Desmin intermediate filaments and tubulin detyrosination stabilize growing microtubules in the cardiomyocyte

Abstract

In heart failure, an increased abundance of post-translationally detyrosinated microtubules stiffens the cardiomyocyte and impedes its contractile function. Detyrosination promotes interactions between microtubules, desmin intermediate filaments, and the sarcomere to increase cytoskeletal stiffness, yet the mechanism by which this occurs is unknown. We hypothesized that detyrosination may regulate the growth and shrinkage of dynamic microtubules to facilitate interactions with desmin and the sarcomere. Through a combination of biochemical assays and direct observation of growing microtubule plus-ends in adult cardiomyocytes, we find that desmin is required to stabilize growing microtubules at the level of the sarcomere Z-disk, where desmin also rescues shrinking microtubules from continued depolymerization. Further, reducing detyrosination (i.e. tyrosination) below basal levels promotes frequent depolymerization and less efficient growth of microtubules. This is concomitant with tyrosination promoting the interaction of microtubules with the depolymerizing protein complex of end-binding protein 1 (EB1) and CAP-Gly domain-containing linker protein 1 (CLIP1/CLIP170). The dynamic growth and shrinkage of tyrosinated microtubules reduce their opportunity for stabilizing interactions at the Z-disk region, coincident with tyrosination globally reducing microtubule stability. These data provide a model for how intermediate filaments and tubulin detyrosination establish long-lived and physically reinforced microtubules that stiffen the cardiomyocyte and inform both the mechanism of action and therapeutic index for strategies aimed at restoring tyrosination for the treatment of cardiac disease.

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Basic Research in Cardiology (2022) 117:53
https://doi.org/10.1007/s00395-022-00962-3
ORIGINAL CONTRIBUTION
Desmin intermediate filaments andtubulin detyrosination stabilize
growing microtubules inthecardiomyocyte
AlexanderK.Salomon1· SaiAungPhyo1,2· NaimaOkami1· JulieHeer1· PatrickRobison1· AlexeyI.Bogush1·
BenjaminL.Prosser1
Received: 12 August 2022 / Revised: 26 September 2022 / Accepted: 17 October 2022 / Published online: 3 November 2022
© The Author(s) 2022
Abstract
In heart failure, an increased abundance of post-translationally detyrosinated microtubules stiffens the cardiomyocyte and
impedes its contractile function. Detyrosination promotes interactions between microtubules, desmin intermediate filaments,
and the sarcomere to increase cytoskeletal stiffness, yet the mechanism by which this occurs is unknown. We hypothesized
that detyrosination may regulate the growth and shrinkage of dynamic microtubules to facilitate interactions with desmin
and the sarcomere. Through a combination of biochemical assays and direct observation of growing microtubule plus-ends
in adult cardiomyocytes, we find that desmin is required to stabilize growing microtubules at the level of the sarcomere
Z-disk, where desmin also rescues shrinking microtubules from continued depolymerization. Further, reducing detyrosina-
tion (i.e. tyrosination) below basal levels promotes frequent depolymerization and less efficient growth of microtubules.
This is concomitant with tyrosination promoting the interaction of microtubules with the depolymerizing protein complex
of end-binding protein 1 (EB1) and CAP-Gly domain-containing linker protein 1 (CLIP1/CLIP170). The dynamic growth
and shrinkage of tyrosinated microtubules reduce their opportunity for stabilizing interactions at the Z-disk region, coinci-
dent with tyrosination globally reducing microtubule stability. These data provide a model for how intermediate filaments
and tubulin detyrosination establish long-lived and physically reinforced microtubules that stiffen the cardiomyocyte and
inform both the mechanism of action and therapeutic index for strategies aimed at restoring tyrosination for the treatment
of cardiac disease.
Keywords Cardiomyocyte· Desmin· Microtubule tyrosination· Microtubule dynamics· Microtubule-associated proteins
Abbreviations
MAP Microtubule-associated protein
PTM Post-translational modification
MCAK Mitotic centromere-associated kinesin
TTL Tubulin tyrosine ligase
E331Q Catalytically dead TTL
αTAT1 Alpha-tubulin acetyltransferase 1
HDAC6 Histone deacetylase 6
PLA Proximity ligation assay
EB1 End-binding protein 1
EB3 End-binding protein 3
CLIP170 CAP-Gly domain-containing linker protein 1
Introduction
Microtubules are polymers of ⍺- and β-tubulin that are char-
acterized by cyclical transitions between polymerization and
depolymerization, a behavior called dynamic instability
[25]. Tuning this dynamic behavior confers unique func-
tionality to specific sub-populations of microtubules [12].
Control of microtubule dynamics is cell type- and context-
specific and can occur either by modulating polymer addi-
tion or subtraction at the ends or through lateral interaction
with the microtubule [1]. The temporal and spatial control
of dynamics can be tuned by post-translational modification
of tubulin, which in turn affects the biophysical properties
Alexander K. Salomon and Sai Aung Phyo contributed equally to
this work.
* Benjamin L. Prosser
bpros@pennmedicine.upenn.edu
1 Department ofPhysiology, Pennsylvania Muscle Institute,
University ofPennsylvania Perelman School ofMedicine,
Philadelphia, PA19104, USA
2 Department ofGenetics andEpigenetics, University
ofPennsylvania Perelman School ofMedicine, Philadelphia,
PA19104, USA
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Basic Research in Cardiology (2022) 117:53
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of the microtubule and interactions with microtubule-associ-
ated proteins (MAPs) [32]. For example, detyrosination, the
post-translational removal of the C-terminal tyrosine residue
on ⍺-tubulin, has been shown to alter microtubule stabil-
ity through modulating interactions with multiple effector
MAPs [10, 27, 28].
In the cardiomyocyte, microtubules fulfill both canonical
roles in intracellular trafficking and organelle positioning
[7, 33], as well as non-canonical functions matched to the
unique demands of working myocytes. In the interior of the
myocyte, microtubules form a predominantly longitudinal
network that runs perpendicular to the transverse Z-disks
that define the sarcomere, the basic contractile unit of mus-
cle. This microtubule network is required for the delivery of
essential cargo in the myocyte, including ion channels and
membrane proteins required for muscle excitation, as well
as the distribution of RNA and the translational machinery
to maintain and grow new sarcomeres [33, 39]. To perform
this role, microtubules must also withstand the high forces
and changes in cell geometry inherent to cardiac contraction.
To this end, sub-populations of microtubules form physi-
cal connections at the level of the Z-disk that serve as lat-
eral reinforcements along the length of the microtubule [31].
Upon stimulation and sarcomere shortening, these physically
coupled microtubules buckle at short stereotypical wave-
lengths between sarcomeres to resist the change in myocyte
length [31]. Lateral reinforcement has significant mechanical
ramifications, as reinforced microtubules can resist forces
three orders of magnitude greater than isolated microtu-
bules [5, 36]. This viscoelastic resistance, while modest
under normal conditions, becomes particularly problematic
in heart failure, where the proliferation of stably coupled
microtubules stiffens the cardiomyocyte and impairs myo-
cyte motion [8].
