Altered mechanical stress and strain in cardiac myocytes induce modifications in gene expression that affects cardiac remodeling and myocyte contractile function. To study the mechanisms of ...mechanotransduction in cardiomyocytes, probing alterations in mechanics and gene expression has been an effective strategy. However, previous studies are self-limited due to the general use of isolated neonatal rodent myocytes or intact animals. The main goal of this study was to develop a novel tissue culture chamber system for mouse myocardium that facilitates loading of cardiac tissue, while measuring tissue stress and deformation within a physiological environment. Intact mouse right ventricular papillary muscles were cultured in controlled conditions with superfusate at 95% O2/ 5% CO2, and 34 degrees C, such that cell to extracellular matrix adhesions as well as cell to cell adhesions were undisturbed and both passive and active mechanical properties were maintained without significant changes. The system was able to measure the induction of hypertrophic markers (BNP, ANP) in tissue after 2 hrs and 5 hrs of stretch. ANP induction was highly correlated with the diastolic load of the muscle but not with developed systolic load. Load induced ANP expression was blunted in muscles from muscle-LIM protein knockout mice, in which defective mechanotransduction pathways have been predicted.
Understanding mechanisms underlying titin regulation in cardiac muscle function is of critical importance given recent compelling evidence that highlight titin mutations as major determinants of ...human cardiomyopathy. We previously identified a cardiac biomechanical stress-regulated complex at the cardiac-specific N2B region of titin that includes four-and-a-half LIM domain protein-1 (Fhl1) and components of the mitogen-activated protein signaling cascade, which impacted muscle compliance in Fhl1 knock-out cardiac muscle. However, direct regulation of these molecular components in mediating titin N2B function remained unresolved. Here we identify Fhl1 as a novel negative regulator of titin N2B levels and phosphorylation-mediated mechanics. We specifically identify titin N2B as a novel substrate of extracellular signal regulated-kinase-2 (Erk2) and demonstrate that Fhl1 directly interferes with Erk2-mediated titin-N2B phosphorylation. We highlight the critical region in titin-N2B that interacts with Fhl1 and residues that are dependent on Erk2-mediated phosphorylation in situ. We also propose a potential mechanism for a known titin-N2B cardiomyopathy-causing mutation that involves this regulatory complex. These studies shed light on a novel mechanism regulating titin-N2B mechano-signaling as well as suggest that dysfunction of these pathways could be important in cardiac disease states affecting muscle compliance.
Background: Titin is critical for cardiac muscle function; however, limited knowledge exists of mechanisms important for its regulation.
Results: A four-and-a-half LIM domain protein-1/extracellular signal-regulated kinase-2-associated complex modulates titin-N2B levels, phosphorylation, and mechanics.
Conclusion: We reveal new mechanisms underlying titin mechano-signaling.
Significance: We advance our understanding of how titin-associated complexes/mutations can impact cardiac muscle function and disease.
The response of cardiomyocytes to biomechanical stress can determine the pathophysiology of hypertrophic cardiac disease, and targeting the pathways regulating these responses is a therapeutic goal. ...However, little is known about how biomechanical stress is sensed by the cardiomyocyte sarcomere to transduce intracellular hypertrophic signals or how the dysfunction of these pathways may lead to disease. Here, we found that four-and-a-half LIM domains 1 (FHL1) is part of a complex within the cardiomyocyte sarcomere that senses the biomechanical stress-induced responses important for cardiac hypertrophy. Mice lacking Fhl1 displayed a blunted hypertrophic response and a beneficial functional response to pressure overload induced by transverse aortic constriction. A link to the Galphaq (Gq) signaling pathway was also observed, as Fhl1 deficiency prevented the cardiomyopathy observed in Gq transgenic mice. Mechanistic studies demonstrated that FHL1 plays an important role in the mechanism of pathological hypertrophy by sensing biomechanical stress responses via the N2B stretch sensor domain of titin and initiating changes in the titin- and MAPK-mediated responses important for sarcomere extensibility and intracellular signaling. These studies shed light on the physiological regulation of the sarcomere in response to hypertrophic stress.
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Although most studies correlate an increase in systolic stress with pressure overload hypertrophy, there is some evidence suggesting diastolic stress/strain may play the greater role. A ...culture system was developed in which the application of uniaxial stresses to papillary muscles from knockout (KO) mice can induce a hypertrophic response, while simultaneously assessing tissue mechanical properties. After 5 hrs of 15% stretch, muscles express hypertrophic markers ANP & BNP. By comparing the linear regressions of ANP expression verses passive or active stress we found that a higher statistical correlation exists between ANP induction and passive stress than active systolic stress. We compared the development of hypertrophy in KO mice with abnormal diastolic tissue mechanical properties. ANP gene expression in muscles from muscle LIM protein KO mice, a well characterized mouse model having significant diastolic dysfunction, was ~4 times lower when compared to controls. Four & a half LIM protein (FHL1) KO mice exhibited a blunted hypertrophic response and lower ANP levels after 35 days aortic banding. ANP induction by stretch in muscles from FHL1KO mice was also reduced (P<0.05). FHL1KO muscles exhibited greater diastolic tissue compliance, with no change in systolic mechanics. We conclude that changes in passive mechanics correlate with attenuated development of pressure overload hypertrophy in FHL1KO mice.
