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

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.

Transcriptional, Post-Transcriptional, and Post-Translational Mechanisms Rewrite the Tubulin Code During Cardiac Hypertrophy and Failure.

A proliferated and post-translationally modified microtubule network underlies cellular growth in cardiac hypertrophy and contributes to contractile dysfunction in heart failure. Yet how the heart achieves this modified network is poorly understood. Determining how the "tubulin code"-the permutations of tubulin isoforms and post-translational modifications-is rewritten upon cardiac stress may provide new targets to modulate cardiac remodeling. Further, while tubulin can autoregulate its own expression, it is unknown if autoregulation is operant in the heart or tuned in response to stress. Here we use heart failure patient samples and murine models of cardiac remodeling to interrogate transcriptional, autoregulatory, and post-translational mechanisms that contribute to microtubule network remodeling at different stages of heart disease. We find that autoregulation is operant across tubulin isoforms in the heart and leads to an apparent disconnect in tubulin mRNA and protein levels in heart failure. We also find that within 4 h of a hypertrophic stimulus and prior to cardiac growth, microtubule detyrosination is rapidly induced to help stabilize the network. This occurs concomitant with rapid transcriptional and autoregulatory activation of specific tubulin isoforms and microtubule motors. Upon continued hypertrophic stimulation, there is an increase in post-translationally modified microtubule tracks and anterograde motors to support cardiac growth, while total tubulin content increases through progressive transcriptional and autoregulatory induction of tubulin isoforms. Our work provides a new model for how the tubulin code is rapidly rewritten to establish a proliferated, stable microtubule network that drives cardiac remodeling, and provides the first evidence of tunable tubulin autoregulation during pathological progression.

Microtubules orchestrate local translation to enable cardiac growth.

Hypertension, exercise, and pregnancy are common triggers of cardiac remodeling, which occurs primarily through the hypertrophy of individual cardiomyocytes. During hypertrophy, stress-induced signal transduction increases cardiomyocyte transcription and translation, which promotes the addition of new contractile units through poorly understood mechanisms. The cardiomyocyte microtubule network is also implicated in hypertrophy, but via an unknown role. Here, we show that microtubules are indispensable for cardiac growth via spatiotemporal control of the translational machinery. We find that the microtubule motor Kinesin-1 distributes mRNAs and ribosomes along microtubule tracks to discrete domains within the cardiomyocyte. Upon hypertrophic stimulation, microtubules redistribute mRNAs and new protein synthesis to sites of growth at the cell periphery. If the microtubule network is disrupted, mRNAs and ribosomes collapse around the nucleus, which results in mislocalized protein synthesis, the rapid degradation of new proteins, and a failure of growth, despite normally increased translation rates. Together, these data indicate that mRNAs and ribosomes are actively transported to specific sites to facilitate local translation and assembly of contractile units, and suggest that properly localized translation - and not simply translation rate - is a critical determinant of cardiac hypertrophy. In this work, we find that microtubule based-transport is essential to couple augmented transcription and translation to productive cardiomyocyte growth during cardiac stress.

A Balance Between Intermediate Filaments and Microtubules Maintains Nuclear Architecture in the Cardiomyocyte.

Rationale: Mechanical forces are transduced to nuclear responses via the linkers of the nucleoskeleton and cytoskeleton (LINC) complex, which couples the cytoskeleton to the nuclear lamina and associated chromatin. While disruption of the LINC complex can cause cardiomyopathy, the relevant interactions that bridge the nucleoskeleton to cytoskeleton are poorly understood in the cardiomyocyte, where cytoskeletal organization is unique. Furthermore, while microtubules and desmin intermediate filaments associate closely with cardiomyocyte nuclei, the importance of these interactions is unknown. Objective: Here, we sought to determine how cytoskeletal interactions with the LINC complex regulate nuclear homeostasis in the cardiomyocyte. Methods and Results: To this end, we acutely disrupted the LINC complex, microtubules, actin, and intermediate filaments and assessed the consequences on nuclear morphology and genome organization in rat ventricular cardiomyocytes via a combination of super-resolution imaging, biophysical, and genomic approaches. We find that a balance of dynamic microtubules and desmin intermediate filaments is required to maintain nuclear shape and the fidelity of the nuclear envelope and lamina. Upon depletion of desmin (or nesprin [nuclear envelope spectrin repeat protein]-3, its binding partner in the LINC complex), polymerizing microtubules collapse the nucleus and drive infolding of the nuclear membrane. This results in DNA damage, a loss of genome organization, and broad transcriptional changes. The collapse in nuclear integrity is concomitant with compromised contractile function and may contribute to the pathophysiological changes observed in desmin-related myopathies. Conclusions: Disrupting the tethering of desmin to the nucleus results in a loss of nuclear homeostasis and rapid alterations to cardiomyocyte function. Our data suggest that a balance of forces imposed by intermediate filaments and microtubules is required to maintain nuclear structure and genome organization in the cardiomyocyte.

