Decrease of Pdzrn3 is required for heart maturation and protects against heart failure

Heart failure is the final common stage of most cardiopathies. Cardiomyocytes (CM) connect with others via their extremities by intercalated disk protein complexes. This planar and directional organization of myocytes is crucial for mechanical coupling and anisotropic conduction of the electric signal in the heart. One of the hallmarks of heart failure is alterations in the contact sites between CM. Yet no factor on its own is known to coordinate CM polarized organization. We have previously shown that PDZRN3, an ubiquitine ligase E3 expressed in various tissues including the heart, mediates a branch of the Planar cell polarity (PCP) signaling involved in tissue patterning, instructing cell polarity and cell polar organization within a tissue. PDZRN3 is expressed in the embryonic mouse heart then its expression dropped significantly postnatally corresponding with heart maturation and CM polarized elongation. A moderate CM overexpression of Pdzrn3 (Pdzrn3 OE) during the first week of life, induced a severe eccentric hypertrophic phenotype with heart failure. In models of pressure-overload stress heart failure, CM-specific Pdzrn3 knockout showed complete protection against degradation of heart function. We reported that Pdzrn3 signaling induced PKC ζ expression, c-Jun nuclear translocation and a reduced nuclear ß catenin level, consistent markers of the planar non-canonical Wnt signaling in CM. We then show that subcellular localization (intercalated disk) of junction proteins as Cx43, ZO1 and Desmoglein 2 was altered in Pdzrn3 OE mice, which provides a molecular explanation for impaired CM polarization in these mice. Our results reveal a novel signaling pathway that controls a genetic program essential for heart maturation and maintenance of overall geometry, as well as the contractile function of CM, and implicates PDZRN3 as a potential therapeutic target for the prevention of human heart failure.

Blood Volume Expansion, Normovolemia and Clinical Outcomes in Chronic Human Heart Failure - More is Better

Expansion in blood volume (BV) is a well-recognized response to arterial under-filling secondary to impaired cardiac output in heart failure (HF). However, the effectiveness of this response in terms of outcomes remains inadequately understood. Prospective analysis was undertaken in 110 HF patients hospitalized and treated for fluid overload. BVs were measured in a compensated state at hospital discharge using indicator-dilution methodology. Data were analyzed for composite 1-year HF-related mortality/1st re-hospitalization. Despite uniform standard of care marked heterogeneity in BVs was identified across the cohort. The cohort was stratified by BV expansion ≥+25% above normal (51% of cohort), mild-moderate expansion (22%), and normal BV (27%). Kaplan-Meier (K-M) survival estimates and regression analyses revealed BV expansion (≥+25%) to be associated with better event-free survival relative to normal BV (p=0.038). Increased red blood cell mass (RBCm) (RBC polycythemia) was identified in 43% of the overall cohort, and 70% in BV expansion ≥+25%. K-M analysis demonstrated polycythemia to be associated with better outcomes compared with normal RBCm (p<0.002). Persistent BV expansion to include RBC polycythemia is common and, importantly, associated with better clinical outcomes compared to normal total BV or normal RBCm in patients with chronic HF. However, compensatory BV expansion is not a uniform physiologic response to the insult of HF with marked variability in BV profiles despite uniform standard of care diuretic therapy. Therefore, recognizing the variability in volume regulation pathophysiology has implications not only for impact on clinical outcomes and risk stratification, but also potential for informing individualized volume management strategies.

Myocardial Aldosterone Receptor and Aquaporin 1 Up-Regulation Is Associated with Cardiomyocyte Remodeling in Human Heart Failure

