AMPK-α1 or AMPK-α2 Deletion in Smooth Muscles Does Not Affect the Hypoxic Ventilatory Response or Systemic Arterial Blood Pressure Regulation During Hypoxia

The hypoxic ventilatory response (HVR) is markedly attenuated by AMPK-α1 deletion conditional on the expression of Cre-recombinase in tyrosine hydroxylase (TH) expressing cells, precipitating marked increases in apnea frequency and duration. It was concluded that ventilatory dysfunction caused by AMPK deficiency was driven by neurogenic mechanisms. However, TH is transiently expressed in other cell types during development, and it is evident that central respiratory depression can also be triggered by myogenic mechanisms that impact blood supply to the brain. We therefore assessed the effect on the HVR and systemic arterial blood pressure of AMPK deletion in vascular smooth muscles. There was no difference in minute ventilation during normoxia. However, increases in minute ventilation during severe hypoxia (8% O2) were, if affected at all, augmented by AMPK-α1 and AMPK-α2 deletion in smooth muscles; despite the fact that hypoxia (8% O2) evoked falls in arterial SpO2 comparable with controls. Surprisingly, these mice exhibited no difference in systolic, diastolic or mean arterial blood pressure during normoxia or hypoxia. We conclude that neither AMPK-α1 nor AMPK-α2 are required in smooth muscle for the regulation of systemic arterial blood pressure during hypoxia, and that AMPK-α1 deficiency does not impact the HVR by myogenic mechanisms

AMPK and the hypoxic ventilatory response: signal integration at an oxygen-sensing nucleus within the brainstem respiratory network?

The capacity for mammals to regulate breathing is critical to match ventilation to changes in oxygen availability and demand, for example during sleep or ascent to altitude. Acute falls in arterial PO2 activate specialised oxygen-sensing chemoreceptors, which relay this information to the cardiorespiratory centre of the brainstem, which then acts to restore blood gas homeostasis. While the role of the carotid bodies as peripheral chemoreceptors is undisputed, it has been proposed that a central oxygen sensor may exist. However, this view remains controversial. The work in this thesis built on recent findings which showed that the AMP-activated protein kinase (AMPK), a ubiquitously expressed cellular energy sensor, within catecholaminergic (tyrosine hydroxylase (TH)-expressing) cells is critical to the proper regulation of breathing during acute hypoxia. It was found that targeted deletion of AMPK within this subpopulation of respiratory neurons of the mouse brainstem led to decreases in ventilation and more frequent and prolonged apnoeas. The fact that this ventilatory deficit was hypoxia-specific and rescued by addition of hypercapnia showed that the capacity for neuronal activation was not affected per se, because central and peripheral chemosensors contributing to the hypercapnic response also express TH. Importantly, the activity of the carotid bodies in response to hypoxia remained fully intact, indicating that the respiratory deficits originated centrally. In light of these findings, the experiments carried out in this thesis further investigated how the deletion of both AMPK catalytic subunits in catecholaminergic cells (TH AMPK-α1/α2 dKO) impacts on hypoxic ventilation and where within the brainstem neuronal activation may be affected. By using whole-body plethysmography, I show that AMPK within the brainstem respiratory network is not only critical for appropriate ventilatory adjustments during acute hypoxia, but also during prolonged periods of low oxygen availability. Following the severe hypoventilation observed at the onset of hypoxia, ventilation remained attenuated relative to controls, even during periods lasting up to an hour. Moreover, some TH AMPK-α1/α2 dKO mice lost all respiratory rhythmogenesis after 20-30min of hypoxia. In those mice where respiratory rhythmogenesis was retained, it was found to be interrupted at regular intervals by high frequency periods of spontaneous apnoeas. Immunohistochemical analyses of the brainstems of TH AMPK-α1/α2 dKO mice revealed a comparable number of catecholaminergic cells relative to controls, supporting the conclusion of a functional deficit within these neurons rather than hypo- or hyperplasia. This was further corroborated by the identification of a specific area within the brainstem that showed significant reductions in immediate early gene expression (cFos) and thus neuronal activation during hypoxia. Importantly, cardiovascular responses to hypoxia were unaltered in TH AMPK-α1/α2 dKO mice relative to controls, indicating that this identified brainstem nucleus may separately control the respiratory and the cardiovascular responses to hypoxia through AMPK-dependent pathways. Finally, directing AMPK deletion to the adrenergic subset of catecholaminergic neurons revealed that the ventilatory deficit originated within the noradrenergic cells of the caudal brainstem, consistent with the location of the identified area of cFos deficiency. In summary, AMPK-dependent signalling is required within a specific group of noradrenergic neurons within the caudal brainstem to ensure appropriate signal integration and transduction during hypoxia in order to appropriately regulate ventilatory adaptations and protect from hypoventilation and respiratory instability. Therefore, AMPK may be a potential new therapeutic target to protect from sleep-disordered breathing associated with metabolic syndrome-related disorders and ascent to altitude

