George E. Taffet

TAK1 is activated in the myocardium after pressure overload and is sufficient to provoke heart failure in transgenic mice.

The transforming-growth-factor-β-activated kinase TAK1 is a member of the mitogen-activated protein kinase kinase kinase family, which couples extracellular stimuli to gene transcription. The in vivo function of TAK1 is not understood. Here, we investigated the potential involvement of TAK1 in cardiac hypertrophy. In adult mouse myocardium, TAK1 kinase activity was upregulated 7 days after aortic banding, a mechanical load that induces hypertrophy and expression of transforming growth factor β. An activating mutation of TAK1 expressed in myocardium of transgenic mice was sufficient to produce p38 mitogen-activated protein kinase phosphorylation in vivo, cardiac hypertrophy, interstitial fibrosis, severe myocardial dysfunction, ‘fetal’ gene induction, apoptosis and early lethality. Thus, TAK1 activity is induced as a delayed response to mechanical stress, and can suffice to elicit myocardial hypertrophy and fulminant heart failure.

Hemodynamic Regulation of Tumor Necrosis Factor-α Gene and Protein Expression in Adult Feline Myocardium

Tumor necrosis factor-alpha (TNF-alpha) mRNA and protein biosynthesis were examined in adult feline myocardium in the presence and absence of superimposed hemodynamic pressure overloading. A brief period of hemodynamic pressure overloading ex vivo resulted in de novo TNF-alpha mRNA expression within 30 minutes and de novo TNF-alpha protein production within 60 minutes; neither TNF-alpha mRNA nor protein was detected in hearts perfused at normal perfusion pressures. Moreover, TNF-alpha mRNA and protein biosynthesis were observed in myocyte and nonmyocyte cell types in the pressure-overloaded hearts. To determine whether a simple passive stretch of the myocardium was a sufficient stimulus for TNF-alpha biosynthesis, we examined TNF-alpha mRNA expression in stretched and unstretched papillary muscles. This study showed that myocardial stretch was a sufficient stimulus for the induction of TNF-alpha mRNA biosynthesis. The functional significance of the intramyocardial production of TNF-alpha was determined by examining cell motion in isolated contracting cardiac myocytes treated with superfusates from pressure-overloaded and control hearts. These studies showed that cell motion was depressed in myocytes treated with superfusates from the pressure-overloaded hearts but was normal with the superfusates from the control hearts. Finally, hemodynamic pressure overloading in vivo under physiological conditions was also shown to result in de novo intramyocardial TNF-alpha mRNA biosynthesis. In conclusion, this study constitutes the initial demonstration that the adult mammalian myocardium elaborates biologically active TNF-alpha, both ex vivo and in vivo, in response to hemodynamic pressure overloading.

Essential Role of Smad3 in Infarct Healing and in the Pathogenesis of Cardiac Remodeling

Background— Postinfarction cardiac repair is regulated through timely activation and repression of inflammatory pathways, followed by transition to fibrous tissue deposition and formation of a scar. The transforming growth factor-&bgr;/Smad3 pathway is activated in healing infarcts and may regulate cellular events critical for the inflammatory and the fibrotic responses. Methods and Results— We examined the effects of Smad3 gene disruption on infarct healing and the pathogenesis of cardiac remodeling. In the absence of injury, Smad3-null hearts had comparable function to and similar morphology as wild-type hearts. Smad3-null animals had suppressed peak chemokine expression and decreased neutrophil recruitment in the infarcted myocardium but showed timely repression of inflammatory gene synthesis and resolution of the inflammatory infiltrate. Although myofibroblast density was higher in Smad3-null infarcts, interstitial deposition of collagen and tenascin-C in the remodeling myocardium was markedly reduced. Compared with wild-type animals, Smad3−/− mice exhibited decreased dilative remodeling and attenuated diastolic dysfunction; however, infarct size was comparable between groups. Transforming growth factor-&bgr;-mediated induction of procollagen type III and tenascin-C in isolated cardiac fibroblasts was dependent on Smad3, which suggests that decreased fibrotic remodeling in infarcted Smad3-null hearts may be due to abrogation of the profibrotic transforming growth factor-&bgr; responses. Conclusions— Smad3 loss does not alter the time course of resolution of inflammation in healing infarcts, but it prevents interstitial fibrosis in the noninfarcted myocardium and attenuates cardiac remodeling. Thus, the Smad3 cascade may be a promising therapeutic target for the treatment of myocardial infarction.

