Microtubule forces drive nuclear damage in LMNA cardiomyopathy

Daria Amiad Pavlov; Julie Heffler; Carmen Suay-Corredera; Mohammad Dehghany; Kaitlyn M Shen; Noam Zuela-Sopilniak; Rani Randell; Keita Uchida; Rajan Jain; Vivek Shenoy; Jan Lammerding; Benjamin Prosser

doi:10.1101/2024.02.10.579774

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[Preprint]. 2025 Jun 2:2024.02.10.579774. [Version 5] doi: 10.1101/2024.02.10.579774

Microtubule forces drive nuclear damage in LMNA cardiomyopathy

Daria Amiad Pavlov ^1,^*, Julie Heffler ^2,^*, Carmen Suay-Corredera ¹, Mohammad Dehghany ³, Kaitlyn M Shen ⁴, Noam Zuela-Sopilniak ², Rani Randell ¹, Keita Uchida ¹, Rajan Jain ⁴, Vivek Shenoy ³, Jan Lammerding ^2,^#, Benjamin Prosser ^1,^#

PMCID: PMC11212868 PMID: 38948795

Abstract

Nuclear homeostasis requires a balance of forces between the cytoskeleton and nucleus. Mutations in the LMNA gene, which encodes the nuclear envelope proteins lamin A/C, disrupt this balance by weakening the nuclear lamina. This results in nuclear damage in contractile tissues and ultimately muscle disease. Intriguingly, disrupting the LINC complex that connects the cytoskeleton to the nucleus has emerged as a promising strategy to ameliorate LMNA-associated cardiomyopathy. Yet how LINC complex disruption protects the cardiomyocyte nucleus remains unclear. To address this question, we developed an assay to quantify the coupling of cardiomyocyte contraction to nuclear deformation and interrogated its dependence on the nuclear lamina and LINC complex. We found that, surprisingly, the LINC complex was mostly dispensable for transferring contractile strain to the nucleus, and that increased nuclear strain in lamin A/C-deficient cardiomyocytes was not rescued by LINC complex disruption. Instead, LINC complex disruption eliminated the cage of microtubules encircling the nucleus. Disrupting microtubules was sufficient to prevent nuclear damage and rescue cardiac function induced by lamin A/C deficiency. We computationally simulated the stress fields surrounding cardiomyocyte nuclei and show how microtubule forces generate local vulnerabilities that damage lamin A/C-deficient nuclei. Our work pinpoints localized, microtubule-dependent force transmission to the nucleus as a pathological driver and therapeutic target for LMNA-cardiomyopathy.

Graphical Abstract

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Introduction

Cardiac muscle represents an extremely stressful mechanical environment with continuous contraction and relaxation cycles. Accordingly, the cytoskeleton and nucleus uniquely adapt to sense and withstand the mechanical load. In the adult cardiomyocyte, the actin-myosin, microtubule (MT), and desmin intermediate filament networks physically couple to the nucleus via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex that spans the nuclear envelope (NE). The LINC complex interacts directly with the nuclear lamina, a meshwork of A and B-type lamin filaments that provides structural support to the nucleus and contributes to spatial chromatin organization. The LINC complex consists of various Nuclear Envelope Spectrin Repeat (nesprin) protein isoforms at the outer nuclear membrane containing a conserved Klarsicht/ANC-1/Syne Homology (KASH) domain. The KASH domains interact across the luminal space with Sad1p/UNC-84 (SUN) domain-containing proteins SUN1 or SUN2 located at the inner nuclear membrane, which bind to the nuclear lamina and chromatin^1,2. Thus, nuclear homeostasis and integrity depend on the balance of cytoskeletal forces, nuclear resistance, and their proper coupling through the LINC complex and nuclear lamina³.

The importance of this balance in the heart is demonstrated by variants in genes encoding lamins or other NE components that disproportionally affect highly contractile striated muscle tissues ^4,5. Specifically, mutations in the LMNA gene that encodes A-type nuclear lamins (lamin A/C) cause a heterogeneous group of diseases (“laminopathies”) with high prevalence of cardiomyopathies ^6–8. The LMNA N195K variant is associated with dilated cardiomyopathy in patients, and the Lmna^N195K/N195K mouse model leads to heart failure and death within 12 weeks of age ⁹. Specific and effective treatments are currently not available for LMNA cardiomyopathy, and the molecular mechanism of disease pathogenesis remains poorly understood. A prevailing hypothesis points to mechanically induced structural damage to the NE when the nuclear lamina is compromised ^10–15.

Importantly, decoupling the nucleus from the cytoskeleton by disrupting the LINC complex restores cardiac function and extends the lifespan of Lmna cardiomyopathy mice ^16,17. Yet the specific cytoskeletal forces responsible for nuclear damage in cardiomyocytes and the mechanism of protection by LINC complex disruption remain unclear ^10,13,17. This is in part due to the lack of direct tools to measure how cytoskeletal forces are transferred to the nucleus in the relevant physiological and mechanical environment ^4,5,13. Adult cardiomyocytes are often binucleated with nuclei equally spaced in the center of the cardiomyocyte, surrounded by a dense sarcomeric network and perinuclear cage of MTs. This central location prevents direct physical access to probe nuclear stiffness, as often reported in other cell types ¹⁸.

Here we introduce a new assay to quantify sarcomere-nuclear strain coupling in beating, adult cardiomyocytes. We use this assay to interrogate the role of the LINC complex and the cytoskeleton in driving nuclear strain during active cardiomyocyte contraction. We further investigate mechanisms of nuclear damage in Lmna cardiomyopathy and protection from nuclear damage by LINC complex disruption ¹⁹. Surprisingly, we found that sarcomere-nuclear strain coupling is largely preserved upon disruption of the LINC complex. Further, LINC complex disruption did not reduce contraction induced nuclear strain in Lmna-deficient cardiomyocytes. Instead, we observed that LINC complex disruption eliminates the dense cage of MTs normally formed around the nuclei of cardiomyocytes, which reduces MT-associated forces and confers protection from nuclear rupture at the nuclear tips. We found that MT disruption is sufficient to prevent nuclear damage in lamin A/C-deficient cardiomyocytes of mouse and human origin, and via a computational model that simulates the distribution of forces and sites of nuclear vulnerability in the cardiomyocyte. Finally, we demonstrate that in vivo disruption of the MT network is sufficient to preserve cardiac function and extend survival in mice following cardiomyocyte-specific Lmna depletion, further demonstrating the crucial role of MT-associated forces acting on the fragile nuclei in driving disease pathology in Lmna cardiomyopathy.

Results

Sarcomere-nuclear strain coupling in beating, adult cardiomyocytes

Measuring active mechanical signal transfer into the nucleus of mature cardiomyocytes is a missing component of interrogations into cardiac mechanobiology and LMNA cardiomyopathy. We developed a method to quantify sarcomere-nuclear strain coupling during electrically stimulated contractions in adult cardiomyocytes (Supplementary movie S1). We combined real-time measurements of sarcomere length change proximal to the nucleus with high spatiotemporal resolution (90 frames per second) Airyscan 2D imaging of the nucleus during steady-state (1 Hz) contraction (Fig. 1A). Figure 1B demonstrates that during the contraction cycle, sarcomere shortening and relaxation (magenta) are tightly coupled to the shortening and relaxation of nuclear length (cyan), with an opposing increase in nuclear width (orange). Notably, the relative change in sarcomere length (strain) during contraction is not fully transferred to the nucleus, resulting in dampening of nuclear strain (Fig. 1C). For example, at peak systole, for 11.4 ± 0.4 % sarcomere compression, nuclear length decreases only 6.6 ± 0.3 % (Fig. 1D). We generated sarcomere-nuclear strain coupling maps by plotting absolute sarcomere length versus absolute nuclear length during systolic compression and diastolic re-lengthening (Fig. 1E), or the respective sarcomere strain versus nuclear strain (Fig. 1F; unless otherwise specified, in our notation ‘strain’ refers to strain along the contractile axis). The dotted line represents a theoretical scenario where sarcomere strain would be linearly coupled, without loss, to nuclear strain. The upward deviation of the curve from the dotted line indicates that during contraction there is dampening, and not linear elastic coupling, between the sarcomere and nucleus. We can quantify this sarcomere-nuclear strain dampening during both phases of the contractile cycle (Fig. 1G). Systolic dampening is the integrated area under the curve during contraction with respect to the linear relationship (Fig. 1G, left). Diastolic dampening is the integrated area under the curve during re-lengthening with respect to a linear relationship with the intercept fixed at end-systolic strain (Fig. 1G, right). Using this approach, we can dissect how specific perturbations might compromise coupling between the sarcomeres and nucleus during each phase of the contractile cycle.

Fig. 1: — (A) Live isolated adult rat cardiomyocyte stained with SiR-actin and Hoechst 33342 to visualize sarcomeres and DNA, respectively (top). Cardiomyocytes were stimulated at 1 Hz, and the nucleus imaged at 90 fps to follow nuclear deformation during the contraction cycle (bottom – representative time lapse images, see Supplementary movie S1). (B) Sarcomere length, nuclear length, and nuclear width recordings over time for a single contraction cycle. (C) Sarcomere strain, nuclear length strain and nuclear width strain over time. (D) Quantification of peak sarcomere and nuclear compression. (E) Sarcomere-nuclear coupling represented with a plot of nuclear length versus sarcomere length and (F) respective nucleus strain versus sarcomere strain. The latter, dimensionless strain coupling map depicts the dampened strain on the nucleus during systolic compression and diastolic re-lengthening, as marked by the deviation from a linear correlation (dotted line). (G) Sarcomere-nuclear strain dampening during systole quantified from area above linear correlation (left schematic). Diastolic dampening is quantified from area under end systolic linear correlation (right schematic). Data presented as mean ± standard error (SE) for 20 cells from a single representative adult rat heart. Statistical significance determined by two-tailed t-test (***, p < 0.001).

