John A. Schoenhard

Regulation of the PAI-1 promoter by circadian clock components: differential activation by BMAL1 and BMAL2.

Circadian variation in plasminogen activator inhibitor-1 (PAI-1) production likely contributes to increased risk of myocardial infarction and decreased efficacy of thrombolytic therapy during the morning. In this study, we characterize the abilities of fundamental molecular components of intrinsic circadian clocks to regulate the human PAI-1 promoter in transfected endothelial cells. Both CLOCK:BMAL1 and CLOCK:BMAL2 heterodimers activate the PAI-1 promoter through requisite proximal (-565 to -560 bp) and distal (-680 to -675 bp) E-box enhancers. Although the distal E-box overlaps the 4G/5G polymorphism of the PAI-1 promoter, allelic variation at this site does not influence CLOCK:BMAL1-and CLOCK:BMAL2-mediated transactivation. Together, CLOCK:BMAL1 and CLOCK:BMAL2 make additive contributions to PAI-1 gene transcription. While the abilities of these heterodimers to activate gene expression differ by twofold, the susceptibilities of these circadian activators to inhibition by period and cryptochrome proteins are equivalent and redox independent. Given that BMAL1 and BMAL2 differ in their spatiotemporal distributions, such distinctions may allow intrinsic circadian clocks to modulate the amplitudes of their oscillators, while maintaining circadian periodicity. In this way, fundamental circadian clock components may drive circadian variation in PAI-1, which in turn influences the pathogenesis, timing, and treatment of acute atherothrombotic events.

Functional modeling in zebrafish demonstrates that the atrial-fibrillation-associated gene GREM2 regulates cardiac laterality, cardiomyocyte differentiation and atrial rhythm

SUMMARY Atrial fibrillation (AF) is the most common cardiac arrhythmia and carries a significant risk of stroke and heart failure. The molecular etiologies of AF are poorly understood, leaving patients with limited therapeutic options. AF has been recognized as an inherited disease in almost 30% of patient cases. However, few genetic loci have been identified and the mechanisms linking genetic variants to AF susceptibility remain unclear. By sequencing 193 probands with lone AF, we identified a Q76E variant within the coding sequence of the bone morphogenetic protein (BMP) antagonist gremlin-2 (GREM2) that increases its inhibitory activity. Functional modeling in zebrafish revealed that, through regulation of BMP signaling, GREM2 is required for cardiac laterality and atrial differentiation during embryonic development. GREM2 overactivity results in slower cardiac contraction rates in zebrafish, and induction of previously identified AF candidate genes encoding connexin-40, sarcolipin and atrial natriuretic peptide in differentiated mouse embryonic stem cells. By live heart imaging in zebrafish overexpressing wild-type or variant GREM2, we found abnormal contraction velocity specifically in atrial cardiomyocytes. These results implicate, for the first time, regulators of BMP signaling in human AF, providing mechanistic insights into the pathogenesis of the disease and identifying potential new therapeutic targets.

Stem Cell Therapy: Pieces of the Puzzle

Acute ischemic injury and chronic cardiomyopathies can cause irreversible loss of cardiac tissue leading to heart failure. Cellular therapy offers a new paradigm for treatment of heart disease. Stem cell therapies in animal models show that transplantation of various cell preparations improves ventricular function after injury. The first clinical trials in patients produced some encouraging results, despite limited evidence for the long-term survival of transplanted cells. Ongoing research at the bench and the bedside aims to compare sources of donor cells, test methods of cell delivery, improve myocardial homing, bolster cell survival, and promote cardiomyocyte differentiation. This article reviews progress toward these goals.

Male-female differences in the genetic regulation of t-PA and PAI-1 levels in a Ghanaian population.

Tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) directly influence thrombus formation and degradation, and have been identified as risk factors for thromboembolic disease. Prior studies investigated determinants of t-PA and PAI-1 expression, but mainly in Caucasian subjects. The aim of this study was to identify the contributions of genetic and other factors to inter-individual variation in plasma levels of t-PA and PAI-1 in a large-scale population-based sample from urban West Africa. t-PA, PAI-1 and several demographic, anthropometric, and metabolic parameters were measured in 992 residents of Sunyani, the capital of the Brong-Ahafo region of Ghana. In addition, nine gene polymorphisms associated with components of the renin-angiotensin and fibrinolytic systems were determined. We found that BMI, systolic and diastolic blood pressure, total cholesterol, glucose, and triglycerides were all significant predictors of t-PA and PAI-1 in both females and males. In addition, a significant relationship was found between the PAI-1 4G/5G (rs1799768) polymorphism on PAI-1 levels in females, the TPA I/D (rs4646972) polymorphism on t-PA and PAI-1 in males, the renin (rs3730103) polymorphism on t-PA and PAI-1 in males, the ethanolamine kinase 2 (rs1917542) polymorphism on PAI-1 in males, and the renin (rs1464816) polymorphism on t-PA in females and on PAI-1 in males. This study of urban West Africans shows that t-PA and PAI-1 levels are determined by both genetic loci of the fibrinolytic and renin-angiotensin systems and other factors often associated with cardiovascular disease, and that genetic factors differ between males and females.

