Transplantation of Fibroblast Sheets with Blood Mononuclear Cell Culture Exerts Cardioprotective Effects by Enhancing Anti-Inflammation and Vasculogenic Potential in Rat Experimental Autoimmune Myocarditis Model

Fulminant myocarditis causes impaired cardiac function, leading to poor prognosis and heart failure. Cell sheet engineering is an effective therapeutic option for improving cardiac function. Naïve blood mononuclear cells (MNCs) have been previously shown to enhance the quality and quantity of cellular fractions (QQMNCs) with anti-inflammatory and vasculogenic potential using the one culture system. Herein, we investigated whether autologous cell sheet transplant with QQMNCs improves cardiac function in a rat model with experimental autoimmune myocarditis (EAM). Fibroblast sheets (F-sheet), prepared from EAM rats, were co-cultured with or without QQMNCs (QQ+F sheet) on temperature-responsive dishes. QQ+F sheet induced higher expression of anti-inflammatory and vasculogenic genes (Vegf-b, Hgf, Il-10, and Mrc1/Cd206) than the F sheet. EAM rats were transplanted with either QQ+F sheet or F-sheet, and the left ventricular (LV) hemodynamic analysis was performed using cardiac catheterization. Among the three groups (QQ+F sheet, F-sheet, operation control), the QQ+F sheet transplant group showed alleviation of end-diastolic pressure–volume relationship on a volume load to the same level as that in the healthy group. Histological analysis revealed that QQ+F sheet transplantation promoted revascularization and mitigated fibrosis by limiting LV remodeling. Therefore, autologous QQMNC-modified F-sheets may be a beneficial therapeutic option for EAM.

Evaluation of [68Ga]Ga-NODAGA-RGD for PET Imaging of Rat Autoimmune Myocarditis

The 68Gallium-labeled 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid conjugated radiolabelled arginine-glycine-aspartic acid peptide ([68Ga]Ga-NODAGA-RGD) is a positron emission tomography (PET) tracer binding to cell surface receptor αvβ3 integrin that is upregulated during angiogenesis and inflammation. We studied whether αvβ3 targeting PET imaging can detect myocardial inflammation in a rat model of autoimmune myocarditis. To induce myocarditis, rats (n = 8) were immunized with porcine cardiac myosin in complete Freund's adjuvant on days 0 and 7. Control rats (n = 8) received Freund's adjuvant alone. On day 21, in vivo PET/CT imaging with [68Ga]Ga-NODAGA-RGD followed by ex vivo autoradiography and immunohistochemistry were carried out. Inflammatory lesions were detected histologically in the myocardium of 7 out of 8 immunized rats. In vivo PET images showed higher [68Ga]Ga-NODAGA-RGD accumulation in the myocardium of rats with inflammation than the non-inflamed myocardium of control rats (SUVmean 0.4 ± 0.1 vs. 0.1 ± 0.02; P = 0.00006). Ex vivo autoradiography and histology confirmed that [68Ga]Ga-NODAGA-RGD uptake co-localized with inflammatory lesions containing αvβ3 integrin-positive capillary-like structures. A non-specific [68Ga]Ga-DOTA-(RGE)2 tracer showed 76% lower uptake than [68Ga]Ga-NODAGA-RGD in the inflamed myocardium. Our results indicate that αvβ3 integrin-targeting [68Ga]Ga-NODAGA-RGD is a potential PET tracer for the specific detection of active inflammatory lesions in autoimmune myocarditis.

Macrophage Inflammatory Protein-1 Alpha, a Potential Biomarker for Predicting Left Atrial Remodeling in Patients With Atrial Fibrillation

Objectives: Left atrial (LA) remodeling itself is an independent risk factor for ischemic stroke and mortality, with or without atrial fibrillation (AF). Macrophage inflammatory protein-1 alpha (MIP-1α) has been reported to be involved in the induction of autoimmune myocarditis and dilated cardiomyopathy. Little is known about whether MIP-1α can be used to predict LA remodeling, especially in patients with AF.Methods: We prospectively enrolled 78 patients who had received a cardiac implantable electronic device due to sick sinus syndrome in order to define AF accurately. AF was diagnosed clinically before enrollment, according to 12-lead electrocardiography (ECG) and 24-h Holter test in 54 (69%) patients. The serum cytokine levels and the mRNA expression levels of peripheral blood leukocytes were checked and echocardiographic study was performed on the same day within 1 week after the patients were enrolled into the study. The 12-lead ECG and 24-h Holter test were performed on the same day of the patients' enrollment, and the device interrogation was performed every 3 months after enrollment. The enrolled patients were clinically followed up for 1 year.Results: There was no difference in baseline characteristics, cytokine levels and mRNA expression between patients with and without AF. Larger LA volume was positively correlated with higher levels of MIP-1α (r = 0.461, p ≤ 0.001) and the atrial high-rate episodes (AHREs) burden (r = 0.593, p < 0.001), and negatively correlated with higher levels of transforming growth factor (TGF)-β1 (r = −0.271, p = 0.047) and TGF-β3 (r = −0.279, p = 0.041). The higher AHREs burden and MIP-1α level could predict LA volume independently. The mRNA expression of RORC was negatively associated with the MIP-1α level.Conclusions: This study showed that higher MIP-1α was significantly associated with LA remodeling and may have the potentials to predict LA remodeling in terms of a larger LA volume, and that circadian gene derangement might affect the expression of MIP-1α.