The physical coupling of the microtubule to the sar-
comere is tuned by detyrosination. Reversing detyrosina-
tion (i.e. tyrosination) by overexpression of tubulin tyros-
ine ligase (TTL), the enzyme responsible for ligating the
terminal tyrosine residue on detyrosinated tubulin, reduces
sarcomeric buckling and the viscoelastic resistance provided
by microtubules, thus increasing the contractility of failing
myocytes [8]. Detyrosination also governs microtubule-
dependent mechanotransduction that regulates downstream
second messengers and is implicated in myopathic states
[20]. As reversing detyrosination can lower stiffness and
improve myocardial function in patient tissues [8] and ani-
mal models of heart failure [35, 43], it is under pursuit as
a novel therapeutic approach. Yet how detyrosination pro-
motes the interaction of microtubules with the sarcomere to
regulate myocyte viscoelasticity remains poorly understood.
Several observations suggest this interaction may be
mediated at least in part through desmin intermediate fila-
ments that wrap around the Z-disk. Detyrosination promotes
microtubule interaction with intermediate filaments [17, 31],
and in the absence of desmin, microtubules are disorganized
and detyrosination no longer alters myocyte mechanics [31].
Importantly, a recent publication indicates that intermedi-
ate filaments can directly stabilize dynamic microtubules
invitro [34]. However, there has been no investigation into
the effects of desmin or detyrosination on the dynamics of
cardiac microtubules.
Here, using a combination of genetic manipulations, bio-
chemical assays, and direct live-cell observation of dynamic
microtubules, we interrogated the effects of desmin deple-
tion and tubulin tyrosination on microtubule dynamics. We
find that desmin spatially organizes microtubule dynamics,
conferring local stability to both growing and shrinking
microtubules at the sarcomere Z-disk. Additionally, we find
that tyrosinated microtubules are more dynamic and prone
to shrinkage, a characteristic that precludes their ability to
efficiently grow between adjacent sarcomeres and form sta-
bilizing interactions at the Z-disk. These findings provide
insight into the fundamental organizing principles of myo-
cyte cytoarchitecture and inform on how detyrosination can
promote cytoskeletal stability to tune myocyte mechanics.
Methods
AnimalCare
Animal care and procedures were approved and performed
in accordance with the standards set forth by the University
of Pennsylvania Institutional Animal Care and Use Com-
mittee and the Guide for the Care and Use of Laboratory
Animals published by the US National Institutes of Health.
Rat cardiomyocyte isolation andculture
Primary adult ventricular myocytes were isolated from 6-
to 8-week-old Sprague Dawley rats as previously described
[30]. Briefly, rats were anesthetized under isoflurane while
the heart was removed and retrograde perfused on a Lan-
gendorff apparatus with a collagenase solution. The digested
heart was then minced and triturated using a glass pipette.
The resulting supernatant was separated and centrifuged at
300 revolutions per minute to isolate cardiomyocytes that
were resuspended in rat cardiomyocyte media at a den-
sity that ensured adjacent cardiomyocytes did not touch.
Cardiomyocytes were cultured at 37°C and 5% CO2 with
25μmol/L of cytochalasin D. The viability of rat cardiomyo-
cytes upon isolation was typically on the order of 50–75%
rod-shaped, electrically excitable cells, and the survivability
for 48h of culture is > 80% (See Heffler etal.) [18] for our
quantification of cardiomyocyte morphology in culture).
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Rat cardiomyocyte media: medium 199 (Thermo Fisher
115090) supplemented with 1 × Insulin-transferrin-sele-
nium-X (Gibco 51500056), 1μgμl−1 primocin (Invivogen
ant-pm-1), 20mmol/L HEPES at pH 7.4 and 25μmol/L
cytochalasin D.
Fractionation assay offree tubulin
andcold‑sensitive microtubules
Free tubulin was separated from cold-labile microtubules
using a protocol adapted from Tsutsui etal. 1993 and Ostlud
etal. 1979. Isolated rat cardiomyocytes were washed once
with PBS and homogenized with 250ml of the microtubule-
stabilizing buffer using a tissue homogenizer. The homoge-
nate was centrifuged at 100,000g for 15min at 25°C and
the resulting supernatant was stored at −80°C as the free
tubulin fraction. The pellet was resuspended in ice-cold
microtubule destabilizing buffer and incubated at 0°C for
1h. After centrifugation at 100,000g for 15min at 4°C the
supernatant containing the cold-labile microtubule fraction
was stored at −80°C.
Microtubule stabilizing buffer: 0.5mM MgCl2, 0.5mM
EGTA, 10mM Na3PO4, 0.5mM GTP, and 1X protease and
phosphatase inhibitor cocktail (Cell Signaling #5872S) at
pH 6.95.
Microtubule destabilizing buffer: 0.25M sucrose, 0.5mM
MgCl2 10mM Na3PO4, 0.5mM GTP, and 1X protease and
phosphatase inhibitor cocktail (Cell Signaling #5872S) at
pH 6.95.
Western blot
For whole cell protein extraction, isolated rat cardiomyo-
cytes were lysed in RIPA buffer (Cayman #10010263) sup-
plemented with protease and phosphatase Inhibitor cocktail
(Cell Signaling #5872S) on ice for 1h. The supernatant
was collected and combined with 4X loading dye (Li-COR
#928-40004), supplemented with 10% 2-mercaptoethanol,
and boiled for 10min. The resulting lysate was resolved
on SDS-PAGE gel and protein was blotted to nitrocellulose
membrane (Li-COR #926-31902) with a mini Trans-Blot
Cell (Bio-Rad). Membranes were blocked for an hour in
Odyssey Blocking Buffer (TBS) (LI-COR #927-50000) and
probed with corresponding primary antibodies overnight at
4°C. Membranes were rinsed with TBS containing 0.5%
Tween 20 (TBST) three times and incubated with secondary
antibodies TBS supplemented with extra 0.2% Tween 20 for
1h at room temperature. Membranes were washed again
with TBST (0.5% Tween 20) and imaged on an Odyssey
Imager. Image analysis was performed using Image Studio
Lite software (LI-COR). All samples were run in duplicates
and analyzed in reference to GAPDH.
Antibodies andlabels
Acetylated tubulin; mouse monoclonal (Sigma T6793-
100UL); western blot: 1:1000.
Detyrosinated tubulin; rabbit polyclonal (Abcam
ab48389); western blot: 1:1000.
Alpha tubulin; mouse monoclonal, clone DM1A (Cell
Signaling #3873); western blot: 1:1000.
Alpha tubulin; mouse monoclonal, clone DM1A con-
jugated to AlexaFluor (AF) 488 (Cell Signaling #8058S);
immunofluorescence: 1:100.
Beta tubulin; rabbit polyclonal (Abcam ab6046); western
blot: 1:1000.
