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Introduction: Hyperglycemia-mediated cardiac dysfunction is a critical initiator in the development of vascular complications, which, in turn, leads to cardiac fibrosis. In this study, we investigated the role of the Hippo signaling pathway in cardiomyocytes to study the complex signaling network of YAP1/TAZ on fibrotic and vascular inflammatory mediators in hyperglycemic condition. This in-vitro study demonstrated that YAP1/TAZ signaling is highly activated in the hyperglycemic cardiomyocytes. To further investigate the differentially expressed genes that are related to inflammation and fibrosis, RNA-sequencing studies were employed. Methods: To investigate the effects of hyperglycemia-mediated changes in cardiomyocytes, we used human AC16 cells cultured in-vitro under normoglycemic (5 mM D-Glucose) and hyperglycemic (50 mM D-glucose) conditions. After 24-hours of hyperglycemic insult, cells were collected and processed for RNA-seq studies. Furthermore, we also performed Western Blot analysis to evaluate the protein expression of YAP1/TAZ under hyperglycemia induced stress conditions. Results: Our study showed a significant upregulation of the protein expression of the YAP1/TAZ pathway in hyperglycemic cardiomyocytes. RNA-seq studies revealed differentially expressed genes (DEG) in the hyperglycemic condition in comparison with the normoglycemic condition. Among the extracellular matrix proteins, the following ECM and related markers were significantly upregulated including MMP3, TNC, TGF-beta1 and 2, COL4A1, FN1 and FGF-2. Altered expression of inflammatory mediators included the following markers, IL-6, CXCL10, CXCL12, CCL2 and VEGF-C. In addition, the following transcriptional co-activators were also significantly upregulated, including EPHA2 and MYOCD. Conclusions: This study suggests that changes in YAP1/TAZ signaling increases vascular inflammation in response to hyperglycemia. This also leads to increased expression of inflammatory mediators as shown by our results. Thus, the inhibition of the YAP1/TAZ pathway may prevent and improve hyperglycemia associated vascular damage and inflammation. 

Since conventional human cardiac two-dimensional (2D) cell culture and multilayered three-dimensional (3D) models fail in recapitulating cellular complexity and possess inferior translational capacity, we designed and developed a high-throughput scalable 3D bioprinted cardiac spheroidal droplet-organoid model with cardiomyocytes and cardiac fibroblasts that can be used for drug screening or regenerative engineering applications. This study helped establish the parameters for bioprinting and cross-linking a gelatin-alginate-based bioink into 3D spheroidal droplets. A flattened disk-like structure developed in prior studies from our laboratory was used as a control. The microstructural and mechanical stability of the 3D spheroidal droplets was assessed and was found to be ideal for a cardiac scaffold. Adult human cardiac fibroblasts and AC16 cardiomyocytes were mixed in the bioink and bioprinted. Live-dead assay and flow cytometry analysis revealed robust biocompatibility of the 3D spheroidal droplets that supported the growth and proliferation of the cardiac cells in the long-term cultures. Moreover, the heterocellular gap junctional coupling between the cardiomyocytes and cardiac fibroblasts further validated the 3D cardiac spheroidal droplet model. 

Diabetes is a major risk factor for cardiovascular diseases, especially cardiomyopathy, a condition in which the smooth muscles of the heart become thick and rigid, affecting the functioning of cardiomyocytes, the contractile cells of the heart. Uncontrolled elevated glucose levels over time can result in oxidative stress, which could lead to inflammation and altered epigenetic mechanisms. In the current study, we investigated whether hyperglycemia can modify cardiac function by directly affecting these changes in cardiomyocytes. To evaluate the adverse effect of high glucose, we measured the levels of gap junction protein, connexin 43, which is responsible for modulating cardiac electric activities and Troponin I, a part of the troponin complex in the heart muscles, commonly used as cardiac markers of ischemic heart disease. AC16 human cardiomyocyte cells were used in this study. Under hyperglycemic conditions, these cells demonstrated altered levels of connexin 43 and Troponin-I after 24 h of exposure. We also examined hyperglycemia induced changes in epigenetic markers: H3K9me1, Sirtuin-1 (SIRT1), and histone deacetylase (HDAC)-2 as well as in inflammatory and stress-related mediators, such as heat shock protein (HSP)-60, receptor for advanced glycation end products (RAGE), toll-like receptor (TLR)-4, high mobility group box (HMGB)-1 and CXC chemokine receptor (CXCR)-4. Cardiomyocytes exposed to 25mM glucose resulted in the downregulation of HSP60 and SIRT1 after 48 h. We further examined that hyperglycemia mediated the decrease in the gap junction protein CX43, as well as CXC chemokine receptor CXCR4 which may affect the physiological functions of the cardiomyocytes when exposed to high glucose for 24 and 48 h. Upregulated expression of DNA-binding nuclear protein HMGB1, along with changes in histone methylation marker H3K9me1 have demonstrated hyperglycemia-induced damage to cardiomyocyte at 24 h of exposure. Our study established that 24 to 48 h of hyperglycemic exposure could stimulate stress-mediated inflammatory mediators in cardiomyocytes in vitro. These stress-related changes in hyperglycemia-induced cardiomyocytes may further initiate an increase in injury markers which eventually could alter the epigenetic processes. Therefore, epigenetic and inflammatory mechanisms in conjunction with alterations in a downstream signaling pathway could have a direct effect on the functionality of the cardiomyocytes exposed to high glucose during short and long-term exposures.