J. W. de Jong

Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins

OBJECTIVE The alterations in contractile proteins underlying enhanced Ca(2+)-sensitivity of the contractile apparatus in end-stage failing human myocardium are still not resolved. In the present study an attempt was made to reveal to what extent protein alterations contribute to the increased Ca(2+)-responsiveness in human heart failure. METHODS Isometric force and its Ca(2+)-sensitivity were studied in single left ventricular myocytes from non-failing donor (n=6) and end-stage failing (n=10) hearts. To elucidate which protein alterations contribute to the increased Ca(2+)-responsiveness isoform composition and phosphorylation status of contractile proteins were analysed by one- and two-dimensional gel electrophoresis and Western immunoblotting. RESULTS Maximal tension did not differ between myocytes obtained from donor and failing hearts, while Ca(2+)-sensitivity of the contractile apparatus (pCa(50)) was significantly higher in failing myocardium (deltapCa(50)=0.17). Protein analysis indicated that neither re-expression of atrial light chain 1 and fetal troponin T (TnT) nor degradation of myosin light chains and troponin I (TnI) are responsible for the observed increase in Ca(2+)-responsiveness. An inverse correlation was found between pCa(50) and percentage of phosphorylated myosin light chain 2 (MLC-2), while phosphorylation of MLC-1 and TnT did not differ between donor and failing hearts. Incubation of myocytes with protein kinase A decreased Ca(2+)-sensitivity to a larger extent in failing (deltapCa(50)=0.20) than in donor (deltapCa(50)=0.03) myocytes, abolishing the difference in Ca(2+)-responsiveness. An increased percentage of dephosphorylated TnI was found in failing hearts, which significantly correlated with the enhanced Ca(2+)-responsiveness. CONCLUSIONS The increased Ca(2+)-responsiveness of the contractile apparatus in end-stage failing human hearts cannot be explained by a shift in contractile protein isoforms, but results from the complex interplay between changes in the phosphorylation status of MLC-2 and TnI.

The effect of myosin light chain 2 dephosphorylation on Ca2+-sensitivity of force is enhanced in failing human hearts

OBJECTIVE Phosphorylation of the myosin light chain 2 (MLC-2) isoform expressed as a percentage of total MLC-2 was decreased in failing (21.1+/-2.0%) compared to donor (31.9+/-4.8%) hearts. To assess the functional implications of this change, we compared the effects of MLC-2 dephosphorylation on force development in failing and non-failing (donor) human hearts. METHODS Cooperative effects in isometric force and rate of force redevelopment (K(tr)) were studied in single Triton-skinned human cardiomyocytes at various [Ca(2+)] before and after protein phosphatase-1 (PP-1) incubation. RESULTS Maximum force and K(tr) values did not differ between failing and donor hearts, but Ca(2+)-sensitivity of force (pCa(50)) was significantly higher in failing myocardium (Deltap Ca(50)=0.17). K(tr) decreased with decreasing [Ca(2+)], although this decrease was less in failing than in donor hearts. Incubation of the myocytes with PP-1 (0.5 U/ml; 60 min) decreased pCa(50) to a larger extent in failing (0.20 pCa units) than in donor cardiomyocytes (0.10 pCa units). A decrease in absolute K(tr) values was found after PP-1 in failing and donor myocytes, while the shape of the K(tr)-Ca(2+) relationships remained unaltered. CONCLUSIONS Surprisingly, the contractile response to MLC-2 dephosphorylation is enhanced in failing hearts, despite the reduced level of basal MLC-2 phosphorylation. The enhanced response to MLC-2 dephosphorylation in failing myocytes might result from differences in basal phosphorylation of other thin and thick filament proteins between donor and failing hearts. Regulation of Ca(2+)-sensitivity via MLC-2 phosphorylation may be a potential compensatory mechanism to reverse the detrimental effects of increased Ca(2+)-sensitivity and impaired Ca(2+)-handling on diastolic function in human heart failure.

Effect of protein kinase A on calcium sensitivity of force and its sarcomere length dependence in human cardiomyocytes

