Dongmin Kwak

Left Ventricular Failure Produces Profound Lung Remodeling and Pulmonary Hypertension in Mice: Heart Failure Causes Severe Lung Disease

Chronic left ventricular failure causes pulmonary congestion with increased lung weight and type 2 pulmonary hypertension. Understanding the molecular mechanisms for type 2 pulmonary hypertension and the development of novel treatments for this condition requires a robust experimental animal model and a good understanding of the nature of the resultant pulmonary remodeling. Here we demonstrate that chronic transverse aortic constriction causes massive pulmonary fibrosis and remodeling, as well as type 2 pulmonary hypertension, in mice. Thus, aortic constriction-induced left ventricular dysfunction and increased left ventricular end-diastolic pressure are associated with a ⩽5.3-fold increase in lung wet weight and dry weight, pulmonary hypertension, and right ventricular hypertrophy. Interestingly, the aortic constriction-induced increase in lung weight was not associated with pulmonary edema but resulted from profound pulmonary remodeling with a dramatic increase in the percentage of fully muscularized lung vessels, marked vascular and lung fibrosis, myofibroblast proliferation, and leukocyte infiltration. The aortic constriction-induced left ventricular dysfunction was also associated with right ventricular hypertrophy, increased right ventricular end-diastolic pressure, and right atrial hypertrophy. The massive lung fibrosis, leukocyte infiltration, and pulmonary hypertension in mice after transverse aortic constriction clearly indicate that congestive heart failure also causes severe lung disease. The lung fibrosis and leukocyte infiltration may be important mechanisms in the poor clinical outcome in patients with end-stage heart failure. Thus, the effective treatment of left ventricular failure may require additional efforts to reduce lung fibrosis and the inflammatory response.

Metformin Protects Against Systolic Overload–Induced Heart Failure Independent of AMP-Activated Protein Kinase α2

Activation of AMP-activated protein kinase (AMPK)-&agr;2 protects the heart against pressure overload–induced heart failure in mice. Although metformin is a known activator of AMPK, it is unclear whether its cardioprotection acts independently of an AMPK&agr;2-dependent pathway. Because the role of AMPK&agr;1 stimulation on remodeling of failing hearts is poorly defined, we first studied the effects of disruption of both the AMPK&agr;1 and AMPK&agr;2 genes on the response to transverse aortic constriction–induced left ventricular (LV) hypertrophy and dysfunction in mice. AMPK&agr;2 gene knockout significantly exacerbated the degree of transverse aortic constriction–induced LV hypertrophy and dysfunction, whereas AMPK&agr;1 gene knockout had no effect on the degree of transverse aortic constriction–induced LV hypertrophy and dysfunction. Administration of metformin was equally effective in attenuating transverse aortic constriction-induced LV remodeling in both wild-type and AMPK&agr;2 knockout mice, as evidenced by reduced LV and lung weights, a preserved LV ejection fraction, and reduced phosphorylation of mammalian target of rapamycin (p-mTORSer2448) and its downstream target p-p70S6KThr389. These data support the notion that activation of AMPK&agr;1 plays a negligible role in protecting the heart against the adverse effects of chronic pressure overload, and that metformin protects against adverse remodeling through a pathway that seems independent of AMPK&agr;2.

Microtubule Actin Cross-Linking Factor 1 Regulates Cardiomyocyte Microtubule Distribution and Adaptation to Hemodynamic Overload

Aberrant cardiomyocyte microtubule growth is a feature of pressure overload induced cardiac hypertrophy believed to contribute to left ventricular (LV) dysfunction. Microtubule Actin Cross-linking Factor 1 (MACF1/Acf7) is a 600 kd spectraplakin that stabilizes and guides microtubule growth along actin filaments. MACF1 is expressed in the heart, but its impact on cardiac microtubules, and how this influences cardiac structure, function, and adaptation to hemodynamic overload is unknown. Here we used inducible cardiac-specific MACF1 knockout mice (MACF1 KO) to determine the impact of MACF1 on cardiac microtubules and adaptation to pressure overload (transverse aortic constriction (TAC).In adult mouse hearts, MACF1 expression was low under basal conditions, but increased significantly in response to TAC. While MACF1 KO had no observable effect on heart size or function under basal conditions, MACF1 KO exacerbated TAC induced LV hypertrophy, LV dilation and contractile dysfunction. Interestingly, subcellular fractionation of ventricular lysates revealed that MACF1 KO altered microtubule distribution in response to TAC, so that more tubulin was associated with the cell membrane fraction. Moreover, TAC induced microtubule redistribution into this cell membrane fraction in both WT and MACF1 KO mice correlated strikingly with the level of contractile dysfunction (r2 = 0.786, p<.001). MACF1 disruption also resulted in reduction of membrane caveolin 3 levels, and increased levels of membrane PKCα and β1 integrin after TAC, suggesting MACF1 function is important for spatial regulation of several physiologically relevant signaling proteins during hypertrophy. Together, these data identify for the first time, a role for MACF1 in cardiomyocyte microtubule distribution and in adaptation to hemodynamic overload.

