Anne M. Murphy

Animal models of heart failure: a scientific statement from the American Heart Association.

Heart failure (HF) is a leading cause of morbidity and mortality in the United States. Despite a number of important therapeutic advances for the treatment of symptomatic HF,1 the prevalence, mortality, and cost associated with HF continue to grow in the United States and other developed countries. Given the aging of our population and the prevalence of diseases such as diabetes mellitus and hypertension that predispose patients to this syndrome, it is possible that HF prevalence will increase in the next decade. Current treatments primarily slow the progression of this syndrome, and there is a need to develop novel preventative and reparative therapies. Development of these novel HF therapies requires testing of the putative therapeutic strategies in appropriate HF animal models. The purposes of this scientific statement are to define the distinctive clinical features of the major causes of HF in humans and to recommend those distinctive pathological features of HF in humans that should be present in an animal model being used to identify fundamental causes of HF or to test preventative or reparative therapies that could reduce HF morbidity and mortality. HF is a clinical syndrome with primary symptoms including dyspnea, fatigue, exercise intolerance, and retention of fluid in the lungs and peripheral tissues. The causes of HF are myriad, but the common fundamental defect is a decreased ability of the heart to provide sufficient cardiac output to support the normal functions of the tissues because of impaired filling and/or ejection of blood. HF is a significant health burden in both the developed world and in emerging nations. In the United States, over a half million new diagnoses of HF occur each year, and the prevalence is 5.8 million individuals >20 years of age.1 HF has a substantial societal burden, with yearly costs in the United …

Regional cortical white matter reductions in velocardiofacial syndrome: a volumetric MRI analysis

BACKGROUND Velocardiofacial syndrome, caused by a microdeletion on chromosome 22q.11, is associated with craniofacial anomalies, cardiac defects, learning disabilities, and psychiatric disorders. To understand how the 22q.11 deletion affects brain development, this study examined gray and white matter volumes in major lobar brain regions of children with velocardiofacial syndrome relative to control subjects. METHODS Subjects were ten children with velocardiofacial syndrome and ten age- and gender-matched unaffected children. Coronal images were acquired with a 3-D spoiled gradient echo series and partitioned into 124, 1.5-mm contiguous slices. A stereotaxic grid was used to subdivide brain tissue into cerebral lobes, which were segmented into gray, white, and CSF compartments using an algorithm based on intensity values and tissue boundaries. Nonparametric statistics were used to compare lobar volumes of gray and white matter. RESULTS Analyses indicated that children with velocardiofacial syndrome had significantly smaller volumes in nonfrontal, but not frontal, lobar brain regions. Volume reductions affected nonfrontal white matter to a greater extent than nonfrontal gray matter. CONCLUSIONS The presence of white matter reductions may be related to disturbances in myelination or axonal integrity in velocardiofacial syndrome. Further work is required to delineate the nature and extent of white matter anomalies, and to link them to variation in the neurocognitive and neuropsychiatric phenotype of velocardiofacial syndrome.

Frequency- and Afterload-Dependent Cardiac Modulation In Vivo by Troponin I With Constitutively Active Protein Kinase A Phosphorylation Sites

Abstract— Acute &bgr;-adrenergic stimulation enhances cardiac contractility, accelerates muscle relaxation, and amplifies the inotropic and lusitropic response to increased stimulation frequency. These effects are modulated by phosphorylation of calcium handling and myofilament proteins such as troponin I (TnI) by protein kinase A (PKA). To more directly delineate the role of TnI PKA phosphorylation, transgenic mice were generated that overexpress cardiac TnI in which the serine residues normally targeted by PKA are mutated to aspartic acid to mimic constitutive phosphorylation (TnIDD22,23). Native cardiac TnI was near completely replaced in one transgenic line as assessed by in vitro phosphorylation, and this led to reduced calcium sensitivity of myofibrillar MgATPase, as expected. TnIDD22,23 mice had mildly enhanced basal systolic and diastolic function, and displayed marked augmentation of frequency-dependent inotropy and relaxation, with a peak frequency response 2-fold greater in mutants than controls (P <0.005). Increasing afterload prolonged relaxation more in nontransgenic than TnIDD22,23 (P <0.02), whereas contractile responses to afterload were similar between these strains. Isoproterenol treatment eliminated the differential force-frequency and afterload response between TnIDD22,23 and controls. In contrast to in vivo studies, isolated isometric trabeculae from nontransgenic and TnIDD22,23 mice had similar basal, isoproterenol-, and frequency-stimulated function, suggesting that muscle shortening may be important to TnI PKA effects. These results support a novel role for cardiac TnI PKA phosphorylation in the rate-dependent enhancement of systolic and diastolic function in vivo and afterload sensitivity of relaxation. These results have implications for cardiac failure in which force-frequency modulation is blunted and afterload relaxation sensitivity increased in association with diminished PKA TnI phosphorylation.

