Cori Wijaya

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evans blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

An active triple-catalytic hybrid enzyme engineered by linking cyclo-oxygenase isoform-1 to prostacyclin synthase that can constantly biosynthesize prostacyclin, the vascular protector

It remains a challenge to achieve the stable and long‐term expression (in human cell lines) of a previously engineered hybrid enzyme [triple‐catalytic (Trip‐cat) enzyme‐2; Ruan KH, Deng H & So SP (2006) Biochemistry45, 14003–14011], which links cyclo‐oxygenase isoform‐2 (COX‐2) to prostacyclin (PGI2) synthase (PGIS) for the direct conversion of arachidonic acid into PGI2 through the enzyme’s Trip‐cat functions. The stable upregulation of the biosynthesis of the vascular protector, PGI2, in cells is an ideal model for the prevention and treatment of thromboxane A2 (TXA2)‐mediated thrombosis and vasoconstriction, both of which cause stroke, myocardial infarction, and hypertension. Here, we report another case of engineering of the Trip‐cat enzyme, in which human cyclo‐oxygenase isoform‐1, which has a different C‐terminal sequence from COX‐2, was linked to PGI2 synthase and called Trip‐cat enzyme‐1. Transient expression of recombinant Trip‐cat enzyme‐1 in HEK293 cells led to 3–5‐fold higher expression capacity and better PGI2‐synthesizing activity as compared to that of the previously engineered Trip‐cat enzyme‐2. Furthermore, an HEK293 cell line that can stably express the active new Trip‐cat enzyme‐1 and constantly synthesize the bioactive PGI2 was established by a screening approach. In addition, the stable HEK293 cell line, with constant production of PGI2, revealed strong antiplatelet aggregation properties through its unique dual functions (increasing PGI2 production while decreasing TXA2 production) in TXA2 synthase‐rich plasma. This study has optimized engineering of the active Trip‐cat enzyme, allowing it to become the first to stably upregulate PGI2 biosynthesis in a human cell line, which provides a basis for developing a PGI2‐producing therapeutic cell line for use against vascular diseases.

Enhanced Cardiac Function in Gravin Mutant Mice Involves Alterations in the β-Adrenergic Receptor Signaling Cascade

Gravin, an A-kinase anchoring protein, targets protein kinase A (PKA), protein kinase C (PKC), calcineurin and other signaling molecules to the beta2-adrenergic receptor (β2-AR). Gravin mediates desensitization/resensitization of the receptor by facilitating its phosphorylation by PKA and PKC. The role of gravin in β-AR mediated regulation of cardiac function is unclear. The purpose of this study was to determine the effect of acute β-AR stimulation on cardiac contractility in mice lacking functional gravin. Using echocardiographic analysis, we observed that contractility parameters such as left ventricular fractional shortening and ejection fraction were increased in gravin mutant (gravin-t/t) animals lacking functional protein compared to wild-type (WT) animals both at baseline and following acute isoproterenol (ISO) administration. In isolated gravin-t/t cardiomyocytes, we observed increased cell shortening fraction and decreased intracellular Ca2+ in response to 1 µmol/L ISO stimulation. These physiological responses occurred in the presence of decreased β2-AR phosphorylation in gravin-t/t hearts, where PKA-dependent β2-AR phosphorylation has been shown to lead to receptor desensitization. cAMP production, PKA activity and phosphorylation of phospholamban and troponin I was comparable in WT and gravin-t/t hearts both with and without ISO stimulation. However, cardiac myosin binding protein C (cMyBPC) phosphorylation site at position 273 was significantly increased in gravin-t/t versus WT hearts, in the absence of ISO. Additionally, the cardioprotective heat shock protein 20 (Hsp20) was significantly more phosphorylated in gravin-t/t versus WT hearts, in response to ISO. Our results suggest that disruption of gravin’s scaffold mediated signaling is able to increase baseline cardiac function as well as to augment contractility in response to acute β-AR stimulation by decreasing β2-AR phosphorylation and thus attenuating receptor desensitization and perhaps by altering PKA localization to increase the phosphorylation of cMyBPC and the nonclassical PKA substrate Hsp20.

Protein Kinase A and Phosphodiesterase-4D3 Binding to Coding Polymorphisms of Cardiac Muscle Anchoring Protein (mAKAP)

