Streptococcus pneumoniae (the pneumococcus) is the leading cause of community-acquired pneumonia and is now recognized to be a direct contributor to adverse acute cardiac events. During invasive pneumococcal disease, S. pneumoniae can gain access to the myocardium, kill cardiomyocytes, and form bacterium-filled "microlesions" causing considerable acute and long-lasting cardiac damage. While the molecular mechanisms responsible for bacterial translocation into the heart have been elucidated, the initial interactions of heart-invaded S. pneumoniae with cardiomyocytes remain unclear. In this study, we used a model of low multiplicity of S. pneumoniae infection with HL-1 mouse cardiomyocytes to investigate these early events. Using adhesion/invasion assays and immunofluorescent and transmission electron microscopy, we showed that S. pneumoniae rapidly adhered to and invaded cardiomyocytes. What is more, pneumococci existed as intravacuolar bacteria or escaped into the cytoplasm. Pulse-chase assays with BrdU confirmed intracellular replication of pneumococci within HL-1 cells. Using endocytosis inhibitors, bacterial isogenic mutants, and neutralizing antibodies against host proteins recognized by S. pneumoniae adhesins, we showed that S. pneumoniae uptake by cardiomyocytes is not through the well-studied canonical interactions identified for vascular endothelial cells. Indeed, S. pneumoniae invasion of HL-1 cells occurred through clathrin-mediated endocytosis (CME) and independently of choline binding protein A (CbpA)/laminin receptor, CbpA/polymeric immunoglobulin receptor, or cell wall phosphorylcholine/platelet-activating factor receptor. Subsequently, we determined that pneumolysin and streptococcal pyruvate oxidasederived H 2 O 2 production were required for cardiomyocyte killing. Finally, we showed that this cytotoxicity could be abrogated using CME inhibitors or antioxidants, attesting to intracellular replication of S. pneumoniae as a key first step in pneumococcal pathogenesis within the heart. KEYWORDS Cardiac acute events, cardiomyocytes, invasive pneumococcal disease, Streptococcus pneumoniae, facultatively intracellular pathogens, oxidative stress S treptococcus pneumoniae (the pneumococcus) is a Gram-positive bacterium which typically colonizes the nasopharynx asymptomatically (1, 2). However, it is also an opportunistic pathogen, capable of causing a wide spectrum of human diseases, including otitis media, community-acquired pneumonia, bacteremia/sepsis, and meningitis (3-5). In 30% of adults with pneumococcal pneumonia, S. pneumoniae escapes the airway and causes bacteremia/invasive pneumococcal disease (IPD). This typically occurs in those who are Ն65 years of age and/or in some fashion immunocompromised (4). Despite development and use of multiple antipneumococcal vaccines for decades, S. pneumoniae remains a major cause of human morbidity and death worldwide (6).Approximately 20% of adults hospitalized for severe pneumococcal disease experience some sort of major adverse cardiac events (MA...