The immediate effect of HCM causing actin mutants E99K and A230V on actin-Tm-myosin interaction in thin-filament reconstituted myocardium.

Human cardiac actin mutants E99K and A230V were expressed with baculovirus/insect cells and used to reconstitute the thin-filament of bovine cardiac (BVC) muscle fibers, together with tropomyosin (Tm) and troponin (Tn) purified from bovine ventricles. Effects of [Ca(2+)], [ATP], and [phosphate] on tension and its transients were studied at 25°C. In the absence of Tm/Tn, both mutants significantly decreased the tension of actin filament reconstituted fibers (WT: 0.75±0.06 T0, E99K: 0.58±0.04 T0, A230V: 0.58±0.03 T0), where T0 is active tension of native fibers (T0=26.9±1.1kPa, N=41), indicating diminished actin-myosin interactions. However, in the presence of Tm and Tn, WT, E99K, and A230V recovered tension (0.85±0.06 T0, 0.89±0.06 T0, and 0.85±0.05 T0, respectively), demonstrating the compensatory effect of Tm/Tn. Ca(2+) sensitivity (pCa50) increased (5.59±0.02, 5.80±0.03, 5.77±0.03, respectively) and cooperativity (nH) decreased (2.6±0.3, 1.87±0.21, 1.60±0.11, respectively). The kinetic constants of the cross-bridge cycle were deduced using sinusoidal analysis. E99K did not show any significant changes in any of the kinetic constants compared to those of WT. A230V caused a decrease in K1 (ATP association constant), k2 and k-2 (rate constants of the cross-bridge detachment step). The cross-bridge distribution was similar among WT, E99K, and A230V. In conclusion, our experiments demonstrate that the first step of HCM pathogenesis with E99K is increased pCa50 and decreased nH, which result in larger tension during partial activation to cause a diastolic problem. The effect on nH is more severe with A230V. In addition, A230V has a problem of decreased cross-bridge kinetics, which affects the normal functions of the cross-bridge cycle and may contribute to the first step of the HCM pathogenesis.

Using baculovirus/insect cell expressed recombinant actin to study the molecular pathogenesis of HCM caused by actin mutation A331P.

Recombinant WT human cardiac actin (WT actin) was expressed using the baculovirus/insect cell expression system, purified, and used to reconstitute the thin-filament of bovine cardiac muscle fibers, together with bovine cardiac tropomyosin (Tm) and troponin (Tn). Effects of [Ca(2+)], [ATP], [phosphate] and [ADP] on tension and tension transients were studied at 25°C by using sinusoidal analysis, and the results were compared with those of native fibers and fibers reconstituted with purified bovine cardiac actin (BVC actin). In actin filament reconstituted fibers (without Tm/Tn), those reconstituted with WT actin showed exactly the same active tension as those reconstituted with purified BVC actin (WT: 0.75±0.06 T0, N=11; BVC: 0.73±0.07 T0, N=12, where T0 is the tension of original fibers before extraction). After Tm/Tn reconstitution, fibers reconstituted with WT actin generated 0.85±0.06 T0 (N=11) compared to 0.98±0.04 T0 (N=12) recovered by those reconstituted with BVC actin. In the presence of Tm/Tn, WT actin reconstituted fibers showed exactly the same Ca(2+) sensitivity as those of the native fibers and BVC actin reconstituted fibers (pCa50: native fibers: 5.69±0.01, N=10; WT: 5.69±0.02, N=11; BVC: 5.68±0.02, N=12). Sinusoidal analysis showed that the cross-bridge kinetics were the same among native fibers, BVC actin reconstituted fibers and WT actin reconstituted fibers, followed by reconstitution of Tm/Tn. These results demonstrate that baculovirus/insect cell expressed actin has no significant differences from tissue purified actin and can be used for thin-filament reconstitution assays. One hypertrophic cardiomyopathy (HCM) causing actin mutant A331P actin was also expressed and studied similarly, and the results were compared to those of the WT actin. In the reconstituted fibers, A331P significantly decreased the tension both in the absence of Tm/Tn (0.55±0.03 T0, N=13) and in their presence (0.65±0.02 T0, N=13) compared to those of the WT (0.75±0.06 T0 and 0.85±0.06 T0, respectively, N=11). A331P also showed decreased pCa50 (5.57±0.03, N=13) compared to that of WT (5.69±0.02, N=11). The cross-bridge kinetics and its distribution were similar between WT and A331P actin reconstituted fibers, indicating that force/cross-bridge was decreased by A331P. In conclusion, A331P causes a weakened cross-bridge force, which leads to a decreased active tension, reduces left-ventricular ejection fraction, and eventually results in the HCM phenotype.

