Tropomyosin Flexural Rigidity and Single Ca2+ Regulatory Unit Dynamics: Implications for Cooperative Regulation of Cardiac Muscle Contraction and Cardiomyocyte Hypertrophy

Striated muscle contraction is regulated by dynamic and cooperative interactions among Ca2+, troponin, and tropomyosin on the thin filament. While Ca2+ regulation has been extensively studied, little is known about the dynamics of individual regulatory units and structural changes of individual tropomyosin molecules in relation to their mechanical properties, and how these factors are altered by cardiomyopathy mutations in the Ca2+ regulatory proteins. In this hypothesis paper, we explore how various experimental and analytical approaches could broaden our understanding of the cooperative regulation of cardiac contraction in health and disease

Persistence Length of Human Cardiac α-Tropomyosin Measured by Single Molecule Direct Probe Microscopy

α-Tropomyosin (αTm) is the predominant tropomyosin isoform in adult human heart and constitutes a major component in Ca2+-regulated systolic contraction of cardiac muscle. We present here the first direct probe images of WT human cardiac αTm by atomic force microscopy, and quantify its mechanical flexibility with three independent analysis methods. Single molecules of bacterially-expressed human cardiac αTm were imaged on poly-lysine coated mica and their contours were analyzed. Analysis of tangent-angle (θ(s)) correlation along molecular contours, second moment of tangent angles (<θ2(s)>), and end-to-end length (Le-e) distributions respectively yielded values of persistence length (Lp) of 41–46 nm, 40–45 nm, and 42–52 nm, corresponding to 1–1.3 molecular contour lengths (Lc). We also demonstrate that a sufficiently large population, with at least 100 molecules, is required for a reliable Lp measurement of αTm in single molecule studies. Our estimate that Lp for αTm is only slightly longer than Lc is consistent with a previous study showing there is little spread of cooperative activation into near-neighbor regulatory units of cardiac thin filaments. The Lp determined here for human cardiac αTm perhaps represents an evolutionarily tuned optimum between Ca2+ sensitivity and cooperativity in cardiac thin filaments and likely constitutes an essential parameter for normal function in the human heart

Tangent angle correlation analysis shows that L_p of WT human cardiac αTm equals 40.6−45.8 nm.

<p>ln() data obtained from three separate samples independently prepared under identical conditions are plotted as a function of segment length along the molecular contour. The slope of this plot is –1/2L_p. L_p for WT Tm from this analysis were 45.8±0.8 nm (N = 741, R² = 0.99), 43.5±0.8 nm (N = 628, R² = 0.98) and 40.6±0.8 nm (N = 798, R² = 0.98). The variation in the L_p values represents the uncertainty inherent to our experimental setup and the tangent angle correlation analysis.</p

Deposition time study suggests L_p measurements were stable with incubation time of 300 s or longer.

<p>L_p values obtained by tangent angle correlation analysis (blue asterisks, left axis) and the corresponding number of molecules considered (black dots, right axis) were plotted against incubation times. An overestimation of L_p was observed at incubation times below 300 s, which may be due to the shorter incubation time and/or smaller number of molecules available for the analysis. L_p measurements were stable at incubation times above 300 s, where variation was comparable to the inherent uncertainty in our methodology. This implies an incubation time of 300 s was sufficient for surface equilibration of αTm on p-Lys coated mica substrate.</p

Deposition rate of αTm on p-Lys substrate shows the process is diffusion driven and irreversible.

<p>The number ratio between αTm molecules adhered to p-Lys coated mica and in 1 cm³ of the bulk solution, <i>N_s/N_B(t = 0)</i>, increased with incubation time up to 300 s, as shown in both linear (main graph) and logarithmic (inset) scales. 5% of total number of αTm molecules in the bulk solution were deposited on the substrate by the 300 s incubation; the absence of discernible change at longer incubation times of 450 s and 600 s suggests the top layer of the bulk solution was depleted of αTm molecules <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039676#pone.0039676-Rivetti1" target="_blank">[26]</a>. Fitting data from incubation time 10 s to 300 s to Eq. 1 (solid lines) returned estimates of two parameters: exponent parameter <i>p</i> equals 0.49, in close accordance to an irreversible and diffusion driven deposition process <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039676#pone.0039676-Rivetti1" target="_blank">[26]</a>; and diffusion constant parameter <i>D</i> of αTm equals 2.2×10⁻⁷ cm²/s, consistent with previous estimates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039676#pone.0039676-Cantor1" target="_blank">[43]</a>.</p

End-to-end length analysis shows L_p of WT human cardiac αTm consistent with tangent angle correlation analysis.

<p>Normalized end-to-end length (l_e-e) distributions from one of the WT αTm samples incubated on p-Lys coated mica substrate for 600 s (N = 798) fits to the WLC model (Eq. 3). <i>l_p</i> value from the fit was 1.0425±0.0505 (R² = 0.88). Errors were estimated by the jackknife method (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039676#s2" target="_blank">Materials and Methods</a>).</p

AFM images of α-tropomyosin (αTm) molecules.

<p>Wildtype human cardiac αTm was imaged dry on poly-lysine coated mica (A). Collage of 20 αTm molecules (B) show that molecular contours were smooth and continuous. One of the αTm molecules in the collage was processed as described (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039676#pone-0039676-g002" target="_blank">Figure 2</a> C) and overlayed with the x-ray structure of αTm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039676#pone.0039676-Phillips2" target="_blank">[38]</a> on the same scale (B, expanded on right), which is evidence that the AFM images were good representations of single αTm molecules.</p

Image processing procedure to extract the molecular contour of αTm from a typical AFM scan.

<p>An αTm molecule was selected from a typical 512 nm ×512 nm scan (A) and cropped into a smaller image (B). The image was filtered by a Gaussian box-car filter (C), thresholded (D), and skeletonized into a 1-pixel wide connected contour (E, F). A refined skeleton with coordinates defined at sub-pixel precision was generated by fitting the perpendicular height profiles to a Gaussian function (G), which was then fitted with a 5^th order polynomial. The continuous contour defined by the polynomial conformed very well with the shape of the original molecule (H). Contour length (L_c) and end-to-end length (L_e-e) of the molecule shown were 41.7 nm and 38.4 nm, respectively.</p

Summary of L_c, L_e-e, and L_p from tangent angle correlation, second moment and end-to-end length analyses.

*<p>0.01% p-Lys deposition on mica by 30 s incubation.</p>**<p>0.01% p-Lys deposition on mica by 300 s incubation.</p