The phosphoinositide 3-kinase (PI3K) system has a fundamental

Transcription

1 Loss of PIK Enhances camp-dependent MMP Remodeling of the Myocardial N-Cadherin Adhesion Complexes and Extracellular Matrix in Response to Early Biomechanical Stress Danny Guo, Zamaneh Kassiri, Ratnadeep Basu, Fung L. Chow, Vijay Kandalam, Federico Damilano, Wenbin Liang, Seigo Izumo, Emilio Hirsch, Josef M. Penninger, Peter H. Backx, Gavin Y. Oudit Downloaded from by on November, Rationale: Mechanotransduction and the response to biomechanical stress is a fundamental response in heart disease. Loss of phosphoinositide -kinase (PIK), the isoform linked to G protein coupled receptor signaling, results in increased myocardial contractility, but the response to pressure overload is controversial. Objective: To characterize molecular and cellular responses of the PIK knockout (KO) mice to biomechanical stress. Methods and Results: In response to pressure overload, PIK KO mice deteriorated at an accelerated rate compared with wild-type mice despite increased basal myocardial contractility. These functional responses were associated with compromised phosphorylation of Akt and GSK-. In contrast, isolated single cardiomyocytes from banded PIK KO mice maintained their hypercontractility, suggesting compromised interaction with the extracellular matrix as the primary defect in the banded PIK KO mice. -Adrenergic stimulation increased camp levels with increased phosphorylation of CREB, leading to increased expression of camp-responsive matrix metalloproteinases (MMPs), MMP, MT-MMP, and MMP in cardiomyocytes and cardiofibroblasts. Loss of PIK resulted in increased camp levels with increased expression of MMP, MT-MMP, and MMP and increased MMP activation and collagenase activity in response to biomechanical stress. Selective loss of N-cadherin from the adhesion complexes in the PIK KO mice resulted in reduced cell adhesion. The -blocker propranolol prevented the upregulation of MMPs, whereas MMP inhibition prevented the adverse remodeling with both therapies, preventing the functional deterioration in banded PIK KO mice. In banded wild-type mice, long-term propranolol prevented the adverse remodeling and systolic dysfunction with preservation of the N-cadherin levels. Conclusions: The enhanced propensity to develop heart failure in the PIK KO mice is attributable to a camp-dependent upregulation of MMP expression and activity and disorganization of the N-cadherin/ -catenin cell adhesion complex. -Blocker therapy prevents these changes thereby providing a novel mechanism of action for these drugs. (Circ Res. ;7:75-9.) Key Words: myocardial contractility cardiomyopathy signaling pathways heart failure hypertrophy The phosphoinositide -kinase (PIK) system has a fundamental role in cell signaling and is involved in cell survival and growth and modulates myocardial contractility., In the heart, both PIK 5 and PIK, controls distinct aspects of cardiac structure and function. Mechanotransduction plays a fundamental role in cardiac (and vascular) function and it appears to involve interactions between extracellular matrix and intracellular cytoskeletal proteins via cell adhesion complexes which are modulated by both class I A and I B PIKs.,7 Although PIKs and lipid phosphatases can modulate cytoskeletal interactions, stretch can in turn activate Akt/PKB and GSK- activity in both cardiomyocytes and Langendorff-perfused hearts. In cardiac muscle, cell adhesion and the cardiomyocyte stretch sensor machinery play key roles in the complex mechanism leading to human DCM and associated heart failures. 9 Indeed, dilated cardio- Original received November, 9; resubmission received July, ; revised resubmission received September, ; accepted September,. In August, the average time from submission to first decision for all original research papers submitted to Circulation Research was. days. From the Division of Cardiology, Department of Medicine (D.G., F.L.C., G.Y.O.); Mazankowski Alberta Heart Institute (D.G., Z.K., R.B., F.L.C., V.K., G.Y.O.); and Department of Physiology (Z.K., R.B., V.K., G.Y.O.), University of Alberta, Edmonton, Canada; University of Torino (F.D., E.H.), Italy; Department of Physiology (W.L., P.H.B.), University of Toronto, Canada; Beth Israel Deaconess Medical Center (S.I.), Harvard Medical School, Boston Mass; and Institute of Molecular Biotechnology of the Austrian Academy of Sciences (J.M.P.), Vienna. Correspondence to Gavin Y. Oudit, MD, PhD, FRCP(C), Division of Cardiology, Department of Medicine, Mazankowski Alberta Heart Institute, University of Alberta, Edmonton, Alberta, TG S, Canada. gavin.oudit@ualberta.ca American Heart Association, Inc. Circulation Research is available at DOI:./CIRCRESAHA

