Persistent anti-heart autoimmunity causes cardiomyocyte damage in chronic heart failure

Although clinicians and researchers have long appreciated the detrimental effects of excessive acute inflammation after myocardial infarction (MI), less is known about the role of the adaptive immune system in MI complications including heart failure. Yet, abundant cardiac self-antigens released from necrotic cardiomyocytes in a highly inflammatory environment are likely to overwhelm peripheral mechanisms of immunological self-tolerance and adaptive auto-reactivity against the heart may cause ongoing tissue destruction and exacerbate progression to chronic heart failure (CHF). Here, we confirm that the adaptive immune system is indeed persistently active in CHF due to ischemic heart disease triggered by MI in rats. Heart draining mediastinal lymph nodes contain active secondary follicles with mature class-switched IgG2a positive cells, and mature anti-heart auto-antibodies binding to cardiac epitopes are still present in serum as late as 16 weeks after MI. When applied to healthy cardiomyocytes in vitro, humoral factors present in CHF serum promoted apoptosis, cytotoxicity and signs of hypertrophy. These findings directly implicate post-MI autoimmunity as an integral feature of CHF progression, constituting a roadblock to effective regeneration and a promising target for therapeutic intervention.


Post-MI autoimmunity
Sintou et al., 2018 2 range of conditions including ischemic heart disease following myocardial infarction (MI) (1), and a major public health problem affecting 26 million people worldwide (2).
The immune system has been implicated in the early acute immune response to MI, which is crucial for quick tissue repair (3,4). Neutrophils and monocytes/macrophages infiltrating the damaged myocardium in response to danger signals (DAMPs) are an overt sign of early inflammation. However, the post-MI immune response does not cease once acute inflammation has been resolved, and excessive activation of endogenous repair mechanisms may lead to ongoing inflammation, fibrosis, and sustained tissue damage. Cardiac self-antigens released from necrotic cardiomyocytes in a highly inflammatory environment also activate autoreactive B and T cells of the adaptive immune system, which can be long lived and may cause sustained damage to the myocardium (4) (5).
The presence of anti-cardiac auto-antibodies in post-MI serum is well known to clinicians (6) (7). Post-myocardial infarction syndrome was first described in 1956 by Dressler (8) and is now recognized as an adaptive immune response against myocardial proteins (9). However, besides rare cases of clinically obvious extreme manifestations of post-MI autoimmunity, a certain degree of persistent subclinical auto-reactivity can cause ongoing damage to previously healthy myocardial tissue. This may increase susceptibility to subsequent infarcts and development of adverse remodeling and heart failure.
Activated B cells generate tissue-specific auto-antibodies and pro-inflammatory cytokines, which can directly contribute to cardiac dysfunction (37). B cell activation occurs in lymph nodes draining damaged tissue sites. Activated B and T cells with the same antigen specificity interact at the B-T border zone in the lymph node which leads to germinal centers facilitating affinity maturation and class switching to mature IgG isotypes, which convey a range of effector functions (10). Detailed information on the autoantibody isotypes in post-MI and CHF patients is limited, and further research may yield fundamental insights into pathological effects of auto-antibodies present in these patients.
In the present study, we document a highly active adaptive immune system in injured hearts, with anti-cardiac auto-reactivity and humoral factor-mediated cytotoxicity at chronic heart failure stage in the rodent MI model of permanent left anterior descending (LAD) artery ligation. We characterize the serum antibody isotype composition induced by myocardial injury and show a striking degree of immunological activity long after the initial injury. CHF serum creates a cytotoxic and hypertrophic environment for cultured cardiomyocytes, 3 environment may promote heart failure, and constituting a new therapeutic focus for regenerative intervention.

