Ischemic heart disease is the leading cause of death worldwide. Reduced oxygen supply and myocardial hypoxia lead to tissue damage and impairment of the heart function. To the best of our knowledge, the primary functional effects of hypoxia in the multicellular model of living myocardial slices (LMSs) have not been investigated so far. In this study, we analyzed force generation, ultrastructure, gene expression, and proteome changes in rat LMS after 24 h of ex vivo culture in normal and reduced levels of oxygen (O₂). We observed a significant reduction in absolute force and a slowdown of force kinetics as well as an increase in cardiomyocyte apoptosis and myofibrillar and mitochondrial damage, as well as transcriptomic changes. Proteome analysis revealed the deregulation of proteins involved in metabolic processes, hypoxic response, and neutralizing of reactive oxygen species. Our results indicate that hypoxia induces substantial primary changes in heart tissue, which are independent of perfusion and immune responses. Our new LMS model could serve as a screening system for drug development and new mechanistic insights.

Viperid snake venoms contain a group of cytotoxic proteins called Lys49 phospholipase A₂ (PLA₂)-like myotoxins. These proteins lack enzymatic activity, but can still disrupt the sarcolemma and induce cell death. On skeletal and cardiac muscle, PLA₂-like myotoxins induce substantial alterations to the sequence of events in the excitation-contraction coupling, including increased Ca²⁺ influx and consequent hypercontraction. This study aimed to quantify the increase in force development induced by a Lys49 PLA₂-like myotoxin and to assess whether this effect contributes to the overall cytotoxicity induced by the toxin. Force transients generated by mechanically loaded living myocardial slices (LMS) obtained from rat and human hearts were characterized in response to Mt-II, a well-described Lys49 PLA₂-like myotoxin found in the venom of the viperid snake Bothrops asper. Over a 24-hour period, Mt-II initially increased the amplitude of the force transients, which was then replaced by a permanent increase in baseline force. This dual effect may be attributed to progressive sarcolemmal disruption, which initially induces an increase in force development as a consequence of an influx of Ca²⁺. However, at advanced stages of cell damage, this renders the calcium extrusion mechanisms ineffective. The amplitude of the force transients and the baseline force observed during Mt-II exposure were several times greater than the amplitude of the force transients observed before Mt-II exposure. Inhibiting force development by a myosin inhibitor in the presence of Mt-II significantly reduced the release of cytosolic enzymes LDH and CK-MB from the LMS preparations. Therefore, mechanical stress resulting from Ca²⁺ influx and hypercontraction exacerbates Mt-II-induced striated muscle damage.