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p38γ/δ activation alters cardiac electrical activity and predisposes to ventricular arrhythmia

Nature Cardiovascular Research volume 2, pages 1204–1220 (2023)Cite this article

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Abstract

Ventricular fibrillation (VF) is a leading immediate cause of sudden cardiac death. There is a strong association between aging and VF, although the mechanisms are unclear, limiting the availability of targeted therapeutic interventions. Here we found that the stress kinases p38γ and p38δ are activated in the ventricles of old mice and mice with genetic or drug-induced arrhythmogenic conditions. We discovered that, upon activation, p38γ and p38δ cooperatively increase the susceptibility to stress-induced VF. Mechanistically, our data indicate that activated p38γ and p38δ phosphorylate ryanodine receptor 2 (RyR2) disrupt Kv4.3 channel localization, promoting sarcoplasmic reticulum calcium leak, Ito current reduction and action potential duration prolongation. In turn, this led to aberrant intracellular calcium handling, premature ventricular complexes and enhanced susceptibility to VF. Blocking this pathway protected genetically modified animals from VF development and reduced the VF duration in aged animals. These results indicate that p38γ and p38δ are a potential therapeutic target for sustained VF prevention.

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Fig. 1: Immunoblot evaluation of p38γ/δ expression and phosphorylation with aging and in pro-arrhythmogenic models.
Fig. 2: p38γ/δ activation increases the susceptibility to stress-induced VF and a pro-arrhythmic cardiac phosphoproteome profile.
Fig. 3: Identification of RyR2 as a direct target of p38γ/δ.
Fig. 4: p38γ/δ activation due to MKK6 deficiency promotes RyR2 hyperphosphorylation and ventricular arrhythmias.
Fig. 5: MKK6-deficient cardiomyocytes are predisposed to ISO-induced abnormal SCR events.
Fig. 6: Transient outward potassium current is reduced, and APD and QT interval are prolonged, in MKK6 KO mice.
Fig. 7: p38γ deficiency reduces arrhythmia susceptibility and RyR2 phosphorylation in MKK6 KO mice.
Fig. 8: Proposed model for ventricular arrhythmia susceptibility mediated by increased p38γ/δ activity.

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Data availability

All data supporting the findings of this study are included in the main article and associated files. Uncropped immunoblots are provided as Source Data. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE86 partner repository with the dataset identifier PXD045996.

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Acknowledgements

We gratefully acknowledge L. Sen-Martín, J. Alegre-Cebollada (CNIC, Madrid) and L. Carrier (University Medical Center Hamburg-Eppendorf and DZHK, Hamburg) for the cMyBP3-C KO cardiac tissue; D. Roiz-Valle and C. López-Otín (IUOPA; Universidad de Oviedo, Oviedo) for the LmnaG609G/G609G cardiac tissue; and R. J. Davis for the MKK6 KO mice. We thank G. Giovinazzo and the CNIC Pluripotent Cell Technology Unit (CNIC, Madrid) for the hiPSCs. We thank S. Bartlett and F. Chanut (CNIC, Madrid) for English editing, and R. R. Mondragon (University of Michigan, Ann Arbor) for technical support. We are grateful to R. J. Davis (University of Massachusetts Chan Medical School, Worcester), A. Padmanabhan (University of California, San Francisco) and M. Costa and C. López-Otín (IUOPA; Universidad de Oviedo, Oviedo) for critical reading of the manuscript. We thank the staff at the CNIC Genomics and Bioinformatics Units for technical support and help with data analysis and A. C. Silva for help with figure editing and design. This work was funded by a CNIC Intramural Project Severo Ochoa (Expediente 12-2016 IGP) to G.S. and J.J. G.S. is supported by the following projects: PMP21/00057 IMPACT-2021, funded by the Instituto de Salud Carlos III (ISCIII), and PDC2021-121147-I00 and PID2019-104399RB-I00, funded by MCIN/AEI/10.13039/501100011033—all funded by the European Union (FEDER/FSE); ‘Una manera de hacer Europa’/‘El FSE invierte en tu futuro’/Next Generation EU and co-funded by the European Union/Plan de Recuperación, Transformación y Resiliencia (PRTR). R.R.B. is a fellow of the FPU Program (FPU17/03847). B.G.T. was a fellow of the FPI Severo Ochoa CNIC Program (SVP‐2013‐067639) and an American Heart Association Postdoctoral Fellow (18POST34080175). The following grants provided additional funding: Instituto de Salud Carlos III, PDC2021-121147-I00 Convocatoria: Proyectos Prueba de Concepto 2021 Ministerio de Ciencia e Innovación and PID2022-138525OB-I00 Ministerio de Ciencia e Innovación; US National Heart, Lung, and Blood Institute (R01 grant HL122352); Fondos FEDER, Madrid, Spain, and Fundación Bancaria ‘La Caixa (project HR19/52160013) to J.J.; American Heart Association Postdoctoral Fellowship 14POST17820005 to D.P.B.; and MICINN PGC2018-097019-B-I00, ISCIII-SGEFI/ERDF (PRB3-IPT17/0019, ProteoRed), the Fundació Marató TV3 (grant 122/C/2015) and ‘la Caixa’ Banking Foundation (project code HR17-00247) to J.V. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S, funded by MICIN/AEI/10.13039/501100011033).