We next interrogated how cytoskeletal connections to the nucleus regulate sarcomere-nuclear strain coupling during contraction. To disrupt all cytoskeletal interactions with the LINC complex, we utilized a previously validated adenovirus over-expressing a dominant-negative KASH peptide (AdV DN-KASH), which prevents interactions between endogenous nesprin and SUN proteins ^1,3. To disrupt MTs, we used colchicine (colch, 1 μM, overnight) (Fig. 2A). After 48 h AdV transduction of isolated rat cardiomyocytes, we confirmed that AdV DN-KASH disrupted the LINC complex through loss of perinuclear endogenous nesprin 1 (Extended Data Fig. 1A), and that 24 h colchicine treatment resulted in loss of MTs (Extended Data Fig. 1B). Neither acute LINC complex disruption nor MT depolymerization overtly altered sarcomere organization around the nucleus (Extended Data Fig. 1A-B) or resting sarcomere length (Extended Data Fig. 1C).

Fig. 2: — (A) Schematic of cytoskeletal to nucleoskeletal connections and the experimental perturbations. (B) Live 3D super-resolution imaging of resting adult rat cardiomyocyte nuclei (mid-nuclear planes are displayed). Nuclear volume (C) and aspect ratio (D) measurements following AdV DN-KASH or colchicine treatment. For panel (C): AdV empty and AdV DN-KASH (48 h): N = 3, n = 30, DMSO and colch (24 h): N = 3, n = 36. For panel (D): AdV empty and AdV DN-KASH (48 h): N = 3, n = 51, DMSO and colch (24h): N = 3, n = 51. (E-I) Active 2D imaging of electrically stimulated cardiomyocytes. Sarcomere strain (top) and nuclear strain (middle) over time, and sarcomere-nuclear stain coupling (bottom) for (E) AdV DN-KASH and (F) colchicine compared to their respective controls. (G) Snapshots of live, WT cardiomyocyte labeled with SPY-555 tubulin and Hoechst 33342 during diastole and peak systole demonstrating MT cage buckling (arrow) during contraction. (H) Quantification of sarcomere-nuclear strain dampening during the systolic and diastolic phases, for the indicated perturbations (see schematics in Fig. 1G). (I) Integrated nuclear strain over time during the contractile cycle for the indicated perturbations. AdV empty and AdV DN-KASH (48 h): N = 3, n = 51, DMSO and colch (24 h): N = 3, n = 51. Data presented as mean ± SE on individual cells. For panels C, D, H, I individual cells are indicated with open circles and individual animal replicate means are indicated with closed triangles connected by lines between experimental groups. Statistical significance determined by two-tailed t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

We first assessed the effect of acute LINC complex and MT disruption on nuclear morphology via live, 3D super-resolution confocal imaging (AiryScan jDCV) of quiescent (non-contracting) adult rat cardiomyocytes (Fig. 2B). AdV DN-KASH increased nuclear size in all 3 dimensions, leading to a substantial increase in nuclear volume (Fig. 2C). Colchicine treated nuclei displayed elongated morphology with increased nuclear aspect ratio and a subtle decrease in nuclear volume (Fig. 2C-D), in agreement with our earlier report and consistent with a reduction of compression on the nucleus ³.

We then assessed the active sarcomere-nuclear strain coupling via rapid 2D imaging in electrically stimulated (1 Hz) and contracting cardiomyocytes. Upon electrical excitation, peak sarcomere contractility increased subtly with AdV DN-KASH, yet peak nuclear compression decreased slightly (Fig. 2E, top and middle, Extended Data Fig. 1C, left). This indicated a modest decrease in sarcomere-nuclear strain coupling during myocyte contraction, as evident from the upward shift of the AdV DN-KASH curve in the strain coupling map (Fig. 2E bottom), and the significantly increased sarcomere-nuclear dampening during systole (Fig. 2H).

Colchicine treatment moderately increased sarcomere contraction and accelerated sarcomere relaxation (Fig. 2F, top), consistent with MTs normally providing a viscoelastic resistance to sarcomere motion ²⁰. Nuclear compression proportionately increased, but nuclear relaxation did not accelerate to match the faster sarcomere relaxation (Fig. 2F, top and middle). Faster sarcomere vs. nuclear re-lengthening manifests as increased area, or hysteresis, within the diastolic strain coupling curve upon colchicine treatment (Fig. 2F, bottom). Buckling of the MT cage during systole (Fig. 2G, Supplementary movie S2) demonstrates the transmission of contractile forces to the nucleus through the MT cage. This buckling might provide restoring force to match sarcomere and nuclear relaxation rates during diastole ²¹. To examine the cumulative strain on the nucleus during the full contractile cycle, we integrated the nuclear strain over time, and found no statistically significant differences between control cells and cells with LINC complex disruption or colchicine induced MT disruption (Fig. 2I). Together, these results demonstrate that acute, in vitro LINC complex disruption subtly reduces sarcomere-nuclear strain coupling, but neither LINC complex nor MT disruption significantly alter integrated nuclear strain during the contractile cycle. This indicates that nuclear strain during cardiomyocyte contraction is driven primarily by the shortening and re-lengthening of nearby sarcomeres, independent of their connectivity to the nucleus via the LINC complex.

Cardiac-specific, in vivo LINC complex disruption protects from nuclear damage and cardiac dysfunction in Lmna N195K mice.

We next sought to investigate the role of cytoskeletal forces in nuclear damage driven by lamin A/C deficiency or disease-causing Lmna mutations. We utilized the previously described Lmna^N195K/N195K (hereafter ‘Lmna N195K’) mutant mouse model that exhibits heart failure and death by 12 weeks of age ⁹. The Lmna N195K mutation leads to mechanical fragility of the nucleus, with a loss in nuclear stability similar to that observed with complete deletion of lamin A/C ^10,11. Recent studies found that global and cardiac-specific LINC complex disruption improves cardiac function and prolongs lifespan in Lmna N195K and other Lmna mutant mice ^16,17. To probe the cardiomyocyte-specific effects of LINC complex disruption, we used the previously described inducible αMHC MerCreMer DN-KASH mouse model, in which a dominant negative KASH domain of nesprin-2 tagged with GFP is expressed in cardiomyocytes upon tamoxifen treatment (referred to as cardiac-specific DN KASH, or csDN-KASH)¹⁹. We crossed these mice to Lmna N195K mice to generate a Lmna cardiomyopathy model with cardiac-specific inducible LINC complex disruption. Fig. 3A summarizes the different mouse models used in the next several figures. LINC complex disruption was induced by 5 consecutive daily tamoxifen injections at 3–4 weeks of age, followed by cardiomyocyte isolation at 8–9 weeks of age, allowing for 5 weeks of in vivo LINC complex disruption. We confirmed successful csDN-KASH induction following tamoxifen treatment by scoring the appearance of a csDN-KASH-GFP ring that surrounded the nucleus in >95% of cardiomyocytes, consistent with previous estimates of induction efficiency with this model ¹⁹. We confirmed LINC complex disruption upon csDN-KASH induction in cardiomyocytes by the loss of perinuclear nesprin-1 and nesprin-2 (Extended Data Fig. 2A-B). Consistent with previous reports ^16,17, LINC complex disruption significantly extended the lifespan of Lmna N195K mice (Fig. 3B), improved cardiac contractility (Fig. 3C) and structure (Extended Data Fig. 2D), and reduced fibrosis as quantified from cardiac tissue slices (Fig. 3D). Beyond the 10-week time point, cardiac function was largely maintained in Lmna N195K csDN-KASH mice even at 12 weeks of age, when most of the Lmna N195K controls without LINC complex disruption had already died (Extended Data Fig. 2C).

Fig. 3: — (A) Schematic presentation of the mouse models and experimental timelines used in this study. (B) Kaplan-Meier survival plot of the different experimental groups. N=10 mice per genotype. (C) Left ventricular ejection fraction measured by echocardiography, at 10 weeks of age. WT: N = 9, csDN-KASH: N = 15, *Lmna* N195K: N = 9, *Lmna* N195K csDN-KASH: N = 13. Error bar represents mean ± SD. Statistical significance determined by one-way ANOVA with Tukey multiple correction. (D) Representative Picrosirius red--stained heart sections and quantification of % fibrotic area for the indicated groups. N = 3 animal replicates per group. Error bar represents mean ± SD. Statistical significance determined by one-way ANOVA with Tukey multiple correction. (E) Representative images of cardiomyocyte nuclear morphology at 8–9 weeks of age for the studied groups. (F) Quantification of nuclear length, width, and aspect ratio. WT: N = 4, n = 58. csDN-KASH: N = 4, n = 69. *Lmna* N195K: N = 4, n = 55. *Lmna* N195K csDN-KASH: N = 4, n = 59. Data presented as mean ± SE of individual nuclei (open circles) while individual animal replicate means are indicated with closed triangles. (G) Chromatin protrusion from the nucleus in a *Lmna* N195K cardiomyocyte, suggestive of nuclear rupture. Representative mid plane images of a normal *Lmna* N195K nucleus (top) and a nucleus with a chromatin protrusion (bottom) in *Lmna* N195K cardiomyocytes. (H) Quantification of the percentage of nuclei with chromatin protrusions in each genotype by three independent, blinded scorers. Open circles represent the percentage of nuclei with chromatin protrusions quantified by each independent user for each animal. Closed circles correspond to the mean for each animal. Mean ± SE is shown for each genotype. WT: N = 5, n = 223. csDN-KASH: N = 3, n = 75. *Lmna* N195K: N = 5, n = 287. *Lmna* N195K csDN-KASH: N = 3, n = 223. Statistical significance determined by 1-way ANOVA with Bonferroni correction (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

We next assessed resting nuclear morphology using high-resolution imaging of isolated cardiomyocytes from the four mouse groups. We did not observe significant changes in nuclear dimensions between WT and Lmna N195K cardiomyocytes with this approach (Fig. 3E-F). A separate analysis of a larger, albeit lower resolution data set revealed a subtle (11%) but statistically significant increase in nuclear aspect ratio in Lmna N195K cardiomyocytes (Extended Data Fig. 3A).

