Person:

McCully, James

Background

Previous studies have demonstrated that alterations in myocardial structure, consistent with tissue and sarcomere disruption as well as myofibril dissociation, occur after myocardial ischemia and reperfusion. In this study we determine the onset of these structural changes and their contribution to electrical conduction.

Methods

Langendorff perfused rabbit hearts (n = 47) were subjected to 0, 5, 10, 15, 20, 25, and 30 minutes global ischemia, followed by 120 minutes reperfusion. Hemodynamics were recorded and tissue samples were collected for histochemical and immunohistochemical studies. Orthogonal epicardial conduction velocities were measured, with temperature controlled, in a separate group of 10 hearts subjected to 0 or 30 minutes of global ischemia, followed by 120 minutes of reperfusion.

Results

Histochemical and quantitative light microscopy spatial analysis showed significantly increased longitudinal and transverse interfibrillar separation after 15 minutes or more of ischemia (p < 0.05 versus control). Confocal immunohistochemistry and Western blot analysis demonstrated significant reductions (p < .05 versus control) of the intercellular adherens junction protein, N-cadherin, and the active phosphorylated isoform of the principal gap junction protein, connexin 43 at more than 15 minutes of ischemia. Cellular redistribution of connexin 43 was also evidenced on immunohistochemistry. No change in integrin-β1, an extracellular matrix attachment protein, or in epicardial conduction velocity anisotropy was observed.

Conclusions

These data indicate that there are significant alterations in the structural integrity of the myocardium as well as gap and adherens junction protein expression with increasing global ischemia time. The changes occur coincident with previously observed significant decreases in postischemic functional recovery, but are not associated with altered expression of matrix binding proteins or electrical anisotropic conduction.

Objective: Mitochondrial ATP-sensitive potassium channels have been proposed to be myoprotective. The relevance and specificity of this mechanism in cardiac surgery was unknown. The purpose of this study was to examine the effects of the mitochondrial potassium ATP-sensitive channel opener diazoxide on regional and global myocardial protection using a model of acute myocardial infarction. Methods: Pigs (n=19) were placed on total cardiopulmonary bypass and then subjected to 30 min normothermic regional ischemia by snaring the left anterior descending coronary artery (LAD). The aorta was then crossclamped and cold blood Deaconess Surgical Associates cardioplegia (DSA; n=6) or DSA containing 50 μM diazoxide (DZX; n=6) was delivered via the aortic root and the hearts subjected to 30 min hypothermic global ischemia. The crossclamp and snare were removed and the hearts reperfused for 120 min. Results: No significant differences in preload recruitable stroke work relationship, Tau, proximal, distal or proximal/distal coronary flow, regional or global segmental shortening, systolic bulging or post-systolic shortening were observed within or between DSA and DZX hearts during reperfusion. Infarct was present only in the region of LAD occlusion in both DSA and DZX hearts. Infarct size (% of area at risk) was 33.6±2.9% in DSA and was 16.8±2.4% in DZX hearts (P≪0.01 versus DSA). Apoptosis as estimated by TUNEL positive nuclei was 120.3±48.8 in DSA and was significantly decreased to 21.4±5.3 in DZX hearts. Myocardial infarct was located centrally within the area at risk in both DSA and DZX hearts but was significantly increased at borderline zones within the area at risk in DSA hearts. Conclusions: The addition of diazoxide to cardioplegia significantly decreases regional myocardial cell necrosis and apoptosis in a model of acute myocardial infarction and represents an additional modality for achieving myocardial protection.

Background

Previously, we have shown that magnesium supplemented potassium (DSA) cardioplegia and DSA containing diazoxide (DSA+DZX) significantly decrease apoptosis after ischemia. The mechanism for this enhanced cardioprotection was unknown, but we believed that alterations in signal transducers and activators of transcription (STATs) may play a role. To investigate this hypothesis, we examined the effects of DSA and DSA+DZX cardioplegia on STAT1/3 phosphorylation and DNA binding in the in situ blood perfused pig heart model.

