Hypertrophic Cardiomyopathy β-Cardiac Myosin Mutation (P710R) Leads to Hypercontractility by Disrupting Super Relaxed State

Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1,000 mutations, many in β-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreased in vitro motility velocity and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super relaxed state in longer, two-headed myosin constructs, freeing more heads to generate force. Micropatterned human induced pluripotent derived stem cell (hiPSC)–cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrated the measured molecular changes to predict the measured traction forces. These results confirm a key role for regulation of the super relaxed state in driving hypercontractility in HCM with the P710R mutation and demonstrate the value of a multiscale approach in revealing key mechanisms of disease

The Effect of Developmental Heterogeneity and Genetic Variation of Fibroblasts on Cardiac Injury and Repair

Cardiac fibrosis is a pathological process that contributes to adverse cardiac remodeling. It is a consequence of tissue repair processes driven mainly by cardiac fibroblasts (CFbs). In response to stress, CFbs proliferate and secrete extracellular matrix components which, if excessive, leads to scar formation. Scar tissue can interrupt the connections between cardiomyocytes, ultimately compromising the structural integrity and function of the heart. Functional recovery of the myocardium is not only hindered by the formation of fibrotic tissue but also by the irreversible loss of cardiomyocytes. In addition to the key role of CFbs in scar formation, it has been suggested that a subset of CFbs may be the optimal cell source to generate cardiomyocytes through direct reprogramming. Direct cardiac reprogramming of CFbs represents a promising approach that could lead to regeneration of cardiomyocytes from the endogenous fibroblasts while reducing scar tissue formation. Several studies have demonstrated in vivo direct reprogramming of CFbs leads to an improvement in cardiac function and has been shown to be exceedingly more efficient in the context of recent cardiac injury. Despite the prominent role of CFbs in both scar formation, and in the potential generation of new cardiomyocytes through reprogramming, characterization of these cells is still limited. This is mainly due to lack of reliable markers to identify cardiac fibroblasts, their heterogeneity, and the effects of genetic variation when studying these cells in a diverse population. These constraints prompted us to first identify a panel of surface markers to prospectively identify CFbs. We further performed a comprehensive investigation to identify the developmental heterogeneity of CFbs. We then sought to determine whether developmental origin of CFbs may influence their contribution to formation of scar as well as its effect on their direct reprogramming into iCMs. Finally, by studying CFbs from multiple inbred mouse strains and their response to cardiac insult we aimed to investigate the effect of genetic variation in pathogenesis of cardiac fibrosis. To undertake a comprehensive study of CFbs, we established a panel of surface markers that can efficiently isolate the majority of CFbs from the adult mouse heart. We employed lineage tracing, transplantation studies, and parabiosis to show that most adult CFbs are derived from the epicardium, a minority arises from endothelial cells, with no contribution from bone marrow or circulating cells. Intriguingly, developmentally distinct CFbs showed similar proliferation rates, and similar gene expression profiles in response to pressure overload injury. We next sought to determine whether this heterogeneity of CFbs may affect their efficiency to generate cardiomyocytes via direct reprogramming, mainly in the context of injury. Using genetic fate-mapping techniques, transplantation studies and gene expression profiling, we showed that the majority of CFbs originate from a shared mesodermal ancestor as cardiomyocytes while a minority of the CFb population originates from neural crest-derived precursors. We provide compelling evidence that, regardless of their developmental origin, CFbs are able to be successfully converted to functional iCMs through in vitro direct reprogramming. However, CFbs generated iCMs with higher efficiency compared to fibroblasts of extra-cardiac organs of identical developmental origin, emphasizing the importance of the physiological microenvironment on cell fate. Remarkably, cardiac injury induced unique re-expression of early developmental genes in CFbs that corresponded to their developmental origin. Finally, we studied the contribution of CFbs from multiple inbred mouse strains following insult to the heart. Our data showed that despite similar increases in proliferation within the different strains, fibroblast activation is a response that correlates with the extent of scar formation. Additionally, by comparing CFbs from multiple strains, we were able to identify potential pathways as therapeutic targets with latent TGF-b binding protein-2 (LTBP2) as a promising diagnostic marker for fibrosis, with relevance to patients with underlying myocardial fibrosis.Together, our findings suggest that common signaling mechanisms stimulate the pathological response of different CFb populations. However, in the context of direct cardiac reprogramming after injury, the developmental heterogeneity of CFbs may be an essential contributing factor. Our findings also highlight the importance of genetic variation in cardiac fibrosis. Therefore, therapeutic strategies for reducing pathogenic CFbs should target these common pathways instead of targeting fibroblasts of other sources. It may be crucial to study the effects of injury on different CFb subsets for the development of targeted therapies to promote cardiac repair

