The segregation of heterochromatin domains (LADs) at the nuclear periphery by the nuclear lamina, composed by polymerized nuclear Lamin A/C, provides a longstanding paradigm for the control of gene expression and for the mechanisms underlying Lamin-A/C-associated disorders, including progeria and cardiomyopathy. Here, we provide evidence supporting a novel paradigm that Lamin A/C functions as a transcription factor in the nuclear interior. We discovered that Ser22-phosphorylated Lamin A/C (pS22-Lamin A/C), required for lamin depolymerization during mitosis, populated the nuclear interior throughout the cell cycle. pS22-Lamin A/C ChIP-deq demonstrated localization at a large subset of putative active enhancers, not LADs. pS22-Lamin A/C-binding sites were co-occupied by the transcriptional activator c-Jun. In progeria patient-derived fibroblasts, a subset of pS22-Lamin A/C-binding sites were lost whereas new pS22-Lamin A/C-binding sites emerged. New pS22-Lamin A/C binding was accompanied by increased histone acetylation and increased c-Jun binding, whereas loss of pS22-Lamin A/C-binding was accompanied by loss of histone acetylation and c-Jun binding. New pS22-Lamin A/C enhancer binding in progeria was associated with upregulated expression of genes implicated in progeria pathophysiology, including cardiovascular disease. In contrast, alteration of LADs in progeria-patient cells could not explain the observed gene expression changes. These results suggest that Lamin A/C regulates gene expression by enhancer binding in the nuclear interior, independent of its function at the nuclear lamina, providing a new paradigm for the pathogenesis of lamin-associated disorders. pS22-Lamin A/C was also present in the nuclear interior of adult mouse cardiomyocytes. Cardiomyocyte-specific deletion of Lmna encoding Lamin A/C in adult mice caused extensive transcriptional changes in the heart and dilated cardiomyopathy, without apparent reduction of nuclear peripheral Lamin A/C. Disruption of the gene regulatory rather than LAD tethering function of Lamin A/C may underlie the pathogenesis of disorders caused by LMNA mutations, including cardiomyopathy.
Heterozygous loss-of-function mutations in LMNA , encoding nuclear lamina protein Lamin A/C, cause severe adult-onset dilated cardiomyopathy. A prevailing hypothesis posits that LMNA insufficiency causes nuclear envelope structural defects that ultimately cause the disease. However, the mechanisms linking defective nuclear envelopes to cardiomyopathy remain undefined. To determine specific nuclear envelope defects and their consequences, we deleted Lmna in cardiomyocytes in adult mice ( Lmna CKO ). Strikingly, a modest (50%) reduction of Lamin A/C caused widespread localized ruptures of the nuclear envelope in cardiomyocytes (En et al. bioRxiv 2023). The nuclear envelope ruptures did not cause immediate cell death, but accompanied a strong inflammatory response in the heart, prior to fatal cardiomyopathy. We hypothesized that DNA leaked from ruptured nuclei might elicit the cGAS-STING cytosolic DNA sensing pathway of innate immunity. Contrary to this hypothesis, we did not observe cGAS-STING activation in Lmna CKO cardiomyocytes. This lack of cGAS-STING activation was likely due to the near absence of cGAS protein in adult cardiomyocytes. To investigate the mechanism underlying cardiac inflammation in Lmna CKO mice, we conducted time-course single-nucleus RNA-seq. This analysis nominated cardiac fibroblasts as the central mediator of the inflammatory response, receiving ECM-mediated signaling from Lmna CKO cardiomyocytes and recruiting immune cells to the mutant hearts. Finally, we found evidence suggesting that nuclear envelope repair activity counteracts nuclear envelope ruptures in Lmna CKO mice. We found that the envelope ruptured sites co-localized with the ESCRT-III membrane remodeling complex, previously implicated in nuclear envelope repair. We further found that overexpression of DNA-binding protein BANF1 promoted ESCRT-III recruitment to the ruptured sites. We are currently investigating whether facilitated nuclear envelope repair ameliorates cardiomyopathy in Lmna CKO mice. If it does, nuclear envelope repair may be a potential therapeutic strategy for LMNA -related dilated cardiomyopathy.