Methods

Pigs (32 to 42 kg) undergoing total cardiopulmonary bypass underwent left anterior descending coronary artery occlusion for 30 minutes. The aorta was crossclamped and DSA (n = 6) or DSA+DZX (n = 6) cardioplegia was administered, followed by 30 minutes of global ischemia and 120 minutes of reperfusion. Control hearts (n = 3) received cardiopulmonary bypass and sham reperfusion only. Tissue samples from regional and global ischemia zones were harvested and used for Western blot and electrophoretic mobility shift assay.

Results

Regional and global ischemia significantly increase proapoptotic STAT1 tyrosine phoshorylation. This increase is significantly greater in the regional as compared with the global ischemia zone. Tyrosine phosphorylation of antiapoptotic STAT3 is increased in the global ischemic zone but is significantly decreased in the regional ischemic zone and is associated with increased apoptosis. The DSA+DZX significantly increases tyrosine phosphorylation of antiapoptotic STAT3 and DNA binding in the regional ischemia zone and significantly decreases apoptosis.

Conclusions

The addition of diazoxide to DSA cardioplegia significantly decreases apoptosis by significantly increasing tyrosine phosphorylation of STAT3 and its DNA binding and represents an additional modality for enhancing myocardial protection.

Background

Recent studies have demonstrated that aging is associated with reduced tolerance to ischemia and that the aged (not senescent) female heart has greater susceptibility to ischemia as compared with the aged male heart. Previously, we have shown that ischemia can be modulated with cardioplegia in the male heart; however, efficacy in the female heart was unknown.

Methods

In this study, male and female mature (15 to 20 weeks) aged (>32 months) rabbit hearts (n = 134) were subjected to Langendorff perfusion. Control hearts were perfused for 180 minutes. Global ischemia hearts received 30 minutes of equilibrium, 30 minutes of global ischemia, and 120 minutes of reperfusion. Cardioplegia ± diazoxide was infused separately, 5 minutes before global ischemia.

Results

Global ischemia significantly decreased post-ischemic functional recovery and significantly increased infarct size in the mature and aged male and female heart (p < 0.05 versus control). The effects of global ischemia were significantly exacerbated (p < 0.05) in the aged heart as compared with the mature heart. Cardioplegia ± diazoxide significantly increased postischemic functional recovery and significantly decreased infarct size in mature male and female hearts, but these effects were significantly decreased in the aged heart (p < 0.05) and in the aged female as compared with the aged male heart.

Conclusions

Postischemic functional recovery and infarct size are affected by age but not by gender. The cardioprotection afforded by cardioplegia is affected by age and gender with a strong age-by-gender interaction for end-diastolic pressure and infarct size. Our results indicate that currently optimized cardioplegia protocols effective in the male heart are not as efficacious in the aged female heart.

Background Recently, we have shown that the selective opening of mitochondrial ATP-sensitive potassium channels with diazoxide significantly decreases myocardial injury. The purpose of this study was to determine the effects of diazoxide on apoptosis and the mechanisms modulating apoptosis and myocardial injury in a blood-perfused model of acute myocardial infarction.

Methods Pigs (32 to 42 kg) undergoing total cardiopulmonary bypass underwent left anterior descending coronary artery occlusion for 30 minutes. The aorta was cross-clamped and magnesium-supplemented potassium cold-blood cardioplegia (DSA; n = 6) or magnesium-supplemented potassium cardioplegia containing 50 μmol/L diazoxide (DZX; n = 6) was administered, followed by 30 minutes of global ischemia and 120 minutes of reperfusion. Left ventricular tissue samples from DSA and DZX hearts were obtained after reperfusion. Apoptosis was determined by TUNEL, caspase-3 and PARP cleavage, and caspase-3 activity. Bax and bcl-2 levels were determined and tissue morphology was examined by light and transmission electron microscopy.

