Mathis K. Stokke

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.

No Rest for the Weary: Diastolic Calcium Homeostasis in the Normal and Failing Myocardium

Following contraction of the heart, efficient relaxation (diastole) is essential for refilling the ventricles with blood. This review describes how ventricular relaxation is controlled by Ca(2+) homeostasis in cardiac muscle cells and how alterations in Ca(2+) cycling affect diastolic function in the normal and failing heart. These discussions illustrate that the diastolic phase is not simply a period of rest but rather involves highly regulated and dynamic Ca(2+) fluxes.

Ca2+ wave probability is determined by the balance between SERCA2-dependent Ca2+ reuptake and threshold SR Ca2+ 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 β-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 β-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.

Hypokalaemia induces Ca2+ overload and Ca2+ waves in ventricular myocytes by reducing Na+,K+-ATPase α2 activity.

Hypokalaemia is a risk factor for development of ventricular arrhythmias. In rat ventricular myocytes, low extracellular K+ (corresponding to clinical moderate hypokalaemia) increased Ca2+ wave probability, Ca2+ transient amplitude, sarcoplasmic reticulum (SR) Ca2+ load and induced SR Ca2+ leak. Low extracellular K+ reduced Na+,K+‐ATPase (NKA) activity and hyperpolarized the resting membrane potential in ventricular myocytes. Both experimental data and modelling indicate that reduced NKA activity and subsequent Na+ accumulation sensed by the Na+, Ca2+ exchanger (NCX) lead to increased Ca2+ transient amplitude despite concomitant hyperpolarization of the resting membrane potential. Low extracellular K+ induced Ca2+ overload by lowering NKA α2 activity. Triggered ventricular arrhythmias in patients with hypokalaemia may therefore be attributed to reduced NCX forward mode activity linked to an effect on the NKA α2 isoform.

Cre-loxP DNA recombination is possible with only minimal unspecific transcriptional changes and without cardiomyopathy in Tg(αMHC-MerCreMer) mice

Cre-loxP technology for conditional gene inactivation is a powerful tool in cardiovascular research. Induction of gene inactivation can be carried out by per oral or intraperitoneal tamoxifen administration. Unintended transient cardiomyopathy following tamoxifen administration for gene inactivation has recently been reported. We aimed to develop a protocol for tamoxifen-induced gene inactivation with minimal effects on gene transcription and in vivo cardiac function, allowing studies of acute loss of the targeted gene. In mRNA microarrays, 35% of the 34,760 examined genes were significantly regulated in MCM(+/0) compared with wild type. In MCM(+/0), we found a correlation between tamoxifen dose and degree of gene regulation. Comparing one and four intraperitoneal injections of 40 mg·kg(-1)·day(-1) tamoxifen, regulated genes were reduced to 1/5 in the single injection group. Pronounced alteration in protein abundance and acute cardiomyopathy were observed after the four-injection protocols but not the one-injection protocol. For verification of gene inactivation following one injection of tamoxifen, this protocol was applied to MCM(+/0)/Serca2(fl/fl). Serca2 mRNA levels and protein abundance followed the same pattern of decline with one and four tamoxifen injections. The presence of the MCM transgene induced major alterations of gene expression while administration of tamoxifen induced additional but less gene regulation. Thus nonfloxed MCM(+/0) should be considered as controls for mice that carry both a floxed gene of interest and the MCM transgene. One single tamoxifen injection administered to MCM(+/0)/Serca2(fl/fl) was sufficient for target gene inactivation, without acute cardiomyopathy, allowing acute studies subsequent to gene inactivation.