RESULTS:
1) Persistent reactivity of heart-draining lymph nodes during CHF. An MI is a potent trigger for an acute local innate immune reaction, including early infiltration of neutrophils and macrophages (11)(11), but evidence is accumulating that T and B cells are also activated within a week post-MI (12). To investigate the activation state of the adaptive immune system in CHF progression, myocardial infarcts were induced in male Sprague Dawley rats by surgical ligation of the left anterior descending (LAD) coronary artery. Heart-draining mediastinal lymph nodes were isolated at week 2 and week 16 after infarct surgery and analyzed for signs of reactivity. They showed distinct lymphoid follicles containing active germinal centers acutely post-MI (2 weeks) as well as at chronic stage (16 weeks) post-MI. Blood was visible in the medullary sinuses of the week 2 post-MI lymph nodes, indicating that they drained an area of hemorrhage ( Figure 1A), which is in line with the myocardial damage induced by LAD.
Quantification of lymph node size, total number of follicles and the percentage of follicles with germinal centers confirmed an activated state at week 16, albeit decreased compared to week 2 ( Figure 1B). Transcription of genes involved in B cell development and activation, including Rag1, Cd20(MS4a1), Cd81 was upregulated in the spleen 2 weeks post-MI, and some were still elevated over baseline at CHF stage ( Figure 1C). Recombination activating gene (Rag1) was elevated most prominently in both week 2 and week 16, consistent with its role in B cell development and Ig formation. Rag expression is also upregulated in antigen-activated early memory B cells during autoimmune responses (13). Sustained upregulation of Il4 and the Th2associated chemokine receptor Ccr3 together with an increase in Cd40 (Tnfrsf5) but a downregulation of Cd40lg (Tnfsf5) may provide an environment blocking apoptotic cell death and inducing sustained growth and differentiation (14).
2) The CHF antibody repertoire shifts towards mature class-switched isotypes. The presence of active germinal centers in heart-draining lymph nodes during CHF marks ongoing B cell maturation processes. To assess maturity of the CHF antibody repertoire, we characterized the isotype composition of the local and systemic antibodies in response to MI and their potential for pathological effects. Serum was collected 2 and 16 weeks post-MI and anti-heart auto-antibodies at CHF stage, elevation over baseline is evident in all animals.
Notably, inbred Lewis rats of a different genetic background appeared protected from developing anti-heart auto-antibody levels supporting a difference in genetic susceptibility to post-MI autoimmunity (Supplementary Figure 1), most likely due to MHC haplotypes and antigen-depended mechanisms of B cell maturation and antibody production.
A variety of major cardiac proteins, including cardiac myosin and troponin I, are targeted by auto-antibodies post MI (6). To assess binding of auto-antibodies to cardiac structures, CHF and control sera were used to stain frozen sections of healthy rat hearts. Strong staining was observed when using CHF serum, with a pattern resembling cardiomyocyte striations that confirmed auto-antibody binding to the cardiomyocyte contractile apparatus (Figure 3d).

