G. A. Ordway

Mice without myoglobin

Myoglobin, an intracellular haemoprotein expressed in the heart and oxidative skeletal myofibres of vertebrates, binds molecular oxygen and may facilitate oxygen transport from erythrocytes to mitochondria, thereby maintaining cellular respiration during periods of high physiological demand. Here we show, however, that mice without myoglobin, generated by gene-knockout technology, are fertile and exhibit normal exercise capacity and a normal ventilatory response to low oxygen levels (hypoxia). Heart and soleus muscles from these animals are depigmented, but function normally in standard assays of muscle performance invitro across a range of work conditions and oxygen availability. These data show that myoglobin is not required to meet the metabolic requirements of pregnancy or exercise in a terrestrial mammal, and raise new questions about oxygen transport and metabolic regulation in working muscles.

Myoglobin: an essential hemoprotein in striated muscle

SUMMARY Myoglobin is a cytoplasmic hemoprotein, expressed solely in cardiac myocytes and oxidative skeletal muscle fibers, that reversibly binds O2 by its heme residue, a porphyrin ring:iron ion complex. Since the initial discovery of its structure over 40 years ago, wide-ranging work by many investigators has added importantly to our understanding of its function and regulation. Functionally, myoglobin is well accepted as an O2-storage protein in muscle, capable of releasing O2 during periods of hypoxia or anoxia. Myoglobin is also thought to buffer intracellular O2 concentration when muscle activity increases and to facilitate intracellular O2 diffusion by providing a parallel path that augments simple diffusion of dissolved O2. The use of gene targeting and other molecular biological techniques has revealed important new insights into the developmental and environmental regulation of myoglobin and provided additional functions for this hemoprotein such as scavenging nitric oxide and reactive O2 species. These recent findings, coupled with additional emerging technologies and the discovery of other tissue globins, provide a framework for addressing new questions about myoglobin and readdressing old ones.

Transient regulation of c-fos, αB-crystallin, and hsp70 in muscle during recovery from contractile activity

Endurance exercise training increases the oxidative capacity of skeletal muscles, reflecting the induction of genes encoding enzymes of intermediary metabolism. To test the hypothesis that changes in gene expression may be triggered specifically during recovery from contractile activity, we quantified c-fos, αB-crystallin, 70-kDa heat shock protein (hsp70), myoglobin, and citrate synthase RNA in rabbit tibialis anterior muscle during recovery from intermittent (8 h/day), low-frequency (10 Hz) motor nerve stimulation. Recovery from a single 8-h bout of stimulation was characterized by large (>10-fold) transient increases in c-fos, αB-crystallin, and hsp70 mRNA. Similar changes were noted during recovery after 7 or 14 days of stimulation (8 h/day). Myoglobin and citrate synthase mRNA were also induced during recovery, but the changes were of lesser magnitude (2- to 2.5-fold) and were observed only following repeated bouts of muscle activity (7th or 14th day) that promoted sustained (>24 h) increases in these transcripts. These findings indicate that recovery from exercise is associated with specific transient changes in the expression of immediate early and stress protein genes, suggesting that the products of these genes may have specific roles in the remodeling process evoked by repeated bouts of contractile activity.

Protective role of heat stress in burn trauma.

