Remuscularization with triiodothyronine and β1-blocker therapy reverses post-ischemic left ventricular dysfunction and adverse remodeling

Renewal of the myocardium by preexisting cardiomyocytes is a powerful strategy for restoring the architecture and function of hearts injured by myocardial infarction. To advance this strategy, we show that combining two clinically approved drugs, but neither alone, muscularizes the heart through cardiomyocyte proliferation. Specifically, in adult murine cardiomyocytes, metoprolol, a cardioselective β1-adrenergic receptor blocker, when given with triiodothyronine (T3, a thyroid hormone) accentuates the ability of T3 to stimulate ERK1/2 phosphorylation and proliferative signaling by inhibiting expression of the nuclear phospho-ERK1/2-specific phosphatase, dual-specificity phosphatase-5. While short-duration metoprolol plus T3 therapy generates new heart muscle in healthy mice, in mice with myocardial infarction-induced left ventricular dysfunction and pathological remodeling, it remuscularizes the heart, restores contractile function and reverses chamber dilatation; outcomes that are enduring. If the beneficial effects of metoprolol plus T3 are replicated in humans, this therapeutic strategy has the potential to definitively address ischemic heart failure.

To confirm if DUSP5 expression in CMs was specifically regulated by β 1 -AR, we examined the effects of β 1 -AR siRNA or control scrambled siRNA treatment. These siRNAs were administered using the in vivo-jetPEI delivery system 19 . CMs were harvested 64 h after 2 consecutive daily administrations of β 1 -AR siRNA in P35 mice and CMs purified from enzymatically disaggregated LVs using differential centrifugation 19 . This treatment, which reduced CM β 1 -AR expression by 63% (P < 0.001) (Fig. 1C), reduced DUSP5 expression by 87% (relative DUSP5 expression in CMs: 1.0 ± 0.028 and 0.13 ± 0.004 in scrambled siRNA and β 1 -AR siRNA treated mice, respectively; n = 4 mice/group; P < 0.001), indicating that metoprolol-induced inhibition of CM DUSP5 is mediated by β 1 -ARs. β 1 -AR blockade promotes T3-stimulated muscularization of healthy LVs. To test the in vivo effects of T3, with or without β 1 -AR blockade, on CM proliferation, we administered either metoprolol succinate (6 µg/g, i.p., daily) (hereafter referred to as metoprolol) or vehicle to healthy adult mice for 19 days. T3 (2 ng/g, i.p., daily), or vehicle, was then given concomitantly over the last 5 days, between days 14 and 19 of metoprolol therapy (dual therapy with metoprolol and T3 will hereafter be referred to as M + T3 therapy). We then performed two experiments: (1) to determine the effect of this therapy on circulating T3 levels; and (2) to determine the effects of M + T3 therapy on ventricular CM numbers and LV contractile function. Serum T3 levels were determined on the 5th day of T3 therapy; its decay after cessation of T3 therapy was also followed. This analysis showed that 12 h after the 5th dose of T3, serum T3 levels were ~ twofold higher than in control mice that were given vehicle (P < 0.001) ( Fig. 2A). Thereafter, serum T3 levels decayed to baseline values over the next 28 h.
To determine the effect of M + T3 therapy on CM proliferation, we sacrificed the mice at either 14-or 42-days post-therapy (dpt). Relative to untreated mice, or those treated with metoprolol or T3 monotherapy, M + T3 therapy increased the total number of ventricular CMs by > 20% (P < 0.001) by 14 dpt and this increase was sustained over the next 28 days (Fig. 2B). These data indicate that short-term M + T3 therapy stimulates controlled CM proliferation because its proliferative effect is limited to the immediate post-therapy window.
