Honey B. Golden

Integrins and proximal signaling mechanisms in cardiovascular disease

Integrins are heterodimeric cell-surface molecules, which act as the principle mediators of molecular dialog between a cell and its extracellular matrix environment. In addition to their structural functions, integrins mediate signaling from the extracellular space into the cell through integrin-associated signaling and adaptor molecules such as FAK (focal adhesion kinase), ILK (integrin-linked kinase), PINCH (particularly interesting new cysteine-histidine rich protein) and Nck2 (non-catalytic (region of) tyrosine kinase adaptor protein-2). Via these molecules, integrin signaling tightly and cooperatively interacts with receptor tyrosine kinases (RTKs) signaling to regulate survival, proliferation and cell shape as well as polarity, adhesion, migration and differentiation. In the heart and blood vessels, the function and regulation of these molecules can be partially disturbed and thus contribute to cardiovascular diseases such as cardiac hypertrophy and atherosclerosis. In this review, we discuss the primary mechanisms of action and signaling of integrins in the cardiac and vascular system in normal and pathological states, as well as therapeutic strategies for targeting these systems (1).

Rac1 and RhoA differentially regulate angiotensinogen gene expression in stretched cardiac fibroblasts

AIMS Angiotensin II (Ang II) stimulates cardiac remodelling and fibrosis in the mechanically overloaded myocardium. Although Rho GTPases regulate several cellular processes, including myocardial remodelling, involvement in mediating mechanical stretch-induced regulation of angiotensinogen (Ao), the precursor to Ang II, remains to be determined. We, therefore, examined the role and associated signalling mechanisms of Rho GTPases (Rac1 and RhoA) in regulation of Ao gene expression in a stretch model of neonatal rat cardiac fibroblasts (CFs). METHODS AND RESULTS CFs were plated on deformable stretch membranes. Equiaxial mechanical stretch caused significant activation of both Rac1 and RhoA within 2-5 min. Rac1 activity returned to control levels after 4 h, whereas RhoA remained at a high level of activity until the end of the stretch period (24 h). Mechanical stretch initially caused a moderate decrease in Ao gene expression, but was significantly increased at 8-24 h. RhoA had a major role in mediating both the stretch-induced inhibition of Ao at 4 h and the subsequent upregulation of Ao expression at 24 h. β₁ integrin receptor blockade by Tac β₁ expression impaired acute (2 and 15 min) stretch-induced Rac1 activation, but increased RhoA activity. Molecular experiments revealed that Ao gene expression was inhibited by Rac1 through both JNK-dependent and independent mechanisms, and stimulated by RhoA through a p38-dependent mechanism. CONCLUSION These results indicate that stretch-induced activation of Rac1 and RhoA differentially regulates Ao gene expression by modulating p38 and JNK activation.

Stretch-induced regulation of angiotensinogen gene expression in cardiac myocytes and fibroblasts: Opposing roles of JNK1/2 and p38α MAP kinases

The cardiac renin-angiotensin system (RAS) has been implicated in mediating myocyte hypertrophy, remodeling, and fibroblast proliferation in the hemodynamically overloaded heart. However, the intracellular signaling mechanisms responsible for regulation of angiotensinogen (Ao), a substrate of the RAS system, are largely unknown. Here we report the identification of JNK1/2 as a negative, and p38alpha as a major positive regulator of Ao gene expression. Isolated neonatal rat ventricular myocytes (NRVM) and fibroblasts (NRFB) plated on deformable membranes coated with collagen IV, were exposed to 20% equiaxial static-stretch (0-24 h). Mechanical stretch initially depressed Ao gene expression (4 h), whereas after 8 h, Ao gene expression increased in a time-dependent manner. Blockade of JNK1/2 with SP600125 increased basal Ao gene expression in NRVM (10.52+/-1.98 fold, P<0.001) and NRFB (13.32+/-2.07 fold, P<0.001). Adenovirus-mediated expression of wild-type JNK1 significantly inhibited, whereas expression of dominant-negative JNK1 and JNK2 increased basal and stretch-mediated (24 h) Ao gene expression, showing both JNK1 and JNK2 to be negative regulators of Ao gene expression in NRVM and NRFB. Blockade of p38alpha/beta by SB202190 or p38alpha by SB203580 significantly inhibited stretch-induced (24 h) Ao gene expression, whereas expression of wild-type p38alpha increased stretch-induced Ao gene expression in both NRVM (8.41+/-1.50 fold, P<0.001) and NRFB (3.39+/-0.74 fold, P<0.001). Conversely, expression of dominant-negative p38alpha significantly inhibited stretch response. Moreover, expression of constitutively active MKK6b (E) significantly stimulated Ao gene expression in the absence of stretch, indicating that p38 activation alone is sufficient to induce Ao gene expression. Taken together p38alpha was demonstrated to be a positive regulator, whereas JNK1/2 was found to be a negative regulator of Ao gene expression. Prolonged stretch diminished JNK1/2 activation, which was accompanied by a reciprocal increase in p38 activation and Ao gene expression. This suggests that a balance in JNK1/2 and p38alpha activation determines the level of Ao gene expression in myocardial cells.

