Abstract-Three-dimensional cardiac mapping in rabbits with nonischemic cardiomyopathy has shown that ventricular arrhythmias initiate by a nonreentrant mechanism that may be due to triggered activity from delayed afterdepolarizations. Delayed afterdepolarizations are thought to be due to spontaneous release of Ca 2ϩ from the sarcoplasmic reticulum (SR) and consequent activation of an inward Na ϩ /Ca 2ϩ exchange (NaCaX) current. The goal of this study was to determine whether there is enhanced NaCaX gene expression and functional activity that may contribute to nonreentrant activation. Heart failure (HF) was induced in rabbits by combined aortic insufficiency and aortic constriction. HF rabbits had left ventricular enlargement (left ventricular end-diastolic dimension increased from 1.43Ϯ0.03 to 1.97Ϯ0.05 cm) and severely depressed function (fractional shortening reduced from 37% to 26%, PϽ0.02). Heart-to-body weight was increased by 79% in HF. Western blots showed a 93% increase in NaCaX protein in HF (PϽ0.04). NaCaX mRNA (7-kb transcript) was increased by 104% relative to the 18S rRNA in HF. A 14-kb NaCaX transcript was also seen in the HF rabbits, raising total NaCaX mRNA to 2.7-fold compared with controls. Key Words: heart failure Ⅲ Na ϩ /Ca 2ϩ exchange Ⅲ ventricular tachycardia Ⅲ delayed afterdepolarization Ⅲ Ca 2ϩ S udden death in patients with congestive heart failure (HF) is commonly due to lethal ventricular arrhythmias, including ventricular tachycardia (VT) and ventricular fibrillation. 1,2 However, very little is known about the electrophysiologic and molecular mechanisms underlying arrhythmogenesis in HF. An understanding will require 1) development of arrhythmogenic animal models of HF, 2) delineation of the arrhythmic mechanisms in these animal models, 3) validation of these mechanisms by studies in the human heart, and 4) assessment of the function and expression of the channels and carrier proteins that may underlie these arrhythmic mechanisms.There are a considerable number of experimental animal models of HF, 3 but very few are arrhythmogenic. An arrhythmogenic experimental model of nonischemic cardiomyopathy has recently been developed in rabbits, combining aortic insufficiency and aortic constriction. 4,5 These HF rabbits develop severe depression of left ventricular (LV) function, pathologic alterations similar to that in patients with nonischemic cardiomyopathy, and spontaneously-occurring VT. 3 Using 3D cardiac mapping, Pogwizd 5 has demonstrated that the spontaneously-occurring VT in these HF rabbits initiates by a nonreentrant mechanism. Recent 3D mapping studies in failing human hearts at the time of heart transplantation in patients with idiopathic dilated cardiomyopathy have demonstrated that spontaneous and induced VT in these patients also initiates by a focal nonreentrant mechanism. 6 The nature of this nonreentrant mechanism in the myopathic heart is unknown. Vermeulen et al 7 have demonstrated that failing myocardium from rabbits with aortic insufficiency and aortic stenosis demo...

Samarel, Allen M. Costameres, focal adhesions, and cardiomyocyte mechanotransduction. Am J Physiol Heart Circ Physiol 289: H2291-H2301, 2005; doi: 10.1152/ajpheart.00749.2005.-Mechanotransduction refers to the cellular mechanisms by which load-bearing cells sense physical forces, transduce the forces into biochemical signals, and generate appropriate responses leading to alterations in cellular structure and function. This process affects the beat-to-beat regulation of cardiac performance but also affects the proliferation, differentiation, growth, and survival of the cellular components that comprise the human myocardium. This review focuses on the experimental evidence indicating that the costamere and its structurally related structure the focal adhesion complex are critical cytoskeletal elements involved in cardiomyocyte mechanotransduction. Biochemical signals originating from the extracellular matrix-integrin-costameric protein complex share many common features with those signals generated by growth factor receptors. The roles of key regulatory kinases and other muscle-specific proteins involved in mechanotransduction and growth factor signaling are discussed, and issues requiring further study in this field are outlined.focal adhesion kinase; proline-rich tyrosine kinase 2; integrin-linked kinase; protein kinase C; signal transduction; heart THE PROCESS OF MECHANOTRANSDUCTION refers to the cellular mechanisms by which load-bearing cells sense physical forces, transduce the forces into biochemical signals, and generate appropriate responses leading to alterations in cellular structure and function. Physical forces encountered by living cells include membrane stretch, gain and loss of adhesion, and compression due to an increase in pressure. The signal transduction pathways that are activated in response to mechanical forces include many unique components, as well as elements shared by other signaling pathways. Mechanotransduction in cardiomyocytes is particularly complex, in that individual muscle cells both respond to externally applied mechanical forces as well as generate internal loads that are transmitted to adjacent cells and their surrounding extracellular matrix (ECM). Mechanotransduction in both atrial and ventricular cardiomyocytes affects the beat-to-beat regulation of cardiac performance but also profoundly affects the proliferation, differentiation, growth, and survival of the cellular components that comprise the human myocardium. Understanding the cellular and molecular basis for mechanotransduction is therefore important to our overall understanding of cardiac structure and function in the normal and diseased heart.Observational studies conducted during the 1970s addressing the hypertrophic growth response of human myocardium to pathological changes in systolic and diastolic wall stress (42) fostered the development of experimental model systems with which to explore cardiomyocyte mechanotransduction in vivo and in vitro. These model systems now span the breadth of experimental cardiology...