The Effect of Developmental Heterogeneity and Genetic Variation of Fibroblasts on Cardiac Injury and Repair

Cardiac fibrosis is a pathological process that contributes to adverse cardiac remodeling. It is a consequence of tissue repair processes driven mainly by cardiac fibroblasts (CFbs). In response to stress, CFbs proliferate and secrete extracellular matrix components which, if excessive, leads to scar formation. Scar tissue can interrupt the connections between cardiomyocytes, ultimately compromising the structural integrity and function of the heart. Functional recovery of the myocardium is not only hindered by the formation of fibrotic tissue but also by the irreversible loss of cardiomyocytes. In addition to the key role of CFbs in scar formation, it has been suggested that a subset of CFbs may be the optimal cell source to generate cardiomyocytes through direct reprogramming. Direct cardiac reprogramming of CFbs represents a promising approach that could lead to regeneration of cardiomyocytes from the endogenous fibroblasts while reducing scar tissue formation. Several studies have demonstrated in vivo direct reprogramming of CFbs leads to an improvement in cardiac function and has been shown to be exceedingly more efficient in the context of recent cardiac injury. Despite the prominent role of CFbs in both scar formation, and in the potential generation of new cardiomyocytes through reprogramming, characterization of these cells is still limited. This is mainly due to lack of reliable markers to identify cardiac fibroblasts, their heterogeneity, and the effects of genetic variation when studying these cells in a diverse population. These constraints prompted us to first identify a panel of surface markers to prospectively identify CFbs. We further performed a comprehensive investigation to identify the developmental heterogeneity of CFbs. We then sought to determine whether developmental origin of CFbs may influence their contribution to formation of scar as well as its effect on their direct reprogramming into iCMs. Finally, by studying CFbs from multiple inbred mouse strains and their response to cardiac insult we aimed to investigate the effect of genetic variation in pathogenesis of cardiac fibrosis. To undertake a comprehensive study of CFbs, we established a panel of surface markers that can efficiently isolate the majority of CFbs from the adult mouse heart. We employed lineage tracing, transplantation studies, and parabiosis to show that most adult CFbs are derived from the epicardium, a minority arises from endothelial cells, with no contribution from bone marrow or circulating cells. Intriguingly, developmentally distinct CFbs showed similar proliferation rates, and similar gene expression profiles in response to pressure overload injury. We next sought to determine whether this heterogeneity of CFbs may affect their efficiency to generate cardiomyocytes via direct reprogramming, mainly in the context of injury. Using genetic fate-mapping techniques, transplantation studies and gene expression profiling, we showed that the majority of CFbs originate from a shared mesodermal ancestor as cardiomyocytes while a minority of the CFb population originates from neural crest-derived precursors. We provide compelling evidence that, regardless of their developmental origin, CFbs are able to be successfully converted to functional iCMs through in vitro direct reprogramming. However, CFbs generated iCMs with higher efficiency compared to fibroblasts of extra-cardiac organs of identical developmental origin, emphasizing the importance of the physiological microenvironment on cell fate. Remarkably, cardiac injury induced unique re-expression of early developmental genes in CFbs that corresponded to their developmental origin. Finally, we studied the contribution of CFbs from multiple inbred mouse strains following insult to the heart. Our data showed that despite similar increases in proliferation within the different strains, fibroblast activation is a response that correlates with the extent of scar formation. Additionally, by comparing CFbs from multiple strains, we were able to identify potential pathways as therapeutic targets with latent TGF-b binding protein-2 (LTBP2) as a promising diagnostic marker for fibrosis, with relevance to patients with underlying myocardial fibrosis.Together, our findings suggest that common signaling mechanisms stimulate the pathological response of different CFb populations. However, in the context of direct cardiac reprogramming after injury, the developmental heterogeneity of CFbs may be an essential contributing factor. Our findings also highlight the importance of genetic variation in cardiac fibrosis. Therefore, therapeutic strategies for reducing pathogenic CFbs should target these common pathways instead of targeting fibroblasts of other sources. It may be crucial to study the effects of injury on different CFb subsets for the development of targeted therapies to promote cardiac repair

Cardiac Fibrosis Is Associated With Decreased Circulating Levels of Full-Length CILP in Heart Failure.

