Jessica E. Rodríguez

Cardiac muscle ring finger-1 increases susceptibility to heart failure in vivo

Muscle ring finger (MuRF)1 is a muscle-specific protein implicated in the regulation of cardiac myocyte size and contractility. MuRF2, a closely related family member, redundantly interacts with protein substrates and heterodimerizes with MuRF1. Mice lacking either MuRF1 or MuRF2 are phenotypically normal, whereas mice lacking both proteins develop a spontaneous cardiac and skeletal muscle hypertrophy, indicating cooperative control of muscle mass by MuRF1 and MuRF2. To identify the unique role that MuRF1 plays in regulating cardiac hypertrophy in vivo, we created transgenic mice expressing increased amounts of cardiac MuRF1. Adult MuRF1 transgenic (Tg+) hearts exhibited a nonprogressive thinning of the left ventricular wall and a concomitant decrease in cardiac function. Experimental induction of cardiac hypertrophy by transaortic constriction (TAC) induced rapid failure of MuRF1 Tg+ hearts. Microarray analysis identified that the levels of genes associated with metabolism (and in particular mitochondrial processes) were significantly altered in MuRF1 Tg+ hearts, both at baseline and during the development of cardiac hypertrophy. Surprisingly, ATP levels in MuRF1 Tg+ mice did not differ from wild-type mice despite the depressed contractility following TAC. In comparing the level and activity of creatine kinase (CK) between wild-type and MuRF1 Tg+ hearts, we found that mCK and CK-M/B protein levels were unaffected in MuRF1 Tg+ hearts; however, total CK activity was significantly inhibited. We conclude that increased expression of cardiac MuRF1 results in a broad disruption of primary metabolic functions, including alterations in CK activity that leads to increased susceptibility to heart failure following TAC. This study demonstrates for the first time a role for MuRF1 in the regulation of cardiac energetics in vivo.

Muscle ring finger 1 mediates cardiac atrophy in vivo

Pathological cardiac hypertrophy, induced by various etiologies such as high blood pressure and aortic stenosis, develops in response to increased afterload and represents a common intermediary in the development of heart failure. Understandably then, the reversal of pathological cardiac hypertrophy is associated with a significant reduction in cardiovascular event risk and represents an important, yet underdeveloped, target of therapeutic research. Recently, we determined that muscle ring finger-1 (MuRF1), a muscle-specific protein, inhibits the development of experimentally induced pathological; cardiac hypertrophy. We now demonstrate that therapeutic cardiac atrophy induced in patients after left ventricular assist device placement is associated with an increase in cardiac MuRF1 expression. This prompted us to investigate the role of MuRF1 in two independent mouse models of cardiac atrophy: 1) cardiac hypertrophy regression after reversal of transaortic constriction (TAC) reversal and 2) dexamethasone-induced atrophy. Using echocardiographic, histological, and gene expression analyses, we found that upon TAC release, cardiac mass and cardiomyocyte cross-sectional areas in MuRF1(-/-) mice decreased approximately 70% less than in wild type mice in the 4 wk after release. This was in striking contrast to wild-type mice, who returned to baseline cardiac mass and cardiomyocyte size within 4 days of TAC release. Despite these differences in atrophic remodeling, the transcriptional activation of cardiac hypertrophy measured by beta-myosin heavy chain, smooth muscle actin, and brain natriuretic peptide was attenuated similarly in both MuRF1(-/-) and wild-type hearts after TAC release. In the second model, MuRF1(-/-) mice also displayed resistance to dexamethasone-induced cardiac atrophy, as determined by echocardiographic analysis. This study demonstrates, for the first time, that MuRF1 is essential for cardiac atrophy in vivo, both in the setting of therapeutic regression of cardiac hypertrophy and dexamethasone-induced atrophy.

Oral Resveratrol Therapy Inhibits Cancer-Induced Skeletal Muscle and Cardiac Atrophy In Vivo

The mechanism by which cancer mediates muscle atrophy has been delineated in the past 3 decades and includes a prominent role of tumor-derived cytokines, such as IL-6, TNFα, and IL-1. These cytokines interact with their cognate receptors on muscle to activate the downstream transcription factor NF-κB and induce sarcomere proteolysis. Experimentally, inhibiting NF-κB signaling largely prevents cancer-induced muscle wasting, indicating its prominent role in muscle atrophy. Resveratrol, a natural phytoalexin found in the skin of grapes, has recently been shown to inhibit NF-κB in cancer cells, which led us to hypothesize that it might have a protective role in cancer cachexia. Therefore, we investigated whether daily oral resveratrol could protect against skeletal muscle loss and cardiac atrophy in an established mouse model. We demonstrate resveratrol inhibits skeletal muscle and cardiac atrophy induced by C26 adenocarcinoma tumors through its inhibition of NF-κB (p65) activity in skeletal muscle and heart. These studies demonstrate for the first time the utility of oral resveratrol therapy to provide clinical benefit in cancer-induced atrophy through the inhibition of NF-κB in muscle. These findings may have application in the treatment of diseases with parallel pathophysiologies such as muscular dystrophy and heart failure.