The interaction of endothelial cells with extracellular matrix proteins at focal adhesions sites contributes to the integrity of vascular endothelial barrier. Although focal adhesion kinase (FAK) activation is required for the recovery of the barrier function after increased endothelial junctional permeability, the basis for the recovery remains unclear. We tested the hypothesis that FAK activates p190RhoGAP and, thus, negatively regulates RhoA activity and promotes endothelial barrier restoration in response to the permeability-increasing mediator thrombin. We observed that thrombin caused a transient activation of RhoA but a more prolonged FAK activation temporally coupled to the recovery of barrier function. Thrombin also induced tyrosine phosphorylation of p190RhoGAP, which coincided with decrease in RhoA activity. We further showed that FAK was associated with p190RhoGAP, and importantly, recombinant FAK phosphorylated p190RhoGAP in vitro. Inhibition of FAK by adenoviral expression of FRNK (a dominant negative FAK construct) in monolayers prevented p190RhoGAP phosphorylation, increased RhoA activity, induced actin stress fiber formation, and produced an irreversible increase in endothelial permeability in response to thrombin. We also observed that p190RhoGAP was unable to attenuate RhoA activation in the absence of FAK activation induced by FRNK. The inhibition of RhoA by the C3 toxin (Clostridium botulinum toxin) restored endothelial barrier function in the FRNK-expressing cells. These findings in endothelial cells were recapitulated in the lung microcirculation in which FRNK expression in microvessel endothelia increased vascular permeability. Our studies demonstrate that FAK-induced down-modulation of RhoA activity via p190RhoGAP is a crucial step in signaling endothelial barrier restoration after increased endothelial permeability.

Abstract-Reperfusion of cardiac tissue after an ischemic episode is associated with metabolic and contractile dysfunction, including reduced tension development and activation of the Na ϩ -H ϩ exchanger (NHE). Oxygen-derived free radicals are key mediators of reperfusion abnormalities, although the cellular mechanisms involved have not been fully defined. In the present study, the effects of free radicals on mitogen-activated protein (MAP) kinase function were investigated using cultured neonatal rat ventricular myocytes. Acute exposure of spontaneously beating myocytes to 50 mol/L hydrogen peroxide (H 2 O 2 ) caused a sustained decrease in contraction amplitude (80% of control). MAP kinase activity was measured by in-gel kinase assays and Western blot analysis. Acute exposure to H 2 O 2 (100 mol/L, 5 minutes) resulted in sustained MAP kinase activation that persisted for 60 minutes. Catalase, but not superoxide dismutase, completely inhibited MAP kinase activation by H 2 O 2 . Pretreatment with chelerythrine (10 mol/L, 45 minutes), a protein kinase C inhibitor, or genistein (75 mol/L, 45 minutes) or herbimycin A (3 mol/L, 45 minutes), tyrosine kinase inhibitors, caused significant inhibition of H 2 O 2 -stimulated MAP kinase activity (51%, 78%, and 45%, respectively, at 20 minutes). Brief exposure to H 2 O 2 also stimulated NHE activity. This effect was completely abolished by pretreatment with the MAP kinase kinase inhibitor PD 98059 (30 mol/L, 60 minutes). These results suggest that low doses of H 2 O 2 induce MAP kinase-dependent pathways that regulate NHE activity during reperfusion injury.