Disease-Associated Short Tandem Repeats Co-localize with Chromatin Domain Boundaries.

More than 25 inherited human disorders are caused by the unstable expansion of repetitive DNA sequences termed short tandem repeats (STRs). A fundamental unresolved question is why some STRs are susceptible to pathologic expansion, whereas thousands of repeat tracts across the human genome are relatively stable. Here, we discover that nearly all disease-associated STRs (daSTRs) are located at boundaries demarcating 3D chromatin domains. We identify a subset of boundaries with markedly higher CpG island density compared to the rest of the genome. daSTRs specifically localize to ultra-high-density CpG island boundaries, suggesting they might be hotspots for epigenetic misregulation or topological disruption linked to STR expansion. Fragile X syndrome patients exhibit severe boundary disruption in a manner that correlates with local loss of CTCF occupancy and the degree of FMR1 silencing. Our data uncover higher-order chromatin architecture as a new dimension in understanding repeat expansion disorders.

IKK inhibition increases bortezomib effectiveness in ovarian cancer.

Ovarian cancer is associated with increased expression of the pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8), which induces tumor cell proliferation, angiogenesis, and metastasis. Even though bortezomib (BZ) has shown remarkable anti-tumor activity in hematological malignancies, it has been less effective in ovarian cancer; however, the mechanisms are not understood. We have recently shown that BZ unexpectedly induces the expression of IL-8 in ovarian cancer cells in vitro, by IκB kinase (IKK)-dependent mechanism. Here, we tested the hypothesis that IKK inhibition reduces the IL-8 production and increases BZ effectiveness in reducing ovarian tumor growth in vivo. Our results demonstrate that the combination of BZ and the IKK inhibitor Bay 117085 significantly reduces the growth of ovarian tumor xenografts in nude mice when compared to either drug alone. Mice treated with the BZ/Bay 117085 combination exhibit smallest tumors, and lowest levels of IL-8. Furthermore, the reduced tumor growth in the combination group is associated with decreased tumor levels of S536P-p65 NFκB and its decreased recruitment to IL-8 promoter in tumor tissues. These data provide the first in vivo evidence that combining BZ with IKK inhibitor is effective, and suggest that using IKK inhibitors may increase BZ effectiveness in ovarian cancer treatment.

Quantitative analysis of bortezomib-induced IL-8 gene expression in ovarian cancer cells.

Interleukin-8 (IL-8), originally discovered as the neutrophil chemoattractant and inducer of leukocyte-mediated inflammation, contributes to cancer progression through its induction of tumor cell proliferation, survival, and migration. IL-8 expression is increased in many types of advanced cancers, including ovarian cancer, and correlates with poor prognosis. Bortezomib (BZ) is the first FDA-approved proteasome inhibitor that has shown remarkable antitumor activity in multiple myeloma and other hematological malignancies. In solid tumors, including ovarian carcinoma, BZ has been less effective as a single agent; however, the mechanisms remain unknown. We have recently shown that in ovarian cancer cells, BZ greatly increases IL-8 expression, while expression of other NFκB-regulated cytokines, IL-6 and TNF, is unchanged. In this chapter, we describe a protocol that uses real-time qRT-PCR to quantitatively analyze mRNA levels of IL-8 and IL-6 in BZ-treated ovarian cancer cells. The protocol can be easily modified and used for analysis of other cytokines in different cell types.

Proteasome inhibition by bortezomib increases IL-8 expression in androgen-independent prostate cancer cells: the role of IKKα.

Expression of the proinflammatory and proangiogenic chemokine IL-8, which is regulated at the transcriptional level by NF-κB, is constitutively increased in androgen-independent metastatic prostate cancer and correlates with poor prognosis. Inhibition of NF-κB-dependent transcription was used as an anticancer strategy for the development of the first clinically approved 26S proteasome inhibitor, bortezomib (BZ). Even though BZ has shown remarkable antitumor activity in hematological malignancies, it has been less effective in prostate cancer and other solid tumors; however, the mechanisms have not been fully understood. In this article, we report that proteasome inhibition by BZ unexpectedly increases IL-8 expression in androgen-independent prostate cancer PC3 and DU145 cells, whereas expression of other NF-κB-regulated genes is inhibited or unchanged. The BZ-increased IL-8 expression is associated with increased in vitro p65 NF-κB DNA binding activity and p65 recruitment to the endogenous IL-8 promoter. In addition, proteasome inhibition induces a nuclear accumulation of IκB kinase (IKK)α, and inhibition of IKKα enzymatic activity significantly attenuates the BZ-induced p65 recruitment to IL-8 promoter and IL-8 expression, demonstrating that the induced IL-8 expression is mediated, at least partly, by IKKα. Together, these data provide the first evidence, to our knowledge, for the gene-specific increase of IL-8 expression by proteasome inhibition in prostate cancer cells and suggest that targeting both IKKα and the proteasome may increase BZ effectiveness in treatment of androgen-independent prostate cancer.