Restoration of blood flow to ischaemic heart inflicts ischaemia/reperfusion (I/R) injury, which manifests in metabolic and morphological disorders. Klotho is a protein with antioxidative and antiapoptotic activity, and is involved in the regulation of inflammation and fibrosis. The aim of the current research was to determine the role of Klotho in the heart subjected to I/R injury, as well as to study Klotho as a potential cardioprotective agent. Human cardiomyocytes and Wistar rat hearts perfused using Langendorff method subjected to I/R have been used. Hemodynamic parameters of heart function, markers of I/R injury, and gene and protein expression of Klotho were measured. Human cardiomyocytes were also incubated in the presence of recombinant Klotho protein, and the viability of cells was measured. There was a higher expression of Klotho gene and protein synthesis in the cardiomyocytes subjected to I/R injury. The compensatory production and release of Klotho protein from cardiac tissue during I/R were also shown. The treatment of cardiomyocytes subjected to I/R with Klotho protein resulted in increased viability and metabolic activity of cells. Thus, Klotho contributes to compensatory mechanism during I/R, and could be used as a marker of injury and as a potential cardiopreventive/cardioprotective agent.

Background: Acute heart ischemia followed by reperfusion leads to overproduction of reactive oxygen/nitrogen species (ROS/RNS), disrupted expression of nitric oxide synthase (NOS) and unbalanced glucose metabolism. Klotho is a membrane-bound or soluble protein that exerts protective activity in many organs. While Klotho is produced mainly in the kidneys and brain, it has been recently proven that Klotho is expressed in the cardiomyocytes as well. This study aimed to show the influence of the Klotho protein on oxidative/nitrosative stress and metabolic function of the cardiomyocytes subjected to ischemia/reperfusion (I/R) injury. Methods: Human cardiac myocytes underwent in vitro chemical I/R (with sodium cyanide and 2-deoxyglucose), in the presence or absence of the recombinant human Klotho protein. The present study included an investigation of cell injury markers, level of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), level of oxidative/nitrosative stress and metabolic processes of the cardiomyocytes. Results: Administration of Klotho protein resulted in mitigation of injury, decreased level of NOX2 and NOX4, reduced generation of ROS/RNS and hydrogen peroxide (H₂O₂), decreased expression of inducible NOS and limited production of nitrates/nitrites in cells under I/R. Glucose uptake and lactate production in the cardiomyocytes subjected to I/R were normalized after Klotho supplementation. Conclusions: The Klotho protein participates in the regulation of redox balance and supports metabolic homeostasis of the cardiomyocytes and hence, contributes to protection against I/R injury. Background: Acute heart ischemia followed by reperfusion leads to overproduction of reactive oxygen/nitrogen species (ROS/RNS), disrupted expression of nitric oxide synthase (NOS) and unbalanced glucose metabolism. Klotho is a membrane-bound or soluble protein that exerts protective activity in many organs. While Klotho is produced mainly in the kidneys and brain, it has been recently proven that Klotho is expressed in the cardiomyocytes as well. This study aimed to show the influence of the Klotho protein on oxidative/nitrosative stress and metabolic function of the cardiomyocytes subjected to ischemia/reperfusion (I/R) injury. Methods: Human cardiac myocytes underwent in vitro chemical I/R (with sodium cyanide and 2-deoxyglucose), in the presence or absence of the recombinant human Klotho protein. The present study included an investigation of cell injury markers, level of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), level of oxidative/nitrosative stress and metabolic processes of the cardiomyocytes. Results: Administration of Klotho protein resulted in mitigation of injury, decreased level of NOX2 and NOX4, reduced generation of ROS/RNS and hydrogen peroxide (H₂O₂), decreased expression of inducible NOS and limited production of nitrates/nitrites in cells under I/R. Glucose uptake and lactate production in the cardiomyocytes subjected to I/R were normalized after Klotho supplementation. Conclusions: The Klotho protein participates in the regulation of redox balance and supports metabolic homeostasis of the cardiomyocytes and hence, contributes to protection against I/R injury.

