DUSP5 expression in left ventricular cardiomyocytes of young hearts regulates thyroid hormone (T3)-induced proliferative ERK1/2 signaling

Cardiomyocytes of newborn mice proliferate after injury or exposure to growth factors. However, these responses are diminished after postnatal day-6 (P6), representing a barrier to building new cardiac muscle in adults. We have previously shown that exogenous thyroid hormone (T3) stimulates cardiomyocyte proliferation in P2 cardiomyocytes, by activating insulin-like growth factor-1 receptor (IGF-1R)-mediated ERK1/2 signaling. But whether exogenous T3 functions as a mitogen in post-P6 murine hearts is not known. Here, we show that exogenous T3 increases the cardiomyocyte endowment of P8 hearts, but the proliferative response is confined to cardiomyocytes of the left ventricular (LV) apex. Exogenous T3 stimulates proliferative ERK1/2 signaling in apical cardiomyocytes, but not in those of the LV base, which is inhibited by expression of the nuclear phospho-ERK1/2-specific dual-specificity phosphatase, DUSP5. Developmentally, between P7 and P14, DUSP5 expression increases in the myocardium from the LV base to its apex; after this period, it is uniformly expressed throughout the LV. In young adult hearts, exogenous T3 increases cardiomyocyte numbers after DUSP5 depletion, which might be useful for eliciting cardiac regeneration.


Cardiomyocyte isolation for immunocytochemistry and immunoblotting
For immunoblotting and immunocytochemistry, hearts were enzymatically digested as described in Materials and Methods using procedures described earlier 12 . Before making single cell suspensions, atria were excised, and the LVs were divided into apex, mid and base regions, as described below. Cardiac cells of the apex and base were separately disaggregated into single cell suspension. Cardiomyocytes were purified with three low speed centrifugations (18 x g for 4 min at room temperature), which pelleted cardiomyocytes. Supernatant fractions, enriched in non-myocytes, were discarded. The resulting cardiomyocyte preparations were >95% pure.
Aliquots of cardiomyocytes were snap frozen in liquid nitrogen and stored at -80°C for immunoblotting. Additionally, aliquots were fixed with Cytofix (BD Biosciences, 554655) for five min and spread on glass slides for immunocytochemistry.

RT-qPCR
The RT-qPCR protocol used was customized from our earlier studies 12 . Briefly, cardiomyocytes in tubes containing RNAlater stabilization solution at -80 °C, were thawed on ice and then centrifuged at 21,000 x g for 10 min. The RNAlater supernatant fraction was removed and replaced with 240 µl of lysis binding-buffer from the mirVana miRNA Isolation Kit (Thermo Fisher Scientific, AM1560). RNA was purified according to the manufacturer's guidelines.

