Marisol Ruiz-Meana

Translocation of Connexin 43 to the Inner Mitochondrial Membrane of Cardiomyocytes Through the Heat Shock Protein 90–Dependent TOM Pathway and Its Importance for Cardioprotection

We have previously shown that connexin 43 (Cx43) is present in mitochondria, that its genetic depletion abolishes the protection of ischemia- and diazoxide-induced preconditioning, and that it is involved in reactive oxygen species (ROS) formation in response to diazoxide. Here we investigated the intramitochondrial localization of Cx43, the mechanism of Cx43 translocation to mitochondria and the effect of inhibiting translocation on the protection of preconditioning. Confocal microscopy of mitochondria devoid of the outer membrane and Western blotting on fractionated mitochondria showed that Cx43 is located at the inner mitochondrial membrane, and coimmunoprecipitation of Cx43 with Tom20 (Translocase of the outer membrane 20) and with heat shock protein 90 (Hsp90) indicated that it interacts with the regular mitochondrial protein import machinery. In isolated rat hearts, geldanamycin, a blocker of Hsp90-dependent translocation of proteins to the inner mitochondrial membrane through the TOM pathway, rapidly (15 minutes) reduced mitochondrial Cx43 content by approximately one-third in the absence or presence of diazoxide. Geldanamycin alone had no effect on infarct size, but it ablated the protection against infarction afforded by diazoxide. Geldanamycin abolished the 2-fold increase in mitochondrial Cx43 induced by 2 preconditioning cycles of ischemia/reperfusion, but this effect was not associated with reduced protection. These results demonstrate that Cx43 is transported to the inner mitochondrial membrane through translocation via the TOM complex and that a normal mitochondrial Cx43 content is important for the diazoxide-related pathway of preconditioning.

Gap Junction Uncoupler Heptanol Prevents Cell-to-Cell Progression of Hypercontracture and Limits Necrosis During Myocardial Reperfusion

BACKGROUND The objective of this study was to test the hypothesis that chemical interaction through gap junctions may result in cell-to-cell progression of hypercontracture and that this phenomenon contributes to the final extent of reperfused infarcts. METHODS AND RESULTS Cell-to-cell transmission of hypercontracture was studied in pairs of freshly isolated adult rat cardiomyocytes. Hypercontracture induced by microinjection of a solution containing 1 mmol/L Ca2+ and 2% lucifer yellow (LY) was transmitted to the adjacent cell (11 of 11 pairs), and the gap junction uncoupler heptanol (2 mmol/L) prevented transmission in 6 of 8 pairs (P=.003), with a perfect association between passage of the LY and transmission of hypercontracture. In the isolated, perfused rat heart submitted to 30 minutes of hypoxia, addition of heptanol to the perfusion media during the first 15 minutes of reoxygenation had a dose-related protective effect against the oxygen paradox, as demonstrated by a reduction of diastolic pressure and marked recovery of developed pressure (P<.001), as well as less lactate dehydrogenase release during reoxygenation (P<.001) and less contraction band necrosis (P<.001) than controls. In the in situ pig heart submitted to 48 minutes of coronary occlusion, the intracoronary infusion of heptanol during the first 15 minutes of reperfusion at a final concentration of 1 mmol/L limited myocardial shrinkage, reflecting hypercontracture (P<.05), reduced infarct size after 5 hours of reperfusion by 54% (P=.04), and modified infarct geometry with a characteristic fragmentation of the area of necrosis. Heptanol at 1 mmol/L had no significant effect on contractility of nonischemic myocardium. CONCLUSIONS These results demonstrate that hypercontracture may be transmitted to adjacent myocytes through gap junctions and that heptanol may interfere with this transmission and reduce the final extent of myocardial necrosis during reoxygenation or reperfusion. These findings are consistent with the hypothesis tested and open a new approach to limitation of infarct size by pharmacological control of gap junction conductance.

Translating cardioprotection for patient benefit: position paper from the Working Group of Cellular Biology of the Heart of the European Society of Cardiology

Coronary heart disease (CHD) is the leading cause of death and disability worldwide. Despite current therapy, the morbidity and mortality for patients with CHD remains significant. The most important manifestations of CHD arise from acute myocardial ischaemia-reperfusion injury (IRI) in terms of cardiomyocyte death and its long-term consequences. As such, new therapeutic interventions are required to protect the heart against the detrimental effects of acute IRI and improve clinical outcomes. Although a large number of cardioprotective therapies discovered in pre-clinical studies have been investigated in CHD patients, few have been translated into the clinical setting, and a significant number of these have failed to show any benefit in terms of reduced myocardial infarction and improved clinical outcomes. Because of this, there is currently no effective therapy for protecting the heart against the detrimental effects of acute IRI in patients with CHD. One major factor for this lack of success in translating cardioprotective therapies into the clinical setting can be attributed to problems with the clinical study design. Many of these clinical studies have not taken into consideration the important data provided from previously published pre-clinical and clinical studies. The overall aim of this ESC Working Group Cellular Biology of the Heart Position Paper is to provide recommendations for optimizing the design of clinical cardioprotection studies, which should hopefully result in new and effective therapeutic interventions for the future benefit of CHD patients.