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Authors and Affiliations

  1. Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain

    Rafael Romero-Becerra, Francisco M. Cruz, Alfonso Mora, Juan Antonio Lopez, Bárbara González-Terán, Marta León, Maria Elena Rodríguez, Luis Leiva-Vega, David Filgueiras-Rama, Jesús Vázquez, José Jalife & Guadalupe Sabio

  2. CIBER de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain

    Juan Antonio Lopez, David Filgueiras-Rama, Jesús Vázquez & José Jalife

  3. Center for Arrhythmia Research, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA

    Daniela Ponce-Balbuena, Andrew Allan, Roberto Ramos-Mondragón, Guadalupe Guerrero-Serna, Eric N. Jimenez-Vazquez & José Jalife

  4. Gladstone Institutes, San Francisco, CA, USA

    Bárbara González-Terán

  5. Hospital Clínico Universitario San Carlos, Madrid, Spain

    David Filgueiras-Rama

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  1. Rafael Romero-Becerra
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G.S. conceived this project. G.S. and J.J. supervised this project. R.R.-B. performed the primary experiments, acquired and analyzed the data and prepared figures. G.S., J.J. and R.R.-B. designed the study, developed the hypothesis and wrote the paper, with input from all authors. J.A.L. and J.V. performed the phosphoproteomics analysis. R.R.-B. performed the bioinformatic analysis. D.P.-B., A.A., F.M.C.U., R.R.-M, G.G.-S. and E.N.J.-V. performed cardiomyocyte isolation, patch-clamp experiments, optical mapping and in vivo intracardiac catheter burst pacing. A.M., B.G,-T., M.L., M.E.R. and L.L.-V. help to perform some of the experiments and acquired data. D.F.-R. analyzed the ECG recordings and critically revised the paper. J.J. and G.S. led and funded the project.

Corresponding authors

Correspondence to José Jalife or Guadalupe Sabio.

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The authors declare no competing interests.

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Nature Cardiovascular Research thanks Xander Wehrens and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary Handling Editor: Elvira Forte, in collaboration with the Nature Cardiovascular Research team.

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Extended data

Extended Data Fig. 1 p38γ/δ have increased phosphorylation levels in hearts from old female WT mice.

Immunoblot analysis of the phosphorylation and protein levels of p38γ/δ in heart lysates from 2-month-old (n = 5) and 24-month-old (n = 7) female mice. Two-sided unpaired Stundent’s t-test. Data are expressed as mean ± SEM.