Results Apoptosis, as estimated by TUNEL-positive nuclei/3,000 myocardial cells, was 120.3 ± 48.8 in DSA hearts and was significantly decreased to 21.4 ± 5.3 in DZX hearts (p < 0.05 vs control). Caspase-3 and poly-ADP-ribose polymerase cleavage and pro-apoptotic bax protein levels were significantly decreased with diazoxide (p < 0.05 vs DSA). Light and transmission electron microscopy indicated severe disruption of tissue with capillary dilatation, mitochondrial cristae damage, and evidence of increased presence of mitochondrial granules in DSA as compared with DZX hearts.

Conclusions The addition of diazoxide (50 μmol/L) to cardioplegia significantly decreases regional myocardial apoptosis and mitochondrial damage, and provides an additional modality for achieving myocardial protection.

Background

The molecular mechanisms leading to ascending thoracic aortic aneurysms (ATAAs) remain unknown. We hypothesized that alterations in expression levels of specific fibrillar collagens occur during the aneurysmal process.

Methods

Surgical samples from ascending aortas from patients with degenerative ATAAs were subdivided by aneurysm diameter: small, 5 to 6 cm; medium, 6 to 7 cm; and large, greater than 7 cm; and compared with nonaneurysmal aortas (mean diameter, 2.3 cm).

Results

Histology, immunofluorescence, and electron microscopy demonstrated greater disorganization of extracellular matrix constituents in ATAAs as compared with control with an increase in collagen α1(XI) within regions of cystic medial degenerative lesions. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed collagens type V and α1(XI) were significantly and linearly increased in ATAAs as compared with control (p < 0.001). There was no change in the messenger ribonucleic acid (mRNA) expression levels of collagens type I and III. Western blot analysis showed collagens type I and III were significantly decreased and collagens α1(XI) and V were significantly increased and were linearly correlated with the size of the aneurysm (p < 0.001 for both).

Conclusions

These results demonstrate that increased collagen α1(XI) and collagen V mRNA and protein levels are linearly correlated with the size of the aneurysm and provide a potential mechanism for the generation and progression of aneurysmal enlargement.

Background

We have recently shown that the cardioprotection afforded by cardioplegia is affected by age and gender and is less effective in the aged female rabbit heart compared with the aged male rabbit heart. We hypothesized that these differences were due to age and gender-specific modulation of mitochondrial oxygen consumption and mitochondrial free matrix calcium ([Ca2+]Mito) content occurring during early reperfusion.

Methods

To test this hypothesis, 104 male and female rabbit hearts, mature (15 to 20 weeks) and aged (>32 months), were subjected to Langendorff perfusion. Control hearts were perfused for 75 minutes. Global ischemia hearts were underwent 30 minutes of equilibrium, 30 minutes of global ischemia, and 15 minutes of reperfusion. Cardioplegia (potassium/magnesium) ± diazoxide was infused 5 minutes before global ischemia. Mitochondria were isolated from left ventricular tissue and used for the measurement of oxygen consumption and [Ca2+]Mito.

Results

Mitochondrial oxygen consumption was significantly increased in the mature and aged female hearts in all treatment groups (p < 0.001 versus male). Cardioplegia ± diazoxide modulated mitochondrial oxygen consumption, but these effects were significantly decreased in the aged heart and in the female heart (p < 0.001 each versus male). Cardioplegia (potassium/magnesium) significantly decreased [Ca2+]Mito (p < 0.001 versus global ischemia) in aged but not mature hearts. The addition of diazoxide to potassium/magnesium significantly decreased [Ca2+]Mito in mature and aged males (p < 0.001 versus potassium/magnesium) but not in females.

Conclusions

These results demonstrate that mitochondrial oxygen consumption and [Ca2+]Mito are modulated by age and gender and play an important role in the differences observed between mature and aged male and female response to global ischemia and the cardioprotection afforded by cardioplegia ± diazoxide.