2 7 Circulation Research November, Downloaded from by on November, Non-standard Abbreviations and Acronyms ANF BNP DIDA ECM ERK GSK KO LV MHC MMP phospho PIK PIK DN PIK KD PIK KO PLN TIMP aortic banding atrial natriuretic factor B-type natriuretic peptide,5 -dideoxyadenosine extracellular matrix extracellular signal-regulated kinase glycogen synthase kinase knockout left ventricular myosin heavy chain matrix metalloproteinase phosphorylated phosphoinositide -kinase phosphoinositide -kinase dominant negative phosphoinositide -kinase kinase dead phosphoinositide -kinase knockout phospholamban tissue inhibitor of matrix metalloproteinases wild type myopathy in humans is associated with marked remodeling of extracellular matrix and cell adhesion complexes. Loss of p, the catalytic subunit of the PIK complex, leads to enhanced camp generation resulting from a loss of phosphodiesterase activity, resulting in enhanced Ca cycling and attributable primarily to increased phosphorylation (and inhibition) of phospholamban (PLN) and increased sarcoplasmic reticulum Ca stores.,, Using the aortic banding () model, we showed that despite these biochemical changes, PIK KO mice show enhanced susceptibility to early biomechanical stress. Our results provide a crucial link between PIK and the cellular responses to biomechanical stress, with a loss of p resulting in elevated matrix metalloproteinase (MMP) expression and activity, and correlates with a selective loss of N-cadherin from cell adhesion complexes. Importantly, these adverse cellular changes are not observed in the PIK DN and in PIK kinase dead (kda) mutant mice in response to pressure overload. Inhibition of camp levels with propranolol and MMP inhibition provided significant rescue of the dilated cardiomyopathy in banded PIK KO mice. Chronic -blocker therapy also resulted in prevention of adverse myocardial remodeling in wild-type () mice. These results illustrate the importance of cell adhesion and extracellular matrix in the response to biomechanical stress even in the setting of enhanced Ca cycling and increased myocardial contractility. Methods An expanded Methods section is available in the Online Data Supplement at Experimental Animals PIK KO, PIK DN, and PIK KD mice in C57Bl/ background have been described previously.,, All experiments were performed in accordance with institutional guidelines and Canadian Council on Animal Care. Only male mice of to 9 weeks of age and littermate C57Bl/ controls were used. For the propranolol experiments, mice were treated with propranolol in their drinking water (. g/l) to deliver 5 mg/kg per day; propranolol was withdrawn day before the echocardiographic and hemodynamic measurements. The broad-spectrum MMP inhibitor PD79 (Pfizer Inc) was administered orally by daily gavage as described previously. Because of the rapid onset of ventricular dilation in PIK KO mice, PD79 treatment ( mg/kg per day) began days before and continued until mice were euthanized. Aortic Banding The protocol was used as a means of pressure overload and biomechanical stress as described previously., Briefly, the descending aortic arch was accessed via a left thoracotomy and the descending thoracic aorta was surgically constricted to generate a transstenotic pressure gradient of 5 to mm Hg. Histology, Hydroxyproline, and TUNEL Assays For heart morphometry, hearts were arrested with KCl, fixed with % buffered formalin, and embedded in paraffin. Myocardial interstitial fibrosis was determined as collagen volume fraction using confocal microscopy on picrosirius red stained m thick sections pretreated with PMA. Collagen content was also determined using the hydroxyproline assay as previously reported. In situ DNA fragmentation was labeled using the TUNEL assay (ApopTag Plus kit; Oncor, Gaithersburg, Md). Echocardiography and Hemodynamic Measurements Echocardiographic assessments and invasive hemodynamic measurements were carried out as described previously.,, Isolated Cardiomyocyte Contractility Left ventricular (LV) cardiomyocyte contractility was carried out as described previously. Isolation and Culture of Adult Cardiomyocytes and Fibroblasts The protocol used for isolation and culture of adult mouse cardiomyocytes and fibroblasts was modified from O Connell et al (see Online Methods). 5 Measurement of camp Levels The camp levels in isolated adult ventricular cardiomyocytes and LV myocardial tissue were measured using the camp competitive enzyme immunoassay system (GE Healthcare Amersham Biosciences). Cell Adhesion Assay The ECM-based cell adhesion assay was carried out using a colorimetric-based assay (CytoSelect -Well Cell Adhesion Assay; Cell Biolabs Inc). TaqMan Real-Time PCR, Western Blot Analyses, and Membrane Fractionation, Gelatin Zymography, and Collagenase Activity Assay RNA expression levels were quantified with TaqMan RT-PCR using I Prism 77 sequence detection system as described previously (see Online Table I for primers and probes).,7 Western blot analyses, gelatin zymography, and collagenase activity were performed as described previously (see Online Methods).,7 For the in vitro analysis of N-cadherin cleavage, ng of human recombinant N-cadherin Fc Chimera protein (R&D) was incubated with,,, nmol/l MMP (R&D) or MT-MMP (R&D).

3 Guo et al Role of PIK in Biomechanical Stress 77 A B Downloaded from by on November, ANF/S (AU) C PSR E TUNEL Staining week weeks weeks am Week Weeks weeks BNP/S (AU) weeks weeks Confocal Fluorescence Microscopy Formalin-fixed sections were permeabilized with ice-cold methanol for minutes, blocked with goat serum and incubated with primary antibodies (anti N-cadherin [rabbit] and anti -catenin [mouse], :5, Abcam) overnight at C in a humidified chamber. After several washes, Texas red conjugated (for cadherin) or FITCconjugated (for catenin) secondary antibodies were incubated for -minute at room temperature. The sections were evaluated with a Zeiss LSM5 confocal microscope and associated software. Statistical Analysis One-way ANOVA was used to test for overall significance, followed by the Student Newman Keuls test for multiple comparison testing between the various groups. All statistical analyses were performed with the SPSS software (version.). Data are presented as means SEM; n refers to the sample size. Results am Week Weeks Accelerated Development of Ventricular Dilation and Pathological Hypertrophy in PIK KO Mice in Response to Biomechanical Stress In response to pressure overload, mice developed concentric remodeling with increased wall thickness and reduction in chamber size at and weeks following (Figure A). In contrast, LV size dilated rapidly in PIK KO mice characteristic of eccentric remodeling at and weeks CVF (%) week weeks G H I D F apoptotic myocytes (%) week βmhc/s (AU) Hydroxyproline (ug/mg) apoptotic myocytes (%) weeks weeks am Week Weeks weeks Figure. Development of early-onset dilated cardiomyopathy in response to biomechanical stress in PIK KO mice. A and B, Four-chamber trichrome-stained heart sections showing concentric hypertrophy in mice (A) with ventricular dilation and eccentric remodeling in PIK KO mice (B) in response to after. C through F, Increased interstitial fibrosis shown using picrosirius red staining (C, left) and quantification of the collagen volume fraction (CVF) (D) with lack of an increase in apoptosis based on TUNEL staining (E, left) and quantified as percentage of apoptotic nuclei (F, right) (P.; n 5 per group). G through I, Expression profile of hypertrophy disease markers showing early and markedly increased expression of ANF (G), BNP (H), and MHC (I) in banded PIK KO mice compared with mice. n 5 for sham groups and n 7 for groups. AU indicates arbitrary unit. P.5 compared with the group. following as shown by trichrome-stained four-chamber views (Figure B). The early development of dilated cardiomyopathy in the banded PIK KO mice was associated with increased interstitial myocardial fibrosis at weeks after as illustrated by picrosirius red staining (Figure C) and quantified as collagen volume fraction (Figure D). Increased myocardial fibrosis in banded PIK KO mice was confirmed by the biochemical assessment of hydroxyproline content (Figure D) and occurred without a measurable increase in apoptosis (Figure E and F). In banded PIK KO mice, mrna expression profile showed greater myocardial expression of the disease markers, atrial natriuretic factor (ANF) (Figure G), BNP (Figure H), and MHC (Figure I) with morphometric features of hypertrophy (Online Table I). Activation of several signaling pathways is a key mediator of the response to biomechanical stress.,9 The increased phosphorylation of extracellular signal-regulated kinase (ERK)/ (Figure A) and Akt (Figure B) were reduced in banded PIK KO mice compared with banded mice after week of pressure overload. However, following weeks of pressure overload, the degree of ERK/ phosphorylation was equivalent in and PIK KO mice (Figure A). The GSK- system is an important downstream mediator of PIK/Akt signaling,, and consistent with a loss of Akt

