Omecamtiv Mecarbil, a Cardiac Myosin Activator, Increases Ca2+ Sensitivity in Myofilaments With a Dilated Cardiomyopathy Mutant Tropomyosin E54K.

Apart from transplant, there are no satisfactory therapies for the severe depression in contractility in familial dilated cardiomyopathy (DCM). Current heart failure treatments that act by increasing contractility involve signaling cascades that alter calcium homeostasis and induce arrhythmias. Omecamtiv mecarbil is a promising new inotropic agent developed for heart failure that may circumvent such limitations. Omecamtiv is a direct cardiac myosin activator that promotes and prolongs the strong myosin-actin binding conformation to increase the duration of systolic elastance. We tested the effect of omecamtiv on Ca(2+) sensitivity of myofilaments of a DCM mouse model containing a tropomyosin E54K mutation. We compared tension and ATPase activity of detergent-extracted myofilaments with and without treatment with 316 nM omecamtiv at varying pCa values. When transgenic myofilaments were treated with omecamtiv, the pCa50 for activation of tension increased from 5.70 ± 0.02 to 5.82 ± 0.02 and ATPase activity increased from 5.73 ± 0.06 to 6.07 ± 0.04. This significant leftward shift restored Ca(2+) sensitivity to levels no longer significantly different from controls. Proteomic studies lacked changes in sarcomeric protein phosphorylation. Our data demonstrate that omecamtiv can potentially augment cardiac contractility in DCM by increasing Ca(2+) sensitivity. The use of direct myosin activators addresses functional defects without incurring the adverse side effects of Ca(2+)-dependent treatments.

Impact of anesthesia and storage on posttranslational modifications of cardiac myofilament proteins.

Although high fidelity measurements of posttranslational modifications (PTMs) of cardiac myofilament proteins exist, important issues remain regarding basic techniques of sample acquisition and storage. We investigated the effects of anesthetic regimen and sample storage conditions on PTMs of major ventricular sarcomeric proteins. Mice were anesthetized with pentobarbital (Nembutal), ketamine/xylazine mixture (Ket/Xyl), or tribromoethanol (Avertin), and the ventricular tissue was prepared and stored for 1, 7, 30, 60, or 90 days at -80°C. Myofilament protein phosphorylation and glutathionylation were analyzed by Pro-Q Diamond stain and Western blotting, respectively. With up to 7 days of storage, phosphorylation of troponin T was stable for samples from mice anesthetized with either Nembutal or Ket/Xyl but not Avertin; while myosin-binding protein C (MyBP-C) phosphorylation was reduced at 7 days with Nembutal and Ket/Xyl, though generally not significant until 90 days. Tropomyosin and regulatory myosin light chain phosphorylation were stable for up to 7 days regardless of the anesthetic regimen employed. In the case of Troponin I, by 7 days of storage there was a significant fall in phosphorylation across all anesthetics. Storage of samples from 30 to 90 days resulted in a general decrease in myofilament phosphorylation independent of the anesthetic. S-glutathionylation of MyBP-C presented a trend in reduced glutathionylation from days 30-90 in all anesthetics, with only day 90 being statistically significant. Our findings suggest that there are alterations in PTMs of major myofilament proteins from both storage and anesthetic selection, and that storage beyond 30 days will likely result in distortion of data.

Desensitization of myofilaments to Ca2+ as a therapeutic target for hypertrophic cardiomyopathy with mutations in thin filament proteins.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a common genetic disorder caused mainly by mutations in sarcomeric proteins and is characterized by maladaptive myocardial hypertrophy, diastolic heart failure, increased myofilament Ca(2+) sensitivity, and high susceptibility to sudden death. We tested the following hypothesis: correction of the increased myofilament sensitivity can delay or prevent the development of the HCM phenotype. METHODS AND RESULTS: We used an HCM mouse model with an E180G mutation in α-tropomyosin (Tm180) that demonstrates increased myofilament Ca(2+) sensitivity, severe hypertrophy, and diastolic dysfunction. To test our hypothesis, we reduced myofilament Ca(2+) sensitivity in Tm180 mice by generating a double transgenic mouse line. We crossed Tm180 mice with mice expressing a pseudophosphorylated cardiac troponin I (S23D and S24D; TnI-PP). TnI-PP mice demonstrated a reduced myofilament Ca(2+) sensitivity compared with wild-type mice. The development of pathological hypertrophy did not occur in mice expressing both Tm180 and TnI-PP. Left ventricle performance was improved in double transgenic compared with their Tm180 littermates, which express wild-type cardiac troponin I. Hearts of double transgenic mice demonstrated no changes in expression of phospholamban and sarcoplasmic reticulum Ca(2+) ATPase, increased levels of phospholamban and troponin T phosphorylation, and reduced phosphorylation of TnI compared with Tm180 mice. Moreover, expression of TnI-PP in Tm180 hearts inhibited modifications in the activity of extracellular signal-regulated kinase and zinc finger-containing transcription factor GATA in Tm180 hearts. CONCLUSIONS: Our data strongly indicate that reduction of myofilament sensitivity to Ca(2+) and associated correction of abnormal relaxation can delay or prevent development of HCM and should be considered as a therapeutic target for HCM.

