Hind Lal

GSK-3α is a central regulator of age-related pathologies in mice

Aging is regulated by conserved signaling pathways. The glycogen synthase kinase-3 (GSK-3) family of serine/threonine kinases regulates several of these pathways, but the role of GSK-3 in aging is unknown. Herein, we demonstrate premature death and acceleration of age-related pathologies in the Gsk3a global KO mouse. KO mice developed cardiac hypertrophy and contractile dysfunction as well as sarcomere disruption and striking sarcopenia in cardiac and skeletal muscle, a classical finding in aging. We also observed severe vacuolar degeneration of myofibers and large tubular aggregates in skeletal muscle, consistent with impaired clearance of insoluble cellular debris. Other organ systems, including gut, liver, and the skeletal system, also demonstrated age-related pathologies. Mechanistically, we found marked activation of mTORC1 and associated suppression of autophagy markers in KO mice. Loss of GSK-3α, either by pharmacologic inhibition or Gsk3a gene deletion, suppressed autophagy in fibroblasts. mTOR inhibition rescued this effect and reversed the established pathologies in the striated muscle of the KO mouse. Thus, GSK-3α is a critical regulator of mTORC1, autophagy, and aging. In its absence, aging/senescence is accelerated in multiple tissues. Strategies to maintain GSK-3α activity and/or inhibit mTOR in the elderly could retard the appearance of age-related pathologies.

Glycogen Synthase Kinase-3β Regulates Post–Myocardial Infarction Remodeling and Stress-Induced Cardiomyocyte Proliferation In Vivo

Rationale: Numerous studies have proposed that glycogen synthase kinase (GSK)-3&bgr; is a central regulator of the hypertrophic response of cardiomyocytes. However, all of this work has relied on overexpression of GSK-3&bgr;, expression of constitutively active mutants, or small molecule inhibitors with documented off-target effects. Genetic loss of function approaches have not been used in the adult mouse because germ-line deletion of GSK-3&bgr; is embryonic-lethal. Objective: This study was designed to define the role played by GSK-3&bgr; in pressure overload (PO)-induced hypertrophy and remodeling following myocardial infarction (MI). Methods and Results: We used a mouse model that allows inducible, cardiomyocyte-specific deletion of GSK-3&bgr; in the adult knockout. Surprisingly, we find that knockout mice exposed to PO induced by thoracic aortic constriction exhibit a normal hypertrophic response. Thus, in contrast to virtually all prior published studies, GSK-3&bgr; appears to play at most a minor role in the hypertrophic response to PO stress. However, GSK-3&bgr; does regulate post-MI remodeling because the GSK-3&bgr; knockouts had less left ventricular dilatation and better-preserved left ventricular function at up to 8 weeks post-MI despite demonstrating significantly more hypertrophy in the remote myocardium. Deletion of GSK-3&bgr; also led to increased cardiomyocyte proliferation following PO and MI. Conclusions: Deletion of GSK-3&bgr; protects against post-MI remodeling and promotes stress-induced cardiomyocyte proliferation in the adult heart. These studies suggest that inhibition of GSK-3&bgr; could be a strategy to both prevent remodeling and to promote cardiac regeneration in pathological states.

Prevention of liver cancer cachexia-induced cardiac wasting and heart failure

AIMS Symptoms of cancer cachexia (CC) include fatigue, shortness of breath, and impaired exercise capacity, which are also hallmark symptoms of heart failure (HF). Herein, we evaluate the effects of drugs commonly used to treat HF (bisoprolol, imidapril, spironolactone) on development of cardiac wasting, HF, and death in the rat hepatoma CC model (AH-130). METHODS AND RESULTS Tumour-bearing rats showed a progressive loss of body weight and left-ventricular (LV) mass that was associated with a progressive deterioration in cardiac function. Strikingly, bisoprolol and spironolactone significantly reduced wasting of LV mass, attenuated cardiac dysfunction, and improved survival. In contrast, imidapril had no beneficial effect. Several key anabolic and catabolic pathways were dysregulated in the cachectic hearts and, in addition, we found enhanced fibrosis that was corrected by treatment with spironolactone. Finally, we found cardiac wasting and fibrotic remodelling in patients who died as a result of CC. In living cancer patients, with and without cachexia, serum levels of brain natriuretic peptide and aldosterone were elevated. CONCLUSION Systemic effects of tumours lead not only to CC but also to cardiac wasting, associated with LV-dysfunction, fibrotic remodelling, and increased mortality. These adverse effects of the tumour on the heart and on survival can be mitigated by treatment with either the β-blocker bisoprolol or the aldosterone antagonist spironolactone. We suggest that clinical trials employing these agents be considered to attempt to limit this devastating complication of cancer.

