David L. Hoffman

Effect of combined therapy with sodium fluoride, vitamin D and calcium in osteoporosis☆

Abstract Fluoride administration in both man and animals has been shown to stimulate new bone formation. However, the bone is poorly mineralized, and osteomalacia and secondary hyperparathyroidism frequently occur. In this study we investigated the effect of variable levels of fluoride and calcium intake, accompanied by vitamin D, on osteoporosis in eleven patients treated for one year. Bone biopsies indicated an increase in new bone formation in all patients receiving 45 mg of sodium fluoride per day, whereas 600 mg of calcium per day prevented both osteomalacia and any increase in bone resorption. In order to restore bone mass in osteoporotic subjects without producing roentgenographic or microscopic evidence of fluorosis, a therapeutic regimen of 50 mg of sodium fluoride and at least 900 mg of calcium per day and 50,000 units of vitamin D twice weekly is recommended.

Short- and long-term effects of estrogen and synthetic anabolic hormone in postmenopausal osteoporosis

In 29 women with postmenopausal osteoporosis, the proportion of total bone surface undergoing resorption or formation was evaluated by microradiography of iliac crest biopsy samples before and after short-term (2(1/2)-4 months) and long-term (26-42 months for estrogen and 9-15 months for anabolic hormone) treatment. After estrogen administration, values for bone-resorbing surfaces decreased, although less prominently after long-term than after short-term therapy. The magnitude of this decrease was positively correlated with the pretreatment value for bone-resorbing surfaces (P < 0.001). When the pretreatment value for bone-resorbing surfaces was used as a covariable, estrogen and anabolic hormone appeared to be equally effective. For bone-forming surfaces, short-term therapy with either hormone had no effect but long-term therapy significantly decreased the values. Serum immunoreactive parathyroid hormone (IPTH) increased significantly after estrogen therapy; the change in IPTH was inversely related to the change in serum calcium (P < 0.001, sign test). We conclude that the primary effect of sex hormones in postmenopausal osteoporosis is to decrease the increased level of bone resorption, perhaps by decreasing the responsiveness of bone to endogenous parathyroid hormone. However, this favorable effect, at least in part, is negated after long-term treatment by a secondary decrease in bone formation. Our data are consistent with the concept that the maximal benefit that can be derived from sex hormone therapy in postmenopausal osteoporosis is arrest or slowing of the progession of bone loss.

The Permeability Transition Pore Controls Cardiac Mitochondrial Maturation and Myocyte Differentiation

Although mature myocytes rely on mitochondria as the primary source of energy, the role of mitochondria in the developing heart is not well known. Here, we find that closure of the mitochondrial permeability transition pore (mPTP) drives maturation of mitochondrial structure and function and myocyte differentiation. Cardiomyocytes at embryonic day (E) 9.5, when compared to E13.5, displayed fragmented mitochondria with few cristae, a less-polarized mitochondrial membrane potential, higher reactive oxygen species (ROS) levels, and an open mPTP. Pharmacologic and genetic closing of the mPTP yielded maturation of mitochondrial structure and function, lowered ROS, and increased myocyte differentiation (measured by counting Z bands). Furthermore, myocyte differentiation was inhibited and enhanced with oxidant and antioxidant treatment, respectively, suggesting that redox-signaling pathways lie downstream of mitochondria to regulate cardiac myocyte differentiation.

Oxygen sensitivity of mitochondrial reactive oxygen species generation depends on metabolic conditions

The mitochondrial generation of reactive oxygen species (ROS) plays a central role in many cell signaling pathways, but debate still surrounds its regulation by factors, such as substrate availability, [O2] and metabolic state. Previously, we showed that in isolated mitochondria respiring on succinate, ROS generation was a hyperbolic function of [O2]. In the current study, we used a wide variety of substrates and inhibitors to probe the O2 sensitivity of mitochondrial ROS generation under different metabolic conditions. From such data, the apparent Km for O2 of putative ROS-generating sites within mitochondria was estimated as follows: 0.2, 0.9, 2.0, and 5.0 μm O2 for the complex I flavin site, complex I electron backflow, complex III QO site, and electron transfer flavoprotein quinone oxidoreductase of β-oxidation, respectively. Differential effects of respiratory inhibitors on ROS generation were also observed at varying [O2]. Based on these data, we hypothesize that at physiological [O2], complex I is a significant source of ROS, whereas the electron transfer flavoprotein quinone oxidoreductase may only contribute to ROS generation at very high [O2]. Furthermore, we suggest that previous discrepancies in the assignment of effects of inhibitors on ROS may be due to differences in experimental [O2]. Finally, the data set (see supplemental material) may be useful in the mathematical modeling of mitochondrial metabolism.

