Distinct Mechanisms for Increased Cardiac Contraction Through Selective Alteration of Either Myosin or Troponin Activity

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SUMMARY
Modulation of sarcomere contractility represents a new therapeutic opportunity for the treatment of heart failure by directly targeting the thick and thin filament proteins of the sarcomere to increase cardiac muscle contraction. This study compared the effect of 2 small molecules (M and T) that selectively alter myosin thick filament (M) or troponin thin filament (T) activity on overall cardiac muscle mechanics. This study revealed key differences related to the mechanism utilized by M and T to increase contractile force generation and suggests that targeting different proteins within the sarcomere may result in differentiating therapeutic pro-  5,6 Molecules that directly target sarcomere protein activity represent a new therapeutic class for the treatment of HF that aim to improve cardiac muscle contraction. Sarcomeres, the basic functional unit of striated and cardiac muscle, facilitate muscle contraction through the movement of the actin thin filament over the myosin thick filament. This process is coupled to myosin ATPase activity and regulated both by changes in intracellular calcium concentrations and by post-translational modifications to the sarcomere proteins. [7][8][9][10][11] Omecamtiv mecarbil (OM) improves contractile performance by stimulating myosin. [12][13][14] OM increases myosin ATPase activity, allowing for increased contractile performance in cardiac muscle. 13 shown to reduce left ventricular (LV) diameter and improve cardiac function, and resulted in a modest but significant reduction in the time to first event in the composite endpoint of cardiovascular death or HF events, respectively. [15][16][17][18] HF is a heterogenous disease, and efforts are currently underway to better understand if a subgroup of patients within GALACTIC-HF exhibited a greater therapeutic benefit with OM to identify those best suited to receive this medicine. 15 Several post-translational modifications to the sarcomere resulting in abnormal contractile performance are associated with HF. 19,20 These modifications impede cross-bridge cycling, the process by which myosin binds to and releases actin during muscle contraction. Cardiac myosin binding protein-C (cMyBP-C) is a sarcomere protein that has multiple functional roles that influence contraction. When dephosphorylated, it promotes the super relaxed state (SRX), or less active state of myosin; however, when phosphorylated, it facilitates sarcomere contraction by activating actin thin filaments. 21 Its phosphorylation is essential for normal cardiac function. 9,[22][23][24][25] cMyBP-C consists of 8 immunoglobulin-and 3 fibronectin-like domains, C0-C10, and is found in repeating stripes throughout the C-zone of the sarcomere. 26 The importance of cMyBP-C is highlighted by the fact that allelic variants in cMyBP-C are highly prevalent in hypertrophic cardiomyopathy and implicated to an extent in dilated cardiomyopathy. [27][28][29][30] The phosphorylation of serine residues at 273, 282, and 302 within the m-domain of cMyBP-C are essential for normal cardiac function. 26,31,32 cMyBP-C is 50% dephosphorylated in failing hearts regardless of disease etiology. [33][34][35] Additionally, the ablation of cMyBP-C phosphorylation leads to pathological hypertrophy in animal models. 36 The phosphorylation of cMyBP-C destabilizes SRX, a myosin head configuration that is inactive and not actively undergoing crossbridge cycling. Phosphorylation of cMyBP-C therefore results in increased contractility by decreasing the proportion of myosin heads in SRX, whereas dephosphorylation has a stabilizing effect on SRX, resulting in decreased contractility. 37 Given the impact this post-translational modification has on cross-bridge cycling and HF, questions remain regarding how this Here, we used a transgenic mouse model in which phosphorylation sites at serines 273, 282, and 302 within the m-domain of cMyBP-C were replaced with the phospho-null residue alanine (AAA). 36,38,39 The AAA transgenic mice expressed 40% cMyBP-C AAA and displayed no changes in morbidity or mortality, but displayed depressed cardiac contractility, altered sarcomere structure, and upregulation of transcripts associated with a hypertrophic response. 40 Transgenic mice in which the phosphorylation sites were replaced with a phospho-mimetic residue, aspartate (DDD), had no phenotype, consistent with the observation that w90% of cMyBP-C was at least monophosphorylated in healthy hearts. 26  The decay in fluorescence was fitted to a custom defined 2-state exponential decay curve to yield the biphasic exchange of mANT-ATP with ATP in accordance with decrease in fluorescence:

RECONSTITUTED SARCOMERE MYOSIN ATPase
ASSAY. The myosin ATPase activity in a reconstituted sarcomere were performed based on the previous study. 48 In short, myosin, 49 tropomyosin, 50 actin, 51 and troponin complex 52 were isolated and purified from bovine left ventricle (cardiac), rabbit back muscle (fast skeletal), and bovine masseter tissue (slow skeletal). All muscle tissues were obtained from Pel-Freez Biologicals. The purified myosin was further subjected to a-chymotrypsin digestion to yield soluble myosin S1 heads. 49 The active myosin S1 heads were purified by adding equimolar ATP and F-actin to soluble myosin S1 heads, further centrifuging the dead myosin S1-actin complex at 10,000 revolutions/min in 4 C. 53 The reconstituted sarcomere consisted of myosin S1 of 1 muscle origin and coupled with thin filament (actin, troponin, and tropomyosin) of same or other muscle origin in PM12  Figure S1A). M is a structural analog of OM, and it is therefore not surprising that it exhibited a similar selectivity profile to OM. 54 Similarly, T is a structural analog of CK-136 and was anticipated to exhibit a similar selectivity profile. 55 For T, we saw activity that tracked with the source of troponin. T increased myosin ATPase activity in the reconstituted assays containing cardiac thin filaments as well as in reconstituted assays containing cardiac troponin irrespective of the source of myosin or tropomyosin (Supplemental Figures S1B and S1C). A modest degree   Figure 1B). For AAA isolated myofibrils, the steady-state ATPase activity rate was determined to be 0.013 AE 9 Â 10 À4 AU/min for M and 0.009 AE 4 Â 10 À4 AU/min for T. For DDD isolated myofibrils, the steady-state ATPase activity rate was 0.029 AE 3 Â 10 À4 AU/min for M and 0.021 AE 5 Â 10 À4 AU/min for T ( Figures 1C and 1D, Table 1).