Integrins and proximal signaling mechanisms in cardiovascular disease

Integrins are heterodimeric cell-surface molecules, which act as the principle mediators of molecular dialog between a cell and its extracellular matrix environment. In addition to their structural functions, integrins mediate signaling from the extracellular space into the cell through integrin-associated signaling and adaptor molecules such as FAK (focal adhesion kinase), ILK (integrin-linked kinase), PINCH (particularly interesting new cysteine-histidine rich protein) and Nck2 (non-catalytic (region of) tyrosine kinase adaptor protein-2). Via these molecules, integrin signaling tightly and cooperatively interacts with receptor tyrosine kinases (RTKs) signaling to regulate survival, proliferation and cell shape as well as polarity, adhesion, migration and differentiation. In the heart and blood vessels, the function and regulation of these molecules can be partially disturbed and thus contribute to cardiovascular diseases such as cardiac hypertrophy and atherosclerosis. In this review, we discuss the primary mechanisms of action and signaling of integrins in the cardiac and vascular system in normal and pathological states, as well as therapeutic strategies for targeting these systems (1).

The GSK-3 Family as Therapeutic Target for Myocardial Diseases

Glycogen synthase kinase-3 (GSK-3) is one of the few signaling molecules that regulate a truly astonishing number of critical intracellular signaling pathways. It has been implicated in several diseases including heart failure, bipolar disorder, diabetes mellitus, Alzheimer disease, aging, inflammation, and cancer. Furthermore, a recent clinical trial has validated the feasibility of targeting GSK-3 with small molecule inhibitors for human diseases. In the current review, we will focus on its expanding role in the heart, concentrating primarily on recent studies that have used cardiomyocyte- and fibroblast-specific conditional gene deletion in mouse models. We will highlight the role of the GSK-3 isoforms in various pathological conditions including myocardial aging, ischemic injury, myocardial fibrosis, and cardiomyocyte proliferation. We will discuss our recent findings that deletion of GSK-3α specifically in cardiomyocytes attenuates ventricular remodeling and cardiac dysfunction after myocardial infarction by limiting scar expansion and promoting cardiomyocyte proliferation. The recent emergence of GSK-3β as a regulator of myocardial fibrosis will also be discussed. We will review our recent findings that specific deletion of GSK-3β in cardiac fibroblasts leads to fibrogenesis, left ventricular dysfunction, and excessive scarring in the ischemic heart. Finally, we will examine the underlying mechanisms that drive the aberrant myocardial fibrosis in the models in which GSK-3β is specifically deleted in cardiac fibroblasts. We will summarize these recent results and offer explanations, whenever possible, and hypotheses when not. For these studies we will rely heavily on our models and those of others to reconcile some of the apparent inconsistencies in the literature.

Cancer Genetics and the Cardiotoxicity of the Therapeutics

Cancer genomics has focused on the discovery of mutations and chromosomal structural rearrangements that either increase susceptibility to cancer or support the cancer phenotype. Protein kinases are the most frequently mutated genes in the cancer genome, making them attractive therapeutic targets for drug design. However, the use of some of the kinase inhibitors (KIs) has been associated with toxicities to the heart and vasculature, including acute coronary syndromes and heart failure. Herein we discuss the genetic basis of cancer, focusing on mutations in the kinase genome (kinome) that lead to tumorigenesis. This will allow an understanding of the real and potential power of modern cancer therapeutics. The underlying mechanisms that drive the cardiotoxicity of the KIs are also examined. The preclinical models for predicting cardiotoxicity, including induced pluripotent stem cells and zebrafish, are reviewed, with the hope of eventually being able to identify problematic agents before their use in patients. Finally, the use of biomarkers in the clinic is discussed, and newer strategies (i.e., metabolomics and enhanced imaging strategies) that may allow earlier and more accurate detection of cardiotoxicity are reviewed.

GSK-3α directly regulates β-adrenergic signaling and the response of the heart to hemodynamic stress in mice

The glycogen synthase kinase-3 (GSK-3) family of serine/threonine kinases consists of 2 highly related isoforms, alpha and beta. Although GSK-3beta has an important role in cardiac development, much remains unknown about the function of either GSK-3 isoform in the postnatal heart. Herein, we present what we believe to be the first studies defining the role of GSK-3alpha in the mouse heart using gene targeting. Gsk3a(-/-) mice over 2 months of age developed progressive cardiomyocyte and cardiac hypertrophy and contractile dysfunction. Following thoracic aortic constriction in young mice, we observed enhanced hypertrophy that rapidly transitioned to ventricular dilatation and contractile dysfunction. Surprisingly, markedly impaired beta-adrenergic responsiveness was found at both the organ and cellular level. This phenotype was reproduced by acute treatment of WT cardiomyocytes with a small molecule GSK-3 inhibitor, confirming that the response was not due to a chronic adaptation to LV dysfunction. Thus, GSK-3alpha appears to be the central regulator of a striking range of essential processes, including acute and direct positive regulation of beta-adrenergic responsiveness. In the absence of GSK-3alpha, the heart cannot respond effectively to hemodynamic stress and rapidly fails. Our findings identify what we believe to be a new paradigm of regulation of beta-adrenergic signaling and raise concerns given the rapid expansion of drug development targeting GSK-3.