Tyrosinated tubulin; mouse monoclonal (Sigma T9028-
0.2ML); immunofluorescence: 1:1000.
Anti-sarcomeric alpha-actinin; mouse monoclonal, clone
EA-53 (Abcam ab9465); western blot, PLA: 1:1000.
Desmin; rabbit polyclonal (ThermoFisher PA5-16705);
western blot, immunofluorescence: 1: 1000.
Desmin; mouse monoclonal, clone D33 (Agilent Tech-
nologies M076029-2); western blot, PLA; 1:500.
EB1; rabbit polyclonal (Sigma E3406-200UL); western
blot, PLA: 1:400.
CLIP170; mouse monoclonal, clone F-3 (Santa Cruz
sc-28325); immunofluorescence, PLA: 1:100.
GAPDH; mouse monoclonal (VWR GenScript A01622-
40); western blot: 1:1000.
Goat anti-mouse AF 488 (Life Technologies A11001);
immunofluorescence: 1:1000.
Goat anti-rabbit AF 565 (Life Technologies A11011);
immunofluorescence: 1:1000.
IRDye 680RD Donkey anti-Mouse IgG (H + L) (LI-COR
926-68072); western blot: 1:10,000.
IRDye 800CW Donkey anti-Rabbit IgG (H + L) (LI-COR
926-32213); western blot: 1:10,000.
Duolink InSitu PLA probe Anti-Rabbit PLUS, Donkey
anti-Rabbit IgG (H + L) (Sigma DUO92002); PLA: 1:5 (as
per manufacturer’s protocol).
Duolink InSitu PLA probe Anti-Mouse MINUS, Donkey
anti-Mouse IgG (H + L) (Sigma DUO92004); PLA: 1:5 (as
per manufacturer’s protocol).
Microtubule dynamics byEB3
Isolated rat cardiomyocytes were infected with an adeno-
virus containing an EB3-GFP construct [37]. After 48h,
cells were imaged on an LSM Zeiss 880 inverted Airys-
can confocal microscope using a 40X oil 1.4 numerical
aperture objective. Cells expressing EB3-GFP only at the
tip were imaged for four minutes at a rate of 1fps. Files
were blinded, Gaussian blurred, and Z-compressed using
Image J (National Institutes of Health) to generate kymo-
graphs. The number of catastrophes, rescues, and pauses
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Basic Research in Cardiology (2022) 117:53
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were recorded per kymograph in addition to the manual trac-
ing of microtubule runs to quantify the time, distance, and
velocity of microtubule growth or shrinkage. We refer to the
entire kymograph as the microtubule ‘track’ that is made up
of individual growth and shrinkage events we call ‘runs’.
Catastrophe and rescue frequency were calculated per cell by
dividing the number of catastrophes or rescues by total time
spent in growth or shrinkage time, respectively. Catastro-
phes and rescues occurring specifically on or off the Z-disk
were normalized by the total time of microtubule growth
and shrinkage. Experimental values were normalized to their
respective control cells (Null for TTL and E331Q, or shScrm
for shDes) acquired from the same animals. A minimum of
3 separate cell isolations were performed for each group.
Immunofluorescence
To stain for desmin, cardiomyocytes were fixed in pre-
chilled 100% methanol for 8min at −20°C. Cells were
washed 4 × then blocked with Sea Block Blocking Buffer
(Abcam #166951) for at least 1h followed by antibody incu-
bation in Sea Block for 24–48h. Incubation was followed by
washing 3 × with Sea Block, then incubated with secondary
antibody for 1h at RT. Fixed cells were mounted using Pro-
long Diamond (Thermo #P36961).
To stain for CLIP170, cardiomyocytes were glued to
cleaned coverglass (Electron Microscopy Sciences 72222-
01) using MyoTak (IonOptix). The cardiomyocytes on
coverslips were fixed in 4% paraformaldehyde (Electron
Microscopy Sciences 15710) for 10min at RT, followed
by 2 washes in PBS, and then permeabilized using 0.25%
Triton in PBS for 10min at RT. Cells were washed 3 × then
blocked with Sea Block Blocking Buffer for at least 1h fol-
lowed by antibody incubation in Sea Block for 24. Incuba-
tion was followed by washing 3 × with PBS, then incubated
with secondary antibody in Sea Block for 1h at RT. Fixed
cells were mounted using Prolong Diamond.
We used ImageJ to calculate the percent area fraction of
desmin or the mean integrated density of CLIP170. An ROI
was drawn to include the entire cell boundary. To calculate %
area for desmin, we identified the percent fractional coverage
of a fluorescence signal over a manually identified threshold
for each image as described previously [8]. The mean inte-
grated density data for CLIP170 was collected directly from
ImageJ output using unthresholded max-intensity projected
images (3 images per cell) of individual cells.
Buckling analysis
Adult rat cardiomyocytes were isolated as previously
described [30] and infected with adenovirus carrying the
microtubule-binding protein EMTB chimerically fused
to 3 copies of GFP. The purpose of this construct was to
label microtubules fluorescently for imaging. The cells
were allowed 48h to express the construct. To interrogate
microtubule buckling amplitude and wavelength, cells were
induced to contract at 1Hz 25V and imaged during the
contraction. For analysis, images were blinded, and a micro-
tubule was located that could be followed during the contrac-
tion. The backbone was manually traced at rest and during
its peak of the contraction and the ROI was saved. The ROI
was then analyzed using a macro that rotated so that the
ROI had the peak of contraction 90 degrees to the axis of
contraction to protect from aliasing errors. The program then
calculated the distance between the axis of the ROI and its
peak and calculated the peak (amplitude) and the width (half
wavelength).
Nanoindentation
Nanoindentation was performed using nanoindenter (Piuma
Chiaro, Optics11, The Netherlands) as previously described
[9]. Briefly, isolated adult rat cardiomyocytes were attached
to glass bottom dishes coated with MyoTak13 in normal
Tyrode’s solution containing 140mmol/L NaCl, 0.5mmol/L
MgCl2, 0.33 mmol/L NaH2PO4, 5mmol/L HEPES,
5.5mmol/L glucose, 1mmol/L CaCl2, 5mmol/L KCl, pH to
7.4 at room temperature. A spherical nano-indentation probe
with a radius of 3.05μm and stiffness of 0.026N m−1 was
used. Myocytes were indented to a depth of 1.5–3.5μm with
velocities of 0.1, 0.25, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0,
100.0, and 150.0μm s−1. The tip was held in this indenta-
tion depth for 1s and retracted over 2s. The Young’s moduli
were calculated automatically by the software by fitting the
force versus indentation curve to the Hertz equation.
Electron microscopy
Transmission electron microscopy images were collected as
previously described [18]. Images at 7500× were rotated so
the cells were parallel to the longitudinal axis. ROIs were
generated between adjacent Z-disks to quantify sarcomere
spacing and the angle relative to 90°.