Double Stranded RNA-Dependent Protein Kinase Deficiency Protects the Heart from Systolic Overload-Induced Congestive Heart Failure

Background— Double-stranded RNA–dependent protein kinase (PKR) is a eukaryotic initiation factor 2&agr; kinase that inhibits mRNA translation under stress conditions. PKR also mediates inflammatory and apoptotic signaling independently of translational regulation. Congestive heart failure is associated with cardiomyocyte hypertrophy, inflammation, and apoptosis, but the role of PKR in left ventricular hypertrophy and the development of congestive heart failure has not been examined. Methods and Results— We observed increased myocardial PKR expression and translocation of PKR into the nucleus in humans and mice with congestive heart failure. To determine the impact of PKR on the development of congestive heart failure, PKR knockout and wild-type mice were exposed to pressure overload produced by transverse aortic constriction. Although heart size increased similarly in wild-type and PKR knockout mice after transverse aortic constriction, PKR knockout mice exhibited very little pulmonary congestion, well-preserved left ventricular ejection fraction and contractility, and significantly less myocardial fibrosis compared with wild-type mice. Bone marrow–derived cells from wild-type mice did not abolish the cardiac protective effect observed in PKR knockout mice, whereas bone marrow–derived cells from PKR knockout mice had no cardiac protective effect in wild-type mice. Mechanistically, PKR knockout attenuated transverse aortic constriction–induced tumor necrosis factor-&agr; expression and leukocyte infiltration and lowered cardiac expression of proapoptotic factors (Bax and caspase-3), so that PKR knockout hearts were more resistant to transverse aortic constriction–induced cardiomyocyte apoptosis. PKR depletion in isolated cardiomyocytes also conferred protection against tumor necrosis factor-&agr;– or lipopolysaccharide-induced apoptosis. Conclusion— PKR is a maladaptive factor upregulated in hemodynamic overload that contributes to myocardial inflammation, cardiomyocyte apoptosis, and the development of congestive heart failure.

Endoplasmic Reticulum Stress Sensor Protein Kinase R–Like Endoplasmic Reticulum Kinase (PERK) Protects Against Pressure Overload–Induced Heart Failure and Lung Remodeling

Studies have reported that development of congestive heart failure is associated with increased endoplasmic reticulum stress. Double stranded RNA-activated protein kinase R–like endoplasmic reticulum kinase (PERK) is a major transducer of the endoplasmic reticulum stress response and directly phosphorylates eukaryotic initiation factor 2&agr;, resulting in translational attenuation. However, the physiological effect of PERK on congestive heart failure development is unknown. To study the effect of PERK on ventricular structure and function, we generated inducible cardiac-specific PERK knockout mice. Under unstressed conditions, cardiac PERK knockout had no effect on left ventricular mass, or its ratio to body weight, cardiomyocyte size, fibrosis, or left ventricular function. However, in response to chronic transverse aortic constriction, PERK knockout mice exhibited decreased ejection fraction, increased left ventricular fibrosis, enhanced cardiomyocyte apoptosis, and exacerbated lung remodeling in comparison with wild-type mice. PERK knockout also dramatically attenuated cardiac sarcoplasmic reticulum Ca2+-ATPase expression in response to aortic constriction. Our findings suggest that PERK is required to protect the heart from pressure overload–induced congestive heart failure.Studies have reported that development of congestive heart failure (CHF) is associated with increased endoplasmic reticulum (ER) stress. Double stranded RNA activated protein kinase-like endoplasmic reticulum kinase (PERK) is a major transducer of the ER stress response and directly phosphorylates eIF2α, resulting in translational attenuation. However, the physiological effect of PERK on CHF development is unknown. In order to study the effect of PERK on ventricular structure and function, we generated inducible cardiac specific PERK knockout (KO) mice. Under unstressed conditions, cardiac PERK KO had no effect on left ventricular mass, or its ratio to body weight, cardiomyocyte size, fibrosis, or left ventricular function. However, in response to chronic transverse aortic constriction, PERK KO mice exhibited decreased ejection fraction, increased left ventricular fibrosis, enhanced cardiomyocyte apoptosis and exacerbated lung remodeling in comparison to wild type mice. PERK KO also dramatically attenuated cardiac sarcoplasmic reticulum Ca++-ATPase expression in response to aortic constriction. Our findings suggest that PERK is required to protect the heart from pressure overload-induced CHF.