Abstract-Protein kinase C (PKC) ⑀ and PKC␦ translocation in neonatal rat ventricular myocytes (NRVMs) is accompanied by subsequent activation of the ERK, JNK, and p38 MAPK cascades; however, it is not known if either or both novel PKCs are necessary for their downstream activation. Use of PKC inhibitors to answer this question is complicated by a lack of isoenzyme specificity, and the fact that many PKC inhibitors stimulate JNK and p38 MAPK activity. Therefore, replication-defective adenoviruses (Advs) encoding constitutively active (ca) mutants of PKC⑀ and PKC␦ were used to test if either or both of these PKCs are sufficient to activate ERKs, JNKs, and/or p38 MAPK in NRVMs. Adv-caPKC⑀ infection (1 to 25 multiplicities of viral infection (MOI); 4 to 48 hours) increased total PKC⑀ levels in a time-and dose-dependent manner, with maximal expression observed 8 hours after Adv infection. Adv-caPKC⑀ induced a time-and dose-dependent increase in phosphorylated p42 and p44 ERKs, as compared with a control Adv encoding ␤-galactosidase (Adv-ne␤gal). Maximal ERK phosphorylation occurred 8 hours after Adv infection. In contrast, JNK was only minimally activated, and p38MAPK was relatively unaffected. Adv-caPKC␦ infection (1 to 25 MOI, 4 to 48 hours) increased total PKC␦ levels in a similar fashion. Adv-caPKC␦ (5 MOI) induced a 29-fold increase in phosphorylated p54 JNK, and a 15-fold increase in phosphorylated p38MAPK 24 hours after Adv infection. In contrast, p42 and p44 ERK were only minimally activated. Whereas neither Adv induced NRVM hypertrophy, Adv-caPKC␦, but not Adv-caPKC⑀, induced NRVM apoptosis. We conclude that the novel PKCs differentially regulate MAPK cascades and apoptosis in an isoenzyme-specific and time-dependent manner. here is now substantial evidence to indicate a critical role for protein kinase C (PKC) activation in coordinating specific aspects of cardiomyocyte hypertrophy. 1 Previous studies have also implicated PKCs as potential upstream regulators of the mitogen-activated protein kinases (MAPK), which are involved in both hypertrophic signal transduction, 2 as well as apoptosis. 3 However, cardiomyocytes express several PKC isoenzymes that are differentially activated by various stimuli. For instance, the hypertrophic agonists endothelin-1 (ET) and phenylephrine (PE) caused the membrane translocation of PKC⑀, and to a lesser extent PKC␦, in cultured neonatal rat ventricular myocytes (NRVMs). 4,5 ETinduced PKC⑀ and PKC␦ translocation was accompanied by subsequent activation of all three MAPK cascades. [5][6][7][8][9] In contrast, electrical stimulation of contraction induced a similar degree of cardiomyocyte hypertrophy, but predominantly activated PKC␦ rather than PKC⑀. 10 Electrical pacing was also associated with a rapid increase in JNK 10,11 and p38 MAPK 12 activities, but ERKs were not significantly activated. 10,11 None of these hypertrophic stimuli induced the membrane translocation of PKC␣, the major Ca 2ϩ -dependent, phorbol-ester-sensitive PKC in NRVMs. 4,5,10 Although PKC⑀ has been imp...

Targeting kinases is central to drug-based cancer therapy but remains challenging because the drugs often lack specificity, which may cause toxic side effects. Modulating side effects is difficult because kinases are evolutionarily and hence structurally related. The lack of specificity of the anticancer drug imatinib enables it to be used to treat chronic myeloid leukemia, where its target is the Bcr-Abl kinase, as well as a proportion of gastrointestinal stromal tumors (GISTs), where its target is the C-Kit kinase. However, imatinib also has cardiotoxic effects traceable to its impact on the C-Abl kinase. Motivated by this finding, we made a modification to imatinib that hampers Bcr-Abl inhibition; refocuses the impact on the C-Kit kinase; and promotes inhibition of an additional target, JNK, a change that is required to reinforce prevention of cardiotoxicity. We established the molecular blueprint for target discrimination in vitro using spectrophotometric and colorimetric assays and through a phage-displayed kinase screening library. We demonstrated controlled inhibitory impact on C-Kit kinase in human cell lines and established the therapeutic impact of the engineered compound in a novel GIST mouse model, revealing a marked reduction of cardiotoxicity. These findings identify the reengineered imatinib as an agent to treat GISTs with curbed side effects and reveal a bottom-up approach to control drug specificity.

