D. Kuppuswamy - Academia.edu (original) (raw)

Papers by D. Kuppuswamy

Molecular and Cellular Biology, 1999

Src family kinases are implicated in cellular proliferation and transformation. Terminally differ... more Src family kinases are implicated in cellular proliferation and transformation. Terminally differentiated myocytes have lost the ability to proliferate, indicating the existence of a down-regulatory mechanism(s) for these mitogenic kinases. Here we show that feline cardiomyocyte lysate contains thermostable components that inhibit c-Src kinase in vitro. This inhibitory activity, present predominantly in heart tissue, involves two components acting combinatorially. After purification by sequential chromatography, one component was identified by mass and nuclear magnetic resonance spectroscopies as 5′-AMP, while the other was identified by peptide sequencing as a small heat shock protein (sHSP). 5′-AMP and to a lesser extent 5′-ADP inhibit c-Src when combined with either HSP-27 or HSP-32. Other HSPs, including αB-crystallin, HSP-70, and HSP-90, did not exhibit this effect. The inhibition, observed preferentially on Src family kinases and independent of the Src tyrosine phosphorylation...

Journal of cardiovascular pharmacology, Jan 14, 2015

Connective tissue growth factor (CTGF) is a fibrogenic cytokine that promotes fibrosis in various... more Connective tissue growth factor (CTGF) is a fibrogenic cytokine that promotes fibrosis in various organs. In the heart, both cardiomyocytes (CM) and cardiac fibroblasts (CFb) have been reported as a source of CTGF expression, aiding cardiac fibrosis. Since the mammalian target of rapamycin (mTOR) forms two distinct complexes, mTORC1 and mTORC2, and plays a central role in integrating biochemical signals for protein synthesis and cellular homeostasis, we explored its role in CTGF expression in adult feline CM. CM were stimulated with 10 μM phenylephrine (PE), 200 nM angiotensin (Ang) or 100 nM insulin for 24 h. PE and Ang, but not insulin, caused an increase in CTGF mRNA expression with the highest expression observed with PE. Inhibition of mTOR with torin1 but not rapamycin significantly enhanced PE-stimulated CTGF expression. Furthermore, silencing of Raptor and Rictor using shRNA adenoviral vectors to suppress mTORC1 and mTORC2, respectively, or blocking phosphatidylinositol 3-kin...

PloS one, 2015

Reactive cardiac fibrosis resulting from chronic pressure overload (PO) compromises ventricular f... more Reactive cardiac fibrosis resulting from chronic pressure overload (PO) compromises ventricular function and contributes to congestive heart failure. We explored whether nonreceptor tyrosine kinases (NTKs) play a key role in fibrosis by activating cardiac fibroblasts (CFb), and could potentially serve as a target to reduce PO-induced cardiac fibrosis. Our studies were carried out in PO mouse myocardium induced by transverse aortic constriction (TAC). Administration of a tyrosine kinase inhibitor, dasatinib, via an intraperitoneally implanted mini-osmotic pump at 0.44 mg/kg/day reduced PO-induced accumulation of extracellular matrix (ECM) proteins and improved left ventricular geometry and function. Furthermore, dasatinib treatment inhibited NTK activation (primarily Pyk2 and Fak) and reduced the level of FSP1 positive cells in the PO myocardium. In vitro studies using cultured mouse CFb showed that dasatinib treatment at 50 nM reduced: (i) extracellular accumulation of both collagen...