Altered mechanical stresses and strains in cardiac myocytes can induce modifications in gene expression that can affect cardiac remodeling and myocyte contractile function. Most studies of myocyte ...mechanotransduction use isolated neonatal rat myocytes. To study the genetics of these pathways it is helpful to be able to probe alterations in gene expression in intact muscle from genetically engineered mice. We have developed a tissue culture system that facilitates straining of cardiac tissue, while measuring its force within a physiological environment. The system was developed to house intact right ventricular papillary muscles, such that cell to extracellular matrix adhesions as well as cell to cell adhesions, which influence cardiac remodeling, were undisturbed. The tissue chamber is isolated from the external environment and provides control of O2 supply, temperature, and superfusate delivery. Isolated papillary muscles are suspended within the chamber in modified M199 cell culture media, between a micromanipulator attached to a linear voltage displacement transducer and a force transducer. Through this mechanism, the diastolic and systolic mechanics of papillary muscles were studied and hypertrophic markers can be induced in specimens for a period up to 12 hours. By quantifying mRNA levels of hypertrophic markers (BNP, ELK1, ANP) we monitored the development of hypertrophy within normal specimens and within specimens obtained from FHL1 knock out mice that may have dysregulated hypertrophic or biochemical signaling. Our results revealed that the system is capable of maintaining tissue viability, measuring tissue mechanics, and induces hypertrophic markers (ANP, BNP) in specimens in the acute phase of development (2-5 hours). Compared to wild type specimens, specimens deficient in FHL1 were more compliant and had a blunted response to mechanical load induced hypertrophy. We concluded that FHL1 has dual functions in modulating the passive mechanics of myocardial tissue and hypertrophic signaling of the heart.
Thesis (Ph. D.)--University of California, San Diego, 2008.
Title from first page of PDF file (viewed October 3, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical ...references.
This review focuses on mechanoelectric feedback and mechanotransduction in cardiac cells and stem-cell derived cardiomyocyte progenitor cells. Topics include: methods to apply mechanical stimuli to ...isolated cells and tissues; methods for patterned growth of cells; effects of stretch and shear stress on cellular function and tissue electrophysiology; regulation of structural and junctional proteins by stretch; the role of the cytoskeleton in mechanotransduction and heart failure; signaling pathways involved in mechanotransduction and load-induced hypertrophic responses; and the role of substrate stiffness in stem cell differentiation and maturation of excitation-contraction coupling.
•Both radiation-induced and neurofibromatosis-associated MPNSTs have poorer prognosis than sporadic MPNSTs.•Complete resection of the tumor is a significant prognostic factor for MPNST.•Surgery with ...adjuvant radiotherapy is related to improved local control in patients with positive surgical margins.
Malignant peripheral nerve sheath tumors (MPNST) may be sporadic or associated with neurofibromatosis or prior radiation. MPNST may behave aggressively with a high rate of local recurrence and distant metastasis.
In an IRB approved protocol, we reviewed the clinical characteristics, treatment, and outcomes of 280 patients treated for MPNST at Massachusetts General Hospital (MGH) between 1960 and 2016.
There were 138 men and 142 women with a median age of 41 (range: 3–95) years. Tumors were classified as neurofibromatosis-associated (nfMPNST, n = 77), radiation-induced (rMPNST, n = 21), or sporadic (sMPNST, n = 182) MPNST. The median time to development of rMPNST from prior radiation was 15 years. With a median follow-up of 43.1 months, the median overall survival (OS) was 65.3 months. Older age, nfMPNST, rMPNST, increased tumor size, lymph node involvement, metastatic disease, intermediate to high grade, radiotherapy alone, and R2 resection were related to worse OS, whereas surgery with radiotherapy was associated with improved OS. Among the 251 patients without metastasis, nfMPNST, rMPNST, and increased tumor size were correlated with worse metastasis-free survival; nfMPNST, radiotherapy alone, and R1/R2 resection were associated with local recurrence, whereas surgery with adjuvant radiotherapy was related to improved local control in patients with R1/R2 resection.
Both radiation-induced and neurofibromatosis-associated MPNSTs have poorer prognosis than sporadic MPNSTs. Complete resection of the tumor is a significant prognostic factor for MPNST. The addition of radiotherapy after surgery should be considered especially when the surgical margins are positive.
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