Soumitra Roy

Calcium signaling phenomena in heart diseases: a perspective.

Ca2+ is a major intracellular messenger and nature has evolved multiple mechanisms to regulate free intracellular (Ca2+)i level in situ. The Ca2+ signal inducing contraction in cardiac muscle originates from two sources. Ca2+ enters the cell through voltage dependent Ca2+ channels. This Ca2+ binds to and activates Ca2+ release channels (ryanodine receptors) of the sarcoplasmic reticulum (SR) through a Ca2+ induced Ca2+ release (CICR) process. Entry of Ca2+ with each contraction requires an equal amount of Ca2+ extrusion within a single heartbeat to maintain Ca2+ homeostasis and to ensure relaxation. Cardiac Ca2+ extrusion mechanisms are mainly contributed by Na+/Ca2+ exchanger and ATP dependent Ca2+ pump (Ca2+-ATPase). These transport systems are important determinants of (Ca2+)i level and cardiac contractility. Altered intracellular Ca2+ handling importantly contributes to impaired contractility in heart failure. Chronic hyperactivity of the β-adrenergic signaling pathway results in PKA-hyperphosphorylation of the cardiac RyR/intracellular Ca2+ release channels. Numerous signaling molecules have been implicated in the development of hypertrophy and failure, including the β-adrenergic receptor, protein kinase C, Gq, and the down stream effectors such as mitogen activated protein kinases pathways, and the Ca2+ regulated phosphatase calcineurin. A number of signaling pathways have now been identified that may be key regulators of changes in myocardial structure and function in response to mutations in structural components of the cardiomyocytes. Myocardial structure and signal transduction are now merging into a common field of research that will lead to a more complete understanding of the molecular mechanisms that underlie heart diseases. Recent progress in molecular cardiology makes it possible to envision a new therapeutic approach to heart failure (HF), targeting key molecules involved in intracellular Ca2+ handling such as RyR, SERCA2a, and PLN. Controlling these molecular functions by different agents have been found to be beneficial in some experimental conditions.

Role of protein kinase C in NADPH oxidase derived O2*(-)-mediated regulation of KV-LVOCC axis under U46619 induced increase in [Ca2+]i in pulmonary smooth muscle cells.

Treatment of bovine pulmonary smooth muscle cells with the TxA(2) mimetic, U46619 stimulated [Ca(2+)](i), which was inhibited upon pretreatment with apocynin (NADPH oxidase inhibitor). Pretreatment with cromakalim (K(V) channel opener) or nifedepine (L-VOCC inhibitor) inhibited U46619 induced increase in [Ca(2+)](i), indicating a role of K(V)-LVOCC axis in this scenario. Neither cromakalim nor nifedepine inhibited U46619 induced increase in NADPH oxidase activity, suggesting that the NADPH oxidase activation is proximal to the K(V)-LVOCC axis in the cells. Pretreatment with calphostin C (PKC inhibitor) markedly reduced U46619 induced increase in NADPH oxidase activity and [Ca(2+)](i) in the cells. Calphostin C pretreatment also markedly reduced p(47phox) phosphorylation and translocation to the membrane and association with p(22phox), a component of Cyt.b(558) of NADPH oxidase in the membrane. Overall, PKC plays an important role in NADPH oxidase derived O(2)(-)-mediated regulation of K(V)-LVOCC axis leading to an increase in [Ca(2+)](i) by U46619 in the cells.

Role of PKCα-p(38)MAPK-G(i)α axis in NADPH oxidase derived O(2)(·-)-mediated activation of cPLA(2) under U46619 stimulation in pulmonary artery smooth muscle cells.

We have recently reported that treatment of bovine pulmonary artery smooth muscle cells with the thromboxane A(2) mimetic, U46619 stimulated NADPH oxidase derived O(2)(·-) level, which subsequently caused marked increase in [Ca(2+)](i)[17]. Herein, we demonstrated that O(2)(·-)-mediated increase in [Ca(2+)](i) stimulates an aprotinin sensitive proteinase activity, which proteolytically activates PKC-α under U46619 treatment to the cells. The activated PKC-α then phosphorylates p(38)MAPK and that subsequently caused G(i)α phosphorylation leading to stimulation of cPLA(2) activity in the cell membrane.

Role of protein kinase C in phospholemman mediated regulation of α2β1 isozyme of Na+/K+-ATPase in caveolae of pulmonary artery smooth muscle cells

We have recently reported that α(2)β(1) and α(1)β(1) isozymes of Na(+)/K(+)-ATPase (NKA) are localized in the caveolae whereas only the α(1)β(1) isozyme of NKA is localized in the non-caveolae fraction of pulmonary artery smooth muscle cell membrane. It is well known that different isoforms of NKA are regulated differentially by PKA and PKC, but the mechanism is not known in the caveolae of pulmonary artery smooth muscle cells. Herein, we examined whether this regulation occurs through phospholemman (PLM) in the caveolae. Our results suggest that PKC mediated phosphorylation of PLM occurs only when it is associated with the α(2) isoform of NKA, whereas phosphorylation of PLM by PKA occurs when it is associated with the α(1) isoform of NKA. To investigate the mechanism of regulation of α(2) isoform of NKA by PKC-mediated phosphorylation of PLM, we have purified PLM from the caveolae and reconstituted into the liposomes. Our result revealed that (i) in the reconstituted liposomes phosphorylated PLM (PKC mediated) stimulate NKA activity, which appears to be due to an increase in the turnover number of the enzyme; (ii) phosphorylated PLM did not change the affinity of the pump for Na(+); and (iii) even after phosphorylation by PKC, PLM still remains associated with the α(2) isoform of NKA.