Source data

Extended Data Fig. 2 Cardiac arrhythmia phosphoproteome network in cTnT-p38γ*/δ* hearts.

a, Phosphorylation levels of p38γ/δ assessed by immunoblot in heart lysates from 24-month-old WT (n = 3) and cTnT-p38γ*/δ* (n = 3) mice. Two-sided unpaired Student’s t-test. b, Percentage of atrial fibrillation (AF) inducibility by S1-S2 protocol 1 in the absence (basal) and presence (ISO) of isoproterenol (i.p. 5 mg/kg) in 2-month-old cTnT-GFP (n = 5) and cTnT-p38γ*/δ* (n = 5) male mice. Each dot shows the percentage of AF induced in each mouse by the S1-S2 protocol 1. 2-way ANOVA followed by Sidak’s post-test. c, Percentage of VT/VF inducibility by S1-S2 protocol 1 in cTnT-GFP (n = 9), cTnT-p38γ* (n = 9), cTnT-p38δ* (n = 9) and cTnT-p38γ*/δ* (n = 8) mice in the absence (basal) and presence (ISO) of isoproterenol (i.p. 5mg/kg). Each dot shows the percentage of VT/VF induced in each mouse by the S1-S2 protocol 1. 2-way ANOVA followed by Tukey’s post-test. Data are expressed as mean ± SEM. d, Clinical Pathology Enrichment (Tox Functions) of the differentially hyperphosphorylated proteins (z-value difference>0; limma two-sided t-test p-value<0.05) from cTnT-p38γ*/δ* hearts non-corrected phosphoproteome compared to controls, as determined by Ingenuity Pathway Analysis. The vertical dashed line indicates the threshold of -Log10 (Two-sided Fisher’s exact test p-value) =1.3. e, Interaction network of DPhos proteins involved in the category ‘Cardiac Arrhythmia’ shown in (d), based on STRING database, and visualized in Cytoscape. The node colors indicate the differential abundance (inner circle) and phosphorylation state (outer circle) of the proteins in cTnT-p38γ*/δ* vs cTnT-GFP hearts. Small numbers pointing to segments of the outer circles indicate the position of the phosphosite in the sequence of the protein. The edge colors indicate the STRING interaction score between proteins. Only significant (z-value difference > 0; limma two-sided t-test p-value < 0.05) phosphopeptides are indicated. Proteins are defined by their gene name for simplicity. f, Chord diagram of the phosphoproteins found in the category ‘Cardiac Arrhythmia’shown in (d), and the 5 most significant pathologies in this category to which the phosphoproteins are assigned. Proteins are identified by their gene name for simplicity. Data are expressed as mean ± SEM.

Source data

Extended Data Fig. 3 p38γ/δ activity is related to phosphorylation of RyR2 at p-Ser p38 motif.

a, Representative blot of two independent in vitro phosphorylation kinase assays of RyR2 isolated from mice hearts on P-Ser p38 motif by active-recombinant p38α, p38β, p38γ, p38δ, and p38γ and p38δ together. b, Immunoblot analysis of RyR2 phosphorylation at P-Ser p38 motif in heart lysates from 2-month-old (n=4) and 24-month-old (n=3) WT mice. Two-sided unpaired Student’s t-test. Data are expressed as mean ± SEM.

Source data

Extended Data Fig. 4 p38γ/δ activation due to MKK6 deficiency promotes atrial fibrillation and also ventricular arrhythmias independently of the sex.

a, Percentage of atrial fibrillation (AF) inducibility by S1-S2 protocol 1 in the absence (basal) and presence (ISO) of isoproterenol (i.p. 5 mg/kg) in 5-month-old WT (n = 5) and MKK6 KO (n = 6) male mice. b, Percentage of VT/VF inducibility by S1-S2 protocol 1 in WT (n = 4) and MKK6 KO (n = 3) female mice in the absence (basal) and presence (ISO) of isoproterenol (i.p. 5mg/kg). Each dot shows the percentage of AF (a) or VT/VF (b) induced in each mouse by the S1-S2 protocol 1. 2-way ANOVA followed by Sidak’s post-test. Dara are expressed as mean ± SEM.

