Enhancing calstabin binding to ryanodine receptors improves cardiac and skeletal muscle function in heart failure

Abstract

Abnormalities in intracellular calcium release and reuptake are responsible for decreased contractility in heart failure (HF). We have previously shown that cardiac ryanodine receptors (RyRs) are protein kinase A-hyperphosphorylated and depleted of the regulatory subunit calstabin-2 in HF. Moreover, similar alterations in skeletal muscle RyR have been linked to increased fatigability in HF. To determine whether restoration of calstabin binding to RyR may ameliorate cardiac and skeletal muscle dysfunction in HF, we treated WT and calstabin-2-/- mice subjected to myocardial infarction (MI) with JTV519. JTV519, a 1,4-benzothiazepine, is a member of a class of drugs known as calcium channel stabilizers, previously shown to increase calstabin binding to RyR. Echocardiography at 21 days after MI demonstrated a significant increase in ejection fraction in WT mice treated with JTV519 (45.8 +/- 5.1%) compared with placebo (31.1 +/- 3.1%; P < 0.05). Coimmunoprecipitation experiments revealed increased amounts of calstabin-2 bound to the RyR2 channel in JTV519-treated WT mice. However, JTV519 did not show any of these beneficial effects in calstabin-2-/- mice with MI. Additionally, JTV519 improved skeletal muscle fatigue in WT and calstabin-2-/- mice with HF by increasing the binding of calstabin-1 to RyR1. The observation that treatment with JTV519 improved cardiac function in WT but not calstabin-2-/- mice indicates that calstabin-2 binding to RyR2 is required for the beneficial effects in failing hearts. We conclude that JTV519 may provide a specific way to treat the cardiac and skeletal muscle myopathy in HF by increasing calstabin binding to RyR.

Fig. 1.

Increased cardiac function and reverse remodeling in mice treated with JTV519 3 weeks after MI. (A) Kaplan–Meier survival curve for WT and calstabin-2^-/- mice after permanent coronary artery ligation resulting in MI. (B) Quantification of M-mode echocardiograms showing improved ejection fraction (EF) in JTV519-treated WT mice compared with placebo. *, P < 0.05. (C and D) Pressure–volume loop quantifications showing improved cardiac function in JTV519-treated WT compared with calstabin-2^-/- mice. dP/dt_max, slope of the maximum derivative of change in systolic pressure over time; Ped, end-systolic pressure; *, P < 0.05.

Fig. 2.

Effect of JTV519 on calstabin-2 affinity to RyR2 in mice with HF. (A) Equivalent amounts of RyR2 were immunoprecipitated with an antibody against RyR2 (top blot). Representative immunoblots (A) and bar graphs (B and C) show the amount of PKA phosphorylation of RyR2 at Ser-2808 (B) and the amount of calstabin-2 (C) bound to RyR2 from WT or calstabin-2^-/- mice treated with JTV519 or placebo. Animals were treated with JTV519 by implantable osmotic pumps (0.5 mg·kg^-1·h^-1 for 28 days after MI).

Fig. 3.

Normalized RyR2 channel gating in JTV519-treated WT mice with HF. RyR2 channels isolated from hearts 28 days after MI showing normal (low) P_o in WT mice treated with JTV519. Representative single-channel tracings are shown at 150 nM Ca²⁺. (Left) Channel openings are upward, the dash indicates the full level of channel opening (4 pA), the dotted lines indicate subconductance levels, and “c” indicates the closed state of the channels. (Right) For the amplitude histograms, amplitude is represented on the x axis and “events” indicates the number of channel openings. P_o, T_o, and T_c values correspond to the representative tracings shown; average data for all channels were measured as indicated in the text.

Fig. 4.

Increased calstabin-1 binding to RyR1 in soleus muscle after treatment with JTV519. (A) Equivalent amounts of RyR2 were immunoprecipitated with an antibody against RyR1 (top blot). Representative immunoblots (A) and bar graphs (B and C) show the amount of PKA phosphorylation of RyR1 at Ser-2844 (B) and the amount of calstabin-1 bound to RyR1 (C) from WT or calstabin-2^-/- mice treated with JTV519 or placebo. Mice were treated with JTV519 by implantable osmotic pumps (0.5 mg·kg^-1·h^-1 for 28 days after MI).

Fig. 5.

Reduced fatigability and normalized RyR1 single-channel gating in HF mice treated with JTV519. (A) Soleus muscles from JTV519-treated WT mice with HF are more resistant to fatigue compared with placebo. Representative fatigue time tracing is shown for WT and calstabin-2^-/- mice treated with JTV519 or placebo. (B) Bar graph shows mean (±SEM) time to fatigue. *, P <0.05. (C) Representative RyR1 single-channel tracings are shown at 150 nM Ca²⁺. Treatment with JTV519 of mice with HF normalized RyR1 channel gating in skeletal muscle. (Left) Channel openings are upward, the dash indicates the full level of channel opening (4 pA), the dotted lines indicate subconductance levels, and “c” indicates the closed state of the channels. (Right) For the amplitude histograms, amplitude is represented on the x axis, and “events” indicates the number of channel openings. P_o, T_o, and T_c values correspond to the representative tracings shown; average data for all channels were measured as indicated in the text.