Objective:This study was designed to determine whether cutaneous burn injury up-regulated expression of myocardial heat shock protein (HSP)70 and to determine a potential cardioprotective role of inducible HSP70 (iHSP70) in postburn myocardial contractile function. Design:Experimental study. Setting:Research laboratory. Subjects:Adult Hartley guinea pigs. Interventions:The first set of studies determined whether heat stress (increasing body temperature to 42°C for 20 mins) in adult Hartley guinea pigs would increase expression of myocardial iHSP70. Measurements and Main Results:Our model of heat stress increased expression of inducible HSP in the myocardium (Western blot), and this response persisted 1, 2, 4, and 24 hrs after the initial heat stress. We then determined whether burn trauma over 40% total body surface area (TBSA) increased myocardial expression of iHSP70. Time-matched sham and burned guinea pigs were killed 1, 2, 4, 12, 18, or 24 hrs postburn, and hearts were used either to examine myocardial iHSP70 expression by Western blot or to determine myocardial contractile function (Langendorff). Burn trauma produced a two-fold increase in myocardial iHSP70 that was evident as early as 1 hr postburn and persisted 24 hrs postburn; increased iHSP70 expression occurred despite only a modest and transient increase in body temperature after burn trauma. We then determined whether heat shock stress before burn trauma provided a protective or detrimental effect on cardiac function. Body temperature was increased to 42°C for 20 mins, animals were allowed to recover, and body temperature returned to baseline; burn trauma was then produced (40% TBSA) either 1, 2, 4, or 24 hrs after the initial heat stress. Myocardial contraction and relaxation deficits were evident after burn trauma alone; however, heat stress 1 hr before burn trauma improved left ventricular developed pressure and positive or negative maximum change in pressure in time and shifted left ventricular function curves upward and leftward from those calculated for burn in the absence of heat stress, indicating improved ventricular performance. Increasing the time between the initial heat stress and burn injury decreased the cardioprotective effects of heat stress. Thus, organ protection was evident only when the time period between the initial heat stress and the second insult was brief (1 hr). Conclusions:Our finding that the amount of myocardial iHSP70 remained constantly elevated after heat stress while the cardio-protective effect afforded by a prior heat stress declined with time suggested that the initial heat stress evoked several compensatory/adaptive mechanisms that may include modulation of autonomic nervous system responses, changes in metabolic function, modulation of pro/anti-inflammatory cytokine responses, and heat stress-related alterations in antioxidant capacity.

Activation of gastric afferent fibers increases coronary arterial resistance in anesthetized dogs

Stimulation of chemically sensitive receptors in the stomach with capsaicin is known to reflexly increase heart rate, arterial pressure, left ventricular contractility, and systemic vascular resistance. What is not known, however, is if activating these gastric afferents can also evoke reflex changes in coronary arterial resistance. Therefore, in 24 chloralose-anesthetized dogs, we used a Gregg cannula and a constant flow preparation to assess left circumflex coronary arterial (LCCA) resistance while stimulating chemically sensitive gastric receptors with capsaicin. Capsaicin (60 micrograms), applied topically to the serosal surface of the stomach, produced significant (P less than 0.05) increases in heart rate (8 +/- 2 bpm), mean arterial pressure (22 +/- 1 mm Hg), LCCA pressure (7 +/- 1 mm Hg), and LCCA resistance (0.30 +/- 0.04 mm Hg/ml/min). These responses were still present after bilateral thoracic vagotomy, but were eliminated by bilateral thoracic splanchnicotomy. alpha-adrenergic blockade eliminated and beta-adrenergic blockade enhanced the increases in LCCA pressure and resistance that were evoked by capsaicin. Thus, stimulating chemically sensitive receptors in the stomach can reflexly increase coronary arterial resistance by alpha-adrenergic vasoconstriction. The possibility exists, therefore, that physiological or pathological events which activate gastric afferents can affect reflexly the coronary circulation.

Exercise function and biochemical properties of β-adrenergic receptors of canine heart

We tested the hypothesis that the biochemical properties of cardiac beta‐adrenergic receptors (βAR), as determined by standard in vitro methods, limit physiological responsiveness of dogs to PAR stimulation during peak exercise. Accordingly, we measured oxygen consumption, left ventricular dP/dt at 40 mmHg developed pressure, heart rate, and cardiac output during maximal dynamic exercise in 13 adult mongrel dogs. These studies were followed by biochemical analysis of PAR in membrane homogenates of left ventricle from the same animals using saturation binding and agonist competition curves for [125I] cyanopindolol. There was a substantial range in both biochemical and physiological variables within this population of normal dogs. However, there were no significant correlations between biochemical descriptors of cardiac PAR and any physiological variables measured during exercise. These results suggest that receptor‐effector coupling and maximal cardiac performance during intense sympathetic stimulation of peak exercise are limited by factors other than the biochemical properties of cardiac PAR.

Molecular regulation of the training response: New techniques to study old questions

Exercise training provides a powerful tool with which to study the adaptive capacity of tissues, organ systems, and organisms. A rich literature from a number of disciplines gives strong evidence that exercise training is perhaps the best chronic physiological stress for the study of human or animal function. It is a stress the allows one to look not only at how individual systems respond but also how they interact in providing an overall response. Although much is known about the adaptations to exercise training, we are only beginning to understand molecular mechanisms that regulate the responses to training. This summary paper will present some basic concepts regarding these molecular mechanisms and their role in the training response in skeletal muscle, followed by some examples of how these new techniques are being used to study old questions.