International Journal of Molecular Sciences Article Cardiac-Specific Overexpression of ERRγ in Mice Induces Severe Heart Dysfunction and Early Lethality Jaime Lasheras 1,† , Rosario Pardo 1,† , Marc Velilla 1,† , Marcos Poncelas 2 , Núria Salvatella 1 , Rafael Simó 3,4 , Marisol Ruiz-Meana 2 , Mònica Zamora 5, * and Josep A. Villena 1,4, * 1 Laboratory of Metabolism and Obesity, Vall d’Hebron—Institut de Recerca, Universitat Autònoma de Barcelona, 08035 Barcelona, Spain; jaime.lasheras@vhir.org (J.L.); rosario.pardo@vhir.org (R.P.); marc.velilla@vhir.org (M.V.); salvatella.n@gmail.com (N.S.) 2 Laboratory of Experimental Cardiology, Vall d’Hebron—Institut de Recerca, Universitat Autònoma de Barcelona, 08035 Barcelona, Spain; poncelas@gmail.com (M.P.); marisol.ruizmeana@vhir.org (M.R.-M.) 3 Unit of Diabetes and Metabolism, Vall d’Hebron—Institut de Recerca, Universitat Autònoma de Barcelona, 08035 Barcelona, Spain; rafael.simo@vhir.org 4 CIBER on Diabetes and Associated Metabolic Diseases (CIBERDEM), 28029 Madrid, Spain 5 Institut d’Investigacions Biomediques August Pi i Sunyer, University of Barcelona, 08036 Barcelona, Spain * Correspondence: MZAMORAV@hospitalclinic.org (M.Z.); josep.villena@vhir.org (J.A.V.) † These authors contributed equally to this work. Abstract: Proper cardiac function depends on the coordinated expression of multiple gene networks related to fuel utilization and mitochondrial ATP production, heart contraction, and ion transport. Key transcriptional regulators that regulate these gene networks have been identified. Among   them, estrogen-related receptors (ERRs) have emerged as crucial modulators of cardiac function by regulating cellular metabolism and contraction machinery. Consistent with this role, lack of Citation: Lasheras, J.; Pardo, R.; ERRα or ERRγ results in cardiac derangements that lead to functional maladaptation in response to Velilla, M.; Poncelas, M.; Salvatella, N.; Simó, R.; Ruiz-Meana, M.; increased workload. Interestingly, metabolic inflexibility associated with diabetic cardiomyopathy Zamora, M.; Villena, J.A. has been recently associated with increased mitochondrial fatty acid oxidation and expression Cardiac-Specific Overexpression of of ERRγ, suggesting that sustained expression of this nuclear receptor could result in a cardiac ERRγ in Mice Induces Severe Heart pathogenic outcome. Here, we describe the generation of mice with cardiac-specific overexpression Dysfunction and Early Lethality. Int. of ERRγ, which die at young ages due to heart failure. ERRγ transgenic mice show signs of dilated J. Mol. Sci. 2021, 22, 8047. https:// cardiomyopathy associated with cardiomyocyte hypertrophy, increased cell death, and fibrosis. Our doi.org/10.3390/ijms22158047 results suggest that ERRγ could play a role in mediating cardiac pathogenic responses. Academic Editor: María Galán Keywords: cardiac dysfunction; estrogen-related receptors; transgenic mice Received: 5 July 2021 Accepted: 24 July 2021 Published: 28 July 2021 1. Introduction Publisher’s Note: MDPI stays neutral Proper cardiac function depends on the coordinated expression of multiple gene with regard to jurisdictional claims in networks that ensure functional adaption of the heart to changing demands on workload published maps and institutional affil- in response to physiological stimuli, such as exercise and pregnancy. The heart also adapts iations. to the pathological stress imposed by increased hemodynamic loads, such as hypertension, via hypertrophic growth to maintain adequate contractility and blood supply to tissues. However, if such pathogenic stimuli persist, the heart may undergo a maladaptive re- modeling that eventually leads to heart failure, a major cause of death and disability in Copyright: © 2021 by the authors. modern society. Licensee MDPI, Basel, Switzerland. Programs related to fuel utilization and mitochondrial ATP production, as well as This article is an open access article those associated with contraction, are critical for heart maturation and functional adapta- distributed under the terms and tion. During perinatal maturation of the heart, a metabolic shift from glucose utilization by conditions of the Creative Commons means of glycolysis towards fatty acid oxidation (FAO) in mitochondria occurs to warrant Attribution (CC BY) license (https:// the ATP production necessary to meet increased energy demands [1]. Such metabolic shift creativecommons.org/licenses/by/ is accompanied by an intense mitochondriogenic process that endows cardiomyocytes 4.0/). with a remarkable number of mitochondria equipped with all the enzymatic machinery Int. J. Mol. Sci. 2021, 22, 8047. https://doi.org/10.3390/ijms22158047 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 8047 2 of 14 for efficient ATP production [2,3]. This metabolic reprograming during the perinatal pe- riod is paralleled by a complete remodeling of components of the contractile machinery characterized by a decrease in the fetal isoforms of sarcomeric proteins (i.e., β-MHC or TNNI1) and an increase of adult proteins isoforms (i.e., α-MHC, TNNI3, MYL2, etc.) [4]. In contrast, pathogenic cardiac remodeling in adult individuals is often associated with a reduction in mitochondrial FAO, increased glycolysis, and re-expression of fetal forms of sarcomeric proteins. Numerous studies over the last two decades have contributed to uncover the network of key transcriptional regulators that tightly control metabolism and mitochondrial biogene- sis during cardiac development, maturation, and physiological adaptation. Members of the peroxisome proliferator-activated receptor γ co-activator-1 (PGC-1) family of co-activators, PGC-1α and PGC-1β, have emerged as master regulators of cardiac energy metabolism. Although individual deletion of either Ppargc1a or Ppargc1b genes in rodent models does not lead to significant cardiac alterations due to the functional redundancy [5–7], their rele- vance in the control of cardiac energetics is demonstrated in mice simultaneously devoid of both PGC-1 co-activators specifically in cardiac cells, which die early after birth from heart failure [3]. Nonetheless, individual deletion of Ppargc1a or Ppargc1b accelerates heart failure in response to increased workload imposed by transverse aortic constriction [8,9]. Several hormone nuclear receptors have been identified as critical mediators of the action of PGC-1 co-activators on cardiac gene expression, including hormone thyroid receptor (TR) [10,11], peroxisome