Human Myocardium Research Articles

1173Anti-heart and anti-intercalated disk autoantibodies: possible novel biomarkers of cardiac sarcoidosis

Abstract Background Sarcoidosis is an immune-mediated disease; cardiac involvement, a granulomatous form of myocarditis, is under-recognised and prognostically relevant, as it can present with significant morbidity and mortality. Anti-heart autoantibodies (AHA) and anti-intercalated disk autoantibodies (AIDA) are reliable autoimmune markers in non-sarcoidosis myocarditis forms. Purpose The aim of this study was to assess the potential role of serum AHA and AIDA in cardiac sarcoidosis. Methods This is a cross-sectional study on a series of 29 patients with biopsy proven extra-cardiac sarcoidosis and with biopsy-proven or clinically suspected cardiac involvement, who were tested for AHA and AIDA. Patients were recruited in two recruiting tertiary centres, in USA and Italy. AHA and AIDA were detected by indirect immunofluorescence on human myocardium and skeletal muscle. Controls included sera from patients with non-inflammatory cardiac disease (NICD) (n=160), with ischemic heart failure (IHF) (n=141) and normal blood donors (NBD) (n=270). Results The frequencies of AHA and of AIDA were higher in sarcoidosis (86%; 62%) than in NICD (8%; 4%), IHF (7%; 2%), NBD (9%; 0%) (p=0.0001; p=0.0001 respectively). Sensitivity and specificity were: 86% and 92% for positive AHA and 62% and 98% for positive AIDA, respectively (see figure). Figure 1 Conclusions The detection of serum AHA and AIDA in biopsy-proven or clinically suspected cardiac sarcoidosis supports the involvement of heart-specific autoimmunity in the majority of our cases and may provide a novel non invasive diagnostic marker.

4967Effects of atrial fibrillation on the human ventricle

Abstract Background The consequence of normofrequent atrial fibrillation (AF) on the ventricle remains largely unknown. Methods and results To elucidate the effects of arrhythmic excitation on human ventricular myocardium we performed contractility experiments using ventricular trabecula from patients with heart failure (HF). Normofrequent AF was simulated using arrhythmic (60 bpm, 40% R-R interval variability) or rhythmic field stimulation (60 bpm). Within 8h of arrhythmic stimulation, human specimen showed an impaired systolic force, while diastolic tension increased pathologically (n=5–7 each/7 HF patients, Fig. 1). The characterization of the ventricular (in-vivo) phenotype in patients with AF was performed by utilizing ventricular myocardium from patients with sinus rhythm (SR) and from patients with AF in the absence of HF (compensated hypertrophy, EF>50%, matched clinical characteristics, LV myocardium obtained from aortic valve replacement surgery). Histological investigation showed increased levels of interstitial fibrosis in myocardium from patients with AF compared to SR (n=10 patients each). Studies of cellular Ca-homeostasis (epifluorescence microscopy, Fura-2) were performed using isolated human ventricular cardiomyocytes. While systolic Ca-transient amplitude (0.5 Hz) was preserved in ventricular cardiomyocytes from patients with AF, we found a significantly prolonged Ca-elimination time (RT80) by 22.0±7.7% and a trend towards increased diastolic Ca-levels (n=17–23 cells/4 patients each). This finding may be explained by a decrease in SERCA2a activity (ksys-kCaff, n=10–12/4 each) and an enhanced phospholamban expression in Western Blot experiments (n=5 patients each). For the standardized investigation of the involved targets/mechanisms mediating the pathological changes upon arrhythmic excitation, we utilized human induced pluripotent stem cell cardiomyocytes (iPSC-CM) from healthy donors for chronic arrhythmic culture stimulation (24h). Arrhythmic paced iPSC-CM showed no changes in systolic Ca-transient amplitude (0.5 Hz), whereas diastolic Ca-levels were increased, which fits nicely to the finding of disturbed trabeculae diastolic function (n=15 cells each). In patch clamp experiments, arrhythmic paced cells showed no alterations of resting membrane potential, upstroke velocity, action-potential amplitude or -duration (n=7–9 cells each). Protein expression levels of key Ca-handling proteins in iPSC-CM as well as regulated genes are already under investigation. Conclusion This study demonstrates that arrhythmic ventricular excitation deteriorates human myocardial contractility early in HF. In biopsies from patients with preserved EF, chronic AF was associated with increased levels of interstitial fibrosis and pathological diastolic Ca-handling, which could be causally confirmed in chronically arrhythmic paced iPSC-CM. Therefore, this study provides first mechanistic characterization of AF mediated effects on the human ventricle.