Fibronectin fragments (FN-f), including the 110-kDa fragment that binds the ␣ 5 ␤ 1 integrin, stimulate collagenase-3 (MMP-13) production and cartilage destruction. In the present study, treatment of chondrocytes with the 110-kDa FN-f or an activating antibody to the ␣ 5 ␤ 1 integrin was found to increase tyrosine autophosphorylation (Tyr-402) of the proline-rich tyrosine kinase-2 (PYK2) without significant change in autophosphorylation (Tyr-397) of focal adhesion kinase (FAK). The tyrosine kinase inhibitor tyrphostin A9, shown previously to block a PYK2-dependent pathway, blocked the FN-f-stimulated increase in MMP-13, whereas tyrphostin A25 did not. FN-f-stimulated PYK2 phosphorylation and MMP-13 production was also blocked by reducing intracellular calcium levels. Adenovirally mediated overexpression of wild type but not mutant PYK2 resulted in increased MMP-13 production. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate stimulated PYK2 phosphorylation and MMP-13 production. MMP-13 expression stimulated by either phorbol 12-myristate 13-acetate or FN-f was blocked by PKC inhibitors including the PKC␦ inhibitor rottlerin. Furthermore, PKC␦ translocation from cytosol to membrane was noted within 5 min of stimulation with FN-f. Immortalized human chondrocytes, transiently transfected with MMP-13 promoter-luciferase reporter constructs, showed increased promoter activity after FN-f treatment that was inhibited by co-transfection with either of two dominant negative mutants of PYK2 (Y402F and K457A). No inhibition was seen after cotransfection with wild type PYK2, a dominant negative of FAK (FRNK) or empty vector plasmid. FN-f-stimulated MMP-13 promoter activity was also inhibited by chemical inhibitors of ERK, JNK, and p38 mitogen-activated protein (MAP) kinases or by co-transfection of dominant negative MAP kinase mutant constructs. These studies have identified a novel pathway for the MAP kinase regulation of MMP-13 production which involves FN-f stimulation of the ␣ 5 ␤ 1 integrin and activation of the nonreceptor tyrosine kinase PYK2 by PKC, most likely PKC␦. Matrix metalloproteinases (MMPs)1 are expressed by a number of different cell types and play a key role in diverse processes ranging from morphogenesis to tumor invasion to tissue remodeling (reviewed in Ref. 1). Because of their potent ability to degrade the extracellular matrix and the undesirable consequences of excess matrix degradation, the production and activation of MMPs must be tightly regulated. In articular cartilage, excess production of MMPs results in destruction of the cartilage matrix (2-4). MMP-13 (collagenase-3) is a very potent degrader of type II collagen (5), the major collagen type in cartilage, and overexpression of MMP-13 in a transgenic mouse model was found to reproduce the joint changes characteristic of osteoarthritis (6). A better understanding of the cellular mechanisms responsible for regulating MMP expression could lead to the development of novel therapies aimed at controlling excess matrix degradation...

Abstract-Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in adhesion-dependent signal transduction. FAK is highly expressed in cultured neonatal rat ventricular myocytes (NRVMs) and undergoes tyrosine autophosphorylation in response to cell adhesion, stretch, and growth factor stimulation. We previously showed that inhibition of FAK phosphorylation by adenovirally mediated overexpression of FRNK (the autonomously expressed C-terminal domain of FAK) prevented endothelin-1 (ET)-induced NRVM hypertrophy. One question raised by these studies was whether FRNK localized to focal adhesions and displaced FAK from sites required for downstream signaling. Therefore, we constructed a replication-defective adenovirus encoding a GFP-FRNK fusion protein (Adv-GFP-FRNK) and examined its effects on NRVM cytoarchitecture and signaling. Uninfected NRVMs contained small amounts of endogenous FRNK. NRVMs infected with Adv-GFP-FRNK expressed much larger amounts of a 66-/68-kDa protein that localized to costameres and focal adhesions. GFP-FRNK overexpression suppressed basal and ET-induced FAK phosphorylation and also inhibited ET-induced phosphorylation of PYK2, the other member of the FAK family of nonreceptor protein tyrosine kinases. In contrast, GFP-FRNK overexpression did not prevent ET-induced ERK, JNK, or p70S6K phosphorylation. Furthermore, GFP-FRNK resulted in the loss of detectable FAK and paxillin in focal adhesions, which was accompanied by reduced levels of total paxillin and, ultimately, cell detachment and apoptosis. We conclude that FRNK functions as a dominant-negative inhibitor of adhesion-dependent signaling by displacing FAK from focal adhesions and interfering with the anchorage of NRVMs that is necessary for cell survival, a process known as anoikis. Costamere-like structures containing integrins are found in cultured neonatal and adult cardiac myocytes. 2 Cultured cardiomyocytes also form focal adhesions (similar to focal adhesions assembled by adherent nonmuscle cells in culture) containing ␤ 1 -integrins and vinculin. Their organization is highly regulated by peptide growth factors and externally applied or intrinsically generated mechanical load. [3][4][5][6] In addition to their structural role, focal adhesions and costameres are major sites for the localization of cell signaling molecules that are activated during cardiomyocyte hypertrophy. 7-9 Furthermore, disruption of normal cardiomyocyte anchorage during the transition from hypertrophy to early failure leads to apoptosis in an experimental model of ascending aortic coarctation. 10 Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that is localized to focal adhesions and costameres in cardiomyocytes. FAK undergoes autophosphorylation at a specific tyrosine residue (Y397) in response to cell adhesion, integrin clustering, and growth factor stimulation. 11 In addition, a homologous protein, known as FRNK (FAK-related non-kinase), is also a product of the FAK gene but is autonomously expressed under the cont...