Since an increase in CM numbers improves LV contractile function 33 , we studied the effect of M + T3 therapy on LV mass and contractile function (ejection fraction (EF)), between the end of therapy and 14 dpt, using M-mode echocardiography. While M + T3 therapy did not change body weight (Fig. 2C), LV mass and EF increased by ~ 18% (P < 0.05) and 28% (P < 0.0001), respectively (Fig. 2D,E); these parameters were not altered by metoprolol or T3 monotherapy. At 14 dpt, hearts were 13% larger in M + T3-treated mice than in those treated with metoprolol alone (heart-to-body weight ratios were 5.36 ± 0.23 and 4.73 ± 0.05 mg/g in M + T3-and metoprolol-treated mice, respectively; n = 6 mice/group; P < 0.05), but CMs were 14% smaller (CM areas were 2100 ± 31 and 2400 ± 91 µm 2 in M + T3-and metoprolol-treated mice, respectively; n = 5 mice/group; ~ 200 isolated CMs were sampled from each heart; P < 0.01). Thus, M + T3 therapy muscularizes the heart by increasing CM endowment, which increases LV contractility. M + T3 therapy reverses severe LV dysfunction in post-MI mice. To evaluate the effects of M + T3 therapy in a model of ischemic cardiomyopathy, we induced a MI in C57Bl/6 mice by permanent coronary artery ligation. C57Bl/6 mice have a relatively low frequency of mononuclear diploid CMs in the naive adult heart and the extent of endogenous cellular regeneration and functional recovery after a MI is relatively low 34 . In these mice, echocardiographic assessments at 30 days post-injury (dpi) revealed LV dysfunction relative to baseline LV function (LVEF ~ 65%), which ranged from modest (LVEF > 50%) to extremely severe (LVEF < 15%). The American College of Cardiology defines severe LV dysfunction as > 30-percentage point decrease in LVEF 5 (https:// www. acc. org/ tools-and-pract ice-suppo rt/ clini cal-toolk its/ heart-failu re-pract ice-solut ions/ left-ventr icular-eject ion-fract ion-lvef-asses sment-outpa tient-setti ng).
There is variability in the coronary arterial anatomy of C57BL/6 mice 35 . We selected mice with a 1-month post-MI LVEF between 15 and 30% (that is, a > 35-percentage point decrease from baseline)-prior work suggests that for large infarcts, there is a negative linear relationship between infarct size and LVEF 36  www.nature.com/scientificreports/ randomly assigned these post-MI mice to treatment groups in a blinded fashion to minimize between-group differences in area of risk after coronary occlusion. We then conducted a pilot study comparing the effects of M + T3 therapy versus metoprolol monotherapy to determine if the immediate benefit (at 30 days dpt) of therapy was consistent with extensive regenerative repair (which we arbitrarily considered to be a > 70% restoration of pre-MI LVEF) and to determine the sample size for the subsequent study.
Twenty-two mice with LVEFs between 15 and 30% at 1-month post-MI were used for the pilot study (Supplementary Fig. S2A,B); their LV volumes at end-diastole were elevated twofold compared with pre-MI baseline values (50 ± 2 µl and 101 ± 6 µl at baseline and at 30 dpi, respectively; P < 0.0001, using a paired t-test) indicating that the MI injury resulted in substantial pathological LV remodeling. These mice were then given either metoprolol for 19 days with the addition of T3 over the final 5 days of metoprolol therapy, or only metoprolol monotherapy for 19 days. At 30 days after M + T3 therapy, LVEF increased by 28-percentage points, representing an almost complete reversal of the MI-induced 36-percentage point decrease in LVEF (that is, 78% restoration in LV contractile function) ( Supplementary Fig. S2A). By contrast, LVEF did not change significantly after shortterm metoprolol monotherapy ( Supplementary Fig. S2B). Based on these data, we estimated that a sample size of 4 in each group should have 90% power to show a 25-perentage point increase in post-therapy LVEF at a two-sided significance of 0.01.
We then randomized an additional 16 mice with post-MI LVEFs ranging between 15 and 30%, in a blinded fashion, into 4 groups. In addition to a M + T3 therapy group (metoprolol and T3 given as described above), 3 control groups were given either metoprolol for 19 days, T3 monotherapy for 5 days, or no therapy. We studied the initial effects of the treatments at 30 dpt and over the next 120 days (Fig. 3A). M + T3 therapy produced an ~ 30-percentage point increase in LVEF at 30 dpt that persisted throughout the 150 dpt observation period (Fig. 3B); it also rapidly and stably reversed the ~ fourfold post-MI increase in LV end-systolic volume (Fig. 3C). LVEF and end-systolic volume independently predict worsening outcomes in heart failure patients 37 . In contrast to M + T3 therapy, relative to untreated controls, LVEF and end-systolic volume remained unchanged with either metoprolol or T3 monotherapy (Fig. 3B,C) or with no therapy. These data show that M + T3 therapy stably reverses LV systolic dysfunction in mice with severe preexisting post-MI heart failure.
