Background: The proliferative capacity of cardiomyocytes in adult mammalian hearts is far too low to replace the cells that are lost to myocardial infarction. Both cardiomyocyte proliferation and myocardial regeneration can be improved via the overexpression of a constitutively active variant of YAP5SA (Yes‐associated protein, 5SA [active] mutant), but persistent overexpression of proliferation‐inducing genes could lead to hypertrophy and arrhythmia, whereas off‐target expression in fibroblasts and macrophages could increase fibrosis and inflammation. Methods and Results: Transient overexpression of YAP5SA or GFP (green fluorescent protein; control) was targeted to cardiomyocytes via our cardiomyocyte‐specific modified mRNA translation system ( ^YAP5SA CM‐SMRTs or ^GFP CM‐SMRTs, respectively). YAP5SA‐cardiomyocyte specificity was confirmed via in vitro experiments in cardiomyocytes and cardiac fibroblasts that had been differentiated from human induced‐ pluripotent stem cells and in human umbilical‐vein endothelial cells, and the regenerative potency of ^YAP5SA CM‐SMRTs was evaluated in a mouse myocardial infarction model. In cultured human induced‐pluripotent stem cells‐cardiomyocytes, YAP was abundantly expressed for 3 days after ^YAP5SA CM‐SMRTs administration and was accompanied by increases in the expression of markers for proliferation, before declining to near‐background levels after day 7, whereas GFP fluorescence remained high from days 1 to 3 after ^GFP CM‐SMRTs treatment and then slowly declined. GFP fluorescence was also observed in human induced‐pluripotent stem cells‐cardiac fibroblasts and human umbilical‐vein endothelial cells on day 1 after ^GFP CM‐SMRTs administration but declined substantially by day 3. In the mouse myocardial infarction model, echocardiographic assessments of left‐ventricular ejection fraction and fractional shortening were significantly greater, whereas infarct sizes were significantly smaller in ^YAP5SA CM‐SMRTs–treated mice than in vehicle‐treated control animals, and ^YAP5SA CM‐SMRTs appeared to promote cardiomyocyte proliferation. Conclusions: The CM‐SMRTs can be used to transiently and specifically overexpress YAP5SA in cardiomyocytes, and this treatment strategy significantly promoted cardiomyocyte proliferation and myocardial regeneration in a mouse myocardial infarction model.

Background: ELMSAN1 (ELM2‐SANT domain‐containing scaffolding protein 1) is a newly identified scaffolding protein of the MiDAC (mitotic deacetylase complex), playing a pivotal role in early embryonic development. Studies on Elmsan1 knockout mice showed that its absence results in embryo lethality and heart malformation. However, the precise function of ELMSAN1 in heart development and formation remains elusive. To study its potential role in cardiac lineage, we employed human‐induced pluripotent stem cells (hiPSCs) to model early cardiogenesis and investigated the function of ELMSAN1. Methods and Results: We generated ELMSAN1 ‐deficient hiPSCs through knockdown and knockout techniques. During cardiac differentiation, ELMSAN1 depletion inhibited pluripotency deactivation, decreased the expression of cardiac‐specific markers, and reduced differentiation efficiency. The impaired expression of genes associated with contractile sarcomere structure, calcium handling, and ion channels was also noted in ELMSAN1 ‐deficient cardiomyocytes derived from hiPSCs. Additionally, through a series of structural and functional assessments, we found that ELMSAN1 ‐null hiPSC cardiomyocytes are immature, exhibiting incomplete sarcomere Z‐line structure, decreased calcium handling, and impaired electrophysiological properties. Of note, we found that the cardiac‐specific role of ELMSAN1 is likely associated with histone H3K27 acetylation level. The transcriptome analysis provided additional insights, indicating maturation reduction with the energy metabolism switch and restored cell proliferation in ELMSAN1 knockout cardiomyocytes. Conclusions: In this study, we address the significance of the direct involvement of ELMSAN1 in the differentiation and maturation of hiPSC cardiomyocytes. We first report the impact of ELMSAN1 on multiple aspects of hiPSC cardiomyocyte generation, including cardiac differentiation, sarcomere formation, calcium handling, electrophysiological maturation, and proliferation.

Peripheral arterial disease (PAD) is estimated to affect at least 10 million people in the United States and over 200 million individuals worldwide. The clinical manifestations of PAD range from impaired mobility and pain with activity to chronic limb-threatening ischemia with risk of eventual tissue loss. Despite the significant global prevalence and debilitating consequences of the disease, non-option patients remain largely undertreated. Recognizing that ECs are well-known for their therapeutic role in enhancing perfusion and neovascularization in mouse models of critical limb ischemia, we hypothesize that a synergy of ECs with SMCs may achieve better recovery of perfusion and neovascularization than ECs or SMCs alone. In this study, we compared the therapeutic efficacy of hiPSC-ECs, hiPSC-SMCs, and hiPSC-ECs + hiPSC-SMCs for the treatment of critical limb disease using a NOD-SCID mouse model of hind limb ischemia (HLI). A total of 1 × 10⁶ hiPSC-ECs, 1 × 10⁶ hiPSC-SMCs, or 0.5 × 10⁶ hiPSC-ECs + 0.5 × 10⁶ hiPSC-SMCs were intramuscularly injected into the ischemic limb of the mouse on day 3 after HLI induction. Laser Doppler was recorded on day 0 before, days 3 (i.e., before cell administration) and 31 after HLI induction. Compared with only basal medium injection, hiPSC-ECs alone most effectively improved perfusion (63.8% after normalized non-ischemic limb, p < 0.001) and neovascularization (total vessel density = 180.9/magnification, p < 0.001) followed by co-administration of hiPSC-ECs + hiPSC-SMCs (perfusion = 59%, p = 0.008 and total vessel density = 170.5, p = 0.002) compared with basal medium injection (perfusion = 39.7% and total vessel density = 107.2), while hiPSC-SMCs administration achieved mild to moderate improvement (perfusion = 48.9% and total vessel density = 159.5, p = 0.01). When profiles of secreted growth factors or cytokines were determined using a protein array, 34 growth factors or cytokines involved in angiogenesis were highly enriched (at least >50% increase) in a hiPSC-ECs conditioned medium, including basic fibroblast growth factor and vascular endothelial growth factor, while there were 14 in hiPSC-SMCs conditioned medium. In conclusion, our results demonstrate that hiPSC-ECs alone most effectively improved perfusion and neovascularization, followed by co-administration of hiPSC-ECs + hiPSC-SMCs, which may be due to the abundance of growth factors and cytokines secreted by hiPSC-ECs. Future studies are warranted to evaluate the therapeutic efficacy of hiPSC-EC and hiPSC-SMC co-transplantation in aged animals with metabolic and cardiovascular comorbidities.