Cardiac function is compromised in patients with elevated blood cobalt levels secondary to metal-on-metal hip implants

Elevated blood cobalt secondary to metal-on-metal (MoM) hip arthroplasties has been shown to be a risk factor for developing cardiovascular complications including cardiomyopathy. Published case reports document cardiomyopathy in patients with blood cobalt levels as low as 13µg/l. Clinical studies have found conflicting evidence of cobalt-induced cardiomyopathy in patients with MoM hips. The extent of cardiovascular injury, measured by global longitudinal strain (GLS), in patients with elevated blood cobalt levels has not previously been examined. Sixteen patients with documented blood cobalt ion levels above 13µg/l were identified and matched with eight patients awaiting hip arthroplasty with no history of cobalt implants. Patients underwent echocardiogram assessment including GLS. Patients with MoM hip arthroplasties had a mean blood cobalt level of 29µg/l compared to 0.01µg/l in the control group. There was no difference or correlation in EF, left ventricular (LV) end systolic dimension, LV end diastolic dimension, fractional shortening, ventricular wall thickness or E/e’ ratio. However, GLS was significantly reduced in patients with MoM hip arthroplasties compared to those without (-15.2% v -18%, (MoM v control) p= 0.0125). Pearson correlation demonstrated that GLS is significantly correlated with blood cobalt level (r= 0.8742, p=0.0009). This study has demonstrated reduced cardiac function in the presence of normal EF as assessed by GLS in patients with elevated cobalt above 13µg/l. As GLS is a more sensitive measure of systolic function than EF, routine echocardiogram assessment including GLS should be performed in all patients with MoM hip arthroplasties and elevated blood cobalt

The LKB1-AMPK-α1 signaling pathway triggers hypoxic pulmonary vasoconstriction downstream of mitochondria

International audienceHypoxic pulmonary vasoconstriction (HPV), which aids ventilation-perfusion matching in the lungs, is triggered by mechanisms intrinsic to pulmonary arterial smooth muscles. The unique sensitivity of these muscles to hypoxia is conferred by mitochondrial cytochrome c oxidase subunit 4 isoform 2, the inhibition of which has been proposed to trigger HPV through increased generation of mitochondrial reactive oxygen species. Contrary to this model, we have shown that the LKB1-AMPK-α1 signaling pathway is critical to HPV. Spectral Doppler ultrasound revealed that deletion of the AMPK-α1 catalytic subunit blocked HPV in mice during mild (8% O2) and severe (5% O2) hypoxia, whereas AMPK-α2 deletion attenuated HPV only during severe hypoxia. By contrast, neither of these genetic manipulations affected serotonin-induced reductions in pulmonary vascular flow. HPV was also attenuated by reduced expression of LKB1, a kinase that activates AMPK during energy stress, but not after deletion of CaMKK2, a kinase that activates AMPK in response to increases in cytoplasmic Ca2+ Fluorescence imaging of acutely isolated pulmonary arterial myocytes revealed that AMPK-α1 or AMPK-α2 deletion did not affect mitochondrial membrane potential during normoxia or hypoxia. However, deletion of AMPK-α1, but not of AMPK-α2, blocked hypoxia from inhibiting KV1.5, the classical "oxygen-sensing" K+ channel in pulmonary arterial myocytes. We conclude that LKB1-AMPK-α1 signaling pathways downstream of mitochondria are critical for the induction of HPV, in a manner also supported by AMPK-α2 during severe hypoxia

Cobalt-induced cardiomyopathy - do circulating cobalt levels matter?

Elevated levels of circulating cobalt ions have been linked with a wide range of systemic complications including neurological, endocrine and cardiovascular symptoms. Case reports of patients with elevated blood cobalt ions have described significant cardiovascular complications including cardiomyopathy. However, correlation between the actual level of circulating cobalt and extent of cardiovascular injury has not previously been performed. This review examines evidence from the literature for a link between elevated blood cobalt levels secondary to metal-on-metal (MoM) hip arthroplasties and cardiomyopathy. Correlation between low, moderate and high blood cobalt with cardiovascular complications has been considered. Elevated blood cobalt at levels over 250µg/l have been shown to be a risk factor for developing systemic complications and published case reports document cardiomyopathy, cardiac transplantation and death in patients with severely elevated blood cobalt ions. However, it is not clear that there is a hard cut off value and cardiac dysfunction may occur at lower levels. Clinical and laboratory research has found conflicting evidence of cobalt induced cardiomyopathy in patients with MoM hips. Further work needs to be done to clarify the link between severely elevated blood cobalt ions and cardiomyopathy

Effects of acute and chronic cobalt treatment on adult rat cardiomyocyte calcium handling