Int. J. Biol. Sci., 2008

Growth, survival and cytoskeletal rearrangement of cardiomyocytes are critical for cardiac hypert... more Growth, survival and cytoskeletal rearrangement of cardiomyocytes are critical for cardiac hypertrophy. Signal transducer and activator of transcription-3 (STAT3) activation is an important cardioprotective factor associated with cardiac hypertrophy. Although STAT3 activation has been reported via signaling through Janus Kinase 2 (JAK2) in several cardiac models of hypertrophy, the importance of other nonreceptor tyrosine kinases (NTKs) has not been explored. Utilizing an in vivo feline right ventricular pressure-overload (RVPO) model of hypertrophy, we demonstrate that in 48 h pressure-overload (PO) myocardium, STAT3 becomes phosphorylated and redistributed to detergent-insoluble fractions with no accompanying JAK2 activation. PO also caused increased levels of phosphorylated STAT3 in both cytoplasmic and nuclear fractions. To investigate the role of other NTKs, we used our established in vitro cell culture model of hypertrophy where adult feline cardiomyocytes are embedded three-dimensionally (3D) in type-I collagen and stimulated with an integrin binding peptide containing an Arg-Gly-Asp (RGD) motif that we have previously shown to recapitulate the focal adhesion complex (FAC) formation of 48 h RVPO. RGD stimulation of adult cardiomyocytes in vitro caused both STAT3 redistribution and activation that were accompanied by the activation and redistribution of c-Src and the TEC family kinase, BMX, but not JAK2. However, infection with dominant negative c-Src adenovirus was unable to block RGD-stimulated changes on either STAT3 or BMX. Further analysis in vivo in 48 h PO myocardium showed the presence of both STAT3 and BMX in the detergent-insoluble fraction with their complex formation and phosphorylation. Therefore, these studies indicate a novel mechanism of BMX-mediated STAT3 activation within a PO model of cardiac hypertrophy that might contribute to cardiomyocyte growth and survival.

The FASEB Journal, 2009

Identifying the molecular mechanisms activated in compensatory hypertrophy and absent during deco... more Identifying the molecular mechanisms activated in compensatory hypertrophy and absent during decompensation will provide molecular targets for prevention of heart failure. We have previously shown enhanced ubiquitination (Ub) during the early growth period of pressure overload (PO) hypertrophy near intercalated discs of cardiomyocytes, where integrins are important for mechanotransduction. In this study, we tested the role of integrins upstream of Ub, whether enhanced Ub contributes to survival signaling in early PO, and if loss of this mechanism could lead to decreased ventricular function. The study used a beta(3) integrin (-/-) mouse and a wild-type mouse as a control for in vivo PO by transverse aortic constriction (TAC) and for cultured cardiomyocytes in vitro, stimulated with the integrin-activating peptide RGD. We demonstrate beta(3) integrin mediates transient Ub of targeted proteins during PO hypertrophy, which is necessary for cardiomyocyte survival and to maintain ventricular function. Prosurvival signaling proceeds by initiation of NF-kappaB transcription of the E3 ligase, cIAP1. In PO beta(3)(-/-) mice, absence of this mechanism correlates with increased TUNEL staining and decreased ventricular mass and function by 4 wk. This is the first study to show that a beta(3) integrin/Ub/NF-kappaB pathway contributes to compensatory hypertrophic growth.

PLoS ONE, 2012

The adhesion receptor b3 integrin regulates diverse cellular functions in various tissues. As b3 ... more The adhesion receptor b3 integrin regulates diverse cellular functions in various tissues. As b3 integrin has been implicated in extracellular matrix (ECM) remodeling, we sought to explore the role of b3 integrin in cardiac fibrosis by using wild type (WT) and b3 integrin null (b32/2) mice for in vivo pressure overload (PO) and in vitro primary cardiac fibroblast phenotypic studies. Compared to WT mice, b32/2 mice upon pressure overload hypertrophy for 4 wk by transverse aortic constriction (TAC) showed a substantially reduced accumulation of interstitial fibronectin and collagen. Moreover, pressure overloaded LV from b32/2 mice exhibited reduced levels of both fibroblast proliferation and fibroblast-specific protein-1 (FSP1) expression in early time points of PO. To test if the observed impairment of ECM accumulation in b32/2 mice was due to compromised cardiac fibroblast function, we analyzed primary cardiac fibroblasts from WT and b32/2 mice for adhesion to ECM proteins, cell spreading, proliferation, and migration in response to platelet derived growth factor-BB (PDGF, a growth factor known to promote fibrosis) stimulation. Our results showed that b32/2 cardiac fibroblasts exhibited a significant reduction in cell-matrix adhesion, cell spreading, proliferation and migration. In addition, the activation of PDGF receptor associated tyrosine kinase and non-receptor tyrosine kinase Pyk2, upon PDGF stimulation were impaired in b32/2 cells. Adenoviral expression of a dominant negative form of Pyk2 (Y402F) resulted in reduced accumulation of fibronectin. These results indicate that b3 integrin-mediated Pyk2 signaling in cardiac fibroblasts plays a critical role in PO-induced cardiac fibrosis.