TNF provokes cardiomyocyte apoptosis and cardiac remodeling through activation of multiple cell death pathways

Transgenic mice with cardiac-restricted overexpression of secretable TNF (MHCsTNF) develop progressive LV wall thinning and dilation accompanied by an increase in cardiomyocyte apoptosis and a progressive loss of cytoprotective Bcl-2. To test whether cardiac-restricted overexpression of Bcl-2 would prevent adverse cardiac remodeling, we crossed MHCsTNF mice with transgenic mice harboring cardiac-restricted overexpression of Bcl-2. Sustained TNF signaling resulted in activation of the intrinsic cell death pathway, leading to increased cytosolic levels of cytochrome c, Smac/Diablo and Omi/HtrA2, and activation of caspases -3 and -9. Cardiac-restricted overexpression of Bcl-2 blunted activation of the intrinsic pathway and prevented LV wall thinning; however, Bcl-2 only partially attenuated cardiomyocyte apoptosis. Subsequent studies showed that c-FLIP was degraded, that caspase-8 was activated, and that Bid was cleaved to t-Bid, suggesting that the extrinsic pathway was activated concurrently in MHCsTNF hearts. As expected, cardiac Bcl-2 overexpression had no effect on extrinsic signaling. Thus, our results suggest that sustained inflammation leads to activation of multiple cell death pathways that contribute to progressive cardiomyocyte apoptosis; hence the extent of such programmed myocyte cell death is a critical determinant of adverse cardiac remodeling.

Telomere attrition and Chk2 activation in human heart failure

The “postmitotic” phenotype in adult cardiac muscle exhibits similarities to replicative senescence more generally and constitutes a barrier to effective restorative growth in heart disease. Telomere dysfunction is implicated in senescence and apoptotic signaling but its potential role in heart disorders is unknown. Here, we report that cardiac apoptosis in human heart failure is associated specifically with defective expression of the telomere repeat- binding factor TRF2, telomere shortening, and activation of the DNA damage checkpoint kinase, Chk2. In cultured cardiomyocytes, interference with either TRF2 function or expression triggered telomere erosion and apoptosis, indicating that cell death can occur via this pathway even in postmitotic, noncycling cells; conversely, exogenous TRF2 conferred protection from oxidative stress. In vivo, mechanical stress was sufficient to down-regulate TRF2, shorten telomeres, and activate Chk2 in mouse myocardium, and transgenic expression of telomerase reverse transcriptase conferred protection from all three responses. Together, these data suggest that apoptosis in chronic heart failure is mediated in part by telomere dysfunction and suggest an essential role for TRF2 even in postmitotic cells.

Targeted deletion of ROCK1 protects the heart against pressure overload by inhibiting reactive fibrosis