In contrast, LINC complex disruption (csDN-KASH) resulted in substantial elongation of WT cardiomyocyte nuclei (36% increase in nuclear aspect ratio) and even more extreme nuclear elongation in Lmna N195K mice (Lmna N195K csDN-KASH), which exhibited a 63% increase in nuclear aspect ratio (Fig. 3E-F). This nuclear elongation occurred independent of any corresponding changes in cellular length, width, or aspect ratio (Extended Data Fig. 3B), instead indicating an altered balance between cytoskeletal forces acting on the nucleus and internal nuclear resistance ³. Notably, the increased nuclear aspect ratio due to in vivo LINC complex disruption mirrored the alterations in nuclear morphology upon colchicine treatment (Fig. 2D, Fig. 3F), suggesting it may arise from a reduction in MT compressive forces.

In addition, we observed increased nuclear fragility and NE rupture in Lmna N195K vs. WT nuclei, scored as chromatin that appeared to spill out of the nucleus, with the nuclear lamina only partially enclosing the protrusion (Fig. 3G, Extended data Fig 3C). In agreement with a recent report of nuclear ruptures in cardiac specific Lmna KO mice ¹⁵, we observed chromatin protrusions in mature cardiomyocytes exclusively at the tips of nuclei. Using blinded scoring, we identified chromatin protrusions in 20% of nuclei from Lmna N195K cardiomyocytes (Fig. 3H, see Methods for details on scoring criteria), similar to recent results reported in lamin A/C-depleted cardiomyocytes ¹⁵. Importantly, in vivo LINC complex disruption rescued NE rupture events in Lmna N195K mice back to levels of WT controls (Fig. 3H). Taken together, these findings confirm the protective effect of LINC complex disruption on lifespan and cardiac function in Lmna N195K mice, and further demonstrate that LINC complex disruption leads to a marked protection from NE ruptures.

In vivo LINC complex disruption does not reduce nuclear strain during myocyte contraction

A prevailing hypothesis for NE ruptures in laminopathies is that fragile nuclei are more susceptible to mechanical damage induced by forceful sarcomeric contractions ¹³. Therefore, decoupling the nucleus from the cytoskeleton might reduce contractility-induced nuclear damage ¹⁶. To probe this hypothesis, we first asked whether Lmna N195K cardiomyocyte nuclei indeed undergo more strain during the contractile cycle. Representative images of Lmna N195K nuclei at diastole and peak systole are depicted in Fig. 4A. Consistent with a more deformable nucleus ¹⁰, Lmna N195K cardiomyocytes showed increased nuclear compression during sarcomere contraction (Extended Data Fig. 4A) and nuclear re-lengthening that lagged behind sarcomere re-lengthening. This resulted in a downward shift in the strain coupling curve (Fig. 4B), increased diastolic sarcomere-nuclear dampening (Fig. 4G), and a significant increase in integrated nuclear strain over the contractile cycle in Lmna N195K cardiomyocytes (Fig. 4H). These data provide the first direct evidence for increased nuclear strain upon contraction of mutant Lmna mature cardiomyocytes.

Fig. 4: — (A) Representative snapshots of *Lmna* N195K cardiomyocyte nuclei during diastole and peak systole. (B) Increased nuclear compression as evidenced by a downward shift in the laminopathy strain coupling curve. (C) Representative snapshots of cardiac specific LINC complex disruption in WT cardiomyocyte nuclei during diastole and peak systole with (D) no change in active strain coupling. (E) Representative snapshots of cardiac specific LINC complex disruption in *Lmna* N195K cardiomyocyte nuclei during diastole and peak systole with (F) no change in active strain coupling. Data presented as mean ± SE (G) Quantification of sarcomerenuclear strain dampening during the systolic and diastolic phases, for the indicated groups (see schematics in Fig. 1G). (H) Integrated nuclear strain over time during the contractile cycle for the indicated groups. WT: N = 4, n = 58. *Lmna* N195K: N = 4, n = 69. WT veh: N = 4, n = 81. csDN-KASH: N = 4, n = 69. *Lmna* N195K veh: N = 4, n = 55. *Lmna* N195K csDN-KASH: N = 4, n = 59. Data presented as mean ± SE of individual cells (open circles) while individual animal replicate means are indicated with closed triangles. Statistical significance determined by two-tailed t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

We next hypothesized that LINC complex disruption reduces the increased nuclear strain in Lmna N195K cardiomyocytes. Representative images of nuclei from csDN-KASH and Lmna N195K csDN-KASH mice at diastole and peak systole are depicted in Fig. 4C and 4E, respectively. Surprisingly, nuclear compression was not reduced in csDN-KASH cardiomyocytes (Extended Data Fig. 4B), and the overall strain coupling curve (Fig. 4D) and sarcomere-nuclear dampening (Fig. 4G) remained unchanged. These data further indicate that an intact LINC complex is not required for transferring the majority of sarcomeric strain into the nucleus during myocyte contraction. Consistently, neither nuclear compression (Extended Data Fig. 4C), sarcomere-nuclear dampening (Fig. 4G), nor active strain coupling (Fig. 4F) were altered in Lmna N195K csDN-KASH myocytes compared to Lmna N195K controls. The increased integrated nuclear strain in Lmna N195K mutants was not rescued by in vivo LINC complex disruption (Fig.4H). These results indicate that although the more deformable Lmna N195K nuclei undergo increased strain during the contractile cycle, this effect is not rescued by LINC complex disruption, yet LINC complex disruption reduces nuclear damage and improves cardiac function and overall survival. These findings suggests that LINC complex disruption confers cardioprotection through an alternative mechanism, independent of reducing nuclear strain during cardiomyocyte contraction.

In vivo LINC complex disruption eliminates the perinuclear microtubule cage.

We next explored alternative sources of mechanical stress that could damage Lmna deficient nuclei and be reduced by LINC complex disruption. We hypothesized that nuclear elongation upon csDN-KASH might be a consequence of reduced MT compression on the nucleus. Consistently, we observed almost complete elimination of the perinuclear MT cage (measured as perinuclear to cytosolic α-tubulin enrichment) upon csDN-KASH induction in WT and Lmna N195K cardiomyocytes (Fig. 5A-B). The dense perinuclear MT cage in cardiomyocytes was specifically enriched at nuclear tips (Extended Data Fig. 5A), which might contribute to increased mechanical forces at this location. This appeared noteworthy given that chromatin protrusions in Lmna N195K cardiomyocytes were exclusively observed at nuclear tips (Fig. 3G, Extended data Fig 3C).

Fig. 5: — (A) Representative mid-plane immunofluorescence images of the MT network (α-tubulin), with a zoom in on a merge with labeled nuclei (Hoechst) in the WT, csDN-KASH, *Lmna* N195K, and *Lmna* N195K csDN-KASH adult mouse cardiomyocytes. (B) Quantification of perinuclear MT enrichment defined as perinuclear (PN) to cytoplasmic (Cyt) α-tubulin ratio (illustrated on the image on the right). Data presented as mean ± SE of individual nuclei (open circles) while individual animal replicate means are indicated with closed triangles. Statistical significance determined by one-way ANOVA with Bonferroni correction (*, p < 0.05; **, p < 0.01; ***, p < 0.001). (C) Nuclear aspect ratio (AR) as a function of perinuclear MT enrichment. Open circles represent individual nuclei from the respective groups. ‘Loess’ smoothing trace depicted with continuous black line and gray error area. Biphasic piecewise linear regression fit is indicated with a dashed black line. (D) Super resolution Airyscan jDCV mid plane image of a *Lmna* N195K cardiomyocyte nucleus (Hoechst) with the dense MT network (α-tubulin) penetrating the chromatin protrusion. WT: N = 3, n = 85. csDN-KASH: N = 3, n = 71. *Lmna* N195K: N = 3, n = 70. *Lmna* N195K csDN-KASH: N = 3, n = 75.

We leveraged intracellular heterogeneity to check if the nuclear aspect ratio depended on the levels of perinuclear MT enrichment. We observed a biphasic relationship between nuclear aspect ratio and the perinuclear MT cage (Fig. 5C). A ‘Loess’ smoothing algorithm applied to the pooled data from all experimental groups (Fig. 5C continuous black trace with gray error area) revealed a deflection point at ‘MT cage enrichment’ = 1.9. This deflection point was further verified by fitting a piecewise linear regression (dashed black trace) which indicated a negative correlation between nuclear aspect ratio and MT cage enrichment below 1.9 (slope = –2.1, p < 0.001). In contrast, the slope above this deflection point was flat (p > 0.05) indicating constant nuclear aspect ratio for MT cage enrichment ≥ 1.9. Together, these observations support the hypothesis that nuclear elongation upon in vivo LINC complex disruption is likely driven by decoupling of the MT network from the nucleus, with even greater elongation in more deformable Lmna N195K nuclei.

We thus hypothesized that nuclear damage in Lmna N195K nuclei is driven by perinuclear MT-dependent forces. In support of this hypothesis, super-resolution images of the MT network in Lmna N195K cardiomyocytes showed MTs encaging the protruded chromatin at the nuclear tip (Fig. 5D). Desmin intermediate filaments normally help balance compressive MT forces on the nucleus in mature cardiomyocytes ³; thus desmin deficiency could potentially contribute to such nuclear damage. However, we did not observe any decrease in the levels of desmin or alterations in the organization of the desmin network between the groups (Extended Data Fig. 5B-C). These data suggest that nuclear fragility, and not desmin deficiency, facilitates MT-based nuclear damage in the Lmna N195K cardiomyocytes.