NF-κB Inhibition Protects against Tumor-Induced Cardiac Atrophy in Vivo

Cancer cachexia is a severe wasting syndrome characterized by the progressive loss of lean body mass and systemic inflammation. It occurs in approximately 80% of patients with advanced malignancy and is the cause of 20% to 30% of all cancer-related deaths. The mechanism by which striated muscle loss occurs is the tumor release of pro-inflammatory cytokines, such as IL-1, IL-6, and TNF-α. These cytokines interact with their cognate receptors on muscle cells to enhance NF-κB signaling, which then mediates muscle loss and significant cardiac dysfunction. Genetic inhibition of NF-κB signaling has demonstrated its predominant role in skeletal muscle loss. Therefore, we tested two novel drugs designed to specifically inhibit NF-κB by targeting the IκB kinase (IKK) complex: Compound A and NEMO binding domain (NBD) peptide. Using an established mouse model of cancer cachexia (C26 adenocarcinoma), we determined how these drugs affected the development of tumor-induced cardiac atrophy and function. Echocardiographic and histological analysis revealed that both Compound A and NBD inhibit cardiac NF-κB activity and prevent the development of tumor-induced systolic dysfunction and atrophy. This protection was independent of any effects of the tumor itself (Compound A) or tumor-secreted cytokines (NBD). This study identifies for the first time, to our knowledge, that drugs targeting the IKK complex are cardioprotective against cancer cachexia-induced cardiac atrophy and systolic dysfunction, suggesting therapies that may help reduce cardiac-associated morbidities found in patients with advanced malignancies.

Familial hypertrophic cardiomyopathy: Basic concepts and future molecular diagnostics

Familial hypertrophic cardiomyopathies (FHC) are the most common genetic heart diseases in the United States, affecting nearly 1 in 500 people. Manifesting as increased cardiac wall thickness, this autosomal dominant disease goes mainly unnoticed as most affected individuals are asymptomatic. Up to 1-2% of children and adolescents and 0.5-1% adults with FHC die of sudden cardiac death, making it critical to quickly and accurately diagnose FHC to institute therapy and potentially reduce mortality. However, due to the heterogeneity of the genetic defects in mainly sarcomere proteins, this is a daunting task even with current diagnostic methods. Exciting new methods utilizing high-throughput microarray technology to identify FHC mutations by a method known as array-based resequencing has recently been described. Additionally, next generation sequencing methodologies may aid in improving FHC diagnosis. In this review, we discuss FHC pathophysiology, the rationale for testing, and discuss the limitations and advantages of current and future diagnostics.

Muscle ring finger 1 and muscle ring finger 2 are necessary but functionally redundant during developmental cardiac growth and regulate E2F1-mediated gene expression in vivo

Muscle ring finger (MuRF) proteins have been implicated in the transmission of mechanical forces to nuclear cell signaling pathways through their association with the sarcomere. We recently reported that MuRF1, but not MuRF2, regulates pathologic cardiac hypertrophy in vivo. This was surprising given that MuRF1 and MuRF2 interact with each other and many of the same sarcomeric proteins experimentally.

MuRF1 activity is present in cardiac mitochondria and regulates reactive oxygen species production in vivo

