Gerald W. Dorn

β 1 -adrenergic receptor polymorphisms confer differential function and predisposition to heart failure

Catecholamines stimulate cardiac contractility through β1-adrenergic receptors (β1-ARs), which in humans are polymorphic at amino acid residue 389 (Arg/Gly). We used cardiac-targeted transgenesis in a mouse model to delineate mechanisms accounting for the association of Arg389 with human heart failure phenotypes. Hearts from young Arg389 mice had enhanced receptor function and contractility compared with Gly389 hearts. Older Arg389 mice displayed a phenotypic switch, with decreased β-agonist signaling to adenylyl cyclase and decreased cardiac contractility compared with Gly 389 hearts. Arg389 hearts had abnormal expression of fetal and hypertrophy genes and calcium-cycling proteins, decreased adenylyl cyclase and Gαs expression, and fibrosis with heart failure This phenotype was recapitulated in homozygous, end-stage, failing human hearts. In addition, hemodynamic responses to β-receptor blockade were greater in Arg389 mice, and homozygosity for Arg389 was associated with improvement in ventricular function during carvedilol treatment in heart failure patients. Thus the human Arg389 variant predisposes to heart failure by instigating hyperactive signaling programs leading to depressed receptor coupling and ventricular dysfunction, and influences the therapeutic response to β-receptor blockade.

Cardiac-specific Overexpression of Mouse Cardiac Calsequestrin Is Associated with Depressed Cardiovascular Function and Hypertrophy in Transgenic Mice

Calsequestrin is a high capacity Ca2+-binding protein in the sarcoplasmic reticulum (SR) lumen. To elucidate the functional role of calsequestrin in vivo, transgenic mice were generated that overexpressed mouse cardiac calsequestrin in the heart. Overexpression (20-fold) of calsequestrin was associated with cardiac hypertrophy and induction of a fetal gene expression program. Isolated transgenic cardiomyocytes exhibited diminished shortening fraction (46%), shortening rate (60%), and relengthening rate (60%). The Ca2+ transient amplitude was also depressed (45%), although the SR Ca2+storage capacity was augmented, as suggested by caffeine application studies. These alterations were associated with a decrease in L-type Ca2+ current density and prolongation of this channel’s inactivation kinetics without changes in Na+-Ca2+ exchanger current density. Furthermore, there were increases in protein levels of SR Ca2+-ATPase, phospholamban, and calreticulin and decreases in FKBP12, without alterations in ryanodine receptor, junctin, and triadin levels in transgenic hearts. Left ventricular function analysis in Langendorff perfused hearts and closed-chest anesthetized mice also indicated depressed rates of contraction and relaxation of transgenic hearts. These findings suggest that calsequestrin overexpression is associated with increases in SR Ca2+ capacity, but decreases in Ca2+-induced SR Ca2+ release, leading to depressed contractility in the mammalian heart.

Gq Signaling in Cardiac Adaptation and Maladaptation

Accumulating evidence suggests that cardiac responses to a number of circulating or locally released humoral factors contribute to adaptive responses after hemodynamic stress or myocardial injury. In particular, hormones such as angiotensin II, endothelin 1, norepinephrine and prostaglandin F2 alpha which bind to and activate cardiomyocyte membrane receptors coupled to the Gq class of GTP binding proteins have been implicated in the development and ultimate decompensation of cardiac hypertrophy. Herein we summarize recent developments in cultured cardiomyocyte and transgenic mouse systems which are defining the phenotypes resulting from Gq signaling events in cardiomyocytes, and which are elucidating the critical downstream mediators. Postulated roles for protein kinase C, p38 MAP kinase and jun-N terminal kinase are discussed in relation to Gq-mediated cardiomyocyte hypertrophy and apoptotic signaling. The evidence to date suggests that molecular targeting of Gq or its effectors has the potential to modify cardiac adaptive and maladaptive responses to stress or injury.