Role of PKC-α in NF-κB–MT1-MMP–mediated activation of proMMP-2 by TNF-α in pulmonary artery smooth muscle cells

We sought to evaluate the mechanism(s) associated with pro matrix metalloprotease 2 (proMMP-2) activation in bovine pulmonary artery smooth muscle cells. Preincubation of cells with anti-TNFR1 antibody prevented tumour necrosis factor-α (TNF-α)-induced proMMP-2 activation and increase in membrane type 1 matrix metalloprotease (MT1-MMP) expression as well as inhibition of tissue inhibitor of metalloproteinase 2 (TIMP-2) expression, indicating the role of TNFR1 receptor during TNF-α stimulation. Anti-MT1-MMP antibody abrogated proMMP-2 activation by TNF-α-stimulated cell membrane, suggesting the involvement of MT1-MMP in proMMP-2 activation. Induction of MT1-MMP expression in response to TNF-α occurs via activation of nuclear factor (NF)-κB on inhibitory κB kinase (IKK) activation and subsequently phosphorylation/degradation of IκB-α. Inhibition of protein kinase C (PKC)-α activity by Go6976 and PKC-α siRNA prevented TNF-α-induced IKK activity, IκB-α phosphorylation/degradation and NF-κB activation. Inhibition of PKC-α activity also prevented TNF-α-induced MT1-MMP expression and proMMP-2 activation as well as down regulation of TIMP-2 expression. Inhibition of IκB-α phosphorylation by PS-1145, an IKK selective inhibitor, prevented TNF-α-induced increase in MT1-MMP expression and proMMP-2 activation, which although did not alter inhibition of TIMP-2 expression. Overall, we unravelled a hitherto unknown mechanism of the involvement of PKC-α in proMMP-2 activation and inhibition of TIMP-2 expression by NF-κB-MT1-MMP-dependent and -independent pathway, respectively, during TNF-α stimulation in pulmonary artery smooth muscle cells.

Role of PKCα−p38MAPK−Giα axis in peroxynitrite-mediated inhibition of β-adrenergic response in pulmonary artery smooth muscle cells

In the context of cross-talk between transmembrane signaling pathways, we studied the loci within the β-adrenergic receptor/G protein/adenyl cyclase system at which PKC exerts regulatory effects of peroxynitrite (ONOO(-)) on isoproterenol stimulated adenyl cyclase activity in pulmonary artery smooth muscle cells. Treatment of the cells with ONOO(-) stimulated PKC-α activity and that subsequently increased p(38)MAPK phosphorylation. Pretreatment with Go6976 (PKC-α inhibitor) and SB203580 (p(38)MAPK inhibitor) eliminated ONOO(-) caused inhibition on isoproterenol stimulated adenyl cyclase activity. Pretreatment with Go6976, but not SB203580, prevented ONOO(-) induced increase in PKC-α activity. Studies using genetic inhibitors of PKC-α (PKC-α siRNA) and p(38)MAPK (p(38)MAPK siRNA) also corroborated the findings obtained with their pharmacological inhibitors in eliminating the attenuation of ONOO(-) effect on isoproterenol stimulated adenyl cyclase activity. This inhibitory effect of ONOO(-) was found to be eliminated upon pretreatment of the cells with pertussis toxin thereby pointing to a G(i) dependent mechanism. This hypothesis was reinforced by G(i)α phosphorylation as well as by the observation of the loss of the ability of Gpp(NH)p (a measure of G(i) mediated response) to stimulate adenyl cyclase activity upon ONOO(-) treatment to the cells. We suggest the existence of a pertussis toxin sensitive G protein (G(i))-mediated mechanism in isoproterenol stimulated adenyl cyclase activity, which is regulated by PKCα-p(38)MAPK axis dependent phosphorylation of its α-subunit (G(i)α) in the pulmonary artery smooth muscle cells.

