The acetyl group deficit at the onset of contraction in ischaemic canine skeletal muscle

Abstract

Considerable debate surrounds the identity of the precise cellular site(s) of inertia that limit the contribution of mitochondrial ATP resynthesis towards a step increase in workload at the onset of muscular contraction. By detailing the relationship between canine gracilis muscle energy metabolism and contractile function during constant-flow ischaemia, in the absence (control) and presence of pyruvate dehydrogenase complex activation by dichloroacetate, the present study examined whether there is a period at the onset of contraction when acetyl-coenzyme A (acetyl-CoA) availability limits mitochondrial ATP resynthesis, i.e. whether a limitation in mitochondrial acetyl group provision exists. Secondly, assuming it does exist, we also aimed to identify the mechanism by which dichloroacetate overcomes this "acetyl group deficit". No increase in pyruvate dehydrogenase complex activation or acetyl group availability occurred during the first 20 s of contraction in the control condition, with strong trends for both acetyl-CoA and acetylcarnitine to actually decline (indicating the existence of an acetyl group deficit). Dichloroacetate increased resting pyruvate dehydrogenase complex activation, acetyl-CoA and acetylcarnitine by approximately 20-fold (P < 0.01), approximately 3-fold (P < 0.01) and approximately 4-fold (P < 0.01), respectively, and overcame the acetyl group deficit at the onset of contraction. As a consequence, the reliance upon non-oxidative ATP resynthesis was reduced by approximately 40 % (P < 0.01) and tension development was increased by approximately 20 % (P < 0.05) following 5 min of contraction. The present study has demonstrated, for the first time, the existence of an acetyl group deficit at the onset of contraction and has confirmed the metabolic and functional benefits to be gained from overcoming this inertia.

Figure 1. Experimental overview

In protocol 1, biopsy samples were taken at rest, and after 20, 60 and 300 s of contraction. In protocol 2, biopsy samples were taken at rest, and after 10, 40 and 180 s of contraction. In protocol 2, stimulation was maintained for a further 2 min after the final biopsy sampling to generate comparable isometric force data between the two protocols. PBS, physiologically buffered saline; DCA, dichloroacetate.

Figure 2. Effect of pretreatment with DCA on isometric tension development in the canine gracilis muscle

Isometric tension development (percentage of peak) during 5 min of ischaemic muscle contraction following pretreatment with saline (CON, ○; n = 6) or DCA (•; n = 6). Results are expressed as means ± s.e.m. No difference in peak tension (which always occurred at 40 s) existed between groups. * Significantly different from the corresponding CON value (P < 0.05).

Figure 3. Effect of pretreatment with DCA on levels of the active form of the pyruvate dehydrogenase complex (PDCa) during ischaemic contractions

Levels of PDCa at rest and during 5 min of ischaemic contraction following pretreatment with saline (CON, ○; n = 6) or DCA (•; n = 6). PDCa is expressed as mmol acetyl-CoA min⁻¹ (kg wet muscle)⁻¹ at 37 °C. Results are expressed as means ± s.e.m. *Significantly different from corresponding CON value (P < 0.05); †significantly different from rest within the same trial (P < 0.05).

Figure 4. Effect of pretreatment with DCA on concentrations of acetylcarnitine and free carnitine in the canine gracilis muscle during ischaemic contractions

Acetylcarnitine (A) and free carnitine (B) concentration at rest and during 5 min of ischaemic contraction following pretreatment with saline (CON, ○; n = 6) or DCA (•; n = 6). Results are expressed as means ± s.e.m. with units of mmol (kg dry muscle)⁻¹. *Significantly different from the corresponding CON value (P < 0.05); †significantly lower than rest within the same group (P = 0.08); ‡ different from 20 s within same group (P < 0.05).

Figure 5. Effect of pretreatment with DCA on concentrations of acetyl-CoA and free CoASH in the gracilis muscle during ischaemic contractions

Acetyl-CoA (A) and free CoASH (B) concentrations at rest and during 5 min of ischaemic contraction following pretreatment with saline (CON, ○; n = 6) or DCA (•; n = 6). Results are expressed as means ± s.e.m. with units of μmol (kg dry muscle)⁻¹. *Significantly different from the corresponding CON value (P < 0.05); †significantly lower than rest within the same group (P = 0.06); ‡significantly lower than rest within the same group (P < 0.05).

Figure 6. Effect of pretreatment with DCA on the rate of ATP production from substrate-level phosphorylation (SLP) at various times during ischaemic muscle contraction

Rate of ATP production from SLP between rest and 1 min, 1 min and 3 min, and 3 min and 5 min of ischaemic skeletal muscle contraction following pretreatment with saline (CON, □; n = 6) or DCA (▪; n = 6). Results are expressed as means ± s.e.m. with units of mmol ATP equivalents min⁻¹ (kg dry muscle)⁻¹. *Significantly different from the corresponding CON value (P < 0.05).