proliferator-activated receptor α (PPARα) [12,13], and estrogen-related receptors (ERRs). Interestingly, PPARα knockout mice show decreased FAO capacity and higher susceptibility to progressive deterioration of cardiac function during a work overload challenge [14]. A crucial role for ERRs in the adaptive changes of cardiac metabolism and function has also been demonstrated. Indeed, it has been shown that loss of ERRα leads to pathologic ventricular remodeling and dysfunction in response to chronic pressure overload. The development of this pathological condition in mice devoid of ERRα is associated with reduced expression of mitochondrial genes and impaired ATP production [15]. On the other hand, the role of ERRγ is especially relevant during cardiac development. Indeed, mice lacking ERRγ die early after birth from car- diac defects associated with severe alterations in mitochondrial oxidative metabolism [16]. Beyond bioenergetics, both ERRα and ERRγ have proven to be essential for cardiac contrac- tion by regulating the expression of sarcomeric proteins during postnatal developmental maturation [17]. Whereas reduced expression of ERRs has been associated with impaired cardiac ener- getics and adaptability to imposed work overload, the potential protective role of increased ERRs activity has not been investigated. Here, we present a new mouse model of cardiac- specific overexpression of ERRγ. We show that sustained overexpression of ERRγ in cardiac tissue unexpectedly leads to premature death as a result of severe cardiac dysfunction. 2. Results 2.1. Transgenic Mice with Cardiac-Specific Overexpression of ERRγ Show Premature Lethality To investigate the metabolic and functional implications of sustained expression of high levels of ERRγ in heart, we generated transgenic mice that overexpress a flag-tagged version of human ERRγ under the heart-specific promoter of the α-MHC gene (Figure 1A), hereafter referred as hERRγ-Tg mice. Fourteen mice carrying the α-MHC-2xFlag-hERRγ transgene were obtained, but only four of these founders were able to transmit the transgene to the next generation. Analysis of the Flag-hERRγ mRNA expression by PCR revealed that only three of the transgenic mouse lines (named L3, L6, and L7) exhibited cardiac-specific expression of the transgene, whereas line L10 showed a preferential expression of the transgene in heart but detectable expression in almost all other tissues analyzed (Figure 1B, left panel). Cardiac-specific expression of the Flag-hERRγ protein was confirmed in lines L3, L6, and L7 by detecting the chimeric protein with a Flag antibody (Figure 1B, right panel). The three cardiac-specific transgenic lines exhibit different levels of Flag-hERRγ cardiac-specific expression of the transgene, whereas line L10 showed a preferential ex- pression of the transgene in heart but detectable expression in almost all other tissues an- Int. J. Mol. Sci. 2021, 22, 8047 alyzed (Figure 1B, left panel). Cardiac-specific expression of the Flag-hERRγ protein was 3 of 14 confirmed in lines L3, L6, and L7 by detecting the chimeric protein with a Flag antibody (Figure 1B, right panel). The three cardiac-specific transgenic lines exhibit different levels of Flag-hERRγ protein, L3 being the line with higher expression levels, L6 with interme- protein, L3 being the line with higher expression levels, L6 with intermediate expression diate expression levels, and L7 with lower expression levels of the protein (Figure 1C). levels, and L7 with lower expression levels of the protein (Figure 1C). Figure 1. Generation of a mouse model of cardiac-specific overexpression of ERRγ (hERRγ-Tg). (A) A transgene Figure that contains 1. Generation the cDNA of a mouse modelencoding for a flag-tagged of cardiac-specific version of overexpression of ERRγ hERRγ under the control (hERRγ-Tg). (A) of the α-MHC A transgene promoter that contains thewas usedencoding cDNA to generate for amice that specifically flag-tagged version ofoverexpress hERRγ under ERRγtheincontrol the heart. (B)α-MHC of the Three lines of hERRγ-Tg promoter was usedmiceto were generateobtained, firstspecifically mice that by selectingoverexpress mice that specifically ERRγ in the transmitted heart. (B) the Three lines of to transgene hERRγ-Tg mice were their offspring, and obtained, first by that then by ensuring selecting thosemice mousethat specifically lines expressed transmitted the transgene the specifically transgene in to the their offspring, heart, andmRNA both at the then by andensuring that (C) protein level. those mouse Western blotlines (left expressed the panel) comparing transgene specifically the expression in of levels thethe heart, both at the Flag-hERRγ mRNA protein andin levels protein hearts level. of the(C) Western three blot (left transgenic linespanel) obtained. comparing the expression levels of the Flag-hERRγ protein levels in hearts of the three transgenic Quantification of Flag-hERRγ protein expression in the heart of hERRγ-Tg mice (right panel). Data lines obtained. Quantification of Flag-hERRγ protein expression in the heart of hERRγ-Tg mice are mean ± SEM of three animals/line; ## p = 0.01. # indicates the statistical significance of the (right panel). Data are mean ± SEM of three animals/line; ## p = 0.01. # indicates the statistical signif- comparison icance between L3 of the comparison and L6;L3 between n.s. andindicates L6; n.s. nonspecific bands. bands. indicates nonspecific Soon after establishing the three hERRγ-Tg lines, we observed an unusual lethality in Soon after establishing the three hERRγ-Tg lines, we observed an unusual lethality young mice of lines L3 and L6, which expressed higher levels of Flag-hERRγ protein in the in young mice of lines L3 and L6, which expressed higher levels of Flag-hERRγ protein in heart, but not in the low expresser line L7. Indeed, post-weaning analysis of mice survival the heart, but not in the low expresser line L7. Indeed, post-weaning analysis of mice sur- over a period of 30 weeks showed that the median survival was severely decreased in vival over a period of 30 weeks showed that the median survival was severely decreased L3 and L6 hERRγ-Tg mice, in both males and females (Figure 2). The estimated median in L3 and L6 hERRγ-Tg mice, in both males and females (Figure 2). The estimated median survival was 8 weeks for males and 19 weeks for females of the L3 line (Figure 2A), survival was 8 weeks for males and 19 weeks for females of the L3 line (Figure 2A), and 9 and 9 weeks for both males and females of the L6 line (Figure 2B). No differences in the weeks for both survival ratemales andWt between females of the L6 line and hERRγ-Tg mice(Figure of the 2B). low No differences expresser in the L7 line weresurvival observed rate(Figure between Wt and hERRγ-Tg mice of the low expresser L7 line were observed (Figure 2C). 