Background: Abnormal aldosterone signaling is a recognized source of cardiovascular damage. Its influence on cardiomyocyte structure, function, and hormonal receptors when associated with heart failure is still unreported. Methods: Twenty-six consecutive patients with heart failure (LVEF < 40%) and normal coronaries and valves underwent left ventricular endomyocardial biopsy (EMB) for evaluation of myocardial substrate. Biopsy samples were processed for histology, electron microscopy, immunohistochemistry, and Western blot analysis of myocardial aldosterone receptor and aquaporin-1 correlated with plasma aldosterone (AD) and renin activity (PRA). Eight patients with virus-negative inflammatory cardiomyopathy (ICM) had a control EMB after 6 months of immunosuppressive therapy and recovery of cardiac function with re-evaluation of cardiomyocyte structure and receptor expression. Results: EMB in addition to the diagnosis of myocarditis (15 cases), dilated cardiomyopathy CM (6), alcohol CM (2), and diabetic CM (3) showed vacuolar degeneration and cloudy swelling of cardiomyocytes corresponding at electron microscopy to ions and water accumulation into cytosol, membrane-bound vesicles, nucleus, and other organelles, and was associated with an increased AD, PRA, and myocardial expression of aldosterone receptor (2.6 fold) and aquaporin 1 (2.7 fold). In the 8 patients recovered from ICM, cardiomyocyte diameter reduced with disappearance of intracellular vacuoles and normalization of cytosol, nucleus, and cell organelles’ electron-density, along with down-regulation of aldosterone receptor and aquaporin-1. Conclusion: Human heart failure is associated with overexpression of myocardial aldosterone receptor and aquaporin-1. These molecular changes are paralleled by intracellular water overloading and cardiomyocyte swelling and dysfunction. Cardiac recovery is accompanied by down-regulation of hormonal receptors and normalization of cell structure and composition.

PDE1 Inhibition Modulates Ca v 1.2 Channel to Stimulate Cardiomyocyte Contraction

Rationale: cAMP activation of PKA (protein kinase A) stimulates excitation-contraction (EC) coupling, increasing cardiac contractility. This is clinically achieved by β-ARs (β-adrenergic receptor) stimulation or PDE3i (inhibition of phosphodiesterase type-3), although both approaches are limited by arrhythmia and chronic myocardial toxicity. PDE1i (Phosphodiesterase type-1 inhibition) also augments cAMP and enhances contractility in intact dogs and rabbits. Unlike β-ARs or PDE3i, PDE1i-stimulated inotropy is unaltered by β-AR blockade and induces little whole-cell Ca 2+ (intracellular Ca 2+ concentration; [Ca 2+ ] i ) increase. Positive inotropy from PDE1i was recently reported in human heart failure. However, mechanisms for this effect remain unknown. Objective: Define the mechanism(s) whereby PDE1i increases myocyte contractility. Methods and Results: We studied primary guinea pig myocytes that express the PDE1C isoform found in larger mammals and humans. In quiescent cells, the potent, selective PDE1i (ITI-214) did not alter cell shortening or [Ca 2+ ] i , whereas β-ARs or PDE3i increased both. When combined with low-dose adenylate cyclase stimulation, PDE1i enhanced shortening in a PKA-dependent manner but unlike PDE3i, induced little [Ca 2+ ] i rise nor augmented β-ARs. β-ARs or PDE3i reduced myofilament Ca 2+ sensitivity and increased sarcoplasmic reticulum Ca 2+ content and phosphorylation of PKA-targeted serines on TnI (troponin I), MYBP-C (myosin binding protein C), and PLN (phospholamban). PDE1i did not significantly alter any of these. However, PDE1i increased Ca v 1.2 channel conductance similarly as PDE3i (both PKA dependent), without altering Na + -Ca 2+ exchanger current density. Cell shortening and [Ca 2+ ] i augmented by PDE1i were more sensitive to Ca v 1.2 blockade and to premature or irregular cell contractions and [Ca 2+ ] i transients compared to PDE3i. Conclusions: PDE1i enhances contractility by a PKA-dependent increase in Ca v 1.2 conductance with less total [Ca 2+ ] i increase, and no significant changes in sarcoplasmic reticulum [Ca 2+ ], myofilament Ca 2+ -sensitivity, or phosphorylation of critical EC-coupling proteins as observed with β-ARs and PDE3i. PDE1i could provide a novel positive inotropic therapy for heart failure without the toxicities of β-ARs and PDE3i.

