Multiple forms of cMyBP-C in the whole animal
Western blots of lysed rat ventricle quickly frozen immediately after euthanasia were produced using antibodies against C0C2, C5 and C8C9 fragments of cMyBP-C. Each blot had a dense band with an apparent molecular weight of 145 kD () and, in some cases, a faint suggestion of a band at 130 kD in the anti-C5 and anti-C8C9 blots (). During euthanasia of a rat there is a large release of catecholamines that increases phosphorylation of cMyBP-C. This release of catecholamines can be blocked by intraperitoneal injection of the β adrenergic blocker, propranolol. When 10 μg/kg of propranolol was injected into the peritoneal cavity 20 min before euthanasia to block the action of the catecholamine, Western blots with anti-C5 and anti-C8C9 produced two clear, separate bands, one at 145 kD and a second at 130 kD (). There was no staining of a 130-kD band with anti-C0C2. The density of the single 145-kD band stained with anti-C0C2 was significantly reduced by the propranolol injection, suggesting that some cMyBP-C existed in a form not stained by that antibody. In every one of 17 experiments the second band disappeared after washing out the propranolol and incubating the intact trabecula in 0.1 μM isoproterenol for 15 min (). These results indicate that under normal physiological conditions two different forms of cMyBP-C under adrenergic control can exist in vivo and the conversion of one to the other can occur within minutes.
Figure 3. Western blots of rat heart using three antibodies. Lane 1, quickly frozen tissue from a heart of a control animal (had not received propranolol); lane 2, quickly frozen tissue from a heart that had received propranolol before euthanasia; and lane 3, quickly (more ...)
Beta adrenergic agonists cannot cause phosphorylation of cMyBP-C in intact cardiac cells unless one phosphate is already present (McClellan et al., 2001
). A similar restriction exists on the conversion of the 130- to the 145-kD form by β adrenergic agonists. 0.1 μM isoproterenol was added to the bathing solutions of trabeculae that had been soaked in 1.0 or 2.5 mM Ca for 2 h to produce, respectively, low and moderately high levels of phosphorylated cMyBP-C (McClellan et al., 2001
). The drug increased the density of the 145-kD band where a major fraction (>60%) of cMyBP-C was phosphorylated before the addition of the drug and had much less effect where only a minor fraction of the cMyBP-C (<20%) was phosphorylated (unpublished data).
The 130-kD protein was not a proteolytic fragment of cMyBP-C. The N-terminal sequence of the 130-kD band (PEPGKRPVSA), identified by Edman degradation, was the same as the sequence in human, mouse, and dog cMyBP-C except for substitution of arginine for lysine at position 6, both of which carry a negative charge (xPASy database). It was not possible to sequence the N terminus of the 145-kD band because the protein appeared to be blocked to Edman degradation. Proteolysis of C10 and/or C9 at the C terminus was an unlikely cause of the more rapid migration on the gel because of the following: (a) anti-C8C9 still recognized the 130-kD form and anti-C0C2 did not; (b) rapid reversibility of the migration pattern; and (c) the rapid change in density of the 145- and 130-kD bands after exposure to isoproterenol. The increase in the density of the 145-kD bands and the decrease of the 130-kD bands occurred too rapidly for in vitro protein synthesis. Restoration of cMyBP-C after the proteolysis that occurs during transient ischemia requires ~10 d (Decker et al., 2005
). Very small changes in a protein can produce changes in its rate of migration on SDS gels that normally would indicate a larger change in molecular weight (Bottinelli et al., 1998
; Katori et al., 2004
). This is particularly true of phosphorylation (Burden and Sullivan, 1994
Effect of [Ca] on the two forms of cMyBP-C
The density of both bands in gels from resting trabeculae varied with the concentration of Ca in the superfusion solution and the duration of incubation ( and ; n = 16; P < 0.05). Trabeculae frozen within 3 min of euthanasia produced the same single band as quickly frozen ventricles. After 2 or more hours at rest in 1.0 mM Ca (during which bound catecholamine was washed out), Western blots of trabeculae produced the same bands as hearts from propranolol-injected animals. Trabeculae soaked at rest in 7.5 mM Ca for up to 6 h had little or no 130-kD form and a constant density of their 145-kD form (). Decreasing Ca concentration from 7.5 to 1.0 mM produced a 130-kD band and decreased the 145-kD band. Raising Ca from 1.0 to 7.5 inhibited further change in the two bands, but it did not significantly reverse the changes that had already occurred in 1.0 mM (n = 4, P < 0.05).
