Perturbed atrial calcium handling in an ovine model of heart failure: potential roles for reductions in the L-type calcium current.

Heart failure (HF) is commonly associated with reduced cardiac output and an increased risk of atrial arrhythmias particularly during ß-adrenergic stimulation. The aim of the present study was to determine how HF alters systolic Ca(2+) and the response to ß-adrenergic (ß-AR) stimulation in atrial myocytes. HF was induced in sheep by ventricular tachypacing and changes in intracellular Ca(2+) concentration studied in single left atrial myocytes under voltage and current clamp conditions. The following were all reduced in HF atrial myocytes; Ca(2+) transient amplitude (by 46% in current clamped and 28% in voltage clamped cells), SR dependent rate of Ca(2+) removal (kSR, by 32%), L-type Ca(2+) current density (by 36%) and action potential duration (APD90 by 22%). However, in HF SR Ca(2+) content was increased (by 19%) when measured under voltage-clamp stimulation. Inhibiting the L-type Ca(2+) current (ICa-L) in control cells reproduced both the decrease in Ca(2+) transient amplitude and increase of SR Ca(2+) content observed in voltage-clamped HF cells. During ß-AR stimulation Ca(2+) transient amplitude was the same in control and HF cells. However, ICa-L remained less in HF than control cells whilst SR Ca(2+) content was highest in HF cells during ß-AR stimulation. The decrease in ICa-L that occurs in HF atrial myocytes appears to underpin the decreased Ca(2+) transient amplitude and increased SR Ca(2+) content observed in voltage-clamped cells.

Age-related divergent remodeling of the cardiac extracellular matrix in heart failure: collagen accumulation in the young and loss in the aged.

The incidence of heart failure (HF) increases with age. This study sought to determine whether aging exacerbates structural and functional remodeling of the myocardium in HF. HF was induced in young (~18 months) and aged sheep (>8 years) by right ventricular tachypacing. In non-paced animals, aging was associated with increased left ventricular (LV) end diastolic internal dimensions (EDID, P<0.001), reduced fractional shortening (P<0.01) and an increase in myocardial collagen content (P<0.01). HF increased EDID and reduced fractional shortening in both young and aged animals, although these changes were more pronounced in the aged (P<0.05). Age-associated differences in cardiac extracellular matrix (ECM) remodeling occurred in HF with collagen accumulation in young HF (P<0.001) and depletion in aged HF (P<0.05). MMP-2 activity increased in the aged control and young HF groups (P<0.05). Reduced tissue inhibitor of metalloproteinase (TIMP) expression (TIMPs 3 and 4, P<0.05) was present only in the aged HF group. Secreted protein acidic and rich in cysteine (SPARC) was increased in aged hearts compared to young controls (P<0.05) while serum procollagen type I C-pro peptide (PICP) was increased in both young failing (P<0.05) and aged failing (P<0.01) animals. In conclusion, collagen content of the cardiac ECM changes in both aging and HF although; whether collagen accumulation or depletion occurs depends on age. Changes in TIMP expression in aged failing hearts alongside augmented collagen synthesis in HF provide a potential mechanism for the age-dependent ECM remodeling. Aging should therefore be considered an important factor when elucidating cardiac disease mechanisms.

In the RyR2(R4496C) mouse model of CPVT, ß-adrenergic stimulation induces Ca waves by increasing SR Ca content and not by decreasing the threshold for Ca waves.

RATIONALE: mutations of the ryanodine receptor (RyR) cause catecholaminergic polymorphic ventricular tachycardia (CPVT). These mutations predispose to the generation of Ca waves and delayed afterdepolarizations during adrenergic stimulation. Ca waves occur when either sarcoplasmic reticulum (SR) Ca content is elevated above a threshold or the threshold is decreased. Which of these occurs in cardiac myocytes expressing CPVT mutations is unknown. OBJECTIVE: we tested whether the threshold SR Ca content is different between control and CPVT and how it relates to SR Ca content during ß-adrenergic stimulation. METHODS AND RESULTS: ventricular myocytes from the RyR2 R4496C(+/-) mouse model of CPVT and wild-type (WT) controls were voltage-clamped; diastolic SR Ca content was measured and compared with the Ca wave threshold. The results showed the following. (1) In 1 mmol/L [Ca(2+)](o), ß-adrenergic stimulation with isoproterenol (1µmol/L) caused Ca waves only in R4496C. (2) SR Ca content and Ca wave threshold in R4496C were lower than those in WT. (3) ß-Adrenergic stimulation increased SR Ca content by a similar amount in both R4496C and WT. (4) ß-Adrenergic stimulation increased the threshold for Ca waves. (5) During ß-adrenergic stimulation in R4496C, but not WT, the increase of SR Ca was sufficient to reach threshold and produce Ca waves. CONCLUSIONS: in the R4496C CPVT model, the RyR is leaky, and this lowers both SR Ca content and the threshold for waves. ß-Adrenergic stimulation produces Ca waves by increasing SR Ca content and not by lowering threshold.

