Myosin heavy chain and cardiac troponin T damage is associated with impaired myofibrillar ATPase activity contributing to sarcomeric dysfunction in Ca2+-paradox rat hearts.

This study aimed to explore the potential contribution of myofibrils to contractile dysfunction in Ca2+-paradox hearts. Isolated rat hearts were perfused with Krebs-Henseleit solution (Control), followed by Ca2+-depletion, and then Ca2+-repletion after Ca2+-depletion (Ca2+-paradox) by Langendorff method. During heart perfusion left ventricular developed pressure (LVDP), end-diastolic pressure (LVEDP), rate of pressure development (+ dP/dt), and pressure decay (-dP/dt) were registered. Control LVDP (127.4 ± 6.1 mmHg) was reduced during Ca2+-depletion (9.8 ± 1.3 mmHg) and Ca2+-paradox (12.9 ± 1.3 mmHg) with similar decline in +dP/dt and -dP/dt. LVEDP was increased in both Ca2+-depletion and Ca2+-paradox. Compared to Control, myofibrillar Ca2+-stimulated ATPase activity was decreased in the Ca2+-depletion group (12.08 ± 0.57 vs. 8.13 ± 0.19 µmol Pi/mg protein/h), besides unvarying Mg2+ ATPase activity, while upon Ca2+-paradox myofibrillar Ca2+-stimulated ATPase activity was decreased (12.08 ± 0.57 vs. 8.40 ± 0.22 µmol Pi/mg protein/h), but Mg2+ ATPase activity was increased (3.20 ± 0.25 vs. 7.21 ± 0.36 µmol Pi/mg protein/h). In force measurements of isolated cardiomyocytes at saturating [Ca2+], Ca2+-depleted cells had lower rate constant of force redevelopment (k tr,max, 3.85 ± 0.21) and unchanged active tension, while those in Ca2+-paradox produced lower active tension (12.12 ± 3.19 kN/m2) and k tr,max (3.21 ± 23) than cells of Control group (25.07 ± 3.51 and 4.61 ± 22 kN/m2, respectively). In biochemical assays, α-myosin heavy chain and cardiac troponin T presented progressive degradation during Ca2+-depletion and Ca2+-paradox. Our results suggest that contractile impairment in Ca2+-paradox partially resides in deranged sarcomeric function and compromised myofibrillar ATPase activity as a result of myofilament protein degradation, such as α-myosin heavy chain and cardiac troponin T. Impaired relaxation seen in Ca2+-paradoxical hearts is apparently not related to titin, rather explained by the altered myofibrillar ATPase activity.

Heme-induced contractile dysfunction in human cardiomyocytes caused by oxidant damage to thick filament proteins.

Intracellular free heme predisposes to oxidant-mediated tissue damage. We hypothesized that free heme causes alterations in myocardial contractility via disturbed structure and/or regulation of the contractile proteins. Isometric force production and its Ca(2+)-sensitivity (pCa50) were monitored in permeabilized human ventricular cardiomyocytes. Heme exposure altered cardiomyocyte morphology and evoked robust decreases in Ca(2+)-activated maximal active force (Fo) while increasing Ca(2+)-independent passive force (F passive). Heme treatments, either alone or in combination with H2O2, did not affect pCa50. The increase in F passive started at 3 µM heme exposure and could be partially reversed by the antioxidant dithiothreitol. Protein sulfhydryl (SH) groups of thick myofilament content decreased and sulfenic acid formation increased after treatment with heme. Partial restoration in the SH group content was observed in a protein running at 140 kDa after treatment with dithiothreitol, but not in other proteins, such as filamin C, myosin heavy chain, cardiac myosin binding protein C, and α-actinin. Importantly, binding of heme to hemopexin or alpha-1-microglobulin prevented its effects on cardiomyocyte contractility, suggesting an allosteric effect. In line with this, free heme directly bound to myosin light chain 1 in human cardiomyocytes. Our observations suggest that free heme modifies cardiac contractile proteins via posttranslational protein modifications and via binding to myosin light chain 1, leading to severe contractile dysfunction. This may contribute to systolic and diastolic cardiac dysfunctions in hemolytic diseases, heart failure, and myocardial ischemia-reperfusion injury.

Myeloperoxidase impairs the contractile function in isolated human cardiomyocytes.

