Disruption of Ca2+i Homeostasis and Connexin 43 Hemichannel Function in the Right Ventricle Precedes Overt Arrhythmogenic Cardiomyopathy in Plakophilin-2-Deficient Mice

Joon-Chul Kim; Marta Perez-Hernandez; Francisco J. Alvarado; Svetlana R. Maurya; Jerome Montnach; Yandong Yin; Mingliang Zhang; Xianming Lin; Carolina Vasquez; Adriana Heguy; Feng-Xia Liang; Sun-Hee Woo; Gregory E. Morley; Eli Rothenberg; Alicia Lundby; Hector H. Valdivia; Marina Cerrone; Mario Delmar

doi:10.1161/circulationaha.119.039710

Disruption of Ca²⁺_iHomeostasis and Connexin 43 Hemichannel Function in the Right Ventricle Precedes Overt Arrhythmogenic Cardiomyopathy in Plakophilin-2-Deficient Mice

Joon-Chul Kim, Marta Perez-Hernandez, Francisco J. Alvarado, Svetlana R. Maurya, Jerome Montnach, Yandong Yin, Mingliang Zhang, Xianming Lin, Carolina Vasquez, Adriana Heguy, Feng-Xia Liang, Sun-Hee Woo, Gregory E. Morley, Eli Rothenberg, Alicia Lundby, Hector H. Valdivia, Marina Cerrone, Mario Delmar

Molecular Cardiology and Membrane Proteins

25 Citations (Scopus)

Abstract

Background: Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy. A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. Methods: We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice (PKP2cKO) 14 days post-tamoxifen injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. Results: Most properties of PKP2cKO left ventricular myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca2+ transients, increased Ca2+ in the cytoplasm and sarcoplasmic reticulum, increased frequency of spontaneous Ca2+ release events (sparks) even at comparable sarcoplasmic reticulum load, and dynamic Ca2+ accumulation in mitochondria. We also observed early- and delayed-after transients in RV myocytes and heightened susceptibility to arrhythmias in Langendorff-perfused hearts. In addition, ryanodine receptor 2 in PKP2cKO-RV cells presented enhanced Ca2+ sensitivity and preferential phosphorylation in a domain known to modulate Ca2+ gating. RNAseq at 14 days post-tamoxifen showed no relevant difference in transcript abundance between RV and left ventricle, neither in control nor in PKP2cKO cells. Instead, we found an RV-predominant increase in membrane permeability that can permit Ca2+ entry into the cell. Connexin 43 ablation mitigated the membrane permeability increase, accumulation of cytoplasmic Ca2+, increased frequency of sparks and early stages of RV dysfunction. Connexin 43 hemichannel block with GAP19 normalized [Ca2+](i) homeostasis. Similarly, protein kinase C inhibition normalized spark frequency at comparable sarcoplasmic reticulum load levels. Conclusions: Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca2+ dysregulation) that precedes the cardiomyopathy; this is, at least in part, mediated by a Connexin 43-dependent membrane conduit and repressed by protein kinase C inhibitors. Given that asymmetric Ca2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

Original language	English
Book series	Circulation
Volume	140
Issue number	12
Pages (from-to)	1015-1030
ISSN	0009-7322
DOIs	https://doi.org/10.1161/circulationaha.119.039710
Publication status	Published - 17 Sept 2019

Keywords

arrhythmogenic right ventricular cardiomyopathy
connexin43
plakophilin 2
right ventricle
sudden death

Access to Document

10.1161/circulationaha.119.039710

CIRCULATIONAHA.119.039710Final published version, 2.85 MB

https://www.ahajournals.org/doi/pdf/10.1161/CIRCULATIONAHA.119.039710

Cite this

Kim, J-C., Perez-Hernandez, M., Alvarado, F. J., Maurya, S. R., Montnach, J., Yin, Y., Zhang, M., Lin, X., Vasquez, C., Heguy, A., Liang, F-X., Woo, S-H., Morley, G. E., Rothenberg, E., Lundby, A., Valdivia, H. H., Cerrone, M., & Delmar, M. (2019). Disruption of Ca²⁺_iHomeostasis and Connexin 43 Hemichannel Function in the Right Ventricle Precedes Overt Arrhythmogenic Cardiomyopathy in Plakophilin-2-Deficient Mice. Circulation, 140(12), 1015-1030. https://doi.org/10.1161/circulationaha.119.039710

Disruption of Ca²⁺_iHomeostasis and Connexin 43 Hemichannel Function in the Right Ventricle Precedes Overt Arrhythmogenic Cardiomyopathy in Plakophilin-2-Deficient Mice. / Kim, Joon-Chul; Perez-Hernandez, Marta; Alvarado, Francisco J. et al.

