CDK5 activity is upregulated in response to hypoxia.

<p>CDK5 was immunoprecipitated from EC after exposure to hypoxia (1% O₂, 5% CO₂ and balance nitrogen) at durations of 6, 12, and 18 hours and incubated with histone H1. Reactions were subjected to SDS-PAGE and probed for phospho-histone H1 as an indirect measure of CDK5 kinase activity. Western blot analysis showed the levels of phospho-histone H1 compared with total H1 levels increased significantly in a hypoxia exposure duration dependent manner compared to normoxia (A). Densitometric analysis demonstrated significant increase in CDK5 activity comparing normoxia versus hypoxia for 18 hours duration (B). The protein levels of CDK5 did not change significantly for all tested time points (6, 12, and 18 hours) comparing normoxic and hypoxic conditions as demonstrated by Western blot (C) and densitometric analysis of the 18 hour time point (D). n = 4 for each group.</p

Hypoxia-induced hyperactivation of XOR is dependent on a phosphorylation event.

<p>XOR activity was measured in lysates from EC exposed to hypoxia (1% O₂, 5% CO₂ and balance nitrogen) or normoxia (21% oxygen, 5% CO₂, and balance nitrogen) for 24 hours followed by treatment with vehicle (DMSO) or alkaline phosphatase (Alk Phos). XOR activity in both normoxic and hypoxic conditions is significantly reduced in cell extracts treated with Alk Phos (A). n = 3–5 for each condition. This hypoxia-induced increase in XOR activity is independent of changes in protein expression as demonstrated by Western blot and densitometric analysis (B). n = 3 for each group.</p

Hypoxia upregulates protein levels of the regulatory subunits p35 and p25 but not CDK5.

<p>Hypoxia augments CDK5 activity without significant increase in protein expression. Cell lysates from EC exposed to hypoxia (1% O_2, 5% CO₂ and balance nitrogen) compared to normoxia for 18 hours have increased protein levels of the CDK5 regulatory subunits p35 and p25 as demonstrated by Western blot (A) and densitometric analysis (B). n = 3–5 for each group. Also shown is a decreased protein level of the cleaved fragment p10, a known inhibitor of CDK5 activity (A and B).</p

CDK5 and its regulatory subunit p35 are necessary for hypoxia induced hyperactivation of XOR activity.

<p>Using siRNA directed against p35/CDK5r1, we demonstrated effective knockdown of p35 by Western blot (inset) compared to the negative control siRNA. XOR activity measured in EC transfected with p35/CDK5r1 siRNA and exposed to hypoxia (1% O₂, 5% CO₂ and balance nitrogen) for 24 hours revealed effective suppression of the upregulation of XOR activity (A). n = 3–5 for each group. In parallel, cell lysates from EC exposed to hypoxia (1% O₂, 5% CO₂ and balance nitrogen) or normoxia for 24 hours in the presence of the CDK5 inhibitor olomoucine (100 μM) or vehicle (DMSO) were measured for XOR activity as described in Methods. XOR activity is significantly elevated after exposure to hypoxia in the cells pretreated with vehicle (DMSO), which is abrogated by olomoucine pretreatment (B). n = 6 for each condition. In parallel experiments, EC were transfected with a plasmid overexpressing the kinase inactive mutant CDK5 (D144N) or an empty vector prior to exposure to hypoxia or normoxia for 24 hours. Effective transfection was demonstrated by Western blot probing for the HA-tag (inset). XOR activity was significantly increased in EC transfected with an empty vector and exposed to hypoxia compared to the dominant negative CDK5 construct (C). n = 3–5 for each group. CDK5 was effectively knocked down in EC with the specific CDK5 siRNA (inset). CDK5 siRNA significantly prevented the upregulation of XOR activity after exposure to hypoxia (1% O₂, 5% CO₂ and balance nitrogen) for 24 hours compared to negative control siRNA (D). n = 3–5 for each group.</p

CDK5 is sufficient to upregulate XOR activity in a cell free reaction.

