Disruption of myoglobin in mice induces multiple compensatory mechanisms

Abstract

Myoglobin may serve a variety of functions in muscular oxygen supply, such as O(2) storage, facilitated O(2) diffusion, and myoglobin-mediated oxidative phosphorylation. We studied the functional consequences of a myoglobin deficiency on cardiac function by producing myoglobin-knockout (myo(-/-)) mice. To genetically inactivate the myoglobin gene, exon 2 encoding the heme binding site was deleted in embryonic stem cells via homologous recombination. Myo(-/-) mice are viable, fertile, and without any obvious signs of functional limitations. Hemoglobin concentrations were significantly elevated in myo(-/-) mice. Cardiac function and energetics were analyzed in isolated perfused hearts under resting conditions and during beta-adrenergic stimulation with dobutamine. Myo(-/-) hearts showed no alteration in contractile parameters either under basal conditions or after maximal beta-adrenergic stimulation (200 nM dobutamine). Tissue levels of ATP, phosphocreatine ((31)P-NMR), and myocardial O(2) consumption were not altered. However, coronary flow [6.4 +/- 1.3 ml.min(-1).g(-1) [wild-type (WT)] vs. 8.5 +/- 2.4 ml.min(-1).g(-1) [myo(-/-)] [and coronary reserve [17.1 +/- 2.1 (WT) vs. 20.8 +/- 1.1 (myo(-/-) ml. min(-1).g(-1) were significantly elevated in myo(-/-) hearts. Histological examination revealed that capillary density also was increased in myo(-/-) hearts [3,111 +/- 400 mm(-2) (WT) vs. 4,140 +/- 140 mm(-2) (Myo(-/-)]. These data demonstrate that disruption of myoglobin results in the activation of multiple compensatory mechanisms that steepen the pO(2) gradient and reduce the diffusion path length for O(2) between capillary and the mitochondria; this suggests that myoglobin normally is important for the delivery of oxygen.

Figure 1

Targeting strategy and molecular verification of myoglobin disruption. (a) Structures of the WT and mutated alleles (mut) and the targeting vector are shown. (Restriction sites: B, BamHI; Bg, BglII; HIII, HindIII; HcII, HincII; Xb, XbaI. neoR, neomycin resistance gene). (b) Southern blot analysis of BglII-digested DNA from WT (+/+), heterozygous (+/−), and homozygously mutated (−/−) mice. The BamHI-HindII-fragment indicated in a was used as a probe. Hybridizing fragments of 2.9 kb (WT allele) and 12 kb (mutated allele) were detected. (c) Northern blot analysis of cardiac RNA isolated from the three genotypes. (d) SDS/PAGE analysis of protein patterns of myo^−/− and WT hearts. Two-hundred micrograms of cardiac proteins were separated on a 12.5% SDS gel and were stained with Coomassie brilliant blue. Five micrograms of horse myoglobin were loaded as control. (e) Morphology of myoglobin-deficient hearts. WT (left) and myo^−/− hearts (right) were perfused free of blood and were photographed.

Figure 2

Analysis of cardiac function and energetics of isolated perfused WT and myo^−/− hearts under basal conditions. Bars show the means ± SD for n = 8 hearts from WT (black bars) and myo^−/− hearts (gray bars). ∗, statistically significant (P < 0.05). LVP, left ventricular pressure; dP/dt_max, rate of maximal pressure development; MVO₂, oxygen consumption.

Figure 3

Analysis of cardiac function and energetics of isolated perfused WT and myo^−/− hearts under β-adrenergic stimulation. Hearts were stimulated by coronary application of 50 or 200 nM dobutamine. Symbols show means ± SD for n = 8 hearts from WT (black) and myo^−/− hearts (open symbols). ∗, statistically significant (P < 0.05). For abbreviations see Fig. 2.

Figure 4

Coronary flow reserve in WT and myo^−/− hearts. Maximal coronary flow of WT and myo^−/−hearts was determined as peak flow of reactive hyperemia elicited by 20 s of coronary occlusion or as maximal vasodilation in response to intracoronary application of 1 μM adenosine. Bars show means ± SD for n = 6 WT (black bars) and myo^−/− hearts (gray bars). ∗, statistically significant (P < 0.05).

Figure 5

Analysis of capillary densities in WT and myo^−/− hearts. Representative micrographs for WT (left) and myo^−/− hearts (right) are shown together with the quantitative data for capillary density and capillary distance. Bars represent means ± SD for 6 WT (black bars) and 6 myo^−/− hearts (gray bars). ∗∗, statistically significant (P < 0.01).