Naturally secreted nanovesicles, known as exosomes, play important roles in stem cell-mediated cardioprotection. We have previously demonstrated that atorvastatin (ATV) pretreatment improved the cardioprotective effects of mesenchymal stem cells (MSCs) in a rat model of acute myocardial infarction (AMI). The aim of this study was to investigate if exosomes derived from ATV-pretreated MSCs exhibit more potent cardioprotective function in a rat model of AMI and if so to explore the underlying mechanisms. Exosomes were isolated from control MSCs (MSC-Exo) and ATV-pretreated MSCs (MSC^ATV-Exo) and were then delivered to endothelial cells and cardiomyocytes in vitro under hypoxia and serum deprivation (H/SD) condition or in vivo in an acutely infarcted Sprague-Dawley rat heart. Regulatory genes and pathways activated by ATV pretreatment were explored using genomics approaches and functional studies. In vitro, MSC^ATV-Exo accelerated migration, tube-like structure formation, and increased survival of endothelial cells but not cardiomyocytes, whereas the exosomes derived from MSC^ATV-Exo-treated endothelial cells prevented cardiomyocytes from H/SD-induced apoptosis. In a rat AMI model, MSC^ATV-Exo resulted in improved recovery in cardiac function, further reduction in infarct size and reduced cardiomyocyte apoptosis compared to MSC-Exo. In addition, MSC^ATV-Exo promoted angiogenesis and inhibited the elevation of IL-6 and TNF-α in the peri-infarct region. Mechanistically, we identified lncRNA H19 as a mediator of the role of MSC^ATV-Exo in regulating expression of miR-675 and activation of proangiogenic factor VEGF and intercellular adhesion molecule-1. Consistently, the cardioprotective effects of MSC^ATV-Exo was abrogated when lncRNA H19 was depleted in the ATV-pretreated MSCs and was mimicked by overexpression of lncRNA H19. Exosomes obtained from ATV-pretreated MSCs have significantly enhanced therapeutic efficacy for treatment of AMI possibly through promoting endothelial cell function. LncRNA H19 mediates, at least partially, the cardioprotective roles of MSC^ATV-Exo in promoting angiogenesis.

Background: Bone marrow mesenchymal stem cells (MSCs) are among the most common cell types to be used and studied for cardiac regeneration. Low survival rate and difficult retention of delivered MSCs in infarcted heart remain as major challenges in the field. Co-delivery of stem cell-derived exosomes (Exo) is expected to improve the recruitment and survival of transplanted MSCs. Methods: Exo was isolated from MSCs and delivered to an acute myocardial infarction (AMI) rat heart through intramyocardial injection with or without intravenous infusion of atrovastatin-pretreated MSCs on day 1, day 3, or day 7 after infarction. Echocardiography was performed to evaluate cardiac function. Histological analysis and ELISA test were performed to assess angiogenesis, SDF-1, and inflammatory factor expression in the infarct border zone. The anti-apoptosis effect of Exo on MSCs was evaluated using flow cytometry and Hoechst 33342 staining assay. Results: We found that intramyocardial delivery of Exo followed by MSC transplantation (in brief, Exo+MSC treatment) into MI hearts further improved cardiac function, reduced infarct size, and increased neovascularization when compared to controls treated with Exo or MSCs alone. Of note, comparing the three co-transplanting groups, intramyocardially injecting Exo 30 min after AMI combined with MSCs transplantation at day 3 after AMI achieved the highest improvement in heart function. The observed enhanced heart function is likely due to an improved microenvironment via Exo injection, which is exemplified as reduced inflammatory responses and better MSC recruitment and retention. Furthermore, we demonstrated that pre-transplantation injection of Exo enhanced survival of MSCs and reduced their apoptosis both in vitro and in vivo. Conclusions: Combinatorial delivery of exosomes and stem cells in a sequential manner effectively reduces scar size and restores heart function after AMI. This approach may represent as an alternative promising strategy for stem cell-based heart repair and therapy.

