Levosimendan improves calcium sensitivity of diaphragm muscle fibres from a rat model of heart failure

Abstract

Background and purpose: Diaphragm muscle weakness occurs in patients with heart failure (HF) and is associated with exercise intolerance and increased mortality. Reduced sensitivity of diaphragm fibres to calcium contributes to diaphragm weakness in HF. Here we have investigated the ability of the calcium sensitizer levosimendan to restore the reduced calcium sensitivity of diaphragm fibres from rats with HF.

Experimental approach: Coronary artery ligation in rats was used as an animal model for HF. Sham-operated rats served as controls. Fifteen weeks after induction of HF or sham operations animals were killed and muscle fibres were isolated from the diaphragm. Diaphragm fibres were skinned and activated with solutions containing incremental calcium concentrations and 10 µM levosimendan or vehicle (0.02% DMSO). Developed force was measured at each calcium concentration, and force-calcium concentration relationships were plotted.

Key results: Calcium sensitivity of force generation was reduced in diaphragm muscle fibres from HF rats, compared with fibres from control rats (P < 0.01). Maximal force generation was ∼25% lower in HF diaphragm fibres than in control fibres (P < 0.05). Levosimendan significantly increased calcium sensitivity of force generation in diaphragm fibres from HF and control rats, without affecting maximal force generation.

Conclusions and implications: Levosimendan enhanced the force generating capacity of diaphragm fibres from HF rats by increasing the sensitivity of force generation to calcium concentration. These results provide strong support for testing the effect of calcium sensitizers on diaphragm muscle weakness in patients with HF.

Figure 1

Dose-response effects of levosimendan on sub-maximal force generating capacity of rat soleus fibres. Incremental levosimendan concentrations were added to rat soleus fibres (n = 10) during sub-maximal activation at pCa 6.5. Force generation increases with rising levosimendan concentration, with a significant effect at 10 µM.

Figure 2

The effect of levosimendan on force-calcium characteristics of diaphragm muscle fibres from rats with heart failure and control rats. Diaphragm fibres were exposed to 10 µM levosimendan or vehicle activating solutions. Isometric force generation in response to solutions with incremental calcium concentrations was determined. The force-pCa relations of diaphragm fibres treated with levosimendan showed increased sensitivity to calcium force-pCa relations of diaphragm fibres treated with vehicle, for both slow (Figure 2A) and fast (Figure 2B) fibres. *P < 0.05, significant effect of levosimendan; two-way repeated measures anova.

Figure 3

Values of pCa₅₀, an index for calcium sensitivity of force generation, from control and HF diaphragm fibres exposed to either 10 µM levosimendan or vehicle solution. Calcium sensitivity was lower in HF fibres than in control fibres. Diaphragm fibres treated with levosimendan displayed higher calcium sensitivity than diaphragm fibres treated with vehicle, for both slow (Figure 3A) and fast (Figure 3B) fibres. *P < 0.05 compared with control vehicle-treated fibres; #P < 0.05 compared with vehicle treated HF fibres. pCa₅₀ = −¹⁰log[Ca²⁺] at which 50% of maximal force is generated.

Figure 4

Maximal force generation of control diaphragm fibres and HF diaphragm fibres exposed to 10 µM levosimendan or vehicle solution. HF diaphragm fibres generated significantly lower maximal forces than control fibres, in both slow and fast fibres (Figure 4A and B, respectively). Maximal force generation was not significantly different between levosimendan and vehicle-treated HF fibres. *P < 0.05 compared with vehicle-treated control fibres.