4 7 Circulation Research November, A p-erk / ERK / wk wk p-erk / / ERK / (AU) am wk B p-akt (S7) Akt wk wk p-akt (S7) / Akt (AU) C p-gsk-α GSK-α wk wk p-gsk-α / GSK-α (AU) Downloaded from by on November, D E p-gsk-β GSK-β p-fak FAK wk wk wk wk p-gsk-β / GSK-β (AU) p-fak / FAK (AU) Figure. Altered signaling in response to biomechanical stress in PIK KO mice. A and B, Western blot analysis (left) and quantification (right) showing increased phosphorylation of the mitogen-activated protein kinase ERK/ (A) with loss of serine 7 Akt phosphorylation (B) in the banded PIK KO mice. C through E, Western blot analysis (left) and quantification (right) showing preservation of glycogen synthase kinase (GSK- ) (C) and GSK- (D) phosphorylation in banded mice but with a loss of phosphorylation in the banded PIK KO mice, whereas earlier phosphorylation of focal adhesion kinase (FAK) occurs in the banded PIK KO mice (E). n 5 per group. AU indicates arbitrary unit. P.5 compared with the corresponding sham group; #P.5 compared with sham # signaling, phosphorylated (phospho) GSK- (Figure C) and phospho GSK- (Figure D) were not affected initially at week and then showed a drastic reduction at weeks after in PIK KO mice. Interestingly, baseline level of phospho GSK- was significantly greater in PIK KO compared with mice (Figure D). The phosphorylation of focal adhesion kinase (FAK) is widely accepted as a fundamental response in myocardial mechanical stretch and hypertrophy. In the banded PIK KO mice, increased phospho- FAK occurred at an earlier stage compared with mice consistent with an aberrant response to biomechanical stress (Figure E). These results show that PIK KO mice devel-

5 Guo et al Role of PIK in Biomechanical Stress 79 Downloaded from by on November, oped a rapid onset of adverse myocardial remodeling and pathological hypertrophy in response to biomechanical stress with resultant alteration in the activation of mechanosensitive and Akt-dependent signaling cascades. Uncoupling Between In Vivo Myocardial Contractility and Single-Cardiomyocyte Contractility in Pressure-Overloaded PIK KO Mice To examine whether these differences in myocardial remodeling in and PIK KO mice result in functional alteration, we used transthoracic echocardiography to assess heart function. LV size rapidly dilated with a marked and progressive decrement in systolic function at and weeks in PIK KO mice (Figure A through E; Online Table II). In marked contrast, banded mice developed increased wall thickness with reduced ventricular size and preserved systolic function (Figure A through D; Online Table II). We used invasive hemodynamic measurements to further characterize these functional alterations which showed greater elevation in LV end diastolic pressure (Online Table II) and reduced myocardial contractility as assessed by dp/dt max (Figure D; Online Table II) and dp/dt min (Figure E; Online Table II) in banded PIK KO mice. The early and marked decompensation in banded PIK KO mice despite enhanced Ca cycling and increased basal myocardial contractility (Figure ; Online Table II),, suggests that myocardial cell adhesion is compromised. As such, we hypothesize that isolated cardiomyocyte contractility would remain elevated in banded PIK KO mice. Increased phosphorylation of PLN is a critical target of the elevated camp levels in PIK KO mice.,, Western blot analyses confirmed increased serine- phosphorylation of PLN in PIK KO compared with mice, which was preserved in response to pressure overload (Figure F). Analysis of camp levels in the LV from PIK KO mice confirmed elevated basal levels of camp which were maintained in response to week of after (Figure G). Indeed, isolated cardiomyocytes from banded PIK KO mice showed increased cell shortening (Figure H) and rate of contractility (Figure I) compared with cardiomyocytes obtained from banded mice. Whereas ECM-based cell adhesion was intact in the PIK KO cardiomyocytes under baseline conditions (sham), there was a drastic loss of adhesive properties to collagen IV and laminin in response to pressure overload (Figure J). In contrast, cardiomyocyte adhesion from mice remained intact following pressure overload (Figure J). These results show that isolated cardiomyocytes maintain an increased contractility despite the early deterioration in whole heart systolic function providing strong evidence that the primary defect in banded PIK KO mice is a disorganized extracellular matrix and/or cell adhesion. Selective Upregulation of MMP and MT-MMP and MMP Inhibition Mediated Rescue of Pressure-Overloaded PIK KO Mice Adverse remodeling of the extracellular matrix by MMPs is a key determinant of the response to biomechanical stress. MMP (gelatinase A) and MMP (collagenase-) genes contain promoter regions encoding a camp-response element (CRE), which binds CRE binding protein (CREB) and mediates camp-dependent increase in the synthesis of MMP 5 and MMP, whereas MT-MMP expression is also positively regulated by camp. 7 We hypothesize that elevated camp levels in the setting of increased biomechanical stress synergistically increase MMP expression and/or activity in banded PIK KO mice. Indeed, both MMP (Figure A) and MT-MMP (Figure B) myocardial mrna expression increased within week of after and persisted at weeks in PIK KO compared with mice, whereas MMP expression was drastically increased in PIK KO mice at weeks after (Figure C). In contrast, non camp-responsive MMPs such as MMP9 (gelatinase B) (Figure D) and MMP (collagenase-) (data not shown) showed no differential change in expression in response to after. To provide a more definitive connection between camp and MMP expression/activity, we also examined the expression of these MMPs in banded PIK DN mice which also develop an early dilated cardiomyopathy and in banded PIK KD mice, which lack PIK signaling without elevation in camp levels. Consistent with our hypothesis, myocardial expression levels of MMP (Figure A), MT-MMP (Figure B), and MMP (Figure C) were not increased in PIK DN and PIK KD mice at and weeks after. Next, we examined the changes in MMP expression in cultured adult ventricular cardiomyocyte and cardiofibroblast fractions in response to the activation of camp signaling using the -adrenergic agonist, isoproterenol. The levels of camp in cardiomyocytes increase significantly in response to isoproterenol stimulation preventable by -adrenergic blockade using propranolol (5 mol/l) and specific adenylate cyclase inhibition using,5 -dideoxyadenosine (DIDA) ( mol/l; Figure E). Western blot analysis showed an early and marked increase in serine- CREB phosphorylation in cultured cardiomyocytes and cardiofibroblasts (Figure F) and shown quantitatively (Figure G) in response to -adrenergic receptor stimulation. Consistent with activation of CREB, mrna levels of camp-responsive MMPs following hours of stimulation with nmol/l isoproterenol showed that MMP (Figure H), MT-MMP (Figure I), and MMP (Figure J) all showed a significant rise in mrna expression in cultured adult cardiomyocytes. Although the basal expressions of MMP and MT-MMP were greater in cultured adult cardiofibroblasts, MMPs also showed a similar increase in response to -adrenergic stimulation in these cells (Figure H through J). Importantly, the corresponding increase in mrna expression of MMP, MT-MMP, and MMP in both cardiomyocytes and cardiofibroblasts were suppressed by both propranolol and DIDA (Figure H through J). We next assessed for direct biochemical evidence for increased MMP activity in the PIK KO mice in response to pressure overload. Myocardial collagenase activity showed a significant increase at and weeks after in the PIK KO mice compared with banded mice, which was suppressible by the MMP inhibitor PD79 (Figure 5A), whereas gelatin zymography showed selective activation of