M + T3 therapy induces renewal by preexisting CMs. We then tested the hypothesis that, in hearts with severe ischemic injury, M + T3 therapy stimulates CM renewal from preexisting CMs. We sought evidence of such renewal using multicolor lineage tracing in double-transgenic Myh6-MerCreMer::Rosa26fs-Confetti mice 38 , an approach that allows the simultaneous evaluation of the proliferative trajectories of many LV CMs (Fig. 4A). In these mice, Cre recombinase activation upon 4-hydroxytamoxifen (4-HT) administration causes the Confetti construct to recombine, which randomly labels CMs with red fluorescent protein (RFP), yellow fluorescent protein (YFP), green fluorescent protein (GFP) or cyan fluorescent protein (CFP) (Fig. 4B). The administration of 4-HT at a previously determined dose (1.5 µg/g, i.p.) 26 to these mice causes recombination- www.nature.com/scientificreports/ driven RFP and YFP expression in CMs, as determined by confocal microscopy, at frequencies that minimize the occurrence of monochromatic CM clusters 26 , a prerequisite for multicolor analysis of discrete CM replication events. However, at this low 4-HT dose, GFP-and CFP-positive CMs are not observed 26 . Eleven days after we gave 4-HT to Myh6-MerCreMer::Rosa26fs-Confetti mice, we subjected them to MI and, upon confirmation of injury at 30 dpi (LVEF < 30% but > 15%), we administered M + T3 therapy or, as a control, T3 monotherapy, as before, and imaged the hearts at 15 dpt (Fig. 4A). Because T3 increases transcription at the Mhy6 promoter, it has the potential to increase Cre expression. To obviate this confounding effect, we used T3 monotherapy as a con-  To visualize the near-term morphological consequences of M + T3-stimulated CM proliferation, we examined gross anatomy and trichrome-stained tissue sections of post-MI LVs before therapy and then at 15 dpt; scar tissue being shown in blue and viable myocardium in red (Fig. 5A,B). Before therapy (at 30 dpi), a thin scar was evident between the LV apex and the mid-apical PW (halfway between the apex and the mid-papillary muscle) (Fig. 5A, green arrows). At 15 dpt, new myocardium appeared to extend from the apical tip of the intraventricular septum (IVS) toward the LV mid-apical PW (Fig. 5B, red arrowheads). The gross anatomy of an uninjured age-matched control is shown for comparison (Fig. 5C). Together, these data indicate the rapidity of regenerative repair, which involves remuscularization of the scarred LV, and is evident as early as 15 dpt.
Long-term consequences of M + T3 therapy on the LV structure and function. Heart failure is a progressive disease that is characterized by cellular (for example, CM hypertrophy), as well as regional and global structural and functional changes resulting from a loss of viable myocardium and increased wall stress [39][40][41] . We, therefore, addressed the ability of M + T3 therapy to reverse some of these processes in hearts with severe preexisting ischemic injury. At 150 dpt, assessments of gross LV morphology and visualization of fibrosis and viable myocardium (using trichrome staining) indicated the formation of a new myocardium surrounding a fibrous core (Fig. 6A, right and Fig. 6B, right), which is a remnant of the scarred LV PW observed at 30 dpi (Fig. 5A). Quantitative measurements, at 150 days after M + T3 therapy, indicated that the amount of viable myocardium per unit length of the LV mid-apical PW was > 20-fold higher than in untreated mice (P < 0.001) (Fig. 6C). Using immunohistochemical markers of CMs (cardiac troponin T, cTnT) and cell wall (wheat germ agglutinin, WGA), we show that the remuscularized myocardium of M + T3 therapy-treated post-MI mice have ~ tenfold greater number of CMs than those of untreated mice (P = 0.027) (Fig. 6D-F). To examine the extent of angiogenesis, we compared the capillary densities of the remuscularized LV PW with those of uninjured hearts, using high power microscopy. We found that the capillary-to-CM ratio was similar between these mouse groups (Supplementary Fig. S3), suggesting that the process of rebuilding heart muscle increases angiogenesis to match CM proliferation. We also determined collagen deposition (blue area in trichrome stained sections) (Fig. 6B) in LV mid-apical PW segments, which showed that M + T3 decreased scar area (by 64%), but this change was not statistically significant (relative collagen areas/unit PW length were: 1.0 ± 0.3 and 0.36 ± 0.06 in 150 dpt vehicle and M + T3 therapy groups, respectively; n = 3/group; P = 0.11).