In: Circulation, Vol. 140, No. 12, 17.09.2019, p. 1015-1030.

Research output: Contribution to journal › Journal article › Research › peer-review

Kim, J-C, Perez-Hernandez, M, Alvarado, FJ, Maurya, SR, Montnach, J, Yin, Y, Zhang, M, Lin, X, Vasquez, C, Heguy, A, Liang, F-X, Woo, S-H, Morley, GE, Rothenberg, E, Lundby, A, Valdivia, HH, Cerrone, M & Delmar, M 2019, 'Disruption of Ca²⁺_iHomeostasis and Connexin 43 Hemichannel Function in the Right Ventricle Precedes Overt Arrhythmogenic Cardiomyopathy in Plakophilin-2-Deficient Mice', Circulation, vol. 140, no. 12, pp. 1015-1030. https://doi.org/10.1161/circulationaha.119.039710

@article{1969ea683dbe4699bd1e4fb3207af984,

title = "Disruption of Ca2+i Homeostasis and Connexin 43 Hemichannel Function in the Right Ventricle Precedes Overt Arrhythmogenic Cardiomyopathy in Plakophilin-2-Deficient Mice",

abstract = "Background: Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy. A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. Methods: We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice (PKP2cKO) 14 days post-tamoxifen injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. Results: Most properties of PKP2cKO left ventricular myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca2+ transients, increased Ca2+ in the cytoplasm and sarcoplasmic reticulum, increased frequency of spontaneous Ca2+ release events (sparks) even at comparable sarcoplasmic reticulum load, and dynamic Ca2+ accumulation in mitochondria. We also observed early- and delayed-after transients in RV myocytes and heightened susceptibility to arrhythmias in Langendorff-perfused hearts. In addition, ryanodine receptor 2 in PKP2cKO-RV cells presented enhanced Ca2+ sensitivity and preferential phosphorylation in a domain known to modulate Ca2+ gating. RNAseq at 14 days post-tamoxifen showed no relevant difference in transcript abundance between RV and left ventricle, neither in control nor in PKP2cKO cells. Instead, we found an RV-predominant increase in membrane permeability that can permit Ca2+ entry into the cell. Connexin 43 ablation mitigated the membrane permeability increase, accumulation of cytoplasmic Ca2+, increased frequency of sparks and early stages of RV dysfunction. Connexin 43 hemichannel block with GAP19 normalized [Ca2+](i) homeostasis. Similarly, protein kinase C inhibition normalized spark frequency at comparable sarcoplasmic reticulum load levels. Conclusions: Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca2+ dysregulation) that precedes the cardiomyopathy; this is, at least in part, mediated by a Connexin 43-dependent membrane conduit and repressed by protein kinase C inhibitors. Given that asymmetric Ca2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.",

keywords = "arrhythmogenic right ventricular cardiomyopathy, connexin43, plakophilin 2, right ventricle, sudden death",

author = "Joon-Chul Kim and Marta Perez-Hernandez and Alvarado, {Francisco J.} and Maurya, {Svetlana R.} and Jerome Montnach and Yandong Yin and Mingliang Zhang and Xianming Lin and Carolina Vasquez and Adriana Heguy and Feng-Xia Liang and Sun-Hee Woo and Morley, {Gregory E.} and Eli Rothenberg and Alicia Lundby and Valdivia, {Hector H.} and Marina Cerrone and Mario Delmar",

year = "2019",

month = sep,

day = "17",

doi = "10.1161/circulationaha.119.039710",

language = "English",

volume = "140",

pages = "1015--1030",

journal = "Circulation. Supplement",

issn = "0009-7322",

publisher = "Lippincott Williams & Wilkins",

number = "12",

}

TY - JOUR

T1 - Disruption of Ca2+i Homeostasis and Connexin 43 Hemichannel Function in the Right Ventricle Precedes Overt Arrhythmogenic Cardiomyopathy in Plakophilin-2-Deficient Mice

AU - Kim, Joon-Chul

AU - Perez-Hernandez, Marta

AU - Alvarado, Francisco J.

