Calcium sensitivity and myofilament lattice structure in titin N2B KO mice

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Abstract

The cellular basis of the Frank–Starling “Law of the Heart” is the length-dependence of activation, but the mechanisms by which the sarcomere detects length changes and converts this information to altered calcium sensitivity has remained elusive. Here the effect of titin-based passive tension on the length-dependence of activation (LDA) was studied by measuring the tension–pCa relation in skinned mouse LV muscle at two sarcomere lengths (SLs). N2B KO myocardium, where the N2B spring element in titin is deleted and passive tension is elevated, was compared to WT myocardium. Myofilament lattice structure was studied with low-angle X-ray diffraction; the myofilament lattice spacing (d1,0) was measured as well as the ratio of the intensities of the 1,1 and 1,0 diffraction peaks (I1,1/I1,0) as an estimate of the degree of association of myosin heads with the thin filaments. Experiments were carried out in skinned muscle in which the lattice spacing was reduced with Dextran-T500. Experiments with and without lattice compression were also carried out following PKA phosphorylation of the skinned muscle. Under all conditions that were tested, LDA was significantly larger in N2B KO myocardium compared to WT myocardium, with the largest differences following PKA phosphorylation. A positive correlation between passive tension and LDA was found that persisted when the myofilament lattice was compressed with Dextran and that was enhanced following PKA phosphorylation. Low-angle X-ray diffraction revealed a shift in mass from thin filaments to thick filaments as sarcomere length was increased. Furthermore, a positive correlation was obtained between myofilament lattice spacing and passive tension and the change in I1,1/I1,0 and passive tension and these provide possible explanations for how titin-based passive tension might regulate calcium sensitivity.

Highlights

► We studied passive tension, length-dependent activation (LDA), and muscle structure. ► A positive correlation between titin-based passive tension and LDA was found. ► Passive tension and myofilament lattice spacing were positively correlated. ► A shift in mass from thin filaments to thick filaments occurred.

Introduction

An important aspect of cardiac adaptation is the immediate systolic pressure increase in response to increased venous return, an effect known as the Frank–Starling mechanism (FSM)2 of the heart [1], [2], [3]. It had been suggested that increased calcium release in response to increased sarcomere length might be an important contributor to the FSM but this now seems unlikely [2], [4] and instead increased calcium sensitivity of force development appears to be the dominant effect [2], [5]. Thus, when sarcomeres are stretched, the myofilaments produce more force for the same level of calcium, i.e. the myofilaments display length-dependent activation (LDA). Various myofilament components are likely to contribute to LDA: the thin-filament based troponin (Tn) complex [6], [7], the thick-filament based proteins cMyBP-C and MLC2 [8], [9], and titin, the third myofilament of the cardiac sarcomere [10], [11], [12], [13], [14], [15]. The titin filament directly senses stretch and interacts with both actin and myosin; titin is, therefore, a good candidate for a major role in the FSM. This study is focused on the role of titin in LDA, using the N2B KO model in which one of the spring elements of titin (the N2B element) is deleted [16]. Titin is a giant filamentous protein that spans the half-sarcomeric distance from Z-disk to M-band [17]. Titin’s I-band region functions as a molecular spring that develops passive tension when sarcomeres are stretched [18] and it is now well accepted that titin is important for the diastolic health of the heart [16], [17], [19], [20]. A role for titin in LDA has been suggested by experiments in which titin was degraded by trypsin and LDA was reduced [10], [21], [22] and also by experiments in which changes in titin-based passive tension due to variable passive stress relaxation [11] or to the variable expression of titin isoforms [13], [23] correlated with changes in LDA. A recent study on a rat model that expresses highly compliant titin isoforms and in which LDA was reduced [15] supports the notion that titin plays an important role in determining calcium sensitivity.

In the aforementioned titin studies, LDA was measured in skinned muscle, which has the advantage that the calcium concentration can be well controlled. However, skinning also results in myofilament lattice spacing expansion [24]. We therefore studied LDA in skinned muscle from WT and N2B KO mice, using both tension–pCa measurements and low angle X-ray diffraction, in which the lattice spacing was reduced with Dextran-T500. We measured the myofilament lattice spacing (d1,0) and the ratio of the intensities of the 1,1 and 1,0 diffraction peaks (I1,1/I1,0) as an estimate of the proximity of myosin heads to the thin filaments. Because the beta-adrenergic tone of cardiac muscle affects LDA [25] and to rule out subtle differences in the PKA phosphorylation status between WT and KO muscle, experiments with and without lattice compression were carried out following PKA phosphorylation of the skinned muscle (see Methods), normalizing thereby the phosphorylation status between WT and N2B KO myocardium. It was found that PKA phosphorylation increases LDA, mainly by lowering calcium sensitivity at the short sarcomere length (1.95 μm) and, under all experimental conditions, calcium sensitivity at the long sarcomere length (2.3 μm) is greatest in N2B KO myocardium. X-ray diffraction revealed that both myofilament lattice spacing and I1,1/I1,0 became smaller as sarcomere length was increased and that the reduction in myofilament lattice spacing and in I1,1/I1,0 scaled with passive tension.

Section snippets

Animal model

Wild type (WT) mice and N2B element-deficient mice (N2B KO) were used in which exon 49 of the titin gene had been deleted [16]. Mice were genotyped as described previously [16], and the results were confirmed by 1% agarose protein gels [26]. Three months old male N2B KO and WT mice were used, and all surgical procedures and skinning protocols were as described previously [27]. Briefly, mice were sacrificed by cervical dislocation, the hearts were removed, and papillary muscles from the left

Results

Skinned muscle strips, stretched to a sarcomere length (SL) of either 1.95 μm or 2.3 μm, were activated at various calcium concentrations (expressed as pCa or −log[Ca2+]), and active tension at each pCa was determined as a fraction of the maximal tension measured at pCa 4.5. Calcium sensitivity was determined from the pCa50 (pCa where active tension is half of the value at pCa 4.5). An example of an experiment is shown in Fig. 1, and average tension–pCa curves are shown in Fig. 2. WT mice were

Discussion

It is well-established that the cellular basis underlying the Frank–Starling relationship is the length-dependence of activation, but the mechanisms by which the sarcomere detects length changes and converts this information in altered calcium sensitivity has remained elusive. Here we studied the effect of titin-based passive tension on the length-dependence of activation by measuring, at two SLs, the tension–pCa relation in skinned mouse LV muscle. We compared WT mice with N2B KO mice that

Acknowledgments

We would like to acknowledge the contributions of Chen-Ching Yuan, Sami Somo, Cynthia Yang and Hsiao Man Hsu who analyzed the X-ray intensity data as well as funding by NIH grant HL62881 (HG), DFG (MG) and an AHA postdoctoral fellowship (EJL). “Use of the Advanced Photon Source, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357. “This project was

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    1

    Present address: Department of Rehabilitation Medicine, College of Medicine, Seoul National University, Seoul, South Korea.

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