We set out to characterize the mechanical effects of myeloperoxidase (MPO) in isolated left-ventricular human cardiomyocytes. Oxidative myofilament protein modifications (sulfhydryl (SH)-group oxidation and carbonylation) induced by the peroxidase and chlorinating activities of MPO were additionally identified. The specificity of the MPO-evoked functional alterations was tested with an MPO inhibitor (MPO-I) and the antioxidant amino acid Met. The combined application of MPO and its substrate, hydrogen peroxide (H2O2), largely reduced the active force (Factive), increased the passive force (Fpassive), and decreased the Ca(2+) sensitivity of force production (pCa50) in permeabilized cardiomyocytes. H2O2 alone had significantly smaller effects on Factive and Fpassive and did not alter pCa50. The MPO-I blocked both the peroxidase and the chlorinating activities, whereas Met selectively inhibited the chlorinating activity of MPO. All of the MPO-induced functional effects could be prevented by the MPO-I and Met. Both H2O2 alone and MPO + H2O2 reduced the SH content of actin and increased the carbonylation of actin and myosin-binding protein C to the same extent. Neither the SH oxidation nor the carbonylation of the giant sarcomeric protein titin was affected by these treatments. MPO activation induces a cardiomyocyte dysfunction by affecting Ca(2+)-regulated active and Ca(2+)-independent passive force production and myofilament Ca(2+) sensitivity, independent of protein SH oxidation and carbonylation. The MPO-induced deleterious functional alterations can be prevented by the MPO-I and Met. Inhibition of MPO may be a promising therapeutic target to limit myocardial contractile dysfunction during inflammation.

Single acute stress-induced progesterone and ovariectomy alter cardiomyocyte contractile function in female rats.

AIM: To assess how ovarian-derived sex hormones (in particular progesterone) modify the effects of single acute stress on the mechanical and biochemical properties of left ventricular cardiomyocytes in the rat. METHODS: Non-ovariectomized (control, n=8) and ovariectomized (OVX, n=8) female rats were kept under normal conditions or were exposed to stress (control-S, n=8 and OVX-S, n=8). Serum progesterone levels were measured using a chemiluminescent immunoassay. Left ventricular myocardial samples were used for isometric force measurements and protein analysis. Ca(2+)-dependent active force (Factive), Ca(2+)-independent passive force (Fpassive), and Ca(2+)-sensitivity of force production were determined in single, mechanically isolated, permeabilized cardiomyocytes. Stress- and ovariectomy-induced alterations in myofilament proteins (myosin-binding protein C [MyBP-C], troponin I [TnI], and titin) were analyzed by sodium dodecyl sulfate gel electrophoresis using protein and phosphoprotein stainings. RESULTS: Serum progesterone levels were significantly increased in stressed rats (control-S, 35.6±4.8 ng/mL and OVX-S, 21.9±4.0 ng/mL) compared to control (10±2.9 ng/mL) and OVX (2.8±0.5 ng/mL) groups. Factive was higher in the OVX groups (OVX, 25.9±3.4 kN/m(2) and OVX-S, 26.3±3.0 kN/m(2)) than in control groups (control, 16.4±1.2 kN/m(2) and control-S, 14.4±0.9 kN/m(2)). Regarding the potential molecular mechanisms, Factive correlated with MyBP-C phosphorylation, while myofilament Ca(2+)-sensitivity inversely correlated with serum progesterone levels when the mean values were plotted for all animal groups. Fpassive was unaffected by any treatment. CONCLUSION: Stress increases ovary-independent synthesis and release of progesterone, which may regulate Ca(2+)-sensitivity of force production in left ventricular cardiomyocytes. Stress and female hormones differently alter Ca(2+)-dependent cardiomyocyte contractile force production, which may have pathophysiological importance during stress conditions affecting postmenopausal women.

Differences in angiotensin convertase enzyme (ACE) activity and expression may contribute to shorter event free period after coronary artery bypass graft surgery.

AIMS: The goal of this study was to investigate the importance of the vascular angiotensin convertase enzyme (ACE) in coronary artery bypass graft surgery (CABG) patients. METHODS: Vascular tissue (distal saphenous vein [n= 163] and/or radial artery [n= 120] segments) and blood samples were collected from CABG patients (n= 81). We studied (i) the potency of angiotensin I (AngI) and angiotensin II (AngII) to evoke vascular contractions; (ii) vascular and plasma ACE concentrations; and (iii) ACE genotype of the patients enrolled. RESULTS: The ratio of the potencies (EC(50) ) of AngII and AngI was significantly lower in radial artery compared to the saphenous vein (0.17 ± 0.03 nM and 0.51 ± 0.14 nM, respectively, P= 0.003), suggesting a 3-fold more effective AngI conversion in saphenous vein samples. Angiotensin constrictions were inhibited with telmisartan and captopril in both saphenous veins and radial arteries. Vascular ACE expression was significantly higher in saphenous vein compared to radial artery (9.7 ± 1.0 ng/mg and 5.3 ± 0.7 ng/mg, respectively, P= 0.01). Serum but no tissue ACE concentration was determined by ACE insertion/deletion polymorphism. Accordingly, no relation was found between serum and tissue ACE expression. CONCLUSION: ACE-inhibitor therapy targeting tissue located ACE may be beneficial to patients with saphenous vein grafts after CABG surgery.