ICaL inhibition prevents arrhythmogenic Ca2+ waves caused by abnormal Ca2+ sensitivity of RyR or SR Ca2+ accumulation

AIMS Arrhythmogenic Ca(2+) waves result from uncontrolled Ca(2+) release from the sarcoplasmic reticulum (SR) that occurs with increased Ca(2+) sensitivity of the ryanodine receptor (RyR) or excessive Ca(2+) accumulation during β-adrenergic stimulation. We hypothesized that inhibition of the L-type Ca(2+) current (I(CaL)) could prevent such Ca(2+) waves in both situations. METHODS AND RESULTS Ca(2+) waves were induced in mouse left ventricular cardiomyocytes by isoproterenol combined with caffeine to increase RyR Ca(2+) sensitivity. I(CaL) inhibition by verapamil (0.5 µM) reduced Ca(2+) wave probability in cardiomyocytes during electrostimulation, and during a 10 s rest period after ceasing stimulation. A separate type of Ca(2+) release events occurred during the decay phase of the Ca(2+) transient and was not prevented by verapamil. Verapamil decreased Ca(2+) spark frequency, but not in permeabilized cells, indicating that this was not due to direct effects on RyR. The antiarrhythmic effect of verapamil was due to reduced SR Ca(2+) content following I(CaL) inhibition. Computational modelling supported that the level of I(CaL) inhibition obtained experimentally was sufficient to reduce the SR Ca(2+) content. Ca(2+) wave prevention through reduced SR Ca(2+) content was also effective in heterozygous ankyrin B knockout mice with excessive SR Ca(2+) accumulation during β-adrenergic stimulation. CONCLUSION I(CaL) inhibition prevents diastolic Ca(2+) waves caused by increased Ca(2+) sensitivity of RyR or excessive SR Ca(2+) accumulation during β-adrenergic stimulation. In contrast, unstimulated early Ca(2+) release during the decay of the Ca(2+) transient is not prevented, and merits further study to understand the full antiarrhythmic potential of I(CaL) inhibition.

Exercise training prevents ventricular tachycardia in CPVT1 due to reduced CaMKII-dependent arrhythmogenic Ca2+ release

AIMS Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is caused by mutations in the cardiac ryanodine receptor (RyR2) that lead to disrupted Ca(2+) handling in cardiomyocytes and ventricular tachycardia. The aim of this study was to test whether exercise training could reduce the propensity for arrhythmias in mice with the CPVT1-causative missense mutation Ryr2-R2474S by restoring normal Ca(2+) handling. METHODS AND RESULTS Ryr2-R2474S mice (RyR-RS) performed a 2 week interval treadmill exercise training protocol. Each exercise session comprised five 8 min intervals at 80-90% of the running speed at maximal oxygen uptake (VO2max) and 2 min active rest periods at 60%. VO2max increased by 10 ± 2% in exercise trained RyR-RS (ET), while no changes were found in sedentary controls (SED). RyR-RS ET showed fewer episodes of ventricular tachycardia compared with RyR-RS SED, coinciding with fewer Ca(2+) sparks and waves, less diastolic Ca(2+) leak from the sarcoplasmic reticulum, and lower phosphorylation levels at RyR2 sites associated with Ca(2) (+)-calmodulin-dependent kinase type II (CaMKII) compared with RyR-RS SED. The CaMKII inhibitor autocamtide-2-related inhibitory peptide and also the antioxidant N-acetyl-l-cysteine reduced Ca(2+) wave frequency in RyR-RS equally to exercise training. Protein analysis as well as functional data indicated a mechanism depending on reduced levels of oxidized CaMKII after exercise training. Two weeks of detraining reversed the beneficial effects of the interval treadmill exercise training protocol in RyR-RS ET. CONCLUSION Long-term effects of interval treadmill exercise training reduce ventricular tachycardia episodes in mice with a CPVT1-causative Ryr2 mutation through lower CaMKII-dependent phosphorylation of RyR2.

Beta-Adrenoceptor Stimulation Reveals Ca2+ Waves and Sarcoplasmic Reticulum Ca2+ Depletion in Left Ventricular Cardiomyocytes from Post-Infarction Rats with and without Heart Failure