Loss of the Eukaryotic Initiation Factor 2α Kinase General Control Nonderepressible 2 Protects Mice From Pressure Overload–Induced Congestive Heart Failure Without Affecting Ventricular Hypertrophy

In response to several stresses, including nutrient deprivation, general control nonderepressible 2 kinase (GCN2) attenuates mRNA translation by phosphorylating eukaryotic initiation factor 2&agr;Ser51. Energy starvation is known to exacerbate congestive heart failure, and eukaryotic initiation factor 2&agr;Ser51 phosphorylation is increased in the failing heart. However, the effect of GCN2 during the evolution of congestive heart failure has not been tested. In this study, we examined the influence of GCN2 expression in response to a cardiac stress by inducing chronic pressure overload with transverse aortic constriction in wild-type and GCN2 knockout mice. Under basal conditions, GCN2 knockout mice had normal left ventricular structure and function, but after transverse aortic constriction, they demonstrated less contractile dysfunction, less increase in lung weight, less increase in lung inflammation and vascular remodeling, and less myocardial apoptosis and fibrosis compared with wild-type mice, despite an equivalent degree of left ventricular hypertrophy. As expected, GCN2 knockout attenuated transverse aortic constriction–induced cardiac eukaryotic initiation factor 2&agr;Ser51 phosphorylation and preserved sarcoplasmic reticulum Ca2+ ATPase expression compared with wild-type mice. Interestingly, the expression of the antiapoptotic protein Bcl-2 was significantly elevated in GCN2 knockout hearts, whereas in isolated neonatal cardiomyocytes, selective knockdown of GCN2 increased Bcl-2 protein expression and enhanced myocyte resistance to an apoptotic stress. Collectively, our data support the notion that GCN2 impairs the ventricular adaptation to chronic pressure overload by reducing Bcl-2 expression and increasing cardiomyocyte susceptibility to apoptotic stimuli. Our findings suggest that strategies to reduce GCN2 activity in cardiac tissue may be a novel approach to attenuate congestive heart failure development.

Increasing Regulatory T Cells With Interleukin-2 and Interleukin-2 Antibody Complexes Attenuates Lung Inflammation and Heart Failure Progression

Congestive heart failure (CHF) is associated with an increase of leukocyte infiltration, proinflammatory cytokines, and fibrosis in the heart and lung. Regulatory T cells (Tregs, CD4+CD25+FoxP3+) suppress inflammatory responses in various clinical conditions. We postulated that expansion of Tregs attenuates CHF progression by reducing cardiac and lung inflammation. We investigated the effects of interleukin-2 (IL-2) plus IL-2 monoclonal antibody clone JES6-1 complexes (IL2/JES6-1) on induction of Tregs, transverse aortic constriction–induced cardiac and lung inflammation, and CHF progression in mice. We demonstrated that end-stage CHF caused a massive increase of lung macrophages and T cells, as well as relatively mild left ventricular (LV) leukocyte infiltration. Administration of IL2/JES6-1 caused an ≈6-fold increase of Tregs within CD4+ T cells in the spleen, lung, and heart of mice. IL2/JES6-1 treatment of mice with existing transverse aortic constriction–induced LV failure markedly reduced lung and right ventricular weight and improved LV ejection fraction and LV end-diastolic pressure. Mechanistically, IL2/JES6-1 treatment significantly increased Tregs; suppressed CD4+ T-cell accumulation; dramatically attenuated leukocyte infiltration, including decreasing CD45+ cells, macrophages, CD8+ T cells, and effector memory CD8+; and reduced proinflammatory cytokine expressions and fibrosis in the lung of mice. Furthermore, IL2/JES6-1 administered before transverse aortic constriction attenuated the development of LV hypertrophy and dysfunction in mice. Our data indicate that increasing Tregs through administration of IL2/JES6-1 effectively attenuates pulmonary inflammation, right ventricular hypertrophy, and further LV dysfunction in mice with existing LV failure, suggesting that strategies to properly expand Tregs may be useful in reducing CHF progression.

CD28/B7 Deficiency Attenuates Systolic Overload-Induced Congestive Heart Failure, Myocardial and Pulmonary Inflammation, and Activated T Cell Accumulation in the Heart and Lungs.

The inflammatory response regulates congestive heart failure (CHF) development. T cell activation plays an important role in tissue inflammation. We postulate that CD28 or B7 deficiency inhibits T cell activation and attenuates CHF development by reducing systemic, cardiac, and pulmonary inflammation. We demonstrated that chronic pressure overload–induced end-stage CHF in mice is characterized by profound accumulation of activated effector T cells (CD3+CD44high cells) in the lungs and a mild but significant increase of these cells in the heart. In knockout mice lacking either CD28 or B7, there was a dramatic reduction in the accumulation of activated effector T cells in both hearts and lungs of mice under control conditions and after transverse aortic constriction. CD28 or B7 knockout significantly attenuated transverse aortic constriction–induced CHF development, as indicated by less increase of heart and lung weight and less reduction of left ventricle contractility. CD28 or B7 knockout also significantly reduced transverse aortic constriction–induced CD45+ leukocyte, T cell, and macrophage infiltration in hearts and lungs, lowered proinflammatory cytokine expression (such as tumor necrosis factor-&agr; and interleukin-1&bgr;) in lungs. Furthermore, CD28/B7 blockade by CTLA4-Ig treatment (250 &mgr;g/mouse every 3 days) attenuated transverse aortic constriction–induced T cell activation, left ventricle hypertrophy, and left ventricle dysfunction. Our data indicate that CD28/B7 deficiency inhibits activated effector T cell accumulation, reduces myocardial and pulmonary inflammation, and attenuates the development of CHF. Our findings suggest that strategies targeting T cell activation may be useful in treating CHF.

