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Background: Heart failure with preserved ejection fraction (HFPEF) is increasingly common but the established heart failure (HF) drugs are not effective. The underlying cellular mechanisms are incompletely understood. Therefore we investigated cardiomyocyte function and intracellular Ca2+ homeostasis in a model of HFPEF. Methods: Young male Wistar rats were subjected to subtotal nephrectomy (NXT) or sham operation (SOP). Serial blood/urine samples, echocardiography and pressure-volume loops at 8 and 24 weeks were performed. After sacrifice, left ventricular (LV) hypertrophy and NCX function (Caffeine induced Ca2+ transient, TAU) and protein expression (Western blot) were determined. Cardiomyocyte function (Ca2+ transients, sarcoplasmic reticulum (SR) diastolic Ca2+ leak (Ca2+ sparks) and SR Ca2+ content; Fluo4-AM) were quantified in isolated LV cardiomyocytes without and with the NCX inhibitor SEA0400 (300nM). Results: NXT rats showed stable compensated renal impairment and significantly hypertrophied LV at 8 weeks with a further increase after 24 weeks. LV systolic function (EF, dP/dt) was preserved. End diastolic pressure (EDP) volume relationship was markedly shifted left- and upwards and lung weight were significantly increased, indicating HFPEF with pulmonary congestion. LV cardiomyocytes from NXT showed no significant differences in amplitudes of Ca2+ transients. However, time for early (50%) relaxation of the Ca2+ transients at 8 weeks were significantly prolonged with a further increase after 24 weeks (RT50 17.2±2.9 and 30.8±2.7 vs. 27.6±1.8 and 41.8±2.6 ms; n≥20; p< 0.05). TAU was significantly prolonged at 8 and 24 weeks indicating reduced NCX forward mode activity, while NCX protein expression was upregulated. At 8 weeks, Ca2+ spark frequency tended to be increased (p=0.07) while SR Ca2+ content was unchanged. SEA0400 accelerated Ca2+ transient decay but did not affect Ca2+ spark frequency. At 24 weeks, Ca2+ spark frequency was increased (4.3±0.7 vs. 11.5±1.8 sparks/s/μm3; n≥20; p

We present a one-dimensional mathematical model to explain changes in regional radial myocardial deformation during ischaemia. The model makes use of cellular contraction properties of normal and ischaemic myocytes extracted from an animal model of induced chronic, regional ischaemia of the posterior wall. Simulations of the model are compared to the deformation patterns observed in the experimental animal models of regional ischaemia.

Background: Renal impairment and heart failure with preserved ejection fraction (HFPEF) are often associated but the underlying mechanisms are largely unresolved. Therefore we investigated cardiomyocyte function in a rat model with compensated renal failure and diastolic dysfunction. Methods: Fifty-six young male Wistar rats were subjected to subtotal nephrectomy (NXT) or sham operation (SOP) and observed up to 24 weeks. Urine and blood samples for calculating the glomerular filtration rate (GFR) and echocardiography were performed during disease progression. Invasive hemodynamics (P/V-loops), morphometry, protein expression analysis and Ca2+ transients (confocal and ratiometric) as well as cell shortening (CS) were studied in isolated left ventricle (LV) cardiomyocytes in final experiments at 8 and 24 weeks. Results: Following NXT rats develop compensated renal failure (GFR 22±2 and 16±2 vs. 32±2 and 28±3 ml/min/m2 at 8 and 24 weeks, resp. p

