Cardiac Specific Overexpression of Transcription Factor EB (TFEB) in Normal Hearts Induces Pathologic Cardiac Hypertrophy and Lethal Cardiomyopathy

TFEB promotes lysosomal biogenesis, autophagy, and lysosomal exocytosis. The present study characterized the consequence of inducible TFEB overexpression in cardiomyocytes in vivo. We generated cardiomyocyte-specific doxycycline inducible (Tet off) mice to achieve spatial and temporal control of TFEB overexpression, by crossing TFEB transgenic mice with mice harboring the tTA transgene (TFEB/tTA). Two weeks after doxycycline removal, an 8-fold increase in TFEB protein expression was observed in transgenic hearts. Heart weight normalized to tibia length was increased by 2.5-fold following TFEB overexpression (TFEB/tTA), characterized by induction of markers of pathological hypertrophy, such as Nppa, Nppb and Acta1, progressive contractile dysfunction and cardiac dilatation. Overexpression of TFEB resulted in premature death, associated with high degree AV block. Reversal of TFEB overexpression normalized cardiac structure and function. Mitochondrial respiration and ATP levels were preserved after 2-weeks of TFEB induction, despite reduced mitochondrial (OXPHOS) protein expression, mtDNA content, and altered mitochondrial morphology. Signaling through mTOR was induced in TFEB/tTA mice, and when inhibited by rapamycin treatment for 4 weeks, partially offset left ventricular dysfunction. Transcriptome analysis revealed early suppression of mitochondrial metabolic pathways, induction of fibrosis and altered calcium signaling. MCOLN1, a lysosomal calcium release channel, the calcineurin target RCAN1.4, and the mitochondrial calcium uniporter (MCU) were strikingly induced in TFEB/tTA mice. In summary, persistent overexpression of TFEB at high levels (8-fold protein upregulation) in cardiomyocytes promotes pathologic cardiac hypertrophy via suppression of mitochondrial bioenergetic pathways and activation of pro-fibrotic and calcium regulatory pathways.

Abstract 15863: Macrophage Foxp1 is a Regulator of Pathologic Cardiac Hypertrophy

Introduction: Pathologic cardiac hypertrophy is a maladaptive response to neurohormonal and hemodynamic stress that is a hallmark of human heart failure. While inflammation has been implicated in pathologic hypertrophy, the molecular mechanisms underlying innate immune dysregulation in this disease process are incompletely defined. We have previously demonstrated that the forkhead transcription factor Foxp1 controls monocyte differentiation and suppresses inflammatory activation of macrophages. In this study, we hypothesized that monocyte/macrophage Foxp1 regulates pathologic cardiac hypertrophy. Methods: Macrophage-specific Foxp1 over-expressing (macFoxp1tg=anti-inflammatory) vs. non-tg controls, as well as macrophage-specific Foxp1 knockdown (macFoxp1ko=pro-inflammatory) vs. Cre-control male mice were subject to chronic angiotensin II (AII) infusion (1.8 mcg/kg/min via subcutaneous osmotic mini-pump) for 4 weeks. Results: AII-mediated cardiac hypertrophy (heart mass and cardiomyocyte cross-sectional area), left ventricular (LV) systolic dysfunction, LV dilation, interstitial fibrosis and macrophage (Mac-3+ cells) accumulation were significantly attenuated in macFoxp1tg mice compared to non-tg controls. In contrast, AII-mediated cardiac hypertrophy, LV systolic dysfunction and cavity dilation were significantly exacerbated in macFoxp1ko mice compared to Cre controls. There were no differences in systemic blood pressure between these groups, corroborating a load-independent role for macrophage Foxp1 in cardiac hypertrophy. Conclusion: These studies identify macrophage Foxp1 as a novel negative regulator of pathologic cardiac hypertrophy in vivo. Modulation of Foxp1 signaling may provide a novel strategy for prevention and treatment of heart failure.