2C). Int.Int. J. Mol. Sci. J. Mol. 2021,22, Sci.2021, 22, 8047 x FOR PEER REVIEW 4 of 14 4 of 14 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 14 Survival Figure2.2.Survival Figure estimates estimates of of Flag-hERRγ Flag-hERRγ transgenic transgenic lines. lines. Survival Survival of both of both malesmales and females from and females eacheach from of the three of the cardiac-specific three hERRγ-Tgmouse cardiac-specific hERRγ-Tg mouse transgenic transgenic lineslines was estimated was estimated by performing by perform- Figure ing 2. Survival estimates a Kaplan–Meier a Kaplan–Meier analysis of analysis forFlag-hERRγ for which which transgenic thethe log rank log testlines. rank was Survival test used. was of both L3 (A), used. males L6 (A), L3 (B), L6 and and females L7 (C); (B), 80 L7 and to (C); 80 to from each of the 202 animals perthree cardiac-specific transgenic hERRγ-Tg line and sex were usedmouse transgenic for the analysis.lines was estimated by perform- 202 animals per transgenic line and sex were used for the analysis. ing a Kaplan–Meier analysis for which the log rank test was used. L3 (A), L6 (B), and L7 (C); 80 to 202 2.2.animals per transgenic line Mice Overexpressing Highand sex were Levels usedExhibit of ERRγ for the Cardiac analysis.Functional Defects 2.2. Mice Overexpressing High Levels of ERRγ Exhibit Cardiac Functional Defects Because the overexpression of hERRγ in Tg mice was restricted to the heart, we in- 2.2. Mice Because the overexpression Overexpressing High Levels of of ERRγ hERRγ in Cardiac Exhibit Tg miceFunctional was restricted Defects to the heart, we inves- vestigated if the premature lethality observed was due to a cardiac defect. Echocardio- tigated Becauseif thethepremature overexpression lethality observed of hERRγ in Tgwasmice duewastorestricted a cardiactodefect. the heart, Echocardiographic we in- graphic analysis of male mice revealed significant morphological alterations in hERRγ-Tg analysis vestigated of if male the mice premature revealed lethality significant observed wasmorphological due to a cardiac mice of the L3 and L6 lines (Figure 3). Indeed, a substantial increase in the end-diastolic alterations defect. in hERRγ-Tg Echocardio- mice of graphicthe L3 and analysis L6 of lines male mice(Figure revealed3). Indeed, significant a substantial morphological left ventricular internal diameter (LVEDd) and end-systolic left ventricular internal diam-increase alterations in inthe end-diastolic hERRγ-Tg left ventricular mice of the L3 internal and L6 diameter lines (Figure (LVEDd) 3). Indeed, anda end-systolic substantial left increase eter (LVEDs) was observed in mice of lines L3 and L6 (Figure 3A,B), which show the high- ventricular in the internal end-diastolic diameter left est ventricular (LVEDs) levels ofwas internal observed Flag-hERRγ diameter in in themice(LVEDd) heart. and ofMoreover, lines L3end-systolic and leftdecrease L6 (Figure a significant ventricular 3A,B),in which theinternal diam- show interventric- the highest eter ular (LVEDs) levels was wallobserved of Flag-hERRγ septum thickness ininthemice (SWT) of heart. waslines L3 observed Moreover, only and L6a (Figure 3A,B), significant in mice which of decrease the L6 lineshow theinterventricular in(Figure the high- 3C), est levels of septum whereas no Flag-hERRγ wall thickness changes were in(SWT) the heart. observed wasin Moreover, only a significant observed the thickness ofinthe mice decrease in the of posterior heart the L6 line interventric- wall(Figure (PWT)3C), in whereas ular any no of septum wall the transgenic changes thickness (SWT) lines (Figure were observed was in 3D). only the The observed structural thickness in mice alterations of the of the L6 line observed inwall heart posterior (Figure L33C), lines(PWT) and in any of whereas L6 resultedno changes in an overt were observed in thefunction, thicknessasofevidenced the heart by posterior wall (PWT) in the transgenic lines decline (Figureof3D). cardiacThe structural alterations reduced observed leftin ventricu- lines L3 and L6 any lar of the transgenic ejectioninfraction lines (Figure (EF)decline 3D). and fractional The structural shortening alterations (FS)as(Figure observed 3E,F),by in tightly lines L3 and correlating resulted an overt of cardiac function, evidenced reduced left ventricular L6 resulted with in an overt the expression decline levels of cardiac of the Flag-hERRγ function, as evidenced protein. No evidence by reduced of structuralleft ventricu- or func- ejection lar ejection fraction fraction (EF) (EF) and andin fractional fractional shortening shortening (FS) (FS) the (Figure (Figure 3E,F), 3E,F), tightly tightly levels correlating correlating with tional alterations was found the L7 line, which show lowest expression of the the with expression the expression levels of the Flag-hERRγ protein. No evidence of structural or functional al- transgene among alllevels of the Flag-hERRγ the transgenic protein. No evidence of structural or func- lines generated. terations was found in the L7 line, which show the lowest tional alterations was found in the L7 line, which show the lowest expression levels of the expression levels of the transgene among all transgene the transgenic among all the transgeniclines generated. lines generated. Figure 3. Structural and functional alterations in hearts of hERRγ-Tg mice. Cardiac dimen- sions and function in 8–10-week-old hERRγ-Tg mice were assessed by transthoracic M-mode and two-dimensional echocardiography. (A) LVEDd: left ventricular end-diastolic internal diameter; (B) LVEDs: left ventricular end-systolic internal diameter; (C) SWT: interventricular septum wall thickness; (D) PWT: posterior wall thickness; (E), EF: ejection fraction; (F) FS: fractional shortening). Data are mean ± SEM of 3–4 animals/group; * p = 0.05; ** p = 0.01. Figure 3. Structural and functional alterations in hearts of hERRγ-Tg mice. Cardiac dimensions and function in 8–10-week-old hERRγ-Tg mice were assessed by transthoracic M-mode and two-dimen- sional echocardiography. (A) LVEDd: left ventricular end-diastolic internal diameter; (B) LVEDs: left