Proteomic Analysis of Pharmacological Preconditioning: Novel Protein Targets Converge to Mitochondrial Metabolism Pathways

Ischemic preconditioning is characterized by resistance to ischemia reperfusion injury in response to previous short ischemic episodes, a protective effect that can be mimicked pharmacologically. The underlying mechanism of protection remains controversial and requires greater understanding before it can be fully exploited therapeutically. To investigate the overall effect of preconditioning on the myocardial proteome, isolated rabbit ventricular myocytes were treated with drugs known to induce preconditioning, adenosine or diazoxide (each at 100 &mgr;mol/L for 60 minutes). Their protein profiles were then compared with vehicle-treated controls (n=4 animals per treatment) using a multitiered 2D gel electrophoresis approach. Of 28 significantly altered protein spots, 19 nonredundant proteins were identified (5 spots remained unidentified). The majority of these proteins are involved in mitochondrial energetics, including subunits of tricarboxylic acid cycle enzymes and oxidative phosphorylation complexes. These changes were not indiscriminate, with only a small number of enzymes or complex subunits altered, indicating a very specific and targeted affect of these 2 preconditioning mimetics. Among the changes were shifts in the extent of posttranslational modification of 4 proteins. One of these, the adenosine-induced phosphorylation of the ATP synthase β subunit, was fully characterized with the identification of 5 novel phosphorylation sites. This proteomics approach provides an overall assessment of the cellular response to pharmacological treatment with adenosine and diazoxide and identifies a distinct subset of enzymes and protein complex subunit that may underlie the preconditioned phenotype.

Multiple Reaction Monitoring to Identify Site-Specific Troponin I Phosphorylated Residues in the Failing Human Heart

Background— Human cardiac troponin I is known to be phosphorylated at multiple amino acid residues by several kinases. Advances in mass spectrometry allow sensitive detection of known and novel phosphorylation sites and measurement of the level of phosphorylation simultaneously at each site in myocardial samples. Methods and Results— On the basis of in silico prediction and liquid chromatography/mass spectrometry data, 14 phosphorylation sites on cardiac troponin I, including 6 novel residues (S4, S5, Y25, T50, T180, S198), were assessed in explanted hearts from end-stage heart failure transplantation patients with ischemic heart disease or idiopathic dilated cardiomyopathy and compared with samples obtained from nonfailing donor hearts (n=10 per group). Thirty mass spectrometry–based multiple reaction monitoring quantitative tryptic peptide assays were developed for each phosphorylatable and corresponding nonphosphorylated site. The results show that in heart failure there is a decrease in the extent of phosphorylation of the known protein kinase A sites (S22, S23) and other newly discovered phosphorylation sites located in the N-terminal extension of cardiac troponin I (S4, S5, Y25), an increase in phosphorylation of the protein kinase C sites (S41, S43, T142), and an increase in phosphorylation of the IT-arm domain residues (S76, T77) and C-terminal domain novel phosphorylation sites of cardiac troponin I (S165, T180, S198). In a canine dyssynchronous heart failure model, enhanced phosphorylation at 3 novel sites was found to decline toward control after resynchronization therapy. Conclusions— Selective, functionally significant phosphorylation alterations occurred on individual residues of cardiac troponin I in heart failure, likely reflecting an imbalance in kinase/phosphatase activity. Such changes can be reversed by cardiac resynchronization.