MuRF1 is a previously reported ubiquitin-ligase found in striated muscle that targets troponin I and myosin heavy chain for degradation. While MuRF1 has been reported to interact with mitochondrial substrates in yeast two-hybrid studies, no studies have identified MuRF1’s role in regulating mitochondrial function to date. In the present study, we measured cardiac mitochondrial function from isolated permeabilized muscle fibers in previously phenotyped MuRF1 transgenic and MuRF1−/− mouse models to determine the role of MuRF1 in intermediate energy metabolism and ROS production. We identified a significant decrease in reactive oxygen species production in cardiac muscle fibers from MuRF1 transgenic mice with increased α-MHC driven MuRF1 expression. Increased MuRF1 expression in ex vivo and in vitro experiments revealed no alterations in the respiratory chain complex I and II function. Working perfusion experiments on MuRF1 transgenic hearts demonstrated significant changes in glucose oxidation. This is an factual error as written; however, total oxygen consumption was decreased. This data provides evidence for MuRF1 as a novel regulator of cardiac ROS, offering another mechanism by which increased MuRF1 expression may be cardioprotective in ischemia reperfusion injury, in addition to its inhibition of apoptosis via proteasome-mediate degradation of c-Jun. The lack of mitochondrial function phenotype identified in MuRF1−/− hearts may be due to the overlapping interactions of MuRF1 and MuRF2 with energy regulating proteins found by yeast two-hybrid studies reported here, implying a duplicity in MuRF1 and MuRF2’s regulation of mitochondrial function.

Muscle RING finger-1 attenuates IGF-I-dependent cardiomyocyte hypertrophy by inhibiting JNK signaling

Recent studies implicate the muscle-specific ubiquitin ligase muscle RING finger-1 (MuRF1) in inhibiting pathological cardiomyocyte growth in vivo by inhibiting the transcription factor SRF. These studies led us to hypothesize that MuRF1 similarly inhibits insulin-like growth factor-I (IGF-I)-mediated physiological cardiomyocyte growth. We identified two lines of evidence to support this hypothesis: IGF-I stimulation of cardiac-derived cells with MuRF1 knockdown 1) exhibited an exaggerated hypertrophy and, 2) conversely, increased MuRF1 expression-abolished IGF-I-dependent cardiomyocyte growth. Enhanced hypertrophy with MuRF1 knockdown was accompanied by increases in Akt-regulated gene expression. Unexpectedly, MuRF1 inhibition of this gene expression profile was not a result of differences in p-Akt. Instead, we found that MuRF1 inhibits total protein levels of Akt, GSK-3β (downstream of Akt), and mTOR while limiting c-Jun protein expression, a mechanism recently shown to govern Akt, GSK-3β, and mTOR activities and expression. These findings establish that MuRF1 inhibits IGF-I signaling by restricting c-Jun activity, a novel mechanism recently identified in the context of ischemia-reperfusion injury. Since IGF-I regulates exercise-mediated physiological cardiac growth, we challenged MuRF1(-/-) and MuRF1-Tg+ mice and their wild-type sibling controls to 5 wk of voluntary wheel running. MuRF1(-/-) cardiac growth was increased significantly over wild-type control; conversely, the enhanced exercise-induced cardiac growth was lost in MuRF1-Tg+ animals. These studies demonstrate that MuRF1-dependent attenuation of IGF-I signaling via c-Jun is applicable in vivo and establish that further understanding of this novel mechanism may be crucial in the development of therapies targeting IGF-I signaling.

Regulation of the calpain and ubiquitin-proteasome systems in a canine model of muscular dystrophy

Introduction: Previous studies have tested the hypothesis that calpain and/or proteasome inhibition is beneficial in Duchenne muscular dystrophy, based largely on evidence that calpain and proteasome activities are enhanced in the mdx mouse. Methods: mRNA expression of ubiquitin‐proteasome and calpain system components were determined using real‐time polymerase chain reaction in skeletal muscle and heart in the golden retriever muscular dystrophy model. Similarly, calpain 1 and 2 and proteasome activities were determined using fluorometric activity assays. Results: We found that less than half of the muscles tested had increases in proteasome activity, and only half had increased calpain activity. In addition, transcriptional regulation of the ubiquitin‐proteasome system was most pronounced in the heart, where numerous components were significantly decreased. Conclusion: This study illustrates the diversity of expression and activities of the ubiquitin‐proteasome and calpain systems, which may lead to unexpected consequences in response to pharmacological inhibition. Muscle Nerve, 2011

Fast square root calculation for DTC magnetic flux estimator

Direct Torque Control (DTC) is a control strategy fully used with induction motors and permanent magnet synchronous motors due to a fast torque response, compared to other traditional control strategies. This paper presents a new technique for calculating the square root, which is the operation that presents the greatest difficulty when developing the DTC strategy on an FPGA. The architecture presented is in VHDL using a structural modeling style and a successive approximation register to calculate digit by digit, the square root of a binary unsigned 32-bit number, which can be easily expanded to a larger number of bits. With this architecture the flux estimator processing time was lowered to 1.12μs, when the lowest time reported was 5μs. This time reduction will allow a dramatic reduction in torque ripple, a common effect when using DTC.