Ribose supplementation alone or with elevated creatine does not preserve high energy nucleotides or cardiac function in the failing mouse heart

Background: Reduced levels of creatine and total adenine nucleotides (sum of ATP, ADP and AMP) are hallmarks of chronic heart failure and restoring these pools is predicted to be beneficial by maintaining the diseased heart in a more favourable energy state. Ribose supplementation is thought to support both salvage and re-synthesis of adenine nucleotides by bypassing the rate-limiting step. We therefore tested whether ribose would be beneficial in chronic heart failure in control mice and in mice with elevated myocardial creatine due to overexpression of the creatine transporter (CrT-OE). Methods and Results: Four groups were studied: sham; myocardial infarction (MI); MI+ribose; MI+CrT-OE+ribose. In a pilot study, ribose given in drinking water was bioavailable, resulting in a two-fold increase in myocardial ribose-5-phosphate levels. However, 8 weeks post-surgery, total adenine nucleotide (TAN) pool was decreased to a similar amount (8–14%) in all infarcted groups irrespective of the treatment received. All infarcted groups also presented with a similar and substantial degree of left ventricular (LV) dysfunction (3-fold reduction in ejection fraction) and LV hypertrophy (32–47% increased mass). Ejection fraction closely correlated with infarct size independently of treatment (r2 = 0.63, p<0.0001), but did not correlate with myocardial creatine or TAN levels. Conclusion: Elevating myocardial ribose and creatine levels failed to maintain TAN pool or improve post-infarction LV remodeling and function. This suggests that ribose is not rate-limiting for purine nucleotide biosynthesis in the chronically failing mouse heart and that alternative strategies to preserve TAN pool should be investigated

Chronic creatine kinase deficiency eventually leads to congestive heart failure, but severity is dependent on genetic background, gender and age

The creatine kinase (CK) energy transport and buffering system supports cardiac function at times of high demand and is impaired in the failing heart. Mice deficient in muscle- and mitochondrial-CK (M/Mt-CK(−/−)) have previously been described, but exhibit an unexpectedly mild phenotype of compensated left ventricular (LV) hypertrophy. We hypothesised that heart failure would develop with age and performed echocardiography and LV haemodynamics at 1 year. Since all previous studies have utilised mice with a mixed genetic background, we backcrossed for >10 generations on to C57BL/6, and repeated the in vivo investigations. Male M/Mt-CK(−/−) mice on the mixed genetic background developed congestive heart failure as evidenced by significantly elevated end-diastolic pressure, impaired contractility, LV dilatation, hypertrophy and pulmonary congestion. Female mice were less severely affected, only showing trends for these parameters. After backcrossing, M/Mt-CK(−/−) mice had LV dysfunction consisting of impaired isovolumetric pressure changes and reduced contractile reserve, but did not develop congestive heart failure. Body weight was lower in knockout mice as a consequence of reduced total body fat. LV weight was not significantly elevated in relation to other internal organs and gene expression of LVH markers was normal, suggesting an absence of hypertrophy. In conclusion, the consequences of CK deficiency are highly dependent on genetic modifiers, gender and age. However, the observation that a primary defect in CK can, under the right conditions, result in heart failure suggests that impaired CK activity in the failing heart could contribute to disease progression. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00395-012-0276-2) contains supplementary material, which is available to authorized users

Accelerating global left-ventricular function assessment in mice using reduced slice acquisition and three-dimensional guide-point modelling

<p>Abstract</p> <p>Background</p> <p>To investigate the utility of three-dimensional guide-point modeling (GPM) to reduce the time required for CMR evaluation of global cardiac function in mice, by reducing the number of image slices required for accurate quantification of left-ventricular (LV) mass and volumes.</p> <p>Methods</p> <p>Five female C57Bl/6 mice 8 weeks post myocardial infarction induced by permanent occlusion of the left coronary artery, and six male control (un-operated) C57Bl/6 mice, were subject to CMR examination under isoflurane anaesthesia. Contiguous short axis (SAX) slices (1 mm thick 7-9 slices) were obtained together with two long axis (LAX) slices in two chamber and four chamber orientations. Using a mathematical model of the heart to interpolate information between the available slices, GPM LV mass and volumes were determined using full slice (all SAX and two LAX), six slice (four SAX and two LAX) and four slice (two SAX and two LAX) analysis protocols. All results were compared with standard manual volumetric analysis using all SAX slices.</p> <p>Results</p> <p>Infarct size was 39.1 ± 5.1% of LV myocardium. No significant differences were found in left ventricular mass and volumes between the standard and GPM full and six slice protocols in infarcted mice (113 ± 10, 116 ± 11, and 117 ± 11 mg respectively for mass), or between the standard and GPM full, six and four slice protocols in control mice, (105 ± 14, 106 ± 10, 104 ± 12, and 105 ± 7 mg respectively for mass). Significant differences were found in LV mass (135 ± 18 mg) and EF using the GPM four slice protocol in infarcted mice (p < 0.05).</p> <p>Conclusion</p> <p>GPM enables accurate analysis of LV function in mice with relatively large infarcts using a reduced six slice acquisition protocol, and in mice with normal/symmetrical left-ventricular topology using a four slice protocol.</p

