Rationale/ Hypothesis: Reactive oxygen species, including lipid hydroperoxides, play a critical role as second messengers in many physiological processes in the body. Heightened reactive oxygen species production at the time of birth imposes an oxidative stress upon the lung, which may trigger postnatal alveologenesis and physiological lung cell apoptosis in the neonatal rat. Methods: Neonatal rats were subcutaneously injected daily with vehicle (corn oil) or diphenyl-phenyl diamine for the first 6 days of life to study alveologenesis and physiological lung cell apoptosis. Add-back experiments were conducted with a prototypic lipid hydroperoxide, t-butyl hydroperoxide. Main Results: Treatment with diphenyl-phenyl diamine resulted in parenchymal thickening, reduced numbers of secondary crests and enlarged air spaces, all consistent with the inhibition of alveologenesis and reduced physiological lung cell apoptosis. Conclusion: Following an oxidative stress at birth, lipid hydroperoxide formation triggers postnatal alveologenesis and physiological lung cell apoptosis in the neonatal rat.

Rationale/ Hypothesis: Reactive oxygen species, including lipid hydroperoxides, play a critical role as second messengers in many physiological processes in the body. Heightened reactive oxygen species production at the time of birth imposes an oxidative stress upon the lung, which may trigger postnatal alveologenesis and physiological lung cell apoptosis in the neonatal rat. Methods: Neonatal rats were subcutaneously injected daily with vehicle (corn oil) or diphenyl-phenyl diamine for the first 6 days of life to study alveologenesis and physiological lung cell apoptosis. Add-back experiments were conducted with a prototypic lipid hydroperoxide, t-butyl hydroperoxide. Main Results: Treatment with diphenyl-phenyl diamine resulted in parenchymal thickening, reduced numbers of secondary crests and enlarged air spaces, all consistent with the inhibition of alveologenesis and reduced physiological lung cell apoptosis. Conclusion: Following an oxidative stress at birth, lipid hydroperoxide formation triggers postnatal alveologenesis and physiological lung cell apoptosis in the neonatal rat.

Irx5 is a homeodomain transcription factor that negatively regulates cardiac fast transient outward K+ currents (Ito,f) via the KV4.2 gene and is thereby a major determinant of the transmural repolarization gradient. While Ito,f is invariably reduced in heart disease and changes in Ito,f can modulate both cardiac contractility and hypertrophy, less is known about a functional role of Irx5, and its relationship with Ito,f, in the normal and diseased heart. Here I show that Irx5 plays crucial roles in the regulation of cardiac contractility and proper adaptive hypertrophy. Specifically, Irx5-deficient (Irx5-/-) hearts had reduced cardiac contractility and lacked the normal regional difference in excitation-contraction with decreased action potential duration, Ca2+ transients and myocyte shortening in sub-endocardial, but not sub-epicardial, myocytes. In addition, Irx5-/- mice showed less cardiac hypertrophy, but increased interstitial fibrosis and greater contractility impairment following pressure overload. A defect in hypertrophic responses in Irx5-/- myocardium was confirmed in cultured neonatal mouse ventricular myocytes, exposed to norepinephrine while being restored with Irx5 replacement. Interestingly, studies using mice virtually lacking Ito,f (i.e. KV4.2-deficient) showed that reduced contractility in Irx5-/- mice was completely restored by loss of KV4.2, whereas hypertrophic responses to pressure-overload in hearts remained impaired when both Irx5 and Ito,f were absent. These findings suggest that Irx5 regulates cardiac contractility in an Ito,f-dependent manner while affecting hypertrophy independent of Ito,f. On the other hand, Irx5-ablation attenuated calcineurin (Cn)-induced hypertrophy in hearts and cultured cardiomyocytes, suggesting that the effect of Irx5 on hypertrophy involves the Cn-NFAT signalling cascade. Biochemical assessments further revealed that Irx5 can positively mediate Cn-NFAT activities as well as Nfatc3 and Gata4 expression, and interacts with Nfatc3 and Gata4, suggesting the formation of a transcription complex for hypertrophic gene regulation. Taken together, these studies have identified Irx5 as a vital cardiac transcription factor, important for contractile function of the heart by regulating Ito,f, and compensatory hypertrophic response to biomechanical stress in the heart by affecting the Cn-NFAT (and Gata4) signaling pathway.