Proximity ligation assay (PLA)
Freshly isolated rat cardiomyocytes were untreated or
treated for 48h with Null, TTL, or E331Q adenoviruses at
37°C with 5% CO2. Once viral construct expressions were
confirmed using the tagged mCherry, the cardiomyocytes
were glued to cleaned coverglass (EMS 72222-01) using
MyoTak (IonOptix). The cardiomyocytes on coverslips
were fixed in 4% paraformaldehyde for 10min at RT, fol-
lowed by 2 washes in PBS, and then permeabilized using
0.25% Triton in PBS for 10min at RT. The samples were
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blocked in Sea Block for 1h at RT and stored at 4°C until
further processing.
The samples were incubated with EB1 and CLIP170 or
alpha-actinin or desmin, primary antibodies overnight at
4°C; the coverslips were then washed in PBS for 15min
at RT. Following immediately, PLA was performed in
humidified chambers using ThermoFisher DuoLink man-
ufacturer’s protocol starting with “Duolink PLA Probe
Incubation.” Briefly, the samples were incubated with
Duolink PLA secondary antibodies followed by ligation
and amplification. Amplification was performed using
Duolink FarRed detection reagents (Sigma DUO92013).
Post-amplified samples were washed and incubated with
alpha-tubulin antibody (DM1A) conjugated to AF 488
(Cell Signaling #8058S) in Sea Block overnight at RT. The
processed samples were washed twice with PBS, and the
coverslips were mounted using ProLong Diamond Anti-
fade Mountant (Thermo Fisher P36961).
Imaging was performed using a Zeiss AiryScan micro-
scope. 6 imaging slices of 0.18mm thickness were sam-
pled for each cell; 10 cells were sampled per group per
experiment (N = 3, n = 30). ImageJ was used to analyze
the images. Microtubules and PLA channels were thresh-
olded and the thresholded images were used to construct
a microtubule-PLA overlap image. An ROI was drawn
to outline the cardiomyocyte border. The raw integrated
intensities of the thresholded microtubule only, PLA only,
and the microtubule-PLA overlap images for each imaging
slice were collected. The microtubule-PLA overlap was
then normalized to microtubule only to account for cellu-
lar and sub-cellular heterogeneity of microtubule density.
The average microtubule-normalized microtubule-PLA
overlap for one cell was calculated and the data set was
constructed by normalizing all values from one experiment
to the average control value of that experiment.
Statistics
Statistical analysis was performed using OriginPro (Ver-
sion 2018 & 2019). Normality was determined by the
Shapiro–Wilk test. For normally distributed data, Two-
sample Student’s T test or one-way ANOVA with post
hoc test was utilized as appropriate. For non-normally
distributed data, the Two-sample Kolmogorov–Smirnov
test or Kruskal–Wallis ANOVA was utilized as appropri-
ate. Specific statistical tests and information on biological
and technical replicates can be found in the figure legends.
Unless otherwise noted, ‘N’ indicates the number of cells
analyzed and ‘n’ indicates the number of microtubule runs.
Results
Dynamic microtubules are stabilized attheZ‑disk
andinteract withdesmin intermediate filaments
To study the dynamics of growing microtubules in mature
cardiomyocytes, we treated adult rat cardiomyocytes with
adenovirus containing GFP-labeled End-Binding Protein
3 (EB3-GFP) to directly visualize the plus-end of grow-
ing microtubules by time-lapse imaging (S. Movie 1). The
dynamic properties of microtubules can be quantified as
events that mark their transitions from growing (polym-
erization) to shrinking (depolymerization) states (Fig.1a).
These events consist of catastrophes (transitions from
growth to shrinkage), rescues (transitions from shrinkage
to growth), and pauses (neither growth nor shrinkage).
Conveniently, EB3-GFP also provided a fainter, non-spe-
cific labeling of the protein-rich Z-disk region, enabling
us to visualize where dynamic events occurred relative to
a sarcomeric marker (Fig.1b).
Under basal conditions, we observed a stark spatial bias
in microtubule dynamic behavior, similar to that previ-
ously observed [13]. The initiation of microtubule growth,
as well as pausing of growth, predominantly occurred at
the level of the Z-disk (Fig.1c). Conversely, catastrophes
predominantly occurred off the Z-disk, while rescue from
catastrophe again occurred more frequently at the Z-disk.
As exemplified in S. Movies 1–2, myocyte microtubules
tend to grow iteratively from one Z-disk to another, often
pausing at each Z-disk region. If a microtubule undergoes
catastrophe before reaching a Z-disk, it tends to shrink to
a previous Z-disk, where rescue is more likely to occur.
These data suggest factors at the Z-disk region strongly
bias microtubule behavior and support the initialization
and stabilization of growing microtubules.
Electron microscopy images of cardiomyocytes help
illustrate the local environment surrounding microtubules
at the nanoscale and suggest nearby elements that may
stabilize microtubules. As seen in Fig.1d, the microtu-
bules running along the long-axis of the myocyte appear
as 25nm diameter tubes coming at the viewer in transverse
sections, with a faint halo surrounding them where their
C-terminal tails project. Microtubules most commonly
run alongside, and not within, the sarcomere-containing
myofibrils, squeezing in the gaps between myofibrils and
the mitochondria or nucleus. Desmin intermediate fila-
ments also occupy some of these gaps, wrapping around
the myofibrils at the level of the Z-disk, and we observe
microtubules bisecting through structures that resemble
intermediate filaments and which surround the myofibrils
at these locations (Fig.1d, right). To orthogonally probe
whether growing microtubules are more likely to interact
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Fig. 1 Dynamic microtubules are stabilized at the Z-disk and pref-
erentially interact with desmin intermediate filaments. a Schematic
of the transition states of microtubule dynamics. b Representative
kymograph from control cardiomyocytes transduced with AdV-EB3-
GFP; black arrows denote Z-disk and colored arrows denote transi-
tion events. c Quantification of initiation, rescue, pause, and catastro-
phe events On and Off the Z-disk in control cardiomyocytes (N = 19
cells, n = 228 events). The bar represents mean ± 1SEM; statistical
significance determined with Two Sample Kolmogorov–Smirnov
Test. d Representative EM images from transverse sections of iso-
lated cardiomyocytes. Microtubules are denoted by white arrows.
In the right-hand panel, the area between the myofibrils is filled by
membranous and filamentous structures consistent with intermedi-
ate filaments, which are bisected by microtubules. e Representative
immunofluorescent images and f quantification of a-actinin-EB1 or
desmin-EB1 PLA interactions in control cardiomyocytes (N = 3 rats,
n = 10 cells per rat). The box represents the 25th and 75th percen-
tiles ± 1SD, bolded line represents the mean; statistical significance
was determined with two-sample Student’s T test
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Basic Research in Cardiology (2022) 117:53
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with the intermediate filament vs. sarcomeric cytoskel-
eton, we utilized proximity ligation assay (PLA) to probe
interactions between the endogenous microtubule plus-
end tracking protein end-binding protein 1 (EB1) and
either sarcomeric a-actinin or the intermediate filament
desmin in adult rat cardiomyocytes. Although a-actinin
is the most abundant protein in the Z-disk and expressed
at substantially higher levels than desmin [8] (S. Fig.1a),
we observed ~ tenfold more abundant PLA puncta in the
desmin-EB1 group compared to a-actinin-EB1, suggest-
ing that the growing end of microtubules are frequently
in close proximity to desmin intermediate filaments at the
Z-disk (Fig.1e, f).