3073Leveraging transcriptome sequencing for detecting novel disease-related pathways using human cardiac sarcoidosis myocardium biopsies

Abstract Background/Introduction Cardiac sarcoidosis (CS) is one of the main causes of poor outcomes in patients with sarcoidosis, a systemic granulomatous disorder of complex etiology including a genetically susceptible host and specific exposure to disease-triggering antigens. Recently, transcriptome analysis using sarcoidosis peripheral monocytes was reported to be useful for exploring genetic susceptibility and novel disease-causing pathways. However, transcriptome sequencing has not been used to explore disease-related genes and pathways directly using human CS myocardial biopsies. Purpose This study aimed to identify transcriptome profiles and novel disease-related pathways of CS by comparing human CS myocardial biopsies with control samples using ribonucleic acid (RNA) sequencing (RNA-Seq). Methods We assessed 30 patients with suspected myocardial disorders who underwent transcatheter endomyocardial biopsies at our University Hospital, Japan. Of those, 7 were clinically diagnosed with CS, 9 with hypertrophic cardiomyopathy (HCM), and 14 with dilated cardiomyopathy (DCM). Messenger RNAs were extracted from cardiac muscle biopsies using the Ovation SoLo RNA-Seq System (NuGEN Technologies), according to the manufacturer's instructions. Sequencing was performed with coverage of approximately 20 million reads per sample using Illumina HiSeq 2000. Sequencing reads were mapped using the STAR 2-pass method with GRCh37 as the reference. The DESeq2 R package (version 3.8) was used for further analyses. Principal component analysis (PCA) on gene expression was conducted for detecting outliers such as non-muscular samples. Differential gene expression analysis was performed between the 7 patients with CS and 23 patients with cardiomyopathy (HCM and DCM, non-CS). Gene Ontology (GO) enrichment analysis was conducted to estimate possible disease-related pathways. Results We successfully sequenced 60 myocardial biopsy samples (original and biological duplicates) from 30 CS patients. Of these, 2 outlier samples shown by the PCA plot were removed, and 58 were used for further analyses. We found 243 genes that were differentially expressed between CS patients and non-CS patients. Top-rated genes were RP11–366M4.8, RELN, S100A6, WASF3and UCHL1. Pathway analysis using GO demonstrated enrichment oflymphocyte activation (P=4.8x10–16), organelle fission (P=6.1x10–14), the M phase of the mitotic cell cycle (P=2.2x10–13), nuclear division (P=2.4x10–13), mitosis (P=2.4x10–13) and T-cell activation pathways (P=1.2x10–12). Conclusions Our differential gene expression and pathway analysis results using human CS myocardial biopsies suggested that lymphocyte activation, specifically the T-cell activation pathway, is linked to CS pathogenesis. Further studies are needed to decipher the role of specific genes related to genetic susceptibility and/or pathways associated with CS occurrence.

KLF15-Wnt–Dependent Cardiac Reprogramming Up-Regulates SHISA3 in the Mammalian Heart

BackgroundThe combination of cardiomyocyte (CM) and vascular cell (VC) fetal reprogramming upon stress culminates in end-stage heart failure (HF) by mechanisms that are not fully understood. Previous studies suggest KLF15 as a key regulator of CM hypertrophy. ObjectivesThis study aimed to characterize the impact of KLF15-dependent cardiac transcriptional networks leading to HF progression, amenable to therapeutic intervention in the adult heart. MethodsTranscriptomic bioinformatics, phenotyping of Klf15 knockout mice, Wnt-signaling-modulated hearts, and pressure overload and myocardial ischemia models were applied. Human KLF15 knockout embryonic stem cells and engineered human myocardium, and human samples were used to validate the relevance of the identified mechanisms. ResultsThe authors identified a sequential, postnatal transcriptional repression mediated by KLF15 of pathways implicated in pathological tissue remodeling, including distinct Wnt-pathways that control CM fetal reprogramming and VC remodeling. The authors further uncovered a vascular program induced by a cellular crosstalk initiated by CM, characterized by a reduction of KLF15 and a concomitant activation of Wnt-dependent transcriptional signaling. Within this program, a so-far uncharacterized cardiac player, SHISA3, primarily expressed in VCs in fetal hearts and pathological remodeling was identified. Importantly, the KLF15 and Wnt codependent SHISA3 regulation was demonstrated to be conserved in mouse and human models. ConclusionsThe authors unraveled a network interplay defined by KLF15–Wnt dynamics controlling CM and VC homeostasis in the postnatal heart and demonstrated its potential as a cardiac-specific therapeutic target in HF. Within this network, they identified SHISA3 as a novel, evolutionarily conserved VC marker involved in pathological remodeling in HF.

Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Preclinical Cancer Drug Cardiotoxicity Testing: A Scientific Statement From the American Heart Association.