ventricular end-systolic internal diameter; (C) SWT: interventricular septum wall thickness; (D) Int. J. Mol. Sci. 2021, 22, 8047 5 of 14 PWT: posterior wall thickness; (E), EF: ejection fraction; (F) FS: fractional shortening). Data are mean ± SEM of 3–4 animals/group; * p = 0.05; ** p = 0.01. Morphological inspectionofofhERRγ-Tg Morphological inspection hERRγ-Tgmice mice revealed revealed enlarged enlarged hearts hearts in those in those ani- animals mals from lines L3 and L6, but not in those of line L7. Histological analysis from lines L3 and L6, but not in those of line L7. Histological analysis of hearts confirmed of hearts con- firmed the echocardiographic the echocardiographic results. results. Indeed,Indeed, compared compared to their toWttheir Wt littermates, littermates, hERRγ-Tg hERRγ- mice Tg mice from line L3 exhibited enlarged ventricular cavities along with from line L3 exhibited enlarged ventricular cavities along with marked thinning of the marked thinning of the ventricular ventricular walls andwalls and interventricular interventricular septum,septum, indicativeindicative of pathological of pathological cardiac cardiac dilation dilation (Figure (Figure 4A). 4A). Although Although less dramatic, less dramatic, a similara similar phenotypephenotype was observed was observed in histo- in histological logical preparations of hERRγ-Tg mice from line L6. In contrast, preparations of hERRγ-Tg mice from line L6. In contrast, in agreement with the low in agreement with the low expression levels of Flag-hERRγ and the echocardiographic results, no expression levels of Flag-hERRγ and the echocardiographic results, no structural differences structural dif- ferences were observed were observed between hERRγ-Tg between hERRγ-Tg mice and mice and Wt littermates Wt littermates of the line ofL7.the line L7. Con- Consistent with sistent with the functional decline observed, a dramatic increase in the expression the functional decline observed, a dramatic increase in the expression of atrial natriuretic of atrial natriuretic peptide (Nppa) peptide was (Nppa) detected was detected in hearts in hearts ofmice of transgenic transgenic from the mice from L3 and L6the L3 but lines, andnot L6 lines, the L7butlinenot the L74B). (Figure line (Figure 4B). Figure 4. Morphological abnormalities in hearts of hERRγ-Tg mice. (A) Histological sections of Figure 4. Morphological abnormalities in hearts of hERRγ-Tg mice. (A) Histological sections of hERRγ-Tg mouse lines L3, L6, and L7 stained with hematoxilin/eosin. (B) mRNA expression of hERRγ-Tg mouse lines L3, L6, and L7 stained with hematoxilin/eosin. (B) mRNA expression of Nppa Nppa was determined by real-time quantitative PCR in cardiac tissue from hERRγ-Tg mice. Data are was determined by real-time quantitative PCR in cardiac tissue from hERRγ-Tg mice. Data are mean mean ± SEM of 3–4 animals/group; * p = 0.05; ** p = 0.01. ± SEM of 3–4 animals/group; * p = 0.05; ** p = 0.01. 2.3. ERRγ Cardiac Overexpression Is Associated with Cardiomyocyte Hypertrophy, Apoptosis, 2.3. ERRγ Fibrosis, Cardiac and Overexpression Altered Is Associated with Cardiomyocyte Hypertrophy, Apoptosis, Gene Expression Fibrosis, and Altered Gene Expression To investigate the cellular effects of ERRγ overexpression potentially involved in To investigate cardiac dysfunction, the wecellular focusedeffects of ERRγ on line overexpression L6, which potentially shows intermediate involved levels of theinFlag- car- diac dysfunction, we focused on line L6, which shows intermediate levels hERRγ protein but still develops an overt cardiac phenotype. Morphometric analysis of the Flag- hERRγ revealedprotein but still an increase in develops an overt cardiac the cross-sectional area ofphenotype. ventricularMorphometric cardiomyocytes analysis from 7re-to vealed an increase 10-week-old in the mice, hERRγ-Tg cross-sectional comparedarea of ventricular to their cardiomyocytes Wt littermates (Figure 5A).from 7– to 10- In addition, apoptosis was increased in hearts of hERRγ-Tg mice, as revealed by the abundance of active caspase-3 positive cells (Figure 5B). Moreover, cardiac dysfunction in hERRγ-Tg mice was also associated with increased fibrosis, as demonstrated by sirius red staining of histological sections of hearts (Figure 5C). week-old hERRγ-Tg mice, compared to their Wt littermates (Figure 5A). In addition, apoptosis was increased in hearts of hERRγ-Tg mice, as revealed by the abundance of active caspase-3 positive cells (Figure 5B). Moreover, cardiac dysfunction in hERRγ-Tg Int. J. Mol. Sci. 2021, 22, 8047 mice was also associated with increased fibrosis, as demonstrated by sirius red staining 6 of 14 of histological sections of hearts (Figure 5C). Figure 5. Cellular alterations in hearts of L6 hERRγ-Tg mice. (A) Cardiomyocyte cross-sectional Figure area 5. Cellular (CSA) wasalterations determinedin hearts using of L6ImageJ the hERRγ-Tg mice. (A) software afterCardiomyocyte staining heartcross-sectional sections with area wheat (CSA) was determined using the ImageJ software after staining heart sections with wheat germ agglutinin (WGA) conjugated with FITC. (B) Apoptosis was estimated by immunodetection germ ag- glutinin (WGA) conjugated with FITC. (B) Apoptosis was estimated by immunodetection with with active caspase-3 (red). (C) Cardiac fibrosis was detected with the sirius red staining. Data ac-are tive caspase-3 (red). (C) Cardiac fibrosis was detected with the sirius red staining. Data are mean ± mean ± SEM of 3–4 animals/group; * p = 0.05. SEM of 3–4 animals/group; * p = 0.05. ERRγ has been shown to regulate the expression of genes related to oxidative metabolism, ERRγ hasthose particularly been related shown to to mitochondrial regulate the expression biogenesisofand genes relatedastowell function, oxidative as genesmetab- related olism, particularly those related to mitochondrial biogenesis and function, to the contractile function of cardiac cells. Therefore, expression of ERRγ target genes as well as genes was related to the assessed contractile in hearts function of young of cardiac hERRγ-Tg and Wt cells. miceTherefore, from lineexpression of ERRγ L6. Surprisingly, target expression genes was assessed in of mitochondrial hearts genes of young related to FAOhERRγ-Tg and Wt (i.e., Acadvl, miceehhadh Acaa2, from line and L6. Surprisingly, Cpt1b) (Figure 6A) expression of mitochondrial genes related to FAO (i.e., Acadvl, and the OxPhos system (i.e., Atp5b, Cox4i1, Cox7a1 and Ndufa4) (Figure 6B) were