6 Circulation Research November, Downloaded from by on November, Figure. Uncoupling between in vivo myocardial contractility and isolated single cardiomyocyte contractility in PIK KO mice in response to early biomechanical stress. A through E, M-mode echocardiograms (A) and in vivo quantitative assessment of heart function showing baseline hypercontractility followed by rapid and marked ventricular dilation (B) and reduction in systolic function based on fractional shortening (C) and invasive hemodynamic measurement of dp/dt max (D) and dp/dt min (E) at week after. LVEDD indicates LV end-diastolic diameter. n for sham groups; n for groups. F through G, Western blot analysis (left) and quantification (right) using myocardial membrane fractions showing increased phospho-pln at baseline and in banded PIK KO mice (F) with elevated myocardial camp levels in PIK KO hearts at baseline and at week after (G). n 5 for all groups. H and I, Single cardiomyocyte contractility measurements showing basal hypercontractility based on percentage (H) and rate of change ( dl/dt) (I) of cell shortening in PIK KO mice, which persists in response to week of pressure overload. n for sham groups; n for groups. J, ECM-based cell adhesion of isolated adult LV cardiomyocytes following week of after showing an increase in fibronectin adhesion in cardiomyocytes and a marked decrease in adhesion to collagen IV and laminin in PIK KO cardiomyocytes. AU indicates arbitrary unit; Col I, collagen I; Col IV, collagen IV; FN, fibronectin; LN, laminin. n for and PIK KO. P.5 compared with corresponding group.

7 Guo et al Role of PIK in Biomechanical Stress A MMP/S (AU) PIKα-DN PIKγ-KD B MT-MMP/S (AU) Downloaded from by on November, camp (pmol/mg) E MMP /S (AU) C MMP/S (AU) am Week Weeks am Week Weeks Cardiomyocyte Cardiofibroblast F p-creb CREB MT-MMP/S (AU) 5 Control D MMP9/S (AU) Cardiomyocyte Cardiofibroblast Cardiomyocyte ISO mins ISO hour Cardiofibroblast Control ISO mins ISO hour H I J Control ISO ISO + Prop ISO + ACi p-creb CREB am Week Weeks am Week Weeks MMP /S (AU) G p-creb/creb (AU) # Control ISO mins ISO hour # # #. Cardiomyocyte Cardiofibroblast Control ISO ISO + Prop ISO + ACi Cardiomyocyte Cardiofibroblast Figure. Upregulation of myocardial matrix metalloproteinase expression in banded PIK KO mice with -adrenergic receptor stimulation of MMPs expression in isolated adult cardiomyocytes and cardiofibroblasts. A through D, Expression profiling of MMPs revealed selective upregulation of myocardial MMP (A), MT-MMP (B), and MMP (C) expression without a change in MMP9 (D) expression in the banded PIK KO mice compared with banded, PIK DN, and PIK -kda mice. P.5 compared with all other groups. E, Normalization of elevated camp levels in cultured adult cardiomyocytes in response to -adrenergic receptor stimulation using isoproterenol ( nmol/l) by -adrenergic receptor blockade using propranolol (5 mol/l) and specific adenylate cyclase inhibition using DIDA ( mol/l); n 5 for each group. F and G, Western blot analyses showing early phosphorylation of campresponse element (CRE) binding protein (CREB) in response to -adrenergic receptor stimulation using isoproterenol ( nmol/l) in cultured adult cardiomyocytes and cardiofibroblasts (F) and shown quantitatively (G). n 5 for each group. #P.5 compared with the control group. H through J, Expression analysis showing that long-term -adrenergic receptor stimulation using isoproterenol ( nmol/l) leads to an upregulation of mrna expression of MMP (H), MT-MMP (I), and MMP (J) in cultured adult cardiomyocytes and cardiofibroblasts, which was prevented by the -adrenergic receptor blocker propranolol (5 mol/l) and specific adenylate cyclase inhibition using DIDA ( mol/l). AU indicates arbitrary unit. n 5 for each group. P.5 compared with all other groups. MMP in banded PIK KO compared with mice (Figure 5B). MMP9 protein levels did not change in banded hearts, whereas at weeks, post- PIK KO hearts showed an increase (Online Figure III) despite minimal increase in mrna levels (Figure D), suggesting a posttranscriptional mechanism for the increased MMP9 levels. Given the lack of an increased MMP expression in banded PIK KD mice, myocardial collagenase activity was not elevated in these

8 Circulation Research November, Downloaded from by on November, A Collagenase activity (AU) C F..5 MT-MMP Ponceau Red E ANF/S (AU)..5. PIKγ-KD am wk wks wk + MMPi MMPi (-) (-) (+) sh wk wks sh wk wks + + MMPi BNP/S (AU) MT-MMP/Ponceau Red (AU) MMPi (-) (-) (+) B 7kDa MMP kda MMP 7kDa MMP kda MMP α-tubulin am week weeks + + MMPi LVEDD (mm) 5 # # TIMP/S (AU) TIMP/S (AU) sh wk wks D MMPi (-) (-) (+) am am LVESD (mm) sh wk wks sh wk wks sh wk wks # # Week Weeks Week Weeks 5 MMPi (-) (-) (+) # # TIMP/S (AU) TIMP/S (AU) MMP (7kDa)/α-tubulin (AU) MMP (kda)/α-tubulin (AU) 5 5 am am FS (%) 5 am week weeks # MMPi (-) (-) (+) Week Weeks Week Weeks Figure 5. Increased collagenase activity, MMP activation, and MT-MMP levels with prevention of the dilated cardiomyopathy by broad-spectrum MMP inhibition in banded PIK KO mice. A, Increased myocardial collagenase activity in banded PIK KO mice suppressible by the MMP inhibitor PD79 ( mg/kg per day). B and C, Gelatin zymography showed increased pro-mmp and active MMP levels in the banded PIK KO mice and Western blot analysis showing increased expression of active MMP ( kda) and pro-mmp (7 kda) (B) and membrane-fractionated MT-MMP (C) in banded PIK KO at and weeks after. n for sham group and groups. P.5 compared with the sham group. D, Expression profile of tissue inhibitors of metalloproteinases (TIMP) showing equivalent upregulation in TIMP without alteration in TIMP, whereas TIMP and TIMP levels were increased in banded PIK KO mice. n for sham group and n for groups. #P.5 compared with the sham group; P.5 compared with the group. E and F, Treatment with the broad-spectrum MMP inhibitor PD79 ( mg/kg per day) prevented the dilated cardiomyopathy and disruption of the extracellular collagen network shown as picrosirius red staining (E) while reversing the upregulation of disease markers, ANF and BNP, ventricular dilation at end-diastole and end-systole, and reduction in systolic performance (F) in banded PIK KO mice. FS indicates fractional shortening; LVEDD, LV end diastolic diameter; LVESD, LV end systolic diameter. n for each group. P.5 compared with all other groups; #P.5 compared with group. AU indicates arbitrary unit.