Impaired ß-adrenergic responsiveness accentuates dysfunctional excitation-contraction coupling in an ovine model of tachypacing-induced heart failure.

Reduced inotropic responsiveness is characteristic of heart failure (HF). This study determined the cellular Ca2+ homeostatic and molecular mechanisms causing the blunted ß-adrenergic (ß-AR) response in HF.We induced HF by tachypacing in sheep; intracellular Ca2+ concentration was measured in voltage-clamped ventricular myocytes. In HF, Ca2+ transient amplitude and peak L-type Ca2+ current (ICa-L) were reduced (to 70 ± 11% and 50 ± 3.7% of control, respectively, P <0.05) whereas sarcoplasmic reticulum (SR) Ca2+ content was unchanged. ß-AR stimulation with isoprenaline (ISO) increased Ca2+ transient amplitude, ICa-L and SRCa2+ content in both cell types; however, the response of HF cells was markedly diminished (P <0.05).Western blotting revealed an increase in protein phosphatase levels (PP1, 158 ± 17% and PP2A, 188 ± 34% of control, P <0.05) and reduced phosphorylation of phospholamban in HF (Ser16, 30 ± 10% and Thr17, 41 ± 15% of control, P <0.05). The ß-AR receptor kinase GRK-2 was also increased in HF (173 ± 38% of control, P <0.05). In HF, activation of adenylyl cyclase with forskolin rescued the Ca2+ transient, SR Ca2+ content and SR Ca2+ uptake rate to the same levels as control cells in ISO. In conclusion, the reduced responsiveness of the myocardium to ß-AR agonists in HF probably arises as a consequence of impaired phosphorylation of key intracellular proteins responsible for regulating the SR Ca2+ content and therefore failure of the systolic Ca2+ transient to increase appropriately during ß-AR stimulation.

Temporal Development of Autonomic Dysfunction in Heart Failure: Effects of Age in an Ovine Rapid-pacing Model.

Heart failure (HF) is predominantly a disease of older adults and characterized by extensive sympatho-vagal imbalance leading to impaired reflex control of heart rate (HR). However, whether aging influences the development or extent of the autonomic imbalance in HF remains unclear. To address this, we used an ovine model of aging with tachypacing-induced HF to determine whether aging affects the chronotropic and inotropic responses to autonomic stimulation and reduction in heart rate variability (HRV) in HF. We find that aging is associated with increased cardiac dimensions and reduced contractility before the onset of tachypacing, and these differences persist in HF. Additionally, the chronotropic response to ß-adrenergic stimulation was markedly attenuated in HF, and this occurred more rapidly in aged animals. By measuring HR during sequential autonomic blockade, our data are consistent with a reduced parasympathetic control of resting HR in aging, with young HF animals having an attenuated sympathetic influence on HR. Time-domain analyses of HR show a reduction in HRV in both young and aged failing animals, although HRV is lowest in aged HF. In conclusion, aging is associated with altered autonomic control and ß-adrenergic responsiveness of HR, and these are exacerbated with the development of HF.

Balanced changes in Ca buffering by SERCA and troponin contribute to Ca handling during ß-adrenergic stimulation in cardiac myocytes.

AIMS: During activation of cardiac myocytes, less than 1% of cytosolic Ca is free; the rest is bound to buffers, largely SERCA, and troponin C. Signalling by phosphorylation, as occurs during ß-adrenergic stimulation, changes the Ca-binding affinity of these proteins and may affect the systolic Ca transient. Our aim was to determine the effects of ß-adrenergic stimulation on Ca buffering and to differentiate between the roles of SERCA and troponin. METHODS AND RESULTS: Ca buffering was studied in cardiac myocytes from mice: wild-type (WT), phospholamban-knockout (PLN-KO), and mice expressing slow skeletal troponin I (ssTnI) that is not protein kinase A phosphorylatable. WT cells showed no change in Ca buffering in response to the ß-adrenoceptor agonist isoproterenol (ISO). However, ISO decreased Ca buffering in PLN-KO myocytes, presumably unmasking the role of troponin. This effect was confirmed in WT cells in which SERCA activity was blocked with the application of thapsigargin. In contrast, ISO increased Ca buffering in ssTnI cells, presumably revealing the effect of an increase in Ca binding to SERCA. CONCLUSIONS: These data indicate the individual roles played by SERCA and troponin in Ca buffering during ß-adrenergic stimulation and that these two buffers effectively counterbalance each other so that Ca buffering remains constant during ß-adrenergic stimulation, a factor which may be physiologically important. This study also emphasizes the importance of taking into account Ca buffering, particularly in disease states where Ca binding to myofilaments or SERCA may be altered.

Ca(2+) wave probability is determined by the balance between SERCA2-dependent Ca(2+) reuptake and threshold SR Ca(2+) content.