The pump function of the LV is compromised progressively by a MI, as was evident by reductions in LVEF (Fig. 3B). This LV dysfunction mostly results from a loss of PW motion between the papilla and the apex. As the LV PW contracts, the LV chamber dimension decreases and its wall thickens. At 30 dpi, fractional shortening and end-systolic PW thickening at the mid-point between the papilla and the apex decreased by ~ 90% and ~ 70%, respectively (P < 0.001, in each case) (Fig. 7A-C). In contrast, 150 days after acute M + T3 therapy, these variables www.nature.com/scientificreports/ were similar to those of uninjured hearts (Fig. 7A-C). Sequential B-mode images (parasternal long axis views) of a representative mouse heart demonstrate the debilitating effect of MI-injury on LV PW motion (Supplementary Videos S1, S2), and then the transformation of a mostly akinetic LV mid-apical PW at 30 dpi to one in which PW motion had been reestablished at 150 dpt (Supplementary Videos S2, S3). Severe heart failure is also characterized by altered LV geometry and chamber dilatation (increased enddiastolic volume). At 30 dpi, LV geometry changed from that of a prolate ellipsoid to being more spherical (Fig. 8A,B), and LV end-diastolic volume increased by > twofold (P < 0.001) (Fig. 8C). In marked contrast, by 150 dpt, the LV chamber was much less dilated (Fig. 8C) and more conical in shape (Figs. 6A and 8B). Thus, M + T3 therapy produced an enduring reversal of MI-induced defects in LV structure.
CM hypertrophy in the spared LV myocardium (that is, the remote zone) is a hallmark of post-MI remodeling. We looked for evidence of CM hypertrophy in the LV remote zone as well as in the remuscularized LV mid-apical PW (Supplementary Fig. S5A). Estimation of CM cross-sectional areas revealed that LV remote zone CMs of M + T3-treated post-MI hearts were ~ 30% smaller than those of untreated post-MI hearts (P < 0.001), but similar in size to those of uninjured hearts (Supplementary Fig. S5B). We also found that the mean cross-sectional area of LV mid-apical PW CMs of M + T3 treated post-MI mice was similar to that of uninjured mice (Supplementary Fig. S5C). Collectively, these findings indicate that M + T3 therapy stably rebuilds the myocardium where previously there was an injury and, in uninjured LV regions, it prevents both CM hypertrophy and increases in LV end-diastolic volume, the hallmarks of pathological remodeling.

Discussion
The key objective of regenerative therapy is to redress severe postischemic heart failure by restoring lost LV myocardium and, thereby, improving contractile function. A more intractable objective is restoration of organ architecture which, in the case of chronic ischemic cardiomyopathy, contributes to increased LV wall stress and results in progressive deterioration of LV function. Another objective is to develop therapies that have high translational potential. Here, we describe the development and use of a therapeutic regimen that fulfils these objectives, which heretofore has remained elusive.
In terms of gross morphology, LV muscularization is an increase in LV myocardial tissue volume or LV mass. But, at the cellular level, LV muscularization can occur in distinct ways: through hypertrophy, hyperplasia or both. Stressors, such as pressure overload, cause CMs to hypertrophy, which is initially compensatory but, long term, such hearts frequently progress to failure 41,42 . In LVs in which CMs can be forced to proliferate without cellular hypertrophy muscularization increases LV contractility 26 . However, excessive stimulation of CM proliferation can be detrimental. For example, overexpression of a constitutively active mutant of ERBB2 not only results in profound CM proliferation, but also dedifferentiation and hypertrophy and, ultimately, increased mortality 17 .