Background. Arterial hypertension (HT) is a serious and prevalent epidemiological factor in the development of coronary artery disease (CAD). Metalloproteinases (MMPs), especially MMP-2 and MMP-9, and their natural endogenous tissue inhibitors (TIMPs) are involved in the pathogenesis of HT and its complications. MMPs are also involved in the development of diabetes (DM), a risk factor for CAD. The aim of the study was to explore the influence of CAD, HT, and DM on changes in plasma levels of MMP-2 and MMP-9 and their inhibitor TIMP-4. Methods and Results. The study involved 70 patients with stable CAD admitted for coronary angiography and 15 healthy subjects. Whole blood samples were collected prior to angiography. MMP-2, MMP-9, and TIMP-4 levels in plasma were estimated using ELISA tests. CAD patients showed a significantly increased level of TIMP-4 and decreased level of MMP-2 in comparison to healthy controls (p=0.011 and p=0.037, respectively). Concentration of MMP-2, MMP-9, and TIMP-4 did not differ in the group with and without hypertension. Patients with DM presented higher MMP-2 level than patients without DM (p<0.001). Multiple regression analysis of the influence of independent variables such as CAD stage, DM, and HT on MMP-2, MMP-9, and TIMP-4 showed that only DM was independently associated with a higher level of MMP-2 (β = 0.42, R² = 0.17, p<0.001). Conclusion. Data showed that patients with CAD presented higher TIMP-4 and lower MMP-2 concentration regardless of HT and DM. HT had no effect on MMP-2, MMP-9, and TIMP-4 levels in serum. DM was independently associated with higher MMP-2 concentration; however, co-occurrence of CAD and DM was associated with the balance in the MMP-2 level. Concentration of MMP-9 did not change significantly in any of the analysed groups.

Ischemia/reperfusion (I/R) injury induces post-translational modifications of myosin light chains (MLCs), increasing their susceptibility to degradation by matrix metalloproteinase 2 (MMP-2). This results in the degradation of ventricular light chains (VLC1) in heart ventricles. The aim of the study was to investigate changes in MLCs content in the mechanism of adaptation to oxidative stress during I/R. Rat hearts, perfused using the Langendorff method, were subjected to I/R. The control group was maintained in oxygen conditions. Lactate dehydrogenase (LDH) activity and reactive oxygen/nitrogen species (ROS/RNS) content were measured in coronary effluents. Atrial light chains (ALC1) and ventricular light chains (VLC1) gene expression were examined using RQ-PCR. ALC1 and VLC1 protein content were measured using ELISA tests. MMP-2 activity was assessed by zymography. LDH activity as well as ROS/RNS content in coronary effluents was higher in the I/R group (p = 0.01, p = 0.04, respectively), confirming heart injury due to increased oxidative stress. MMP-2 activity in heart homogenates was also higher in the I/R group (p = 0.04). ALC1 gene expression and protein synthesis were significantly increased in I/R ventricles (p < 0.01, 0.04, respectively). VLC1 content in coronary effluents was increased in the I/R group (p = 0.02), confirming the increased degradation of VLC1 by MMP-2 and probably an adaptive production of ALC1 during I/R. This mechanism of adaptation to oxidative stress led to improved heart mechanical function.

Ischemia/reperfusion (I/R) injury is accompanied by an increase of matrix metalloproteinase 2 (MMP-2) activity, which degrades heart contractile proteins. The aim of the study was to investigate the effect of MMP-2 small interfering RNA (MMP-2 siRNA) administration on I/R heart. Isolated rat hearts perfused by the Langendorff method were subjected to I/R in the presence or absence of MMP-2 siRNA. The hemodynamic parameters of heart function were monitored. Lactate dehydrogenase (LDH) activity was measured in coronary effluents. Activity and concentration of MMPs in the hearts were measured. Concentration of troponin I (TnI) in coronary effluents was examined as a target for MMP-2 degradation. Recovery of heart mechanical function was reduced after I/R; however, administration of MMP-2 siRNA resulted in restoration of proper mechanical function ( ). LDH activity was decreased after the use of MMP-2 siRNA ( ), providing evidence for reduced cardiac damage. Both MMP-2 and MMP-9 syntheses as well as their activity were inhibited in the I/R hearts after siRNA administration ( ). MMP-2 siRNA administration inhibited TnI release into the coronary effluents ( ). The use of MMP-2 siRNA contributed to the improvement of heart mechanical function and reduction of contractile proteins degradation during I/R; therefore, MMP-2 siRNA may be considered a cardioprotective agent.