Reversibility of adverse, calcineurin-dependent cardiac remodeling

Abstract

Rationale: Studies to dissect the role of calcineurin in pathological cardiac remodeling have relied heavily on murine models, in which genetic gain- and loss-of-function manipulations are initiated at or before birth. However, the great majority of clinical cardiac pathology occurs in adults. Yet nothing is known about the effects of calcineurin when its activation commences in adulthood. Furthermore, despite the fact that ventricular hypertrophy is a well-established risk factor for heart failure, the relative pace and progression of these 2 major phenotypic features of heart disease are unknown. Finally, even though therapeutic interventions in adults are designed to slow, arrest, or reverse disease pathogenesis, little is known about the capacity for spontaneous reversibility of calcineurin-dependent pathological remodeling.

Objective: We set out to address these 3 questions by studying mice engineered to harbor in cardiomyocytes a constitutively active calcineurin transgene driven by a tetracycline-responsive promoter element.

Methods and results: Expression of the mutant calcineurin transgene was initiated for variable lengths of time to determine the natural history of disease pathogenesis, and to determine when, if ever, these events are reversible. Activation of the calcineurin transgene in adult mice triggered rapid and robust cardiac growth with features characteristic of pathological hypertrophy. Concentric hypertrophy preceded the development of systolic dysfunction, fetal gene activation, fibrosis, and clinical heart failure. Furthermore, cardiac hypertrophy reversed spontaneously when calcineurin activity was turned off, and expression of fetal genes reverted to baseline. Fibrosis, a prominent feature of pathological cardiac remodeling, manifested partial reversibility.

Conclusions: Together, these data establish and define the deleterious effects of calcineurin signaling in the adult heart and reveal that calcineurin-dependent hypertrophy with concentric geometry precedes systolic dysfunction and heart failure. Furthermore, these findings demonstrate that during much of the disease process, calcineurin-dependent remodeling remains reversible.

Figure 1. Conditional activation of calcineurin in cardiomyocytes

A: Schematic depiction of the transgenic constructs used to engineer double transgenic tTA/CnA* mice. B, C: Representative immunoblots of LV lysates prepared after 8 weeks of treatment with doxycycline (Dox) or water and probed for calcineurin (CnA) (B) (upper bands represent endogenous calcineurin; lower bands represent mutant calcineurin) or RCAN (C) (lower bands represent RCAN1.4 in different phosphorylation states). Ponceau stains show protein loading in each lane. D: Representative immunoblot of LV lysates probed for RCAN from hearts harvested at different times after removal of doxycycline. tTA, single transgenic for αMHC-tTA only; tTA/CnA, double transgenic for both αMHC-tTA and tetO-CnA*.

Figure 2. Hypertrophy and heart failure develop when doxycycline is withheld from tTA/CnA* mice

A: Experimental design. Double transgenic tTA/CnA* mice maintained on doxycycline (OFF) were used as controls. B: Left ventricular internal diameter (LVID) at end-diastole and end-systole after 16 weeks of calcineurin activation. C: Mean percent fractional shortening measured from M-mode tracings from mice in the OFF (n=10) and ON (n=14) groups over the course of the experiment. Representative M-mode echocardiographic tracings showing left ventricular mechanical activity at study completion. D: Representative images of hearts at study completion (scale bar = 800 µm). E: Mean absolute heart weight, heart weight normalized to body weight (HW/BW), and lung weight normalized to body weight for mice in the OFF (n=10) and ON (n=14) groups at study completion. F: Representative histological images of picrosirius red staining revealing cardiomyocyte size and fibrosis at study completion. Bar = 80 µm. G: Mean cardiomyocyte cross-sectional area measured from hearts in the OFF and ON groups (4 hearts per group; 60 cells measured per heart; bar = 1 SD). *p<0.01. H. Mean percent area of myocardium staining for fibrosis in the OFF and ON groups (6 images per heart, 6 hearts per group).

Figure 2. Hypertrophy and heart failure develop when doxycycline is withheld from tTA/CnA* mice

A: Experimental design. Double transgenic tTA/CnA* mice maintained on doxycycline (OFF) were used as controls. B: Left ventricular internal diameter (LVID) at end-diastole and end-systole after 16 weeks of calcineurin activation. C: Mean percent fractional shortening measured from M-mode tracings from mice in the OFF (n=10) and ON (n=14) groups over the course of the experiment. Representative M-mode echocardiographic tracings showing left ventricular mechanical activity at study completion. D: Representative images of hearts at study completion (scale bar = 800 µm). E: Mean absolute heart weight, heart weight normalized to body weight (HW/BW), and lung weight normalized to body weight for mice in the OFF (n=10) and ON (n=14) groups at study completion. F: Representative histological images of picrosirius red staining revealing cardiomyocyte size and fibrosis at study completion. Bar = 80 µm. G: Mean cardiomyocyte cross-sectional area measured from hearts in the OFF and ON groups (4 hearts per group; 60 cells measured per heart; bar = 1 SD). *p<0.01. H. Mean percent area of myocardium staining for fibrosis in the OFF and ON groups (6 images per heart, 6 hearts per group).