Here we show that M + T3 therapy, given transiently to healthy adult mice, increases ventricular CM endowment. We demonstrate this increase both indirectly and directly. We found that, 14 days after the cessation of M + T3 therapy, LV mass was 18% higher at a time when LV myofiber mean cross-sectional area was 14% lower than that of controls. The most parsimonious explanation of this finding is that the LV myocardium of post-M + T3 hearts has more CMs. The decrease in CM size not only means that M + T3 therapy is not hypertrophyinducing, but results in cell replication because daughter cells are initially smaller than their parent cells. To confirm this conclusion, we directly counted ventricular CMs after enzymatic disaggregation of the heart, which revealed a 20% increase in CM numbers. Importantly, this increase, which was sustained over the following 28 days, cannot be attributed to differences in CM number estimation between M + T3 therapy and control groups resulting from incomplete enzymatic digestion of the myocardium, since we carefully quantitated the efficiency of tissue disaggregation and found it to be over 97% in both control and treated animals.
In healthy adult mice, M + T3 therapy increased LVEF by a remarkable 28% and did not result in any death in the 42 days of post-therapy follow up. Thus, the finding of an increase in CM endowment coupled with an increase in LV mass and contractile function is consistent with M + T3 therapy inducing therapeutic LV muscularization.
We show here that β 1 -AR blockade and T3 therapy increases CM numbers in adult mice. The key finding that β 1 -ARs negatively regulate T3-stimulated phospho-ERK1/2 buildup in CMs, but not the accumulation of its upstream activator MEK1/2, implicates phospho-ERK1/2 dephosphorylation by its specific phosphatase DUSP5 as a β 1 -AR target. Consistent with this notion, we found that β 1 -AR depletion (using siRNA-mediated receptor knockdown) inhibited CM DUSP5 expression and increased T3-stimulated phospho-ERK1/2 in CMs. Also, β 1 -AR blockade reversibly inhibited DUSP5 expression in CMs and synergized with T3 to increase CM endowment. The effects of β 1 -AR blockade were identical to those observed with genetic or pharmacological inhibition of DUSP5, which is necessary and sufficient for suppressing T3-induced CM proliferation and therapeutic LV muscularization 19 www.nature.com/scientificreports/ CM dedifferentiation frequently 44 , but not always 8,18 , accompanies CM proliferation; in cases where dedifferentiation occurs, LV function is depressed 9 . In murine CMs, we found that T3 not only increases the expression of multiple cell cycle-promoting genes, it also increases expression of Mhy6 and Tnni1, key markers of CM differentiation, as well as genes that control oxidative phosphorylation 18 , again markers of mature CMs. These effects, which result from direct genomic actions of T3 24 , may explain why the CM regeneration observed with M + T3 therapy is not associated with a decrease in LV function (Fig. 2E).
Injury-induced cardiac regeneration is observed in adult zebrafish and neonatal mice 16,45 . CMs of these hearts have high proliferative potential that is activated by the injury. But the molecular and cellular signals caused by injury must be short lived because CMs become quiescent after repair is complete. Repairing hearts long after the injury-that is, those with chronic preexisting disease-requires a remuscularization stimulus that is independent of that produced by injury. Because of its effectiveness in enhancing muscularization in healthy animals, we examined the potential for M + T3 therapy to repair hearts long after MI.
We studied therapeutic remuscularization with short-term M + T3 therapy only in hearts with severe injury. We found that short-term M + T3 therapy remuscularizes the thin heavily scarred post-MI LV PW and that its effects are enduring. At 150 dpt, mid-apical viable myocardial area was more than 20-fold higher in M + T3 treated mice than in untreated controls. Histological analysis indicated that these thicker LV PW segments contained ~ tenfold more CMs per unit wall length than those of untreated post-MI hearts (Fig. 6D-F). Because wall stress is inversely proportional to wall thickness, therapeutic remuscularization is expected to reduce regional wall stress, which is an independent predictor of LV remodeling after MI 46 . By 150 dpt, M + T3 therapy had effectively reverse remodeled the heart leading to a normalization of overall LV shape (Fig. 8) and regression of CM hypertrophy (Supplementary Fig. S5). Lack of LV PW motion, or akinesis, is a serious complication of MI. Serial assessments of end-systolic and end-diastolic LV cavity dimensions at the mid-apical level of each heart, between 30 dpi and 150 days post-M + T3 therapy, showed that M + T3 therapy caused a > sixfold increase in regional fractional shortening. Thus, we show concordance between direct (increased viable myocardium in the scarred LV PW and increased regional LV fractional shortening) and indirect (reversal of remote zone CM hypertrophy and reverse-remodeling of the LV chamber) effects of M + T3-induced remuscularization.
