P1596Interaction of CaMKII and NaV1.8 modulates cardiac electrophysiology in human heart failure

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
P Tirilomis ◽  
S Ahmad ◽  
P Bengel ◽  
S Pabel ◽  
L Maier ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cardiac Electrophysiology ◽  
Human Heart ◽  
Association Studies ◽  
Cardiac Conduction ◽  
Epifluorescence Microscopy ◽  
Genome Wide Association Studies ◽  
Human Heart Failure ◽  
Human Cardiomyocytes ◽  
Ventricular Cardiomyocytes

Abstract Introduction In human heart failure, electrical remodeling contributes to the risk of arrhythmia generation. Increased expression of Ca/Calmodulin-dependent protein kinase IIδ (CaMKIIδ) and an enhanced persistent Na current (INaL) have been linked to arrhythmogenesis. CaMKIIδ increases INaL via regulation of sodium channels thereby contributing to arrhythmias through early- and delayed-afterdepolarizations (EADs and DADs). Genome-wide association studies (GWAS) have described the implication of the neuronal sodium channel isoform NaV1.8 (SCN10A) in cardiac electrophysiology showing modulation in cardiac conduction. We showed that the expression of the isoform Nav1.8 is significantly increased in human failing cardiomyocytes and contributes substantially to the enhanced INaL. Purpose We investigated a potential interaction of CaMKIIδ and NaV1.8 and thereby its role in arrhythmia generation and electrophysiology in human and murine failing hearts. Methods Cardiomyocytes were isolated from explanted failing hearts and CaMKIIδ transgenic (TG) mice. We performed immunostainings and co-immunoprecipitation (Co-IP) to show interactions of CaMKIIδ and Nav1.8 in isolated cardiomyocytes and homogenates. Whole-cell patch clamp experiments were conducted in isolated human and murine ventricular cardiomyocytes. Additionally, Ca2+ transients were measured using epifluorescence microscopy with the Ca2+ dye fura-2 (10μmol/L) whereas Ca2+ sparks measurements were performed by using confocal microscopy with the Ca2+ dye fluo-4 (10μmol/L). PF-01247324 is a novel specific NaV1.8 inhibitor (orally bioavailable; 1 μmol/L) and autocamtide inhibitory peptide (AIP, 1 μmol/L) was used to inhibit CaMKIIδ. Results Co-immunoprecipitation experiments revealed an association of CaMKIIδ and Nav1.8 in human homogenates compared to healthy controls. Furthermore, immunohistochemistry stainings in isolated human cardiomyocytes showed a co-localization of CaMKIIδ and NaV1.8 at the intercalated disc and t-tubules. We observed a significant reduction of INaL integral and proarrhythmic SR-Ca2+ spark frequency (CaSpF) after addition of either PF-01247324 or the CaMKIIδ inhibitor AIP in failing human and murine ventricular cardiomyocytes. When PF-01247324 and AIP were added together, the decrease in INaL integral and CaSpF was comparable to PF-01247324 alone in human failing cardiomyocytes. Inhibition of NaV1.8 did not show an effect on Ca2+ transient amplitude or Ca2+ transient decay at different stimulation frequencies in CaMKIIδ TG cardiomyocytes. Conclusion Our results demonstrate the significance of both CaMKIIδ and NaV1.8 in INaL generation and their detrimental interaction. This data suggest that increased CaMKIIδ activity plays a substantial role for the activation of NaV1.8-mediated late sodium current and SR-Ca2+ leak.

scholarly journals Intracellular Dyssynchrony of Diastolic Cytosolic [Ca 2+ ] Decay in Ventricular Cardiomyocytes in Cardiac Remodeling and Human Heart Failure

2013 ◽  
Vol 113 (5) ◽  
pp. 527-538 ◽  
Author(s):  
Felix Hohendanner ◽  
Senka Ljubojević ◽  
Niall MacQuaide ◽  
Michael Sacherer ◽  
Simon Sedej ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cardiac Remodeling ◽  
Human Heart ◽  
Cyclopiazonic Acid ◽  
Cardiomyocyte Hypertrophy ◽  
Human Heart Failure ◽  
Mitochondrial Density ◽  
Human Cardiomyocytes ◽  
End Stage ◽  
The Impact