Figure 4. Western blots with three antibodies against different regions of cMyBP-C. The protocols (duration of soak and Ca concentration) for the various muscles are shown. Note the single bands with quickly frozen trabeculae, and the second band with antibodies (more ...)
Figure 5. (A) Densities of 145- and 130-kD forms plotted as a function of the duration of incubation of the trabeculae in 1.0 or 7.5 mM Ca. Each point represents the mean ± SEM of at least five experiments. The antibody against C5 was used in the Western (more ...)
Density of both bands changed with the duration of the exposure to 1.0 mM Ca ( and ). For 3 h the density of the 130- and 145-kD forms, respectively, increased and decreased, but the combined densities remained constant (). The intensity of the 130-kD band increased to a peak at 3 h, and then declined with further superfusion (). After 3 h, the combined densities decreased (). 2.5 mM Ca produced a similar course, but the magnitude of the changes was smaller.
Proteolysis of the 130-kD Form of cMyBP-C
The sum of densities of the 145- and 130-kD forms remained constant for 3 h regardless of the Ca concentration in the soak solution (). After >3 h in 1 mM Ca, the amount of the 130 kD and the sum of the two densities gradually decreased (). Initially the 145 kD was converted to the 130-kD form, but after 3 h, proteolysis of the 130-kD form became apparent. Lower molecular weight bands stained by the antibodies against cMyBP-C appeared (). Most of the visible smaller peptides were stained by anti-C0C2 and not by anti-C5 or -C8C9, indicating that the proteolysis was probably occurring in the C5 to C10 region, which forms a putative collar around the thick filament.
The ability of anti-C0C2 to stain proteolytic fragments of the 130-kD form of cMyBP-C supports the conclusion from sequencing that the N-terminal region has not been lost in the conversion of the 145- to the 130-kD form. The proteolysis in the C5C9 region has apparently had an effect on the epitope in the C0C2 region enhancing the recognition of the protein by the anti-C0C2, consistent with other data that events in one end of the molecule can influence the nature of the other end of the molecule (McClellan et al., 2004
Differences in Phosphorylation in the Two Forms of cMyBP-C
Anti-phosphate antibody measures total phosphate and cannot distinguish among the three phosphorylation sites. The antibody always stained the 145-kD band and with one exception never stained the 130-kD band (). The extent of antiphosphate antibody staining varied considerably depending on prior treatment. Incubation with 7.5 mM Ca, which prevented the appearance of the 130-kD band, was associated with a high level of phosphorylation. A 2-h soak in 1 mM Ca reduced antiphosphate staining by >50%.
Figure 6. (A) Blots of antibody against C5 (bottom) and phosphorylated PKA substrate (top). The same gel is shown. Lane 1, quickly frozen; lane 2, 4 h in 1.0 mM Ca; lanes 3 and 4, stimulated at 12/min for 30 min in 2.5 mM Ca after 3 h at rest in 2.5 mM Ca; lane (more ...)
Incubation with 10 U of alkaline phosphatase for 4 h reduced phosphorylation to an undetectable level and converted all of the 145-kD form to the 130-kD form (). Incubation with 3 U decreased the phosphate content of cMyBP-C by 64 ± 7% (n = 4, P < 0.05), and converted 23 ± 4% (n = 4, P < 0.05) of the 145- to the 130-kD form. After incubation of a preparation having a low level of phosphorylation of cMyBP-C (as a result of incubation in a 1.0 mM Ca solution) with PKA, the level of phosphorylation and the relative amount of the 145-kD form were increased substantially ().
Effect of Contraction on the Two Forms of cMyBP-C
After 3 h of superfusion at rest in 2.5 mM Ca, several trabeculae were stimulated for 30 min at 12 Hz. The stimulation, which would be expected to raise time-averaged concentration of intracellular Ca, increased antiphosphate staining of the 145-kD band by 42 ± 6% (P < 0.05; n = 5), and produced the only detectable staining of the 130-kD band. A portion of the 130-kD band was converted to the 145-kD band. The relative intensity of the 145-kD band increased by 37 ± 5% (n = 5, P < 0.05) and the intensity of the 130-kD band decreased (). Since it was not possible to stimulate isolated trabeculae at in vivo rates, we could not ascertain whether physiological rates of stimulation would produce greater phosphorylation and transition between the two forms.