It is now well recognized that many lifesaving oncology drugs may adversely affect the heart and cardiovascular system, including causing irreversible cardiac injury that can result in reduced quality of life. These effects, which may manifest in the short term or long term, are mechanistically not well understood. Research is hampered by the reliance on whole-animal models of cardiotoxicity that may fail to reflect the fundamental biology or cardiotoxic responses of the human myocardium. The emergence of human induced pluripotent stem cell-derived cardiomyocytes as an in vitro research tool holds great promise for understanding drug-induced cardiotoxicity of oncological drugs that may manifest as contractile and electrophysiological dysfunction, as well as structural abnormalities, making it possible to deliver novel drugs free from cardiac liabilities and guide personalized therapy. This article briefly reviews the challenges of cardio-oncology, the strengths and limitations of using human induced pluripotent stem cell-derived cardiomyocytes to represent clinical findings in the nonclinical research space, and future directions for their further use.

The way to control the interventricular septum thickness during septal myectomy. An experimental study

Background: At present, there are no methods for intraoperative monitoring of the interventricular septum (IVS) thickness in a stopped and empty heart. This might be an obvious reason for unsatisfactory results after a number of septal myectomies.Aim: To provide an experimental background for the method to control the IVS thickness (that we had proposed) during septal myectomy.Materials and methods: The proposed technique is based on the transillumination method. The experimental models were cadaveric porcine hearts, as well as fragments of the human myocardium removed during septal myectomies. The thickness of the translucent myocardium was estimated depending on the local illumination value at the entrance to the myocardium and the external illumination of the surgical field. We compared the results of 67 septal myectomies performed in the clinic of the Almazov National Medical Research Centre with the results of 35 similar experimental procedures with cadaveric porcine hearts using the proposed way of measurement.Results: A graph of the illumination at the entrance to the myocardium against the thickness of the translucent myocardium was constructed. After conventionally performed septal myectomies the median variation of the myocardial thickness was 4 [3; 6] mm. In the experiment using the proposed control method, the median variation was 1 [1; 2] mm, i.e. significantly less than with the conventional approach (p = 3 x 10-10).Conclusion: The method to control the IVS thickness when performing septal myectomy makes it possible to achieve the required myocardial thickness the resection area with much greater accuracy than with the conventional one.

Dysregulation of H11 Kinase in the Failing Left Ventricular Myocardium

Background: Increased expression of H11 kinase (H11K) has been observed in Left Ventricular (LV) myocardium of failed human hearts. Its potential role as a contributor to the progression of Heart Failure (HF) remains uncertain. In the present study we examined the expression of H11K in cytosolic and mitochondrial fractions of failing human and dog LV myocardium and assessed its interaction with Akt (cell-survival enzyme) and p38MAPK (programmed cell death enzyme). Methods: Total RNA and Sodium-Dodecyl Sulfate (SDS) extracts were prepared from homogenate of LV specimens of 6 dogs with intracoronary microembolization-induced HF, 6 normal (NL) dogs, 7 explanted failed human hearts due to Idiopathic Dilated Cardiomyopathy (IDC), 7 failed human hearts due to Ischemic Cardiomyopathy (ICM) and 7 non-failing human donor hearts (DNR). SDS extracts were also prepared from cytosolic and mitochondrial fractions isolated from LV specimens. Results: H11K mRNA and protein levels normalized to GAPDH increased significantly in LV tissue from ICM and IDC hearts compared to DNR hearts and in HF dogs compared with NL dogs. H11K protein levels increased in cytosolic fractions but decreased in mitochondrial fractions of both failed human and dog hearts compared to DNR hearts and NL dog hearts. Immunoprecipitation studies in specimens from HF dogs showed that H11K cytosolic fractions interacted predominantly with p38MAPK and least with Akt when compared with NL dogs. Conclusions: Enhanced interaction of H11K with p38MAPK in HF can promote cell death thus contributing to progressive LV dysfunction. Therapeutic modalities that restore interaction of H11K with Akt and augment H11K translocation to mitochondria can potentially and partially reverse the progression of LV dysfunction by promoting cell survival.

Future Needs in Mast Cell Biology.

