Heart failure (HF) is characterized by abnormal mitochondrial calcium (Ca2+) handling, energy failure and impaired mitophagy resulting in contractile dysfunction and myocyte death. We have previously shown that the 18-kDa mitochondrial translocator protein of the outer mitochondrial membrane (TSPO) can modulate mitochondrial Ca2+ uptake. Experiments were designed to test the role of the TSPO in a murine pressure-overload model of HF induced by transverse aortic constriction (TAC). Conditional, cardiac-specific TSPO knockout (KO) mice were generated using the Cre-loxP system. TSPO-KO and wild-type (WT) mice underwent TAC for 8 weeks. TAC-induced HF significantly increased TSPO expression in WT mice, associated with a marked reduction in systolic function, mitochondrial Ca2+ uptake, complex I activity and energetics. In contrast, TSPO-KO mice undergoing TAC had preserved ejection fraction, and exhibited fewer clinical signs of HF and fibrosis. Mitochondrial Ca2+ uptake and energetics were restored in TSPO KO mice, associated with decreased ROS, improved complex I activity and preserved mitophagy. Thus, HF increases TSPO expression, while preventing this increase limits the progression of HF, preserves ATP production and decreases oxidative stress, thereby preventing metabolic failure. These findings suggest that pharmacological interventions directed at TSPO may provide novel therapeutics to prevent or treat HF.

Background The limited regenerative capacity of cardiac tissue has long been an obstacle to treating damaged myocardium. Cell-based therapy offers an enormous potential to the current treatment paradigms. However, the efficacy of regenerative therapies remains limited by inefficient delivery and engraftment. Electrotaxis, electrically guided cell movement, has been clinically utilized to improve recovery in a number of tissues, but has not been investigated for treating myocardial damage. Objectives The goal of the study is to test the electrotactic behaviors of several types of cardiac cells. Methods Cardiac Progenitor Cells (CPCs), Cardiac Fibroblasts (CFs), and human induced Pluripotent Stem cell-derived Cardiac Progenitor Cells (hiPSC-CPCs) were used. Results CPCs and CFs electrotax towards the anode of a direct current electric field (EF), while hiPSC-CPCs electrotax toward the cathode. The voltage-dependent electrotaxis of CPCs and CFs requires the presence of serum in the media. Addition of soluble vascular cell adhesion molecule (sVCAM) to serum-free media restores directed migration. We provide evidence that CPC and CF electrotaxis is mediated through phosphatidylinositide 3-kinases (PI3′K) signaling. In addition, Very Late Antigen-4 (VLA4), an integrin and growth factor receptor, is required for electrotaxis and localizes to the anodal edge of CPCs in response to direct current EF. HiPSC-derived CPCs do not express VLA4, migrate toward the cathode in a voltage-dependent manner, and similar to CPCs and CFs require media serum and PI3′K activity for electrotaxis. Conclusion The electrotactic behaviors of these therapeutic cardiac cells may be utilized for improving cell-based therapy for recovering function in damaged myocardium.

Neutrophils and non-muscle myosin light chain kinase (nmMLCK) have been implicated in intestinal microvascular leakage and mucosal hyperpermeability in inflammation and trauma. The aim of this study was to characterize the role of nmMLCK in neutrophil-dependent gut barrier dysfunction following thermal injury, a common form of trauma that typically induces inflammation in multiple organs. Histopathological examination of the small intestine in mice after a full-thickness burn revealed morphological evidence of mucosa inflammation characterized by neutrophil infiltration into the lamina propria, epithelial contraction, and narrow villi with blunt brush borders and loss of goblet cells. Compared to their wild-type counterparts, nmMLCK-/- mice displayed diminished morphological abnormalities. Likewise, intravital microscopic studies showed significant leukocyte adhesion in intestinal microvessels post-burn, a response that was blunted in the absence of nmMLCK. Functionally, thermal injury significantly increased the gut lumen-to-blood transport of FITC-dextran (4 kD), and this hyperpermeability was attenuated by either neutrophil depletion or nmMLCK deficiency. Consistent with the in vivo observations, in vitro assays with Caco-2 epithelial cell monolayers revealed a decrease in transcellular electric resistance coupled with myosin light chain phosphorylation, actomyosin ring condensation, and claudin-1 internalization upon stimulation with fMLP-activated neutrophils. Pretreatment of the cells with the MLCK inhibitor ML-7 prevented the tight junction responses. Taken together, the results suggest that nmMLCK plays an important role in neutrophil-dependent intestinal barrier dysfunction during inflammatory injury.

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Neutrophils and non-muscle myosin light chain kinase (nmMLCK) have been implicated in intestinal microvascular leakage and mucosal hyperpermeability in inflammation and trauma. The aim of this study was to characterize the role of nmMLCK in neutrophil-dependent gut barrier dysfunction following thermal injury, a common form of trauma that typically induces inflammation in multiple organs. Histopathological examination of the small intestine in mice after a full-thickness burn revealed morphological evidence of mucosa inflammation characterized by neutrophil infiltration into the lamina propria, epithelial contraction, and narrow villi with blunt brush borders and loss of goblet cells. Compared to their wild-type counterparts, nmMLCK-/mice displayed diminished morphological abnormalities. Likewise, intravital microscopic studies showed significant leukocyte adhesion in intestinal microvessels post-burn, a response that was blunted in the absence of nmMLCK. Functionally, thermal injury significantly increased the gut lumen-to-blood transport of FITC-dextran (4 kD), and this hyperpermeability was attenuated by either neutrophil depletion or nmMLCK deficiency. Consistent with the in vivo observations, in vitro assays with Caco-2 epithelial cell monolayers revealed a decrease in transcellular electric resistance coupled with myosin light chain phosphorylation, actomyosin ring condensation, and claudin-1 internalization upon stimulation with fMLP-activated neutrophils. Pretreatment of the cells with the MLCK inhibitor ML-7 prevented the tight junction responses. Taken together, the results suggest that nmMLCK plays an important role in neutrophil-dependent intestinal barrier dysfunction during inflammatory injury.