Nebulin (Neb) is associated with the thin filament in skeletal muscle cells, but its functions are not well understood. For this goal, we study skinned slow-twitch soleus muscle fibers from wild-type (Neb+) and conditional Neb knockout (Neb−) mice. We characterize cross-bridge (CB) kinetics and the elementary steps of the CB cycle by sinusoidal analysis during full Ca2+ activation and observe that Neb increases active tension 1.9-fold, active stiffness 2.7-fold, and rigor stiffness 3.0-fold. The ratio of stiffness during activation and rigor states is 62% in Neb+ fibers and 68% in Neb− fibers. These are approximately proportionate to the number of strongly attached CBs during activation. Because the thin filament length is 15% shorter in Neb− fibers than in Neb+ fibers, the increase in force per CB in the presence of Neb is ∼1.5 fold. The equilibrium constant of the CB detachment step (K2), its rate (k2), and the rate of the reverse force generation step (k−4) are larger in Neb+ fibers than in Neb− fibers. The rates of the force generation step (k4) and the reversal detachment step (k−2) change in the opposite direction. These effects can be explained by Le Chatelier’s principle: Increased CB strain promotes less force-generating state(s) and/or detached state(s). Further, when CB distributions among the six states are calculated, there is no significant difference in the number of strongly attached CBs between fibers with and without Neb. These results demonstrate that Neb increases force per CB. We also confirm that force is generated by isomerization of actomyosin (AM) from the AM.ADP.Pi state (ADP, adenosine diphophate; Pi, phosphate) to the AM*ADP.Pi state, where the same force is maintained after Pi release to result in the AM*ADP state. We propose that Neb changes the actin (and myosin) conformation for better ionic and hydrophobic/stereospecific AM interaction, and that the effect of Neb is similar to that of tropomyosin.

The cardiac troponin C (TnC)-A8V mutation is associated with hypertrophic and restrictive cardiomyopathy (HCM and RCM) in human and mice. The residue affected lies in the N-helix, a region known to affect Ca-binding affinity to the N-terminal domain. Here we report on the functional effects of this mutation in skinned papillary muscle fibers from homozygous knock-in TnC-A8V mice. Muscle fibers from left ventricle were activated at 25°C under the ionic conditions of working cardiomyocytes. The pCa-tension relationship showed a 3× increase in Ca-sensitivity and a decrease (0.8×) in cooperativity (n) in mutant fibers. The elementary steps of the cross-bridge (CB) cycle were investigated by sinusoidal analysis. The ATP study revealed that there is no significant change in the affinity of ATP (K) for the myosin head. In TnC-A8V mutant fibers, the CB detachment rate (k) and its equilibrium constant (K) increased (1.5×). The phosphate study revealed that rate constant of the force-generation step (k) decreased (0.5×), reversal step (k) increased (2×), and the phosphate-release step (1/K) increased (2×). Pro-Q Diamond staining of the skinned fibers samples revealed no significant changes in total phosphorylation of multiple sarcomeric proteins. Further investigation using liquid chromatography-tandem mass spectrometry revealed hypophosphorylation of the rod domain of myosin heavy chain in TnC-A8V mutant fibers compared to wild-type. Immunoblotting confirmed the results observed in the mass spectrometry analysis. The results suggest perturbed CB kinetics-possibly caused by changes in the α-myosin heavy chain phosphorylation profile-as a novel mechanism, to our knowledge, by which a mutation in TnC can have rippling effects in the myofilament and contribute to the pathogenesis of HCM/RCM.

In mammalian ventricles, two myosin heavy chain (MHC) isoforms have been identified. Small animals express α-MHC, whereas large animals express β-MHC, which contribute to a large difference in the heart rate. Sprague-Dawley rats possessing ~99% α-MHC were treated with propylthiouracil to result in 100% β-MHC. Papillary muscles were skinned, dissected into small fibers, and used for experiments. To understand the functional difference between α-MHC and β-MHC, skinned-fibers were activated under the intracellular ionic conditions: 5 mM MgATP, 1 mM Mg, 8 mM Pi, 200 mM ionic strength, pH 7.00 at 25 °C. Small amplitude sinusoidal length oscillations were applied in the frequency range 0.13-100 Hz (corresponding time domain: 1.6-1200 ms), and effects of Ca, Pi, and ATP were studied. The results show that Ca sensitivity was slightly less (10-15%) in β-MHC than α-MHC containing fibers. Sinusoidal analysis at pCa 4.66 (full Ca activation) demonstrated that, the apparent rate constants were 2-4× faster in α-MHC containing fibers. The ATP study demonstrated that, in β-MHC containing fibers, K (ATP association constant) was greater (1.7×), k and k (cross-bridge detachment and its reversal rate constants) were smaller (×0.6). The Pi study demonstrated that, in β-MHC containing fibers, k (rate constant of the force-generation step) and k were smaller (0.75× and 0.25×, respectively), resulting in greater K (3×). There were no differences in active tension, rigor stiffness, or K (equilibrium constant of the cross-bridge detachment step). Our study further demonstrated that there were no differences in parameters between fibers obtained from left and right ventricles, but with an exception in K (Pi association constant).

Jojoba oil has been shown to maintain high‐density lipoprotein (HDL) concentration in hypercholesterolemic New Zealand White (NZW) rabbits, suggesting that jojoba oil regulates HDL metabolism and has a protective effect against atherosclerosis, one of the leading causes of death worldwide. We thus hypothesized that jojoba oil decreases the development of atherosclerosis in NZW rabbits. As a preliminary study, five NZW rabbits were fed either a normal chow diet (N), or a normal diet supplemented with either 3% jojoba seed oil (J), 1% cholesterol (C), or 1% cholesterol + 3% jojoba seed oil (CJ) for 7 weeks; each diet group contained one rabbit, except the C‐fed group which contained two. During the study, serum lipoprotein cholesterol concentrations were monitored via enzymatic assays and gel electrophoresis. Liver function was examined via serum albumin measurements. Rabbit aortas were extracted and assessed for the development of atherosclerotic lesions. Results indicated that NZW rabbits responded to the experimental diets as shown in previous studies. N and J‐fed rabbits showed no change in their lipoprotein profiles. C‐ and CJ‐fed rabbits had a decrease in HDL concentration; however, the decrease was less in the CJ rabbits than the C rabbits. Serum albumin measurement indicated proper liver function. Unfortunately, none of the rabbits developed atherosclerotic lesions and the effects on dietary jojoba oil on atherosclerosis development could not be assessed in this study. Despite the negative results, this preliminary study allowed us to better understand what must be done in order to study the original question of interest. In future studies, we plan to ensure atherosclerotic lesion development by conducting a dose response study.

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