MyoMed205 Counteracts Titin Hyperphosphorylation and the Expression of Contraction‐Regulating Proteins in a Rat Model of HFpEF

Figures

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  FIGURE 1. SKM rearrangement occurs earlier in the EDL than in the Sol during HFpEF development. (A) CSA soleus, (B) CSA EDL, as well as (C) maximal specific force and (D) titin phosphorylation of soleus and EDL from ZSF1‐control (con) (represented as scattered line) and ZSF1‐HFpEF rats (HFpEF). Samples were obtained from 6‐, 10‐, 15‐, 20‐ and 32‐week‐old rats. The results are expressed as median ± Min–Max (n = 4–5 per group) **** p < 0.0001 (A, B) or as x‐fold change versus control (scattered line, set to 1.0) ± SEM (n = 4–12 per group) * p < 0.05 versus con (C, D).

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  FIGURE 2. MyoMed205 has antiatrophic effects. CSA of (A) soleus and (B) EDL from ZSF1‐control (con) and untreated ZSF1‐HFpEF rats (HFpEF) as well as treated ZSF1‐HFpEF rats (MyoMed205) at 32 weeks of age. The results are expressed as median ± Min–Max (n = 4–5 per group). **** p < 0.0001.

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  FIGURE 3. Titin hyperphosphorylation in the Sol can be normalized by treatment with MyoMed205. Titin expression, as well as phosphorylation, was quantified via VAGE analysis in skeletal muscle homogenates of Sol obtained from ZSF1‐control (con) (represented as a solid line) and untreated ZSF1‐HFpEF rats (HFpEF) as well as treated ZSF1‐HFpEF rats (MyoMed205) at 32 weeks of age. The results are expressed as x‐fold versus control (solid line, set to 1.0) ± SEM (n = 8–14 per group). Representative stains and VAGE blots are depicted below (c = con, HF = HFpEF, M = MyoMed205). # p < 0.05 versus con, $ p < 0.05 versus HFpEF.

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  FIGURE 4. Titin degradation gets reduced, whereas hyperphosphorylation can be normalized by treatment with MyoMed205 in the EDL. Protein expression, as well as phosphorylation of titin, was quantified via VAGE analysis in SKM homogenates of EDL obtained from ZSF1‐control (con) (represented as a solid line) and untreated ZSF1‐HFpEF rats (HFpEF) as well as treated ZSF1‐HFpEF rats (MyoMed205) at 32 weeks of age. The results are expressed as x‐fold versus control (solid line, set to 1.0) ± SEM (n = 10–14 per group). Representative stains and VAGE blots are depicted below (c = con, HF = HFpEF, M = MyoMed205). # p < 0.05 versus con, $ p < 0.05 versus HFpEF.

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  FIGURE 5. PEVK phosphorylation strongly correlates with functional parameters in the SKM. Correlation analyses between S11878 phosphorylation and total titin phosphorylation in the Sol (A) and the EDL (B). In addition, a correlation between S11878 phosphorylation and SKM CSA (C) and maximal (max.) specific force in the EDL (D) was evident.

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  FIGURE 6. Ubiquitination, but not of MHC, is susceptible to MyoMed205, whereas MLC phosphorylation is significantly decreased only in HFpEF animal. MHC expression as well as ubiquitination of MHC and a 85‐kDa protein (A) and MLC isoform expression and phosphorylation (B) were quantified by WB analysis in skeletal muscle homogenates of EDL, obtained from ZSF1‐control (con) (represented as solid line) and untreated ZSF1‐HFpEF rats (HFpEF) as well as treated ZSF1‐HFpEF rats (MyoMed205) at 32 weeks of age. The results are expressed as x‐fold versus control (solid line, set to 1.0) ± SEM (n = 6–11 per group). Representative stains and blots are depicted below (c = con, HF = HFpEF, M = MyoMed205). # p < 0.05 versus con, $ p < 0.05 versus HFpEF.

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  FIGURE 7. MyoMed205 modulates expression of proteins associated with Ca 2+‐dependent muscle contraction. TnC, TnI, TnT and α‐ and β‐Tm expression was quantified by WB analysis of skeletal muscle homogenates of EDL obtained from ZSF1‐control (con) (represented as solid line) and untreated ZSF1‐HFpEF rats (HFpEF) as well as treated ZSF1‐HFpEF rats (MyoMed205) at 32 weeks of age. The results are expressed as x‐fold versus control (solid line, set to 1.0) ± SEM (n = 10–14 per group). Representative blots are shown below (c = con, HF = HFpEF, M = MyoMed205). # p < 0.05 versus con, $ p < 0.05 versus HFpEF.

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  FIGURE 8. Normalized expression of atrophy markers after treatment with MyoMed205. MuRF1 and GDF8 expression was quantified via WB analysis of skeletal muscle homogenates of EDL obtained from ZSF1‐control (con) (represented as solid line) and untreated ZSF1‐HFpEF rats (HFpEF) as well as treated ZSF1‐HFpEF rats (MyoMed205) at 32 weeks of age. The results are expressed as x‐fold versus control (solid line, set to 1.0) ± SEM (n = 10–14 per group). Representative blots are shown below (c = con, HF = HFpEF, M = MyoMed205). # p < 0.05 versus con, $ p < 0.05 versus HFpEF.