MTs interact with the LINC complex via kinesin motors, and nesprins are required to recruit kinesin motors and MTs to the nuclear periphery ²². We found that LINC complex disruption depletes kinesin-1 from the perinuclear space (Extended Data Fig. 5D), in agreement with earlier reports ^10,17,23. We also observed robust perinuclear kinesin-1 depletion with partial loss of the perinuclear MT cage following 48 h AdV DN-KASH transduction in isolated adult rat cardiomyocytes (Extended Data Fig. 5E-F), confirming our findings in an independent adult cardiomyocyte model. These findings suggest that LINC complex disruption leads to rapid loss of perinuclear kinesin motors and eventual loss of the perinuclear MT cage.

Lmna N195K mutant and lamin A/C-deficient cardiomyocytes have increased NE ruptures linked to perinuclear MT network.

To directly test the role of MTs in nuclear ruptures, we used transgenic mice expressing a fluorescent cGAS-tdTomato reporter ¹⁰ crossed with the Lmna N195K mouse model to allow quantification of NE rupture in live cardiomyocytes. Live imaging of freshly isolated cardiomyocytes revealed cGAS-tdTomato foci in and around the nucleus in the Lmna N195K group that were significantly increased compared to cGAS-tdTomato WT mice (Fig. 6A and Extended Data Fig. 6A). In Lmna N195K cardiomyocytes, cGAS foci were mostly observed at the nuclear tips, where MTs were enriched, regardless of the absence or presence of chromatin protrusions (Fig. 6B).

Fig. 6: — (A) Quantification of number of cGAS foci per nucleus for WT (N = 4, n = 1898) and *Lmna* N195K (N = 4, n = 2915) groups. Bar graphs represent the mean ± 1 SD of the pooled nuclei. Superimposed black triangles represent the replicate means. Statistical significance determined by two-tailed t-test. (B) Immunofluorescence mid plane image of the dense MT network (α-tubulin) on the tip of *Lmna* N195K nucleus, associated with a chromatin protrusion (Hoechst, arrow), and cGAS foci (cGAS-tdTomato) indicative of NE ruptures. (C) Experimental design for live imaging of perinuclear cGAS-tdTomato foci in a *Lmna* N195K mouse model, following in-vitro stimulation in the presence of isoproterenol or colchicine treatment. Representative maximum intensity projection images of nuclei (Hoechst) and perinuclear cGAS-tdTomato foci, from (D) freshly isolated (baseline), and contractility induced (Iso + stim) cardiomyocytes, and (E) DMSO or colchicine treated cardiomyocytes. The 1 μm perinuclear rings used for foci identification are indicated with white lines, and the detected cGAS foci are indicated with arrows. Quantification of the number and area of perinuclear cGAS foci, per nucleus (bottom). N = 6, n = 4869 (baseline), n = 2286 (1 h iso + stim), n = 4820 (24 h DMSO), n = 4263 (24 h colch). Bar graphs represent the mean ± 1 SD of the pooled nuclei. Superimposed black triangles represent the replicate means and connected by lines to their respective treatment conditions. Statistical significance determined by two-tailed t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

We first asked whether augmented systolic actomyosin contractility would lead to additional NE ruptures in cardiomyocytes isolated from cGAS-tdTomato Lmna N195K mice. We electrically stimulated cardiomyocytes for 1 hour at 0.5 Hz in the presence of isoproterenol to increase contractile workload and compared the number of nuclear cGAS-tdTomato foci to that in non-stimulated cells (Fig. 6C-D). Since the cGAS-tdTomato signal could occasionally be detected in the cytosol (Extended Data Fig. 6B), we restricted our analysis to perinuclear cGAS-tdTomato foci immediately adjacent to the nucleus and within 1 μm of the nuclear border (Fig. 6D-E) to account for small chromatin protrusions difficult to detect based on the Hoechst signal. Augmented systolic workload induced by electrical stimulation and isoproterenol affected neither the number nor size of perinuclear cGAS-tdTomato foci (Fig. 6D), indicating that in this context active sarcomere contractility did not promote NE rupture, possibly due to the limited duration of electrical pacing.

We next tested the alternative hypothesis that MT associated forces drive nuclear damage. We disrupted the MT network in quiescent, isolated cardiomyocytes with 1 μM colchicine for 24 h and compared perinuclear cGAS-tdTomato foci to DMSO treated controls (Fig. 6C). Colchicine treatment induced nuclear elongation, consistent with reduced compressive forces (Extended Data Fig. 6C). MT disruption consistently resulted in a decrease in the number of perinuclear cGAS-tdTomato foci, with no change in their size (Fig. 6E). These data suggest that 24 h of MT disruption prevent the formation of new NE ruptures in Lmna N195K cardiomyocytes, without modulating the size of pre-existing ruptures.

We further probed the role of MT forces in a complementary model of LMNA deficient, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), which exhibit mechanically induced DNA damage ^{10,12,13,24,25}. We transfected hiPSC-CMs with LMNAtargeted siRNA (siLMNA) or non-targeted siRNA (siNT) for 5 days and disrupted MTs with colchicine on the last day before cell harvesting (see Experimental Design in Extended Data Fig. 7A). Lamin A/C depletion was confirmed by western blot analysis (Extended Data Fig. 7B) and DNA damage was assessed by immunofluorescence for γH2A.X, an early marker for double-stranded DNA breaks ²⁶. Extended Data Fig. 7C depicts representative nuclei from hiPSC-CMs, demonstrating increased DNA damage in the LMNA depleted nuclei that is significantly reduced upon MT disruption (Extended Data Fig. 7D). Taken together, our findings demonstrate that reducing MT forces in lamin A/C deficient cardiomyocytes protects from nuclear damage.

MT disruption is sufficient to protect from nuclear damage and preserve cardiac function in mice with cardiac specific Lmna depletion.

To extend our in vitro observations and test whether MT disruption can protect from nuclear damage and preserve cardiac function upon lamin A/C deficiency in vivo, we used a mouse model with inducible, cardiomyocyte-specific depletion of lamin A/C (Lmna-cKO) that we recently generated ²⁷. Mice at 10 weeks of age were injected with tamoxifen every other day to induce cardiomyocyte specific Lmna depletion²⁷. Mice were concomitantly injected with increasing doses of colchicine to disrupt MTs (see schematic for experimental design in Fig. 7A, ascending dose protocol and validation from ²⁸). Lamin A/C depletion and MT disruption were confirmed 22 days after tamoxifen induction by western blot analysis (Fig. 7B). At this time point, lamin A/C levels were reduced ~58%, comparable to recent reports ^15,27,29, and consistent with the long half-life of lamin proteins. This depletion mimics the reduced lamin A/C levels seen in multiple human LMNA cardiomyopathy models ^30,31. We performed sequential echocardiography analysis at day 0 (before injections), and at day 11, 22, and 29 post injection. At day 22, we observed significantly reduced cardiac function, measured via LV ejection fraction, in the Lmna-cKO mice (Fig. 7C), as well as indicators of pathological remodeling (LV dilation and hypertrophy, Extended Data Fig. 8A). Importantly, Lmna cKO mice treated with colchicine had fully preserved LV ejection fraction at 22 days (Fig. 7C) and did not show any evidence of chamber dilation or hypertrophy (Extended Data Fig. 8A), demonstrating a significant rescue effect by the MT disruption. Some colchicine-treated mice exhibited reduced cardiac function only at a later point, 29-day post injection, while vehicle-treated Lmna-cKO mice did not survive until that time. This longer survival of Lmna-cKO mice treated with colchicine is further depicted in the overall extended survival curves (Extended Data Fig. 8B).

Fig. 7: — (A) Experimental scheme for concurrent MT depolymerization and cardiomyocyte-specific deletion of *Lmna* in mice. Briefly, mice harboring a cardiomyocyte-specific Cre and *Lmna* floxed sites were injected with 3 tamoxifen (TMX) injections at 10-weeks every other day. In between TMX doses, mice were also injected with a ramp-up of colchicine up to 1 mg/kg every other day until sacrifice. (B) Western blot measuring lamin A/C (left) and α-tubulin (right) protein levels 22 days post first TMX injection (C) Sequential % ejection fraction measured by echocardiography in *Lmna* cKO mice with in vivo microtubule disruption. Data shown for pre-injection (day 0), 11, 22, and 29 days post initial injection. Each data point represents 1 mouse (2–3 left-ventricle regions averaged per mouse). WT + veh: N = 8 (d29 N = 5), WT + colch: N = 9 (d29 N = 6), *Lmna* cKO + veh: N = 6 (*Lmna* cKO + vehicle treated mice do not survive to day 29 post injection), *Lmna* cKO + colch: N = 9 (d29 N = 5). Error bars represent mean ± 1 SD. Statistical significance determined by two-way ANOVA with Tukey multiple correction. (D) Representative cardiac tissue sections of the indicated groups, stained for Wheat Germ Agglutinin (WGA, yellow) and desmin (magenta). Images are maximum intensity projections of 4 μm thick optical sections. Quantification of CM coverage area, inversely related to fibrosis, from WGA channel. Detected CM areas are highlighted on the images with semi-transparent white. Individual blue circles represent images, black triangles represent replicate means. N = 3 mice per group. WT veh: n = 35, WT colch: n = 37, *Lmna* cKO + veh: n=39, *Lmna* cKO + colch: n=38 images. Error bars represent mean ± 1 SE. Statistical significance determined by one-way ANOVA with Bonferroni multiple correction. (E) Representative images of cardiomyocyte nuclei (Hoechst, gray) and surrounding MT network (α-tubulin, cyan) of the indicated groups. A chromatin protrusion at the tip of *Lmna* cKO +veh nucleus is indicated with an arrow, with closely encaging MT network. Images are maximum intensity projections of 4 μm thick optical sections. Quantification of the percentage of nuclei with chromatin protrusions in each group by three independent, blinded scorers. Open circles represent the percentage of nuclei with chromatin protrusions quantified by each independent user for each animal. Closed circles correspond to the mean for each animal. Mean ± SE is shown for each group. N = 3 mice per group. WT + veh: n = 114. WT + colch: n = 102. *Lmna* cKO + veh: n = 112. *Lmna* cKO + colch: n = 113 nuclei. Statistical significance determined by 1-way ANOVA with Bonferroni correction. (F) Representative cardiac tissue sections of the indicated groups, stained for Wheat Germ Agglutinin (WGA, yellow), nuclei (Hoechst, gray), desmin (magenta) and α-tubulin (cyan). Images are maximum intensity projections of 4 μm optical sections. Cardiomyocyte nuclei indicated with arrows. (G) Quantification of cardiomyocyte nuclei aspect ratio (AR), perinuclear (PN), cytoplasmic (Cyt) and PN/Cyt. α-tubulin intensity. N = 3 mice per group. WT veh: n=113. WT colch: n=112. *Lmna* cKO + veh: n = 111. *Lmna* cKO + colch: n = 112 nuclei. Statistical significance determined by 1-way ANOVA with Bonferroni correction. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

To further assess cardiac structure, we analyzed cardiac tissue sections at day 29 post injection (for all groups except Lmna cKO mice treated with vehicle control, which were analyzed at day 22). We observe decreased cardiomyocyte coverage area, indicative of increased fibrosis, in Lmna cKO mice, in agreement with earlier reports ^15,27,29. Moreover, in vivo colchicine treatment partially restored cardiomyocyte coverage area (Fig. 7D).