Inflammation in Human Heart Failure: Major Mediators and Therapeutic Targets

Inflammation has been recognized as a major pathophysiological contributor to the entire spectrum of human heart failure (HF), including HF with reduced ejection fraction, HF with preserved ejection fraction, acute HF and cardiogenic shock. Nevertheless, the results of several trials attempting anti-inflammatory strategies in HF patients have not been consistent or motivating and the clinical implementation of anti-inflammatory treatments for HF still requires larger and longer trials, as well as novel and/or more specific drugs. The present work reviews the different inflammatory mechanisms contributing to each type of HF, the major inflammatory mediators involved, namely tumor necrosis factor alpha, the interleukins 1, 6, 8, 10, 18, and 33, C-reactive protein and the enzymes myeloperoxidase and inducible nitric oxide synthase, and their effects on heart function. Furthermore, several trials targeting these mediators or involving other anti-inflammatory treatments in human HF are also described and analyzed. Future therapeutic advances will likely involve tailored anti-inflammatory treatments according to the patient’s inflammatory profile, as well as the development of resolution pharmacology aimed at stimulating resolution of inflammation pathways in HF.

Myofibril orientation as a metric for characterizing heart disease

Myocyte disarray is a hallmark of cardiomyopathy. However, the relationship between alterations in the orientation of individual myofibrils and myofilaments to disease progression has been largely underexplored. This oversight has predominantly been due to a paucity of methods for objective and quantitative analysis. Here we introduce a novel, less-biased approach to quantify myofibrillar and myofilament orientation in cardiac muscle under near physiological conditions and demonstrate its superiority as compared to conventional histological assessments. Using small-angle X-ray diffraction, we first investigated changes in myofibrillar orientation at increasing sarcomere lengths in permeabilized, relaxed, wildtype mouse myocardium by assessing the angular spread of the 1,0 equatorial reflection (angle sigma). At a sarcomere length (SL) of 1.9 microns, the angle sigma was 0.23 +/- 0.01 rad, decreased to 0.19 +/- 0.01 rad at a SL of 2.1 microns, and further decreased to 0.15 +/- 0.01 rad at a SL of 2.3 microns (p<0.0001). Angle sigma was significantly larger in R403Q (a MYH7 HCM model) porcine myocardium (0.24 +/- 0.01 rad) compared to WT myocardium (0.14 +/- 0.005 rad, p<0.0001) as well as in human heart failure tissue (0.19 +/- 0.006 rad) when compared to non-failing samples (0.17 +/- 0.007 rad, p=0.01). These data indicate that diseased myocardium suffers from greater myofibrillar disorientation compared to healthy controls. Finally, we showed that conventional, histology-based analysis of disarray can be subject to user bias and/or sampling error and lead to false positives. Our method for directly assessing myofibrillar orientation avoids the artifacts introduced by conventional histological methods that directly assess myocyte orientation and only indirectly assess myofibrillar orientation, and provides a precise and objective metric for phenotypically characterizing myocardium. The ability to obtain excellent X-ray diffraction patterns from frozen human myocardium provides a new tool for investigating the structural bases of cardiomyopathies.

Involvement of GPR37L1 in murine blood pressure regulation and human cardiac disease pathophysiology

Multiple mouse lines lacking the orphan G protein-coupled receptor, GPR37L1, have elicited disparate cardiovascular phenotypes. The first published Gpr37l1 knockout mice study (1) indicated a marked elevation in systolic blood pressure (SBP; ~60 mmHg), revealing a potential therapeutic opportunity. The phenotype differs from our independently generated knockout line, where male mice exhibited equivalent baseline blood pressure to wildtype (2, 3). Here, we attempted to reproduce the findings of Min et al. (1) by characterizing the cardiovascular phenotype of both the original knockout and transgenic lines alongside a C57BL/6J control line, using the same method of blood pressure measurement. The present study supports the findings from our independently developed Gpr37l1 knockout line (2, 3), namely that SBP and diastolic blood pressure (DBP) are not different in the original Gpr37l1 knockout male mice (SBP: 130.9±5.3 mmHg; DBP: 90.7±3.0 mmHg) compared to C57BL/6J mice (SBP: 123.1±4.1 mmHg; DBP: 87.0±2.7 mmHg) (2, 3). Instead, we attribute the apparent hypertension of the knockout line described by Min et al. (1) to comparison with a seemingly hypotensive transgenic line (SBP 103.7±5.0 mmHg; DBP 71.9±3.7 mmHg). Additionally, we quantified myocardial GPR37L1 transcript in humans, which was suggested to be downregulated in cardiovascular disease (1). We found that GPR37L1 has very low native transcript levels in human myocardium, and that expression is not different in tissue samples from patients with heart failure compared to gender-matched healthy control tissue. These findings indicate that cardiac GPR37L1 expression is unlikely to contribute to the pathophysiology of human heart failure.