Rationale : Synchronized release of Ca 2+ into the cytosol during each cardiac cycle determines cardiomyocyte contraction. Objective: We investigated synchrony of cytosolic [Ca 2+ ] decay during diastole and the impact of cardiac remodeling. Methods and Results: Local cytosolic [Ca 2+ ] transients (1-µm intervals) were recorded in murine, porcine, and human ventricular single cardiomyocytes. We identified intracellular regions of slow (slowCaR) and fast (fastCaR) [Ca 2+ ] decay based on the local time constants of decay (TAU local ). The SD of TAU local as a measure of dyssynchrony was not related to the amplitude or the timing of local Ca 2+ release. Stimulation of sarcoplasmic reticulum Ca 2+ ATPase with forskolin or istaroxime accelerated and its inhibition with cyclopiazonic acid slowed TAU local significantly more in slowCaR, thus altering the relationship between SD of TAU local and global [Ca 2+ ] decay (TAU global ). Na + /Ca 2+ exchanger inhibitor SEA0400 prolonged TAU local similarly in slowCaR and fastCaR. FastCaR were associated with increased mitochondrial density and were more sensitive to the mitochondrial Ca 2+ uniporter blocker Ru360. Variation in TAU local was higher in pig and human cardiomyocytes and higher with increased stimulation frequency (2 Hz). TAU local correlated with local sarcomere relengthening. In mice with myocardial hypertrophy after transverse aortic constriction, in pigs with chronic myocardial ischemia, and in end-stage human heart failure, variation in TAU local was increased and related to cardiomyocyte hypertrophy and increased mitochondrial density. Conclusions: In cardiomyocytes, cytosolic [Ca 2+ ] decay is regulated locally and related to local sarcomere relengthening. Dyssynchronous intracellular [Ca 2+ ] decay in cardiac remodeling and end-stage heart failure suggests a novel mechanism of cellular contractile dysfunction.

P3829Antiarrhythmic effects of Sacubitrilat (LBQ657) on Ca2+ homeostasis in ventricular cardiomyocytes

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
J Eiringhaus ◽  
C Wuensche ◽  
J Herting ◽  
G Hasenfuss ◽  
S Sossalla ◽  
...  
Keyword(s):  
Heart Failure ◽  
Angiotensin Ii ◽  
Left Ventricular ◽  
Epifluorescence Microscopy ◽  
Transient Kinetics ◽  
Human Heart Failure ◽  
Beneficial Effects ◽  
Ventricular Cardiomyocytes ◽  
Neprilysin Inhibitor ◽  
Neprilysin Inhibition

Abstract Background and objectives Simultaneous inhibition of neprilysin and angiotensin II receptors by sacubitril/valsartan was shown to significantly reduce morbidity and mortality in heart failure patients compared to sole interference with the renin angiotensin system. Beneficial effects of increased levels of natriuretic peptides following neprilysin inhibition have been suggested, whereas direct effects of sacubitrilat on myocardial Ca2+ cycling properties remain elusive. Methods and results Under basal conditions the combination of active neprilysin-inhibitior sacubitrilat (LBQ657) and angiotensin II receptor inhibitor valsartan did not influence diastolic Ca2+ spark frequency (CaSpF) nor arrhythmogenic SR Ca2+ leak in murine ventricular cardiomyocytes (confocal microscopy, n CMs/hearts=80/7 vs. 100/7, P=0.91/0.99). In contrast, sacubitrilat/valsartan treatment significanty reduced CaSpF by 35±9% and SR Ca2+ leak by 45±9% in CMs that had been put under catecholaminergic stress (isoproterenol 10nM, n=81/7 vs. 62/7, P<0.001 both). This effect could be clearly be attributed to the neprilysin inhibitor sacubitrilat as sole sacubitrilat treatment also reduced both parameters by similar degrees (reduction of CaSpF by 57±7% and SR Ca2+ leak by 76±5%; n=101/4 vs. 108/4, P<0.01 both) whereas sole valsartan treatment did not affect diastolic SR Ca2+ leak. Of note, systolic Ca2+ release, SR Ca2+ load and Ca2+ transient kinetics of murine CMs were not compromised upon treatment with sacubitrilat (epifluorescence microscopy, n=41/6 vs. 39/6). Importantly, sacubitrilat/valsartan in combination as well as sacubitrilat alone also reduced diastolic CaSpF and SR Ca2+ leak by 40–74% in human left-ventricular CMs from patients with end-stage heart failure (n=71/8 vs. 78/8, P<0.05). Conclusion This study demonstrates that neprilysin-inhibition directly exerts beneficial effects on Ca2+ homeostasis in human heart failure. We can show for the first time that neprilysin-inhibition by sacubitrilat yields a strong reduction of arrhythmogenic SR Ca2+ leak without affecting systolic Ca2+ release. These effects might contribute to the mortality benefit of sacubitril/valsartan treatment in the PARADIGM Study. Acknowledgement/Funding THF was funded by the Deutsche Forschungsgemeinschaft (DFG) through the SFB 1002 (A11). SS is supported by the Marga und Walter Boll-Stiftung.