To test whether in vivo MT disruption protects cardiomyocyte nuclei from damage, we quantified the percentage of cardiomyocyte nuclei with chromatin protrusions. Similar to the Lmna N195K model and an earlier report,¹⁵ we observed chromatin protrusions specifically at the tips of the Lmna depleted cardiomyocyte nuclei, where the perinuclear MT population is enriched (Fig. 7E top). Consistent with a rapid and severe Lmna-DCM model, we observed ~40% of nuclei with chromatin protrusions at day 22 post injection. Notably, in vivo MT disruption robustly protected cardiomyocyte nuclei from chromatin protrusions and damage at day 29 post injection (Fig. 7F), suggestive of potentially even greater effect at comparable day 22. We further characterized nuclear morphology and cytoskeletal organization from cardiac tissue sections and observed no significant changes in nuclear morphology in Lmna cKO or colchicine treated mice (Fig. 7F-G, Extended Data Fig. 9A). As expected, in vivo colchicine injections significantly reduced the perinuclear and cytoplasmic MT populations, and specifically reduced enrichment of the perinuclear MT cage (Fig. 7F-G). Colchicine treatment also reduced the density of desmin intermediate filaments, in the WT and Lmna cKO mice (Extended Data Fig. 9B). Collectively, these findings are consistent with our observations from the Lmna N195K mutant model and demonstrate that disruption of the perinuclear MT cage protects from nuclear damage and is sufficient to preserve cardiac function in Lmna cKO mice.

Computational model of nuclear damage due to resting cytoskeletal forces.

To better understand the forces that dictate nuclear morphology and damage, we developed a computational finite element (FE) axisymmetric model for the resting, mature cardiomyocyte (Fig. 8A). In this model, we explicitly consider the nucleus, its surrounding MT cage, and the myofibrils (cytoplasm) as components involved in nuclear deformations. The nucleus is further divided into 1) the nucleoplasm, and 2) the NE and its underlying lamina. Experimental observations suggest a complex prestress field in resting cardiomyocytes that results from cytoskeletal forces that govern nuclear shape and stiffness in these rod-shaped cells ^3,32,33. To induce this prestress field in our model, we first consider a (imaginary) cylindrical stress-free configuration for the cardiomyocytes where the nucleus is round, and the sarcomere units are not yet assembled (Fig. 8A, Extended Data Fig. 10 and Methods for more details). We further restrict axial displacement $(u_{z} = 0)$ of the cell ends (Fig. 8A) to mimic the geometric constraints imposed by the myocardial microenvironment ³³ and restoring stresses from titin springs ³⁴. We add an isotropic and homogenous compressive stress field with magnitude $σ_{M T}$ to the perinuclear MT cage to capture the pushing forces mediated by MT polymerization ³ and MT motors (such as kinesin-1) on the nucleus. We assume that in the stress-free configuration, actomyosin fibers are distributed randomly and thus their associated contractility (shown by tensor $ρ_{i j}$ in our model) is initially isotropic and uniform everywhere in the cytoplasm with magnitude $ρ_{0}$ . Note that $ρ_{i j}$ shows the cardiomyocyte contractility at rest (diastole) ³⁴, which differs from its active contractility during systole.

We next increase $ρ_{0}$ and $σ_{M T}$ from zero (stress-free configuration) to reach the physiologically stressed (WT) configuration (Fig. 8A bottom, Fig. 8B). As a result, the cytoplasm contracts in the radial direction while its length remains constant (Supplementary movie S3), leading to the development of an anisotropic stress field within the cell. This stress field triggers stress-activated signaling pathways which result in polarization of actomyosin fibers ^35,36 and assembly of myofibrils in the direction of maximum principal stress ³⁷. Our simulations show that this principal direction is perfectly aligned with the direction of myofibrils in the cardiomyocytes (Fig. 8A bottom and Extended Data Fig. 10B). Furthermore, our simulations show that myofibril maturation imposes significant lateral compressive forces on the nucleus, leading to its elongation in the axial direction (Fig. 8A bottom). Nuclear elongation is however resisted by elastic compression of surrounding MTs and their pushing forces ( $σ_{M T}$ ). Together, these interactions induce the prestress field in the quiescent cardiomyocyte (Extended Data Fig. 10A), even in the absence of cell contractility, although cell contractility might further increase the stress on the nucleus. Our model predicts that maximum and minimum myofibril tensions happen at the short and long tips of the MT cage, respectively, while far from this cage, tension is nearly constant (Fig. 8C). In contrast, maximum and minimum axial MT compression happens at the long and short tips of the nucleus, respectively. The obtained values for diastolic tension of myofibrils $(2 - 3 k P a)$ and axial compression of MTs $(1 - 1.8 k P a)$ are in good agreement with their corresponding experimental estimations ^32–34.

We next explored model predictions for nuclear morphology changes due to LMNA mutations and LINC complex disruption. To simulate the effect of lamin A/C depletion or mutant lamins, we reduce the stiffness of the NE and lamina ^38,39. To simulate LINC complex disruption, we decreased both the stress (Fig. 8B) and the stiffness of the MT cage (Fig. 8F), as supported by the loss perinuclear kinesin-1 (Extended Fig. 5D) and MTs (Fig. 5B) upon LINC complex disruption. Our simulations reveal that LINC complex disruption increases the nuclear aspect ratio, which is further exacerbated upon LMNA depletion, closely matching our experimental findings (see simulated nuclei images and nuclear aspect ratio comparison in Fig. 8D). This result highlights the significant role of MT enrichment around the nucleus in shaping its morphology. To examine this role in more detail, we then varied MT enrichment in our model by changing the cage stress and stiffness (Extended Data Fig. 10D and methods). Our simulations reproduced the experimentally observed biphasic relationship between nuclear aspect ratio and MT enrichment in the perinuclear region (Fig. 8E).

Our simulations also show that for any specified stiffness of the NE and lamina, a critical value for MT enrichment exists beyond which a form of instability emerges at the nuclear tips (Fig. 8F). Interestingly, the appearance of this instability is concomitant with the shift of the location of maximum principal stress (tension) from the middle of the nucleus to its long tips (Fig. 8F). Recent experimental studies ⁴⁰ indicate that the nuclear lamina can become diluted at the nuclear tips, where the Gaussian curvature is high. Consequently, the combination of high tension and low lamina concentration makes nuclear tips the most likely places to form nuclear lamina gaps, ultimately leading to nuclear damage and rupture ^41,42. This is consistent with our experimental observation that chromatin protrusions are always observed at the tips. Together, these simulation results suggest that compressive forces of the MT cage are sufficient to drive nuclear damage in quiescent LMNA-deficient cardiomyocytes.

Finally, we aimed to simulate rescue of the NE rupture in LMNA-deficient cardiomyocytes through LINC complex disruption. After appearance of the instability at the nuclear tips, we simulated LINC complex disruption by decreasing stress and stiffness of the MT cage (Fig. 8B). As a result, the instability disappears, and the nucleus becomes thinner and longer while the location of the maximum principal stress comes back to the middle of the nucleus (Fig. 8F, left). This provides a quantitative explanation for rescue of nuclear damage by LINC complex disruption in LMNA-deficient cardiomyocytes.

Overall, our experimental and modeling data suggest that nuclear elongation is regulated by the balance between cross-sectional myofibril compression and the perinuclear MT cage that provides axial compressive forces to resist elongation (Fig. 8F, right). A compromised lamina leads to redistribution of the maximum principal stress to nuclear tips, which contributes to NE rupture and chromatin protrusions. Decoupling the nucleus from MT-based forces via LINC complex disruption or direct MT disruption redistributes the stress away from the tips, thus protecting the nucleus from new ruptures.

Discussion

This study investigated force transmission into the nucleus during cardiomyocyte contraction and at rest (i.e., non-contracting cells). We describe a novel method to measure active strain transfer from the contracting sarcomeres into the nucleus. Using this and additional approaches, we did not find any evidence to support active sarcomere contractility as a primary driver of nuclear damage in Lmna-cardiomyopathy. Instead, we found that nuclear ruptures depend upon the dense cage of perinuclear MTs and associated kinesin motors. Either LINC complex or MT disruption eliminates MT forces and protects from NE ruptures and DNA damage. We further found that in vivo MT disruption protects from nuclear damage, prevents declining cardiac function, and extends survival in mice with cardiomyocyte-specific lamin A/C depletion. Finally, we developed a computational model that demonstrates redistribution of stresses and local vulnerabilities at the long tips of lamin-deficient nuclei, and restoration of stress distribution upon reduction in MT cage stress and stiffness. In sum, our data support a model where local MT compressive forces, independent of actomyosin contractility, induce damage at sites of nuclear fragility in laminopathic cardiomyocytes. Protection from nuclear ruptures and preservation of cardiac function can thus be achieved by disrupting MT-LINC complex interactions.

