Increased cardiac myosin super-relaxation as an energy saving mechanism in hibernating grizzly bears

Figure 1. Left ventricle myosin biochemical and structural states during hibernation. Thin cardiac strips were isolated from active and hibernating grizzly bears. Mant-ATP chase experiments were carried out to estimate the percentage of myosin heads in the DRX (A; P1) or SRX states (B; P2). The experiments also allowed an estimate of DRX (T1; C) or SRX ATP turnover lifetimes (T2; D). X-ray diffraction experiments were run allowing the calculation of the inter-filament lattice spacing derived from the 1.0 equatorial reflections (d1,0; E), the equatorial intensity ratio (I1,1/I1,0; F), M3 myosin meridional reflection intensity relative to I1,0 (IM3; G) and spacing (SM3; H), M6 myosin meridional reflection intensity relative to I1,0 (IM3; I) and spacing (SM3; J). Dots correspond to averages for individual bears. The unpaired t-test was applied with p < 0.05 as level of significance. (K) is a cartoon summarizing the results. Note that all the cardiac strips tested for this figure were mostly expressing cardiac beta-myosin heavy chain isoform (Supplementary Fig. 3).

Figure 2. Right ventricle myosin super-relaxation during hibernation. Thin cardiac strips were isolated from active and hibernating grizzly bears. Mant-ATP chase experiments were carried out to estimate the percentage of myosin heads in the DRX (A; P1) or SRX states (B; P2). The experiments also allowed an estimate of DRX (T1; C) or SRX ATP turnover lifetimes (T2; D). Dots correspond to means for individual bears. The unpaired t-test was applied with p < 0.05 as level of significance.

Figure 3. Left ventricle myosin phosphorylation during hibernation.(A–C) are the phosphorylation levels of essential sarcomeric proteins, i.e., myosin regulatory light chain (RLC), myosin binding protein C (cMyBP-C) and beta-myosin heavy chain (Beta-MyHC). These were obtained using ProQ Diamond/Sypro Ruby (phosphor-)protein stains. (D–F) depict three significant differences observed in beta-myosin heavy chain phosphorylation through an estimate of the z-scores. Dots correspond to averages for individual bears. The unpaired t-test was applied with p < 0.05 as level of significance. (G) EvoEF calculations of protein stability in both wild type and phosphor-mimetic mutants. ΔGStability indicates the stability score for the protein in its corresponding configuration. ΔΔGStability represents the change in stability in mutant proteins versus the wild-type protein (MYH7). ΔΔGStability of >0 represents an increase in the stability of a mutant versus wild type. (H) is a thick filament representation using https://www.ebi.ac.uk/pdbe/entry/pdb/8q6t/highlighting the region where the unusual phosphorylations were found in the myosin rod region of various filaments.

Figure 4. Left ventricle mitochondrial proteins during hibernation.(A) is a cartoon showing mitochondria main complexes. The following figures represent the relative abundance of key proteins involved in the various complexes of the mitochondria (B–F) using western blotting approach. Dots correspond to averages for individual bears. The unpaired t-test was applied with p < 0.05 as level of significance.