TRPC3 has been demonstrated as a player in structural remodeling of the heart, while its proarrhythmogenic potential is incompletely understood. using a TRPC3 transgenic overexpression mouse model, we examined the impact of TRPC3 expression on cardiac actions of Angiotensin II (AngII) in whole hearts (Langendorff perfusion) and in isolated single ventricular myocytes. At low TRPC3 expression (WT mice) AngII elicited a previously described 3-phasic inotropic response without generating significant arrhythmias. By contrast, at high TRPC3 expression (TRPC3 transgenic mice; TRPC3-TG), AngII induced substantial impairment of cardiac contractile function associated with prominent bigeminal beats and ventricular tachycardia. Calcium imaging experiments revealed a dramatic increase in diastolic Ca2+ levels associated with spontaneous sarcomere shortenings during AngII exposure of TRPC3-TG myocytes. More detailed electrophysiological characterization demonstrated AngII-induced depolarization, distortion of action potential morphology and enhanced frequency of delayed afterdepolarizations (DADs) in TRPC3-TG. This phenotype was not observed in WT mice. Analysis of NCX-tail currents suggested that AngII-induced arrhythmias were based on profound NCX-1 forward mode-associated depolarization. On the contrary, our analysis of Ca2+ handling in TRPC3-TG myocytes at basal conditions clearly indicated suppressed forward-mode NCX-1 action as expected from enhanced Na+ entry at TRPC3- NCX-1 signalplexes. Immunocytochemistry demonstrated a partial disruption of the tight colocalization of TRPC3 and NCX-1 confined to the T-tubuli system during AngII stimulation of TRPC3-TG myocytes. Our results confirm the concept of a close physical and functional coupling between TRPC3 and NCX-1, which results in a digitalis-like modulation of cardiac function by TRPC3 overexression in non-stimulated myocardium. AngII is suggested to disrupt the organization of TRPC3- NCX-1 signalplexes, thereby enabling NCX-1-mediated distortion of AP morphology, DADs and arrhytmogenesis.

In cardiomyocytes, cytosolic Ca removal is mediated by sarcoplasmic reticulum Ca ATPase (SERCA), sarcolemmal Na/Ca exchanger (NCX), plasmalemmal Ca ATPase and mitochondrial Ca uniporter (MCU). We quantified spatiotemporal inhomogeneities in cytosolic Ca removal in normal and diseased hearts and explored underlying mechanisms. Ca transients (1 Hz, Fluo-4 AM) were recorded using confocal microscopy in ventricular cardiomyocytes. Time constant of cytosolic [Ca] decay (tau_loc) was quantified at 1 μm intervals across the cell. In murine cardiomyocytes tau_loc distribution was inhomogeneous, with a maximal difference between cell regions of 237±9 ms and a variation coefficient (CV_tau) of 14±2% (mean±S.E.M., n=10 cells). tau_loc was not releated to the local amplitude or release kinetics of Ca. Coherent regions of fast (fastCaR, tau_loc < mean tau of whole cell) and slow (slowCaR) Ca removal had similar widths (4.6±0.2 vs. 5.1±0.5 im). Forskolin accelerated, and SERCA inhibitor cyclopiazonic acid (CPA) inhibited Ca removal significantly more in slowCaR than in fastCaR. In contrast, NCX-inhibitor SEA0400 slowed cytosolic Ca removal similarly in slowCaR and fastCaR. Also, CV_tau was unchanged in NCX+/- knock-out mice, suggesting no contribution of NCX to dyssynchrony in cytosolic Ca removal. tau_loc distribution was also inhomogenous in pig cardiomyocytes. Ca removal was more dyssynchronous in cardiomyocytes from chronic (4 weeks) ischemic pig myocardium (peri-infarct zone) vs. sham-operated pigs (CV_tau 24±1%vs. 20±1%, n=57 cells/group, p

Rationale: In ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI). Objective: To characterize RyR functional properties in relation to TT proximity, at baseline and after MI. Methods: Myocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca2+ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca2+ release, F>F50 within 20 ms) or their absence (delayed areas). Spontaneous Ca2+ release events during diastole, Ca2+ sparks, reflecting RyR activity and properties, were subsequently assigned to either category. Results: In CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na+/Ca2+ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca2+ influx via Ca2+ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca2+ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca2+ removal by NCX at the membrane was significantly lower in MI. Conclusion: TT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca2+ loss and raise SR Ca2+ content, but may promote Ca2+ waves.

