Normal cardiac contraction in mice lacking the proline-alanine rich region and C1 domain of cardiac myosin binding protein C

Abstract

Cardiac myosin binding protein C (cMyBP-C) is an essential regulator of cross bridge cycling. Through mechanisms that are incompletely understood the N-terminal domains (NTDs) of cMyBP-C can activate contraction even in the absence of calcium and can also inhibit cross bridge kinetics in the presence of calcium. In vitro studies indicated that the proline-alanine rich (p/a) region and C1 domain are involved in these processes, although effects were greater using human proteins compared to murine proteins (Shaffer et al. J Biomed Biotechnol 2010, 2010: 789798). We hypothesized that the p/a and C1 region are critical for the timing of contraction. In this study we tested this hypothesis using a mouse model lacking the p/a and C1 region (p/a-C1(-/-) mice) to investigate the in vivo relevance of these regions on cardiac performance. Surprisingly, hearts of adult p/a-C1(-/-) mice functioned normally both on a cellular and whole organ level. Force measurements in permeabilized cardiomyocytes from adult p/a-C1(-/-) mice and wild type (Wt) littermate controls demonstrated similar rates of force redevelopment both at submaximal and maximal activation. Maximal and passive force and calcium sensitivity of force were comparable between groups as well. Echocardiograms showed normal isovolumetric contraction times, fractional shortening and ejection fraction, indicating proper systolic function in p/a-C1(-/-) mouse hearts. p/a-C1(-/-) mice showed a slight but significant reduction in isovolumetric relaxation time compared to Wt littermates, yet this difference disappeared in older mice (7-8months of age). Moreover, stroke volume was preserved in p/a-C1(-/-) mice, corroborating sufficient time for normal filling of the heart. Overall, the hearts of p/a-C1(-/-) mice showed no signs of dysfunction even after chronic stress with an adrenergic agonist. Together, these results indicate that the p/a region and the C1 domain of cMyBP-C are not critical for normal cardiac contraction in mice and that these domains have little if any impact on cross bridge kinetics in mice. These results thus contrast with in vitro studies utilizing proteins encoding the human p/a region and C1 domain. More detailed insight in how individual domains of cMyBP-C function and interact, across species and over the wide spectrum of conditions in which the heart has to function, will be essential to a better understanding of how cMyBP-C tunes cardiac contraction.

Keywords: Cardiac contraction; Echocardiogram; Mice; Myosin binding protein C; Sarcomere.

Figure 1

Schematic overview of the domains that make up cardiac myosin binding protein C (cMyBP-C). Ig-like domains are represented as ovals and FnIII-like domains as grey rectangles. The C0 and C1 domains are connected by proline-alanine rich region (p/a). A second linker region called the M-domain, or motif, is located between C1 and C2. The M-domain contains phosphorylation sites (asterisks) that modulate the interaction of cMyBP-C with other sarcomeric proteins. Regions of cMyBP-C that binds to other sarcomeric proteins are indicated. The region of interest studied in this work, the p/a region and C1 domain, is indicated by a green box.

Figure 2

Cardiac myosin binding protein C (cMyBP-C) expression in hearts of p/a-C1^−/− and Wt mice. A. Immunostaining with antibodies directed against cMyBP-C (green) and α-actinin (red) demonstrated incorporation of cMyBP-C in doublets both in sarcomeres of Wt and p/a-C1^−/−mice. Image stacks were deconvolved using softWoRx® version 5.5 (GE Healthcare). Appropriate images were flattened into a single projection using Graphic Converter X version 6.7 (Lemke Software GmbH) and the “Merge folder into one Image” command. The images were histogram stretched and false colored in Photoshop CS5.1 (Adobe) with α-actinin in red and cMyBP-C in green. B. Coomassie stain of SDS-page gels or Western blots with antibodies raised against cMyBP-C of cardiac protein homogenates confirmed the presence of a shorter cMyBP-C in p/a-C1^−/− mice. C. Quantification of l of cMyBP-C expression demonstrated similar cMyBP-C expression in p/a-C1^−/− and Wt mice.

Figure 3

A. Force-pCa relationships of p/a-C1^−/− and Wt mice before and after incubation with protein kinase A (PKA) demonstrated no differences in calcium sensitivity between groups. PKA incubation decreased calcium sensitivity in both groups as indicated by a rightward shift of the curve. B. Rate of force redevelopment at different pCa a C. levels of activation showed no significant differences between p/a-C1^−/− and Wt mice. *P<0.05 Wt versus p/a-C1^−/−.

Figure 4

Phosphorylation of cMyBP-C (A, D), cardiac troponin I (cTnI) (B, E) and cardiac troponin T (cTnT) (C, F) was comparable between p/a-C1^−/− mice and Wt mice as determined by ProQ Diamond phosphoprotein stain. Treatment of the mice with propranolol for a week prior to their sacrifice lowered protein phosphorylation (Supplemental figure 1), but did not introduce differences between groups (D, E, F).

Figure 5

Representative echocardiograms from wild type (Wt) and p/a-C1^−/− mice showing a B-mode short axis view (A), a parasternal long axis view (B), and an M-mode view showing traces used to analyze the morphometry and function of the hearts (blue lines) (C).