Molecular effects of the myosin activator omecamtiv mecarbil on contractile properties of skinned myocardium lacking cardiac myosin binding protein-C

Decreased expression of cardiac myosin binding protein-C (cMyBP-C) in the myocardium is thought to be a contributing factor to hypertrophic cardiomyopathy in humans, and the initial molecular defect is likely abnormal cross-bridge (XB) function which leads to impaired force generation, decreased contractile performance, and hypertrophy in vivo. The myosin activator omecamtiv mecarbil (OM) is a pharmacological drug that specifically targets the myosin XB and recent evidence suggests that OM induces a significant decrease in the in vivo motility velocity and an increase in the XB duty cycle. Thus, the molecular effects of OM maybe beneficial in improving contractile function in skinned myocardium lacking cMyBP-C because absence of cMyBP-C in the sarcomere accelerates XB kinetics and enhances XB turnover rate, which presumably reduces contractile efficiency. Therefore, parameters of XB function were measured in skinned myocardium lacking cMyBP-C prior to and following OM incubation. We measured k_tr, the rate of force redevelopment as an index of XB transition from both the weakly- to strongly-bound state and from the strongly- to weakly-bound states and performed stretch activation experiments to measure the rates of XB detachment (k_rel) and XB recruitment (k_df) in detergent-skinned ventricular preparations isolated from hearts of wild-type (WT) and cMyBP-C knockout (KO) mice. Samples from donor human hearts were also used to assess the effects of OM in cardiac muscle expressing a slow β-myosin heavy chain (β-MHC). Incubation of skinned myocardium with OM produced large enhancements in steady-state force generation which were most pronounced at low levels of [Ca^2 +] activations, suggesting that OM cooperatively recruits additional XB’s into force generating states. Despite a large increase in steady-state force generation following OM incubation, parallel accelerations in XB kinetics as measured by k_tr were not observed, and there was a significant OM-induced decrease in k_rel which was more pronounced in the KO skinned myocardium compared to WT skinned myocardium (58% in WT vs. 76% in KO at pCa 6.1), such that baseline differences in k_rel between KO and WT skinned myocardium were no longer apparent following OM-incubation. A significant decrease in the k_df was also observed following OM incubation in all groups, which may be related to the increase in the number of cooperatively recruited XB’s at low Ca^2 +-activations which slows the overall rate of force generation. Our results indicate that OM may be a useful pharmacological approach to normalize hypercontractile XB kinetics in myocardium with decreased cMyBP-C expression due to its molecular effects on XB behavior.     

Hormonal influences on cardiac myosin ATPase activity and myosin isoenzyme distribution

It has been recognized for a long time that changes in hormone secretion can influence cardiac function; however, the biochemical basis for these changes has only recently been clarified. In this review the influences of hormonal status on the contractile protein myosin is discussed. Myosin has a rod-like portion and a globular head and consists of two myosin heavy chains (MHC) and four light chains (LC), two of which are identical. The globular head is the site of an ATP-splitting enzyme, the myosin ATPase, and increases in myosin ATPase activity are closely related to an increased velocity of contraction of the heart. Myosin ATPase activity shows marked response to alterations in thyroid hormone, insulin, glucocorticoid, testosterone and catecholamine levels, but marked animal species differences in this response occur. Thyroid hormone administration to normal rabbits, for example, increases myosin ATPase activity markedly, but the myosin ATPase activity of hyperthyroid rats remains unchanged. In contrast, in hypothyroid rats myosin ATPase activity is markedly decreased but the hypothyroid rabbit shows no such response. These species-related differences in the hormonal response of myosin ATPase activity result from the predominance pattern of specific myosin isoenzymes. In the normal rat heart three myosin isoenzymes, v₁, V₂ and V₃, can be separated electrophoretically. Myosin V₁ predominates (70% of total myosin), and has the highest myosin ATPase activity, whereas in rabbits myosin v₃, which has a lower myosin ATPase activity, is the predominant isomyosin. Thyroid hormone administration to rabbits induces myosin V₁ predominance and therefore increases myosin ATPase activity, whereas in hyperthyroid rats only a small further increase in V₁ predominance can occur. The alterations in myosin isoenzyme predominance and myosin ATPase activity are closely correlated to changes in cardiac contractility. Hormone-induced alterations in myosin isoenzyme predominance are mediated through changes in the formation of two isoforms of myosin heavy chain. Changes in the expression of different myosin heavy chain genes are most likely responsible for the thyroid hormone and insulin-induced alterations in myosin isoenzyme predominance. Investigation of the control of myosin heavy chain formation can provide further insights into the hormonal control of a multigene family as well as broaden our understanding of the molecular events which result in altered cardiac contractility. It is currently unclear if androgens, glucocorticoids and catecholamines influence myosin ATPase activity through changes in myosin isoenzyme predominance resulting from alterations in myosin heavy chain gene expression. Post-translational modifications of myosin heavy chain and light chain polypeptides have also to be considered.     