Patients with cobalt-chromium hip arthroplasties display increased cobalt levels in circulation. Elevated cobalt has been linked to cardiomyopathy, yet the mechanisms underlying cobalt-induced pathology remain unknown. Here, we have examined the effects of acute ( Cardiomyocytes were isolated enzymatically from adult male rats (n=9) and treated acutely for 5min, 1h and 24h with 1, 10 and 100µM CoCl2. After loading with Cal520 AM, calcium transients were measured during electrical pacing at 1Hz and sparks were recorded using confocal microscopy. Chronic cobalt effects were measured in cardiac preparations taken from adult male rats (n=8) injected daily with CoCl2(1mg/kg) for 28 days. A concentration and time-dependent decrease in Ca2+ transient amplitude was evident in cobalt-treated cells compared to controls, with 10μM CoCl2 inducing an amplitude reduction of 15.1±5.4% and 30.0±7.2% at 5min and 1h respectively and 100µM resulting in 25.6±0.05% and 58.8±0.09% reduction at 5min and 1h. Spark frequency was increased with 100µM cobalt relative to control, resulting in 55.4±0.9% and 76.8±1.4% increase at 5min and 1h. RyR2 expression in chronic cobalt-treated ventricular tissue was similar to controls, suggesting RyR2 post-translational modification may account for altered spark frequency. This study demonstrates reduced Ca2+ release and increased RyR2 activation in cardiomyocytes treated acutely with CoCl2. Indications suggest RyR2 phosphorylation may be involved in the negative inotropic effects observed

Image_2_AMPK-α1 or AMPK-α2 Deletion in Smooth Muscles Does Not Affect the Hypoxic Ventilatory Response or Systemic Arterial Blood Pressure Regulation During Hypoxia.tif

<p>The hypoxic ventilatory response (HVR) is markedly attenuated by AMPK-α1 deletion conditional on the expression of Cre-recombinase in tyrosine hydroxylase (TH) expressing cells, precipitating marked increases in apnea frequency and duration. It was concluded that ventilatory dysfunction caused by AMPK deficiency was driven by neurogenic mechanisms. However, TH is transiently expressed in other cell types during development, and it is evident that central respiratory depression can also be triggered by myogenic mechanisms that impact blood supply to the brain. We therefore assessed the effect on the HVR and systemic arterial blood pressure of AMPK deletion in vascular smooth muscles. There was no difference in minute ventilation during normoxia. However, increases in minute ventilation during severe hypoxia (8% O₂) were, if affected at all, augmented by AMPK-α1 and AMPK-α2 deletion in smooth muscles; despite the fact that hypoxia (8% O₂) evoked falls in arterial SpO₂ comparable with controls. Surprisingly, these mice exhibited no difference in systolic, diastolic or mean arterial blood pressure during normoxia or hypoxia. We conclude that neither AMPK-α1 nor AMPK-α2 are required in smooth muscle for the regulation of systemic arterial blood pressure during hypoxia, and that AMPK-α1 deficiency does not impact the HVR by myogenic mechanisms.</p

Cardiac function may be compromised in patients with elevated blood cobalt levels secondary to metal-on-metal hip implants

Aims Elevated blood cobalt levels secondary to metal-on-metal (MoM) hip arthroplasties are a suggested risk factor for developing cardiovascular complications including cardiomyopathy. Clinical studies assessing patients with MoM hips using left ventricular ejection fraction (LVEF) have found conflicting evidence of cobalt-induced cardiomyopathy. Global longitudinal strain (GLS) is an echocardiography measurement known to be more sensitive than LVEF when diagnosing early cardiomyopathies. The extent of cardiovascular injury, as measured by GLS, in patients with elevated blood cobalt levels has not previously been examined. Methods A total of 16 patients with documented blood cobalt ion levels above 13 µg/l (13 ppb, 221 nmol/l) were identified from a regional arthroplasty database. They were matched with eight patients awaiting hip arthroplasty. All patients underwent echocardiography, including GLS, investigating potential signs of cardiomyopathy. Results Patients with MoM hip arthroplasties had a mean blood cobalt level of 29 µg/l (495 nmol/l) compared to 0.01 µg/l (0.2 nmol/l) in the control group. GLS readings were available for seven of the MoM cohort, and were significantly lower when compared with controls (-15.5% vs -18% (MoM vs control); p = 0.025)). Pearson correlation demonstrated that GLS significantly correlated with blood cobalt level (r = 0.8521; p &lt; 0.001). However, there were no differences or correlations for other echocardiography measurements, including LVEF (64.3% vs 63.7% (MoM vs control); p = 0.845). Conclusion This study supports the hypothesis that patients with elevated blood cobalt levels above 13 µg/l in the presence of a MoM hip implant may have impaired cardiac function compared to a control group of patients awaiting hip arthroplasty. It is the first study to use the more sensitive parameter of GLS to assess for any cardiac contractile dysfunction in patients with a MoM hip implant and a normal LVEF. Larger studies should be performed to determine the potential of GLS as a predictor of cardiac complications in patients with MoM arthroplasties