Polymorphisms of the β2-Adrenergic Receptor Determine Exercise Capacity in Patients With Heart Failure

Abstract—The β2-adrenergic receptor (β2AR) exists in multiple polymorphic forms with different characteristics. Their relevance to heart failure (HF) physiology is unknown. Cardiopulmonary exercise testing was performed on 232 compensated HF patients with a defined β2AR genotype. Patients with the uncommon Ile164 polymorphism had a lower peak Vo2 (15.0±0.9 mL · kg−1 · min−1) than did patients with Thr164 (17.9±0.9 mL · kg−1 · min−1, P<0.0001). The percentage achieved of predicted peak Vo2 was also lower in patients with Ile164 (62.3±4.5% versus 71.5±5.1%, P=0.045). The relative risk of a patient having a Vo2 ≤14 mL · kg−1 · min−1 who had Ile164 was 8.0 (P=0.009). Catheterization-based invasive exercise testing revealed depressed changes in the exercise-induced cardiac index, systemic vascular resistance, stroke volume, and Vo2 in patients with Ile164. The polymorphisms at position 16 also impacted exercise capacity: peak Vo2 for Arg16 versus Gly16 was 17.0±0.8 versus 15.6±0.5 mL · kg−1 · min−1, respe...

Transforming growth factor beta in cardiovascular development and function

Transforming growth factor betas (TGFbetas) are pleiotropic cytokines involved in many biological processes. Genetic engineering and tissue explanation studies have revealed specific non-overlapping roles for TGFbeta ligands and their signaling molecules in development and in normal function of the cardiovascular system in the adult. In the embryo, TGFbetas appear to be involved in epithelial-mesenchymal transformations (EMT) during endocardial cushion formation, and in epicardial epithelial-mesenchymal transformations essential for coronary vasculature, ventricular myocardial development and compaction. In the adult, TGFbetas are involved in cardiac hypertrophy, vascular remodeling and regulation of the renal renin-angiotensin system. The evidence for TGFbeta activities during cardiovascular development and physiologic function will be given and areas which need further investigation will be discussed.

Superinhibition of sarcoplasmic reticulum function by phospholamban induces cardiac contractile failure.

To determine whether selective impairment of cardiac sarcoplasmic reticulum (SR) Ca2+ transport may drive the progressive functional deterioration leading to heart failure, transgenic mice, overexpressing a phospholamban Val49 → Gly mutant (2-fold), which is a superinhibitor of SR Ca2+-ATPase affinity for Ca2+, were generated, and their cardiac phenotype was examined longitudinally. At 3 months of age, the increased EC50 level of SR Ca2+ uptake for Ca2+ (0.67 ± 0.09 μm) resulted in significantly higher depression of cardiomyocyte rates of shortening (57%), relengthening (31%), and prolongation of the Ca2+ signal decay time (165%) than overexpression (2-fold) of wild type phospholamban (68%, 64%, and 125%, respectively), compared with controls (100%). Echocardiography also revealed significantly depressed function and impaired β-adrenergic responses in mutant hearts. The depressed contractile parameters were associated with left ventricular remodeling, recapitulation of fetal gene expression, and hypertrophy, which progressed to dilated cardiomyopathy with interstitial tissue fibrosis and death by 6 months in males. Females also had ventricular hypertrophy at 3 months but exhibited normal systolic function up to 12 months of age. These results suggest a causal relationship between defective SR Ca2+ cycling and cardiac remodeling leading to heart failure, with a gender-dependent influence on the time course of these alterations.

Rescue of Contractile Parameters and Myocyte Hypertrophy in Calsequestrin Overexpressing Myocardium by Phospholamban Ablation

Cardiac-specific overexpression of murine cardiac calsequestrin results in depressed cardiac contractile parameters, low Ca2+-induced Ca2+ release from sarcoplasmic reticulum (SR) and cardiac hypertrophy in transgenic mice. To test the hypothesis that inhibition of phospholamban activity may rescue some of these phenotypic alterations, the calsequestrin overexpressing mice were cross-bred with phospholamban-knockout mice. Phospholamban ablation in calsequestrin overexpressing mice led to reversal of the depressed cardiac contractile parameters in Langendorff-perfused hearts or in vivo. This was associated with increases of SR Ca2+ storage, assessed by caffeine-induced Na+-Ca2+ exchanger currents. The inactivation time of the L-type Ca2+ current (I Ca), which has an inverse correlation with Ca2+-induced SR Ca2+ release, and the relation between the peak current density and half-inactivation time were also normalized, indicating a restoration in the ability ofI Ca to trigger SR Ca2+ release. The prolonged action potentials in calsequestrin overexpressing cardiomyocytes also reversed to normal upon phospholamban ablation. Furthermore, ablation of phospholamban restored the expression levels of atrial natriuretic factor and α-skeletal actin mRNA as well as ventricular myocyte size. These results indicate that attenuation of phospholamban function may prevent or overcome functional and remodeling defects in hypertrophied hearts.