Perturbed Length-Dependent Activation in Human Hypertrophic Cardiomyopathy With Missense Sarcomeric Gene Mutations

Rationale: High-myofilament Ca2+ sensitivity has been proposed as a trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) on the basis of in vitro and transgenic mice studies. However, myofilament Ca2+ sensitivity depends on protein phosphorylation and muscle length, and at present, data in humans are scarce. Objective: To investigate whether high myofilament Ca2+ sensitivity and perturbed length-dependent activation are characteristics for human HCM with mutations in thick and thin filament proteins. Methods and Results: Cardiac samples from patients with HCM harboring mutations in genes encoding thick (MYH7, MYBPC3) and thin (TNNT2, TNNI3, TPM1) filament proteins were compared with sarcomere mutation-negative HCM and nonfailing donors. Cardiomyocyte force measurements showed higher myofilament Ca2+ sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA) targets compared with donors. After exogenous PKA treatment, myofilament Ca2+ sensitivity was similar (MYBPC3mut, TPM1mut, sarcomere mutation-negative HCM), higher (MYH7mut, TNNT2mut), or even significantly lower (TNNI3mut) compared with donors. Length-dependent activation was significantly smaller in all HCM than in donor samples. PKA treatment increased phosphorylation of PKA-targets in HCM myocardium and normalized length-dependent activation to donor values in sarcomere mutation-negative HCM and HCM with truncating MYBPC3 mutations but not in HCM with missense mutations. Replacement of mutant by wild-type troponin in TNNT2mut and TNNI3mut corrected length-dependent activation to donor values. Conclusions: High-myofilament Ca2+ sensitivity is a common characteristic of human HCM and partly reflects hypophosphorylation of PKA targets compared with donors. Length-dependent sarcomere activation is perturbed by missense mutations, possibly via posttranslational modifications other than PKA hypophosphorylation or altered protein–protein interactions, and represents a common pathomechanism in HCM.

Nitroxyl increases force development in rat cardiac muscle

Donors of nitroxyl (HNO), the reduced congener of nitric oxide (NO), exert positive cardiac inotropy/lusitropy in vivo and in vitro, due in part to their enhancement of Ca2+ cycling into and out of the sarcoplasmic reticulum. Here we tested whether the cardiac action of HNO further involves changes in myofilament–calcium interaction. Intact rat trabeculae from the right ventricle were mounted between a force transducer and a motor arm, superfused with Krebs–Henseleit (K‐H) solution (pH 7.4, room temperature) and loaded iontophoretically with fura‐2 to determine [Ca2+]i. Sarcomere length was set at 2.2–2.3 μm. HNO donated by Angelis salt (AS; Na2N2O3) dose‐dependently increased both twitch force and [Ca2+]i transients (from 50 to 1000 μm). Force increased more than [Ca2+]i transients, especially at higher doses (332 ± 33%versus 221 ± 27%, P < 0.01 at 1000 μm). AS/HNO (250 μm) increased developed force without changing Ca2+ transients at any given [Ca2+]o (0.5–2.0 mm). During steady‐state activation, AS/HNO (250 μm) increased maximal Ca2+‐activated force (Fmax, 106.8 ± 4.3 versus 86.7 ± 4.2 mN mm−2, n= 7–8, P < 0.01) without affecting Ca2+ required for 50% activation (Ca50, 0.44 ± 0.04 versus 0.52 ± 0.04 μm, not significant) or the Hill coefficient (4.75 ± 0.67 versus 5.02 ± 1.1, not significant). AS/HNO did not alter myofibrillar Mg‐ATPase activity, supporting an effect on the myofilaments themselves. The thiol reducing agent dithiothreitol (DTT, 5.0 mm) both prevented and reversed HNO action, confirming AS/HNO redox sensitivity. Lastly, NO (from DEA/NO) did not mimic AS/HNO cardiac effects. Thus, in addition to reported changes in Ca2+ cycling, HNO also acts as a cardiac Ca2+ sensitizer, augmenting maximal force without altering actomyosin ATPase activity. This is likely to be due to modulation of myofilament proteins that harbour reactive thiolate groups that are targets of HNO.