<p>Recombinant hXOR proteins (wild type T222 versus alanine mutant A222) were incubated in the presence or absence of immunoprecipitated CDK5 with Mg²⁺/ATP and XOR activity was measured using an Amplex red assay. The activity of T222 XOR was increased in the presence of CDK5 and abrogated by olomoucine treatment (A). n = 3–5 for each group. There was no change in XOR activity with the A222 mutant in the presence of CDK5 (B).</p

CDK5 is sufficient to phosphorylate XOR in a cell free reaction.

<p>Human poly-histidine tagged XOR expressed in 293A cells and purified via chromatography was incubated with buffer alone, immunoprecipitated CDK5 with or without olomoucine, and with mouse IgG as a negative control along with Mg²⁺/ATP. Each reaction was resolved on SDS-PAGE and probed with anti-phospho-threonine-proline antibody and anti-XOR antibody. CDK5 was sufficient to phosphorylate XOR at a phospho-threonine-proline site (lane B). The amount of XOR protein in each reaction was uniform as shown on the immunoblot.</p

CDK5 is necessary for hypoxia-induced hyperactivation of XOR in mouse lung.

<p>C57/Bl6 mice were exposed to hypoxia (10% O₂) or room air for 24 hours with designated groups receiving pretreatment with olomoucine (3mg/kg) versus vehicle. Lungs were harvested and homogenized for XOR activity. There was significant hypoxia-induced XOR activation which was dependent on CDK5 activity (A). n = 5 for each group. XOR activation was not dependent on increased protein levels as demonstrated by Western blot and densitometric analysis (B).</p

CDK5 is associated with XOR under normoxic and hypoxic conditions.

<p>Using <i>in silico</i> analysis, we identified a CDK5 consensus motif adjacent to the flavin adenine dinucleotide (FAD) binding domain (A). There was increased XOR phospho-threonine expression in cells exposed to hypoxia compared to normoxia as determined by Western blot analysis (B). n = 4 for each group. CDK5 immunoprecipitated from EC lysates after exposure to hypoxia or normoxia was subjected to SDS-PAGE and probed for XOR and mouse IgG control. Western blot analysis revealed an association in a complex of CDK5 and XOR in both hypoxic and normoxic conditions (C). Densitometric analysis demonstrated no change in XOR/CDK5 protein ratios comparing normoxic and hypoxic conditions (D). n = 3–5 for each condition.</p

Heart Rate Dependence of the Pulmonary Resistance x Compliance (RC) Time and Impact on Right Ventricular Load

<div><p>Background</p><p>The effect of heart rate (HR) and body surface area (BSA) on pulmonary RC time and right ventricular (RV) load is unknown.</p><p>Methods</p><p>To determine the association of HR and BSA with the pulmonary RC time and measures of RV load, we studied three large patient cohorts including subjects with 1) known or suspected pulmonary arterial hypertension (PAH) (n = 1008), 2) pulmonary hypertension due to left heart disease (n = 468), and 3) end-stage heart failure with reduced ejection fraction (n = 150). To corroborate these associations on an individual patient level, we performed an additional analysis using high-fidelity catheters in 22 patients with PAH undergoing right atrial pacing.</p><p>Results</p><p>A faster HR inversely correlated with RC time (p<0.01 for all), suggesting augmented RV pulsatile loading. Lower BSA directly correlated with RC time (p<0.05) although the magnitude of this effect was smaller than for HR. With incremental atrial pacing, cardiac output increased and total pulmonary resistance (TPR) fell. However, effective arterial elastance, its mean resistive component (TPR/heart period; 0.60±0.27 vs. 0.79±0.45;p = 0.048), and its pulsatile component (0.27±0.18 vs 0.39±0.28;p = 0.03) all increased at faster HR.</p><p>Conclusion</p><p>Heart rate and BSA are associated with pulmonary RC time. As heart rate increases, the pulsatile and total load on the RV also increase. This relationship supports a hemodynamic mechanism for adverse effects of tachycardia on the RV.</p></div

Multiple linear regression models for determinants of RC Time in Cohorts A-C.

<p>Multiple linear regression models for determinants of RC Time in Cohorts A-C.</p