AIMS: In this manuscript, we determined the roles of the sarcoendoplasmic reticulum Ca(2+) ATPase 2 (SERCA2) and the ryanodine receptor (RyR) in Ca(2+) wave development during ß-adrenergic stimulation. METHODS AND RESULTS: SERCA2 knockout mice (KO) were used 6 days after cardio-specific gene deletion, with left ventricular SERCA2a abundance reduced by 54 ± 9% compared with SERCA2(flox/flox) controls (FF) (P < 0.05). Ca(2+) waves occurred in fewer KO than FF myocytes (40 vs. 68%, P < 0.05), whereas the addition of isoproterenol (ISO) induced waves in an equal percentage of myocytes (82 vs. 64%). SERCA2-dependent Ca(2+) reuptake was slower in KO (-ISO, KO vs. FF: 15.4 ± 1.2 vs. 21.1 ± 1.4 s(-1), P < 0.05), but equal during ISO (+ISO, KO vs. FF: 21.9 ± 3.3 vs. 27.7 ± 2.7 s(-1)). Threshold SR Ca(2+) content for wave development was lower in KO (-ISO, KO vs. FF: 126.6 ± 10.3 vs. 159.3 ± 7.1 µmol/L, P < 0.05) and was increased by ISO only in FF (+ISO, KO vs. FF: 131.7 ± 8.7 vs. 205.5 ± 20.4 µmol/L, P < 0.05). During ISO, Ca(2+)/calmodulin-dependent kinase II (CaMKII)-dependent phosphorylation of RyR in KO was 217 ± 21% of FF (P < 0.05), and SR Ca(2+) leak indicated higher RyR open probability in KO. CaMKII inhibition decreased Ca(2+) spark frequency in KO by 44% (P < 0.05) but not in FF. Mathematical modelling predicted that increased Ca(2+) sensitivity of RyR in KO could account for increased Ca(2+) wave probability during ISO. CONCLUSIONS: In ventricular cardiomyocytes with reduced SERCA2 abundance, Ca(2+) wave development following ß-adrenergic stimulation is potentiated. We suggest that this is caused by a CaMKII-dependent shift in the balance between SERCA2-dependent Ca(2+) reuptake and threshold SR Ca(2+) content.

Reduced SERCA2 abundance decreases the propensity for Ca2+ wave development in ventricular myocytes.

AIMS: To describe the overall role of reduced sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) for Ca(2+) wave development. METHODS AND RESULTS: SERCA2 knockout [Serca2(flox/flox) Tg(alphaMHC-MerCreMer); KO] mice allowing inducible cardiomyocyte-specific disruption of the Serca2 gene in adult mice were compared with Serca(flox/flox) (FF) control mice. Six days after Serca2 gene disruption, SERCA2 protein abundance was reduced by 53% in KO compared with FF, whereas SERCA2 activity in field-stimulated, Fluo-5F AM-loaded cells was reduced by 42%. Baseline Ca(2+) content of the sarcoplasmic reticulum (SR) and Ca(2+) transient amplitude and rate constant of decay measured in whole-cell voltage-clamped cells were decreased in KO to 75, 81, and 69% of FF values. Ca(2+) waves developed in only 31% of KO cardiomyocytes compared with 57% of FF when external Ca(2+) was raised (10 mM), although SR Ca(2+) content needed for waves to develop was 79% of FF values. In addition, waves propagated at a 15% lower velocity in KO cells. Ventricular extrasystoles (VES) occurred with lower frequency in SERCA2 KO mice (KO: 3 +/- 1 VES/h vs. FF: 8 +/- 1 VES/h) (P < 0.05 for all results). CONCLUSION: Reduced SERCA2 abundance resulted in decreased amplitude and decay rate of Ca(2+) transients, reduced SR Ca(2+) content, and decreased propensity for Ca(2+) wave development.

PAX4 enhances beta-cell differentiation of human embryonic stem cells.

BACKGROUND: Human embryonic stem cells (HESC) readily differentiate into an apparently haphazard array of cell types, corresponding to all three germ layers, when their culture conditions are altered, for example by growth in suspension as aggregates known as embryoid bodies (EBs). However, this diversity of differentiation means that the efficiency of producing any one particular cell type is inevitably low. Although pancreatic differentiation has been reported from HESC, practicable applications for the use of beta-cells derived from HESC to treat diabetes will only be possible once techniques are developed to promote efficient differentiation along the pancreatic lineages. METHODS AND FINDINGS: Here, we have tested whether the transcription factor, Pax4 can be used to drive the differentiation of HESC to a beta-cell fate in vitro. We constitutively over-expressed Pax4 in HESCs by stable transfection, and used Q-PCR analysis, immunocytochemistry, ELISA, Ca(2+) microfluorimetry and cell imaging to assess the role of Pax4 in the differentiation and intracellular Ca(2+) homeostasis of beta-cells developing in embryoid bodies produced from such HESC. Cells expressing key beta-cell markers were isolated by fluorescence-activated cell sorting after staining for high zinc content using the vital dye, Newport Green. CONCLUSION: Constitutive expression of Pax4 in HESC substantially enhances their propensity to form putative beta-cells. Our findings provide a novel foundation to study the mechanism of pancreatic beta-cells differentiation during early human development and to help evaluate strategies for the generation of purified beta-cells for future clinical applications.