Extended Data Fig. 5 ICaL, INa and IK1 in 2-month-old WT and MKK6 KO cardiomyocytes.

a, Representative ICaL traces (left) and ICaL I/V relationship (right) from 2-month-old WT (n = 21) and MKK6 KO (n = 22) cardiomyocytes. Mean peak current densities at -0 mV were -9±0.5 (WT) versus-8±0.6 (MKK6 KO) pA/pF. 2-way ANOVA followed by Sidak’s post-test. Data were collected from 3 independent cardiomyocyte isolation for each condition. b, Representative sodium current traces (left) and INa I/V relationship (right) in WT (n = 22) and MKK6 KO (n = 28) cardiomyocytes. INa densities at -30 mV were similar in both groups (-31±2 pA/pF in WT vs -33±2 pA/pF in MKK6 KO cardiomyocytes). 2-way ANOVA followed by Sidak’s post-test. Data were collected from 5 independent cardiomyocyte isolation for each condition. c, Representative IK1 current traces (left) and IK1 I/V relationship (right) from WT (n = 10) and MKK6 KO (n = 9) cardiomyocytes. Mean peak current densities at -120 mV were -14±1.1 (WT) versus -16±1.1 (MKK6 KO) pA/pF. 2-way ANOVA followed by Sidak’s post-test. Data were collected from 3 independent cardiomyocyte isolation for each condition. d, Resting membrane potential, action potential amplitude, overshoot, and maximum rate of action potential depolarization (dV/dtMax) in WT (n = 26) and MKK6 KO (n = 22) cardiomyocytes. Two-sided unpaired Student’s t-test or Mann-Whitney U test. Data were collected from 4 independent cardiomyocyte isolation for each condition. Data are expressed as mean ± SEM.

Extended Data Fig. 6 Stress-induced arrhythmia burden in 9–10-month-old WT and p38γ KO mice.

a, Percentage of VT/VF inducibility by S1-S2 protocol 1 in 9–10-month-old WT (n = 8) and p38γ KO (n = 5) female mice in the absence (basal) and presence (ISO) of isoproterenol (i.p. 5 mg/kg). Each dot shows the percentage of VT/VF induced in each mouse by the S1-S2 protocol. 2-way ANOVA followed by Tukey’s post test. b, VT/VF duration in WT (n = 7 VT/VF) and p38γ KO (n = 11 VT/VF) female mice in basal condition. Each dot represents the duration of each induced arrhythmia. Two-sided unpaired Student’s t-test. Data are expressed as mean ±SEM.

Supplementary information

Reporting Summary

Supplementary Table 1

Clinical pathologies (Tox Functions) identified in the IPA of differentially hyperphosphorylated proteins (DPhos; Z value difference > 0, two-sided limma t-test P < 0.05) from the non-corrected phosphoproteomics analysis of hearts from 9-week-old WT cTnT-GFP and cTnT-p38γ*/δ* mice. The table includes the P value (two-sided Fisher’s exact test) and the proteins (identified by their gene name) for each category.

Supplementary Table 2

UniProt identifiers, gene names, phosphorylation Z value difference (cTnT-p38γ*/δ* versus cTnT-GFP), two-sided limma t-test P values, phosphopeptide sequences and phosphosites of the proteins belonging to the ‘cardiac arrhythmia’ Tox Function from IPA of the non-corrected phosphoproteomics analysis of hearts from 9-week-old WT cTnT-GFP and cTnT-p38γ*/δ* mice.

Supplementary Table 3

Diseases and functions annotations corresponding to the ‘cardiac arrhythmia’ category from IPA of differentially hyperphosphorylated proteins (DPhos; Z value difference > 0, two-sided limma t-test P < 0.05) of hearts from 9-week-old WT cTnT-GFP and cTnT-p38γ*/δ* mice non-corrected phosphoproteomics analysis. The table includes the disease name, P values (two-sided Fisher’s exact test) for each pathology and corresponding proteins (identified by their gene name).