Journal of Molecular and Cellular Cardiology, 2003

In pressure-overloaded myocardium, our recent study demonstrated cytoskeletal assembly of c-Src a... more In pressure-overloaded myocardium, our recent study demonstrated cytoskeletal assembly of c-Src and other signaling proteins which was partially mimicked in vitro using adult feline cardiomyocytes embedded in three-dimensional (3D) collagen matrix and stimulated with an integrin-binding Arg-Gly-Asp (RGD) peptide. In the present study, we improved this model further to activate c-Src and obtain a full assembly of the focal adhesion complex (FAC), and characterized c-Src localization and integrin subtype(s) involved. RGD dose response experiments revealed that c-Src activation occurs subsequent to its cytoskeletal recruitment and is accompanied by p130Cas cytoskeletal binding and focal adhesion kinase (FAK) Tyr925 phosphorylation. When cardiomyocytes expressing hexahistidine-tagged c-Src via adenoviral gene delivery were used for RGD stimulation, the expressed c-Src exhibited relocation: (i) biochemical analysis revealed c-Src movement from the detergent-soluble to the -insoluble cytoskeletal fraction and (ii) confocal microscopic analysis showed c-Src movement from a nuclear/perinuclear to a sarcolemmal region. RGD treatment also caused sarcolemmal co-localization of FAK and vinculin. Characterization of integrin subtypes revealed that beta3, but not beta1, integrin plays a predominant role: (i) expression of cytoplasmic domain of beta1A integrin did not affect the RGD-stimulated FAC formation and (ii) both pressure-overloaded myocardium and RGD-stimulated cardiomyocytes exhibited phosphorylation of beta3 integrin at Tyr773/785 sites but not beta1 integrin at Thr788/789 sites. Together these data indicate that RGD treatment in cardiomyocytes causes beta3 integrin activation and c-Src sarcolemmal localization, that subsequent c-Src activation is accompanied by p130Cas binding and FAK Tyr925 phosphorylation, and that these events might be crucial for growth and remodeling of hypertrophying adult cardiomyocytes.

Journal of Molecular and Cellular Cardiology, 2006

Ubiquitin conjugation of proteins is critical for cell homeostasis and contributes to both cell s... more Ubiquitin conjugation of proteins is critical for cell homeostasis and contributes to both cell survival and death. Here we studied ubiquitination of proteins in pressure overloaded (PO) myocardium in the context of cardiomyocyte survival. Analysis using a feline right ventricular pressure overload (RVPO) model revealed a robust and transient increase in ubiquitination of proteins present in the Triton X-100-insoluble fraction in 24 to 48 h PO myocardium, and confocal micrographs indicate this increase in ubiquitination occurs subsarcolemmaly near the intercalated disc area of cardiomyocytes. The ubiquitination was accompanied by changes in E3 ligases including Cbl, E6AP, Mdm2 and cIAP in the same period of PO, although atrophy-related E3 ligases, MuRF1 and MuRF3 were unaltered. Furthermore, Cbl displayed a substantial increase in both levels of expression and tyrosine phosphorylation in 48 h PO myocardium. Confocal studies revealed enrichment of Cbl at the intercalated discs of 48 h PO cardiomyocytes, as evidenced by its colocalization with N-cadherin. Although apoptosis was observed in 48 h PO myocardium by TUNEL staining, cardiomyocytes showing ubiquitin staining were not positive for TUNEL staining. Furthermore, 48 h PO resulted in the phosphorylation of inhibitor of nuclear factor kappa B (IκB), suggesting its ubiquitin-mediated degradation and the nuclear localization of NFκB for the expression of specific cell survival factors such as cIAPs. Together these data indicate that increased levels of E3 ligases that regulate cell homeostasis and promote cell survival could ubiquitinate multiple cytoskeletal protein targets and that these events that occur during the early phase of PO may contribute to both cardiomyocyte survival and hypertrophy.

