Dietary iron deficiency induces ventricular dilation, mitochondrial ultrastructural aberrations and cytochrome c release: involvement of nitric oxide synthase and protein tyrosine nitration

Iron deficiency is associated with multiple health problems, including the cardiovascular system. However, the mechanism of action of iron-deficiency-induced cardiovascular damage is unclear. The aim of the present study was to examine the effect of dietary iron deficiency on cardiac ultrastructure, mitochondrial cytochrome c release, NOS (nitric oxide synthase) and several stress-related protein molecules, including protein nitrotyrosine, the p47phox subunit of NADPH oxidase, caveolin-1 and RhoA. Male weanling rats were fed with either control or iron-deficient diets for 12 weeks. Cardiac ultrastructure was examined by transmission electron microscopy. Western blot analysis was used to evaluate cytochrome c, endothelial and inducible NOS, NADPH oxidase, caveolin-1 and RhoA. Protein nitrotyrosine formation was measured by ELISA. Rats fed an iron-deficient diet exhibited increased heart weight and size compared with the control group. Heart width, length and ventricular free wall thickness were similar between the two groups. However, the left ventricular dimension and chamber volume were significantly enhanced in the iron-deficient group compared with controls. Ultrastructural examination revealed mitochondrial swelling and abnormal sarcomere structure in iron-deficient ventricular tissues. Cytochrome c release was significantly enhanced in iron-deficient rats. Protein expression of eNOS (endothelial NOS) and iNOS (inducible NOS), and protein nitrotyrosine formation were significantly elevated in cardiac tissue or mitochondrial extraction from the iron-deficient group. Significantly up-regulated NADPH oxidase, caveolin-1 and RhoA expression were also detected in ventricular tissue of the iron-deficient group. Taken together, these results suggest that dietary iron deficiency may have induced cardiac hypertrophy characterized by aberrant mitochondrial and irregular sarcomere organization, which was accompanied by increased reactive nitrogen species and RhoA expression.

A Langendorf preparation for quick-freezing small hearts

The validity of studies of cell structures at high spatial and temporal resolution depends on the fidelity with which tissue preparation maintains in vivo conditions. Optimal preservation of structural substrates of precisely timed physiological intracellular events is offered by cryopreservation followed by freeze-fracture and freeze-substitution; we have established criteria for gauging that quality of cryopreservation in skeletal and cardiac muscle. Ciyopreservation is indispensable for electron probe x-ray microanalysis (EPXMA) of freeze-dried cryosections. We have developed a Langendorf preparation for small hearts (Figs. 1,2) suitable for use in our quick-freeze device (“Cryopress”; Med-Vac, Inc., St. Louis, MO 63117) to a) investigate the spatial distribution of physiologically important elements (e.g. calcium) during excitation-contraction coupling (ECC), especially in intact avian hearts and, b) assess damage to cardiac ultrastructure that is caused by pathological conditions (e.g. ischemia), rather than by artifacts due to chemical fixation (e.g. membrane damage by glutaraldehyde). In our Langendorf preparation, the tips of hearts can be quick-frozen at optimal freezing conditions, and comparative studies of the hearts of different animal species performed.