Chapter 10 The Interaction of Mitochondrial Membranes with Reactive Oxygen and Nitrogen Species

Abstract Within the context of the overall effects of free radicals (ROS and RNS) on biological membranes, the mitochondrion offers a unique biochemical environment which leads to a number of organelle-specific reactions, This includes a unique complement of membrane lipids and proteins, an alkaline pH in the matrix, specialized systems for the generation and quenching of ROS and RNS, and a tightly controlled redox status. All of these parameters come together in a highly orchestrated system that is essential to the proper functioning of the mitochondrion. This chapter will examine some of the mitochondria-specific aspects of ROS and RNS reactions with membranes, within the context of the many disease processes that originate at the organelle level.

Complex I of the Mitochondrial Electron Transport Chain is Dysfunctional in the Early Embryonic Heart

Proper cardiac function is crucial to ensure embryonic survival. The heart is the first organ to become functional during embryonic development, with the onset of beating at 8 days post fertilization (E8). Within two days of this (E10.5), blood begins to circulate providing nutrients to the developing embryo. Early defects in the embryonic heart can result in embryonic death or severe deformities leading to death during or shortly following birth. Although structural cardiac anomalies rarely cause demise, functional cardiac defects are much more devastating in utero.In the adult heart, mitochondria play an important role in proper function. Mitochondrial dysfunction can result in cardiac dysfunction and eventually death. While mitochondrial function is well studied in the adult heart, which relies on complex I as its primary source of electron entry, little is known about mitochondrial function in the developing embryonic heart.Data generated in this lab show that at the early stages of embryonic development (E9.5) mitochondrial membrane potential (Δψm) is low, and the potential for increased oxidative stress is high. A minimal change in Δψm is observed at E9.5 upon the addition of the complex I inhibitor rotenone. This observation is contrasted in E13.5 myocytes, which exhibit a higher sensitivity of Δψm to rotenone. In concert, these data suggest that at E9.5 complex I of the mitochondrial electron transport chain is non-functional. This study employs established bioenergetic and mitochondrial proteomic techniques, which have been adapted and applied in whole embryonic hearts and cardiomyocytes during cardiac organogenesis (E9.5, E11.5, E13.5) to support the hypothesis that at early stages of embryonic cardiac development, mitochondrial function is limited due to an immature complex I, thus resulting in decreased Δψm and increased potential for oxidative stress.

The Embryonic Mitochondrial Permeability Transition Pore Controls Cardiac Myocyte Mitochondrial Maturation and Differentiation

Little is known about cardiac energetics and mitochondrial function in the embryo, and we hypothesize that the mitochondrial permeability transition pore (mPTP) controls mitochondrial structure and function during embryonic cardiac development and is critical for normal myocyte differentiation and cardiac morphogenesis. To test this hypothesis, we examined mitochondrial structure and function in cultured myocytes and whole heart using light and electron microscopy. Mitochondria of embryonic day (E) 9.5 ventricular myocytes displayed less dense cristae and were shorter in length and less branched. By E13.5, mitochondria had abundant cristae, were longer, branched and networked, and were more closely associated with the contractile apparatus. Functional measurements demonstrated dramatic increases in mitochondrial membrane potential, an increased reliance on complex I, and a decrease in oxidative stress as the heart developed. These structural and functional data suggested an increase in inner mitochondrial membrane permeability, and closure of the mPTP using cyclosporin A or cyclophilin-D null embryos caused premature maturation of mitochondrial structure, mitochondrial function, and myocyte differentiation. Furthermore, long term opening of the mPTP using carboxyatractyloside after E9.5 inhibited mitochondrial maturation and myocyte differentiation. Taken together, these data suggest a critical role of the embryonic mPTP as a mediator of mitochondrial maturation and cardiac differentiation.

Aging and Cardiac Ischemia—Mitochondria and Free Radical Considerations

Acute myocardial infarction (MI, heart attack) kills 200,000 people annually in the United States, and the number 1 risk factor for fatal MI is age. The biological phenomenon underlying MI is ischemia-reperfusion (IR) injury, i.e., damage to tissue that occurs during and immediately after an MI. In this chapter, following a brief primer on mitochondrial ROS generation, and the underlying pathologic mechanisms of mitochondrial dysfunction in IR injury, we then break down the effects of aging on MI risk into three parts. First, changes in risk factors for MI with aging, most of which are external to the heart (e.g., atherosclerosis). Second, changes in cardiac mitochondrial function with age. Third, changes in the response of cardiac mitochondria to MI. Last, the role of mitochondria in protecting the heart from ischemia is introduced, and the possibility that increased MI risk with aging originates from a degeneration of protective signaling pathways is raised. We conclude with an outlook on the therapeutic opportunities, both current and developing, for the treatment of increased MI risk, in aged human populations.