Jennifer Davis

Chronic administration of membrane sealant prevents severe cardiac injury and ventricular dilatation in dystrophic dogs

Duchenne muscular dystrophy (DMD) is a fatal disease of striated muscle deterioration caused by lack of the cytoskeletal protein dystrophin. Dystrophin deficiency causes muscle membrane instability, skeletal muscle wasting, cardiomyopathy, and heart failure. Advances in palliative respiratory care have increased the incidence of heart disease in DMD patients, for which there is no cure or effective therapy. Here we have shown that chronic infusion of membrane-sealing poloxamer to severely affected dystrophic dogs reduced myocardial fibrosis, blocked increased serum cardiac troponin I (cTnI) and brain type natriuretic peptide (BNP), and fully prevented left-ventricular remodeling. Mechanistically, we observed a markedly greater primary defect of reduced cell compliance in dystrophic canine myocytes than in the mildly affected mdx mouse myocytes, and this was associated with a lack of utrophin upregulation in the dystrophic canine cardiac myocytes. Interestingly, after chronic poloxamer treatment, the poor compliance of isolated canine myocytes remained evident, but this could be restored to normal upon direct application of poloxamer. Collectively, these findings indicate that dystrophin and utrophin are critical to membrane stability-dependent cardiac myocyte mechanical compliance and that poloxamer confers a highly effective membrane-stabilizing chemical surrogate in dystrophin/utrophin deficiency. We propose that membrane sealant therapy is a potential treatment modality for DMD heart disease and possibly other disorders with membrane defect etiologies.

Designing Heart Performance by Gene Transfer

The birth of molecular cardiology can be traced to the development and implementation of high-fidelity genetic approaches for manipulating the heart. Recombinant viral vector-based technology offers a highly effective approach to genetically engineer cardiac muscle in vitro and in vivo. This review highlights discoveries made in cardiac muscle physiology through the use of targeted viral-mediated genetic modification. Here the history of cardiac gene transfer technology and the strengths and limitations of viral and nonviral vectors for gene delivery are reviewed. A comprehensive account is given of the application of gene transfer technology for studying key cardiac muscle targets including Ca(2+) handling, the sarcomere, the cytoskeleton, and signaling molecules and their posttranslational modifications. The primary objective of this review is to provide a thorough analysis of gene transfer studies for understanding cardiac physiology in health and disease. By comparing results obtained from gene transfer with those obtained from transgenesis and biophysical and biochemical methodologies, this review provides a global view of cardiac structure-function with an eye towards future areas of research. The data presented here serve as a basis for discovery of new therapeutic targets for remediation of acquired and inherited cardiac diseases.

Molecular networks underlying myofibroblast fate and fibrosis

Fibrotic remodeling is a hallmark of most forms of cardiovascular disease and a strong prognostic indicator of the advancement towards heart failure. Myofibroblasts, which are a heterogeneous cell-type specialized for extracellular matrix (ECM) secretion and tissue contraction, are the primary effectors of the hearts fibrotic response. This review is focused on defining myofibroblast physiology, its progenitor cell populations, and the core signaling network that orchestrates myofibroblast differentiation as a way of understanding the basic determinants of fibrotic disease in the heart and other tissues.

Fibroblast-Specific Genetic Manipulation of p38 Mitogen-Activated Protein Kinase In Vivo Reveals Its Central Regulatory Role in Fibrosis

Background: In the heart, acute injury induces a fibrotic healing response that generates collagen-rich scarring that is at first protective but if inappropriately sustained can worsen heart disease. The fibrotic process is initiated by cytokines, neuroendocrine effectors, and mechanical strain that promote resident fibroblast differentiation into contractile and extracellular matrix–producing myofibroblasts. The mitogen-activated protein kinase p38&agr; (Mapk14 gene) is known to influence the cardiac injury response, but its direct role in orchestrating programmed fibroblast differentiation and fibrosis in vivo is unknown. Methods: A conditional Mapk14 allele was used to delete the p38&agr; encoding gene specifically in cardiac fibroblasts or myofibroblasts with 2 different tamoxifen-inducible Cre recombinase–expressing gene–targeted mouse lines. Mice were subjected to ischemic injury or chronic neurohumoral stimulation and monitored for survival, cardiac function, and fibrotic remodeling. Antithetically, mice with fibroblast-specific transgenic overexpression of activated mitogen-activated protein kinase kinase 6, a direct inducer of p38, were generated to investigate whether this pathway can directly drive myofibroblast formation and the cardiac fibrotic response. Results: In mice, loss of Mapk14 blocked cardiac fibroblast differentiation into myofibroblasts and ensuing fibrosis in response to ischemic injury or chronic neurohumoral stimulation. A similar inhibition of myofibroblast formation and healing was also observed in a dermal wounding model with deletion of Mapk14. Transgenic mice with fibroblast-specific activation of mitogen-activated protein kinase kinase 6–p38 developed interstitial and perivascular fibrosis in the heart, lung, and kidney as a result of enhanced myofibroblast numbers. Mechanistic experiments show that p38 transduces cytokine and mechanical signals into myofibroblast differentiation through the transcription factor serum response factor and the signaling effector calcineurin. Conclusions: These findings suggest that signals from diverse modes of injury converge on p38&agr; mitogen-activated protein kinase within the fibroblast to program the fibrotic response and myofibroblast formation in vivo, suggesting a novel therapeutic approach with p38 inhibitors for future clinical application.