The pathophysiological roles of mast cells are still not fully understood, over 140 years since their description by Paul Ehrlich in 1878. Initial studies have attempted to identify distinct “subpopulations” of mast cells based on a relatively small number of biochemical characteristics. More recently, “subtypes” of mast cells have been described based on the analysis of transcriptomes of anatomically distinct mouse mast cell populations. Although mast cells can potently alter homeostasis, in certain circumstances, these cells can also contribute to the restoration of homeostasis. Both solid and hematologic tumors are associated with the accumulation of peritumoral and/or intratumoral mast cells, suggesting that these cells can help to promote and/or limit tumorigenesis. We suggest that at least two major subsets of mast cells, MC1 (meaning anti-tumorigenic) and MC2 (meaning pro-tumorigenic), and/or different mast cell mediators derived from otherwise similar cells, could play distinct or even opposite roles in tumorigenesis. Mast cells are also strategically located in the human myocardium, in atherosclerotic plaques, in close proximity to nerves and in the aortic valve. Recent studies have revealed evidence that cardiac mast cells can participate both in physiological and pathological processes in the heart. It seems likely that different subsets of mast cells, like those of cardiac macrophages, can exert distinct, even opposite, effects in different pathophysiological processes in the heart. In this chapter, we have commented on possible future needs of the ongoing efforts to identify the diverse functions of mast cells in health and disease.

CGRP derived from cardiac fibroblasts is an endogenous suppressor of cardiac fibrosis.

Aberrant activation of cardiac fibroblasts leads to cardiac fibrosis, and evolving evidences suggest that endogenous bioactive substances derived from cardiac fibroblasts regulate cardiac fibroblasts activation in an autocrine/paracrine manner. Here we first presented evidence that cardiac fibroblasts can synthesize and secrete calcitonin gene-related peptide (CGRP), therefore, this study aimed to investigate the role of cardiac fibroblasts-derived CGRP in cardiac fibroblasts activation and its regulative mechanism. The abundantly expression of CGRP in rat, mouse, and human myocardium allowed us to explore the cellular origin of CGRP, and found that the cardiac CGRP was mainly derived from cardiac fibroblasts. Activating TRPA1 with a specific agonist allyl isothiocyanate promoted the synthesis and secretion of CGRP, as well as intracellular Ca2+. These effects were reversed by TRPA1-specific antagonist HC030031 and Ca2+ chelator BAPTA-AM. TGF-β1 was applied to induce the activation of cardiac fibroblasts, and found that TGF-β1 can increase the mRNA expression and secretion levels of CGRP in cardiac fibroblasts. Either CGRP8-37 (CGRP receptor antagonist) or α-CGRP small interfering RNA (siRNA) aggravated TGF-β1-induced proliferation, differentiation, collagen production, and instigated inflammation in cardiac fibroblasts. Moreover, TGF-β1-induced NF-κB activation including IκBα phosphorylation and p65 nuclear translocation were also promoted by CGRP8-37 and α-CGRP siRNA. NF-κB inhibitor pyrrolidinedithiocarbamate ammonium (PDTC) reversed the effects of CGRP8-37 on NF-κB activation. The promotive effects of CGRP8-37 on TGF-β1-induced activation of cardiac fibroblasts were all reversed by PDTC. Monocrotaline (MCT) induces pulmonary arterial hypertension, progressively leading to right ventricular fibrosis. This model of cardiac fibrosis was developed here to test the potentially beneficial effects of TRPA1 activation in vivo. The non-toxic TRPA1 agonist Cinnamaldehyde (CA) inhibited MCT-induced elevation in right ventricle systolic pressure, RV/LV + S, and right ventricular collagen accumulation, as well as down-regulation of CGRP. CA increased the synthesis and secretion of CGRP, and inhibited TGF-β1-induced activation in cardiac fibroblasts. Our data suggested an autocrine role for cardiac fibroblasts-derived CGRP in suppressing activation of cardiac fibroblasts through inhibiting NF-κB activation. Increasing autocrine CGRP by activating TRPA1 can ameliorate cardiac fibrosis. These findings support the notion that CGRP derived from cardiac fibroblasts is an endogenous suppressor of cardiac fibrosis.

Characterization of the human myocardium by optical coherence tomography.

Imaging of cardiac tissue structure plays a critical role in the treatment and understanding of cardiovascular disease. Optical coherence tomography (OCT) offers the potential to provide valuable, high-resolution imaging of cardiac tissue. However, there is a lack of comprehensive OCT imaging data of the human heart, which could improve identification of structural substrates underlying cardiac abnormalities. The objective of this study was to provide qualitative and quantitative analysis of OCT image features throughout the human heart. Fifty human hearts were acquired, and tissues from all chambers were imaged with OCT. Histology was obtained to verify tissue composition. Statistical differences between OCT image features corresponding to different tissue types and chambers were estimated using analysis of variance. OCT imaging provided features that were able to distinguish structures such as thickened collagen, as well as adipose tissue and fibrotic myocardium. Statistically significant differences were found between atria and ventricles in attenuation coefficient, and between adipose and all other tissue types. This study provides an overview of OCT image features throughout the human heart, which can be used for guiding future applications such as OCT-integrated catheter-based treatments or ex vivo investigation of structural substrates.

Quantitative Expression of RNA from Frozen Organs and Formaldehyde-fixed and Paraffin-embedded Tissues.

