Function In Human Myocardium Research Articles

Inotropic and lusitropic, but not arrhythmogenic, effects of adipocytokine resistin on human atrial myocardium

The adipocytokine resistin is released from epicardial adipose tissue (EAT). Plasma resistin and EAT deposition are independently associated with atrial fibrillation. The EAT secretome enhances arrhythmia susceptibility and inotropy of human myocardium. Therefore, we aimed to determine the effect of resistin on the function of human myocardium and how resistin contributes to the proarrhythmic effect of EAT. EAT biopsies were obtained from 25 cardiac surgery patients. Resistin levels were measured by ELISA in 24-h EAT culture media (n = 8). The secretome resistin concentrations increased over the culture period to a maximal level of 5.9 ± 1.2 ng/mL. Coculture with β-adrenergic agonists isoproterenol (n = 4) and BRL37344 (n = 13) had no effect on EAT resistin release. Addition of resistin (7, 12, 20 ng/mL) did not significantly increase the spontaneous contraction propensity of human atrial trabeculae (n = 10) when given alone or in combination with isoproterenol. Resistin dose-dependently increased trabecula-developed force (maximal 2.9-fold increase, P < 0.0001), as well as the maximal rates of contraction (2.6-fold increase, P = 0.002) and relaxation (1.8-fold increase, P = 0.007). Additionally, the postrest potentiation capacity of human trabeculae was reduced at all resistin doses, suggesting that the inotropic effect induced by resistin might be due to altered sarcoplasmic reticulum Ca2+ handling. EAT resistin release is not modulated by common arrhythmia triggers. Furthermore, exogenous resistin does not promote arrhythmic behavior in human atrial trabeculae. Resistin does, however, induce an acute dose-dependent positive inotropic and lusitropic effect.

PKCα-Specific Phosphorylation of the Troponin Complex in Human Myocardium: A Functional and Proteomics Analysis

AimsProtein kinase Cα (PKCα) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKCα is implicated in heart failure and serves as a potential therapeutic target, however, the exact consequences for contractile function in human myocardium are unclear. This study aimed to investigate the effects of PKCα phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved.Methods and ResultsEndogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (∼69%) with PKCα-treated recombinant human cTn (cTn (DD+PKCα)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out in vitro cross-phosphorylation of the PKA sites by PKCα. Isometric force was measured at various [Ca2+] after exchange. The maximal force (Fmax) in the cTn (DD+PKCα) group (17.1±1.9 kN/m2) was significantly reduced compared to the cTn (DD) group (26.1±1.9 kN/m2). Exchange of endogenous cTn with cTn (DD+PKCα) increased Ca2+-sensitivity of force (pCa50 = 5.59±0.02) compared to cTn (DD) (pCa50 = 5.51±0.02). In contrast, subsequent PKCα treatment of the cells exchanged with cTn (DD+PKCα) reduced pCa50 to 5.45±0.02. Two PKCα-phosphorylated residues were identified with mass spectrometry: Ser198 on cTnI and Ser179 on cTnT, although phosphorylation of Ser198 is very low. Using mass spectrometry based-multiple reaction monitoring, the extent of phosphorylation of the cTnI sites was quantified before and after treatment with PKCα and showed the highest phosphorylation increase on Thr143.ConclusionPKCα-mediated phosphorylation of the cTn complex decreases Fmax and increases myofilament Ca2+-sensitivity, while subsequent treatment with PKCα in situ decreased myofilament Ca2+-sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKCα are phosphorylated in human cTn complex treated with PKCα with a high degree of specificity for Thr143.

The co-application of hypoxic preconditioning and postconditioning abolishes their own protective effect on systolic function in human myocardium

Ischemic preconditioning (IPC) and postconditioning (POC) are well documented to trigger cardioprotection against ischemia/reperfusion (I/R) injury, but the effect oftheir both co-application remains unclear in human heart. The present study sought to assessthe co-application of IPC and POC on fragments of human myocardium in vitro. Muscular trabeculae of the human right atrial were electrically driven in the organbath and subjected to simulated I/R injury - hypoxia/re-oxygenation injury in vitro. To achieveIPC of trabeculae the single brief hypoxia period preceded the applied lethal hypoxia, and to achieve POC triple brief hypoxia periods followed the lethal hypoxia. Additional muscular trabeculae were exposed only to the hypoxic stimulation (Control) or were subjected to the non-hypoxic stimulation (Sham). 10 μM norepinephrine (NE) application ended every experiment to assess viability of trabeculae. The contraction force of the myocardium assessed as a maximal amplitude of systolic peak (%Amax) was obtained during the whole experiment's period. Co-application of IPC and POC resulted in decrease in %Amax during the re-oxygentaionperiod and after NE application, as compared to Control (30.35 ± 2.25 vs. 41.89 ± 2.25, 56.26 ± 7.73 vs. 65.98 ± 5.39, respectively). This was in contrary to the effects observed when IPC and POC were applied separately. The co-application of IPC and POC abolishes the cardioprotection of either intervention alone against simulated I/R injury in fragments of the human right heart atria.