Diastolic dysfunction and thin filament dysregulation resulting from excitation–contraction uncoupling in a mouse model of restrictive cardiomyopathy

Restrictive cardiomyopathy (RCM) has been linked to mutations in the thin filament regulatory protein cardiac troponin I (cTnI). As the pathogenesis of RCM from genotype to clinical phenotype is not fully understood, transgenic (Tg) mice were generated with cardiac specific expression of an RCM-linked missense mutation (R193H) in cTnI. R193H Tg mouse hearts with 15% stoichiometric replacement had smaller hearts and significantly elevated end diastolic pressures (EDP) in vivo. Using a unique carbon microfiber-based assay, membrane intact R193H adult cardiac myocytes generated higher passive tensions across a range of physiologic sarcomere lengths resulting in significant Ca(2+) independent cellular diastolic tone that was manifest in vivo as elevated organ-level EDP. Sarcomere relaxation and Ca(2+) decay was uncoupled in isolated R193H Tg adult myocytes due to the increase in myofilament Ca(2+) sensitivity of tension, decreased passive compliance of the sarcomere, and adaptive in vivo changes in which phospholamban (PLN) content was decreased. Further evidence of Ca(2+) and mechanical uncoupling in R193H Tg myocytes was demonstrated by the biphasic response of relaxation to increased pacing frequency versus the negative staircase seen with Ca(2+) decay. In comparison, non-transgenic myocyte relaxation closely paralleled the accelerated Ca(2+) decay. Ca(2+) transient amplitude was also significantly blunted in R193H Tg myocytes despite normal mechanical shortening resulting in myocyte hypercontractility when compared to non-transgenics. These results identify for the first time that a single point mutation in cTnI, R193H, directly causes elevated EDP due to a myocyte intrinsic loss of compliance independent of Ca(2+) cycling or altered cardiac morphology. The compound influence of impaired relaxation and elevated EDP represents a clinically severe form of diastolic dysfunction similar to the hemodynamic state documented in RCM patients.

Muscular dystrophy in a dish: engineered human skeletal muscle mimetics for disease modeling and drug discovery

Engineered in vitro models using human cells, particularly patient-derived induced pluripotent stem cells (iPSCs), offer a potential solution to issues associated with the use of animals for studying disease pathology and drug efficacy. Given the prevalence of muscle diseases in human populations, an engineered tissue model of human skeletal muscle could provide a biologically accurate platform to study basic muscle physiology, disease progression, and drug efficacy and/or toxicity. Such platforms could be used as phenotypic drug screens to identify compounds capable of alleviating or reversing congenital myopathies, such as Duchene muscular dystrophy (DMD). Here, we review current skeletal muscle modeling technologies with a specific focus on efforts to generate biomimetic systems for investigating the pathophysiology of dystrophic muscle.

Combinatorial Effects of Double Cardiomyopathy Mutant Alleles in Rodent Myocytes: A Predictive Cellular Model of Myofilament Dysregulation in Disease

Inherited cardiomyopathy (CM) represents a diverse group of cardiac muscle diseases that present with a broad spectrum of symptoms ranging from benign to highly malignant. Contributing to this genetic complexity and clinical heterogeneity is the emergence of a cohort of patients that are double or compound heterozygotes who have inherited two different CM mutant alleles in the same or different sarcomeric gene. These patients typically have early disease onset with worse clinical outcomes. Little experimental attention has been directed towards elucidating the physiologic basis of double CM mutations at the cellular-molecular level. Here, dual gene transfer to isolated adult rat cardiac myocytes was used to determine the primary effects of co-expressing two different CM-linked mutant proteins on intact cardiac myocyte contractile physiology. Dual expression of two CM mutants, that alone moderately increase myofilament activation, tropomyosin mutant A63V and cardiac troponin mutant R146G, were shown to additively slow myocyte relaxation beyond either mutant studied in isolation. These results were qualitatively similar to a combination of moderate and strong activating CM mutant alleles αTmA63V and cTnI R193H, which approached a functional threshold. Interestingly, a combination of a CM myofilament deactivating mutant, troponin C G159D, together with an activating mutant, cTnIR193H, produced a hybrid phenotype that blunted the strong activating phenotype of cTnIR193H alone. This is evidence of neutralizing effects of activating/deactivating mutant alleles in combination. Taken together, this combinatorial mutant allele functional analysis lends molecular insight into disease severity and forms the foundation for a predictive model to deconstruct the myriad of possible CM double mutations in presenting patients.