Journal of Cardiovascular Pharmacology, 2010

Although cardiac hypertrophy initially ensues as a compensatory mechanism it often culminates in ... more Although cardiac hypertrophy initially ensues as a compensatory mechanism it often culminates in congestive heart failure. Based on our earlier studies that calpain and β3 integrin play cell death and survival roles, respectively during pressure-overload (PO) hypertrophy, we investigated if the loss of β3 integrin signaling is a potential mechanism for calpain-mediated cardiomyocyte death during PO. β3 Integrin knockout (β3−/−) and wild type (WT) mice were used to induce either moderate or severe PO in vivo for short-term (72 h) and long-term (4 wk) by transverse aortic constriction (TAC). Whereas WT mice showed no changes during moderate PO at both time points, β3−/− mice exhibited both enrichment of the μ-calpain isoform and programmed cell death of cardiomyocytes following 4 wk PO. However, with severe PO that caused increased mortality in both mice groups, cell death was observed in WT mice also. To study calpain's role, calpeptin, a specific inhibitor of calpain, was administered via osmotic mini pump at 2.5 mg/kg/day beginning three days prior to moderate TAC or sham surgery. Calpeptin administration blocked both calpain enrichment and myocardial cell death in the 4 wk PO β3−/− mice. Since β3 integrin contributes to cardioprotective signaling, these studies indicate that the loss of specific integrin function could be a key mechanism for calpain-mediated programmed cell death of cardiomyocytes in PO myocardium.

Journal of Cardiac Failure, 2007

Journal of Biological Chemistry, 2002

p70S6 kinase (S6K1) plays a pivotal role in hypertrophic cardiac growth via ribosomal biogenesis.... more p70S6 kinase (S6K1) plays a pivotal role in hypertrophic cardiac growth via ribosomal biogenesis. In pressure-overloaded myocardium, we show S6K1 activation accompanied by activation of protein kinase C (PKC), c-Raf, and mitogen-activated protein kinases (MAPKs). To explore the importance of the c-Raf/MAPK kinase (MEK)/MAPK pathway, we stimulated adult feline cardiomyocytes with 12-O-tetradecanoylphorbol-13-acetate (TPA), insulin, or forskolin to activate PKC, phosphatidylinositol-3-OH kinase, or protein kinase A (PKA), respectively. These treatments resulted in S6K1 activation with Thr-389 phosphorylation as well as mammalian target of rapamycin (mTOR) and S6 protein phosphorylation. Thr-421/Ser-424 phosphorylation of S6K1 was observed predominantly in TPA-treated cells. Dominant negative c-Raf expression or a MEK1/2 inhibitor (U0126) treatment showed a profound blocking effect only on the TPA-stimulated phosphorylation of S6K1 and mTOR. Whereas p38 MAPK inhibitors exhibited only partial effect, MAPK-phosphatase-3 expression significantly blocked the TPA-stimulated S6K1 and mTOR phosphorylation. Inhibition of mTOR with rapamycin blocked the Thr-389 but not the Thr-421/Ser-424 phosphorylation of S6K1. Therefore, during PKC activation, the c-Raf/MEK/extracellular signal-regulated kinase-1/2 (ERK1/2) pathway mediates both the Thr-421/ Ser-424 and the Thr-389 phosphorylation in an mTORindependent and-dependent manner, respectively. Together, our in vivo and in vitro studies indicate that the PKC/c-Raf/MEK/ERK pathway plays a major role in the S6K1 activation in hypertrophic cardiac growth.

Current Molecular Medicine, 2003

The major research interests in our laboratory are the regulation of replication of mammalian chr... more The major research interests in our laboratory are the regulation of replication of mammalian chromosomes, and the way in which chromosome architecture influences replication and transcription processes. As a model system, we have developed a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) that has amplified a 240 kb sequence from the dihydrofolate reductase (DHFR) locus-1,000 times. The multiple DHFR amplicons are located in three stable, abnormally-banding chromosome regions. Previous in vivo labelling studies on synchronized cells showed that replication initiates preferentially somewhere within a 28 kb zone mapping just downstream from the DHFR gene. This result suggested the presence of a fixed origin in this region, analogous to the origins of microorganisms. However, more recent two-dimensional replicon mapping studies suggest that replication initiates at multiple random sites scattered over the entire 28 kb zone. A model is suggested in which interaction of a-acting factor with a &-acting origin induces melting of a large chromosomal domain prior to the actual polymerization of DNA chains: once the region is melted, DNA chains then initiate anywhere within the single-stranded region.