Conformational transition in the myosin hinge upon activation of muscle.

We have determined the rates of chymotryptic proteolysis of the myosin hinge region in glycerinated rabbit psoas fibers and myofibrils in in rigor-inducing, activating, and relaxing buffers. The time course of formation of light meromyosin (LMM) provides a specific probe for cleavage within the hinge domain. In rigor-inducing and relaxing buffers proteolysis within the hinge is depressed, but on activation LMM is formed at a markedly increased rate, which is dependent on the concentration of MgATP. Peptide bond cleavage occurs at four widely separated sites spanning the length of the hinge domain. Only a trivial amount of proteolysis occurs at the head--rod swivel or within the heavy chain of the head itself (S-1 subunit) in rigor-inducing and relaxing solvents, and we find no significant change on activation. The rate of formation of LMM in rigor-inducing buffer is unchanged by addition of MgADP, Pi, or magnesium adenosine 5'-[beta, gamma-imido]triphosphate or in activating solvent at zero overlap between thick and thin filaments. These results provide evidence for a conformational (helix--coil) transition within the myosin hinge upon activation of skeletal muscle.     

Further studies on the effects of myosin P-light chain phosphorylation on contractile properties of skinned cardiac fibres

Summary We investigated the influence of myosin P-light chain phosphorylation by Ca²⁺-calmodulin dependent myosin light chain kinase (MLCK) on the sensitivity of the tension-pCa relation and maximum unloaded shortening velocity (v _max) of chemically skinned heart fibres of the pig.Submaximum Ca²⁺ stimulation (pCa 5.5) induced 20±5% of the isometric tension achieved at maximum Ca²⁺ activation (pCa 4.3).MLCK-induced myosin P-light chain phosphorylation increased the isometric force development at pCa 5.5 by 40% whereas maximum tension at pCa 4.3 was not affected.Unloaded shortening velocity (v _max) was not altered by myosin P-light chain phosphorylation either at maximum or at submaximum Ca²⁺ concentration, being c. 1.2 muscle length/s at pCa 5.5 and 2.2 muscle length/s at pCa 4.3.The MLCK-induced increase of the myosin P-light chain phosphorylation level was evaluated by determination of³²P-incorporation. Two phosphorylatable myosin P-light chains could be demonstrated.     

The ATPase activity of cardiac myosin from failing and hypertropied hearts.

We have measured the EDTA and Ca²⁺ activated ATPase activity of myosin from hearts of normal dogs, dogs in gross heart failure and dogs with cardiac hypertrophy without failure. Heart failure was caused by systolic and/or diastolic hemodynamic loads. Under one or more assay conditions, depressed ATPase activity was consistently found for myosin from failing hearts. In contrast to myosin from hearts with diastolic loads, myosin from failing hearts with pulmonic stenosis in the presence of Ca²⁺ and 0.1 m KCl showed normal mean ATPase activity with several preparations having elevated activity. Elevated ATPase activities were obtained for myosin from the non-failing, but hypertrophied hearts of dogs with aortic stenosis. The data suggest that the interaction of ionic strength and pH with the ATPase active site of suggest that the interaction of ionic strength and pH with the ATPase active site of myosin from failing hearts is altered, thus modifying the ATPase activity in the presence of Ca²⁺ or EDTA. The altered interaction may be modified by the presence of hypertrophy, resulting in normal or increased activity in the presence of Ca²⁺ and decreased activity in the presence of EDTA.     

