Deletion of hmox1a increases cardiac output in larvae. (A) Scheme of CRISPR/Cas9‐generated 52‐base pair deletion in exon 3 of hmox1a. (B) RT‐qPCR analysis of hmox1a, hmox1b, hmox2a and hmox2b in wild type (WT), heterozygous (HET) and homozygous (KO) hmox1a mutant at 5–6 dpf. The results are presented as fold change compared to WT. RNA was extracted from 5 to 10 pooled larvae. The number of RNA samples used for RT‐qPCR analyses as following: WT n = 5; HET(52del) n = 6; KO(52del) n = 6. (C) Representative images of hmox1a WT, HET(52del) and KO(52del) larvae at 5 dpf showing no obvious gross phenotype. Scale bar: 500 μm. (D–H) Analysis of cardiac function at 5–6 dpf larvae indicating an increase in cardiac output (D) and heart rate (E), and no significant change in stroke volume (F), ejection fraction (G) and end‐diastolic ventricular area normalized to body length (H) in hmox1a KO(52del). WT n = 28; HET(52del) n = 43; KO(52del) n = 30. Data are presented as mean ± SD. One‐way ANOVA with Tukey adjustment for multiple comparisons. *p < 0.05.

Deletion of hmox1a provokes cardiac output and hypertrophy in adults. (A–D) Echocardiography analysis depicting elevated cardiac output in KO(52del) zebrafish compared to their HET sibling (A) with a tendency toward increase in stroke volume (B) and no significant change in heart rate (C) and ejection fraction (D). (E) KO(52del) zebrafish exhibits enlarged end‐diastolic area compared to HET siblings. (F–H) PWD analysis indicating increased peak E wave velocity (F) and E/A ratio (H) without significant change on Peak A wave velocity (G). (I) Deceleration times of E wave from maximum velocity to baseline display no significant difference between the three genotypic groups. (J) KO(52del) zebrafish are lean in comparison with their HET siblings. (K) ZnPPIX treatment leads to enlarged end‐diastolic area in WT zebrafish at 13 dpi. Zebrafish treated with saline display no change at 13 dpi. (L) ZnPPIX treatment induces a tendency toward increase in cardiac output in WT zebrafish at 13 dpi. Zebrafish treated with saline display no change at 13 dpi. (A–J) WT n = 18; HET(52del) n = 25; KO(52del) n = 21. K, L ZnPPIX n = 10; Control n = 10. Data are presented as mean ± SD. One‐way ANOVA with Tukey adjustment for multiple comparisons in A–J. Two‐sample t‐test in K, L. *p < 0.05, **p < 0.01, ***p < 0.001.

Analyses of myocardial interstitial fibrosis, cardiac OXPHOS gene expression and mitochondrial respiration in adult cardiomyocytes. (A) Representative images of acid fuchsin orange G (AFOG) staining of ventricular sections showing accumulation of collagen (blue) in the myocardium (orange). Arrowhead indicates accumulated collagen. (B) Quantification of AFOG staining indicating increased collagen accumulation in the myocardium in KO(52del). WT n = 6; HET(52del) n = 9; KO(52del) n = 7. (C) Representative images of immunohistochemical staining of ventricular sections for Collagen type I (brown). Arrowhead indicates Collagen type I‐positive signal. (D) Quantification of Collagen type I positive area relative to myocardium. WT n = 3; HET(52del) n = 3; KO(52del) n = 3. (E) RT‐qPCR analyses showing downregulation of OXPHOS complex II subunit sdhb in KO(52del) hearts compared to WT controls. sdhb, succinate dehydrogenase iron–sulfur subunit B; The graphs represent the quantification of two individual analyses of RNA extracts from pooled samples of two‐three hearts. Each analysis includes three replicates. WT n = 19; HET(52del) n = 19; KO(52del) n = 19. (F) Representative oxygen consumption (OCR) profile in zebrafish primary cardiomyocytes at basal respiration and after addition of oligomycin (Oligo.), carbonyl cyanide‐4 (trifluoromethoxy) phenylhydrazone (FCCP), followed by a combination of rotenone and antimycin A (Rot./AA). (G) Representative extracellular acidification (ECAR) profile in zebrafish primary cardiomyocytes at basal respiration and after addition of Oligo., FCCP and Rot./AA. (H) Cardiomyocytes from HET(52del) display a decline in the rate of respiration to drive mitochondrial ATP synthesis. (I) Cardiomyocytes from HET(52del) exhibit an increase in ECAR relative to the basal rate upon the addition of Rot./AA. (J) HET(52del) cardiomyocytes show increased non‐mitochondrial respiration. (K) HET(52del) cardiomyocytes show increased spare respiration capacity. (L) HET(52del) cardiomyocytes display increased ECAR relative to the basal rate upon the addition of FCCP. (M) Spare respiration capacity of HET(52del) and WT cardiomyocytes in response to ISO. (N) ECAR relative to the basal rate upon the addition of FCCP in HET(52del) and WT cardiomyocytes in response to ISO. (F–L) WT n = 15; HET(52del) n = 8; KO(52del) n = 7. M, N HET(52del) n = 15; WT n = 15. Data are presented as mean ± SD. One‐way ANOVA with Tukey adjustment for multiple comparisons in B, D, E and H–L. Two‐sample t‐test in M, N. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars: 20 μm.