9 Guo et al Role of PIK in Biomechanical Stress Downloaded from by on November, mice (Figure 5A). Consistent with gelatin zymography, Western blot analysis and quantification (Figure 5B) revealed increased myocardial expression of active MMP ( kda) and pro-mmp (7 kda) in banded PIK KO mice at and weeks after. Similarly, membrane fractionation and analysis of MT-MMP levels showed an earlier and greater increase in MT-MMP levels in banded PIK KO mice (Figure 5C). The activity of MMPs are inhibited by tissue inhibitor of matrix metalloproteinases (TIMPs), TIMP, TIMP, TIMP, and TIMP with TIMP playing a key role in the myocardial response to biomechanical stress., Interestingly, TIMP levels increased similarly, whereas the expression of TIMP did not change in banded and PIK KO mice (Figure 5D). By contrast, increase in TIMP and TIMP expression occurs at -weeks after in PIK KO mice possibly attributable to a negative-feedback response to the increased MMP expression and activities (Figure 5D). Our data provide a crucial link between the adverse myocardial remodeling in banded PIK KO mice and the increase in activation and activity of MMPs. Human recombinant MMP rather than MT-MMP cleaves human recombinant N-cadherin Fc chimera and was inhibited by the MMP inhibitor, PD79 (Online Figure II). We hypothesized that MMP inhibition will lead to a marked protection in banded PIK KO mice. Daily treatment of banded PIK KO mice with the broad-spectrum MMP inhibitor PD79 ( mg/kg per day) prevented the dilated cardiomyopathy, fibrosis, and disruption of the extracellular collagen network (Figure 5E). This also reversed the upregulation of disease markers, ANF and BNP, and ventricular dilation at end-diastole and end-systole, resulting in improved systolic performance in banded PIK KO mice (Figure 5F). These results illustrate a key role of camp-mediated upregulation of MMP and MT-MMP expressions in mediating the adverse myocardial remodeling in pressure-overloaded PIK KO mice. Specific Loss of N-Cadherin From Adhesion Complexes While -Adrenergic Blocker Prevents Upregulation of MMP and Loss of N-Cadherin in Banded PIK KO Mice In addition to the degradation of the ECM, N-cadherin/ catenin cell adhesion complexes are also important targets of an activated MMP system. 9, Western blot analysis of the myocardial membrane fraction in banded and PIK DN mice showed a modest increase in N-cadherin levels (Figure A and B), whereas in banded PIK KO mice, there was a 75% loss of N-cadherin levels (Figure A and B). In contrast, in the banded PIK KO (and PIK DN) mice, levels of -catenin in the heart were significantly increased compared with banded mice (Figure C). The relative preservation of N-cadherin levels in the PIK KD hearts is consistent with a critical role of camp in mediating the loss of N-cadherin from adhesion complexes independent of the PIK lipid kinase activity per se. The quality of Western blot analysis was confirmed by absence of the membranespecific protein (toll-like receptor ) in the cytosolic fraction and absence of the cytosolic-specific protein (caspase-) in the membrane fraction (Online Figure I). Immunofluorescence microscopy confirmed colocalization of N-cadherin and -catenin in the end-to-end and side-to-side connections between cardiomyocytes in banded hearts (Figure D). Consistent with our Western blot analysis, there was a near complete loss of N-cadherin from end-to-end and side-to-side connections from pressure-overloaded PIK KO myocardium (Figure E). Consistent with the ability of PD79 to prevent MMP-mediated cleavage of N-cadherin (Online Figure II) and rescue the dilated cardiomyopathy in banded PIK KO mice (Figure 5), Western blot analysis confirmed reduced loss of membrane-associated N-cadherin (Figure F and G), resulting in more pronounced staining of N-cadherin in the myocardial cell adhesion junctions (Figure H) in banded PIK KO mice treated with PD79. Given the key role of integrin complexes in mediating cell adhesion in the heart,, we also examined changes in integrin levels in response to pressure overload. Western blot analysis of membrane fractionated D and 7B integrins showed increased levels in hearts at and weeks after with a delayed increase in the PIK KO hearts with a significant increase only at weeks after (Online Figure IV). We then tested the hypothesis that elevated camp plays a key in vivo role in the adverse remodeling in banded PIK KO mice by using the nonspecific -adrenergic blocker propranolol at a dose that has been previously shown to normalize elevated camp levels. Propranolol treatment normalizes the mrna expression of MMP, MT-MMP, and MMP (Figure 7A) with similar changes seen in the expression of disease markers, ANF, MHC, and BNP (Figure 7B). Consistent with a reduction in MMP expression, elevated collagenase activity in the banded PIK KO mice was normalized in response to propranolol (Figure 7C) with relative preservation of N-cadherin levels in the myocardial membrane fraction (Figure 7D). These biochemical and cellular changes implies that cardiac function was rescued in banded PIK KO mice treated with propranolol. Indeed, echocardiographic assessment showed a near normalization of the increased LV dilation and improved fractional shortening with invasive hemodynamic parameters showing marked increase in dp/dt max and dp/dt min in the banded PIK KO mice treated with propranolol (Figure 7E). These results highlight a key reversible defect in the N-cadherin system in the PIK KO mice in response to biomechanical stress caused by camp-dependent upregulation of MMP activity. -Blocker Therapy Prevents the Adverse Remodeling in Response to Chronic Pressure Overload The beneficial effects of -blocker therapy in the banded PIK KO mice suggest that blockade may have a similar protective role in a long-term (9 weeks) model of mice. Aortic banding for 9 weeks resulted in marked increase in expression of hypertrophy markers, -skeletal actin, MHC and BNP (Figure A) in association with LV dilation and reduced systolic function (Figure B; Online Figure V). Propranolol treatment prevented the adverse remodeling and systolic dysfunction in response to chronic biomechanical stress (Figure A and B; Online Figure V). Quantitative