Objective Quantitative analysis and comparison of the expression of ribonucleic acid （RNA） from frozen organs and formaldehyde-fixed and paraffin-embedded （FFPE） tissues. Methods Frozen specimens of human brain, myocardium and liver tissues as well as FFPE samples at different postmortem intervals were collected and mass concentration of RNA was extracted and detected. Reverse transcription-quantitative polymerase chain reaction （RT-qPCR） technology was used to analyze the amplification efficiency and relative expression of each RNA marker. Results The mass concentration and integrity of RNA extracted from FFPE samples were relatively low compared with frozen specimens. The amplification efficiency of RNA markers was related with RNA species and the length of amplification products. Among them, glyceraldehyde-3-phosphate dehydrogenase （GAPDH） and β-actin （ACTB） with relatively long amplification products failed to achieve optimal amplification efficiency, whereas 5S ribosomal RNA （5S rRNA） achieved ideal amplification efficiency and showed quite stable expression across various tissues, therefore it was chosen as internal reference marker. The expression quantity of GAPDH and ACTB in frozen specimens with longer postmortem intervals and in FFPE samples with relatively long amplification products was decreased. The expressions of tissue-specific microRNAs （miRNAs）, GAPDH and ACTB with relatively short amplification products had consistency in the same tissues and FFPE samples. Conclusion Through standardizing the RT-qPCR experiment, selecting the appropriate RNA marker and designing primers of appropriate product length, RNA expression levels of FFPE samples can be accurately quantified.

Engineered Cardiac Tissues Generated in the Biowire II: A Platform for Human-Based Drug Discovery.

Recent advances in techniques to differentiate human induced pluripotent stem cells (hiPSCs) hold the promise of an unlimited supply of human derived cardiac cells from both healthy and disease populations. That promise has been tempered by the observation that hiPSC-derived cardiomyocytes (hiPSC-CMs) typically retain a fetal-like phenotype, raising concern about the translatability of the in vitro data obtained to drug safety, discovery, and development studies. The Biowire II platform was used to generate 3D engineered cardiac tissues (ECTs) from hiPSC-CMs and cardiac fibroblasts. Long term electrical stimulation was employed to obtain ECTs that possess a phenotype like that of adult human myocardium including a lack of spontaneous beating, the presence of a positive force-frequency response from 1 to 4 Hz and prominent postrest potentiation. Pharmacology studies were performed in the ECTs to confirm the presence and functionality of pathways that modulate cardiac contractility in humans. Canonical responses were observed for compounds that act via the β-adrenergic/cAMP-mediated pathway, eg, isoproterenol and milrinone; the L-type calcium channel, eg, FPL64176 and nifedipine; and indirectly effect intracellular Ca2+ concentrations, eg, digoxin. Expected positive inotropic responses were observed for compounds that modulate proteins of the cardiac sarcomere, eg, omecamtiv mecarbil and levosimendan. ECTs generated in the Biowire II platform display adult-like properties and have canonical responses to cardiotherapeutic and cardiotoxic agents that affect contractility in humans via a variety of mechanisms. These data demonstrate that this human-based model can be used to assess the effects of novel compounds on contractility early in the drug discovery and development process.

Abstract 933: Integrated Omics Analysis of Diabetic Heart Failure in Human Myocardium

Background: Patients with diabetes mellitus (DM) have a higher risk of heart failure (HF). While metabolic dysregulation has been well-established in HF and DM separately, molecular mechanisms associated with diabetic HF are incompletely understood. Thus, we sought to test the overarching hypothesis that cardiac metabolic programming is distinct in diabetic HF compared to DM and HF individually. Methods: RNA-sequencing and targeted metabolomics profiles were completed on human myocardial tissue from 54 unique individuals received from the Duke Human Heart Biorepository and the University of Colorado. Multi-omics profiles of cardiac tissue from 4 groups were compared: a) controls with no clinical evidence of DM or HF (n = 12), b) DM-only (n = 15), c) HF-only (n = 10), and d) HF+DM (n = 17). Results: After using statistical criteria to prioritize genes, we looked for genes that function together or modulate the same activity. Genes encoding producers of reactive oxygen species (ROS), DUOX2, DUOXA2, and DUOXA1, were differentially upregulated in diabetic-HF, compared to DM or HF alone (FDR HF+DM vs DM-only/HF-only = 6.6 x 10 -7 /2.2 x 10 -4 , 7.7 x 10 -3 /6.7 x 10 -3 , and 3.6 x 10 -6 /0.013, respectively). Modulators of mTOR signaling, ERBB3, ERBB4, and PIK3R3, were also differentially regulated in diabetic-HF (FDR HF+DM vs DM-only/HF-only = 1.4 x 10 -3 /0.010, 0.012/7.4 x 10 -3 , and 0.011/9.3 x 10 -3 , respectively). Gene set enrichment analysis supported our focus on these genes; top enriched gene sets included mTOR signaling and processes related to oxidative stress response. In our metabolomics data, long chain acylcarnitines (AC) and ceramides (Cer) are decreased in diabetic-HF. For example, C20:1 AC can be converted to C20:1 Cer and accumulation of both is diminished in diabetic-HF (FDR = 0.0032 and 0.035, respectively). Conclusions: We found that diabetic-HF was characterized by a distinct omics profile consistent with increased ROS production, diminished AC and Cer accumulation, and modulation of mTOR signaling. The fact that ceramide accumulation has been previously associated with oxidative stress and also linked to ERBB and PI3K pathways suggests that these data may reveal a unique regulation of these pathways in diabetic-HF.