Ultrastructure of Thick Filaments
Isolated, negatively stained thick filaments had different structures depending on which form of cMyBP-C was predominant (). Populations of predominately the 130- or the 145-kD form were produced by using, respectively, 1.0 or 7.5 mM Ca in the soak solutions. Thick filaments with the 130-kD form had disordered myosin heads that were often extended at different angles to the filament backbone (). With the 145-kD form, myosin heads were well ordered, lying uniformly along the backbone of the filament ().
Figure 7. Electron micrographs of a part of two isolated thick filaments. (A) From a trabecula that had been soaked in 1.0 mM Ca to produce dephosphorylated cMyBP-C and a band at 130 kD with anti-C5 or anti-C8C9. Arrowheads point to irregular, extended myosin heads. (more ...)
Loss of MHC Follows Loss of cMyBP-C
Trabeculae were chemically skinned to permit control of the microenvironment of the myofibrils, to detect changes in the composition of the contractile filaments, and to determine whether the cMyBP-C or its proteolytic fragments were bound to myofibrils. Bound protein remained with the skinned cardiomyocytes while protein not bound in the cell interior appeared in the soak solution.
After dissection, intact resting trabeculae were soaked in electrolyte solution containing either 1.0 or 7.5 mM Ca for 2 h to produce cells with respectively low (<20%) and high (>80%) levels of phosphorylation of cMyBP-C. During and immediately after their skinning, there was no loss of myosin or cMyBP-C in the several bathing solutions, indicating that no cMyBP-C or MHC had been removed from the trabeculae. 4 h after skinning, the relaxing solution that had bathed trabeculae from the 7.5 mM Ca group contained 3 ± 0.4% of the total cMyBP-C (P < 0.05) and 1% of myosin heavy chain (MHC; P > 0.05; the reference value for 100% protein was the total amount of each protein in trabeculae after their skinning). There was no statistically significant change in the cMyBP-C or MHC content of the trabeculae. Over the same period in relaxing solution, trabeculae from the 1.0 mM Ca group lost significantly more cMyBP-C and MHC, respectively, 18 ± 3% cMyBP-C and 6 ± 2% MHC (n = 6; P < 0.05). These washouts of cMyBP-C and MHC were accompanied by significant declines in the content of cMyBP-C and MHC in the trabeculae of, respectively, 23 ± 5 and 8 ± 4% (P < 0.05). The total cMyBP-C lost may have been underestimated by this method because washout solutions contained low molecular weight peptides stained by antibodies against regions of cMyBP-C. TNT was not present in any soak solutions, indicating that the instability was limited to the thick filament ().
After extraction of ~20% of the total cMyBP-C, release of MHC began (). Release of MHC always followed, never preceded or accompanied, the initial extraction of cMyBP-C (). Thick filament stability appeared to exist until the filaments had lost at least 20% of cMyBP-C. The rate at which cMyBP-C was extracted was faster when the trabeculae had been exposed to 1.25 mM than to 2.5 or 7.5 mM Ca, but the threshold of cMyBP-C loss at which the loss of MHC began was not significantly different.
Figure 9. (A) Relation between cMyBP-C extracted and MHC lost. Each point represents the mean of at least four different experiments. Lines between points are interpolations and do not represent a specific function. (B) Western blots with anti C0C2 (bottom) and (more ...)
Isolated thick filaments were much less resistant to shear forces after a large fraction of cMyBP-C has been extracted (Kulikovskaya et al., 2003b
). Filaments with 35% of cMyBP-C extracted (using the protocol of Kulikovskaya et al., 2003b
) began to fragment at ~17 dynes/cm2
. Thick filaments with a normal complement of cMyBP-C were able to withstand shear forces at least two to three times higher without fragmenting (). The length of the fragments varied from over 0.8 μm to the dimensions of individual double-headed myosin molecules.
Figure 10. Left, electron micrographs of negatively stained, isolated thick filaments with a normal content of cMyBP-C; right, filaments from a trabecula that had had 36% of its cMyBP-C extracted. Both sets of filaments had been subjected to the same shear force (more ...)
Thick filaments were isolated from trabeculae that had been bathed at rest for 2 h in 1.25 mM Ca solutions to produce a low level of phosphorylation of cMyBP-C (McClellan et al., 2001
; Kulikovskaya et al., 2003b
) and then subjected to the same level of shear force as extracted filaments. These thick filaments fragmented into pieces that were similar to those in the preparation of extracted filaments. As only a limited range of shear force was used, it was not possible to determine whether dephosphorylation of cMyBP-C produces the same degree of filament instability as extraction. (This point is currently being studied using a more quantified level of shear force and a larger population of thick filaments.)