Regulation of Pulmonary Arterial Myosin Phosphatase Activity in Neonatal Circulatory Transition and in Hypoxic Pulmonary Hypertension: A Role for CPI-17

Abstract

Neonatal circulatory transition is dependent upon tightly regulated pulmonary circuit relaxation. Persistent pulmonary hypertension of the newborn (PPHN) is characterized by pulmonary arterial myocyte relaxation failure. We examined the effect of short course (72 hour) in vivo normobaric hypoxia in newborn swine on smooth muscle contractile enzyme activity and regulatory phosphoprotein abundance, in tissue homogenates of 2nd to 4th generation pulmonary arteries. Myosin light chain kinase (MLCK) and phosphatase (MLCP) protein contents were unchanged in hypoxic pulmonary arteries compared to controls. MLCP activity increased in normoxic animals from birth to day 3. This was ablated by hypoxia; phosphatase activity, measured as in vitro myosin light chain dephosphorylation, was decreased significantly (P < 0.005) in the hypoxic group. Inhibitory site phosphorylations of MLCP myosin binding subunit at threonines 696 and 850 were similar in both hypoxic and normoxic subjects, suggesting that downregulation of MLCP in hypoxia does not involve this pathway. However, content of regulatory protein CPI-17 (protein kinase C-related phosphatase inhibitor) increased from birth in hypoxic subjects (P < 0.05); active (phosphorylated) CPI-17 protein abundance declined after birth in normals, but increased in hypoxic arteries (P < 0.05). This corresponded with the decrease in phosphatase activity. We speculate that CPI-17 may play a role in myosin phosphatase upregulation during neonatal circulatory transition, and in hypoxic inhibition of pulmonary phosphatase activity in PPHN.

Trophic action of sympathetic nerves reduces arterial smooth muscle Ca2+sensitivity during early post-natal development in rats

“…The observed phenomenon of the trophic modulation of arterial Ca 2+ sensitivity by sympathetic nerves may have important functional consequences; its failure may result in increased contractility and subsequent cardiovascular disorders (Dakshinamurti et al 2005, Noma et al 2006. The agonist-induced augmentation of myofilament Ca 2+ sensitivity seems to be a major mechanism of vascular smooth muscle contraction in the developing organism.…”

Regulation of Cellular Protein Phosphatase-1 (PP1) by Phosphorylation of the CPI-17 Family, C-kinase-activated PP1 Inhibitors

“…Furthermore, fluctuations in CPI-17 signals reportedly occur under pathological conditions, such as hypertension, asthma, inflammation, and diabetes (40 -45). For example, CPI-17 expression and phosphorylation are up-regulated in hypoxia-induced pulmonary hypertension (40). CPI-17 up-regulation is also found in airway smooth muscle during inflammation and in diabetic bladder smooth muscle (41,45).…”

Persistent pulmonary hypertension results in reduced tetralinoleoyl-cardiolipin and mitochondrial complex II + III during the development of right ventricular hypertrophy in the neonatal pig heart

“…Newborn piglets (Ͻ24 h old, n ϭ 4) were killed on the day of arrival from the farm supplier. Newborn piglets (Ͻ24 h old, n ϭ 4) were exposed to 21% (control) or 10% (PPHN) O 2 for 3 days after birth in a normobaric chamber as previously described (8,23,24). Animals were fed ad libitum with an artificial sow milk replacer obtained commercially from Feedrite (Winnipeg, MB, Canada).…”

“…Animals were euthanized by a pentobarbital overdose and exsanguination, and the heart was removed and placed in oxygenated, cold (4°C) Ca 2ϩ -free Krebs-Henseleit physiological buffer [containing (in mM) 112.6 NaCl, 25 NaHCO3, 1.38 NaH2PO4, 4.7 KCl, 2.46 MgSO4, 7 H2O, and 5.56 dextrose; pH 7.4]. RV afterloading was determined by the relative cardiac weight ratio (blotted tissue weight, RV to LV plus septum) to diagnose the development of PPHN, and arterial PO 2 (PaO 2 ) was measured in femoral arterial samples drawn before euthanasia as previously described (8,23,24). For histological analysis, tissues were fixed in 10% neutral buffered formalin for 24 h and embedded in paraffin.…”