Supplementary Table 4

Mass spectrometry non-corrected phosphoproteomics and proteomics data of hearts lysates from 2-month-old WT mice infected with adeno-associated viruses with cardiac troponin T promoter (AAV-cTnT) driving expression of either constitutively active p38γ/δ kinases (cTnT-p38γ*/δ*, n = 3) or GFP as a control (cTnT-GFP, n = 4).

Supplementary Table 5

Clinical pathologies (Tox Functions) identified in the IPA of differentially hyperphosphorylated proteins (DPhos; Z value difference > 0, two-sided limma t-test P < 0.05) from the corrected phosphoproteomics analysis of hearts from 9-week-old WT cTnT-GFP and cTnT-p38γ*/δ* mice. The table includes the P value (two-sided Fisher’s exact test) and the proteins (identified by their gene name) for each category.

Supplementary Table 6

UniProt identifiers, gene names, phosphorylation Z value difference (cTnT-p38γ*/δ* versus cTnT-GFP), two-sided limma t-test P values, phosphopeptide sequences and phosphosites of the proteins belonging to the ‘cardiac arrhythmia’ Tox Function from IPA of the corrected phosphoproteomics analysis of hearts from 9-week-old WT cTnT-GFP and cTnT-p38γ*/δ* mice.

Supplementary Table 7

Disease and function annotations corresponding to the ‘cardiac arrhythmia’ category from IPA of differentially hyperphosphorylated proteins (DPhos; Z value difference > 0, two-sided limma t-test P < 0.05) of hearts from 9-week-old WT cTnT-GFP and cTnT-p38γ*/δ* mice corrected phosphoproteomics analysis. The table includes the disease name, P values (two-sided Fisher’s exact test) for each pathology and corresponding proteins (identified by their gene name).

Supplementary Table 8

Mass spectrometry corrected phosphoproteomics (phosphorylated peptides abundance corrected by protein expression changes) and proteomics data of hearts lysates from 2-month-old WT mice infected with adeno-associated viruses with cardiac troponin T promoter (AAV-cTnT) driving expression of either constitutively active p38γ/δ kinases (cTnT-p38γ*/δ*, n = 3) or GFP as a control (cTnT-GFP, n = 4).

Supplementary Video 1

Optical mapping phase movies from the surface of the epicardial free wall of the ventricles of each MKK6 KO heart that developed high-frequency rotational activity. Patterns of activation show propagation waves compatible with fibrillatory conduction. Singularity points are also present in correlation with wave breaks in the fibrillatory conduction region. The color bar indicates the phase in the excitation–recovery cycle. Each video corresponds to a different MKK6 KO animal.

Source data

Source Data Fig. 1

Unprocessed, uncropped western blots for Fig. 1.

Source Data Fig. 3

Unprocessed, uncropped western blots for Fig. 3.

Source Data Fig. 4

Unprocessed, uncropped western blots for Fig. 4.

Source Data Fig. 5

Unprocessed, uncropped western blots for Fig. 5.

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Unprocessed, uncropped western blots for Fig. 6.

Source Data Fig. 7

Unprocessed, uncropped western blots for Fig. 7.

Source Data Extended Data Fig./Table 1

Unprocessed, uncropped western blots for Extended Data Fig. 1.

Source Data Extended Data Fig./Table 2.

Unprocessed, uncropped western blots for Extended Data Fig. 2.

Source Data Extended Data Fig./Table 3

Unprocessed, uncropped western blots for Extended Data Fig. 3.

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Romero-Becerra, R., Cruz, F.M., Mora, A. et al. p38γ/δ activation alters cardiac electrical activity and predisposes to ventricular arrhythmia. Nat Cardiovasc Res 2, 1204–1220 (2023). https://doi.org/10.1038/s44161-023-00368-x

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