Anna Raskin

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.

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.

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.

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