ObjectivesThis study sought to explore whether subclinical alterations of sarcoplasmic reticulum (SR) Ca2+ release through cardiac ryanodine receptors (RyR2) aggravate cardiac remodeling in mice carrying a human RyR2R4496C+/– gain-of-function mutation in response to pressure overload.BackgroundRyR2 dysfunction causes increased diastolic SR Ca2+ release associated with arrhythmias and contractile dysfunction in inherited and acquired cardiac diseases, such as catecholaminergic polymorphic ventricular tachycardia and heart failure (HF).MethodsFunctional and structural properties of wild-type and catecholaminergic polymorphic ventricular tachycardia–associated RyR2R4496C+/– hearts were characterized under conditions of pressure overload induced by transverse aortic constriction (TAC).ResultsWild-type and RyR2R4496C+/– hearts had comparable structural and functional properties at baseline. After TAC, RyR2R4496C+/– hearts responded with eccentric hypertrophy, substantial fibrosis, ventricular dilation, and reduced fractional shortening, ultimately resulting in overt HF. RyR2R4496C+/–-TAC cardiomyocytes showed increased incidence of spontaneous SR Ca2+ release events, reduced Ca2+ transient peak amplitude, and SR Ca2+ content as well as reduced SR Ca2+-ATPase 2a and increased Na+/Ca2+-exchanger protein expression. HF phenotype in RyR2R4496C+/–-TAC mice was associated with increased mortality due to pump failure but not tachyarrhythmic events. RyR2-stabilizer K201 markedly reduced Ca2+ spark frequency in RyR2R4496C+/–-TAC cardiomyocytes. Mini-osmotic pump infusion of K201 prevented deleterious remodeling and improved survival in RyR2R4496C+/–-TAC mice.ConclusionsThe combination of subclinical congenital alteration of SR Ca2+ release and pressure overload promoted eccentric remodeling and HF death in RyR2R4496C+/– mice, and pharmacological RyR2 stabilization prevented this deleterious interaction. These findings suggest potential clinical relevance for patients with acquired or inherited gain-of-function of RyR2-mediated SR Ca2+ release.

Diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) via the ryanodine receptor (RyR2) contributes to arrhythmias. Protein kinase A (PKA) and Ca2+/Calmodulin- dependent protein kinase II (CaMKII) have been involved in SR Ca2+ leak by altering RyR2 gating. Under these conditions, JTV519 (K201), a benzothiazepine derivate, has been shown to stabilize the modified RyR2 and reduce diastolic SR Ca2+ leak. We tested, whether JTV519 reduces SR Ca2+ leak induced by Na+- and Ca2+ overload (induced by 100 µM ouabain), i.e. independent of CaMKII signaling. Methods: [Ca2+]i was measured (Fluo4-AM, confocal) in paced murine cardiomyocytes ±JTV519 (1 µM, >1h preincubation). [Ca2+]-transients, diastolic SR Ca2+ leak (Ca2+ spark (SparkF, in s−1∗pL−1) and Ca2+ wave frequency) and SR [Ca2+] (caffein) were measured ±ouabain (OUAB) and KN93 (CaMKII-inhibitor, 1 µM). Phosphorylation of RyR2 (pSer2814, CaMKII site) was quantified by Western blot.Results: With OUAB, total cellular [Ca2+] increased from 3.3±0.3 to 4.3±0.3 (F/F0, mean±SE), SparkF increased from 31±20 to 85±30 (both pl0.05). Ouabain did not increase pSer2814, and KN93 had no effect on elevated SparkF with ouabain (89±24), indicating no contribution of CaMKII to increased SR Ca2+ leak. JTV519 decreased SparkF (25±4), and Ca2+ waves, but also attenuated the increase in SR [Ca2+] in OUAB (F/F0: 5.2±0.3 with JTV+Ouab vs. 7.7±0.6 OUAB, pl0.05). However, matching cells for equal SR [Ca2+] revealed an SR [Ca2+] - independent effect of JTV519 on SparkF. In contrast, propagation speed of Ca2+ waves and the ratio between [Ca2+] transient amplitude and SR [Ca2+] were unchanged.Conclusion: In conditions not related to CaMKII-mediated alterations of RyR2 gating, JTV519 reduces spontaneous SR Ca2+ release, but does not influence propagated Ca2+ release or the fraction of SR Ca2+ released with each beat.

The effect of synthetic parathyroid hormone (PTH)-related peptide [PTHrP(1–34)] on regional myocardial function was studied in 11 anesthetized pigs. Intracoronary infusion of PTHrP (cumulative dose: 14 ± 1 μg) decreased coronary resistance to 33 ± 2% of baseline ( P < 0.05) and regional myocardial function to 90 ± 3% of baseline (not significant). Ischemia-reperfusion alters the activity of several kinases and therefore possibly the myocardial effects of PTHrP. In stunned myocardium, induced by 20-min ischemia and 30-min reperfusion, the dose of PTHrP reducing coronary resistance to a minimum of 29 ± 2% was decreased to 8 ± 2 μg ( P < 0.05). Regional myocardial function was no longer decreased but increased to 132 ± 9% ( P < 0.05). The increase in regional myocardial function during PTHrP was inversely related to baseline function at 30-min reperfusion in vivo ( r = 0.9) as well as in myocytes isolated from stunned pig hearts ( r = 0.7). In isolated rat hearts subjected to 30-min global ischemia followed by 30-min reperfusion, blockade of endogenous PTHrP byd-Trp12-Tyr34-PTH(7–34) attenuated the recovery of left ventricular developed pressure by 30 ± 14% ( P < 0.05). Thus endogenous and exogenous PTHrP impact on the function of stunned myocardium.

