Disruption of Phosphodiesterase 3A Binding to SERCA2 Increases SERCA2 Activity and Reduces Mortality in Mice With Chronic Heart Failure.

BACKGROUND: Increasing SERCA2 (sarco[endo]-plasmic reticulum Ca2+ ATPase 2) activity is suggested to be beneficial in chronic heart failure, but no selective SERCA2-activating drugs are available. PDE3A (phosphodiesterase 3A) is proposed to be present in the SERCA2 interactome and limit SERCA2 activity. Disruption of PDE3A from SERCA2 might thus be a strategy to develop SERCA2 activators. METHODS: Confocal microscopy, 2-color direct stochastic optical reconstruction microscopy, proximity ligation assays, immunoprecipitations, peptide arrays, and surface plasmon resonance were used to investigate colocalization between SERCA2 and PDE3A in cardiomyocytes, map the SERCA2/PDE3A interaction sites, and optimize disruptor peptides that release PDE3A from SERCA2. Functional experiments assessing the effect of PDE3A-binding to SERCA2 were performed in cardiomyocytes and HEK293 vesicles. The effect of SERCA2/PDE3A disruption by the disruptor peptide OptF (optimized peptide F) on cardiac mortality and function was evaluated during 20 weeks in 2 consecutive randomized, blinded, and controlled preclinical trials in a total of 148 mice injected with recombinant adeno-associated virus 9 (rAAV9)-OptF, rAAV9-control (Ctrl), or PBS, before undergoing aortic banding (AB) or sham surgery and subsequent phenotyping with serial echocardiography, cardiac magnetic resonance imaging, histology, and functional and molecular assays. RESULTS: PDE3A colocalized with SERCA2 in human nonfailing, human failing, and rodent myocardium. Amino acids 277-402 of PDE3A bound directly to amino acids 169-216 within the actuator domain of SERCA2. Disruption of PDE3A from SERCA2 increased SERCA2 activity in normal and failing cardiomyocytes. SERCA2/PDE3A disruptor peptides increased SERCA2 activity also in the presence of protein kinase A inhibitors and in phospholamban-deficient mice, and had no effect in mice with cardiomyocyte-specific inactivation of SERCA2. Cotransfection of PDE3A reduced SERCA2 activity in HEK293 vesicles. Treatment with rAAV9-OptF reduced cardiac mortality compared with rAAV9-Ctrl (hazard ratio, 0.26 [95% CI, 0.11 to 0.63]) and PBS (hazard ratio, 0.28 [95% CI, 0.09 to 0.90]) 20 weeks after AB. Mice injected with rAAV9-OptF had improved contractility and no difference in cardiac remodeling compared with rAAV9-Ctrl after aortic banding. CONCLUSIONS: Our results suggest that PDE3A regulates SERCA2 activity through direct binding, independently of the catalytic activity of PDE3A. Targeting the SERCA2/PDE3A interaction prevented cardiac mortality after AB, most likely by improving cardiac contractility.

Exercise Training Stabilizes RyR2-Dependent Ca2+ Release in Post-infarction Heart Failure.

Aim: Dysfunction of the cardiac ryanodine receptor (RyR2) is an almost ubiquitous finding in animal models of heart failure (HF) and results in abnormal Ca2+ release in cardiomyocytes that contributes to contractile impairment and arrhythmias. We tested whether exercise training (ET), as recommended by current guidelines, had the potential to stabilize RyR2-dependent Ca2+ release in rats with post-myocardial infarction HF. Materials and Methods: We subjected male Wistar rats to left coronary artery ligation or sham operations. After 1 week, animals were characterized by echocardiography and randomized to high-intensity interval ET on treadmills or to sedentary behavior (SED). Running speed was adjusted based on a weekly VO2max test. We repeated echocardiography after 5 weeks of ET and harvested left ventricular cardiomyocytes for analysis of RyR2-dependent systolic and spontaneous Ca2+ release. Phosphoproteins were analyzed by Western blotting, and beta-adrenoceptor density was quantified by radioligand binding. Results: ET increased VO2max in HF-ET rats to 127% of HF-SED (P < 0.05). This coincided with attenuated spontaneous SR Ca2+ release in left ventricular cardiomyocytes from HF-ET but also reduced Ca2+ transient amplitude and slowed Ca2+ reuptake during adrenoceptor activation. However, ventricular diameter and fractional shortening were unaffected by ET. Analysis of Ca2+ homeostasis and major proteins involved in the regulation of SR Ca2+ release and reuptake could not explain the attenuated spontaneous SR Ca2+ release or reduced Ca2+ transient amplitude. Importantly, measurements of beta-adrenoceptors showed a normalization of beta1-adrenoceptor density and beta1:beta2-adrenoceptor ratio in HF-ET. Conclusion: ET increased aerobic capacity in post-myocardial infarction HF rats and stabilized RyR2-dependent Ca2+ release. Our data show that these effects of ET can be gained without major alterations in SR Ca2+ regulatory proteins and indicate that future studies should include upstream parts of the sympathetic signaling pathway.

