Engraftment of mesenchymal stem cells (MSCs) derived from adult bone marrow has been proposed as a potential therapeutic approach for postinfarction left ventricular dysfunction. However, limited cell viability after transplantation into the myocardium has restricted its regenerative capacity. In this study, we genetically modified MSCs with an antiapoptotic Bcl-2 gene and evaluated cell survival, engraftment, revascularization, and functional improvement in a rat left anterior descending ligation model via intracardiac injection. Rat MSCs were manipulated to overexpress the Bcl-2 gene. In vitro, the antiapoptotic and paracrine effects were assessed under hypoxic conditions. In vivo, the Bcl-2 gene-modified MSCs (Bcl-2-MSCs) were injected after myocardial infarction. The surviving cells were tracked after transplantation. Capillary density was quantified after 3 weeks. The left ventricular function was evaluated by pressure-volume loops. The Bcl-2 gene protected MSCs against apoptosis. In vitro, Bcl-2 overexpression reduced MSC apoptosis by 32% and enhanced vascular endothelial growth factor secretion by more than 60% under hypoxic conditions. Transplantation with Bcl-2-MSCs increased 2.2-fold, 1.9-fold, and 1.2-fold of the cellular survival at 4 days, 3 weeks, and 6 weeks, respectively, compared with the vector-MSC group. Capillary density in the infarct border zone was 15% higher in Bcl-2-MSC transplanted animals than in vector-MSC treated animals. Furthermore, Bcl-2-MSC transplanted animals had 17% smaller infarct size than vector-MSC treated animals and exhibited functional recovery remarkably. Our current findings support the premise that transplantation of antiapoptotic gene-modified MSCs may have values for mediating substantial functional recovery after acute myocardial infarction.

Our study shows that inhibition of the reverse mode of the NCE, during reperfusion only, protects cardiomyocytes and whole hearts against reperfusion injury.

The cAMP signaling pathway plays an essential role in modulating the apoptotic response to various stress stimuli. Until now, it was attributed exclusively to the activity of the G-protein-responsive transmembrane adenylyl cyclase. In addition to transmembrane AC, mammalian cells possess a second source of cAMP, the ubiquitously expressed soluble adenylyl cyclase (sAC). However, the role of this cyclase in apoptosis was unknown. A mitochondrial localization of this cyclase has recently been demonstrated, which led us to the hypothesis that sAC may play a role in apoptosis through modulation of mitochondria-dependent apoptosis. To prove this hypothesis, apoptosis was induced by simulated in vitro ischemia or by acidosis, which is an important component of ischemia. Suppression of sAC activity with the selective inhibitor KH7 or sAC knockdown by small interfering RNA transfection abolished endothelial apoptosis. Furthermore, pharmacological inhibition or knockdown of protein kinase A, an important cAMP target, demonstrated a significant anti-apoptotic effect. Analysis of the underlying mechanisms revealed (i) the translocation of sAC to mitochondria under acidic stress and (ii) activation of the mitochondrial pathway of apoptosis, i.e. cytochrome c release and caspase-9 cleavage. sAC inhibition or knockdown abolished the activation of the mitochondrial pathway of apoptosis. Analysis of mitochondrial co-localization of Bcl-2 family proteins demonstrated sAC-and protein kinase A-dependent translocation of Bax to mitochondria. Taken together, these results suggest the important role of sAC in modulating the mitochondria-dependent pathway of apoptosis in endothelial cells. Increasing evidence suggests that apoptosis of endothelial cells (EC)3 may be responsible for acute and chronic vascular diseases, e.g. through atherogenesis (1), endothelial dysfunction (2), or thrombosis (3). Within several signaling mechanisms, a cAMP-dependent signaling pathway plays a substantial role in mediating apoptotic cell death induced by various stress factors. Elevation of the cellular cAMP either by forskolin-induced stimulation of the G-protein-responsive transmembrane adenylyl cyclase (tmAC) or by treatment with cAMP analogs has been shown to lead to both induction and suppression of apoptosis in different cell types (4 -7). This discrepancy may be due to differences in cell types and experimental models. Alternatively, a lack of specificity of tmAC-induced signals, especially directed to distant intracellular targets like mitochondria, may be a cause of the discrepancy. Indeed, the classical model of cAMP signaling requires the diffusion of cAMP from plasma membrane-localized tmAC to targets localized throughout the cell. Diffusion of cAMP throughout the cytosol makes it difficult to selectively activate distally localized targets without also activating more proximal targets. Therefore, such diffusion of cAMP would likely diminish specificity, selectivity, and signal strength. This model is further complicated by the presence of p...