甲状腺机能对心肌肌球蛋白ATP酶活性的影响

本实验研究甲状腺机能对动物体重、心重及心肌肌球蛋白 ATP 酶活性的影响。结果表明:甲亢时,动物体重明显下降,心重,心重/体重比值明显升高,兔心肌肌球蛋白 ATP 酶活性显著增加,鼠心肌肌球蛋白 ATP 酶活性未见明显变化;甲减退时,动物体重、心重/体重比值降低,鼠心肌肌球蛋白 ATP 酶活性明显降低,兔心肌肌球蛋白 ATP 酶活性未见明显变化。甲亢组动物心重随肌球蛋白 ATP 酶活性升高而增加,甲减退组动物心重随肌球蛋白 ATP 酶活性的降低而减少,两者呈现出一致性改变。     

Effect of the thyroid state on the enzymatic characteristics of cardiac myosin. A difference in behavior of rat and rabbit cardiac myosin.

The effect of thyroid state on the activity of myosin adenosinetriphosphatase (ATPase) was examined in the rat and the rabbit. Cardiac myosin from thyroxine-treated rabbits showed enzymatic properties characterized by high Ca2plus-activated ATPase activity, low activation energy, lower rate of inactivation at alkaline pH, and no activation by N-ethylmaleimide compared with the same properties in the normal rabbit; thyroidectomy did not affect the enzymatic properties of rabbit cardiac myosin. These findings suggest a difference in the myosin molecule at or near the active site, involving some sulfhydryl groups, between hyperthyroid and euthyroid rabbits. However, rat cardiac myosin showed a pattern of activity in the euthyroid state similar to that of the hyperthyroid rabbit and changed to the euthyroid type after thyroidectomy. These changes were specific for cardiac myosin, since no change was observed in skeletal myosin. It is unlikely that there are major differences in the myosin molecule associated with the two types of activity, since similar proportion and amino acid composition of the subunits of cardiac myosin were observed in the different thyroid states. Thus, we concluded that the administration of thyroxine to the rabbit stimulates the synthesis of new cardiac myosin with altered enzymatic properties and that synthesis of this type of cardiac myosin is maintained by the normal level of thyroid hormone in the rat.     

Biochemical characteristics of human cardiac myosin.

Myosin was purified from the left ventricles of eight patients. Samples of the ventricle were obtained immediately after the death of three patients whereas the rest were obtained from $\text{2}\text{1}\text{2}$ to 20 h after the death. Human cardiac myosin, like the cardiac myosin from other mammalian species has only two light chains. The corresponding molecular weights were 26000 and 20000 for light chain 1 (LC₁) and light chain (LC₂) respectively. In its chemical properties, total sulfhydryl groups and responses to solvents the human cardiac myosin resembles canine cardiac myosin. These studies also show that a delay in the preparations of myosin up to 20 h does not alter its properties. ATPase activities (Ca²⁺, Mg²⁺ and K⁺-EDTA) did not show any correlation with the age of the subject. However the total-SH content was significantly lower in the elderly patients.     

Influence of thyroid hormone administration on myosin ATPase activity and myosin isoenzyme distribution in the heart of diabetic rats

Previous studies have shown that diabetes mellitus leads in rats to a 45% decrease in cardiac Ca++ activated myosin ATPase, a change in myosin isoenzyme distribution and a lowering of plasma T4 and T3 levels. Hypothyroidism causes similar changes in myosin ATPase and myosin isoenzyme distribution. We determined if thyroid hormone administration in physiological replacement dose (0.3 microgram T3/100 g BW) or pharmacological doses (3 micrograms T3/100 g BW and 10 micrograms T4/100 g BW) can normalize myosin ATPase and isoenzyme distribution in diabetic rats. Control animals have a Ca++ myosin ATPase activity of 1.23 +/- 0.14 mumol Pi/mg protein/min and myosin V1 represented 70% and myosin V3 15% of total myosin. Four weeks after streptozotocin administration myosin ATPase was 0.61 +/- 0.14, and myosin V3 represented 67% of total myosin. Administration of 0.3 microgram T3/100 g BW/day for four weeks to diabetic animals resulted in no significant increase in myosin ATPase (0.69 +/- 0.07 mumol Pi/mg protein/min) or in myosin isoenzyme distribution. In contrast, administration of 3 micrograms T3/100 g BW/day or 10 micrograms T4/100 g BW/day for 4 wk led to a normalization of myosin ATPase activity (for T3 1.03 +/- 0.18, for T4 1.06 +/- 0.15). In addition the myosin isoenzyme distribution pattern normalized. These findings may point to a diminished thyroid hormone responsiveness in diabetic rats or could result from diabetes related disturbances of cellular metabolism, which are normalized by pharmacologic doses of thyroid hormone.     