Acaa2, ehhadh and Cpt1b) severely (Figure 6A) and the downregulated in OxPhos hearts ofsystem hERRγ-Tg(i.e., mice. Atp5b,Similarly, Cox4i1, Cox7a1 genes and Ndufa4) related (Figure to other 6B)of aspects were lipid metabolism (i.e., Ppara and Fabp3) were also decreased (Figure 6A). Furthermore,towe severely downregulated in hearts of hERRγ-Tg mice. Similarly, genes related other aspects observed anofexacerbated lipid metabolism increase(i.e., Ppara in the and Fabp3) expression wereencoding of genes also decreased for the(Figure 6A). fetal isoforms Furthermore, we observed an exacerbated increase in the expression of of myosin heavy chain (Myh7) and actin gene (Acta1), whereas adult isoforms of contractile genes encoding forproteins, the fetalsuch isoforms of myosin as titin heavy (Ttn), were chain (Myh7) (Figure downregulated and actin gene (Acta1), whereas adult 6C). isoforms of contractile proteins, such as titin (Ttn), were downregulated (Figure 6C). Int.J. J.Mol. Int. Mol.Sci. Sci. 22,22, 2021, 2021, 8047 PEER REVIEW x FOR 7 7ofof1414 ExpressionofofERRγ Figure6.6.Expression Figure ERRγtarget targetgenes genesininadult adultL6L6hERRγ-Tg hERRγ-Tgmice. mice.mRNA mRNAexpression expressionofofgenes genes related relatedtotolipid lipidmetabolism metabolism(A), (A),OxPhos OxPhossystem system(B), (B),and andsarcomeric sarcomericproteins proteins(C) (C)was wasdetermined determinedbyby real-time real-timequantitative quantitativePCR. PCR.Data Dataare aremean mean± ±SEMSEMof of 3–4 3–4animals/group; animals/group;* p*=p0.05; ** p**=p0.01. = 0.05; = 0.01. InInorder ordertotoshed shedsomesomelight lightononwhether whetherthe thegene geneexpression expressionprofile profileobserved observedininadultadult L6L6hERRγ-Tg hERRγ-Tgmice mice is the the direct directresult resultofofERRγERRγ overexpression overexpression or simply or simply secondary secondary to heart to failure, heart we analyzed failure, we analyzed expression expression of ERRγ-target of ERRγ-target genes in younger genes in younger animals. Interestingly, animals. Interest- at 3 weeks ingly, of age, at 3 weeks hERRγ-Tg of age, hERRγ-Tgmicemice show expression show expressionof OxPhos of OxPhos and andFAOFAO genes similar genes sim-to that ilar to of Wtofmice that (Figure Wt mice 7A).7A). (Figure Still,Still, at this age, at this hERRγ-Tg age, hERRγ-Tg micemice exhibit exhibit very veryhigh highlevels levelsof ofNppa Nppaand andMyh7, Myh7, indicating indicating somesome degree degree of cardiac of cardiac functional functional alteration. On the alteration. Onother hand, the other hearts of 1-day-old mice show a gene expression profile that is, at hand, hearts of 1-day-old mice show a gene expression profile that is, at least in part, com- least in part, compatible with increased patible ERRγ ERRγ with increased transcriptional transcriptionalactivity. Indeed, activity. expression Indeed, expressionof some bona fide of some bonaERRγfide targettarget ERRγ genes,genes, such as those such involved as those in lipidinmetabolism involved lipid metabolism(i.e., Fabp3, (i.e., Acadm) and OxPhos Fabp3, Acadm) and (i.e., Cox6b2), OxPhos appearsappears (i.e., Cox6b2), mildly induced in heartsinofhearts mildly induced hERRγ-Tγ mice, although of hERRγ-Tγ differences mice, although dif-do not reach statistical significance (Figure 7B). Moreover, hearts ferences do not reach statistical significance (Figure 7B). Moreover, hearts from 1-day-old from 1-day-old mice show higher mice showlevels of Myl7 higher (myosin levels of Myl7 light(myosin chain 7)light mRNA, chainwhich is expressed 7) mRNA, which in is hearts of healthy expressed in individuals, whereas the expression of Nppa is almost the same hearts of healthy individuals, whereas the expression of Nppa is almost the same as in Wt as in Wt mice. Altogether, theseAltogether, mice. results suggest these that at early results suggest developmental stages, ERRγ overexpression that at early developmental stages, ERRγ overex-induces a gene expression profile characterized by the induction of pression induces a gene expression profile characterized by the induction of genes in- genes involved in oxidative metabolism. volved However, in oxidative as hERRγ-Tg metabolism. However, mice asgethERRγ-Tg older, heart failure mice develops get older, heartand leadsde- failure to a velops and leads to a reduction in the expression of mitochondrial OxPhos and FAOit reduction in the expression of mitochondrial OxPhos and FAO genes, at the same time as altersat genes, thetheproper same expression of genes time as it alters encoding the proper for contractile expression of genes proteins. encoding for contractile proteins. Int.J.J.Mol. Int. Mol.Sci. Sci.2021, 22,x8047 2021,22, FOR PEER REVIEW 88of of14 14 Figure 7. Expression of ERRγ target genes in young L6 hERRγ-Tg mice. mRNA expression of Figure 7. Expression of ERRγ target genes in young L6 hERRγ-Tg mice. mRNA expression of genes genes related related to sarcomeric to sarcomeric proteins, proteins, OxPhosOxPhos system,system, and and lipid lipid metabolism, metabolism, as well as as well Nppa, Nppa,aswas was deter- determined mined by real-time by real-time quantitative quantitative PCR inPCR in 3-week-old 3-week-old (A) 1-day-old (A) and and 1-day-old (B) hERRγ-Tg (B) hERRγ-Tg micemice and and Wt Wt littermates. littermates. DataData are mean are mean ± SEM± SEM of 3–5ofanimals/group; 3–5 animals/group; ** p = ** p = 0.01. 0.01. 3. Discussion 3. Discussion The results obtained in our study indicate that sustained cardiac overexpression of The results obtained in our study indicate that sustained cardiac overexpression of ERRγ is sufficient to induce a severe cardiomyopathic phenotype that leads to lethality in ERRγ is sufficient to induce a severe cardiomyopathic phenotype that leads to lethality in young transgenic mice. Several studies have demonstrated that members of the ERR family young transgenic mice. Several studies have demonstrated that members of the ERR fam- of orphan nuclear receptors play an important role in the heart, being necessary for proper ily of orphan nuclear receptors play an important role in the heart, being necessary for cardiac maturation and functional adaptation to work overload by regulating the expression proper cardiac maturation and functional adaptation to work overload by regulating the of genetic programs related to energy metabolism and contraction [15–17]. In this sense, expression of genetic programs related to energy metabolism and