Luge Li

Muscle Ring Finger 1, but not Muscle Ring Finger 2, Regulates Cardiac Hypertrophy In Vivo

Muscle ring finger (MuRF) proteins have been implicated in transmitting mechanical forces to cell signaling pathways through their interactions with the giant protein titin. Recent evidence has linked mechanically-induced stimuli with the control of serum response factor activity and localization through MuRF2. This observation is particularly intriguing in the context of cardiac hypertrophy, where serum response factor transactivation is a key event necessary for the induction of cardiac hypertrophy in response to increased afterload. We have previously reported that MuRF1, which is also a titin-associated protein, exerts antihypertrophic activity in vitro. In the present study, we induced cardiac hypertrophy in mice lacking MuRF1 and MuRF2 to distinguish the physiologic role of these divergent proteins in vivo. We identified for the first time that MuRF1, but not MuRF2, plays a key role in regulating the induction of cardiac hypertrophy, likely by its direct interactions with serum response factor. These studies describe for the first time distinct and nonoverlapping functional characteristics of MuRF1 and MuRF2 in response to cardiac stress in vivo.

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.

The Ubiquitin Ligase MuRF1 Protects Against Cardiac Ischemia/Reperfusion Injury by Its Proteasome-Dependent Degradation of Phospho-c-Jun

Despite improvements in interventions of acute coronary syndromes, primary reperfusion therapies restoring blood flow to ischemic myocardium leads to the activation of signaling cascades that induce cardiomyocyte cell death. These signaling cascades, including the mitogen-activated protein kinase signaling pathways, activate cardiomyocyte death in response to both ischemia and reperfusion. We have previously identified muscle ring finger-1 (MuRF1) as a cardiac-specific protein that regulates cardiomyocyte mass through its ubiquitin ligase activity, acting to degrade sarcomeric proteins and inhibit transcription factors involved in cardiac hypertrophy signaling. To determine MuRF1s role in cardiac ischemia/reperfusion (I/R) injury, cardiomyocytes in culture and intact hearts were challenged with I/R injury in the presence and absence of MuRF1. We found that MuRF1 is cardioprotective, in part, by its ability to prevent cell death by inhibiting Jun N-terminal kinase (JNK) signaling. MuRF1 specifically targets JNKs proximal downstream target, activated phospho-c-Jun, for degradation by the proteasome, effectively inhibiting downstream signaling and the induction of cell death. MuRF1s inhibitory affects on JNK signaling through its ubiquitin proteasome-dependent degradation of activated c-Jun is the first description of a cardiac ubiquitin ligase inhibiting mitogen-activated protein kinase signaling. MuRF1s cardioprotection in I/R injury is attenuated in the presence of pharmacologic JNK inhibition in vivo, suggesting a prominent role of MuRF1s regulation of c-Jun in the intact heart.

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

Fenofibrate unexpectedly induces cardiac hypertrophy in mice lacking MuRF1.

The muscle-specific ubiquitin ligase muscle ring finger-1 (MuRF1) is critical in regulating both pathological and physiological cardiac hypertrophy in vivo. Previous work from our group has identified MuRF1s ability to inhibit serum response factor and insulin-like growth factor-1 signaling pathways (via targeted inhibition of cJun as underlying mechanisms). More recently, we have identified that MuRF1 inhibits fatty acid metabolism by targeting peroxisome proliferator-activated receptor alpha (PPARα) for nuclear export via mono-ubiquitination. Since MuRF1-/- mice have an estimated fivefold increase in PPARα activity, we sought to determine how challenge with the PPARα agonist fenofibrate, a PPARα ligand, would affect the heart physiologically. In as little as 3 weeks, feeding with fenofibrate/chow (0.05% wt/wt) induced unexpected pathological cardiac hypertrophy not present in age-matched sibling wild-type (MuRF1+/+) mice, identified by echocardiography, cardiomyocyte cross-sectional area, and increased beta-myosin heavy chain, brain natriuretic peptide, and skeletal muscle α-actin mRNA. In addition to pathological hypertrophy, MuRF1-/- mice had an unexpected differential expression in genes associated with the pleiotropic effects of fenofibrate involved in the extracellular matrix, protease inhibition, hemostasis, and the sarcomere. At both 3 and 8 weeks of fenofibrate treatment, the differentially expressed MuRF1-/- genes most commonly had SREBP-1 and E2F1/E2F promoter regions by TRANSFAC analysis (54 and 50 genes, respectively, of the 111 of the genes >4 and <-4 log fold change; P ≤ .0004). These studies identify MuRF1s unexpected regulation of fenofibrates pleiotropic effects and bridges, for the first time, MuRF1s regulation of PPARα, cardiac hypertrophy, and hemostasis.