Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure.
Loss of T-tubules (TT), sarcolemmal invaginations of cardiomyocytes (CMs), was recently identified as a general heart failure (HF) hallmark. However, whether TT per se or the overall sarcolemma is altered during HF process is still unknown. In this study, we directly examined sarcolemmal surface topography and physical properties using Atomic Force Microscopy (AFM) in living CMs from healthy and failing mice hearts. We confirmed the presence of highly organized crests and hollows along myofilaments in isolated healthy CMs. Sarcolemma topography was tightly correlated with elasticity, with crests stiffer than hollows and related to the presence of few packed subsarcolemmal mitochondria (SSM) as evidenced by electron microscopy. Three days after myocardial infarction (MI), CMs already exhibit an overall sarcolemma disorganization with general loss of crests topography thus becoming smooth and correlating with a decreased elasticity while interfibrillar mitochondria (IFM), myofilaments alignment and TT network were unaltered. End-stage post-ischemic condition (15days post-MI) exacerbates overall sarcolemma disorganization with, in addition to general loss of crest/hollow periodicity, a significant increase of cell surface stiffness. Strikingly, electron microscopy revealed the total depletion of SSM while some IFM heaps could be visualized beneath the membrane. Accordingly, mitochondrial Ca(2+) studies showed a heterogeneous pattern between SSM and IFM in healthy CMs which disappeared in HF. In vitro, formamide-induced sarcolemmal stress on healthy CMs phenocopied post-ischemic kinetics abnormalities and revealed initial SSM death and crest/hollow disorganization followed by IFM later disarray which moved toward the cell surface and structured heaps correlating with TT loss. This study demonstrates that the loss of crest/hollow organization of CM surface in HF occurs early and precedes disruption of the TT network. It also highlights a general stiffness increased of the CM surface most likely related to atypical IFM heaps while SSM died during HF process. Overall, these results indicate that initial sarcolemmal stress leading to SSM death could underlie subsequent TT disarray and HF setting.
CNRS, LAAS, F-31400 Toulouse, France; CNRS, ITAV-USR3505, Toulouse, France; Université de Toulouse, ITAV, LAAS, F-31400 Toulouse France. Electronic address: edague@laas.fr.
Etienne
E
Dague
Institut des Maladies Métaboliques et Cardiovasculaires, INSERM UMR1048, Toulouse, France.
Gaël
G
Genet
CNRS, ITAV-USR3505, Toulouse, France.
Véronique
V
Lachaize
Institut des Maladies Métaboliques et Cardiovasculaires, INSERM UMR1048, Toulouse, France; Department of Histopathology, Centre Hospitalier Universitaire de Toulouse, 31432 Toulouse, France.
Céline
C
Guilbeau-Frugier
INSERM U1046, Université Montpellier 1, Université Montpellier 2, Montpellier, France.
Jérémy
J
Fauconnier
Institut des Maladies Métaboliques et Cardiovasculaires, INSERM UMR1048, Toulouse, France.
Céline
C
Mias
Centre de Microscopie Électronique Appliquée à la Biologie, Faculté de Médecine Rangueil, 31062 Toulouse, France.
Bruno
B
Payré
CNRS, LAAS, F-31400 Toulouse, France; CNRS, IPBS-UMR5089, F-31077 Toulouse, France.
Louise
L
Chopinet
Institute of Life Sciences, Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium.
David
D
Alsteens
Department of Cellular Biology and Morphology, Université de Lausanne, Institut de Physique des Systèmes Biologiques, École Polytechnique Fédérale de Lausanne, Switzerland.
Sandor
S
Kasas
CNRS, ITAV-USR3505, Toulouse, France; Université de Toulouse, ITAV, LAAS, F-31400 Toulouse France.
Childerick
C
Severac
INSERM U1046, Université Montpellier 1, Université Montpellier 2, Montpellier, France.
Jérôme
J
Thireau
Institut des Maladies Métaboliques et Cardiovasculaires, INSERM UMR1048, Toulouse, France.
Christophe
C
Heymes
Institut des Maladies Métaboliques et Cardiovasculaires, INSERM UMR1048, Toulouse, France.
Benjamin
B
Honton
INSERM U1046, Université Montpellier 1, Université Montpellier 2, Montpellier, France.
Alain
A
Lacampagne
Institut des Maladies Métaboliques et Cardiovasculaires, INSERM UMR1048, Toulouse, France; Department of Clinical Pharmacology, Centre Hospitalier Universitaire de Toulouse, F-31432 Toulouse, France.
Atul
A
Pathak
Institut des Maladies Métaboliques et Cardiovasculaires, INSERM UMR1048, Toulouse, France; Department of Clinical Pharmacology, Centre Hospitalier Universitaire de Toulouse, F-31432 Toulouse, France.
Jean-Michel
JM
Sénard
CNRS, ITAV-USR3505, Toulouse, France; Institut des Maladies Métaboliques et Cardiovasculaires, INSERM UMR1048, Toulouse, France. Electronic address: celine.gales@inserm.fr.
Céline
C
Galés
D000818:Animals; D004548:Elasticity; D006333:Heart Failure / Q000473:pathology*; D051379:Mice; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D008929:Mitochondria, Heart / Q000648:ultrastructure*; D032383:Myocytes, Cardiac / Q000648:ultrastructure*; D009210:Myofibrils / Q000648:ultrastructure*; D012508:Sarcolemma / Q000648:ultrastructure*
Atomic Force Microscopy; Cardiomyocytes; Heart failure; Interfibrillar mitochondria; Lateral membrane; Subsarcolemmal mitochondria