Isolation of cardiac myocytes and fibroblasts from neonatal rat pups.

Neonatal rat ventricular myocytes (NRVM) and fibroblasts (FBs) serve as in vitro models for studying fundamental mechanisms underlying cardiac pathologies, as well as identifying potential therapeutic targets. Both cell types are relatively easy to culture as monolayers and can be manipulated using molecular and pharmacological tools. Because NRVM cease to proliferate after birth, and FBs undergo phenotypic changes and senescence after a few passages in tissue culture, primary cultures of both cell types are required for experiments. Below we describe methods that provide good cell yield and viability of primary cultures of NRVM and FBs from 0 to 3-day-old neonatal rat pups.

Modulation of myocardial mitochondrial mechanisms during severe polymicrobial sepsis in the rat.

Background We tested the hypothesis that 5-Hydroxydecanoic acid (5HD), a putative mitoKATP channel blocker, will reverse sepsis-induced cardiodynamic and adult rat ventricular myocyte (ARVM) contractile dysfunction, restore mitochondrial membrane permeability alterations and improve survival. Methodology/Principal Findings Male Sprague-Dawley rats (350–400 g) were made septic using 400 mg/kg cecal inoculum, ip. Sham animals received 5% dextrose water, ip. The Voltage Dependent Anion Channels (VDAC1), Bax and cytochrome C levels were determined in isolated single ARVMs obtained from sham and septic rat heart. Mitochondria and cytosolic fractions were isolated from ARVMs treated with norepinephrine (NE, 10 µmoles) in the presence/absence of 5HD (100 µmoles). A continuous infusion of 5HD using an Alzet pump reversed sepsis-induced mortality when administered at the time of induction of sepsis (−40%) and at 6 hr post-sepsis (−20%). Electrocardiography revealed that 5HD reversed sepsis-induced decrease in the average ejection fraction, Simpsons+m Mode (53.5±2.5 in sepsis and 69.2±1.2 at 24 hr in sepsis+5HD vs. 79.9±1.5 basal group) and cardiac output (63.3±1.2 mL/min sepsis and 79.3±3.9 mL/min at 24 hr in sepsis+5HD vs. 85.8±1.5 mL/min basal group). The treatment of ARVMs with 5HD also reversed sepsis-induced depressed contractility in both the vehicle and NE-treated groups. Sepsis produced a significant downregulation of VDAC1, and upregulation of Bax levels, along with mitochondrial membrane potential collapse in ARVMs. Pretreatment of septic ARVMs with 5HD blocked a NE-induced decrease in the VDAC1 and release of cytochrome C. Conclusion The data suggest that Bax activation is an upstream event that may precede the opening of the mitoKATP channels in sepsis. We concluded that mitoKATP channel inhibition via decreased mitochondrial membrane potential and reduced release of cytochrome C provided protection against sepsis-induced ARVM and myocardial contractile dysfunction.