Mechanoregulation of Myofibroblast Fate and Cardiac Fibrosis

During myocardial infarction, myocytes die and are replaced by a specialized fibrotic extracellular matrix, otherwise known as scarring. Fibrotic scarring presents a tremendous hemodynamic burden on the heart, as it creates a stiff substrate, which resists diastolic filling. Fibrotic mechanisms result in permanent scarring which often leads to hypertrophy, arrhythmias, and a rapid progression to failure. Despite the deep understanding of fibrosis in other tissues, acquired through previous investigations, the mechanisms of cardiac fibrosis remain unclear. Recent studies suggest that biochemical cues as well as mechanical cues regulate cells in myocardium. However, the steps in myofibroblast transdifferentiation, as well as the molecular mechanisms of such transdifferentiation in vivo, are poorly understood. This review is focused on defining myofibroblast physiology, scar mechanics, and examining current findings of myofibroblast regulation by mechanical stress, stiffness, and topography for understanding fibrotic disease dynamics.

Cyclic Stiffness Modulation of Cell-Laden Protein-Polymer Hydrogels in Response to User-Specified Stimuli Including Light

Although mechanical signals presented by the extracellular matrix are known to regulate many essential cell functions, the specific effects of these interactions, particularly in response to dynamic and heterogeneous cues, remain largely unknown. Here, a modular semisynthetic approach is introduced to create protein–polymer hydrogel biomaterials that undergo reversible stiffening in response to user‐specified inputs. Employing a novel dual‐chemoenzymatic modification strategy, fusion protein‐based gel crosslinkers are created that exhibit stimuli‐dependent intramolecular association. Linkers based on calmodulin yield calcium‐sensitive materials, while those containing the photosensitive light, oxygen, and voltage sensing domain 2 (LOV2) protein give phototunable constructs whose moduli can be cycled on demand with spatiotemporal control about living cells. These unique materials are exploited to demonstrate the significant role that cyclic mechanical loading plays on fibroblast‐to‐myofibroblast transdifferentiation in 3D space. The moduli‐switchable materials should prove useful for studies in mechanobiology, providing new avenues to probe and direct matrix‐driven changes in 4D cell physiology.

Putting the Brakes on Hypertensive Remodeling: An ATF3 Mechanism of Myofibroblast Restraint.

Article, see p 2041 Heart disease remains the leading cause of death in this country and, for the first time in 30 years, the Centers for Disease Control and Prevention reported that cardiac-related deaths are on the rise. A primary contributor to these numbers is hypertensive heart disease, which is characterized by myocyte hypertrophy and widespread fibrosis. Fibrotic remodeling increases both cardiac arrhythmogenicity and myocardial stiffness that cause a rapid progression toward failure. Thus, decades of research have been directed toward identifying the basis for cardiac remodeling with an eye toward short circuiting this maladaptive process. In doing so, the field has largely focused on cardiac myocytes, but the recent emergence of targeted genetic approaches for manipulating cardiac cells has revealed vital roles for nonmyocytes in mediating cardiac plasticity. Fibroblasts have gained traction in this respect, as demonstrated by the surge of evidence shifting their role from a passive player responding to cardiac myocyte dysfunction to a primary component of the disease process.1,2 Building on this theme, the findings from Li et al,3 which are featured in this issue of Circulation , underscore the importance of revisiting previously defined remodeling signals in a more targeted cellular context. This study was focused on uncovering new transcriptional mechanisms for cardiac remodeling, and with RNAseq analysis they identified 22 transcription factors that were differentially expressed in whole heart lysates from 2 independent mouse models of hypertensive heart disease: chronic angiotensin II (AngII) infusion and pressure overload (transverse aortic constriction). Within this overlapping subgroup, activating transcription factor 3 (ATF3) had the greatest increase in expression and was the most consistently upregulated of the 22 candidate factors. ATF3 has long been identified as a global stress-response mechanism because it is rarely expressed in healthy tissues, but is induced by a wide …