Abstract
Cardiac dysfunction is a common complication of severe influenza virus infection, but whether this occurs due to direct infection of cardiac tissue or indirectly through systemic lung inflammation remains unclear. To test the etiology of this aspect of influenza disease, we generated a novel recombinant heart-attenuated influenza virus via genome incorporation of target sequences for miRNAs expressed in cardiomyocytes. Compared with control virus, mice infected with miR-targeted virus had significantly reduced heart viral titers, confirming cardiac attenuation of viral replication. However, this virus was fully replicative in the lungs and induced similar systemic inflammation and weight loss compared to control virus. The miR-targeted virus induced fewer cardiac conduction irregularities and significantly less fibrosis in mice lacking interferon-induced transmembrane protein 3 (IFITM3), which serve as a model for influenza-associated cardiac pathology. We conclude that robust virus replication in the heart is required for pathology, even when lung inflammation is severe.
INTRODUCTION
Seasonal influenza virus remains a major contributor to human mortality, and the potential for emergence of new pandemic strains is an ever-present worldwide concern (1–3). In addition to the lung damage traditionally associated with these infections, influenza virus can also cause or exacerbate cardiac dysfunction (4–10). Ample evidence exists for the role of cardiac dysfunction in influenza-associated morbidity and mortality, including the following: (i) Myocarditis is observed in a substantial portion of hospitalized influenza patients (11–13), (ii) heart damage at autopsy has been reported for fatal seasonal influenza cases (13–16), (iii) severe cardiac damage was described in nearly all patients who died from infection with the 1918 pandemic influenza virus (17), and (iv) cardiac events increase annually during flu season, especially among the unvaccinated (18, 19). Despite the implications for public health, little is known about the underlying mechanisms by which influenza virus causes heart pathology (11–16).There is a debate within the clinical literature as to whether influenza virus directly or indirectly causes cardiac complications (6–11). Although live virus has been detected in human and nonhuman primate heart samples, direct infection of the heart has rarely been investigated (20–24). Instead, current dogma states that severely infected lungs produce a cytokine storm with systemic cardiotoxic inflammation, which indirectly drives cardiac dysfunction (6–11, 25). Attempts to resolve this fundamental question have been hindered by the lack of tractable animal model systems for influenza-mediated cardiac pathology (26, 27).Laboratory mouse strains generally show minimal cardiac dysfunction upon influenza virus infection, even with high doses of virus (26–28). Overcoming this obstacle, we recently reported that mice lacking the interferon-induced transmembrane protein 3 (IFITM3) suffer from severe cardiac electrical dysfunction and fibrosis upon influenza virus infection, thus providing a long-sought model for influenza-associated cardiac complications (29). IFITM3 is an innate immunity protein that blocks the fusion of viruses with cell membranes, and deficiencies in IFITM3 are among the only known genetic risk factors in humans for developing severe influenza (30–36). We observed that severe influenza virus–induced cardiac pathology in IFITM3 knockout (KO) mice correlates with markedly increased and sustained viral loads in heart tissue when compared with rapid virus clearance in wild-type (WT) mice (29). These results suggested a direct role for influenza virus replication in the heart in driving cardiac dysfunction, but the observed cardiac phenomena could not be decoupled from the severe lung infection and heightened inflammation that also occurs in IFITM3 KO mice (29, 32, 37).Here, we sought to address the fundamental question of whether severe lung infection is sufficient to drive influenza-associated cardiac dysfunction, or whether virus replication in heart cells is required. To decouple lung inflammation and the direct infection of cardiomyocytes that both occur in IFITM3 KO mice, we designed, rescued, and validated a recombinant influenza virus that is attenuated for replication in cardiomyocytes while being fully replication competent and inflammatory in the lungs. We accomplished this cardiomyocyte attenuation via incorporation of target sites for muscle-specific microRNAs (miRNAs) miR133b and miR206 into the viral genome in a manner similar to previous engineering of a virus with blunted replication specifically in hematopoietic cells (38–44). miRNAs are short (<25 nucleotides), noncoding RNAs that interact with complementary sequences of target RNAs to suppress their translation or target them for degradation (45, 46). While some miRNAs are expressed ubiquitously, others are limited to specific tissue or cell types, which allows for tissue-specific gene silencing (45, 46). Using this novel heart-attenuated influenza virus, we found that severe lung inflammation during influenza virus infection, even in highly infected IFITM3 KO mice, was not sufficient to drive cardiac dysfunction in the absence of virus replication in cardiomyocytes. Thus, direct infection and replication of influenza virus in cardiomyocytes is a primary determinant of cardiac pathology associated with severe influenza.