P2Y12 Inhibition in Murine Myocarditis Results in Reduced Platelet Infiltration and Preserved Ejection Fraction

Previous mouse studies have shown the increased presence of platelets in the myocardium during early stages of myocarditis and their selective detection by MRI. Here, we aimed to depict early myocarditis using molecular contrast-enhanced ultrasound of activated platelets, and to evaluate the impact of a P2Y12 receptor platelet inhibition. Experimental autoimmune myocarditis was induced in BALB/c mice by subcutaneous injection of porcine cardiac myosin and complete Freund adjuvant (CFA). Activated platelets were targeted with microbubbles (MB) coupled to a single-chain antibody that binds to the “ligand-induced binding sites” of the GPIIb/IIIa-receptor (=LIBS-MB). Alongside myocarditis induction, a group of mice received a daily dose of 100 g prasugrel for 1 month. Mice injected with myosin and CFA had a significantly deteriorated ejection fraction and histological inflammation on day 28 compared to mice only injected with myosin. Platelets infiltrated the myocardium before reduction in ejection fraction could be detected by echocardiography. No selective binding of the LIBS-MB contrast agent could be detected by either ultrasound or histology. Prasugrel therapy preserved ejection fraction and significantly reduced platelet aggregates in the myocardium compared to mice without prasugrel therapy. Therefore, P2Y12 inhibition could be a promising early therapeutic target in myocarditis, requiring further investigation.

Ethyl Pyruvate Ameliorates Experimental Autoimmune Myocarditis

Ethyl pyruvate (EP) has profound anti-inflammatory and immunomodulatory properties. Here, its effects were determined on experimental autoimmune myocarditis (EAM) induced in mice by heart-specific myosin-alpha heavy chain peptide immunization. EP was applied intraperitoneally, daily, starting with the immunization. Severity of EAM was determined by histological assessment of immune cell infiltrates into the heart. Cells were phenotypically characterized by flow cytometry. Concentration of cytokines in cell culture supernatants and sera was determined by ELISA. EP reduced the infiltration of immune cells into the heart and lessened heart inflammation. Smaller number of total immune cells, as well as of CD11b+ and CD11c+ cells were isolated from the hearts of EP-treated mice. A reduced number of antigen-presenting cells, detected by anti-CD11c, MHC class II and CD86 antibodies, as well as of T helper (Th)1 and Th17 cells, detected by anti-CD4, IFN-γ and IL-17 antibodies, was determined in mediastinal lymph nodes draining the heart, in parallel. In the spleen, only the number of CD11c+ cells were reduced, but not of the other examined populations, thus implying limited systemic effect of EP. Reduced production of IFN-γ and IL-17 by myosin-alpha heavy chain peptide-restimulated cells of the lymph nodes draining the site of immunization was observed in EP-treated mice. Our results clearly imply that EP restrains autoimmunity in EAM. Therapeutic application of EP in the treatment of myocarditis in humans should be addressed in the forthcoming studies.

Serum Organ-Specific Anti-Heart and Anti-Intercalated Disk Autoantibodies as New Autoimmune Markers of Cardiac Involvement in Systemic Sclerosis: Frequency, Clinical and Prognostic Correlates

Background: Heart involvement (HInv) in systemic sclerosis (SSc) may relate to myocarditis and is associated with poor prognosis. Serum anti-heart (AHA) and anti-intercalated disk autoantibodies (AIDA) are organ and disease-specific markers of isolated autoimmune myocarditis. We assessed frequencies, clinical correlates, and prognostic impacts of AHA and AIDA in SSc. Methods: The study included consecutive SSc patients (n = 116, aged 53 ± 13 years, 83.6% females, median disease duration 7 years) with clinically suspected heart involvement (symptoms, abnormal ECG, abnormal troponin I or natriuretic peptides, and abnormal echocardiography). All SSc patients underwent CMR. Serum AHA and AIDA were measured by indirect immunofluorescence in SSc and in control groups of non-inflammatory cardiac disease (NICD) (n = 160), ischemic heart failure (IHF) (n = 141), and normal blood donors (NBD) (n = 270). AHA and AIDA status in SSc was correlated with baseline clinical, diagnostic features, and outcome. Results: The frequency of AHA was higher in SSc (57/116, 49%, p < 0.00001) than in NICD (2/160, 1%), IHF (2/141, 1%), or NBD (7/270, 2.5%). The frequency of AIDA was higher (65/116, 56%, p < 0.00001) in SSc than in NICD (6/160, 3.75%), IHF (3/141, 2%), or NBD (1/270, 0.37%). AHAs were associated with interstitial lung disease (p = 0.04), history of chest pain (p = 0.026), abnormal troponin (p = 0.006), AIDA (p = 0.000), and current immunosuppression (p = 0.01). AHAs were associated with death (p = 0.02) and overall cardiac events during follow-up (p = 0.017). Conclusions: The high frequencies of AHA and AIDA suggest a high burden of underdiagnosed autoimmune HInv in SSc. In keeping with the negative prognostic impact of HInv in SSc, AHAs were associated with dismal prognosis.