ABSTRACT LMNA encodes nuclear lamin A/C that tethers lamina-associated heterochromatin domains (LADs) to the nuclear periphery. Point mutations in LMNA cause degenerative disorders including the premature aging disorder Hutchinson-Gilford progeria, but the mechanisms are unknown. We report that Ser22-phosphorylated Lamin A/C (pS22-Lamin A/C) was localized to the interior of the nucleus in human fibroblasts throughout the cell cycle. pS22-Lamin A/C interacted with a specific subset of putative active enhancers, not LADs, primarily at locations co-bound by the transcriptional activator c-Jun. In progeria-patient fibroblasts, a subset of pS22-Lamin A/C-binding sites were lost whereas new pS22-Lamin A/C-binding sites emerged in normally quiescent loci. These new pS22-Lamin A/C-binding sites displayed increased histone acetylation and c-Jun binding, implying increased enhancer activity. The genes near these new binding sites, implicated in clinical components of progeria including carotid artery diseases, hypertension, and cardiomegaly, were upregulated in progeria. These results suggest that Lamin A/C regulates gene expression by direct enhancer binding in the nuclear interior. Disruption of the gene regulatory rather than LAD function of Lamin A/C presents a novel mechanism for disorders caused by LMNA mutations including progeria. HIGHLIGHTS pS22-Lamin A/C is present in the nuclear interior throughout interphase. pS22-Lamin A/C associates with active enhancers, not lamina-associated domains. pS22-Lamin A/C-genomic binding sites are co-bound by the transcriptional activator c-Jun. New pS22-Lamin A/C binding in progeria accompanies upregulation of disease-related genes.
Nuclear envelope (NE) ruptures are emerging observations in Lamin-related dilated cardiomyopathy, an adult-onset disease caused by loss-of-function mutations in Lamin A/C, a nuclear lamina component. Here, we test a prevailing hypothesis that NE ruptures trigger the pathological cGAS-STING cytosolic DNA-sensing pathway using a mouse model of Lamin cardiomyopathy. The reduction of Lamin A/C in cardio-myocyte of adult mice causes pervasive NE ruptures in cardiomyocytes, preceding inflammatory transcription, fibrosis, and fatal dilated cardiomyopathy. NE ruptures are followed by DNA damage accumulation without causing immediate cardiomyocyte death. However, cGAS-STING-dependent inflammatory signaling remains inactive. Deleting cGas or Sting does not rescue cardiomyopathy in the mouse model. The lack of cGAS-STING activation is likely due to the near absence of cGAS expression in adult cardiomyocytes at baseline. Instead, extracellular matrix (ECM) signaling is activated and predicted to initiate pro-inflammatory communication from Lamin-reduced cardiomyocytes to fibroblasts. Our work nominates ECM signaling, not cGAS-STING, as a potential inflammatory contributor in Lamin cardiomyopathy.
