speed, and extent of muscle contraction. In people with HCM, the two most common genes affected by HCM mutations are the myosin heavy chain gene (MYH7), the gene that encodes for the motor protein β-myosin heavy chain (the sarcomeric protein that splits ATP to generate force), and the cardiac myosin binding protein-C gene (MYBPC3), a gene that encodes for the closely related structural and regulatory protein, cardiac myosin binding protein-C (cMyBP-C). To date, the two mutations linked to HCM in domestic cats (one each in Maine Coon and Ragdoll breeds) also occur in MYBPC3 (Meurs et al., 2005, 2007). This is a review of the genetics of HCM in both humans and domestic cats that focuses on the aspects of human genetics that are germane to veterinarians and on all aspects of feline HCM genetics.

Razumova, M. V., Shaffer, J. F., Tu, A., Flint, G. V., Regnier, M., & Harris, S. P. (2006). Effects of the N-terminal domains of myosin binding protein-C in an in vitro motility assay: Evidence for long-lived cross-bridges. The Journal of biological chemistry, 281(47), 35846-54.

Myosin binding protein-C (MyBP-C) is a thick-filament protein whose precise function within the sarcomere is not known. However, recent evidence from cMyBP-C knock-out mice that lack MyBP-C in the heart suggest that cMyBP-C normally slows cross-bridge cycling rates and reduces myocyte power output. To investigate possible mechanisms by which cMyBP-C limits cross-bridge cycling kinetics we assessed effects of recombinant N-terminal domains of MyBP-C on the ability of heavy meromyosin (HMM) to support movement of actin filaments using in vitro motility assays. Here we show that N-terminal domains of cMyBP-C containing the MyBP-C "motif," a sequence of approximately 110 amino acids, which is conserved across all MyBP-C isoforms, reduced actin filament velocity under conditions where filaments are maximally activated (i.e. either in the absence of thin filament regulatory proteins or in the presence of troponin and tropomyosin and high [Ca2+]). By contrast, under conditions where thin filament sliding speed is submaximal (i.e. in the presence of troponin and tropomyosin and low [Ca2+]), proteins containing the motif increased filament speed. Recombinant N-terminal proteins also bound to F-actin and inhibited acto-HMM ATPase rates in solution. The results suggest that N-terminal domains of MyBP-C slow cross-bridge cycling kinetics by reducing rates of cross-bridge detachment.