Circulation Research, 2002

Cellular Signalling, 2013

Our earlier work showed that mammalian target of rapamycin (mTOR) is essential to the development... more Our earlier work showed that mammalian target of rapamycin (mTOR) is essential to the development of various hypertrophic responses, including cardiomyocyte survival. mTOR forms two independent complexes, mTORC1 and mTORC2, by associating with common and distinct cellular proteins. Both complexes are sensitive to a pharmacological inhibitor, torin1, although only mTORC1 is inhibited by rapamycin. Since mTORC2 is known to mediate the activation of a prosurvival kinase, Akt, we analyzed whether mTORC2 directly mediates Akt activation or whether it requires the participation of another prosurvival kinase, PKCε (epsilon isoform of protein kinase-C). Our studies reveal that treatment of adult feline cardiomyocytes in vitro with insulin results in Akt phosphorylation at S473 for its activation which could be augmented with rapamycin but blocked by torin1. Silencing the expression of Rictor (rapamycin-insensitive companion of mTOR), an mTORC2 component, with a sh-RNA in cardiomyocytes lowers both insulin-stimulated Akt and PKCε phosphorylation. Furthermore, phosphorylation of PKCε and Akt at the critical S729 and S473 sites respectively was blocked by torin1 or Rictor knockdown but not by rapamycin, indicating that the phosphorylation at these specific sites occurs downstream of mTORC2. Additionally, expression of DN-PKCε significantly lowered the insulin-stimulated Akt S473 phosphorylation, indicating an upstream role for PKCε in the Akt activation. Biochemical analyses also revealed that PKCε was part of Rictor but not Raptor (a binding partner and component of mTORC1). Together, these studies demonstrate that mTORC2 mediates prosurvival signaling in adult cardiomyocytes where PKCε functions downstream of mTORC2 leading to Akt activation.

American Journal of Physiology-Heart and Circulatory Physiology, 2009

Cardiac pathology, such as myocardial infarction (MI), activates intracellular proteases that oft... more Cardiac pathology, such as myocardial infarction (MI), activates intracellular proteases that often trigger programmed cell death and contribute to maladaptive changes in myocardial structure and function. To test whether inhibition of calpain, a Ca2+-dependent cysteine protease, would prevent these changes, we used a mouse MI model. Calpeptin, an aldehydic inhibitor of calpain, was intravenously administered at 0.5 mg/kg body wt before MI induction and then at the same dose subcutaneously once per day. Both calpeptin-treated ( n = 6) and untreated ( n = 6) MI mice were used to study changes in myocardial structure and function after 4 days of MI, where end-diastolic volume (EDV) and left ventricular ejection fraction (EF) were measured by echocardiography. Calpain activation and programmed cell death were measured by immunohistochemistry, Western blotting, and TdT-mediated dUTP nick-end labeling (TUNEL). In MI mice, calpeptin treatment resulted in a significant improvement in EF [E...

American Journal of Physiology-Heart and Circulatory Physiology, 2008

Calpain activation is linked to the cleavage of several cytoskeletal proteins and could be an imp... more Calpain activation is linked to the cleavage of several cytoskeletal proteins and could be an important contributor to the loss of cardiomyocytes and contractile dysfunction during cardiac pressure overload (PO). Using a feline right ventricular (RV) PO model, we analyzed calpain activation during the early compensatory period of cardiac hypertrophy. Calpain enrichment and its increased activity with a reduced calpastatin level were observed in 24- to 48-h-PO myocardium, and these changes returned to basal level by 1 wk of PO. Histochemical studies in 24-h-PO myocardium revealed the presence of TdT-mediated dUTP nick-end label (TUNEL)-positive cardiomyocytes, which exhibited enrichment of calpain and gelsolin. Biochemical studies showed an increase in histone H2B phosphorylation and cytoskeletal binding and cleavage of gelsolin, which indicate programmed cardiomyocyte cell death. To test whether calpain inhibition could prevent these changes, we administered calpeptin (0.6 mg/kg iv)...

Journal of Biological Chemistry, 2003

PLoS ONE, 2010

The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The ... more The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While a-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of b-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, b-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO) model, we measured the level and distribution of b-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of b-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET) or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin) of b-actin. To determine the localization and dynamics of b-actin, we adenovirally expressed GFP-tagged b-actin in isolated adult cardiomyocytes. The ectopically expressed b-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP) measurements of b-actin dynamics revealed that b-actin at the Z-discs is constantly being exchanged with b-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while b-actin overexpression improved cardiomyocyte contractility, immunoneutralization of b-actin resulted in a reduced contractility suggesting that b-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of b-actin in the adult cardiomyocyte and reinforce its usefulness in measuring cardiac cytoskeletal rearrangement during hypertrophic stimulation.