Ventricular myocyte hypertrophy is an important compensatory growth response to pressure overload. However, pathophysiological cardiac hypertrophy is accompanied by reactive fibrosis and remodeling. The Rho kinase family, consisting of ROCK1 and ROCK2, has been implicated in cardiac hypertrophy and ventricular remodeling. However, these previous studies relied heavily on pharmacological inhibitors, and not on gene deletion. Here we used ROCK1 knockout (ROCK1−/−) mice to investigate role of ROCK1 in the development of ventricular remodeling induced by transverse aortic banding. We observed that ROCK1 deletion did not impair compensatory hypertrophic response induced by pressure overload. However, ROCK1−/− mice exhibited reduced perivascular and interstitial fibrosis, which was observed at 3 wk but not at 1 wk after the banding. The reduced fibrosis in the myocardium of ROCK1−/− mice was closely associated with reduced expression of a variety of extracellular matrix (ECM) proteins and fibrogenic cytokines such as TGFβ2 and connective tissue growth factor. This inhibitory effect of ROCK1 deletion on pathophysiological induction of fibrogenic cytokines was further confirmed in the myocardium of transgenic mice with cardiomyocyte‐specific overexpression of Gαq. Thus, these results indicate that ROCK1 contributes to the development of cardiac fibrosis and induction of fibrogenic cytokines in cardiomyocytes in response to pathological stimuli. Zhang, Y.‐M., Bo, J., Taffet, G. E., Chang, J., Shi, J., Reddy, A. K., Michael, L. H., Schneider, M. D., Entman, M. L., Schwartz, R. J., Wei, L. Targeted deletion of ROCK1 protects the heart against pressure overload by inhibiting reactive fibrosis. FASEB J. 20, 916–925 (2006)

Impaired vascular contractility and blood pressure homeostasis in the smooth muscle α-actin null mouse

The smooth muscle (SM) α‐actin gene activated during the early stages of embryonic cardiovascular development is switched off in late stage heart tissue and replaced by cardiac and skeletal α‐actins. SM α‐actin also appears during vascular development, but becomes the single most abundant protein in adult vascular smooth muscle cells. Tissue‐specific expression of SM α‐actin is thought to be required for the principal force‐generating capacity of the vascular smooth muscle cell. We wanted to determine whether SM α‐actin gene expression actually relates to an actin isoforms function. Analysis of SM α‐actin null mice indicated that SM α‐actin is not required for the formation of the cardiovascular system. Also, SM α‐actin null mice appeared to have no difficulty feeding or reproducing. Survival in the absence of SM α‐actin may result from other actin isoforms partially substituting for this isoform. In fact, skeletal α‐actin gene, an actin isoform not usually expressed in vascular smooth muscle, was activated in the aortas of these SM α‐actin null mice. However, even with a modest increase in skeletal α‐actin activity, highly compromised vascular contractility, tone, and blood flow were detected in SM α‐actin‐defective mice. This study supports the concept that SM α‐actin has a central role in regulating vascular contractility and blood pressure homeostasis, but is not required for the formation of the cardiovascular system.—Schildmeyer, L. A., Braun, R., Taffet, G., DeBiasi, M., Burns, A. E., Bradley, A., and Schwartz, R. J. Impaired vascular contractility and blood pressure homeostasis in the smooth muscle α‐actin null mouse. FASEB J. 14, 2213–2220 (2000)

Mice with the R176Q cardiac ryanodine receptor mutation exhibit catecholamine-induced ventricular tachycardia and cardiomyopathy

Mutations in the cardiac ryanodine receptor 2 (RyR2) have been associated with catecholaminergic polymorphic ventricular tachycardia and a form of arrhythmogenic right ventricular dysplasia. To study the relationship between RyR2 function and these phenotypes, we developed knockin mice with the human disease-associated RyR2 mutation R176Q. Histologic analysis of hearts from RyR2R176Q/+ mice revealed no evidence of fibrofatty infiltration or structural abnormalities characteristic of arrhythmogenic right ventricular dysplasia, but right ventricular end-diastolic volume was decreased in RyR2R176Q/+ mice compared with controls, indicating subtle functional impairment due to the presence of a single mutant allele. Ventricular tachycardia (VT) was observed after caffeine and epinephrine injection in RyR2R176Q/+, but not in WT, mice. Intracardiac electrophysiology studies with programmed stimulation also elicited VT in RyR2R176Q/+ mice. Isoproterenol administration during programmed stimulation increased both the number and duration of VT episodes in RyR2R176Q/+ mice, but not in controls. Isolated cardiomyocytes from RyR2R176Q/+ mice exhibited a higher incidence of spontaneous Ca2+ oscillations in the absence and presence of isoproterenol compared with controls. Our results suggest that the R176Q mutation in RyR2 predisposes the heart to catecholamine-induced oscillatory calcium-release events that trigger a calcium-dependent ventricular arrhythmia.