contraction [15–17]. In whole-body deletion of ERRγ results in premature death within 48 h after birth [16]. this sense, whole-body deletion of ERRγ results in premature death within 48 h after birth These mice exhibit an abnormal ventricular depolarization and delayed cardiomyocyte [16]. These mice exhibit an abnormal ventricular depolarization and delayed cardiomyo- repolarization, in part due to reduced expression of the sodium channel SCN5A. In addition cyte repolarization, in part due to reduced expression of the sodium channel SCN5A. In to altered current conduction, ERRγ null mice also show a decrease in the expression of addition to altered mitochondrial current genes, conduction, particularly ERRγ of genes null mice related to thealso showsystem. OxPhos a decrease in the ex- Therefore, the pression of mitochondrial genes, particularly of genes related to the limited ATP production resulting from impaired mitochondrial function may have also OxPhos system. Therefore, contributed the tolimited ATPdysfunction the cardiac production observed resulting infrom nullimpaired neonates mitochondrial [16]. However,function whether may have also contributed to the cardiac dysfunction observed the neonatal lethality of whole-body ERRγ knockout mice is the direct in null neonates result of[16]. the How- severe ever, whether cardiac the neonatal alterations observed lethality ofmice in these whole-body has beenERRγ knockout questioned by mice is the studies usingdirect re- rodent sult of the severe cardiac alterations observed in these mice has been questioned models with specific deletion of the Esrrg gene in the heart. Indeed, cardiac-specific ERRγ by stud- ies using rodent knockout mice, models despitewith specific showing deletion of a decrease in the the Esrrg gene inofthe expression heart. Indeed,genes, mitochondrial car- diac-specific exhibit normal ERRγ knockout survival ratesmice, and despite no grossshowing cardiac aalterations, decrease inindicating the expression of mito- that postnatal chondrial cardiac lossgenes, exhibit of ERRγ normal does survival not bring rates about and nophenotype a lethal gross cardiac [18].alterations, In contrast indicating to ERRγ, that ERRα postnatal seems tocardiac loss of ERRγ be dispensable does not for proper bring cardiac about a lethal maturation, and phenotype ERRα null mice [18]. develop In con- trast to ERRγ, ERRα seems to be dispensable for proper cardiac maturation, normally, without any apparent cardiac defect [19]. Nevertheless, loss of ERRα accelerates and ERRα null mice develop normally, without any apparent cardiac defect [19]. Nevertheless, the functional and energetic defects associated with increased pressure overload of adult loss of ERRα mice accelerates [15], pointing the functional towards and energetic a relevant defects role of ERRα in associated with increased cardiac adaptation pres- to increased Int. J. Mol. Sci. 2021, 22, 8047 9 of 14 workload. Altogether, these findings rather suggest a functional redundancy between ERRα and ERRγ. Such functional compensation is in agreement with the high degree of overlap existing between the target genes of both ERRs [20] and recent studies showing that simultaneous postnatal loss of ERRα and ERRγ in the heart induce severe alterations in cardiac function, tightly associated with altered expression of mitochondrial genes, reduced mitochondrial biogenesis, and altered expression of contractile proteins [17,18]. Considering the deleterious effects described as a result of cardiac loss of ERRγ, one would expect that overexpression of ERRγ in the heart should either cause no harm to mice, or even improve cardiac metabolism by increasing the expression of genes encoding for mitochondrial or adult contractile proteins. To our surprise, sustained cardiac overexpres- sion of ERRγ led to a severe cardiomyopathic phenotype characterized by severe structural and metabolic derangements, including reduced expression of mitochondrial genes and increased expression of the fetal forms of contractile proteins. Considering that genes encoding for mitochondrial and adult sarcomeric proteins are well-known targets of ERRγ, the suppressed expression of these genes seems to be secondary to the development of cardiomyopathy, rather than the direct regulatory effect of ERRγ on gene expression. In this regard, it is noteworthy to remark that the pathophysiology of heart failure is characterized by a metabolic remodeling distinguished by a depression of energy metabolism, in particu- lar, reduced FAO and OxPhos gene expression and impaired mitochondrial biogenesis [1], similar to what occurs in our hERRγ-Tg model. Also supporting the notion that reduced expression of genes related to oxidative metabolism in adult hERRγ-Tg mice is secondary to the progression of heart failure, the gene expression profile in younger transgenic mice is compatible with an expression profile resulting from increased ERRγ transcriptional activity, which includes increased mRNA levels of genes involved in mitochondrial ox- idative phosphorylation, lipid metabolism, and postnatal contractile machinery. Still, we cannot absolutely rule out the possibility that the decrease in the expression of ERRγ bona fide targets observed in adult hERRγ-Tg mice was the result of co-activator squelching due to the very high levels of Flag-hERRγ protein. The hERRγ-Tg mice displayed ventricular dilation and heart failure, demonstrating that sustained increase in the levels of ERRγ protein is sufficient to induce cardiac dys- function. In support of this hypothesis, we have recently reported that heart dysfunction in obese diabetic db/db mice is characterized by a gene expression profile featuring in- creased expression of OxPhos, FAO, and lipid metabolism genes, as well as alterations in the expression of contractile proteins, including an increase in the expression of fetal isoforms of sarcomeric proteins [21]. Interestingly, a computational analysis conducted in order to find potential transcriptional regulators responsible for this expression profile found ERRγ as a major regulatory node [21]. The capacity of ERRγ to drive the expression profile and metabolic derangements found in hearts of db/db mice was demonstrated in cultured neonatal cardiomyocytes, in which adenoviral-mediated overexpression of a Flag-hERRγ chimeric protein was sufficient to induce cardiomyocyte hypertrophy, increase mitochondrial gene expression, and enhance FAO [21]. Similar to our studies, Kwon