In this study we tested our previous hypothesis that ischemia is a multifactorial injurious event involving all components of the myocyte simultaneously. This hypothesis was based on ultrastructural findings and was now tested again by protein analysis of sarcolemmal structural proteins and of markers of transcriptional and translational activities. This knowledge may help to clarify the cellular mechanisms involved in progression of acute ischemic myocardial injury and reperfusion. Therefore, we investigated all three intracellular/extracellular linkage systems of the sarcolemma using antibodies against dystrophin, beta-dystroglycan, gamma-sarcoglycan, vinculin, beta1-integrin, laminin, and spectrin. In addition, antibodies were used to evaluate membrane permeability (albumin), transcriptional efficacy (non-snRNP splicing factor SC-35), and translational capacity (phosphorylated p70 ribosomal protein S6 kinase). Tissue samples were obtained from a canine model of regional myocardial ischemia (90 min or 4.5 h) with or without reperfusion. Immunoconfocal microscopy and Western blotting revealed that the rank order of sensitivity was the following: dystrophin, beta-dystroglycan, gamma-sarcoglycan, vinculin, spectrin, integrin and laminin. Different levels of dystrophin loss indicate reversible/irreversible injury as established by albumin uptake and electron microscopy. Dystrophin depletion closely coincided with generally depressed transcription and translation. These changes occurred simultaneously in a time-dependent manner and persisted during reperfusion. In conclusion, damage of the different structural proteins results in membrane destabilization and disruption of the contractile apparatus from the sarcolemma. These changes, concomitantly associated with disturbances in transcription and translation, are major mechanisms determining the transition to irreversibility of myocardial ischemic injury and confirm our hypothesis that ischemia is a multifactorial injurious event involving all components of the cardiac myocyte.

Tako-Tsubo cardiomyopathy (TTC) is characterized by a transient contractile dysfunction, but its specific pathomechanism remains unknown. Thus, we performed a systematic expression profiling of genes by microarray analysis in the acute phase and after functional recovery. We studied 3 female patients presenting with TTC. Complementary RNA was isolated from left ventricular biopsies taken in the acute phase (group A) and after functional recovery (group B). It was profiled for gene expression using cDNA microarrays. Functionally related genes were determined with the Gene Set Enrichment Analysis (GSEA) bioinformatic tool. Validation of selected genes was performed by means of real-time PCR and immunohistochemistry. In group A, different functional gene sets, such as Nrf2-induced genes, triggered by oxidative stress, and protein biosynthesis were significantly overrepresented among the upregulated targets. Increased transcription of GPX1, CAT, RPS6, and eIF4E was confirmed by RT-PCR. The targets of the Akt/PKB signaling showed significant upregulation in both groups. Immunohistochemistry showed that the downstream targets NF-kappaB and BcL-X(L) are upregulated and activated. Gene sets involved in energy metabolism (oxidative phosphorylation, mitochondrial genes) showed no differences in group A but were overexpressed in group B. This study demonstrated a significant contribution of oxidative stress to the pathomechanism of TTC; it is possibly triggered by excess catecholamine. Increased protein biosynthesis and an activated cell survival cascade can be interpreted as potential compensatory mechanisms. After functional recovery, processes involved in energy metabolism play a pivotal role, thereby potentially contributing to the normalization of contractile function.