Molecular Signaling Mechanisms of Myocardial Stretch: Implications for Heart Disease

With every heartbeat, myocardial cells are subjected to substantial mechanical stretch. Stretch is a potent stimulus for growth, differentiation, migration, remodeling and gene expression. Mechanical load is a major cause of cardiac hypertrophy. Since the initial observation of stretch-induced growth, our understanding of this complex field has been steadily growing, but remains incomplete. The mechanisms by which myocardial cells convert mechanical stimuli into biochemical signals that result in physiologic and pathological changes remain to be completely understood. Integrins, caveolae and focal adhesions have been shown to have important mechanosensing roles in cardiac myocytes. Downstream effectors activated by mechanosensors include guanine-nucleotide binding proteins (G-proteins), mitogen-activated protein (MAP) kinases, Janus-associated kinase/signal transducers and activators of transcription (JAK/Stat), protein kinase C (PKC) and protein kinase B/Akt pathways. Multiple levels of crosstalk exist between these pathways. Early studies have implicated most of these pathways in cardiac injury and growth response, however, more recent advancements in the development of kinase-specific inhibitors and genetically-engineered animal models have revealed significant new insights. Recent studies suggest that acute mechanical stretch activates protective pathways including c-jun N-terminal kinase (JNK) and Akt as a tolerance response, rather than injury-related signaling cascades such as p38 MAP kinase. However, chronic stretch/mechanical load creates an imbalance that favors the injury related pathway by an unknown mechanism in the myocardium. The following chapter provides an overview of the fundamental processes of stretch-activated mechano-signaling in myocardial cells, and recent advances in our understanding of this increasingly important field.

Production of Spontaneously Beating Neonatal Rat Heart Tissue for Calcium and Contractile Studies

Neonatal rat ventricular myocytes (NRVM) and fibroblasts (FB) serve as in vitro models for studying fundamental mechanisms underlying cardiac pathologies, as well as identifying potential therapeutic targets. Typically, these cell types are separated using Percoll density gradient procedures. Cells located between the Percoll bands (interband cells [IBCs]), which contain less mature NRVM and a variety of non-myocytes, including coronary vascular smooth muscle cells and endothelial cells (ECs), are routinely discarded. However, we have demonstrated that IBCs readily attach to extracellular matrix-coated coverslips, plastic culture dishes, and deformable membranes to form a 2-dimensional cardiac tissue layer which quickly develops spontaneous contraction within 24 h, providing a robust coculture model for the study of cell-to-cell signaling and contractile studies. Below, we describe methods that provide good cell yield and viability of IBCs during isolation of NRVM and FB obtained from 0- to 3-day-old neonatal rat pups. Basic characterization of IBCs and methods for use in intracellular calcium and contractile experiments are also presented. This method maximizes the use of cells obtained from neonatal rat hearts.

Caveolin and β1-integrin coordinate angiotensinogen expression in cardiac myocytes.

BACKGROUND The cardiac renin-angiotensin system (RAS) has been implicated in mediating myocyte hypertrophy and remodeling, although the biochemical mechanisms responsible for regulating the local RAS are poorly understood. Caveolin-1 (Cav-1)/Cav-3 double-knockout mice display cardiac hypertrophy, and in vitro disruption of lipid rafts/caveolae using methyl-β-cyclodextrin (MβCD) abolishes cardiac protection. METHODS In this study, neonatal rat ventricular myocytes (NRVM) were used to determine whether lipid rafts/caveolae may be involved in the regulation of angiotensinogen (Ao) gene expression, a substrate of the RAS system. RESULTS Treatment with MβCD caused a time-dependent upregulation of Ao gene expression, which was associated with differential regulation of mitogen-activated protein (MAP) kinases ERK1/2, p38 and JNK phosphorylation. JNK was highly phosphorylated shortly after MβCD treatment (2-30 min), whereas marked activation of ERK1/2 and p38 occurred much later (2-4h). β1D-Integrin was required for MβCD-induced activation of the MAP kinases. Pharmacologic inhibition of ERK1/2 and JNK enhanced MβCD-induced Ao gene expression, whereas p38 blockade inhibited this response. Adenovirus-mediated expression of wild-type p38α enhanced MβCD-induced Ao gene expression; conversely expression of dominant negative p38α blocked the stimulatory effects of MβCD. Expression of Cav-3 siRNA stimulated Ao gene expression, whereas overexpression of Cav-3 was inhibitory. Cav-1 and Cav-3 expression levels were found to be positively regulated by p38, but unaffected by ERK1/2 and JNK. CONCLUSION Collectively, these studies indicate that lipid rafts/caveolae couple to Ao gene expression through a mechanism that involves β1-integrin and the differential actions of MAP kinase family members.