scholarly journals Absence of Functional Nav1.8 Channels in Non-diseased Atrial and Ventricular Cardiomyocytes

2019 ◽  
Vol 33 (6) ◽  
pp. 649-660 ◽  
Author(s):  
Simona Casini ◽  
Gerard A. Marchal ◽  
Makiri Kawasaki ◽  
Fransisca A. Nariswari ◽  
Vincent Portero ◽  
...  
Keyword(s):  
Action Potential ◽  
Cardiac Electrophysiology ◽  
Sodium Current ◽  
Association Studies ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Left Ventricular ◽  
Resting Membrane Potential ◽  
Genome Wide Association Studies ◽  
Ventricular Cardiomyocytes

Abstract Purpose Several studies have indicated a potential role for SCN10A/NaV1.8 in modulating cardiac electrophysiology and arrhythmia susceptibility. However, by which mechanism SCN10A/NaV1.8 impacts on cardiac electrical function is still a matter of debate. To address this, we here investigated the functional relevance of NaV1.8 in atrial and ventricular cardiomyocytes (CMs), focusing on the contribution of NaV1.8 to the peak and late sodium current (INa) under normal conditions in different species. Methods The effects of the NaV1.8 blocker A-803467 were investigated through patch-clamp analysis in freshly isolated rabbit left ventricular CMs, human left atrial CMs and human-induced pluripotent stem cell-derived CMs (hiPSC-CMs). Results A-803467 treatment caused a slight shortening of the action potential duration (APD) in rabbit CMs and hiPSC-CMs, while it had no effect on APD in human atrial cells. Resting membrane potential, action potential (AP) amplitude, and AP upstroke velocity were unaffected by A-803467 application. Similarly, INa density was unchanged after exposure to A-803467 and NaV1.8-based late INa was undetectable in all cell types analysed. Finally, low to absent expression levels of SCN10A were observed in human atrial tissue, rabbit ventricular tissue and hiPSC-CMs. Conclusion We here demonstrate the absence of functional NaV1.8 channels in non-diseased atrial and ventricular CMs. Hence, the association of SCN10A variants with cardiac electrophysiology observed in, e.g. genome wide association studies, is likely the result of indirect effects on SCN5A expression and/or NaV1.8 activity in cell types other than CMs.

4964Loss-of-spen disturbs cardiac conduction in zebrafish mediated by impaired connexin43 function

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
M Rattka ◽  
S Westphal ◽  
A Pott ◽  
M Kessler ◽  
K Weinmann ◽  
...  
Keyword(s):  
Heart Failure ◽  
In Situ Hybridization ◽  
Ventricular Fibrillation ◽  
Western Blot ◽  
Association Studies ◽  
Severe Heart Failure ◽  
Transcriptional Regulator ◽  
Cardiac Conduction ◽  
Genome Wide Association Studies