Coupling of the Na+/K+-ATPase to Ankyrin B controls Na+/Ca2+ exchanger activity in cardiomyocytes.

AIMS: Ankyrin B (AnkB) is an adaptor protein that assembles Na+/K+-ATPase (NKA) and Na+/Ca2+ exchanger (NCX) in the AnkB macromolecular complex. Loss-of-function mutations in AnkB cause the AnkB syndrome in humans, characterized by ventricular arrhythmias and sudden cardiac death. It is unclear to what extent NKA binding to AnkB allows regulation of local Na+ and Ca2+ domains and hence NCX activity. METHODS AND RESULTS: To investigate the role of NKA binding to AnkB in cardiomyocytes, we synthesized a disruptor peptide (MAB peptide) and its AnkB binding ability was verified by pulldown experiments. As opposed to control, the correlation between NKA and NCX currents was abolished in adult rat ventricular myocytes dialyzed with MAB peptide, as well as in cardiomyocytes from AnkB+/- mice. Disruption of NKA from AnkB (with MAB peptide) increased NCX-sensed cytosolic Na+ concentration, reduced Ca2+ extrusion through NCX, and increased frequency of Ca2+ sparks and Ca2+ waves without concomitant increase in Ca2+ transient amplitude or SR Ca2+ load, suggesting an effect in local Ca2+ domains. Selective inhibition of the NKAα2 isoform abolished both the correlation between NKA and NCX currents and the increased rate of Ca2+ sparks and waves following NKA/AnkB disruption, suggesting that an AnkB/NKAα2/NCX domain controls Ca2+ fluxes in cardiomyocytes. CONCLUSION: NKA binding to AnkB allows ion regulation in a local domain, and acute disruption of the NKA/AnkB interaction using disruptor peptides lead to increased rate of Ca2+ sparks and waves. The functional effects were mediated through the NKAα2 isoform. Disruption of the AnkB/NKA/NCX domain could be an important pathophysiological mechanism in the AnkB syndrome.

PDE3 inhibition by C-type natriuretic peptide-induced cGMP enhances cAMP-mediated signaling in both non-failing and failing hearts.

We have previously shown that the natriuretic peptide receptor B (NPR-B) agonist C-type natriuretic peptide (CNP) enhances cyclic adenosine 3´,5´-monophosphate (cAMP)-mediated signaling in failing hearts, through cyclic guanosine 3´,5´-monophosphate (cGMP)-mediated phosphodiesterase (PDE) 3 inhibition. As several signaling pathways are importantly changed in failing hearts, it could not be taken for granted that this crosstalk would be the same in non-failing hearts. Thus, we wanted to clarify to which extent this effect of CNP occurred also in non-failing hearts. Inotropic and lusitropic responses were measured in muscle strips and cGMP levels, localized cAMP levels, cAMP-PDE activity and mRNA levels were analyzed in isolated cardiomyocytes from left ventricles of non-failing and failing rat hearts. CNP increased cGMP and enhanced ß1- and ß2-adrenoceptor-mediated inotropic and ß1-adrenoceptor-mediated lusitropic responses, in non-failing and failing hearts. The NPR-A agonist brain natriuretic peptide (BNP) increased cGMP, but did not affect inotropic or lusitropic responses, indicating different compartmentation of cGMP from the two natriuretic peptide receptors. cAMP-PDE activity of PDE3 was concentration-dependently inhibited by cGMP with the same potency and to the same extent in non-failing and failing cardiomyocytes. CNP enhanced ß1-adrenoceptor-induced cAMP increase in living cardiomyocytes in the absence, but not in the presence of a PDE3 inhibitor indicating involvement of PDE3. In summary, CNP sensitizes cAMP-mediated signaling in non-failing as in failing hearts, via NPR-B-mediated increase of cGMP that inhibits the cAMP-PDE activity of PDE3.

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.

Slow Ca²âº sparks de-synchronize Ca²âº release in failing cardiomyocytes: evidence for altered configuration of Ca²âº release units?