10 Circulation Research November, A B C.. N-cadherin β-catenin Coomassie Blue N-cadherin G β-catenin Co-localization H N-cadherin Co-localization + MMPi N-cadherin Coomassie Blue + MMPi N-Cadherin (AU). β-catenin. N-cadherin Co-localization..... KO PI Kγ - T MMPi (-) (-) (+) W Downloaded from by on November, F PI K γkd PI K γko T αdn W PI K β-catenin.5. E... D am W T PI K αdn PI K γkd PI K γko PIKγ-KD β-catenin (AU) PIKα-DN N-Cadherin (AU) Figure. Selective loss of N-cadherin from myocardial membrane fraction and the cell adhesion complexes in the myocardium from banded PIK KO mice. A through C, Western blot analysis of the myocardial membrane fraction (A) and quantification (B) showing a selective loss of N-cadherin levels without alteration in -catenin levels (C) in banded PIK KO mice, whereas the level of N-cadherin increases in banded and PIK DN mice. P.5 compared with the corresponding sham group. D and E, Immunofluorescence microscopy of the myocardial -catenin and N-cadherin proteins showing end-to-end connections (top) and side-to-side connections (bottom) in banded (D) and PIK KO (E) mice with a distinct loss of N-cadherin in the banded PIK KO mice. F through H, Western blot analysis of the myocardial membrane fraction (F) and quantification (G) and immunofluorescence microscopy of myocardial -catenin and N-cadherin proteins (H) showing MMP inhibition prevents loss of N-cadherin in banded PIK KO mice. AU indicates arbitrary unit. n 5 for all groups. P.5 compared with all other groups. assessment of cardiac systolic function using echocardiography showed a marked improvement in fractional shortening and reduction in LV end-diastolic dimension (Online Figure V). Similarly, invasive hemodynamic measurement revealed restoration of an elevated LV end-diastolic pressure and normalization of dp/dtmax and dp/dtmin in response to chronic -blocker therapy (Figure C). These functional changes correlated with a reduction of MMP and MT-

11 Guo et al Role of PIK in Biomechanical Stress 5 Downloaded from by on November, Figure 7. The nonspecific -receptor blocker propranolol (5 mg/kg per day) prevents the molecular, biochemical, and functional deterioration in response to biomechanical stress in PIK KO mice. A and B, Expression profiling showing that treatment with propranolol prevents the increased expression of MMPs including MMP, MT-MMP, and MMP (A) and disease markers such as ANF, MHC, and BNP (B) in banded PIK KO mice. P.5 compared with all other groups. C and D, Suppression of the increased myocardial collagenase activity (C) with Western blot analysis and quantification (D) showing restoration of the membrane N-cadherin levels in banded PIK KO mice. P.5 compared with all other groups. E, Reduction in LV dilation and preservation of systolic function in response to treatment with propranolol in banded PIK KO mice based on echocardiographic parameters, LV end diastolic diameter (LVEDD) and fractional shortening (FS), and hemodynamic assessment, dp/dt max and dp/dt min. AU indicates arbitrary unit. n for all groups. P.5 compared with all other groups.

12 Circulation Research November, Downloaded from by on November, Figure. Chronic -blocker prevents pressure overload induced adverse remodeling and preserves systolic function in mice. A, Expression profile of hypertrophy disease markers showing markedly increased expression of -skeletal actin, MHC, and BNP in banded mice at 9 weeks, which was dramatically reduced by treatment with propranolol. n 5 per group. B, Echocardiographic assessment of systolic function with M-mode echocardiograms showing a marked reduction in LV dilation and increase in fractional shortening in response to therapy with propranolol. C, Invasive hemodynamic assessment showing marked reduction in LVEDP and prevention of the deterioration in myocardial contractility based on dp/dt max and dp/dt min in response to chronic -blocker therapy. n for sham and n for groups. D, Expression of MMPs showing a marked increase in MMP and MT-MMP expression without a differential effect on MMP- levels and a normalization in response to chronic -blocker therapy (n 5 per group). E, Western blot analysis revealed a greater increase in myocardial N-cadherin levels with equivalent increase in -catenin levels, leading to a relative preservation of the N-cadherin/ -catenin ratio in response to chronic -blocker therapy. AU indicates arbitrary unit. n 5 per group. P.5 compared with all other groups; #P.5 compared with 9 weeks; P.5 compared with sham group.

