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Mitochondrial function is critical to myocardial ischemia/reperfusion injury and cardioprotection. The measurement of mitochondrial function in isolated mitochondria requires cardiac specimens of about 300 mg and is therefore only possible at the end of an animal experiment or during cardiosurgical interventions in humans. As an alternative, mitochondrial function can be measured in permeabilized myocardial tissue (PMT) specimens of about 2-5 mg, which are retrieved by sequential biopsies in animal experiments and during cardiac catheterization in humans. We attempted to validate measurements of mitochondrial respiration from PMT by comparison to those from isolated mitochondria of left ventricular myocardium from anesthetized pigs undergoing 60 min coronary occlusion and 180 min reperfusion. Mitochondrial respiration was normalized to the content of mitochondrial marker proteins (cytochrome c oxidase 4 (COX4), citrate synthase and manganese-dependent superoxide dismutase). When normalized to COX4, mitochondrial respiration measurements in PMT and isolated mitochondria agreed well in Bland-Altman plots (bias score: -0.03 nmol/min/COX4; 95% confidence interval: 6.31 nmol/min/COX4 and -6.37 nmol/min/COX4) and correlated well (slope of 0.77 and Pearson R of 0.87). Mitochondrial dysfunction by ischemia/reperfusion was equally reflected in PMT and isolated mitochondria (44% and 48% reduction of ADP-stimulated complex I respiration). Also in isolated human right atrial trabeculae, simulation of ischemia/reperfusion injury by exposure to 60 min hypoxia and 10 min reoxygenation reduced mitochondrial ADP-stimulated complex I respiration by 37% in PMT. In conclusion, mitochondrial function measurements in permeabilized cardiac tissue can substitute for that in isolated mitochondria to reflect mitochondrial dysfunction following ischemia/reperfusion.

The leech-derived hirudins and hirudin-like factors (HLFs) share a common molecule structure: a short N-terminus, a central globular domain, and an elongated C-terminal tail. All parts are important for function. HLF6 and HLF7 were identified in the Asian medicinal leech, Hirudinaria manillensis. The genes of both factors encode putative splice variants that differ in length and composition of their respective C-terminal tails. In either case, the tails are considerably shorter compared to hirudins. Here we describe the functional analyses of the natural splice variants and of synthetic variants that comprise an altered N-terminus and/or a modified central globular domain. All natural splice variants of HLF6 and HLF7 display no detectable thrombin-inhibitory potency. In contrast, some synthetic variants effectively inhibit thrombin, even with tails as short as six amino acid residues in length. Our data indicate that size and composition of the C-terminal tail of hirudins and HLFs can vary in a great extent, yet the full protein may still retain the ability to inhibit thrombin.

Background In patients with acute myocardial infarction, the one‐year mortality remains at 7‐15% despite successful interventional reperfusion. Thus, innovative treatment strategies beyond reperfusion are needed. In isolated perfused rat hearts, triiodothyronine (T3), when given during early reperfusion in a supraphysiological concentration, improves left ventricular function and reduces infarct size (Basic Res Cardiol 2021;116:27). Aim To investigate whether or not such acute, non‐genomic effects of T3 also protect human myocardium. Methods We isolated right atrial trabeculae from patients undergoing routine coronary artery bypass grafting and investigated the effect of T3 on contractile function during hypoxia/reoxygenation. Trabeculae (≥ 3 mm length, ≤ 1 mm diameter) were isolated in cardioplegic buffer and transferred to Tyrode buffer. Electrically initiated (1 Hz) developed force of contraction (mN/mm2) was recorded for 10 min at baseline (95% O2 – 5% CO2, glucose). Hypoxia/reoxygenation (60 min/30 min) was induced by changing the buffer gas supply and composition and by increasing the stimulation rate (95% N2 – 5% CO2, choline chloride, 3 Hz) and returning back to baseline. T3 (500 µg/l) or NaOH (as solvent control), respectively, was administered A) during hypoxia and reoxygenation (n=11 vs. n=10) or B) 15 min before and during reoxygenation (n=12 vs. n=14). Results At baseline, before T3 or NaOH treatment, the contractile function of trabeculae was comparable (mN/mm2: 15.4±3.5, 13.8±4.4 in protocol A, and 14.5±4.1, 13.7±4.0 in protocol B, respectively). T3 improved the contractile function during reoxygenation, irrespectively of whether given continuously (A) or just before and during reoxygenation (B; see Figure). Conclusion T3 protects patient myocardium: contractile function is improved during reoxygenation in right atrial trabeculae. This bioassay will now permit an identification of causally involved cardioprotective signaling molecules of such non‐genomic protection in human tissue by T3.