Protein kinase C α and ε phosphorylation of troponin and myosin binding protein C reduce Ca2+ sensitivity in human myocardium

Previous studies indicated that the increase in protein kinase C (PKC)-mediated myofilament protein phosphorylation observed in failing myocardium might be detrimental for contractile function. This study was designed to reveal and compare the effects of PKCα- and PKCε-mediated phosphorylation on myofilament function in human myocardium. Isometric force was measured at different [Ca2+] in single permeabilized cardiomyocytes from failing human left ventricular tissue. Activated PKCα and PKCε equally reduced Ca2+ sensitivity in failing cardiomyocytes (ΔpCa50 = 0.08 ± 0.01). Both PKC isoforms increased phosphorylation of troponin I- (cTnI) and myosin binding protein C (cMyBP-C) in failing cardiomyocytes. Subsequent incubation of failing cardiomyocytes with the catalytic subunit of protein kinase A (PKA) resulted in a further reduction in Ca2+ sensitivity, indicating that the effects of both PKC isoforms were not caused by cross-phosphorylation of PKA sites. Both isozymes showed no effects on maximal force and only PKCα resulted in a modest significant reduction in passive force. Effects of PKCα were only minor in donor cardiomyocytes, presumably because of already saturated cTnI and cMyBP-C phosphorylation levels. Donor tissue could therefore be used as a tool to reveal the functional effects of troponin T (cTnT) phosphorylation by PKCα. Massive dephosphorylation of cTnT with alkaline phosphatase increased Ca2+ sensitivity. Subsequently, PKCα treatment of donor cardiomyocytes reduced Ca2+ sensitivity (ΔpCa50 = 0.08 ± 0.02) and solely increased phosphorylation of cTnT, but did not affect maximal and passive force. PKCα- and PKCε-mediated phosphorylation of cMyBP-C and cTnI as well as cTnT decrease myofilament Ca2+ sensitivity and may thereby reduce contractility and enhance relaxation of human myocardium.

Functional effects of protein kinase C-mediated myofilament phosphorylation in human myocardium

In human heart failure beta-adrenergic-mediated protein kinase A (PKA) activity is down-regulated, while protein kinase C (PKC) activity is up-regulated. PKC-mediated myofilament protein phosphorylation might be detrimental for contractile function in cardiomyopathy. This study was designed to reveal the effects of PKC on myofilament function in human myocardium under basal conditions and upon modulation of protein phosphorylation by PKA and phosphatases. Isometric force was measured at different [Ca(2+)] in single permeabilized cardiomyocytes from non-failing and failing human left ventricular tissue. Basal phosphorylation of myofilament proteins and the influence of PKC, PKA, and phosphatase treatments were analyzed by one- and two-dimensional gel electrophoresis, Western immunoblotting, and ELISA. Troponin I (TnI) phosphorylation at the PKA sites was decreased in failing compared to non-failing hearts and correlated well with myofilament Ca(2+) sensitivity (pCa(50)). Incubation with the catalytic domain of PKC slightly decreased maximal force under basal conditions, but not following PKA and phosphatase pretreatments. PKC reduced Ca(2+) sensitivity to a larger extent in failing (DeltapCa(50)=0.19+/-0.03) than in non-failing (DeltapCa(50)=0.08+/-0.01) cardiomyocytes. This shift was reduced, though still significant, when PKC was preceded by PKA, while PKA following PKC did not further decrease pCa(50). Protein analysis indicated that PKC phosphorylated PKA sites in human TnI and increased phosphorylation of troponin T, while myosin light chain phosphorylation remained unaltered. In human myocardium PKC-mediated myofilament protein phosphorylation only has a minor effect on maximal force development. The PKC-mediated decrease in Ca(2+) sensitivity may serve to improve diastolic function in failing human myocardium in which PKA-mediated TnI phosphorylation is decreased.

Selective mitochondrial KATP channel opening controls human myocardial preconditioning: Too much of a good thing?

