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Frequency of mononuclear diploid cardiomyocytes underlies natural variation in heart regeneration

Abstract

Adult mammalian cardiomyocyte regeneration after injury is thought to be minimal. Mononuclear diploid cardiomyocytes (MNDCMs), a relatively small subpopulation in the adult heart, may account for the observed degree of regeneration, but this has not been tested. We surveyed 120 inbred mouse strains and found that the frequency of adult mononuclear cardiomyocytes was surprisingly variable (>7-fold). Cardiomyocyte proliferation and heart functional recovery after coronary artery ligation both correlated with pre-injury MNDCM content. Using genome-wide association, we identified Tnni3k as one gene that influences variation in this composition and demonstrated that Tnni3k knockout resulted in elevated MNDCM content and increased cardiomyocyte proliferation after injury. Reciprocally, overexpression of Tnni3k in zebrafish promoted cardiomyocyte polyploidization and compromised heart regeneration. Our results corroborate the relevance of MNDCMs in heart regeneration. Moreover, they imply that intrinsic heart regeneration is not limited nor uniform in all individuals, but rather is a variable trait influenced by multiple genes.

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Figure 1: Variation in mononuclear diploid cardiomyocyte content predicts outcome after injury.
Figure 2: Mononuclear diploid cardiomyocytes proliferate after injury.
Figure 3: Tnni3k influences mononuclear diploid cardiomyocyte population size and cellular regenerative capacity after injury.
Figure 4: Overexpression of Tnni3k induces polyploidization and impairs regeneration in zebrafish hearts.

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Acknowledgements

M.P. was funded by CIRM Training Grant TG2-01161, an award from the University of Southern California's Provost Office and NHLBI NRSA 1F32HL124932. Zebrafish work was supported in part by a Doerr Family Foundation award provided to M.P. and L.B. S.R.K. was supported by K08HL121191. Purchase and husbandry of HMDP strains was supported by grants HL123295 and HL114437 to A.J.L. and grant NS083265 to T.M.

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Authors and Affiliations

Authors

Contributions

M.P., L.B., B.V.H., P.G. and A.S. conducted laboratory analyses. C.D.R. contributed bioinformatics insight and analysis, and M.D. provided FISH probes. S.P. and T.I.F. provided Tnni3k conditional mice. M.D., Y.H., Y.Y. and H.S. gave technical guidance. H.A. and A.J.L. provided insight into the use of the HMDP, and A.J.L. provided most of the HMDP mice. J.G.C., C.-L.L., T.M. and A.J.L. provided conceptual advice. S.R.K. read and interpreted echocardiographic data and provided conceptual advice. The overall project was conceived by, data were interpreted by, and the manuscript was written by M.P. and H.M.S.

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Correspondence to Henry M Sucov.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Parameters and consequences of mononuclear cardiomyocyte frequency.

(a) Correlation of the frequency of mononuclear cardiomyocytes to the frequency of cardiomyocytes with three or four nuclei (multinucleated) across 120 HMDP mouse strains. P = 1.1 x 10-4. See Supplementary Table 1 for a full list of strains and values. (b-e) Left ventricular anterior wall thickness (mm) (b), stroke volume (μL) (c), left ventricular systolic volume (μL) (d), and left ventricular diastolic volume (μL) (e) at baseline, 3 days and 1 month after injury. Strain A (n = 5), SWR (n = 8), C57Bl/6 (n = 4), and SJL (n = 7). *P < 0.05; #P < 0.01; ##P < 0.001. Complete statistics for echocardiography parameters can be found in Supplementary Table 3. (f) Example image of trichrome stain at mid-papillary view 1 month after injury. Right panel is a digital cut-out of the left ventricle with the blue scar selected (yellow outline) by the color thresholding in FIJI imaging software. Scale bar represents 1 mm. (g) Orthogonal view of a confocal z-stack showing a cardiac Troponin C (cTnC, green), phospho-Histone H3 (pHH3, red) cardiomyocyte (white arrowhead). Scale bar represents 20 μm. (h) Quantification of percent of pHH3+ CM nuclei in the infarction border zone of the left ventricle for strain A (n = 3) and C57Bl/6 (n = 3) mice. P value is a two-tailed Student t-test. All error bars represent s.e.m.

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Supplementary Figure 2 Relationships of Tnni3k to mononuclear cardiomyocyte frequency.

(a) Dot plot of 120 HMDP strains distributed across three alleles of Tnni3k. Allele 1 is a C at rs49812611 resulting in a splice mutation and reduced expression (n = 60 strains). Allele 2 is a T at rs49812611 with wild-type expression (n = 56 strains). Allele 3 is T at rs49812611 but has a separate polymorphism that results in reduced expression (see main text) (n = 4 strains). (b) Western blot for Tnni3k across 4 strains of the HMDP. C57Bl/6 has allele 2, A has allele 1, and SJL and SWR both have allele 3. (c-f) Ejection fraction (c), stroke volume (d), left ventricular wall thickness (e), and left ventricular volume in diastole and systole (f) measured by echocardiography on Tnni3k knockout (KO, n = 5) and wild-type (WT, n = 5) mice at baseline, 3 days post coronary artery ligation and 28 days post ligation. (g) Confocal image of WGA stain (green) and cardiomyocyte autofluorescence (red). Scale bar represents 50 μm. (h) Quantification of the average cardiomyocyte area at one month post-infarction in WT (n = 6) and KO (n = 7) animals. (i) Quantification of the scar area 1 month after injury represented as a percent of the total left ventricle in WT (n = 6) and KO (n = 7) animals. (j) RT-PCR for mouse Tnni3k, zebrafish tnni3k and zebrafish eef1g in mouse heart and control and Tnni3k transgenic fish. (k) Western blot for Tnni3k in C57Bl/6 mice across developmental ages. Error bars are s.e.m.

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Patterson, M., Barske, L., Van Handel, B. et al. Frequency of mononuclear diploid cardiomyocytes underlies natural variation in heart regeneration. Nat Genet 49, 1346–1353 (2017). https://doi.org/10.1038/ng.3929

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