Elevated spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) Ca2+ release channel (ryanodine receptor-RyR2) due to a gain-of-function of the RyR2 defect contributes to contractile dysfunction and arrhythmias in heart failure (HF). However, whether increased diastolic SR Ca2+ release can promote HF is yet unclear.RyR2R4496C+/- mice associated with an increased SR Ca2+ leak and wild-type (WT) littermates underwent surgery with (TAC) or without (Sham) transverse aortic constriction. Transthoracic echocardiography was performed 1 and 3 weeks post-surgery. Hearts and lungs were dissected, weighed and normalized to tibia length. Gross morphology and collagen content were examined in paraffin-embedded heart slices stained with hematoxylin/eosin and picrosirius red, respectively. Arrhythmias were monitored (24h) with radiotelemetry. Data are presented as mean±S.E.M.RyR2R4496C+/--Sham hearts exhibited no gross baseline changes in ventricular structure and function and fibrosis compared to WT-Sham. 1 week after TAC, WT-TAC mice showed concentric left ventricular (LV) hypertrophy with preserved ejection fraction. In contrast, RyR2R4496C+/--TAC mice exhibited eccentric hypertrophy and significant deterioration of phenotypic changes associated with the transition to HF, such as chamber dilation (LV end-diastolic diameter;WT:-1.4±3%,RyR2R4496C+/-:19.7±3.6%,P

Chronic beta-adrenergic stimulation leads to heart failure (HF). In mice overexpressing beta1-adrenoceptors (TG), increased diastolic Ca load in cardiomyocytes at early age is pivotal for the development of HF. The mechanisms underlying intracellular Ca dysregulation are unclear. We examined cytosolic Ca transients (Fluo4-AM, field stimulation), Na-Ca-exchanger (NCX) function and protein expression, cytosolic Na (SBFI) and T-tubular structures (Di8-ANEPPS) in cardiomyocytes from young (8-16 wks) TG mice and wildtype (WT) littermates.Results: Systolic [Ca] amplitude was unchanged, time to peak [Ca] (140±5 vs. 127±3 ms) and [Ca] decay (time constant, tau, 223±16 vs. 182±9 ms) were significantly prolonged in TG vs. WT. Diastolic Ca leak from the SR (quantified as tetracaine-sensitive change in diastolic [Ca] or diastolic Ca spark frequency) was unchanged. However, cytosolic Ca removal by NCX during coffein application was significantly slower (tau, 3683±337 in TG vs. 2304±272 ms in WT), indicating reduced forward mode NCX activity. NCX protein expression was unchanged. Preliminary results indicate increased cytosolic [Na] in young TG. Furthermore, confocal line scans revealed delayed (> 15 ms until half-maximal) systolic Ca release in 24.7±2.6 (TG) vs. 4.6±1.4% (WT) of the intracellular regions (n=32 and 31 cells, resp., p

The Wnt/frizzled signalling pathways play a key role in cardiogenesis and in adult hearts during cardiac remodelling. We investigated whether the expression of Wnt-11 and its anticipated downstream signalling components is altered in human failing hearts and whether Wnt-11 acutely affects Ca2+ transients in adult murine cardiomyocytes.Left ventricles (LV) homogenates of human non-failing hearts (NF, n=4) and end-stage failing hearts with ischemic (ICM, n=5) and dilated (DCM, n=5) cardiomyopathy were used for western blotting with antibodies against Wnt-11, Fzd-4 and Fzd-7 (Wnt receptors) and Dvl-1 (downstream cytoplasmic regulator). Expression of proteins was normalized to GAPDH and is given as a fraction of average protein expression in NF. Ca2+ transients were measured during confocal line scan imaging with Fluo-4 in isolated murine adult cardiomyocytes after 5 (ncells=20) and 25 minutes (ncells=18) incubation with recombinant Wnt-11 (2 µg/ml). Untreated cells served as controls (ncells=27).Wnt-11 expression in LV in ICM (0.85 ± 0.05) and DCM (0.91 ± 0.34) was not significantly changed when compared to NF. However, the expression of Wnt-11 receptors was highly increased: Fzd-4 in ICM (6.25 ± 1.23; p