13 Guo et al Role of PIK in Biomechanical Stress 7 Downloaded from by on November, MMP expression in the absence of a differential effect on MMP expression (Figure D). Western blot analysis showed a higher level of membrane-associated N-cadherin with an equivalent increase in -catenin levels resulting in preservation of the myocardial N-cadherin/ -catenin ratio (Figure E) in response to chronic -blocker therapy. Our results highlight a novel mechanism of chronic -blocker therapy in preventing the adverse myocardial remodeling in pressure overload induced heart failure. Discussion The response to biomechanical stress is a fundamental response in heart disease and plays a key adaptive role in response to a pressure overload state characteristic of hypertension and valvular stenosis. Mechanotransduction plays a fundamental role in cardiac structure and function and involves a concerted interaction between extracellular matrix, intracellular cytoskeletal proteins, and cell adhesion complexes. 7, Advanced heart failure in response to myocardial injury including biomechanical stress leads to impaired Ca cycling and strategies aimed at enhancing Ca cycling are currently being developed as therapies for heart failure.,5 Loss of PIK increases basal camp levels, enhances SERCA function and Ca cycling, and increases basal myocardial contractility.,, In the heart, loss of PIK prevents phosphorylation of Akt in response to G protein coupled receptor agonists,,, and PIK KO mice are resistant to the pathological effects of -adrenergic stimulation. Despite these biochemical changes, PIK KO mice show enhanced susceptibility to biomechanical stress,,7 and we provide direct evidence that the primary defect is a compromised cell-adhesion/extracellular matrix system linked to elevated camp levels. In contrast, in PIK KD mice, which have normal myocardial camp levels, there is no upregulation of MMPs with preservation of N-cadherin levels consistent with the ability of these mice to maintain normal systolic function in response to early biomechanical stress. The PIK/PTEN system may also have a more direct role in the cardiac response to biomechanical stress.,7,, Whereas PIK and lipid phosphatases can modulate cytoskeletal interactions, stretch can in turn activate Akt/PKB and GSK- activity. Indeed, loss of PTEN prevents pressure overload induced heart failure, whereas loss of Akt/PKB leads to a rapid onset of ventricular dilation and systolic dysfunction in response to pressure overload. Consistent with a critical role of PIK in pathological G protein coupled receptor signaling,,, phosphorylation of ERK/ is impaired in response to early biomechanical stress in the PIK KO mice. However, this differential response is loss at weeks after, which is likely driven by severe disruption of the ECM and/or cell adhesion leading to G protein coupled receptor independent activation of ERK/ in the PIK KO mice. Despite equivalent basal phospho-akt levels in and PIK KO hearts, basal phospho GSK- was increased in the PIK KO mice, likely mediated by PKA 9 given the chronic elevation of myocardial camp levels in these mice. The diverse downstream effects of elevated basal phospho GSK- may have contributed to the adverse remodeling in the PIK KO mice. Elevated camp levels in response to increased biomechanical stress leads to increased MMP expression and increased active (cleaved) MMP and collagenase activity, leading to adverse myocardial remodeling. Activation of pro-mmp occurs by proteolytic cleavage of the N-terminal propeptide and requires two MT-MMP molecules in association with TIMP., In addition to degrading various components of the ECM, increased MMP activity can also adversely modify cell cell adhesion complexes. Cardiomyocyte cell adhesion complexes provide an important mechanism by which cardiomyocytes (and cardiofibroblasts) are anchored to the extracellular matrix while allowing force transmission to the intracellular cytoskeletal network.,, In particular, we have shown that increased MMP expression and activity can regulate N-cadherin function through proteolytic degradation. 9,, Cardiac-specific loss of N-cadherin in the heart leads to a dilated cardiomyopathy attributable to loss of the integrity of cell adhesion junctions. 5 Because of their homophilic binding and adhesive specificities, N-cadherin/catenin complex is required for cadherin-mediated cell adhesion and linkage to the actin cytoskeleton. The delayed increase in membrane D and 7B integrins in the banded PIK KO hearts may have also contributed to the early onset of dilated cardiomyopathy., In addition, MT-MMP is a potent collagenase that also targets other ECM components such as fibronectin, laminin, and integrins 7, while activating MMP (collagenase-), thereby amplifying the collagen-degradation process.,9 Our results are consistent with the conclusion that elevated camp (and its downstream effects) is the primary driver of the adverse remodeling in banded PIK KO mice rather than loss of PIK signaling per se. Our findings may help to explain the cardiomyopathy in experimental models with enhanced -adrenergic signaling (and camp levels) 5,5 and lack of a protection against pressure overload and tumor necrosis factor induced heart failure despite enhanced Ca cycling in the PLN knockout mice. 5,5 Indeed, whereas Ca transients in cardiomyocytes were normalized in tumor necrosis factor induced cardiomyopathy in a PLN-null background, global systolic function remained depressed and unchanged. 5 We propose that enhancing cell cell adhesion and cell ECM interaction will promote the salutary effects of enhanced intracellular Ca cycling on whole heart function and booster the therapeutic potential of normalizing intracellular Ca cycling in patients with heart failure. Increased sympathetic nervous system activity and -adrenergic receptor signaling are key aspects of the pathophysiology of heart failure 5 and in catecholamine-mediated cardiomyopathies. 55,5 -Adrenergic receptor blockers improve clinical outcomes in patients with chronic heart failure. 5 In contrast, agents that increase myocardial camp such as PDE inhibitors are associated with adverse outcomes and increased mortality in patients with heart failure. 57 Our data support a novel role of -adrenergic blocker in reducing MMP expression and/or activity and preservation of cell adhesion, thereby curtailing adverse myocardial remodeling.