The use of a cardioselective β 1 -AR blocker, together with T3, to remuscularize MI-injured LVs would suggest that preexisting CMs are a source of new CMs; CMs abundantly express β 1 -ARs 30 . We sought evidence to support this contention using lineage tracing, the technique of choice to identify progeny of single cells 47 . With these studies, specificity is provided by the use of a Myh6 promoter to drive tamoxifen-dependent Cre recombinase expression, which results in monochromatic fluorescent labeling of CMs with a fluorescent protein. Moreover, by using low-dose 4-HT, random occurrence of monochromatic CM clusters is minimized. The additional use of WGA labeling, which identifies cell borders in tissue sections, distinguishes one CM from another.
Using Myh6-MerCreMer::Rosa26fs-Confetti mice, we found that M + T3 therapy increases the frequency of LV monochromatic CM clusters by 12-fold. Such clusters, which are mostly non-existent after control therapy, signify the conversion of labeled CMs into adjacently-positioned, similarly labeled daughters; that is, replication of preexisting CMs, which was observed in all post-MI LV regions. Our finding that β 1 -AR depletion potentiates T3-stimulated expression of ECT2 in CMs (by > 12-fold)-ECT2 facilitates hepatocyte cytokinesis during liver regeneration 48 -could explain the efficacy of M + T3 therapy in stimulating CM replication.
MI induces a surge in CM death over the first 6-24 h of ischemic injury 49 . T3 therapy, instituted at the time of ischemic injury, has been shown to stimulate cardioprotective prosurvival pathways 50 . We do not believe, however, that this action of T3 has an important role in the beneficial effects of M + T3 regenerative therapy in the setting of preexisting heart failure, because in our studies M + T3 therapy was initiated one month after MI, where levels of CM apoptosis are negligible 49 . It is perhaps for this reason that the marked increase in viable myocardium in the mid-apical LV PW was associated with a much smaller (and non-significant) decrease in scar area. However, it will be important to evaluate if late cardiac remuscularization therapy with M + T3 can be complimented with early T3 monotherapy to limit scar formation ab initio.
Effective cardiomyogenesis-that is, the process leading to the formation of myocardium-also involves neovascularization, reinnervation and restoration of the extracellular matrix 51 . It will be important to study the effect of M + T3 therapy on these processes and to determine their role in the regenerative response observed  Supplementary Fig. S4A-E, show other examples of gross anatomy of post-MI heart, including those shown in "A", that were untreated or given T3 monotherapy, metoprolol monotherapy or M + T3 therapy. (C) Quantitative analysis of viable myocardium in the LV mid-apical PW at 150 dpt. Data represent values from 3 independent hearts per group. ***P < 0.001. (D,E) Representative longitudinal tissue section (7 µm thick) images, representing 0.2 mm-long views of the LV posterior wall at the mid-apical region, from an untreated post-MI mouse (A) and from a post-MI mouse after M + T3 therapy (B). These hearts are from mice followed for 150 dpt. CMs are visualized using cardiac troponin T (cTnT) staining (red) and cell boundaries are outlined by wheat-germ agglutinin (WGA) staining (white). WGA-staining also indicates areas of scar tissue and fibrosis. In these longitudinal images of the LV posterior wall, the LV cavity is on the left side of the tissue section. (F) Number of CMs (CMs) in 0.2 mm-long tissue section views of the LV mid-apical wall from post-MI untreated and post-MI M + T3-treated hearts. Data represent values from 3-4 independent hearts per group. An unpaired 2-sided student's t-test was used to compare differences between groups. Data are shown as individual values and mean ± SEM. www.nature.com/scientificreports/ with this therapy. Here, we examined capillaries as a part of the cardiac regenerative response and found that the CM-to-capillary ratio was near normal after M + T3 therapy. This suggests that, post-M + T3 therapy, neoangiogenesis was commensurate with LV remuscularization. This may not be surprising, given that CMs elaborate angiogenic factors, such as vascular endothelial growth factor 52 .