Cardiac Fibroblast Glycogen Synthase Kinase-3β Regulates Ventricular Remodeling and Dysfunction in Ischemic Heart

Background— Myocardial infarction–induced remodeling includes chamber dilatation, contractile dysfunction, and fibrosis. Of these, fibrosis is the least understood. After myocardial infarction, activated cardiac fibroblasts deposit extracellular matrix. Current therapies to prevent fibrosis are inadequate, and new molecular targets are needed. Methods and Results— Herein we report that glycogen synthase kinase-3&bgr; (GSK-3&bgr;) is phosphorylated (inhibited) in fibrotic tissues from ischemic human and mouse heart. Using 2 fibroblast-specific GSK-3&bgr; knockout mouse models, we show that deletion of GSK-3&bgr; in cardiac fibroblasts leads to fibrogenesis, left ventricular dysfunction, and excessive scarring in the ischemic heart. Deletion of GSK-3&bgr; induces a profibrotic myofibroblast phenotype in isolated cardiac fibroblasts, in post–myocardial infarction hearts, and in mouse embryonic fibroblasts deleted for GSK-3&bgr;. Mechanistically, GSK-3&bgr; inhibits profibrotic transforming growth factor-&bgr;1/SMAD-3 signaling via interactions with SMAD-3. Moreover, deletion of GSK-3&bgr; resulted in the significant increase of SMAD-3 transcriptional activity. This pathway is central to the pathology because a small-molecule inhibitor of SMAD-3 largely prevented fibrosis and limited left ventricular remodeling. Conclusions— These studies support targeting GSK-3&bgr; in myocardial fibrotic disorders and establish critical roles of cardiac fibroblasts in remodeling and ventricular dysfunction.

A novel cardioprotective p38-MAPK/mTOR pathway

Despite intensive study, the mechanisms regulating activation of mTOR and the consequences of that activation in the ischemic heart remain unclear. This is particularly true for the setting of ischemia/reperfusion (I/R) injury. In a mouse model of I/R injury, we observed robust mTOR activation, and its inhibition by rapamycin increased injury. Consistent with the in-vivo findings, mTOR activation was also protective in isolated cardiomyocytes exposed to two models of I/R. Moreover, we identify a novel oxidant stress-activated pathway regulating mTOR that is critically dependent on p38-MAPK and Akt. This novel p38-regulated pathway signals downstream through REDD1, Tsc2, and 14-3-3 proteins to activate mTOR and is independent of AMPK. The protective role of p38/Akt and mTOR following oxidant stress is a general phenomenon since we observed it in a wide variety of cell types. Thus we have identified a novel protective pathway in the cardiomyocyte involving p38-mediated mTOR activation. Furthermore, the p38-dependent protective pathway might be able to be selectively modulated to enhance cardio-protection while not interfering with the inhibition of the better-known detrimental p38-dependent pathways.

Rac1 and RhoA differentially regulate angiotensinogen gene expression in stretched cardiac fibroblasts

AIMS Angiotensin II (Ang II) stimulates cardiac remodelling and fibrosis in the mechanically overloaded myocardium. Although Rho GTPases regulate several cellular processes, including myocardial remodelling, involvement in mediating mechanical stretch-induced regulation of angiotensinogen (Ao), the precursor to Ang II, remains to be determined. We, therefore, examined the role and associated signalling mechanisms of Rho GTPases (Rac1 and RhoA) in regulation of Ao gene expression in a stretch model of neonatal rat cardiac fibroblasts (CFs). METHODS AND RESULTS CFs were plated on deformable stretch membranes. Equiaxial mechanical stretch caused significant activation of both Rac1 and RhoA within 2-5 min. Rac1 activity returned to control levels after 4 h, whereas RhoA remained at a high level of activity until the end of the stretch period (24 h). Mechanical stretch initially caused a moderate decrease in Ao gene expression, but was significantly increased at 8-24 h. RhoA had a major role in mediating both the stretch-induced inhibition of Ao at 4 h and the subsequent upregulation of Ao expression at 24 h. β₁ integrin receptor blockade by Tac β₁ expression impaired acute (2 and 15 min) stretch-induced Rac1 activation, but increased RhoA activity. Molecular experiments revealed that Ao gene expression was inhibited by Rac1 through both JNK-dependent and independent mechanisms, and stimulated by RhoA through a p38-dependent mechanism. CONCLUSION These results indicate that stretch-induced activation of Rac1 and RhoA differentially regulates Ao gene expression by modulating p38 and JNK activation.