Desmin stabilizes growing andshrinking
microtubules attheZ‑disk
We next directly interrogated the role of desmin in regulat-
ing microtubule stability by adenoviral delivery of shRNA
to acutely deplete desmin (desmin KD) in cardiomyocytes.
Complementing our previous validation of this construct by
western blotting [18], we measured a 40–50% reduction in
desmin expression after 48h of desmin KD (S. Fig.1b). We
first interrogated the effect on microtubule stability using a
modified subcellular fractionation assay from Fasset etal.
[15] that allowed us to separate free tubulin from polymer-
ized tubulin in the dynamic (i.e. cold-sensitive) microtu-
bule pool (Fig.2a). Acute desmin depletion resulted in an
increased free to polymerized ratio in the dynamic micro-
tubule pool (Fig.2b,c, S, Fig.1c), suggesting that desmin
coordinates the stability of dynamic microtubules. We next
quantified microtubule acetylation and detyrosination, mark-
ers of long-lived microtubules, and found that both were
decreased in desmin KD myocytes, without alterations
in whole cell tubulin content (Fig.2b, c), suggesting that
desmin normally helps maintain microtubule stability.
Next, we directly quantified plus-end microtubule dynam-
ics by EB3-GFP upon desmin depletion. Blind quantification
of global event frequency revealed that desmin depletion
modestly increased the frequency of catastrophes while
more robustly reducing both the frequency of rescues and
pauses (Fig.2d, e). As seen in S. Movies 3–4, upon desmin
depletion (S. Movies 4) microtubule growth still initiated at
the Z-disk, but the iterative, longitudinal growth from one
Z-disk to another seen in control cells (S. Movies 3) was
lost. Instead, microtubules often grew past Z-disk regions
without pausing, and following catastrophe they were less
likely to be rescued at the previous Z-disk (Fig.2d, f). Inter-
rogation of where dynamic events occurred in relation to the
Z-disk revealed that desmin depletion specifically increased
the number of catastrophes that occurred on the Z-disk while
reducing the number of catastrophes that occurred off the
Z-disk (Fig.2f). More strikingly, desmin depletion markedly
reduced the number of pauses and rescues that occur spe-
cifically on the Z-disk, while not affecting pause or rescue
behavior elsewhere (Fig.2f). Together, these results indicate
that desmin spatially coordinates microtubule dynamics and
stabilizes both the growing and shrinking microtubule at
the Z-disk.
Cardiomyocytes from global, desmin germ-line knockout
mice are characterized by misaligned and degenerated sar-
comeres with a disorganized microtubule network [6, 31].
Gross restructuring of the myofilaments could affect micro-
tubule dynamics due to a change in the physical environment
that is permissive to microtubule growth, for example by
increasing the spacing between Z-disks of adjacent myofila-
ments. To assess if our comparatively brief desmin depletion
altered myofilament spacing or alignment, we performed
quantitative measurements on electron micrographs from
desmin KD cardiomyocytes. Blind analysis indicated that
this relatively short-term desmin depletion did not detect-
ably alter myofilament spacing or alignment (S. Figure2),
consistent instead with a direct stabilizing effect of desmin
intermediate filaments on the microtubule network.
We next interrogated the functional consequences of
this reduced microtubule stability driven by desmin deple-
tion. As a reduction in detyrosinated microtubules and
their association with the Z-disk is associated with reduced
cardiomyocyte viscoelasticity [31], we hypothesized that
desmin-depleted myocytes would be less stiff. To test this,
we performed transverse nanoindentation of cardiomyo-
cytes and quantified Young’s modulus of the myocyte over
a range of indentation rates. Desmin depletion specifically
reduced the rate-dependent viscoelastic stiffness of the myo-
cyte without significantly altering rate-independent elastic
stiffness (S. Fig.3a, b). Reduced viscoelasticity is consist-
ent with reduced transient interactions between dynamic
cytoskeletal filaments.
To directly test if the reduction in desmin alters micro-
tubule buckling between sarcomeres, we performed a
semi-automated, blind analysis of microtubule buckling, as
in our previous work [31]. In control cells, most microtu-
bules buckle in a clear sinusoidal pattern with a wavelength
corresponding to the distance of a contracted sarcomere
(~ 1.5–1.9µm) (S. Fig.3c, d) (S. Movie 5). Upon desmin
depletion, fewer polymerized microtubules were observed
in general, with more chaotic deformations and organization
upon contraction (S. Movie 6). For microtubules that did
buckle, we observed reductions in the amplitude of buckles
(S. Fig.3d) and the proportion of microtubules that buck-
led at wavelengths corresponding to the distance between
1 and 2 sarcomeres (1.5–1.9 or 3.0–3.8µm, respectively)
(S. Fig.3e, f). Combined, these results are consistent with
desmin coordinating the physical tethering and lateral rein-
forcement of detyrosinated microtubules at the cardiomyo-
cyte Z-disk to regulate myocyte viscoelasticity.
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Basic Research in Cardiology (2022) 117:53
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Fig. 2 Desmin stabilizes dynamic microtubules at the Z-disk. a Over-
view of the cell fractionation assay adapted from Fassett etal. [15]
that allows for the separation of free tubulin and polymerized micro-
tubules within the dynamic tubulin pool. b Representative western
blot and c quantification of α-tubulin in free and dynamic microtubule
fractions (top) or of total dTyr-tubulin, α-tubulin, and acetylated tubu-
lin in the whole-cell lysate (bottom) from control (Scram) or Desmin
knock-down (Des KD) cardiomyocytes (N = 3 rats, n = 5 WB techni-
cal lanes for dtyr and 6 for acetyl and tubulin fractions). d Represent-
ative EB3-GFP kymograph from Scram (top) or Des KD (bottom)
cardiomyocytes. e Quantification of catastrophe, pause, and rescue
event frequencies and f event locations in Scram or Des KD cardio-
myocytes (N = cells, n = events). The bar represents mean ± 1SEM;
statistical significance for C was determined with two-sample Stu-
dent’s T test, and for E and F was determined with two-sample Kol-
mogorov–Smirnov test
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Tyrosination alters thedynamics ofthemicrotubule
network
Next, we sought to determine the effect of detyrosination on
the dynamics of the cardiomyocyte microtubule network.