M AND T INCREASED CARDIOMYOCYTE CELL
SHORTENING. To determine if M and T improved cell contractility, we measured cell shortening using cardiomyocytes isolated from tWt, AAA, or DDD transgenic lines with an IonOptix system. Preliminary work found that it required 3-to 4-fold less T relative to M to increase bovine myofibril turnover. For simplicity, we have referred to the concentrations for M and T as 1Â and 3Â throughout the paper, where 1Â and 3Â are 3.2 mmol/L and 9.6 mmol/L for M, whereas 1Â and 3Â are 1 mmol/L and 3 mmol/L for T.
Isolated cardiomyocytes were pulsed through an electric field to induce contraction. The resting baseline sarcomere length was significantly shorter for cardiomyocytes isolated from AAA and DDD transgenic mice compared with tWt (Supplemental Figure S2). When we compared cells with a resting sarcomere length below the median to cells with a resting sarcomere length at or above the median for each genotype and treatment, we did not observe any obvious or consistent trend, suggesting that cells below the median sarcomere length were less responsive to treatment or influenced by genotype (Supplemental Figure S3). Consistent with the changes in steady-state ATPase activity, both compounds increased contraction relative to baseline contraction across all 3 genotypes. M increased percentage contraction, or cell shortening, in tWt (11.98%   Table 2). The 1Â and 3Â dose of M did not result in a significant increase in F max in tWt (47.7    Figure 4C, Table 2). This suggests that the acto-myosin cross-bridges do not break as easily, and a proportion are bound for a prolonged period.
Another observation that supports the impediment for detachment of myosin from actin thin filaments was made during the washout step of calcium in skinned papillary muscle fibers. The force generated in muscle fibers at maximal pCa 4.5 rapidly decays when exchanged with pCa 9.0 (Supplemental Figure S4C, black). Here, treatment with T slowed force decay relative to untreated fibers, and M   Values are group mean AE SEM. Statistical analyses were performed in all groups using a 1-way analysis of variance, Tukey's multiple comparison test. Data represents 5 fibers analyzed per animal per compound treatment isolated from 12-to 14-week-old mice. The pCa50 values were obtained for each individual fiber by fitting each of the fiber data to log(agonist) vs normalized response-variable slope model. The ktr values were obtained for each fiber by fitting the curve to 1 phase association curve. The errors were obtained by comparing the fitted parameters for each fiber. a P < 0.001 tWt vs AAA for the equivalent dose. b P < 0.001 tWt vs DDD for the equivalent dose. c P < 0.05 for control vs 1Â and 3Â M. d P < 0.001 for control vs 1Â and 3Â T. e P < 0.01 for control vs 1Â and 3Â M. f P < 0.05 for control vs 1Â and 3Â T. g P < 0.01 for control vs 1Â and 3Â T. h P < 0.001 for M vs T for the equivalent dose. i P < 0.01 tWt vs AAA for the equivalent dose. j P < 0.001 and k P < 0.01 AAA vs DDD for the equivalent dose. l P < 0.001 for control vs 1Â and 3Â M.
Abbreviations as in Table 1.   Table 3). In contrast, T did not significantly increase the mANT-ATP slow phase exchange rate ( Figures 5B and 5C, Table 3). Changes to the proportion of muscle fibers with fast mANT-ATP exchange were inversely related to the observed changes in the phase for slow mANT-ATP exchange (Supplemental Figure S5A, Supplemental Table S1).  Table S1).

DISCUSSION
This study evaluated 2 new small molecules that target 2 different sarcomere proteins to increase    propose that M has not led to a higher proportion of myosin heads in SRX but has slowed the rate of mANT-ATP exchange in a manner that is similar to its previously reported ability to slow the rate of phosphate release. In this context, we speculate that M has not altered the myosin head confirmation but has altered the affinity of mANT-ATP for myosin thereby impeding the exchange rate. Alternatively, it remains possible that CK-138 alters the proportion of myosin heads in SRX in a manner that is fundamentally different relative to OM. However, it is worth noting that CK-138 and OM are structurally very similar to one another. The only difference is at a single position where a hydrogen atom is replaced with fluorine. 54 We feel that this substitution is unlikely to lead to such a large change in activity between OM and CK-138, given that a comparison of the 2 molecules, to date, suggest that they exhibit a similar activity profile with OM being more potent.
Our data indicate that both molecules sensitize the cardiac muscle to calcium resulting in an in- can improve cardiac performance in cMyBP-C KO mice. 61 Neither small molecule targets cMyBP-C directly. Therefore, the small molecules are not anticipated to evoke full activation of the thick filament.
Additionally, translatability is always a concern.
The studies were performed primarily on mice, where MYH6 is the dominant form of cardiac myosin, whereas humans express the MYH7 variant. M is a surrogate of OM, and OM has been shown to be MYH7 selective. 65 It, therefore, seems reasonable to infer that M may be exhibit more robust activity in humans. Finally, it remains to be seen how a molecule that alters troponin activity to increase cardiac contraction will perform in the clinic. Our data collectively suggest that targeting troponin may result in a differentiating therapeutic profile.