Influence of the hinge region on complement activation, C1q binding, and segmental flexibility in chimeric human immunoglobulins.

We have characterized a series of genetically engineered chimeric human IgG3 and IgG4 anti-dansyl (DNS) antibodies with identical antibody-combining sites but different hinge region amino acid compositions to determine how the hinge region influences Fab fragment segmental flexibility, C1q binding, and complement activation. Our data support the correlation between "upper hinge" length and Fab segmental flexibility; moreover, we confirm that a hinge region is essential for C1q binding and complement activation. However, the hinge length by itself is not sufficient for complement activity in IgG molecules. We have demonstrated that the IgG4 hinge, which imparts restricted segmental flexibility, reduces the ability of IgG3 molecules to activate complement. We also find that the IgG3 hinge region, which imparts greater segmental motion, is not sufficient to create complement activation activity in IgG4 anti-DNS antibodies. Finally, we conclude that (i) segmental motion is correlated with "upper hinge" length, (ii) hinge length and segmental flexibility is not enough to alter complement binding and activation, and (iii) segmental flexibility does not correlate with proficiency to activate the complement cascade.     

Deletions within a hinge region of a specific DNA-binding protein.

Many proteins are organized as a set of compact functional domains connected by flexible, exposed segments of the polypeptide chain. To study one of these connector regions, we isolated a series of functional in-frame deletions in the central portion of a specific DNA-binding protein, the LexA repressor of Escherichia coli. These mutant proteins fell into two main classes: those with small deletions of two to eight amino acids functioned as repressor about as well as did wild type, while those with large deletions of 17-22 amino acids functioned well only at considerably higher concentrations. The mutant proteins were resistant to the specific cleavage reaction that triggers the SOS response. These data suggest that the conformation of the hinge region in LexA protein is important for cleavage. By contrast, the hinge plays a topological role in repressor function, connecting the two functional halves of the protein; in the SOS response, this function of the hinge is inactivated by cleavage, leading to inactivation of the repressor.     

Influence of hypertension on cardiac contractile response of human erythrocyte-derived depressing factor in ventricular myocytes

BACKGROUND: Erythrocyte-derived depressing factor (EDDF), a novel hypotensive factor purified from human erythrocytes, elicits endothelium-dependent vasorelaxation by reducing intracellular Ca2+ in vascular smooth muscle cells. However, its cardiac response is unknown. OBJECTIVE: This study was designed to examine the cardiac contractile response of EDDF under both normotensive and hypertensive conditions. METHODS: Ventricular myocytes were isolated from adult male spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) normotensive rats. Mechanical properties were evaluated using an IonOptix MyoCam system and intracellular Ca2+ was measured with fura-2 fluorescence. Myocytes were electrically stimulated to contract at 0.5 Hz. The contractile properties analyzed included peak shortening (PS), time-to-PS (TPS), time-to-90% re-lengthening (TR(90)), maximal velocity of shortening/re-lengthening (+/- dl/dt), fura-fluorescence intensity change (DeltaFFI), and fura-fluorescence decay rate (tau). RESULTS: SHR rats displayed significantly elevated blood pressure. EDDF (10-9-10-4 g/ml) did not affect PS, TPS, TR(90), DeltaFFI and tau but depressed +/- dl/dt at higher doses in WKY myocytes. However, EDDF depressed PS, +/- dl/dt and DeltaFFI, shortened TPS without affecting TR(90) and tau in SHR myocytes. Pretreatment of the myocytes with the nitric oxide synthase inhibitor Nvarpi-nitro-l-arginine methyl ester (l-NAME) did not affect the EDDF-induced inhibition of PS and +/- dl/dt in SHR myocytes but unmasked an EDDF-induced negative response in WKY myocytes. CONCLUSIONS: These data indicate that EDDF may participate in the modulation of cardiac contractile function under hypertensive, but not normotensive, conditions. The cardiac depressive effect of EDDF is unlikely due to release of nitric oxide, as suggested in vascular smooth muscles.     