Analysis of the molecular pathogenesis of cardiomyopathy-causing cTnT mutants I79N, ΔE96, and ΔK210.

Three troponin T (TnT) mutants that cause hypertrophic, restrictive, and dilated cardiomyopathy (I79N, ΔE96, and ΔK210, respectively), were examined using the thin-filament extraction/reconstitution technique. Effects of Ca(2+), ATP, phosphate, and ADP concentrations on force and its transients were studied at 25°C. Maximal Ca(2+) tension (THC) and Ca(2+)-activatable tension (Tact), respectively, were similar among I79N, ΔE96, and WT, whereas ΔK210 led to a significantly lower THC (∼20% less) and Tact (∼25% less) than did WT. In pCa solution containing 8 mM Pi and ionic strength adjusted to 200 mM, the Ca(2+) sensitivity (pCa50) of I79N (5.63 ± 0.02) and ΔE96 (5.60 ± 0.03) was significantly greater than that of WT (5.45 ± 0.04), but the pCa50 of ΔK210 (5.54 ± 0.04) remained similar to that of WT. Five equilibrium constants were deduced using sinusoidal analysis. All three mutants showed significantly lower K0 (ADP association constant) and larger K4 (equilibrium constant of force generation step) relative to the corresponding values for WT. I79N and ΔK210 were associated with a K2 (equilibrium constant of cross-bridge detachment step) significantly lower than that of ΔE96 and WT. These results demonstrated that at pCa 4.66, the force/cross-bridge is ∼18% less in I79N and ∼41% less in ΔK210 than that in WT. These results indicate that the molecular pathogenesis of the cardiac TnT mutation-related cardiomyopathies is different for each mutation.

Exercise leads to unfavourable cardiac remodelling and enhanced metabolic homeostasis in obese mice with cardiac and skeletal muscle autophagy deficiency.

Autophagy is stimulated by exercise in several tissues; yet the role of skeletal and cardiac muscle-specific autophagy on the benefits of exercise training remains incompletely understood. Here, we determined the metabolic impact of exercise training in obese mice with cardiac and skeletal muscle disruption of the Autophagy related 7 gene (Atg7h&mKO). Muscle autophagy deficiency did not affect glucose clearance and exercise capacity in lean adult mice. High-fat diet in sedentary mice led to endoplasmic reticulum stress and aberrant mitochondrial protein expression in autophagy-deficient skeletal and cardiac muscles. Endurance exercise training partially reversed these abnormalities in skeletal muscle, but aggravated those in the heart also causing cardiac fibrosis, foetal gene reprogramming, and impaired mitochondrial biogenesis. Interestingly, exercise-trained Atg7h&mKO mice were better protected against obesity and insulin resistance with increased circulating fibroblast growth factor 21 (FGF21), elevated Fgf21 mRNA and protein solely in the heart, and upregulation of FGF21-target genes involved in thermogenesis and fatty acid oxidation in brown fat. These results indicate that autophagy is essential for the protective effects of exercise in the heart. However, the atypical remodelling elicited by exercise in the autophagy deficient cardiac muscle enhances whole-body metabolism, at least partially, via a heart-brown fat cross-talk involving FGF21.