Myocarditis is an inflammatory disease of the heart muscle most commonly caused by viral infection and often maintained by autoimmunity. Virus-induced tissue damage triggers chemokine production and, subsequently, immune cell infiltration with pro-inflammatory and pro-fibrotic cytokine production follows. In patients, the overall inflammatory burden determines the disease outcome. Following the aim to define specific molecules that drive both immunopathology and/or autoimmunity in inflammatory heart disease, here we report on increased expression of colony stimulating factor 1 (CSF-1) in patients with myocarditis. CSF-1 controls monocytes originating from hematopoietic stem cells and subsequent progenitor stages. Both, monocytes and macrophages are centrally involved in mediating tissue damage and fibrotic scarring in the heart. CSF-1 influences monocytes via engagement of CSF-1 receptor, and it is also produced by cells of the mononuclear phagocyte system themselves. Based on this, we sought to modulate the virus-triggered inflammatory response in an experimental model of Coxsackievirus B3-induced myocarditis by silencing the CSF-1 axis in myeloid cells using nanoparticle-encapsulated siRNA. siCSF-1 inverted virus-mediated immunopathology as reflected by lower troponin T levels, a reduction of accumulating myeloid cells in heart tissue and improved cardiac function. Importantly, pathogen control was maintained and the virus was efficiently cleared from heart tissue. Since viral heart disease triggers heart-directed autoimmunity, in a second approach we investigated the influence of CSF-1 upon manifestation of heart tissue inflammation during experimental autoimmune myocarditis (EAM). EAM was induced in Balb/c mice by immunization with a myocarditogenic myosin-heavy chain-derived peptide dissolved in complete Freund's adjuvant. siCSF-1 treatment initiated upon established disease inhibited monocyte infiltration into heart tissue and this suppressed cardiac injury as reflected by diminished cardiac fibrosis and improved cardiac function at later states. Mechanistically, we found that suppression of CSF-1 production arrested both differentiation and maturation of monocytes and their precursors in the bone marrow. In conclusion, during viral and autoimmune myocarditis silencing of the myeloid CSF-1 axis by nanoparticle-encapsulated siRNA is beneficial for preventing inflammatory tissue damage in the heart and preserving cardiac function without compromising innate immunity's critical defense mechanisms.

Rationale: Rapid reperfusion is the most effective treatment for attenuating cardiac injury caused by myocardial ischemia. Yet, reperfusion itself elicits damage to the myocardium through incompletely understood mechanisms, known as ischemia/reperfusion (I/R) injury. The myocardium adapts to I/R by changes in gene expression, which determines the cellular response to reperfusion. Protein translation is a key component of gene expression. However, it is unknown how regulation of translation contributes to cardiac gene expression in response to reperfusion and whether it can be targeted to mitigate I/R injury. Methods: To examine translation and its impact on gene expression in response to I/R we assessed protein synthesis at different timepoints after ischemia and reperfusion in vitro and in vivo. Pharmacological inhibitors were used to dissect the underlying molecular mechanisms of translational control. Transient inhibition of protein synthesis was undertaken to decipher the effects of the translational response to reperfusion on cardiac function and inflammation. Cell-type-specific ribosome profiling was performed in mice subjected to I/R to determine the impact of translation on the regulation of gene expression in cardiomyocytes. Results: Reperfusion increased translation rates from a previously suppressed state during ischemia in cardiomyocytes, which was associated with the induction of cell death. In vivo, I/R resulted in strong activation of translation in the myocardial border zone. Detailed analysis revealed that the upregulation of translation is mediated by eIF4F complex formation, which was specifically mediated by the mTORC1-4EBP1-eIF4F axis. Short-term pharmacological inhibition of eIF4F complex formation by 4EGI-1 or rapamycin, respectively, attenuated translation, reduced infarct size and improved long-term cardiac function after myocardial infarction. Cardiomyocyte-specific ribosome profiling identified that reperfusion damage increased translation of mRNA networks in cardiomyocytes associated with cardiac inflammation and cell infiltration. Transient inhibition of the mTORC1-4EBP1-eIF4F axis decreased the expression of proinflammatory transcripts such as Ccl2, thereby reducing Ly6Chi monocyte infiltration and myocardial inflammation. Conclusions: Myocardial reperfusion induces protein synthesis in the border zone which contributes to I/R injury by rapidly translating a specific maladaptive mRNA network that mediates immune cell infiltration and inflammation. Transient inhibition of the mTORC1-4EBP1-eIF4F signaling axis during reperfusion attenuates this proinflammatory translational response, protects against I/R injury and improves long-term cardiac function after myocardial infarction.