OBJECTIVE We investigated whether the Frank-Starling mechanism is absent or preserved in end-stage failing human myocardium and if phosphorylation of contractile proteins modulates its magnitude through the sarcomere length-dependence of calcium sensitivity of isometric force development. METHODS The effect of phosphorylation of troponin I and C-protein by the catalytic subunit of protein kinase A (3 microg/ml; 40 min at 20 degrees C) was studied in single Triton-skinned human cardiomyocytes isolated from donor and end-stage failing left ventricular myocardium at sarcomere lengths measured at rest of 1.8, 2.0 and 2.2 microm. Isometric force development was studied at various free-calcium concentrations before and after protein kinase A incubation at 15 degrees C (pH 7.1). RESULTS Maximal isometric tension at 2.2 microm amounted to 39.6+/-10.4 and 33.7+/-3.5 kN/m2 in donor and end-stage failing cardiomyocytes, respectively. The midpoints of the calcium sensitivity curves (pCa50) of donor and end-stage failing hearts differed markedly at all sarcomere lengths (mean delta pCa50=0.22). A reduction in sarcomere length from 2.2 to 1.8 microm caused reductions in maximum isometric force to 64% and 65% and in pCa50 by 0.10 and 0.08 pCa units in donor and failing cardiomyocytes, respectively. In donor tissue, the effect of protein kinase A treatment was rather small, while in end-stage failing myocardium it was much larger (delta pCa50=0.24) irrespective of sarcomere length. CONCLUSIONS The data obtained indicate that the Frank-Starling mechanism is preserved in end-stage failing myocardium and suggest that sarcomere length dependence of calcium sensitivity and the effects of phosphorylation of troponin I and C-protein are independent.

Myocardial substrate utilization and hemodynamics following repeated coronary flow reduction in pigs.

SummaryThe effect of repeated local ischemia and reperfusion on myocardial metabolism and ventricular performance was studied in 12 open-chested pigs fasted overnight. Myocardial ischemia was induced by reduction of the flow in the left anterior descending coronary artery to 40% of control during 30 min. After 35 min of reperfusion a second 30-min occlusion period was started, again followed by a 35-min reperfusion period. At the end of both reperfusion periods coronary flow and coronary resistance had returned to control values. During control there was lactate uptake, but no significant uptake of glucose, free fatty acids (FFA), triglycerides, glycerol and inosine. During the first occlusion period the heart released lactate and inosine, and used glucose and FFA. At the end of the first reperfusion period lactate uptake approached control values, but inosine was still released by 10 of the 12 animals. In the second ischemic period, glucose and FFA were again taken up. Lactate and inosine were released, but the production was much smaller than during the first occlusion period. Depletion of myocardial glycogen and high-energy phosphates could be responsible for this quantitatively different response. Necrosis may have played a role, although enzyme release was minimal and only observed after the second occlusion period.Heart rate, peripheral resistance and ventricular filling pressure were virtually unchanged throughout the course of the experiments. Maximum rate of fall of left ventricular pressure (min LVdP/dt) decreased during ischemia and did not recover during reperfusion. Changes in min LVdP/dt and cardiac output were more closely related than changes in max LVdP/dt and cardiac output.This model cannot be used for the study of interventions during myocardial ischemia in which the animal serves as its own control.ZusammenfassungDer Effekt von wiederholter lokaler Ischämie und Reperfusion auf den myokardialen Energiestoffwechsel und Ventrikelfunktion wurde bei 12 narkotisierten Schweinen mit geöffnetem Thorax studiert. Die Schweine hatten 24 Stunden gefastet. Die myokardiale Ischämie wurde verursacht durch eine Reduktion der Blutdurchströmung in der linken Anterior Descending Koronar Arterie bis 40% vom Anfangswert während 30 Minuten. Eine zweite Reduktion wurde nach 35 Minuten Reperfusion angefangen. Die zweite Reduktion folgte wieder durch eine 35 Minuten dauernde Reperfusion. Am Ende der beiden Reperfusionen der Koronardurchströmung und Koronarwiderstand hatten sie wieder Anfangswerte angenommen. Während der Kontrolle gab es Laktat-Aufnahme, aber keine Aufnahme von Glukose, Freie Fettsäure (FFA), Triglyzeride, Glyzerol und Inosine. Während der ersten Okklusions-Periode wurde aus dem Herzen Lactat und Inosine freigemacht, es wurde Glukose und FFA aufgenommen. Am Ende der ersten Reperfusion wurde Laktat wieder aufgenommen, aber Inosine wurde noch immer an 10 von 12 Schweinen freigemacht. In der zweiten Okklusions-Periode gab es wieder Glukose und FFA-Aufnahme. Laktat und Inosine wurden freigemacht, aber die Produktion war viel kleiner als in der ersten Okklusions-Periode. Erschöpfung von myokardialen Glykogen und energiereichen Phosphaten können dafür verantwortlich sein. Nekrose könnte auch eine Rolle gespielt haben, obschon Enzym-Abgaben sehr gering waren und nur nach der zweiten Okklusions-Periode gefunden wurden. Herzfrequenz, Peripherewiderstand und ventrikulärer Füllungsdruck blieben so gut wie unverändert. Während der Experimente wurde die maximale Druckabfallgeschwindigkeit (minLVdP/dt) weniger, während Ischämie sich bei Reperfusion nicht herstellte. Veränderungen in minLVdP/dt und Herzminutenvolumen hatten einen höheren Korrelations-Koeffizient, als maxLVdP/dt und Herzminutenvolumen. Dieses Modell kann nicht für die Studien von Interventionen während myokardialer Ischämie benutzt werden.

Calcium sensitivity of force in human ventricular cardiomyocytes from donor and failing hearts.