Background: Paradoxically, patients with noninsulin-dependent diabetes mellitus experience a higher cardiovascular mortality rate than patients with insulin-dependent diabetes mellitus. We have shown that KATP channel inhibition, with oral sulfonylureas, prevents myocardial preconditioning and may explain the paradox of cardiovascular death in patients with noninsulin-dependent diabetes mellitus. Cardiac preconditioning is an attractive protective strategy against any elective ischemia/reperfusion (I/R) injury. The relationship between the KATP channels and human myocardial preconditioning has not previously been elucidated. Methods: Human atrial trabeculae were harvested, placed in organ baths, and paced (1 Hz). Developed force was recorded during simulated 37°C I/R (30/45 or 45/60 minutes). Before I/R, trabeculae were treated transiently with a selective mitochondrial KATP channel opener for 5 minutes, followed by a 10-minute washout, or were exposed to the channel opener throughout ischemia. Recovery of function is expressed as percentage of baseline developed force. Conserved creatine kinase activity (units per gram of wet tissue) was measured at the end of reperfusion as an indicator of cellular protection. Results: Transient mitochondrial KATP channel opening provided protection from both I/R insults. Surprisingly, there was no protection afforded by continuous mitochondrial KATP channel opening. Conclusions: Transient selective mitochondrial KATP channel opening protects both viability and function of human myocardium against I/R injury, although prolonged opening of the mitochondrial KATP channel does not. These results reinforce the concept of preconditioning as a transient event that must be completed before the onset of ischemia. (Surgery 2000;128:368-73.)

Adenosine Reduces Cardiac TNF-α Production and Human Myocardial Injury Following Ischemia-Reperfusion

Myocardial tumor necrosis factor-α (TNF-α) is an autocrine contributor to myocardial dysfunction and cardiomyocyte death in ischemia-reperfusion injury (I/R), sepsis, chronic heart failure, and cardiac allograft rejection. Cardiac resident macrophages and cardiomyocytes themselves produce TNF-α. In this regard, adenosine (ADO) has been reported to reduce macrophage TNF-α production. Our purposes were to determine whether (1) I/R induces rat myocardial TNF-α production; (2) ADO decreases ischemia-induced rat myocardial TNF-α production; (3) ADO functionally protects human myocardium against I/R; and (4) TNF-α-binding protein (TNFBP; p55) confers similar protection when substituted for ADO pretreatment. To study this, human atrial trabeculae were obtained during cardiac surgery and suspended in organ baths, paced at 1 Hz, and force development was recorded during I/R (45/120 min) with or without ADO pretreatment (125 μM × 10 min), or TNFBP (1 μg/ml) during I/R. Isolated rat hearts were perfused using the Langendorff method undergoing I/R (20/40 min) with or without ADO pretreatment (125 μM × 2 min) and rat myocardial expression of TNF-α was assessed by ELISA. Results demonstrated that I/R increased rat myocardial TNF-α levels from 324 ± 36 to 902 ± 77 pg/g (P< 0.05; ANOVA and Bonferroni/Dunn) and decreased human myocardial developed force (DF) to 18 ± 2% of baseline (%BDF;P< 0.05). ADO pretreatment decreased ischemia-induced rat myocardial TNF-α production (356 ± 107 pg/g;P< 0.05) and increased postischemic DF of human myocardium to 39 ± 3% BDF (P< 0.05). Further substantiating the link between ischemia-induced TNF-α production and injury, TNFBP administration similarly improved post-I/R function of human myocardium (55 ± 5% BDF;P< 0.05 vs. I/R alone). We conclude that (1) I/R induces rat myocardial TNF-α production; (2) ADO pretreatment decreases I/R-induced rat myocardial TNF-α production; (3) ADO improves human myocardial function; (4) TNFBP confers similar protection; and (5) inhibition/ neutralization of TNF-α represents a novel strategy for protecting human myocardium against ischemia and reperfusion injury.

Merits and limitations of radionuclide approaches to viability and future developments.

Radionuclide imaging approaches have proved useful for the noninvasive identification of a potentially reversible impairment of contractile function in human myocardium. Foremost among these approaches are (1) the thallium 201 uptake, redistribution, and reinjection technique and (2) the evaluation of blood flow and metabolism with positron emission tomography (PET). Both general approaches appear equally accurate in predicting the postrevascularization outcome of regional contractile function. In patients with severely depressed left ventricular function who are likely to benefit most from viability assessments, the available evidence suggests that the metabolic approach with PET outperforms the more conventional approach with 201Tl. Several investigations have suggested that PET can identify those patients with a low probability of long-term survival, as well as patients in whom revascularization will reduce mortality rates, improve global left ventricular function, and relieve related symptoms of congestive heart failure. Moreover, several new radionuclide techniques are currently emerging. They probe key points of viable myocardium, such as residual oxidative metabolism, fatty acid uptake and oxidation, membrane function, and cellular homeostasis. Although initial observations have been encouraging, further validation and especially testing in those patients in whom viability assessment is clinically critical will be required. These new approaches may also offer new insights into the mechanisms of reversible contractile dysfunction in the human myocardium.