Samantha Harris
Co-Chair, ABBS Program
Member of the Graduate Faculty
Professor, BIO5 Institute
Professor, Biomedical Engineering
Professor, Cellular and Molecular Medicine
Professor, Physiological Sciences - GIDP
Professor, Physiology
Primary Department
(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

Kolb, J., Li, F., Methawasin, M., Adler, M., Escobar, Y. N., Nedrud, J., Pappas, C. T., Harris, S. P., & Granzier, H. (2016). Thin filament length in the cardiac sarcomere varies with sarcomere length but is independent of titin and nebulin. Journal of molecular and cellular cardiology, 97, 286-94.
BIO5 Collaborators
Hendrikus L Granzier, Samantha Harris

Thin filament length (TFL) is an important determinant of the force-sarcomere length (SL) relation of cardiac muscle. However, the various mechanisms that control TFL are not well understood. Here we tested the previously proposed hypothesis that the actin-binding protein nebulin contributes to TFL regulation in the heart by using a cardiac-specific nebulin cKO mouse model (αMHC Cre Neb cKO). Atrial myocytes were studied because nebulin expression has been reported to be most prominent in this cell type. TFL was measured in right and left atrial myocytes using deconvolution optical microscopy and staining for filamentous actin with phalloidin and for the thin filament pointed-end with an antibody to the capping protein Tropomodulin-1 (Tmod1). Results showed that TFLs in Neb cKO and littermate control mice were not different. Thus, deletion of nebulin in the heart does not alter TFL. However, TFL was found to be ~0.05μm longer in the right than in the left atrium and Tmod1 expression was increased in the right atrium. We also tested the hypothesis that the length of titin's spring region is a factor controlling TFL by studying the Rbm20(ΔRRM) mouse which expresses titins that are ~500kDa (heterozygous mice) and ~1000kDa (homozygous mice) longer than in control mice. Results revealed that TFL was not different in Rbm20(ΔRRM) mice. An unexpected finding in all genotypes studied was that TFL increased as sarcomeres were stretched (~0.1μm per 0.35μm of SL increase). This apparent increase in TFL reached a maximum at a SL of ~3.0μm where TFL was ~1.05μm. The SL dependence of TFL was independent of chemical fixation or the presence of cardiac myosin-binding protein C (cMyBP-C). In summary, we found that in cardiac myocytes TFL varies with SL in a manner that is independent of the size of titin or the presence of nebulin.

Chow, M. L., Shaffer, J. F., Harris, S. P., & Dawson, J. F. (2014). Altered interactions between cardiac myosin binding protein-C and α-cardiac actin variants associated with cardiomyopathies. Archives of biochemistry and biophysics, 550-551, 28-32.

The two genes most commonly associated with mutations linked to hypertrophic or dilated cardiomyopathies are β-myosin and cardiac myosin binding protein-C (cMyBP-C). Both of these proteins interact with cardiac actin (ACTC). Currently there are 16 ACTC variants that have been found in patients with HCM or DCM. While some of these ACTC variants exhibit protein instability or polymerization-deficiencies that might contribute to the development of disease, other changes could cause changes in protein-protein interactions between sarcomere proteins and ACTC. To test the hypothesis that changes in ACTC disrupt interactions with cMyBP-C, we examined the interactions between seven ACTC variants and the N-terminal C0C2 fragment of cMyBP-C. We found there was a significant decrease in binding affinity (increase in Kd values) for the A331P and Y166C variants of ACTC. These results suggest that a change in the ability of cMyBP-C to bind actin filaments containing these ACTC protein variants might contribute to the development of disease. These results also provide clues regarding the binding site of the C0C2 fragment of cMyBP-C on F-actin.

Luther, P. K., Bennett, P. M., Knupp, C., Craig, R., Padrón, R., Harris, S. P., Patel, J., & Moss, R. L. (2008). Understanding the organisation and role of myosin binding protein C in normal striated muscle by comparison with MyBP-C knockout cardiac muscle. Journal of molecular biology, 384(1), 60-72.