Abstract 100: Integrated Cardiac Metabolism In End-Stage Human Heart Failure

Heart failure affects millions of people worldwide with mortality near 50% within five years. This disease is characterized by widespread cardiac and systemic metabolic changes, but a comprehensive evaluation of metabolism in failing human hearts is lacking. Here, we provide a comprehensive depiction of cardiac and systemic metabolic changes in 89 explanted failing and non-failing human hearts through integration of plasma and cardiac tissue metabolomics, genome-wide RNAseq, and proteomic data. The data confirm a profound bioenergetic defect in end-stage human heart failure and demonstrate extensive changes in metabolic homeostasis. The data indicate a substantial defect in fatty acid (FA) use in failing hearts, in particular unsaturated FAs. Reduction of FAs and acyl-carnitines in failing tissue in contrast to concomitant elevations in plasma suggest a defect in import of FAs into the cell, rather than a defect in FA oxidation. Intermediates of glycolysis, the pentose phosphate pathway, and glycogen synthesis are all similarly reduced, as is expression of GLUT1, indicating diminished glucose uptake. However, there was no significant change in tissue pyruvate content, suggesting an increase in lactate utilization. The data suggest increased flux of pyruvate into mitochondria, likely promoting pyruvate oxidation but not pyruvate carboxylation. Blunted anabolic pyruvate flux, in turn, likely leads to insufficient TCA cycle intermediates. Ketone levels were increased in both failing tissue and plasma, as previously reported. The phospholipid content of failing human hearts is greatly increased in both failing tissue and plasma. Nucleotide synthesis pathways also appear to be reprogrammed, with a notable decrease in adenosine metabolism, specifically. Together, these data indicate widespread change in the local cardiac and greater systemic metabolic landscape in severe human heart failure.

Abstract P499: Histone H4K20 Trimethylation Is Differentially Regulated In Heart Disease

It has been well established that many cardiac pathologies result from dynamic changes in gene expression and conversely that modulating key epigenetic factors in murine models is capable of preventing or abrogating ischemic injury and pathological remodeling. One epigenetic mechanism is the post-translational modification of histones, which are reversibly methylated on lysine (K) residues and can accept up to three methyl groups (Me1, Me2 and Me3). In the heart, significant changes in global levels of histone H3K4Me3 and H3K9Me3 have been previously reported to be upregulated and downregulated, respectively, during hypertrophy and failure in mice. However, the majority of post-translational modifications on histones have never been examined to quantify global abundance in the heart during disease. In particular, histone H4K20Me3 is important in heterochromatin formation and gene repression in non-cardiac cells but has never been evaluated in the heart. Therefore, we utilized cardiac tissue from three animal models of cardiac stress and employed western blotting and mass spectrometry to quantify the global abundance of total histone H4 and H4K20 methylation. We specifically evaluated tissue from mice subjected to LAD ligation, transverse aortic banding and isoproterenol infusion (via mini-osmotic pump). In addition, we also utilized primary neonatal cardiomyocytes treated with the hypertrophic agonist phenylephrine to quantify H4K20 methylation. Our data show that global levels of histone H4K20Me3 are differentially regulated in some models of cardiac dysfunction, but not all (i.e. isoproterenol infusion). In addition, we measured the abundance of histone methyltransferases and demethylases (via western blotting and qPCR) which are responsible for adding or removing this methyl mark in mouse cardiac tissue, and compared this to published data from human heart failure patients. These analyses allowed us to identify two enzymes, the methyltransferase Smyd5 and demethylase KDM7B, which are also differentially expressed in cardiac tissue during disease. Together these results are the first analysis of histone H4K20 methylation in the heart and suggest a novel role for this methylation site in the pathophysiology of cardiovascular disease.