Mouse Cardiac Myocytes from C57BL/6

Background Doxorubicin (DOX) cardiotoxicity is a major complication of cancer therapy and involves macrophage-driven inflammation and myocardial remodeling. The macropha... More

Background Doxorubicin (DOX) cardiotoxicity is a major complication of cancer therapy and involves macrophage-driven inflammation and myocardial remodeling. The macrophage surface protein Mac-2 (galectin-3) is upregulated in cardiac injury, but its role in regulating macrophage function and downstream injury pathways remains undefined. Methods We used CRISPR/Cas9-engineered Mac-2-null macrophages to evaluate chemotaxis, cytokine gene expression, and lysosomal stress signaling in vitro. To examine paracrine injury mechanisms, we performed co-culture assays with cardiomyocytes and fibroblasts. In vivo, we studied homozygous Mac-2-mutant mice and used CD45.1/CD45.2 bone marrow transplantation with lineage tracking to define hematopoietic versus stromal contributions to DOX-induced inflammation, apoptosis, fibrosis, and systolic dysfunction. Results Doxorubicin induced Mac-2 and inflammatory transcripts (Il6, Tnf, Ccl2) in wild-type macrophages, whereas Mac-2 knockout reduced DOX uptake, chemotaxis, and cytokine induction. In co-culture, DOX-treated WT macrophages increased caspase-3/7 activity in cardiomyocytes and phospho-TFEB in fibroblasts, both attenuated with Mac-2 deletion. In vivo, Mac-2-null mice exhibited less cardiac inflammation, apoptosis, and fibrosis with preserved systolic function and reduced mortality. Bone marrow transplantation demonstrated that hematopoietic Mac-2 suppressed cardiac Tfeb and upregulated Sqstm1 and Tgfb1, enhancing inflammatory and apoptotic responses, whereas Mac-2-deficient marrow restored Tfeb, limited Sqstm1/Tgfb1, and protected cardiac function. Conclusions Mac-2 promotes DOX-induced cardiac injury by facilitating inflammatory activation in macrophages, driving fibroblast lysosomal stress via TFEB and SQSTM1, and augmenting caspase-3–associated apoptosis in cardiomyocytes. Loss of Mac-2 in hematopoietic cells reduces inflammation, fibrosis, and systolic dysfunction in vivo. Less

Aims: Pathological cardiac fibrosis and hypertrophy are common features of left ventricular remodeling that often progress to heart failure (HF). Endothelial cells (ECs) ... More

Aims: Pathological cardiac fibrosis and hypertrophy are common features of left ventricular remodeling that often progress to heart failure (HF). Endothelial cells (ECs) are the most abundant non-myocyte cells in adult mouse heart. Simvastatin, a strong inducer of Krüppel-like Factor 2 (Klf2) in ECs, ameliorates pressure overload induced maladaptive cardiac remodeling and dysfunction. This study aims to explore the detailed molecular mechanisms of the anti-remodeling effects of simvastatin. Methods and Results: RGD-magnetic-nanoparticles were used to endothelial specific delivery of siRNA and we found absence of simvastatin's protective effect on pressure overload induced maladaptive cardiac remodeling and dysfunction after in vivo inhibition of EC-Klf2. Mechanism studies showed that EC-Klf2 inhibition reversed the simvastatin-mediated reduction of fibroblast proliferation and myofibroblast formation, as well as cardiomyocyte size and cardiac hypertrophic genes, which suggested that EC-Klf2 might mediate the anti-fibrotic and anti-hypertrophy effects of simvastatin. Similar effects were observed after Klf2 inhibition in cultured ECs. Moreover, Klf2 regulated its direct target gene TGFβ1 in ECs and mediated the protective effects of simvastatin, and inhibition of EC-Klf2 increased the expression of EC-TGFβ1 leading to simvastatin losing its protective effects. Also, EC-Klf2 was found to regulate EC-Foxp1 and loss of EC-Foxp1 attenuated the protective effects of simvastatin similar to EC-Klf2 inhibition. Conclusions: We conclude that cardiac microvasculature ECs are important in the modulation of pressure overload induced maladaptive cardiac remodeling and dysfunction, and the endothelial Klf2-TGFβ1 or Klf2-Foxp1-TGFβ1 pathway mediates the preventive effects of simvastatin. This study demonstrates a novel mechanism of the non-cholesterol lowering effects of simvastatin for HF prevention. Keywords: Heart failure (HF), Maladaptive cardiac remodeling (cardiac fibrosis and hypertrophy), HMG-CoA reductase inhibitors, Simvastatin, Vascular endothelial cells (ECs), Krüppel-like Factor 2 (Klf2), Transforming growth factor-beta 1 (TGFβ1), Forkhead Box P1 (Foxp1) Less

The hypoxic conditions induced by reduced blood flow decreases oxygen availability in target tissues. Cellular hypoxia leads to mitochondrial dysfunction, decreased energ... More

The hypoxic conditions induced by reduced blood flow decreases oxygen availability in target tissues. Cellular hypoxia leads to mitochondrial dysfunction, decreased energy production, and increased production of reactive oxygen species. To determine the alteration in expression of mitochondrial genes after hypoxia in cardiomyocytes, we developed a rodent mitochondrial gene chip (RoMitoChip). The chip had 1088 probe sets including 46 probe sets representing 37 mouse mitochondrial DNA transcripts and the remaining probe sets representing mouse nuclear genes contributing to the mitochondrial structure and function. Mouse cardiomyocytes isolated from neonatal C57BL/6 mice that were subjected to hypoxia (1% oxygen) for different time intervals demonstrated a dichotomy in the expression profile of tRNA and mRNA transcripts. We report a total of 483 signature genes that were altered by hypoxia in the cardiac myocytes and related to mitochondrial structure and function. This includes 23 transcripts on mitochondrial DNA. Pathway analysis demonstrated predominant changes in the expression of genes involved in oxidative phosphorylation, glucose and fatty acid metabolism, and apoptosis. The most upregulated genes after 24 h of hypoxia included hypoxia-inducible factor 1, alpha subunit, inducible genes Bnip3, Pdk1, and Aldoc. Whereas Bnip3 is important in the cardiomyocyte death pathway, Pdk1 enzyme is critical in conserving mitochondrial function by diverting metabolic intermediates to glycolysis. This study identifies the participation of two important pathways, cell death and glycolytic, and two key proteins, Bnip3 and Pdk1, playing critical roles in these pathways in cardiomyocytes after severe hypoxia. Less