Tamás Ivanics

Early cardiac dysfunction is rescued by upregulation of SERCA2a pump activity in a rat model of metabolic syndrome

Various components of metabolic syndrome associate with cardiac intracellular calcium ( Cai 2+ ) mishandling, a precipitating factor in the development of heart failure. We aimed to provide a thorough description of early stage Cai 2+ ‐cycling alterations in the fructose‐fed rat, an experimental model of the disorder, where insulin resistance, hypertension and dyslipidaemia act cooperatively on the heart.

Time related changes in calcium handling in the isolated ischemic and reperfused rat heart

The main aim of this study was to assess the kinetics of intracellular free calcium (Ca2+i) handling by isolated rat hearts rendered ischemic for 30 min followed by 30 min of reperfusion analyzing the upstroke and downslope of the Ca2+i transient. Changes in mechanical performance and degradation of membrane phospholipids – estimated by tissue arachidonic acid content – were correlated with Ca2+i levels of the heart. The fluorescence ratio technique was applied to estimate Ca2+i. The disappearance of mechanical activity of the heart preceded that of the Ca2+i transient in the first 2 min of ischemia. The slope of upstroke of the Ca2+i transient, reflecting Ca2+ release, decreased by 60%, while the duration of the downslope of the transient, reflecting Ca2+ sequestration, expressed a significant prolongation (105 ± 17 vs. 149 ± 39 msec) during the first 3 min of ischemia. At about 20 min of ischemia end-diastolic pressure expressed a 3.5-fold increase (contracture) when the fluorescence ratio showed a 2-fold elevation. Reperfusion was accompanied with a further precipitous increase in end-diastolic pressure, while resting Ca2+i remained at end-ischemic levels. Increases in the arachidonic acid (AA) content of the ischemic and postischemic hearts were proportional to Ca2+i levels. In summary, the present findings indicate that both calcium release and removal are hampered during the early phase of ischemia. Moreover, a critical level of Ca2+i and a critical duration of ischemia may exist to provoke contracture of the heart. Upon reperfusion the hearts show membrane phospholipid degradation and signs of stunning exemplified by elevated AA levels, partial recovery of Ca2+i handling and sustained depression of mechanical performance.

Hearts of surviving MLP-KO mice show transient changes of intracellular calcium handling

The muscle Lim protein knock-out (MLP-KO) mouse model is extensively used for studying the pathophysiology of dilated cardiomyopathy. However, explanation is lacking for the observed long survival of the diseased mice which develop until adulthood despite the gene defect, which theoretically predestines them to early death due to heart failure. We hypothesized that adaptive changes of cardiac intracellular calcium (Cai2+) handling might explain the phenomenon. In order to study the progression of changes in cardiac function and Cai2+ cycling, myocardial Cai2+-transients recorded by Indo-1 surface fluorometry were assessed with concomitant measurement of hemodynamic performance in isolated Langendorff-perfused hearts of 3- and 9-month old MLP-KO animals. Hearts were challenged with β-agonist isoproterenol and the sarcoplasmic reticular Ca2+-ATPase (SERCA2a) inhibitor cyclopiazonic acid (CPA). Cardiac mRNA content and levels of key Ca2+ handling proteins were also measured. A decline in lusitropic function was observed in 3-month old, but not in 9-month old MLP-KO mice under unchallenged conditions. β-adrenergic responses to isoproterenol were similar in all the studied groups. The CPA induced an increase in end-diastolic Cai2+-level and a decrease in Ca2+-sequestration capacity in 3-month old MLP-KO mice compared to age-matched controls. This unfavorable condition was absent at 9 months of age. SERCA2a expression was lower in 3-month old MLP-KO than in the corresponding controls and in 9-month old MLP-KO hearts. Our results show time-related recovery of hemodynamic function and an age-dependent compensatory upregulation of Cai2+ handling in hearts of MLP-KO mice, which most likely involve the normalization of the expression of SERCA2a in the affected hearts.

Depressed calcium cycling contributes to lower ischemia tolerance in hearts of estrogen-deficient rats.

ObjectiveEstrogens enhance ischemia tolerance (IT) in the myocardium, the mechanism of which remains unclear. We investigated the effects of long-term estrogen deprivation on the intracellular calcium (Ca2+i) transient of the heart and its possible influence on IT. MethodsHearts of ovariectomized (OVX) and sham-operated (control) adult female rats (some receiving estrogen therapy) were studied 10 weeks after surgical operation: control (n = 8), OVX (n = 10), sham-operated estrogen-substituted (n = 7), and ovariectomized estrogen-substituted (n = 9). In vivo heart function was assessed by echocardiography, whereas Ca2+i transients were recorded, concomitantly with left ventricular pressure and coronary flow, by Indo-1 surface fluorometry in isolated Langendorff-perfused hearts. Isolated hearts were subjected to a 30-minute global ischemia–30-minute reperfusion protocol. Left ventricular expression of myocardial sarcoendoplasmic reticulum Ca2+-ATPase (SERCA2a), phospholamban (PLB), and Ser16-phosphorylated PLB was measured. ResultsOvariectomy did not influence resting cardiac function in vivo or ex vivo. However, Ca2+ removal was slower. During ischemia, Ca2+i elevation and ischemic contracture were more pronounced after ovariectomy. Postischemic restitution of inotropic function (developed pressure; +dP/dtmax) and lusitropic function (−dP/dtmax) and Ca2+i transient recovery (amplitude; ±dCa2+i/dtmax) were decreased in OVX hearts. Sarcoendoplasmic reticulum Ca2+-ATPase expression was unaltered, whereas PLB and Ser16-phosphorylated PLB levels were higher after ovariectomy. All effects of ovariectomy were restored by estrogen therapy. ConclusionsOvariectomy impairs myocardial Ca2+ removal by increasing the expression of the SERCA2a inhibitor PLB. Defective Ca2+ transport causes ischemic Ca2+i overload and insufficient postischemic recovery of Ca2+i transients, which entail depressed hemodynamic restitution. Protection of intact Ca2+ cycling in the myocardium by estrogens plays a major role in enhancing IT.

