The Role Of Cardiac Membrane PIP₂ In The Development Of Cardiac Hypertrophy

Phosphatidylinositol-4,5-bisphosphate (PIP₂) is a minor phospholipid component of cell membranes. PIP₂ is enriched in the inner leaflet of the plasma membrane, where it anchors and modulates a number of important signaling proteins and cytoskeleton. PIP₂ is an important second messenger that regulates diversity of cellular activities, such as modulation of endocytosis, exocytosis, cell migration, vesicle trafficking and focal adhesion formation, angiogenesis, gene expression, and activity of ion channels.Cardioactive hormones and other physiologically relevant interventions can modulate cardiac PIP₂. One such modulation, carried by Gq coupled receptors, is expected to hydrolyze membrane PIP₂. However, some published works suggest that the cardiac PIP₂ is not decreased but even increased in the guinea pig heart after stimulated with agonists for Gq coupled receptors such as phenylephrine and endothelin-1. Thus it seems that the hydrolysis in localized fashion does not mean the overall decline of PIP₂ in cell, suggesting that the metabolism of membrane phospholipid is a complicated issue. Stimulation of GPCR can induce hydrolysis of PIP₂ into D-myo-inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). In the downstream signaling conduction, DAG and IP₃-induced Ca²⁺ are able to activate protein kinase C (PKC), which affects a variety of cellular events, including activating expression of some hypertrophy associated genes in cardiomyocytes. It will be interesting to know the relationship between these cellular events and the level of membrane PIP₂.In this study, we measured cardiac PIP₂ level under actions of norepinephrine (NE) and angiotension II (Ang II), which activate Gq/PLC pathway. The relationship between the development of cardiac hypertrophy in cultured cardiomyocytes and hypertrophy animal models and cardiac PIP₂ levels were studied. The possible mechanism for the role of cardiac PIP₂ level in cardiac hypertrophy was also investigated.1 The effects of NE and Ang II on the levels of PIP₂ in cardiomyocytes.Objective: To study the levels of PIP₂ in cultured adult rat cardiomyocytes after challenged with NE and Ang II.Methods:(1)Using thin layer chromatography (TLC) and lipid-protein overlay assay (LPOA) to measure the change of PIP₂ in cardiomyocytes after treated with NE (5μM) and Ang II (3μM)(.2)The effects of NE and Ang II on PIP₂ of cardiomyocytes were studied in the presence of PLC inhibitors (U73122 and U73343), PKC inhibitors (Bis-1 and staurosporine), PI4K inhibitor (wortmannin) and Ca²⁺ chelators (EGTA and BAPTA-am).Results:(1)Activation of GPCR with NE (5μM) or Ang II(3μM) caused ²0% augment of PIP₂ level in primary cultured cardiomyocytes from adult rats.(2)Inhibition of PLC, PKC, PI4K and chelating of Ca²⁺ blocked the effects of NE or Ang II on PIP₂; furthermore, NE or Ang II decreased total PIP₂ level in cardiomyocytes pretreated with BAPTA-am.Conclusion: Although activation of Gq coupled GPCR is expected to hydrolyze membrane PIP₂, it dose not reduce but increase the total PIP₂ level in cardiomyocytes; this increased PIP₂ level may be resulted from an increased synthesis of PIP₂ initiated by signaling molecules of the GPCR pathways, including Ca²⁺.2 Changes of PIP₂ level during the process of cardiac hypertrophyObjective: To study the relationship between changes of PIP₂ level and the development of the cardiac hypertrophy.Methods:(1)PIP₂ level in cardiomyocytes was measured after treated with NE for the time of 20min, 1h, 2h, 24h and 48h, and with Ang II for the time of 2h, 24h, 48h and 72h.(2)The expression level of mRNA forα-MHC and c-fos was measured and used for indications of development of cardiac hypertrophy; the effects of NE, in the absence or presence of wortmannin, on the expression level ofα-MHC and c-fos mRMA were also studied.(3)Western blot was used to study the effect of Ang II on phosphorylation state of Akt in cardiomyocytes.(4)Lipid-protein overlay assay was used to measure the changes of total PIP₂ from rat hearts with hypertrophy induced either with isopreternol or with swimming excise.Results:(1)The total PIP₂ level of cardiac myocytes increased initially when treated with NE or Ang II. However, the PIP₂ level fall back to normal or lower than normal level when treated with NE or Ang II for longer time (48h-72h).(2)NE treatment decreased gene expression ofα-MHC and increased gene expression of c-fos, indication of hyperophy. Pretreatment of wortmannin enhanced the effects of NE onα-MHC but did not affect the effects of NE on c-fos.(3)Western blot study show that Ang II incubation increased the Akt phosphorylation of the cardiomyocytes(.4)The PIP₂ level in the hearts from the Iso-induced but not from the swimming-induced rat cardiac hypertrophic model was significantly decreased.Conclusion: The increased synthesis of membrane PIP₂ level in hearts is a positive response to hypertrophic stimulation, which may underlie the compensatory maintenance of cardiac function in the early stage of cardiac hypertrophy.