Sepsis is an infection-induced severe inflammatory disorder that leads to multiple organ failure. Amongst organs affected, myocardial depression is believed to be a major contributor to septic death. While it has been identified that large amounts of circulating pro-inflammatory cytokines are culprit for triggering cardiac dysfunction in sepsis, the underlying mechanisms remain obscure. Additionally, recent studies have shown that exosomes released from bacteria-infected macrophages are pro-inflammatory. Hence, we examined in this study whether blocking the generation of exosomes would be protective against sepsis-induced inflammatory response and cardiac dysfunction. To this end, we pre-treated RAW264.7 macrophages with GW4869, an inhibitor of exosome biogenesis/release, followed by endotoxin (LPS) challenge. In vivo, we injected wild-type (WT) mice with GW4869 for 1 h prior to endotoxin treatment or cecal ligation/puncture (CLP) surgery. We observed that pre-treatment with GW4869 significantly impaired release of both exosomes and pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in RAW264.7 macrophages. At 12 h after LPS treatment or CLP surgery, WT mice pretreated with GW4869 displayed lower amounts of exosomes and pro-inflammatory cytokines in the serum than control PBS-injected mice. Accordingly, GW4869 treatment diminished the sepsis-induced cardiac inflammation, attenuated myocardial depression and prolonged survival. Together, our findings indicate that blockade of exosome generation in sepsis dampens the sepsis-triggered inflammatory response and thereby, improves cardiac function and survival.

Mesenchymal stem cells (MSCs) have been shown to elicit cardio-protective effects in sepsis. However, the underlying mechanism remains obscure. While recent studies have indicated that miR-223 is highly enriched in MSC-derived exosomes, whether exosomal miR-223 contributes to MSC-mediated cardio-protection in sepsis is unknown. In this study, loss-of-function approach was utilized, and sepsis was induced by cecal ligation and puncture (CLP). We observed that injection of miR-223-KO MSCs at 1 h post-CLP did not confer protection against CLP-triggered cardiac dysfunction, apoptosis and inflammatory response. However, WT-MSCs were able to provide protection which was associated with exosome release. Next, treatment of CLP mice with exosomes released from miR-223-KO MSCs significantly exaggerated sepsis-induced injury. Conversely, WT-MSC-derived-exosomes displayed protective effects. Mechanistically, we identified that miR-223-KO exosomes contained higher levels of Sema3A and Stat3, two known targets of miR-223 (5p & 3p), than WT-exosomes. Accordingly, these exosomal proteins were transferred to cardiomyocytes, leading to increased inflammation and cell death. By contrast, WT-exosomes encased higher levels of miR-223, which could be delivered to cardiomyocytes, resulting in down-regulation of Sema3A and Stat3. These data for the first time indicate that exosomal miR-223 plays an essential role for MSC-induced cardio-protection in sepsis.

Decreased heat shock protein (Hsp) expression in type 1 and type 2 diabetes has been implicated as a primary factor contributing to diabetes-induced organ damage. We recently showed that diabetic cardiomyocytes could release detrimental exosomes, which contain lower levels of Hsp20 than normal ones. To investigate whether such detrimental exosomes could be modified in cardiomyocytes by raising Hsp20 levels to become protective, we used a transgenic (TG) mouse model with cardiac-specific overexpression of Hsp20. TG and control wild-type (WT) mice were injected with streptozotocin (STZ) to induce diabetes. We observed that overexpression of Hsp20 significantly attenuated STZ-caused cardiac dysfunction, hypertrophy, apoptosis, fibrosis, and microvascular rarefaction. Moreover, Hsp20-TG cardiomyocytes exhibited an increased generation/secretion of exosomes by direct interaction of Hsp20 with Tsg101. Of importance, exosomes derived from TG cardiomyocytes encased higher levels of Hsp20, p-Akt, survivin, and SOD1 than WT exosomes and protected against in vitro hyperglycemia-triggered cell death, as well as in vivo STZ-induced cardiac adverse remodeling. Last, blockade of exosome generation by GW4869 remarkably offset Hsp20-mediated cardioprotection in diabetic mice. Our results indicate that elevation of Hsp20 in cardiomyocytes can offer protection in diabetic hearts through the release of instrumental exosomes. Thus, Hsp20-engineered exosomes might be a novel therapeutic agent for diabetic cardiomyopathy.

Recent studies have shown that myocardial ischemia/reperfusion (I/R)-induced necrosis can be controlled by multiple genes. In this study, we observed that both strands (5p and 3p) of miR-223 were remarkably dysregulated in mouse hearts upon I/R. Precursor miR-223 (pre-miR-223) transgenic mouse hearts exhibited better recovery of contractile performance over reperfusion period and lesser degree of myocardial necrosis than wild type hearts upon ex vivo and in vivo myocardial ischemia. Conversely, pre-miR-223 knock-out (KO) mouse hearts displayed opposite effects. Furthermore, we found that the RIP1/RIP3/MLKL necroptotic pathway and inflammatory response were suppressed in transgenic hearts, whereas they were activated in pre-miR-223 KO hearts upon I/R compared with wild type controls. Accordingly, treatment of pre-miR-223 KO mice with necrostatin-1s, a potent necroptosis inhibitor, significantly decreased I/R-triggered cardiac necroptosis, infarction size, and dysfunction. Mechanistically, we identified two critical cell death receptors, TNFR1 and DR6, as direct targets of miR-223-5p, whereas miR-223-3p directly suppressed the expression of NLRP3 and IκB kinase α, two important mediators known to be involved in I/R-induced inflammation and cell necroptosis. Our findings indicate that miR-223-5p/-3p duplex works together and cooperatively inhibits I/R-induced cardiac necroptosis at multiple layers. Thus, pre-miR-223 may constitute a new therapeutic agent for the treatment of ischemic heart disease.