Our active strain coupling approach provides the first demonstration of sarcomere-nuclear strain coupling in electrically stimulated, mature cardiomyocytes. Earlier studies report nuclear strain in response to passive stretch in non-muscle cells ^1,43, or spontaneously contracting immature cardiomyocytes ^32,44 but provided inconsistent insights on cytoskeletal-nucleoskeletal coupling. We report physiologically relevant sarcomere strains of >10%, and high temporal resolution for instantaneous strain coupling with the nucleus, providing amplitude and kinetics for systolic and diastolic phases.

This approach demonstrates that the strain on the nucleus in mature cardiomyocytes is tightly coupled to sarcomere strain during stimulated contraction, but with dampening of nuclear strain amplitude. Surprisingly, this active coupling is only mildly disturbed following perturbation of MTs or the LINC complex. These findings are in agreement with the dispensability of nesprin-1’s actin binding domain on striated muscle structure and function in adult mice ²³. These results suggests that the central location of cardiomyocyte nuclei facilitates nuclear deformation largely by the surrounding myofibrils that squeeze on the nuclei during contraction, independent of direct physical connection via LINC complexes.

Our study incorporates two complementary approaches to disrupt the LINC complex: 1) acutely in isolated rat cardiomyocytes (48 hrs AdV DN-KASH), and 2) chronically for 5 weeks in mouse hearts in vivo (csDN-KASH). In contrast to the reduced active nuclear strain upon acute LINC complex disruption, chronic in vivo perturbation did not elicit the same effect. This may be due to differential cytoskeletal and nuclear remodeling in response to rapid in vitro vs. chronic in vivo LINC complex disruption. In support of this concept, acute AdV-DN KASH expression led to nuclear expansion in all dimensions and a modest reduction of perinuclear MTs (enrichment ~1.7-fold, Ext. Data Fig. 5E). Alternatively, chronic csDN-KASH led to more complete reduction of the MT cage (enrichment ~1.4-fold, Fig. 5B) and marked nuclear elongation. Greater elongation upon csDN-KASH is consistent with the relationship between MT cage enrichment and nuclear aspect ratio measured in Fig. 5C. The lack of expansion in the nuclear short axis upon in vivo csDN-KASH could arise from several factors, including but not limited to, greater nuclear confinement from myofilaments in vivo. Regardless, csDN-KASH nuclei show similarly unaltered strain coupling as with acute MT disruption, consistent with MT disruption driving the csDN-KASH phenotype. Overall, these findings are instructive of nuclear responses to robust and acute LINC complex disruption versus a more chronic and perhaps therapeutically relevant disruption in vivo.

Our strain coupling analysis demonstrates a ~30% increase in integrated nuclear strain in Lmna N195K cardiomyocytes (Fig. 4H). Such increased strain during each contractile cycle may (or may not) be associated with nuclear damage. However, cardiac-specific LINC complex disruption preserves cardiac function and protects Lmna N195K nuclei from ruptures independent of reducing active nuclear strain, suggesting increased contractile nuclear strain is not the dominant driver of nuclear damage. In support, increased contractile load did not increase nuclear ruptures (cGAS-tdTomato foci) in laminopathic cardiomyocytes in vitro. However, these results are limited by the short stimulation time (1 hour), and further investigation utilizing chronic alteration in actomyosin contractility is required to conclude on its possible involvement in laminopathy associated NE ruptures in vivo.

Alternatively, our experimental and modeling results demonstrate that MT compressive forces regulate nuclear morphology in the adult cardiomyocyte. During maturation, cardiomyocytes undergo uniaxial elongation, and the centrally located nuclei are squeezed by aligning myofibrils, causing overall nuclear elongation ³³. The dense perinuclear MT cage is also formed during maturation and provides balancing compressive forces to the nucleus, with a denser concentration at the myofibril void spaces along the longitudinal nuclear tips (Extended Data Fig. 1B). Our analysis demonstrates that nuclear elongation is restricted by the perinuclear MT cage, that when reduced below a critical value (Fig. 5C) leads to nuclear elongation. Consistent with softer and more deformable Lmna mutant nuclei, we observe significantly elongated nuclei in LINC complex disrupted (perinuclear MT depleted) Lmna N195K cardiomyocytes compared to WT controls. The dramatically elongated nuclei in Lmna N195K csDN-KASH mice (where nuclear ruptures are reduced and cardiac function is improved) indicate that elongation of soft nuclei is indeed a common feature of Lmna cardiomyopathy, but nuclear elongation itself is not sufficient to drive pathological progression.

When comparing WT and Lmna N195K cardiomyocytes with an intact LINC complex (and comparable levels of MT enrichment), we observed no statistically significant difference in nuclear elongation. This differs from earlier reports on elongated nuclei in alternative laminopathy mouse models ^7,8. This discrepancy can be explained by differences in cytoskeletal remodeling or disease severity between the models but might also be partly influenced by imaging resolution and selection criteria. Here we employed high resolution imaging that allows identification of nuclei with chromatin protrusions, which we excluded from nuclear shape analysis (20% in Lmna N195K group). Indeed, we observe mild (11%) but statistically significant elongation of Lmna N195K nuclei on a larger data set with lower resolution imaging and no excluded nuclei (Extended Data Fig. 2B). Further, our observation of chromatin protrusions and nuclear ruptures at the tips of the nuclei, without significant nuclear elongation, agree with recent reports from Lmna-depleted mature cardiomyocytes correlating nuclear ruptures with phenotype severity ^15,29.

We demonstrate the loss of perinuclear MTs and interacting kinesin-1 motors upon cardiac-specific LINC complex disruption in a cardiac laminopathy model, concomitant with a reduction in NE damage. We previously demonstrated a protective role of kinesin-1 depletion in Lmna-deficient skeletal myotubes, where MT motors are required for nuclear migration during myotube maturation ¹⁰. In maturing skeletal muscle myotubes, nuclear damage is driven by cytoskeletal forces associated with kinesin-1 dependent nuclear movement. However, the cardiomyocyte nuclei remain centrally located in maturation, and the damage in Lmna-compromised nuclei appears to be driven by compressive forces from the perinuclear MT cage. Despite this mechanistic distinction, convergent strategies that disrupt kinesin’s interaction with the nucleus warrant further examination for striated muscle laminopathies.

Computational modeling provides additional insights into the subcellular forces underlying nuclear damage and rescue. The model incorporates tensile forces from maturing myofibrils, stiffness of the nuclear lamina, and combined stress and stiffness from the MT cage and associated motors. Simulations demonstrate the redistribution of overall nuclear stress to the long nuclear tips in laminopathic nuclei with intact MT forces, localizing a site of nuclear vulnerability. The localized stress at the nuclear tip is restored to the center of the nucleus when both MT stiffness and stress are reduced, concomitant with nuclear elongation. This provides an important quantitative framework to support the sufficiency of resting, perinuclear MT forces for nuclear damage upon lamina compromise.

We incorporated an in vivo mouse model with inducible, cardiomyocyte-specific Lmna depletion to test the potential of MT disruption in protecting compromised nuclei and preventing deterioration of cardiac function. This rapid and aggressive Lmna cardiomyopathy model presents significant cardiac dilation and reduced function within 3 weeks, with reduction in lamin A/C levels comparable to reports from LMNA cardiomyopathy patients ²⁷. Our cellular analysis identified chromatin protrusions in ~40% of cardiomyocyte nuclei, in agreement with recent studies utilizing this approach that reported nuclear abnormalities and ruptures preceding reduced cardiac function ^15,29. Accordingly, our experimental design induced concomitant Lmna depletion and MT depolymerization. In support of our findings in Lmna N195K mice, we identify chromatin protrusions at the tips of the elongated cardiomyocyte nuclei, closely encaged by the perinuclear MTs. These perinuclear MTs can be significantly reduced by in vivo colchicine administration, thus protecting the nuclei from damage, improving cardiac function and remodeling, and extending lifespan in Lmna depleted mice. Despite the reduced perinuclear MT cage, we did not observe significant nuclear elongation in the WT or Lmnadepleted nuclei treated with colchicine, which might be attributed to distinct force profiles acting on the nucleus in vivo vs. ex vivo, and/or distinct cytoskeletal remodeling in the faster and more severe Lmna-depletion model, compared to longer and milder Lmna N195K model with LINC complex disruption. The latter notion is partially supported by colchicine-induced reduction in cytoplasmic desmin (Extended Data Fig. 9B). While these results demonstrate the therapeutic potential of targeting MTs in cardiomyocytes in patients with LMNA cardiomyopathy, we have not directly tested the ability of MT disruption to rescue cardiac function in already developed cardiomyopathy. Moreover, the tolerable colchicine doses in mice are two orders of magnitude higher than the tolerable dose in humans. Therefore, transferring this potential safely to the clinic will require perturbing specific MT interactions with the nucleus, with doses that will not interfere with other crucial functions of MTs in the cell.