O-Linked GlcNAc Modification of Cardiac Myofilament Proteins. A Novel Regulator of Myocardial Contractile Function

In addition to O-phosphorylation, O-linked modifications of serine and threonine by &bgr;-N-acetyl-d-glucosamine (GlcNAc) may regulate muscle contractile function. This study assessed the potential role of O-GlcNAcylation in cardiac muscle contractile activation. To identify specific sites of O-GlcNAcylation in cardiac myofilament proteins, a recently developed methodology based on GalNAz-biotin labeling followed by dithiothreitol replacement and light chromatography/tandem mass spectrometry site mapping was adopted. Thirty-two O-GlcNAcylated peptides from cardiac myofilaments were identified on cardiac myosin heavy chain, actin, myosin light chains, and troponin I. To assess the potential physiological role of the GlcNAc, force–[Ca2+] relationships were studied in skinned rat trabeculae. Exposure to GlcNAc significantly decreased calcium sensitivity (pCa50), whereas maximal force (Fmax) and Hill coefficient (n) were not modified. Using a pan-specific O-GlcNAc antibody, it was determined that acute exposure of myofilaments to GlcNAc induced a significant increase in actin O-GlcNAcylation. This study provides the first identification of O-GlcNAcylation sites in cardiac myofilament proteins and demonstrates their potential role in regulating myocardial contractile function.

C-Terminal Truncation of Cardiac Troponin I Causes Divergent Effects on ATPase and Force Implications for the Pathophysiology of Myocardial Stunning

Abstract— Myocardial stunning is a form of reversible myocardial ischemia/reperfusion injury associated with systolic and diastolic contractile dysfunction. In the isolated rat heart model, myocardial stunning is characterized by specific C-terminal proteolysis of the myofilament protein, troponin I (cTnI) that yields cTnI1-193. To determine the effect of this particular C-terminal truncation of cTnI, without the confounding factor of other stunning-induced protein modifications, a series of solution biochemical assays has been undertaken using the human homologue of mouse/rat cTnI1-193, cTnI1-192. Affinity chromatography and actin sedimentation experiments detected little, or no, difference between the binding of cTnI (cTnI1-209) and cTnI1-192 to actin-tropomyosin, troponin T, or troponin C. Both cTnI and cTnI1-192 inhibit the actin-tropomyosin–activated ATPase activity of myosin subfragment 1 (S1), and this inhibition is released by troponin C in the presence of Ca2+. However, cTnI1-192, when reconstituted as part of the troponin complex (cTn1-192), caused a 54±11% increase in the maximum Ca2+-activated actin-tropomyosin-S1 ATPase activity, compared with troponin reconstituted with cTnI (cTn). Furthermore, cTn1-192 increased Ca2+ sensitivity of both the actin-tropomyosin-activated S1 ATPase activity and the Ca2+-dependent sliding velocity of reconstituted thin filaments, in an in vitro motility assay, compared with cTn. In an in vitro force assay, the actin-tropomyosin filaments bearing cTn1-192 developed only 76±4% (P <0.001) of the force obtained with filaments composed of reconstituted cTn. We suggest that cTnI proteolysis may contribute to the pathophysiology of myocardial stunning by altering the Ca2+-sensing and chemomechanical properties of the myofilaments.

Chronic Heart Failure in Congenital Heart Disease: A Scientific Statement from the American Heart Association

### Introduction The past 60 years have brought remarkable advancements in the diagnosis and treatment of congenital heart disease (CHD). Early diagnosis and improvements in cardiac surgery and interventional cardiology have resulted in unprecedented survival of patients with CHD, even those with the most complex lesions. Despite remarkable success in treatments, many interventions are palliative rather than curative, and patients often develop cardiac complications, including heart failure (HF). HF management in the setting of CHD is challenged by the wide range of ages at which HF occurs, the heterogeneity of the underlying anatomy and surgical repairs, the wide spectrum of HF causes, the lack of validated biomarkers for disease progression, the lack of reliable risk predictors or surrogate end points, and the paucity of evidence demonstrating treatment efficacy. The purposes of this statement are to review the literature pertaining to chronic HF in CHD and to elucidate important gaps in our knowledge, emphasizing the need for specific studies of HF mechanisms and improving outcomes for those with HF. In this document, the definition of CHD severity is the definition common in CHD documents, including the American College of Cardiology (ACC)/American Heart Association (AHA) guidelines1 for the management of adults with CHD (Table 11–3). The definition of HF corresponds to that found in the multiple guidelines on diagnosis and management of HF. Although nuances and specific details may be controversial,4 the broad definition from the Heart Failure Society of America guidelines states the following: “In physiologic terms, HF is a syndrome characterized by either or both pulmonary and systemic venous congestion and/or inadequate peripheral oxygen delivery, at rest or during stress, caused by cardiac dysfunction.”5 The definition of chronic HF in this document concurs with that of the European Society of Cardiology guidelines, which emphasize chronic HF …