Figure 2. Hypertrophy and heart failure develop when doxycycline is withheld from tTA/CnA* mice

A: Experimental design. Double transgenic tTA/CnA* mice maintained on doxycycline (OFF) were used as controls. B: Left ventricular internal diameter (LVID) at end-diastole and end-systole after 16 weeks of calcineurin activation. C: Mean percent fractional shortening measured from M-mode tracings from mice in the OFF (n=10) and ON (n=14) groups over the course of the experiment. Representative M-mode echocardiographic tracings showing left ventricular mechanical activity at study completion. D: Representative images of hearts at study completion (scale bar = 800 µm). E: Mean absolute heart weight, heart weight normalized to body weight (HW/BW), and lung weight normalized to body weight for mice in the OFF (n=10) and ON (n=14) groups at study completion. F: Representative histological images of picrosirius red staining revealing cardiomyocyte size and fibrosis at study completion. Bar = 80 µm. G: Mean cardiomyocyte cross-sectional area measured from hearts in the OFF and ON groups (4 hearts per group; 60 cells measured per heart; bar = 1 SD). *p<0.01. H. Mean percent area of myocardium staining for fibrosis in the OFF and ON groups (6 images per heart, 6 hearts per group).

Figure 3. Calcineurin activation in the adult heart triggers pathological hypertrophy

A: Experimental design. Single transgenic tTA mice were used as controls. B: Mean fractional shortening calculated from M-mode tracings for tTA (n=9) and tTA/CnA* (n=11) mice at study completion. C: Mean LV mass, LV posterior wall thickness (LVPWd), and LV internal diameter in diastole (LVIDd) measured from 2D echocardiographic images at study completion. D: Mean heart weight normalized to body weight (HW/BW) measured at necropsy. E: Representative immunoblot of LV lysates from tTA, tTA/CnA*, and ON/OFF hearts probed for βMHC. F: Mean mRNA levels of βMHC, BNP, ANF, and SERCA2a determined by real-time RT-PCR from RNA isolated from tTA (gray bars; n=6) and tTA/CnA* (black bars, n=8) hearts. mRNA levels are calculated relative to cyclophilin mRNA and normalized to control levels. *p < 0.05 ON: double-transgenic tTA/CnA mice; OFF: single transgenic tTA mice. ON/OFF: double-transgenic tTA/CnA mice after doxycycline was withdrawn for 8 weeks and then restored for 8 weeks.

Figure 3. Calcineurin activation in the adult heart triggers pathological hypertrophy

A: Experimental design. Single transgenic tTA mice were used as controls. B: Mean fractional shortening calculated from M-mode tracings for tTA (n=9) and tTA/CnA* (n=11) mice at study completion. C: Mean LV mass, LV posterior wall thickness (LVPWd), and LV internal diameter in diastole (LVIDd) measured from 2D echocardiographic images at study completion. D: Mean heart weight normalized to body weight (HW/BW) measured at necropsy. E: Representative immunoblot of LV lysates from tTA, tTA/CnA*, and ON/OFF hearts probed for βMHC. F: Mean mRNA levels of βMHC, BNP, ANF, and SERCA2a determined by real-time RT-PCR from RNA isolated from tTA (gray bars; n=6) and tTA/CnA* (black bars, n=8) hearts. mRNA levels are calculated relative to cyclophilin mRNA and normalized to control levels. *p < 0.05 ON: double-transgenic tTA/CnA mice; OFF: single transgenic tTA mice. ON/OFF: double-transgenic tTA/CnA mice after doxycycline was withdrawn for 8 weeks and then restored for 8 weeks.

Figure 4. Calcineurin-induced ventricular hypertrophy precedes systolic dysfunction, and fetal gene activation is delayed after calcineurin activation

A: Mean LV mass calculated from serial 2D echocardiographic images of tTA (n=9) and tTA/CnA* (n=9) mice recorded after removing doxycycline. B: Mean LV posterior wall thickness at end-diastole (LVPWd) by serial echocardiography after removing doxycycline. C: Mean percent fractional shortening from serial M-mode tracings obtained after removing doxycycline. D: Mean LV internal diameter in diastole (LVIDd) measured from serial 2D echocardiographic images obtained after removing doxycycline. E,F: Quantitative real-time RT-PCR measurements of mRNA levels of indicated genes from RNA isolated from selected hearts at (E) one week and (F) eight weeks after removing doxycycline. mRNA levels are calculated relative to cyclophilin mRNA and normalized to control levels. *p < 0.01. ON: double-transgenic tTA/CNA mice; OFF: single transgenic tTA mice.