Abstract 100: Dual Activation of PKA and PKG by PDE1 Inhibition Facilitates Proteasomal Degradation of Misfolded Proteins and Protects Against Proteinopathy-Based HFpEF

No current treatment is intended to target cardiac proteotoxicity or can reduce mortality of heart failure with preserved ejection fraction (HFpEF), a prevalent form of heart failure (HF). Selective degradation of misfolded proteins by the ubiquitin-proteasome system (UPS) is vital to the cell. Proteasome impairment is recently implicated in HF genesis. Activation of the cGMP-protein kinase G (PKG) or the cAMP-protein kinase A (PKA) pathways facilitates proteasome functioning. Phosphodiesterase 1 (PDE1) hydrolyzes both cyclic nucleotides and accounts for the majority of PDE activities in human myocardium. Here we report the preclinical therapeutic efficacy and a new mechanism of action of PDE1 inhibition (IC86430) for cardiac proteinopathy caused by Arg120Gly missense mutant αB-crystallin (CryAB R120G ). In mice expressing GFPdgn, an inverse reporter of UPS proteolytic activity, IC86430 treatment increased myocardial 26S proteasome activities and substantially decreased GFPdgn protein levels. Myocardial PDE1A expression was highly upregulated in CryAB R120G mice. HFpEF was detected in CryAB R120G mice at 4 months; IC86430 treatment initiated at this stage markedly attenuated HFpEF, substantially delayed mouse premature death, increased myocardial levels of Ser14-phosphorylated Rpn6 (the primary proteasome target of PKA), and reduced the steady state level of the misfolded CryAB species in these mice. In cultured cardiomyocytes, IC86430 treatment increased proteasome activities and accelerated proteasomal degradation of GFPu and CryAB R120G in a PKA- and PKG- dependent manner. We conclude that PDE1 inhibition induces PKA- and PKG-mediated promotion of proteasomal degradation of misfolded proteins in cardiomyocytes and effectively treats HFpEF caused by CryAB R120G ; hence, PDE1 inhibition represents a potentially new therapeutic strategy for HFpEF and heart disease with increased proteotoxic stress.

Abstract 479: Cardiomyopathy Phenotypes Observed in Human Engineered Heart Tissue Depend on Functional Maturation

Human iPSC-based models of cardiomyopathies have become increasingly prevalent. These models enable the study of genotype-phenotype relationships within a human context and have already led to new insights. A potential limitation of iPSC-derived cardiomyocytes (iPSC-CMs) is their varying degrees of functional maturation with respect to electrophysiology, calcium handling, and contractility. Variable levels of maturity may explain how different groups investigating hypertrophic cardiomyopathy (HCM) mutations in iPSC cardiomyocytes have arrived at conflicting results. Nevertheless, whether or not advanced functional maturation truly impacts disease phenotype in iPSC cardiomyocyte models remains an open question. Using a novel protocol that combines pacing and physiological media in a three-dimensional engineered heart tissue (EHT) derived from decellularized myocardium, we are able to mature human engineered heart tissues within three weeks to recapitulate key functional characteristics of adult heart tissue, such as a robustly positive force-frequency response, a strong post-rest potentiation, potent calcium handling and excitation-contraction handling, and mature isometric twitches that closely match that of intact adult human ventricular myocardium. Using our platform, we investigated the effect of maturation on the functional consequence of a classic variant (E62Q) in the thin filament protein alpha-tropomyosin (TPM1). Initial characterization of TPM1 E62Q in EHTs show a hypercontractile phenotype compared to its isogenic wild-type line, with limited changes in calcium handling. Preliminary data on maturing TPM1 E62Q EHTs show that additional hallmarks of disease may develop with maturation such as arrhythmogenicity, hypertrophy, and fibrosis. In our work, we develop a method for advanced maturation of hiPSC-CMs in EHTs and show the dependence on maturation status of progressive hallmarks of HCM in human EHTs compared to EHTs grown under conditions standard for the field.