Trials, tribulations and speculation! Report from the 7th Biennial Hatter Cardiovascular Institute Workshop

The 7th biennial Hatter Cardiovascular Institute Workshop, comprising 21 leading basic science and clinical experts, was held in South Africa in August 2012 to discuss the current cutting edge status of cardioprotection and the application of cardioprotective modalities in the clinical management of myocardial ischaemia/reperfusion injury in the context of acute coronary syndromes and cardiac surgery. The meeting, chaired by Professor Derek Yellon and Professor Lionel Opie, was run to a format of previous Hatter Cardiovascular workshops with data presented by proponents followed by discussion and debate by the faculty.

Effect of inhibition of Na+/Ca2+ exchanger at the time of myocardial reperfusion on hypercontracture and cell death

OBJECTIVE There is recent evidence that Ca(2+) influx via reverse mode Na(+)/Ca(2+) exchange (NCX) at the time of reperfusion can contribute to cardiomyocyte hypercontracture. However, forward NCX is essential for normalization of [Ca(2+)](i) during reperfusion, and its inhibition may be detrimental. This study investigates the effect of NCX inhibition with KB-R7943 at the time of reperfusion on cell viability. METHODS The effect of several concentrations of KB-R7943 added at reperfusion was studied in Fura-2 loaded quiescent cardiomyocytes submitted to 40 min of simulated ischemia (NaCN 2 mM, pH 6.4), and in rat hearts submitted to 60 min of ischemia. [Ca(2+)](i) and cell length were monitored in myocytes, and functional recovery and LDH release in isolated hearts. From these experiments an optimal concentration of KB-R7943 was identified and tested in pigs submitted to 48 min of coronary occlusion and 2 h of reperfusion. RESULTS In myocytes, KB-R7943 at concentrations up to 15 microM reduced [Ca(2+)](i) rise and the probability of hypercontracture during re-energization (P<0.01). Nevertheless, in rat hearts, the effects of KB-R7943 applied during reperfusion after 60 min of ischemia depended on concentration and timing of administration. During the first 5 min of reperfusion, KB-R7943 (0.3-30 microM) induced a dose-dependent reduction in LDH release (half-response concentration 0.29 microM). Beyond 6 min of re-flow, KB-R7943 had no effect on LDH release, except at concentrations > or = 15 microM, which increased LDH. KB-R7943 at 5 microM given during the first 10 min of reflow reduced contractile dysfunction (P=0.011), LDH release (P=0.019) and contraction band necrosis (P=0.014) during reperfusion. Intracoronary administration of this concentration during the first 10 min of reperfusion reduced infarct size by 34% (P=0.033) in pigs submitted to 48 min of coronary occlusion. CONCLUSIONS These results are consistent with the hypothesis that during initial reperfusion NCX activity results in net reverse mode operation contributing to Ca(2+) overload, hypercontracture and cell death, and that NCX inhibition during this phase is beneficial. Beyond this phase, NCX inhibition may impair forward mode-dependent Ca(2+) extrusion and be detrimental. These findings may help in the design of therapeutic strategies against lethal reperfusion injury, with NCX as the target.

Calcium-mediated cell death during myocardial reperfusion

Reperfusion may induce additional cell death in patients with acute myocardial infarction receiving primary angioplasty or thrombolysis. Altered intracellular Ca(2+) handling was initially considered an essential mechanism of reperfusion-induced cardiomyocyte death. However, more recent studies have demonstrated the importance of Ca(2+)-independent mechanisms that converge on mitochondrial permeability transition (MPT) and are shared by cardiomyocytes and other cell types. This article analyses the importance of Ca(2+)-dependent cell death in light of these new observations. Altered Ca(2+) handling includes increased cytosolic Ca(2+) levels, leading to activation of calpain-mediated proteolysis and sarcoplasmic reticulum-driven oscillations; this can induce hypercontracture, but also MPT due to the privileged Ca(2+) transfer between sarcoplasmic reticulum and mitochondria through cytosolic Ca(2+) microdomains. In the opposite direction, permeability transition can worsen altered Ca(2+) handling and favour hypercontracture. Ca(2+) appears to play an important role in cell death during the initial minutes of reperfusion, particularly after brief periods of ischaemia. Developing effective and safe treatments to prevent Ca(2+)-mediated cardiomyocyte death in patients with transient ischaemia, by targeting Ca(2+) influx, intracellular Ca(2+) handling, or Ca(2+)-induced cell death effectors, is an unmet challenge with important therapeutic implications and large potential clinical impact.

The role of Na+/H+ exchange in ischemia-reperfusion

In ischemia the cytosol of cardiomyocytes acidifies; this is reversed upon reperfusion. One of the major pHi-regulating transport systems involved is the Na+/H+ exchanger. Inhibitors of the Na+/H+ exchanger have been found to more effectively protect ischemic-reperfused myocardium when administered before and during ischemia than during reperfusion alone. It has been hypothesized that the protection provided by pre-ischemic administration is due to a reduction in Na+ and secondary Ca2+ influx. Under reperfusion conditions Na+/H+ exchange inhibition also seems protective since it prolongs intracellular acidosis which can prevent hypercontracture. In detail, however, the mechanisms by which Na+/H+ exchange inhibition provides protection in ischemic-reperfused myocardium are still not fully identified.