and collaborators reported that ERRγ overexpression in cultured cardiomyocytes leads to cellu- lar hypertrophy in a Gata-4-dependent manner [22]. Importantly, this study also reported that patients with hypertrophic cardiomyopathy exhibit high content of ERRγ protein in the heart [22], supporting the notion that elevated levels of ERRγ could be associated with cardiac disease also in humans. ERRγ is not the only transcriptional regulator that plays a dual role in cardiac patho- physiology. Indeed, PPARα has been shown to have a protective role by maintaining normal cardiac function under basal conditions and preventing hypertrophic growth in response to pressure overload [12,13]. However, elevated cardiac levels of PPARα have been found in a pathological context, such as in diabetic individuals [23–25]. It has been suggested that elevated levels of PPARα in diabetic hearts contribute to the development of metabolic and functional alterations that characterize diabetic cardiomyopathy by pro- moting cardiac steatosis and lipotoxicity through the activation of the expression of genes Int. J. Mol. Sci. 2021, 22, 8047 10 of 14 related to fatty acid uptake and oxidation. The pathogenic activity of PPARα has been unequivocally demonstrated in transgenic mice with cardiac-specific overexpression of PPARα, which, similar to what we have described for ERRγ, develop ventricular hy- pertrophy and systolic dysfunction [24,25] and exhibit delayed postischemic recovery of cardiac function [26]. Analogously, chronic treatment with PPARα agonists impairs heart function, at least in part, by attenuating cardiomyocyte respiratory capacity [27]. A dual, either protective or pathogenic, role has also been described for PGC-1α, a transcriptional co-activator of many nuclear receptors, including PPARα and ERRγ [28]. Although mice lacking PGC-1α do not exhibit any gross cardiac morphological or functional alteration under basal non-stimulated conditions [5], when subjected to increase workload by trans- verse aortic constriction, these mice develop significant heart hypertrophy and accelerated heart failure [8]. However, cardiac overexpression of PGC-1α, far from improving cardiac function, has been shown to lead to severe myocardiopathy and early lethality [29–31]. Similar to PGC-1α, cardiac deficiency of PGC-1β leads to heart hypertrophy and functional failure only when mice are subjected to pressure overload [9]. As for ERRs, the redundant role of PGC-1α and PGC-1β in controlling mitochondria biogenesis in cardiomyocytes could have contributed to the absence of a cardiac phenotype under basal conditions. This notion is supported by the early lethality resulting from the severely impaired contractile function exhibited by mice with simultaneous deficiency of both PGC-1α and PGC-1β in the heart [32]. The effects of sustained overexpression of PGC-1β in the heart have not been yet elucidated. The contribution of ERRγ to human cardiac development, function, or pathogenesis remains to be precisely defined. Whereas a study reported that the expression of ERRγ, as well as that of ERRα and PGC-1α, is decreased in cardiac tissue of patients with heart failure [33], another described an increase in ERRγ protein in patients with cardiac hy- pertrophy [22]. These apparently contradictory data are somehow in line with the data obtained from genetically engineered mouse models, in which reduced expression of ERRγ accelerated the progression of cardiac dysfunction during embryonic development or in response to increased cardiac overload [16–18], whereas, as demonstrated in the present study, sustained overexpression induces severe heart failure. In view of these findings, the use of drugs that modulate ERRγ activity for the treatment of cardiac diseases in humans seems plausible. In support of this notion, it has been shown that treatment of cultured cardiomyocytes with the ERRγ inverse agonist GSK-5182 prevents hypertrophy induced by phenylephrine [22]. Still, the therapeutic use of ERRγ modulators in humans—and ani- mal models—is hampered by their limited bioavailability and specificity. Additionally, an important aspect that needs to be taken into account when considering the use of ERRγ syn- thetic ligands as therapeutic drugs is to limit their use within a defined therapeutic window, since both sustained activation or inhibition of ERRγ could lead to cardiac dysfunction. In summary, despite the crucial role of ERRγ in cardiac development and functional adaption to increased workload, our study shows that sustained elevated levels of ERRγ, far from improving cardiac function, have a detrimental effect on the heart, promoting the development of cardiomyopathy and heart failure. Our results, together with evidence provided by other studies, point towards the axis PGC-1/ERR/PPARα as a crucial regula- tory circuit whose fine-tuning is essential for proper cardiac function. Alterations of this regulatory axis that result in either a sustained reduction or increase in the transcriptional activity of any of its components can lead to cardiac dysfunction. Taken together, these studies indicate that pharmacological modulation of the PGC-1/ERR/PPARα should be carefully considered, since either sustained activation or inhibition may lead to undesirable harmful cardiac effects. 4. Materials and Methods 4.1. Generation of Cardiac-Specific ERRγ Transgenic Mice To generate transgenic mice with cardiac overexpression of ERRγ, the coding sequence of human ERRγ (hERRγ) containing two Flag epitopes in the 50 region were amplified Int. J. Mol. Sci. 2021, 22, 8047 11 of 14 by PCR using a pcDNA3.1-2xFlag-hERRγ vector as a template (described in [21]) and primers 50 -ACTGTCGACGCCACCATGGATTACAAGGATGACGACGAT-30 (forward) and 50 -ACTGTCGACTTAGCAGACCTTGGCCTCAAACATTTC-30 (reverse). The resulting fragment was digested with SalI restriction enzyme and cloned into the SalI site of the pBSII-SK-αMHC that contains a 5.7 kb of mouse genomic DNA comprising the 30 end of the β-cardiac MHC gene, the intergenic region, and the 50 end of the α-cardiac MHC gene, ensuring cardiac-specific expression of the 2xFlag-hERRγ cDNA [34]. A ≈7.4 kb BamHI digestion fragment was obtained, purified, and microinjected into the pronuclei of fertilized mouse eggs (C57BL/6J mice). Embryos were implanted into pseudo-pregnant foster females at the Mouse Mutant Core Facility of the Institute for Research in Biomedicine (IRB, Barcelona, Spain). Transgenic mice were identified by PCR. For this, DNA was purified from tail clips and used to amplify a 537 bp fragment of the transgene using the following primers: 50 - GCAGGGAAGTGGTGGTGTAGG-30 (forward) and 50 -ACCCAGAAGCGATGTCACCAC- 30 (reverse). Mice were housed in a temperature-controlled environment at 21 ◦ C, subjected to a 12 h/12 h light/dark cycle and fed a standard diet (2018 Tecklad Global 18% Protein Rodent Diet, 18 Kcal from fat, Harlan Laboratories). 