Abstract 336: The Effect Of Phosphodiesterase 9a Inhibition In 2 Mouse Models Of Diastolic Dysfunction

Low myocardial cGMP-PKG activity is associated with increased cardiomyocyte stiffness in HFpEF. cGMP is mainly hydrolyzed by PDE (phosphodiesterases)5a and PDE9a. Importantly, PDE9a protein expression was reported to be upregulated in human HFpEF myocardium and chronic administration of PDE9a inhibitor reversed pre-established cardiac hypertrophy and systolic dysfunction in mice subjected to TAC (Transverse Aortic Constriction). We evaluated whether PDE9a inhibition had a beneficial effect on diastolic dysfunction (DD). We investigated two mouse models of DD: 1) TAC-DOCA (TAC with deoxycorticosterone acetate (DOCA) supplementation) and 2) LepR db/db , a metabolically induced DD models. Mice were subjected to subcutaneous implantation with osmotic minipumps containing either PD9a inhibitor (PF-4449613) or vehicle for 4 wks. PDE9a transcript was not upregulated in myocardium of either TAC-DOCA or LepR db/db mice. PV analysis revealed that in TAC-DOCA mice, inhibition of PDE9a resulted in reduced LV diastolic stiffness, but impairment of systolic function observed as ventricular-vascular decoupling. In LepR db/db mice, cardiomyocyte stiffness was slightly but significantly reduced in PDE9a inhibitor treated animals. No differences in myocardial fibrosis or cardiac morphometry were observed. The effect of acute PDE9a inhibition was investigated in intact cardiomyocytes isolated from LVs of TAC-DOCA mice. Cellular work loop force measurement was conducted before and after administration of PDE9a inhibitor combined with ANP (Atrial Natriuretic Peptide) for 15 mins. Cardiomyocytes from TAC-DOCA had high diastolic stiffness compared to sham hearts, however, no stiffness reduction was observed after acute inhibitor treatment. Conclusion: Chronic inhibition of PDE9a ameliorates LV diastolic stiffness, but suppresses systolic function in TAC-DOCA induced DD mice. The LV stiffness reduction is in part due to cardiomyocyte stiffness reduction. The advantage of PDE9a inhibition on diastolic function might be more evident, with less systolic suppression, in hearts with PDE9a upregulation.

Abstract 850: Metabolic Landscape of Human Heart Failure

Heart failure is characterized by insufficient pumping to match the needs of the body. Heart failure is thought to be associated with dysfunctional mitochondria and metabolic alterations, but there is little to no direct evidence in humans. The heart metabolizes a variety of substrates for energy production, including fatty acids, glucose, ketones, and branch chain amino acids (BCAAs). In the healthy heart, fatty acids are the main fuel source, and oxidation is balanced with energy need. In heart failure, it is asserted that fatty acid oxidation is suppressed, and glucose utilization is increased, but this has not been directly measured in humans. If true, this metabolic switch toward glucose may contribute to mechanical insufficiency by disrupting the balance between metabolism and energetic need, thus representing a potential area for therapeutic intervention in heart failure. Prior studies were largely performed in animal models, and results are often inconsistent across models and species. Challenges have constrained the use of human myocardium, including costly and cumbersome techniques for in vivo assessment; limited availability of high-quality human myocardial tissue; and low yields and short durability of isolated human cardiomyocytes. We addressed these challenges by taking advantage of human explanted hearts available via the Penn Heart Transplant program to investigate the metabolic differences between failing and healthy human hearts. We performed metabolomics analysis using mass spectrometry on myocardium from 18 failing and 20 nonfailing human hearts. Of the 334 metabolites measured, 99 were significantly different between groups by Wilcoxon test and FDR correction. Metabolite set enrichment analysis revealed that the most enriched pathways included the glycerol phosphate shuttle, ammonia recycling, methylhistidine metabolism, and glutathione metabolism. The mitochondrial electron transport chain, BCAA catabolism, purine metabolism, the urea cycle, gluconeogenesis, glycolysis, ketone body metabolism, and the pentose phosphate pathway were also included in the top 50 most enriched pathways. These data will guide future targeted metabolic studies in human heart failure.