Activation of proMMP-2 by U46619 occurs via involvement of p 38 MAPK-NFκB-MT1MMP signaling pathway in pulmonary artery smooth muscle cells

Abstract We investigated the mechanism by which TxA2 mimetic, U46619, activates proMMP-2 in bovine pulmonary artery smooth muscle cells. Our study showed that treatment of the cells with U46619 caused an increase in the expression and subsequently activation of proMMP-2 in the cells. Pretreatment with p38MAPK inhibitor, SB203580; and NF-κB inhibitor, Bay11-7082 inhibited the expression and activation of proMMP-2 induced by U46619. U46619 also induced increase in MT1-MMP expression, which was inhibited upon pretreatment with SB203580 and Bay11-7082. U46619 treatment to the cells stimulated p38MAPK activity as well as NF-κB activation by IκB-α phosphorylation, translocation of NF-κBp65 subunit from cytosol to nucleus and subsequently, by increasing its DNA-binding activity. Induction of NF-κB activation seems to be mediated through IKK, as transfection of cells with either IKKα or IKKβ siRNA prevented U46619-induced phosphorylation of IκB-α and NF-κBp65 DNA-binding activity. U46619 treatment to the cells also downregulated the TIMP-2 level. Pretreatment of the cells with SB203580 and Bay11-7082 did not show any discernible change in TIMP-2 level by U46619. Overall, U46619-induced activation of proMMP-2 is mediated via involvement of p38MAPK-NFκB-MT1MMP signaling pathway with concomitant downregulation of TIMP-2 expression in bovine pulmonary artery smooth muscle cells.

Role of TGF-β1 and TNF-α in IL-1β mediated activation of proMMP-9 in pulmonary artery smooth muscle cells: involvement of an aprotinin sensitive protease.

We investigated the role of TGF-β1 and TNF-α in mediating the effect of IL-1β in activating proMMP-9 and proMMP-2, and the involvement of an aprotinin sensitive protease in this scenario in bovine pulmonary artery smooth muscle cells. IL-1β induces TGF-β1 mediated stimulation of 92kDa proMMP-9 and 72kDa proMMP-2 mRNA and protein expression; whereas, the elevated level of TNF-α promotes activation of proMMP-9 and proMMP-2. Interestingly, TNF-α induced activation of proMMP-9 appeared to be mediated via a 43kDa aprotinin sensitive protease. TNF-α inhibited aprotinin and TIMP-1 mRNA and protein expression, which apparently facilitated the proteolytic conversion of proMMP-9 to MMP-9 with the involvement of the aprotinin sensitive protease. The aprotinin sensitive protease did not activate proMMP-2 under IL-1β stimulation, albeit a marked inhibition of TIMP-2 mRNA and protein expression were elicited by TNF-α. Thus, IL-1β induced stimulation of the two progelatinases occurs via different mechanisms.

Identification, purification and partial characterization of a 70 kDa inhibitor protein of Na+/K+-ATPase from cytosol of pulmonary artery smooth muscle

AIMS We sought to identify, purify and partially characterize a protein inhibitor of Na(+)/K(+)-ATPase in cytosol of pulmonary artery smooth muscle. MAIN METHODS (i) By spectrophotometric assay, we identified an inhibitor of Na(+)/K(+)-ATPase in cytosolic fraction of pulmonary artery smooth muscle; (ii) the inhibitor was purified by a combination of ammonium sulfate precipitation, diethylaminoethyl (DEAE) cellulose chromatography, hydroxyapatite chromatography and gel filtration chromatography; (iii) additionally, we have also purified Na(+)/K(+)-ATPase alpha(2)beta(1) and alpha(1)beta(1) isozymes for determining some characteristics of the inhibitor. KEY FINDINGS We identified a novel endogenous protein inhibitor of Na(+)/K(+)-ATPase having an apparent mol mass of approximately 70kDa in the cytosolic fraction of the smooth muscle. The IC(50) value of the inhibitor towards the enzyme was determined to be in the nanomolar range. Important characteristics of the inhibitor are as follows: (i) it showed different affinities toward the alpha(2)beta(1) and alpha(1)beta(1) isozymes of the Na(+)/K(+)-ATPase; (ii) it interacted reversibly to the E(1) site of the enzyme; (iii) the inhibitor blocked the phosphorylated intermediate formation; and (iv) it competitively inhibited the enzyme with respect to ATP. CD studies indicated that the inhibitor causes an alteration of the conformation of the enzyme. The inhibition study also suggested that the DHPC solubilized Na(+)/K(+)-ATPase exists as (alphabeta)(2) diprotomer. SIGNIFICANCE The inhibitor binds to the Na(+)/K(+)-ATPase at a site different from the ouabain binding site. The novelty of the inhibitor is that it acts in an isoform specific manner on the enzyme, where alpha(2) is more sensitive than alpha(1).

Matrix Metalloprotease-2 in the Development and Progression of Cardiovascular Diseases

Matrix metalloproteases (MMPs) are a family of proteolytic enzymes that are regulated by a variety of signals that mediate changes in extracellular matrix (ECM). MMPs are important in the progression of cardiovascular diseases. MMP activation modifies the plaque architecture and may also be involved in the process of plaque rupture. MMPs participate in cardiac remodeling following myocardial infarction and in the development of cardiomyopathy. Among the MMPs, MMP-2 is one of the most ubiquitous members of the MMP family and is expressed in all cells of the heart. In the past two decades, there has been tremendous progress in understanding the role of MMP-2 in the development of cardiovascular pathology. In this review, we discuss the implications of MMP-2 in the progression and development of different types of cardiovascular diseases such as atherosclerosis, myocardial infarction, cardiomyopathy, and heart failure.