4.2. Gene Expression Analysis For gene expression analysis, RNA was isolated from heart or other tissues with Trizol (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. cDNA was synthesized from 400 ng of RNA with SuperScript II reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) and oligo dT primers. Gene expression was assessed by real-time quantitative PCR (qPCR) using gene-specific primers and SYBR green (Thermo Fisher Scientific, Waltham, MA, USA) in an ABI PRISM 7500 Sequence Detection System (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA expression was calculated according to the 2−∆∆CT threshold cycle method, using cyclophilin A as a reference gene, as previously described [35]. The tissue-specific expression of the 2xFlag-hERRγ transgene was achieved by conven- tional PCR using cDNA obtained from total RNA by reverse transcription as a template and specific primers 50 -GGTTCAGCCAGCCAAAAAGCC-30 (forward) and 50 -CAGGGACAGCGTGGAGAAGCC-30 (reverse) that allow the detection of hERRγ. The resulting 204 bp amplicon was resolved in a 2% agarose gel. 4.3. Western Blot Protein extracts from hearts and other tissues were prepared in homogenization buffer (25 mM Tris-HCl pH = 7.5, 150 mM NaCl, 1% sodium deoxycholate, 1% Nonidet P-40, 0.1% Triton X-100, 1% SDS, 10 mM NaF, 1 mM Na3 VO4 ) supplemented with protease inhibitors. Forty µg of proteins were resolved by 12% SDS-PAGE, transferred to a PVDF membrane, and probed with specific antibodies against Flag epitope (Merck, Darmstat, Germany) or α-tubulin (Cell Signaling Technology, Danvers, MA, USA). 4.4. Histological Analysis For histological analysis, the hearts were rapidly removed, washed in saline, and fixed in formalin overnight. After sequential dehydration in ethanol solutions of increasing concentrations, whole hearts were embedded in paraffin. Then, 5–8 µm heart sections were obtained and stained with hematoxylin/eosin for gross morphological examination. To detect cardiac fibrosis, heart sections were stained with sirius red, which specifically stains collagen fibers. To measure the cardiomyocyte cross-sectional area, paraffin sections of hearts were stained with wheat germ agglutinin conjugated with TRITC and DAPI. Pictures of three different fields of each heart were randomly taken (n = 3 mice/group), and the area of at least 900 cells/heart was measured with the ImageJ Software. Int. J. Mol. Sci. 2021, 22, 8047 12 of 14 4.5. Apoptosis Cell apoptosis was determined in frozen sections of mouse hearts by immunohisto- chemistry, as previously described [21]. Briefly, after dissection, hearts were rapidly frozen in OCT compound, and 8–10 µm sections were obtained in a Leica CM-3050-S Cryostat (Leica Microsystems, Wetzlar, Germany). To detect apoptotic cells, sections were first incubated with a rabbit polyclonal antibody against active caspase-3 (Abcam, Cambridge, UK), and subsequently incubated with an anti-rabbit goat secondary antibody conjugated with the Alexa Fluor 594 dye (Abcam, Cambridge, UK) and DAPI. To quantify the number of apoptotic cells, 8–10 fields from each heart were randomly taken (at least 800 cells/heart, n = 3 mice/group), and total and apoptotic cells counted. 4.6. Echocardiography Cardiac function and dimensions were assessed by transthoracic M-mode and two- dimensional echocardiography in mice under light anesthesia (0.5–1% isofluorane) using a Vivid Q portable ultrasound system with an ILS 12 MHz transducer (GE Healthcare, Bosston, MA, USA). M-mode images of four different cardiac cycles from each animal were analyzed to assess cardiac function. The ejection fraction (EF), left ventricular end-diastolic internal diameter (LVEDd), left ventricular end-systolic internal diameter (LVEDs), inter- ventricular septum wall thickness (SWT), posterior wall thickness (PWT), and fractional shortening (FS) were blindly measured. 4.7. Statistical Analysis Results are expressed as mean ± SEM. The statistical significance of differences between experimental groups was assessed using an unpaired Student’s t-test or a one-way analysis of the variance (ANOVA) followed by Tukey post hoc test. For the Kaplan–Meier survival analysis, the log rank test was used. Differences were considered significant when p < 0.05. Author Contributions: J.L., R.P., M.V., M.P., N.S. and M.Z. performed research and analyzed data; R.S., M.R.-M. and M.Z. reviewed and edited the drafted manuscript; M.Z. and J.A.V. supervised the project, analyzed data, and wrote the manuscript; J.A.V. and M.Z. conceived the study and acquired funding. All authors have read and agreed to the published version of the manuscript. Funding: Research was funded by “Fundació la Marató de TV3” (# 082610 to JAV) and by Instituto de Salud Carlos III (PI19/00167 to MZ, co-funded by European Regional Development Fund -FEDER- “A way to make Europe”). Institutional Review Board Statement: All animal procedures were performed in accordance with the EU Directive 2010/63/EU for animal experimentation and the Institutional Guidelines for the Ethical Care and Treatment of Animals, and were approved by the Animal Experimentation and Ethics Committee of the Vall d’Hebron Research Institute (12/11, 66/17, 68/16 CEEA). Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: The authors are thankful to J. Robbins (Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA) for providing the vector containing the α-MHC promoter. Conflicts of Interest: The authors declare no conflict of interest. References 1. Lopaschuk, G.D.; Jaswal, J.S. Energy Metabolic Phenotype of the Cardiomyocyte during Development, Differentiation, and Postnatal Maturation. J. Cardiovasc. Pharmacol. 2010, 56, 130–140. [CrossRef] [PubMed] 2. Piquereau, J.; Novotova, M.; Fortin, D.; Garnier, A.; Ventura-Clapier, R.; Veksler, V.; Joubert, F. Postnatal Development of Mouse Heart: Formation of Energetic Microdomains: Energetic Maturation of Postnatal Mouse Heart. J. Physiol. 2010, 588, 2443–2454. [CrossRef] Int. J. Mol. Sci. 2021, 22, 8047 13 of 14 3. 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