Abstract In failing human myocardium changes occur, in particular, in isoform composition and phosphorylation level of the troponin T (TnT) and troponin I (TnI) subunits of the actin filament and the myosin light chains (MLC-1 and -2), but it is unclear to what extent they influence cardiac performance. This overview concentrates on the relation between contractile function, contractile protein composition and phosphorylation levels in small biopsies from control (donor) hearts, from biopsies obtained during open heart surgery (NYHA Class I – IV) and from end-stage failing (explanted, NYHA class IV) hearts. Furthermore, attention is paid to the effect of the catalytic subunit of protein kinase A on isometric force development in single Triton-skinned human cardiomyocytes isolated from donor and end-stage failing left ventricular myocardium at different resting sarcomere lengths. A reduction in sarcomere length from 2.2 to 1.8 μm caused reductions in maximum isometric force by approximately 35 % both in donor and in failing cardiomyocytes. The midpoints of the calcium sensitivity curves (pCa50) of donor and end-stage failing hearts differed markedly at all sarcomere lengths (mean ΔpCa50 = 0.22). Our findings indicate that 1) TnI phosphorylation contributes to the differences in calcium sensitivity between donor and end-stage failing hearts, 2) human ventricular myocardium is heterogeneous with respect of the phosphorylation of TnT, MLC-2 and the isoform distribution of MLC-1 and MLC-2, and 3) the Frank-Starling mechanism is preserved in end-stage failing myocardium.

Myosin Light Chain Composition in Non-Failing Donor and End-Stage Failing Human Ventricular Myocardium

The increased Ca(2+)-responsiveness in end-stage human heart failure cannot be attributed to contractile protein isoform changes, but rather is the complex resultant of changes in degree of phosphorylation of VLC-2 and TnI. Despite the decreased basal level of VLC-2 phosphorylation the response to VLC-2 dephosphorylation is enhanced in failing myocytes, which might result from differences in endogenous phosphorylation of thin and thick filament proteins between donor and failing hearts. Taken together decreased VLC-2 phosphorylation in end-stage human heart failure might represent a compensatory process leading to an improvement of myocardial contractility by opposing the detrimental effects of increased Ca(2+)-responsiveness of force and impaired Ca(2+)-handling on diastolic function.

Developmental differences in myocardial ATP metabolism.

Little is known about postnatal changes in myocardial purine metabolism. We therefore studied how ATP catabolism was affected by hypothermia and ischaemia in neonatal and adult hearts. Hypothermia during ischaemia protected isolated adult and newborn hearts against ATP decline. Reperfusion after normothermic ischaemia resulted in higher ATP levels in newborn hearts with less release of ATP-catabolites. During normoxia adult hearts released mainly urate (80% of total purine release), while newborns released mainly hypoxanthine (64%). During early reperfusion adult and newborn hearts released mainly inosine (50-60%). The very low xanthine oxidase activity in the neonatal heart could be an important factor in the observed ATP preservation during reperfusion.

Balance of Purine Nucleotides and Catabolites in the Isolated Ischemic Rat Heart

Myocardial contraction depends fully on purine nucleotides (ATP) and creatine phosphate (CrP). The oxygen dependent formation and the breakdown of ATP are in a delicately regulated balance. During ischemia oxygen supply is lowered, ATP-levels fall and the heart releases catabolites of purine nucleotides, probably to maintain an adequately high energy charge. Released are uric acid (UA), xanthine (X), hypoxanthine (Hx), inosine (Ino) and adenosine (Ado). Amongst these, adenosine is very important because of its coronary vasodilating properties (1). Maintenance of adequate levels of purine nucleotides occurs not only by (slow) de novo synthesis, but also by phosphorylation of Ado and salvage of Hx.

Ischemic preconditioning—do we need more (pharmacological) experiments?

ConclusionPreconditioning against myocardial ischemia is potentially a powerful method to protect the human heart. It is obvious that only limited possibilities, e.g., during heart surgery, exist to use short periods of ischemia to elicit the effect. A pharmacological approach, using adenosine A1/A3 analogs or K+ATP channel openers, seems logical. In view of the gaps in our knowledge on the mechanisms involved, we believe it is prudent to continue basic research in this field before studying ‘preconditioning drugs’ clinically on a larger scale.

Uridine and Purine Nucleoside Phosphorylase Activity in Human and Rat Heart

Literature data on cardiac pyrimidine metabolism are scarce, contrasting our knowledge on myocardial purine metabolism. However, degradation of pyrimidine nucleotides in the ischemic myocardium seems to parallel catabolism of adenylates1. More knowledge about pyrimidine nucleotide breakdown is useful because of 1) the regulatory role of pyrimidine derivatives2; 2) the interconversions of anticancer drugs by pyrimidine metabolizing enzymes3,4; 3) the potential diagnostic value of pyrimidine catabolites.