Myosin binding protein C (MyBP-C) is a component of the thick filament of striated muscle. The importance of this protein is revealed by recent evidence that mutations in the cardiac gene are a major cause of familial hypertrophic cardiomyopathy. Here we investigate the distribution of MyBP-C in the A-bands of cardiac and skeletal muscles and compare this to the A-band structure in cardiac muscle of MyBP-C-deficient mice. We have used a novel averaging technique to obtain the axial density distribution of A-bands in electron micrographs of well-preserved specimens. We show that cardiac and skeletal A-bands are very similar, with a length of 1.58+/-0.01 mum. In normal cardiac and skeletal muscle, the distributions are very similar, showing clearly the series of 11 prominent accessory protein stripes in each half of the A-band spaced axially at 43-nm intervals and starting at the edge of the bare zone. We show by antibody labelling that in cardiac muscle the distal nine stripes are the location of MyBP-C. These stripes are considerably suppressed in the knockout mouse hearts as expected. Myosin heads on the surface of the thick filament in relaxed muscle are thought to be arranged in a three-stranded quasi-helix with a mean 14.3-nm axial cross bridge spacing and a 43 nm helix repeat. Extra "forbidden" meridional reflections, at orders of 43 nm, in X-ray diffraction patterns of muscle have been interpreted as due to an axial perturbation of some levels of myosin heads. However, in the MyBP-C-deficient hearts these extra meridional reflections are weak or absent, suggesting that they are due to MyBP-C itself or to MyBP-C in combination with a head perturbation brought about by the presence of MyBP-C.

van Dijk, S. J., Kooiker, K. B., Mazzalupo, S., Yang, Y., Kostyukova, A. S., Mustacich, D. J., Hoye, E. R., Stern, J. A., Kittleson, M. D., & Harris, S. P. (2016). The A31P missense mutation in cardiac myosin binding protein C alters protein structure but does not cause haploinsufficiency. Archives of biochemistry and biophysics.

Mutations in MYBPC3, the gene encoding cardiac myosin binding protein C (cMyBP-C), are a major cause of hypertrophic cardiomyopathy (HCM). While most mutations encode premature stop codons, missense mutations causing single amino acid substitutions are also common. Here we investigated effects of a single proline for alanine substitution at amino acid 31 (A31P) in the C0 domain of cMyBP-C, which was identified as a natural cause of HCM in cats. Results using recombinant proteins showed that the mutation disrupted C0 structure, altered sensitivity to trypsin digestion, and reduced recognition by an antibody that preferentially recognizes N-terminal domains of cMyBP-C. Western blots detecting A31P cMyBP-C in myocardium of cats heterozygous for the mutation showed a reduced amount of A31P mutant protein relative to wild-type cMyBP-C, but the total amount of cMyBP-C was not different in myocardium from cats with or without the A31P mutation indicating altered rates of synthesis/degradation of A31P cMyBP-C. Also, the mutant A31P cMyBP-C was properly localized in cardiac sarcomeres. These results indicate that reduced protein expression (haploinsufficiency) cannot account for effects of the A31P cMyBP-C mutation and instead suggest that the A31P mutation causes HCM through a poison polypeptide mechanism that disrupts cMyBP-C or myocyte function.

Mun, J. Y., Kensler, R. W., Harris, S. P., & Craig, R. (2016). The cMyBP-C HCM variant L348P enhances thin filament activation through an increased shift in tropomyosin position. Journal of molecular and cellular cardiology, 91, 141-7.

Mutations in cardiac myosin binding protein C (cMyBP-C), a thick filament protein that modulates contraction of the heart, are a leading cause of hypertrophic cardiomyopathy (HCM). Electron microscopy and 3D reconstruction of thin filaments decorated with cMyBP-C N-terminal fragments suggest that one mechanism of this modulation involves the interaction of cMyBP-C's N-terminal domains with thin filaments to enhance their Ca(2+)-sensitivity by displacement of tropomyosin from its blocked (low Ca(2+)) to its closed (high Ca(2+)) position. The extent of this tropomyosin shift is reduced when cMyBP-C N-terminal domains are phosphorylated. In the current study, we have examined L348P, a sequence variant of cMyBP-C first identified in a screen of patients with HCM. In L348P, leucine 348 is replaced by proline in cMyBP-C's regulatory M-domain, resulting in an increase in cMyBP-C's ability to enhance thin filament Ca(2+)-sensitization. Our goal here was to determine the structural basis for this enhancement by carrying out 3D reconstruction of thin filaments decorated with L348P-mutant cMyBP-C. When thin filaments were decorated with wild type N-terminal domains at low Ca(2+), tropomyosin moved from the blocked to the closed position, as found previously. In contrast, the L348P mutant caused a significantly larger tropomyosin shift, to approximately the open position, consistent with its enhancement of Ca(2+)-sensitization. Phosphorylated wild type fragments showed a smaller shift than unphosphorylated fragments, whereas the shift induced by the L348P mutant was not affected by phosphorylation. We conclude that the L348P mutation causes a gain of function by enhancing tropomyosin displacement on the thin filament in a phosphorylation-independent way.