We previously demonstrated that cardiac myosin can use 2-deoxy-ATP (dATP) as an energy substrate, that it enhances contraction and relaxation with minimal effect on calcium-handling properties in vitro, and that contractile enhancement occurs with only minor elevation of cellular [dATP]. Here, we report the effect of chronically enhanced dATP concentration on cardiac function using a transgenic mouse that overexpresses the enzyme ribonucleotide reductase (TgRR), which catalyzes the rate-limiting step in de novo deoxyribonucleotide biosynthesis. Hearts from TgRR mice had elevated left ventricular systolic function compared with wild-type (WT) mice, both in vivo and in vitro, without signs of hypertrophy or altered diastolic function. Isolated cardiomyocytes from TgRR mice had enhanced contraction and relaxation, with no change in Ca 2+ transients, suggesting targeted improvement of myofilament function. TgRR hearts had normal ATP and only slightly decreased phosphocreatine levels by 31 P NMR spectroscopy, and they maintained rate responsiveness to dobutamine challenge. These data demonstrate long-term (at least 5-mo) elevation of cardiac [dATP] results in sustained elevation of basal left ventricular performance, with maintained β-adrenergic responsiveness and energetic reserves. Combined with results from previous studies, we conclude that this occurs primarily via enhanced myofilament activation and contraction, with similar or faster ability to relax. The data are sufficiently compelling to consider elevated cardiac [dATP] as a therapeutic option to treat systolic dysfunction.metabolism | inotropy | cross-bridge cycling A wide variety of cardiac pathologies such as myocardial infarct, dilated cardiomyopathy, and congestive heart failure involve reduced systolic function of ventricles that often results from altered ATP-mediated actin-myosin (cross-bridge) cycling (1-4). The schematic shown in Fig. 1 outlines the basic components of this chemomechanical cycle that are critical to understand how cardiomyocytes use energy to develop tension and shortening: (i) ATP bound to detached myosin is hydrolyzed to ADP and inorganic phosphate (Pi); (ii) myosin binds to actin and undergoes the power-stroke associated with Pi release, tension development, and shortening; (iii) ADP is released from myosin; and (iv) ATP binds and precipitates myosin detachment from actin.We (5-8) and others (9-17) have reported that striated muscle myosin can use most naturally occurring nucleotides to support cross-bridge cycling and contraction to varying degrees. Although most are not as effective as ATP, we found that 2-deoxy-ATP (dATP) is more effective than ATP as a substrate for contraction of demembranated cardiac muscle, augmenting both force and shortening at all levels of Ca 2+ -mediated contractile activation (5-8). Our detailed biochemical and mechanical analysis suggests that dATP increases the rates of both myosin binding and product release (steps 2 and 3 in Fig. 1) (6). More recently, we reported that adenoviral overexpres...

Impaired systolic function, resulting from acute injury or congenital defects, leads to cardiac complications and heart failure. Current therapies slow disease progression but do not rescue cardiac function. We previously reported that elevating the cellular 2 deoxy-ATP (dATP) pool in transgenic mice via increased expression of ribonucleotide reductase (RNR), the enzyme that catalyzes deoxy-nucleotide production, increases myosin-actin interaction and enhances cardiac muscle contractility. For the current studies, we initially injected wild-type mice retro-orbitally with a mixture of adeno-associated virus serotype-6 (rAAV6) containing a miniaturized cardiac-specific regulatory cassette (cTnT(455)) composed of enhancer and promotor portions of the human cardiac troponin T gene (TNNT2) ligated to rat cDNAs encoding either the Rrm1 or Rrm2 subunit. Subsequent studies optimized the system by creating a tandem human RRM1-RRM2 cDNA with a P2A self-cleaving peptide site between the subunits. Both rat and human Rrm1/Rrm2 cDNAs resulted in RNR enzyme overexpression exclusively in the heart and led to a significant elevation of left ventricular (LV) function in normal mice and infarcted rats, measured by echocardiography or isolated heart perfusions, without adverse cardiac remodeling. Our study suggests that increasing RNR levels via rAAV-mediated cardiac-specific expression provide a novel gene therapy approach to potentially enhance cardiac systolic function in animal models and patients with heart failure.

Myosin activation is a viable approach to treat systolic heart failure. We previously demonstrated that striated muscle myosin is a promiscuous ATPase that can use most nucleoside triphosphates as energy substrates for contraction. When 2-deoxy ATP (dATP) is used, it acts as a myosin activator, enhancing cross-bridge binding and cycling. In vivo, we have demonstrated that elevated dATP levels increase basal cardiac function and rescues function of infarcted rodent and pig hearts. Here we investigate the molecular mechanism underlying this physiological effect. We show with molecular dynamics simulations that the binding of dADP.Pi (dATP hydrolysis products) to myosin alters the structure and dynamics of the nucleotide binding pocket, myosin cleft conformation, and actin binding sites, which collectively yield a myosin conformation that we predict favors weak, electrostatic binding to actin. In vitro motility assays at high ionic strength were conducted to test this prediction and we found that dATP increased motility. These results highlight alterations to myosin that enhance cross-bridge formation and reveal a potential mechanism that may underlie dATP-induced improvements in cardiac function.

NOWAKOWSKI, S. G., S. J. SWOAP, AND N. J. SANDSTROM. A single bout of torpor in mice protects memory processes. PHYSIOL BEHAV 00(0) 000-000, 2008. -Memory consolidation is the process by which new and labile information is stabilized as long-term memory. Consolidation of spatial memories is thought to involve the transfer of information from the hippocampus to cortical regions. While the hypometabolic and hypothermic state of torpor dramatically changes hippocampal connectivity, little work has considered the functional consequences of these changes. The present study examines the role of a single bout of shallow torpor in the process of memory consolidation in mice. Adult female C57Bl/6NHSD mice were trained on the Morris Water Maze (MWM) task. Immediately following acquisition, the mice were exposed to one of four experimental manipulations for 24 hours: fasted at an ambient temperature of 19°C, fasted at 29°C, allowed free access to food at 19°C, or allowed free access to food at 29°C. Mice fasted at 19°C entered a bout of torpor as assessed by core body temperature while none of the mice in the other conditions did so. Spatial biases were then assessed with a probe trial in the MWM. During the probe trial, mice that had entered torpor and mice that were fed at 29°C spent twice as much time in the prior target platform location than mice that were fed at 19°C and those that were fasted at 29°C. These findings demonstrate that, while food restriction or cool ambient temperature independently disrupt memory processes, together they cause physiological changes including the induction of a state of torpor that results in functional preservation of the memory process.

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