In this era when percutaneous coronary interventions being widely used as primary therapy for occlusive myocardial disease, myocardial damage is now more commonly due to ischemia-reperfusion injury than to permanent coronary artery occlusion injury 53 . However, even with the widespread use of primary percutaneous intervention, ~ 15-25% patients are not successfully reperfused in a timely manner 40 . The key objective of our study was to determine the effect of remuscularization in hearts with severe LV dysfunction and extensive LV remodeling. Long-term remodeling is more robustly seen after MI due to permanent coronary artery occlusion, reducing the sample size needed to detect differences between groups 40 . We note that M + T3 therapy did not repair the LV PW immediately distal to the occlusion. It will be important to determine if ischemia-reperfusion injury is more completely repaired with M + T3 therapy than permanent coronary occlusion injury.
In summary, using a murine model of ischemic cardiomyopathy with marked cardiac remodeling and severely impaired LV function, we show that short-term T3 therapy together with β 1 -AR blockade remuscularizes the heavily scarred LV PW, which results in an enduring improvement in LV structure and contractile function.
Potential for translation. In the United States, the long-acting β 1 -AR blocker metoprolol succinate is specifically approved for the treatment of heart failure due to structural heart disease 54 . Thus, in many heart failure patients on metoprolol therapy, future regenerative therapy might simply involve the addition of short-duration T3 therapy. A recent Phase II randomized clinical trial has examined the effect of long-term (6-month) T3 therapy in patients with a prior MI and low-T3 syndrome 55 . While the therapy was considered safe and reduced regional contractile dysfunction, subgroup analysis of patients receiving β 1 -AR blocker therapy was not performed. It is difficult to extrapolate findings from this clinical trial to the translational value of M + T3 therapy because the investigators of this clinical trial used T3 therapy to reverse the low-T3 syndrome, but not to elevate circulating T3 levels beyond the normal range which, in mice, is necessary for inducing CM proliferation. Given the findings here, short-duration T3 therapy, at doses that elevate circulating T3 beyond the normal range in heart failure patients receiving metoprolol therapy, may prove useful in permanently reversing LV dysfunction and therefore warrants further clinical investigation.

Materials and methods
A detailed description of the experimental procedures related to cardiomyocyte isolation for number determination, immunocytochemical studies with antibodies, histology and immunoblotting is provided in the Supplementary Information section.
Animal care and use. Mice were housed under pathogen-free conditions in a facility approved by the American Association for the Accreditation of Laboratory Animal Care. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Emory University. We confirm that all experiments were performed in accordance with IACUC guidelines and regulations. We also confirm that the study is reported in accordance with ARRIVE guidelines. C57Bl/6 wild type (Jackson Laboratory, 000664) male mice were used for the studies reported here. We performed cardiomyocyte (CM) lineage tracing studies as previously described 19,26 (21,28) using Rosa26fs-Confetti [B6.129P2-Gt(ROSA)26Sortm1(CAG-Brainbow2.1)Cle/J, Jack-   19 show that 5 doses of T3 (2 ng/g, daily, for 5 days; i.p.) sustain ERK1/2 activation in CMs, which is necessary for in vivo CM proliferation; fewer doses of T3 did not consistently or robustly stimulate CM proliferation when given in combination with DUSP5 depletion in CMs (data not shown). Moreover, while i.p. T3 administration (2 ng/g) increased circulating T3 levels, the extent of this increase differed between operators. For this reason all drug administrations were performed by one investigator (L. T.); in these studies 2 ng/g T3 increased circulating T3 levels by about twofold (e.g., Fig. 2A). Metoprolol was injected daily (6 µg/g body weight) for 14 days before the start of T3 and then continued for another 5 days in combination with T3. The rationale for this metoprolol dosing protocol is based on our studies showing nearly complete suppression of DUSP5 expression in CMs using this protocol (Supplementary Fig. S1); higher doses of metoprolol have also been used to selectively block the β 1 -AR 56   www.nature.com/scientificreports/ based on dose titration studies 26 , minimizes replication-independent occurrence of monochromatic clusters by ensuring minimal labeling of CMs (~ 1% of the heart) 26 . β 1 -AR-specific siRNA (sc-29581, Santa Cruz Biotechnology) or scrambled siRNA (control) was administered using in vivo-jetPEI, (VWR, 89,129-960). β 1 -AR siRNA (100 ng) was dissolved in 1 ml of the in vivo-jetPEI:10% glucose mixture and was injected 100 μl per mouse via i.p. route (10 ng/mouse). β 1 -AR siRNA was a pool of 2 different siRNA duplexes.