To reduce detyrosination, we utilized adenoviral delivery of
TTL into isolated adult rat cardiomyocytes [31]. TTL binds
and tyrosinates tubulin in a 1:1 complex, and this binding
leads to tubulin sequestration. Hence, to separate the effects
of tubulin tyrosination from tubulin sequestration, we uti-
lized adenoviral delivery of TTL-E331Q (E331Q), a verified
catalytically dead mutant of TTL that binds and sequesters
tubulin but does not tyrosinate [9]. We have previously con-
firmed that TTL overexpression under identical conditions
reduces detyrosination below 25% of initial levels, while
TTL-E331Q does not significantly affect detyrosination lev-
els with similar overexpression [9]. To specifically quan-
tify the effects of reducing detyrosination on the dynamic
microtubule population, we fractionated free and polymer-
ized tubulin as outlined above (Fig.2a). Expression of TTL,
but not E331Q, resulted in significantly less detyrosinated
tubulin in the dynamic microtubule pool (Fig.3a). Further,
only TTL expression shifted tubulin away from the polymer-
ized fraction towards the free tubulin fraction, resulting in
an increased ratio of free:polymerized tubulin (Fig.3a, S.
Fig.4a). This suggests that tyrosination affects the cycling
of tubulin within the dynamic microtubule pool. If indeed
tyrosinated microtubules are more dynamic, then levels of
acetylation, a canonical marker of long-lived microtubules
[40], should also be decreased by TTL. Consistent with this,
TTL, but not E331Q, led to a robust reduction in levels of
microtubule acetylation, suggesting that tyrosination reduces
microtubule lifetime in the cardiomyocyte (Fig.3b).
As acetylation itself is linked to microtubule stability
[14, 42], the TTL-dependent change in the dynamic micro-
tubule pool (Fig.3b) could be directly related to tyrosina-
tion, or it could be a secondary effect due to the reduction in
acetylation. To discriminate between these two hypotheses,
we directly modulated acetylation. To this end, we devel-
oped adenoviral constructs encoding histone deacetylase
6 (HDAC6) and α tubulin acetyltransferase 1 (αTAT1).
HDAC6 expression reduced total microtubule acetylation
to 25% of initial levels (Fig.3c) and αTAT1 expression
increased acetylation 12-fold (Fig.3c). Because αTAT1 has
been shown to modulate microtubule dynamics independent
of enzymatic activity [19], we also used a pharmacological
inhibitor of HDAC6, Tubastatin A (TubA) to increase acet-
ylation through an orthogonal approach (Fig.3c). Having
validated robust tools to modulate acetylation, we next deter-
mined the effect of acetylation on the dynamic microtubule
pool utilizing the same fractionation assay. Neither increas-
ing nor decreasing acetylation altered the free:polymerized
tubulin ratio (Fig.3d, S. Fig.4b). Given that modulating
tyrosination altered levels of acetylation (Fig.3c), we also
asked whether this relationship was reciprocal. However,
whole-cell levels of detyrosination were largely unaffected
by modulating acetylation (Fig.3e), except for a modest
increase with HDAC6 expression that may be related to
HDAC6 association with microtubules increasing their sta-
bility and availability for detyrosination [2]. Together, these
results suggest tyrosination directly alters cardiomyocyte
microtubule stability, independent of corresponding changes
in acetylation.
Tyrosination promotes catastrophe ofgrowing
microtubules
Next, to precisely quantify the effects of tyrosination on
the dynamics of individual microtubules, we overexpressed
either Null, TTL, or E331Q viruses in conjunction with
EB3-GFP in adult rat cardiomyocytes. Although EB inter-
action is thought to be unaffected by microtubule detyrosi-
nation [27], we first wanted to validate that EB3 labeling of
microtubules did not systematically differ with TTL expres-
sion. EB3 fluorescence intensity along the length and at the
tip of the microtubule was unchanged in control, TTL, or
E331Q expressing cells (S. Fig.4c), indicating that EB3
expression or labeling of microtubules was not altered by
our experimental interventions.
As seen in S. Movie 7, microtubules in TTL-expressing
cells still initiated growth at the Z-disk, but often had shorter
runs and underwent catastrophe before reaching a subse-
quent Z-disk. Consistently, TTL overexpression significantly
increased the frequency of catastrophes, while reducing the
frequency of pausing (Fig.4a, b). E331Q expression did not
alter event frequency compared to control cells (S. Movie
8), suggesting a tyrosination-specific effect on microtu-
bule dynamics (S. Fig.4d). Further examination of spatial
dynamics revealed that the effect of TTL on microtubule
breakdown was agnostic to subcellular location; TTL simi-
larly increased the number of catastrophes both on and off
the Z-disk. In contrast, TTL reduced the number of pauses
specifically on the Z-disk (Fig.4c). As a readout of inef-
ficient growth, TTL increased the tortuosity of microtubule
trajectories, defined as the ratio of growth distance to net
growth (Fig.4d). Combined, the lack of stabilization at the
Z-disk and more frequent catastrophes resulted in tyrosi-
nated microtubules depolymerizing ~ fivefold as often before
successfully crossing a Z-disk when compared to either null
or E331Q expressing cells (Fig.4d). In sum, this data indi-
cates that tyrosination increases the stochastic transition to
microtubule breakdown irrespective of subcellular location
and that tyrosinated microtubules inefficiently navigate suc-
cessive sarcomeres with fewer stabilizing interactions at the
Z-disk.
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Basic Research in Cardiology (2022) 117:53
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Fig. 3 TTL reduces microtubule stability through its tyrosinase activ-
ity. a Representative western blot (top) and quantification (bottom) of
α-tubulin and detyrosinated (dTyr) tubulin in free and cold-sensitive
dynamic microtubule fractions from adult rat cardiomyocytes treated
with null, TTL, or TTL-E331Q adenoviruses; detyrosinated tubulin
values are normalized to α-tubulin in cold-sensitive fraction (N = 4
rats, n = 8 WB technical lanes). b Representative western blot (top)
and quantification (bottom) of α-tubulin and acetylated tubulin in
whole-cell lysate from null, TTL, or E331Q expressing cardiomyo-
cytes (N = 3 rats, n = 6 WB technical lanes). c Validation of HDAC6
and αTAT1 constructs and Tubastatin A (TubA) treatment. Repre-
sentative western blot (top) and quantification (bottom) of a-tubulin
and acetylated tubulin in whole-cell lysate from adult rat cardiomyo-
cytes treated with null, HDAC6, or αTAT1 adenoviruses, or DMSO
or 1 mM TubA treatment overnight (N = 3 rats, n = 6 WB technical
lanes). d Representative western blot (top) and quantification (bot-
tom) of α-tubulin and acetylated tubulin, in free and polymerized
dynamic fractions. Lysates from cardiomyocytes were infected with
null, HDAC6, or αTAT1 adenoviruses, or DMSO or 1 mM TubA
overnight (N = 3 rats, n = 6 WB technical lanes). e Representa-
tive western blot (top) and quantification (bottom) of α-tubulin and
detyrosinated tubulin in whole-cell lysate from adult rat cardiomyo-
cytes treated with null, HDAC6, or αTAT1 adenoviruses, or DMSO
or 1 mM TubA treatment overnight (N = 4 rats, n = 8 WB technical
lanes). The bar represents mean ± 1SEM; statistical significance for
(a) and (b) was determined with one-way ANOVA with post hoc test,
and for (c) to (e) was determined with Two-sample Student’s T test
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Basic Research in Cardiology (2022) 117:53
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To summarize how our different interventions (tyrosina-
tion, desmin depletion) affected the spatial organization
of microtubule behavior, we took the ratio of events that
occurred on vs. off the Z-disk and performed a log2 trans-
form, calculating a “Z-disk bias” for each type of dynamic
event (Fig.4e). Of note, this metric only reflects the spa-
tial bias of events, not their frequencies. TTL reduced the
preference for microtubule pausing at the Z-disk but did
not affect the spatial preference of rescues, catastrophes,
or initiations. Desmin depletion, on the other hand, virtu-
ally eliminated the typical Z-disk bias for pauses, rescues,
or fewer catastrophes. Initiations had a strong Z-disk bias
regardless of intervention, which likely reflects nucleating
events from microtubule organizing centers at Golgi out-
posts proximal to the Z-disk that are not affected by these
manipulations [26].