Tissue- and agonist-specific regulation of human and murine plasminogen activator inhibitor-1 promoters in transgenic mice

Summary.  Numerous studies have described regulatory factors and sequences that control transcriptional responses in vitro. However, there is a paucity of information on the qualitative and quantitative regulation of heterologous promoters using transgenic strategies. In order to investigate the physiological regulation of human plasminogen activator inhibitor type‐1 (hPAI‐1) expression in vivo compared to murine PAI‐1 (mPAI‐1) and to test the physiological relevance of regulatory mechanisms described in vitro, we generated transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the proximal −2.9 kb of the hPAI‐1 promoter. Transgenic animals were treated with Ang II, TGF‐β1 and lipopolysaccharide (LPS) to compare the relative activation of the human and murine PAI‐1 promoters. Ang II increased EGFP expression most effectively in brain, kidney and spleen, while mPAI‐1 expression was quantitatively enhanced most prominently in heart and spleen. TGF‐β1 failed to induce activation of the hPAI‐1 promoter but potently stimulated mPAI‐1 in kidney and spleen. LPS administration triggered robust expression of mPAI‐1 in liver, kidney, pancreas, spleen and lung, while EGFP was induced only modestly in heart and kidney. These results indicate that the transcriptional response of the endogenous mPAI‐1 promoter varies widely in terms of location and magnitude of response to specific stimuli. Moreover, the physiological regulation of PAI‐1 expression likely involves a complex interaction of transcription factors and DNA sequences that are not adequately replicated by in vitro functional studies focused on the proximal −2.9 kb promoter.

The clinical pharmacology of eplerenone.

Eplerenone is an aldosterone receptor antagonist indicated for the treatment of hypertension and congestive heart failure. Eplerenone contains an epoxy group, which offers greater mineralocorticoid receptor specificity. It is an effective antihypertensive that has been shown to reduce morbidity and mortality in individuals with left ventricular dysfunction post myocardial infarction. Studies are continuing to determine whether the benefit of mineralocorticoid receptor blockade in advanced congestive heart failure is also observed when eplerenone treatment is initiated in earlier stages of the disease. The most common side effect is hyperkalemia necessitating close monitoring in individuals with diabetes and proteinuria, heart failure or in those who are taking moderate CYP450 3A4 inhibitors. It is category B in pregnancy.

BMP Antagonist Gremlin 2 Limits Inflammation After Myocardial Infarction

RATIONALE We have recently shown that the bone morphogenetic protein (BMP) antagonist Gremlin 2 (Grem2) is required for early cardiac development and cardiomyocyte differentiation. Our initial studies discovered that Grem2 is strongly induced in the adult heart after experimental myocardial infarction (MI). However, the function of Grem2 and BMP-signaling inhibitors after cardiac injury is currently unknown. OBJECTIVE To investigate the role of Grem2 during cardiac repair and assess its potential to improve ventricular function after injury. METHODS AND RESULTS Our data show that Grem2 is transiently induced after MI in peri-infarct area cardiomyocytes during the inflammatory phase of cardiac tissue repair. By engineering loss- (Grem2(-/-)) and gain- (TG(Grem2)) of-Grem2-function mice, we discovered that Grem2 controls the magnitude of the inflammatory response and limits infiltration of inflammatory cells in peri-infarct ventricular tissue, improving cardiac function. Excessive inflammation in Grem2(-/-) mice after MI was because of overactivation of canonical BMP signaling, as proven by the rescue of the inflammatory phenotype through administration of the canonical BMP inhibitor, DMH1. Furthermore, intraperitoneal administration of Grem2 protein in wild-type mice was sufficient to reduce inflammation after MI. Cellular analyses showed that BMP2 acts with TNFα to induce expression of proinflammatory proteins in endothelial cells and promote adhesion of leukocytes, whereas Grem2 specifically inhibits the BMP2 effect. CONCLUSIONS Our results indicate that Grem2 provides a molecular barrier that controls the magnitude and extent of inflammatory cell infiltration by suppressing canonical BMP signaling, thereby providing a novel mechanism for limiting the adverse effects of excessive inflammation after MI.

Arrhythmogenic Munchausen syndrome culminating in caffeine-induced ventricular tachycardia.

We report a patient with numerous abnormal electrocardiograms (ECGs) in both inpatient and outpatient settings. Our patient both simulated and stimulated her arrhythmias with an ECG rhythm generator and intentional caffeine intoxication. To our knowledge, this is the first report of caffeine overdose for arrhythmogenesis.