Cardiac Specific Overexpression of Transglutaminase II (Gh) Results in a Unique Hypertrophy Phenotype Independent of Phospholipase C Activation

Tissue type transglutaminase (TGII, also known as Gh) has been considered a multifunctional protein, with both transglutaminase and GTPase activity. The role of the latter function, which is proposed as a coupling mechanism between α1-adrenergic receptors and phospholipase C (PLC), is not well defined. TGII was overexpressed in transgenic mice in a cardiac specific manner to delineated relevant signaling pathways and their consequences in the heart. Cardiac transglutaminase activity in the highest expressing line was ∼37-fold greater than in nontransgenic lines. However, in vivo signaling to PLC, as assessed by inositol phosphate turnover in [3H]myoinositol organ bath atrial preparations, was not increased in the TGII mice at base line or in response to α1-adrenergic receptor stimulation; nor was protein kinase Cα (PKCα) or PKCε activity enhanced in the TGII transgenic mice. This is in contrast to mice moderately (∼5-fold) overexpressing Gαq, where inositol phosphate turnover and PKC activity were found to be clearly enhanced. TGII overexpression resulted in a remodeling of the heart with mild hypertrophy, elevated expression of β-myosin heavy chain and α-skeletal actin genes, and diffuse interstitial fibrosis. Resting ventricular function was depressed, but responsiveness to β-agonist was not impaired. This set of pathophysiologic findings is distinct from that evoked by overexpression of Gαq. We conclude that TGII acts in the heart primarily as a transglutaminase, and modulation of this function results in unique pathologic sequelae. Evidence for TGII acting as a G-protein-like transducer of receptor signaling to PLC in the heart is not supported by these studies.

Mechanisms of Pharmacogenomic Effects of Genetic Variation within the Cardiac Adrenergic Network in Heart Failure

One of the goals of pharmacogenomics is the use of genetic variants to predict an individuals response to treatment. Although numerous candidate and genome-wide associations have been made for cardiovascular response-outcomes, little is known about how a given polymorphism imposes the phenotype. Such mechanisms are important, because they tie the observed human response to specific signaling alterations and thus provide cause-and-effect relationships, aid in the design of hypothesis-based clinical studies, can help to devise workaround drugs, and can reveal new aspects of the pathophysiology of the disease. Here we discuss polymorphisms within the adrenergic receptor network in the context of heart failure and β-adrenergic receptor blocker therapy, where multiple approaches to understand the mechanism have been undertaken. We propose a comprehensive series of studies, ranging from transfected cells, transgenic mice, and ex vivo and in vitro human studies as a model approach to explore mechanisms of action of pharmacogenomic effects and extend the field beyond observational associations.

Response of a human megakaryocytic cell line to thrombin: increase in intracellular free calcium and mitogen release.

The CHRF-288-11 cell line has been previously shown to exhibit properties consistent with a megakaryocytic origin. The response of these cells to thrombin has now been investigated. Thrombin treatment of CHRF-288-11 cells results in both an increase in intracellular free calcium levels and secretion of mitogenic activity and beta-thromboglobulin. Cell viability is not affected. The mitogenic activity released from the cells is due primarily to the presence of basic fibroblast growth factor. Immunohistochemical data indicate a packaging of basic fibroblast growth factor into granular structures. Trypsin and phorbol 12-myristate 13-acetate also initiate release of mitogenic activity from this cell line, whereas under non-stirred conditions collagen and ADP do not. Through measurements of intracellular calcium levels it was determined that thrombin pretreatment of cells ablates a further response to thrombin, but does not block an increase in intracellular calcium levels due to trypsin. This suggests that these two agonists may act through different mechanisms. The thrombin-induced release reaction is inhibited almost completely by the reagents hirudin and dipyridamole, and only partially by indomethacin. These data indicate that the CHRF-288-11 cell line should provide an excellent model system in which to study the packaging of factors into granules which undergo regulated release.