Cardiac fibrosis is a pathological process associated with various forms of heart failure. This study identified latent transforming growth factor-β binding protein 2, cartilage oligomeric matrix protein, and cartilage intermediate layer protein 1 as potential biomarkers for cardiac fibrosis. All 3 encoded proteins showed increased expression in fibroblasts after transforming growth factor-β stimulation in vitro and localized specifically to fibrotic regions in vivo. Of the 3, only the full-length cartilage intermediate layer protein 1 showed a significant decrease in circulating levels in patients with heart failure compared with healthy volunteers. Further studies on these 3 proteins will lead to a better understanding of their biomarker potential for cardiac fibrosis

Generation of Nkx2-5/CreER transgenic mice for inducible Cre expression in developing hearts.

Nkx2-5 is a homeobox-containing transcriptional regulator that serves as one of the earliest markers of cardiac lineage commitment. To study the role of Nkx2-5-expressing progenitors at specific time points in cardiac development, we have generated a novel and inducible NKX2-5 mouse line by knocking in a CreER cassette into the Nkx2-5 genomic locus, while preserving the endogenous Nkx2-5 gene to avoid haploinsufficiency. We evaluated the specificity and efficiency of CreER activity after 4-OHT injection by crossing Nkx2-5CreER/+ mice with a Rosa26tdT/+ reporter strain. Our immunohistochemistry results confirmed Cre-induced tdTomato expression specifically in cells expressing Nkx2-5. These cells were mainly cardiomyocytes and were observed in the embryonic heart as early as day 9.5. Additionally, quantitative polymerase chain reaction on postnatal hearts showed enriched expression of Nkx2-5 in isolated tdTomato-expressing cells. No tdTomato expression was observed in Nkx2-5CreER/+ ;Rosa26tdT/+ mice in the absence of 4-OHT, confirming the inducible nature of CreER activity. The Nkx2-5/CreER mouse model described in this article will serve as an invaluable tool to trace myocardial lineage and to temporally induce genetic manipulation in a selective population of cardiac progenitors during embryonic development and in the adult heart

Developmental Heterogeneity of Cardiac Fibroblasts Does Not Predict Pathological Proliferation and Activation

RationaleFibrosis is mediated partly by extracellular matrix-depositing fibroblasts in the heart. Although these mesenchymal cells are reported to have multiple embryonic origins, the functional consequence of this heterogeneity is unknown.ObjectiveWe sought to validate a panel of surface markers to prospectively identify cardiac fibroblasts. We elucidated the developmental origins of cardiac fibroblasts and characterized their corresponding phenotypes. We also determined proliferation rates of each developmental subset of fibroblasts after pressure overload injury.Methods and resultsWe showed that Thy1(+)CD45(-)CD31(-)CD11b(-)Ter119(-) cells constitute the majority of cardiac fibroblasts. We characterized these cells using flow cytometry, epifluorescence and confocal microscopy, and transcriptional profiling (using reverse transcription polymerase chain reaction and RNA-seq). We used lineage tracing, transplantation studies, and parabiosis to show that most adult cardiac fibroblasts derive from the epicardium, a minority arises from endothelial cells, and a small fraction from Pax3-expressing cells. We did not detect generation of cardiac fibroblasts by bone marrow or circulating cells. Interestingly, proliferation rates of fibroblast subsets on injury were identical, and the relative abundance of each lineage remained the same after injury. The anatomic distribution of fibroblast lineages also remained unchanged after pressure overload. Furthermore, RNA-seq analysis demonstrated that Tie2-derived and Tbx18-derived fibroblasts within each operation group exhibit similar gene expression profiles.ConclusionsThe cellular expansion of cardiac fibroblasts after transaortic constriction surgery was not restricted to any single developmental subset. The parallel proliferation and activation of a heterogeneous population of fibroblasts on pressure overload could suggest that common signaling mechanisms stimulate their pathological response

Developmental heterogeneity of cardiac fibroblasts does not predict pathological proliferation and activation.