Abstract 377: KLF15-TCF7L2-dependent Cardiac Transcriptional Reprogramming Induces Cardiomyocyte and Vascular Cell Remodeling in the Mammalian Heart

Integrative biochemical and omic-based approaches have refined our understanding of how cells integrate the Wnt signal at the chromatin level to yield specific cellular responses. However, this aspect remains unexplored in the heart. Since Wnt/β-catenin signaling activation is a hallmark in pathological cardiac remodeling, we aimed to characterize the specific Wnt cardiac transcriptional network regulation, amenable to therapeutic intervention in the adult heart. In the adult heart, we found the transcription factor KLF15 occupying regulatory regions of tissue remodeling genes containing Wnt transcriptional activator, TCF7L2, binding sites, which are silenced in the healthy myocardium but are active during pathological remodeling. Supporting KLF15 repressive roles, its loss resulted in cardiac TCF7L2 activation, maladaptive reprograming and failure in vivo . We demonstrated that KLF15 possess transcriptional age-specific repressive functions controlling Wnt signaling, cardiomyocyte de-differentiation and vascular cell (VC) remodeling. Employing different transgenic mouse models we further identified a cooperative program inducing aberrant VC remodeling, caused by a reduction of KLF15 with a concomitant TCF7L2 activation in cardiomyocytes. Furthermore, we characterized a cardiac specific Wnt transcriptional inhibitory complex consisting of KLF15 directly interacting with β-catenin and TCF7L2 and identified the amino acids critical for these interactions. Next, using a CRISPR/Cas9-mediated approach we generated KLF15 knockout (KO) hESC lines differentiated into functional cardiomyocytes and used for engineering human myocardium (EHM) generation. KLF15 KO EHMs showed activation of TCF7L2-dependent transcription as well as impaired function in comparison to control lines, recapitulating the Klf15 KO mouse phenotype. Altogether, we uncover an exquisite evolutionary conserved cardiac specific regulation mediated by KLF15 on Wnt signaling in myocardium offering a basis for designing highly specific pharmacological intervention for controlling Wnt cardiac-specific gene activation to prevent irreversible heart failure. We also underscore the significance of KLF15-Wnt dynamics in VC remodeling of the adult heart.

Cardiac contractile dysfunction and protein kinase C-mediated myofilament phosphorylation in disease and aging.

Increases in protein kinase C (PKC) are associated with diminished cardiac function, but the contribution of downstream myofilament phosphorylation is debated in human and animal models of heart failure. The current experiments evaluated PKC isoform expression, downstream cardiac troponin I (cTnI) S44 phosphorylation (p-S44), and contractile function in failing (F) human myocardium, and in rat models of cardiac dysfunction caused by pressure overload and aging. In F human myocardium, elevated PKCα expression and cTnI p-S44 developed before ventricular assist device implantation. Circulatory support partially reduced PKCα expression and cTnI p-S44 levels and improved cellular contractile function. Gene transfer of dominant negative PKCα (PKCαDN) into F human myocytes also improved contractile function and reduced cTnI p-S44. Heightened cTnI phosphorylation of the analogous residue accompanied reduced myocyte contractile function in a rat model of pressure overload and in aged Fischer 344 × Brown Norway F1 rats (≥26 mo). Together, these results indicate PKC-targeted cTnI p-S44 accompanies cardiac cellular dysfunction in human and animal models. Interfering with PKCα activity reduces downstream cTnI p-S44 levels and partially restores function, suggesting cTnI p-S44 may be a useful target to improve contractile function in the future.

Impact of heart rate on cross-bridge cycling kinetics in failing and nonfailing human myocardium.

The force-frequency relationship (FFR) is an important regulatory mechanism that increases the force-generating capacity as well as the contraction and relaxation kinetics in human cardiac muscle as the heart rate increases. In human heart failure, the normally positive FFR often becomes flat, or even negative. The rate of cross-bridge cycling, which has been reported to affect cardiac output, could be potentially dysregulated and contribute to blunted or negative FFR in heart failure. We recently developed and herein use a novel method for measuring the rate of tension redevelopment. This method allows us to obtain an index of the rate of cross-bridge cycling in intact contracting cardiac trabeculae at physiological temperature and assess physiological properties of cardiac muscles while preserving posttranslational modifications representative of those that occur in vivo. We observed that trabeculae from failing human hearts indeed exhibit an impaired FFR and a reduced speed of relaxation kinetics. However, stimulation frequencies in the lower spectrum did not majorly affect cross-bridge cycling kinetics in nonfailing and failing trabeculae when assessed at maximal activation. Trabeculae from failing human hearts had slightly slower cross-bridge kinetics at 3 Hz as well as reduced capacity to generate force upon K+ contracture at this frequency. We conclude that cross-bridge kinetics at maximal activation in the prevailing in vivo heart rates are not majorly impacted by frequency and are not majorly impacted by disease.NEW & NOTEWORTHY In this study, we confirm that cardiac relaxation kinetics are impaired in filing human myocardium and that cross-bridge cycling rate at resting heart rates does not contribute to this impaired relaxation. At high heart rates, failing myocardium cross-bridge rates are slower than in nonfailing myocardium.