A dominant ENU screen produced mouse line 7238 with large platelets. Sequence capture and Next Generation sequencing identified a mutation in Myh9 at Q1443L [1]. Mice were tested for aspects of MYH9-Related Disease (MYH9RD), a rare human condition caused by mutations within MYH9; macrothrombocytopenia and neutrophil inclusions are found in almost all cases, while deafness, cataracts and renal disease have variable penetrance and severity. Myh9Q1443L/+ and Myh9Q1443L/Q1443L animals have neutrophil inclusions [1] and increased cataracts at 2, 6 and 12 months; Myh9Q1443L/Q1443L animals at 12 months have changes in kidney output [2]. Immunofluoresence showed changes in protein expression in glomeruli at two months. This is the first ENU mouse model identified by a sequence capture mechanism, and the first mouse line to produce a point mutation within the Myh9 gene [1,2]. This mouse models MYH9RD, and is an invaluable tool to understand the role of this protein, and to determine mechanisms underlying this disease.

Rationale: Activation of the platelet-derived growth factor receptors-α and -β (PDGF-Rα and -Rβ) is critical in the formation of secondary crests/septa during alveologenesis, and its regulation has been found to be disrupted in rat lung injury models. Objective: To determine whether the process of secondary septation can occur after transient pharmacologic inhibition of PDGF-R action during postnatal days (P)1 – 7 in rats. Hypothesis: The initial process of secondary crest formation is time-limited and, if missed, will result in a permanent loss of alveoli. Methods: Imatinib mesylate, a PDGF-R inhibitor, was injected intraperitoneally from P1 – 7. Pups were sacrificed on P2, 4, 8, 14, 28 and 65 for studies of alveolar development. Main results: The injection of imatinib inhibited PDGF-R action, resulting in a permanent decrease in alveolar number in treated rats. Conclusions: Inhibition of secondary septation during the first 7 days of life resulted in a decrease in alveolar number lasting into early adult life. This is consistent with a critical time window for secondary septation, which, if disrupted, results in long-term adverse effects on lung development.

Rationale: Activation of the platelet-derived growth factor receptors-α and -β (PDGF-Rα and -Rβ) is critical in the formation of secondary crests/septa during alveologenesis, and its regulation has been found to be disrupted in rat lung injury models. Objective: To determine whether the process of secondary septation can occur after transient pharmacologic inhibition of PDGF-R action during postnatal days (P)1 – 7 in rats. Hypothesis: The initial process of secondary crest formation is time-limited and, if missed, will result in a permanent loss of alveoli. Methods: Imatinib mesylate, a PDGF-R inhibitor, was injected intraperitoneally from P1 – 7. Pups were sacrificed on P2, 4, 8, 14, 28 and 65 for studies of alveolar development. Main results: The injection of imatinib inhibited PDGF-R action, resulting in a permanent decrease in alveolar number in treated rats. Conclusions: Inhibition of secondary septation during the first 7 days of life resulted in a decrease in alveolar number lasting into early adult life. This is consistent with a critical time window for secondary septation, which, if disrupted, results in long-term adverse effects on lung development.

A dominant ENU screen produced mouse line 7238 with large platelets. Sequence capture and Next Generation sequencing identified a mutation in Myh9 at Q1443L [1]. Mice were tested for aspects of MYH9-Related Disease (MYH9RD), a rare human condition caused by mutations within MYH9; macrothrombocytopenia and neutrophil inclusions are found in almost all cases, while deafness, cataracts and renal disease have variable penetrance and severity. Myh9Q1443L/+ and Myh9Q1443L/Q1443L animals have neutrophil inclusions [1] and increased cataracts at 2, 6 and 12 months; Myh9Q1443L/Q1443L animals at 12 months have changes in kidney output [2]. Immunofluoresence showed changes in protein expression in glomeruli at two months. This is the first ENU mouse model identified by a sequence capture mechanism, and the first mouse line to produce a point mutation within the Myh9 gene [1,2]. This mouse models MYH9RD, and is an invaluable tool to understand the role of this protein, and to determine mechanisms underlying this disease.