Deletion of hmox1a restrains cardiomyocyte proliferation and downregulates antioxidative genes and haemoglobin genes in adult zebrafish. (A) Representative immunohistochemical staining of Pcna (in green) and Mef2 (in meganne) in ventricular sections. Arrow indicates Pcna⁺ non‐cardiomyocytes. Arrowhead indicates Pcna⁺ cardiomyocytes. Pcna, proliferating cell nuclear antigen; Mef2, myocyte‐specific enhancer factor 2. (B) Quantification of Pcna⁺ cardiomyocytes relative to total cardiomyocytes. (C) Quantification of Pcna⁺ cardiac cells per mm² ventricular area. Three hearts from each genotypic group were selected. Two sections from each heart were stained. The number of stain‐positive signals for Pcna and Mef2 from ventricular areas was quantified with ImageJ. (D–G) RT‐qPCR analyses indicating downregulation of antioxidative genes, txnrd3 (D) and sod2 (E), and the transcription regulators of antioxidative and antihypertrophic signalling, foxo3a (F) and sirt3 (G), in KO(52del) hearts. txnrd3, thioredoxin reductase 3; sod2, superoxide dismutase 2; foxo3a, forkhead box O3a; sirt3, sirtuin 3. The graphs represent the quantification of two individual analyses of RNA extracts from pooled samples of two‐three hearts. Each analysis includes three replicates. WT n = 19; HET(52del) n = 19; KO(52del) n = 19. (H–J) RT‐qPCR analyses showing downregulation of erythropoietin receptor epor (H), and adult haemoglobin genes hbαa1 (I) and hbβa1 (J) in kidney lacking hmox1a. epor, erythropoietin receptor; hbαa1, haemoglobin alpha adult‐1; hbβa1, haemoglobin beta adult‐1. The graphs represent the quantification of two individual analyses of RNA extracts from pooled samples of two kidneys. Each analysis includes three replicates. WT n = 10; HET(52del) n = 10; KO(52del) n = 10. Data are presented as mean ± SD. One‐way ANOVA with Tukey adjustment for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars: 20 μm.

Hypoxia enhances cardiac output in HET(52del) larvae. (A) Representative immunoblots of β‐tubulin and Gapdh in WT, HET(52del), and KO(52del) at normoxic and hypoxic (3% O₂ for 24 h) conditions. Gapdh serves as an internal control. Norm. normoxia; Hypox. Hypoxia. (B) RT‐qPCR analyses indicate that hypoxia induces upregulation of epoa and hmox1a, but not hmox1b, in WT larvae. (C–G) Hypoxia enhances ejection fraction (C) in WT and HET(52del) larvae, and cardiac output (D) and stroke volume (E) in HET(52del) larvae, but not in KO(52del), compared to normoxia. Hypoxia has no effect on heart rate (F) or diastolic area (G) in the three genotypic groups. WT, normoxia n = 15, hypoxia n = 9; HET(52del), normoxia n = 19, hypoxia n = 10; KO(52del), normoxia n = 16, hypoxia n = 10. Data are presented as mean ± SD. Two‐sample t‐test. *p < 0.05, **p < 0.01, ***p < 0.001.

ISO deteriorates cardiac function in HET(52del) adults. (A) ISO treatment results in reduced cardiac output in WT and HET(52del), but not in KO(52del), compared to vehicle‐treated controls. (B) ISO treatment leads to reduced stroke volume in HET(52del), not in WT and KO(52del) compared to vehicle‐treated controls. (C, D) ISO treatment has no significant effect on heart rate (C) or ejection fraction (D) in the three genotypic groups compared to respective vehicle controls. (E, F) ISO treatment leads to reduced end‐diastolic (E) and end‐systolic area (F) in HET(52del), not in WT and KO(52del) compared to vehicle‐treated controls. WT, vehicle n = 10, ISO n = 8; HET(52del), vehicle n = 10, ISO n = 7; KO(52del), vehicle n = 9, ISO n = 8. Data are presented as mean ± SD. Two‐sample t‐test. * p < 0.05, ** p < 0.01, *** p < 0.001.

ISO upregulates cardiac hmox1 paralogs and increases cardiac cell proliferation in HET(52del) adults. (A–D) RT‐qPCR analyses of the expression of hmox1 homologues in ISO‐induced zebrafish hearts compared to vehicle‐treated controls. The results are presented as fold change compared to vehicle‐treated WT hearts. WT, vehicle n = 6, ISO n = 4; HET(52del), vehicle n = 6, ISO n = 4; KO(52del), vehicle n = 5, ISO n = 4. (E–G) Representative immunohistochemical staining of Pcna (in green) and Mef2 (in meganne) in ventricular sections from vehicle‐ or ISO‐treated zebrafish in each genotypic group. Arrow indicates Pcna⁺ non‐cardiomyocytes. Arrowhead indicates Pcna⁺ cardiomyocytes. (H, I) Quantification of Pcna⁺ cardiac cells per mm² ventricular area (H) and Pcna⁺ cardiomyocytes relative to cardiomyocytes (I). Three hearts from each genotypic group were chosen. Two sections from each heart were stained. The number of stain‐positive signals for Pcna and Mef2 from ventricular areas was quantified with ImageJ. Data are presented as mean ± SD. Two‐sample t‐test. *p < 0.05, **p < 0.01.