Influence of a constitutive increase in myofilament Ca(2+)-sensitivity on Ca(2+)-fluxes and contraction of mouse heart ventricular myocytes.

Chronic increases in myofilament Ca(2+)-sensitivity in the heart are known to alter gene expression potentially modifying Ca(2+)-homeostasis and inducing arrhythmias. We tested age-dependent effects of a chronic increase in myofilament Ca(2+)-sensitivity on induction of altered alter gene expression and activity of Ca(2+) transport systems in cardiac myocytes. Our approach was to determine the relative contributions of the major mechanisms responsible for restoring Ca(2+) to basal levels in field stimulated ventricular myocytes. Comparisons were made from ventricular myocytes isolated from non-transgenic (NTG) controls and transgenic mice expressing the fetal, slow skeletal troponin I (TG-ssTnI) in place of cardiac TnI (cTnI). Replacement of cTnI by ssTnI induces an increase in myofilament Ca(2+)-sensitivity. Comparisons included myocytes from relatively young (5-7months) and older mice (11-13months). Employing application of caffeine in normal Tyrode and in 0Na(+) 0Ca(2+) solution, we were able to dissect the contribution of the sarcoplasmic reticulum Ca(2+) pump (SR Ca(2+)-ATPase), the Na(+)/Ca(2+) exchanger (NCX), and "slow mechanisms" representing the activity of the sarcolemmal Ca(2+) pump and the mitochondrial Ca(2+) uniporter. The relative contribution of the SR Ca(2+)-ATPase to restoration of basal Ca(2+) levels in younger TG-ssTnI myocytes was lower than in NTG (81.12±2.8% vs 92.70±1.02%), but the same in the older myocytes. Younger and older NTG myocytes demonstrated similar contributions from the SR Ca(2+)-ATPase and NCX to restoration of basal Ca(2+). However, the slow mechanisms for Ca(2+) removal were increased in the older NTG (3.4±0.3%) vs the younger NTG myocytes (1.4±0.1%). Compared to NTG, younger TG-ssTnI myocytes demonstrated a significantly bigger contribution of the NCX (16±2.7% in TG vs 6.9±0.9% in NTG) and slow mechanisms (3.3±0.4% in TG vs 1.4±0.1% in NTG). In older TG-ssTnI myocytes the contributions were not significantly different from NTG (NCX: 4.9±0.6% in TG vs 5.5±0.7% in NTG; slow mechanisms: 2.5±0.3% in TG vs 3.4±0.3% in NTG). Our data indicate that constitutive increases in myofilament Ca(2+)-sensitivity alter the relative significance of the NCX transport system involved in Ca(2+)-homeostasis only in a younger group of mice. This modification may be of significance in early changes in altered gene expression and electrical stability hearts with increased myofilament Ca-sensitivity.

The ß-arrestin-biased ligand TRV120023 inhibits angiotensin II-induced cardiac hypertrophy while preserving enhanced myofilament response to calcium.

In the present study, we compared the cardioprotective effects of TRV120023, a novel angiotensin II (ANG II) type 1 receptor (AT1R) ligand, which blocks G protein coupling but stimulates ß-arrestin signaling, against treatment with losartan, a conventional AT1R blocker in the treatment of cardiac hypertrophy and regulation of myofilament activity and phosphorylation. Rats were subjected to 3 wk of treatment with saline, ANG II, ANG II + losartan, ANG II + TRV120023, or TRV120023 alone. ANG II induced increased left ventricular mass compared with rats that received ANG II + losartan or ANG II + TRV120023. Compared with saline controls, ANG II induced a significant increase in pCa50 and maximum Ca(2+)-activated myofilament tension but reduced the Hill coefficient (nH). TRV120023 increased maximum tension and pCa50, although to lesser extent than ANG II. In contrast to ANG II, TRV120023 increased nH. Losartan blocked the effects of ANG II on pCa50 and nH and reduced maximum tension below that of saline controls. ANG II + TRV120023 showed responses similar to those of TRV120023 alone; compared with ANG II + losartan, ANG II + TRV120023 preserved maximum tension and increased both pCa50 and cooperativity. Tropomyosin phosphorylation was lower in myofilaments from saline-treated hearts compared with the other groups. Phosphorylation of cardiac troponin I was significantly reduced in ANG II + TRV120023 and TRV120023 groups versus saline controls, and myosin-binding protein C phosphorylation at Ser(282) was unaffected by ANG II or losartan but significantly reduced with TRV120023 treatment compared with all other groups. Our data indicate that TRV120023-related promotion of ß-arrestin signaling and enhanced contractility involves a mechanism promoting the myofilament response to Ca(2+) via altered protein phosphorylation. Selective activation of ß-arrestin-dependent pathways may provide advantages over conventional AT1R blockers.