Actin-activated adenosine triphosphatase activity of native and N-ethylmaleimide-modified cardiac myosin from normal and thyrotoxic rabbits.

The Ca2+-ATPase activity of cardiac myosin is increased in thyrotoxic animals. However, the physiological significance of this observation is uncertain since, in living muscle, Mg-ATP is hydrolyzed by myosin under the stimulating influence of actin. In this study, we have compared the actin-activated ATPase activity of myosin from euthyroid (myosin-N) and thyrotoxic (myosin-T) rabbits and the derivatives of myosin-N and myosin-T formed by blocking the most rapidly reacting class of thiols (SH1) with N-ethylmaleimide (NEM). Also, we have studied the activity of these myosins in the presence of a complex of troponin and tropomyosin that confers calcium sensitivity on the system. Vmax for the actin-activated ATPase of myosin-T was about 168% greater than for myosin N. The apparent dissociation constant for actin, Kapp, for myosin-T was about 42% of the normal value. After NEM modification, Vmax and Kapp for NEM-modified myosin-T and myosin-N decreased, becoming essentially the same for both myosins. In the presence of troponin-tropomyosin complex, the actin-activated ATPase of myosin-T exhibited calcium sensitivity that could be reduced by thiol modification. These results suggest that the SH1 thiols or the region near these groups are important to the actin-activated ATPase of myosin-N and are essential to the increased activity of myosin-T. Also, they suggest that the changes in the enzymatic properties of myosin induced by thyroxine may be responsible for altering the contractile properties of the heart.     

Rapid helix--coil transitions in the S-2 region of myosin.

Temperature-jump studies on the long S-2 fragment (100,000 daltons) isolated from myosin show that this structure can undergo alpha-helix--random coil transitions in a time range approximating the cycle time of a crossbridge. Two relaxation times are observed after temperature jumps of 5 degrees C over the range 35--55 degrees C, one in the submillisecond (tau f) and the other in the millisecond (tau s) time ranges. Both processes exhibit maxima near the midpoint of the helix--coil transition (tm = 45 +/- 2 degrees C) as determined by optical rotation melt experiments. Similar results were observed for the low temperature transition (tm = 45 degrees C) of the myosin rod. Viscosity studies reveal that the S-2 particles has significant flexibility at physiological temperature. Results are considered in terms of the Huxley--Simmons and helix--coil transition models for force generation in muscle.     

Characterization of human cardiac myosin heavy chain genes.

We have isolated and analyzed the structure of the genes coding for the alpha and beta forms of the human cardiac myosin heavy chain (MYHC). Detailed analysis of four overlapping MYHC genomic clones shows that the alpha-MYHC and beta-MYHC genes constitute a total length of 51 kilobases and are tandemly linked. The beta-MYHC-encoding gene, predominantly expressed in the normal human ventricle and also in slow-twitch skeletal muscle, is located 4.5 kilobases upstream of the alpha-MYHC-encoding gene, which is predominantly expressed in normal human atrium. We have determined the nucleotide sequences of the beta form of the MYHC gene, which is 100% homologous to the cardiac MYHC cDNA clone (pHMC3). It is unlikely that the divergence of a few nucleotide sequences from the cardiac beta-MYHC cDNA clone (pHMC3) reported in a MYHC cDNA clone (pSMHCZ) from skeletal muscle is due to a splicing mechanism. This finding suggests that the same beta form of the cardiac MYHC gene is expressed in both ventricular and slow-twitch skeletal muscle. The promoter regions of both alpha- and beta-MYHC genes, as well as the first four coding regions in the respective genes, have also been sequenced. The sequences in the 5'-flanking region of the alpha- and beta-MYHC-encoding genes diverge extensively from one another, suggesting that expression of the alpha- and beta-MYHC genes is independently regulated.