14 Circulation Research November, Downloaded from by on November, Sources of Funding This work was supported by the Heart and Stroke Foundation of Canada (to G.Y.O.), Canadian Institute for Health Research (grant to G.Y.O. and grant 79 to Z.K.), and the Alberta Innovates Health Solutions (to G.Y.O. and Z.K.). None. Disclosures References. Oudit GY, Sun H, Kerfant BG, Crackower MA, Penninger JM, Backx PH. The role of phosphoinositide- kinase and PTEN in cardiovascular physiology and disease. J Mol Cell Cardiol. ;7:9 7.. Oudit GY, Penninger JM. Cardiac regulation by phosphoinositide -kinases and PTEN. Cardiovasc Res. 9;:5.. Crackower MA, Oudit GY, Kozieradzki I, Sarao R, Sun H, Sasaki T, Hirsch E, Suzuki A, ioi T, Irie-Sasaki J, Sah R, Cheng HY, Rybin VO, Lembo G, Fratta L, Oliveira-dos-Santos AJ, Benovic JL, Kahn CR, Izumo S, Steinberg SF, Wymann MP, Backx PH, Penninger JM. Regulation of myocardial contractility and cell size by distinct PIK-PTEN signaling pathways. Cell. ;: McMullen JR, ioi T, Zhang L, Tarnavski O, erwood MC, Kang PM, Izumo S. Phosphoinositide -kinase(palpha) plays a critical role for the induction of physiological, but not pathological, cardiac hypertrophy. Proc Natl Acad Sci U S A. ;: Inuzuka Y, Okuda J, Kawashima T, Kato T, Niizuma S, Tamaki Y, Iwanaga Y, Yoshida Y, Kosugi R, Watanabe-Maeda K, Machida Y, Tsuji S, Aburatani H, Izumi T, Kita T, ioi T. Suppression of phosphoinositide -kinase prevents cardiac aging in mice. Circulation. 9;: Patrucco E, Notte A, Barberis L, Selvetella G, Maffei A, Brancaccio M, Marengo S, Russo G, Azzolino O, Rybalkin SD, Silengo L, Altruda F, Wetzker R, Wymann MP, Lembo G, Hirsch E. PIKgamma modulates the cardiac response to chronic pressure overload by distinct kinasedependent and -independent effects. Cell. ;: Sugden PH. Ras, Akt, and mechanotransduction in the cardiac myocyte. Circ Res. ;9: Baba HA, Stypmann J, Grabellus F, Kirchhof P, Sokoll A, Schafers M, Takeda A, Wilhelm MJ, Scheld HH, Takeda N, Breithardt G, Levkau B. Dynamic regulation of MEK/Erks and Akt/GSK-beta in human end-stage heart failure after left ventricular mechanical support: myocardial mechanotransduction-sensitivity as a possible molecular mechanism. Cardiovasc Res. ;59: Knoll R, Hoshijima M, Hoffman HM, Person V, Lorenzen-Schmidt I, Bang ML, Hayashi T, iga N, Yasukawa H, Schaper W, McKenna W, Yokoyama M, Schork NJ, Omens JH, McCulloch AD, Kimura A, Gregorio CC, Poller W, Schaper J, Schultheiss HP, Chien KR. The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy. Cell. ;: Kerfant BG, Gidrewicz D, Sun H, Oudit GY, Penninger JM, Backx PH. Cardiac sarcoplasmic reticulum calcium release and load are enhanced by subcellular camp elevations in PIKgamma-deficient mice. Circ Res. 5;9:79.. Oudit GY, Crackower MA, Eriksson U, Sarao R, Kozieradzki I, Sasaki T, Irie-Sasaki J, Gidrewicz D, Rybin VO, Wada T, Steinberg SF, Backx PH, Penninger JM. Phosphoinositide -kinase gamma-deficient mice are protected from isoproterenol-induced heart failure. Circulation. ;: Kassiri Z, Oudit GY, Sanchez O, Dawood F, Mohammed FF, Nuttall RK, Edwards DR, Liu PP, Backx PH, Khokha R. Combination of tumor necrosis factor-alpha ablation and matrix metalloproteinase inhibition prevents heart failure after pressure overload in tissue inhibitor of metalloproteinase- knock-out mice. Circ Res. 5;97: 9.. Oudit GY, Kassiri Z, Zhou J, Liu QC, Liu PP, Backx PH, Dawood F, Crackower MA, Scholey JW, Penninger JM. Loss of PTEN attenuates the development of pathological hypertrophy and heart failure in response to biomechanical stress. Cardiovasc Res. ;7: Bradshaw AD, Baicu CF, Rentz TJ, Van Laer AO, Boggs J, Lacy JM, Zile MR. Pressure overload-induced alterations in fibrillar collagen content and myocardial diastolic function: role of secreted protein acidic and rich in cysteine (SPARC) in post-synthetic procollagen processing. Circulation. 9;9:9. 5. O Connell TD, Rodrigo MC, Simpson PC. Isolation and culture of adult mouse cardiac myocytes. Methods Mol Biol. 7;57:7 9.. Kandalam V, Basu R, Abraham T, Wang X, Soloway PD, Jaworski DM, Oudit GY, Kassiri Z. TIMP deficiency accelerates adverse postmyocardial infarction remodeling because of enhanced MT-MMP activity despite lack of MMP activation. Circ Res. ;: Zhong JC, Basu R, Guo D, Chow FL, Byrns S, uster M, Loibner H, Wang X, Penninger JM, Kassiri Z, Oudit GY. Angiotensin converting enzyme suppresses pathological hypertrophy, myocardial fibrosis and cardiac dysfunction. Circulation. ;: Sussman MA, McCulloch A, Borg TK. Dance band on the Titanic: biomechanical signaling in cardiac hypertrophy. Circ Res. ;9: Heineke J, Molkentin JD. Regulation of cardiac hypertrophy by intracellular signalling pathways. Nat Rev Mol Cell Biol. ;7:59.. Matsuda T, Zhai P, Maejima Y, Hong C, Gao S, Tian B, Goto K, Takagi H, Tamamori-Adachi M, Kitajima S, Sadoshima J. Distinct roles of GSK-alpha and GSK-beta phosphorylation in the heart under pressure overload. Proc Natl Acad Sci U S A. ;5: Romer LH, Birukov KG, Garcia JG. Focal adhesions: paradigm for a signaling nexus. Circ Res. ;9:.. Oudit GY, Kassiri Z. Role of PI kinase gamma in excitation-contraction coupling and heart disease. Cardiovasc Hematol Disord Drug Targets. 7;7:95.. Spinale FG. Myocardial matrix remodeling and the matrix metalloproteinases: influence on cardiac form and function. Physiol Rev. 7;7: 5.. Sands WA, Palmer TM. Regulating gene transcription in response to cyclic AMP elevation. Cell Signal. ;:. 5. Melnikova VO, Mourad-Zeidan AA, Lev DC, Bar-Eli M. Plateletactivating factor mediates MMP- expression and activation via phosphorylation of camp-response element-binding protein and contributes to melanoma metastasis. J Biol Chem. ;:9 9.. Quinn CO, Rajakumar RA, Agapova OA. Parathyroid hormone induces rat interstitial collagenase mrna through Ets- facilitated by cyclic AMP response element-binding protein and Ca( )/calmodulin-dependent protein kinase II in osteoblastic cells. J Mol Endocrinol. ;5:7. 7. ankavaram UT, Lai WC, Netzel-Arnett S, Mangan PR, Ardans JA, Caterina N, Stetler-Stevenson WG, Birkedal-Hansen H, Wahl LM. Monocyte membrane type -matrix metalloproteinase. Prostaglandindependent regulation and role in metalloproteinase- activation. J Biol Chem. ;7: Kassiri Z, Khokha R. Myocardial extra-cellular matrix and its regulation by metalloproteinases and their inhibitors. Thromb Haemost. 5;9: Ho AT, Voura EB, Soloway PD, Watson KL, Khokha R. MMP inhibitors augment fibroblast adhesion through stabilization of focal adhesion contacts and up-regulation of cadherin function. J Biol Chem. ;7: 5.. Covington MD, Burghardt RC, Parrish AR. Ischemia-induced cleavage of cadherins in NRK cells requires MT-MMP (MMP-). Am J Physiol Renal Physiol. ;9:F F5.. Ross RS, Borg TK. Integrins and the myocardium. Circ Res. ;: 9.. Babbitt CJ, ai SY, Harpf AE, Pham CG, Ross RS. Modulation of integrins and integrin signaling molecules in the pressure-loaded murine ventricle. Histochem Cell Biol. ;: 9.. Jamora C, Fuchs E. Intercellular adhesion, signalling and the cytoskeleton. Nat Cell Biol. ;:E E.. Chien KR, Ross J Jr, Hoshijima M. Calcium and heart failure: the cycle game. Nat Med. ;9: Jaski BE, Jessup ML, Mancini DM, Cappola TP, Pauly DF, Greenberg B, Borow K, Dittrich H, Zsebo KM, Hajjar RJ. Calcium upregulation by percutaneous administration of gene therapy in cardiac disease (CUPID Trial), a first-in-human phase / clinical trial. J Card Fail. 9;5: 7.. Naga Prasad SV, Esposito G, Mao L, Koch WJ, Rockman HA. Gbetagamma-dependent phosphoinositide -kinase activation in hearts with in vivo pressure overload hypertrophy. J Biol Chem. ;75: Oudit GY, Kassiri Z, Crackower MA, Kozieradzki I, Penninger J, Backx PH. Loss of PI kinase gamma leads to an accelerated progression to dilated cardiomyopathy in response to pressure overload. Circulation. ;(Suppl IV):IV-.