Tyrosination increases EB1 andCLIP170 association
onmicrotubules
Next, we wanted to determine why tyrosinated microtu-
bules exhibit increased catastrophe frequencies. Several
pieces of evidence suggest that the tyrosinated or detyrosi-
nated status of the microtubule alone is likely insufficient to
alter microtubule dynamics [21, 41], but instead the PTM
exerts its effect by governing the interaction of stabilizing/
destabilizing MAPs with the microtubule [10, 28]. There
are two prominent examples of tyrosination altering inter-
actions with depolymerizing effector proteins in the litera-
ture. First, mitotic centromere-associated kinesin (MCAK/
Kif2C) is a depolymerizing MAP that preferentially binds
and depolymerizes tyrosinated microtubules [28]. Second, a
recent invitro reconstitution study indicates that tyrosination
Fig. 4 Tyrosinated microtubules are more dynamic. a Representa-
tive kymographs from cardiomyocytes treated with EB3-GFP plus
null, TTL, or E331Q adenoviruses. b Quantification of catastrophe
and pause event frequencies and c event locations in cardiomyo-
cytes treated with EB3-GFP plus null, TTL, or E331Q adenoviruses
(N = cells, n = events). d Gross measurements of microtubule dynam-
ics. (Left) Tortuosity, the distance a microtubule grows divided
by its displacement, & (right) number of catastrophes in relation to
the number of successful Z-disk crossing in cardiomyocytes treated
with EB3-GFP plus null, TTL, or E331Q adenoviruses. e Z-disc bias
score (log2 transformation of the ratio of events that occurred On vs.
Off the Z-disk) for all experimental conditions. The bar represents
mean ± 1SEM; statistical significance was determined with Kruskal–
Wallis ANOVA with post hoc test
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Basic Research in Cardiology (2022) 117:53
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promotes the binding of CLIP170 on microtubule plus ends,
which synergizes with EB1 to increase the frequency of
catastrophes [10]. This mechanism has not been examined in
cells. Due to its low abundance in the post-mitotic cardiomy-
ocyte, our attempts to detect and knock down MCAK levels
were unreliable; we thus hypothesized that tyrosination may
promote the interaction of EB1 and CLIP170 on microtu-
bules to promote their destabilization and catastrophe.
To test this hypothesis, we utilized a PLA to test whether
EB1 and CLIP170 interactions on cardiac microtubules
were guided by tyrosination. We first performed control
assays to ensure the specificity of this PLA assay and ask
whether EB1-CLIP170 interactions are observed on intact
microtubules. No PLA puncta were observed when primary
antibodies against EB1 or CLIP170 were excluded from
the PLA assay (S. Fig.5a). Further, the majority of EB1-
CLIP170 interactions co-localized directly on super-resolved
microtubules (Fig.5a) indicating that interactions occur pri-
marily on the polymerized microtubule. We next evaluated
whether this interaction was sensitive to tyrosination. First,
we ensured that global levels of EB1 or CLIP170 were not
changing due to TTL or E331Q expression (Fig.5b, c). We
then quantified specific interactions of EB1-CLIP170 that
were occurring on microtubules by thresholding the micro-
tubule and PLA images, quantifying the fractional area
covered by their overlap, and normalizing that area to the
microtubule coverage in the same image plane (S. Fig.5b).
As shown in Fig.5d, TTL increased the number of EB1-
CLIP170 interactions per microtubule area by ~ fourfold
relative to control or E331Q transduced cardiomyocytes
(Fig.5d), despite unchanging levels of EB1 or CLIP170.
As this interaction has been demonstrated to be sufficient
to robustly increase the catastrophe frequency of dynamic
microtubules [10], we conclude that tyrosination destabilizes
cardiac microtubules at least in part by promoting increased
association with the destabilizing effector complex of EB1
and CLIP170.
Discussion
In this paper we identify that (1) desmin intermediate fila-
ments structure and stabilize growing microtubules; (2)
microtubule tyrosination promotes destabilizing interactions
with EB1 + CLIP170; (3) the catastrophe-prone nature of
tyrosinated microtubules precludes their ability to faith-
fully traverse and be stabilized at successive Z-disks. When
combined with recent invitro studies using reconstituted
microtubules and intermediate filaments [10, 34], our in
cellulo findings provide a molecular model for how chang-
ing levels of desmin and detyrosination may synergistically
control cytoskeletal stability in the heart. These findings also
provide guidance for strategies that target the tyrosination
cycle for the treatment of heart failure.
This study represents the first direct observation that
tyrosination increases the dynamics of cardiac microtubules.
A recent report provides compelling evidence to support the
long-standing belief that altered dynamicity does not arise
from tyrosination/detyrosination itself, but instead through
PTM-dependent changes in the recruitment of effector pro-
teins [10, 21, 27]. The C-terminal tyrosine on unstructured
tubulin tails is likely insufficient to influence lateral contacts
between tubulin dimers in the microtubule lattice that confer
stability. Yet the removal of the large hydrophobic tyrosine
residue, and the subsequent exposure of acidic residues, will
alter hydrophobic and electrostatic interactions on the outer
surface [28] of the polymerized microtubule. Through such
a mechanism, tyrosination can promote microtubule dynam-
ics via increased interaction with destabilizing MAPs, or
through decreased interaction with stabilizing MAPs.