Direct reprogramming of fibroblasts to alternative cell fates by forced expression of transcription factors offers a platform to explore fundamental molecular events governing cell fate identity. The discovery and study of induced cardiomyocytes (iCMs) not only provides alternative therapeutic strategies for heart disease but also sheds lights on basic biology underlying CM fate determination. The iCM field has primarily focused on early transcriptome and epigenome repatterning, whereas little is known about how reprogramming iCMs remodel, erase, and exit the initial fibroblast lineage to acquire final cell identity. Here, we show that autophagy-related 5 (Atg5)–dependent autophagy, an evolutionarily conserved self-digestion process, was induced and required for iCM reprogramming. Unexpectedly, the autophagic factor Beclin1 (Becn1) was found to suppress iCM induction in an autophagy-independent manner. Depletion of Becn1 resulted in improved iCM induction from both murine and human fibroblasts. In a mouse genetic model, Becn1 haploinsufficiency further enhanced reprogramming factor–mediated heart function recovery and scar size reduction after myocardial infarction. Mechanistically, loss of Becn1 up-regulated Lef1 and down-regulated Wnt inhibitors, leading to activation of the canonical Wnt/β-catenin signaling pathway. In addition, Becn1 physically interacts with other classical class III phosphatidylinositol 3-kinase (PI3K III) complex components, the knockdown of which phenocopied Becn1 depletion in cardiac reprogramming. Collectively, our study revealed an inductive role of Atg5-dependent autophagy as well as a previously unrecognized autophagy-independent inhibitory function of Becn1 in iCM reprogramming.

Direct conversion of cardiac fibroblasts into induced cardiomyocytes (iCMs) by forced expression of defined factors holds great potential for regenerative medicine by offering an alternative strategy for treatment of heart disease. Successful iCM conversion can be achieved by minimally using three transcription factors, Mef2c (M), Gata4(G), and Tbx5 (T). Despite increasing interest in iCM mechanistic studies using MGT(polycistronic construct with optimal expression of M,G and T), the reprogramming efficiency varies among different laboratories. Two main Mef2c isoforms (isoform2, Mi2 and isoform4, Mi4) are present in heart and are used separately by different labs, for iCM reprogramming. It is currently unknown if differently spliced isoform of Mef2c contributes to varied reprogramming efficiency. Here, we used Mi2 and Mi4 together with Gata4 and Tbx5 in separate vectors or polycistronic vector, to convert fibroblasts to iCMs. We found that Mi2 can induce higher reprogramming efficiency than Mi4 in MEFs. Addition of Hand2 to MGT retroviral cocktail or polycistronic Mi2-GT retroviruses further enhanced the iCM conversion. Overall, this study demonstrated the isoform specific effects of Mef2c, during iCM reprogramming, clarified some discrepancy about varied efficiency among labs and might lead to future research into the role of alternative splicing and the consequent variants in cell fate determination.

Aims: To determine the associations of long-term SBP (LT-SBP) levels with clinical outcomes and health-related quality of life in heart failure with preserved ejection fraction (HFpEF). Methods and results: We analyzed participants from the Treatment of Preserved Cardiac Function Heart Failure with an Aldosterone Antagonist (TOPCAT) study with available different SBP measurements from different follow-ups (n = 3310). LT-SBP was the mean SBP value from 4-week measurement to the last one. The outcome measures are all-cause mortality and a composite of heart failure readmission or all-cause mortality and the Kansas City Cardiomyopathy Questionnaire (KCCQ) overall summary score. To determine the associations of LT-SBP and outcomes, we used adjusted Cox proportional hazards models and restricted cubic spline models. After multivariable adjustment, LT-SBP of 120–129 and 130–139 mmHg were associated with a lower risk of mortality (hazard ratio 0.66, 95% CI 0.51–0.87, P = 0.003; hazard ratio 0.68, 95% CI 0.51–0.90, P = 0.007, respectively); LT-SBP of 100–119 mmHg had similar risk of mortality (hazard ratio 0.96, 95% CI 0.72–1.28, P = 0.778) compared with LT-SBP of at least 140 mmHg. There was U-shaped relationship between LT-SBP and all-cause mortality (P < 0.001) with nadir risk occurring around 123 mmHg. Similar relationships were observed between LT-SBP and composite end point of heart failure readmission or all-cause mortality. The adjusted mean improvement in KCCQ score was significantly higher in the 120–129 mmHg group than in the at least 140 mmHg group beginning from the 12-month follow-up visit without significant differences in other groups. Conclusion: Among patients with HFpEF, long-term control of SBP level at 120–129 mmHg is independently associated with the highest risk reduction of all-cause mortality and improvement of KCCQ score. Future randomized clinical trials need to specifically evaluate optimal SBP treatment goals in patients with HFpEF.