Effects of Na+/H+ exchange inhibition and cytosolic acidosis on reoxygenated adult rat ventricular cardiomyocytes were investigated. Cells were incubated in anoxic media at pH 6.4 until pCa of < or = 5, intracellular pH (pHi) of 6.5, and cytosolic [Na+] of 50 mM were reached. On reoxygenation, medium pH was changed to 7.4 to activate Na+/H+ exchange. In one group, 20 microM HOE-694, an inhibitor of Na+/H+ exchange, was added. With or without HOE-694, cytosolic Ca2+ and Na+ returned to control levels within 10 min of reoxygenation. In the absence of HOE-694, the pHi renormalized (to 7.2) within 8 min, but irreversible hypercontracture and transient Ca2+ oscillations were observed. In the presence of HOE-694, pHi stayed acidotic (at 6.5), hypercontracture was prevented, and Ca2+ oscillations were attenuated. When the Na+ pump was inhibited with 0.1 mM ouabain, even partial recovery of Ca2+ control became impossible unless HOE-694 was added. Our conclusions are 1) activation of Na+/H+ exchange does not impair recovery of cytosolic Na+ and Ca2+ control unless activity of the sarcolemmal Na+ pump is critically reduced, and 2) due to prolongation of cytosolic acidosis, inhibition of Na+/H+ exchange protects against reoxygenation-induced hypercontracture and cytosolic Ca2+ oscillations.

These results suggest a key role of sAC in SI-induced mitochondrial Bax translocation and activation of the mitochondrial pathway of apoptosis in adult cardiomyocytes.

While both cord blood and marrow CD133+ cells have some beneficial effects on post-injury angiogenesis and survival, only marrow cells appear to improve myocardial contractility.

Uncontrolled release of Ca2+ from the sarcoplasmic reticulum (SR) contributes to the reperfusion-induced cardiomyocyte injury, e.g. hypercontracture and necrosis. To find out the underlying cellular mechanisms of this phenomenon, we investigated whether the opening of mitochondrial permeability transition pores (MPTP), resulting in ATP depletion and reactive oxygen species (ROS) formation, may be involved. For this purpose, isolated cardiac myocytes from adult rats were subjected to simulated ischemia and reperfusion. MPTP opening was detected by calcein release and by monitoring the ΔΨm. Fura-2 was used to monitor cytosolic [Ca2+]i or mitochondrial calcium [Ca2+]m, after quenching the cytosolic compartment with MnCl2. Mitochondrial ROS [ROS]m production was detected with MitoSOX Red and mag-fura-2 was used to monitor Mg2+ concentration, which reflects changes in cellular ATP. Necrosis was determined by propidium iodide staining. Reperfusion led to a calcein release from mitochondria, ΔΨm collapse and disturbance of ATP recovery. Simultaneously, Ca2+ oscillations occurred, [Ca2+]m and [ROS]m increased, cells developed hypercontracture and underwent necrosis. Inhibition of the SR-driven Ca2+ cycling with thapsigargine or ryanodine prevented mitochondrial dysfunction, ROS formation and MPTP opening. Suppression of the mitochondrial Ca2+ uptake (Ru360) or MPTP (cyclosporine A) significantly attenuated Ca2+ cycling, hypercontracture and necrosis. ROS scavengers (2-mercaptopropionyl glycine or N-acetylcysteine) had no effect on these parameters, but reduced [ROS]m. In conclusion, MPTP opening occurs early during reperfusion and is due to the Ca2+ oscillations originating primarily from the SR and supported by MPTP. The interplay between Ca2+ cycling and MPTP promotes the reperfusion-induced cardiomyocyte hypercontracture and necrosis. Mitochondrial ROS formation is a result rather than a cause of MPTP opening.

Background The tight junction proteins claudin-2 and claudin-10a form paracellular cation and anion channels, respectively, and are expressed in the proximal tubule. However, the physiological role of claudin-10a in the kidney has been unclear. Methods To investigate the physiologic role of claudin-10a, we generated claudin-10a-deficient mice; confirmed successful knockout by Southern blot, Western blot, and immunofluorescence staining; and analyzed urine and serum of knockout and wild-type animals. We also used electrophysiologic studies to investigate the functionality of isolated proximal tubules, and studied compensatory regulation by pharmacologic intervention, RNA sequencing analysis, Western blot, immunofluorescence staining, and respirometry. Results Mice deficient in claudin-10a were fertile and without overt phenotypes. Upon knockout, claudin-10a was replaced by claudin-2 in all proximal tubule segments. Electrophysiology showed conversion from paracellular anion preference to cation preference and a loss of paracellular Cl- over HCO3- preference. As a consequence, there was tubular retention of calcium and magnesium, higher urine pH, and mild hypermagnesemia. A comparison of other urine and serum parameters under control conditions and sequential pharmacologic transport inhibition, as well as unchanged fractional lithium excretion, suggested compensative measures in proximal and distal tubular segments. Changes in proximal tubular oxygen handling and differential expression of genes regulating fatty acid metabolism indicated proximal tubular adaptation. Western blot and immunofluorescence revealed alterations in distal tubular transport. Conclusions Claudin-10a is the major paracellular anion channel in the proximal tubule and its deletion causes calcium and magnesium hyperreabsorption by claudin-2 redistribution. Transcellular transport in proximal and distal segments and proximal tubular metabolic adaptation compensate for loss of paracellular anion permeability.