Local insulin-like growth factor I expression induces physiologic, then pathologic, cardiac hypertrophy in transgenic mice

In the present study we determined the long‐term effects of persistent, local insulin‐like growth factor I (IGF‐I) expression on cardiac function in the SIS2 transgenic mouse. Cardiac mass/tibial length was increased in SIS2 mice by 10 wk of age; this cardiac hypertrophy became more pronounced later in life. Peak aortic outflow velocity, a correlate of cardiac output, was increased at 10 wk in SIS2 mice but was decreased at 52 wk. 72 wk SIS2 mouse hearts exhibited wide variability in the extent of cardiac hypertrophy and enlargement of individual cardiac myofibers. Sirius red staining revealed increased fibrosis in 72 wk SIS2 hearts. Persistent local IGF‐I expression is sufficient to initially induce an analog of physiological cardiac hypertrophy in which peak aortic outflow velocity is increased relative to controls in the absence of any observed detrimental histological changes. However, this hypertrophy progresses to a pathological condition characterized by decreased systolic performance and increased fibrosis. Our results confirm the shortterm systolic performance benefit of increased IGF‐I, but our demonstration that IGF‐I ultimately diminishes systolic performance raises doubt about the therapeutic value of chronic IGF‐I administration. Considering these findings, limiting temporal exposure to IGF‐I seems the most likely means of delivering IGF‐Is potential benefits while avoiding its deleterious side effects.—Delaughter, M. C., Taffet, G. E., Fiorotto, M. L., Entman, M. L., Schwartz, R. J. Local insulin‐like growth factor I expression induces physiologic, then pathologic, cardiac hypertrophy in transgenic mice. FASEB J. 13, 1923–1929 (1999)

Monocytic fibroblast precursors mediate fibrosis in angiotensin-II-induced cardiac hypertrophy

Angiotensin-II (Ang-II) is an autacoid generated as part of the pathophysiology of cardiac hypertrophy and failure. In addition to its role in cardiac and smooth muscle contraction and salt retention, it was shown to play a major role in the cardiac interstitial inflammatory response and fibrosis accompanying cardiac failure. In this study, we examined a model of Ang-II infusion to clarify the early cellular mechanisms linking interstitial fibrosis with the onset of the tissue inflammatory response. Continuous infusion of Ang-II resulted in increased deposition of collagen in the heart. Ang-II infusion also resulted in the appearance of distinctive small, spindle-shaped, bone marrow-derived CD34(+)/CD45(+) fibroblasts that expressed collagen type I and the cardiac fibroblast marker DDR2 while structural fibroblasts were CD34(-)/CD45(-). Genetic deletion of monocyte chemoattractant protein (MCP)-1 (MCP-1-KO mice) prevented the Ang-II-induced cardiac fibrosis and the appearance of CD34(+)/CD45(+) fibroblasts. Real-time PCR in Ang-II-treated hearts revealed a striking induction of types I and III collagen, TGF-beta1, and TNF mRNA expression; this was obviated in Ang-II-infused MCP-1-KO hearts. In both wild-type and MCP-1-KO mice, Ang-II infusion resulted in cardiac hypertrophy, increased systolic function and hypertension which were not significantly different between the WT and MCP-1-KO mice over the 6-week course of infusion. In conclusion, the development of Ang-II-induced non-adaptive fibrosis in the heart required induction of MCP-1, which modulated the uptake and differentiation of a CD34(+)/CD45(+) fibroblast precursor population. In contrast to the inflammatory and fibrotic response, the hemodynamic response to Ang-II was not affected by MCP-1 in the first 6weeks.