Figure 4. Calcineurin-induced ventricular hypertrophy precedes systolic dysfunction, and fetal gene activation is delayed after calcineurin activation

A: Mean LV mass calculated from serial 2D echocardiographic images of tTA (n=9) and tTA/CnA* (n=9) mice recorded after removing doxycycline. B: Mean LV posterior wall thickness at end-diastole (LVPWd) by serial echocardiography after removing doxycycline. C: Mean percent fractional shortening from serial M-mode tracings obtained after removing doxycycline. D: Mean LV internal diameter in diastole (LVIDd) measured from serial 2D echocardiographic images obtained after removing doxycycline. E,F: Quantitative real-time RT-PCR measurements of mRNA levels of indicated genes from RNA isolated from selected hearts at (E) one week and (F) eight weeks after removing doxycycline. mRNA levels are calculated relative to cyclophilin mRNA and normalized to control levels. *p < 0.01. ON: double-transgenic tTA/CNA mice; OFF: single transgenic tTA mice.

Figure 4. Calcineurin-induced ventricular hypertrophy precedes systolic dysfunction, and fetal gene activation is delayed after calcineurin activation

A: Mean LV mass calculated from serial 2D echocardiographic images of tTA (n=9) and tTA/CnA* (n=9) mice recorded after removing doxycycline. B: Mean LV posterior wall thickness at end-diastole (LVPWd) by serial echocardiography after removing doxycycline. C: Mean percent fractional shortening from serial M-mode tracings obtained after removing doxycycline. D: Mean LV internal diameter in diastole (LVIDd) measured from serial 2D echocardiographic images obtained after removing doxycycline. E,F: Quantitative real-time RT-PCR measurements of mRNA levels of indicated genes from RNA isolated from selected hearts at (E) one week and (F) eight weeks after removing doxycycline. mRNA levels are calculated relative to cyclophilin mRNA and normalized to control levels. *p < 0.01. ON: double-transgenic tTA/CNA mice; OFF: single transgenic tTA mice.

Figure 5. Impaired cardiac function recovers after activated calcineurin is extinguished

A: Experimental design. Double transgenic mice maintained on doxycycline were used as controls. B: Mean percent fractional shortening measured from serial M-mode tracings of tTA/CnA* mice in the ON/OFF group (n=16). Dotted lines represent mean percent fractional shortening in the OFF (n=10) and ON groups (n=14). *p < 0.01. C: Mean HW/BW at study completion. *p < 0.01 for ON vs OFF; † p < 0.01 for ON/OFF vs ON.

Figure 6. Pathological hypertrophy regresses after activated calcineurin is extinguished

Fractional shortening (A) and LV mass by echocardiography (B) for ON/OFF mice (n=11) before and after restarting doxycycline. Squares represent means. C: Representative immunoblot of LV lysates probed for RCAN from treatment groups as listed. Ponceau stain shows protein loading for each lane. D: Mean absolute heart weight and HW/BW for OFF (n=9) and ON (n=11) mice in study A and OFF (n=4) and ON/OFF (n=6) mice in study B. † p < 0.05 for ON/OFF (study B) vs. ON (study A) E: Mean mRNA levels of βMHC, BNP, and SERCA2a determined by real-time RT-PCR from RNA isolated from OFF (gray bars; n=4) and ON/OFF (black bars, n=6) hearts. mRNA levels are calculated relative to cyclophilin mRNA and normalized to control levels. F: Mean mRNA levels of MuRF3 determined by real-time RT-PCR from RNA isolated from OFF, ON, and ON/OFF hearts at 8 weeks (after removing doxycycline) or 16 weeks (after removing and then restarting doxycycline). ON: tTA/CnA* mice with Dox removed for 8 weeks; ON/OFF: tTA/CnA* mice with Dox removed for 8 weeks then restarted for 8 weeks; OFF: single transgenic tTA mice served as controls.

Figure 6. Pathological hypertrophy regresses after activated calcineurin is extinguished

Fractional shortening (A) and LV mass by echocardiography (B) for ON/OFF mice (n=11) before and after restarting doxycycline. Squares represent means. C: Representative immunoblot of LV lysates probed for RCAN from treatment groups as listed. Ponceau stain shows protein loading for each lane. D: Mean absolute heart weight and HW/BW for OFF (n=9) and ON (n=11) mice in study A and OFF (n=4) and ON/OFF (n=6) mice in study B. † p < 0.05 for ON/OFF (study B) vs. ON (study A) E: Mean mRNA levels of βMHC, BNP, and SERCA2a determined by real-time RT-PCR from RNA isolated from OFF (gray bars; n=4) and ON/OFF (black bars, n=6) hearts. mRNA levels are calculated relative to cyclophilin mRNA and normalized to control levels. F: Mean mRNA levels of MuRF3 determined by real-time RT-PCR from RNA isolated from OFF, ON, and ON/OFF hearts at 8 weeks (after removing doxycycline) or 16 weeks (after removing and then restarting doxycycline). ON: tTA/CnA* mice with Dox removed for 8 weeks; ON/OFF: tTA/CnA* mice with Dox removed for 8 weeks then restarted for 8 weeks; OFF: single transgenic tTA mice served as controls.