Autoimmune Myocarditis and Arrhythmogenic Mitral Valve Prolapse: An Unexpected Overlap Syndrome

Background: both myocarditis and mitral valve prolapse (MVP) are known uncommon causes of ventricular arrhythmias in young patients. Aim: to report the first clinical case of endomyocardial biopsy (EMB)-proven autoimmune myocarditis and associated arrhythmogenic MVP in a patient with recurrent ventricular fibrillation (VF) episodes. Methods: myocarditis was diagnosed both by cardiac magnetic resonance (CMR) and EMB. Arrhythmogenic MVP was documented by transthoracic echocardiogram, CMR, and electroanatomical mapping of the trigger premature ventricular contractions (PVCs). Results: a 22-year-old woman underwent immunosuppressive therapy after EMB-proven diagnosis of autoimmune myocarditis with VF onset and early implantable cardioverter defibrillator (ICD) placement. Three years later, she experienced two VF recurrences and persistent PVCs, despite no signs of myocarditis recurrence. An echocardiogram revealed bileaflet MVP with high arrhythmic risk features. Finally, electroanatomical mapping and ablation of the trigger PVC were successfully performed. Conclusion: in patients with recurrent VF episodes despite evidence-based medical treatment for myocarditis, MVP should be considered as an alternative arrhythmogenic substrate, and warrants early ICD implant and PVC-targeted therapy.

Activated myofibroblasts promote cardiac hypertrophy and systolic dysfunction independently of cardiac fibrosis in experimental autoimmune myocarditis

Background/Introduction Heart-specific inflammation – myocarditis is a common cause dilated cardiomyopathy which is characterized by pathological tissue remodeling, ventricular stiffening, cardiomyopathy and heart failure. In experimental autoimmune myocarditis (EAM) susceptible mice immunized with alpha myosin heavy chain (αMyHC) and complete Freund's adjuvant (CFA) develop acute myocarditis driven by autoreactive CD4+ T cells that is followed by progressive fibrosis, cardiomyopathy and systolic dysfunction. Purpose The aim of the study was to investigate the role of cardiac fibroblasts and myofibroblasts in myocarditis and post-inflammatory dilated cardiomyopathy in mouse model of EAM. Methods EAM was induced in BALB/c mice by immunization with αMyHC/CFA. We used reporter mice expressing EGFP under collagen type I promoter (Coll-EGFP) and RFP under a control of α-smooth muscle actin (αSMA) promoter (αSMA-RFP) and transgenic αSMA-TK mice with ganciclovir-inducible ablation of proliferating myofibroblasts. Cardiac cells were quantified using flow cytometry. Cardiac fibroblasts (CD45-CD31-EGFP+) were sorted from healthy and myocarditis-positive (day 21) mice using BD FACSAria™ II Cell Sorter and analyzed for the whole genome transcriptomics by RNA sequencing. Echocardiography was performed on Vevo 2100 Imaging System. Cardiac fibrosis was assessed by Trichrome Massons's staining and hydroxyproline assay, whereas cardiac hypertrophy by analysing cross-sectional cardiomyocyte area. Profibrotic gene expression was assessed by qRT-PCR. Results The total number of cardiac fibroblasts (CD45-CD31-EGFP+) and the subset of myofibroblasts (CD45-CD31-EGFP+RFP+) remained unchanged at inflammatory (d21) and fibrotic stages (d40). Analysis of differentially expressed genes (min. 2x fold change, p value &lt;0.05) pointed out activation of immune processes (mainly chemokine production), response to stress, cytoskeletal and extracellular matrix re-organization in cardiac fibroblasts in response to myocarditis. αSMA-TK mice treated with ganciclovir (from day 21) showed comparable percent of fibrotic area, but significantly reduced heart weight, decreased cardiomyocyte hypertrophy and improved ejection fraction and cardiac output at day 40 comparing to PBS-treated mice. Ganciclovir-treated mice showed also attenuated cardiac Acta2 and Srf but markedly enhanced Mmp2 expression. Conclusions In EAM model cardiac fibroblasts actively participate in proinflammatory and profibrotic responses, while activated myofibroblasts contribute to dilated cardiomyopathy development independently of cardiac fibrosis. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Science Centre (Poland)