Mathematical modelling of the calcium-left ventricular pressure relationship in the intact diabetic rat heart

Aim:  The objective was to characterize cross‐bridge kinetics from the cytoplasmic calcium ion concentration ([Ca2+]i) and the left ventricular pressure (LVP) in the early‐stage diabetic rat heart under baseline conditions and upon β‐adrenergic stimulation.

Quantitative assessment of [Ca2+]ilevels in rat skeletal muscle in vivo

Intracellular free Ca2+ concentration ([Ca2+]i) plays an essential role in physiological regulatory processes and common pathological conditions. Better understanding of these phenomena is still hampered by problems encountered in the quantitative assessment of [Ca2+]i changes, especially in blood-perfused organs. This study demonstrates that the ratiometric fluorescence technique can be adapted for quantitative in vivo [Ca2+]i determinations. The rat spinotrapezius muscle was topically loaded with indo 1-AM and imaged by a cooled digital camera. Ratio images were calculated in small regions (100 micrometers x 100 micrometers) practically devoid of large vessels in the resting state, after 30 min of ischemia, 20 min of reperfusion, or ionomycin or manganate treatments. When we assumed an average [Ca2+]i of 100 nM in the resting blood-perfused muscle, ischemia increased [Ca2+]i to approximately 200 nM. During reperfusion [Ca2+]i decreased to approximately 140 nM. Ionomycin induced an increase in [Ca2+]i to well above 750 nM. Manganate reduced Ca2+-dependent fluorescence to virtually zero. Our main conclusion is that changes in [Ca2+]i can be monitored and quantitatively determined in vivo.Intracellular free Ca2+ concentration ([Ca2+]i) plays an essential role in physiological regulatory processes and common pathological conditions. Better understanding of these phenomena is still hampered by problems encountered in the quantitative assessment of [Ca2+]ichanges, especially in blood-perfused organs. This study demonstrates that the ratiometric fluorescence technique can be adapted for quantitative in vivo [Ca2+]ideterminations. The rat spinotrapezius muscle was topically loaded with indo 1-AM and imaged by a cooled digital camera. Ratio images were calculated in small regions (100 μm × 100 μm) practically devoid of large vessels in the resting state, after 30 min of ischemia, 20 min of reperfusion, or ionomycin or manganate treatments. When we assumed an average [Ca2+]iof 100 nM in the resting blood-perfused muscle, ischemia increased [Ca2+]ito ∼200 nM. During reperfusion [Ca2+]idecreased to ∼140 nM. Ionomycin induced an increase in [Ca2+]ito well above 750 nM. Manganate reduced Ca2+-dependent fluorescence to virtually zero. Our main conclusion is that changes in [Ca2+]ican be monitored and quantitatively determined in vivo.

Identification of a switching model of calcium cycling in isolated rat hearts

So far, the processes involved in regulation of intracellular calcium (Ca/sub i//sup 2+/) in cardiomyocytes have been mainly studied through biochemical and isolated cell analysis. Here, we present a novel technique to model and identify cardiac Ca/sub i//sup 2+/-cycling under physiologically relevant conditions in the intact beating heart. Ca/sub i//sup 2+/ was measured using fluorescence techniques in ex vivo perfused rat hearts. For analysis, we developed a parametric mathematical model, switching between active and inactive calcium release. The kinetic parameters of the two submodes of the model were computed using a recently developed technique from hybrid system identification. Application of the method to control and isoproterenol-stimulated hearts resulted in parameter values within a physiologically reliable range.

System identification to analyse changed kinetics of SERCA in intact rat heart

Abstract A mechanistic-based model has been derived of calcium handling in the intact heart. This model incorporates the quantitatively most important processes involved in beat-to-beat calcium homeostasis. Based on a priori physiological information the model has been reduced to yield (kinetic) parameters that could be estimated using time-series data of the free calcium concentration in the sarcoplasma. Observations of the dynamics of the overall system were translated into the underlying mechanisms. Experiments in which the most important calcium extrusion pump (Sarcoplasmic Reticulum Ca 2+ - ATPase, SERCA) was disturbed have been successfully analysed and interpreted using model and identification.