RationaleFibrosis is mediated partly by extracellular matrix-depositing fibroblasts in the heart. Although these mesenchymal cells are reported to have multiple embryonic origins, the functional consequence of this heterogeneity is unknown.ObjectiveWe sought to validate a panel of surface markers to prospectively identify cardiac fibroblasts. We elucidated the developmental origins of cardiac fibroblasts and characterized their corresponding phenotypes. We also determined proliferation rates of each developmental subset of fibroblasts after pressure overload injury.Methods and resultsWe showed that Thy1(+)CD45(-)CD31(-)CD11b(-)Ter119(-) cells constitute the majority of cardiac fibroblasts. We characterized these cells using flow cytometry, epifluorescence and confocal microscopy, and transcriptional profiling (using reverse transcription polymerase chain reaction and RNA-seq). We used lineage tracing, transplantation studies, and parabiosis to show that most adult cardiac fibroblasts derive from the epicardium, a minority arises from endothelial cells, and a small fraction from Pax3-expressing cells. We did not detect generation of cardiac fibroblasts by bone marrow or circulating cells. Interestingly, proliferation rates of fibroblast subsets on injury were identical, and the relative abundance of each lineage remained the same after injury. The anatomic distribution of fibroblast lineages also remained unchanged after pressure overload. Furthermore, RNA-seq analysis demonstrated that Tie2-derived and Tbx18-derived fibroblasts within each operation group exhibit similar gene expression profiles.ConclusionsThe cellular expansion of cardiac fibroblasts after transaortic constriction surgery was not restricted to any single developmental subset. The parallel proliferation and activation of a heterogeneous population of fibroblasts on pressure overload could suggest that common signaling mechanisms stimulate their pathological response

Genetic Regulation of Fibroblast Activation and Proliferation in Cardiac Fibrosis.

BackgroundGenetic diversity and the heterogeneous nature of cardiac fibroblasts (CFbs) have hindered characterization of the molecular mechanisms that regulate cardiac fibrosis. The Hybrid Mouse Diversity Panel offers a valuable tool to examine genetically diverse cardiac fibroblasts and their role in fibrosis.MethodsThree strains of mice (C57BL/6J, C3H/HeJ, and KK/HlJ) were selected from the Hybrid Mouse Diversity Panel and treated with either isoproterenol (ISO) or saline by an intraperitoneally implanted osmotic pump. After 21 days, cardiac function and levels of fibrosis were measured by echocardiography and trichrome staining, respectively. Activation and proliferation of CFbs were measured by in vitro and in vivo assays under normal and injury conditions. RNA sequencing was done on isolated CFbs from each strain. Results were analyzed by Ingenuity Pathway Analysis and validated by reverse transcription-qPCR, immunohistochemistry, and ELISA.ResultsISO treatment in C57BL/6J, C3H/HeJ, and KK/HlJ mice resulted in minimal, moderate, and extensive levels of fibrosis, respectively (n=7-8 hearts per condition). Isolated CFbs treated with ISO exhibited strain-specific increases in the levels of activation but showed comparable levels of proliferation. Similar results were found in vivo, with fibroblast activation, and not proliferation, correlating with the differential levels of cardiac fibrosis after ISO treatment. RNA sequencing revealed that CFbs from each strain exhibit unique gene expression changes in response to ISO. We identified Ltbp2 as a commonly upregulated gene after ISO treatment. Expression of LTBP2 was elevated and specifically localized in the fibrotic regions of the myocardium after injury in mice and in human heart failure patients.ConclusionsThis study highlights the importance of genetic variation in cardiac fibrosis by using multiple inbred mouse strains to characterize CFbs and their response to ISO treatment. Our data suggest that, although fibroblast activation is a response that parallels the extent of scar formation, proliferation may not necessarily correlate with levels of fibrosis. In addition, by comparing CFbs from multiple strains, we identified pathways as potential therapeutic targets and LTBP2 as a marker for fibrosis, with relevance to patients with underlying myocardial fibrosis