4) CHF serum creates a cytotoxic and hypertrophic environment for cardiomyocytes.
The action of mature class-switched auto-antibodies has been implicated in immune-mediated tissue damage through complement-or cell-mediated cytotoxicity (15). To assess potential cell-independent pathological effects of CHF serum on cardiomyocytes, primary adult cardiomyocytes were isolated from healthy rats and treated with CHF and control sera for 24 hours. We observed significant cytotoxicity with CHF serum (Figure 4a), and an increase in  (23). Depending on specificity and isotype, anticardiac auto-antibodies in CHF can therefore result in cardiac injury in a variety of ways (24). They mediate physiological damage by cross reacting with β1-adrenergic receptors, which play a role in heart contractility via sympathetic stimulation. The activation of β1-adrenergic receptors by auto-antibodies results in excessive stimulation which can induce left ventricular hypertrophy and pathological remodeling (25). Most notably, antibodies of IgG2 isotype including IgG2a and IgG2b play well established pathogenic role in autoimmune diseases such as systemic lupus erythematosus (SLE) (26). As we reported previously for an Resiquimodinduced SLE model, anti-cardiac antibodies of IgG2a and IgG2b isotype are present in circulation and deposited in the hearts (27). IgG auto-antibodies activate the complement cascade, which culminates in formation of the cytolytic membrane attack complex (MAC) comprising of complement C5b-9. The MAC forms a trans-membrane pore leading to necrotic death of the target cell (28). At sub-lytic concentrations however, MAC can induce caspase activation and apoptosis (29). Complement is accepted as a mediator of additional damage during acute MI and after reperfusion (30)(31), but more subtle sub-lytic activity has also been implicated in development of dilated cardiomyopathy. C5b-9 correlates with myocardial immunoglobulin deposition and expression of TNF-α (40). In vitro, C5b-9 attack on cardiomyocytes induces nuclear factor (NF)-κB activation as well as transcription, synthesis, and secretion of TNF-α by the cardiomyocytes themselves (32). NF-κB activation and TNF-α both induce cardiomyocyte hypertrophy (33) (34) in vitro, and the presence of immune cells able to respond to auto-antibody deposition in vivo will further increase the number of pathological pathways that contribute to CHF. In addition, as others have observed, an increase in Myh7 upon CHF serum stimulation of HL1-6 cardiomyocytes, without corresponding increase of Nppa and Nppb, may still reflect detrimental effects (22). Notably, transgenic mice overexpressing Myh7 had more progressive and severe cardiac damage than their wildtype counterparts (20), suggesting that an increase in Myh7 alone is sufficient to cause cardiac deterioration. Additional cardiomyocyte responses induced by cell-free serum containing autoantibodies, namely necrotic cell death, activation of caspase 3 as well as upregulation of MyH7, are likely mediated by direct complement-induced effects.
In summary, we show that long after the initially triggering MI, CHF serum still carries the ability to induce pathophysiological changes in healthy cardiomyocytes. This confirms that post-MI immune auto-reactivity is indeed a crucial contributing factor to adverse remodeling Therapies may include immunomodulation to achieve an appropriate balance between inflammatory and regulatory immune cell populations and most importantly restore immunological tolerance to the heart. A large number of clinical trials have attempted to improve post-MI outcome by immunomodulation, with a strong focus so far on the innate immune system and short term readouts (35). The CANTOS trial using Canakinumab, a monoclonal antibody that neutralizes IL-1β, was the most recent attempt of immunomodulatory therapy aiming to prevent secondary infarcts in post-MI patients with elevated inflammatory profile (36). The beneficial effects of cardiomyocyte antigen-specific tolerogenic dendritic cells (DC) on post-MI function and remodelling in mice (37)   Adult rat cardiomyocyte isolation: Adult rat cardiomyocytes were isolated as described previously (40). Briefly, hearts were excised and placed in ice cold Krebs-Henseleit (KH) Buffer (119mM NaCl, 4.7mM KCl, 0.94mM MgSO4, 1mM CaCl2, 1.2mM KH2PO4, 25mM NaHCO3, 11.5mM glucose; 95% O2, 5% CO2), cannulated via the aorta and perfused with KH at 37 o C using a Langendorff apparatus. When blood was cleared from the coronary circulation, the KH buffer was switched to a low calcium (LoCa2+) buffer (12-15µM CaCl2, Keynes, UK) or a Zeiss Axio Observer inverted microscope and processed using the public domain software ImageJ (NIH; http://rsb.info.nih.gov) (41).

ELISA:
The ELISA protocol for detection of rat anti-heart auto-antibodies was optimized using mouse, rat, pig lysate and titration of serum concentration to achieve the best possible background to signal ratio (Supplementary Figure 4).

COMPETING INTERESTS
The authors declare no competing financial interests.

MATERIALS & CORRESPONDENCE.
Please address correspondence to Dr Susanne Sattler s.sattler@imperial.ac.uk.   10um NE used as positive control to assess HL1-6 response to a hypertrophic stimulus. Data analysis was performed using the 2 -∆∆Ct method using beta-2 microglobulin as internal reference gene to achieve a fold change over control, n=3/group, technical triplicates / group, values represent mean +/-s.d., two-tailed unpaired Student's t-test. *P<0.05, **P<0.005.