AU - Maurya, Svetlana R.

AU - Montnach, Jerome

AU - Yin, Yandong

AU - Zhang, Mingliang

AU - Lin, Xianming

AU - Vasquez, Carolina

AU - Heguy, Adriana

AU - Liang, Feng-Xia

AU - Woo, Sun-Hee

AU - Morley, Gregory E.

AU - Rothenberg, Eli

AU - Lundby, Alicia

AU - Valdivia, Hector H.

AU - Cerrone, Marina

AU - Delmar, Mario

PY - 2019/9/17

Y1 - 2019/9/17

N2 - Background: Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy. A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. Methods: We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice (PKP2cKO) 14 days post-tamoxifen injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. Results: Most properties of PKP2cKO left ventricular myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca2+ transients, increased Ca2+ in the cytoplasm and sarcoplasmic reticulum, increased frequency of spontaneous Ca2+ release events (sparks) even at comparable sarcoplasmic reticulum load, and dynamic Ca2+ accumulation in mitochondria. We also observed early- and delayed-after transients in RV myocytes and heightened susceptibility to arrhythmias in Langendorff-perfused hearts. In addition, ryanodine receptor 2 in PKP2cKO-RV cells presented enhanced Ca2+ sensitivity and preferential phosphorylation in a domain known to modulate Ca2+ gating. RNAseq at 14 days post-tamoxifen showed no relevant difference in transcript abundance between RV and left ventricle, neither in control nor in PKP2cKO cells. Instead, we found an RV-predominant increase in membrane permeability that can permit Ca2+ entry into the cell. Connexin 43 ablation mitigated the membrane permeability increase, accumulation of cytoplasmic Ca2+, increased frequency of sparks and early stages of RV dysfunction. Connexin 43 hemichannel block with GAP19 normalized [Ca2+](i) homeostasis. Similarly, protein kinase C inhibition normalized spark frequency at comparable sarcoplasmic reticulum load levels. Conclusions: Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca2+ dysregulation) that precedes the cardiomyopathy; this is, at least in part, mediated by a Connexin 43-dependent membrane conduit and repressed by protein kinase C inhibitors. Given that asymmetric Ca2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

AB - Background: Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy. A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. Methods: We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice (PKP2cKO) 14 days post-tamoxifen injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. Results: Most properties of PKP2cKO left ventricular myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca2+ transients, increased Ca2+ in the cytoplasm and sarcoplasmic reticulum, increased frequency of spontaneous Ca2+ release events (sparks) even at comparable sarcoplasmic reticulum load, and dynamic Ca2+ accumulation in mitochondria. We also observed early- and delayed-after transients in RV myocytes and heightened susceptibility to arrhythmias in Langendorff-perfused hearts. In addition, ryanodine receptor 2 in PKP2cKO-RV cells presented enhanced Ca2+ sensitivity and preferential phosphorylation in a domain known to modulate Ca2+ gating. RNAseq at 14 days post-tamoxifen showed no relevant difference in transcript abundance between RV and left ventricle, neither in control nor in PKP2cKO cells. Instead, we found an RV-predominant increase in membrane permeability that can permit Ca2+ entry into the cell. Connexin 43 ablation mitigated the membrane permeability increase, accumulation of cytoplasmic Ca2+, increased frequency of sparks and early stages of RV dysfunction. Connexin 43 hemichannel block with GAP19 normalized [Ca2+](i) homeostasis. Similarly, protein kinase C inhibition normalized spark frequency at comparable sarcoplasmic reticulum load levels. Conclusions: Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca2+ dysregulation) that precedes the cardiomyopathy; this is, at least in part, mediated by a Connexin 43-dependent membrane conduit and repressed by protein kinase C inhibitors. Given that asymmetric Ca2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

KW - arrhythmogenic right ventricular cardiomyopathy

KW - connexin43

KW - plakophilin 2

KW - right ventricle

KW - sudden death

U2 - 10.1161/circulationaha.119.039710

DO - 10.1161/circulationaha.119.039710

M3 - Journal article

C2 - 31315456

SN - 0009-7322

VL - 140

SP - 1015

EP - 1030

JO - Circulation. Supplement

JF - Circulation. Supplement

IS - 12

ER -