Murine model of ischemic cardiac injury. MI injury was induced by permanent ligation of the left anterior descending artery. Surgeries were performed under aseptic techniques. Body temperature was maintained using warm-water blankets. Under aseptic conditions and anesthesia, mice were orally intubated using polyethylene-60 (PE-60) tubing. The tubing was connected to a rodent ventilator (MiniVent Type 845), which was set at a tidal volume of 235 µl at a rate of 105 breaths/min. A side port on the ventilator was used to supplement 100% oxygen with 2% isoflurane. The surgery was performed using a Leica surgical microscope to aid in the visualization of the mouse left anterior descending artery. The heart was accessed through a left thoracotomy and the left anterior descending artery was exposed by opening the pericardium. The suture was positioned 2 mm below the left atrial appendage to permanently ligate left anterior descending artery. The lungs were then fully inflated, and the chest wall and skin incision were sutured in layers and the thoracic cavity was closed as described 57 . Buprenorphine (sustained release (SR)-LAB, ZooPharm) was administered subcutaneously (0.5 mg/kg) at the time of surgery to provide post-operative analgesia (releases over 72 h). Mice were kept warm until recovery.
Seventy-seven mice (including C57BL6 and Myh6-MerCreMer::Rosa26fs-Confetti mice) were subjected to MI surgery. Of these mice 10 died between the time of surgery and 30 dpi. Fifteen post-MI mice were excluded from the study because their LVEFs, at 30 dpi, were either > 35% or < 15%.
Echocardiography. Cardiac function was evaluated by transthoracic echocardiography performed on mice sedated with isoflurane (Piramal Critical Care) using Vevo 3100 (VisualSonics) and analyzed using the Vevo Lab software (VisualSonics). Measurements for the LVEF, end-systolic and end-diastolic volumes were calculated from the long axis as previously described 26 . Measurements for the LV PW at end-systole and end-diastole were calculated from the short axis, representing the mid-apical plane. Movies, showing LV contraction, were made using B-mode views on the parasternal long axis. Before taking images, anesthesia was adjusted to ensure heart rates of mice were between 450 and 500 beats/min. Quantification and statistical analysis. All data are presented as the mean ± standard error of the mean (SEM). For immunoblotting experiments 4 independent biological replicates were used in each experiment. Mice were randomized to different drug treatment groups using a preselect criteria of 15-30% LVEF. Mice with EF over or under this range were not included for drug randomization. No data points were excluded from any analysis. Most of the experiments were replicated at least once. Number of biological replicates for all experiments are mentioned in relevant figure legends. Investigators were not blinded to drug administration and outcome assessment. Statistical significance of data was determined using Grahpad Prism 9. The tests included: two-way ANOVA, followed by Sidak's multiple comparisons test; one-way ANOVA followed by Tukey's multiple comparisons test; and unpaired 2-sided Student's t-test for comparisons involving 2 groups. F-test was used for estimation of variance when comparing two groups and Brown-Forsythe test was used for estimation of variance when multiple groups were compared using one-way ANOVA. Where described, sample size estimation was conducted using nQuery (Statsols). Differences at P < 0.05 were considered significant.

Data availability
RNA-Seq, microarray, microRNA microarray or genome wide association studies were not performed for this manuscript. Any other data and resources generated for this manuscript are available upon reasonable request from the corresponding authors.