Abstract 104: Protein Kinase GIα Functions in the Cardiac Myocyte as a Tonic Inhibitor of Pathologic Cardiac Hypertrophy In Vivo

Objectives: We and others previously demonstrated that activation of the NO-cGMP-Protein Kinase G (PKG) pathway inhibits cardiac hypertrophy and remodeling in vivo. However, it remains untested whether PKG specifically in the cardiac myocyte (CM) mediates these effects. We therefore tested the hypothesis that PKGIα inhibits pathologic cardiac hypertrophy through a specific role in the CM. Methods and Results: We created and characterized mice with CM-restricted excision of PKGIα. Mice were generated in which the PKGI exon 1 (specific for the Iα isoform) was flanked by loxP sites. We crossed these PKGIα fl/fl mice with αMHC-Cre mice which constitutively express Cre recombinase selectively in the CM. The resultant PKGIα fl/fl / αMHC-Cre+/- mice were compared with PKGIα fl/fl / αMHC-Cre-/- littermate controls (termed PKG CMKO and Ctrl, respectively). By age 3 months (n=5 per genotype), male PKG CMKO mice developed atrial and LV hypertrophy compared with Ctrl littermates PKG CMKO atrial weight/tibia length 0.33 ± 0.03 mg/mm vs 0.22 ± .01 in Ctrl, P <0.05; PKG CMKO LV/TL 5.0 ± 0.2 mg/mm vs 4.1 ± 0.4 in Ctrl, P <0.05). LV CM cross sectional area also increased in the 3 month old PKG CMKO mice (9445 ± 282 pixels PKG CMKO vs 8273 ± 213 in Ctrl, n>400 cells/genotype, 5 hearts per genotype; P <0.001). The systolic index end systolic elastance was decreased in 3 month old PKG CMKO mice (PKG CMKO 3.1 ± 0.4 mmHg/μ l vs 6.1 ± 1.0 in Ctrl-; P <0.05). Importantly, blood pressure did not differ between genotypes. By age 6 months, PKG CMKO mice developed early mortality (3 of 4 PKG CMKO males died at 6 months of age vs 0 of 4 Ctrl males). Total heart and atrial weights of male mice (n=3 PKG CMKO , 4 Ctrl) increased in PKG CMKO mice (heart weight/tibia length 10.8 ± 1.7 mg/mm in PKG CMKO vs 7.1 ± 0.6 in Ctrl; P <0.05; atrial weight/tibia length 2.3 ± 1.1 mg/mm in PKG CMKO vs 0.30 ± 0.1 Ctrl, P <0.05). LV fractional shortening percentage, recorded at 6 months age, trended lower in the PKG CMKO mice as well (35 ± 3% PKG CMKO vs 44 ± 4% Ctrl, P 0.09). Conclusions: These data provide the first evidence that PKGIα functions in the CM as a tonic inhibitor of age-dependent pathologic hypertrophy, supporting further study of PKGIα as a therapeutic target in the prevention and treatment of LV remodeling and congestive heart failure.

Abstract 276: Cyclin D2 Is a Critical Mediator of Exercise-Induced Cardiac Hypertrophy

Exercise training activates a number of hypertrophic signaling pathways that can be distinct from those activated by pathologic stimuli. However, there must be some overlap in those pathways underlying myocyte cell growth. Using a number of mouse genetic models, we investigated the role of several molecules implicated in pathologic cardiac hypertrophy for their cardiac responses to exercise. We used three-month-old female transgenic mice expressing the anti-hypertrophic molecules, cardiac-specific constitutively active glycogen synthase kinase-3β (caGSK-3β), an inhibitor of Ca 2+ -calmodulin-dependent protein kinase (CaMKII Inh), and doubly transgenic mice expressing both caGSK-3β and CaMKII inhibition (caGSK-3β/CaMKII Inh). We also studied the exercise responsiveness of mice expressing the pro-hypertrophic cardiac-specific activated (myr)Akt. MAPK/ERK kinase kinase-1 (MEKK1) has been shown to be essential for pathologic cardiac hypertrophy and we therefore studied the requirement of MEKK1 for exercise-induced cardiac growth. Cell cycle regulators such as cyclin D2 have been shown to be required for pathologic cardiac hypertrophy; therefore we studied cyclin D2 null mice. Mice were divided into sedentary and 21 days of voluntary exercise on a cage wheel. Across the seven different mouse models, exercise capacity was similar with regards to running duration, distance, and speed. Importantly, we analyzed the impact of exercise on cardiac hypertrophy by measuring heart weight-to-body weight (HW/BW) ratios of sedentary and exercised mice. While exercise had no impact on body weight, heart weight increased significantly in all mouse models except the cyclin D2 -/- mice. Overall there was a 3.5-fold range of percent increase in HW/BW ratios from the highest (caGSK-3β) to the lowest (cyclin D2 -/- ). In conclusion, genetic manipulation of these hypertrophic signaling pathways has little impact on exercise performance and only the loss of cyclin D2 attenuates exercise-induced cardiac growth. These data establish cyclin D2 as an important regulator of physiological hypertrophy and underscore the differences in pathologic and physiologic cardiac hypertrophy.