Narcolepsy is a disabling sleep disorder that is characterized by persistent sleepiness, and cataplexy – an involuntary loss of waking muscle tone. Cataplexy and narcolepsy are caused by the loss of hypocretin containing neurons in the hypothalamus. However, it is hypothesized that dopamine is also involved in sleep and motor control and plays a role in cataplexy. This study investigated how manipulating dopamine affected sleep and cataplexy in narcoleptic mice devoid of hypocretin. We used d-amphetamine to increase endogenous dopamine levels and quinpirole (D2 agonist) to agonize D2 receptor sites. Amphetamine promoted wakefulness while decreasing sleep in wild-type mice, but was less effective in narcoleptic mice. Amphetamine also reduced cataplexy as well as sleep attacks (an indicator of sleepiness) in narcoleptic mice. Quinpirole had no effect on sleep or wakefulness; however, it potently increased cataplexy without affecting sleep attacks in narcoleptic mice.

In this thesis I investigated the neural circuits that trigger cataplexy in mice. Specifically, I first addressed the theory that cataplexy is a REM sleep disorder. I then investigated a role for the noradrenergic and dopaminergic systems in murine cataplexy. Finally, I addressed the role of the amygdala in triggering cataplexy. From this work several specific conclusions can be drawn: 1. Cataplexy does not share a common executive mechanism with REM sleep, although the two may share a common mechanism that generates muscle atonia. Muscle tone during REM sleep and cataplexy is similar, however increasing REM sleep pressure does not increase cataplexy and positive affective stimuli that can increase cataplexy tend to decrease REM sleep. 2. Systemic manipulation of dopamine receptors can modulate cataplexy without affecting behavioral state. Specifically, manipulation of D2-like dopamine receptors at specific doses can modulate cataplexy while having no affect on sleep-wake state or sleep attacks, and manipulation of D1-like receptors potently affects sleep-wake state and sleep attacks without affecting cataplexy. 3. Systemic modulation of noradrenergic activity in orexin KO mice is sufficient to modulate cataplexy. Specifically, activation of excitatory α1 receptors reduces the occurrence of cataplexy while blockade of these receptors exacerbates it. 4. Withdrawal of an endogenous α1-mediated noradrenergic drive from motor neurons during wakefulness contributed to the loss of muscle tone during cataplexy. Re-establishing this excitatory drive exogenously alleviated cataplexy-dependant muscle atonia. 5. The amygdala is a critical part of the neural mechanism that triggers cataplexy in orexin KO mice. Ablation of the amygdala resulted in significant decreases in both baseline cataplexy and emotionally-induced cataplexy. The amygdala may trigger cataplexy through direct projections to brainstem areas that regulate muscle atonia.

Narcolepsy is a disabling sleep disorder that is characterized by persistent sleepiness, and cataplexy – an involuntary loss of waking muscle tone. Cataplexy and narcolepsy are caused by the loss of hypocretin containing neurons in the hypothalamus. However, it is hypothesized that dopamine is also involved in sleep and motor control and plays a role in cataplexy. This study investigated how manipulating dopamine affected sleep and cataplexy in narcoleptic mice devoid of hypocretin. We used d-amphetamine to increase endogenous dopamine levels and quinpirole (D2 agonist) to agonize D2 receptor sites. Amphetamine promoted wakefulness while decreasing sleep in wild-type mice, but was less effective in narcoleptic mice. Amphetamine also reduced cataplexy as well as sleep attacks (an indicator of sleepiness) in narcoleptic mice. Quinpirole had no effect on sleep or wakefulness; however, it potently increased cataplexy without affecting sleep attacks in narcoleptic mice.