Currently, a wide range of experimental models is available to study cardiac pathogenesis in laminopathies, including Lmna-depletion mouse models, Lmna-mutant knockins representing several identified patient mutations, and human iPSC-derived cardiomyocyte (iPSC-CM) models ^45,46. In the present study, we span the above models and demonstrate the importance of studying mature cardiomyocytes with their unique cytoskeletal and nucleoskeletal organization, to identify drivers of nuclear damage in the pathogenesis of laminopathy-associated cardiomyopathies. Although a powerful tool, the human iPSC-CM model represents immature cardiomyocytes. Despite evidence for LMNA induced DNA damage, they lack nuclear ruptures likely due to an immature cytoskeleton and nucleoskeleton, and as a result exhibit rounder nuclear morphology with altered stress distribution on the nucleus, which limits the utility of this model to study LMNA associated nuclear damage ^12,25,47,48. Within the mature mouse models, systemic or cardiomyocyte specific Lmna depletion is the most aggressive and rapidly progressing DCM model. Mouse models with specific mutations such as the Lmna N195K and Lmna H222P present variable severity phenotypes, but ultimately all lead to progressive DCM with contractile disfunction and fibrosis. Nuclear structure abnormalities are a common theme in all mature Lmna cardiomyocytes, but the wide spectrum of nuclear abnormalities, and accompanied cytoskeletal remodeling, complicate mechanistic insights on disease pathogenesis. Elongated nuclei morphology is a feature of Lmna H222P model ^8,49, which also displays decreased acetylated MT population ^50,51. In comparison, here we report chromatin protrusions in Lmna N195K nuclei, which are elongated (and protected from protrusions) upon elimination of perinuclear MT cage. Moreover, we observe even higher chromatin protrusion rate in the Lmna depletion model, in agreement with a recent report¹⁵. Importantly, in vivo disruption of MTs is sufficient to protect from the severe nuclear damage with lamin A/C depletion supporting a unifying mechanism for MT forces in driving nuclear damage in Lmna cardiomyopathy. Since nuclear stability is only mildly impaired in Lmna H222P muscle cells, unlike in Lmna N195K and Lmna deletion models ¹⁰, it will be imperative to test whether LINC complex or MT disruption also improves cardiac function or survival in the Lmna H222P animals. Additionally, further characterization of the MT network in the Lmna H222P model will be beneficial to identify potential adaptive cytoskeleton remodeling mechanisms that might decrease the rate of DCM progression in this model.

Limitations of the current study:

This study focused on the role of cytoskeletal forces as a pathological driver in Lmna cardiomyopathy. While the role of the nuclear lamina in chromatin organization and gene expression was outside the scope of this study, it is also hypothesized to participate in the molecular pathogenesis of laminopathies ^14,52,53. The dual role of lamin A/C in providing nuclear integrity but also organizing chromatin at the nuclear periphery, which in turn affects nuclear mechanical stability, points to potentially integrated effects of structural stability and chromatin organization in driving LMNA pathology ⁵⁴. While our study implicates resting MT forces, but not actomyosin contractility in laminopathy nuclear damage, further studies with chronically increased contractility are required to rule out potential involvement in contractility-driven damage to the nucleus, particularly in an in vivo setting. Further, the AdV empty construct, that was used as a control for acute AdV DN-KASH transductions, does not rule out other potential functions of KASH overexpression at the NE. Our cardiac function analysis in Lmna N195K csDN-KASH mice demonstrates preserved cardiac function at 10- and 12-week time points. Since these mice eventually perish at 17 weeks, we cannot rule out non-cardiac causes as contributors to mortality. Similarly, we observe extended survival for Lmna cKO mice treated with colchicine; however, colchicine treatment by itself is toxic and contributes over time to the mortality of the animals, confounding the analysis. Due to the long-term toxicity of colchicine treatment, we were unable to test its efficacy in improving cardiac function or survival in the Lmna N195K model, which would require extended treatment periods. Given these limitations of colchicine treatment, we primarily consider these studies as proof-of-principal studies to demonstrate the crucial role of the perinuclear MT cage in the pathogenesis of LMNA cardiomyopathies and as a potential therapeutic target. For clinical applications, though, we postulate that more refined treatment approaches would be needed instead of complete MT disruption.

In summary, our study introduces a novel method to investigate active sarcomere-nuclear strain coupling in the primary beating cardiomyocyte and implicates compressive MT forces in nuclear damage in laminopathy. We conclude that targeting specific interactions between MTs and the LINC complex should be pursued as a potential cardioprotective strategy in LMNA cardiomyopathy.

Extended Data

Extended Data Fig. 3: — (A) Quantification of nuclear aspect ratio in cardiomyocytes isolated from WT and *Lmna* N195K mice carrying cGAS-tdTomato reporter. Red lines represent mean of pooled nuclei, and black triangles correspond to individual replicate means. WT cGAS-tdTomato: N = 4, n = 1898. *Lmna* N195K cGAS-tdTomato: N = 4, n = 2915. (B) Isolated cardiomyocyte cell length, width and cell aspect ratio for the indicated mouse models. WT: N = 3, n = 133. csDN-KASH: N = 3, n = 105. *Lmna* N195K: N = 3, n = 113. *Lmna* N195K csDN-KASH: N = 3, n = 143. Data presented as mean ± SE. Statistical significance determined by 1-way ANOVA with Bonferroni correction (*, p < 0.05, **, p < 0.01, ***, p < 0.001). (C) NE rupture in *Lmna* N195K cardiomyocytes manifested by chromatin protrusion from the nucleus, with partial lamina coverage. Representative single confocal image of the nucleus is shown.

Extended Data Fig. 4: — Sarcomere length (SL), nuclear length (NL), and nuclear width (NW) recordings over time during stimulated contraction of freshly isolated cardiomyocytes 8–9 weeks of age. Sarcomere strain and nuclear strain over time are plotted below. (A) *Lmna* N195K (red) and WT littermate controls (black). (B) Cardiac specific LINC complex disruption (gray) and WT vehicle controls (black). (C) *Lmna* N195K with cardiac specific LINC complex disruption (blue) and littermate *Lmna* N195K controls (black). (D) NL versus SL coupling plots for the corresponding experimental groups. WT: N = 4, n = 58. *Lmna* N195K: N = 4, n = 69. WT veh: N = 4, n = 81. csDN-KASH: N = 4, n = 69. *Lmna* N195K veh: N = 4, n = 55. *Lmna* N195K csDN-KASH: N = 4, n = 59. Data presented as mean ± SE.

Extended Data Fig. 5: — (A) Quantification α-tubulin enrichment at the nuclear tips and sides in the indicated mouse model cardiomyocytes. (B) Representative mid-plane immunofluorescence images of desmin intermediate filament network surrounding the nuclei (Hoechst) of the indicated groups. (C) Desmin western blot and quantification, normalized to H3, for the indicated groups. (D) Representative mid-plane immunofluorescence images of kinesin-1 and quantification of perinuclear kinesin-1 enrichment, defined as PN to Cyt kinesin-1 ratio. WT: N = 2, n = 46. csDN-KASH: N = 2, n = 51. *Lmna* N195K: N = 2, n = 50. *Lmna* N195K csDN-KASH: N = 2, n = 59. (E) Representative nuclear mid-plane images of adult rat cardiomyocytes labeled with α-tubulin and Hoechst (left), following 48h of adenoviral LINC complex disruption. Quantification of perinuclear α-tubulin enrichment is shown on the right. AdV empty and AdV DN-KASH: N = 2, n = 29. (F) Representative nuclear mid-plane images of kinesin-1 in the entire adult rat cardiomyocyte following 48h of adenoviral LINC complex disruption (left). Quantification of kinesin-1 intensity separately at the perinucleus and the cytoplasm is shown on the right. AdV empty: N = 2, n = 78, AdV DN-KASH: N = 2, n = 80. Data presented as mean ± SE. Statistical significance determined by two-tailed t-test (*, p < 0.05, **, p < 0.01, ***, p < 0.001).

Extended Data Fig. 6: — (A) Full cell views of representative maximum intensity projection images labeled for nuclei (Hoechst) and cGAS-tdTomato (red), from freshly isolated cGAS-tdTomato WT, and cGAS-tdTomato *Lmna* N195K mouse models. The middle panel shows a zoom in on the nucleus and the perinuclear region used for cGAS foci quantification. (B) (C) Full cell views of representative maximum intensity projection images labeled for nuclei (Hoechst) and cGAS-tdTomato (red) for the indicated experimental groups. (D) Nuclear aspect ratio (AR) for the experimental groups of cGAS-tdTomato *Lmna* N195K model. N = 6, n = 4869 (baseline), n = 2286 (1 h iso + stim), n = 4820 (24 h DMSO), n = 4263 (24 h colch). Red lines represent the puled nuclei mean for each experimental group. Superimposed black triangles represent the replicate means and connected by lines to their respective treatment conditions. Statistical significance determined by 1-way ANOVA with Bonferroni correction (*, p < 0.05, **, p < 0.01, ***, p < 0.001).

Extended Data Fig. 7: — (A) Experimental design for the examination of DNA damage in hiPSC-derived cardiomyocytes upon transfection with non-targeted siRNA (siNT) or LMNA-targeted siRNA (siLMNA), and subsequent DMSO or colchicine (colch) treatment. (B) Western blots, separate for replicate 1 and pooled lysates from replicates 2/3 showing the siRNA-mediated lamin A/C knock-down in hiPSC-derived cardiomyocytes. GAPDH was used as a loading control. (C) Representative mid-plane images of nuclei (Hoechst) and γH2A.X foci, from the four experimental groups of hiPSC-derived cardiomyocytes. Hoechst was used to outline the nuclei (white line), and the detected γH2AX foci are highlighted in yellow. (D) Quantification of mean nuclear γH2A.X intensity and γH2A.X foci fraction of nuclear volume coverage (top). Quantification of the normalized number and volume of γH2A.X foci (bottom). Each replicate is normalized to the mean value of the NT DMSO group. Individual nuclei presented as open circles with mean ± SE of the pooled nuclei. Closed triangles depict replicate means connected by lines to their respective experimental conditions. N = 3, n = 199 (NT DMSO), N = 3, n = 197 (NT colch), N = 3, n = 192 (siLMNA DMSO), N = 3, n = 203 (siLMNA colch). Statistical significance determined by one-way ANOVA with Bonferroni correction (*, p < 0.05, **, p < 0.01, ***, p < 0.001).