Abnormal cellular Ca2+ handling contributes to both contractile dysfunction and arrhythmias in heart failure. Reduced Ca2+ transient amplitude due to decreased sarcoplasmic reticulum Ca2+ content is a common finding in heart failure models. However, heart failure models also show increased propensity for diastolic Ca2+ release events which occur when sarcoplasmic reticulum Ca2+ content exceeds a certain threshold level. Such Ca2+ release events can initiate arrhythmias. In this study we aimed to investigate if both of these aspects of altered Ca2+ homeostasis could be found in left ventricular cardiomyocytes from rats with different states of cardiac function six weeks after myocardial infarction when compared to sham-operated controls. Video edge-detection, whole-cell Ca2+ imaging and confocal line-scan imaging were used to investigate cardiomyocyte contractile properties, Ca2+ transients and Ca2+ waves. In baseline conditions, i.e. without beta-adrenoceptor stimulation, cardiomyocytes from rats with large myocardial infarction, but without heart failure, did not differ from sham-operated animals in any of these aspects of cellular function. However, when exposed to beta-adrenoceptor stimulation, cardiomyocytes from both non-failing and failing rat hearts showed decreased sarcoplasmic reticulum Ca2+ content, decreased Ca2+ transient amplitude, and increased frequency of Ca2+ waves. These results are in line with a decreased threshold for diastolic Ca2+ release established by other studies. In the present study, factors that might contribute to a lower threshold for diastolic Ca2+ release were increased THR286 phosphorylation of Ca2+/calmodulin-dependent protein kinase II and increased protein phosphatase 1 abundance. In conclusion, this study demonstrates both decreased sarcoplasmic reticulum Ca2+ content and increased propensity for diastolic Ca2+ release events in ventricular cardiomyocytes from rats with heart failure after myocardial infarction, and that these phenomena are also found in rats with large myocardial infarctions without heart failure development. Importantly, beta-adrenoceptor stimulation is necessary to reveal these perturbations in Ca2+ handling after a myocardial infarction.

Ca2+-clock-dependent pacemaking in the sinus node is impaired in mice with a cardiac specific reduction in SERCA2 abundance

Background: The sarcoplasmic reticulum Ca2+-ATPase (SERCA2) pump is an important component of the Ca2+-clock pacemaker mechanism that provides robustness and flexibility to sinus node pacemaking. We have developed transgenic mice with reduced cardiac SERCA2 abundance (Serca2 KO) as a model for investigating SERCA2s role in sinus node pacemaking. Methods and Results: In Serca2 KO mice, ventricular SERCA2a protein content measured by Western blotting was 75% (P < 0.05) lower than that in control mice (Serca2 FF) tissue. Immunofluorescent labeling of SERCA2a in ventricular, atrial, sinus node periphery and center tissue sections revealed 46, 45, 55, and 34% (all P < 0.05 vs. Serca2 FF) lower labeling, respectively and a mosaic pattern of expression. With telemetric ECG surveillance, we observed no difference in basal heart rate, but the PR-interval was prolonged in Serca2 KO mice: 49 ± 1 vs. 40 ± 1 ms (P < 0.001) in Serca2 FF. During exercise, heart rate in Serca2 KO mice was elevated to 667 ± 22 bpm, considerably less than 780 ± 17 bpm (P < 0.01) in Serca2 FF. In isolated sinus node preparations, 2 mM Cs+ caused bradycardia that was equally pronounced in Serca2 KO and Serca2 FF (32 ± 4% vs. 29 ± 5%), indicating no change in the pacemaker current, If. Disabling the Ca2+-clock with 2 μM ryanodine induced bradycardia that was less pronounced in Serca2 KO preparations (9 ± 1% vs. 20 ± 3% in Serca2 FF; P < 0.05), suggesting a disrupted Ca2+-clock. Mathematical modeling was used to dissect the effects of membrane- and Ca2+-clock components on Serca2 KO mouse heart rate and sinus node action potential. Computer modeling predicted a slowing of heart rate with SERCA2 downregulation and the heart rate slowing was pronounced at >70% reduction in SERCA2 activity. Conclusions: Serca2 KO mice show a disrupted Ca2+-clock-dependent pacemaker mechanism contributing to impaired sinus node and atrioventricular node function.

Lower than expected burden of premature ventricular contractions impairs myocardial function

We aimed to explore the burden of frequent premature ventricular contractions (PVCs) associated with myocardial dysfunction in patients with outflow tract arrhythmia (OTA). We hypothesized that this threshold is lower than the previously suggested threshold of 24 000 PVCs/24 h (24%PVC) when systolic function is assessed by strain echocardiography. Furthermore, we aimed to characterize OTA patients with malignant arrhythmic events.