Protein kinase A (PKA) substrate phosphorylation is facilitated through its co-localization with its signaling partner by A-kinase anchoring proteins (AKAPs). mAKAP (muscle-selective AKAP) localizes PKA and its substrates such as phosphodiesterase-4D3 (PDE4D3), ryanodine receptor, and protein phosphatase 2A (PP2A) to the sarcoplasmic reticulum and perinuclear space. The genetic role of mAKAP, in modulating PKA/PDE4D3 molecular signaling during cardiac diseases, remains unclear. The purpose of this study was to examine the effects of naturally occurring mutations in human mAKAP on PKA and PDE4D3 signaling. We have recently identified potentially important human mAKAP coding non-synonymous polymorphisms located within or near key protein binding sites critical to β-adrenergic receptor signaling. Three mutations (P1400S, S2195F, and L717V) were cloned and transfected into a mammalian cell line for the purpose of comparing whether those substitutions disrupt mAKAP binding to PKA or PDE4D3. Immunoprecipitation study of mAKAP-P1400S, a mutation located in the mAKAP-PDE4D3 binding site, displayed a significant reduction in binding to PDE4D3, with no significant changes in PKA binding or PKA activity. Conversely, mAKAP-S2195F, a mutation located in mAKAP-PP2A binding site, showed significant increase in both binding propensity to PKA and PKA activity. Additionally, mAKAP-L717V, a mutation flanking the mAKAP-spectrin repeat domain, exhibited a significant increase in PKA binding compared to wild type, but there was no change in PKA activity. We also demonstrate specific binding of wild-type mAKAP to PDE4D3. Binding results were demonstrated using immunoprecipitation and confirmed with surface plasmon resonance (Biacore-2000); functional results were demonstrated using activity assays, Ca(2+) measurements, and Western blot. Comparative analysis of the binding responses of mutations to mAKAP could provide important information about how these mutations modulate signaling.

Mesp1 Marked Cardiac Progenitor Cells Repair Infarcted Mouse Hearts

Mesp1 directs multipotential cardiovascular cell fates, even though it’s transiently induced prior to the appearance of the cardiac progenitor program. Tracing Mesp1-expressing cells and their progeny allows isolation and characterization of the earliest cardiovascular progenitor cells. Studying the biology of Mesp1-CPCs in cell culture and ischemic disease models is an important initial step toward using them for heart disease treatment. Because of Mesp1’s transitory nature, Mesp1-CPC lineages were traced by following EYFP expression in murine Mesp1Cre/+; Rosa26EYFP/+ ES cells. We captured EYFP+ cells that strongly expressed cardiac mesoderm markers and cardiac transcription factors, but not pluripotent or nascent mesoderm markers. BMP2/4 treatment led to the expansion of EYFP+ cells, while Wnt3a and Activin were marginally effective. BMP2/4 exposure readily led EYFP+ cells to endothelial and smooth muscle cells, but inhibition of the canonical Wnt signaling was required to enter the cardiomyocyte fate. Injected mouse pre-contractile Mesp1-EYFP+ CPCs improved the survivability of injured mice and restored the functional performance of infarcted hearts for at least 3 months. Mesp1-EYFP+ cells are bona fide CPCs and they integrated well in infarcted hearts and emerged de novo into terminally differentiated cardiac myocytes, smooth muscle and vascular endothelial cells.

Force development and intracellular Ca2+ in intact cardiac muscles from gravin mutant mice ☆

Abstract Gravin (AKAP12) is an A‐kinase‐anchoring‐protein that scaffolds protein kinase A (PKA), &bgr;2‐adrenergic receptor (&bgr;2‐AR), protein phosphatase 2B and protein kinase C. Gravin facilitates &bgr;2‐AR‐dependent signal transduction through PKA to modulate cardiac excitation‐contraction coupling and its removal positively affects cardiac contraction. Trabeculae from the right ventricles of gravin mutant (gravin‐t/t) mice were employed for force determination. Simultaneously, corresponding intracellular Ca2+ transient ([Ca2+]i) were measured. Twitch force (Tf)‐interval relationship, [Ca2+]i‐interval relationship, and the rate of decay of post‐extrasysolic potentiation (Rf) were also obtained. Western blot analysis were performed to correlate sarcomeric protein expression with alterations in calcium cycling between the WT and gravin‐t/t hearts. Gravin‐t/t muscles had similar developed force compared to WT muscles despite having lower [Ca2+]i at any given external Ca2+ concentration ([Ca2+]o). The time to peak force and peak [Ca2+]i were slower and the time to 75% relaxation was significantly prolonged in gravin‐t/t muscles. Both Tf‐interval and [Ca2+]i‐interval relations were depressed in gravin‐t/t muscles. Rf, however, did not change. Furthermore, Western blot analysis revealed decreased ryanodine receptor (RyR2) phosphorylation in gravin‐t/t hearts. Gravin‐t/t cardiac muscle exhibits increased force development in responsiveness to Ca2+. The Ca2+ cycling across the SR appears to be unaltered in gravin‐t/t muscle. Our study suggests that gravin is an important component of cardiac contraction regulation via increasing myofilament sensitivity to calcium. Further elucidation of the mechanism can provide insights to role of gravin if any in the pathophysiology of impaired contractility.