Norepinephrine induces systolic failure and inhibits antiapoptotic genes in a polymicrobial septic rat model

AIMS We examined the effect of norepinephrine (NE) infusion on left ventricular function and apoptotic genes during progression of polymicrobial sepsis. METHODS Male Sprague-Dawley rats (350-400 g) were made septic by intraperitoneal (i.p.) administration of 200mg/kg cecal inoculum. Sham animals received 5% dextrose water, i.p. Echocardiography was performed at baseline, 3 days and 7 days post-sepsis/sham. NE (0.6 μgkg(-1)h(-1)) was infused for 2h, before the end of day 3 of echocardiography. At the end of day 7, rats were euthanized and heart tissues harvested for isolation of total RNA. PCR was performed using RT(2) profiler™ PCR array PARN-012 (Rat apoptosis array; SuperArray, MD) using RT(2) Real-Time™ SYBR Green PCR master mix PA-012. KEY FINDINGS NE-infusion resulted in a significant decrease in the left ventricular ejection fraction (EF) (62.56±2.07 from the baseline 71.11±3.23, p<0.05) and fractional shortening (FS) (39.90±2.64 from the sham group 54.41±2.19, p<0.05) at 7 days post-sepsis, respectively. Super Array data revealed that during sepsis, tumor necrosis factor (TNF-α) (2.85±0.07 fold, p<0.0001), anti-apoptotic molecules, Prok2 (16.07±0.48 fold, p<0.0001) and interleukin-10 (IL-10) (23.5±0.57 fold, p<0.0001) were up regulated at day 1. At 7-days post-sepsis, CD40l g (2.49±0.54 fold, p<0.08) and Birc1b (17.8±0.58 fold, p<0.0001) were up regulated compared to the sham, 1 and 3-days post-sepsis groups. SIGNIFICANCE The data suggest that upregulation of a series of pro-apoptotic molecules could be responsible for systolic and diastolic dysfunction during 3 and 7 days post sepsis.

Anthrax lethal toxin induces acute diastolic dysfunction in rats through disruption of the phospholamban signaling network

BACKGROUND Anthrax lethal toxin (LT), secreted by Bacillus anthracis, causes severe cardiac dysfunction by unknown mechanisms. LT specifically cleaves the docking domains of MAPKK (MEKs); thus, we hypothesized that LT directly impairs cardiac function through dysregulation of MAPK signaling mechanisms. METHODS AND RESULTS In a time-course study of LT toxicity, echocardiography revealed acute diastolic heart failure accompanied by pulmonary regurgitation and left atrial dilation in adult Sprague-Dawley rats at time points corresponding to dysregulated JNK, phospholamban (PLB) and protein phosphatase 2A (PP2A) myocardial signaling. Using isolated rat ventricular myocytes, we identified the MEK7-JNK1-PP2A-PLB signaling axis to be important for regulation of intracellular calcium (Ca(2+)(i)) handling, PP2A activation and targeting of PP2A-B56α to Ca(2+)(i) handling proteins, such as PLB. Through a combination of gain-of-function and loss-of-function studies, we demonstrated that over-expression of MEK7 protects against LT-induced PP2A activation and Ca(2+)(i) dysregulation through activation of JNK1. Moreover, targeted phosphorylation of PLB-Thr(17) by Akt improved sarcoplasmic reticulum Ca(2+)(i) release and reuptake during LT toxicity. Co-immunoprecipitation experiments further revealed the pivotal role of MEK7-JNK-Akt complex formation for phosphorylation of PLB-Thr(17) during acute LT toxicity. CONCLUSIONS Our findings support a cardiogenic mechanism of LT-induced diastolic dysfunction, by which LT disrupts JNK1 signaling and results in Ca(2+)(i) dysregulation through diminished phosphorylation of PLB by Akt and increased dephosphorylation of PLB by PP2A. Integration of the MEK7-JNK1 signaling module with Akt represents an important stress-activated signalosome that may confer protection to sustain cardiac contractility and maintain normal levels of Ca(2+)(i) through PLB-T(17) phosphorylation.