Connexin43 in cardiomyocyte mitochondria contributes to mitochondrial potassium uptake

AIMS Connexin43 is present at the inner membrane of cardiomyocyte mitochondria (mCx43), but its function remains unknown. METHODS AND RESULTS In this study we verified the presence of mCx43 by a mass spectrometry-based proteomic approach in purified mitochondrial preparations from mouse myocardium and determined by western blot analysis that the C-terminus of mCx43 is oriented towards the intermembrane space. Cross-linking studies with dimethylsuberimidate indicated the presence of Cx43 hexamers in mitochondrial membranes. The contribution of Cx43 to both mitochondrial dye uptake and K(+) flux was assessed in wild-type mice using hemichannel blockers and Cx43KI32 mice in which Cx43 had been replaced by Cx32. Uptake of the Cx43 hemichannel-permeant dye Lucifer Yellow was reduced in mitochondria from wild-type mice by two hemichannel blockers (carbenoxolone and heptanol) and in Cx43KI32 compared with wild-type mice. Mitochondrial K(+) influx (PBFI fluorescence) was decreased in digitonin-permeabilized cardiomyocytes from Cx32 mutants compared with wild-type mice, and addition of the Cx43 hemichannel blocker 18alpha-glycyrrhetinic acid had an inhibitory effect on mitochondrial K(+) influx in wild-type cardiomyocytes, but not in cardiomyocytes from Cx32 mutants. CONCLUSION These results indicate that mCx43 contributes to mitochondrial K(+) flux in cardiomyocytes, potentially by forming hemichannel-like structures.

Prevention of ischemic rigor contracture during coronary occlusion by inhibition of Na+–H+ exchange

OBJECTIVE To determine the effect of Na(+)-H+ exchange blockade on ischemic rigor contracture and reperfusion-induced hypercontracture. METHODS Thirty-six pigs were submitted to 55 min of coronary occlusion and 5 h reperfusion. Myocardial segment length analysis with ultrasonic microcrystals was used to detect ischemic rigor (reduction in passive segment length change) and hypercontracture (reduction in end-diastolic length). RESULTS Pretreatment with the new, highly selective Na(+)-H+ exchange inhibitor HOE642 before occlusion reduced ischemic rigor (P < 0.05), attenuated segment shrinkage (P < 0.05) during subsequent reperfusion, dramatically reduced infarct size (P < 0.0001) and attenuated arrhythmias (P < 0.01). Inhibition of Na(+)-H+ exchange only during reperfusion by means of direct intracoronary infusion of HOE642 into the area at risk prevented reperfusion arrhythmias but had no effect on final infarct size, while treatment with intravenous HOE642 immediately before reperfusion had no detectable effects. CONCLUSION These results indicate that inhibition of Na(+)-H+ exchange during ischemia is necessary to limit myocardial necrosis secondary to transient coronary occlusion, and that this action could by mediated by a protective effect against ischemic contracture. Inhibition of Na(+)-H+ exchange only during reperfusion has a partial and transient beneficial effect, but only when the inhibitor reaches the area at risk before reflow.

Endonuclease G is a novel determinant of cardiac hypertrophy and mitochondrial function.

Left ventricular mass (LVM) is a highly heritable trait and an independent risk factor for all-cause mortality. So far, genome-wide association studies have not identified the genetic factors that underlie LVM variation, and the regulatory mechanisms for blood-pressure-independent cardiac hypertrophy remain poorly understood. Unbiased systems genetics approaches in the rat now provide a powerful complementary tool to genome-wide association studies, and we applied integrative genomics to dissect a highly replicated, blood-pressure-independent LVM locus on rat chromosome 3p. Here we identified endonuclease G (Endog), which previously was implicated in apoptosis but not hypertrophy, as the gene at the locus, and we found a loss-of-function mutation in Endog that is associated with increased LVM and impaired cardiac function. Inhibition of Endog in cultured cardiomyocytes resulted in an increase in cell size and hypertrophic biomarkers in the absence of pro-hypertrophic stimulation. Genome-wide network analysis unexpectedly implicated ENDOG in fundamental mitochondrial processes that are unrelated to apoptosis. We showed direct regulation of ENDOG by ERR-α and PGC1α (which are master regulators of mitochondrial and cardiac function), interaction of ENDOG with the mitochondrial genome and ENDOG-mediated regulation of mitochondrial mass. At baseline, the Endog-deleted mouse heart had depleted mitochondria, mitochondrial dysfunction and elevated levels of reactive oxygen species, which were associated with enlarged and steatotic cardiomyocytes. Our study has further established the link between mitochondrial dysfunction, reactive oxygen species and heart disease and has uncovered a role for Endog in maladaptive cardiac hypertrophy.