019 The time course of inorganic phosphate release and actomyosin ATPase rate in permeabilised cardiac trabeculae

Abstract

Guanidinoacetate N-methyltransferase (GAMT) catalyses the final step of creatine biosynthesis such that GAMT 2/2 mice have undetectable levels of phosphocreatine and creatine and accumulation of the precursor (phospho-)guanidinoacetate (PGA). Like phosphocreatine, PGA acts as an energy reservoir, but energy transfer via creatine kinase is 100 times slower. We hypothesised that reduced energy transfer would be detrimental following myocardial infarction (MI). Methods GAMT 2/2 and wild-type controls received coronary artery ligation or sham operation (n5104), with 3D echocardiography and left ventricular haemodynamics after 6 weeks. Results Sham GAMT 2/2 mice had reduced pressure-generating capacity compared with wild-type (wt), with left ventricular systolic pressure and dP/dt max both significantly lower and impaired contractile reserve. Despite this, there was no significant difference in post-MI survival between GAMT 2/2 and wt. Both GAMT 2/2 and wt infarct groups exhibited left ventricular dilatation compared with sham controls, and systolic and diastolic function was also severely impaired. However, there was no significant difference between GAMT 2/2 and wt infarct groups for left ventricular systolic pressure, left ventricular end-diastolic pressure, dP/dt max , or Tau, nor for end-diastolic and endsystolic volumes or ejection fraction. Left ventricular/body weight increased by 30% in GAMT 2/2 and 27% in wt, indicating a similar degree of left ventricular hypertrophy in response to MI. Conclusions Loss of energy transfer in GAMT 2/2 mice was not detrimental to left ventricular remodelling, haemodynamics and survival post-MI. As acute reduction of energy transfer in the rat infarct model dramatically reduces survival, this strongly suggests that significant compensatory processes occur in GAMT 2/2 mice as a result of creatine loss during early life. The rate of inorganic phosphate (P i ) release, and therefore the crossbridge ATPase rate, was determined in permeabilised rat trabeculae. Contraction was elicited by laser-flash photolysis of NPE-caged ATP, and time-resolved P i release was monitored using MDCC-PBP, a coumarin-labelled phosphate binding protein, which fluoresces upon P i binding. The ATPase rate during the first turnover of the total crossbridges (assuming 150 mM myosin heads) was 23/s. The rate decreased to a steady state of 4/s after the eighth turnover (0.5-0.6 s after activation). This rate is comparable to published values of 3-10/ s, made ;15 s after activation using a NADH-linked enzyme assay of ADP release. The advantage of using MDCC-PBP is that the control of mechanochemical coupling can be examined from the onset of force production. Force production and P i release were simulated using a seven-step scheme. Force was attributed to the states in the sequence A.M.ADP.P i $A.M.ADP1$A.M.ADP2, with strain sensitivity incorporated into the isomerisation of A.M.ADP. The A.M.ADP.P i and A.M.ADP2 states populated rapidly as force was increasing. In contrast, the preisomerisation A.M.ADP1 accu...