In heart failure, cardiomyocytes exhibit slowing of the rising phase of the Ca(2+) transient which contributes to the impaired contractility observed in this condition. We investigated whether alterations in ryanodine receptor function promote slowing of Ca(2+) release in a murine model of congestive heart failure (CHF). Myocardial infarction was induced by left coronary artery ligation. When chronic CHF had developed (10 weeks post-infarction), cardiomyocytes were isolated from viable regions of the septum. Septal myocytes from SHAM-operated mice served as controls. Ca(2+) transients rose markedly slower in CHF than SHAM myocytes with longer time to peak (CHF=152 ± 12% of SHAM, P<0.05). The rise time of Ca(2+) sparks was also increased in CHF (SHAM=9.6 ± 0.6 ms, CHF=13.2 ± 0.7 ms, P<0.05), due to a sub-population of sparks (≈20%) with markedly slowed kinetics. Regions of the cell associated with these slow spontaneous sparks also exhibited slowed Ca(2+) release during the action potential. Thus, greater variability in spark kinetics in CHF promoted less uniform Ca(2+) release across the cell. Dyssynchronous Ca(2+) transients in CHF additionally resulted from T-tubule disorganization, as indicated by fast Fourier transforms, but slow sparks were not associated with orphaned ryanodine receptors. Rather, mathematical modeling suggested that slow sparks could result from an altered composition of Ca(2+) release units, including a reduction in ryanodine receptor density and/or distribution of ryanodine receptors into sub-clusters. In conclusion, our findings indicate that slowed, dyssynchronous Ca(2+) transients in CHF result from alterations in Ca(2+) sparks, consistent with rearrangement of ryanodine receptors within Ca(2+) release units.

I(CaL) inhibition prevents arrhythmogenic Ca(2+) waves caused by abnormal Ca(2+) sensitivity of RyR or SR Ca(2+) 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 ß-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 ß-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 ß-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.

Cre-loxP DNA recombination is possible with only minimal unspecific transcriptional changes and without cardiomyopathy in Tg(alphaMHC-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.

Natriuretic peptides increase beta1-adrenoceptor signalling in failing hearts through phosphodiesterase 3 inhibition.

AIMS: Whereas natriuretic peptides increase cGMP levels with beneficial cardiovascular effects through protein kinase G, we found an unexpected cardio-excitatory effect of C-type natriuretic peptide (CNP) through natriuretic peptide receptor B (NPR-B) stimulation in failing cardiac muscle and explored the mechanism. METHODS AND RESULTS: Heart failure was induced in male Wistar rats by coronary artery ligation. Contraction studies were performed in left ventricular muscle strips. Cyclic nucleotides were measured by radio- and enzyme immunoassay. Apoptosis was determined in isolated cardiomyocytes by Annexin-V/propidium iodide staining and phosphorylation of phospholamban (PLB) and troponin I was measured by western blotting. Stimulation of NPR-B enhanced beta1-adrenoceptor (beta1-AR)-evoked contractile responses through cGMP-mediated inhibition of phosphodiesterase 3 (PDE3). CNP enhanced beta1-AR-mediated increase of cAMP levels to the same extent as the selective PDE3 inhibitor cilostamide and increased beta1-AR-stimulated protein kinase A activity, as demonstrated by increased PLB and troponin I phosphorylation. CNP promoted cardiomyocyte apoptosis similar to inhibition of PDE3 by cilostamide, indicative of adverse effects of NPR-B signalling in failing hearts. CONCLUSION: An NPR-B-cGMP-PDE3 inhibitory pathway enhances beta(1)-AR-mediated responses and may in the long term be detrimental to the failing heart through mechanisms similar to those operating during treatment with PDE3 inhibitors or during chronic beta-adrenergic stimulation.

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.

Sodium accumulation promotes diastolic dysfunction in end-stage heart failure following Serca2 knockout.

Alterations in trans-sarcolemmal and sarcoplasmic reticulum (SR) Ca(2+) fluxes may contribute to impaired cardiomyocyte contraction and relaxation in heart failure. We investigated the mechanisms underlying heart failure progression in mice with conditional, cardiomyocyte-specific excision of the SR Ca(2+)-ATPase (SERCA) gene. At 4 weeks following gene deletion (4-week KO) cardiac function remained near normal values. However, end-stage heart failure developed by 7 weeks (7-week KO) as systolic and diastolic performance declined. Contractions in isolated myocytes were reduced between 4- and 7-week KO, and relaxation was slowed. Ca(2+) transients were similarly altered. Reduction in Ca(2+) transient magnitude resulted from complete loss of SR Ca(2+) release between 4- and 7-week KO, due to loss of a small remaining pool of SERCA2. Declining SR Ca(2+) release was partly offset by increased L-type Ca(2+) current, which was facilitated by AP prolongation in 7-week KO. Ca(2+) entry via reverse-mode Na(+)-Ca(2+) exchange (NCX) was also enhanced. Up-regulation of NCX and plasma membrane Ca(2+)-ATPase increased Ca(2+) extrusion rates in 4-week KO. Diastolic dysfunction in 7-week KO resulted from further SERCA2 loss, but also impaired NCX-mediated Ca(2+) extrusion following Na(+) accumulation. Reduced Na(+)-K(+)-ATPase activity contributed to the Na(+) gain. Normalizing [Na(+)] by dialysis increased the Ca(2+) decline rate in 7-week KO beyond 4-week values. Thus, while SERCA2 loss promotes both systolic and diastolic dysfunction, Na(+) accumulation additionally impairs relaxation in this model. Our observations indicate that if cytosolic Na(+) gain is prevented, up-regulated Ca(2+) extrusion mechanisms can maintain near-normal diastolic function in the absence of SERCA2.