Om wild-type (WT) concentrations to a .4-fold increase due to variability in transgene mRNA expression. The Tg46 strain was used for this study as its distribution of creatine levels is closer to the target range (88?42 nmol/mg protein, i.e. 20?00 above wildtype). Control groups consisted of a random mixture of WT littermates from CrT-OE mice and C57BL/6J 58-49-1 obtained from a commercial breeder (Harlan, UK). Mice were kept in a specific pathogen free environment, with controlled humidity and temperature (20uC to 22uC) and 12 hours light-dark cycle.Group S consisted of WT mice, which underwent sham surgery and were not fed ribose; Group MI consisted of WT mice, which underwent myocardial infarction (MI) surgery and were given normal drinking water; Group MI+R consisted of WT mice, which underwent MI surgery and were given ribose in drinking water (10 (w/v) D-ribose solution); Group MI+R+C consisted of CrT-OE mice, which underwent MI surgery and were given ribose in drinking water (10 (w/v) D-ribose solution).Pilot Study 1: TAN Pool in the Failing Mouse HeartFemale adult C57BL/6J mice underwent coronary artery ligation or sham surgery (n = 7 in each group). Four weeks following surgery, TAN pool was measured in homogenatesCrT-OE mice underwent 1H-MRS examination to pre-select animals with moderately elevated myocardial creatine (88?142 nmol/mg prot). At least one week later, WT and CrT-OE mice had coronary artery ligation surgery to induce chronic myocardial infarction as previously described in detail by Lygate [27]. Mice received 0.27 mg of buprenorphine subcutaneously for pain relief and ribose supplementation was started 24 hours postsurgery. An echocardiogram was performed ,4 weeks following surgery under isoflurane anaesthesia using a Visualsonics Vevo2100 to exclude animals with very small or no infarcts. Eight weeks after surgery, surviving mice had cine-MRI to measure infarct size and assess LV remodelling. One week later, haemodynamic function was assessed by LV catheterisation under isoflurane anaesthesia in closed-chest, spontaneously breathing mice using a mikro-tip pressure catheter (SPR-839, Millar instruments, Houston, Texas) as previously described [28]. Measurements were taken at baseline and following maximal stimulation with 16 ng/gBW/min dobutamine. Hearts wereRibose Treatment in Chronic Murine Heart Failureexcised, rinsed in ice-cold heparinised saline then whole hearts freeze-clamped in liquid nitrogen for measurement of creatine and high-energy phosphates by HPLC [26]. Mice with infarct size below 25 of LV circumference were excluded as these animals do not develop the hallmarks of heart failure [27]. At the end of the study, some mice were excluded in order to retrospectively match groups for the same range of infarct sizes. This is essential to allow inter-group comparison of LV function and remodelling parameters, which are highly dependent on the extent of myocardial injury.In vivo Magnetic ResonanceAll experiments were carried out on a 9.4 T (400 MHz) MR system (Agilent Technologies) using a quadrature-driven birdcage resonator (Rapid Biomedical). For high-resolution magnetic resonance cine imaging (cine MRI), 8?0 short-axis slices, covering the heart from base to apex, were acquired using a cardiac-triggered and 78919-13-8 respiration-gated fast low-angle-shot sequence with the following parameters: field of view (25.6 mm)2, matrix size 2566256, echo time (TE)/repetition time TR) = 1.79/ 4.6 ms, 15u sinc excitation pulse.Om wild-type (WT) concentrations to a .4-fold increase due to variability in transgene mRNA expression. The Tg46 strain was used for this study as its distribution of creatine levels is closer to the target range (88?42 nmol/mg protein, i.e. 20?00 above wildtype). Control groups consisted of a random mixture of WT littermates from CrT-OE mice and C57BL/6J obtained from a commercial breeder (Harlan, UK). Mice were kept in a specific pathogen free environment, with controlled humidity and temperature (20uC to 22uC) and 12 hours light-dark cycle.Group S consisted of WT mice, which underwent sham surgery and were not fed ribose; Group MI consisted of WT mice, which underwent myocardial infarction (MI) surgery and were given normal drinking water; Group MI+R consisted of WT mice, which underwent MI surgery and were given ribose in drinking water (10 (w/v) D-ribose solution); Group MI+R+C consisted of CrT-OE mice, which underwent MI surgery and were given ribose in drinking water (10 (w/v) D-ribose solution).Pilot Study 1: TAN Pool in the Failing Mouse HeartFemale adult C57BL/6J mice underwent coronary artery ligation or sham surgery (n = 7 in each group). Four weeks following surgery, TAN pool was measured in homogenatesCrT-OE mice underwent 1H-MRS examination to pre-select animals with moderately elevated myocardial creatine (88?142 nmol/mg prot). At least one week later, WT and CrT-OE mice had coronary artery ligation surgery to induce chronic myocardial infarction as previously described in detail by Lygate [27]. Mice received 0.27 mg of buprenorphine subcutaneously for pain relief and ribose supplementation was started 24 hours postsurgery. An echocardiogram was performed ,4 weeks following surgery under isoflurane anaesthesia using a Visualsonics Vevo2100 to exclude animals with very small or no infarcts. Eight weeks after surgery, surviving mice had cine-MRI to measure infarct size and assess LV remodelling. One week later, haemodynamic function was assessed by LV catheterisation under isoflurane anaesthesia in closed-chest, spontaneously breathing mice using a mikro-tip pressure catheter (SPR-839, Millar instruments, Houston, Texas) as previously described [28]. Measurements were taken at baseline and following maximal stimulation with 16 ng/gBW/min dobutamine. Hearts wereRibose Treatment in Chronic Murine Heart Failureexcised, rinsed in ice-cold heparinised saline then whole hearts freeze-clamped in liquid nitrogen for measurement of creatine and high-energy phosphates by HPLC [26]. Mice with infarct size below 25 of LV circumference were excluded as these animals do not develop the hallmarks of heart failure [27]. At the end of the study, some mice were excluded in order to retrospectively match groups for the same range of infarct sizes. This is essential to allow inter-group comparison of LV function and remodelling parameters, which are highly dependent on the extent of myocardial injury.In vivo Magnetic ResonanceAll experiments were carried out on a 9.4 T (400 MHz) MR system (Agilent Technologies) using a quadrature-driven birdcage resonator (Rapid Biomedical). For high-resolution magnetic resonance cine imaging (cine MRI), 8?0 short-axis slices, covering the heart from base to apex, were acquired using a cardiac-triggered and respiration-gated fast low-angle-shot sequence with the following parameters: field of view (25.6 mm)2, matrix size 2566256, echo time (TE)/repetition time TR) = 1.79/ 4.6 ms, 15u sinc excitation pulse.