Cardiomyocyte dimethylarginine dimethylaminohydrolase-1 (DDAH1) plays an important role in attenuating ventricular hypertrophy and dysfunction

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthases that limits nitric oxide bioavailability. Dimethylarginine dimethylaminohydrolase-1 (DDAH1) exerts a critical role for ADMA degradation and plays an important role in NO signaling. In the heart, DDAH1 is observed in endothelial cells and in the sarcolemma of cardiomyocytes. While NO signaling is important for cardiac adaptation to stress, DDAH1 impact on cardiomyocyte homeostasis is not clear. Here we used the MerCreMer-LoxP model to specifically disrupt cardiomyocyte DDAH1 expression in adult mice to determine the physiological impact of cardiomyocyte DDAH1 under basal conditions and during hypertrophic stress imposed by transverse aortic constriction (TAC). Under control conditions, cardiomyocyte-specific DDAH1 knockout (cDDAH KO) had no detectable effect on plasma ADMA and left ventricular (LV) hypertrophy or function in adult or aging mice. In response to TAC, DDAH1 levels were elevated 2.5-fold in WT mice, which exhibited no change in LV or plasma ADMA content and moderate LV hypertrophy and LV dysfunction. In contrast, cDDAH1 KO mice exposed to TAC showed no increase in LV DDAH1 expression, slightly increased LV tissue ADMA levels, no increase in plasma ADMA, but significantly exacerbated LV hypertrophy, fibrosis, nitrotyrosine production, and LV dysfunction. These findings indicate cardiomyocyte DDAH1 activity is dispensable for cardiac function under basal conditions, but plays an important role in attenuating cardiac hypertrophy and ventricular remodeling under stress conditions, possibly through locally confined regulation of subcellular ADMA and NO signaling.

Endoplasmic reticulum stress sensor PERK protects against pressure overload induced heart failure and lung remodeling

Studies have reported that development of congestive heart failure is associated with increased endoplasmic reticulum stress. Double stranded RNA-activated protein kinase R–like endoplasmic reticulum kinase (PERK) is a major transducer of the endoplasmic reticulum stress response and directly phosphorylates eukaryotic initiation factor 2&agr;, resulting in translational attenuation. However, the physiological effect of PERK on congestive heart failure development is unknown. To study the effect of PERK on ventricular structure and function, we generated inducible cardiac-specific PERK knockout mice. Under unstressed conditions, cardiac PERK knockout had no effect on left ventricular mass, or its ratio to body weight, cardiomyocyte size, fibrosis, or left ventricular function. However, in response to chronic transverse aortic constriction, PERK knockout mice exhibited decreased ejection fraction, increased left ventricular fibrosis, enhanced cardiomyocyte apoptosis, and exacerbated lung remodeling in comparison with wild-type mice. PERK knockout also dramatically attenuated cardiac sarcoplasmic reticulum Ca2+-ATPase expression in response to aortic constriction. Our findings suggest that PERK is required to protect the heart from pressure overload–induced congestive heart failure.Studies have reported that development of congestive heart failure (CHF) is associated with increased endoplasmic reticulum (ER) stress. Double stranded RNA activated protein kinase-like endoplasmic reticulum kinase (PERK) is a major transducer of the ER stress response and directly phosphorylates eIF2α, resulting in translational attenuation. However, the physiological effect of PERK on CHF development is unknown. In order to study the effect of PERK on ventricular structure and function, we generated inducible cardiac specific PERK knockout (KO) mice. Under unstressed conditions, cardiac PERK KO had no effect on left ventricular mass, or its ratio to body weight, cardiomyocyte size, fibrosis, or left ventricular function. However, in response to chronic transverse aortic constriction, PERK KO mice exhibited decreased ejection fraction, increased left ventricular fibrosis, enhanced cardiomyocyte apoptosis and exacerbated lung remodeling in comparison to wild type mice. PERK KO also dramatically attenuated cardiac sarcoplasmic reticulum Ca++-ATPase expression in response to aortic constriction. Our findings suggest that PERK is required to protect the heart from pressure overload-induced CHF.