Cardiomyopathies caused by mutations in LMNA, encoding nuclear Lamin A/C, are highly malignant and prevalent. How LMNA mutations cause cardiomyopathies remains unknown. We characterized cellular, molecular, and pathological evolution of mouse models of LMNA -related cardiomyopathy and provide evidence for a model in which nuclear rupture generates nuclear-localized proinflammatory signaling as a candidate molecular mechanism underlying disease pathogenesis. We observed that cardiomyocyte-specific, tamoxifen-inducible deletion of Lmna in adult mice ( Lmna CMKO ) caused a gradual reduction of Lamin A/C protein at the nuclear lamina, reflecting the slow turnover of Lamin A/C. A modest reduction of Lamin A/C in Lmna CMKO was sufficient to cause extensive fibrosis, reduced ejection fraction, and chamber dilation by 3 weeks after Lmna gene deletion. Lmna CMKO cardiomyocytes exhibited localized rupture of the nuclear envelope 2 weeks prior to the development of fibrosis and reduction of ejection fraction. Nuclear rupture in Lmna CMKO was immediately followed by an extensive upregulation of pro-inflammatory gene expression programs. We hypothesized that nuclear rupture might expose nuclear DNA to the cytoplasm thereby activating the pro-inflammatory cGas-STING cytosolic DNA sensing pathway. However, we did not observe localization of the cytosolic DNA sensor cGas to cytoplasmic DNA protruded from the ruptured nuclei in Lmna CMKO cardiomyocytes. Instead, we found that HMGB1, a potent proinflammatory protein normally sequestered in the nucleus, was released from the ruptured nuclei in Lmna CMKO cardiomyocytes. Mass spectrometry identified a strong interaction between Lamin A/C and HMGB1 in normal human fibroblast cells. Our data suggested that Lamin A/C tethers HMGB1 to the nuclear periphery by direct interaction and that reduction of Lamin A/C unleashes HMGB1 to the cytoplasm upon nuclear rupture. Future work will examine the hypothesis that cytoplasmic HMGB1 triggers pathogenic sterile inflammation leading to dilated cardiomyopathies in Lmna CMKO mice. In conclusion, we identified the nuclear rupture-induced cytoplasmic release of HMGB1 as a candidate mechanism underlying LMNA -related cardiomyopathies.
Nuclear envelope (NE) ruptures are emerging observations in Lamin-related dilated cardiomyopathy, an adult-onset disease caused by loss-of-function mutations in Lamin A/C, a nuclear lamina component. Here, we tested a prevailing hypothesis that NE ruptures trigger pathological cGAS-STING cytosolic DNA-sensing pathway, using a mouse model of Lamin-cardiomyopathy. Reduction of Lamin A/C in cardiomyocytes of adult mice caused pervasive NE ruptures in cardiomyocytes, preceding inflammatory transcription, fibrosis, and fatal dilated cardiomyopathy. NE ruptures were followed by DNA damage accumulation without causing immediate cardiomyocyte death. However, cGAS-STING-dependent inflammatory signaling remained inactive. Deleting
Lamin A/C (encoded by the LMNA gene) are the components of the nuclear lamina, a protein meshwork providing the structural integrity to the nuclear membrane. Dilated cardiomyopathy (DCM) caused by LNNA gene mutations is the second most common familial DCM in adults and associated with severe prognosis and high mortality. Despite decades of investigations into cell autonomous impacts of LMNA mutations, LMNA-DCM lacks effective treatments except for heart transplantation. Here, we identify cell non-autonomous effects of LMNA mutations and demonstrate the efficacy of targeting immune activities in mouse models of LMNA-DCM. We induced Lmna gene deletion specifically in cardiomyocytes in adult mice and tracked molecular, cellular, and physiological alterations in the heart at a high temporal resolution. We identified frequent localized nuclear envelope rupture, with the concomitant exposure of nuclear DNA to the cytoplasm, as the earliest direct consequence of Lamin A/C protein reduction in cardiomyocytes. Surprisingly, nuclear envelope rupture did accompany cardiomyocyte cell death or extensive transcriptional changes or cytosolic DNA sensing pathway activation within the cardiomyocytes. Instead, it coincided with extensive proinflammatory signaling activation within the cardiac macrophage population. This was followed by extensive fibrosis and structural and functional alterations of the heart, culminating in DCM and death. Treatment of LMNA-DCM mice with the immunosuppressant Dexamethasone suppressed fibrosis, profoundly improved the cardiac structure and function, and conferred a two-fold lifespan extension after Lmna gene deletion. This work suggested that immune activation is the major pathological component of LMNA-DCM and an effective target for treating LMNA-DCM patients.