As a case in point, tyrosination increases the affinity of
the depolymerizing kinesin MCAK for the microtubule,
decreasing microtubule stability [28]. The low abundance
of MCAK in the cardiomyocyte motivated interrogation
into alternative stabilizing or destabilizing effector pro-
teins. Tyrosination is also known to impact the recruitment
of plus-end tip proteins (+TIPs), such as CLIP170 and p150
glued [27], which can tune microtubule dynamics through
either direct or indirect effects. +TIP proteins can couple the
growing microtubule plus end to subcellular targets through
a search and capture mechanism [22, 24]. While +TIP inter-
action with a target often stabilizes searching microtubules,
Chen etal. recently found that the tyrosination-dependent
recruitment of the +TIP CLIP170 paradoxically led to a syn-
ergistic interaction with EB1 that selectively reduced the
stability of tyrosinated microtubules, increasing their catas-
trophe frequency. Here we find that in cardiomyocytes, while
tyrosination does not affect the global levels of either EB1 or
CLIP170 (Fig.5b, c), it robustly increases the frequency of
their interaction on microtubules (Fig.5c, d), concomitant
with increased frequency of catastrophe (Fig.4b). While
this does not rule out other potentially destabilizing effects
of tyrosination, it provides one mechanism for the increased
dynamicity/decreased stability of tyrosinated microtubules.
We also identified that the intermediate filament desmin
provides structure to the growing microtubule network by
stabilizing both growing and shrinking microtubules at the
cardiomyocyte Z-disk. What is the mechanism of desmin-
dependent stabilization? A recent elegant invitro study
using reconstituted vimentin intermediate filaments and
microtubules indicates that intermediate filaments are suffi-
cient to stabilize growing microtubules through electrostatic
and hydrophobic interactions [34]. Dynamic microtubules
interacting with intermediate filaments reduce catastro-
phes and promote rescues, in strong accordance with our in
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Basic Research in Cardiology (2022) 117:53
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Page 13 of 16 53
cellulo findings. While MAPs may also be involved in mod-
ulating microtubule-intermediate filament interactions, this
direct effect is sufficient to explain the primary phenotypes
we observe upon desmin depletion (i.e. increased catastro-
phes, reduced pausing, and a loss of rescues at the Z-disk).
Desmin stabilization of growing microtubules would provide
a longer-lived microtubule substrate to facilitate reinforcing
interactions, such as those previously documented between
desmin and the microtubule through Kinesin-1 [23] or mem-
bers of the plakin family of cytoskeletal cross-linkers [16].
Fig. 5 Tyrosination promotes EB1 and CLIP170 interactions on car-
diomyocyte microtubules. a Representative AiryScan Joint Deconvo-
luted immunofluorescent images of EB1-CLIP170 PLA interactions
in adult rat cardiomyocytes treated with null, TTL, or TTL-E331Q
adenoviruses. b Representative western blot (top) and quantification
(bottom) of EB1 in whole-cell lysate from adult rat cardiomyocytes
treated with null, TTL, or E331Q adenoviruses for 48 h (N = 3 rat,
n = 3 WB technical lanes). c Representative immunofluorescent
images (left) and quantification (right) of CLIP170 in adult rat car-
diomyocytes treated with null, TTL, or E331Q adenoviruses for 48h
(N = 3 rats, n = 10 cells per rat). d Quantification of EB1-CLIP170
PLA interactions in adult rat cardiomyocytes treated with null, TTL,
or TTL-E331Q adenoviruses (N = 3 rats, n = 10 cells per rat). The bar
represents mean ± 1SEM, and the middle line in the box graph rep-
resents mean ± 1SEM; statistical significance for (b) was determined
with one-way ANOVA with post hoc test, and for (c) and (d) was
determined with Kruskal–Wallis ANOVA with post hoc test
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Basic Research in Cardiology (2022) 117:53
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Desmin-mediated frictional interaction along the length of
the microtubule may also lead to the loss of tubulin dimers
at sites of frictional contact; these lattice defects are replaced
by GTP-tubulin, which upon microtubule catastrophe can
function as a rescue site [3]. Future work should examine
whether there is evidence of microtubule damage and repair
at sites of intermediate filament-microtubule interaction.
While multiple mechanisms may contribute, these lateral
interactions between microtubules and intermediate fila-
ments govern microtubule mechanical behavior upon com-
pressive loading of microtubules [36], allowing desmin to
orchestrate microtubule buckling and its viscoelastic contri-
bution to the cardiomyocyte.
Combined with past and current work, we propose a
unifying model for microtubule-intermediate filament
interactions in the cardiomyocyte and how they contribute
to myocardial mechanics (S. Fig.6). Detyrosinated micro-
tubules, with less frequent depolymerization, experience
more chance interactions with intermediate filaments at
the Z-disk. The altered surface chemistry of detyrosinated
microtubules may also strengthen the electrostatic interac-
tions with intermediate filaments and additional cross-link-
ing proteins. The periodic, lateral reinforcement of micro-
tubules increases their stability, leading to longer-lived
microtubules and providing a dynamic cross-link with the
sarcomere, increasing the viscoelastic resistance to myocyte
motion and the ability of microtubules to bear and trans-
duce mechanical stress. Increased microtubule lifetimes also
promote microtubule acetylation, which itself increases the
ability of microtubules to withstand mechanical stress [29]
and increases myocyte viscoelasticity [11]. In the setting
of heart disease, the increased abundance of both desmin
intermediate filaments and detyrosinated microtubules thus
promotes a feed-forward substrate for enhanced mecha-
notransduction and myocardial stiffening. Therapeutic strate-
gies that selectively re-tyrosinate the network to basal levels
may thus reduce myocardial stiffening in heart failure via
restoring dynamicity to cardiac microtubules. However, our
work also cautions that complete re-tyrosination of cardiac
microtubules may excessively destabilize the network, which
would likely compromise myocyte homeostasis over time.
Encouragingly, previous literature suggests that only a par-
tial reversal of detyrosination levels is sufficient to lower
myocardial stiffness and improve myocyte mechanics in the
setting of heart failure [8, 9, 35, 43]. Together with past lit-
erature, this work thus helps illustrate a potential therapeutic
index for re-tyrosination strategies through novel insight into
the regulation of microtubule dynamics.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s00395- 022- 00962-3.
Acknowledgements The authors thank Matthew Caporizzo for provid-
ing the script used to quantify IF fractional coverage, Tim McKinsey
for the HDAC6 and ⍺TAT1 constructs, and Keita Uchida for assisting
with PLA image analysis. Funding for this work was provided by the
National Institute of Health (NIH) R01s-HL133080 and HL149891 to
B. Prosser, by the Foundation Leducq Research Grant no. 20CVD01
to B. Prosser, and by the Center for Engineering Mechanobiology to
B. Prosser through a grant from the National Science Foundation’s
Science and Technology program: 15-48571.
Author contributions Conceptualization: AKS, SAP, NO, and BLP;
methodology: AKS, SAP, and PR; formal analysis and investigation:
AKS, SAP, NO, JH, PR, and AIB; writing—original draft preparation:
AKS, SAP, NO, and BLP; writing—review and editing: AKS, SAP,
NO, JH, PR, AIB, and BLP; funding acquisition: BLP; supervision:
BLP.
Declarations
Conflict of interest The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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