Extended Data Fig. 8: — Sequential echocardiography measurements in cardiac specific *Lmna* depleted mice with in-vivo microtubule disruption. Data shown for pre-injection (day 0), 11, 22, and 29 days post initial injection. Herat rate (HR), stroke volume (SV), % fractional shortening (FS), cardiac output (CO), end-systolic and end-diastolic left ventricular diameters (Diameter, s and d), end-systolic and end-diastolic volume (ESV, EDV), corrected left-ventricular mass (LV mass), end-systolic and end-diastolic left-ventricle anterior and posterior wall thickness (LVAW, s and d, LVPW s and d). WT + veh: N = 8 (day 29: N = 5), WT + colch: N = 9 (day 29: N = 6), *Lmna* cKO + veh: N = 6 (*Lmna* cKO + vehicle treated mice do not survive to day 29 post injection), *Lmna* cKO + colch: N = 9 (day 29: N = 5). Error bars represent mean ± 1 SD. B) Kaplan-Meier survival plots of the different experimental groups. Male: WT+veh: N = 2, WT+colch: N = 2, *Lmna* cKO+veh: N = 5, *Lmna* cKO+colch: N = 5. Female: WT+veh: N = 3, WT+colch: N = 2, *Lmna* cKO+veh: N = 5, *Lmna* cKO+colch: N = 6. Statistical significance determined by two-way ANOVA with Tukey multiple correction. (*, p < 0.05, **, p < 0.01, ***, p < 0.001)

Extended Data Fig. 9: — (A) Quantification of cardiomyocyte nuclear length, width, and area. (B) Quantification of perinuclear (PN), cytoplasmic (Cyt) and PN/Cyt. desmin intensity from maximum-intensity-projection images of cardiac tissue sections for the indicated groups. N=3 mice per group. WT veh: n = 113. WT colch: n = 112. *Lmna* cKO (MUT) + veh: n = 111. *Lmna* cKO + colch: n = 112 nuclei. Statistical significance determined by 1-way ANOVA with Bonferroni correction. (*, p < 0.05, **, p < 0.01, ***, p < 0.001)

Extended Data Fig. 10: — (A) Simulation results depicting stress distribution and average myosin motor density in the cardiomyocyte cytoplasm under physiological conditions. Radial stress component, Tangential stress component and average motor density defined as $(ρ_{11} + ρ_{22} + ρ_{33}) / 3$ . (B) Model predictions for the maximum principal (maximum tensile) stress direction in the cytoplasm. (C) In vivo % volume changes relative to WT conditions. (D) Variations of the MT cage stiffness and active stress to simulate MT enrichment around the nucleus. (E) Simulation results for stress distribution in the microtubule cage in WT conditions. Radial stress component (Pa), Tangential stress component (Pa) and hydrostatic pressure (Pa).

Supplementary Material

Supplement 1

Supplementary Movie S1: Electrically stimulated (1 Hz) adult rat cardiomyocyte labeled with SiR-actin and Hoechst to visualize sarcomeres and DNA, respectively.

Download video file^{(17.3MB, mp4)}

Supplement 2

Supplementary Movie S2: Electrically stimulated (1 Hz) adult rat cardiomyocyte labeled with SPY-555 tubulin and Hoechst demonstrating MT cage buckling during contraction.

Download video file^{(1.9MB, mp4)}

Supplement 3

Supplementary Movie S3: Cardiomyocyte model simulation following assembly of myofibrils (increased myofibril prestress with constant cytoplasm length) demonstrating development of anisotropic stress field within the cell with overall cellular radial compression and nuclear elongation.

Download video file^{(4MB, mp4)}

Supplement 4

NIHPP2024.02.10.579774v5-supplement-4.pdf^{(356.2KB, pdf)}

Acknowledgements

Funding for this work was provided by the National Institute of Health (NIH) R01s-HL133080 and HL149891 to B.P., R01 HL082792, R01 AR084664, and R35 GM153257 to J.L., and R35 HL16663 to R.J., the Foundation Leducq Research Grant no. 20CVD01 to B.P. and J.L., the Foundation Leducq Research Grant no. 24CVD03 to J.L., and by the Center for Engineering Mechanobiology to R.J., B.P., and V.S. through a grant from the National Science Foundation’s Science and Technology program: 15-48571. Funding was also provided by the National Science Foundation (CBET 1715606 and URoL-2022048 to J.L.), and the Volkswagen Foundation (A130142 to J.L.), as well as a Fleming Research Fellowship to J.H. We thank the Bridge Position Program, Weizmann Institute of Science for support to D.A.P. We thank R. Rotkopf from the Life Sciences Core Facility, Weizmann Institute, for advice on statistical analysis. We thank Justin Bi for his help as a blinded scorer in the chromatin protrusion analysis. The Lammerding lab thanks the Mills family for their generous support and the Cornell CARE staff, Ern Hwei Hannah Fong and Alexander Sopilniak-Mints for their invaluable contribution to the maintenance of our animal colony.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplement 1

Supplementary Movie S1: Electrically stimulated (1 Hz) adult rat cardiomyocyte labeled with SiR-actin and Hoechst to visualize sarcomeres and DNA, respectively.

Download video file^{(17.3MB, mp4)}

Supplement 2

Supplementary Movie S2: Electrically stimulated (1 Hz) adult rat cardiomyocyte labeled with SPY-555 tubulin and Hoechst demonstrating MT cage buckling during contraction.

Download video file^{(1.9MB, mp4)}

Supplement 3

Download video file^{(4MB, mp4)}

Supplement 4

NIHPP2024.02.10.579774v5-supplement-4.pdf^{(356.2KB, pdf)}

[R1] 1.Lombardi M. L. et al. The interaction between nesprins and sun proteins at the nuclear envelope is critical for force transmission between the nucleus and cytoskeleton. J. Biol. Chem. 286, 26743–26753 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R2] 2.Zhou C., Rao L., Shanahan C. M. & Zhang Q. Nesprin-1/2: roles in nuclear envelope organisation, myogenesis and muscle disease. Biochem. Soc. Trans. 46, 311–320 (2018). [DOI] [PubMed] [Google Scholar]

[R3] 3.Heffler J. et al. A Balance Between Intermediate Filaments and Microtubules Maintains Nuclear Architecture in the Cardiomyocyte. Circ. Res. 126, e10–e26 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R4] 4.Janin A. & Gache V. Nesprins and Lamins in Health and Diseases of Cardiac and Skeletal Muscles. Front. Physiol. 0, 1277 (2018). [DOI] [PMC free article] [PubMed]

[R5] 5.Ross J. A. & Stroud M. J. THE NUCLEUS: Mechanosensing in cardiac disease. Int. J. Biochem. Cell Biol. 137, 106035 (2021). [DOI] [PubMed] [Google Scholar]

[R6] 6.Crasto S., My I. & Di Pasquale E. The Broad Spectrum of LMNA Cardiac Diseases: From Molecular Mechanisms to Clinical Phenotype. Front. Physiol. 11, 761 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R7] 7.Nikolova V. et al. Defects in nuclear structure and function promote dilated cardiomyopathy in lamin A/C–deficient mice. J. Clin. Invest. 113, 357–369 (2004). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R8] 8.Muchir A., Shan J., Bonne G., Lehnart S. E. & Worman H. J. Inhibition of extracellular signalregulated kinase signaling to prevent cardiomyopathy caused by mutation in the gene encoding A-type lamins. Hum. Mol. Genet. 18, 241–247 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R9] 9.Mounkes L. C., Kozlov S. V., Rottman J. N. & Stewart C. L. Expression of an LMNA-N195K variant of A-type lamins results in cardiac conduction defects and death in mice. Hum. Mol. Genet. 14, 2167–2180 (2005). [DOI] [PubMed] [Google Scholar]

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PERMALINK

This is a preprint.

Microtubule forces drive nuclear damage in LMNA cardiomyopathy

Daria Amiad Pavlov

Julie Heffler

Carmen Suay-Corredera

Mohammad Dehghany

Kaitlyn M Shen

Noam Zuela-Sopilniak

Rani Randell

Keita Uchida

Rajan Jain

Vivek Shenoy

Jan Lammerding

Benjamin Prosser

Abstract

Graphical Abstract

Introduction

Results

Sarcomere-nuclear strain coupling in beating, adult cardiomyocytes

Fig. 1: Active sarcomere-nuclear strain coupling in beating cardiomyocytes.

Fig. 2: Distinct effects of LINC complex disruption and MT depolymerization on resting nuclear morphology and active sarcomere-nuclear strain coupling.

Cardiac-specific, in vivo LINC complex disruption protects from nuclear damage and cardiac dysfunction in Lmna N195K mice.

Fig. 3: Cardiac specific LINC complex disruption extends lifespan, improves cardiac function and protects against nuclear ruptures in Lmna N195K cardiomyopathy.

In vivo LINC complex disruption does not reduce nuclear strain during myocyte contraction

Fig. 4: Increased active nuclear strain in Lmna cardiomyopathy is not restored by cardiac specific in vivo LINC complex disruption.

In vivo LINC complex disruption eliminates the perinuclear microtubule cage.

Fig. 5: In vivo LINC complex disruption eliminates perinuclear MT cage leading to nuclear elongation.

Lmna N195K mutant and lamin A/C-deficient cardiomyocytes have increased NE ruptures linked to perinuclear MT network.

Fig. 6: MT disruption protects from nuclear damage in Lmna N195K cardiomyocytes.

MT disruption is sufficient to protect from nuclear damage and preserve cardiac function in mice with cardiac specific Lmna depletion.

Fig. 7: MT disruption preserves cardiac function and protects from nuclear damage in cardiac specific Lmna depleted (Lmna cKO) mice.

Computational model of nuclear damage due to resting cytoskeletal forces.

Fig. 8: A computational model to explain nuclear damage in resting LMNA mutated cardiomyocytes and its rescue through LINC complex disruption.

Discussion

Limitations of the current study:

Extended Data

Extended Data Fig. 1:

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Extended Data Fig. 7:

Extended Data Fig. 8:

Extended Data Fig. 9:

Extended Data Fig. 10:

Supplementary Material

Acknowledgements

References

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