Abstract Introduction Genome-wide association studies identified SPLIT ENDS (SPEN) gene as an interesting candidate in the pathogenesis of heart failure. SPEN is known to be of importance in transcriptional regulation in several pathways and knockout in mice results in severe cardiac structural abnormalities and premature intrauterine lethality. Moreover, SPEN was associated with human 1.36 syndrome and is located in the critical region for congenital heart defects. Hence, spen seems to be of importance in cardiac physiology, but not much is known about its biologic function in the heart. Purpose The aim of our study was to further deepen the insights on spen's role in cardiac pathology. Methods Morpholino-mediated gene knockdown is an excellent method to approach in vivo function of cardiac candidate genes in zebrafish. We conducted spen-specific knockdown experiments and first analyzed the emerging heart failure and arrhythmia phenotype. Next, we assessed possible target genes of the transcriptional regulator spen by gene expression profiling and verified our results by quantitative real-time PCR (qPCR), Western Blot, in situ hybridization and further knockdown- and rescue-experiments. Results Spen-deficiency results in severe heart failure and arrhythmia in zebrafish. While in the first 24 hpf (hours post fertilization) spen-morphant embryos develop normally, they exhibit reduced contractility and bradycardia at 48 hpf. From 60 hpf onwards impaired spen function is associated with electrophysiological abnormalities, closely resembling human second grade av block as well as atrial and ventricular fibrillation. Transcriptional profiling of pooled spen-morphant hearts revealed connexin43 (cx43) to be severely downregulated. This finding was verified by qPCR, Western Blot analysis and in situ hybridization experiments. Consequently, we hypothesized cx43 to be transcriptionally controlled by spen and that inadequate function of spen lowers cx43 levels, thereby causing cardiac conduction abnormalities. Subsequently, we performed cx43-knockdown, which phenocopied the spen-morphants phenotype. Importantly, all loss-of function experiments could be rescued by overexpression of wild type cx43 mRNA. Moreover, sensitizing via double injection of sub-phenotypic concentrations of spen- and cx43-morpholinos resulted in a supra-additive effect, validating molecular crosstalk between spen and cx43 on the signaling level. Compared to unaffected controls, over 90% of sensitized embryos display the arrhythmia phenotype and show atrial- and ventricular fibrillation already at 48 hpf. Conclusion Our results demonstrate that the transcriptional regulator spen controls the transcription of connexin43 and that impairment of this mechanism leads to severe cardiac arrhythmia in zebrafish.

Abstract 13: Suppressing Microtubule Detyrosination Reduces Stiffness and Improves Contractility in Human Cardiomyocytes

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Benjamin L Prosser ◽  
Kenneth Bedi ◽  
Matthew Caporizzo ◽  
Christina Chen ◽  
Patrick Robison ◽  
...  
Keyword(s):  
Heart Failure ◽  
High Speed ◽  
Human Heart ◽  
Contractile Function ◽  
Super Resolution ◽  
Tensile Tests ◽  
Mechanical Resistance ◽  
Human Heart Failure ◽  
Human Cardiomyocytes ◽  
End Stage

The microtubule contribution to myocyte mechanics has been a controversial topic over the years. Utilizing high-speed, super-resolution imaging, we were recently able to directly observe microtubule behavior in working myocytes (Robison et al., Science 2016). Strikingly, we found that microtubules buckle like springs between sarcomeric attachment points, providing a mechanical resistance that limits sarcomere shortening and stretch. Further, we identified that post-translational “detyrosination” of microtubules regulates their attachment to the sarcomere, and thus the microtubule contribution to both passive and active mechanics. Here we present new data identifying microtubule detyrosination as a compelling therapeutic target for the treatment of human heart failure. Using quantitative mass spectrometry, we have probed the cytoskeletal changes that occur during the progression of human heart failure in over 40 patient samples at different stages and etiologies of disease. We find that progressive upregulation and stabilization of the structural cytoskeleton, particularly microtubules and intermediate filaments, is a robust hallmark of human heart failure. Next, we have performed detailed biophysical studies on isolated myocytes from explanted failing and non-failing human hearts. Using advanced imaging, single myocyte tensile tests and atomic force microscopy (e.g. Prosser et al., Science 2011; Robison et al. Science 2016), we have interrogated the contribution of detyrosinated microtubules to the active and passive mechanics of human myocytes. We find that by reducing microtubule detyrosination, we can robustly improve contractile function. Suppressing detyrosination significantly lowers passive stiffness at physiologic rates, while robustly improving contraction velocity, fractional shortening, and relaxation speed. Of note, the improvement in mechanics correlates with the severity of disease, as myocytes from end-stage patients show greater benefits than those from non-failing or compensated hypertrophic hearts. In conclusion, our work demonstrates pre-clinical efficacy for suppressing detyrosinated microtubules to improve myocyte mechanics in human heart failure.

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