Human Signaling Scaffold Protein (mAKAP) Binding Kinetics to PKA and Phosphodiesterase (PDE4DE): Implications for a Possible Role in Heart Failure

Heart failure is a leading cause of morbidity and mortality in the USA. There are several therapeutic agents available for heart failure management. In particular, agents that block beta-adrenergic receptor improve mortality rate among heart failure patients by enhancing cardiac function. Beta-adrenergic receptor stimulation signals through protein kinase A (PKA) dependent phosphorylation, partly by binding to A-kinase anchoring proteins, influencing calcium homeostasis. In particular, mAKAP (muscle-selective A-kinase anchoring protein) is targeted to specific intracellular compartments resulting in localization of PKA with its substrates as well as to bind with ryanodine receptors and phosphodiesterase-4D3 (PDE4DE). The signal transduction complex formed by the scaffold protein mAKAP at the perinuclear envelop of striated myocytes contains cAMP specific binding protein PDE4D3 which is responsible for cAMP signaling termination. Agents that modify PKA signaling would be expected to mediate an altered inotropic response. From different genomic databases, we have recently identified fifteen human mAKAP coding non-synonymous polymorphisms located within or near key protein binding sites critical to beta-adrenergic receptors signaling. Seven of these mutants were cloned for the purpose of comparing whether those substitution disrupt mAKAP binding to either the PKA binding domain R2alpha or the phosphodiesterase PDE4DE. Using surface plasmon resonance (Biacore 2000) we demonstrate specific binding of wild type mAKAP to PDE4DE. Experiments were run in triplicate and as twofold serial dilutions to explore the kinetics of the interaction and analyzed using Scrubber2 with a 1:1 Langmuir model. Comparative analysis of the binding responses of mutations to mAKAP could provide important information about how these mutations modulate signaling.

An active triple-catalytic hybrid enzyme engineered by linking COX-1 to prostacyclin synthase that can constantly biosynthesize prostacyclin, the vascular protector

It remains a challenge to achieve the stable and long‐term expression (in human cell lines) of a previously engineered hybrid enzyme [triple‐catalytic (Trip‐cat) enzyme‐2; Ruan KH, Deng H & So SP (2006) Biochemistry45, 14003–14011], which links cyclo‐oxygenase isoform‐2 (COX‐2) to prostacyclin (PGI2) synthase (PGIS) for the direct conversion of arachidonic acid into PGI2 through the enzyme’s Trip‐cat functions. The stable upregulation of the biosynthesis of the vascular protector, PGI2, in cells is an ideal model for the prevention and treatment of thromboxane A2 (TXA2)‐mediated thrombosis and vasoconstriction, both of which cause stroke, myocardial infarction, and hypertension. Here, we report another case of engineering of the Trip‐cat enzyme, in which human cyclo‐oxygenase isoform‐1, which has a different C‐terminal sequence from COX‐2, was linked to PGI2 synthase and called Trip‐cat enzyme‐1. Transient expression of recombinant Trip‐cat enzyme‐1 in HEK293 cells led to 3–5‐fold higher expression capacity and better PGI2‐synthesizing activity as compared to that of the previously engineered Trip‐cat enzyme‐2. Furthermore, an HEK293 cell line that can stably express the active new Trip‐cat enzyme‐1 and constantly synthesize the bioactive PGI2 was established by a screening approach. In addition, the stable HEK293 cell line, with constant production of PGI2, revealed strong antiplatelet aggregation properties through its unique dual functions (increasing PGI2 production while decreasing TXA2 production) in TXA2 synthase‐rich plasma. This study has optimized engineering of the active Trip‐cat enzyme, allowing it to become the first to stably upregulate PGI2 biosynthesis in a human cell line, which provides a basis for developing a PGI2‐producing therapeutic cell line for use against vascular diseases.

An active triple-catalytic hybrid enzyme engineered by linking cyclo-oxygenase isoform-1 to prostacyclin synthase that can constantly biosynthesize prostacyclin, the vascular protector: Prostacyclin-synthesizing protein with COX-1 and PGIS properties

It remains a challenge to achieve the stable and long‐term expression (in human cell lines) of a previously engineered hybrid enzyme [triple‐catalytic (Trip‐cat) enzyme‐2; Ruan KH, Deng H & So SP (2006) Biochemistry45, 14003–14011], which links cyclo‐oxygenase isoform‐2 (COX‐2) to prostacyclin (PGI2) synthase (PGIS) for the direct conversion of arachidonic acid into PGI2 through the enzyme’s Trip‐cat functions. The stable upregulation of the biosynthesis of the vascular protector, PGI2, in cells is an ideal model for the prevention and treatment of thromboxane A2 (TXA2)‐mediated thrombosis and vasoconstriction, both of which cause stroke, myocardial infarction, and hypertension. Here, we report another case of engineering of the Trip‐cat enzyme, in which human cyclo‐oxygenase isoform‐1, which has a different C‐terminal sequence from COX‐2, was linked to PGI2 synthase and called Trip‐cat enzyme‐1. Transient expression of recombinant Trip‐cat enzyme‐1 in HEK293 cells led to 3–5‐fold higher expression capacity and better PGI2‐synthesizing activity as compared to that of the previously engineered Trip‐cat enzyme‐2. Furthermore, an HEK293 cell line that can stably express the active new Trip‐cat enzyme‐1 and constantly synthesize the bioactive PGI2 was established by a screening approach. In addition, the stable HEK293 cell line, with constant production of PGI2, revealed strong antiplatelet aggregation properties through its unique dual functions (increasing PGI2 production while decreasing TXA2 production) in TXA2 synthase‐rich plasma. This study has optimized engineering of the active Trip‐cat enzyme, allowing it to become the first to stably upregulate PGI2 biosynthesis in a human cell line, which provides a basis for developing a PGI2‐producing therapeutic cell line for use against vascular diseases.