Immunoblotting
The immunoblotting protocol used was customized from our earlier studies 12 . Briefly, whole cell cardiomyocyte lysates were generated by re-suspending cardiomyocytes in 250 µl of RIPA buffer (Cell Signaling, 9806S) supplemented with phosphatase inhibitor cocktail 2 and 3 (Sigma-Aldrich, P5726-1ML and P0044-1ML), 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich, 93482-50ML-F) and protease inhibitor cocktail (Roche, 11697498001); cardiomyocytes were lysed by sonication and then pelleted by centrifugation at 21,000 x g for 30 min. The resulting supernatant fractions were aliquoted into fresh Eppendorf tubes and then snap-frozen in liquid nitrogen. Aliquots, stored at -80 °C, were allowed to thaw on ice immediately before use. Initially, a 5 to 10 µl aliquot (~20 μg protein) was mixed with an equal volume of 2x Laemmli sample buffer (Bio-Rad, 1610737), heated for 5 min at 95-99 °C and then immediately cooled on ice for 5 min. The samples were then centrifuged briefly before fractionation by SDS-polyacrylamide gel (12-18%) electrophoresis (SDS-PAGE), which was performed at 200 volts for 5 min, and then at 150 volts for 30 min to 2 h. The resolved proteins were then transferred to a PVDF membrane by electroblotting (Turbo Transfer; Bio-Rad).
Depending upon the molecular weight of the proteins or protein complexes, the transfer time on Turbo Transfer was varied for high (11 min), average (7 min) and low (5 min) molecular weight proteins. After transfer, all blots were pre-blocked for 30-60 min with Superblock (Thermo Fisher Scientific, 37536). Initially, the samples were probed with a GAPDH antibody. Based on the intensity of GAPDH, the volume of each sample loaded was adjusted to normalize the amount of GAPDH per sample. Membranes were probed with the target protein-specific primary antibody. For quantitative analysis, the membrane was then stripped and re-probed with GAPDH to ensure that loading was normalize for each sample. For stripping, the membrane was washed twice with 1x Tris-buffered saline (TBS, Thermo Fisher Scientific, BP2471-1) five min each and then incubated with Restore Western Blot Stripping Buffer (Thermo Fisher Scientific, 21059) for 5-15 min and then washed again twice with 1x TBS and pre-blocked with Superblock (Thermo Fisher Scientific, 37536) for 1 h before incubating with GAPDH antibody. Primary antibodies (see below) were also diluted in Superblock and incubated with the membranes for 2 h at 22 °C, or overnight at 4 °C, followed by horseradish peroxidase (HRP)-labeled secondary antibody (1:10,000) for 45 min at 22 °C. The signals were detected using Super Signal West Dura Detection Reagent (Thermo Fisher Scientific, 34075) and images captured on a Bio-Rad GelDoc system equipped with a CCD camera. Quantification was performed by densitometry using the ImageLab program (Bio-Rad).
For studies involving nuclear protein quantification, nuclear and cytoplasmic fractions were separated from cardiomyocytes using NE-PER Nuclear Cytoplasmic Kit (Thermo Fisher Scientific, 78833) as per the manufacturer's protocol. Nuclear and cytoplasmic fractions were then similarly resuspended in RIPA buffer and stored and analyzed as described above.
For studies involving developmental quantification of cardiac specific proteins, we generated tissue lysates from ventricular apical, mid or basal myocardial section at each of the postnatal ages specified.
The husbandry of the mice used in these studies is detailed above. We harvested hearts at each postnatal day between P1 to P17 and snap-froze them in liquid nitrogen. The ventricular apex, mid or base was then separated and after adding 200 µl of RIPA buffer (Cell Signaling Technology, 9806S) supplemented with phosphatase inhibitor cocktail 2 and 3 (Sigma-Aldrich, P5726-1ML and P0044-1ML), 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich, 93482-50ML-F) and protease inhibitor cocktail (Roche, 11697498001) the samples were homogenized using a Polytron PT 1200E handheld homogenizer (Kinematica). The resulting lysate was centrifuged at 21,000 xg for 30 min, the supernatant fraction harvested and aliquoted into fresh Eppendorf tubes and then snap-frozen in liquid nitrogen. These samples were subsequently fractionated by SDS-PAGE and analyzed as detailed above for cardiomyocyte lysates.
For studies involving different cardiac regions, the heart was divided into atria, RV and LV.
In some studies, the LV was further subdivided into apex, mid and base using a sharp scalpel to cut the hearts into pieces of equal width from the apex. On average, the fraction of LV myocardium in LV apex, mid and base was approximately 18%, 38% and 44%, respectively.
These regions were then immediately snap frozen in liquid nitrogen and subsequently used to prepare lysates, as above.
Antibodies used for immunoblots are detailed in Supplementary Table S1. Most of these antibodies are profiled in 1DegreeBio and were additionally validated in siRNA knockdown studies. Most of these antibodies are profiled in 1DegreeBio and were also validated using siRNA. Immunoblot shows expression levels of indicated proteins in whole lysates obtained from ventricular CMs of 5-week-old mice. Quantitative data is shown next to the immunoblots. Data are mean ± SEM. Comparisons between protein levels in CMs and non-myocytes were made using a 2-sided Student's t-test. ***P < 0.001.

Full unedited gel for Figure 1B
Cyclin A2 50kDa

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Cyclin B1 58kDa

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Cyclin B1 58kDa Atria RV LV Full unedited gel for Figure 1B (over exposed) Atria RV LV Full unedited gel for Figure 1B

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Cyclin D1 34kDa

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Cyclin A2 50kDa

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Cyclin D1 34kDa

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Cyclin A2 50kDa Apex Base

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Cyclin B1 58kDa Full unedited gel for Figure 4D GAPDH 37kDa Full unedited gel for Figure 4D DUSP-5 42kDa

A B A B A B A B A B A B A B A B A B A B
Full unedited gel for Figure 6A A= apex B=base

A B A B A B A B A B A B A B A B A B A B
Full unedited gel for Figure 6A A= apex B=base