An apparent paradox in smooth muscle biology is the ability of unphosphorylated myosin to maintain a filamentous structure in the presence of ATP in vivo, whereas unphosphorylated myosin filaments are depolymerized in vitro in the presence of ATP. This suggests that additional uncharacterized factors are required for the stabilization of myosin filaments in the presence of ATP. We report here that an abundant smooth muscle protein forms sedimentable complexes with unphosphorylated smooth muscle myosin, partially reverses the depolymerizing effect of ATP on unphosphorylated myosin, and promotes the assembly of minifilaments as revealed by electron microscopy. This protein is called kinase-related protein (KRP) because it is derived from a gene within the gene for myosin light chain kinase (MLCK) and has an amino acid sequence identical to the carboxyl-terminal domain of MLCK. Consistent with the results with purified KRP, deletion of the KRP domain within MLCK results in a diminished ability of MLCK to interact with unphosphorylated myosin. KRP binds to the heavy meromyosin fragment of myosin but not to myosin rod or fragments lacking the hinge region and light chains. Altogether, these results suggest that KRP may play a critical role in stabilizing unphosphorylated myosin filaments and that the KRP domain of MLCK may be important for subcellular targeting to filaments.

Type 2 diabetes mellitus (T2DM) is a socially important disease with only symptomatic therapy developed due to lack of knowledge about its pathogenesis and underlying mechanism. Insulin resistance (IR) is the first link of T2DM pathogenesis and results in decrease of ability of insulin to stimulate glucose uptake by target cells. Development of IR involves genetic predisposition, excessive nutrition, stress, obesity or chronic inflammation due to disruption of insulin signaling within cells. Molecular mechanisms and markers of IR are characterized rather poorly, which prevents early diagnosis and creation of preventive therapy. Euglycemic clamp test is still a golden standard for IR diagnosis in clinic. Hyperglycemia is a distant consequence of IR in which damaging effect of oxidative and carbonyl stress is realized and diagnosis of T2DM is stipulated. Molecular chaperones and small heat-shock proteins have a protective effect at the early stages of T2DM pathogenesis, preventing development of reticulum stress and apoptosis. Endothelial dysfunction is related to T2DM and its cardiovascular complications, however, it is unknown on which stage of pathogenesis these changes occur and what are their molecular inductors. Finally, transcriptional activity and adipogenic differentiation play an important role in formation of new fat depots from predecessor cells and activation of brown and beige fat demonstrating hypolipidemic and hypoglycemic properties. The aim of this study was investigation of pathophysiological mechanisms of development of IR and endothelial dysfunction, role of transcription factor Prep1 and small heat shock proteins, evaluation of novel methods of diagnostics of IR and therapeutic potential of brown and beige fat, determination of biotargets for new antidiabetic drugs.

Hyperlipidemia manifested by high blood levels of free fatty acids (FFA) and lipoprotein triglycerides is critical for the progression of type 2 diabetes (T2D) and its cardiovascular complications via vascular endothelial dysfunction. However, attempts to assess high FFA effects in endothelial culture often result in early cell apoptosis that poorly recapitulates a much slower pace of vascular deterioration in vivo and does not provide for the longer-term studies of endothelial lipotoxicity in vitro. Here, we report that palmitate (PA), a typical FFA, does not impair, by itself, endothelial barrier and insulin signaling in human umbilical vein endothelial cells (HUVEC), but increases NO release, reactive oxygen species (ROS) generation, and protein labeling by malondialdehyde (MDA) hallmarking oxidative stress and increased lipid peroxidation. This PA-induced stress eventually resulted in the loss of cell viability coincident with loss of insulin signaling. Supplementation with 5-aminoimidazole-4-carboxamide-riboside (AICAR) increased endothelial AMP-activated protein kinase (AMPK) activity, supported insulin signaling, and prevented the PA-induced increases in NO, ROS, and MDA, thus allowing to maintain HUVEC viability and barrier, and providing the means to study the long-term effects of high FFA levels in endothelial cultures. An upgraded cell-based model reproduces FFA-induced insulin resistance by demonstrating decreased NO production by vascular endothelium.

Background. Nitric oxide can successfully compete with oxygen for sites of electron‐transport chain in conditions of myocardial hypoxia. These features may prevent excessive oxidative stress occurring in cardiomyocytes during sudden hypoxia‐reoxygenation. Aim. To study the action of the potent stable NO donor dinitrosyl iron complex with glutathione (Oxacom®) on the recovery of myocardial contractile function and Ca²⁺ transients in cardiomyocytes during hypoxia‐reoxygenation. Results. The isolated rat hearts were subjected to 30 min hypoxia followed by 30 min reoxygenation. The presence of 30 nM Oxacom in hypoxic perfusate reduced myocardial contracture and improved recovery of left ventricular developed pressure partly due to elimination of cardiac arrhythmias. The same Oxacom concentration limited reactive oxygen species generation in hypoxic cardiomyocytes and increased the viability of isolated cardiomyocytes during hypoxia from 12 to 52% and after reoxygenation from 0 to 40%. Oxacom prevented hypoxia‐induced elevation of diastolic Ca²⁺ level and eliminated Ca²⁺ transport alterations manifested by slow Ca²⁺ removal from the sarcoplasm and delay in cardiomyocyte relaxation. Conclusion. The potent stable NO donor preserved cardiomyocyte integrity and improved functional recovery at hypoxia‐reoxygenation both in the isolated heart and in cardiomyocytes mainly due to preservation of Ca²⁺ transport. Oxacom demonstrates potential for cardioprotection during hypoxia‐reoxygenation.