Refined approach for quantification of in vivo ischemia-reperfusion injury in the mouse heart

Cardiac ischemia-reperfusion experiments in the mouse are important in vivo models of human disease. Infarct size is a particularly important scientific readout as virtually all cardiocirculatory pathways are affected by it. Therefore, such measurements must be exact and valid. The histological analysis, however, remains technically challenging, and the resulting quality is often unsatisfactory. For this report we have scrutinized each step involved in standard double-staining histology. We have tested published approaches and challenged their practicality. As a result, we propose an improved and streamlined protocol, which consistently yields high-quality histology, thereby minimizing experimental noise and group sizes

Left ventricular morphology and function derived from MRI 8 weeks post myocardial infarction.

<p>Group S are untreated wild-type sham-operated mice; Group MI are untreated wild-type infarcted mice; Group MI+R are wild-type infarcted mice treated with ribose; Group MI+C+R are infarcted creatine transporter overexpressing mice treated with ribose. Infarcted groups were matched for infarct size (A). Left ventricular remodelling and function was measured by cine-MRI (B–F). Data are reported as mean ± SD. *** denotes p<0.001 (1-way ANOVA with Bonferroni’s correction).</p

Morphometry and myocardial biochemistry 8 weeks after myocardial infarction.

<p>All values are mean ± SD. Comparisons were made by one-way ANOVA with Bonferroni’s post-hoc test.</p>*<p>denotes p<0.05,</p>**<p>p<0.01,</p>***<p>p<0.001 vs group S and ^#p<0.05,</p>###<p>p<0.001 vs group MI.</p

Oral ribose treatment increases ribose-5-phosphate levels in the heart.

<p>Myocardial ribose-5-phosphate levels following administration of ribose (10% w/v) in drinking water for seven weeks. Control n = 5, ribose n = 4, mean ± SD, ** denotes p<0.01.</p

Left ventricular haemodynamic parameters 8 weeks post myocardial infarction.

<p>Group S are untreated wild-type sham-operated mice; Group MI are untreated wild-type infarcted mice; Group MI+R are wild-type infarcted mice treated with ribose; Group MI+C+R are infarcted creatine transporter overexpressing mice treated with ribose. Heart rate (A), LV end-systolic and end-diastolic pressures (B–C) and maximal and minimal rates of pressure change (D–E) under baseline non-stimulated conditions. Differences analysed by one-way ANOVA with Bonferroni’s correction. ** denotes p<0.01 and *** p<0.001 versus group S. There was no difference between any of the infarcted groups. Panels F–H show heart rate and maximal and minimal rates of pressure change before and after stimulation with dobutamine (16 ng/g BW/min). Effect of genotype and dobutamine assessed by two-way ANOVA with post-hoc Bonferroni’s correction. ^ denotes p<0.05, ^^ p<0.01, ^^^ p<0.001 for dobutamine (black bars) vs baseline (white bars) and * denotes p<0.05, ** p<0.01, *** p<0.001 versus sham at the same dobutamine dose. Data are mean ± SD.</p

Factors influencing ejection fraction by correlation analysis.

<p>Ejection fraction assessed by MRI 8 weeks after myocardial infarction correlated well with infarct size (A), but not with myocardial total adenine nucleotides (B), or myocardial creatine levels (C). Correlation analysis and linear regression is for all groups analysed together. Group MI are untreated wild-type infarcted mice; Group MI+R are wild-type infarcted mice treated with ribose; Group MI+C+R are infarcted creatine transporter overexpressing mice treated with ribose.</p